Effective weight control via an implanted self-powered vagus nerve stimulation device


Abstract

In vivo vagus nerve stimulation holds great promise in regulating food intake for obesity treatment. Here we present an implanted vagus nerve stimulation system that is battery-free and spontaneously responsive to stomach movement. The vagus nerve stimulation system comprises a flexible and biocompatible nanogenerator that is attached on the surface of stomach. It generates biphasic electric pulses in responsive to the peristalsis of stomach. The electric signals generated by this device can stimulate the vagal afferent fibers to reduce food intake and achieve weight control. This strategy is successfully demonstrated on rat models. Within 100 days, the average body weight is controlled at 350 g, 38% less than the control groups. This work correlates nerve stimulation with targeted organ functionality through a smart, self-responsive system, and demonstrated highly effective weight control. This work also provides a concept in therapeutic technology using artificial nerve signal generated from coordinated body activities.

Introduction

Obesity resulted from ingesting calories in excess of normal biological requirement is a major risk for a large number of chronic diseases, including cardiovascular disease1,2, diabetes mellitus1,3, chronic kidney disease1, gallbladder diseases4, certain cancers3,5, musculoskeletal disorders6, and even genetic variation7. Treatment of obesity imposes an enormous economic burden on the global healthcare system8,9,10. According to a recent global survey, over 710 million people worldwide, including 107.7 million children and 603.7 million adults, are plagued by obesity problems, and about 4 million people died of overweight- or obesity-related diseases in 201510. Common approaches for treating obesity include non-surgical and surgical treatments. Daily physical exercise and taking weight-loss drugs are common non-surgical weight reduction regimens, but there is a high potential of weight rebound or side effects from drugs11. Current bariatric surgical procedures such as gastric bypass, biliopancreatic diversion, and sleeve gastrectomy have demonstrated a significant impact on weight loss, but these procedures are invasive with the potential of serious complications12,13,14. The rising healthcare standards demand new obesity treatment strategies that are effective, easy to operate, and have less side effects.

Neuromodulation, as a non-destructive and reversible therapeutic strategy, can manipulate body functions by stimulating or influencing neurophysiological signals through the neural networks to achieve therapeutic purpose15,16. It has been known for a century that the vagus nerve (tenth cranial), a mixed parasympathetic nerve containing both afferent and efferent nerve fibers, acts as a signal bridge to transport information between the brain (the center of the nervous system) and the body (head, neck, thorax, and abdomen)17,18,19. Recent breakthroughs in neuromodulation for body weight control have provided potential opportunities for therapeutic interventions and brought renewed promises and vitality to the development of new anti-obesity strategies. A number of studies have demonstrated that pulsed electrical stimulations on vagus nerve could induce multiple physiologic functions related to food intake, energy metabolism, and glycemic control, which can result in appreciable weight loss20,21,22. An implantable vagus nerve stimulation (VNS) device for weight control was recently approved by Food and Drug Administration and commercialized20,23. Major concerns of current electrical stimulation are potential compensation mechanisms that blunt physiological responses14 and vicinity tissue damage that induces adverse effects17,24,25. In addition, the electrical system is bulky and complicated in operation. All the electrical stimulations need to be programed externally and the device needs to be charged periodically13,26. How to achieve real-time-responsive and self-sustainable stimulation remains a major challenge for this promising weight control strategy.

In this work, we present a correlated VNS system that is battery free and automatically generates electrical stimulations in correlation to stomach movement. A flexible nanogenerator device is developed to be attached to the stomach surface and produce biphasic electrical pulses in response to the peristalsis of stomach. The electric signals can stimulate the vagal afferent fibers to reduce food intake and eventually achieve weight control. We successfully demonstrated this strategy on rats and achieved 38% weight loss in as short as 15 days without further rebound, exceeding all current electrical stimulation approaches. This work provided an effective weight control strategy that is self-responsive, battery free, and directly correlating food intake to stomach movements.

Results

Development and working principle of the VNS device

The correlated VNS system for weight control is designed following the principle depicted in Fig. 1a. The stomach motion is used as the sole source to generate pulsed voltage signals, which in response will stimulate the vagus nerves to reduce food intake. This self-responsive function is enabled by a triboelectric nanogenerator (TENG)27,28,29,30,31 attached on the surface of stomach, which generates biphasic electric pulses when the stomach is in peristalsis. Here, a bilateral VNS is implemented by wrapping the two gold (Au) leads around the anterior vagus nerves (AVNs) and posterior vagus nerves (PVNs) at the proximity of the gastroesophageal junction (Fig. 1b). The AVNs and PVNs were ~6 mm apart and could be clearly observed via multiple staining images (Fig. 1c, Supplementary Figure 1). Connecting at this position could provide a focused stimulation to the small unmyelinated C fibers and avoid stimulating fibers that join the trunk from the heart and lungs32.

Fig. 1
Fig. 1

The correlated vagus nerve stimulation (VNS) system and its biocompatibility. a Operation principle of the correlated VNS system schematically showing the pathway for biphasic electric signal generation and VNS. b An implanted VNS device with Au leads being connected to anterior and posterior vagal trunks. c Hematoxylin–eosin (H&E) staining of the implanted tissues (transverse section). Areas within the blue and red boxes are enlarged view of the anterior (red) and posterior (blue) vagus nerves (scale bar = 100 μm). d A packaged VNS device. e, f Fluorescence images of stained 3T3 cells that were cultured on a regular cell culture dish (e) and on the surface of a packaged device (f). g Comparison of normalized cell viability for 4 days showing excellent biocompatibility of the packaged device (n = 3 for each group). All data in g are presented as mean ± s.d.

To ensure the mechanical robustness and flexibility of implanted devices and to avoid potential erosion in the physiological environment, the entire VNS device was packaged by a multilayer film composed of polyimide, polydimethylsiloxane (PDMS), and ecoflex. Au leads were connected to the tips of Cu wire electrodes and partially exposed for electrical signal transmission (Fig. 1d, fabrication details are included in Supplementary Figure 2a and b, which was described in Supplementary Note 1). The TENG was able to generate reasonably high voltage and current output under normal contact-separation motions, with an optimal output power of ~40 µW at an external load of 20MΩ (Supplementary Figure 2c, d and e). Based on the impedance of the vagus nerve, the stimulation voltage was found to be around 200 mV (Supplementary Figure 2d). Similar outputs were obtained from various displacement motions, suggesting that the TENG was able to respond to complex stomach motions (Supplementary Figure 3). To confirm the biocompatibility of the packaged VNS device, mouse fibroblast 3T3 cells were cultured on the encapsulated device surface and in a reference cultural dish for 4 days to examine and compare the cell attachment, proliferation, and morphology. Cells in both media exhibited similar density and equivalent morphology. No dead or distorted cells were observed from the encapsulation material surface (Supplementary Figure 4). The fluorescent staining results showed that the 3T3 cells can spread and form intact cytoarchitecture in both groups (Fig. 1e, f). In addition, 3-{4,5-dimethylthiazol-2-thiazolyl}−2,5-diphenyl-2H-tetrazolium bromide (MTT) assay revealed that the relative viability of 3T3 cells on encapsulation material was more than 98% within 4 days, comparable to the cells cultured in the culture dish (Fig. 1g). These results confirmed that the encapsulated device is non-cytotoxic and biocompatible.

When the stomach is under peristalsis stomach33,34, the corresponding motion cycle of the triboelectric layers in the VNS device is depicted in Fig. 2a (i)–(iv). As the stomach is distended, the two triboelectric layers are pushed into contact, where oppositely charged surfaces are created based on their different electron affinity (stage i). The following contraction of the stomach pulls the bottom electrode layer (BEL) layer away from the polymer layer, and thus drives electrons flowing from the top electrode layer (TEL) electrode to the BEL through the two connections with the vagus nerve (stage ii). When the stomach is fully contracted, the triboelectric layers are fully separated, where maximum charge transfer is reached and the net current through the nerve drops back to zero (stage iii). The BEL layer is then brought back toward the polymer layer in the following stomach distention (phase iv), resulting in an opposite current flow through the vagus nerve until the stomach reaches the original distended stage (i) again. The recorded voltage output within one cycle at the frequency of 0.05 Hz is shown in Fig. 2b and corresponding stages are marked along the curve. This contributes to the cyclic alternating electrical signals as the stomach continues peristalsis.

Fig. 2
Fig. 2

Working principle and voltage signal of the vagus nerve stimulation device. a Schematics of the working principle of VNS device under different stomach motion stages. b A typical single-cycle voltage biphasic signal corresponding to the four stages of stomach movement at a frequency of 0.05 Hz. c Voltage signal measured in PBS solution under different agitation frequency when the VNS device was connected to an external load with the same impedance of the implanted area. d Voltage signal measured when the VNS device was implanted and connected to vagus nerves. e Long-term stability test of the VNS device, where the device was removed from the rats and the voltage was measured on an external load of 0.3MΩ. f Electrophysiological signals recorded from rats without implantation and with an active implanted VNS device on the same day, and 7 and 15 days post implantation. g Enlarged view of one group of electrophysiological signal highlighted in the dotted box in f

To investigate how the implanted VNS device functioned in response to stomach movements, voltage signals were first measured between the two Au leads. The stomach was arbitrarily deformed by cyclically pressurizing it via a gavage needle at a series of frequencies of 0.05, 0.1, 0.5, 1.0, 1.5, and 2.0 Hz (Fig. 2c). Correspondingly, the voltage signals were also measured when the two Au leads were connected to the vagus nerve in a rat’s body (Fig. 2d and Supplementary Movie 1). Both voltage signals exhibited similar amplitudes ranging from 0.05 to 0.12 V. It should be noted that the recorded voltage was lower than the actual operational voltage due to the finite internal impedance of the measurement system (1MΩ). Higher voltage outputs were recorded from higher frequency. While the stomach deformations (i.e., pressure change) remained constant, higher voltage signal could be attributed to a higher displacement rate, suggesting faster stomach motion is favorable for more intense stimulation. The implanted devices were removed from the rats 1 day, 7 days, 15 days, 4 weeks, 8 weeks, and 12 weeks after implantation, and their voltage output was measured accordingly. All the devices showed a good structural integrity without any observable defects (Supplementary Figure 6). The nearly unchanged voltage amplitude confirmed good stability and durability of the VNS device in the biological environment (Fig. 2e).

Electrophysiological signals were measured from the cervical vagal trunk on the same day, and 7 and 15 days after implantation (Supplementary Figure 7). As shown in Fig. 2f, when there was no external stimulation, a regular electrophysiological signal from the vagus nerve can be detected. The voltage amplitude was ~0.5 mV with 11 electric pulses per signal group (Fig. 2g). When the VNS device was activated, the amplitude increased to ~0.8 mV, and the number of electric pulse per group increased to 18–19. This measurement clearly showed that the VNS device can stimulate vagus nerves effectively. Similar electrophysiological signals could be detected at different time points post implantation, which evidenced the vagus nerves were effectively stimulated by the VNS device during the implantation period. Electrophysiological signals were further measured in response to a range of stimulation voltage from 50 to 740 mV. The stimulated state of the vagus nerve was detected as the voltage from VNS device was above 100 mV. The signal intensity from the vagus nerve increased monotonically following the stimulation voltage (Supplementary Figure 8, Supplementary Note 2).

Biocompatibility and biosafety of implanted VNS device

Rats with the VNS device implanted on stomach and the Au leads connected to the vagus nerve were defined as the VNS-active group. Small animal computed tomography (CT) was used to produce three-dimensional (3D) x-ray images of representative rat models as a function of implantation time to investigate the implantation stability when the rat was under normal daily activity (Fig. 3a, Supplementary Figure 9, and Movie 2). The high contrast spot (Au is a good contrast agent for CT) inside the rat was the implanted VNS device. No position shifting was observed during the entire 12 weeks of implantation period. This high stability could be attributed to the good biocompatibility of the packaged VNS device, which was observed being completely imbedded possibly by omentum and tightly fixed to the stomach surface post study (Supplementary Figure 10). The right panel of Fig. 3a shows an enlarged CT image of the implantation area, where the Au leads and exposed tip (with much brighter contrast) can be clearly identified, wrapping around at the vagus nerve region. In contrast to the VNS group, the sham group had the same VNS device implanted on stomach but without Au leads connecting the Cu wire electrodes to the vagus nerve (the completely packaged Cu wires were still placed at the same vicinity of the vagus nerve, Fig. 3b). 3D CT images showed the same stable VNS implantation in the sham group. The enlarged image revealed the insulated leads exhibiting a uniformly low contrast. As a comparison, rats in the laparotomy (Lap) group, which rats were subjected to surgery but without the VNS device implantation, were also imaged and showed only the skeleton of the rats (Fig. 3c). The Sham, Lap, and Intact (rats without any operation) groups are defined as the control groups.

Fig. 3
Fig. 3

Computed tomography (CT) 3D projection images and hematology data. ac Serial CT images over time of the VNS group, Sham group, and Lap group, respectively. Schematics on the left show the setup of each group. A series of CT images (coronal and sagittal) show a representative rat for each group at different time points. The enlarged views of the implantation site are shown at the end. di Hematology results of all four groups over time (n = 3 for each group). d Blood glucose (GLU) levels. e Infection-related lymphocytes (LYM) levels. f Hematopoietic function-related red blood cell (RBC) levels. g Hepatological function-related alanine aminotransferase (ALT) levels. h Renal function-related creatinine (CRE) levels. i Electrolyte metabolism-related calcium (Ca) levels. j H&E stains of vital organs (heart, lung, liver, spleen, kidney, bowel, stomach, and esophagus) at different time points (1 day, 7 days and 15 days) post implantation. All data in di are presented as mean ± s.d.

Whole blood and chemical analysis were performed on the four groups of rats for biosafety assessment during the implantation period. Compared to the hematology data in intact group, the blood glucose (GLU) concentration (Fig. 3d) in VNS and Sham group decreased at week 2 due to reduced food intake after surgery, and eventually recovered to the normal levels. The indicators of infection such as lymphocytes (Fig. 3e), hematopoietic function such as red blood cell (RBC) (Fig. 3f) and hemoglobin (Supplementary Figure 11a), hepatological function such as alanine aminotransferase (ALT) (Fig. 3g) and albumin (Supplementary Figure 11b), renal function such as creatinine (Fig. 3h) and blood urea nitrogen (Supplementary Figure 11c), and electrolyte metabolism such as calcium (Ca) (Fig. 3i) and phosphorus (Supplementary Figure 11d) all remained steady during the entire implantation period. In general, all the blood testing results were within the normal range shortly after the device implantation and did not show any abnormality35,36, suggesting that the VNS device is highly hemocompatible. The comprehensive blood analyses, together with the imaging results, confirmed that implanting the VNS device on stomach surface did not cause any measurable adverse effect to the rats. All rats with the VNS implantation exhibited normal daily behaviors, which were not different from the intact groups (Supplementary Movie 3).

Pathological tests were conducted on most vital organs, including heart, lung, liver, spleen, kidney, bowel, stomach, and esophagus. Hematoxylin and eosin (H&E) staining were collected from these organs at different time points (1 day, 7 days, and 15 days) post implantation. All the organs showed no deformation and abnormal lymphatic cell invasion (Fig. 3j), which further confirmed that all the rats were in a good health condition, and the VNS device had no systemic side effects. Histological analysis of the vagus nerves 15 days after implantation showed no signs of nerve cell shape change or invasion of inflammation cells (Supplementary Figure 12). This revealed the vagus nerves were not damaged by connecting the VNS device.

Weight control by implanted VNS device

The weight control performance was first examined in the four groups of rats (VNS, Sham, Lap, and Intact) that were fed and grown under the same conditions. The average initial weight of rats was 250 g, and their body weight and food intake were monitored on a daily basis. After 100 days, the body size of the VNS group was significantly smaller than all three control groups (Fig. 4a). The recorded body weight and corresponding daily food intake over time are shown in Fig.  4b, c, respectively. Since the implantation surgery was conducted on the seventh day of this study, all four groups exhibited the same body growth trend and the same amount of food consumption over the first week, indicating that all rats were under the identical growth conditions and their results were comparable. Immediately after the surgery, the VNS and Sham groups (both had VNS implanted) exhibited an obvious weight loss. Accordingly, their food intake was also largely reduced likely due to the VNS device attachment. The Lap group, which had the same surgical procedure/opening, did not show any abnormity in weight change or food intake when compared to the intact group, suggesting that surgery itself had minimal impact on weight control. As the rat’s body adapted to the VNS device implantation, the amount of food intake of the Sham group quickly recovered to the same level as the other two control groups after ~15 days of implantation. As a result, the average body weight of the Sham group bounced back after the initial 2–3 days, and increased following the same trend of the other two control groups (Lap and Intact). After 60 days, all three control groups exhibited a very close body weight of 535 ± 18 g (Sham), 538 ± 32 g (Lap), and 538 ± 32 g (Intact) (n = 6 for each control group), confirming that neither surgery nor simple stomach attachment had any effect on weight control. On the contrary, although the food intake of the VNS group recovered as well after the initial reduction and reached a steady level after ~15 days, the daily consumption of food was only ~2/3 of those consumed by the other control groups. Therefore, the average body weight of the VNS group exhibited a much slower growth rate. It reached a steady value of 350 ± 23 g (n = 6), about 63% of the other three control groups. Box plots were implemented to provide a statistic analysis of the final body weight (on the day of sacrifice, Fig. 4d) and food intake (on the last day before euthanasia, Fig. 4e). The differences between the VNS group and three control groups were statistically significant (P < 0.001) for both body weight and food intake, while the differences between all three control groups were not (P > 0.2). Such comparison clearly revealed that spontaneous nerve stimulation by the implanted VNS device had obvious impact on weight control.

Fig. 4
Fig. 4

Weight control during the weight-gaining growth stage of rats. a Representative images of body size of the VNS group (n = 6) and the control groups (Sham, Lap, and Intact group, n = 6 for each group). b Average body weight of rats in different groups over time (implantation was performed after a week of observation period under normal conditions). c Rat’s food intake over time in different groups. d Final body weight of rats in different groups. e Final daily food intake in steady state of rats in different groups. f Representative images of white adipose tissue (epididymal fat pad and perirenal fat pad) of the VNS group and the control group. g Adipose tissue weight of rats in different groups. h Epididymal fat pad/body weight ratio (EBR) of rats in different groups. i Percentage of weight loss over time (black dots) in comparison to the reported results by chronic electric stimulation with a rectangular waveform (voltage, frequency, and pulse duration are also shown). All data in b, c, and g are presented as mean ± s.d. In d, e, and h (box plots), dots are the mean, center lines are the median, box limits are the lower quartile (Q1) and upper quartile (Q3), and whiskers are the most extreme data points that are no more than 1.5× (Q3–Q1) from the box limits. Statistical analysis was performed by two-tailed unpaired Student’s t tests. n.s., non-significant (P > 0.05); *P < 0.05, **P < 0.01, and ***P < 0.001

All the rats were sacrificed after the 100-day weight control study for anatomical examinations. Similarly, the anatomical adipose tissues (epididymal fat pad and perirenal fat pad, representative of the body fat level)37 in the VNS group were significantly smaller than the control groups (Fig. 4f). The average weight of the epididymal fat pad and perirenal fat pad were only 4.01 and 1.36 g, 58 and 67% smaller than the control groups, respectively (Fig. 4g). The epididymal fat pad/body weight ratio (EBR) was calculated by dividing the fat pad weight by the total body weight. Average EBR was maintained at 1.14% in the VNS group, significantly lower than the control groups which all exhibited EBR of ~1.7% (Fig. 4h). By comparing the weight difference between the VNS and control groups, our correlated VNS system rapidly achieved 35% weight loss within 18 days and maintained a weight-loss ratio as high as 38% for remaining study period (75 days), which largely exceeded other reported electrical stimulation approaches based on similar rat models38,39,40,41,42,43 (Fig. 4i).

To further exploit the capability of the VNS system for weight loss, the same implantation surgery and analyses were conducted on grown adult rats that have been fed for 7 weeks and reached a steady average body weight of ~500 g. Little body weight gain was observed for the intact group during the 70-day study period, while the VNS group exhibited an obvious body size reduction (Fig. 5a). Similar as the previous study, the Lap group exhibited no difference when compared to the intact group, while the Sham group quickly recovered to the same levels after the initial drop in both body weight and food intake (Fig. 5b, c, respectively). Food intake of the VNS group exhibited a much slower recovery rate and eventually remained at the steady value that was ~2/3 of the control groups. Accordingly, the average body weight of the VNS group exhibited a steep drop over the first 25 days after implantation, followed by a small recovery and stabilized at ~400 g. The final body weight (Fig. 5d) and food intake (Fig. 5e) showed significant differences between the VNS group and control groups, and no significant difference were found among the control groups. The final average body weight was controlled at 410 ± 17 g (n = 4), significantly smaller than the control groups (Sham: 575 ± 22 g; Lap: 569 ± 39 g; and Intact: 574 ± 48 g, n = 4 for each control group). Significant differences were also found in the adipose tissue sizes (Fig. 5f) and weight (Fig. 5g). The final EBR was controlled at 1.26% in the VNS group, while the three control groups maintained a much high value from 1.69% to 1.77% (Fig. 5h). The calculated weight-loss percentage peaked at 38% at day 29 and gradually reached a stable 28% (Fig. 5i).

Fig. 5
Fig. 5

Weight loss of fully grown adult rats after implantation of the VNS device. a Representative images of rats in the VNS group (n = 4) and the control groups (n = 4 for each control group). b Average body weight of rats in different groups over time (implantation was performed after 7 days of observation under normal conditions). c Rat’s food intake in different groups over time. d Final body weight of rats in different groups. e Final daily food intake in steady state of rats in different groups. f Representative images of white adipose tissue (epididymal fat pad and perirenal fat pad) of the VNS group and the control group (scale bar = 2 cm). g Adipose tissue weight of rats in different groups. h Epididymal fat pad/body weight ratio (EBR) of rats in different groups. i Percentage of weight loss over time after implantation of the VNS device. All data in b, c, and g are presented as mean ± s.d. In d, e, and h (box plots), dots are the mean, center lines are the median, box limits are the lower quartile (Q1) and upper quartile (Q3), and whiskers are the most extreme data points that are no more than 1.5× (Q3–Q1) from the box limits. Statistical analysis was performed by two-tailed unpaired Student’s t tests. n.s., non-significant (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001

Discussion

In this work, we present a correlated VNS system as an effective therapeutic strategy for obesity, which provided correlated nerve stimulation signal in response to stomach peristalsis. The VNS device was built based on a flexible TENG that was attached to the stomach wall of rats and could generate biphasic electric pulses when the stomach wall moved. The TENG electrodes were directly connected to the vagus nerve, where the stomach motion-generated voltage signals stimulated the vagus nerve to reduce food intake. We envision that this correlated stimulation could provide less amount but more targeted stimulation so that the nerves might be more responsive to the stimulation, and thus more effective to control food intake. The VNS device exhibited excellent biocompatibility without any signs of side effects from the whole blood and chemical analysis. CT and hematology indicators revealed the implantation was very stable and remained at the same position during the entire testing period. The weight control performance was examined and compared among the VNS, Sham, Lap, and Intact groups of rats that were fed and grown under the same conditions. From the weight-gain test, the average body weight and EBR can be controlled at 350 g and 1.14% in the VNS group, compared to 559–564 g and 1.65–1.69% in the other three control groups with insignificant differences. The VNS system rapidly achieved 35% weight loss within 18 days, which was maintained 38% during the remaining 75-day testing period. From the weight-loss test, the average body weight and EBR was controlled at 410 g and 1.26% in the VNS group, compared to 570–575 g and 1.69–1.77% in the other three control groups. Rats in the VNS groups were also able to recover their normal weight pattern immediately after the implanted VNS devices were removed (Supplementary Figure 13, Supplementary Note 3). Our correlated VNS system demonstrates outstanding weight control results, which largely outperformed other reported chronic microchip VNS systems based on similar rat models. In addition, this correlated VNS is battery free and less invasive compared to the bariatric surgical strategies (e.g., gastric bypass, biliopancreatic diversion or sleeve gastrectomy) for weight control. For future clinical trials, a switch may be needed by the VNS device to control the treatment. This could be achieved by integrating a shutter switch to the electrical wires from the VNS device, as it has been proved that a disconnected device has no impact to food intake and weight change. More broadly, this development demonstrated a successful example of a self-responsive and real-time peripheral neuromodulation mechanism that may be more effective for achieving therapeutic purpose.

Methods

Device fabrication and encapsulation

Polyimide film (50 μm) was used as the core package layer that was proven to be biocompatible and corrosion resistant for bio-implanted devices44,45. Casting and curing a pre-polymer to PDMS (15:1 PDMS; Dow Corning, USA) covers the entire device as the shell package (1 mm) to enhance the leakproof and ensure good structural flexibility and stability. To further increase the flexibility to closely fit the non-planar surfaces of stomach and maintain sensitivity in response to stomach motions, a layer of ecoflex (200 μm) was coated onto the surface as another shell structure of the device46,47,48. The PTFE surface was treated by reactive ionic etching to introduce nanostructured features to enhance the electrical output49 (Supplementary Figure 2a). The overall VNS device dimensions are approximately 16 × 12 × 2.5 (L × W × T) mm3 and the weight was measured to be only ~0.8 g.

Electrical characterization of VNS devices

The electrical performance of all implanted VNS devices were measure by a portable oscilloscope (Agilent, DSO1012A, internal impedance is 1MΩ). The voltage signals shown in Fig. 2b, d were measured directly from an implanted VNS device where the two Au leads were connected to the vagus nerve. The stomach motions were induced by injecting water into the stomach using a gavage needle through the mouth and the injection volume difference was 2 mL. The voltage signal shown in Fig. 2c was measured by pressing the VNS devices in phosphate-buffered saline (PBS) solution under different frequency when the VNS device was connected to an external load with the same impedance of the implanted area. Voltage signals in Fig. 2e and Supplementary Figure 3 were measured by the pressing the VNS devices at frequency of 4 Hz when connected to an external load of 0.3MΩ, the same as the impedance of the vagus nerve. The voltage and current output of TENGs as a function of load (10Ω to 200MΩ) was measured by a Stanford Research Systems model SR 560 low-noise preamplifier (internal impedance is 100MΩ). The impedance of implanted VNS device was characterized from 0.01 to 10,000 Hz using an Autolab PGSTAT302N station (Supplementary Figure 5).

Electrophysiological properties of vagus nerve

A Sprague–Dawley rat was anesthetized and its right cervical vagal trunk was carefully exposed (Supplementary Figure 7a). A pair of bipolar platinum hook electrodes was then placed under the right vagal nerve immediately. The exposed nerve tissue was covered with warm (37 °C) paraffin oil. The electrical signals were recorded and analyzed by a Cambridge Electronic Design (CED) 1401 interface (Cambridge, UK) with Spike 2 software to monitor the change the electrical signal of the VNS device (Supplementary Figure 7b and c). The VNS device was implanted in rat’s body with Au leads being connected to anterior and PVNs. The implanted VNS devices were activated by gently pressing the abdomen of the rats at a frequency of 1 Hz. In addition, VNS devices with different sizes were fabricated to study the response of vagus nerve in corresponding to the amplitude of stimulation voltage (Supplementary Figure 8).

Animals and diets

All animal experiments were conducted under a protocol approved by the University of Wisconsin Institutional Animal Care and Use Committee. Seven- and eight-week-old male Sprague–Dawley rats were acquired from Envigo (New Jersey, USA). All rats were housed in separated cages at a temperature-controlled room (22 °C) with a 12-h light/dark cycle with free access to water and Purina PMI-certified rodent chow 5002 (LabDiet, MO, USA).

Food intake and body weight

Body weight and food intake was recorded at 8:00 p.m. every other day. The daily food intake was determined from the difference in food quality between each measurement and divided by two. All rats were deprived of food for 12 h before surgical implantation and blood test. Percentage of weight loss (Pweight loss) over time in Figs. 4i and 5i was calculated according to the formula: Pweight loss = (WIntactWVNS)/WIntact × 100%, where WIntact and WVNS represent the average weight of the Intact group and the VNS group, respectively.

Histological staining of vagus nerve and vital organs

Tissue slices of the bottom of esophagus and its surrounding tissue were prepared. H&E staining, immunohistochemical (Supplementary Figure 1a), and immunofluorescent (Supplementary Figure 1b) staining using anti-S-100 rabbit anti-rat poly-antibody50 showed clearly the anterior vagal trunk and posterior vagal trunk distributed on both sides of the esophagus. Vital organs including heart, lung, liver, spleen, kidney, bowel, stomach, and esophagus were retrieved from rats for H&E staining after euthanasia at different time points (1 day, 7 days, and 15 days) post implantation. In addition, vagus nerves were re-evaluated after the device being implanted for 15 days, and the additional H&E staining results were shown in Supplementary Figure 12.

Cell morphology and immunofluorescence staining

After 3T3 cells were cultured on encapsulation or cell plates in 24-well plates, cell morphology was observed directly using an inverted optical microscope (Nikon Eclipse Ti-U, Japan). The cytoskeleton and nucleus were stained with Texas Red-X phalloidin (591/608 nm) and blue fluorescent Hoechst (352/461 nm) (Thermo Fisher Scientific), respectively. The samples were fixed with 2–4% formaldehyde for 15 min and then rinsed three times with pre-warmed PBS. The samples were incubated with Texas Red-X phalloidin (100 nM) and Hoechst (50 nM) for 30 min at 37 °C. After staining, cells were rinsed with pre-warmed buffer for three times and imaged using a Nikon A1RS confocal microscope.

MTT assay

After 3T3 cells were cultured on the packaging film on 24-well plates, MTT assay (Thermo Fisher scientific) was performed to examine cell proliferation. After incubation at 37 °C in a humidified atmosphere with 5% CO2 for up to 4 days, 100 μL of MTT solution was added to each well. After 4-h incubation, the medium was removed and dimethyl sulfoxide (500 μL/well) was added to dissolve the precipitated fomazan. The optical density (n = 3) of the solution was evaluated using a microplate spectrophotometer at a wavelength of 490 nm.

Device implantation

In brief, anesthesia was induced by inhalation of 2–5% isoflurane and maintained with 2% isoflurane. Following anesthesia, rats were fixed in supine position. The abdomen of rats was scrubbed with iodine scrub, and then alcohol prior to surgery. An incision of 2–5 cm was made on the left upper abdomen of rats. The device was placed beside the stomach. The anterior and posterior vagus nerves were identified and separated from the gastroesophageal junction. For the VNS group, Au wires were placed in contact with the nerves and secured with sterile surgical tape. For the sham group, the device was implanted with insulated electrodes connecting to the vagus nerves. Afterwards, the muscle and skin were sutured layer by layer and the stitches were removed 2 weeks later. The implantation procedure is shown step by step in Supplementary Figure 14. The entire surgery lasted approximately 15 min.

CT scan

CT whole-body scan was performed to evaluate the position and integrity of the VNS device post implantation, which can generate 3D images and reconstruct a high-definition 2D projection image of rats with a resolution of up to 100 µm. In brief, rats were placed in the prone position after anesthesia and scanned by an Inveon micro positron emission tomography/CT scanner (Siemens Medical Solutions, USA) at 1 day, 2 weeks, 6 weeks, and 12 weeks post implantation. CT images were reconstructed and presented as 3D projection or slices.

Hematology data

Whole-blood and chemical analysis were performed for safety assessment pre-implantation and 2, 6, and 12 weeks post implantation. Blood was drawn from the tail vein of the rats and various tests were performed using an Abaxis VetScan HM5 Hematology Analyzer (Abaxis, USA) and VS2 Blood Chemistry Analyzer (Abaxis, USA). No centrifugation or other treatment were needed.

Anatomic examination and adipose tissue collection

After rats were euthanized at the end of the study, an incision was made on the abdomen. The stomach along with the VNS device were taken out for further analysis. In addition, epididymal fat pad and perirenal fat pad were removed and weighed for further analysis (Supplementary Figure 15).

Statistical analysis

For the final body weight, food intake, and adipose tissue weight, statistical analysis was performed by two-tailed unpaired Student’s t tests. n.s., non-significant (P > 0.05); *P < 0.05, **P < 0.01, ***P < 0.001. In box plots, dot is the mean, center line is the median, box limits are the lower quartile (Q1) and upper quartile (Q3), and whiskers are the most extreme data points that are no more than 1.5× (Q3–Q1) from the box limits.

Data availability

The authors declare that all data supporting the findings of this study are available within the Article and its Supplementary Information. The raw data generated in this study are available from the corresponding author upon reasonable request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

This publication was supported by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Numbers R01EB021336 and P30CA014520. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

Author notes

  1. These authors contributed equally: Guang Yao, Lei Kang.

Affiliations

  1. Department of Materials Science and Engineering, University of Wisconsin-Madison, Madison, WI, 53706, USA

    • Guang Yao
    • , Jun Li
    • , Yin Long
    •  & Xudong Wang
  2. State Key Laboratory of Electronic Thin films and Integrated Devices, University of Electronic Science and Technology of China, Chengdu, Sichuan, 610054, People’s Republic of China

    • Guang Yao
    • , Yin Long
    •  & Yuan Lin
  3. Department of Radiology, University of Wisconsin-Madison, Madison, WI, 53705, USA

    • Lei Kang
    • , Hao Wei
    • , Carolina A. Ferreira
    •  & Weibo Cai
  4. Department of Nuclear Medicine, Peking University First Hospital, Beijing, 100034, People’s Republic of China

    • Lei Kang
  5. University of Wisconsin Carbone Cancer Center, Madison, WI, 53705, USA

    • Justin J. Jeffery

Source:Nature

Honey bee Royalactin unlocks conserved pluripotency pathway in mammals


Abstract

Royal jelly is the queen-maker for the honey bee Apis mellifera, and has cross-species effects on longevity, fertility, and regeneration in mammals. Despite this knowledge, how royal jelly or its components exert their myriad effects has remained poorly understood. Using mouse embryonic stem cells as a platform, here we report that through its major protein component Royalactin, royal jelly can maintain pluripotency by activating a ground-state pluripotency-like gene network. We further identify Regina, a mammalian structural analog of Royalactin that also induces a naive-like state in mouse embryonic stem cells. This reveals an important innate program for stem cell self-renewal with broad implications in understanding the molecular regulation of stem cell fate across species.

Introduction

The regulation of self-renewal and differentiation potential in mouse embryonic stem cells (mESCs) occurs through complex transcriptional networks orchestrated by conserved transcription factors1,2. Although differences exist in the specific signaling pathways that control self-renewal and lineage development, culture conditions that allow for derivation and maintenance of stem cells have been identified3,4,5,6,7,8. In particular, use of two small molecule inhibitors targeting MAPK/ERK Kinase (Mek) and glycogen synthase kinase-3 (GSK3) in addition to Leukemia inhibitory factor (LIF) in serum-free media permitted derivation of germline-competent ESCs that resemble the mature mouse inner cell mass (ICM)9. However, recent findings suggest that prolonged Mek1/2 suppression may have detrimental effects on the epigenetic and genetic integrity of mESCs, effectively limiting their developmental potential10,11. As such, additional methods of maintaining mESCs in an ICM state are required.

Though best-known as an epigenetic driver of queen development in A. mellifera12, the functional component of royal jelly, Major Royal Jelly Protein 1 (MRJP1, also known as Royalactin), has been shown to modulate biological function in a broad range of species12,13,14,15. Indeed, conservation of increased growth stimulation and cellular proliferation phenotypes in response to MRJP1 has been observed in murine hepatocytes16,17,18. While this indicates a functionally important role for this royal jelly protein in regulating cell state and fate, the full scope of its effects has not yet been well characterized19.

In this study, we identify Royalactin as a potent activator of a pluripotency gene network through modulation of chromatin accessibility, that maintains mESC self-renewal in the absence of LIF. Royalactin cultured cells also occupy a more naive ground state capable of generating chimeric animals with germline transmission. Finally, we identify a mammalian structural analog of Royalactin possessing similar functional capacity, uncovering a molecular conservation that supports distinct evolutionary processes.

Results

Royalactin maintains mESC self-renewal and pluripotency

As mESCs provide a powerful model with which to study cellular regulation through pluripotency and differentiation programs, we first employed this model to dissect Royalactin’s mechanisms of action on mammalian cells. Upon LIF withdrawal (serum/–LIF), mESCs previously cultured in serum/+LIF media readily demonstrated a differentiated morphology as expected (Fig. 1a). Surprisingly, addition of Royalactin in the absence of LIF (serum/–LIF + Royalactin) for multiple passages resulted in dose-dependent formation of undifferentiated colonies that demonstrated similar morphology to those grown in the presence of LIF (Fig. 1a, Supplementary Figure 1a). Gene expression profiles of the cells grown in serum/–LIF + Royalactin further demonstrated a high degree of similarity relative to those cultured in the presence of LIF (Fig. 1b, Supplementary Figure 1b).

Fig. 1
Fig. 1

Royalactin maintains stemness in murine embryonic stem cells. a Representative images of J1 and R1 mESCs cultured in serum/+LIF, serum/−LIF, or serum/−LIF + Royalactin for 10 and 20 passages. After LIF withdrawal, mESCs rapidly differentiated, whereas cells cultured with Royalactin supported self-renewal with negligible differentiation. Scale bar, 200 μm. b Quantitative expression of pluripotency and differentiation-associated genes from a. Data are means ± SD (n = 2). c Mice bearing mESC-derived teratomas from J1 mESCs cultured three passages in +LIF and −LIF + Royalactin demonstrated retained pluripotency, and on high magnification (400×) produced differentiated ectodermal, mesodermal, and endodermal tissues. Scale bar, 80 μm. d RNA-seq log2-fold change values in transcript level of all genes in serum/+LIF or serum/–LIF + Royalactin J1 mESCs (passage 10) relative to serum/–LIF. e GO term analysis of differentially expressed genes from d. f ATAC-seq activity in J1 mESCs at passage 10. Each column is a sample, each row is an element. Samples and elements are organized by unsupervised k-means clustering. g GO term analysis of differentially accessible regions from f. h Representative images of Stat3, Esrrb, and Tfcp2l1 knockdown in J1 mESCs with serum/+LIF and serum/−LIF + Royalactin conditions and qPCR analysis of pluripotency and differentiation-associated genes from the same cells. Data are means ± SD (n = 2). Scale bar, 200 μm. RylA Royalactin

Having observed a robust stemness-maintenance effect of Royalactin in vitro, we next examined whether mESCs treated with Royalactin in serum/–LIF retain embryonic identity and developmental potential in vivo. Indeed, mESCs grown in serum/–LIF + Royalactin were grafted subcutaneously and gave rise within 6 weeks to large multi-differentiated teratomas (Fig. 1c). Collectively, these results indicate that Royalactin can functionally maintain self-renewal and pluripotency in mESCs.

Royalactin modulates chromatin and pluripotent networks

In the interest of understanding Royalactin’s effects on the transcriptome, RNA-seq analyses of serum/+LIF, serum/–LIF, and serum/–LIF + Royalactin cells were conducted. These analyses revealed a strong enrichment for canonical pluripotency genes and a suppression of lineage-specific genes in serum/–LIF + Royalactin cells at levels similar to those in mESCs cultured in serum/+ LIF (Fig. 1d). Gene Ontology (GO) term analysis of all genes differentially expressed in the presence of Royalactin revealed strong enrichment for genes involved with proliferation and stemness in the upregulated gene set, and an overrepresentation of developmental processes in the downregulated gene set (Fig. 1e). Similarly, analysis of chromatin accessibility using the assay for transposase-accessible chromatin using sequencing (ATAC-seq)20 of mESCs cultured in serum/–LIF + Royalactin and serum/+LIF conditions revealed similar patterns of increases in ATAC-seq signal relative to mESCs grown in serum/–LIF conditions (Fig. 1f), specifically at promoters (TSS; 14234 total peaks with 7373 gaining accessibility and 6861 losing accessibility; Supplementary Figure 1c), traditional enhancers (TE; 5356 total peaks with 2571 gaining accessibility and 2785 losing accessibility; Supplementary Figure 1d), and super enhancer regions (SEs; 127 of 231 elements gaining accessibility; Supplementary Figure 1e). As expected, high correlation was observed between ATAC-seq changes and RNA-seq experiments (Supplementary Figure 1c–e), with functional annotation revealing that the ATAC-seq changes were located near genes associated with pluripotency, metabolism, and differentiation (Fig. 1g). Furthermore, motif enrichment analysis revealed that TFs such as KLF5, KLF4, and SOX2 bound at high frequency to the Royalactin-upregulated SE constituents (Supplementary Figure 1f). Collectively, this suggested that regulatory regions are sensitive to Royalactin culture conditions and cause subsequent changes in gene expression.

In order to gain a molecular understanding of patterns of gene expression and identify candidate regulators of pluripotency in response to Royalactin, a transcriptional network analysis was performed that identified Stat3, Tfcp2l1, Esrrb, and Nanog as the most significant nodes (Supplementary Figure 1g). Subsequent experimentation revealed a dose-dependent effect for Royalactin in stimulating phospho-Stat3 activation concomitant with other pluripotency factors (Supplementary Figure 1h), which was sustained after 10 and 20 passages (Supplementary Figure 1i). In addition, knockdown of these transcription factors greatly diminished the mESC response to Royalactin, with the most significant effects being observed following Stat3 knockdown (Fig. 1h). As these findings suggested that Royalactin triggers activation of a Stat3-driven LIF-independent pathway on mESC self-renewal, further analysis of gene expression profiles from serum/+LIF and serum/–LIF + Royalactin mESCs were compared to identify 519 genes that are specifically activated in response to Royalactin (Supplementary Figure 1j). GO term analysis showed enrichment of metabolic and biosynthetic processes (Supplementary Figure 1k), reminiscent of mESCs cultured without serum in the presence of inhibitors targeting mitogen-activated protein kinase kinase and GSK3 (2i)21. Global expression profiles from serum/–LIF + Royalactin and 2i-cultured cells also clustered together by principal component analysis away from serum/+LIF-cultured cells (Supplementary Figure 1l). GO term enrichment analysis found that genes involved in basic metabolism, transcription, and development were responsible for this separation (Supplementary Figure 1m). Collectively, this suggested that Royalactin may be driving ground-state pluripotency in mESCs.

Royalactin treated mESCs mimic ground-state pluripotency

We next sought to test the hypothesis that Royalactin was driving a ground-state-like pluripotency state in mESCs. As expected, mESCs cultured in 2i + LIF media maintained pluripotency and sustained expression of a Rex1 GFP pluripotency marker22, while those in 2i base media without inhibitors (0i) readily differentiated (Fig. 2a, b, Supplementary Figure 2). Remarkably, addition of Royalactin in the absence of inhibitors (0i + Royalactin) for multiple passages maintained undifferentiated GFP positive colonies with similar gene expression profiles to 2i + LIF cultured cells (Fig. 2a, b, Supplementary Figure 2). In addition, injection of 0i + Royalactin cultured cells into mouse blastocysts generated chimeric animals with germline transmission, highlighting the robust effects of this protein in vivo (Fig. 2c, Supplementary Table 1).

Fig. 2
Fig. 2

Royalactin drives a ground-state-like pluripotency state in mESCs. a Representative images of J1 and R1 mESCs cultured in serum-free media in the presence (2i + LIF) or absence (0i) of MAPKKi, GSK3i, and LIF for 10 and 20 passages. mESCs rapidly differentiated in 0i, whereas cells cultured with Royalactin (0i + Royalactin) supported self-renewal with negligible differentiation. Scale bar, 200 μm. b Quantitative expression of pluripotency and differentiation-associated genes from a. Data are means ± SD (n = 2). c Chimeras with germline transmission formed by CGR 8.8 mESCs treated for ten passages with 0i + Royalactin. d RNA-seq log2 fold change values in transcript level of all genes in 2i or 0i + Royalactin (0i + RylA) J1 mESCs (passage 10) relative to 0i. e GO term analysis of the differentially expressed genes from d. f J1 mESCs cultured in serum/+LIF, serum/−LIF + Royalactin, 2i + LIF, and 0i + Royalactin for ten passages are projected onto the first two principal components. All genes with mean normalized read counts larger than 10 were considered for principal component analysis (PCA). g Distribution of genes contributing to principal component 1 (PC1) in f, and GO enrichment analysis of genes most strongly contributing to PC1 separation. RylA Royalactin

RNA-seq and GO-term analyses further demonstrated marked similarities in enriched genes between 2i + LIF and 0i + Royalactin cells (Fig. 2d, e), and global expression profiles from 2i + LIF, 0i + Royalactin, serum/+LIF, serum/–LIF + Royalactin, and serum/–LIF cells demonstrated a clear clustering by principal component analysis of 0i + Royalactin cells nearer those cultured in 2i + LIF than those cultured in serum/+LIF (Fig. 2f). GO term enrichment analysis found that genes involved in basic metabolism, transcription, and development were responsible for this separation (Fig. 2g). These data suggest that Royalactin treatment is accompanied by a profound metabolic reprogramming resembling the 2i + LIF state, mimicking the environment of the mature mouse ICM.

Identification of Royalactin mammalian analog

We next wondered whether a homolog of Royalactin existed in mammals. Sensitive searches of sequence databases using iterative PSI-BLAST23, as well as aiming HHPRED sequence and structural profiles against the human and mouse proteomes24 did not reveal any Royalactin orthologs. However, the latter computational tool revealed that Royalactin is distantly related to an existing structure in the PDB database25, a secreted salivary gland protein (SGP) from the sand fly, L. longipalpis (PDB ID: 3Q6K)26. We then used this structure––a six-bladed β-propeller fold with no additional domains—as an accurate template for MODELLER27, yielding a high confidence model for the Royalactin fold (Fig. 3a). The resulting superposition of Royalactin and SGP sequences was then used to seed new and more precise HHPRED scans of the human proteome, in search of a possible structural and functional analog of the Royalactin/SGP β-propeller fold. Fitting the description of a secreted, single domain chain, with a predicted 6-bladed β-propeller architecture, only one protein, the provisionally named NHL Repeat Containing 3 (NHLRC3), arose as a potential candidate, with striking fold similarity to the Royalactin model (Fig. 3a). Although no known function of NHLRC3 has been identified to date, single-cell RNA-seq analyses of early mouse embryos revealed that it is expressed starting in E4.5 embryos, and that its expression increases steadily thereafter (Supplementary Figure 3a). To elucidate whether it served a functional purpose in stemness maintenance in mESCs similar to that observed with Royalactin, recombinant mouse NHLRC3 was added to mESC culture in the presence of serum/–LIF (serum/–LIF + NHLRC3) as well as 0i (0i + NHLRC3). As seen with Royalactin, NHLRC3 maintained mESCs in an undifferentiated state in both culture conditions for multiple passages (Fig. 3b, c, Supplementary Figure 3b, c), with expected changes in gene expression (Fig. 3d, e). Additionally, injection of 0i + NHLRC3 cultured cells into mouse blastocysts generated chimeric animals with germline transmission, highlighting the robust effects of this protein in vivo (Supplementary Figure 4, Supplementary Table 1). Thus, NHLRC3 appears to be a mammalian pluripotency maintenance factor, whose existence demonstrates a remarkable conservation of macromolecular structure and function. We renamed NHLRC3 as Regina due to its conservation of functions with those of Royalactin and the queenmaker Royal Jelly.

Fig. 3
Fig. 3

The mammalian structural analog of Royalactin maintains naive and ground-state pluripotency in mouse embryonic stem cells. a Computational modeling predicts the structure of Royalactin (left), allowing for identification of a highly structurally analogous protein, NHLRC3 (center). Superimposition of these models (right) demonstrates striking similarity between them. b Representative images of J1 and R1 mESCs cultured in serum/+LIF, serum/–LIF, or serum/−LIF + NHLRC3 for 10 and 20 passages. After LIF withdrawal, mESCs rapidly differentiated, whereas cells cultured with NHLRC3 supported self-renewal with negligible differentiation. Scale bar, 200 μm. c Representative images of J1 and R1 mESCs cultured in serum-free media in presence (2i + LIF) or absence (0i) of MAPKKi, GSK3i, and LIF for 10 and 20 passages. mESCs rapidly differentiated in 0i, whereas cells cultured with NHLRC3 (0i + NHLRC3) supported self-renewal with negligible differentiation. Scale bar, 200 μm. d Quantitative expression of pluripotency and differentiation-associated genes from b. Data are means ± SD (n = 2). e Quantitative expression of pluripotency and differentiation-associated genes from c. Data are means ± SD (n = 2)

In summary, our results demonstrate an unexpected capacity for Royalactin as a pluripotency factor that confers self-renewal and promotes emergence of the naive pluripotent gene regulatory network, and identify Regina as a factor that can promote ground-state pluripotency in mESCs. A better understanding of the interaction of Royalactin and Regina with genetic pathways and biochemical processes conserved in evolution, and how the different pluripotency networks function independently or synergistically to maintain stem cell self-renewal, will advance our efforts to better control stem cell fate, and provide a platform for dissection of the pluripotent state. Our findings and the discovery of Regina thus support the intriguing idea that profound molecular conservation underlies even the most evolutionarily novel traits. Future work in dissecting the mechanistic action of Royalactin in mammalian cells, including further characterization of its mammalian structural analog Regina, will shed new light on mammalian pluripotency and provide additional means to enhance optimal maintenance and derivation of ESCs for therapeutic application and regenerative medicine.

Methods

Embryonic stem cell culture

J1 and R1 mESCs (gift from Howard Y. Chang) were grown on 0.2% gelatin-coated (Sigma G6144) tissue culture plates. Cells were maintained for a minimum of ten passages in mESC serum medium containing KnockOut™ D-MEM (Life Technologies 10829), 15% HyClone™ fetal bovine serum (FBS; Thermo Scientific™ SH30396.03), 100 U/ml Penicillin-Streptomycin (P/S; Life Technologies 15140), 1% MEM non-essential amino acids (Life Technologies 11140), 1% GlutaMAX™ (Life Technologies 35050), and 0.1 mM 2-mercaptoethanol (Life Technologies 21985). Mouse leukemia inhibitory factor (LIF; Millipore ESG1107; 1000 U/mL), purified Royalactin (0.5 mg/mL), or purified NHLRC3 (0.125 mg/mL) were added to culture as indicated. Cells were passaged every 2 to 3 days using Trypsin-EDTA (0.25%; Life Technologies 25200) and seeded at 15,000 cells/cm2. Media and protein were changed daily.

For cultures under 2i conditions, J1, R1, and Rex1-GFP mESCs were grown on Poly-L-ornithine (Sigma-Aldrich)/Laminin (Fisher Scientific 23017–015) coated plates and CGR8.8 mESCs were grown on Matrigel (Corning 354277) plates coated according to manufacturer’s specifications. All cells were maintained a minimum of ten passages in serum-free media containing: 1:1 Neurobasal:DMEM-F12 base (Thermo Scientific 21103049), 1% Glutamax, 1% high insulin N2 (Thermo Scientific 17502001), 1% B27 supplement (Thermo Scientific 12587001), 0.1 mM 2-mercaptoethanol, 1% Penicillin-Streptomycin, 1% MEM non-essential amino acids, 1% sodium pyruvate (Thermo Scientific 11360070). Mouse leukemia inhibitory factor (LIF; Millipore ESG1107; 1000 U/mL), 1 μM PD0325901 (Selleckchem), 3 μM CHIR99021 (Selleckchem), purified Royalactin (0.5 mg/mL), or purified NHLRC3 (0.125 mg/mL) were added to cultures as indicated. Cells were passaged every 2 to 3 days using Accutase (Stemcell Technologies 07920) and seeded at 15,000 cells/cm2. Media and protein were changed daily.

Production of recombinant Royalactin and NHLRC3

FLAG-MRJP1-His (Genbank ID# NM_001011579.1) was cloned into LakePharma’s proprietary antibiotic selection vector and transfected by electroporation into suspension CHO parental cells. The FLAG-MRJP1-His stable cell line was generated after 2 weeks of antibiotic selection period. Purification of FLAG-MRJP1-His was achieved by a two-step chromatography method. Conditioned media was centrifuged, filtered, and loaded onto an anion exchange chromatography (AEX) resin pre-equilibrated with 20 mM Tris pH 7.5. FLAG-MRJP1-His was eluted by increasing sodium chloride concentration and fractions containing the protein were pooled together. This sample was further polished by a second immobilized metal (Ni) affinity chromatography (IMAC) step using increasing concentrations of imidazole for elution. SDS-PAGE was performed for each fraction and the ones containing FLAG-MRJP1-His were pooled together for dialysis. The final formulation for FLAG-MRJP1-His was 200 mM NaCl in 30 mM HEPES pH 7.0.

For production of supernatants containing recombinant NHLRC3, suspension CHO cells were seeded at 350,000 cells/mL into 90% CD OptiCHO Medium (Thermo Scientific 12681011) containing 6mM L-glutamine (Thermo Scientific 25030–081) and 10% CHO CD EfficientFeed (Thermo Scientific A1023401). CHO cells were grown at 37 °C for 5 days, shaking constantly. The cell suspension was centrifuged at 10,000 × g for 40 min and the supernatant run through a 0.22 μm filter. Presence of concentrated NHLRC3 was verified by western blot. The filtered supernatant was concentrated 35-fold and used directly in cell culture assays.

RNA extraction and quantitative PCR

Total RNA was isolated using TRIzol® (Life Technologies) and RNeasy Kit (QIAGEN) according to the manufacturer’s protocol. cDNA was made with Superscript VILO (Life Technologies). All primers (Supplementary Table 2) were tested for efficiency and single products confirmed. qPCR analyses were performed on the Light Cycler 480II (Roche).

Lentiviral expression and viral production

pLKO vectors were a gift of Alejandro Sweet-Cordero. N103 vector was a gift of Howard Y. Chang. Sequence verified constructs were used to produce lentivirus using pRSV (Addgene plasmid #12253), pMD2.G (Addgene plasmid #12259), and pMDLg/pRRE (Addgene plasmid #12251). 293Ts were maintained in G418 (Sigma-Aldrich G8168). Plasmids were transfected using Lipofectamine 2000 following manufacturer’s protocol (Thermo-Fisher Scientific 11668). After 12 h, media was changed to viral production media (DMEM, 10% FBS, 1% P/S, 20 mM HEPES). After 48 h, media was collected, spun to remove cell debris, and lentiviral-containing supernatant was added to Lenti-X™ Concentrator (CloneTech). Following incubation at 4 ˚C for >4 h, the mixture was spun at 500 × g for 45 min, the pellet resuspended in mESC media, and frozen at −80 ˚C.

Lentiviral transduction of mESCs

mESCs were plated at a density of 30,000/cm2. After 12 h, virus was added with 6 µg/mL polybrene. Media was changed 12 h later and puromycin selection began 48 h post-transduction.

Cell culture for teratoma formation assay

J1 mESCs were cultured in serum-free media (as described above) with addition of 1000 U/mL mouse LIF, 1 µM PD0325901, and 3 µM CHIR99021, or 0.5 mg/mL Royalactin for three passages. Cells were grown in suspension on Corning Costar Ultra-Low attachment plates (Sigma-Aldrich). Wells were seeded in duplicate at a cell density of 100,000/well, and media and protein were changed daily. To split, colonies were first pelleted by centrifugation at 1500 × g, suspended in TrypLE (Life Technologies), incubated at 37 °C for 5 min, diluted with PBS (Life Technologies) and pelleted. Cells were counted and re-seeded at a density of 100,000/mL.

Teratoma generation and histopathology

All animal studies were conducted in accordance with Stanford University animal use guidelines and were approved by the Administrative Panel on Laboratory Animal Care (APLAC). J1 mESCs were mixed with Matrigel (BD 356237) prior to being subcutaneously injected into 8-week-old female SCID/Beige mice (Charles River) on each flank. Four weeks after injection, the mice were euthanized and the teratomas were harvested. All animal studies were approved by Stanford University IACUC guidelines. For histological analysis, slides were stained with hematoxylin and eosin (H&E) following manufacturer’s instructions. Analyses were performed by a board-certified veterinary pathologist.

Chimera experiments

CGR 8.8 mESCs were grown in serum-free media (as described above) with addition of 1000 U/mL mouse LIF, 1 µM PD0325901, and 3 µM CHIR99021 (2i + LIF), 0i + Royalactin, or 0i + NHLRC3 (as noted) for ten passages. Media was changed daily. Cells were passaged using Accutase (Stemcell Technologies 07920) and suspended in M2 media for injection.

Protein extraction and western blot analysis

Cellular extracts were prepared using lysis buffer containing 50 mM Tris HCL (pH 7.5), 250 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, Halt™ Protease, and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific). Extracts were run on a 4–12% Bis-Tris gel (Novex) and transferred onto PVDF membranes. Blots were blocked in 5% milk PBS-T (TBS for phospho-specific) for 1 h at room temperature followed by overnight incubation at 4 ˚C with primary antibodies. HRP-conjugated secondary antibodies were used at 1:10,000. Antibodies used in this study include Nanog (ReproCELL, RCAB001P, 1:1000), Klf2 (Millipore, 09–280, 1:1000), Esrrb (Perseus Proteomics, PP-H6705–00, 1:500), Stat3 (Cell Signaling, 124H6, 1:1000), pStat3 Y705 (Cell Signaling, D3A7, 1:1000), Sox2 (Santa Cruz, sc-17320, 1:250), Tfcp2l1 (R&D, AF5726, 1:250), NHLRC3 (OriGene, TA336106, 1:500), anti-HA (Cell Signaling, C29F4, 1:1000), and Actin-HRP (Santa Cruz, sc-1616, 1:2500). Uncropped scans of the most important blots are included in Supplementary Figure 5.

RNA-seq library construction

RNA was extracted using TRIzol and purified on column with the RNeasy Mini Kit (Qiagen). Ribosomal RNA was depleted with the Ribo-Zero Gold rRNA Removal Kit (Illumina). RNA was lyophilized, suspended in 10 μL of water and fragmented to an average size of 200 base pairs using the Ambion RNA Fragmentation Kit (AM8740), and purified using Zymo clean and concentrator 5 columns. 3′ Phosphorylation, adapter ligation, reverse transcription, immunoprecipitation, circularization, amplification, and PAGE separation were performed using the FAST-iCLIP library construction method as previously described28. The quality of the libraries, including size distribution and molarity, was assessed on a BioAnalyzer High Sensitivity DNA chip (Agilent). The libraries were then multiplexed and sent for sequencing on an Illumina NextSeq 400 High Output machine for 1 × 75 cycles. Sequencing data deposited under GEO GSE81799.

ATAC-seq

ATAC-seq was performed on 50,000 J1 mESCs20. Cells were grown in serum mESC media (as described above) in serum/+LIF, serum/–LIF, or serum/–LIF + Royalactin for ten passages. Cells were washed with PBS (Life Technologies), trypsinized with Trypsin-EDTA (0.25%), quenched with serum mESC media, washed with PBS, before nuclear isolation with NP-40. Nuclei were resuspended in a transposase reaction mix containing 25µL 2× TD buffer, 2.5 µL Transposase (Illumina) and 22.5 µL of nuclease free water with sequencing adapters. Final libraries were purified on column using the QIAquick PCR Purification Kit (Qiagen) per the manufacturer’s protocol as well as with Agencourt AMPure XP beads (Beckman Coulter) to remove any remaining free adapters. The quality of the libraries, including size distribution and molarity, was assessed on a BioAnalyzer High Sensitivity DNA chip. Libraries were then multiplexed and sent for deep sequencing on the Illumina HiSeq 2500 machine for 2 × 50 cycles. Sequencing data deposited under GEO GSE81799.

RNA-seq data analysis

Reads were aligned to the mouse reference genome (build mm9) using Tophat. A maximum of a default 2 mismatches was allowed for read alignment. Gene counts were calculated using the HTSeq-count utility29 and used as an input for differential gene expression analysis with DESeq version 1.20.030. Genes with a p-value of 0.05, as well as a fold change of 2 were selected for further analysis. Validation of top differentially regulated genes was performed with quantitative reverse transcription polymerase chain reaction. Further network analysis on differentially significant genes was performed using NetworkAnalyst31. For RNA-seq analysis, GO terms were obtained using DAVID and its default parameters.

PCA analysis was performed using samples as indicated. The genes that led to the maximum amount of variance (PC1) were selected and GO terms obtained using the GO Consortium. Samples from different libraries were normalized using shifted log of normalized counts using DESeq. The ‘plotPCA’ function, which is a part of DESeq2, was used to construct the PCA plots.

ATAC-seq data analysis

Reads were aligned to the mouse reference genome (build mm9) using Bowtie. The ATAC-seq regions were divided into separate analyses: correlation with closest TSS, correlation with 5356 traditional enhancer regions present in the mm9 genome, and correlation with 361 super-enhancer regions discovered for the mm9 genome28. The ATAC-seq signals for serum/+LIF, serum/–LIF, and serum/–LIF + Royalactin after ten passages were compared using DESeq, and the results are represented in the heatmaps. The heatmaps for TSS regions, traditional enhancers, super-enhancers, and differential peaks were produced using unsupervised clustering methods, which used the normalized signal values obtained by quantile normalization, to extract transitions between two states: upregulated and downregulated. The differential peaks between serum/–LIF + Royalactin and serum/–LIF were used for correlation with RNA-seq. The peaks were filtered on the basis of a p-value threshold of 0.05 as well as fold change. Boxplots were produced using the ‘BOXPLOT’ function in R. p-value was calculated using the student’s t-test.

GO terms for peaks differentially expressed in serum/–LIF and serum/–LIF + Royalactin was performed using GREAT. The significant GO terms were filtered to only include GO terms associated with pluripotency and GO terms associated with metabolism. For pluripotency related GO terms, biological processes including morphogenesis, development, proliferation, and stem cell processes were analyzed. For metabolic GO terms, biological processes that were related to metabolism and biosynthetic processes were chosen. Motif analysis for the differential peak lists was performed using HOMER with all differential peaks used as background.

Structural modeling and Royalactin analog identification

As implemented at the MPI Toolkit (http://toolkit.tuebingen.mpg.de), HHPRED enables sensitive searches of sequence and structural databases through the assembly of profile Hidden Markov Models (HMMs) from a seed sequence, with multiple iterations of Hhblits (a more sensitive and faster program than PSI-BLAST) and PSIPRED (a very accurate secondary structure prediction program)24. The detection of a six-bladed β-propeller fold for Royalactin from the top salivary gland protein (PDB ID: 3Q6K) HHPRED hit was accompanied by a significant score of 177.4, an E-value of 6e−28, and a 28% amino acid identity from the structure-guided overlap of the mature, 413 residue honeybee Royalactin, with the 381 amino acid sand fly SGP. This structural alignment was used by MODELLER27 to guide a secure template-guided three-dimensional model of Royalactin (with a VERIFY3D score of 119.7)32, and also to nucleate a more sensitive search by HHPRED for a structurally analogous protein (to the greater Royalactin/SGP family) in the human and mouse proteomes. This latter screen, filtered by the signal peptide, single domain, and six-bladed β-propeller fold constraints common to Royalactin and SGPs, yielded NHLRC3 (Uniprot IDs: Q5JS37 and Q8CCH2, for the human and mouse orthologs, respectively) as the sole analog candidate. A three-dimensional model of the NHLRC3 β-propeller was then built by MODELLER on the best six-bladed β-propeller template recognized by HHPRED, Peptidyl-alpha-hydroxyglycine alpha-Amidating Lyase (PDB ID: 3FVZ; at a significant score of 125.6, E value of 1.7e-18, and amino acid identity of 24%). The comparison and visualization of Royalactin and NHLRC3 structural models were in turn performed by PyMOL (www.pymol.org). We recognize that β-propeller folds in general (irrespective of ‘blade’ number) are consistently used as interaction scaffolds and preferred binding platforms in the cell33. The structural models are available upon request.

Source:Nature

Rise of the Clones


Study identifies inherited and acquired mutations that drive precancerous blood condition

leukemia cells

Leukemia cells.

A new study led by researchers at Harvard Medical School and the Harvard T.H. Chan School of Public Health has identified some of the first known inherited genetic variants that significantly raise a person’s likelihood of developing clonal hematopoiesis, an age-related white blood cell condition linked with higher risk of certain blood cancers and cardiovascular disease.

The findings, published online July 11 in Nature, should help illuminate several questions about clonal hematopoiesis: how it arises, why it occurs in more than 10 percent of people over 65 and how the genome we inherit influences the mutations we acquire later in life.

The condition, uncovered in a series of studies over the past 10 years, is marked by the accumulation of genetically abnormal white blood cells, which may become cancerous or contribute to inflammation in atherosclerotic plaques.

“Clonal hematopoiesis is increasingly appreciated to be an important biomarker of risk for future illness, but we haven’t known what brings it about,” said the study’s co-senior author, Steven McCarroll, the Dorothy and Milton Flier Professor of Biomedical Science and Genetics at HMS and director of genetics at the Stanley Center for Psychiatric Research at the Broad Institute of MIT and Harvard.

“These findings reveal specific sequences of genetic events—some inherited, others acquired—that give rise to these abnormal blood cells,” he said.

The study also reaches the surprising conclusion that inherited genetic variants and acquired mutations are more connected than previously understood.

Acquired mutations are believed to occur randomly over time, appearing spontaneously or after exposure to damaging agents such as ultraviolet light. However, the team found examples where inherited variants led to the appearance of specific acquired mutations later in life or gave cells with such mutations a growth advantage over other cells.

“Conceptually one of the most intriguing things to come out of this work is the blurring of that distinction between genetic inheritance and acquired mutations,” said McCarroll. “Inherited alleles turn out to have powerful influences on what was previously thought to be a more capricious process.”

Out of proportion

Normally, each of the body’s 10,000 to 20,000 hematopoietic, or blood-forming, stem cells contributes roughly the same number of mature blood cells to the body’s total. The result: a pool of hundreds of billions of blood cells with many different parents.

Clonal hematopoiesis occurs when a single stem cell acquires mutations that cause it to produce far more than its share of new cells, including white blood cells. Over many years, the mutants out-compete normal blood cells, either proliferating more rapidly or surviving longer. Instead of the typical 1/10,000th, or 0.0001 percent, of a person’s total white cell count, the progeny of a single mutated stem cell might make up 2 percent, or 20 percent, or more than 90 percent of a person’s white blood cells. These genetically dominant blood cells are called clones.

Previous research from McCarroll’s lab and others showed that only some clones cause trouble. For example, only about 10 percent of people with clonal hematopoiesis go on to develop blood cancer. Even so, that risk is 10 times higher than that in the general U.S. population.

The team set out to learn which parts of the genome tend to be mutated in clones, which mutations are most harmful, and how clones arise and expand their numbers.

To do so, co-first authors Po-Ru Loh, assistant professor of medicine at HMS and Brigham and Women’s Hospital, and Giulio Genovese, senior computational biologist in the McCarroll lab, developed a mathematical approach that let them identify clones early on, when they accounted for as little as 1 percent of a person’s white blood cells. Previous methods lacked the precision to detect clones unless they had expanded to at least 15 to 20 percent of white blood cells.

Armed with their new technique, Loh and Genovese analyzed DNA from the blood of 151,000 people who’d donated samples to the UK Biobank.

Not so random after all

The increased level of sensitivity allowed the team to find clones in more than 8,000 participants, many of whom had acquired similar mutations.

To the researchers’ surprise, participants with similar acquired mutations often shared a rare, inherited variant nearby. Further investigation confirmed that this was far from a coincidence; the inherited variants had powerful effects on whether people acquired those other mutations later in life.

“When Giulio suggested searching for influences of inherited genetic variants, I never expected to turn up anything interesting,” said Loh. “When I first saw the results, the associations were so strong I wondered if they were a bug in the code.”

The researchers were then able to figure out the specific ways that the inherited variants made people vulnerable to developing clones.

The inherited variants and acquired mutations typically appeared in the same part of the genome. Some inherited variants made certain spots on chromosomes more vulnerable to future mutation. Others created easy ways for future mutations to increase the rate at which cells proliferate.

In some cases, an inherited variant inactivated one copy of a gene that normally protects against cancer. Later on, an acquired mutation inactivated the other copy.

“These are examples of what cancer geneticists call the two-hit model, where the inherited allele is the first hit and then the subsequent acquired mutation is the second hit,” said McCarroll. “It’s still not cancer, but having many, many white blood cells with that combination of mutations almost certainly puts one in a more vulnerable place.”

Another inherited variant inactivated one copy of a gene that promotes cell growth. This flummoxed the researchers at first, since the variant appeared to protect against aggressive cell growth or cancer. But many of the people who inherited this variant later acquired a mutation that replaced the inactivated gene with the full-strength copy inherited from the other parent. Cells with the acquired mutation then out-competed other cells.

The variants the team uncovered are rare, and inheriting one doesn’t guarantee that a person will develop clonal hematopoiesis. However, certain variants did make acquiring clones with a specific mutation much more likely—conferring up to a 50 percent chance, compared to the normal risk of well under 1 percent.

The researchers even found instances where multiple family members who inherited the same variant went on to develop clones with the same mutation.

The authors believe their findings are likely not a fluke.

“I think it’s safe to predict that these are early examples of a phenomenon we’ll see again and again,” said McCarroll.

The demographics of clones

Some acquired mutations were more common in women, others in men. Although clones in general are much more common in older people, two acquired mutations appeared across all ages, suggesting they arise from developmental rather than age-related processes.

The discoveries invite further efforts to understand the nature and consequences of each mutation.

“Although it’s been possible to say that, on average, clones might increase the risk of blood cancer tenfold, that doesn’t mean every specific clone does,” said McCarroll. “A key direction is to go from talking generically about clones to knowing each clone’s history and risk profile based on its specific mutations and frequency in the blood.”

As information builds, researchers will be able to better assess the risk of each clone and try to develop environmental or medical interventions might slow the growth of clones and avert disease, McCarroll said.

26 Powerful Lessons to Learn from Nature


26 Powerful Lessons to Learn from Nature

“Nature does not hurry, yet everything is accomplished.” ~ Lao Tzu

I recently had the honor to hear the empowering Gabrielle Bernstein and Kris Carr speak at the Crazy Sexy Miracles lecture in NYC. The evening was filled with many “AHA moments”  but one thing that stood out, in particular, was Kris Carr’s wise suggestion, “If you struggle with mastering patience, acceptance or any lesson, look to nature as your teacher. Kris said “Ask how the stars do it? How does the ocean do it? How do the birds do it?”  Therein you will find an illustration and answer of how you should handle your issue. That struck a strong cord in my heart because I’ve had many deeply connected moments with nature and animals where time stands still and I feel one with a higher energy, yet I never thought to look to nature for answers.

It’s the moment you see a beautiful cloud formation while driving, taking in the magnitude of colors during a sunset, seeing autumn foliage, watching flocks of birds migrating or deeply looking into the eyes of your pet. I began to ponder what we are suppose to learn and what other messages I missed by our silent teachers. As I began to become present with nature, these are some of the humbling lessons and answers by tuning into nature and animals.

26 Powerful Lessons to Learn from Nature

1. Trees

As seasons change, we are guided to learn acceptance and non-resistance. A green leaf doesn’t resist turning red when autumn approaches. Trees don’t resist leaves falling when winter arrives. They stand deeply rooted in the ground, with their vulnerability out in the open and branches spread wide, surrendering to the Universe. Do what you will with me, I trust it is for my highest good.

2. Ocean

The vast ocean can’t exist without each particle of water. Each human being plays its part in humanity. We are all one small part of the greater whole.

3. Birds

Birds soaring through the sky represents the limitless freedom and potential available to us if we release our fears. Taking off to fly for the first time can be scary and bring about feelings of fear. Without taking the risk of the first flight, we won’t find the internal freedom we desire. We must dare to take our feet off the ground, spread our wings and soar.

4. Pets

Pets teach us more about love than any person or thing. We understand the true nature of unconditional love without expectations. The true nature of forgiveness is forgetting and letting go of grudges. We learn uninhibited, unreserved affection by giving our full attention. Understanding love is a feeling and doesn’t require words. Love is felt in the heart by making eye connection, being in someone’s presence and through physical touch.

5. Ants and Bees

The community of bees and ants all participate together to benefit all those in their community. We each have our own calling that is best performed by us. Each part is necessary for a functioning family, community, nation and world. Embrace your special responsibility, share it proudly with the world, and always do your best.

6. Bamboo and the Maple Tree

Who said that the bamboo is more beautiful than the maple tree and maple tree is more valuable than the bamboo because it gives out maple syrup? Does the bamboo feel jealous of the maple tree because it is bigger and its leaves change color? The idea of trees comparing themselves to others is ridiculous as should humans comparing themselves to one another. We must compare our growth to who we were yesterday not to the growth of another. Everyone is incomparably unique.

7. Flocks of Birds

I’ve never seen two birds run into each other when they are flying in a flock. Why is that if they never talk to each other? True communication doesn’t always need words. Body language, sensing others` energy and tone can say much more than the actual words we speak. At all times we are communicating through our thoughts and the energy we dispel. Be mindful of your thoughts as the energy behind them affect others and the world.

8. Night Sky

Darkness is necessary to appreciate the light. We need to experience the opposite of what we want so we can appreciate and experience the thing we desire.

9. Sky

No matter what storms are passing, know it is always transient because beyond the clouds, the sky is always blue and the sun is always shining.

10. Rain

Water is required to cleanse negativity in the world and allow a space of clarity. It is through showering and soaking in a tub, that we clear our bodies from the stagnant, negative energy of yesterday and replenish our positive energy. Shower with the intent of cleansing your body, spirit and mind.

11. Clouds

The sky is the backdrop of our mind. The clouds with different formations, speeds and heights represent the frequency, types and speed of our thoughts. As clouds, our thoughts too shall pass. Glide through your thoughts like birds glides through clouds. Don’t resist the clouds, fly through them.

12. Stars

Stars bring beauty and light in the darkness. Instead of succumbing to the darkness of the world, be one of the radiant stars that shines their bright inner light. As we inspire others to be stars, we can light up the night sky with our intentional beams of star light.

13. Wind

Not all things that exist can be seen or heard. Some things need to be felt. Don’t be limited to your 5 senses. Use your intuition and develop the practice of believing in the things you feel.

14. Sunrise and Sunset

The breathtaking colors of a sunrise and sunset show us that colors vibrate energy and have the power to elicit certain emotions and feelings. Be mindful about the colors you surround yourself with.

15. Animals

Zebras do not look at tigers and wish they could hunt like tigers. Accept yourself as you are, know your weaknesses and strengths and embrace your unique beauty and gifts.

16. Preys

Animals who are prey don’t over analyze and plan in advance the ways they are going to outsmart a predator in the future. When the threat approaches their fight or flight kicks in, when the threat is gone, they go back to grazing without a thought in mind about the predator. Don’t dwell in a space of fear of the future and regret of the past when the threat doesn’t exist. That’s the breeding ground of stress, anxiety and regret.

17. Gardens

Have faith in tomorrow. We plant seeds of hope today, nourish them with love and attention with the faith that our labor will result in fruits in the future. We can’t impatiently force a garden to grow on our terms. A seed will sprout into a plant when the time is right. A fruit will fall from the tree when it is ripe and ready. They grow not because they are forced to, because they let go and allow divine energy and timing to run its course. Be persistent, patient and have trust in divine timing.

18. Natural Disasters

Our earth absorbs the negative energy humans expel as do our bodies. There are times when the earth and our bodies need to recalibrate and dispel the negative energy we absorbed. Mental breakdowns and hitting rock bottom will bring chaos, change and discomfort, but it can be the most positive, life-changing event. Sometimes, we need to be brought to our knees to remember what we are grateful for and start on a new life path.

19. Mountains

Stand firm, poised and majestic like a mountain. Regardless of the external situations life will bring you, remain strong like the mountains do when faced with avalanches, rain storms, and water erosion. Your emotional guidance system should be tough like a rooted mountain, immune to the actions and reactions of others.

20. Flowers

We all carry a different fragrance, color and beauty for the world to enjoy. Flowers don’t discriminate who they share their beauty and fragrance with. They share with all friends, strangers and enemies. True compassion and love comes from sharing your beauty with all you meet.

21. Snakes

It’s necessary to shed your own skin and personality to allow an improved and better version of ourselves to emerge

22. Gravity

The Universe has its own sets of laws that are not man-made and trumps any rule, law or limiting belief set by man. Figure out the Universal laws and make sure you are working with them and not against them.

23. Flow of Water

As we set sail in our life, we take sail through a calm stream. As our dreams get bigger, we are guided to a river with faster currents and more opportunities. Eventually, for our dreams to be realized, we must end up in the vast ocean. We won’t always have the protection of the river banks as our safety net. To achieve our dreams we have to lose sight of the land and sail into open waters, where there are unlimited possibilities for our dreams to manifest. Anything and everything is possible.

24. Butterflies

Butterflies symbolize our entire life cycle metamorphosis. Life is short and from the moment of birth we are constantly changing our form, inside and out. Don’t resist change. Some of the most beautiful wisdom and changes occur as you grow older and transform from a caterpillar to a butterfly. Appreciate each phase of your life before you transform to a new cycle.

25. Streams

There is always a natural undercurrent to water. We have the choice to either flow with the current of life or paddle against the stream. We dispel our energy, creativity and time working against the flow of the Universe. Throw your paddles in the water and let your boat take course in the natural direction of the current. You are being guided to go in the direction you are meant to go.

26. Weather

Just like weather forecasts, nothing is certain in life. We can’t control and prepare for everything. On days when there is suppose to be sunshine, the rain may unexpectedly fall. Don’t let your mood be affected by the weather. Looking to nature and animals, we see beauty and wisdom in the simple and ordinary. We easily take this beautiful world and its many messages and lessons for granted. Don’t wait for extraordinary moments to take your breath away, look to nature and bring that beauty into all that you do and every moment of your life. Tending a garden, folding laundry, consoling your child having a tantrum or cooking a meal- all regular tasks take on a sacred quality when we perform them with the total involvement, acceptance and love.

What is one lesson nature has taught you? You can share it with us in the comment section below 🙂

The scientist who predicted ice-sheet collapse — 50 years ago


A seminal 1968 study warned of the demise of the West Antarctic Ice Sheet.
Antarctic glacier

It’s 50 years since scientists first suggested that the West Antarctic Ice Sheet could melt away.

Fifty years ago, many scientists were looking up. In 1968, the Russians sent the first animals to orbit the Moon (including a couple of tortoises), and NASA’s Apollo programme kicked into gear to produce the first views of Earth from space. But in Antarctica, John Mercer was looking down — and he was concerned about what he saw.

That year, the late Mercer, a glaciologist at Ohio State University in Columbus, first warned about the potential for rapid sea-level rise from melting ice caps. His landmark paper drew on fieldwork at the Reedy Glacier, which feeds into West Antarctica’s Ross Sea (J. H. Mercer Int. Assoc. Sci. Hydrol. Symp. 79, 217–225; 1968). Geological evidence from a former lake, located at an altitude of 1,400 metres in the Transantarctic Mountains, suggested that the area was once awash with open water and floating icebergs. Mercer took that as evidence that the entire West Antarctic Ice Sheet had once melted away.

The paper was an intriguing synthesis of the science of the times. Using multiple lines of evidence, Mercer sought to explain how sea levels could have risen by 6 metres in the previous interglacial period, around 120,000 years ago. The melting of Greenland or the East Antarctic Ice Sheet could not explain it, because both are located on solid earth and would respond relatively slowly to warming. By contrast, much of the West Antarctic Ice Sheet is grounded well below sea level. That makes it a “uniquely vulnerable and unstable body of ice”, Mercer wrote.

Many credit a 1974 paper by Johannes Weertman, a geophysicist at Northwestern University in Evanston, Illinois, with providing a technical explanation for how such a massive ice sheet could disintegrate (J. Weertman J. Glaciol. 13, 3–11; 1974). And the late Bob Thomas, a NASA glaciologist, spent years investigating and explaining how floating ice shelves acted as corks, stemming the flow of land-bound glaciers into the sea. But Mercer still deserves credit for sounding the alarm.

It took a while for the idea to take hold. Advanced numerical ice-sheet models developed in the late 1980s tended to downplay the risk of rapid ice loss from western Antarctica, and the Intergovernmental Panel on Climate Change suggested in its 1995 report that Antarctica as a whole was stable. But evidence to the contrary mounted: the massive Larsen A and B ice shelves collapsed in 1995 and 2002, respectively, followed by a major rift in Larsen C in 2017. In 2014, a team of scientists declared that the loss of ice in the Amundsen Sea Embayment had accelerated and appeared “unstoppable”.

The future of the ice sheet, which holds enough water to boost global sea levels by more than three metres, is now at the top of the Antarctic research agenda. Scientists are still scouring the world for palaeoclimate records to pin down past sea-level change, modellers are refining their calculations and fieldwork continues apace. As early as next month, the US National Science Foundation and the UK National Environmental Research Council are expected to jointly announce the recipients of a US$25-million fund for research on the future of the Thwaites glacier, which flows into the Amundsen Sea. Satellite measurements indicate that melting there has doubled in the past several years, and now accounts for roughly 10% of the global sea-level rise.

In a 1978 paper in Nature, Mercer updated his arguments in clear and elegant terms. “A disquieting thought is that if the present highly simplified climatic models are even approximately correct,” he wrote, “this deglaciation may be part of the price that must be paid in order to buy enough time for industrial civilisation to make the changeover from fossil fuels to other sources of energy” (J. H. Mercer Nature 271, 321–325; 1978).

That thought still rings frighteningly true. Thus far, the 2015 Paris climate agreement, which commits the world to limiting warming to 1.5–2 °C, remains intact, despite the objections of US President Donald Trump. But grand commitments aside, the governments of the world, and by extension the citizens that they represent, have yet to demonstrate that they are up to the task of reducing greenhouse-gas emissions quickly enough to avert the most disastrous consequences.

Fifty years is the blink of an eye in geological terms, but it is long enough for science to raise its voice. It might feel like pushing against the tide, but researchers have to keep making the point that strong action on emissions could still prevent the worst. Without it, significant sea-level rise will become a certainty. In the long run, higher oceans could well become one of humanity’s most obvious self-inflicted wounds.

 

Source:Nature

How warp-speed evolution is transforming ecology


Darwin thought evolution was too slow to change the environment on observable timescales. Ecologists are discovering that he was wrong.
T. bartmani stick insect on plant

The coloration of stick insects such as this Timema bartmani help it to hide, but might also affect the local ecology.

It took Timothy Farkas less than a week to catch and relocate 1,500 stick insects in the Santa Ynez mountains in southern California. His main tool was an actual stick.

“It feels kind of brutish,” says Farkas. “You just pick a stick up off the ground and beat the crap out of a bush.” That low-tech approach dislodged hordes of stick insects that the team easily plucked off the dirt.

On this hillside outside Santa Barbara, there are two kinds of bush that the stick insect (Timema cristinae) inhabits. The creature comes in two corresponding colorations: green and striped. Farkas and his fellow ecologists knew that the stick insects had evolved to blend in with their surroundings. But the researchers wanted to see whether they could turn this relationship around, so that an evolved trait — camouflage — would affect the organism’s ecology.

To find out, the team relocated mixtures of green and striped insects to different plants, so that some insects’ coloration clashed with their new home. Suddenly maladapted, these insects became targets for hungry birds, and that caused a domino effect1. Birds drawn to bushes with mismatched stick insects stuck around to eat other residents, such as caterpillars and beetles, stripping some plants clean. “That this evolutionary force can cause local extinction is striking,” says Farkas, an ecologist at the University of New Mexico in Albuquerque. “It affects the entire community.” All this happened because of an out-of-place evolutionary trait.

Ecologists have generally ignored evolution when studying their systems; they thought it was impossible to test whether such a slow process could change ecosystems on observable timescales. But they have come to realize that evolution can happen more quickly than they assumed, and a wave of studies has capitalized on this idea to observe evolution and ecology in unison.

Such eco-evolutionary dynamics could be important for understanding how new populations emerge, or for predicting when one might go extinct. Experiments suggest that evolutionary changes alter some ecosystems just as much as shifts in more-conventional ecological elements, such as the amount of light reaching a habitat. “Eco-evolutionary dynamics is the dragon lots of people are chasing right now,” says Troy Simon, an ecologist at the University of Georgia in Athens.

Rapid evolution can sometimes offset some of the detrimental effects of a warming climate and other known drivers of change; in other cases, it can worsen those effects. Even for the most common processes, such as changes in population size or food chains, ecologists must take evolution into consideration, researchers say. “Everybody realized rapid evolution was occurring everywhere,” says evolutionary ecologist Andrew Hendry of McGill University in Montreal, Canada.

Darwin in reverse

It all goes back to Charles Darwin’s finches. When the naturalist visited Ecuador’s Galapagos Islands in 1835, he documented some variation in the beaks of finches living on different islands and eating different foods. Years after the voyage, he hinted in his Journal of Researches that this variation suggested a tight relationship between the birds’ ecology and their evolution.

Darwin never imagined seeing this in action, because he thought that evolution occurs only at the “long lapse of ages”. But by the late 1990s, ecologists had started to realize that evolution could be observed within a few generations of a given species — a timescale that they could work with.

Organisms that live and die quickly provided some of the early data demonstrating how evolution influences ecology. A key study2 published in 2003 focused on algae and rotifers, microscopic predators that feed on algae; both species can tick through up to 20 generations in the course of a couple of weeks. The study mixed the organisms together in tanks and showed that when algae evolve rapidly, they throw off normal predator–prey population dynamics.

Usually, the two species play out a cycle between ‘boom’ and ‘bust’. The algal population grows; the rotifers then gobble them up and their own population explodes. When the predators have depleted the algae, their numbers crash. The algae then rebound and the pattern starts again. But when the researchers introduced different algal varieties — seeding some genetic diversity — the algae began to evolve rapidly and the cycle changed completely. The algal population remained elevated for longer, and the rotifers’ own boom was abnormally delayed because the new algae were more resistant to predation.

Similar studies in aphids3 and water fleas4 have confirmed that rapid evolution can affect characteristics of populations, such as how fast they grow. These ecological changes can alter future rounds of evolution and selection. Seeing such rapid evolution in action has changed ecologists’ picture of what they thought was a predictable and fundamental ecological process, and showed how important it is to consider evolution when studying how populations interact. “Everything about ecology has to be re-examined in light of the fact that evolution is more important than we thought,” says Stephen Ellner, an ecologist at Cornell University in Ithaca, New York. “This changes everything.”

Fake lakes

After these initial lab studies, ecologists started to think bigger. Experiments conducted indoors at small scales can’t reproduce the intricacies of natural ecosystems, so researchers have been testing their ideas in grander, less artificial set-ups.

Working out whether eco-evolutionary dynamics affect the real world is one of the field’s biggest challenges, says Rebecca Best, an evolutionary ecologist at Northern Arizona University in Flagstaff, because so many uncontrollable factors can affect wild ecosystems.

She has found a middle ground by incorporating natural elements into a tightly controlled experiment. At a site overlooking Lake Lucerne in Switzerland, she and her team set up 50 miniature lakes: large plastic tanks each holding 1,000 litres of water, plus a slurry of sediment, plant life, algae, invertebrates and water collected from three lakes — Geneva, Constance and Lucerne. Once these ‘mesocosms’ were settled, with plankton reproducing and plants taking root, the team introduced into each tank one of two genetically distinct lineages of adult threespine sticklebacks (Gasterosteus aculeatus): one lineage from Lake Constance and the other from Lake Geneva. A few weeks later, the researchers removed the fish and replaced them with a mixture of lab-raised juveniles from both locations, plus some hybrids of the two lineages.

They found5 that how the adults had manipulated their environments affected the survival of the next generation of fish (see ‘Fishy feedback’). If the adult fish removed prey of a certain size, for example, younger fish that shared characteristics with the adults — in this case, mouth size — went hungry. Juveniles that were different from the former occupants fared better. The study showed that the traits of the adult fish shaped the environment for the next generation — enough to dictate the evolutionary trajectory of those that followed.

Best says that her mesocosm experiments are more sophisticated and realistic than lab studies, but less easy to control. Ideally, she says, the team would run the experiment in the field, but that would come with its own obstacles, such as having to factor in the evolution of other species in the ecosystem, or the risk of events such as extreme storms.

Experiments such as Best’s are “vastly easier and more controlled than anything you can do in nature”, Hendry says. But they might not reflect what happens in real ecosystems. “That’s the watershed moment we’re at right now. Does this actually play out in the real world?”

In the messy real world, it can be difficult to pinpoint the impact of a single feature, either an ecological attribute (such as rainfall) or an evolutionary one (such as a change in camouflage).

A few intrepid ecologists are trying anyway. Last year, a study6 on guppies in Trinidad demonstrated that the fish’s evolution can drive an ecological change as strongly as an environmental factor: the amount of light available.

The study focused on two populations of guppies (Poecilia reticulata) in the northern part of the island. Their habitats differ in several ecological characteristics, including how much shade they receive from the forest canopy, which affects how many algae grow in the streams.

The team moved populations of guppies — which differed in evolved traits such as body proportions and colour — between eight rivers in the watershed, and measured the canopy above the water. In some of the study sites, introducing a new kind of guppy altered algal populations as much as allowing 20% more light to stream onto the water did. Even a natural ecosystem, say the researchers, is a product of evolution as well as ecology.

This experiment did use a more natural setting than many others, but Trinidadian guppies are ecological celebrities that have appeared in hundreds of studies, and the rivers they inhabit have been highly manipulated already. Researchers want to know whether the forces at work in the guppy populations also play out in species that are not necessarily famous for evolutionary dynamics, says McGill ecologist Gregor Fussmann. “We need systems that are generic,” he says.

Lizard limbs

That’s exactly what Thomas Schoener, an evolutionary ecologist at the University of California, Davis, and his team have set out to do with two populations of lizard in the Bahamas. Their project is part of an ongoing multigenerational study, begun in 1977. They have been attempting to simulate accelerated evolution by catching curly-tailed lizards (Leiocephalus carinatus) and moving them to a string of tiny islands inhabited by brown anoles (Anolis sagrei), to see how the ecosystems change as a result.

Curly-tails are natural predators of the smaller brown anole, so when the team first moved the curly-tails onto islands with the anoles, populations of the latter dropped7. Spider populations increased when anoles — their main predator — took a hit, and the excess spiders then ate more springtail insects (Collembola). Researchers spotted surviving anoles fleeing to the trees to escape their new predator, and that triggered damage to plants. The team knew from previous work8 that anoles adapt fairly quickly to tree climbing by favouring shorter-limbed offspring.

Curly-tailed lizard in Cuba

A curly tailed lizard (Leiocephalus carinatus).Credit: Dov Makabaw Cuba/Alamy

But then something unexpected happened. Hurricane Irene hit the islands in 2011, followed by Hurricane Sandy in 2012. Populations of both anoles and curly-tailed lizards crashed. On some islands, anoles were completely wiped out after the storm.

“The hurricanes are a mixed blessing because on the one hand, they give us all kinds of interesting data about disturbance,” Schoener says. “But on the other hand, it can slow down what might be a normal progression of evolution.”

The team has managed to keep its project on track, and is observing evolutionary changes in leg length and the lizards’ re-colonization of the islands after the hurricane.

Surprisingly, the anoles that survived the storm have longer limbs than the pre-hurricane population7 — the opposite of the team’s prediction, but perhaps better for holding on to branches tightly during a storm. The team has just received funding to study how this evolutionary change will affect the ecosystem.

The hurricanes certainly complicated Schoener’s study, but other researchers appreciate the unplanned intervention because it provides a chance to study the consequences of real events and watch the lizards recolonize the islands. Even in the absence of a natural disaster, any number of dynamics could also change the course of an organism’s evolution, says Best. “Those potential interactions are going on for everything in the ecosystem.”

She and others say there is plenty more to do, both in the lab and in more-elaborate field studies. Some researchers want to add genetic data to their work, to understand what is driving evolution in the first place. This would tell them whether a particular trait — growth rate, for example — is truly heritable and evolving, rather than a characteristic that can be directly affected by an animal’s environment. Genomic data could also help to find hidden characteristics — those harder to observe than body size or growth rate — that might affect ecology.

In a study9 of algae and rotifers, Lutz Becks, an evolutionary ecologist at the Max Planck Institute for Evolutionary Biology in Plön, Germany, and his colleagues watched several cycles in which populations waxed and waned as the algae clumped together and dispersed. But when the team looked at individual genes underlying clumping behaviour, they found that their expression varied wildly from one cycle to the next, even though the clumping looked the same. They have since observed co-evolution of three species at once — algae, rotifers and a virus — and found10 that the rotifers slowed the rate at which the algae and virus co-evolved. The team plans to repeat this type of experiment, analysing genome data to see how specific details of the algal and viral genes change over time. “We’d like to get to a point where we can actually predict what genomic architecture might be needed for rapid evolution,” says Becks.

Rapid evolution can offset — at least partially — the damaging effects of climate change and other ecological disturbances. In 2011, for instance, a group led by Ellner reanalysed11 35 years of data from dormant eggs of Daphnia water fleas, exhumed from a sediment core in Lake Constance. The data represented periods before, during and after a time when the lake was affected by blooms of cyanobacteria, a microbe with low nutritional value for Daphnia. The team found that as the Daphnia’s food became less nutritious, juvenile fleas grew poorly and ended up as smaller adults. But after several generations, evolutionary changes caused the growth rate of juveniles to return to normal. And the adults regained some of their lost stature, although they didn’t reach the same size as they had before the blooms. The researchers suggest that rapid evolution is likely to occur most often when the environment is changing, but the effects are hidden because they pull in opposite directions. “Evolution is going to be part of how the biosphere responds to climate change,” Ellner says.

Farkas has these questions about evolution and ecology at the front of his mind as he beats the bushes around Santa Barbara and sorts his stick insects. He and his team are planning even more elaborate schemes. They want to catch a full feedback cycle unfolding — ecology affecting evolution affecting ecology once more — all while collecting genetic data. “Comparing how large these effects of evolution will be and understanding when and where evolution is happening is going to be important,” says Farkas. “To me, it’s the final frontier. But it’s going to take a really long time.”

 

Source: Nature

“Talk to the Trees” – A Simple Exercise to Develop Your Communication with Nature


Nature has lessons for those who listen. Communicating, interacting, and listening to nature is easier than we think. In fact, it’s natural for us to do so. Nature is  aware and sensitive to our feelings, thoughts, and emotions, and trees are able to sense our energy and communicate with us — as long as we are open to the possibility.

Science has proven that trees can talk to one another, but just in a different way than we do. Resource sharing happens throughout the forest. Old trees help seedlings, and healthy trees will help sick trees to recover. And we can communicate with them too. This is why it is so important to honour and respect all plant life, no matter where they are in their life cycle. They all play a very important role — and there is a great deal we can learn, if we take the time to communicate with them, one to one.

 

“Talk to the Trees” — A Communication Exercise

Try placing your hands upon a tree, or sit beneath it, or lie in the grass and stare up into its branches.

Relax and bring a smile to your face. Smiling is a simple way to relax our energy and open us up to the peaceful, loving energy of Nature.

You could try asking, “Is there anything that you would like me to know?”

You do not have to speak out loud, as our thoughts are energy and trees can hear them.

You may hear the tree answer, or you may just get a feeling from the tree and you will know how it feels.

Nature tends to communicate through telepathy, which is mostly received in pictures, so if you close your eyes, you may see pictures and you will feel like you are watching a movie.

And you can communicate by showing pictures of what you are feeling and thinking.

Of course, you can tell the tree anything that you would like it to know.

Use your imagination, as this is how Nature communicates with us.

I like to find faces in the trees as well. Sometimes there will only be an eye, but sometimes I can find a whole face hidden within the bark.

It really does feel easy to communicate when you can see a face staring back at you. And it doesn’t have to be a human face. Sometimes it is the shape of an animal face that I see.

And as I have come to know, “The older the tree, the more faces you see.” But if you don’t find a face, it does not matter. All life can communicate with us if we are open. I also like to place my hands on the trees and share my healing energy with them.

Simply imagine your energy, healing and radiating, like a glowing ball in the palms of your hands. Focus on this feeling and then feel it flowing from you and being shared with the tree. It often becomes easier to communicate with them after I do this because the pathways of communication are more open.

Simply taking a walk among the trees, spending time with them, and being open to hearing them is enough to hear what they are saying.

When we approach nature with sincerity, we are capable of communication.

I hope that you have found this helpful! I would love to hear from you. I can be contacted via my website www.jessieklassen.com. And while you’re there, feel free to subscribe my free “weeklyish” newsletter where I share advice, wisdom, and lessons that I have learned from living my life close to Nature.

Much love and many blessings,

Jessie

The Sapling: An Inspiring Story From the Trees

By Jessie Klassen…

Learn how to communicate with Nature while enjoying fun activities and energy exercises that will encourage spiritual growth, self-confidence, and awareness in you and your child while developing a close relationship with Nature.

In “The Sapling”, author Jessie Klassen offers an inspiring story from the Trees for the children of Earth, with vivid, full colour Nature illustrations that will appeal to younger children and provide valuable Life lessons that will grow with your child — just like a Tree! Full colour demonstrations easily display dozens of activities and exercises for you and your child to enjoy.

How Forest Bathing Gave Me A Whole New Perspective On the Idea of “Natural Healing”


A foray into the world of talking to trees helped me become not just more mindful, but more adventurous.
Forest-Sunlight-Person-Walking

“Think of a question you have about your life. Now find a tree and place your hands on the bark. Feel the tree. Listen to the tree. Don’t leave until you have your answer.”

This was a literal thing that was said to me during my first “forest bath”—a mindfulness-based hike I was hoping wouldn’t be too hippie-dippy. Spoiler alert: it was. Rather than some profound pearl of wisdom from Mother Nature herself, all I could hear was the sound of my own voice inside my head saying, What the hell are you doing here? Do you think they’ll notice if you leave?

As a health and wellness journalist, I like cold, hard, clinical science. I abstain from any activity involving incense, I have absolutely zero desire to try Ayahuasca. But I’m also curious, and I’m a sucker for a good hike, which is how I ended up non-ironically hugging a tree with 10 strangers.

“Forest bathing” may sound like new age-y nonsense, but the core philosophy is really about how being outdoors can facilitate calming of the mind and body.

A forest bath, I’d learned after I’d RSVP’d to a friend’s event, involves no literal bathing. (Thank god, since the idea of stripping down like a wood nymph would have crossed way too many lines for me. To say nothing of the splinters!) Called shinrin-yoku in Japan where the practice was first created, forest bathing is simply the practice of mindfully walking through a natural space—touching the bark on the trees, inhaling the cool mossy scent of the dirt, feeling the splashes of sunlight peeking through the leaves and falling on your skin. It’s a literal manifestation of stopping to smell the roses.

“Mindfulness in nature, or forest bathing, provides an opportunity to calm the mind and body, while also being supported by nature,” Nina Smiley, Ph.D., director of mindfulness programing at the postcard-worthy Mohonk Mountain House in upstate New York, and author of Mindfulness in Nature, told me much later.

In theory, it sounded lovely. In practice, it meant I was talking to trees. I was instructed to “place” all my anxieties on a leaf and then cast it away, a little ship of worries now owned by the breeze. I munched on vegan chocolate bark made with flowers foraged from the trail. I worried increasingly about my relationship to reality with every step.

After we’d sat in a circle in a meadow reflecting on Mother Nature’s messages and closed the experience mindfully, I high-tailed it to find the nearest Uber, pizza, and glass of wine. My dip into the world of forest bathing had brought me way the hell out of my comfort zone.

The transformation of my mood after forest bathing was not a single epiphany so much as a slow, natural healing process that enhanced my ability to be mindful in otherwise chaotic situations.

https://www.instagram.com/p/BZEkQuBDanX/embed/captioned/?cr=1&v=2&wp=685#%7B%22ci%22%3A0%2C%22os%22%3A173469.50500000003%7D

But in the days following, I started noticing some odd after-bath effects: a calmer response to emails that would have normally thrown me into a tailspin of anxiety, an awareness of the dozens of shades of green in the tree outside my window, a sense of how the air in San Francisco where I live always smells marine and deliciously briney, even in its more urban center.

Then there was a slowly awakening sense of curiosity in new things, even a desire for adventure. One of the main themes of the bath was focusing on the idea that nature is a powerful and pervasive force—no matter what’s going on in our busy, social media-saturated lives, Mama Nature will always have our backs. We meditated on being empowered by that—taking more risks and following our inner compasses. Coincidentally or not, I found myself doing more of that in the weeks after the forest bath, saying yes to more things out of my comfort zone and ditching some of the narrow-minded hang ups that had kept me from trying new things (like any restaurant that put flowers on the menu, the meditation class at my yoga studio, or the makeup counter at Whole Foods).

When I stopped to reflect several weeks later, I realized my new age-y encounter with the wood nymphs had actually made a pretty significant mark.

Though it freaked me out, I wanted more. So I scheduled a hike with Smiley—a true expert in forest bathing—on her turf at Mohonk just before peak foliage season (picture hiking trails with views so good you can almost see all the way to Manhattan and a crystal clear lake that looks like it was taken from a Wes Anderson film) to ask her about how a skeptic like me could be more open-minded about adopting some of the tenets of forest bathing.

“It has long been known that nature nurtures,” she told me. “The interest in forest bathing speaks to the desire for ways to calm, center, and strengthen the body, mind, and spirit. Minds are saturated with information overload, and many feel the need to be constantly multi-tasking and digitally connected, creating an addiction to busyness.” Guilty.

With an expert guide, I hit the trail with a different set of priorities (and a formal request to skip any hugging of and/or talking to trees), namely to slow down and use all of my senses to really take in each moment rather than focus on getting a workout or a view, like I normally would on a hike. “The vibe of forest bathing is very different,” Smiley says. “Once you’re outdoors, forest bathing is only a mindful breath away. Once you understand how to do this—and how it feels to calm the body and clear the mind—you can do this walking down a city street, appreciating nature in the middle of an urban setting.”

It turns out that forest bathing doesn’t actually require a forest at all, just an outdoor space where I can be fully present.

Three hours later, I was stepping off a bus in midtown Manhattan at rush hour—a place I’d usually hug a thousand trees to avoid. Normally, I’d grab a cab and get the f**k out, but in the spirit of a little mindful adventure, I decided to walk through the heart of the city and practice some of what Smiley was preaching. I was surprised to find there’s a shocking number of flowers in Times Square—on an early fall night, with the right mindset, it’s almost a lovely stroll.

At first, my hang up with forest bathing was the new age-y nature of the idea—and to be clear, I’m still skeptical about the whole getting answers from trees thing. But the surprising sense of being more open-minded and adventurous about integrating natural healing into my wellness routine and—at the risk of sounding too hippie-dippy—my life, made me a convert. “Bringing the principles of forest bathing into everyday life means understanding that being fully present in the moment is a powerful way to enhance well-being,” Smiley says. If that means opening my eyes and mind wide enough to find a moment of serenity even in the concrete jungle, I’m willing to at least try to see the forest for the trees.

Machine learning predicts the look of stem cells.


 No two stem cells are identical, even if they are genetic clones. This stunning diversity is revealed today in an enormous publicly available online catalogue of 3D stem cell images. The visuals were produced using deep learning analyses and cell lines altered with the gene-editing tool CRISPR. And soon the portal will allow researchers to predict variations in cell layouts that may foreshadow cancer and other diseases.

The Allen Cell Explorer, produced by the Allen Institute for Cell Science in Seattle, Washington, includes a growing library of more than 6,000 pictures of induced pluripotent stem cells (iPS) — key components of which glow thanks to fluorescent markers that highlight specific genes.

The Cell Explorer complements ongoing projects by several groups that chart the uniqueness of single cells at the level of DNA, RNA and proteins. Rick Horwitz, director of the Allen Institute for Cell Science, says that the institute’s images may hasten progress in stem cell research, cancer research and drug development by revealing unexpected aspects of cellular structure. “You can’t predict the outcome of a football game if you know stats on all the players but have never watched a game.”

Looking skin deep

The project began about a year ago with adult skin cells that had been reprogrammed into an embryonic-like, undifferentiated state. Horwitz and his team then used CRISPR–Cas9 to insert tags in genes to make structures within the cells glow. The genes included those that code for proteins that highlight actin filaments, which help cells to move and maintain their shape. It quickly became clear that the cells, which were all genetic clones from the same parent cell, varied in the placement, shape and number of their components, such as mitochondria and actin fibres.

Computer scientists analysed thousands of the images using deep learning programs and found relationships between the locations of cellular structures. They then used that information to predict where the structures might be when the program was given just a couple of clues, such as the position of the nucleus. The program ‘learned’ by comparing its predictions to actual cells.

The deep learning algorithms are similar to those that companies use to predict people’s preferences, Horwitz says. “If you buy a chainsaw at Amazon, it might then show you chain oil and plaid shirts.”

The 3D interactive tool based on this deep learning capability should go live later this year. At the moment, the site shows a preview of how it will work using side-by-side comparisons of predicted and actual images.

Benjamin Freedman, a cell biologist at the University of Washington in Seattle, looks forward to playing with the Cell Explorer’s predictive function once the Allen Institute team has taught their algorithm to recognize more iPS cells that have been changed genetically or chemically. For example, Freedman says he could delete a gene related to kidney disease in one of the fluorescently tagged stem cells from the Allen Institute and see how the mutation affects the glowing structure. Then he could use the site’s modelling tool to determine how other cellular components might be altered. “Ultimately,” Freedman says, “we want to understand processes at the cellular level that cause disease in the kidney as a whole.”

Filling in the holes

In the coming months, Allen Institute researchers will update the site with images of stem cells at different stages of cell division, and as they transform into distinct cell types, such as heart and kidney cells. Catching cells at different time points can be crucial to identifying fundamental processes, says Horwitz.

Structural differences in the DNA (purple) and cellular membrane (blue) of genetically identical stem cells.

The Allen Institute’s visual emphasis on stem cells dovetails with a number of efforts to catalogue other aspects of cells. For example, the London-based charity Cancer Research UK is creating interactive virtual-reality models of breast cancer cells in tumours. And an international effort called the Human Cell Atlas seeks to define all human cell types in terms of their molecular profiles, including DNA sequences, RNA transcripts and proteins.

Aviv Regev, a computational biologist at the Broad Institute in Cambridge, Massachusetts, who is working on the Human Cell Atlas, says that the Allen Cell Explorer complements her project by focusing on the look of cellular features as opposed to how genes, RNA and proteins interact within the cell. “The community is accepting that there are a lot of differences between cells that we thought were the same until recently,” she says, “so now we’re taking an unbiased approach to learn about pieces in the puzzle we didn’t know existed before.”

Source:Nature

Lurking HIV detected by scientists in a major biomarker discovery.


Scientists have discovered a unique protein that gives away the presence of inactive HIV in the body.

Sniffing out these hidden caches of the virus is something researchers have been trying to do for decades. Now that we have a lead, the finding could speed up research on a cure.

 Thanks to modern antiretroviral therapies, for many people, HIV is not the death sentence it once was. But we still don’t have a reliable way of permanently flushing it out of someone’s system.

Drugs can keep the virus in check, but unfortunately HIV has a major weapon – it stows away in secret reservoirs in the immune system. There it lies dormant until conditions are more suitable to re-emerge.

That’s why people infected with HIV have to spend a lifetime on expensive drugs, because the virus can take only weeks to come back from its latent state if drug treatment is stopped.

Those nasty secret reservoirs HIV creates are located in long-lived immune cells known as resting T cells. Because the virus hijacks these cells and integrates its genetic material into the DNA of the patient, it makes reservoir T cells extremely hard to track down.

Now a team of French scientists has managed to achieve this important milestone in HIV research by discovering a biomarker that exists only on the surface of T cells that harbour the latent virus.

“Since 1996, the dream has been to kill these nasty cells in hiding, but we had no way to do it because we had no way to recognise them,” says virologist Monsef Benkirane from University of Montpellier in France.

 Benkirane’s team discovered that a specific protein, called CD32a, hangs out on the surface of T cells with a latent HIV infection, but is not found on uninfected T cells, or even T cells with active HIV.

This is huge. Having CD32a as a biomarker for HIV reservoirs means scientists have a better chance to track them down in a patient’s blood. This paves the way for more research into the mechanisms that allow HIV to create such reservoirs in the first place.

Armed with such knowledge, scientists could then find ways to actually get rid of these HIV nests for good.

The team first detected the protein in a lab-made model of HIV infection, before moving on to test it as a biomarker in actual blood samples from 12 people who live with HIV and are receiving treatment.

They separated T cells with CD32a from other T cells in the blood samples, and found that the cells with this particular protein indeed had latent HIV harboured inside them.

Unfortunately, it’s not a smoking gun in every case, since the protein was found only on about half of all latently infected T cells.

Douglas Richman from University of California San Diego, who wasn’t involved in the research, writes that “the eradication of latent HIV would require a much greater reduction in the number of latently infected cells in the body.”

But it’s an extremely encouraging first step in the long search for a marker that could help us track down the nasty virus once it goes into hiding.

Tony Fauci, director of the US National Institute of Allergies and Infectious Disease, told Nature that a good next step would be to replicate the findings in more blood samples from a larger variety of patients who have the virus.

It’s still way too soon to say that we’re on the path to an actual HIV cure, but the news is super-exciting to researchers who have been hammering away at this problem for decades.

“I really hope this is correct,” says Fauci. “The fact that this work has been done by such competent investigators, and the data looks good, makes me optimistic.”

In a world where HIV continues to be a major health issue, this discovery indeed gives cause for optimism.

About 36.7 million people around the world live with HIV, but only 17 million have access to antiretroviral therapy, according to data from the US CDC.

The scientists have already filed a patent for the diagnostic and therapeutic use of the new biomarker.

Soource:Nature.

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