Inducible nitric oxide synthase is a major intermediate in signaling pathways for the survival of plasma cells.

While a number of extrinsic factors are known to promote the survival of plasma cells (PCs), the signaling intermediates involved remain poorly characterized. Here we identified inducible nitric oxide synthase (iNOS) as an intermediate that supported the survival of PCs. PCs deficient in iNOS (Nos2(-/-) PCs) showed enhanced death in vitro, after transfer into congenic adoptive hosts, and in chimeras made with wild-type and Nos2(-/-) bone marrow. The iNOS-mediated protection involved activation of protein kinase G and modulation of endoplasmic reticulum stress components. Activation of caspases was also diminished. We found that iNOS was required for PCs to respond to some prosurvival mediators associated with bone marrow stromal cells and that at least one mediator, interleukin 6, fed directly into this pathway by inducing iNOS.

G protein gamma subunit, a hidden master regulator of GPCR signaling.

Heterotrimeric G proteins (αßγ subunits) that are activated by G protein-coupled receptors (GPCRs) mediate the biological responses of eukaryotic cells to extracellular signals. The α subunits and the tightly bound ßγ subunit complex of G proteins have been extensively studied and shown to control the activity of effector molecules. In contrast, the potential roles of the large family of γ subunits have been less studied. In this review, we focus on present knowledge about these proteins. Induced loss of individual γ subunit types in animal and plant models result in strikingly distinct phenotypes indicating that γ subtypes play important and specific roles. Consistent with these findings, downregulation or upregulation of particular γ subunit types result in various types of cancers. Clues about the mechanistic basis of γ subunit function have emerged from imaging the dynamic behavior of G protein subunits in living cells. This shows that in the basal state, G proteins are not constrained to the plasma membrane but shuttle between membranes and on receptor activation ßγ complexes translocate reversibly to internal membranes. The translocation kinetics of ßγ complexes varies widely and is determined by the membrane affinity of the associated γ subtype. On translocating, some ßγ complexes act on effectors in internal membranes. The variation in translocation kinetics determines differential sensitivity and adaptation of cells to external signals. Membrane affinity of γ subunits is thus a parsimonious and elegant mechanism that controls information flow to internal cell membranes while modulating signaling responses.

An optogenetic model reveals cell shape regulation through FAK and fascin.

Cell shape regulation is important, but the mechanisms that govern shape are not fully understood, in part due to limited experimental models in which cell shape changes and underlying molecular processes can be rapidly and non-invasively monitored in real time. Here, we used an optogenetic tool to activate RhoA in the middle of mononucleated macrophages to induce contraction, resulting in a side with the nucleus that retained its shape and a non-nucleated side that was unable to maintain its shape and collapsed. In cells overexpressing focal adhesion kinase (FAK; also known as PTK2), the non-nucleated side exhibited a wide flat morphology and was similar in adhesion area to the nucleated side. In cells overexpressing fascin, an actin-bundling protein, the non-nucleated side assumed a spherical shape and was similar in height to the nucleated side. This effect of fascin was also observed in fibroblasts even without inducing furrow formation. Based on these results, we conclude that FAK and fascin work together to maintain cell shape by regulating adhesion area and height, respectively, in different cell types. This article has an associated First Person interview with the first author of the paper.

Retroperitoneal Cyst of Mullerian Type.

Retroperitoneal mullerian cysts are rare, benign neoplastic cyst of urogenital subtype. They are usually asymptomatic and may present with symptoms if they grow considerably in size with pressure over the adjacent organ or follow infection, hemorrhage or rupture. Histologically, these cyst are lined with benign ciliated columnar epithelium. We present the case of a 30-year-old female with history of abdominal distension and epigastric pain. The mass excised was in retroperitoneal space and microscopic examination revealed benign cyst of mullerian origin.

Association of Different Biochemical and Hemodynamic Characteristic with Type 2 Diabetes Mellitus and Hypertension in Nephrolithiasis Patients.

Background Although Nephrolithiasis is a common condition caused by a wide variety of metabolic or environmental disturbances, its being one of the major factor of morbidity. Incidence of kidney stone disease (KSD) is highly affected by metabolic disorders and change in blood pressure and glucose. Objective To find out association of different biochemical and hemodynamic parameters with various glycemic status and hypertension in kidney stone disease. Method A cross sectional study was conducted in patients diagnosed as nephrolithiasis by using re¬nal ultrasonography and underwent nephrectomy between January 2019 to January 2021 in Shahid Dharmabhakta National Transplant Centre (SDNTC). A total of 100 subjects with 60 male and 40 females were enrolled. Glycemic status was categorized based on criteria of American Diabetes Association (ADA) and hypertension was defined as BP ≥ 140/90 mm Hg in right arm supine position. All biochemical and hemodynamic profile was carried out following standard protocol. Result Out of 100 patients enrolled, pre-diabetes accounted for 31% followed by diabetes (4%). However, hypertension comprised of 66% in total subjects. Serum urea, cholesterol and triglyceride level were found to be increased by 84.6%, 67.7% and 64.7% respectively in diabetes followed by increase of 3.9%, 19.5% and 3.1% respectively in prediabetes when compared to normal glycemic condition in nephrolithiasis subjects. Serum fasting blood glucose, creatinine and uric acid level was observed significantly higher (p=0.003, p=0.004, p < 0.001 respectively) in hypertensive patients. Duration of hospital stay was also seen positively correlated with hypertension. Conclusion Not only diabetes, prediabetes also manifests the increased risk of kidney stone disease along with hypertension. There is significant impairment in renal function and lipid profile based on diabetes mellitus and hypertension. Early identifying these systemic diseases, different biochemical and hemodynamic parameters and proper treatment accordingly may minimize risk and prevent serious complication in these patients.

Perception of Medical Students Regarding TU-IOM MBBS Curriculum and Teaching Learning Methods in Nepal.

Background The present Bachelor of Medicine and Bachelor of Surgery (MBBS) curriculum under Tribhuvan University - Institute of Medicine (TU-IOM) was last revised twelve-years back. Though the curriculum was built upon internationally approved recommendations on curriculum design, it is ineffectively practiced in most medical schools of Nepal with major focus on didactic teaching-learning. The curriculum, hence, needs effective implementation and revision. Objective To identify the strengths, weaknesses, and areas of improvement in the medical curriculum through student-based feedback and outline the possibility of incorporating newer evidence-based teaching-learning methodologies in Nepal. Method This is a descriptive and cross-sectional study. With appropriate ethical approval, a questionnaire was developed and disseminated virtually to all medical students of Nepal under TU from MBBS fourth year onwards. The questionnaire comprised of Likert and close-ended questions. The data analysis was followed after receiving the filled questionnaire through Google forms. Result A total of 337 respondents participated in the study. The most effectively implemented components out of the SPICES model were Integrated learning (I) and Communitybased learning (C), with 73.89% and 68.84% responses. There were 94.7% (319) students who favored the incorporation of research in the core curriculum. Only 34.2% (115) students found PowerPoint lectures, the most utilized form of teachinglearning in Nepal, as engaging. The respondents (84.6%) showed a high degree of readiness to incorporate newer evidence-based teaching-learning tools such as flipped learning, blended learning, and peer-to-peer learning. Conclusion This study shows that effective interventions must be rethought on various aspects of the curriculum, taking students' feedback on the table while considering curricular revision.

Appendiceal Neuroendocrine Tumor Mimicking Acute Appendicitis.

Appendiceal carcinoids are the most frequent tumors arising from the appendix, comprising between 32 and 57% of all the appendiceal tumors. The gross appearance of the appendix showed perforation at the tip with 30 ml of periappendicular collection. On histopathological examination, carcinoid tumor on the tip of appendix was found with tumor cells arranged in tubules, acini and nests infiltrating the muscularis propria and sub serosa. Perineural and vascular invasion was not seen. Immunohistochemistry for Synaptophysin was positive with Ki-67 labelling index of 2%. We present a case of appendiceal neuroendocrine tumor with the chief complaint of acute appendicitis.

Correlation between Sonoelastographic, Doppler and Histopathological Findings in Chronic Kidney Disease Patients in Tertiary Care Centre.

Background Ultrasound (USG) with Doppler examination of intrarenal vessels is the imaging modality of choice employed in patients with renal failure and is commonly performed early in the clinical course. The pulsatility index (PI) and the resistive index (RI) of downstream renal artery have been found to correlate with renal vascular resistance, filtration fraction and effective renal plasma flow in chronic renal failure. Pathological process in any tissues alters their elastic properties which can be assessed non-invasively through newer technique like elastography. Objective To correlate the findings obtained by sonoelastographic, doppler and histopathological studies in chronic kidney disease patients. Method Study was done in 146 patients referred to Department of Radiodiagnosis and Imaging, TUTH for native renal biopsy. Renal sonographic morphology (length, echogenicity, cortical thickness), Sonoelastography (Young's modulus) and Doppler parameters (peak systolic velocity, resistive index) were assessed. The grading of estimated GFR (eGFR) was calculated based on chronic kidney disease (CKD) criteria. Result Among 146 patients, 63 (43.2%) were females and 83 (56.8%) were males. Maximum patients were in age group of 41-50 years (25.3%) followed by age group 51-60 years (24%). Mean age of patient was 42.06±14.70 for males and 39.57±12.54 females. Maximum mean Young's modulus was seen in eGFR stage G1 with 46.57±19.51 kPa followed by in stage G3a with 36.46±10.01 kPa and observed to be statistically non-significant (p=0.172). However, statistical significance difference was noted between the resistive index and elastographic measurement of Young's modulus (r=0.462, p=0.0001). Minimum mean cortical thickness was seen in eGFR stage G5 with 4.42±1.48 mm followed by stage G4 with 5.57±1.24 mm (p= 0.0001). Cortical thickness is decreasing as eGFR stage was increasing in our study (p=0.0001). Resistive index is increasing with decrease in renal size (r=-0.202, p=0.015). Conclusion Ultrasonography along with doppler study and elastography have limited role in diagnosing the pathology of chronic kidney disease, however, it has significant role in the disease progression.

Pharmaceutical Aspects and Clinical Evaluation of COVID-19 Vaccines.

COVID-19, the disease caused by the novel severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), was first detected in December 2019 and has since morphed into a global pandemic claiming over 2.4 million human lives and severely impacting global economy. The race for a safe and efficacious vaccine was thus initiated with government agencies as well as major pharmaceutical companies as frontrunners. An ideal vaccine would activate multiple arms of the adaptive immune system to generate cytotoxic T cell responses as well as neutralizing antibody responses, while avoiding pathological or deleterious immune responses that result in tissue damage or exacerbation of the disease. Developing an effective vaccine requires an inter-disciplinary effort involving virology, protein biology, biotechnology, immunology and pharmaceutical sciences. In this review, we provide a brief overview of the pathology and immune responses to SARS-CoV-2, which are fundamental to vaccine development. We then summarize the rationale for developing COVID-19 vaccines and provide novel insights into vaccine development from a pharmaceutical science perspective, such as selection of different antigens, adjuvants, delivery platforms and formulations. Finally, we review multiple clinical trial outcomes of novel vaccines in terms of safety and efficacy.

Lipid Profile and Ultrasonographic Grading in Alcoholic and Non Alcoholic Fatty Liver Patients.

Background Fatty liver disease (FLD) is a common and major chronic liver disease. It has been implicated that patients have disorders of lipid metabolism and are involved in the pathogenesis of fatty liver. Hence, it was designed to observe the association between lipid profile and fatty liver disease. Objective This study was undertaken to evaluate the association of lipid profile status, hemoglobin and albumin levels with fatty liver disease patients diagnosed based on ultrasonography (USG). Method This Cross-sectional study was undertaken in the Department of Internal Medicine with the collaboration of the Department of Radiology and Department of Biochemistry, Universal College of Medical Sciences-Teaching Hospital (UCMS-TH), Bhairahawa, Nepal from March 2019 to February 2020 in a total of 100 patients diagnosed with fatty liver disease by ultrasonography. The fasting blood was collected for lipid profile and carried out in the automated analyzer following standard protocol. Result In 100 cases, the male to female ratio was 1.8:1. Fifty six percent of the total cases presented with alcoholic fatty liver disease (AFLD) while the remaining 44% with nonalcoholic fatty liver disease (NAFLD). The spectrum of lipid abnormality was observed with increased total cholesterol (TC), Low Density Lipoprotein (LDL), increased triglycerides (TG), Very Low Density Lipoprotein (VLDL) in alcoholic fatty liver disease cases as compared to nonalcoholic fatty liver disease cases. However, it has been observed that TG/HDL and Non-HDL/HDL were higher in nonalcoholic fatty liver disease as compared to alcoholic fatty liver disease. Moreover, a statistically significant difference was observed in HDL between AFLG2 and NAFLG2 (p-value: 0.012). Conclusion Dyslipidemia and decreased HDL have been implicated in fatty liver diseases. USG in conjunction with Non-HDL/HDL, TG/HDL, hemoglobin, and albumin can be useful in early screening and monitoring of dyslipidemia in fatty liver patients.

Cartilaginous Choristoma of Tonsil: A hidden clinical entity.

Choristoma is a tumor like mass which is an ectopic rest of normal tissue due to embryological developmental defect. The presence of choristoma in tonsil is extremely rare. On histopathological examination, mature hyaline cartilage were found surrounded by lymphoid follicles. We present a case of cartilaginous choristoma with the complaint of recurrent tonsillitis.

Surveillance for pathology associated with cancer on endoscopy (SPACE): criteria to identify high-risk gastric polyps in familial adenomatous polyposis.

BACKGROUND AND AIMS: Gastric cancer is an extracolonic manifestation of familial adenomatous polyposis (FAP) and is associated with high-risk gastric polyps. There are no known endoscopic criteria to identify these high-risk polyps. Our aim was to develop endoscopic criteria to identify high-risk polyps on endoscopy in FAP. METHODS: We prospectively collected 150 gastric polyps in consecutive patients undergoing surveillance EGD at the Cleveland Clinic. Pictures were taken of each polyp under narrow-band imaging and high-definition white light. In an exploratory phase, 5 endoscopists developed consensus criteria using the images to distinguish high-risk (pyloric gland adenoma, tubular adenoma, hyperplastic) from low-risk (fundic gland with low-grade or no dysplasia) polyps. In the assessment phase, endoscopists were blinded to polyp pathology and used the criteria to predict the individual polyp risk category. To measure diagnostic accuracy, we reported the mean sensitivity, specificity, and interrater agreement (κ). RESULTS: Consensus criteria were developed based on 16 low-risk and 9 high-risk polyps. The final 149 polyps consisted of 128 low-risk and 22 high-risk polyps (1 polyp was excluded from analysis). Using the criteria, the 5 endoscopists distinguished high- from low-risk polyps with a mean sensitivity and specificity of 79% (16.3%) and 78.8% (10.8%), respectively. The κ coefficient was .45, indicating moderate agreement. CONCLUSIONS: We developed endoscopic criteria to distinguish between high- and low-risk polyps associated with gastric cancer in FAP. The criteria provide guidance to endoscopists in targeting high-risk polyps while surveying the stomach of patients with proximal gastric polyposis.

Colonic Decompression Reduces Proximal Acute Colonic Pseudo-obstruction and Related Symptoms.

BACKGROUND: Colonoscopic decompression is performed in inpatients for management of acute colonic pseudo-obstruction. Evidence for its efficacy is limited to small descriptive studies published before the use of neostigmine for acute colonic pseudo-obstruction. Furthermore, therapeutic end points were not defined. OBJECTIVE: The aim was to compare the effectiveness of colonic decompression with standard medical therapy (supportive and pharmacologic therapy) to standard medical therapy alone. DESIGN: This is a retrospective, propensity-matched study. SETTING: The study was conducted at a tertiary care center. PATIENTS: Inpatients with first diagnosis of acute colonic pseudo-obstruction between 2000 and 2016 were selected. INTERVENTIONS: The intervention group received colonic decompression as well as supportive and/or pharmacologic therapy. The control group did not receive colonic decompression. MAIN OUTCOME MEASURES: The primary outcome was the resolution of overall colonic dilation on imaging 48 hours following colonic decompression or the initiation of standard medical therapy alone. Secondary outcomes included symptom improvement, colonic segment diameter percentage change, perforation, 30-day readmission, and all-cause mortality. RESULTS: The standard medical therapy and colonic decompression groups included 61 and 83 patients. Of the patients who underwent colonic decompression, 47.7% had complete resolution of acute colonic pseudo-obstruction versus 19.9% of patients who underwent standard medical therapy (p < 0.001). There were no significant differences in mid or distal colon diameter reduction between groups. The 30-day readmission rate was 15.7% in the colonic decompression group versus 26.2% in the standard medical therapy group. No immediate adverse events were noted in either group. Thirty-day all-cause mortality was 8.4% for the colonic decompression group and 14.8% in the standard medical therapy group. LIMITATIONS: The study was a retrospective review on a highly comorbid population. CONCLUSIONS: Colonic decompression is effective compared to standard medical therapy alone for proximal colonic dilation or symptoms associated with acute colonic pseudo-obstruction. On segmental analysis, colonic decompression does not provide any additional benefit over standard medical therapy in improving transverse or distal colonic dilation. See Video Abstract at http://links.lww.com/DCR/B32. LA DESCOMPRESIÓN COLÓNICA REDUCE LA PSEUDOOBSTRUCCIÓN COLÓNICA AGUDA PROXIMAL Y LOS SÍNTOMAS RELACIONADOS.: La descompresión colonica se realiza en pacientes hospitalizados para el tratamiento de la pseudoobstrucción colónica aguda. La evidencia de su eficacia se limita a pequeños estudios descriptivos antes del uso de neostigmina para la pseudoobstrucción colónica aguda. Además, los puntos finales terapéuticos no se definieron.El objetivo fue comparar la efectividad de la descompresión colónica mas el tratamiento médico estándar (tratamiento de apoyo y farmacológico) contra el tratamiento médico estándar solamente.Este es un estudio retrospectivo de propensión coincidente.El estudio se realizó en un centro de atención de tercer nivel.Pacientes hospitalizados con diagnóstico de pseudoobstrucción colónica aguda entre 2000 y 2016.El grupo de intervención recibió descompresión colónica, así como tratamiento de apoyo o farmacológica. El grupo control no recibió descompresión colónica.La medida de resultado primaria fue la resolución de la dilatación colónica general en la imagen 48 horas después de la descompresión colónica o el inicio del tratamiento médico estándar solo. Los resultados secundarios incluyeron mejoría de los síntomas, cambio porcentual en el diámetro del segmento colónico, perforación, reingreso a los 30 días y mortalidad por cualquier causa.La terapia médica estándar y los grupos de descompresión colónica incluyeron 61 y 83 pacientes, respectivamente. El 47,7% de los pacientes con descompresión colónica tuvieron una resolución completa de la pseudoobstrucción colónica aguda frente al 19,9% de los pacientes con terapia médica estándar (p < 0,001). No hubo diferencias significativas en la reducción del diámetro del colon medio o distal entre los grupos. La tasa de reingreso a los 30 días fue del 15,7% en el grupo de descompresión colónica frente al 26,2% en el grupo de tratamiento médico estándar. No se observaron eventos adversos inmediatos en ninguno de los dos grupos. La mortalidad por cualquier causa a los 30 días fue del 8.4% para la descompresión del colon y del 14.8% en los grupos de terapia médica estándar.El estudio fue una revisión retrospectiva en una población altamente comórbida.La descompresión colónica es efectiva en comparación con el tratamiento médico estándar solo para la dilatación del colon proximal o los síntomas asociados con la pseudoobstrucción colónica aguda. En el análisis segmentario, la descompresión colónica no proporciona ningún beneficio adicional sobre el tratamiento médica estándar para mejorar la dilatación colónica transversal o distal. Vea el resumen del video en http://links.lww.com/DCR/B32.

Exosomes Represent an Immune Suppressive T Cell Checkpoint in Human Chronic Inflammatory Microenvironments.

Background: T cells present in chronic inflammatory tissues such as nasal polyps (from chronic rhinosinusitis patients) have been demonstrated to be hypo-responsive to activation via the TCR, similar to tumor-specific T cells in multiple different human tumor microenvironments. While immunosuppressive exosomes have been known to contribute to the failure of the tumor-associated T cells to respond optimally to activation stimuli, it is not known whether they play a similar role in chronic inflammatory microenvironments. In the current study, we investigate whether exosomes derived from chronic inflammatory microenvironments contribute to the immune suppression of T cells. Methods: Exosomes were isolated by ultracentrifugation and characterized by size and composition using nanoparticle tracking analysis, scanning electron microscopy, antibody arrays and flow exometry. Immunosuppressive ability of the exosomes was measured by quantifying its effect on activation of T cells, using nuclear translocation of NFκB as an activation endpoint. Results: Exosomes were isolated and characterized from two different types of chronic inflammatory tissues - nasal polyps from chronic rhinosinusitis patients and synovial fluid from rheumatoid arthritis patients. These exosomes arrest the activation of T cells stimulated via the TCR. This immune suppression, like that which is seen in tumor microenvironments, is dependent in part upon a lipid, ganglioside GD3, which is expressed on the exosomal surface. Conclusion: Immunosuppressive exosomes present in non-malignant chronic inflammatory tissues represent a new T cell checkpoint, and potentially represent a novel therapeutic target to enhance the response to current therapies and prevent disease recurrences.

Sialic Acid-Dependent Inhibition of T Cells by Exosomal Ganglioside GD3 in Ovarian Tumor Microenvironments.

The tumor microenvironment is rendered immunosuppressive by a variety of cellular and acellular factors that represent potential cancer therapeutic targets. Although exosomes isolated from ovarian tumor ascites fluids have been previously reported to induce a rapid and reversible T cell arrest, the factors present on or within exosomes that contribute to immunosuppression have not been fully defined. In this study, we establish that GD3, a ganglioside expressed on the surface of exosomes isolated from human ovarian tumor ascites fluids, is causally linked to the functional arrest of T cells activated through their TCR. This arrest is inhibited by Ab blockade of exosomal GD3 or by the removal of GD3+ exosomes. Empty liposomes expressing GD3 on the surface also inhibit the activation of T cells, establishing that GD3 contributes to the functional arrest of T cells independent of factors present in exosomes. Finally, we demonstrate that the GD3-mediated arrest of the TCR activation is dependent upon sialic acid groups, because their enzymatic removal from exosomes or liposomes results in a loss of inhibitory capacity. Collectively, these data define GD3 as a potential immunotherapeutic target.

Seasonal Variation in Helminth Parasites of Snakeheads Channa Punctatus and Channa Striatus (Perciformes: Channidae) in Uttar Pradesh, India.

Snakehead fishes are widely consumed throughout South East Asia, China and India because of their good taste of meat and high nutrient values such as presence of prostaglandins, thromboxane and Omega-6 fatty acid. Parasitic infection constitutes significant economic loss in fish production. The aim of this work was to study the seasonal variation of helminths in snakeheads. In the presented study, a three-year survey has been performed. A total of 1013 individuals of Channa punctatus and 247 individuals of Channa striatus were examined. A total of 3783 helminths were collected, with an average of 3.02 helminths/fish. 43.50 % individuals of C. punctatus and 59.10 % of C. striatus were found to be infected with acanthocephalans, trematodes, nematodes and cestodes per year. The prevalence and mean abundance of Pallisentis sp. was at its peak in summer. However the prevalence of trematodes, nematodes and cestodes was at peak during autumn. Mean abundance of nematodes was at peak in summer. Interestingly, the males were found more infected as compared to the females and the infection rate in males peaked in summer. In comparison to other weight groups, medium size hosts (21 - 40 g) were found more consistently infected. Thus the results indicate that there are seasonal variations in parasitic helminths infecting C. punctatus and C. striatus which also depend upon sex and weight. These variations may be attributed to various environmental and biological factors including parasite life cycle and immune level of host.

Dynamics of sustained use and abandonment of clean cooking systems: study protocol for community-based system dynamics modeling.

BACKGROUND: More than 3 billion of the world's population are affected by household air pollution from relying on unprocessed solid fuels for heating and cooking. Household air pollution is harmful to human health, climate, and environment. Sustained uptake and use of cleaner cooking technologies and fuels are proposed as solutions to this problem. In this paper, we present our study protocol aimed at understanding multiple interacting feedback mechanisms involved in the dynamic behavior between social, ecological, and technological systems driving sustained use or abandonment of cleaner cooking technologies among the rural poor in India. METHODS/DESIGN: This study uses a comparative case study design to understand the dynamics of sustained use or abandonment of cleaner cooking technologies and fuels in four rural communities of Rajasthan, India. The study adopts a community based system dynamics modeling approach. We describe our approach of using community based system dynamics with rural communities to delineate the feedback mechanisms involved in the uptake and sustainment of clean cooking technologies. We develop a reference mode with communities showing the trend over time of use or abandonment of cleaner cooking technologies and fuels in these communities. Subsequently, the study develops a system dynamics model with communities to understand the complex sub-systems driving the behavior in these communities as reflected in the reference mode. We use group model building techniques to facilitate participation of relevant stakeholders in the four communities and elicit a narrative describing the feedback mechanisms underlying sustained adoption or abandonment of cleaner cooking technologies. DISCUSSION: In understanding the dynamics of feedback mechanisms in the uptake and exclusive use of cleaner cooking systems, we increase the likelihood of dissemination and implementation of efficacious interventions into everyday settings to improve the health and wellbeing of women and children most affected by household air pollution. The challenge is not confined to developing robust technical solutions to reduce household air pollution and exposure to improve respiratory health, and prevent associated diseases. The bigger challenge is to disseminate and implement cleaner cooking technologies and fuels in the context of various social, behavioral, and economic constraints faced by poor households and communities. TRIAL REGISTRATION: The Institutional Review Board of Washington University in St. Louis has exempted community based system dynamics modeling from review.

Optically triggering spatiotemporally confined GPCR activity in a cell and programming neurite initiation and extension.

G-protein-coupled receptor (GPCR) activity gradients evoke important cell behavior but there is a dearth of methods to induce such asymmetric signaling in a cell. Here we achieved reversible, rapidly switchable patterns of spatiotemporally restricted GPCR activity in a single cell. We recruited properties of nonrhodopsin opsins--rapid deactivation, distinct spectral tuning, and resistance to bleaching--to activate native Gi, Gq, or Gs signaling in selected regions of a cell. Optical inputs were designed to spatiotemporally control levels of second messengers, IP3, phosphatidylinositol (3,4,5)-triphosphate, and cAMP in a cell. Spectrally selective imaging was accomplished to simultaneously monitor optically evoked molecular and cellular response dynamics. We show that localized optical activation of an opsin-based trigger can induce neurite initiation, phosphatidylinositol (3,4,5)-triphosphate increase, and actin remodeling. Serial optical inputs to neurite tips can refashion early neuron differentiation. Methods here can be widely applied to program GPCR-mediated cell behaviors.

Optical control demonstrates switch-like PIP3 dynamics underlying the initiation of immune cell migration.

There is a dearth of approaches to experimentally direct cell migration by continuously varying signal input to a single cell, evoking all possible migratory responses and quantitatively monitoring the cellular and molecular response dynamics. Here we used a visual blue opsin to recruit the endogenous G-protein network that mediates immune cell migration. Specific optical inputs to this optical trigger of signaling helped steer migration in all possible directions with precision. Spectrally selective imaging was used to monitor cell-wide phosphatidylinositol (3,4,5)-triphosphate (PIP3), cytoskeletal, and cellular dynamics. A switch-like PIP3 increase at the cell front and a decrease at the back were identified, underlying the decisive migratory response. Migration was initiated at the rapidly increasing switch stage of PIP3 dynamics. This result explains how a migratory cell filters background fluctuations in the intensity of an extracellular signal but responds by initiating directionally sensitive migration to a persistent signal gradient across the cell. A two-compartment computational model incorporating a localized activator that is antagonistic to a diffusible inhibitor was able to simulate the switch-like PIP3 response. It was also able simulate the slow dissipation of PIP3 on signal termination. The ability to independently apply similar signaling inputs to single cells detected two cell populations with distinct thresholds for migration initiation. Overall the optical approach here can be applied to understand G-protein-coupled receptor network control of other cell behaviors.

Optimizing optogenetic constructs for control over signaling and cell behaviours.

Optogenetic tools have recently been developed that enable dynamic control over the activities of select signaling proteins. They provide the unique ability to rapidly turn signaling events on or off with subcellular control in living cells and organisms. This capability is leading to new insights into how the spatial and temporal coordination of signaling events governs dynamic cell behaviours such as migration and neurite outgrowth. These tools can also be used to dissect a protein's signaling functions at different organelles. Here we review the properties of photoreceptors from diverse organisms that have been leveraged to control signaling in mammalian cells. We emphasize recent engineering approaches that have been used to create optogenetic constructs with optimized spectral, kinetic, and signaling properties for controlling cell behaviours.