Publications by authors named "Sarah Stanley"

89 Publications

Mapping of UV-C dose and SARS-CoV-2 viral inactivation across N95 respirators during decontamination.

Sci Rep 2021 10 13;11(1):20341. Epub 2021 Oct 13.

The UC Berkeley - UCSF Graduate Program in Bioengineering, University of California Berkeley, 308B Stanley Hall, Mailcode 1762, Berkeley, CA, 94720, USA.

During public health crises like the COVID-19 pandemic, ultraviolet-C (UV-C) decontamination of N95 respirators for emergency reuse has been implemented to mitigate shortages. Pathogen photoinactivation efficacy depends critically on UV-C dose, which is distance- and angle-dependent and thus varies substantially across N95 surfaces within a decontamination system. Due to nonuniform and system-dependent UV-C dose distributions, characterizing UV-C dose and resulting pathogen inactivation with sufficient spatial resolution on-N95 is key to designing and validating UV-C decontamination protocols. However, robust quantification of UV-C dose across N95 facepieces presents challenges, as few UV-C measurement tools have sufficient (1) small, flexible form factor, and (2) angular response. To address this gap, we combine optical modeling and quantitative photochromic indicator (PCI) dosimetry with viral inactivation assays to generate high-resolution maps of "on-N95" UV-C dose and concomitant SARS-CoV-2 viral inactivation across N95 facepieces within a commercial decontamination chamber. Using modeling to rapidly identify on-N95 locations of interest, in-situ measurements report a 17.4 ± 5.0-fold dose difference across N95 facepieces in the chamber, yielding 2.9 ± 0.2-log variation in SARS-CoV-2 inactivation. UV-C dose at several on-N95 locations was lower than the lowest-dose locations on the chamber floor, highlighting the importance of on-N95 dose validation. Overall, we integrate optical simulation with in-situ PCI dosimetry to relate UV-C dose and viral inactivation at specific on-N95 locations, establishing a versatile approach to characterize UV-C photoinactivation of pathogens contaminating complex substrates such as N95s.
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http://dx.doi.org/10.1038/s41598-021-98121-6DOI Listing
October 2021

Practical considerations for Ultraviolet-C radiation mediated decontamination of N95 respirator against SARS-CoV-2 virus.

PLoS One 2021 12;16(10):e0258336. Epub 2021 Oct 12.

Department of Molecular and Cell Biology, University of California, Berkeley, California, United States of America.

Decontaminating N95 respirators for reuse could mitigate shortages during the COVID-19 pandemic. Although the United States Center for Disease Control has identified Ultraviolet-C irradiation as one of the most promising methods for N95 decontamination, very few studies have evaluated the efficacy of Ultraviolet-C for SARS-CoV-2 inactivation. In addition, most decontamination studies are performed using mask coupons that do not recapitulate the complexity of whole masks. We sought to directly evaluate the efficacy of Ultraviolet-C mediated inactivation of SARS-CoV-2 on N95 respirators. To that end we created a portable UV-C light-emitting diode disinfection chamber and tested decontamination of SARS-CoV-2 at different sites on two models of N95 respirator. We found that decontamination efficacy depends on mask model, material and location of the contamination on the mask. Our results emphasize the need for caution when interpreting efficacy data of UV-C decontamination methods.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0258336PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8509861PMC
October 2021

SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA.

PLoS Biol 2021 Oct 11;19(10):e3001425. Epub 2021 Oct 11.

Biophysics Graduate Group, University of California, Berkeley, California, United States of America.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (COVID-19), a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry (CLMS) identified a region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. We also identified small molecules that alter the formation of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.
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http://dx.doi.org/10.1371/journal.pbio.3001425DOI Listing
October 2021

The B.1.427/1.429 (epsilon) SARS-CoV-2 variants are more virulent than ancestral B.1 (614G) in Syrian hamsters.

bioRxiv 2021 Aug 25. Epub 2021 Aug 25.

As novel SARS-CoV-2 variants continue to emerge, it is critical that their potential to cause severe disease and evade vaccine-induced immunity is rapidly assessed in humans and studied in animal models. In early January 2021, a novel variant of concern (VOC) designated B.1.429 comprising 2 lineages, B.1.427 and B.1.429, was originally detected in California (CA) and shown to enhance infectivity in vitro and decrease antibody neutralization by plasma from convalescent patients and vaccine recipients. Here we examine the virulence, transmissibility, and susceptibility to pre-existing immunity for B 1.427 and B 1.429 in the Syrian hamster model. We find that both strains exhibit enhanced virulence as measured by increased body weight loss compared to hamsters infected with ancestral B.1 (614G), with B.1.429 causing the most body weight loss among all 3 lineages. Faster dissemination from airways to parenchyma and more severe lung pathology at both early and late stages were also observed with B.1.429 infections relative to B.1. (614G) and B.1.427 infections. In addition, subgenomic viral RNA (sgRNA) levels were highest in oral swabs of hamsters infected with B.1.429, however sgRNA levels in lungs were similar in all three strains. This demonstrates that B.1.429 replicates to higher levels than ancestral B.1 (614G) or B.1.427 in the upper respiratory tract (URT) but not in the lungs. In multi-virus in-vivo competition experiments, we found that epsilon (B.1.427/B.1.429) and gamma (P.1) dramatically outcompete alpha (B.1.1.7), beta (B.1.351) and zeta (P.2) in the lungs. In the URT gamma, and epsilon dominate, but the highly infectious alpha variant also maintains a moderate size niche. We did not observe significant differences in airborne transmission efficiency among the B.1.427, B.1.429 and ancestral B.1 (614G) variants in hamsters. These results demonstrate enhanced virulence and high relative fitness of the epsilon (B.1.427/B.1.429) variant in Syrian hamsters compared to an ancestral B.1 (614G) strain.

Author Summary: In the last 12 months new variants of SARS-CoV-2 have arisen in the UK, South Africa, Brazil, India, and California. New SARS-CoV-2 variants will continue to emerge for the foreseeable future in the human population and the potential for these new variants to produce severe disease and evade vaccines needs to be understood. In this study, we used the hamster model to determine the epsilon (B.1.427/429) SARS-CoV-2 strains that emerged in California in late 2020 cause more severe disease and infected hamsters have higher viral loads in the upper respiratory tract compared to the prior B.1 (614G) strain. These findings are consistent with human clinical data and help explain the emergence and rapid spread of this strain in early 2021.
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http://dx.doi.org/10.1101/2021.08.25.457626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8404898PMC
August 2021

Optical Clearing and 3D Analysis Optimized for Mouse and Human Pancreata.

Bio Protoc 2021 Aug 5;11(15):e4103. Epub 2021 Aug 5.

Diabetes, Obesity, and Metabolism Institute, Division of Endocrinology, Diabetes, and Bone Disease, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

The pancreas is a heavily innervated organ, but pancreatic innervation can be challenging to comprehensively assess using conventional histological methods. However, recent advances in whole-mount tissue clearing and 3D rendering techniques have allowed detailed reconstructions of pancreatic innervation. Optical clearing is used to enhance tissue transparency and reduce light scattering, thus eliminating the need to section the tissue. Here, we describe a modified version of the optical tissue clearing protocol iDISCO+ (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) optimized for pancreatic innervation and endocrine markers. The protocol takes 13-19 days, depending on tissue size. In addition, we include protocols for imaging using light sheet and confocal microscopes and for 3D segmentation of pancreatic innervation and endocrine cells using Imaris.
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http://dx.doi.org/10.21769/BioProtoc.4103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8376577PMC
August 2021

Screening a Library of FDA-Approved and Bioactive Compounds for Antiviral Activity against SARS-CoV-2.

ACS Infect Dis 2021 08 15;7(8):2337-2351. Epub 2021 Jun 15.

School of Public Health, Division of Infectious Diseases and Vaccinology, University of California, Berkeley, Berkeley, California 94720, United States.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in over 168 million cases and 3.4 million deaths to date, while the number of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clinical compounds holds the potential for rapid identification of drugs effective against SARS-CoV-2. Here, we utilized a library of FDA-approved and well-studied preclinical and clinical compounds to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compounds that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compounds with anti-inflammatory activity, and compounds targeting host pathways such as kinases and proteases critical for SARS-CoV-2 replication. We identified seven compounds not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compound screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.
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http://dx.doi.org/10.1021/acsinfecdis.1c00017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8231672PMC
August 2021

The Innate Immune Response to Infection.

Annu Rev Immunol 2021 04 26;39:611-637. Epub 2021 Feb 26.

Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA; email:

Infection with causes >1.5 million deaths worldwide annually. Innate immune cells are the first to encounter , and their response dictates the course of infection. Dendritic cells (DCs) activate the adaptive response and determine its characteristics. Macrophages are responsible both for exerting cell-intrinsic antimicrobial control and for initiating and maintaining inflammation. The inflammatory response to infection is a double-edged sword. While cytokines such as TNF-α and IL-1 are important for protection, either excessive or insufficient cytokine production results in progressive disease. Furthermore, neutrophils-cells normally associated with control of bacterial infection-are emerging as key drivers of a hyperinflammatory response that results in host mortality. The roles of other innate cells, including natural killer cells and innate-like T cells, remain enigmatic. Understanding the nuances of both cell-intrinsic control of infection and regulation of inflammation will be crucial for the successful development of host-targeted therapeutics and vaccines.
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http://dx.doi.org/10.1146/annurev-immunol-093019-010426DOI Listing
April 2021

Virtual reality in specialist palliative care: a feasibility study to enable clinical practice adoption.

BMJ Support Palliat Care 2021 Feb 17. Epub 2021 Feb 17.

Palliative Care Unit, University of Liverpool, Liverpool, UK.

Background: The use of virtual reality (VR) is increasing in palliative care. However, despite increasing interest in VR, there is little evidence of how this technology can be implemented into practice.

Aims: This paper aims to: (1) explore the feasibility of implementing VR therapy, for patients and caregivers, in a hospital specialist inpatient palliative care unit and a hospice, and (2) to identify questions for organisations, to support VR adoption in palliative care.

Methods: The Samsung Gear VR system was used in a hospital specialist palliative inpatient unit and a hospice. Patients and caregivers received VR distraction therapy and provided feedback of their experience. Staff completed a feedback questionnaire to explore their opinion of the usefulness of VR in palliative care. A public engagement event was conducted, to identify questions to support implementation of VR in palliative care settings.

Results: Fifteen individuals (12 (80%) patients and 3 (20%) caregivers) participated. All had a positive experience. No adverse effects were reported. Ten items were identified for organisations to consider ahead of adoption of VR in palliative care. These were questions about: the purpose of VR; intended population; supporting evidence; session duration; equipment choice; infection control issues; content choice; setting of VR; person(s) responsible for delivery and the maintenance plan.

Conclusions: It is feasible to use VR therapy in palliative care; however, further evidence about its efficacy and effectiveness is needed. Palliative care practitioners considering VR use should carefully consider several factors, to ensure that this technology can be used safely and effectively in clinical practice.
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http://dx.doi.org/10.1136/bmjspcare-2020-002327DOI Listing
February 2021

Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.

PLoS One 2021 3;16(2):e0246647. Epub 2021 Feb 3.

Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California, United States of America.

Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited new waves of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. This study evaluates several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, isopropanol precipitation is shown to provide an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, direct addition of NP swab samples to RT-qPCRs is evaluated without an RNA extraction step. A simple, inexpensive swab collection solution suitable for direct addition is validated using contrived swab samples. Third, an open-source master mix for RT-qPCR is described that permits detection of viral RNA in NP swab samples with a limit of detection of approximately 50 RNA copies per reaction. Quantification cycle (Cq) values for purified RNA from 30 known positive clinical samples showed a strong correlation (r2 = 0.98) between this homemade master mix and commercial TaqPath master mix. Lastly, end-point fluorescence imaging is found to provide an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, open-source methods has the potential to reduce the time and expense of COVID-19 testing.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0246647PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857565PMC
February 2021

Rapid, point-of-care molecular diagnostics with Cas13.

medRxiv 2021 Apr 5. Epub 2021 Apr 5.

Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.

Rapid nucleic acid testing is a critical component of a robust infrastructure for increased disease surveillance. Here, we report a microfluidic platform for point-of-care, CRISPR-based molecular diagnostics. We first developed a nucleic acid test which pairs distinct mechanisms of DNA and RNA amplification optimized for high sensitivity and rapid kinetics, linked to Cas13 detection for specificity. We combined this workflow with an extraction-free sample lysis protocol using shelf-stable reagents that are widely available at low cost, and a multiplexed human gene control for calling negative test results. As a proof-of-concept, we demonstrate sensitivity down to 40 copies/μL of SARS-CoV-2 in unextracted saliva within 35 minutes, and validated the test on total RNA extracted from patient nasal swabs with a range of qPCR Ct values from 13-35. To enable sample-to-answer testing, we integrated this diagnostic reaction with a single-use, gravity-driven microfluidic cartridge followed by real-time fluorescent detection in a compact companion instrument. We envision this approach for Diagnostics with Coronavirus Enzymatic Reporting (DISCoVER) will incentivize frequent, fast, and easy testing.
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http://dx.doi.org/10.1101/2020.12.14.20247874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7755151PMC
April 2021

Repeated hypoglycemia remodels neural inputs and disrupts mitochondrial function to blunt glucose-inhibited GHRH neuron responsiveness.

JCI Insight 2020 11 5;5(21). Epub 2020 Nov 5.

Diabetes, Obesity and Metabolism Institute.

Hypoglycemia is a frequent complication of diabetes, limiting therapy and increasing morbidity and mortality. With recurrent hypoglycemia, the counterregulatory response (CRR) to decreased blood glucose is blunted, resulting in hypoglycemia-associated autonomic failure (HAAF). The mechanisms leading to these blunted effects are only poorly understood. Here, we report, with ISH, IHC, and the tissue-clearing capability of iDISCO+, that growth hormone releasing hormone (GHRH) neurons represent a unique population of arcuate nucleus neurons activated by glucose deprivation in vivo. Repeated glucose deprivation reduces GHRH neuron activation and remodels excitatory and inhibitory inputs to GHRH neurons. We show that low glucose sensing is coupled to GHRH neuron depolarization, decreased ATP production, and mitochondrial fusion. Repeated hypoglycemia attenuates these responses during low glucose. By maintaining mitochondrial length with the small molecule mitochondrial division inhibitor-1, we preserved hypoglycemia sensitivity in vitro and in vivo. Our findings present possible mechanisms for the blunting of the CRR, significantly broaden our understanding of the structure of GHRH neurons, and reveal that mitochondrial dynamics play an important role in HAAF. We conclude that interventions targeting mitochondrial fission in GHRH neurons may offer a new pathway to prevent HAAF in patients with diabetes.
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http://dx.doi.org/10.1172/jci.insight.133488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710320PMC
November 2020

A 3D atlas of the dynamic and regional variation of pancreatic innervation in diabetes.

Sci Adv 2020 Oct 9;6(41). Epub 2020 Oct 9.

Diabetes, Obesity, and Metabolism Institute, Division of Endocrinology, Diabetes and Bone Diseases, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Understanding the detailed anatomy of the endocrine pancreas, its innervation, and the remodeling that occurs in diabetes can provide new insights into metabolic disease. Using tissue clearing and whole-organ imaging, we identified the 3D associations between islets and innervation. This technique provided detailed quantification of α and β cell volumes and pancreatic nerve fibers, their distribution and heterogeneity in healthy tissue, canonical mouse models of diabetes, and samples from normal and diabetic human pancreata. Innervation was highly enriched in the mouse endocrine pancreas, with regional differences. Islet nerve density was increased in nonobese diabetic mice, in mice treated with streptozotocin, and in pancreata of human donors with type 2 diabetes. Nerve contacts with β cells were preserved in diabetic mice and humans. In summary, our whole-organ assessment allows comprehensive examination of islet characteristics and their innervation and reveals dynamic regulation of islet innervation in diabetes.
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http://dx.doi.org/10.1126/sciadv.aaz9124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7557000PMC
October 2020

SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA.

bioRxiv 2021 Mar 29. Epub 2021 Mar 29.

Biophysics Graduate Group, University of California, Berkeley CA.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes COVID-19, a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. Phase separation is driven, in part, by hydrophobic and electrostatic interactions. While the N protein forms spherical assemblies with unstructured RNA, it forms asymmetric condensates with viral RNA strands that contain secondary structure elements. Cross-linking mass spectrometry identified a region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. We also identified small molecules that alter the formation of N protein condensates. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.
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http://dx.doi.org/10.1101/2020.09.14.295824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523105PMC
March 2021

Acromelic dysplasias: how rare musculoskeletal disorders reveal biological functions of extracellular matrix proteins.

Ann N Y Acad Sci 2021 04 2;1490(1):57-76. Epub 2020 Sep 2.

Leni & Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai, New York, New York.

Acromelic dysplasias are a group of rare musculoskeletal disorders that collectively present with short stature, pseudomuscular build, stiff joints, and tight skin. Acromelic dysplasias are caused by mutations in genes (FBN1, ADAMTSL2, ADAMTS10, ADAMTS17, LTBP2, and LTBP3) that encode secreted extracellular matrix proteins, and in SMAD4, an intracellular coregulator of transforming growth factor-β (TGF-β) signaling. The shared musculoskeletal presentations in acromelic dysplasias suggest that these proteins cooperate in a biological pathway, but also fulfill distinct roles in specific tissues that are affected in individual disorders of the acromelic dysplasia group. In addition, most of the affected proteins directly interact with fibrillin microfibrils in the extracellular matrix and have been linked to the regulation of TGF-β signaling. Together with recently developed knockout mouse models targeting the affected genes, novel insights into molecular mechanisms of how these proteins regulate musculoskeletal development and homeostasis have emerged. Here, we summarize the current knowledge highlighting pathogenic mechanisms of the different disorders that compose acromelic dysplasias and provide an overview of the emerging biological roles of the individual proteins that are compromised. Finally, we develop a conceptual model of how these proteins may interact and form an "acromelic dysplasia complex" on fibrillin microfibrils in connective tissues of the musculoskeletal system.
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http://dx.doi.org/10.1111/nyas.14465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921208PMC
April 2021

Uncovering a possible role of reactive oxygen species in magnetogenetics.

Sci Rep 2020 08 4;10(1):13096. Epub 2020 Aug 4.

Department of Chemical and Biological Engineering, and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.

Recent reports have shown that intracellular, (super)paramagnetic ferritin nanoparticles can gate TRPV1, a non-selective cation channel, in a magnetic field. Here, we report the effects of differing field strength and frequency as well as chemical inhibitors on channel gating using a Ca-sensitive promoter to express a secreted embryonic alkaline phosphatase (SEAP) reporter. Exposure of TRPV1-ferritin-expressing HEK-293T cells at 30 °C to an alternating magnetic field of 501 kHz and 27.1 mT significantly increased SEAP secretion by ~ 82% relative to control cells, with lesser effects at other field strengths and frequencies. Between 30-32 °C, SEAP production was strongly potentiated 3.3-fold by the addition of the TRPV1 agonist capsaicin. This potentiation was eliminated by the competitive antagonist AMG-21629, the NADPH oxidase assembly inhibitor apocynin, and the reactive oxygen species (ROS) scavenger N-acetylcysteine, suggesting that ROS contributes to magnetogenetic TRPV1 activation. These results provide a rational basis to address the heretofore unknown mechanism of magnetogenetics.
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http://dx.doi.org/10.1038/s41598-020-70067-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403421PMC
August 2020

Repurposing erectile dysfunction drugs tadalafil and vardenafil to increase bone mass.

Proc Natl Acad Sci U S A 2020 06 8;117(25):14386-14394. Epub 2020 Jun 8.

The Mount Sinai Bone Program, Icahn School of Medicine at Mount Sinai, New York, NY 10029.

We report that two widely-used drugs for erectile dysfunction, tadalafil and vardenafil, trigger bone gain in mice through a combination of anabolic and antiresorptive actions on the skeleton. Both drugs were found to enhance osteoblastic bone formation in vivo using a unique gene footprint and to inhibit osteoclast formation. The target enzyme, phosphodiesterase 5A (PDE5A), was found to be expressed in mouse and human bone as well as in specific brain regions, namely the locus coeruleus, raphe pallidus, and paraventricular nucleus of the hypothalamus. Localization of PDE5A in sympathetic neurons was confirmed by coimmunolabeling with dopamine β-hydroxylase, as well as by retrograde bone-brain tracing using a sympathetic nerve-specific pseudorabies virus, PRV152. Both drugs elicited an antianabolic sympathetic imprint in osteoblasts, but with net bone gain. Unlike in humans, in whom vardenafil is more potent than tadalafil, the relative potencies were reversed with respect to their osteoprotective actions in mice. Structural modeling revealed a higher binding energy of tadalafil to mouse PDE5A compared with vardenafil, due to steric clashes of vardenafil with a single methionine residue at position 806 in mouse PDE5A. Collectively, our findings suggest that a balance between peripheral and central actions of PDE5A inhibitors on bone formation together with their antiresorptive actions specify the osteoprotective action of PDE5A blockade.
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http://dx.doi.org/10.1073/pnas.2000950117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321982PMC
June 2020

The ADAMTS/Fibrillin Connection: Insights into the Biological Functions of ADAMTS10 and ADAMTS17 and Their Respective Sister Proteases.

Biomolecules 2020 04 12;10(4). Epub 2020 Apr 12.

Orthopaedic Research Laboratories, Leni & Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Secreted a disintegrin-like and metalloprotease with thrombospondin type 1 motif (ADAMTS) proteases play crucial roles in tissue development and homeostasis. The biological and pathological functions of ADAMTS proteases are determined broadly by their respective substrates and their interactions with proteins in the pericellular and extracellular matrix. For some ADAMTS proteases, substrates have been identified and substrate cleavage has been implicated in tissue development and in disease. For other ADAMTS proteases, substrates were discovered in vitro, but the role of these proteases and the consequences of substrate cleavage in vivo remains to be established. Mutations in and cause Weill-Marchesani syndrome (WMS), a congenital syndromic disorder that affects the musculoskeletal system (short stature, pseudomuscular build, tight skin), the eyes (lens dislocation), and the heart (heart valve abnormalities). WMS can also be caused by mutations in fibrillin-1 (), which suggests that ADAMTS10 and ADAMTS17 cooperate with fibrillin-1 in a common biological pathway during tissue development and homeostasis. Here, we compare and contrast the biochemical properties of ADAMTS10 and ADAMTS17 and we summarize recent findings indicating potential biological functions in connection with fibrillin microfibrils. We also compare ADAMTS10 and ADAMTS17 with their respective sister proteases, ADAMTS6 and ADAMTS19; both were recently linked to human disorders distinct from WMS. Finally, we propose a model for the interactions and roles of these four ADAMTS proteases in the extracellular matrix.
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http://dx.doi.org/10.3390/biom10040596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226509PMC
April 2020

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA.

J Vis Exp 2020 02 12(156). Epub 2020 Feb 12.

Orthopaedic Research Laboratories, Leni & Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai;

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in C2C12 myoblasts can be applied to study the role of these proteins in skeletal muscle development. Here, we describe a protocol to deplete the ECM protein ADAMTSL2 as an example, using small-hairpin (sh) RNA in C2C12 cells. Following transfection of shRNA plasmids, stable cells were batch-selected using puromycin. We further describe the maintenance of these cell lines and the phenotypic analysis via mRNA expression, protein expression, and C2C12 differentiation. The advantages of the method are the relatively fast generation of stable C2C12 knockdown cells and the reliable differentiation of C2C12 cells into multinucleated myotubes upon depletion of serum in the cell culture medium. Differentiation of C2C12 cells can be monitored by bright field microscopy and by measuring the expression levels of canonical marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC) indicating the progression of C2C12 myoblast differentiation into myotubes. In contrast to the transient knockdown of genes with small-interfering (si) RNA, genes that are expressed later during C2C12 differentiation or during myotube maturation can be targeted more efficiently by generating C2C12 cells that stably express shRNA. Limitations of the method are a variability in the knockdown efficiencies, depending on the specific shRNA that may be overcome by using gene knockout strategies based on CRISPR/Cas9, as well as potential off-target effects of the shRNA that should be considered.
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http://dx.doi.org/10.3791/60824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553128PMC
February 2020

Evaluation of a digitally-enabled care pathway for acute kidney injury management in hospital emergency admissions.

NPJ Digit Med 2019 31;2:67. Epub 2019 Jul 31.

3Department of Applied Health Research, University College London, 1-19 Torrington Place, London, WC1E 7HB UK.

We developed a digitally enabled care pathway for acute kidney injury (AKI) management incorporating a mobile detection application, specialist clinical response team and care protocol. Clinical outcome data were collected from adults with AKI on emergency admission before (May 2016 to January 2017) and after (May to September 2017) deployment at the intervention site and another not receiving the intervention. Changes in primary outcome (serum creatinine recovery to ≤120% baseline at hospital discharge) and secondary outcomes (30-day survival, renal replacement therapy, renal or intensive care unit (ICU) admission, worsening AKI stage and length of stay) were measured using interrupted time-series regression. Processes of care data (time to AKI recognition, time to treatment) were extracted from casenotes, and compared over two 9-month periods before and after implementation (January to September 2016 and 2017, respectively) using pre-post analysis. There was no step change in renal recovery or any of the secondary outcomes. Trends for creatinine recovery rates (estimated odds ratio (OR) = 1.04, 95% confidence interval (95% CI): 1.00-1.08,  = 0.038) and renal or ICU admission (OR = 0.95, 95% CI: 0.90-1.00,  = 0.044) improved significantly at the intervention site. However, difference-in-difference analyses between sites for creatinine recovery (estimated OR = 0.95, 95% CI: 0.90-1.00,  = 0.053) and renal or ICU admission (OR = 1.06, 95% CI: 0.98-1.16,  = 0.140) were not significant. Among process measures, time to AKI recognition and treatment of nephrotoxicity improved significantly ( < 0.001 and 0.047 respectively).
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http://dx.doi.org/10.1038/s41746-019-0100-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669220PMC
July 2019

Implementation of a Digitally Enabled Care Pathway (Part 1): Impact on Clinical Outcomes and Associated Health Care Costs.

J Med Internet Res 2019 07 15;21(7):e13147. Epub 2019 Jul 15.

Royal Free London NHS Foundation Trust, London, United Kingdom.

Background: The development of acute kidney injury (AKI) in hospitalized patients is associated with adverse outcomes and increased health care costs. Simple automated e-alerts indicating its presence do not appear to improve outcomes, perhaps because of a lack of explicitly defined integration with a clinical response.

Objective: We sought to test this hypothesis by evaluating the impact of a digitally enabled intervention on clinical outcomes and health care costs associated with AKI in hospitalized patients.

Methods: We developed a care pathway comprising automated AKI detection, mobile clinician notification, in-app triage, and a protocolized specialist clinical response. We evaluated its impact by comparing data from pre- and postimplementation phases (May 2016 to January 2017 and May to September 2017, respectively) at the intervention site and another site not receiving the intervention. Clinical outcomes were analyzed using segmented regression analysis. The primary outcome was recovery of renal function to ≤120% of baseline by hospital discharge. Secondary clinical outcomes were mortality within 30 days of alert, progression of AKI stage, transfer to renal/intensive care units, hospital re-admission within 30 days of discharge, dependence on renal replacement therapy 30 days after discharge, and hospital-wide cardiac arrest rate. Time taken for specialist review of AKI alerts was measured. Impact on health care costs as defined by Patient-Level Information and Costing System data was evaluated using difference-in-differences (DID) analysis.

Results: The median time to AKI alert review by a specialist was 14.0 min (interquartile range 1.0-60.0 min). There was no impact on the primary outcome (estimated odds ratio [OR] 1.00, 95% CI 0.58-1.71; P=.99). Although the hospital-wide cardiac arrest rate fell significantly at the intervention site (OR 0.55, 95% CI 0.38-0.76; P<.001), DID analysis with the comparator site was not significant (OR 1.13, 95% CI 0.63-1.99; P=.69). There was no impact on other secondary clinical outcomes. Mean health care costs per patient were reduced by £2123 (95% CI -£4024 to -£222; P=.03), not including costs of providing the technology.

Conclusions: The digitally enabled clinical intervention to detect and treat AKI in hospitalized patients reduced health care costs and possibly reduced cardiac arrest rates. Its impact on other clinical outcomes and identification of the active components of the pathway requires clarification through evaluation across multiple sites.
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http://dx.doi.org/10.2196/13147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693300PMC
July 2019

Implementation of a Digitally Enabled Care Pathway (Part 2): Qualitative Analysis of Experiences of Health Care Professionals.

J Med Internet Res 2019 07 15;21(7):e13143. Epub 2019 Jul 15.

Department of Applied Health Research, University College London, London, United Kingdom.

Background: One reason for the introduction of digital technologies into health care has been to try to improve safety and patient outcomes by providing real-time access to patient data and enhancing communication among health care professionals. However, the adoption of such technologies into clinical pathways has been less examined, and the impacts on users and the broader health system are poorly understood. We sought to address this by studying the impacts of introducing a digitally enabled care pathway for patients with acute kidney injury (AKI) at a tertiary referral hospital in the United Kingdom. A dedicated clinical response team-comprising existing nephrology and patient-at-risk and resuscitation teams-received AKI alerts in real time via Streams, a mobile app. Here, we present a qualitative evaluation of the experiences of users and other health care professionals whose work was affected by the implementation of the care pathway.

Objective: The aim of this study was to qualitatively evaluate the impact of mobile results viewing and automated alerting as part of a digitally enabled care pathway on the working practices of users and their interprofessional relationships.

Methods: A total of 19 semistructured interviews were conducted with members of the AKI response team and clinicians with whom they interacted across the hospital. Interviews were analyzed using inductive and deductive thematic analysis.

Results: The digitally enabled care pathway improved access to patient information and expedited early specialist care. Opportunities were identified for more constructive planning of end-of-life care due to the earlier detection and alerting of deterioration. However, the shift toward early detection also highlighted resource constraints and some clinical uncertainty about the value of intervening at this stage. The real-time availability of information altered communication flows within and between clinical teams and across professional groups.

Conclusions: Digital technologies allow early detection of adverse events and of patients at risk of deterioration, with the potential to improve outcomes. They may also increase the efficiency of health care professionals' working practices. However, when planning and implementing digital information innovations in health care, the following factors should also be considered: the provision of clinical training to effectively manage early detection, resources to cope with additional workload, support to manage perceived information overload, and the optimization of algorithms to minimize unnecessary alerts.
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http://dx.doi.org/10.2196/13143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693304PMC
July 2019

Robotic technology for palliative and supportive care: Strengths, weaknesses, opportunities and threats.

Palliat Med 2019 09 28;33(8):1106-1113. Epub 2019 Jun 28.

7 Department of Computer Science, University of Liverpool, Liverpool, UK.

Background: Medical robots are increasingly used for a variety of applications in healthcare. Robots have mainly been used to support surgical procedures, and for a variety of assistive uses in dementia and elderly care. To date, there has been limited debate about the potential opportunities and risks of robotics in other areas of palliative, supportive and end-of-life care.

Aim: The objective of this article is to examine the possible future impact of medical robotics on palliative, supportive care and end-of-life care. Specifically, we will discuss the strengths, weaknesses, opportunities and threats (SWOT) of this technology.

Methods: A SWOT analysis to understand the strengths, weaknesses, opportunities and threats of robotic technology in palliative and supportive care.

Results: The opportunities of robotics in palliative, supportive and end-of-life care include a number of assistive, therapeutic, social and educational uses. However, there are a number of technical, societal, economic and ethical factors which need to be considered to ensure meaningful use of this technology in palliative care.

Conclusion: Robotics could have a number of potential applications in palliative, supportive and end-of-life care. Future work should evaluate the health-related, economic, societal and ethical implications of using this technology. There is a need for collaborative research to establish use-cases and inform policy, to ensure the appropriate use (or non-use) of robots for people with serious illness.
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http://dx.doi.org/10.1177/0269216319857628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691596PMC
September 2019

HIF-1α as a central mediator of cellular resistance to intracellular pathogens.

Curr Opin Immunol 2019 10 20;60:111-116. Epub 2019 Jun 20.

Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, CA, United States; School of Public Health, Division of Infectious Diseases and Vaccinology, University of California, Berkeley, CA, United States. Electronic address:

Hypoxia-inducible transcription factor-1α (HIF-1α) was originally identified as a master regulator of cellular responses to hypoxia. More recently, HIF-1α has emerged as a critical regulator of immune cell function that couples shifts in cellular metabolism to cell type-specific transcriptional outputs. Activation of macrophages with inflammatory stimuli leads to induction of the metabolic program aerobic glycolysis and to HIF-1α stabilization, which reinforce one another in a positive feedback loop that helps drive macrophage activation. This activation of aerobic glycolysis and HIF-1α is important both for production of inflammatory cytokines, such as IL-1β, and for cell intrinsic control of infection. Here, we review the importance of HIF-1α for control of bacterial, fungal, and protozoan intracellular pathogens, highlighting recent findings that reveal mechanisms by which HIF-1α is activated during infection and how HIF-1α coordinates antimicrobial responses of macrophages.
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http://dx.doi.org/10.1016/j.coi.2019.05.005DOI Listing
October 2019

Bioelectrical impedance vector analysis (BIVA) as a method to compare body composition differences according to cancer stage and type.

Clin Nutr ESPEN 2019 04 1;30:59-66. Epub 2019 Mar 1.

Palliative Care Institute Liverpool, University of Liverpool, Liverpool, United Kingdom; Academic Palliative and End of Life Care Centre, Royal Liverpool and Broadgreen University NHS Hospitals Trust, Liverpool, United Kingdom.

Background & Aims: Bioelectrical impedance vector analysis (BIVA) is a non-invasive method of measuring human body composition. This offers the potential to evaluate nutritional and hydration states in cancer. Analysis of BIVA data using the z-score method (the number of standard deviations away from the mean value of the reference group) has the potential to facilitate comparisons between different cancer types. The aim of this study was to use the BIVA Reactance (R)/Reactance (Xc) z-score method to evaluate body composition differences in cancer, using data from previously published BIVA studies.

Methods: Previous studies using BIVA in cancer were identified from the literature. Bioimpedance measurements were analysed using the BIVA RXc z-score graph. The mean impedance vectors from the studied populations were transformed into standard deviates (with respect to the mean and standard deviation of the reference populations). Body composition was classified according to vector placement (i.e. normal, athletic, cachectic, oedematous and dehydrated).

Results: Seven male and three cancer female populations were evaluated. Body composition was classified as normal for the majority (n = 5), followed by cachexia (n = 4) and athletic (n = 1) respectively. Variation in body composition for the studied populations appeared to be related to gender, disease type and severity.

Conclusions: The BIVA RXc z-score method has potential to evaluate body composition differences between cancer groups. This method can study body composition, according to cancer type, stage, gender and ethnicity. Limitations of the method relate to issues concerning the appropriate use of reference populations and variability between bioimpedance analysers. Better body composition assessment has the potential to personalise therapeutic, nutritional and hydration management. Further work is essential to facilitate in-depth evaluation in these areas, in order to achieve meaningful use of BIVA in clinical practice.
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http://dx.doi.org/10.1016/j.clnesp.2019.02.006DOI Listing
April 2019

Central Mechanisms of Glucose Sensing and Counterregulation in Defense of Hypoglycemia.

Endocr Rev 2019 06;40(3):768-788

Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, University of Minnesota, Minneapolis, Minnesota.

Glucose homeostasis requires an organism to rapidly respond to changes in plasma glucose concentrations. Iatrogenic hypoglycemia as a result of treatment with insulin or sulfonylureas is the most common cause of hypoglycemia in humans and is generally only seen in patients with diabetes who take these medications. The first response to a fall in glucose is the detection of impending hypoglycemia by hypoglycemia-detecting sensors, including glucose-sensing neurons in the hypothalamus and other regions. This detection is then linked to a series of neural and hormonal responses that serve to prevent the fall in blood glucose and restore euglycemia. In this review, we discuss the current state of knowledge about central glucose sensing and how detection of a fall in glucose leads to the stimulation of counterregulatory hormone and behavior responses. We also review how diabetes and recurrent hypoglycemia impact glucose sensing and counterregulation, leading to development of impaired awareness of hypoglycemia in diabetes.
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http://dx.doi.org/10.1210/er.2018-00226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505456PMC
June 2019

Electromagnetic Regulation of Cell Activity.

Cold Spring Harb Perspect Med 2019 05 1;9(5). Epub 2019 May 1.

Laboratory of Molecular Genetics, Rockefeller University, New York, New York 10065.

The ability to observe the effects of rapidly and reversibly regulating cell activity in targeted cell populations has provided numerous physiologic insights. Over the last decade, a wide range of technologies have emerged for regulating cellular activity using optical, chemical, and, more recently, electromagnetic modalities. Electromagnetic fields can freely penetrate cells and tissue and their energy can be absorbed by metal particles. When released, the absorbed energy can in turn gate endogenous or engineered receptors and ion channels to regulate cell activity. In this manner, electromagnetic fields acting on external nanoparticles have been used to exert mechanical forces on cell membranes and organelles to generate heat and interact with thermally activated proteins or to induce receptor aggregation and intracellular signaling. More recently, technologies using genetically encoded nanoparticles composed of the iron storage protein, ferritin, have been used for targeted, temporal control of cell activity in vitro and in vivo. These tools provide a means for noninvasively modulating gene expression, intracellular organelles, such as endosomes, and whole-cell activity both in vitro and in freely moving animals. The use of magnetic fields interacting with external or genetically encoded nanoparticles thus provides a rapid noninvasive means for regulating cell activity.
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http://dx.doi.org/10.1101/cshperspect.a034322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6496334PMC
May 2019

Remote control of glucose-sensing neurons to analyze glucose metabolism.

Am J Physiol Endocrinol Metab 2018 09 29;315(3):E327-E339. Epub 2018 May 29.

Diabetes, Obesity, and Metabolism Institute, Icahn School of Medicine at Mount Sinai , New York, New York.

The central nervous system relies on a continual supply of glucose, and must be able to detect glucose levels and regulate peripheral organ functions to ensure that its energy requirements are met. Specialized glucose-sensing neurons, first described half a century ago, use glucose as a signal and modulate their firing rates as glucose levels change. Glucose-excited neurons are activated by increasing glucose concentrations, while glucose-inhibited neurons increase their firing rate as glucose concentrations fall and decrease their firing rate as glucose concentrations rise. Glucose-sensing neurons are present in multiple brain regions and are highly expressed in hypothalamic regions, where they are involved in functions related to glucose homeostasis. However, the roles of glucose-sensing neurons in healthy and disease states remain poorly understood. Technologies that can rapidly and reversibly activate or inhibit defined neural populations provide invaluable tools to investigate how specific neural populations regulate metabolism and other physiological roles. Optogenetics has high temporal and spatial resolutions, requires implants for neural stimulation, and is suitable for modulating local neural populations. Chemogenetics, which requires injection of a synthetic ligand, can target both local and widespread populations. Radio- and magnetogenetics offer rapid neural activation in localized or widespread neural populations without the need for implants or injections. These tools will allow us to better understand glucose-sensing neurons and their metabolism-regulating circuits.
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http://dx.doi.org/10.1152/ajpendo.00469.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171010PMC
September 2018

STING-Activating Adjuvants Elicit a Th17 Immune Response and Protect against Mycobacterium tuberculosis Infection.

Cell Rep 2018 05;23(5):1435-1447

Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, Berkeley, CA 94720, USA; School of Public Health, Division of Infectious Disease and Vaccinology, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address:

There are a limited number of adjuvants that elicit effective cell-based immunity required for protection against intracellular bacterial pathogens. Here, we report that STING-activating cyclic dinucleotides (CDNs) formulated in a protein subunit vaccine elicit long-lasting protective immunity to Mycobacterium tuberculosis in the mouse model. Subcutaneous administration of this vaccine provides equivalent protection to that of the live attenuated vaccine strain Bacille Calmette-Guérin (BCG). Protection is STING dependent but type I IFN independent and correlates with an increased frequency of a recently described subset of CXCR3-expressing T cells that localize to the lung parenchyma. Intranasal delivery results in superior protection compared with BCG, significantly boosts BCG-based immunity, and elicits both Th1 and Th17 immune responses, the latter of which correlates with enhanced protection. Thus, a CDN-adjuvanted protein subunit vaccine has the capability of eliciting a multi-faceted immune response that results in protection from infection by an intracellular pathogen.
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http://dx.doi.org/10.1016/j.celrep.2018.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003617PMC
May 2018

Diabetes: Peripheral nerve modulation to treat metabolic disease.

Authors:
Sarah A Stanley

Nat Rev Endocrinol 2018 04 2;14(4):193-194. Epub 2018 Mar 2.

Diabetes, Obesity and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

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http://dx.doi.org/10.1038/nrendo.2018.21DOI Listing
April 2018
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