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Abstract Breastfeeding is the most complete nutritional method of feeding infants, but several impediments affect the decision to breastfeed, including questions of drug safety for medications needed during lactation. Despite recent FDA guidance, few labels provide clear dosing advice during lactation. Physiologically based pharmacokinetic modeling (PBPK) is well suited to mechanistically explore pharmacokinetics and dosing paradigms to fill gaps in the absence of extensive clinical studies and complement existing real‐world data. For lactation‐focused PBPK (Lact‐PBPK) models, information on system parameters (e.g., expression of drug transporters in mammary epithelial cells) is sparse. The breast cancer resistance protein (BCRP) is expressed on the apical side of mammary epithelial cells where it actively transports drugs/substrates into milk (reported milk: plasma ratios range from 2 to 20). A critical review of BCRP and its role in lactation was conducted. Longitudinal changes in BCRP mRNA expression have been identified in women with a maximum reached around 5 months postpartum. Limited data are available on the ontogeny of BCRP in infant intestine; however, data indicate lower BCRP abundance in infants compared to adults. Current status of incorporation of drug transporter information in Lact‐PBPK models to predict active secretion of drugs into breast milk and consequential exposure of breast‐fed infants is discussed. In addition, this review highlights novel clinical tools for evaluation of BCRP activity, namely a potential non‐invasive BCRP biomarker (riboflavin) and liquid biopsy that could be used to quantitatively elucidate the role of this transporter without the need for administration of drugs and to inform Lact‐PBPK models.

Abstract Background Diabetic foot ulcers (DFU) are a costly complication of diabetes mellitus (DM), with significant implications for the patient and the healthcare professionals that treat them. The primary objective of this study was to evaluate if there were improved healing rates in patients with a DFU that were taking a statin medication compared to those patients with a DFU who were not taking a statin medication. Secondary outcomes assessed were correlations with wound healing or statin use on data obtained from retrospective chart review. Methods A case–control series was performed to obtain appropriate demographic information, comorbid conditions, laboratory values, and physical examination findings. From the time of presentation with DFU, these patients were followed for 12 weeks to evaluate for healing. Healing was defined as full epithelialization of the DFU with no further drainage. Wound healing and statin use correlation testing was then done for collected variables and each cohort. Chi square and Pearson correlation were then performed to identify any significant correlations. All p-values were two-sided, and findings were considered statistically significant at p

Introduction Osteomyelitis is associated with significant morbidity, including amputation. There are limited data on long-term amputation rates following an osteomyelitis diagnosis. We sought to determine the incidence of amputation in patients with osteomyelitis over 2 years.Research design and methods Observational cohort study of 1186 inpatients with osteomyelitis between 2004 and 2015 and stratified by osteomyelitis location status to evaluate the impact on amputation, mortality rates, readmission data, and inpatient days.Results Persons with diabetes had 3.65 times greater probability of lower extremity amputation (p

Abstract The phase distribution model, proposed by Atkinson and Begg in 1990, has been widely used for predicting breastmilk‐to‐plasma drug concentration ratio. However, misrepresentations of the equations have been noted in recent publications. In this perspective, we revisit the derivation of the equations and provide an R/Shiny interface for the model with a view to helping scientists in this field acquire in‐depth understanding of the theoretical background and implementation of the model.

Abstract Introduction Oral cancer patients suffer severe chronic and mechanically-induced pain at the site of the cancer. Our clinical experience is that oral cancer patients report new sensitivity to spicy foods. We hypothesized that in cancer patients, mechanical and chemical sensitivity would be greater when measured at the cancer site compared to a contralateral matched normal site. Methods We determined mechanical pain thresholds (MPT) on the right and left sides of the tongue of 11 healthy subjects, and at the cancer and contralateral matched normal site in 11 oral cancer patients in response to von Frey filaments in the range of 0.008 to 300 g (normally not reported as painful). We evaluated chemical sensitivity in 13 healthy subjects and seven cancer patients, who rated spiciness/pain on a visual analog scale in response to exposure to six paper strips impregnated with capsaicin (0–10 mM). Results Mechanical detection thresholds (MDT) were recorded for healthy subjects, but not MPTs. By contrast, MPTs were measured at the site of the cancer in oral cancer patients (7/11 patients). No MPTs were measured at the cancer patients’ contralateral matched normal sites. Measured MPTs were correlated with patients’ responses to the University of California Oral Cancer Pain Questionnaire. Capsaicin sensitivity at the site of the cancer was evident in cancer patients by a leftward shift of the cancer site capsaicin dose-response curve compared to that of the patient’s contralateral matched normal site. We detected no difference in capsaicin sensitivity on the right and left sides of tongues of healthy subjects. Conclusions Mechanical and chemical sensitivity testing was well tolerated by the majority of oral cancer patients. Sensitivity is greater at the site of the cancer than at a contralateral matched normal site.

Abstract Oral cancer patients report sensitivity to spicy foods and liquids. The mechanism responsible for chemosensitivity induced by oral cancer is not known. We simulate oral cancer-induced chemosensitivity in a xenograft oral cancer mouse model using two-bottle choice drinking and conditioned place aversion assays. An anatomic basis of chemosensitivity is shown in increased expression of TRPV1 in anatomically relevant trigeminal ganglion (TG) neurons in both the xenograft and a carcinogen (4-nitroquinoline 1-oxide)-induced oral cancer mouse models. The percent of retrograde labeled TG neurons that respond to TRPV1 agonist, capsaicin, is increased along with the magnitude of response as measured by calcium influx, in neurons from the cancer models. To address the possible mechanism of TRPV1 sensitivity in tongue afferents, we study the role of PAR2, which can sensitize the TRPV1 channel. We show co-expression of TRPV1 and PAR2 on tongue afferents and using a conditioned place aversion assay, demonstrate that PAR2 mediates oral cancer-induced, TRPV1-evoked sensitivity in an oral cancer mouse model. The findings provide insight into oral cancer-mediated chemosensitivity.

The mechanism of CGRP-evoked peripheral pain is unclear. Here, the authors show that the CGRP-mediated neuronal/Schwann cell pathway mediates allodynia associated with neurogenic inflammation, contributing to the algesic action of CGRP in mice.

Purpose: Lower extremity amputation resulting from diabetic foot ulcer, with neuropathic and/or ischemic etiologies, remains a devastating and costly complication of diabetes mellitus. This study evaluated changes in care delivery of diabetic foot ulcer patients during the COVID-19 pandemic. A longitudinal assessment evaluating the ratio of major lower extremity amputation to minor lower extremity amputations after implementation of novel strategies to combat access restrictions was compared to the pre-COVID-19 era. Methods: The ratio of major to minor lower extremity amputation (i.e. the high-to-low ratio) was assessed at two academic institutions, the University of Michigan, and University of Southern California, in a population of patients with diabetes who had direct access to multidisciplinary foot care clinics in the 2 years prior to the pandemic and the first 2 years of the COVID-19 pandemic. Results: Patient characteristics and volumes including patients with diabetes and those with a diabetic foot ulcer were similar between eras. In addition, inpatient diabetic foot-related admissions were similar, but were suppressed by government shelter in placed mandates and subsequent COVID-19 variants surges (e.g. delta, omicron). In the control group, the Hi-Lo ratio increased every 6 months by an average of 11.8%. Meanwhile, following STRIDE implementation during the pandemic, the Hi-Lo ratio reduced by (−)11% ( p

Oral cancer patients suffer pain at the site of the cancer. Calcitonin gene related polypeptide (CGRP), a neuropeptide expressed by a subset of primary afferent neurons, promotes oral cancer growth. CGRP also mediates trigeminal pain (migraine) and neurogenic inflammation. The contribution of CGRP to oral cancer pain is investigated in the present study. The findings demonstrate that CGRP-immunoreactive (-ir) neurons and neurites innervate orthotopic oral cancer xenograft tumors in mice. Cancer increases anterograde transport of CGRP in axons innervating the tumor, supporting neurogenic secretion as the source of CGRP in the oral cancer microenvironment. CGRP antagonism reverses oral cancer nociception in preclinical oral cancer pain models. Single-cell RNA-sequencing is used to identify cell types in the cancer microenvironment expressing the CGRP receptor components, receptor activity modifying protein 1 Ramp1 and calcitonin receptor like receptor (CLR, encoded by Calcrl). Ramp1 and Calcrl transcripts are detected in cells expressing marker genes for Schwann cells, endothelial cells, fibroblasts and immune cells. Ramp1 and Calcrl transcripts are more frequently detected in cells expressing fibroblast and immune cell markers. This work identifies CGRP as mediator of oral cancer pain and suggests the antagonism of CGRP to alleviate oral cancer pain.

Induction of CD4 T follicular helper (Tfh) cells is important for antibody responses to viral infections. Here, the authors show in a rhesus macaque model of mild COVID-19 that SARS-CoV-2 infection results in transient accumulation of proliferating Tfh cells with a Th1 profile in peripheral blood and generation of germinal center Tfh cells specific for viral proteins.

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable but low levels of antiviral antibodies after infusion. In comparison to the control animals, CCP-treated animals had similar levels of viral RNA in upper and lower respiratory tract secretions, similar detection of viral RNA in lung tissues by in situ hybridization, but lower amounts of infectious virus in the lungs. CCP-treated animals had a moderate, but statistically significant reduction in interstitial pneumonia, as measured by comprehensive lung histology. Thus overall, therapeutic benefits of CCP were marginal and inferior to results obtained earlier with monoclonal antibodies in this animal model. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses.

Two brief biodiversity surveys were conducted near Camp Lemonnier, Djibouti, in May 2014 and February 2016, and to the Day Forest (Forêt du Day) in February 2016. Our complete avian checklist of 143 species, with 96 species preserved as museum specimens is presented here. We document nesting for Spur-winged Lapwing Vanellus spinosus (Linnaeus, 1758), the occurrence of Crimson-rumped Waxbill Estrilda rhodopyga Sundevall, 1850, and confirm Little Tern Sternula albifrons (Pallas, 1764) in Djibouti. We comment on observations for the Critically Endangered Djibouti Spurfowl Pternistis ochropectus (Dorst & Jouanin 1952), and the Endangered Egyptian Vulture Neophron percnopterus (Linnaeus, 1758), and a previously noted Fringillidae (Crithagra sp.) Swainson, 1827 from the Day Forest. Hybridization between Somali Sparrow Passer castanopterus Blythi,1885 and House Sparrow Passer domesticus (Linnaeus, 1758) is documented with voucher specimens. Although these surveys and taxa lists represent brief inventories for the areas visited, they provide a foundation and reference for future work in this understudied region in the Horn of Africa.

Abstract Background Stereotyped sunning behaviour in birds has been hypothesized to inhibit keratin-degrading bacteria but there is little evidence that solar irradiation affects community assembly and abundance of plumage microbiota. The monophyletic New World vultures (Cathartiformes) are renowned for scavenging vertebrate carrion, spread-wing sunning at roosts, and thermal soaring. Few avian species experience greater exposure to solar irradiation. We used 16S rRNA sequencing to investigate the plumage microbiota of wild individuals of five sympatric species of vultures in Guyana. Results The exceptionally diverse plumage microbiotas (631 genera of Bacteria and Archaea) were numerically dominated by bacterial genera resistant to ultraviolet (UV) light, desiccation, and high ambient temperatures, and genera known for forming desiccation-resistant endospores (phylum Firmicutes, order Clostridiales). The extremophile genera Deinococcus (phylum Deinococcus-Thermus) and Hymenobacter (phylum, Bacteroidetes), rare in vertebrate gut microbiotas, accounted for 9.1% of 2.7 million sequences (CSS normalized and log2 transformed). Five bacterial genera known to exhibit strong keratinolytic capacities in vitro (Bacillus, Enterococcus, Pseudomonas, Staphylococcus, and Streptomyces) were less abundant (totaling 4%) in vulture plumage. Conclusions Bacterial rank-abundance profiles from melanized vulture plumage have no known analog in the integumentary systems of terrestrial vertebrates. The prominence of UV-resistant extremophiles suggests that solar irradiation may play a significant role in the assembly of vulture plumage microbiotas. Our results highlight the need for controlled in vivo experiments to test the effects of UV on microbial communities of avian plumage.

Abstract Background The prevalence of diabetes mellitus continues to rise. Diabetic foot ulcers with osteomyelitis are a diabetes-related complication presenting a significant burden to this cohort. A cure to diabetic foot osteomyelitis remains elusive and standard of care has failed to improve outcomes. To advance research and better patient outcomes, the authors offer specific guidance with terminology to enhance operative dictations which may improve surgical practice and guide treatment. Methods A consecutive review of podiatric surgical dictations for inpatient diabetic foot osteomyelitis within a tertiary care facility was performed. Surgical descriptors of bone were standardized: density, anatomic structure, vascular thrombosis, color, and draining sinus. Correlations between the five categories and histopathological results were performed after kappa analysis for interrater reliability was performed. Results Kappa coefficient demonstrated high inter-reliability of surgical findings. This suggests potential agreement amongst surgeons performing similar procedures. It was also found that specific bone descriptors had moderate to strong correlation with clean histopathologic bone margins when biopsied. This further suggests that the use of standardized terms may help guide definitive therapy. Conclusions The authors suggest a standardized approach which includes consistent descriptors of intraoperative bone. With use of standardized terms, vague and blanket descriptors are eliminated. This has potential to improve understanding of changes within bone as a result of infection and diabetes. Early and improved communication of intraoperative findings will enhance the multidisciplinary approach. This could potentially lead to changes in diabetic foot management and may limit hospital waste waiting for final cultures and pathology reports.

ABSTRACT Human clinical studies investigating use of convalescent plasma (CP) for treatment of coronavirus disease 2019 (COVID-19) have produced conflicting results. Outcomes in these studies may vary at least partly due to different timing of CP administration relative to symptom onset. The mechanisms of action of CP include neutralizing antibodies but may extend beyond virus neutralization to include normalization of blood clotting and dampening of inflammation. Unresolved questions include the minimum therapeutic titer in the CP units or CP recipient as well as the optimal timing of administration. Here, we show that treatment of macaques with CP within 24 h of infection does not reduce viral shedding in nasal or lung secretions compared to controls and does not detectably improve any clinical endpoint. We also demonstrate that CP administration does not impact viral sequence diversity in vivo, although the selection of a viral sequence variant in both macaques receiving normal human plasma was suggestive of immune pressure. Our results suggest that CP, administered to medium titers, has limited efficacy, even when given very early after infection. Our findings also contribute information important for the continued development of the nonhuman primate model of COVID-19. These results should inform interpretation of clinical studies of CP in addition to providing insights useful for developing other passive immunotherapies and vaccine strategies. IMPORTANCE Antiviral treatment options for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain very limited. One treatment that was explored beginning early in the pandemic (and that is likely to be tested early in future pandemics) is plasma collected from people who have recovered from coronavirus disease 2019 (COVID-19), known as convalescent plasma (CP). We tested if CP reduces viral shedding or disease in a nonhuman primate model. Our results demonstrate that administration of CP 1 day after SARS-CoV-2 infection had no significant impact on viral loads, clinical disease, or sequence diversity, although treatment with normal human plasma resulted in selection of a specific viral variant. Our results demonstrate that passive immunization with CP, even during early infection, provided no significant benefit in a nonhuman primate model of SARS-CoV-2 infection.

Summary: Anti-viral monoclonal antibody (mAb) treatments may provide immediate but short-term immunity from coronavirus disease 2019 (COVID-19) in high-risk populations, such as people with diabetes and the elderly; however, data on their efficacy in these populations are limited. We demonstrate that prophylactic mAb treatment blocks viral replication in both the upper and lower respiratory tracts in aged, type 2 diabetic rhesus macaques. mAb infusion dramatically curtails severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-mediated stimulation of interferon-induced chemokines and T cell activation, significantly reducing development of interstitial pneumonia. Furthermore, mAb infusion significantly dampens the greater than 3-fold increase in SARS-CoV-2-induced effector CD4 T cell influx into the cerebrospinal fluid. Our data show that neutralizing mAbs administered preventatively to high-risk populations may mitigate the adverse inflammatory consequences of SARS-CoV-2 exposure.

Physiologically based pharmacokinetic (PBPK) modeling has a number of applications, including assessing drug–drug interactions (DDIs) in polymorphic populations, and should be iteratively refined as science progresses. The Simcyp Simulator is annually updated and version 21 included updates to hepatic and intestinal CYP2C19 enzyme abundance, including addition of intermediate and rapid metabolizer phenotypes and changes to the ultra-rapid metabolizer enzyme abundance, with implications for population clearance and DDI predictions. This work details verification of the updates with sensitive CYP2C19 substrates, omeprazole and lansoprazole, using available clinical data from literature. Multiple assessments were performed, including recovery of areas under the concentration-time curve (AUC) and Cmax from compiled datasets for each drug, recovery of victim DDI ratios with CYP2C19 and/or CYP3A4 inhibition and recovery of relative exposure between phenotypes. Simulated data were within respective acceptance criteria for >80% of omeprazole AUC values, >70% of lansoprazole AUC and Cmax, >60% of AUC and Cmax DDI ratios and >80% of exposure ratios between different phenotypes. Recovery of omeprazole Cmax was lower (>50–70% within 2-fold) and possibly attributed to the variety of formulations used in the clinical dataset. Overall, the results demonstrated that the updated data used to parameterize CYP2C19 phenotypes reasonably described the pharmacokinetics of omeprazole and lansoprazole in genotyped or phenotyped individuals.

There is an urgent need for effective therapeutic interventions against SARS-CoV-2, including new variants that continue to arise. Neutralizing monoclonal antibodies have shown promise in clinical studies. We investigated the therapeutic efficacy of a combination of two potent monoclonal antibodies, C135-LS and C144-LS that carry half-life extension mutations, in the rhesus macaque model of COVID-19. Twelve young adult macaques (three groups of four animals) were inoculated intranasally and intra-tracheally with a high dose of SARS-CoV-2 and 24 hours later, treated intravenously with a high (40 mg/kg) or low (12 mg/kg) dose of the C135-LS and C144-LS antibody combination, or a control monoclonal antibody. Animals were monitored for 7 days. Compared to the control animals, animals treated with either dose of the anti-SARS-CoV-2 antibodies showed similarly improved clinical scores, lower levels of virus replication in upper and lower respiratory tract, and significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. In conclusion, this study provides proof-of-concept in support of further clinical development of these monoclonal antibodies against COVID-19 during early infection.

Abstract Background Diabetes mellitus continues to be a rising concern in the United States. It affects an estimated 9.4% of the population and approximately 1.5 million Americans are diagnosed annually. Approximately 85% of diabetic foot ulcers are associated with diabetic peripheral neuropathy and an infected diabetic foot ulcer is often the first sign of diabetes. There are countless studies within the literature that investigate how insensate feet and the manifestation of a foot ulcer further decrease quality of life and increase risk for mortality. Literature focuses on gait and kinematics that contribute to the formation of a diabetic foot ulcer. While pressure and shear forces are etiologic factors that may lead to the formation of diabetic foot ulcers, the position of the foot while driving an automobile has been ignored as a possible risk factor. Case presentation The clinical case will describe the events of healing a neuropathic diabetic foot ulcer beyond the standard of care treatment plan. It is one of the first case reports to describe vehicle ergonomics as an etiologic factor contributing to a diabetic foot ulcer. Once the patient becomes aware of the unnecessary source of pressure, education and care is provided to manage this likely source of daily pressure to the neuropathic foot. Conclusion The article emphasizes the importance of a complete assessment, including nontraditional factors, which may lead to diabetic complications.

Abstract Background Current knowledge about seasonal variation in the gut microbiota of vertebrates is limited to a few studies based on mammalian fecal samples. Seasonal changes in the microbiotas of functionally distinct gut regions remain unexplored. We investigated seasonal variation (summer versus winter) and regionalization of the microbiotas of the crop, ventriculus, duodenum, cecum, and colon of the greater sage-grouse (Centrocercus urophasianus), an avian folivore specialized on the toxic foliage of sagebrush (Artemesia spp.) in western North America. Results We sequenced the V4 region of the 16S rRNA gene on an Illumina MiSeq and obtained 6,639,051 sequences with a median of 50,232 per sample. These sequences were assigned to 457 bacterial and 4 archaeal OTUs. Firmicutes (53.0%), Bacteroidetes (15.2%), Actinobacteria (10.7%), and Proteobacteria (10.1%)were the most abundant and diverse phyla. Microbial composition and richness showed significant differences among gut regions and between summer and winter. Gut region explained almost an order of magnitude more variance in our dataset than did season or the gut region × season interaction. The effect of season was uneven among gut regions. Microbiotas of the crop and cecum showed the greatest seasonal differences. Conclusions Our data suggest that seasonal differences in gut microbiota reflect seasonal variation in the microbial communities associated with food and water. Strong differentiation and uneven seasonal changes in the composition and richness of the microbiota among functionally distinct gut regions demonstrate the necessity of wider anatomical sampling for studies of composition and dynamics of the gut microbiota.

Abstract We propose a new mechanism of sensory modulation through cutaneous dopaminergic signalling. We hypothesize that dopaminergic signalling contributes to differential cutaneous sensitivity in darker versus lighter pigmented humans and mouse strains. We show that thermal and mechanical cutaneous sensitivity is pigmentation dependent. Meta-analyses in humans and mice, along with our own mouse behavioural studies, reveal higher thermal sensitivity in pigmented skin relative to less-pigmented or albino skin. We show that dopamine from melanocytes activates the D1-like dopamine receptor on primary sensory neurons. Dopaminergic activation increases expression of the heat-sensitive TRPV1 ion channel and reduces expression of the mechanically-sensitive Piezo2 channel; thermal threshold is lower and mechanical threshold is higher in pigmented skin.

Oral cancer patients report severe function-induced pain; severity is greater in females. We hypothesize that a neutrophil-mediated endogenous analgesic mechanism is responsible for sex differences in nociception secondary to oral squamous cell carcinoma (SCC). Neutrophils isolated from the cancer-induced inflammatory microenvironment contain β-endorphin protein and are identified by the Ly6G+ immune marker. We previously demonstrated that male mice with carcinogen-induced oral SCC exhibit less nociceptive behavior and a higher concentration of neutrophils in the cancer microenvironment compared to female mice with oral SCC. Oral cancer cells secrete granulocyte colony stimulating factor (G-CSF), a growth factor that recruits neutrophils from bone marrow to the cancer microenvironment. We found that recombinant G-CSF (rG-CSF, 5 μg/mouse, intraperitoneal) significantly increased circulating Ly6G+ neutrophils in the blood of male and female mice within 24 h of administration. In an oral cancer supernatant mouse model, rG-CSF treatment increased cancer-recruited Ly6G+ neutrophil infiltration and abolished orofacial nociceptive behavior evoked in response to oral cancer supernatant in both male and female mice. Local naloxone treatment restored the cancer mediator-induced nociceptive behavior. We infer that rG-CSF-induced Ly6G+ neutrophils drive an endogenous analgesic mechanism. We then evaluated the efficacy of chronic rG-CSF administration to attenuate oral cancer-induced nociception using a tongue xenograft cancer model with the HSC-3 human oral cancer cell line. Saline-treated male mice with HSC-3 tumors exhibited less oral cancer-induced nociceptive behavior and had more β-endorphin protein in the cancer microenvironment than saline-treated female mice with HSC-3 tumors. Chronic rG-CSF treatment (2.5 μg/mouse, every 72 h) increased the HSC-3 recruited Ly6G+ neutrophils, increased β-endorphin protein content in the tongue and attenuated nociceptive behavior in female mice with HSC-3 tumors. From these data, we conclude that neutrophil-mediated endogenous opioids warrant further investigation as a potential strategy for oral cancer pain treatment.

The human retina contains three classes of cone photoreceptors each sensitive to different portions of the visual spectrum: long (L), medium (M) and short (S) wavelengths. Color information is computed by downstream neurons that compare relative activity across the three cone types. How cone signals are combined at a cellular scale has been more difficult to resolve. This is especially true near the fovea, where spectrally-opponent neurons in the parvocellular pathway draw excitatory input from a single cone and thus even the smallest stimulus projected through natural optics will engage multiple color-signaling neurons. We used an adaptive optics microstimulator to target individual and pairs of cones with light. Consistent with prior work, we found that color percepts elicited from individual cones were predicted by their spectral sensitivity, although there was considerable variability even between cones within the same spectral class. The appearance of spots targeted at two cones were predicted by an average of their individual activations. However, two cones of the same subclass elicited percepts that were systematically more saturated than predicted by an average. Together, these observations suggest both spectral opponency and prior experience influence the appearance of small spots.

Protein-restricted (PR), high-carbohydrate diets improve metabolic health in rodents, yet the precise dietary components that are responsible for these effects have not been identified. Furthermore, the applicability of these studies to humans is unclear. Here, we demonstrate in a randomized controlled trial that a moderate PR diet also improves markers of metabolic health in humans. Intriguingly, we find that feeding mice a diet specifically reduced in branched-chain amino acids (BCAAs) is sufficient to improve glucose tolerance and body composition equivalently to a PR diet via metabolically distinct pathways. Our results highlight a critical role for dietary quality at the level of amino acids in the maintenance of metabolic health and suggest that diets specifically reduced in BCAAs, or pharmacological interventions in this pathway, may offer a translatable way to achieve many of the metabolic benefits of a PR diet.

The incidence of oral cancer in the United States is increasing, especially in young people and women. Patients with oral cancer report severe functional pain. Using a patient cohort accrued through the New York University Oral Cancer Center and immune-competent mouse models, we identify a sex difference in the prevalence and severity of oral cancer pain. A neutrophil-mediated endogenous analgesic mechanism is present in male mice with oral cancer. Local naloxone treatment potentiates cancer mediator-induced orofacial nociceptive behavior in male mice only. Tongues from male mice with oral cancer have significantly more infiltrating neutrophils compared to female mice with oral cancer. Neutrophils isolated from the cancer-induced inflammatory microenvironment express beta-endorphin and met-enkephalin. Furthermore, neutrophil depletion results in nociceptive behavior in male mice. These data suggest a role for sex-specific, immune cell-mediated endogenous analgesia in the treatment of oral cancer pain.

Two supplements were awarded to the New York University Health Sciences Libraries from the National Library of Medicine's informationist grant program. These supplements funded research support in a number of areas, including data management and bioinformatics, two fields that the library had recently begun to explore. As such, the supplements were of particular value to the library as a testing ground for these newer services.This paper will discuss a supplement received in support of a grant from the National Institute of Dental and Craniofacial Research (PI: Brian Schmidt) on the role of proteases and peptides in cancer pain. A number of barriers were preventing the research team from maximizing the efficiency and effectiveness of their work. A critical component of the research was to identify which proteins, from among hundreds identified in collected samples, to include in preclinical testing. This selection involved laborious and prohibitively time-consuming manual searching of the literature on protein function. Additionally, the research team encompassed ten investigators working in two different cities, which led to issues around the sharing and tracking of both data and citations.The supplement outlined three areas in which the informationists would assist the researchers in overcoming these barriers: 1) creating an automated literature searching system for protein function discovery, 2) introducing tools and associated workflows for sharing citations, and 3) introducing tools and workflows for sharing data and specimens.

Functional analysis of a clinical microbiome facilitates the elucidation of mechanisms by which microbiome perturbation can cause a phenotypic change in the patient. The direct approach for the analysis of the functional capacity of the microbiome is via shotgun metagenomics. An inexpensive method to estimate the functional capacity of a microbial community is through collecting 16S rRNA gene profiles then indirectly inferring the abundance of functional genes. This inference approach has been implemented in the PICRUSt and Tax4Fun software tools. However, those tools have important limitations since they rely on outdated functional databases and uncertain phylogenetic trees and require very specific data pre-processing protocols. Here we introduce Piphillin, a straightforward algorithm independent of any proposed phylogenetic tree, leveraging contemporary functional databases and not obliged to any singular data pre-processing protocol. When all three inference tools were evaluated against actual shotgun metagenomics, Piphillin was superior in predicting gene composition in human clinical samples compared to both PICRUSt and Tax4Fun (p

The pharmacological effect of opioids originates, at the cellular level, by their interaction with the μ-opioid receptor (mOR) resulting in the regulation of voltage-gated Ca2+ channels and inwardly rectifying K+ channels that ultimately modulate the synaptic transmission. Recently, an alternative six trans-membrane helix isoform of mOR, (6TM-mOR) has been identified, but its function and signaling are still largely unknown. Here, we present the structural and functional mechanisms of 6TM-mOR signaling activity upon binding to morphine. Our data suggest that despite the similarity of binding modes of the alternative 6TM-mOR and the dominant seven trans-membrane helix variant (7TM-mOR), the interaction with morphine generates different dynamic responses in the two receptors, thus, promoting the activation of different mOR-specific signaling pathways. We characterize a series of 6TM-mOR-specific cellular responses, and observed that they are significantly different from those for 7TM-mOR. Morphine stimulation of 6TM-mOR does not promote a cellular cAMP response, while it increases the intracellular Ca2+ concentration and reduces the cellular K+ conductance. Our findings indicate that 6TM-mOR has a unique contribution to the cellular opioid responses. Therefore, it should be considered as a relevant target for the development of novel pharmacological tools and medical protocols involving the use of opioids.

OBJECTIVES/SPECIFIC AIMS: We hypothesize that both NGF and TNF-α contribute to oral cancer pain by upregulating pro-nociceptive inflammatory cytokines. METHODS/STUDY POPULATION: In total, 48 oral cancer patients were evaluated and their pain scores were measured using a validated oral cancer pain questionnaire. Presence of perineural invasion (PNI) was identified from patients’ pathology reports. We utilized The NIH Cancer Genome Atlas (TCGA) Head and Neck Cancer cohort to investigate the association between pain and genes related to NGF, TNF-α, and their receptors (TRKA, P75, TNF-α receptor 1, and TNF-α receptor 2) in oral cancer samples by employing PNI as a surrogate for pain. Demographic characteristics, clinical characteristics, and genes were analyzed using logistic regression models. A xenograft cancer pain model was created by inoculating human oral cancer cells (HSC-3) into the mouse hind paw. Mice (n=6 per group) were treated with anti-NGF alone, anti-TNF-α alone, a combination of anti-NGF and anti-TNF-α, or PBS (vehicle control). Nociceptive behaviors were measured using an electronic paw withdrawal assay. Paw volume was measured using a plethysmometer. Cytokines in the paw tissues were measured using a multiplex assay kit with 28 cytokines. RESULTS/ANTICIPATED RESULTS: Oral cancer patients with PNI report significantly more pain compared with patients without PNI in our patient cohort (p

Individual bacteria and shifts in the composition of the microbiome have been associated with human diseases including cancer. To investigate changes in the microbiome associated with oral cancers, we profiled cancers and anatomically matched contralateral normal tissue from the same patient by sequencing 16S rDNA hypervariable region amplicons. In cancer samples from both a discovery and a subsequent confirmation cohort, abundance of Firmicutes (especially Streptococcus) and Actinobacteria (especially Rothia) was significantly decreased relative to contralateral normal samples from the same patient. Significant decreases in abundance of these phyla were observed for pre-cancers, but not when comparing samples from contralateral sites (tongue and floor of mouth) from healthy individuals. Weighted UniFrac principal coordinates analysis based on 12 taxa separated most cancers from other samples with greatest separation of node positive cases. These studies begin to develop a framework for exploiting the oral microbiome for monitoring oral cancer development, progression and recurrence.

Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) reduces survival. In this study we hypothesized that methylation of key genes mediates cisplatin resistance. We determined whether a demethylating drug, decitabine, could augment the anti-proliferative and apoptotic effects of cisplatin on SCC-25/CP, a cisplatin-resistant tongue SCC cell line. We showed that decitabine treatment restored cisplatin sensitivity in SCC-25/CP and significantly reduced the cisplatin dose required to induce apoptosis. We then created a xenograft model with SCC-25/CP and determined that decitabine and cisplatin combination treatment resulted in significantly reduced tumor growth and mechanical allodynia compared to control. To establish a gene classifier we quantified methylation in cancer tissue of cisplatin-sensitive and cisplatin-resistant HNSCC patients. Cisplatin-sensitive and cisplatin-resistant patient tumors had distinct methylation profiles. When we quantified methylation and expression of genes in the classifier in HNSCC cells in vitro, we showed that decitabine treatment of cisplatin-resistant HNSCC cells reversed methylation and gene expression toward a cisplatin-sensitive profile. The study provides direct evidence that decitabine restores cisplatin sensitivity in in vitro and in vivo models of HNSCC. Combination treatment of cisplatin and decitabine significantly reduces HNSCC growth and HNSCC pain. Furthermore, gene methylation could be used as a biomarker of cisplatin-resistance.

The purposes of this study were to evaluate for differences in phenotypic and genotypic characteristics in women who did and did not develop lymphedema (LE) following breast cancer treatment. Breast cancer patients completed a number of self-report questionnaires. LE was evaluated using bioimpedance spectroscopy. Genotyping was done using a custom genotyping array. No differences were found between patients with (n = 155) and without LE (n = 387) for the majority of the demographic and clinical characteristics. Patients with LE had a significantly higher body mass index, more advanced disease and a higher number of lymph nodes removed. Genetic associations were identified for four genes (i.e., lymphocyte cytosolic protein 2 (rs315721), neuropilin-2 (rs849530), protein tyrosine kinase (rs158689), vascular cell adhesion molecule 1 (rs3176861)) and three haplotypes (i.e., Forkhead box protein C2 (haplotype A03), neuropilin-2 (haplotype F03), vascular endothelial growth factor-C (haplotype B03)) involved in lymphangiogensis and angiogenesis. These genetic associations suggest a role for a number of lymphatic and angiogenic genes in the development of LE following breast cancer treatment.

The interaction of proteins at cellular interfaces is critical for many biological processes, from intercellular signaling to cell adhesion. For example, the selectin family of adhesion receptors plays a critical role in trafficking during inflammation and immunosurveillance. Quantitative measurements of binding rates between surface-constrained proteins elicit insight into how molecular structural details and post-translational modifications contribute to function. However, nano-scale transport effects can obfuscate measurements in experimental assays. We constructed a biophysical simulation of the motion of a rigid microsphere coated with biomolecular adhesion receptors in shearing flow undergoing thermal motion. The simulation enabled in silico investigation of the effects of kinetic force dependence, molecular deformation, grouping adhesion receptors into clusters, surface-constrained bond formation, and nano-scale vertical transport on outputs that directly map to observable motions. Simulations recreated the jerky, discrete stop-and-go motions observed in P-selectin/PSGL-1 microbead assays with physiologic ligand densities. Motion statistics tied detailed simulated motion data to experimentally reported quantities. New deductions about biomolecular function for P-selectin/PSGL-1 interactions were made. Distributing adhesive forces among P-selectin/PSGL-1 molecules closely grouped in clusters was necessary to achieve bond lifetimes observed in microbead assays. Initial, capturing bond formation effectively occurred across the entire molecular contour length. However, subsequent rebinding events were enhanced by the reduced separation distance following the initial capture. The result demonstrates that vertical transport can contribute to an enhancement in the apparent bond formation rate. A detailed analysis of in silico motions prompted the proposition of wobble autocorrelation as an indicator of two-dimensional function. Insight into two-dimensional bond formation gained from flow cell assays might therefore be important to understand processes involving extended cellular interactions, such as immunological synapse formation. A biologically informative in silico system was created with minimal, high-confidence inputs. Incorporating random effects in surface separation through thermal motion enabled new deductions of the effects of surface-constrained biomolecular function. Important molecular information is embedded in the patterns and statistics of motion.