Autonomous and self-sustained circadian oscillators displayed in human islet cells

Aims/hypothesis: Following on from the emerging importance of the pancreas circadian clock on islet function and the development of type 2 diabetes in rodent models, we aimed to examine circadian gene expression in human islets. The oscillator properties were assessed in intact islets as well as in beta cells. Methods: We established a system for long-term bioluminescence recording in cultured human islets, employing lentivector gene delivery of the core clock gene Bmal1 (also known as Arntl)-luciferase reporter. Beta cells were stably labelled using a rat insulin2 promoter fluorescent construct. Single-islet/cell oscillation profiles were measured by combined bioluminescence-fluorescence time-lapse microscopy. Results: Human islets synchronised in vitro exhibited self-sustained circadian oscillations of Bmal1-luciferase expression at both the population and single-islet levels, with period lengths of 23.6 and 23.9h, respectively. Endogenous BMAL1 and CRY1 transcript expression was circadian in synchronised islets over 48h, and antiphasic to REV-ERBα (also known as NR1D1), PER1, PER2, PER3 and DBP transcript circadian profiles. HNF1A and PDX1 exhibited weak circadian oscillations, in phase with the REV-ERBα transcript. Dispersed islet cells were strongly oscillating as well, at population and single-cell levels. Importantly, beta and non-beta cells revealed oscillatory profiles that were well synchronised with each other. Conclusions/interpretation: We provide for the first time compelling evidence for high-amplitude cell-autonomous circadian oscillators displayed in human pancreatic islets and in dispersed human islet cells. Moreover, these clocks are synchronised between beta and non-beta cells in primary human islet cell culture

Autonomous and self-sustained circadian oscillators displayed in human islet cells

Aims/hypothesis: Following on from the emerging importance of the pancreas circadian clock on islet function and the development of type 2 diabetes in rodent models, we aimed to examine circadian gene expression in human islets. The oscillator properties were assessed in intact islets as well as in beta cells. Methods: We established a system for long-term bioluminescence recording in cultured human islets, employing lentivector gene delivery of the core clock gene Bmal1 (also known as Arntl)-luciferase reporter. Beta cells were stably labelled using a rat insulin2 promoter fluorescent construct. Single-islet/cell oscillation profiles were measured by combined bioluminescence-fluorescence time-lapse microscopy. Results: Human islets synchronised in vitro exhibited self-sustained circadian oscillations of Bmal1-luciferase expression at both the population and single-islet levels, with period lengths of 23.6 and 23.9h, respectively. Endogenous BMAL1 and CRY1 transcript expression was circadian in synchronised islets over 48h, and antiphasic to REV-ERBα (also known as NR1D1), PER1, PER2, PER3 and DBP transcript circadian profiles. HNF1A and PDX1 exhibited weak circadian oscillations, in phase with the REV-ERBα transcript. Dispersed islet cells were strongly oscillating as well, at population and single-cell levels. Importantly, beta and non-beta cells revealed oscillatory profiles that were well synchronised with each other. Conclusions/interpretation: We provide for the first time compelling evidence for high-amplitude cell-autonomous circadian oscillators displayed in human pancreatic islets and in dispersed human islet cells. Moreover, these clocks are synchronised between beta and non-beta cells in primary human islet cell culture

Development of and Access to Products for Neglected Diseases

INTRODUCTION: Prior research on neglected disease drug development suggested inadequate funding was responsible for relatively few new approvals. In response, significantly more resources have been allocated towards development of drugs targeting neglected diseases. Our objective was to reassess drug development between 1975 and 1999, evaluate progress in neglected disease drug development since 2000, and explain how increased numbers of approvals are a necessary but insufficient condition to improving access. METHODS: To assess numbers of approvals targeting neglected diseases, we employed two distinct methodologies: First, to revisit numbers published in Trouiller et al. (2002) we used their method to count marketed new chemical entities (NCEs) between 1975 and 1999. Second, using the G-Finder report as a benchmark, we identified which diseases are currently considered "neglected" to tally approvals in the 1975-1999 and 2000-2009 periods. Searching PharmaProjects and IMS R&D Focus databases as well as websites from numerous drug regulatory agencies, we identified new drug approvals and indications. Also, we examined the World Health Organization's (WHO) Essential Drug List (EDL) to see which drugs and indications were on the list. FINDINGS: Upon recount, using Trouiller et al. methodology, we found that between 1975 and 1999 more NCEs (n = 32) targeting tropical diseases and tuberculosis were approved than reported in Trouiller et al. (n = 16). Using the G-Finder method of defining neglected diseases, we found 46 new drug approvals between 1975 and 1999. WHO included 85% of these drugs on the EDL. In the period 2000 to May 2009, despite much greater funding, only 26 new drugs and vaccines for neglected diseases were marketed. Of these, WHO placed 50% on the EDL. CONCLUSIONS: Product approvals for neglected diseases have increased, though progress has been uneven, with malaria appearing to benefit most in the short run from increased funding, while less success has been booked in other disease categories. Uneven progress suggests funding could be better targeted, particularly with regard to neglected diseases that have hitherto received scant attention. In addition, policymakers should focus on other aspects related to access. Besides drug development, there are the issues of EDL listing, architecture, availability, affordability, and adoption

Rhythmic Diurnal Gene Expression in Human Adipose Tissue From Individuals Who Are Lean, Overweight, and Type 2 Diabetic

OBJECTIVE Previous animal studies suggest a functional relationship between metabolism, type 2 diabetes, and the amplitude of daily rhythms in white adipose tissue (WAT). However, data interpretation is confounded by differences in genetic background and diet or limited sampling points. We have taken the novel approach of analyzing serial human WAT biopsies across a 24-h cycle in controlled laboratory conditions.RESEARCH DESIGN AND METHODS Lean (n = 8), overweight/obese (n = 11), or overweight/obese type 2 diabetic (n = 8) volunteers followed a strict sleep-wake and dietary regimen for 1 week prior to the laboratory study. They were then maintained in controlled light-dark conditions in a semirecumbent posture and fed hourly during wake periods. Subcutaneous WAT biopsies were collected every 6 h over 24 h, and gene expression was measured by quantitative PCR.RESULTS Lean individuals exhibited significant (P 0.05) of increased body weight or type 2 diabetes on rhythmic gene expression.CONCLUSIONS The robust nature of these rhythms and their relative phasing indicate that WAT now can be considered as a peripheral tissue suitable for the study of in vivo human rhythms. Comparison of data between subject groups clearly indicates that obesity and type 2 diabetes are not related to the amplitude of rhythmic WAT gene expression in humans maintained under controlled conditions. Diabetes 60:1577-1581, 201

A critical comparison of integral projection and matrix projection models for demographic analysis

Structured demographic models are among the most common and useful tools in population biology. However, the introduction of integral projection models (IPMs) has caused a profound shift in the way many demographic models are conceptualized. Some researchers have argued that IPMs, by explicitly representing demographic processes as continuous functions of state variables such as size, are more statistically efficient, biologically realistic, and accurate than classic matrix projection models, calling into question the usefulness of the many studies based on matrix models. Here, we evaluate how IPMs and matrix models differ, as well as the extent to which these differences matter for estimation of key model outputs, including population growth rates, sensitivity patterns, and life spans. First, we detail the steps in constructing and using each type of model. Second, we present a review of published demographic models, concentrating on size-based studies, which shows significant overlap in the way IPMs and matrix models are constructed and analyzed. Third, to assess the impact of various modeling decisions on demographic predictions, we ran a series of simulations based on size-based demographic data sets for five biologically diverse species. We found little evidence that discrete vital rate estimation is less accurate than continuous functions across a wide range of sample sizes or size classes (equivalently bin numbers or mesh points). Most model outputs quickly converged with modest class numbers (≥10), regardless of most other modeling decisions. Another surprising result was that the most commonly used method to discretize growth rates for IPM analyses can introduce substantial error into model outputs. Finally, we show that empirical sample sizes generally matter more than modeling approach for the accuracy of demographic outputs. Based on these results, we provide specific recommendations to those constructing and evaluating structured population models. Both our literature review and simulations question the treatment of IPMs as a clearly distinct modeling approach or one that is inherently more accurate than classic matrix models. Importantly, this suggests that matrix models, representing the vast majority of past demographic analyses available for comparative and conservation work, continue to be useful and important sources of demographic information.Support for this work was provided by NSF awards 1146489, 1242558, 1242355, 1353781, 1340024, 1753980, and 1753954, 1144807, 0841423, and 1144083. Support also came from USDA NIFA Postdoctoral Fellowship (award no. 2019-67012-29726/project accession no. 1019364) for R. K. Shriver; the Swiss Polar Institute of Food and Agriculture for N. I. Chardon; the ICREA under the ICREA Academia Programme for C. Linares; and SERDP contract RC-2512 and USDA National Institute of Food and Agriculture, Hatch project 1016746 for A .M. Louthan. This is Contribution no. 21-177-J from the Kansas Agricultural Experiment Station

Effects of nonantibiotic feed additives on performance, nutrient retention, gut pH, and intestinal morphology of broilers fed different levels of energy.

An experiment was conducted to determine the effects of different feed additives on performance, nutrient retention, gut pH, and intestinal morphology of broilers fed different levels of energy. This study was a 4 × 2 factorial arrangement of 4 feed additive programs (a basal diet without any feed additive as the control, the basal diet with added organic acid, the basal diet with added prebiotic, and the basal diet with added probiotic) with recommended levels (3,150 and 3,200 kcal of ME/kg of diet for the starter and finisher diets, respectively) or low levels (90% of recommended) of energy. A total of 640 one-day-old male and female broiler chicks were randomly assigned to 8 treatments. Each treatment consisted of 4 replicate floor pens of 20 birds each. Starter and finisher diets were fed from 1 to 21 d and 21 to 42 d of age, respectively. Dietary levels of other nutrients, housing, and general management practices were similar for all treatments. Dietary inclusion of additives had no significant effects on broiler performance, intestinal villus height, crypt depth, gut pH, and dietary AME. Birds fed the low-energy diets were heavier but had inferior FCR compared with those fed the recommended-energy diets. Prebiotic and organic acid significantly (P < 0.05) improved protein digestibility. The recommended-energy diets significantly (P < 0.05) increased AME and protein digestibility. No interactions were observed for the measured parameters

Circadian tumor infiltration and function of CD8+ T cells dictate immunotherapy efficacy.

The quality and quantity of tumor-infiltrating lymphocytes, particularly CD8 &lt;sup&gt;+&lt;/sup&gt; T cells, are important parameters for the control of tumor growth and response to immunotherapy. Here, we show in murine and human cancers that these parameters exhibit circadian oscillations, driven by both the endogenous circadian clock of leukocytes and rhythmic leukocyte infiltration, which depends on the circadian clock of endothelial cells in the tumor microenvironment. To harness these rhythms therapeutically, we demonstrate that efficacy of chimeric antigen receptor T cell therapy and immune checkpoint blockade can be improved by adjusting the time of treatment during the day. Furthermore, time-of-day-dependent T cell signatures in murine tumor models predict overall survival in patients with melanoma and correlate with response to anti-PD-1 therapy. Our data demonstrate the functional significance of circadian dynamics in the tumor microenvironment and suggest the importance of leveraging these features for improving future clinical trial design and patient care