Electrospun Fiber Surface Roughness Modulates Human Monocyte-Derived Macrophage Phenotype.

Injuries to fibrous connective tissues have very little capacity for self-renewal and exhibit poor healing after injury. Phenotypic shifts in macrophages play a vital role in mediating the healing response, creating an opportunity to design immunomodulatory biomaterials which control macrophage polarization and promote regeneration. In this study, electrospun poly(-caprolactone) fibers with increasing surface roughness (SR) were produced by increasing relative humidity and inducing vapor-induced phase separation during the electrospinning process. The impact of surface roughness on macrophage phenotype was assessed using human monocyte-derived macrophages in vitro and in vivo using B6.Cg-Tg(Csf1r-EGFP)1Hume/J (MacGreen) mice. In vitro experiments showed that macrophages cultured on mesh with increasing SR exhibited decreased release of both pro- and anti-inflammatory cytokines potentially driven by increased protein adsorption and biophysical impacts on the cells. Further, increasing SR led to an increase in the expression of the pro-regenerative cell surface marker CD206 relative to the pro-inflammatory marker CD80. Mesh with increasing SR were implanted subcutaneously in MacGreen mice, again showing an increase in the ratio of cells expressing CD206 to those expressing CD80 visualized by immunofluorescence. SR on implanted biomaterials is sufficient to drive macrophage polarization, demonstrating a simple feature to include in biomaterial design to control innate immunity.

Coxsackievirus B infection invokes unique cell-type specific responses in primary human pancreatic islets.

Coxsackievirus B (CVB) infection has long been considered an environmental factor precipitating Type 1 diabetes (T1D), an autoimmune disease marked by loss of insulin-producing ß cells within pancreatic islets. Previous studies have shown CVB infection negatively impacts islet function and viability but do not report on how virus infection individually affects the multiple cell types present in human primary islets. Therefore, we hypothesized that the various islet cell populations have unique transcriptional responses to CVB infection. Here, we performed single-cell RNA sequencing on human cadaveric islets treated with either CVB or poly(I:C), a viral mimic, for 24 and 48 hours. Our global analysis reveals CVB differentially induces dynamic transcriptional changes associated with multiple cell processes and functions over time whereas poly(I:C) promotes an immune response that progressively increases with treatment duration. At the single-cell resolution, we find CVB infects all islet cell types at similar rates yet induces unique cell-type specific transcriptional responses with ß, α, and ductal cells having the strongest response. Sequencing and functional data suggest that CVB negatively impacts mitochondrial respiration and morphology in distinct ways in ß and α cells, while also promoting the generation of reactive oxygen species. We also observe an increase in the expression of the long-noncoding RNA MIR7-3HG in ß cells with high viral titers and reveal its knockdown reduces gene expression of viral proteins as well as apoptosis in stem cell-derived islets. Together, these findings demonstrate a cell-specific transcriptional, temporal, and functional response to CVB infection and provide new insights into the relationship between CVB infection and T1D.

Impact of particle size of cereals and soyabean meal on the intestinal development of weanling pigs and growth performance after an enteric challenge with F4-positive enterotoxigenic E. coli.

The objectives were to determine the interactive effect of particle size of soyabean meal (SBM) and whole wheat, barley and wheat bran (CER) on growth performance of weanling pigs after an enterotoxigenic Escherichia coli F4 challenge (Experiment 1) and on gastrointestinal (GIT) development immediately after weaning (Experiment 2). Experiment 1 consisted of 192 pigs (24 ± 3 days of age; 7.4 ± 1.1 kg weaning bodyweight [BW]) selected for Escherichia coli (E. coli) F4 susceptibility. Pigs were given an oral E. coli inoculum at postweaning day 7, to induce an enteric health challenge. Experiment 2 consisted of 40 pigs (24 ± 3 days of age; 7.2 ± 1.0 kg weaning BW) that were killed on postweaning day 8 or 9, to determine the effects of particle size on GIT development and functionality. Four experimental diets were used in a 2 × 2 factorial design: (1) coarse CER and coarse SBM, (2) coarse CER and fine SBM (CERcSBMf), (3) fine CER and coarse SBM, or (4) fine CER and fine SBM (CERfSBMf). Results showed no interaction between SBM and CER coarseness on growth performance, GIT development and functionality. Diarrhoea incidence was higher (p < 0.05) for CERfSBMf during the 2 weeks following the E. coli challenge compared to the other diets. Daily gain and feed intake during this period were higher (p < 0.05) for pigs fed CERc compared to CERf. Empty stomach weight tended to be greater by 8% (p = 0.09) for CERc compared to CERf. Gastric protein (p = 0.05) and starch (p = 0.04) disappearances were greater for SBMf compared to SBMc. Thus, CERcSBMf resulted in the best growth performance and lowest diarrhoea incidence during the 2 weeks following the E. coli challenge, which may be explained by changes in stomach functionality but not by changes in other parts of the GIT.

Characterization and quantification of in-vitro equine bone resorption in 3D using µCT and deep learning-aided feature segmentation.

High cyclic strains induce formation of microcracks in bone, triggering targeted bone remodeling, which entails osteoclastic resorption. Racehorse bone is an ideal model for studying the effects of high-intensity loading, as it is subject to focal formation of microcracks and subsequent bone resorption. The volume of resorption in vitro is considered a direct indicator of osteoclast activity but indirect 2D measurements are used more often. Our objective was to develop an accurate, high-throughput method to quantify equine osteoclast resorption volume in µCT 3D images. Here, equine osteoclasts were cultured on equine bone slices and imaged with µCT pre- and postculture. Individual resorption events were then isolated and analyzed in 3D. Modal volume, maximum depth, and aspect ratio of resorption events were calculated. A convolutional neural network (CNN U-Net-like) was subsequently trained to identify resorption events on post-culture µCT images alone, without the need for pre-culture imaging, using archival bone slices with known resorption areas and paired CTX-I biomarker levels in culture media. 3D resorption volume measurements strongly correlated with both the CTX-I levels (p < 0.001) and area measurements (p < 0.001). Our 3D analysis shows that the shapes of resorption events form a continuous spectrum, rather than previously reported pit and trench categories. With more extensive resorption, shapes of increasing complexity appear, although simpler resorption cavity morphologies (small, rounded) remain most common, in acord with the left-hand limit paradigm. Finally, we show that 2D measurements of in vitro osteoclastic resorption are a robust and reliable proxy.

A static glucose-stimulated insulin secretion (sGSIS) assay that is significantly predictive of time to diabetes reversal in the human islet bioassay.

INTRODUCTION: Static incubation (static glucose-stimulated insulin secretion, sGSIS) is a measure of islet secretory function. The Stimulation Index (SI; insulin produced in high glucose/insulin produced in low glucose) is currently used as a product release criterion of islet transplant potency. RESEARCH DESIGN AND METHODS: Our hypothesis was that the Delta, insulin secreted in high glucose minus insulin secreted in low glucose, would be more predictive. To evaluate this hypothesis, sGSIS was performed on 32 consecutive human islet preparations, immobilizing the islets in a slurry of Sepharose beads to minimize mechanical perturbation. Simultaneous full-mass subrenal capsular transplants were performed in chemically induced diabetic immunodeficient mice. Logistic regression analysis was used to determine optimal cut-points for diabetes reversal time and the Fisher Exact Test was used to assess the ability of the Delta and the SI to accurately classify transplant outcomes. Receiver operating characteristic curve analysis was performed on cut-point grouped data, assessing the predictive power and optimal cut-point for each sGSIS potency metric. Finally, standard Kaplan-Meier-type survival analysis was conducted. RESULTS: In the case of the sGSIS the Delta provided a superior islet potency metric relative to the SI.ConclusionsThe sGSIS Delta value is predicitive of time to diabetes reversal in the full mass human islet transplant bioassay.

NADPH Oxidase 2-Derived Reactive Oxygen Species Promote CD8+ T Cell Effector Function.

Oxidants participate in lymphocyte activation and function. We previously demonstrated that eliminating the activity of NADPH oxidase 2 (NOX2) significantly impaired the effectiveness of autoreactive CD8+ CTLs. However, the molecular mechanisms impacting CD8+ T cell function remain unknown. In the present study, we examined the role of NOX2 in both NOD mouse and human CD8+ T cell function. Genetic ablation or chemical inhibition of NOX2 in CD8+ T cells significantly suppressed activation-induced expression of the transcription factor T-bet, the master transcription factor of the Tc1 cell lineage, and T-bet target effector genes such as IFN-γ and granzyme B. Inhibition of NOX2 in both human and mouse CD8+ T cells prevented target cell lysis. We identified that superoxide generated by NOX2 must be converted into hydrogen peroxide to transduce the redox signal in CD8+ T cells. Furthermore, we show that NOX2-generated oxidants deactivate the tumor suppressor complex leading to activation of RheB and subsequently mTOR complex 1. These results indicate that NOX2 plays a nonredundant role in TCR-mediated CD8+ T cell effector function.

Variability in feed intake the first days following weaning impacts gastrointestinal tract development, feeding patterns, and growth performance in nursery pigs.

The present study investigated the effects of voluntary feed intake (FI) the first days after weaning on gastrointestinal development and protein fermentation the first week after weaning and growth performance and feeding patterns during the nursery phase. A total of 144 mixed-sex weaned pigs (24 ±â€…2 d old; 7.2 ±â€…0.8 kg body weight [BW]) were allocated to 12 pens with 12 pigs/pen. Each pen was equipped with an electronic feeding station for monitoring individual FI during a 40-d study. Pigs were classified based on their cumulative FI over the initial 3 d after weaning (FId1-3) being above or below their pen median FId1-3 (high = 919 ±â€…244 g or low = 507 ±â€…222 g FId1-3). Similarly, weaning BW classes (BW0; high = 7.72 ±â€…0.59 kg or low = 6.62 ±â€…0.88 kg BW) were created to study interactions with FId1-3. Two female pigs with either a high or a low FId1-3 per pen (n = 24) were selected for sampling at d6 and were used to study gastrointestinal development and fermentation products in the small intestine. Feeding patterns per day, FI, and growth performance were measured individually. Low FId1-3 pigs had lower (P < 0.05) daily FI during d0 to d8, d8 to d15, and d22 to d28, BW on d15, d22, d29, and d40, and average daily gain during d0 to d8, d22 to d29, and d29 to d40 compared to high FId1-3. High FId1-3 pigs increased (P < 0.05) the number of visits to the feeder between d1 to d13 and d31 to d35, and the time spent per visit only for d1 to d4 (P < 0.05). The daily rate of FI (g/min) was higher (P < 0.05) for High FId1-3 pigs on d6, d8, d9, and d10, and again several days later (d20 to d39). In addition, the high FId1-3 × high BW0 interaction improved daily FI during d18 to d40 compared to low FId1-3 × low BW0 class (P < 0.05). For the sampling on d6, low FId1-3 pigs had a lighter small intestine, colon, and pancreas, and reduced villi length, smaller villi surface area, and a lower number of goblet cells size in jejunum (P < 0.05), while concentrations of lactic acid, histamine, and cadaverine in small intestinal content were increased (P < 0.05). In conclusion, pigs with high FId1-3 became faster eaters with higher FI and growth rates toward the second half of the nursery, which was similar and additive for pigs with higher weaning BW. High FId1-3 was also associated with greater development of the gastrointestinal tract and a reduced protein fermentation 1-wk after weaning.

Poor adaptation to solid feed after weaning is often associated with a reduced digestive function and growth in nursery pigs. The reasons driving an early acceptance of feed and its consequences are still largely unknown. We investigated the effects of high and low feed intake between d1-3 after weaning on gastrointestinal development and morphometrics 1-wk after weaning and growth performance and feeding patterns in the nursery phase. The results showed that pigs with a high initial feed intake (increased number of visits to the feeder and time spent per visit early after weaning), consumed feed faster throughout the nursery resulting in higher intakes early and late in the nursery but not for the intermediate period. Higher weaning body weight was also associated with improved feed intake and growth from d17 onwards, which was an additional but independent effect of the early feed intake effect. Besides, pigs with high feed intake between d1 and d3 after weaning had heavier empty gastrointestinal organs, improved intestinal wall morphometrics, and reduced protein fermentation in the small intestine 1-wk after weaning.

GABA and Combined GABA with GAD65-Alum Treatment Alters Th1 Cytokine Responses of PBMCs from Children with Recent-Onset Type 1 Diabetes.

Type 1 diabetes (T1D) is an autoimmune disease culminating in the destruction of insulin-producing pancreatic cells. There is a need for the development of novel antigen-specific strategies to delay cell destruction, including combinatorial strategies that do not elicit systemic immunosuppression. Gamma-aminobutyric acid (GABA) is expressed by immune cells, ß-cells, and gut bacteria and is immunomodulatory. Glutamic-acid decarboxylase 65 (GAD65), which catalyzes GABA from glutamate, is a T1D autoantigen. To test the efficacy of combinatorial GABA treatment with or without GAD65-immunization to dampen autoimmune responses, we enrolled recent-onset children with T1D in a one-year clinical trial (ClinicalTrials.gov NCT02002130) and examined T cell responses. We isolated peripheral blood mononuclear cells and evaluated cytokine responses following polyclonal activation and GAD65 rechallenge. Both GABA alone and GABA/GAD65-alum treatment inhibited Th1 cytokine responses over the 12-month study with both polyclonal and GAD65 restimulation. We also investigated whether patients with HLA-DR3-DQ2 and HLA-DR4-DQ8, the two highest-risk human leukocyte antigen (HLA) haplotypes in T1D, exhibited differences in response to GABA alone and GABA/GAD65-alum. HLA-DR4-DQ8 patients possessed a Th1-skewed response compared to HLA-DR3-DQ2 patients. We show that GABA and GABA/GAD65-alum present an attractive immunomodulatory treatment for children with T1D and that HLA haplotypes should be considered.

Impact of L-Arginine and L-Glutamine supplementation on growth performance and immune status in weanling pigs challenged with Escherichia coli F4.

Arginine (ARG) and Glutamine (GLN) have been reported to play significant roles in protein metabolism, immunity, and intestinal health in weanling pigs. The present study investigated the independent and interactive effect of supplementing ARG and GLN on pigs immune status and growth performance following an Escherichia coli F4 challenge. A total of 240 mixed-sex pigs (24 ±â€…2 d old; 7.3 ±â€…0.1 kg BW) were used in a 42-d experiment after selection for E. coli F4 susceptibility. The pigs were group-housed (3 pigs per pen), and pens were randomly assigned to five experimental treatments (N = 16 pens per treatment). Experimental treatments were: 1) a wheat-barley-soybean meal-based basal diet (CTRL), 2) a basal diet with 2500 mg/kg zinc oxide (ZnO), 3) a basal diet + 0.5% Glutamine (0.5% GLN), 4) basal diet + 0.5% Arginine (0.5% ARG), and 5) basal diet with 0.5% Glutamine + 0.5% Arginine (0.5% GLN + ARG). All Pigs were inoculated with E. coli F4 on days 7, 8, and 9 post-weaning. Rectal swabs were taken from each pig and plated on blood agar plates for E. coli F4 presence. Blood and fecal samples were taken to determine the acute phase response and selected fecal biomarkers for the immune response. Growth performance and fecal scores were recorded. Fecal swabs resulted in no positive pig for E. coli F4 before inoculation and 73.3% positive postinoculation. Diarrhea incidence during days 7 to 14 was significantly lower for the ZnO treatment (P < 0.05). The haptoglobin level on day 3 was lower than days 10 and 20, irrespective of treatment (P < 0.05). The albumin level was lower on day 20 compared to days 3 and 10 (P < 0.05). There was no treatment effect on albumin levels regardless of sampling day (P > 0.05). The PigMAP was lowest on day 3 and highest on day 10 (P < 0.05). We did not observe significant treatment differences (P > 0.05) in myeloperoxidase and calprotectin. Pancreatitis-associated protein was higher in the ZnO (P = 0.001) treatment than in the other treatments. Fecal IgA tended (P = 0.10) to be higher in the ZnO and 0.5% ARG treatments. There were no performance differences, except during days 0 to 7, where the ZnO treatment was lower in average daily gain and average daily feed intake (P < 0.001), while feed efficiency (G:F) FE was similar across treatments. In summary, no improved performance was observed with either ARG, glutamate, or both. The immune response results showed that the E. coli F4 challenge may have exacerbated the acute phase response; hence, the benefits of dietary treatments did not go beyond immune repair and reduction in inflammation.

The supplementation of functional amino acids such as arginine and glutamine has been reported to improve growth performance in weanling pigs. However, during periods of enteric Escherichia coli challenge, results have been inconsistent. Here, we investigated whether independent or combined arginine and glutamine supplementation could improve performance and immune response under an E. coli F4 challenge. The results showed no performance improvements but an improvement in the acute phase response and immune response status. Thus, the benefit of supplemental arginine and glutamine may be prioritized towards building an immune response rather than growth under conditions of an enteric immune challenge.

Nanothin Conformal Coating with Poly(N-vinylpyrrolidone) and Tannic Acid (PVPON/TA) Preserves Murine and Human Pancreatic Islets Function.

Beta cell replacement therapies can restore glycemic control to select individuals living with type 1 diabetes. However, the obligation of lifelong immunosuppression restricts cell therapies from replacing exogenous insulin administration. Encapsulation strategies can reduce the inherent adaptive immune response; however, few are successfully translated into clinical testing. Herein, we evaluated if the conformal coating of islets with poly(N-vinylpyrrolidone) (PVPON) and tannic acid (TA) (PVPON/TA) could preserve murine and human islet function while conferring islet allograft protection. In vitro function was evaluated using static glucose-stimulated insulin secretion, oxygen consumption rates, and islet membrane integrity. In vivo function was evaluated by transplanting human islets into diabetic immunodeficient B6.129S7-Rag1tm1Mom/J (Rag-/-) mice. The immunoprotective capacity of the PVPON/TA-coating was assessed by transplanting BALB/c islets into diabetic C57BL/6 mice. Graft function was evaluated by non-fasting blood glucose measurements and glucose tolerance testing. Both coated and non-coated murine and human islets exhibited indistinguishable in vitro potency. PVPON/TA-coated and control human islets were able to restore euglycemia post-transplant. The PVPON/TA-coating as monotherapy and adjuvant to systemic immunosuppression reduced intragraft inflammation and delayed murine allograft rejection. This study demonstrates that PVPON/TA-coated islets may be clinically relevant as they retain their in vitro and in vivo function while modulating post-transplant immune responses.

Pancreatic islet cell type-specific transcriptomic changes during pregnancy and postpartum.

Pancreatic ß-cell mass expands during pregnancy and regresses in the postpartum period in conjunction with dynamic metabolic demands on maternal glucose homeostasis. To understand transcriptional changes driving these adaptations in ß-cells and other islet cell types, we performed single-cell RNA sequencing on islets from virgin, late gestation, and early postpartum mice. We identified transcriptional signatures unique to gestation and the postpartum in ß-cells, including induction of the AP-1 transcription factor subunits and other genes involved in the immediate-early response (IEGs). In addition, we found pregnancy and postpartum-induced changes differed within each endocrine cell type, and in endothelial cells and antigen-presenting cells within islets. Together, our data reveal insights into cell type-specific transcriptional changes responsible for adaptations by islet cells to pregnancy and their resolution postpartum.

Supplementing oat hulls to the diet of suckling piglets altered their intestinal tract and colonic microbiota development.

Current study evaluated the effect of a fine and coarsely ground insoluble dietary fibre source on the gastrointestinal development of suckling pigs. Oat hulls (OH) were selected as a model feedstuff, rich in cellulose, lignin, and insoluble dietary fibre. Three experimental supplemental diets were formulated: a finely ground, low fibre and nutrient dense diet served as control (CON). For the 2 high fibre diets, 15% heat-treated starch in CON was exchanged with OH, either finely (OH-f) or coarsely ground (OH-c). Litters of 10 primi- and multiparous sows (mean litter size 14.6 ± 0.84) were used. Within a litter, experimental diets were allotted to triplets of 4 piglets. From approximately 12 d of age, piglets' individual feed intakes were recorded 2 times per day when separated from their dam for 70 min. Piglets could suckle with their dam for the remainder of the day. On d 24 and 25, from the total pool of 120 piglets, seven healthy well-eating piglets per treatment were selected for post-mortem evaluation, resulting in 14 replicates per treatment. Consumption of OH-c and OH-f did not impede clinical health and production performance of piglets. The full stomach weights tended to be greater for OH-c compared to OH-f whereas CON was intermediate (P = 0.083). Supplementing OH significantly increased ileal villus height and caecal dry matter concentration (P < 0.05). For the colon, OH increased its length, contents weight, short-chain fatty acid concentration and reduced total bacterial count as well as γ-proteobacteria count and proportion (P < 0.05). The OH-c treatment specifically increased full gastrointestinal tract weight and caecum contents weight compared to piglets fed CON and OH-f. Furthermore, OH-c reduced colonic crypt depth when compared to OH-f (P = 0.018). In conclusion, supplementing OH to a diet for suckling piglets exerted subtle developmental effects on gastrointestinal morphology and colonic microbial community. These effects were largely independent from the particle size of the OH.

Localized cytotoxic T cell-associated antigen 4 and antioxidant islet encapsulation alters macrophage signaling and induces regulatory and anergic T cells to enhance allograft survival.

The loss of functional ß-cell mass is a hallmark of type 1 diabetes. Islet transplantation represents a promising alternative approach, but immune-mediated graft destruction remains a major challenge. We sought to use islet encapsulation technologies to improve graft survival and function without systemic immunosuppression. We hypothesized islet encapsulation with nanothin coatings consisting of tannic acid (TA), an antioxidant; poly(N-vinylpyrrolidone) (PVPON), a biocompatible polymer; and cytotoxic T cell-associated antigen 4 immunoglobulin (CTLA-4-Ig), an inhibitory immune receptor, will elicit localized immunosuppression to prolong islet allograft function and suppress effector T cell responses. In the absence of systemic immunosuppression, we demonstrated (PVPON/TA/CTLA-4-Ig)-encapsulated NOD.Rag islet grafts maintain function significantly longer than control IgG-containing (PVPON/TA/IgG) and nonencapsulated controls after transplantation into diabetic C57BL/6 mice. This protection coincided with diminished proinflammatory macrophage responses mediated by signal transducer and activator of transcription 1 signaling, decreased proinflammatory T cell effector responses, and CTLA-4-Ig-specific concomitant increases in anergic CD4+ T cells and regulatory T cells. Our results provide evidence that conjugation of CTLA-4-Ig to (PVPON/TA) coatings can suppress T cell activation, enhance regulatory T cell populations, prolong islet allograft survival, and induce localized immunosuppression after transplantation.

Deletion of Tbc1d4/As160 abrogates cardiac glucose uptake and increases myocardial damage after ischemia/reperfusion.

BACKGROUND: Type 2 Diabetes mellitus (T2DM) is a major risk factor for cardiovascular disease and associated with poor outcome after myocardial infarction (MI). In T2DM, cardiac metabolic flexibility, i.e. the switch between carbohydrates and lipids as energy source, is disturbed. The RabGTPase-activating protein TBC1D4 represents a crucial regulator of insulin-stimulated glucose uptake in skeletal muscle by controlling glucose transporter GLUT4 translocation. A human loss-of-function mutation in TBC1D4 is associated with impaired glycemic control and elevated T2DM risk. The study's aim was to investigate TBC1D4 function in cardiac substrate metabolism and adaptation to MI. METHODS: Cardiac glucose metabolism of male Tbc1d4-deficient (D4KO) and wild type (WT) mice was characterized using in vivo [18F]-FDG PET imaging after glucose injection and ex vivo basal/insulin-stimulated [3H]-2-deoxyglucose uptake in left ventricular (LV) papillary muscle. Mice were subjected to cardiac ischemia/reperfusion (I/R). Heart structure and function were analyzed until 3 weeks post-MI using echocardiography, morphometric and ultrastructural analysis of heart sections, complemented by whole heart transcriptome and protein measurements. RESULTS: Tbc1d4-knockout abolished insulin-stimulated glucose uptake in ex vivo LV papillary muscle and in vivo cardiac glucose uptake after glucose injection, accompanied by a marked reduction of GLUT4. Basal cardiac glucose uptake and GLUT1 abundance were not changed compared to WT controls. D4KO mice showed mild impairments in glycemia but normal cardiac function. However, after I/R D4KO mice showed progressively increased LV endsystolic volume and substantially increased infarction area compared to WT controls. Cardiac transcriptome analysis revealed upregulation of the unfolded protein response via ATF4/eIF2α in D4KO mice at baseline. Transmission electron microscopy revealed largely increased extracellular matrix (ECM) area, in line with decreased cardiac expression of matrix metalloproteinases of D4KO mice. CONCLUSIONS: TBC1D4 is essential for insulin-stimulated cardiac glucose uptake and metabolic flexibility. Tbc1d4-deficiency results in elevated cardiac endoplasmic reticulum (ER)-stress response, increased deposition of ECM and aggravated cardiac damage following MI. Hence, impaired TBC1D4 signaling contributes to poor outcome after MI.

Diet and stomach characteristics of feral piglets (Sus scrofa): Implications for farmed piglets.

Evaluation of the diet of the pig (Sus scrofa) in natural settings may provide new views on diet optimization for growth and development of commercially raised piglets under farm conditions. A field study was conducted to gain insight in the diet and stomach characteristics of feral piglets. Forty animals (body weight: 4.6 ± 1.37 kg) were collected from the Bahía Samborombón (Buenos Aires, Argentina). Stomachs were weighed after storage in formalin and the particle size distribution of their contents was determined by wet sieving. Diet items present in their stomachs were classified and their proportional weight and relative abundance was calculated. Based on their dentition, 5, 16 and 19 piglets were approximately 1, 3-6 and 6-16 weeks of age respectively. Vegetable matter (mainly 'leaves and stems') was predominantly present in 39 animals. It represented on average 83 ± 36.4% of total stomach contents by weight. The stomachs of 12 piglets contained curd and represented on average 16 ± 35.1% by weight. Other diet items were less abundant or absent. The proportion of stomach particles retained were 24%, 13%, 22%, 13% and 28% for sieves with mesh sizes of 2000, 1000, 420, 210 and <210 µm respectively. For comparison, we used data of farmed piglets of similar age and fed a nutrient-dense, finely ground diet. Feral piglets' relative empty stomach weights increased with age (p < 0.050), whereas this was not the case for farmed piglets. Relative stomach contents weight increased significantly with age only for farmed piglets (p < 0.050). We infer from our data that feral suckling piglets consumed a variety of non-milk items, mainly consisting of vegetable material with a coarse particle size from their first week in life onwards. Their diet is associated with an enhanced stomach development compared to those of farmed piglets.

MDA5-dependent responses contribute to autoimmune diabetes progression and hindrance.

Type 1 diabetes (T1D) is an autoimmune disease resulting in pancreatic ß cell destruction. Coxsackievirus B3 (CVB3) infection and melanoma differentiation-associated protein 5-dependent (MDA5-dependent) antiviral responses are linked with T1D development. Mutations within IFIH1, coding for MDA5, are correlated with T1D susceptibility, but how these mutations contribute to T1D remains unclear. Utilizing nonobese diabetic (NOD) mice lacking Ifih1 expression (KO) or containing an in-frame deletion within the ATPase site of the helicase 1 domain of MDA5 (ΔHel1), we tested the hypothesis that partial or complete loss-of-function mutations in MDA5 would delay T1D by impairing proinflammatory pancreatic macrophage and T cell responses. Spontaneous T1D developed in female NOD and KO mice similarly, but was significantly delayed in ΔHel1 mice, which may be partly due to a concomitant increase in myeloid-derived suppressor cells. Interestingly, KO male mice had increased spontaneous T1D compared with NOD mice. Whereas NOD and KO mice developed CVB3-accelerated T1D, ΔHel1 mice were protected partly due to decreased type I IFNs, pancreatic infiltrating TNF+ macrophages, IFN-γ+CD4+ T cells, and perforin+CD8+ T cells. Furthermore, ΔHel1 MDA5 protein had reduced ATP hydrolysis compared with wild-type MDA5. Our results suggest that dampened MDA5 function delays T1D, yet loss of MDA5 promotes T1D.

Getting clues from nature: the impact of grass hay on suckling piglets' gastrointestinal growth and colonic microbiota.

Introduction: The effect of dietary fiber on pig production has been extensively evaluated. Inspired by observations of the diet of wild, young piglets, this study aimed to examine the possibility of feeding grass hay to suckling piglets besides concentrated creep feed. Methods: The sow-nursed piglets in this study were divided into two groups based on balanced sow parities. The control group (CON, n = 7 sows) only received a regular, concentrated creep feed, while the treatment piglets (GH, n = 8 sows) were also provided with chopped grass hay from 2 days of age until weaning (28 days). At weaning, one piglet with a median weight was selected from each litter for post-mortem evaluation. Subsequently, six pigs around median weight per sow were grouped into nursery pens and monitored for their feed intake and body weight gain until 9 weeks of age. Results and discussion: Piglets in GH consumed, on average, 57 g of grass hay per piglet during the entire lactation period. The emptied weight of the small and large intestine was significantly greater in GH (280 vs. 228 g, 88.8 vs. 79.3 g, respectively, p < 0.05), and the length of the large intestine was stimulated by the grass hay (164 vs. 150 cm, p < 0.05). Morphologically, the villus height in the jejunum was higher in GH (p < 0.05). In the large intestine, the crypt depth of the mid-colon was lower in GH. Moreover, the short-chain fatty acid (SCFA) concentrations in the cecum were increased in GH compared to CON (1,179 vs. 948 µmol/g dry matter, p < 0.05), whereas in the colon, SCFA concentrations were lower in CON (341 vs. 278 µmol/g dry matter, p < 0.05). There was no major impact of grass hay inclusion on the colonic microbiota composition. Only a trend was observed for a lower inverse of the classical Simpson (InvSimpon) index and a higher abundance of Lactobacillus genera in GH. After weaning, no significant differences in feed intake and body weight gain were observed. In conclusion, supplementing the grass hay to suckling piglets led to alterations in intestinal morphology, increased SCFA fermentation in proximal sections of large intestine, stimulation of gastrointestinal tract growth, and subtle modifications in colonic microbiota.

A randomized trial of oral gamma aminobutyric acid (GABA) or the combination of GABA with glutamic acid decarboxylase (GAD) on pancreatic islet endocrine function in children with newly diagnosed type 1 diabetes.

Gamma aminobutyric acid(GABA) is synthesized by glutamate decarboxylase(GAD) in ß-cells. Regarding Type 1 diabetes(T1D), animal/islet-cell studies found that GABA promotes insulin secretion, inhibits α-cell glucagon and dampens immune inflammation, while GAD immunization may also preserve ß-cells. We evaluated the safety and efficacy of oral GABA alone, or combination GABA with GAD, on the preservation of residual insulin secretion in recent-onset T1D. Herein we report a single-center, double-blind, one-year, randomized trial in 97 children conducted March 2015 to June 2019(NCT02002130). Using a 2:1 treatment:placebo ratio, interventions included oral GABA twice-daily(n = 41), or oral GABA plus two-doses GAD-alum(n = 25), versus placebo(n = 31). The primary outcome, preservation of fasting/meal-stimulated c-peptide, was not attained. Of the secondary outcomes, the combination GABA/GAD reduced fasting and meal-stimulated serum glucagon, while the safety/tolerability of GABA was confirmed. There were no clinically significant differences in glycemic control or diabetes antibody titers. Given the low GABA dose for this pediatric trial, future investigations using higher-dose or long-acting GABA formulations, either alone or with GAD-alum, could be considered, although GABA alone or in combination with GAD-alum did nor preserve beta-cell function in this trial.

Effect of capillary fluid flow on single cancer cell cycle dynamics, motility, volume and morphology.

From primary tumours and disseminating to secondary organs, cancer cells experience a wide variety of fluid flow profiles when passing through blood vessels or the lymphatic system before extravasation. Sinusoidal capillaries are a common site for extravasation. Therefore, we aim to investigate how metastatic cancer cells react to a biophysical cue such as capillary fluid flow by quantifying its effect on metastatic cell cycle progression, motility, cell and nuclear volume, and morphology. We use MDA-MB-231 breast cancer cells genetically modified with fluorescent ubiquitination-based cell cycle indicator 2 (FUCCI2) as a model system. Single cells are trapped using a microfluidic device and exposed to different laminar flows. Quantitative time-lapse imaging in both 2D epifluorescence and 3D confocal microscopy is performed using in-house software FUCCItrack. In addition, 3D time-lapse with cell and nuclear segmentation is performed with a deep learning approach to streamline the image processing of big datasets. We show that at a single cell level, faster fluid flow leads to a shorter S/G2/M phase and an overall shorter cell cycle, as well as increase in cell motility irrespective of the flow direction. 3D time-lapse confocal imaging of MDA-FUCCI2 single cells reveals the evolution of cell and nuclear volume and morphology as a function of a specific cell cycle phase. Both cell and nuclear volume increase linearly over time. Cell morphology elongates more strongly during the S/G2/M phase, whereas the nuclear shape remains constant. Under the highest flow conditions, only during the S/G2/M phase can we observe a more elongated nucleus, while the cell sphericity remains similar to the control. Collectively, this data, together with the deep learning approach, provides new insights into the potential impact of fluid flow at a single cell level.

Olfactory Dysfunction Predicts Frailty and Poor Postoperative Outcome in Older Patients Scheduled for Elective Non-Cardiac Surgery.

OBJECTIVES: Frailty has been suggested to take part in the recently demonstrated link between olfactory dysfunction and overall mortality risk. Preoperative assessment of frailty is essential to detect the most vulnerable patients scheduled for surgery. The aim of this study was to evaluate whether olfactory dysfunction is a reliable predictor of preoperative frailty and postoperative outcome. DESIGN: This was a single-center prospective observational study conducted between July and October 2020 in Brussels, Belgium. SETTING AND PARTICIPANTS: 155 preoperative patients aged from 65 years old and scheduled for elective non-cardiac surgery. MEASUREMENTS: Olfactory function was examined using the Sniffin' Sticks 12-item identification test. Frailty was assessed using the Edmonton Frail Scale (EFS) and handgrip strength. The clock drawing test (CDT) from the EFS was also analyzed separately to evaluate cognitive function. Patients were followed for postoperative complications and mortality over one year. RESULTS: Olfactory dysfunction was significantly associated with the EFS score, anosmic patients having a higher median EFS score than normosmic patients (6[4-7] vs 4[2-5], p = .025). Anosmic patients had an increased odds of being frail after adjusting for possible confounding factors (OR: 6.19, 95% CI: 1.65-23.20, p = .007) and were more at risk of poor postoperative outcome (including complications and death) (OR: 4.33, 95% CI: 1.28-14.67, p = .018). CONCLUSIONS: Olfactory dysfunction is associated with preoperative frailty determined by the EFS and with poor post-surgical outcome at one-year.