RFX6 haploinsufficiency predisposes to diabetes through impaired beta cell function.

AIMS/HYPOTHESIS: Regulatory factor X 6 (RFX6) is crucial for pancreatic endocrine development and differentiation. The RFX6 variant p.His293LeufsTer7 is significantly enriched in the Finnish population, with almost 1:250 individuals as a carrier. Importantly, the FinnGen study indicates a high predisposition for heterozygous carriers to develop type 2 and gestational diabetes. However, the precise mechanism of this predisposition remains unknown. METHODS: To understand the role of this variant in beta cell development and function, we used CRISPR technology to generate allelic series of pluripotent stem cells. We created two isogenic stem cell models: a human embryonic stem cell model; and a patient-derived stem cell model. Both were differentiated into pancreatic islet lineages (stem-cell-derived islets, SC-islets), followed by implantation in immunocompromised NOD-SCID-Gamma mice. RESULTS: Stem cell models of the homozygous variant RFX6-/- predictably failed to generate insulin-secreting pancreatic beta cells, mirroring the phenotype observed in Mitchell-Riley syndrome. Notably, at the pancreatic endocrine stage, there was an upregulation of precursor markers NEUROG3 and SOX9, accompanied by increased apoptosis. Intriguingly, heterozygous RFX6+/- SC-islets exhibited RFX6 haploinsufficiency (54.2% reduction in protein expression), associated with reduced beta cell maturation markers, altered calcium signalling and impaired insulin secretion (62% and 54% reduction in basal and high glucose conditions, respectively). However, RFX6 haploinsufficiency did not have an impact on beta cell number or insulin content. The reduced insulin secretion persisted after in vivo implantation in mice, aligning with the increased risk of variant carriers to develop diabetes. CONCLUSIONS/INTERPRETATION: Our allelic series isogenic SC-islet models represent a powerful tool to elucidate specific aetiologies of diabetes in humans, enabling the sensitive detection of aberrations in both beta cell development and function. We highlight the critical role of RFX6 in augmenting and maintaining the pancreatic progenitor pool, with an endocrine roadblock and increased cell death upon its loss. We demonstrate that RFX6 haploinsufficiency does not affect beta cell number or insulin content but does impair function, predisposing heterozygous carriers of loss-of-function variants to diabetes. DATA AVAILABILITY: Ultra-deep bulk RNA-seq data for pancreatic differentiation stages 3, 5 and 7 of H1 RFX6 genotypes are deposited in the Gene Expression Omnibus database with accession code GSE234289. Original western blot images are deposited at Mendeley ( https://data.mendeley.com/datasets/g75drr3mgw/2 ).

Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis.

Primary defects in folding of mutant proinsulin can cause dominant-negative proinsulin accumulation in the endoplasmic reticulum (ER), impaired anterograde proinsulin trafficking, perturbed ER homeostasis, diminished insulin production, and ß-cell dysfunction. Conversely, if primary impairment of ER-to-Golgi trafficking (which also perturbs ER homeostasis) drives misfolding of nonmutant proinsulin-this might suggest bi-directional entry into a common pathological phenotype (proinsulin misfolding, perturbed ER homeostasis, and deficient ER export of proinsulin) that can culminate in diminished insulin storage and diabetes. Here, we've challenged ß-cells with conditions that impair ER-to-Golgi trafficking, and devised an accurate means to assess the relative abundance of distinct folded/misfolded forms of proinsulin using a novel nonreducing SDS-PAGE/immunoblotting protocol. We confirm abundant proinsulin misfolding upon introduction of a diabetogenic INS mutation, or in the islets of db/db mice. Whereas blockade of proinsulin trafficking in Golgi/post-Golgi compartments results in intracellular accumulation of properly-folded proinsulin (bearing native disulfide bonds), impairment of ER-to-Golgi trafficking (regardless whether such impairment is achieved by genetic or pharmacologic means) results in decreased native proinsulin with more misfolded proinsulin. Remarkably, reversible ER-to-Golgi transport defects (such as treatment with brefeldin A or cellular energy depletion) upon reversal quickly restore the ER folding environment, resulting in the disappearance of pre-existing misfolded proinsulin while preserving proinsulin bearing native disulfide bonds. Thus, proper homeostatic balance of ER-to-Golgi trafficking is linked to a more favorable proinsulin folding (as well as trafficking) outcome.

Intra-oral Drainage of Submandibular Abscess: A Minimally Invasive Technique. A Prospective Study.

PURPOSE: To evaluate the efficacy of intraoral drainage of isolated submandibular space abscess as a minimally invasive surgical technique compared to the standard trans-cervical approach. PATIENTS AND METHODS: This prospective study included 40 subjects with isolated submandibular space abscesses. They were randomly divided into 2 equal groups: trans-cervical surgical drainage (group A) and intra-oral surgical drainage (group B). The included data were demographics, repeated surgery requirement, postsurgical hospitalization duration, formation of scar, and complications. RESULTS: Intraoral drainage (Group B) reduced the mean operative time by 15.25 min (P < 0.001) compared with trans-cervical incision (Group A). No considerable difference was found between the 2 groups in regarding hospitalization postoperatively. No weakness in marginal mandibular nerve was found in both groups. Three patients only have a cervical scar in a group (B) who required external drainage due to recollection. No recurrence was detected in a group (A). CONCLUSION: The current study demonstrated that isolated submandibular abscesses can be successfully managed with an intraoral drainage modality, and it is a better option than the trans-cervical approach regarding better cosmetic outcome and shorter operative time.

Primate-specific ZNF808 is essential for pancreatic development in humans.

Identifying genes linked to extreme phenotypes in humans has the potential to highlight biological processes not shared with all other mammals. Here, we report the identification of homozygous loss-of-function variants in the primate-specific gene ZNF808 as a cause of pancreatic agenesis. ZNF808 is a member of the KRAB zinc finger protein family, a large and rapidly evolving group of epigenetic silencers which target transposable elements. We show that loss of ZNF808 in vitro results in aberrant activation of regulatory potential contained in the primate-specific transposable elements it represses during early pancreas development. This leads to inappropriate specification of cell fate with induction of genes associated with liver identity. Our results highlight the essential role of ZNF808 in pancreatic development in humans and the contribution of primate-specific regions of the human genome to congenital developmental disease.

YouTube's recommendation algorithm is left-leaning in the United States.

With over two billion monthly active users, YouTube currently shapes the landscape of online political video consumption, with 25% of adults in the United States regularly consuming political content via the platform. Considering that nearly three-quarters of the videos watched on YouTube are delivered via its recommendation algorithm, the propensity of this algorithm to create echo chambers and deliver extremist content has been an active area of research. However, it is unclear whether the algorithm may exhibit political leanings toward either the Left or Right. To fill this gap, we constructed archetypal users across six personas in the US political context, ranging from Far Left to Far Right. Utilizing these users, we performed a controlled experiment in which they consumed over eight months worth of videos and were recommended over 120,000 unique videos. We find that while the algorithm pulls users away from political extremes, this pull is asymmetric, with users being pulled away from Far Right content stronger than from Far Left. Furthermore, we show that the recommendations made by the algorithm skew left even when the user does not have a watch history. Our results raise questions on whether the recommendation algorithms of social media platforms in general, and YouTube, in particular, should exhibit political biases, and the wide-reaching societal and political implications that such biases could entail.

Perception, performance, and detectability of conversational artificial intelligence across 32 university courses.

The emergence of large language models has led to the development of powerful tools such as ChatGPT that can produce text indistinguishable from human-generated work. With the increasing accessibility of such technology, students across the globe may utilize it to help with their school work-a possibility that has sparked ample discussion on the integrity of student evaluation processes in the age of artificial intelligence (AI). To date, it is unclear how such tools perform compared to students on university-level courses across various disciplines. Further, students' perspectives regarding the use of such tools in school work, and educators' perspectives on treating their use as plagiarism, remain unknown. Here, we compare the performance of the state-of-the-art tool, ChatGPT, against that of students on 32 university-level courses. We also assess the degree to which its use can be detected by two classifiers designed specifically for this purpose. Additionally, we conduct a global survey across five countries, as well as a more in-depth survey at the authors' institution, to discern students' and educators' perceptions of ChatGPT's use in school work. We find that ChatGPT's performance is comparable, if not superior, to that of students in a multitude of courses. Moreover, current AI-text classifiers cannot reliably detect ChatGPT's use in school work, due to both their propensity to classify human-written answers as AI-generated, as well as the relative ease with which AI-generated text can be edited to evade detection. Finally, there seems to be an emerging consensus among students to use the tool, and among educators to treat its use as plagiarism. Our findings offer insights that could guide policy discussions addressing the integration of artificial intelligence into educational frameworks.

Aberrant metabolite trafficking and fuel sensitivity in human pluripotent stem cell-derived islets.

Pancreatic islets regulate blood glucose homeostasis through the controlled release of insulin; however, current metabolic models of glucose-sensitive insulin secretion are incomplete. A comprehensive understanding of islet metabolism is integral to studies of endocrine cell development as well as diabetic islet dysfunction. Human pluripotent stem cell-derived islets (SC-islets) are a developmentally relevant model of human islet function that have great potential in providing a cure for type 1 diabetes. Using multiple 13C-labeled metabolic fuels, we demonstrate that SC-islets show numerous divergent patterns of metabolite trafficking in proposed insulin release pathways compared with primary human islets but are still reliant on mitochondrial aerobic metabolism to derive function. Furthermore, reductive tricarboxylic acid cycle activity and glycolytic metabolite cycling occur in SC-islets, suggesting that non-canonical coupling factors are also present. In aggregate, we show that many facets of SC-islet metabolism overlap with those of primary islets, albeit with a retained immature signature.

Inactivated pentavalent vaccine against mycoplasmosis and salmonellosis for chickens.

Mycoplasma and Salmonella are serious pathogens threaten the poultry industry. This study aimed to prepare and evaluate an inactivated pentavalent vaccine targeting bacteria, including Salmonella enterica serovar Typhimurium (ST), Salmonella enterica serovar Enteritidis (SE), Salmonella enterica serovar Kentucky (SK), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS), from locally isolated strains. The prepared vaccine was adjuvanted with Montanide ISA70 oil and then tested for safety, sterility, and potency. The vaccine efficacy was evaluated in 110 specific pathogen-free, 1-day-old chicks, which were divided into three groups as follows: 1) vaccinated group (50 birds), which was subdivided into five subgroups of ten birds each; 2) control positive (challenged) group (50 birds), which was subdivided into five subgroups of ten birds each; and 3) control negative (blank) group, which included ten birds. Chicks in group 1 were administered the first dose of vaccine at 7 d of age followed by a booster dose after 3 wk. At 3 wk after booster vaccination, the chicks who were administered the booster dose were challenged and kept under observation until the end of the experiment when the chicks were approximately 10 wk. Details of clinical symptoms, daily mortality, weights, and postmortem lesions; serum samples; cloacal swabs; and nasal swabs were collected during the experiment. The humoral immune response to the prepared pentavalent vaccine was assessed using enzyme-linked immunosorbent assay. Our findings revealed that the prepared vaccine showed high protective antibody titers against Salmonella and Mycoplasma with 100% efficacy and no mortalities (100% survival rate) were recorded in vaccinated and challenged birds. The vaccine reduced both clinical signs and bacterial shedding post challenge in vaccinated birds in comparison with control positive group. The prepared vaccine did not affect the body weight gain of the vaccinated birds in comparison with control negative birds. The current study concluded that locally manufactured inactivated pentavalent vaccine offers protection to birds and could be employed as an effective tool along with biosecurity measures to overcome mycoplasmosis and salmonellosis in layer and breeder chicken farms in Egypt.

The type 1 diabetes gene TYK2 regulates ß-cell development and its responses to interferon-α.

Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of insulin producing pancreatic ß-cells. One of the genes associated with T1D is TYK2, which encodes a Janus kinase with critical roles in type-Ι interferon (IFN-Ι) mediated intracellular signalling. To study the role of TYK2 in ß-cell development and response to IFNα, we generated TYK2 knockout human iPSCs and directed them into the pancreatic endocrine lineage. Here we show that loss of TYK2 compromises the emergence of endocrine precursors by regulating KRAS expression, while mature stem cell-islets (SC-islets) function is not affected. In the SC-islets, the loss or inhibition of TYK2 prevents IFNα-induced antigen processing and presentation, including MHC Class Ι and Class ΙΙ expression, enhancing their survival against CD8+ T-cell cytotoxicity. These results identify an unsuspected role for TYK2 in ß-cell development and support TYK2 inhibition in adult ß-cells as a potent therapeutic target to halt T1D progression.

Differentiating functional human islet-like aggregates from pluripotent stem cells.

We present here a robust and reliable protocol by which to differentiate pancreatic islet-like aggregates (SC-islets) from human pluripotent stem cells. The 7-stage protocol mimics developmental patterning factors that induce endocrine lineage formation and spans monolayer, microwell, and aggregate suspension culture. The SC-islets demonstrate dynamic glucose-sensitive insulin secretion and an endocrine cell composition similar to those of primary human islets. SC-islets generated using this optimized protocol are suitable for in vitro modeling of islet cell pathophysiology and therapeutic applications. For complete details on the use and execution of this protocol, please refer to Balboa et al. (2022).

Islet Gene View-a tool to facilitate islet research.

Characterization of gene expression in pancreatic islets and its alteration in type 2 diabetes (T2D) are vital in understanding islet function and T2D pathogenesis. We leveraged RNA sequencing and genome-wide genotyping in islets from 188 donors to create the Islet Gene View (IGW) platform to make this information easily accessible to the scientific community. Expression data were related to islet phenotypes, diabetes status, other islet-expressed genes, islet hormone-encoding genes and for expression in insulin target tissues. The IGW web application produces output graphs for a particular gene of interest. In IGW, 284 differentially expressed genes (DEGs) were identified in T2D donor islets compared with controls. Forty percent of DEGs showed cell-type enrichment and a large proportion significantly co-expressed with islet hormone-encoding genes; glucagon (<i>GCG</i>, 56%), amylin (<i>IAPP</i>, 52%), insulin (<i>INS</i>, 44%), and somatostatin (<i>SST</i>, 24%). Inhibition of two DEGs, <i>UNC5D</i> and <i>SERPINE2</i>, impaired glucose-stimulated insulin secretion and impacted cell survival in a human ß-cell model. The exploratory use of IGW could help designing more comprehensive functional follow-up studies and serve to identify therapeutic targets in T2D.

Evaluation of cell-mediated immunity of E.coli nanovaccines in chickens.

Nanovaccine is a revolutionary type of immunizations for various diseases that is simple to manufacture and administer. As a result, we are working to develop innovative nanovaccines against E. coli, which is capable of causing disease both inside and outside of its predilection sites, causing respiratory and systemic disease (colibacillosis).Colibacillosis is a global disease that significantly affects poultry production. The present study aims to evaluate in vivo cell-mediated immunity against a chitosan-nanovaccine from E. coli serogroups O1 and O78 to aid in limiting colibacillosis in chicken. Two hundred specific pathogen-free (SPF) three weeks old broiler chickens were used and divided into five groups: the first group inoculated with the outer membrane and flagellar antigen (OF), the second group inoculated with chitosan capsulated-outer membrane protein-flagellar antigen (CSC-O-F), the third group inoculated with chitosan loaded-outer membrane protein-flagellar antigen (CSL-O-F), the fourth group was vaccinated with (CSL-O-F-M) adjuvanted with Montanide ISA 71 RVG, and the fifth group was left as unvaccinated control. The immune response was measured by ELISA, lymphocyte proliferation test, and challenge test. The duration of immunity was also studied. The CSL-O-F-M had the highest antibody titer in an ELISA test using the O1 strain, and the CSC-O-F had the highest antibody titer in an ELISA test using the O78 strain. For both O1 and O78 strains, the CSL-O-F-M had the strongest cell-mediated immune response, which was validated by the challenge test and duration study. We recommend producing nanovaccines (CSL-O-F-M) from E.coli O1 and O78 strains as a new manufacturing vaccine based on the demonstrated results. Because it produces highly effective humoral and cell-mediated immune responses, this novel vaccine may be useful in reducing the risk of colibacillosis.

Orange palpebral spots: A case presentation.

Orange palpebral spots are described as bilateral, ovoid, poorly defined orange-yellow macules on the superior eyelid and are predominantly reported in Caucasian populations. Previous reports have found correlations with melatonin incontinence secondary to trauma, lipofuscin accumulation in patients with superficial fatty tissue and palpebral thinness, and vitamin E, carotenoid and beta-cryptoxanthin levels. We present, to our knowledge, the first case of orange palpebral spots reported in the United Kingdom, in a patient with a background of atopy, significant sun exposure, bilateral cataracts and retinal detachment. The 59-year-old male initially presented with a dorsal nasal lesion with the differential: basal cell/trichoblastic carcinoma. During his excisional Mohs surgery, bilateral orange-yellow discolourations of the superior palpebrae were noted. The history was not significant for consumption of dietary sources of pigmentation, such as carotenoids, xanthophylls and vitamin E - found in green leafy vegetables and nut oils, respectively. The age of onset was unknown. A diagnostic skin punch biopsy was suggestive of orange palpebral spots and showed thinning of the epidermis, high-situated superficial and mature fat cells, with minimal pigment incontinence and perivascular lymphocytic infiltration. In addition, solar elastoses were identified on histology. After review in our local clinic-pathological meeting and of the published literature, a diagnosis of orange palpebral spots was given. The pathogenesis of orange palpebral spots remains to be elucidated. The role of sun exposure as a contributing factor to the generation of orange palpebral spots is therefore discussed.

Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells.

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.

PTPN2 Regulates the Interferon Signaling and Endoplasmic Reticulum Stress Response in Pancreatic ß-Cells in Autoimmune Diabetes.

Type 1 diabetes (T1D) results from autoimmune destruction of ß-cells in the pancreas. Protein tyrosine phosphatases (PTPs) are candidate genes for T1D and play a key role in autoimmune disease development and ß-cell dysfunction. Here, we assessed the global protein and individual PTP profiles in the pancreas from nonobese mice with early-onset diabetes (NOD) mice treated with an anti-CD3 monoclonal antibody and interleukin-1 receptor antagonist. The treatment reversed hyperglycemia, and we observed enhanced expression of PTPN2, a PTP family member and T1D candidate gene, and endoplasmic reticulum (ER) chaperones in the pancreatic islets. To address the functional role of PTPN2 in ß-cells, we generated PTPN2-deficient human stem cell-derived ß-like and EndoC-ßH1 cells. Mechanistically, we demonstrated that PTPN2 inactivation in ß-cells exacerbates type I and type II interferon signaling networks and the potential progression toward autoimmunity. Moreover, we established the capacity of PTPN2 to positively modulate the Ca2+-dependent unfolded protein response and ER stress outcome in ß-cells. Adenovirus-induced overexpression of PTPN2 partially protected from ER stress-induced ß-cell death. Our results postulate PTPN2 as a key protective factor in ß-cells during inflammation and ER stress in autoimmune diabetes.

Preparation and evaluation of a lateral flow immunochromatographic nanogold diagnostic kit for brucellosis in sheep.

Background and Aim: Brucellosis is a zoonotic disease with a worldwide distribution. It has a serious impact on the health of humans and animals, along with a negative impact on the economy. This study aimed to prepare and evaluate the diagnostic performance of a lateral flow immunochromatographic test (LFIT) nanogold diagnostic kit for detecting brucellosis in sheep. Materials and Methods: A rapidly developed LFIT, in which lipopolysaccharide conjugates with nanogold molecules, was placed on the conjugate pad. One hundred ovine serum samples were tested to detect Brucella antibodies (Ab) using the prepared lateral flow immunochromatography assay (LFA) kit and Rose Bengal test. The evaluation of specificity, sensitivity, and accuracy for LFIT and Rose Bengal plate test was conducted using the P04310-10 IDEXX brucellosis ovine/caprine Ab enzyme-linked immunosorbent assay (ELISA) test (gold standard). Results: The lower amount of Brucella Ab in the ovine serum samples was detected and was 1.58 S/P ratio ELISA titer/100 µL using LFIT and with Rose Bengal to detect 1.86 S/P ratio ELISA. The results showed that the developed LFIT had high specificity with no cross-reactivity with other tested bacteria. The calculated sensitivity, specificity, and accuracy of LFIT and Rose Bengal test using the P04310-10 IDEXX brucellosis ovine/caprine Ab ELISA test (gold standard) were 74% and 89%, 81% and 59%, and 76.9% and 66%, respectively. Conclusion: The present results showed interesting results implying that the LFIA strip test could be used as a substantial diagnostic tool for field screening ovine Brucella as an essential step in the control of brucellosis. However, further studies for the validation of the present findings are necessary.

In vitro beta-cell killing models using immune cells and human pluripotent stem cell-derived islets: Challenges and opportunities.

Type 1 diabetes (T1D) is a disease of both autoimmunity and ß-cells. The ß-cells play an active role in their own demise by mounting defense mechanisms that are insufficient at best, and that can become even deleterious in the long term. This complex crosstalk is important to understanding the physiological defense mechanisms at play in healthy conditions, their alterations in the T1D setting, and therapeutic agents that may boost such mechanisms. Robust protocols to develop stem-cell-derived islets (SC-islets) from human pluripotent stem cells (hPSCs), and islet-reactive cytotoxic CD8+ T-cells from peripheral blood mononuclear cells offer unprecedented opportunities to study this crosstalk. Challenges to develop in vitro ß-cell killing models include the cluster morphology of SC-islets, the relatively weak cytotoxicity of most autoimmune T-cells and the variable behavior of in vitro expanded CD8+ T-cells. These challenges may however be highly rewarding in light of the opportunities offered by such models. Herein, we discuss these opportunities including: the ß-cell/immune crosstalk in an islet microenvironment; the features that make ß-cells more sensitive to autoimmunity; therapeutic agents that may modulate ß-cell vulnerability; and the possibility to perform analyses in an autologous setting, i.e., by generating T-cell effectors and SC-islets from the same donor.

Inhibition of PHLPP1/2 phosphatases rescues pancreatic ß-cells in diabetes.

Pancreatic ß-cell failure is the key pathogenic element of the complex metabolic deterioration in type 2 diabetes (T2D); its underlying pathomechanism is still elusive. Here, we identify pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1/2) as phosphatases whose upregulation leads to ß-cell failure in diabetes. PHLPP levels are highly elevated in metabolically stressed human and rodent diabetic ß-cells. Sustained hyper-activation of mechanistic target of rapamycin complex 1 (mTORC1) is the primary mechanism of the PHLPP upregulation linking chronic metabolic stress to ultimate ß-cell death. PHLPPs directly dephosphorylate and regulate activities of ß-cell survival-dependent kinases AKT and MST1, constituting a regulatory triangle loop to control ß-cell apoptosis. Genetic inhibition of PHLPPs markedly improves ß-cell survival and function in experimental models of diabetes in vitro, in vivo, and in primary human T2D islets. Our study presents PHLPPs as targets for functional regenerative therapy of pancreatic ß cells in diabetes.