Scalable Generation of 3D Pancreatic Islet Organoids from Human Pluripotent Stem Cells in Suspension Bioreactors.

We describe a scalable method for the robust generation of 3D pancreatic islet-like organoids from human pluripotent stem cells using suspension bioreactors. Our protocol involves a 6-stage, 20-day directed differentiation process, resulting in the production of 104-105 organoids. These organoids comprise α- and ß-like cells that exhibit glucose-responsive insulin and glucagon secretion. We detail methods for culturing, passaging, and cryopreserving stem cells as suspended clusters and for differentiating them through specific growth media and exogenous factors added in a stepwise manner. Additionally, we address quality control measures, troubleshooting strategies, and functional assays for research applications.

T-cell acute lymphoblastic leukemia progression is supported by inflammatory molecules including hepatocyte growth factor.

T-cell acute lymphoblastic leukemia (T-ALL) is a malignant hematological disorder characterized by an increased proliferation of immature T lymphocytes precursors. T-ALL treatment includes chemotherapy with strong side effects, and patients that undergo relapse display poor prognosis. Although cell-intrinsic oncogenic pathways are well-studied, the tumor microenvironment, like inflammatory cellular and molecular components is less explored in T-ALL. We sought to determine the composition of the inflammatory microenvironment induced by T-ALL, and its role in T-ALL progression. We show in two mouse T-ALL cell models that T-ALLs enhance blood neutrophils and resident monocytes, accompanied with a plasmatic acute secretion of inflammatory molecules. Depleting neutrophils using anti-Ly6G treatment or resident monocytes by clodronate liposomes treatment does not modulate plasmatic inflammatory molecule secretion and mice survival. However, inhibiting the secretion of inflammatory molecules by microenvironment with NECA, an agonist of adenosine receptors, diminishes T-ALL progression enhancing mouse survival. We uncovered Hepatocyte Growth Factor (HGF), T-ALL-driven and the most decreased molecule with NECA, as a potential therapeutic target in T-ALL. Altogether, we identified a signature of inflammatory molecules that can potentially be involved in T-ALL evolution and uncovered HGF/cMET pathway as important to target for limiting T-ALL progression.

Atg8/LC3 controls systemic nutrient surplus signaling in flies and humans.

Organisms experience constant nutritional flux. Mechanisms at the interface of opposing nutritional states-scarcity and surplus-enable organismal energy homeostasis. Contingent on nutritional stores, adipocytes secrete adipokines, such as the fat hormone leptin, to signal nutrient status to the central brain. Increased leptin secretion underlies metabolic dysregulation during common obesity, but the molecular mechanisms regulating leptin secretion from human adipocytes are poorly understood. Here, we report that Atg8/LC3 family proteins, best known for their role in autophagy during nutrient scarcity, play an evolutionarily conserved role during nutrient surplus by promoting adipokine secretion. We show that in a well-fed state, Atg8/LC3 promotes the secretion of the Drosophila functional leptin ortholog unpaired 2 (Upd2) and leptin from human adipocytes. Proteomic analyses reveal that LC3 directs leptin to a secretory pathway in human cells. We identified LC3-dependent extracellular vesicle (EV) loading and secretion (LDELS) as a required step for leptin release, highlighting a unique secretory route adopted by leptin in human adipocytes. In Drosophila, mutations to Upd2's Atg8 interaction motif (AIM) result in constitutive adipokine retention. Atg8-mediated Upd2 retention alters lipid storage and hunger response and rewires the bulk organismal transcriptome in a manner conducive to starvation survival. Thus, Atg8/LC3's bidirectional role in nutrient sensing-conveying nutrient surplus and responding to nutrient deprivation-enables organisms to manage nutrient flux effectively. We posit that decoding how bidirectional molecular switches-such as Atg8/LC3-operate at the nexus of nutritional scarcity and surplus will inform therapeutic strategies to tackle chronic metabolic disorders.

Multiple variants of the type VII secretion system in Gram-positive bacteria.

Type VII secretion systems (T7SS) are found in bacteria across the Bacillota and Actinomycetota phyla and have been well described in Staphylococcus aureus, Bacillus subtilis, and pathogenic mycobacteria. The T7SS from Actinomycetota and Bacillota share two common components, a membrane-bound EccC/EssC ATPase and EsxA, a small helical hairpin protein of the WXG100 family. However, they also have additional phylum-specific components, and as a result they are termed the T7SSa (Actinomycetota) and T7SSb (Bacillota), respectively. Here, we identify additional organizations of the T7SS across these two phyla and describe eight additional T7SS subtypes, which we have named T7SSc-T7SSj. T7SSd is found exclusively in Actinomycetota including the Olselnella and Bifodobacterium genus, whereas the other seven are found only in Bacillota. All of the novel subtypes contain the canonical ATPase (TsxC) and the WXG100-family protein (TsxA). Most of them also contain a small ubiquitin-related protein, TsxB, related to the T7SSb EsaB/YukD component. Protein kinases, phosphatases, and forkhead-associated (FHA) proteins are often encoded in the novel T7SS gene clusters. Candidate substrates of these novel T7SS subtypes include LXG-domain and RHS proteins. Predicted substrates are frequently encoded alongside genes for additional small WXG100-related proteins that we speculate serve as cosecretion partners. Collectively our findings reveal unexpected diversity in the T7SS in Gram-positive bacteria.

Measures of insulin resistance and beta cell function before and after treatment of HCV infection.

The association between chronic HCV infection and type 2 diabetes mellitus (T2DM) has been established; however, there is limited research on ß-cell function particularly in the pre-diabetic population. Here, we evaluated indices of ß-cell function and insulin sensitivity across the spectrum from normal glucose tolerance to T2DM in individuals with and without chronic hepatitis C (CHC), and the effects of antiviral treatments on these variables. A total of 153 non-cirrhotic, non-fibrotic CHC patients with a BMI < 25 were enrolled in the study. Among them, 119 were successfully treated with either direct acting antiviral (DAA) drugs or pegylated interferon/ribavirin (IFN/RBV) anti-HCV therapy. Fasting state- and oral glucose tolerance test (OGTT)-derived indexes were used to evaluate ß-cell function and insulin sensitivity. Among all subjects, 19 (13%) had T2DM and 21% exhibited pre-diabetes including 8% isolated impaired fasting glucose (IFG) and 13% combined IFG and impaired glucose tolerance (IGT). Early and total insulin secretion adjusted for the degree of insulin resistance were decreased in prediabetic CHC patients compared to HCV-uninfected individuals. Viral eradication through DAA or IFN/RBV therapy demonstrated positive impacts on insulin sensitivity and ß-cell function in CHC patients who achieved sustained virologic response (SVR), regardless of fasting or OGTT state. These findings emphasize the role of HCV in the development of ß-cell dysfunction, while also suggesting that viral eradication can improve insulin secretion, reverse insulin resistance, and ameliorates glycemic control. These results have important implications for managing prediabetic CHC patients and could prevent diabetes-related clinical manifestations and complications.

Identification of homologs of the Chlamydia trachomatis effector CteG reveals a family of Chlamydiaceae type III secreted proteins that can be delivered into host cells.

Chlamydiae are a large group of obligate endosymbionts of eukaryotes that includes the Chlamydiaceae family, comprising several animal pathogens. Among Chlamydiaceae, Chlamydia trachomatis causes widespread ocular and urogenital infections in humans. Like many bacterial pathogens, all Chlamydiae manipulate host cells by injecting them with type III secretion effector proteins. We previously characterized the C. trachomatis effector CteG, which localizes at the host cell Golgi and plasma membrane during distinct phases of the chlamydial infectious cycle. Here, we show that CteG is a Chlamydiaceae-specific effector with over 60 homologs phylogenetically categorized into two distinct clades (CteG I and CteG II) and exhibiting several inparalogs and outparalogs. Notably, cteG I homologs are syntenic to C. trachomatis cteG, whereas cteG II homologs are syntenic among themselves but not with C. trachomatis cteG. This indicates a complex evolution of cteG homologs, which is unique among C. trachomatis effectors, marked by numerous events of gene duplication and loss. Despite relatively modest sequence conservation, nearly all tested CteG I and CteG II proteins were identified as type III secretion substrates using Yersinia as a heterologous bacterial host. Moreover, most of the type III secreted CteG I and CteG II homologs were delivered by C. trachomatis into host cells, where they localized at the Golgi region and cell periphery. Overall, this provided insights into the evolution of bacterial effectors and revealed a Chlamydiaceae family of type III secreted proteins that underwent substantial divergence during evolution while conserving the capacity to localize at specific host cell compartments.

Rare correlation of somatic PRKACA mutations with pregnancy-associated aldosterone- and cortisol-producing adenomas: a case report and literature review.

BACKGROUND: Somatic mutations have been observed to induce aldosterone-producing adenomas (APAs). These may be accelerated during pregnancy. Somatic PRKACA mutations are common in cortisol-producing adenomas (CPAs). However, their role in APAs, particularly aldosterone- and cortisol-producing adenomas (A/CPAs), is not well understood. This study aims to investigate the association between PRKACA mutations and the accelerated development of A/CPAs during pregnancy. CASE PRESENTATION: A patient with primary aldosteronism (PA) associated with severe Cushing's syndrome (CS) underwent surgical resection of an adrenal tumor one year after delivery. Pathologic examination revealed an adrenocortical adenoma characterized primarily by zona glomerulosa hyperplasia. Somatic mutation analysis revealed the presence of the somatic PRKACA mutation, which was validated as a deleterious mutation by various computational databases. Immunohistochemical results showed positive staining for cytochrome P450 family 11 subfamily B member 1 (CYP11B1), cytochrome P450 family 11 subfamily B member 2 (CYP11B2), and luteinizing hormone/chorionic gonadotropin receptor (LHCGR). Our study included a review of 20 previously documented cases of aldosterone- and cortisol-producing adenomas (A/CPAs), two of which were concurrently positive for both CYP11B1 and CYP11B2, consistent with our findings. CONCLUSION: Somatic mutations in PRKACA may correlate with the upregulation of LHCGR, which synergistically drives the accelerated growth of co-secretion tumors during pregnancy, thereby exacerbating disease progression.

Surrogate measures of first-phase insulin secretion versus reference methods intravenous glucose tolerance test and hyperglycemic clamp: a systematic review and meta-analyses.

INTRODUCTION: In this systematic review, we investigated the diagnostic accuracy of surrogate measures of insulin secretion based on fasting samples and the oral glucose tolerance test (OGTT). The first phase of insulin secretion was calculated using two gold standard methods; the hyperglycemic clamp (HGC) test and intravenous glucose tolerance test (IVGTT). RESEARCH DESIGN AND METHODS: We conducted searches in the PubMed, Cochrane Central, and Web of Science databases, the last of which was conducted at the end of June 2021. Studies were included that measured first-phase insulin secretion in adults using both a gold-standard reference method (either HGC or IVGTT) and one or more surrogate measures from either fasting samples, OGTT or a meal-tolerance test. QUADAS-2, a revised tool for the quality assessment of diagnostic accuracy studies, was used for quality assessment. Random-effects meta-analyses were performed to examine the correlation between first-phase measured with gold standard and surrogate methods. RESULTS: A total of 33 articles, encompassing 5362 individuals with normal glucose tolerance, pre-diabetes or type 2 diabetes, were included in our systematic review. Homeostatic model assessment (HOMA)-beta and Insulinogenic Index 30 (IGI(30)) were the surrogate measures validated in the largest number of studies (17 and 13, respectively). HOMA-beta's pooled correlation to the reference methods was 0.48 (95% CI 0.40 to 0.56) The pooled correlation of IGI to the reference methods was 0.61 (95% CI 0.54 to 0.68). The surrogate measures with the highest correlation to the reference methods were Kadowaki (0.67 (95% CI 0.61 to 0.73)) and Stumvoll's first-phase secretion (0.65 (95% CI 0.58 to 0.71)), both calculated from an OGTT. CONCLUSIONS: Surrogate measures from the first 30 min of an OGTT capture the first phase of insulin secretion and are a good choice for epidemiological studies. HOMA-beta has a moderate correlation to the reference methods but is not a measure of the first phase specifically. PROSPERO REGISTRATION NUMBER: The meta-analysis was registered at PROSPERO (Id: CRD42020169064) before inclusion started.

Proteomic predictors of individualized nutrient-specific insulin secretion in health and disease.

Population-level variation and mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized. We defined prototypical insulin secretion responses to three macronutrients in islets from 140 cadaveric donors, including those with type 2 diabetes. The majority of donors' islets exhibited the highest insulin response to glucose, moderate response to amino acid, and minimal response to fatty acid. However, 9% of donors' islets had amino acid responses, and 8% had fatty acid responses that were larger than their glucose-stimulated insulin responses. We leveraged this heterogeneity and used multi-omics to identify molecular correlates of nutrient responsiveness, as well as proteins and mRNAs altered in type 2 diabetes. We also examined nutrient-stimulated insulin release from stem cell-derived islets and observed responsiveness to fat but not carbohydrate or protein-potentially a hallmark of immaturity. Understanding the diversity of insulin responses to carbohydrate, protein, and fat lays the groundwork for personalized nutrition.

The type II secretion system as an underappreciated and understudied mediator of interbacterial antagonism.

Interbacterial antagonism involves all major phyla, occurs across the full range of ecological niches, and has great significance for the environment, clinical arena, and agricultural and industrial sectors. Though the earliest insight into interbacterial antagonism traces back to the discovery of antibiotics, a paradigm shift happened when it was learned that protein secretion systems (e.g., types VI and IV secretion systems) deliver toxic "effectors" against competitors. However, a link between interbacterial antagonism and the Gram-negative type II secretion system (T2SS), which exists in many pathogens and environmental species, is not evident in prior reviews on bacterial competition or T2SS function. A current examination of the literature revealed four examples of a T2SS or one of its known substrates having a bactericidal activity against a Gram-positive target or another Gram-negative. When further studied, the T2SS effectors proved to be peptidases that target the peptidoglycan of the competitor. There are also reports of various bacteriolytic enzymes occurring in the culture supernatants of some other Gram-negative species, and a link between these bactericidal activities and T2SS is suggested. Thus, a T2SS can be a mediator of interbacterial antagonism, and it is possible that many T2SSs have antibacterial outputs. Yet, at present, the T2SS remains relatively understudied for its role in interbacterial competition. Arguably, there is a need to analyze the T2SSs of a broader range of species for their role in interbacterial antagonism. Such investigation offers, among other things, a possible pathway toward developing new antimicrobials for treating disease.

ACTH-producing adrenocortical carcinoma: an exceedingly rare diagnosis.

BACKGROUND: Adrenocortical carcinoma is a very rare endocrinopathy that has a poor prognosis and is frequently associated with ACTH-independent Cushing's syndrome. Despite having an adrenocortical carcinoma, our patient surprisingly had an ACTH-dependent Cushing's syndrome. CASE REPORT: A 26-year-old female presented with Cushing's syndrome and an abdominal mass. Imaging studies revealed an adrenal mass consistent with a high-grade malignancy. Laboratory workup showed hypercortisolism, hyperandrogenism, and hypokalemia with normal levels of metanephrines. Unexpectedly, her ACTH levels were remarkably elevated. The pathological analysis of a tumor sample was conclusive for adrenocortical carcinoma with immunopositivity for ACTH. CONCLUSIONS: Our patient suffered from an adrenocortical carcinoma that was ectopically producing ACTH. This case emphasizes that physicians should have a broad-minded approach when evaluating cases of rare endocrine malignancies.

Cryo-EM structure of the R388 plasmid conjugative pilus reveals a helical polymer characterized by an unusual pilin/phospholipid binary complex.

Bacterial conjugation is a process by which DNA is transferred unidirectionally from a donor cell to a recipient cell. It is the main means by which antibiotic resistance genes spread among bacterial populations. It is crucially dependent upon the elaboration of an extracellular appendage, termed "pilus," by a large double-membrane-spanning secretion system termed conjugative "type IV secretion system." Here we present the structure of the conjugative pilus encoded by the R388 plasmid. We demonstrate that, as opposed to all conjugative pili produced so far for cryoelectron microscopy (cryo-EM) structure determination, the conjugative pilus encoded by the R388 plasmid is greatly stimulated by the presence of recipient cells. Comparison of its cryo-EM structure with existing conjugative pilus structures highlights a number of important differences between the R388 pilus structure and that of its homologs, the most prominent being the highly distinctive conformation of its bound lipid.

A quantitative pipeline to assess secretion of human leptin coding variants reveals mechanisms underlying leptin deficiencies.

The hormone leptin, primarily secreted by adipocytes, plays a crucial role in regulating whole-body energy homeostasis. Homozygous loss-of-function mutations in the leptin gene (LEP) cause hyperphagia and severe obesity, primarily through alterations in leptin's affinity for its receptor or changes in serum leptin concentrations. Although serum concentrations are influenced by various factors (e.g., gene expression, protein synthesis, stability in the serum), proper delivery of leptin from its site of synthesis in the endoplasmic reticulum via the secretory pathway to the extracellular serum is a critical step. However, the regulatory mechanisms and specific machinery involved in this trafficking route, particularly in the context of human LEP mutations, remain largely unexplored. We have employed the Retention Using Selective Hooks (RUSH) system to elucidate the secretory pathway of leptin. We have refined this system into a medium-throughput assay for examining the pathophysiology of a range of obesity-associated LEP variants. Our results reveal that leptin follows the default secretory pathway, with no additional regulatory steps identified prior to secretion. Through screening of leptin variants, we identified three mutations that lead to proteasomal degradation of leptin and one variant that significantly decreased leptin secretion, likely through aberrant disulfide bond formation. These observations have identified novel pathogenic effects of leptin variants, which can be informative for therapeutics and diagnostics. Finally, our novel quantitative screening platform can be adapted for other secreted proteins.

Selective depletion of Campylobacter jejuni via T6SS dependent functionality: an approach for improving chickens gut health.

The targeted depletion of potential gut pathogens is often challenging because of their intrinsic ability to thrive in harsh gut environments. Earlier, we showed that Campylobacter jejuni (C. jejuni) exclusively uses the Type-VI Secretion System (T6SS) to target its prey such as Escherichia coli (E. coli), and phenotypic differences between T6SS-negative and T6SS-positive C. jejuni isolates toward bile salt sensitivity. However, it remains unclear how the target-driven T6SS functionality prevails in a polymicrobial gut environment. Here, we investigated the fate of microbial competition in an altered gut environment via bacterial T6SS using a T6SS-negative and -positive C. jejuni or its isogenic mutant of the hemolysin-coregulated protein (hcp). We showed that in the presence of bile salt and prey bacteria (E. coli), T6SS-positive C. jejuni experiences enhanced intracellular stress leading to cell death. Intracellular tracking of fluorophore-conjugated bile salts confirmed that T6SS-mediated bile salt influx into C. jejuni can enhance intracellular oxidative stress, affecting C. jejuni viability. We further investigated whether the T6SS activity in the presence of prey (E. coli) perturbs the in vivo colonization of C. jejuni. Using chickens as primary hosts of C. jejuni and non-pathogenic E. coli as prey, we showed a marked reduction of C. jejuni load in chickens cecum when bile salt solution was administered orally. Analysis of local antibody responses and pro-inflammatory gene expression showed a reduced risk of tissue damage, indicating that T6SS activity in the complex gut environment can be exploited as a possible measure to clear the persistent colonization of C. jejuni in chickens.

Traumatic brain injury inducing swift transition from syndrome of inappropriate antidiuretic hormone secretion to central diabetes insipidus: a case report.

Heavy traumatic brain injury (TBI) may lead to the manifestation of either syndrome of inappropriate secretion of antidiuretic hormones (SIADH) or central diabetes insipidus (CDI). We present a case of TBI where SIADH transformed into CDI within a remarkably short timeframe. A previously healthy 4-yr-old boy was admitted to our hospital with hyponatremia and elevated urinary sodium level on the day following a traumatic head injury. Within 150 min after initiating SIADH treatment, a significant increase in urine volume and a decrease in urinary sodium levels were observed. Therefore, the treatment plan was modified to include desmopressin. By the 5th day of admission, the urine volume gradually stabilized and normalized without the need for further desmopressin treatment. Mild TBI can give rise to various conditions that may undergo rapid changes. Closely monitoring serum and urine electrolytes, along with urine volume, is imperative for the administration of appropriate and timely treatment.

Hypertonicity during a rapid rise in D-glucose mediates first-phase insulin secretion.

Introduction: Biphasic insulin secretion is an intrinsic characteristic of the pancreatic islet and has clinical relevance due to the loss of first-phase in patients with Type 2 diabetes. As it has long been shown that first-phase insulin secretion only occurs in response to rapid changes in glucose, we tested the hypothesis that islet response to an increase in glucose is a combination of metabolism plus an osmotic effect where hypertonicity is driving first-phase insulin secretion. Methods: Experiments were performed using perifusion analysis of rat, mouse, and human islets. Insulin secretion rate (ISR) and other parameters associated with its regulation were measured in response to combinations of D-glucose and membrane-impermeable carbohydrates (L-glucose or mannitol) designed to dissect the effect of hypertonicity from that of glucose metabolism. Results: Remarkably, the appearance of first-phase responses was wholly dependent on changes in tonicity: no first-phase in NAD(P)H, cytosolic calcium, cAMP secretion rate (cAMP SR), or ISR was observed when increased D-glucose concentration was counterbalanced by decreases in membrane-impermeable carbohydrates. When D-glucose was greater than 8 mM, rapid increases in L-glucose without any change in D-glucose resulted in first-phase responses in all measured parameters that were kinetically similar to D-glucose. First-phase ISR was completely abolished by H89 (a non-specific inhibitor of protein kinases) without affecting first-phase calcium response. Defining first-phase ISR as the difference between glucose-stimulated ISR with and without a change in hypertonicity, the peak of first-phase ISR occurred after second-phase ISR had reached steady state, consistent with the well-established glucose-dependency of mechanisms that potentiate glucose-stimulated ISR. Discussion: The data collected in this study suggests a new model of glucose-stimulated biphasic ISR where first-phase ISR derives from (and after) a transitory amplification of second-phase ISR and driven by hypertonicity-induced rise in H89-inhibitable kinases likely driven by first-phase responses in cAMP, calcium, or a combination of both.

Endoplasmic reticulum exit sites are segregated for secretion based on cargo size.

TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion.

Proteolytic cleavage of Golgi glycosyltransferases by SPPL3 and other proteases and its implications for cellular glycosylation: (special issue title: New aspects of glycosyltransferases).

Glycosylation of proteins and lipids is of fundamental importance in multicellular eukaryotes. The vast diversity of glycan structures observed is generated in the Golgi apparatus by the concerted activity of >100 distinct enzymes, which include glycosyltransferases and other glycan-modifying enzymes. Well-known for decades, the majority of these enzymes is released from the Golgi apparatus and subsequently secreted into the extracellular space following endoproteolytic cleavage, but the underlying molecular mechanisms and the physiological implications have remained unexplored. This review will summarize our current knowledge of Golgi enzyme proteolysis and secretion and will discuss its conceptual implications for the regulation of cellular glycosylation and the organization of the Golgi apparatus. A particular focus will lie on the intramembrane protease SPPL3, which recently emerged as key protease facilitating Golgi enzyme release and has since been shown to affect a multitude of glycosylation-dependent physiological processes.

The association between circulating phenylalanine and the temporal risk of impaired insulin markers in gestational diabetes mellitus.

Background: We aimed to contrast plasma amino acid concentrations in pregnant women with Gestational Diabetes Mellitus (GDM) to those without, to analyze the link between plasma amino acid concentrations, GDM, insulin resistance, and insulin secretion at 24-28 weeks of gestation. Methods: The research employed a retrospective case-control study design at a single center. Basic demographic and laboratory data were procured from the hospital's case system. The study encompassed seventy women without gestational diabetes mellitus (GDM) and thirty-five women with GDM matched in a 1-to-2 ratio for age and pre-pregnancy BMI. Utilizing high-performance liquid chromatography-mass spectrometry (HPLC-MS), peripheral fasting plasma amino acid concentrations in these women, during mid-pregnancy, were duly measured. We carefully evaluated the significant differences in the quantitative data between the two groups and developed linear regression models to assess the independent risk factors affecting insulin resistance and insulin secretion. Results: Significant variations in insulin secretion and resistance levels distinguished GDM Group from the non-GDM group at three distinct time points, alongside relatively elevated serum Glycosylated Hemoglobin (HbA1c) levels. Triglycerides (TG) were also significantly increased in those with GDM during adipocytokine observations. Apart from glutamic acid and glutamine, the concentrations of the remaining 16 amino acids were notably increased in GDM patients, including all branched chain amino acids(BCAAs) and aromatic amino acids(AAAs). Ultimately, it was ascertained that fasting serum phenylalanine levels were independent risk factors affecting insulin resistance index and insulin secretion at various phases. Conclusions: Various fasting serum amino acid levels are markedly increased in patients with GDM, specifically phenylalanine, which may play role in insulin resistance and secretion.

Trafficking of adhesion and aggregation-modulating proteins during the early stages of Dictyostelium development.

The social amoeba Dictyostelium discoideum has been studied for close to a century to better understand conserved cellular and developmental processes. The life cycle of this model eukaryote is composed of a unicellular growth phase and a multicellular developmental phase that is induced by starvation. When starved, individual cells undergo chemotactic aggregation to form multicellular mounds that develop into slugs. Terminal differentiation of cells within slugs forms fruiting bodies, each composed of a stalk that supports a mass of viable spores that germinate and restart the life cycle when nutrients become available. Calcium-dependent cell adhesion protein A (CadA) and countin (CtnA) are two proteins that regulate adhesion and aggregation, respectively, during the early stages of D. discoideum development. While the functions of these proteins have been well-studied, the mechanisms regulating their trafficking are not fully understood. In this study, we reveal pathways and cellular components that regulate the intracellular and extracellular amounts of CadA and CtnA during aggregation. During growth and starvation, CtnA localizes to cytoplasmic vesicles and punctae. We show that CtnA is glycosylated and this post-translational modification is required for its secretion. Upon autophagy induction, a signal peptide for secretion facilitates the release of CtnA from cells via a pathway involving the µ subunit of the AP3 complex (Apm3) and the WASP and SCAR homolog, WshA. Additionally, CtnA secretion is negatively regulated by the D. discoideum orthologs of the human non-selective cation channel mucolipin-1 (Mcln) and sorting receptor sortilin (Sort1). As for CadA, it localizes to the cell periphery in growth-phase and starved cells. The intracellular and extracellular amounts of CadA are modulated by autophagy genes (atg1, atg9), Apm3, WshA, and Mcln. We integrate these data with previously published findings to generate a comprehensive model summarizing the trafficking of CadA and CtnA in D. discoideum. Overall, this study enhances our understanding of protein trafficking during D. discoideum aggregation, and more broadly, provides insight into the multiple pathways that regulate protein trafficking and secretion in all eukaryotes.