Impaired Secretion Research Articles

Adipose Signals Regulating Distal Organ Health and Disease.

Excessive adiposity in obesity is a significant risk factor for development of type 2 diabetes (T2D), nonalcoholic fatty liver disease, and other cardiometabolic diseases. An unhealthy expansion of adipose tissue (AT) results in reduced adipogenesis, increased adipocyte hypertrophy, adipocyte hypoxia, chronic low-grade inflammation, increased macrophage infiltration, and insulin resistance. This ultimately culminates in AT dysfunction characterized by decreased secretion of antidiabetic adipokines such as adiponectin and adipsin and increased secretion of proinflammatory prodiabetic adipokines including RBP4 and resistin. This imbalance in adipokine secretion alters the physiological state of AT communication with target organs including pancreatic β-cells, heart, and liver. In the pancreatic β-cells, adipokines are known to have a direct effect on insulin secretion, gene expression, cell death, and/or dedifferentiation. For instance, impaired secretion of adipsin, which promotes insulin secretion and β-cell identity, results in β-cell failure and T2D, thus presenting a potential druggable target to improve and/or preserve β-cell function. The cardiac tissue is affected by both the classic white AT-secreted adipokines and the newly recognized brown AT (BAT)-secreted BATokines or lipokines that alter lipid deposition and ventricular function. In the liver, adipokines affect hepatic gluconeogenesis, lipid accumulation, and insulin sensitivity, underscoring the importance of adipose-liver communication in the pathogenesis of nonalcoholic fatty liver disease. In this perspective, we outline what is currently known about the effects of individual adipokines on pancreatic β-cells, liver, and the heart.

ALKBH5 is a prognostic factor and promotes the angiogenesis of glioblastoma

Despite numerous reports indicating the significant impact of RNA modification on malignant glioblastoma (GBM) cell behaviors such as proliferation, invasion and therapy efficacy, its specific involvement in glioblastoma (GBM) angiogenesis is remains unclear and is currently under investigation. In this study, we aimed to investigate the relevance between RNA modification regulators and GBM angiogenesis. Our study employed bioinformatic analyses, including Gene Set Enrichment Analysis (GSEA), differential expression analysis, and Kaplan–Meier survival analysis, to identify regulators of angiogenesis-associated RNA modification (RM). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were applied to identify the enrichment of angiogenesis associated signatures in ALKBH5-high expression GBMs. We also utilized Western blot to verify the upregulation of ALKBH5 in clinical GBM samples. By a series of in vitro and in vivo assays, including plasmid transfection, wound healing, transwell invasion test, tube formation, RT-qPCR, ELISA assays and xenograft mice model, we validated the angiogenesis regulation ability of ALKBH5 in GBM. The N6-methyladenosine (m6A) modification “erase” ALKBH5 emerged as a candidate regulator associated with angiogenesis, demonstrating elevated expression and robust prognostic predictive ability in GBM patients. We also revealed enrichment of vasculature development biological process in GBMs with high ALKBH5 expression. Subsequently, we validated the elevated the expression of ALKBH5 in clinical GBM and paired adjacent tissues through western blot. Additionally, we knocked down the expression of ALKBH5 using sh-RNAs in U87 GBM cells to access the angiogenesis induction ability in U87 cells. In vitro experiments, Human Umbilical Vein Endothelial Cells (HUVECs) were used to perform wound healing, transwell migration and tube formation analysis, results indicated that ALKBH5 knock-down of U87 cells could decrease the pro-angiogenesis ability of U87 GBM cells. Further validation of our bioinformatic findings confirmed that ALKBH5 knockdown impaired VEGFA secretion in both in vitro and in vivo settings in U87 cells. These results comprehensively affirm the crucial role of ALKBH5 in regulating GBM-induced angiogenesis, both in vitro and in vivo. ALKBH5 not only emerges as a promising prognostic factor for GBM patients, but also plays a pivotal role in sustaining GBM progression by promoting angiogenesis.

Exhausted signature and regulatory network of NK cells in myasthenia gravis.

NK cells are dysfunctional in myasthenia gravis (MG), but the mechanism is unclear. This study aims to measure associations and underlying mechanisms between the NK cells and the development of MG. Twenty healthy controls (HCs) and 53 MG patients who did not receive glucocorticoids and immunosuppressants were collected. According to the Myasthenia Gravis Foundation of America (MGFA) classification, MG patients were categorized into MGFA I group (n = 18) and MGFA II-IV group (n = 35). Flow cytometry, cell sorting, ELISA, mRNA-sequencing, RT-qPCR, western blot, and cell culture experiments were performed to evaluate the regulatory mechanism of exhausted NK cells. Peripheral NK cells in MGFA II-IV patients exhibit exhausted phenotypes than HCs, marked by the dramatic loss of total NK cells, CD56dimCD16- NK cells, elevated PD1 expression, reduced NKG2D expression, impaired cytotoxic activity (perforin, granzyme B, CD107a) and cytokine secretion (IFN-γ). Plasma IL-6 and IL-21 are elevated in MG patients and mainly derived from the aberrant expansion of monocytes and Tfh cells, respectively. IL-6/IL-21 cooperatively induced NK-cell exhausted signature via upregulating SOCS2 and inhibiting the phosphorylation of STAT5. SOCS2 siRNA and IL-2 supplement attenuated the IL-6/IL-21-mediated alteration of NK-cell phenotypes and function. Inhibition of IL-6/IL-21/SOCS2/STAT5 pathway and recovery of NK-cell ability to inhibit autoimmunity may be a new direction in the treatment of MG.

Classification of Diabetes Using Machine Learning Technics

Diabetes is one of the most widespread chronic diseases worldwide. More than 400 million people in the world suffer from it, and their number is increasing significantly every year. The continents of the world, especially Asia and Africa, are experiencing a high rate of infection due to a number of factors and environmental factors. Related to diet and healthy habits used in many developing countries. Diabetes is considered one of the most serious chronic diseases that affect the human body due to impaired secretion of the hormone insulin, because the pancreas is unable to secrete a sufficient amount of insulin, or when the cells of the human body do not accept insulin and don't use it. Effective, causing a sudden drop or rise in blood sugar levels. It has a huge impact on human health and type 2 diabetes is the most common form in humans as many factors contribute to its spread, such as genetic factors, poor diet and lack of regular exercise. In this paper we used the machine learning technical to classification of diabetes such as logistic regression, random forest, decision tree, vector classification, KNN and Naïve bayes and the accuracy was 77.66%, 76.33%, 98.66%, 75.33%, 80.66% and 80% The rest of the general standards used in classification are mentioned in the results of this paper.

Mortality in patients with adrenal insufficiency: a protocol for a systematic review and meta-analysis

IntroductionAdrenal insufficiency (AI) is a rare disorder characterised by an impaired secretion of glucocorticoids from the adrenal glands. Treatment strategies for AI have developed over time with reduced glucocorticoid replacement...

The Impact of Chronic Stress Related to COVID-19 on Eating Behaviors and the Risk of Obesity in Children and Adolescents.

During the COVID-19 pandemic, an increase in the incidence of overweight and obesity in children was observed. It appears that unhealthy food choices, an unbalanced diet, and a sedentary lifestyle, as well as experiencing stress related to the pandemic, may be contributing to this disturbing trend. Chronic stress is a significant factor contributing to eating disorders and obesity in youngsters, involving medical, molecular, and psychological elements. Individuals under chronic stress often focus on appearance and weight, leading to negative body image and disrupted relationships with food, resulting in unhealthy eating behaviors. Chronic stress also impacts hormonal balance, reducing the satiety hormone leptin and elevating the appetite-stimulating hormone ghrelin, fostering increased hunger and uncontrolled snacking. Two systems, the hypothalamic-pituitary-adrenal axis and the sympathetic system with the adrenal medulla, are activated in response to stress, causing impaired secretion of noradrenaline and cortisol. Stress-related obesity mechanisms encompass oxidative stress, neuroinflammation, insulin resistance, and neurohormonal and neurotransmission disorders. Stress induces insulin resistance, elevating obesity risk by disrupting blood sugar regulation and fat storage. Stress also affects the gut microbiome, potentially influencing chronic inflammation and metabolic processes linked to obesity. In conclusion, chronic stress is a multifaceted risk factor for eating disorders and obesity in children, necessitating a comprehensive understanding of effective preventive and intervention strategies amid the escalating prevalence of childhood overweight and obesity.

Insulin release from isolated cat islets of Langerhans

Feline diabetes mellitus is a common endocrine disease with increasing prevalence. It shows similarities with human type 2 diabetes and is characterized by insulin resistance and deficient insulin secretion. Moreover, cats and humans belong to the very few species that form amyloid depositions in the pancreatic islets. However, little is known about cat islet function and no studies have addressed insulin secretion from isolated islets ex vivo. The aim of this study was to establish a protocol for isolation of islets of Langerhans from pancreata of cats euthanized due to disease, and to evaluate insulin secretion responses to various physiological and pharmacological stimuli. Collagenase digestion of pancreatic tissue from 13 non-diabetic cats and two cats with diabetic ketoacidosis yielded individual islets surrounded by a layer of exocrine tissue that was reduced after two days in culture. Histological examination showed islet amyloid in pancreatic biopsies from most non-diabetic and in one diabetic cat. Islets from non-diabetic cats cultured at 5.5 mM glucose responded with increased insulin secretion to 16.7 mM glucose, 30 mM K+ and 20 µM of the sulfonylurea glipizide (2-3 times basal secretion at 3 mM glucose). The glucagon-like peptide-1 receptor agonist exendin-4 (100 nM) had no effect under basal conditions but potentiated glucose-triggered insulin release. Only one of nine islet batches from diabetic cats released detectable amounts of insulin, which was enhanced by exendin-4. Culture of islets from non-diabetic cats at 25 mM glucose impaired secretion both in response to glucose and K+ depolarization. In conclusion, we describe a procedure for isolation of islets from cat pancreas biopsies and demonstrate that isolated cat islets secrete insulin in response to glucose and antidiabetic drugs. The study provides a basis for future ex vivo studies of islet function relevant to the understanding of the pathophysiology and treatment of feline diabetes.

Pubertal attainment and Leydig cell function following pediatric hematopoietic stem cell transplantation: a three-decade longitudinal assessment.

Impaired testosterone secretion is a frequent sequela following hematopoietic stem cell transplantation (HSCT) in pediatrics, but long-term longitudinal trendlines of clinical and biochemical findings are still scanty. Monocentric, retrospective analysis. Male patients transplanted <18 years between 1992 and 2021, surviving ≥2 years after HSCT and showing, upon enrollment, clinical and biochemical signs consistent with pubertal onset and progression were included. Clinical and biochemical data collected every 6-12 months were recorded. Of 130 patients enrolled, 56% were prepubertal, while 44% were peri-/postpubertal upon HSCT. Overall, 44% showed spontaneous progression into puberty and normal gonadal profile, while the remaining experienced pubertal arrest (1%), isolated increase of FSH (19%), compensated (23%) or overt (13%) hypergonadotropic hypogonadism. Post-pubertal testicular volume (TV) was statistically smaller among patients still pre-pubertal upon HSCT (p 0.049), whereas no differences were recorded in adult testosterone levels. LH and testosterone levels showed a specular trend between 20 and 30 years, as a progressive decrease in sexual steroids was associated with a compensatory increase of the luteinizing hormone. A variable degree of gonadal dysfunction was reported in 85%, 51%, 32% and 0% of patients following total body irradiation- (TBI), busulfan-, cyclophosphamide- and treosulfan-based regimens, respectively. TBI and busulfan cohorts were associated with the lowest probability of gonadal event-free course (p<0.0001), while it achieved 100% following treosulfan. A statistically greater gonadotoxicity was detected after busulfan than treosulfan (p 0.024). Chemo-only regimens were associated with statistically larger TV (p <0.001), higher testosterone (p 0.008) and lower gonadotropin levels (p <0.001) than TBI. Accordingly, the latter was associated with a 2-fold increase in the risk of gonadal failure compared to busulfan (OR 2.34, CI 1.08-8.40), whereas being pre-pubertal upon HSCT was associated with a reduced risk (OR 0.15, CI 0.08-0.30). a) patients pre-pubertal upon HSCT showed a reduced risk of testicular endocrine dysfunction, despite smaller adult TV; b) patients showed downwards trend in testosterone levels after full pubertal attainment, despite a compensatory increase in LH; c) treosulfan was associated to a statistically lower occurrence of hypogonadism than busulfan, with a trend towards larger TV, higher testosterone levels and lower gonadotropins.

The Impact of Maternal Obesity on Adipose Progenitor Cells.

The concept of Developmental Origin of Health and Disease (DOHaD) postulates that adult-onset metabolic disorders may originate from suboptimal conditions during critical embryonic and fetal programming windows. In particular, nutritional disturbance during key developmental stages may program the set point of adiposity and its associated metabolic diseases later in life. Numerous studies in mammals have reported that maternal obesity and the resulting accelerated growth in neonates may affect adipocyte development, resulting in persistent alterations in adipose tissue plasticity (i.e., adipocyte proliferation and storage) and adipocyte function (i.e., insulin resistance, impaired adipokine secretion, reduced thermogenesis, and higher inflammation) in a sex- and depot-specific manner. Over recent years, adipose progenitor cells (APCs) have been shown to play a crucial role in adipose tissue plasticity, essential for its development, maintenance, and expansion. In this review, we aim to provide insights into the developmental timeline of lineage commitment and differentiation of APCs and their role in predisposing individuals to obesity and metabolic diseases. We present data supporting the possible implication of dysregulated APCs and aberrant perinatal adipogenesis through epigenetic mechanisms as a primary mechanism responsible for long-lasting adipose tissue dysfunction in offspring born to obese mothers.

Autoimmune Thyroid Disease in Psoriasis Patients: A Case-Control Study.

Psoriasis is a common immunologically mediated inflammatory disease characterized by skin inflammation, epidermal hyperplasia, an increased risk of painful and destructive arthritis, cardiovascular morbidity, and psychosocial challenges. Some autoimmune diseases are mediated by stimulating or blocking auto-antibodies. Auto-antibodies may act as antagonists and bind to hormone receptors, blocking receptor function. It may result in impaired secretion of mediators and gradual dysfunction of the affected organ, e.g., Graves disease and myasthenia gravis. This study was planned to evaluate the association between anti-thyroid peroxidase antibody (anti-TPO Ab) and anti-thyroglobulin antibody (anti-TG Ab) as biochemical markers in 30 clinically diagnosed psoriasis patients. This hospital-based, epidemiological case-control study was conducted in the Department of Biochemistry in collaboration with the Department of Dermatology, Venereology, and Leprology at Bhagat Phool Singh Government Medical College for Women, Khanpur Kalan, Sonepat, Haryana, India. Thirty subjects diagnosed clinically with psoriasis and an equal number of age-matched controls with no known autoimmune disease from the outpatient department were also enrolled. The following hormonal tests, i.e., thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), free thyroxine (FT4), and antibodies, anti-TPOAb and anti-TG Ab, were performed. The study period was one year. The data thus obtained was analyzed using SPSS Statistics version 26.0 (IBM Corp. Released 2019. IBM SPSS Statistics for Windows, Version 26.0. Armonk, NY: IBM Corp). The significance level (p-value) was taken as <0.05. The mean age of psoriasis subjects was 37.83±12.89 years compared to 36.91±12.32 years in the control group and was found to be non-significant (p=0.432), reflecting a similar age distribution. A male preponderance was observed in the present study, where the psoriasis group consisted of 80% males and 20% females, while the control group had 60% males and 40% females. All six psoriasis patients diagnosed with autoimmune thyroid disease (AITD) were euthyroid at the time of enrollment, compared to only one control subject in a subclinical hypothyroid state. The mean values of anti-TPO Ab were 30.93±41.26 IU/mL in psoriasis patients and 11.39±28.42 IU/mL in the control group (p=0.001), while the mean values of anti-TG Ab were 11.21±27.69 IU/mL in psoriatic subjects and 2.49±9.05 IU/mL in the control group (p=0.004). No significant correlation between AITD and psoriasis was found when both parameters were analyzed statistically for correlation; even when one marker was considered, no significant correlation was found. The odds ratio was calculated to find an association between the disease and thyroid autoimmunity. The odds ratio was estimated to be 2.25 for psoriasis and the control group, with a confidence interval of 95% (0.77-6.59) and a p-value of 0.139, which was not statistically significant. Psoriasis, a dermatological disorder, has been seen as related to AITD. The role of early detection of anti-thyroid antibodies, i.e., anti-TPO Ab and anti-TG Ab, can be of prognostic value in AITD and psoriasis.

#187 : The Correlation Between Insulin Resistance with Serum Androgens Levels in Lean Women With and Without PCOS

Background and Aims: Polycystic Ovarian Syndrome (PCOS) is a multifactorial disease resulting from the interaction of multiple predisposing genetic variations and environmental factors. Insulin resistance and hyperinsulinemia contribute to hyperandrogenism and impaired gonadotropin secretion by reducing sex hormone binding globulin (SHBG) levels and increasing testosterone bioavailability. However, this insulin resistance is not always present in PCOS patients, especially in lean PCOS patients. This study aims to determine whether insulin resistance correlates with serum androgens levels. Method: We collected 25 samples from lean women with PCOS, and 25 samples from lean women without PCOS. Peripheral whole blood samples will be taken from all samples. Data collected include Anthropometric data, Hormonal profile (using ELISA method), and HOMA-IR value. Results: The result showed that median(min-max) ages for patients was 25(17-34) years. In lean women with PCOS, there was a positive relationship between HOMA-IR with FAI (p=0.017, r=0.472) and negative relationship with SHBG (p=0.001, r=-0.610). Also, in lean women without PCOS, there were no correlation between HOMA-IR with FAI, and HOMA-IR with SHBG (p&gt;0.05) Conclusion: This study shows that there was a correlation between insulin resistance and some serum androgens in lean women with PCOS, but there was no correlation between HOMA-IR with FAI, and HOMA-IR with SHBG in lean women without PCOS. Therefore, the results of this study are hoped to explain the correlation of insulin resistance with serum androgens in lean PCOS patients and can be the basis for further research to find the right solution for the treatment of PCOS patients.

Extracellular Vesicle Trafficking Regulates Hematopoietic Stem and Progenitor Cell Fitness

Among regulatory pathways critical to hematopoietic stem and progenitor cell (HSPC) function, sphingolipid metabolism has recently been recognized for its role in safeguarding essential HSPC properties of self-renewal and lineage fate. In particular, de novo ceramide (Cer) synthesis appears to regulate a coordinated stress response that results in enhanced self-renewal of human HSPCs. However, the fundamental molecular mechanisms underlying these regenerative stress responses have yet to be uncovered and broadly validated. Moreover, alternative synthetic pathways regulating Cer and sphingomyelin (SM) levels may serve additional regulatory roles in HSPCs. Here we target neutral sphingomyelinase-2 (nSMase2/SMPD3)-dependent SM hydrolysis through pharmacologic (GW4869) and genetic (Cas9-mediated) interruption, and similarly confirm an adaptive ATF4-mediated integrated stress response (ISR). Functionally, transient inhibition led to durable improvements in HSPC reconstitution after primary (&amp;gt;8 fold at steady-state, p&amp;lt;0.001) and secondary (&amp;gt;20 fold at steady-state, p&amp;lt;0.001) transplantation. Surprisingly, lipidomic profiling of GW4869-treated HSPCs demonstrated neither global Cer depletion nor SM accumulation, suggesting an alternative mechanism underlying the observed gains in HSPC fitness. Sphingolipid metabolism also regulates the biogenesis of select subclasses of secreted extracellular vesicles (EVs). To segregate metabolic regulation of HSPCs by sphingolipids from their role in EV formation and secretion, we began to systematically modulate and compare several core EV biogenesis pathways by Cas9/ribonucleoprotein-mediated knockout (KO) of SMPD3, CD63 and TSG101 in HSPC ex vivo expansion cultures . Similar to pharmacologic nSMase2 inhibition, loss of all 3 genes suppressed EV secretion, whereas only depletion of SMPD3 or ESCRT protein TSG101, but not the tetraspanin protein CD63, led to ISR activation in HSPCs (p&amp;lt;0.05). As a functional corollary, clonogenicity and long-term transplantation outcomes were enhanced by KO of SMPD3 (1.5-fold; p&amp;lt;0.01)and TSG101 (2-fold; p&amp;lt;0.001), but reduced after CD63-KO (0.6-fold; p&amp;lt;0.05). One function of EVs is the export of protein and nucleic acid cargo from the cell. We reasoned the observed differences in stress response and HSPC fitness may reflect uniquely altered secretomes. Quantitative proteomic analysis of the vesicle secretome across KO cells revealed impaired secretion of mitochondrial (n=160, FDR=8.3E-22) and oxidative stress-related (n=82, FDR=2.8E-12) proteins from SMPD3- and TSG101- KO, but not CD63-KO HSPCs, highlighting the heterogeneity and specificity of EV cargo trafficking. We validated this observation by pharmacologic blockade of SMPD3 with GW4869, which similarly led to transient decreases in mitochondrial DNA (0.36-fold, p&amp;lt;0.01) and protein (0.6-fold, p&amp;lt;0.05) export. Finally, we confirmed that impaired mitochondrial cargo secretion into EVs (mtEV) after SMPD3 or TSG101, but not CD63, depletion led to transient accumulation of mitochondrial-sourced reactive oxygen species (p&amp;lt;0.01) that specifically triggered the observed ISR activation and downstream mTOR inhibition. Our study reveals a novel mechanism by which secretory cargo trafficking into EVs guards HSPC homeostasis and fitness. We further demonstrate that transient modulation of this pathway, wherein EV cargo retention activates a molecularly-defined stress response pathway, can durably improve long-term transplantation outcomes. Sphingolipid biology inextricably links cellular metabolism and EV biogenesis, making it likely that prior descriptions of Cer-dependent adaptive stress response involve modulation of EV secretion. Finally, our work underscores the high degree of specificity that governs EV secretion in regulating intrinsic cell function. In aggregate, we show that differential EV biogenesis pathways regulate HSPC fate and fitness, with important translational implications in safeguarding stemness and regenerative capacity during ex vivo expansion and therapeutic transplantation.

Dectin-2 is critical for phagocyte function and resistance to Paracoccidioides brasiliensis in mice.

Germline-encoded pattern recognition receptors, particularly C-type lectin receptors (CLRs), are essential for phagocytes to sense invading fungal cells. Among CLRs, Dectin-2 (encoded by Clec4n) plays a critical role in the antifungal immune response as it recognizes high-mannose polysaccharides on the fungal cell wall, triggering phagocyte functional activities and ultimately determining adaptive responses. Here, we assessed the role of Dectin-2 on the course of primary Paracoccidioides brasiliensis systemic infection in mice with Dectin-2-targeted deletion. Paracoccidioides brasiliensis constitutes the principal etiologic agent of paracoccidioidomycosis, the most prominent invasive mycosis in Latin American countries. The deficiency of Dectin-2 resulted in shortened survival rates, high lung fungal burden, and increased lung pathology in mice infected with P. brasiliensis. Consistently, dendritic cells (DCs) from mice lacking Dectin-2 infected ex vivo with P. brasiliensis showed impaired secretion of several proinflammatory and regulatory cytokines, including TNF-α, IL-1β, IL-6, and IL-10. Additionally, when cocultured with splenic lymphocytes, DCs were less efficient in promoting a type 1 cytokine pattern secretion (i.e., IFN-γ). In macrophages, Dectin-2-mediated signaling was required to ensure phagocytosis and fungicidal activity associated with nitric oxide production. Overall, Dectin-2-mediated signaling is critical to promote host protection against P. brasiliensis infection, and its exploitation might lead to the development of new vaccines and immunotherapeutic approaches.

Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma.

Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.

Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma

Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.

Effect of GH Deficiency Caused by Nonfunctioning Pituitary Masses on Serum C-reactive Protein Levels.

GH supplementation for GH deficiency (GHD) has been reported to decrease high-sensitivity C-reactive protein (hs-CRP), an inflammatory marker; however, the association between GHD and hs-CRP remains unclear. We aimed to clarify the impact of impaired GH secretion due to pituitary masses on hs-CRP levels. We retrospectively examined the association between GH secretion, assessed using GH-releasing peptide-2, and serum hs-CRP levels before and a year after the pituitary surgery in patients with nonfunctioning pituitary neuroendocrine tumor or Rathke cleft cyst. Among 171 patients, 55 (32%) presented with severe GHD (peak GH response to GH-releasing peptide-2 < 9 ng/mL). Serum hs-CRP levels were significantly higher in patients with severe GHD than in those without (P < .001) and significantly correlated with the peak GH (r = -0.50, P < .001). Multiple regression analyses showed that the peak GH significantly and negatively predicted hs-CRP levels (β = -0.345; 95% CI, -0.533 to -0.158) and the lowest quartile of the peak GH (<5.04 ng/mL) were significantly associated with increase in hs-CRP levels (exp [β] = 1.840; 95% CI, 1.209 to 2.801), after controlling for other anterior hormones and metabolic parameters. Postoperative change in the peak GH (N = 60) significantly predicted change in hs-CRP levels (β = -0.391; 95% CI, -0.675 to -0.108), independent of alterations in other anterior hormones and metabolic parameters. The inverse association between GH secretion and hs-CRP levels highlights the protective role of GH in the increase in hs-CRP.

Quantity and Functional Exhaustion of Mucosal-Associated Invariant T Cells in Acute Myeloid Leukemia: Friends or Foes?

Abstract Introduction Acute Myeloid Leukemia (AML) is a hematological malignancy affecting hematopoietic progenitor cell differentiation, proliferation, and cell cycle progression, possibly involving disturbances in host immune function. Comprehensive understanding of the immune system composition and function in AML patients is crucial for developing novel immunotherapeutic strategies. Mucosa-Associated Invariant T cells (MAITs), as a subset of unconventional T cells, bridge innate and adaptive immunity and have been predominantly studied in the context of mucosa-related cancers. However, their role in AML remains largely unexplored. Investigating the association between MAITs and AML is of significant importance for understanding the immune escape mechanisms in AML, developing novel immunotherapeutic approaches, and predicting patient prognosis. Methods This study aimed to delineate the longitudinal immune profile of MAITs during AML pathogenesis and treatment, which may contribute to understanding immunological changes of MAITs in AML and predicting patient prognosis. In this prospective study, we collected peripheral blood or bone marrow samples from 131 AML patients, including 99 newly diagnosed cases, 18 in remission, and 14 in relapse, as well as 69 healthy controls. We performed multi-parameter flow cytometry for phenotypic and functional analysis of MAITs and explored their relationship with disease features, progression, and prognosis. Results We initially demonstrated that MAITs reflected immunological defects in AML patients. MAITs showed signs of depletion in both frequency and absolute count at initial diagnosis (p&amp;lt;0.0001) (Fig. 1A), especially in the CD8 + MAITs subset. The frequency of MAITs reflected AML cell genetics, tumor burden, disease status, and treatment responsiveness (Fig. 1B). AML patients exhibited reduced frequency of effector memory MAITs (p&amp;lt;0.0001) and increased frequency of terminal differentiated MAITs (p&amp;lt;0.0001) in peripheral blood (Fig. 1C), indicating altered reactivity of MAIT cells to antigen stimulation. MAITs displayed a state of high activation and even exhaustion, as evidenced by upregulated PD-1 expression. Impaired secretion of Th1-type cytokines and increased secretion of Th17-type cytokines (Fig. 1D), granzyme B, and perforin were observed in MAITs, while MAITs also shifted towards secreting tumor-promoting cytokines such as IL-8. Lower frequency of MAITs was correlated with inferior Overall Survival (OS) and Progression-Free Survival (PFS) (Fig. 1E). Multivariate analysis identified MAITs frequency &amp;lt;2.12% as an independent prognostic factor affecting OS. Conclusion Our study highlights the role of MAITs in immunological defects in Acute Myeloid Leukemia and reveals their functional impairment and enhanced tumor-promoting effects. The frequency of MAITs in peripheral blood reflects the genetic features, tumor burden, disease status, and treatment responsiveness of AML patients. Lower frequency of MAITs is associated with inferior OS and PFS, serving as a prognostic indicator for AML.

Characterization of novel PNLIP variants in congenital pancreatic lipase deficiency

Background/objectivesStudies of a rare homozygous missense mutation identified in two brothers diagnosed with congenital pancreatic lipase deficiency (CPLD) provided the first definitive evidence linking CPLD with missense mutations in the gene of PNLIP. Herein, we investigated the molecular basis for the loss-of-function in the three novel PNLIP variants (c.305G > A, p.(W102∗); c.562C > T, p.(R188C); and c.1257G > A, p.(W419∗)) associated with CPLD. MethodsWe characterized three novel PNLIP variants in transfected cells by assessing their secretion, intracellular distribution, and markers of endoplasmic reticulum (ER) stress. ResultsAll three variants had secretion defects. Notably, the p.R188C and p.W419∗ variants induced misfolding of PNLIP and accumulated as detergent-insoluble aggregates resulting in elevated BiP at both protein and mRNA levels indicating increased ER stress. ConclusionsAll three novel PNLIP variants cause a loss-of-function through impaired secretion. Additionally, the p.R188C and p.W419∗ variants may induce proteotoxicity through misfolding and potentially increase the risk for pancreatic acinar cell injury.

T3SS chaperone of the CesT family is required for secretion of the anti-sigma factor BtrA in Bordetella pertussis

ABSTRACT Bordetella pertussis is a Gram-negative, strictly human re-emerging respiratory pathogen and the causative agent of whooping cough. Similar to other Gram-negative pathogens, B. pertussis produces the type III secretion system, but its role in the pathogenesis of B. pertussis is enigmatic and yet to be elucidated. Here, we combined RNA-seq, LC-MS/MS, and co-immunoprecipitation techniques to identify and characterize the novel CesT family T3SS chaperone BP2265. We show that this chaperone specifically interacts with the secreted T3SS regulator BtrA and represents the first non-flagellar chaperone required for the secretion of an anti-sigma factor. In its absence, secretion but not production of BtrA and most T3SS substrates is severely impaired. It appears that the role of BtrA in regulating T3SS extends beyond its activity as an antagonist of the sigma factor BtrS. Predictions made by artificial intelligence system AlphaFold support the chaperone function of BP2265 towards BtrA and outline the structural basis for the interaction of BtrA with its target BtrS. We propose to rename BP2265 to BtcB for the Bordetella type III chaperone of BtrA. In addition, the absence of the BtcB chaperone results in increased expression of numerous flagellar genes and several virulence genes. While increased production of flagellar proteins and intimin BipA translated into increased biofilm formation by the mutant, enhanced production of virulence factors resulted in increased cytotoxicity towards human macrophages. We hypothesize that these phenotypic traits result indirectly from impaired secretion of BtrA and altered activity of the BtrA/BtrS regulatory node.

SAT229 Symptomatic Hypocalcemia In The Setting Of Crohn’s Disease With Severe Hypomagnesemia

Abstract Disclosure: K. Cuan: None. T. Reisman: None. Introduction: People with Crohn’s Disease (CD) are at risk for various nutritional deficiencies due to decreased oral intake, malabsorption, and increased intestinal losses. CD often causes a negative calcium balance, which does not usually cause hypocalcemia because the bones serve as a reservoir for calcium. However, symptomatic hypocalcemia may occur if a concurrent severe hypomagnesemic state is present. The factors resulting in hypocalcemia in patients with magnesium deficiency are impaired PTH secretion, PTH resistance, and vitamin D deficiency. Clinical Case: A 33-year-old man with CD and rheumatoid arthritis presented following a seizure with additional symptoms of hand cramping and facial twitching. He had previously undergone three small bowel resections and had a history of hypocalcemia due to malabsorption. Although this patient had previously been admitted due to a hypocalcemic seizure, he did not start oral calcium supplements at that time. No issues with magnesium were identified previously. On this encounter, he was found to have a corrected calcium (cCa) of 5.3 mg/dL, undetectably low magnesium at &amp;lt;0.7 mg/dL, normal phosphorus, 25-hydroxyvitamin D at 3.5 ng/mL, and an elevated intact PTH (iPTH) at 169 pg/mL. Aggressive intravenous calcium and magnesium repletion improved cCa to 7.4 mg/dL and magnesium to 2 mg/dL with no further seizure activity. He was discharged on calcium carbonate, calcitriol, and ergocalciferol. A month later, he presented to our endocrinology clinic asymptomatic but with a cCa of 6 mg/dL and magnesium at &amp;lt;0.7 mg/dL. Further biochemical workup revealed normal phosphorus, 25-hydroxyvitamin D at 7.9 ng/mL, and an inappropriately normal iPTH at 49 pg/mL. He was readmitted to the hospital where intravenous repletion improved cCa to 8.1 mg/dL and magnesium to 1.9 mg/dL. His recurrent hypomagnesemia was attributed to malabsorption. An increased dose of calcitriol and oral magnesium were added to his discharge medication regimen, in addition to his ergocalciferol and calcium carbonate. Three days later, he had a cCa level of 7.1 mg/dL as an outpatient. Conclusion: Patients with CD often have nutrient deficiencies, especially if they have had surgical resection within the intestinal tract. Our patient initially developed hypocalcemia from malabsorption, which was then exacerbated by the severely hypomagnesemic state. During his initial hospital encounter, the elevated iPTH level indicated PTH resistance. On his second encounter, the normal iPTH level suggested hypoparathyroidism. Given the relationship between magnesium and calcium homeostasis, addressing the hypomagnesemia eventually aided in achieving stabilization of serum calcium levels. Thus, in patients who are at risk for malabsorption and have recurrent symptomatic hypocalcemia, it is prudent to check magnesium levels as it can affect long term management of hypocalcemia. Presentation: Saturday, June 17, 2023