Search

The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-Å resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.

IntroductionFemale reproductive function depends on a choreographed sequence of hormonal secretion and action, where specific stresses such as inflammation exert profound disruptions. Specifically, acute LPS-induced inflammation inhibits gonadotropin production and secretion from the pituitary, thereby impacting the downstream production of sex hormones. These outcomes have only been observed in acute inflammatory stress and little is known about the mechanisms by which chronic inflammation affects reproduction. In this study we seek to understand the chronic effects of LPS on pituitary function and consequent luteinizing and follicle stimulating hormone secretion.MethodsA chronic inflammatory state was induced in female mice by twice weekly injections with LPS over 6 weeks. Serum gonadotropins were measured and bulk RNAseq was performed on the pituitaries from these mice, along with basic measurements of reproductive biology.ResultsSurprisingly, serum luteinizing and follicle stimulating hormone was not inhibited and instead we found it was increased with repeated LPS treatments.DiscussionAnalysis of bulk RNA-sequencing of murine pituitary revealed paracrine activation of TGFβ pathways as a potential mechanism regulating FSH secretion in response to chronic LPS. These results provide a framework with which to begin dissecting the impacts of chronic inflammation on reproductive physiology.

Extracellular vesicles (EV) are a heterogeneous group of lipid particles excreted by cells. They play an important role in regeneration, development, inflammation, and cancer progression, together with the extracellular matrix (ECM), which they constantly interact with. In this review, we discuss direct and indirect interactions of EVs and the ECM and their impact on different physiological processes. The ECM affects the secretion of EVs, and the properties of the ECM and EVs modulate EVs' diffusion and adhesion. On the other hand, EVs can affect the ECM both directly through enzymes and indirectly through the modulation of the ECM synthesis and remodeling by cells. This review emphasizes recently discovered types of EVs bound to the ECM and isolated by enzymatic digestion, including matrix-bound nanovesicles (MBV) and tissue-derived EV (TiEV). In addition to the experimental studies, computer models of the EV-ECM-cell interactions, from all-atom models to quantitative pharmacology models aiming to improve our understanding of the interaction mechanisms, are also considered. Application of extracellular vesicles in tissue engineering is an actively developing area. Vesicles not only affect cells themselves but also interact with the matrix and change it. The matrix also influences both cells and vesicles. In this review, different possible types of interactions between vesicles, matrix, and cells are discussed. Furthermore, the united EV-ECM system and its regulation through the cellular activity are presented. [Display omitted] [ABSTRACT FROM AUTHOR]

Conjugative dissemination of mobile genetic elements (MGEs) among bacteria is initiated by assembly of the relaxosome at the MGE's origin‐of‐transfer (oriT) sequence. A critical but poorly defined step of relaxosome assembly involves recruitment of the catalytic relaxase to its DNA strand‐specific nicking site within oriT. Here, we present evidence by AlphaFold modeling, affinity pulldowns, and in vivo site‐directed photocrosslinking that the TraK Ribbon–Helix–Helix DNA‐binding protein recruits TraI to oriT through a dynamic interaction in which TraI's C‐terminal unstructured domain (TraICTD) wraps around TraK's C‐proximal tetramerization domain. Upon relaxosome assembly, conformational changes disrupt this contact, and TraICTD instead self‐associates as a prerequisite for relaxase catalytic functions or substrate engagement with the transfer channel. These findings delineate key early‐stage processing reactions required for conjugative dissemination of a model MGE. [ABSTRACT FROM AUTHOR]

Food incompatibility (Virudhaahar) is described elaborately in Ayurveda health science. Various types of food incompatibilities with the examples of combination are mentioned in the Ayurveda literatures like in Charaka Samhita and Susruta Samhita but these type of food combinations are not practiced in today's era. Today with the change in lifestyle, food processing methods and the combination of food articles are also modified. So, there is a need to review and identify new food incompatibilities, which are currently practiced as per Ayurvedic perspectives. Such food combinations can prove harmful, which may be imparting its untoward effects on the immune system, cellular metabolism, hormone secretions, and hence, the need to acknowledge this health issue arise. The unwanted effect of wrong combinations of food is not limited up to gastrointestinal tract only but may hamper the major systems of the body as well. Thereby, unwanted side effects can emerge inside the body when two or more types of foods are consumed together. This review article is an endeavour to compile and critically analyse the researches reporting the incompatible food effects with their probable pathogenesis and to identify new food incompatibilities, which are currently practiced. [ABSTRACT FROM AUTHOR]

Key message: The VAMP721, VAMP722, SYP121, SYP122 and SNAP33 SNAREs are required in the Arabidopsis stigma for pollen hydration, further supporting a role for vesicle trafficking in the stigma's pollen responses. In the Brassicaceae, the process of accepting compatible pollen is a key step in successful reproduction and highly regulated following interactions between the pollen and the stigma. Central to this is the initiation of secretion in the stigma, which is proposed to provide resources to the pollen for hydration and germination and pollen tube growth. Previously, the eight exocyst subunit genes were shown to be required in the Arabidopsis stigma to support these pollen responses. One of the roles of the exocyst is to tether secretory vesicles at the plasma membrane for membrane fusion by the SNARE complex to enable vesicle cargo release. Here, we investigate the role of Arabidopsis SNARE genes in the stigma for pollen responses. Using a combination of different knockout and knockdown SNARE mutant lines, we show that VAMP721, VAMP722, SYP121, SYP122 and SNAP33 are involved in this process. Significant disruptions in pollen hydration were observed following pollination of wildtype pollen on the mutant SNARE stigmas. Overall, these results place the Arabidopsis SNARE complex as a contributor in the stigma for pollen responses and reaffirm the significance of secretion in the stigma to support the pollen–stigma interactions. [ABSTRACT FROM AUTHOR]

Aim: To perform a direct, double‐blind, randomised, crossover comparison of subcutaneous and intravenous glucagon‐like peptide‐1 (GLP‐1) in hyperglycaemic subjects with type 2 diabetes naïve to GLP‐1‐based therapy. Materials and Methods: Ten fasted, hyperglycaemic subjects (1 female, age 63 ± 10 years [mean ± SD], glycated haemoglobin 73.5 ± 22.0 mmol/mol [8.9% ± 2.0%], both mean ± SD) received subcutaneous GLP‐1 and intravenous saline, or intravenous GLP‐1 and subcutaneous saline. Infusion rates were doubled every 120 min (1.2, 2.4, 4.8 and 9.6 pmol·kg−1·min−1 for subcutaneous, and 0.3, 0.6, 1.2 and 2.4 pmol·kg−1·min−1 for intravenous). Plasma glucose, total and intact GLP‐1, insulin, C‐peptide, glucagon and gastrointestinal symptoms were evaluated over 8 h. The results are presented as mean ± SEM. Results: Plasma glucose decreased more with intravenous (by ~8.0 mmol/L [144 mg/dL]) than subcutaneous GLP‐1 (by ~5.6 mmol/L [100 mg/dL]; p < 0.001). Plasma GLP‐1 increased dose‐dependently, but more with intravenous than subcutaneous for both total (∆max 154.2 ± 3.9 pmol/L vs. 85.1 ± 3.8 pmol/L; p < 0.001), and intact GLP‐1 (∆max 44.2 ± 2.2 pmol/L vs. 12.8 ± 2.2 pmol/L; p < 0.001). Total and intact GLP‐1 clearance was higher for subcutaneous than intravenous GLP‐1 (p < 0.001 and p = 0.002, respectively). The increase in insulin secretion was greater, and glucagon was suppressed more with intravenous GLP‐1 (p < 0.05 each). Gastrointestinal symptoms did not differ (p > 0.05 each). Conclusions: Subcutaneous GLP‐1 administration is much less efficient than intravenous GLP‐1 in lowering fasting plasma glucose, with less stimulation of insulin and suppression of glucagon, and much less bioavailability, even at fourfold higher infusion rates. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to examine changes in salivary immunoglobulin A (s-IgA) secretion at different intensities or durations of acute exercise. Methods: Twelve healthy untrained young males were included in randomized crossover trials in Experiment 1 (cycling exercise for 30 min at a work rate equivalent to 35%, 55%, and 75% maximal oxygen uptake [ V ˙ O2max]) and Experiment 2 (cycling exercise at 55% V ˙ O2max intensity for 30, 60, and 90 min). Saliva samples were collected at baseline, immediately after, and 60 min after each exercise. Results: Experiment 1: The percentage change in the s-IgA secretion rate in the 75% V ˙ O2max trial was significantly lower than that in the 55% V ˙ O2max trial immediately after exercise (− 45.7%). The percentage change in the salivary concentration of cortisol, an s-IgA regulating factor, immediately after exercise significantly increased compared to that at baseline in the 75% V ˙ O2max trial (+ 107.6%). A significant negative correlation was observed between the percentage changes in saliva flow rate and salivary cortisol concentration (r = − 0.52, P < 0.01). Experiment 2: The percentage change in the s-IgA secretion rate in the 90-min trial was significantly lower than that in the 30-min trial immediately after exercise (−37.0%). However, the percentage change in salivary cortisol concentration remained the same. Conclusion: Our findings suggest that a reduction in s-IgA secretion is induced by exercise intensity of greater than or equal to 75% V ˙ O2max for 30 min or exercise duration of greater than or equal to 90 min at 55% V ˙ O2max in healthy untrained young men. [ABSTRACT FROM AUTHOR]

This study compares oil and mucilage idioblasts occurring together in the vegetative organs of Ocotea pulchella, a Lauraceae species. Our focus is specifically on the ontogeny and developmental cytology of these secretory cells. Both types of idioblasts originate from solitary cells located in the fundamental meristem, underlying the protodermis. The growth of both types of idioblasts is asynchronous, with the oil idioblasts developing first, but their initiation is restricted to the early stages of organ development. Mucilaginous idioblasts occur exclusively in the palisade parenchyma, while oil idioblasts are scattered throughout the mesophyll, midrib, and petiole of the leaves. The lamellar secretion of mucilage idioblasts is mostly made up of polysaccharides, while the secretion of oil idioblasts is made up of terpenes and lipids. Cupule occurred only in the oil idioblasts, while suberized layers occurred in both types of cells. We found that immature oil idioblasts that are close to each other fuse; mature mucilage idioblasts have labyrinthine walls arranged in a reticulate pattern; the cells close to the oil idioblasts have a pectin protective layer; and the oil idioblasts have a sheath of phenolic cells. In contrast to previous reports, the two types of secretory idioblasts were recognized during the early stages of their development. The results emphasize the importance of combining optical and electron microscopy methods to observe the ontogenetic, histochemical and ultrastructural changes that occur during the development of the secretory idioblasts. This can help us understand how secreting cells store their secretions and how their walls become specialized. [ABSTRACT FROM AUTHOR]

Background: Arginine vasopressin (AVP) has been reported to regulate insulin secretion and glucose homeostasis in the body. Previous study has shown that AVP and its receptor V1bR modulate insulin secretion via the hypothalamic-pituitary-adrenal axis. AVP has also been shown to enhance insulin secretion in islets, but the exact mechanism remains unclear. Results: In our study, we unexpectedly discovered that AVP could only stimulates insulin secretion from islets, but not β cells, and AVP-induced insulin secretion could be blocked by V1bR selective antagonist. Single-cell transcriptome analysis identified that V1bR is only expressed by the α cells. Further studies indicated that activation of the V1bR stimulates the α cells to secrete glucagon, which then promotes glucose-dependent insulin secretion from β cells in a paracrine way by activating GLP-1R but not GCGR on these cells. Conclusions: Our study revealed a crosstalk between α and β cells initiated by AVP/V1bR and mediated by glucagon/GLP-1R, providing a mechanism to develop new glucose-controlling therapies targeting V1bR. [ABSTRACT FROM AUTHOR]

Regulated cell death in response to microbial infection plays an important role in immune defense and is triggered by pathogen disruption of essential cellular pathways. Gram-negative bacterial pathogens in the Yersinia genus disrupt NF-κB signaling via translocated effectors injected by a type III secretion system, thereby preventing induction of cytokine production and antimicrobial defense. In murine models of infection, Yersinia blockade of NF-κB signaling triggers cell-extrinsic apoptosis through Receptor Interacting Serine-Threonine Protein Kinase 1 (RIPK1) and caspase-8, which is required for bacterial clearance and host survival. Unexpectedly, we find that human macrophages undergo apoptosis independently of RIPK1 in response to Yersinia or chemical blockade of IKKβ. Instead, IKK blockade led to decreased cFLIP expression, and overexpression of cFLIP contributed to protection from IKK blockade-induced apoptosis in human macrophages. We found that IKK blockade also induces RIPK1 kinase activity-independent apoptosis in human T cells and human pancreatic cells. Altogether, our data indicate that, in contrast to murine cells, blockade of IKK activity in human cells triggers a distinct apoptosis pathway that is independent of RIPK1 kinase activity. These findings have implications for the contribution of RIPK1 to cell death in human cells and the efficacy of RIPK1 inhibition in human diseases. Author summary: Programmed cell death is critical for organismal homeostasis and for host defense against microbial infection. Gram-negative bacteria of the genus Yersinia cause diseases ranging from plague (Y. pestis) to severe gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica). Studies in murine models have demonstrated that all pathogenic Yersinia disrupt pro-inflammatory cell signaling, which triggers cell death in murine macrophages. This cell death is mediated by the kinase RIPK1, and RIPK1-mediated cell death is essential for host defense and survival. Although murine studies have provided insight into mechanisms that regulate host defense during Yersinia infection, there are important differences between human and murine immune systems regarding expression and presence of key proteins. Thus, how human cells respond to Yersinia infection remains poorly understood. Here, we report that, in contrast to murine systems, Yersinia infection or chemical blockade of immune signaling in human cells induces a distinct apoptotic cell death that is entirely independent of RIPK1 kinase activity. RIPK1 is implicated in a wide range of systemic disorders and is currently being targeted in clinical trials. Our study provides insight into human-specific cell death signaling and host defense mechanisms, which has implications for understanding human immune responses to infection and therapeutic approaches for treating human diseases. [ABSTRACT FROM AUTHOR]

Starvation- or stress-induced phosphatidylinositol 3-phosphate (PtdIns3P/PI3P) production at the endoplasmic reticulum (ER) subdomains organizes phagophore assembly and autophagosome formation. Coat protein complex II (COPII) vesicles budding from ER exit site (ERES) also contribute to autophagosome formation. Whether any PtdIns3P phosphatase functions at ERES to inhibit macroautophagy/autophagy is unknown. Here we report Myotubularin 2 (MTM2) of Arabidopsis as a PtdIns3P phosphatase that localizes to ERES and negatively regulates autophagy. MTM2 binds PtdIns3P with its PH-GRAM domain in vitro and acts toward PtdIns3P in vivo. Transiently expressed MTM2 colocalizes with ATG14b, a subunit of the phosphatidylinositol 3-kinase (PtdIns3K) complex, and overexpression of MTM2 blocks autophagic flux and causes over-accumulation of ATG18a, ATG5, and ATG8a. The mtm2 mutant has higher levels of autophagy and is more tolerant to starvation, whereas MTM2 overexpression leads to reduced autophagy and sensitivity to starvation. The phenotypes of mtm2 are suppressed by ATG2 mutation, suggesting that MTM2 acts upstream of ATG2. Importantly, MTM2 does not affect the endosomal functions of PtdIns3P. Instead, MTM2 specifically colocalizes with COPII coat proteins and is cradled by the ERES-defining protein SEC16. MTM2 interacts with SEC23A with its phosphatase domain and inhibits COPII-mediated protein secretion. Finally, a role for MTM2 in salt stress response is uncovered. mtm2 resembles the halophyte Thellungiella salsuginea in its efficient vacuolar compartmentation of Na+, maintenance of chloroplast integrity, and timely regulation of autophagy-related genes. Our findings reveal a balance between PtdIns3P synthesis and turnover in autophagosome formation, and provide a new link between autophagy and COPII function.Abbreviations: ATG: autophagy related; BFA: brefeldin A; BiFC: bimolecular fluorescence complementation; CHX: cycloheximide; ConA: concanamycin A; COPII: coat protein complex II; ER: endoplasmic reticulum; ERES: ER exit site; MS: Murashige and Skoog; MTM: myotubularin; MVB: multivesicular body; PAS: phagophore assembly site; PI: phosphoinositide; TEM: transmission electron microscopy; WT: wild-type. [ABSTRACT FROM AUTHOR]

The Clostridia produce and secrete Large Clostridial Glucosylating Toxins (LCGTs) responsible for disease symptoms, but the secretion mechanism is largely unknown. Recently, a holin-like protein was shown to be essential for toxin secretion. Holins, typically bacteriophage-specific proteins, are part of the holin-endo(lysin) system that releases phage progeny. To determine if the clostridia also use a lysin, we investigated two conserved putative lysins, M7404_01910 and M7404_02200, in the release of the LCGTs TcdA and TcdB from a Clostridioides difficile ribotype 027 strain, M7404. Sequence analysis and structural modelling indicates that both proteins are related to N-acetylmuramoyl-l-alanine amidases, similar to CD27L, a lysin from the C. difficile phage ΦCD27. Disruption of these genes reveal that only M7404_02200 contributes to toxin secretion and does so in a non-lytic fashion. Peptidoglycan hydrolysis assays show that recombinant M7404_02200 is an active peptidoglycan amidase, confirming its role in TcdA and TcdB secretion in C. difficile M7404. Identification of an N-acetylmuramoyl-l-alanine amidase in the ribotype 027 C. difficile strain M7404, involved in the active secretion of the large clostridial glucosylating toxins, TcdA and TcdB. [ABSTRACT FROM AUTHOR]

Stress impairs fertility, at least in part, via inhibition of gonadotropin secretion. Luteinizing hormone (LH) is an important gonadotropin that is released in a pulsatile pattern in males and in females throughout the majority of the ovarian cycle. Several models of stress, including acute metabolic stress, suppress LH pulses via inhibition of neurons in the arcuate nucleus of the hypothalamus that coexpress kisspeptin, neurokinin B, and dynorphin (termed KNDy cells) which form the pulse generator. The mechanism for inhibition of KNDy neurons during stress, however, remains a significant outstanding question. Here, we investigated a population of catecholamine neurons in the nucleus of the solitary tract (NTS), marked by expression of the enzyme dopamine beta-hydroxylase (DBH), in female mice. First, we found that a subpopulation of DBH neurons in the NTS is activated (express c-Fos) during metabolic stress. Then, using chemogenetics, we determined that activation of these cells is sufficient to suppress LH pulses, augment corticosterone secretion, and induce sickness-like behavior. In subsequent studies, we identified evidence for suppression of KNDy cells (rather than downstream signaling pathways) and determined that the suppression of LH pulses was not dependent on the acute rise in glucocorticoids. Together these data support the hypothesis that DBH cells in the NTS are important for regulation of neuroendocrine and behavioral responses to stress. [ABSTRACT FROM AUTHOR]

The present study investigated the associations of serum gamma-glutamyl transferase (GGT), a marker of fatty liver and oxidative stress, and ALT/AST, a marker of fatty liver, with percentage trunk fat and postload glucose, insulin resistance, and β-cell function in middle-aged Japanese individuals, whose BMI averaged < 23.0 kg/m2. Pancreatic β-cell function was assessed using the disposition index calculated by a product of the insulinogenic index (IGI) and Matsuda insulin sensitivity index, a biomarker of early-phase glucose-stimulated insulin secretion and whole-body insulin sensitivity, respectively. Multivariate linear regression analyses revealed that the disposition index was associated inversely with GGT independently of percentage trunk fat, homeostasis model assessment insulin resistance (HOMA-IR), a marker of insulin resistance, and Matsuda index. When IGI was included instead of the disposition index, IGI (inversely) and HOMA-IR were associated with GGT independently of percentage trunk fat and Matsuda index. When the area under the glucose concentration curve (AUCg) during an oral glucose tolerance test was included instead of the disposition index, AUCg and HOMA-IR emerged as independent determinants of GGT. ALT/AST was associated with HOMA-IR alone. Results suggest a different pathophysiologic basis between GGT and ALT/AST in predicting diabetic risk in non-obese Japanese. [ABSTRACT FROM AUTHOR]

Plants respond to pathogen exposure by activating the expression of a group of defense-related proteins known as Pathogenesis-Related (PR) proteins, initially discovered in the 1970s. These PR proteins are categorized into 17 distinct families, denoted as PR1-PR17. Predominantly secreted, most of these proteins execute their defensive roles within the apoplastic space. Several PR proteins possess well-defined enzymatic functions, such as β-glucanase (PR2), chitinases (PR3, 4, 8, 11), proteinase (PR7), or RNase (PR10). Enhanced resistance against pathogens is observed upon PR protein overexpression, while their downregulation renders plants more susceptible to pathogen infections. Many of these proteins exhibit antimicrobial activity in vitro, and due to their compact size, some are classified as antimicrobial peptides. Recent research has unveiled that phytopathogens, including nematodes, fungi, and phytophthora, employ analogous proteins to bolster their virulence and suppress plant immunity. This raises a fundamental question: how can these conserved proteins act as antimicrobial agents when produced by the host plant but simultaneously suppress plant immunity when generated by the pathogen? In this hypothesis, we investigate PR proteins produced by pathogens, which we term "PR-like proteins," and explore potential mechanisms by which this class of virulence factors operate. Preliminary data suggests that these proteins may form complexes with the host's own PR proteins, thereby interfering with their defense-related functions. This analysis sheds light on the intriguing interplay between plant and pathogen-derived PR-like proteins, providing fresh insights into the intricate mechanisms governing plant-pathogen interactions. [ABSTRACT FROM AUTHOR]

C-Mannosyl tryptophan (CMW), a unique glycosylated amino acid, is considered to be produced by degradation of C-mannosylated proteins in living organism. Although protein C-mannosylation is involved in the folding and secretion of substrate proteins, the pathophysiological function in the hematological system is still unclear. This study aimed to assess CMW in the human hematological disorders. The serum CMW levels of 94 healthy Japanese workers were quantified using hydrophilic interaction liquid chromatography. Platelet count was positively correlated with serum CMW levels. The clinical significance of CMW in thrombocytosis of myeloproliferative neoplasms (T-MPN) including essential thrombocythemia (ET) were investigated. The serum CMW levels of the 34 patients with T-MPN who presented with thrombocytosis were significantly higher than those of the 52 patients with control who had other hematological disorders. In patients with T-MPN, serum CMW levels were inversely correlated with anemia, which was related to myelofibrosis (MF). Bone marrow biopsy samples were obtained from 18 patients with ET, and serum CMW levels were simultaneously measured. Twelve patients with bone marrow fibrosis had significantly higher CMW levels than 6 patients without bone marrow fibrosis. Collectively, these results suggested that CMW could be a novel biomarker to predict MF progression in T-MPN. [ABSTRACT FROM AUTHOR]

The use of acute carbon monoxide inhalation (COi) and hot water immersion (HWI) are of growing interest as interventions to stimulate erythropoietin (EPO) production. However, whether EPO production is further augmented when combining these stressors and whether there are sex differences in this response are poorly understood. Therefore, we measured circulating EPO concentration in response to acute COi and HWI independently and in combination and determined whether the responses were altered by sex. Participants completed three study visits—COi, HWI, and combined COi and HWI—separated by 1 week in a randomized, balanced, crossover design. Renal blood velocity was measured during all interventions, and carboxyhaemoglobin was measured during and after COi. Serum samples were analysed every hour for 6 h post‐intervention for EPO concentration. HWI decreased renal blood velocity (46.2 cm/s to 36.2 cm/s) (P < 0.0001), and COi increased carboxyhaemoglobin (1.5%–12.8%) (P < 0.0001) without changing renal blood velocity (46.4–45.2 cm/s) (P = 0.4456). All three interventions increased peak EPO concentration from baseline (COi: 6.02–9.74 mIU/mL; HWI: 6.80–11.10 mIU/mL; COi + HWI: 6.71–10.91 mIU/mL) (P = 0.0048) and to the same extent (P = 0.3505). On average, females increased EPO while males did not in response to COi (females: 6.17 mIU/mL; males: 1.27 mIU/mL) (P = 0.0010), HWI (females: 6.47 mIU/mL; males: 2.14 mIU/mL) (P = 0.0104), and COi and HWI (females: 6.65 mIU/mL; males: 1.76 mIU/mL) (P = 0.0256). These data emphasize that combining these interventions does not augment EPO secretion and that these interventions may work better in females. [ABSTRACT FROM AUTHOR]

Background: Chronic cough in children is a challenging symptom for clinicians. So, we aimed in this study to evaluate the diagnostic role of flexible bronchoscope in differentiating between the underlying causes of chronic wet cough and chronic cough associated with wheeze. Methods: This was a prospective cross-sectional study conducted on children referred to Tanta University Hospitals and Ain-Shams University Hospitals between January 2021 and January 2023, presenting with a primary complaint of chronic cough lasting more than 4 weeks. The children were further classified into two groups: the first group included children with chronic wet cough not associated with wheezing (Cohort A) and the second group included children with chronic cough associated with wheezing (Cohort B). Results: The study enrolled 64 children. During clinical evaluation, 25 (39.1%) children had a chronic cough without wheezing and 39 (60.9%) had a chronic wheezy cough. Bronchoscopic examination findings indicated a notable disparity between the two groups of patients with chronic cough (p=0.006). Among Cohort A patients, the most prevalent bronchoscopic observation was purulent inflammatory secretions in 16 cases (64.0%), followed by congenital airway anomalies in 3 cases (12.0%). Conversely, Cohort B patients exhibited congenital airway anomalies as the primary finding in 14 cases (35.9%), followed by purulent secretions in 7 cases (17.9%). Conclusion: Flexible bronchoscopy is a valuable and safe tool for diagnosing chronic cough in children. It helped differentiate between the underlying causes of chronic cough in children with and without wheezing. [ABSTRACT FROM AUTHOR]

The methylotrophic yeast, Pichia pastoris (P. pastoris ; syn. Komagataella spp.), known for its ability to grow to high cell densities, its strong and tightly regulated promoters, and mammalian liked secretion pathway, has been widely used as a robust system to secrete heterologous proteins. The α-mating factor (MF) secretion signal leader from Saccharomyces cerevisiae (S. cerevisiae) is currently the most successfully used secretion signal sequence in the P. pastoris system. In this study, the secretion efficiency mediated by the α-MF secretion signal leaders from Komagataella pastoris (K. pastoris) and Komagataella phaffii (K. phaffii) was assessed using Enhanced Green Fluorescent Protein (EGFP) as a reporter. The results indicated that the secretion efficiency associated with the α-MF secretion signal leaders from K. pastoris and K. phaffii was notably lower in comparison to the α-MF secretion signal leader from S. cerevisiae. Further research indicated that N-linked glycosylation of the α-MF secretion signal leader enhanced the secretion of EGFP. Disruption of calnexin impaired the secretion of EGFP mediated by the N-linked glycosylated α-MF secretion signal leader, without affecting EGFP secretion mediated by the non-N-linked glycosylation α-MF secretion signal leader. The N-linked glycosylated of the α-MF secretion signal leader reduced the unfolded protein response (UPR) in the endoplasmic reticulum (ER). The enhancement of EGFP secretion by the N-linked glycosylated α-MF secretion signal leader might be achieved through the acceleration of proper folding of glycoproteins by the molecular chaperone calnexin. This study enhances the understanding of protein secretion in P. pastoris , specifically highlighting the influence of N-linked glycosylation on secretion efficiency, and could have implications for the production of recombinant proteins in bioengineering and biotechnological applications in P. pastoris. • α-MF secretion signal leaders from K. pastoris and K. phaffii exhibit lower secretion efficiency compared to that from S. cerevisiae in the P. pastoris system. • N-linked glycosylation on the α-MF secretion signal leader enhances protein secretion. • N-linked glycosylation on the α-MF secretion signal leader reduces ER stress and enhanced protein folding mediated by calnexin. [ABSTRACT FROM AUTHOR]

Aim Methods Results Conclusions To investigate the effect of G protein‐coupled receptor 55 (GPR55) deletion on glucose homeostasis and islet function following diet‐induced obesity.GPR55−/− and wild‐type (WT) mice were fed ad libitum either standard chow (SC) or a high‐fat diet (HFD) for 20 weeks. Glucose and insulin tolerance tests were performed at 9/10 and 19/20 weeks of dietary intervention. Insulin secretion in vivo and dynamic insulin secretion following perifusion of isolated islets were also determined, as were islet caspase‐3/7 activities and β‐cell 5‐bromo‐20‐deoxyuridine (BrdU) incorporation.GPR55−/− mice fed a HFD were more susceptible to diet‐induced obesity and were more glucose intolerant and insulin resistant than WT mice maintained on a HFD. Islets isolated from HFD‐fed GPR55−/− mice showed impaired glucose‐ and pcacahorbol 12‐myristate 13‐acetate‐stimulated insulin secretion, and they also displayed increased cytokine‐induced apoptosis. While there was a 5.6 ± 1.6‐fold increase in β‐cell BrdU incorporation in the pancreases of WT mice fed a HFD, this compensatory increase in β‐cell proliferation in response to the HFD was attenuated in GPR55−/− mice.Under conditions of diet‐induced obesity, GPR55−/− mice show impaired glucose handling, which is associated with reduced insulin secretory capacity, increased islet cell apoptosis and insufficient compensatory increases in β‐cell proliferation. These observations support that GPR55 plays an important role in positively regulating islet function. [ABSTRACT FROM AUTHOR]

Export of secretory cargoes from the endoplasmic reticulum (ER) requires COPII proteins, which were first identified for their ability to coat small vesicles that bud from the ER. Recent data indicate that COPII proteins can also organize into a collar at the necks of tubules, as well as phase-separate into liquid-like condensates. Thus, COPII assemblies seem to be tailored to accommodate variations in the size and quantities of cargo secreted. [ABSTRACT FROM AUTHOR]

Background Secondary hypogonadism (SH) is common in men with Cushing's syndrome (CS), but its impact on comorbidities is largely unknown and longitudinal data are scarce. If SH also affects men with mild autonomous cortisol secretion (MACS) is unknown. Methods We included 30 treatment-naïve adult men with CS and 17 men with MACS diagnosed since 2012. Hypogonadism was diagnosed based on total testosterone (TT) concentrations < 10.4 nmol/L and age-specific cut-offs. Outcomes were compared to age- and BMI-matched controls. In 20 men in remission of CS, a longitudinal analysis was conducted at 6, 12, and 24 months. Results Men with CS had significantly lower concentrations of TT, bioavailable T, and free T compared to controls (P <.0001) with lowest concentrations in ectopic CS. Likewise, TT was lower in men with MACS compared to controls. At baseline, 93% of men with CS and 59% of men with MACS had SH. Testosterone correlated negatively with late night salivary cortisol and serum cortisol pre- and post-1 mg dexamethasone suppression test. Following successful surgery, TT increased significantly (P =.001), normalising within 6 months. Despite normalisation, several RBC parameters remained lower in men with CS even 2 years after successful surgery. Conclusions Secondary hypogonadism is common in men with CS and MACS but usually reversible after successful surgery. The persisting changes observed in RBC parameters need to be further investigated in larger cohorts and longer follow-up durations. [ABSTRACT FROM AUTHOR]

Plants and insects have co-existed for almost 400 million years and their interactions can be beneficial or harmful, thus reflecting their intricate co-evolutionary dynamics. Many herbivorous arthropods cause tremendous crop loss, impacting the agro-economy worldwide. Plants possess an arsenal of chemical defenses that comprise diverse secondary metabolites that help protect against harmful herbivorous arthropods. In response, the strategies that herbivores use to cope with plant defenses can be behavioral, or molecular and/or biochemical of which salivary secretions are a key determinant. Insect salivary secretions/oral secretions (OSs) play a crucial role in plant immunity as they contain several biologically active elicitors and effector proteins that modulate plants' defense responses. Using this oral secretion cocktail, insects overcome plant natural defenses to allow successful feeding. However, a lack of knowledge of the nature of the signals present in oral secretion cocktails has resulted in reduced mechanistic knowledge of their cellular perception. In this review, we discuss the latest knowledge on herbivore oral secretion derived elicitors and effectors and various mechanisms involved in plant defense modulation. Identification of novel herbivore-released molecules and their plant targets should pave the way for understanding the intricate strategies employed by both herbivorous arthropods and plants in their interactions. [ABSTRACT FROM AUTHOR]

Several inflammatory cytokines bind to the allosteric site (site 2) and allosterically activate integrins. Site 2 is also a binding site for 25-hydroxycholesterol, an inflammatory lipid mediator, and is involved in inflammatory signaling (e.g., TNF and IL-6 secretion) in addition to integrin activation. FGF2 is pro-inflammatory and pro-thrombotic, and FGF1, homologous to FGF2, has anti-inflammatory and anti-thrombotic actions, but the mechanism of these actions is unknown. We hypothesized that FGF2 and FGF1 bind to site 2 of integrins and regulate inflammatory signaling. Here, we describe that FGF2 is bound to site 2 and allosterically activated β3 integrins, suggesting that the pro-inflammatory action of FGF2 is mediated by binding to site 2. In contrast, FGF1 bound to site 2 but did not activate these integrins and instead suppressed integrin activation induced by FGF2, indicating that FGF1 acts as an antagonist of site 2 and that the anti-inflammatory action of FGF1 is mediated by blocking site 2. A non-mitogenic FGF1 mutant (R50E), which is defective in binding to site 1 of αvβ3, suppressed β3 integrin activation by FGF2 as effectively as WT FGF1. [ABSTRACT FROM AUTHOR]

Summary: The appressorium of phytopathogenic fungi is a specific structure with a crucial role in plant cuticle penetration. Pathogens with melanized appressoria break the cuticle through cell wall melanization and intracellular turgor pressure. However, in fungi with nonmelanized appressorium, the mechanisms governing cuticle penetration are poorly understood. Here we characterize Row1, a previously uncharacterized appressoria‐specific protein of Ustilago maydis that localizes to membrane and secretory vesicles.Deletion of row1 decreases appressoria formation and plant penetration, thereby reducing virulence. Specifically, the Δrow1 mutant has a thicker cell wall that is more resistant to glucanase degradation. We also observed that the Δrow1 mutant has secretion defects.We show that Row1 is functionally conserved at least among Ustilaginaceae and belongs to the Row family, which consists of five other proteins that are highly conserved among Basidiomycota fungi and are involved in U. maydis virulence. We observed similarities in localization between Row1 and Row2, which is also involved in cell wall remodelling and secretion, suggesting similar molecular functions for members of this protein family.Our data suggest that Row1 could modify the chitin‐glucan matrix of the fungal cell wall and may be involved in unconventional protein secretion, thereby promoting both appressoria maturation and penetration. [ABSTRACT FROM AUTHOR]

Aim: MicroRNAs (miRNAs) regulate β‐cell function, and β‐cell mitochondria and insulin secretion are perturbed in diabetes. We aimed to identify key miRNAs regulating β‐cell mitochondrial metabolism and novel β‐cell miRNA‐mitochondrial pathways. Methods: TargetScan (http://www.targetscan.org/) was used to predict if 16 miRNAs implicated in β‐cell function target 27 cis‐eGenes implicated in mitochondrial activity. The expression of candidate miRNAs and insulin secretion after 24 and 1 h pre‐incubation in 2.8, 11.1‐ and 16.7‐mM glucose was measured in clonal INS‐1 832/13 β‐cells. MiR‐29 silenced INS‐1 832/13 cells were assessed for insulin secretion (glucose, pyruvate, and K+), target cis‐eGene expression (Ndufv3 and Ndufa10 components of mitochondrial complex I (CI)), OXPHOS (CI‐V) protein expression, and mitochondrial OXPHOS respiration/activity. The expression of differentially expressed miR‐29 miRNAs was evaluated in Goto‐Kakizaki (GK) rat, db/db mouse and type 2 diabetic (T2D) human islets, as well as NMRI mouse islets cultured under glucolipotoxic conditions. Results: MiR‐29, miR‐15 and miR‐124 were predicted to regulate ~20 cis‐eGenes, while miR‐29 alone was predicted to regulate ≥12 of these in rat and human species. MiR‐29 expression and insulin secretion were reduced in INS‐1 832/13 cells after 24 h in elevated glucose. MiR‐29 knockdown increased all tested insulin secretory responses, Nudfv3, Ndufa10, complex I and II expression, and cellular mitochondrial OXPHOS. MiR‐29 expression was reduced in db/db islets but increased in GK rat and T2D human islets. Conclusion: We conclude miR‐29 is a key miRNA in regulating β‐cell mitochondrial metabolism and insulin secretion via underlying miR‐29‐OXPHOS complex pathways. Furthermore, we infer reduced miR‐29 expression compensatorily enhances insulin secretion under glucotoxicity. [ABSTRACT FROM AUTHOR]

Five host-defense peptides (figainin 2PL, hylin PL, raniseptin PL, plasticin PL, and peptide YL) were isolated from norepinephrine-stimulated skin secretions of the banana tree dwelling frog Boana platanera (Hylidae; Hylinae) collected in Trinidad. Raniseptin PL (GVFDTVKKIGKAVGKFALGVAKNYLNS.NH 2) and figainin 2PL (FLGTVLKLGKAIAKTVVPMLTNAMQPKQ. NH 2) showed potent and rapid bactericidal activity against a range of clinically relevant Gram-positive and Gram-negative ESKAPE + pathogens and Clostridioides difficile. The peptides also showed potent cytotoxic activity (LC 50 values < 30 μM) against A549, MDA-MB-231 and HT29 human tumor-derived cell lines but appreciably lower hemolytic activity against mouse erythrocytes (LC 50 = 262 ± 14 μM for raniseptin PL and 157 ± 16 μM for figainin 2PL). Hylin PL (FLGLIPALAGAIGNLIK.NH 2) showed relatively weak activity against microorganisms but was more hemolytic. The glycine-leucine-rich peptide with structural similarity to the plasticins (GLLSTVGGLVGGLLNNLGL.NH 2) and the non-cytotoxic peptide YL (YVPGVIESLL.NH 2) lacked antimicrobial and cytotoxic activities. Hylin PL, raniseptinPL and peptide YL stimulated the rate of release of insulin from BRIN-BD11 clonal β-cells at concentrations ≥100 nM. Peptide YL was the most effective (2.3-fold increase compared with basal rate at 1 μM concentration) and may represent a template for the design of a new class of incretin-based anti-diabetic drugs. [Display omitted] • Figainin 2 PL and raniseptin PL show potent antimicrobial activity against ESKAPE pathogens. • Figainin 2 PL and raniseptin PL are weakly hemolytic. • Figainin 2 PL, raniseptin PL and hylin PL are cytotoxic to human tumor-derived cells. • Raniseptin PL, hylin PL, plasticin-PL and peptide YL stimulate insulin release from BRIN-BD11 cells. [ABSTRACT FROM AUTHOR]

Enterohepatic circulation (EHC) is a complex process where drugs undergo secretion and reabsorption from the intestinal lumen multiple times, resulting in pharmacokinetic profiles with multiple peaks. The impact of EHC on area under the curve (AUC) has been a topic of extensive debate, questioning the suitability of conventional AUC estimation methods. Moreover, a universal model for accurately estimating AUC in EHC scenarios is lacking. To address this gap, we conducted a simulation study evaluating five empirical models under various sampling strategies to assess their performance in AUC estimation. Our results identify the most suitable model for EHC scenarios and underscore the critical role of meal-based sampling strategies in accurate AUC estimation. Additionally, we demonstrate that while the trapezoidal method performs comparably to other models with a large number of samples, alternative models are essential when sample numbers are limited. These findings not only illuminate how EHC influences AUC but also pave the way for the application of empirical models in real-world drug studies. [ABSTRACT FROM AUTHOR]

OBJECTIVE: We evaluated the effect of long-term intensive metabolic control with hybrid closed-loop (CL) on residual C-peptide secretion and glucose control compared with standard insulin therapy in youth with type 1 diabetes over 48 months. RESEARCH DESIGN AND METHODS: Following the 24-month primary phase of a multicenter, randomized, parallel trial of 96 newly diagnosed youth aged 10 to 16.9 years, participants were invited to an extension phase using treatment allocated at randomization. They continued with hybrid CL using the Cambridge algorithm or standard insulin therapy (control) until 48 months after diagnosis. Analysis was by intention-to-treat. RESULTS: At 24 months after diagnosis, 81 participants (mean ± SD age 14 ± 2 years) continued in the extension phase (47 CL, 34 control). There was no difference in fasting C-peptide corrected for fasting glucose at 48 months between groups (CL: 5 ± 9 vs. control: 6 ± 14 pmol/L per mmol/L; mean adjusted difference −2 [95% CI −7, 4; P = 0.54]). Central laboratory HbA1c remained lower in the CL group by 0.9% (10 mmol/mol [95% CI 0.2, 1.5; 3, 17 mmol/mol); P = 0.009). Time in target range of 3.9 to 10.0 mmol/L was 12 percentage points (95% CI 3, 20; P = 0.008) higher in the CL group compared with control. There were 11 severe hypoglycemic events (6 CL, 5 control) and 7 diabetic ketoacidosis events (3 CL, 4 control) during the extension phase. CONCLUSIONS: Improved glycemic control was sustained over 48 months after diagnosis with CL insulin delivery compared with standard therapy in youth with type 1 diabetes. This did not appear to confer a protective effect on residual C-peptide secretion. [ABSTRACT FROM AUTHOR]

Cell secretion repairs tissue damage and restores homeostasis throughout adult life. The extracellular heat shock protein-90alpha (eHsp90α) has been reported as an exosome cargo and a potential driver of wound healing. However, neither the mechanism of secretion nor the genetic evidence for eHsp90α in wound healing has been substantiated. Herein, we show that tissue injury causes massive deposition of eHsp90α in tissues and secretion of eHsp90α by cells. Sequential centrifugations of conditioned medium from relevant cell lines revealed the relative distributions of eHsp90α in microvesicle, exosome and trypsin-sensitive supernatant fractions to be approximately <2%, <4% and >95%, respectively. Establishing the cell-number-to-interstitial-fluid-volume (CIF) ratio for the microenvironment of human tissues as 1 × 109 cells: 1 mL interstitial fluid enabled us to predict the corresponding tissue concentrations of eHsp90α in these fractions as 3.74 μg/mL, 5.61 μg/mL and 178 μg/mL. Remarkably, the 178 μg/mL eHsp90α matches the previously reported 100–300 μg/mL of recombinant eHsp90α whose topical application promotes maximum wound healing in animal models. More importantly, we demonstrate that two parallel secretory autophagy-regulating gene families, the autophagy-regulating (AR) genes and the Golgi reassembly-stacking protein (GRASP) genes work together to mediate the secretion of the physiological concentration of eHsp90α to promote wound healing. Thus, utilization of the CIF ratio-based extrapolation method may enable investigators to rapidly predict biomarker targets from cell-conditioned-medium data. [ABSTRACT FROM AUTHOR]

Odorant receptors (ORs) and taste receptors (TRs) are expressed primarily in the nose and tongue in which they transduce electrical signals to the brain. Advances in deciphering the dietary component-sensing mechanisms in the nose and tongue prompted research on the role of gut chemosensory cells. Acting as the pivotal interface between the body and dietary cues, gut cells "smell" and "taste" dietary components and metabolites by taking advantage of chemoreceptors—ORs and TRs, to maintain physiological homeostasis. Here, we reviewed this novel field, highlighting the latest discoveries pertinent to gut ORs and TRs responding to dietary components, their impacts on gut hormone secretion, and the mechanisms involved. Recent studies indicate that gut cells sense dietary components including fatty acid, carbohydrate, and phytochemical by activating relevant ORs, thereby modulating GLP-1, PYY, CCK, and 5-HT secretion. Similarly, gut sweet, umami, and bitter receptors can regulate the gut hormone secretion and maintain homeostasis in response to dietary components. A deeper understanding of the favorable influence of dietary components on gut hormone secretion via gut ORs and TRs, coupled with the facts that gut hormones are involved in diverse physiological or pathophysiological phenomena, may ultimately lead to a promising treatment for various human diseases. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are the most widely used for therapeutic antibody production. In cell line development, engineering secretion processes such as folding-related protein upregulation is an effective way of constructing cell lines with high recombinant protein productivity. However, there have been few studies on the transport of recombinant proteins between the endoplasmic reticulum (ER) and the Golgi apparatus. In this study, Sar1A, a protein involved in COPII vesicle formation, was focused on to improve antibody productivity by enhancing COPII vesicle-mediated antibody transport from the ER to the Golgi apparatus, and to clarify its effect on the secretion process. The constructed Sar1A-overexpressing CHO cell lines were batch-cultured, in which they showed an increased specific antibody production rate. The intracellular antibody accumulation and the specific localization of the intracellular antibodies were investigated by chase assay using a translation inhibitor and observed by immunofluorescence-based imaging analysis. The results showed that Sar1A overexpression reduced intracellular antibody accumulation, especially in the ER. The effects of the engineered antibody transport on the antibody's glycosylation profile and the unfolded protein response (UPR) pathway were analyzed by liquid chromatography-mass spectrometry and UPR-related gene expression evaluation, respectively. Sar1A overexpression lowered glycan galactosylation and induced a stronger UPR at the end of the batch culture. Sar1A overexpression enhanced the antibody productivity of CHO cells by modifying their secretion process. This approach could also contribute to the production of not only monoclonal antibodies but also other therapeutic proteins that require transport by COPII vesicles. [ABSTRACT FROM AUTHOR]

The majority of incidentally discovered adrenal tumours are benign adrenocortical adenomas and the prevalence of adrenocortical adenomas is around 1–7% on cross-sectional abdominal imaging. These can be non-functioning adrenal tumours or they can be associated with autonomous cortisol secretion on a spectrum that ranges from rare clinically overt adrenal Cushing syndrome to the much more prevalent mild autonomous cortisol secretion (MACS) without signs of Cushing syndrome. MACS is diagnosed (based on an abnormal overnight dexamethasone suppression test) in 20–50% of patients with adrenal adenomas. MACS is associated with cardiovascular morbidity, frailty, fragility fractures, decreased quality of life and increased mortality. Management of MACS should be individualized based on patient characteristics and includes adrenalectomy or conservative follow-up with treatment of associated comorbidities. Identifying patients with MACS who are most likely to benefit from adrenalectomy is challenging, as adrenalectomy results in improvement of cardiovascular morbidity in some, but not all, patients with MACS. Of note, diagnosis and management of patients with bilateral MACS is especially challenging. Current gaps in MACS clinical practice include a lack of specific biomarkers diagnostic of MACS-related health outcomes and a paucity of clinical trials demonstrating the efficacy of adrenalectomy on comorbidities associated with MACS. In addition, little evidence exists to demonstrate the efficacy and safety of long-term medical therapy in patients with MACS. Mild autonomous cortisol secretion from benign adrenocortical adenomas (usually diagnosed incidentally) is associated with cardiometabolic risk and other comorbidities, but without the signs of overt Cushing syndrome. This Review outlines the mechanisms, complications and comorbidities, diagnosis and management of mild autonomous cortisol secretion. Key points: Mild autonomous cortisol secretion (MACS) is diagnosed based on the 1 mg overnight dexamethasone test and is found in 20–50% of patients with adrenal adenomas lacking signs and symptoms of Cushing syndrome. Patients with adrenal adenomas show distinct changes in the steroid and global metabolome, which correlate with the degree of cortisol excess across MACS and Cushing syndrome. MACS is associated with an increased likelihood of having cardiovascular risk factors and an increased risk of mortality. Management of MACS must be individualized based on patient characteristics; the options range from adrenalectomy to long-term follow-up and conservative management of comorbidities. Post-operative adrenal insufficiency is seen in around 50% of patients with MACS who undergo unilateral adrenalectomy. [ABSTRACT FROM AUTHOR]

BACKGROUND: Endotracheal suctioning causes discomfort, is associated with adverse effects, and is resource-demanding. An artificial secretion removal method, known as an automated cough, has been developed, which applies rapid, automated deflation, and inflation of the endotracheal tube cuff during the inspiratory phase of mechanical ventilation. This method has been evaluated in the hands of researchers but not when used by attending nurses. The aim of this study was to explore the efficacy of the method over the course of patient management as part of routine care. METHODS: This prospective, longitudinal, interventional study recruited 28 subjects who were intubated and mechanically ventilated. For a maximum of 7 d and on clinical need for endotracheal suctioning, the automatic cough procedure was applied. The subjects were placed in a pressure-regulated ventilation mode with elevated inspiratory pressure, and automated cuff deflation and inflation were performed 3 times, with this repeated if deemed necessary. Success was determined by resolution of the clinical need for suctioning as determined by the attending nurse. Adverse effects were recorded. RESULTS: A total of 84 procedures were performed. In 54% of the subjects, the artificial cough procedure was successful on > 70% of occasions, with 56% of all procedures considered successful. Ninety percent of all the procedures were performed in subjects who were spontaneously breathing and on pressure-support ventilation with peak inspiratory pressures of 20 cm H2O. Rates of adverse events were similar to those seen in the application of endotracheal suctioning. CONCLUSIONS: This study solely evaluated the efficacy of an automated artificial cough procedure, which illustrated the potential for reducing the need for endotracheal suctioning when applied by attending nurses in routine care. [ABSTRACT FROM AUTHOR]

Dietary carbohydrates raise blood glucose levels, and limiting carbohydrate intake improves glycemia in patients with type 2 diabetes. Low carbohydrate intake (< 25 g) allows the body to utilize fat as its primary fuel. As a consequence of increased fatty acid oxidation, the liver produces ketones to serve as an alternative energy source. β-Hydroxybutyrate (βHB) is the most abundant ketone. While βHB has a wide range of functions outside of the pancreas, its direct effects on islet cell function remain understudied. We examined human islet secretory response to acute racemic βHB treatment and observed increased insulin secretion at a low glucose concentration of 3 mM. Because βHB is a chiral molecule, existing as both R and S forms, we further studied insulin and glucagon secretion following acute treatment with individual βHB enantiomers in human and C57BL/6J mouse islets. We found that acute treatment with R-βHB increased insulin secretion and decreased glucagon secretion at physiological glucose concentrations in both human and mouse islets. Proteomic analysis of human islets treated with R-βHB over 72 hours showed altered abundance of proteins that may promote islet cell health and survival. Collectively, our data show that physiological concentrations of βHB influence hormone secretion and signaling within pancreatic islets. [ABSTRACT FROM AUTHOR]

Background Nephrin is a transmembrane protein with well-established signaling roles in kidney podocytes, and a smaller set of secretory functions in pancreatic β cells are implicated in diabetes. Nephrin signaling is mediated in part through its 3 cytoplasmic YDxV motifs, which can be tyrosine phosphorylated by high glucose and β cell injuries. Although in vitro studies demonstrate these phosphorylated motifs can regulate β cell vesicle trafficking and insulin release, in vivo evidence of their role in this cell type remains to be determined. Methods To further explore the role of nephrin YDxV phosphorylation in β cells, we used a mouse line with tyrosine to phenylalanine substitutions at each YDxV motif (nephrin-Y3F) to inhibit phosphorylation. We assessed islet function via primary islet glucose-stimulated insulin secretion assays and oral glucose tolerance tests. Results Nephrin-Y3F mice successfully developed pancreatic endocrine and exocrine tissues with minimal structural differences. Unexpectedly, male and female nephrin-Y3F mice showed elevated insulin secretion, with a stronger increase observed in male mice. At 8 months of age, no differences in glucose tolerance were observed between wild-type (WT) and nephrin-Y3F mice. However, aged nephrin-Y3F mice (16 months of age) demonstrated more rapid glucose clearance compared to WT controls. Conclusion Taken together, loss of nephrin YDxV phosphorylation does not alter baseline islet function. Instead, our data suggest a mechanism linking impaired nephrin YDxV phosphorylation to improved islet secretory ability with age. Targeting nephrin phosphorylation could provide novel therapeutic opportunities to improve β cell function. [ABSTRACT FROM AUTHOR]

CD11c, FcRL5, or T‐bet are commonly expressed by B cells expanding during inflammation, where they can make up >30% of mature B cells. However, the association between the proteins and differentiation and function in the host response remains largely unclear. We have assessed the co‐expression of CD11c, T‐bet, and FcRL5 in an in vitro B‐cell culture system to determine how stimulation via the BCR, toll‐like receptor 9 (TLR9), and different cytokines influence CD11c, T‐bet, and FcRL5 expression. We observed different expression dynamics for all markers, but a largely overlapping regulation of CD11c and FcRL5 in response to BCR and TLR9 activation, while T‐bet was strongly dependent on IFN‐γ signaling. Investigating plasma cell differentiation and APC functions, there was no association between marker expression and antibody secretion or T‐cell help. Rather the functions were associated with TLR9‐signalling and B‐cell‐derived IL‐6 production, respectively. These results suggest that the expression of CD11c, FcRL5, and T‐bet and plasma cell differentiation and improved APC functions occur in parallel and are regulated by similar activation signals, but they are not interdependent. [ABSTRACT FROM AUTHOR]

Background: Personalized medicine, an emerging approach, considers individual differences to tailor treatments optimally. Persian medicine (PM) incorporates individual differences through Mizāj (temperament). A comprehensive understanding of each Mizaj type's characteristics is essential in designing standardized diagnostic tools. Objectives: This study aims to explore the attributes of one of the most significant Mizaj groups, namely cold-wet Mizaj, and its subtypes, including cold-wet Su-e-Mizaj (dystemperament), as well as phlegm dominance in PM sources. Methods: Persian medicine literature, including the Canon of Medicine, was reviewed for relevant concepts. Reliable scientific databases, including PubMed, Scopus, and MagIran, were searched using keywords such as Mizaj, temperament, Su-e-Mizaj, dystemperament, cold-wet dystemperament, phlegm, and Balgham. The obtained results were studied and categorized. Results: Out of the 196 articles obtained related to Mizaj, 16 investigated indices. No studies were found on the indices of coldwet Mizaj and phlegm dominance separately. PM sources exhibit high congruence in describing the characteristics of cold-wet Mizaj. Specifically, emphasis is placed on characteristics such as coldness and softness of the skin to the touch, high fat mass, pale and white complexion, excessive body secretions, weakness and slowness of physical and physiological actions, low vitality, timidity, poor memory, and slow-wittedness. In cold-wet dystemperament, symptoms are more severe, and in the presence of phlegm, a feeling of heaviness is also mentioned as a specific symptom. Conclusions: By compiling indices of cold-wet Mizaj/dystemperament and phlegm dominance, this study can contribute to the development of standardized PM questionnaires, facilitating a personalized approach to the diagnosis and treatment of diseases. [ABSTRACT FROM AUTHOR]

Most animals have large amounts of the special substance melatonin, which is controlled by the light/dark cycle in the suprachiasmatic nucleus. According to what is now understood, the gastrointestinal tract (GIT) and other areas of the body are sites of melatonin production. According to recent studies, the GIT and adjacent organs depend critically on a massive amount of melatonin. Not unexpectedly, melatonin's many biological properties, such as its antioxidant, anti-inflammatory, pro-apoptotic, anti-proliferative, anti-metastasis, and antiangiogenic properties, have drawn the attention of researchers more and more. Because melatonin is an antioxidant, it produces a lot of secretions in the GIT's mucus and saliva, which shields cells from damage and promotes the development of certain GIT-related disorders. Melatonin's ability to alter cellular behavior in the GIT and other associated organs, such as the liver and pancreas, is another way that it functions. This behavior alters the secretory and metabolic activities of these cells. In this review, we attempted to shed fresh light on the many roles that melatonin plays in the various regions of the gastrointestinal tract by focusing on its activities for the first time. [ABSTRACT FROM AUTHOR]

Background: Cellular retinoic acid binding protein 1 (CRABP1) mediates rapid, non-canonical activity of retinoic acid (RA) by forming signalosomes via protein-protein interactions. Two signalosomes have been identified previously: CRABP1-MAPK and CRABP1-CaMKII. Crabp1 knockout (CKO) mice exhibited altered exosome profiles, but the mechanism of CRABP1 action was unclear. This study aimed to screen for and identify novel CRABP1 signalosomes that could modulate exosome secretion by using a combinatorial approach involving biochemical, bioinformatic and molecular studies. Methods: Immunoprecipitation coupled with mass spectrometry (IP-MS) identified candidate CRABP1-interacting proteins which were subsequently analyzed using GO Term Enrichment, Functional Annotation Clustering; and Pathway Analysis. Gene expression analysis of CKO samples revealed altered expression of genes related to exosome biogenesis and secretion. The effect of CRABP1 on exosome secretion was then experimentally validated using CKO mice and a Crabp1 knockdown P19 cell line. Results: IP-MS identified CRABP1-interacting targets. Bioinformatic analyses revealed significant association with actin cytoskeletal dynamics, kinases, and exosome secretion. The effect of CRABP1 on exosome secretion was experimentally validated by comparing circulating exosome numbers of CKO and wild type (WT) mice, and secreted exosomes from WT and siCRABP1-P19 cells. Pathway analysis identified kinase signaling and Arp2/3 complex as the major pathways where CRABP1-signalosomes modulate exosome secretion, which was validated in the P19 system. Conclusion: The combinatorial approach allowed efficient screening for and identification of novel CRABP1-signalosomes. The results uncovered a novel function of CRABP1 in modulating exosome secretion, and suggested that CRABP1 could play roles in modulating intercellular communication and signal propagation. [ABSTRACT FROM AUTHOR]

The colonic epithelium is comprised of three-dimensional crypts (3D) lined with mucus secreted by a heterogeneous population of goblet cells. In this study, we report the formation of a long-lived, and self-renewing replica of human 3D crypts with a mucus layer patterned in the X–Y–Z dimensions. Primary colon cells were cultured on a shaped scaffold under an air–liquid interface to yield architecturally accurate crypts with a mucus bilayer (605 ± 180 μm thick) possessing an inner (149 ± 50 μm) and outer (435 ± 111 μm) region. Lectins with distinct carbohydrate-binding preferences demonstrated that the mucus in the intercrypt regions was chemically distinct from that above and within the crypts replicating in vivo chemical patterning. Constitutive mucus secretion ejected beads from crypt lumens in 8–10 days, while agonist-stimulated secretion increased mucus thickness by 17-fold in 8 h. The tissue was long-lived, > 50 days, the longest time assessed. In conclusion, the in vitro mucus replicated key physiology of the human mucus, including the bilayer (Z) structure and intercrypt-crypt (X–Y) zones, constitutive mucus flow, spatially complex chemical attributes, and mucus secretion response to stimulation, with the potential to reveal local and global determinants of mucus function and its breakdown in disease. [ABSTRACT FROM AUTHOR]

Background: The immunogenicity of the antigen-recognition domains of chimeric antigen receptor (CAR)-T cells leads to immune responses that may compromise the antitumor effects of the adoptively transferred T cells. Herein, we attempt to humanize a CD19-specific VHH (named H85) using in silico techniques and investigate the impact of antigen-recognition domain humanization on CAR expression and density, cytokine secretion, and cytolytic reactivity of CAR-T cells based on the humanized VHH. Methods: H85 was humanized (named HuH85), and then HuH85 was compared with H85 in terms of conformational structure, physicochemical properties, antigenicity and immunogenicity, solubility, flexibility, stability, and CD19-binding capacity using in silico techniques. Next, H85CAR-T cells and HuH85CAR-T cells were developed and CAR expression and surface density were assessed via flow cytometry. Ultimately, the antitumor reactivity and secreted levels of IFN-γ, IL-2, and TNF-α were assessed following the co-cultivation of the CAR-T cells with Ramos, Namalwa, and K562 cells. Results: In silico findings demonstrated no negative impacts on HuH85 as a result of humanization. Ultimately, H85CAR and HuH85CAR could be surface-expressed on transduced T cells at comparable levels as assessed via mean fluorescence intensity. Moreover, H85CAR-T cells and HuH85CAR-T cells mediated comparable antitumor effects against Ramos and Namalwa cells and secreted comparable levels of IFN-γ, IL-2, and TNF-α following co-cultivation. Conclusion: HuH85 can be used to develop immunotherapeutics against CD19-associated hematologic malignancies. Moreover, HuH85CAR-T cells must be further investigated in vitro and in preclinical xenograft models of CD19+ leukemias and lymphomas before advancing into clinical trials. [ABSTRACT FROM AUTHOR]

Musk is an important animal product, but the musk secretion mechanism of forest musk deer (Moschus berezovskii) is still unclear. The musk synthesis process in forest musk deer is extremely complex, and many raw materials are directly or indirectly derived from forest musk deer blood. In this study, metabolomics was used to analyze the blood of forest musk deer in secretory and non-secretory phases for the first time, aim at explaining the secretion mechanism from the perspective of blood metabolism. We found that P450-related, choline-related, axonal regeneration and other pathways and related metabolites were significantly enriched during the musk secretion of forest musk deer. These pathways and metabolites related to P450 and choline in blood may have important implications for the mechanism of musk secretion in forest musk deer, because blood components were closely related to musk components and could provide raw materials for musk synthesis in musk gland cells. [ABSTRACT FROM AUTHOR]

Major constituents of the plant cell walls are structural proteins that belong to the hydroxyproline-rich glycoprotein (HRGP) family. Leucine-rich repeat extensin (LRX) proteins contain a leucine-rich domain and a C-terminal domain with repetitive Ser–Pro3–5 motifs that are potentially to be O -glycosylated. It has been demonstrated that pollen-specific LRX8–LRX11 from Arabidopsis thaliana are necessary to maintain the integrity of the pollen tube cell wall during polarized growth. In HRGPs, including classical extensins (EXTs), and probably in LRXs, proline residues are converted to hydroxyproline by prolyl-4-hydroxylases (P4Hs), thus defining novel O -glycosylation sites. In this context, we aimed to determine whether hydroxylation and subsequent O -glycosylation of Arabidopsis pollen LRXs are necessary for their proper function and cell wall localization in pollen tubes. We hypothesized that pollen-expressed P4H4 and P4H6 catalyze the hydroxylation of the proline units present in Ser–Pro3–5 motifs of LRX8–LRX11. Here, we show that the p4h4-1 p4h6-1 double mutant exhibits a reduction in pollen germination rates and a slight reduction in pollen tube length. Pollen germination is also inhibited by P4H inhibitors, suggesting that prolyl hydroxylation is required for pollen tube development. Plants expressing pLRX11::LRX11-GFP in the p4h4-1 p4h6-1 background show partial re-localization of LRX11–green fluorescent protein (GFP) from the pollen tube tip apoplast to the cytoplasm. Finally, immunoprecipitation-tandem mass spectrometry analysis revealed a decrease in oxidized prolines (hydroxyprolines) in LRX11–GFP in the p4h4-1 p4h6-1 background compared with lrx11 plants expressing pLRX11::LRX11-GFP. Taken together, these results suggest that P4H4 and P4H6 are required for pollen germination and for proper hydroxylation of LRX11 necessary for its localization in the cell wall of pollen tubes. [ABSTRACT FROM AUTHOR]

The African claw-toed frog, Xenopus laevis, is a well-established laboratory model for the biology of vertebrate oogenesis, fertilisation, and development at embryonic, larval, and metamorphic stages. For ovulation, X. laevis females are usually injected with chorionic gonadotropin, whereupon they lay typically hundreds to thousands of eggs in a day. After being rested for a minimum of three months, animals are re-used. The literature suggests that adult females can lay much larger numbers of eggs in a short period. Here, we compared the standard "single ovulation" protocol with a "double ovulation" protocol, in which females were ovulated, then re-ovulated after seven days and then rested for three months before re-use. We quantified egg number, fertilisation rate (development to cleavage stage), and corticosterone secretion rate as a measure of stress response for the two protocol groups over seven 3-month cycles. We found no differences in egg number-per-ovulation or egg quality between the groups and no long-term changes in any measures over the 21-month trial period. Corticosterone secretion was elevated by ovulation, similarly for the single ovulation as for the first ovulation in the double-ovulation protocol, but more highly for the second ovulation (to a level comparable to that seen following shipment) in the latter. However, both groups exhibited the same baseline secretion rates by the time of the subsequent cycle. Double ovulation is thus transiently more stressful/demanding than single ovulation but within the levels routinely experienced by laboratory X. laevis. Noting that "stress hormone" corticosterone/cortisol secretion is linked to physiological processes, such as ovulation, that are not necessarily harmful to the individual, we suggest that the benefits of a doubling in egg yield-per-cycle per animal without loss of egg quality or signs of acute or long-term harm may outweigh the relatively modest and transient corticosterone elevation we observed. The double ovulation protocol therefore represents a potential new standard practice for promoting the "3Rs" (animal use reduction, refinement and replacement) mission for Xenopus research. [ABSTRACT FROM AUTHOR]

Cytokine polyfunctionality is a well-established concept in immune cells, especially T cells, and their ability to concurrently produce multiple cytokines has been associated with better immunological disease control and subsequent effectiveness during infection and disease. To date, only little is known about the secretion dynamics of those cells, masked by the widespread deployment of mainly time-integrated endpoint measurement techniques that do not easily differentiate between concurrent and sequential secretion. Here, we employed a single-cell microfluidic platform capable of resolving the secretion dynamics of individual PBMCs. To study the dynamics of poly-cytokine secretion, as well as the dynamics of concurrent and sequential polyfunctionality, we analyzed the response at different time points after ex vivo activation. First, we observed the simultaneous secretion of cytokines over the measurement time for most stimulants in a subpopulation of cells only. Second, polyfunctionality generally decreased with prolonged stimulation times and revealed no correlation with the concentration of secreted cytokines in response to stimulation. However, we observed a general trend towards higher cytokine secretion in polyfunctional cells, with their secretion dynamics being distinctly different from mono-cytokine-secreting cells. This study provided insights into the distinct secretion behavior of heterogenous cell populations after stimulation with well-described agents and such a system could provide a better understanding of various immune dynamics in therapy and disease. [ABSTRACT FROM AUTHOR]

Background: Exposure to antigens is crucial for child immune system development, aiding disease prevention and promoting infant health. Some common food antigen proteins are found in human breast milk. However, it is unclear whether gluten antigens linked to celiac disease (CD) are transmitted through breast milk, potentially impacting the development of the infant's immune system. Objective: This study aimed to analyze the passage of gluten immunogenic peptides (GIP) into human breast milk. We evaluated the dynamics of GIP secretion after lactating mothers adopted a controlled gluten-rich diet. Methods: We prospectively enrolled 96 non-CD and 23 CD lactating mothers, assessing total proteins and casein in breast milk, and GIP levels in breast milk and urine. Subsequently, a longitudinal study was conducted in a subgroup of 12 non-CD lactating mothers who adopted a controlled gluten-rich diet. GIP levels in breast milk and urine samples were assayed by multiple sample collections over 96 hours. Results: Analysis of a single sample revealed that 24% of non-CD lactating mothers on a regular unrestricted diet tested positive for GIP in breast milk, and 90% tested positive in urine, with significantly lower concentrations in breast milk. Nevertheless, on a controlled gluten-rich diet and the collection of multiple samples, GIP were detected in 75% and 100% of non-CD participants in breast milk and urine, respectively. The transfer dynamics in breast milk samples were long-enduring and GIP secretion persisted from 0 to 72 h. In contrast, GIP secretion in urine samples was limited to the first 24 h, with inter-individual variations. In the cohort of CD mothers, 82.6% and 87% tested negative for GIP in breast milk and urine, respectively. Conclusions: This study definitively established the presence of GIP in breast milk, with substantial inter-individual variations in secretion dynamics. Our findings provide insights into distinct GIP kinetics observed in sequentially collected breast milk and urine samples, suggesting differential gluten metabolism patterns depending on the organ or system involved. Future research is essential to understand whether GIP functions as sensitizing or tolerogenic agents in the immune system of breastfed infants. [ABSTRACT FROM AUTHOR]

Simple Summary: Plexiform neurofibromas in neurofibromatosis type 1 (pNF1), characterized by aggressive growth and local invasiveness, are comprised primarily of Schwann cell-derived tumor cells (Nf1−/−) and the tumor microenvironment (TME). Fibroblasts (Nf1+/−), the most abundant cell type in the TME, are known to significantly contribute to pNF1 formation, but their precise role in the subsequent tumor progression is poorly understood. Using our established three-dimensional (3D) culture models with patient-derived pNF1 cells and fibroblasts, we observed that the fibroblast-derived secretome stimulates the growth and local invasiveness of pNF1 spheroids. Depletion of small extracellular vesicles (sEVs) from the fibroblast secretome completely eliminated the paracrine effect on pNF1 spheroid growth. These results suggest that targeting paracrine interactions between pNF1 tumor cells and fibroblasts may offer a potential therapeutic approach for reducing pNF1 tumor progression. Plexiform neurofibromas (PNs) occur in about a half of neurofibromatosis type 1 (NF1) patients and have garnered significant research attention due to their capacity for growth and potential for malignant transformation. NF1 plexiform neurofibroma (pNF1) is a complex tumor composed of Schwann cell-derived tumor cells (Nf1−/−) and the tumor microenvironment (TME). Although it has been widely demonstrated that the TME is involved in the formation of neurofibromas, little is known about the effects of the TME on the subsequent progression of human pNF1. Elucidating the molecular interactions between tumor cells and the TME may provide new therapeutic targets to reduce the progression of pNF1. In the present study, we focused on the contributions of fibroblasts, the most abundant cell types in the TME, to the growth of pNF1. To simulate the TME, we used a three-dimensional (3D) coculture model of immortalized pNF1 tumor cells (Nf1−/−) and primary fibroblasts (Nf1+/−) derived from pNF1 patients. We performed live-cell imaging of 3D/4D (3D in real-time) cultures through confocal microscopy followed by 3D quantitative analyses using advanced imaging software. The growth of pNF1 spheroids in 3D cocultures with fibroblasts was significantly greater than that of pNF1 spheroids in 3D monocultures. An increase in the growth of pNF1 spheroids also occurred when they were cultured with conditioned media (CM) from fibroblasts. Moreover, fibroblast-derived CM increased the invasive outgrowth and further local invasion of pNF1 spheroids. Interestingly, when small extracellular vesicles (sEVs) were depleted from the fibroblast-derived CM, the stimulation of the growth of pNF1 spheroids was lost. Our results suggest that fibroblast-derived sEVs are a therapeutic target for reducing the growth of pNF1. [ABSTRACT FROM AUTHOR]

Currently, it is widely accepted that the type III secretion system (T3SS) serves as the transport platform for bacterial virulence factors, while flagella act as propulsion motors. However, there remains a noticeable dearth of comparative studies elucidating the functional disparities between these two mechanisms. Entomopathogenic nematode symbiotic bacteria (ENS), including Xenorhabdus and Photorhabdus, are Gram-negative bacteria transported into insect hosts by Steinernema or Heterorhabdus. Flagella are conserved in ENS, but the T3SS is only encoded in Photorhabdus. There are few reports on the function of flagella and the T3SS in ENS, and it is not known what role they play in the infection of ENS. Here, we clarified the function of the T3SS and flagella in ENS infection based on flagellar inactivation in X. stockiae (flhDC deletion), T3SS inactivation in P. luminescens (sctV deletion), and the heterologous synthesis of the T3SS of P. luminescens in X. stockiae. Consistent with the previous results, the swarming movement of the ENS and the formation of biofilms are dominated by the flagella. Both the T3SS and flagella facilitate ENS invasion and colonization within host cells, with minimal impact on secondary metabolite formation and secretion. Unexpectedly, a proteomic analysis reveals a negative feedback loop between the flagella/T3SS assembly and the type VI secretion system (T6SS). RT-PCR testing demonstrates the T3SS's inhibition of flagellar assembly, while flagellin expression promotes T3SS assembly. Furthermore, T3SS expression stimulates ribosome-associated protein expression. [ABSTRACT FROM AUTHOR]