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UNLABELLED: During pilus assembly within the Gram-positive bacterial envelope, membrane-bound sortase enzymes sequentially crosslink specific pilus protein monomers through their cell wall sorting signals (CWSS), starting with a designated tip pilin, followed by the shaft made of another pilin, ultimately anchoring the fiber base pilin to the cell wall. To date, the molecular determinants that govern pilus tip assembly and the underlying mechanism remain unknown. Here, we addressed this in the model organism Actinomyces oris. This oral microbe assembles a pathogenically important pilus (known as type 2 fimbria) whose shafts, made of FimA pilins, display one of two alternate tip pilins-FimB or the coaggregation factor CafA-that share a markedly similar CWSS. We demonstrate that swapping the CWSS of CafA with that of FimB produces a functional hybrid, which localizes at the pilus tip and mediates polymicrobial coaggregation, whereas alanine-substitution of the conserved FLIAG motif within the CWSS hampers these processes. Remarkably, swapping the CWSS of the normal cell wall-anchored glycoprotein GspA with that of CafA promotes the assembly of hybrid GspA at the FimA pilus tip. Finally, exchanging the CWSS of the Corynebacterium diphtheriae shaft pilin SpaA with that of CafA leads to the FLIAG motif-dependent localization of the heterologous pilus protein SpaA at the FimA pilus tip in A. oris. Evidently, the CWSS and the FLIAG motif of CafA are both necessary and sufficient for its destination to the cognate pilus tip specifically assembled by a designated sortase in the organism. IMPORTANCE: Gram-positive pili, whose precursors harbor a cell wall sorting signal (CWSS) needed for sortase-mediated pilus assembly, typically comprise a pilus shaft and a tip adhesin. How a pilin becomes a pilus tip, nevertheless, remains undetermined. We demonstrate here in Actinomyces oris that the CWSS of the tip pilin CafA is necessary and sufficient to promote pilus tip assembly, and this functional assembly involves a conserved FLIAG motif within the CWSS. This is evidenced by the fact that an A. oris cell-wall anchored glycoprotein, GspA, or a heterologous shaft pilin from Corynebacterium diphtheriae, SpaA, engineered to have the CWSS of CafA in place of their CWSS, localizes at the pilus tip in a process that requires the FLIAG motif. Our findings provide the molecular basis for sortase-catalyzed pilus tip assembly that is very likely employed by other Gram-positive bacteria and potential bioengineering applications to display antigens at controlled surface distance.

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.

ATG5 plays a pivotal role in membrane Atg8ylation, influencing downstream processes encompassing canonical autophagy and noncanonical processes. Remarkably, genetic ablation of ATG5 in myeloid cells leads to an exacerbated pathological state in murine models of tuberculosis, characterized by an early surge in mortality much more severe when compared to the depletion of other components involved in Atg8ylation or canonical autophagy. This study shows that in the absence of ATG5, but not other core canonical autophagy factors, endolysosomal organelles display a lysosomal hypersensitivity phenotype when subjected to damage. This is in part due to a compromised recruitment of ESCRT proteins to lysosomes in need of repair. Mechanistically, in the absence of ATG5, the ESCRT protein PDCD6IP/ALIX is sequestered by the alternative conjugate ATG12-ATG3, contributing to excessive exocytic processes while not being available for lysosomal repair. Specifically, this condition increases secretion of extracellular vesicles and particles, and leads to excessive degranulation in neutrophils. Our findings uncover unique functions of ATG5 outside of the autophagy and Atg8ylation paradigm. This finding is of in vivo relevance for tuberculosis pathogenesis as modeled in mice.Abbreviations: Atg5: autophagy related 5; ESCRT: endosomal sorting complex required for transport; EVPs: extracellular vesicles and particles; FPR1: formyl peptide receptor 1; LyHYP: lysosomal hypersensitivity phenotype; LysoIP: lysosome immunopurification; Mtb: Mycobacterium tuberculosis; ORF3a: open reading frame 3a protein; PDCD6IP/ALIX: programmed cell death 6 interacting protein; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2, TFEB: transcription factor EB.

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.

Many species use chemical messengers to communicate a remarkable range of information. Mice appear to make particular use of chemical messengers, including effects on estrous cycling and initiation, pregnancy, aggression, stress and of course attraction. Behavioral studies have helped identify several candidate messengers, or pheromones, that mediate attraction in mice. One question is whether attractive chemical messengers induced a physical vaginal secretory response. The preparation hypothesis posits that increased vaginal secretion would lubricate and protect the vagina in response to the prospect of imminent coitus, but this has been difficult to assess experimentally, particularly in mice. We developed a rapid, sensitive, minimally invasive method of quantifying vaginal moisture in mice and used this model to test vaginal secretory responses to male bedding. We report that female mice experience an increase in vaginal moisture after exposure to male, but not female, bedding. This response is induced by either physical or airborne exposure to male urine, to preputial gland extract, and to the preputial gland-derived pheromone alpha/beta farnesenes. This vaginal response is diurnally regulated, seen only during their active phase. The response is sensitive to the estrous phase, with a clear response during estrus but not during metestrus. We conclude that mice may serve as a model for aspects of vaginal function and that this assay will be readily applicable to other small animals. The identification of a pheromone-mediated vaginal secretory response offers a window into the regulation of the vaginal environment and the neurobiology of sexual responses in mice. [ABSTRACT FROM AUTHOR]

Background: Since 1982, recombinant insulin has been used as a substitute for pancreatic insulin from animals. However, increasing demand in medical and food industries warrants the development of more efficient production methods. In this study, we aimed to develop a novel and efficient method for insulin production using a yeast secretion system. Methods: Here, insulin C-peptide was replaced with a hydrophilic fusion partner (HL18) containing an affinity tag for the hypersecretion and easy purification of proinsulin. The HL18 fusion partner was then removed by in vitro processing with the Kex2 endoprotease (Kex2p), and authentic insulin was recovered via affinity chromatography. To improve the insulin functions, molecular chaperones of the host strain were reinforced via the constitutive expression of HAC1. Results: The developed method was successfully applied for the expression of cow, pig, and chicken insulins in yeast. Moreover, biological activity of recombinant insulins was confirmed by growth stimulation of cell line. Conclusions: Therefore, replacement of the C-peptide of insulin with the HL18 fusion partner and use of Kex2p for in vitro processing of proinsulin guarantees the economic production of animal insulins in yeast. [ABSTRACT FROM AUTHOR]

Insulin treatment should be introduced in patients with slowly progressive type 1 diabetes (SPIDDM; definite), according to the revised diagnostic criteria of SPIDDM (2023). In contrast, SPIDDM (probable) patients are in a non‐insulin‐dependent state; therefore, a more flexible treatment can be considered, although sulfonylurea agents should be avoided. Insulin treatment has been shown to maintain endogenous insulin secretion capacity in SPIDDM (probable); however, this does not mean that all SPIDDM (probable) patients should use insulin from the early phase. Dipeptidyl peptidase‐4 inhibitors and biguanides might be the treatment of choice for SPIDDM (probable), but no evidence exists for other hypoglycemic agents. In any case, careful monitoring of the endogenous insulin secretion capacity should be carried out, and if a decrease in insulin secretion capacity is suspected, a change in treatment should be considered to prevent progression to an insulin‐dependent state. [ABSTRACT FROM AUTHOR]

Extraoral bitter taste receptors offer intriguing potential for modulating metabolism and the gut-brain axis through dietary interventions. Our understanding of these receptors is limited, and data on their effects on ageing are scarce. The complexity conveyed by their high diversity, low expression levels and species-dependent variability challenges our comprehension. We used real-time PCR to examine the relative abundance of multiple TAS2R across different segments of gastrointestinal mucosa in four human cohorts and related them to enteroendocrine secretions at the colon site. TAS2R14 exhibited the highest expression levels in all analyzed tissues. In contrast, TAS2R39, -38 and -42 consistently exhibited lower expression levels. Ageing was found to upregulate TAS2R4, -5, -13, -20 and GLP-1 mRNA in the descending colon. Stimulating TAS2R14 in Hutu-80 cells induced GLP-1 secretion, while stimulating TAS2R5 modulated GLP-1 and PYY secretion. Given the modifications TAS2R agonists may undergo along the GIT, as well as the distinctive expression patterns and possible functional roles of TAS2R receptors along the intestinal tract, our findings suggest the viability of a targeted strategy aimed at enhancing specific functions to improve health outcomes. This study offers valuable insights into the intricate interplay between bitter taste receptors, gut physiology and potential dietary interventions. [ABSTRACT FROM AUTHOR]

Enhancing the secretion of recombinant proteins, particularly non-enzymatic proteins that predominate in food and pharmaceutic protein products, remains a significant challenge due to limitations in high-throughput screening methods. This study addresses this bottleneck by establishing a yeast surface display system in the food-grade microorganism Kluyveromyces lactis, enabling efficient display of model target proteins on the yeast cell surface. To assess its potential as a universal high-throughput screening tool for enhanced non-enzymatic protein secretion, we evaluated the consistency between protein display levels and secretion efficiency under the influence of various genetic factors. Our results revealed a strong correlation between these two properties. Furthermore, screening in a random mutagenesis library successfully identified a mutant with improved secretion. These findings demonstrate the potential of the K. lactis surface display system as a powerful and universal tool for high-throughput screening of strains with superior non-enzymatic protein secretion capacity. We believe this study could pave the way for efficient large-scale production of heterologous food and therapeutic proteins in industries. Key points: • A YSD (yeast surface display) system was established in Kluyveromyces lactis • This system enables high-throughput screening of non-enzymatic protein secretion • This technology assists industrial production of food and therapeutic proteins [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]

The tree frog is a prominent amphibian among terrestrial vertebrates known for its ability to adhere to various surfaces through the capillary forces of water in the microchannels between micropillars on its disc‐shaped toe pads, a phenomenon known as wet adhesion. However, the secretion pattern of mucus on the attachment surface of living tree frog toe pads and the distribution of active mucus pores (AMPs) have not yet been fully elucidated. In this study, we utilized synchrotron X‐ray micro‐computed tomography and interference reflection microscopy to obtain the spatial distribution of the entire population of ventral mucus glands on the toe pads of living tree frogs and the real‐time mucus secretion patterns from the ventral mucus pores on the contact surface under different environmental conditions. We observed that the number and secretion frequency of AMPs on the toe pad are regulated according to environmental conditions. Such dynamic mucus secretion on the tree frog's toe pad could contribute to the understanding of capillary force regulation for wet adhesion and the development of adhesive surfaces by mimicking the mucus‐secreting toe pad. [ABSTRACT FROM AUTHOR]

α-Galactosidase (α-GAL) is a class of hydrolase that releases galactose from galacto-oligosaccharides and synthetic substrates such as pNPG. In this study, the production of α-GAL by Actinoplanes utahensis B1 in submerged fermentation was enhanced by using statistical methods. The effects of temperature, pH, and inoculum percentage on enzyme secretion were optimized using BBD of RSM. The optimized process was scaled up from the shake flask to the laboratory scale (5 L) and to pilot scale (30 L) using KLa based scale-up strategy. By using BBD, a maximum yield of 62.5 U/mL was obtained at a temperature of 28 °C, a pH of 6.9, and an inoculum of 6.4%. Scale-up was performed successfully and achieved a yield of 74.4 U/mL and 76.8 U/mL in laboratory scale and pilot scale fermenters. The TOST was performed to validate the scale-up strategy and the results showed a confidence level of 95% for both scales indicating the perfect execution of scale-up procedure. Through the implementation of BBD and scale-up strategy, the overall enzyme yield has been significantly increased to 76%. This is the first article to explore the scale-up of α-GAL from the A. utahensis B1 strain and provide valuable insights for industrial applications. [ABSTRACT FROM AUTHOR]

Secreted protein Reelin is implicated in neuropsychiatric disorders and its supplementation ameliorates neurological symptoms in mouse disease models. Recombinant human Reelin protein may be useful for the treatment of human diseases, but its properties remain uncharacterized. Here, we report that full-length human Reelin was well secreted from transfected cells and was able to induce Dab1 phosphorylation. Unexpectedly, the central fragment of human Reelin was much less secreted than that of mouse Reelin. Three residues in the sixth Reelin repeat contributed to the secretion inefficiency, and their substitutions with mouse residues increased the secretion without affecting its biological activity. Our findings help efficient production of human Reelin protein for the supplementation therapy. [ABSTRACT FROM AUTHOR]

In assisted fertility protocols, in vitro culture conditions mimic physiological conditions to preserve gametes in the best conditions. After collection, oocytes are maintained in a culture medium inside the incubator until in vitro fertilization (IVF) is performed. This time outside natural and physiological conditions exposes oocytes to an oxidative stress that renders in vitro aging. It has been described that in vitro aging produces a spontaneous cortical granule (CG) release decreasing the fertilization rate of oocytes. Nevertheless, this undesirable phenomenon has not been investigated, let alone prevented. In this work, we characterized the spontaneous CG secretion in in vitro aged oocytes. Using immunofluorescence indirect, quantification, and functional assays, we showed that the expression of regulatory proteins of CG exocytosis was affected. Our results demonstrated that in vitro oocyte aging by 4 and 8 h altered the expression and localization of alpha-SNAP and reduced the expression of NSF and Complexin. These alterations were prevented by supplementing culture medium with dithiothreitol (DTT), which in addition to having a protective effect on those proteins, also had an unexpected effect on the actin cytoskeleton. Indeed, DTT addition thickened the cortical layer of fibrillar actin. Both DTT effects, together, prevented the spontaneous secretion of CG and recovered the IVF rate in in vitro aged oocytes. We propose the use of DTT in culture media to avoid the spontaneous CG secretion and to improve the success rate of IVF protocols in in vitro aged oocytes. • DTT restores the normal cortical granules density in in vitro aged oocytes • DTT has a protective effect on alpha-SNAP, NSF, and Complexin • In vitro aged oocytes respond to SrCl 2 as control oocytes in presence of DTT • DTT increases the polymerization of F-actin in the cortical region of mouse oocytes • DTT recovers the in vitro fertilization rate in in vitro aged oocytes. [ABSTRACT FROM AUTHOR]

SNAP25 plays an essential role in the glucose‐stimulated insulin secretion (GSIS) of pancreatic β‐cells. Carbohydrate response element‐binding protein (ChREBP) is an important transcription factor in β‐cells and, in this study, we aimed to explore whether ChREBP regulates SNAP25 expression in β‐cells. We show that diabetic Goto‐Kakizaki (GK) rats exhibited impaired insulin secretion and hyperglycemia, along with decreased SNAP25 expression and ChREBP phosphorylation in islets. SNAP25 knockdown decreased GSIS in β‐cells, while SNAP25 overexpression increased GSIS in β‐cells. Activation or overexpression of ChREBP led to reduced SNAP25 expression and subsequent suppression of GSIS. Conversely, ChREBP knockdown mitigated the reduction in SNAP25 expression caused by high glucose. Mechanistically, the activation of ChREBP by high glucose increased its occupancy and decreased the level of H3K4me3 at the Snap25 promoter. Our findings reveal the novel regulatory mechanisms of SNAP25 expression in β‐cells and suggest that SNAP25 may be involved in the regulation of β‐cell secretory function controlled by ChREBP. [ABSTRACT FROM AUTHOR]

Hepatic proteomes are intricately controlled through biosynthesis, extracellular secretion, and intrahepatic degradation. Autophagy governs lysosome‐mediated intrahepatic degradation and the hepatic proteome. When autophagy is impaired, it leads to the accumulation of intrahepatic proteins, causing proteinopathy. This study investigates whether autophagy can modulate the hepatic proteome non‐degradatively. Utilizing conditional, inducible, and hepatotoxin models of hepatic autophagy impairment, we assessed the overall hepatic proteome expression using Coomassie brilliant blue (CBB) staining and liquid chromatography–tandem mass spectrometry (LC/MS). We pinpointed and confirmed four specific hepatic proteins—Cps1, Ahcy, Ca3, and Gstm1—that were selectively modified in autophagy‐deficient livers. Expression of Cps1, Ahcy, and Ca3 were significantly reduced, while Gstm1 expression increased in livers with autophagy impairment. Interestingly, these changes in hepatic protein levels were not due to defective autophagic degradation but were associated with alterations in mRNA transcript levels. Moreover, as a result of autophagic dysfunction, sustained activation of the nuclear erythroid‐derived 2‐like 2 (Nrf2) transcription factor, transcriptionally regulated the mRNA levels of these proteins. Our findings indicate that autophagy can influence hepatic proteins not solely via traditional degradative routes but also through non‐degradative transcriptional processes by modulating Nrf2. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) serve as crucial mediators of cell-to-cell communication in normal physiology as well as in diseased states; they have been largely studied in regard to their role in cancer progression. However, the mechanisms by which their biogenesis and secretion are regulated by metabolic or endocrine factors remain unknown. Here, we delineate a mechanism by which EV secretion is regulated by a cholesterol metabolite, 27-hydroxycholesterol (27HC), where treatment of myeloid immune cells (RAW 264.7 and J774A.1) with 27HC impairs lysosomal homeostasis, leading to shunting of multivesicular bodies (MVBs) away from lysosomal degradation, toward secretion as EVs. This altered lysosomal function is likely caused by mitochondrial dysfunction and subsequent increase in reactive oxygen species (ROS). Interestingly, cotreatment with a mitochondria-targeted antioxidant rescued the lysosomal impairment and attenuated the 27HC-mediated increase in EV secretion. Overall, our findings establish how a cholesterol metabolite regulates EV secretion and paves the way for the development of strategies to regulate cancer progression by controlling EV secretion. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 initiates infection in the conducting airways, where mucociliary clearance inhibits pathogen penetration. However, it is unclear how mucociliary clearance impacts SARS-CoV-2 spread after infection is established. To investigate viral spread at this site, we perform live imaging of SARS-CoV-2 infected differentiated primary human bronchial epithelium cultures for up to 12 days. Using a fluorescent reporter virus and markers for cilia and mucus, we longitudinally monitor mucus motion, ciliary motion, and infection. Infected cell numbers peak at 4 days post infection, forming characteristic foci that tracked mucus movement. Inhibition of MCC using physical and genetic perturbations limits foci. Later in infection, mucociliary clearance deteriorates. Increased mucus secretion accompanies ciliary motion defects, but mucociliary clearance and vectorial infection spread resume after mucus removal, suggesting that mucus secretion may mediate MCC deterioration. Our work shows that while MCC can facilitate SARS-CoV-2 spread after initial infection, subsequent MCC decreases inhibit spread, revealing a complex interplay between SARS-CoV-2 and MCC. Becker et al. use live imaging of SARS-CoV-2 infected airway epithelium cultures to demonstrate a dual role for mucociliary clearance in viral spread. Initially, virus goes with the mucus flow, facilitating local spread; later, increased mucus secretion and ciliary motion defects limit spread. [ABSTRACT FROM AUTHOR]

Pathogenic serotypes of Vibrio cholerae, transmitted through contaminated water and food, are responsible for outbreaks of cholera, an acute diarrheal disease. While the cholera toxin is the primary virulence factor, V. cholerae also expresses other virulence factors, such as the tripartite toxin MakABE that is secreted via the bacterial flagellum. These three proteins are co-expressed with two accessory proteins, MakC and MakD, whose functions remain unknown. Here, we present the crystal structures of MakC and MakD, revealing that they are similar in both sequence and structure but lack other close structural relatives. Our study further investigates the roles of MakC and MakD, focusing on their impact on the expression and secretion of the components of the MakABE tripartite toxin. Through deletion mutant analysis, we found that individual deletions of makC or makD do not significantly affect MakA expression or secretion. However, the deletion of both makC and makD impairs the expression of MakB, which is directly downstream, and decreases the expression of MakE, which is separated from makCD by two genes. Conversely, MakA, encoded by the makA gene located between makB and makE, is expressed normally but its secretion is impaired. Additionally, our findings indicate that MakC, in contrast to MakD, exhibits strong interactions with other proteins. Furthermore, both MakC and MakD were observed to be localized within the cytosol of the bacterial cell. This study provides new insights into the regulatory mechanisms affecting the Mak protein family in V. cholerae and highlights the complex interplay between gene proximity and protein expression. [ABSTRACT FROM AUTHOR]

Loss of ARID1A has been reported to drive the progression of lung adenocarcinoma, yet the underlying mechanism remains elusive. In this study, we performed secretome analysis to identify the key secreted proteins regulating lung adenocarcinoma progression. We showed that the VASN level was significantly elevated in the conditioned medium from ARID1A-depleted A549 and H1299 cells. Restoration of ARID1A in ARID1A-depleted lung adenocarcinoma cells prevented the upregulation and secretion of VASN. Clinical analysis demonstrated a negative correlation between ARID1A and VASN expression in ARID1A-mutated lung adenocarcinomas. The patients with ARID1A-mutated lung adenocarcinoma had significantly higher concentrations of serum VASN than healthy controls. Moreover, serum VASN concentrations were associated with TNM stage, lymph node metastasis, and overall survival of the patients with ARID1A-mutated lung adenocarcinoma. Functional studies indicated that VASN overexpression potentiated the proliferation, invasion, and tumorigenesis of lung adenocarcinoma cells. Antibody neutralization of VASN suppressed the aggressiveness of ARID1A-depleted lung adenocarcinoma cells both in vitro and in vivo. Addition of recombinant VASN protein promoted the proliferation and invasion of lung adenocarcinoma cells. Additionally, knockdown of Notch1 blocked the aggressive phenotype induced by recombinant VASN protein. In conclusion, our data uncover the role of VASN in mediating the progression of ARID1A-depleted lung adenocarcinoma and highlight VASN as a promising therapeutic target for this disease. [ABSTRACT FROM AUTHOR]

Introduction: Human peripheral blood mononuclear cells (hPBMCs) are widely used in fundamental research and clinical applications as studying their responses to in vitro activation is an effective way to uncover functional alterations and disease associated phenotypes. However, the availability of samples in large numbers at a specific time and location remains challenging, hence they often might preferably be collected and cryopreserved for later analysis. While the effect of cryopreservation on viability and cell surface expression is well established, changes in activity and cytokine secretion still lead to conflicting results as it is often measured in bulk or within the cells. Methods: Here, we used our platform for dynamic single-cell multiplexed cytokine secretion measurement and compared it to a traditional intracellular cytokine staining to quantify the effect of cryopreservation on cytokine secretion and expression of individual hPBMCs. Results: Following stimulation with LPS or anti-CD3/CD28 antibodies for up to 36 or 72 h incubation, we observed distinct alterations in cytokine responses due to cryopreservation when comparing to fresh samples, but also remarkable consistencies for some cytokines and parameters. In short, the frequencies of cytokine-secreting cells in cryopreserved samples were lower for IL-6 (LPS), IL1-b (CD3/CD28) and IFN-g (CD3/CD28), while the frequency and dynamics of IL-8 secretion were strongly impacted in all cases. We observed a large disconnect between cytokine expression and secretion for TNF-a, where the expression dramatically increased after cryopreservation, but actual secretion was, in comparison, remarkably stable. The polyfunctionality of single cells was altered by cryopreservation in specific co-secreting populations led by the effects on IL-6 or IL-8 secretion. Among immune cells, cryopreservation seemed to affect lymphocytes and monocytes differently as effects appeared early on in lymphocytes while generally observed in later time points in monocytes. Conclusion: Together, this study offers an in-depth quantitative insight into the biological behavior of immune cells in response to cryopreservation and stimulation, further providing some insights into conflicting results in the literature as well as guidelines for researchers planning to assess cytokine-secreting from frozen hPBMCs in immunological research or clinical applications. [ABSTRACT FROM AUTHOR]

As a powerful eukaryotic expression vector, the baculovirus expression vector system (BEVS) is widely applied to the production of heterogeneous proteins for research and pharmaceutical purposes, while optimization of BEVS remains a work in progress for membrane or secreted protein expression. In this study, the impact of the signal peptide (SP) derived from Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 protein on protein expression, secretion, and the endoplasmic reticulum-associated degradation (ERAD) pathway were investigated in BmN cells and BEVS. Transient expression studies in BmN cells revealed that SP alters the localization and expression levels of recombinant proteins, reducing intracellular accumulation while enhancing secretion efficiency. Quantitative analysis demonstrated that SP-mediated secretion was markedly higher compared to controls, albeit with lower total expression levels. Further exploration into SP-mediated ERAD pathway activation showed increased expression of BiP and other ERAD-associated genes (PDI, UFD1, S1P, and ASK1), correlating with higher SP-driven protein expression levels. RNA interference (RNAi) experiments elucidated that knockdown of ERAD-associated genes enhances both the secretion efficiency of SP-guided proteins and the infectivity of BmNPV. Particularly, interference with BiP demonstrated the most pronounced effect on protein secretion enhancement. Viral infection experiments further supported these findings, showing upregulated ERAD-associated genes during BmNPV infection, indicating their role in viral protein processing and infectivity. In conclusion, this study elucidates the complex interplay between SP-mediated protein secretion, ERAD pathway activation, and viral infectivity in BmNPV-infected cells. These insights suggest strategies for optimizing recombinant protein production and viral protein processing in baculovirus expression systems, with potential implications for biotechnological and biomedical applications. Further research could refine our understanding and manipulation of protein secretion pathways in insect cell-based expression systems. [ABSTRACT FROM AUTHOR]

Background: Mesenchymal stromal cell derived extracellular vesicles (MSC-EVs) are a promising therapeutic for neuroinflammation. MSC-EVs can interact with microglia, the resident immune cells of the brain, to exert their immunomodulatory effects. In response to inflammatory cues, such as cytokines, microglia undergo phenotypic changes indicative of their function e.g. morphology and secretion. However, these changes in response to MSC-EVs are not well understood. Additionally, no disease-relevant screening tools to assess MSC-EV bioactivity exist, which has further impeded clinical translation. Here, we developed a quantitative, high throughput morphological profiling approach to assess the response of microglia to neuroinflammation- relevant signals and whether this morphological response can be used to indicate the bioactivity of MSC-EVs. Results: Using an immortalized human microglia cell-line, we observed increased size (perimeter, major axis length) and complexity (form factor) upon stimulation with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Upon treatment with MSC-EVs, the overall morphological score (determined using principal component analysis) shifted towards the unstimulated morphology, indicating that MSC-EVs are bioactive and modulate microglia. The morphological effects of MSC-EVs in TNF-α /IFN-γ stimulated cells were concomitant with reduced secretion of 14 chemokines/cytokines (e.g. CXCL6, CXCL9) and increased secretion of 12 chemokines/cytokines (e.g. CXCL8, CXCL10). Proteomic analysis of cell lysates revealed significant increases in 192 proteins (e.g. HIBADH, MEAK7, LAMC1) and decreases in 257 proteins (e.g. PTEN, TOM1, MFF) with MSC-EV treatment. Of note, many of these proteins are involved in regulation of cell morphology and migration. Gene Set Variation Analysis revealed upregulation of pathways associated with immune response, such as regulation of cytokine production, immune cell infiltration (e.g. T cells, NK cells) and morphological changes (e.g. Semaphorin, RHO/Rac signaling). Additionally, changes in microglia mitochondrial morphology were measured suggesting that MSC-EV modulate mitochondrial metabolism. Conclusion: This study comprehensively demonstrates the effects of MSC-EVs on human microglial morphology, cytokine secretion, cellular proteome, and mitochondrial content. Our high-throughput, rapid, low-cost morphometric approach enables screening of MSC-EV batches and manufacturing conditions to enhance EV function and mitigate EV functional heterogeneity in a disease relevant manner. This approach is highly generalizable and can be further adapted and refined based on selection of the disease-relevant signal, target cell, and therapeutic product. [ABSTRACT FROM AUTHOR]

Leukotriene B4 (LTB4) is an inflammatory lipid produced in response to pathogens that is critical for initiating the inflammatory cascade needed to control infection. However, during plague, Yersinia pestis inhibits the timely synthesis of LTB4 and subsequent inflammation. Using bacterial mutants, we previously determined that Y. pestis inhibits LTB4 synthesis via the action of the Yop effector proteins that are directly secreted into host cells through a type 3 secretion system (T3SS). Here, we show that the T3SS is the primary pathogen associated molecular pattern (PAMP) required for production of LTB4 in response to both Yersinia and Salmonella. However, we also unexpectantly discovered that T3SS-mediated LTB4 synthesis by neutrophils and macrophages require the activation of two distinctly different host signaling pathways. We identified that phagocytosis and the NLRP3/CASP1 inflammasome significantly impact LTB4 synthesis by macrophages but not neutrophils. Instead, the SKAP2/PLC signaling pathway is required for T3SS-mediated LTB4 production by neutrophils. Finally, while recognition of the T3SS is required for LTB4 production, we also discovered that a second unrelated PAMP-mediated signal activates the MAP kinase pathway needed for synthesis. Together, these data demonstrate significant differences in the host factors and signaling pathways required by macrophages and neutrophils to quickly produce LTB4 in response to bacteria. Moreover, while macrophages and neutrophils might rely on different signaling pathways for T3SS-dependent LTB4 synthesis, Y. pestis has evolved virulence mechanisms to counteract this response by either leukocyte to inhibit LTB4 synthesis and colonize the host. Author summary: The production of inflammatory lipid mediators by the host is essential for timely inflammation in response to invasion by bacterial pathogens. Therefore, defining how immune cells recognize pathogens and rapidly produce these lipids is essential for us to understand how our immune system effectively controls infection. In this study, we discovered that the host signaling pathways required for leukotriene B4 (LTB4) synthesis differ between neutrophils and macrophages, highlighting important differences in how immune cells respond to infection. Together, these data represent a significant improvement in our understanding of how neutrophils and macrophages rapidly react to bacteria and provide new insights into how Yersinia pestis manipulates leukocytes to evade immune recognition to cause disease. [ABSTRACT FROM AUTHOR]

Human salivary amylase secretion increases in response to stress; the activity has been reported to rise significantly with high‐intensity exercise. The human salivary amylase gene (AMY1) has copy number variation, with the copy number correlating with salivary amylase activity. However, the relationship between individual AMY1 copy number and salivary amylase activity in response to exercise remains unclear. In this study, we investigated AMY1 copy number and fluctuations in amylase activity in 42 healthy university students (25 males and 17 females). Participants engaged in intermittent round‐trip interval training on a basketball court. Saliva samples were collected pre‐ and post‐exercise to measure amylase activity. DNA was extracted from the oral mucosa, and AMY1 copy number was quantified using RT‐PCR. Results showed a significant increase in amylase activity postexercise. Additionally, amylase activity pre‐ and post‐exercise was positively correlated with AMY1 copy number. The generalize linear model showed that the exercise‐induced increase in amylase activity per AMY1 gene was negatively related to the AMY1 copy number and aerobic fitness. Gender has no effect on amylase activity. These results suggest a different mechanism for the constitutive and exercise‐induced amylase secretion, while aerobic fitness may be independently involved in the secretion. [ABSTRACT FROM AUTHOR]

GPR119 agonists are being developed to safeguard the function of pancreatic β‐cells, especially in the context of non‐alcoholic fatty pancreas disease (NAFPD) that is closely associated with β‐cell dysfunction. This study aims to employ a drug repurposing strategy to screen GPR119 agonists and explore their potential molecular mechanisms for enhancing β‐cell function in the context of NAFPD. MIN6 cells were stimulated with palmitic acid (PA), and a NAFPD model was established in GPR119−/− mice fed with a high‐fat diet (HFD). Terazosin, identified through screening, was utilized to assess its impact on enhancing β‐cell function via the MST1‐Foxo3a pathway and mitophagy. Terazosin selectively activated GPR119, leading to increased cAMP and ATP synthesis, consequently enhancing insulin secretion. Terazosin administration improved high blood glucose, obesity, and impaired pancreatic β‐cell function in NAFPD mice. It inhibited the upregulation of MST1‐Foxo3a expression in pancreatic tissue and enhanced damaged mitophagy clearance, restoring autophagic flux, and improving mitochondrial quantity and structure in β‐cells. Nevertheless, GPR119 deficiency negated the positive impact of terazosin on pancreatic β‐cell function in NAFPD mice and abolished its inhibitory effect on the MST1‐Foxo3a pathway. Terazosin activates GPR119 on the surface of pancreatic β‐cells, enhancing mitophagy and alleviating β‐cell dysfunction in the context of NAFPD by suppressing the MST1‐Foxo3a signalling pathway. Terazosin could be considered a priority treatment for patients with concomitant NAFPD and hypertension. [ABSTRACT FROM AUTHOR]

Plant economics, the way plants allocate and utilize resources, affect multiple soil processes through interactions with root and associated microbial communities. However, the interplay between plant economics and microbial ecological strategies remains poorly understood, which is crucial for integrated manipulation of plant‐ and microbe‐mediated functions in mitigating climate change and sustaining soil health. We used a field experiment with 11 cover crop species grown monocultures in the same base soil to test whether microbial ecological strategies are associated with plant economic strategies and if their interactions are linked to soil functions. A principal component analysis (PCA) was performed on root and leaf traits to identify the loadings of cover crop species on the plant trait space. Metagenomic analysis of rhizosphere microbial communities was conducted to infer their ecological strategies based on genetically encoded community‐aggregated traits. We found a synchronous relationship between the conservation gradient of plant economic strategies and the trade‐offs in microbial ecological strategies. Conservative plant strategists, such as Lolium multiflorum, Triticum turgidum and Brassica juncea, fostered microbial communities characterized by high growth yield potentials (Y‐strategies). This included increased microbial carbon fixation pathways, citrate cycle, ribosome and valine, leucine and isoleucine biosynthesis. As a result, microbial metabolic efficiency improved, shown by higher microbial biomass carbon content and a lower metabolic quotient (qCO2), led to enhanced soil organic carbon accumulation. In comparison, acquisitive plants like Astragalus sinicus, Vicia villosa, Trifolium incarnatum and Medicago sativa stimulated microbial resource‐acquisition strategies (A‐strategies). This included enhanced bacterial chemotaxis, secretion systems, biotin metabolism and cell motility pathways, which in turn increased soil exoenzyme activity and accelerated soil nitrogen mineralization. Consequently, these species enhanced soil nitrogen availability and had substantial feedbacks on subsequent main crop productivity. Synthesis. This study demonstrates how plant economic strategies influence the balance between different microbial ecological strategies, specifically the trade‐offs in Y‐ and A‐strategies. These interactions exert control over carbon and nitrogen dynamics in the soil ecosystem. The findings provide insights for implementing nature‐based solutions to improve agroecosystem management practices. [ABSTRACT FROM AUTHOR]

B cells play a key role in humoral immune responses by producing antibodies. Although there are numerous research on memory B cells definition markers and cytokines on B cell development, different studies have yielded contradictory conclusions due to species studied, the different cells and stimulating agents used. In the current study, we conducted a detailed characterization of B cells in human CBMCs, PBMCs and tonsil, including expression of Igs, activation and memory markers. Furthermore, we found that considerable amounts of IgA and IgG were expressed by CD27− B cells. These "Atypical" memory B cells corresponded to approximately 50% of IgG+ and IgA+B cells in blood, this proportion even reached 90% in tonsil. In addition, we investigated the effect of IL-21 and TGF-β1 on the membrane-bound form and secreted form of Igs using PBMCs and purified blood B cells. There were actual differences between the effect of cytokines on Igs secretion and surface expression. Our study will be helpful to advance the knowledge and understanding of humoral memory. [ABSTRACT FROM AUTHOR]

The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) is a large, multisubunit complex that exports a vast array of substrates into eukaryotic host cells. DotO, a distant homolog of the T4ASS ATPase VirB4, associates with the bacterial inner membrane despite lacking hydrophobic transmembrane domains. Employing a genetic approach, we found DotO's membrane association is mediated by three inner-membrane Dot/Icm components, IcmT, and a combined DotJ-DotI complex (referred to as DotJI). Although deletion of icmT or dotJI individually does not affect DotO's membrane association, the simultaneous inactivation of all three genes results in increased amounts of soluble DotO. Nevertheless, deleting each receptor separately profoundly affects positioning of DotO, disrupting its link with the Dot/Icm complex at the bacterial poles, rendering the receptors nonredundant. Furthermore, a collection of dotO point mutants that we isolated established that DotO's N-terminal domain interacts with the membrane receptors and is involved in dimerization, whereas DotO's C-terminal ATPase domain primarily contributes to the protein's formation of oligomers. Modeling data revealed the complex interaction between DotO and its receptors is responsible for formation of DotO's unique "hexamer of dimers" configuration, which is a defining characteristic of VirB4 family members. [ABSTRACT FROM AUTHOR]

Objectives Methods Results Conclusions This systematic review and meta‐analysis aimed to evaluate the diagnostic ability and examine the efficacy of countermeasures to adverse events of mucosal incision‐assisted biopsy (MIAB) for gastric subepithelial tumors (SETs).We performed a literature search and identified 533 relevant articles. Eleven articles, including 339 lesions, were ultimately used in the meta‐analysis. The primary end‐point was the pathological diagnostic rate of MIAB for gastric SETs, and the secondary end‐point was the incidence of adverse events. The efficacy of acid secretion inhibitors in preventing postoperative bleeding and that of local injection before incision to prevent perforation were also examined.Nine studies were conducted in Japan and two in South Korea, of which only two were prospective studies. The pooled pathological diagnostic rate of MIAB for gastric SETs was 87.8% (95% confidence interval [CI] 80.2–94.0; I2 = 68.7%). The adverse event rate of the pooled population was 0.2% (95% CI 0–1.4; I2 = 0%). The acid secretion inhibitors significantly reduced postoperative bleeding (odds ratio 0.06, 95% CI 0.01–0.66, P = 0.02). Perforation occurred in 0% and 2.6% of the local and nonlocal injection cohorts, respectively, and the pathological diagnostic rates were 50% and 66.7%, respectively.MIAB is a reliable technique with a favorable diagnostic rate and few adverse events. Acid secretion inhibitors may effectively prevent postoperative bleeding; however, the efficacy of local injection remains unclear. This technique could be an option for tissue sampling in gastric SETs. [ABSTRACT FROM AUTHOR]

Plant cell walls are essential for growth. The cell wall hemicellulose xyloglucan (XyG) is produced in the Golgi apparatus before secretion. Loss of the Arabidopsis galactosyltransferase MURUS3 (MUR3) decreases XyG d -galactose side chains and causes intracellular aggregations and dwarfism. It is unknown how changing XyG synthesis can broadly impact organelle organization and growth. We show that intracellular aggregations are not unique to mur3 and are found in multiple mutant lines with reduced XyG D -galactose side chains. mur3 aggregations disrupt subcellular trafficking and induce formation of intracellular cell-wall-like fragments. Addition of d -galacturonic acid onto XyG can restore growth and prevent mur3 aggregations. These results indicate that the presence, but not the composition, of XyG side chains is essential, likely by ensuring XyG solubility. Our results suggest that XyG polysaccharides are synthesized in a highly substituted form for efficient secretion and then later modified by cell-wall-localized enzymes to fine-tune cell wall properties. [Display omitted] • A reduction in xyloglucan (XyG) side chains causes intracellular aggregations • Intracellular aggregations form during cell expansion and disrupt secretion • An alternate XyG side chain restores secretion but not cell wall composition Hoffmann and McFarlane show that the plant cell wall polysaccharide xyloglucan requires side chains for effective secretion following its biosynthesis in the Golgi apparatus, as production of side-chain-deficient xyloglucan causes intracellular aggregations of proteins and polysaccharides. These results highlight the importance of polysaccharide structure for efficient secretion to the cell wall. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests an essential function for autophagy in unconventional protein secretion (UPS). However, despite its relevance for the secretion of aggregate-prone proteins, the mechanisms of secretory autophagy in neurons have remained elusive. Here we show that the lower motoneuron disease-associated guanine exchange factor Plekhg5 drives the UPS of Sod1. Mechanistically, Sod1 is sequestered into autophagosomal carriers, which subsequently fuse with secretory lysosomal-related organelles (LROs). Exocytosis of LROs to release Sod1 into the extracellular milieu requires the activation of the small GTPase Rab26 by Plekhg5. Deletion of Plekhg5 in mice leads to the accumulation of Sod1 in LROs at swollen presynaptic sites. A reduced secretion of toxic ALS-linked SOD1G93A following deletion of Plekhg5 in SOD1G93A mice accelerated disease onset while prolonging survival due to an attenuated microglia activation. Using human iPSC-derived motoneurons we show that reduced levels of PLEKHG5 cause an impaired secretion of ALS-linked SOD1. Our findings highlight an unexpected pathophysiological mechanism that converges two motoneuron disease-associated proteins into a common pathway. Unconventional protein secretion emerges as an important mechanism in aggregate-prone protein removal. Here, the authors demonstrate that Plekhg5 mediates the unconventional secretion of Sod1 by presynaptic secretory autophagy. [ABSTRACT FROM AUTHOR]

VemP is a secretory protein in the Vibrio species that monitors cellular protein-transport activity through its translation arrest, allowing expression of the downstream secD2-secF2 genes in the same operon, which encode components of the protein translocation machinery. When cellular proteintransport function is fully active, secD2/F2 expression remains repressed as VemP translation arrest is canceled immediately. The VemP arrest cancellation occurs on the SecY/E/G translocon in a late stage in the translocation process and requires both trans factors, SecD/F and PpiD/YfgM, and a cis element, Arg-85 in VemP; however, the detailed molecular mechanism remains elusive. This study aimed to elucidate how VemP passing through SecY specifically monitors SecD/F function. Genetic and biochemical studies showed that SecY is involved in the VemP arrest cancellation and that the arrested VemP is stably associated with a specific site in the protein-conducting pore of SecY. VemP-Bla reporter analyses revealed that a short hydrophobic segment adjacent to Arg-85 plays a critical role in the regulated arrest cancellation with its hydrophobicity correlating with the stability of the VemP arrest. We identified Gln-65 and Pro-67 in VemP as novel elements important for the regulation. We propose a model for the regulation of the VemP arrest cancellation by multiple cis elements and trans factors with different roles. [ABSTRACT FROM AUTHOR]

During vigorous physical activity, contracting muscles secrete a variety of immunomodulatory and metabolic factors called myokines that perform a variety of functions. Foremost among these is interleukin 6 (IL‐6), which increases fatty acid mobilization and stimulates anti‐inflammatory cytokine release. Despite being well characterized in vigorous exercise contexts, whether IL‐6 is secreted during moderate‐intensity activities such as walking is unclear. This is especially pertinent as regular walking is one of the oldest and most common forms of physical activity and comes with a variety of health benefits, which may be coordinated in part by IL‐6 signaling. To test the hypothesis that IL‐6 release occurs during evolutionarily normal physical activity contexts like long distance walking, we performed a dose–response experiment to test the effect of walking duration on IL‐6 secretion. Thirteen participants completed four moderate‐intensity walking trials (55% HRmax) of varying durations (30 min, 1 h, 2 h, and 3 h) in a randomized order with intervening washout periods of at least 1 week. Using a linear mixed effects model, we found that IL‐6 levels modestly increased during only the 2 h and 3 h walking trials. These results suggest that small frequent increases in IL‐6 signaling may be an important mechanism underlying the health benefits of regular walking. Furthermore, this finding raises the possibility that IL‐6 secretion is an adaptation to fuel physical activity, especially during periods of negative energy balance. [ABSTRACT FROM AUTHOR]

Patients with salt‐wasting congenital adrenal hyperplasia (SW‐CAH) usually show pronounced impairment of aldosterone secretion and, therefore, also require mineralocorticoid replacement. While a lot of research and discussion focusses on the glucocorticoid therapy in SW‐CAH to replace the missing cortisol and to control adrenal androgen excess, very little research is dealing with mineralocorticoid replacement. However, recent data demonstrated an increased cardiovascular risk in adult CAH patients urging to reflect also on the current mineralocorticoid replacement therapy. In this review, we explain the role and function of the mineralocorticoid receptor, its ligands and inhibitors and its relevance for the therapy of patients with SW‐CAH. We performed an extensive literature search and present data on mineralocorticoid therapy in SW‐CAH patients as well as clinical advice how to monitor and optimise mineralocorticoid replacement therapy. [ABSTRACT FROM AUTHOR]

2-deoxy-D-glucose (2DG) is a glycolysis and protein N-glycosylation inhibitor with promising anti-tumor and immunomodulatory effects. However, 2DG can also suppress T cell function, including IFN-γ secretion. Few human T cell studies have studied low-dose 2DG, which can increase IFN-γ in a Jurkat clone. We therefore investigated 2DG's effect on IFN-γ in activated human T cells from PBMCs, with 2DG treatment commenced either concurrently with activation or 48 h after activation. Concurrent 2DG treatment decreased IFN-γ secretion in a dose-dependent manner. However, 2DG treatment of pre-activated T cells had a hormetic effect on IFN-γ, with 0.15–0.6 mM 2DG (achievable in vivo) increasing and >2.4 mM 2DG reducing its secretion. In contrast, IL-2 levels declined monotonously with increasing 2DG concentration. Lower 2DG concentrations reduced PD-1 and increased CD69 expression regardless of treatment timing. The absence of increased T-bet or Eomes expression or IFNG transcription suggests another downstream mechanism. 2DG dose-dependently induced the unfolded protein response, suggesting a possible role in increased IFN-γ secretion, possibly by increasing the ER folding capacity for IFN-γ via increased chaperone expression. Overall, low-dose, short-term 2DG exposure could potentially improve the T cell anti-tumor response. [ABSTRACT FROM AUTHOR]

Simple Summary: Three-dimensional (3D) models enable simplified and controlled in vitro replication of tumor microenvironment features, making them easier to study. In the current work, we developed 3D hydrogels based on gelatin plus silk fibroin with tunable mechanical properties, incorporating vitronectin (a glycoprotein related to tumor aggressiveness) into the scaffolds. Digital analysis was applied to monitor Ewing sarcoma cell line growth and morphology in response to the 3D model composition and different culture times, opening the possibility of its use to study other proteins of interest and tumor types. Our findings highlight the heterogeneity of cell line responses to their microenvironment, underscoring the enormous potential of these hydrogels as mechanodrug-testing platforms and for studying cell protein secretion as an initial step in liquid biopsy biomarker research. Ewing sarcoma (ES) is an aggressive bone and soft-tissue pediatric cancer. High vitronectin (VN) expression has been associated with poor prognosis in other cancers, and we aimed to determine the utility of this extracellular matrix glycoprotein as a biomarker of aggressiveness in ES. Silk fibroin plus gelatin–tyramine hydrogels (HGs) were fabricated with and without cross-linked VN and cultivated with A673 and PDX73 ES cell lines for two and three weeks. VN secretion to culture media was assessed using ELISA. Morphometric analysis was applied for phenotypic characterization. VN release to culture media was higher in 3D models than in monolayer cultures, and intracellular, intercellular, and pericluster presence was also observed. A673-HGs showed lower density of clusters but a proportion of larger clusters than PDX73-HGs, which presented low cluster circularity. The cluster density of A673-HGs without added VN was higher than with added VN and slightly lower in the case of PDX73-HGs. Furthermore, a culture time of three weeks provided no benefits in cluster growth compared to two weeks, especially in A673-HGs. These advances in 3D modeling and digital quantification pave the way for future studies in ES and other cancers to deepen understanding about intra- and intercellular heterogeneity and anti-adhesion VN therapies. [ABSTRACT FROM AUTHOR]

Congenital hypogonadotropic hypogonadism (CHH) is a rare reproductive disorder caused by deficient secretion or action of gonadotropin‐releasing hormone (GnRH) and is a hormonally treatable form of male infertility. Both pulsatile GnRH treatment and combined gonadotropin therapy effectively induce spermatogenesis in 75%–80% of males with CHH, albeit the ejaculate does not usually approach normal semen parameters by WHO criteria. This is in some contrast to the cumulative fertility outcomes in females with CHH on gonadotropin treatment that are indistinguishable from those of reproductively normal females. Emerging data provide insights into early life determinants of male fertility (i.e., minipuberty), and research has identified key predictors of outcomes for fertility‐inducing treatment in men with CHH. Such developments provide mounting evidence for tailoring approaches to maximize fertility potential in CHH, although there is no clear consensus to date on the optimal approach to fertility‐inducing treatment. This review provides an up‐to‐date review on the current evidence underpinning therapeutic approaches for inducing spermatogenesis in males with CHH. In the absence of evidence‐based clinical guidelines, this synthesis of current evidence provides guidance for clinicians working with males with CHH seeking fertility. [ABSTRACT FROM AUTHOR]

Senescent cells produce a Senescence‐Associated Secretory Phenotype (SASP) that involves factors with diverse and sometimes contradictory activities. One key SASP factor, interleukin‐6 (IL‐6), has the potential to amplify cellular senescence in the SASP‐producing cells in an autocrine action, while simultaneously inducing proliferation in the neighboring cells. The underlying mechanisms for the contrasting actions remain unclear. We found that the senescence action does not involve IL‐6 secretion nor the interaction with the receptor expressed in the membrane but is amplified through an intracrine mechanism. IL‐6 sustains intracrine senescence interacting with the intracellular IL‐6 receptor located in anterograde traffic specialized structures, with cytosolic DNA, cGAS‐STING, and NFκB activation. This pathway triggered by intracellular IL‐6 significantly contributes to cell‐autonomous induction of senescence and impacts in tumor growth control. Inactivation of IL‐6 in somatotrophic senescent cells transforms them into strongly tumorigenic in NOD/SCID mice, while re‐expression of IL‐6 restores senescence control of tumor growth. The intracrine senescent IL‐6 pathway is further evidenced in three human cellular models of therapy‐induced senescence. The compartmentalization of the intracellular signaling, in contrast to the paracrine tumorigenic action, provides a pathway for IL‐6 to sustain cell‐autonomous senescent cells, driving the SASP, and opens new avenues for clinical consideration to senescence‐based therapies. [ABSTRACT FROM AUTHOR]

The 3‐level features of Latent fingerprints (LFPs) are pivotal in linking suspects with problematic or incomplete fingerprint data, constituting one of the most robust forms of evidence. Aggregation‐induced emission (AIE) materials, known for their heightened emissive properties in high aggregation states, provide a robust and powerful approach for developing techniques for 3‐level details of LFPs. So far, tremendous efforts have been devoted to solving high background fluorescence interference during the 3‐level LFPs visualization process. This review commences with an overview of 3‐level features and summarizes the interaction and mechanism of visualizing LFPs involving AIE materials and LFPs secretions. Moreover, it encompasses strategies to enhance the contrast and brightness of 3‐level LFPs images. We highlight the mechanism of AIE′s physicochemical properties and their effects on the visualization of 3‐level LFPs. Additionally, we provide insights into potential challenges and opportunities in this emerging field. [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. What is the central question of the study?Do acute heat stress and carbon monoxide inhalation alone and in combination increase erythropoietin, and are there sex differences in erythropoietin?What is the main finding and its importance?All interventions increased erythropoietin, but combining the two interventions did not further augment erythropoietin. While erythropoietin increased in females in response to all interventions, there was greater erythropoietin variability in males and therefore, on average, males did not increase erythropoietin. This suggests that combining heat and hypoxic stressors does not further augment erythropoietin concentrations but these interventions may have greater efficacy in females. [ABSTRACT FROM AUTHOR]

Aim: To investigate the effect of G protein‐coupled receptor 55 (GPR55) deletion on glucose homeostasis and islet function following diet‐induced obesity. Methods: 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. Results: 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. Conclusions: 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]

Objective: The intestinal luminal pH profile varies from stomach to rectum and becomes disrupted in diseases. However, little is known about the pH dependence of incretin hormone secretion, with most in vitro studies having failed to consider this modulatory factor or having used nonphysiological buffer systems. Here, we report the extracellular pH (pHe) dependence of glucagon‐like peptide‐1 (GLP‐1) exocytosis from L cells. Methods: The pHe dependence of GLP‐1 release from GLUTag cells and murine ex vivo primary gut cultures was detected by ELISA. GLP‐1 release was measured over a range of pHe under a physiological (CO2/HCO3−) buffering regime and in its absence (HEPES buffer). The relationship between intracellular pH (pHi) and pHe was mapped given that at least some component of pH sensitivity is likely to be intracellular. Results: GLP‐1 secretion from L cells was pHe‐dependent and stimulated under alkaline conditions. In the absence of glucose or extracellular calcium, secretion remained at a pHe‐insensitive baseline. pHi followed changes in pHe, but the relationship was offset to more alkaline levels in the absence of CO2/HCO3− buffer and became shallower if [Cl−] changes that normally accompany [HCO3−] changes were compensated iso‐osmotically with gluconate. Conclusions: GLP‐1 secretion is sensitive to pHe and the buffer present. Exploiting this mechanism therapeutically may benefit patients with obesity. [ABSTRACT FROM AUTHOR]

Lamellar bodies (LBs) are tissue-specific lysosome-related organelles in type II alveolar cells that are the main site for the synthesis, storage, and secretion of pulmonary surfactants. Defects in pulmonary surfactants lead to a variety of respiratory and immune-related disorders. LB biogenesis is closely related to their function, but the underlying regulatory mechanism is largely unclear. Here, we found that deficiency of HPS6, a subunit of BLOC-2 (biogenesis of lysosome-related organelles complex-2), led to a reduction of the steady-state concentration of vacuolar-type H+-ATPase and an increase in the luminal pH of LBs. Furthermore, we observed increased LB size, accumulated surfactant proteins, and altered lipid profiling of lung tissue and BAL fluid due to HPS6 deficiency. These findings suggest that HPS6 regulates the distribution of vacuolar-type H+-ATPase on LBs to maintain its luminal acidity and LB homeostasis. This may provide new insights into the LB pathology. [ABSTRACT FROM AUTHOR]

Sebaceous glands (SG) are essential for maintaining skin integrity, as their lipid-rich secretion (sebum) lubricates and protects the epidermis and hairs. In addition, these glands have an emerging role in immunomodulation and may affect whole-body energy metabolism, besides being an appealing model for research in topics as lipogenesis, stem cell biology and tumorigenesis. In spite of the increasing interest in studying SGs pathophysiology, sebocyte cell–cell and cell–matrix adhesion processes have been only superficially examined, and never in a systematic way. This is regrettable considering the key role of cellular adhesion in general, the specific expression pattern of indivdual junctional complexes, and the reports of structural changes in SGs after altered expression of adhesion-relevant proteins. Here, we review the available information on structural and functional aspects of cell–cell and cell–matrix junctions in sebocytes, and how these processes change under pathological conditions. This information will contribute for better understanding sebocyte differentiation and sebum secretion, and may provide hints for novel therapeutic strategies for skin diseases. [ABSTRACT FROM AUTHOR]

An emerging theme in Parkinson's disease (PD) is the propagation of α-synuclein pathology as the disease progresses. Research involving the injection of preformed α-synuclein fibrils (PFFs) in animal models has recapitulated the pathological spread observed in PD patients. At the cellular and molecular levels, this intercellular spread requires the translocation of α-synuclein across various membrane barriers. Recent studies have identified subcellular organelles and protein machineries that facilitate these processes. In this review, we discuss the proposed pathways for α-synuclein intercellular transmission, including unconventional secretion, receptor-mediated uptake, endosome escape and nanotube-mediated transfer. In addition, we advocate for a rigorous examination of the evidence for the localization of α-synuclein in extracellular vesicles. [ABSTRACT FROM AUTHOR]

Salicylic acid (SA) is widely renowned for its efficacy as a beneficial ingredient for skincare, especially for acne and uneven skin texture. The salicylic acid (SA) niosome formulation combined with the essential component of oleoresin from Dipterocarpus alatus Roxb. ex G. Don or Yang‐Na (ODA) was developed and investigated for its physical characteristics, biological effects, and stability. The findings demonstrated that SA combined with ODA in the niosome formulation F4 enhanced the entrapment efficiency of SA, as well as the physical properties and stability of the formulation. Furthermore, the release pattern of this combined formulation indicated sustained release of SA. The permeation of SA was higher in the presence of ODA compared to SA‐niosome formulations without ODA. Moreover, this F4 could downregulate the secretion of iNOS, COX‐2, and TNF‐α including anti‐Propionibacterium acnes activities. Consequently, the incorporation of ODA into the niosome formulation has the potential to improve the entrapment efficiency of SA, facilitating controlled release and enhancing permeation, nitric oxide inhibition capabilities, and anti-P. acnes activity. Therefore, F4 has the potential to be developed as a topical product for the combined treatment of inflammation and P. acnes‐associated conditions in the future. [ABSTRACT FROM AUTHOR]

ABSTRACT Aims/Introduction Materials and Methods Results Conclusions We previously showed that glucokinase haploinsufficiency improves the glucose tolerance of db/db mice by preserving pancreatic β‐cell mass and function. In the present study, we aimed to determine the effects of glucokinase haploinsufficiency on the β‐cell mass and function of long‐term high‐fat, high‐sucrose (HFHS) diet‐fed mice.Four‐week‐old male glucokinase haploinsufficient (Gck+/−) mice and 4‐week‐old male wild‐type (Gck+/+) mice (controls) were each divided into two groups: an HFHS diet‐fed group and a normal chow‐fed group, and the four groups were followed until 16, 40 or 60 weeks‐of‐age. Their glucose tolerance, glucose‐stimulated insulin secretion and β‐cell mass were evaluated. In addition, islets were isolated from 40‐week‐old mice, and the expression of key genes was compared.Gck+/−HFHS mice had smaller compensatory increases in β‐cell mass and glucose‐stimulated insulin secretion than Gck+/+HFHS mice, and their glucose tolerance deteriorated from 16 to 40 weeks‐of‐age. However, their β‐cell mass and glucose‐stimulated insulin secretion did not decrease between 40 and 60 weeks‐of‐age, but rather, tended to increase, and there was no progressive deterioration in glucose tolerance. The expression of Aldh1a3 in pancreatic islets, which is high in several models of diabetes and is associated with an impairment in β‐cell function, was high in Gck+/+HFHS mice, but not in Gck+/−HFHS mice.Glucokinase haploinsufficiency prevents the progressive deterioration of pancreatic β‐cell mass/function and glucose tolerance in long‐term HFHS diet‐fed mice. [ABSTRACT FROM AUTHOR]

[Objective] To study the chemical composition and differences between male and female odor gland secretions of Plautia stali metathorax. [Method] A solid-phase microextraction device coupled with gas chromatography-mass spectrometry (GC-MS) was used to determine and analyze the chemical composition of the metathorax odor gland secretions of the female and male insects of Plautia stali. [Result] There were a total of 38 compounds in the metathorax odor gland secretions of Plautia stali.Among them, 35 compounds were detected in the metathorax odor gland secretions of the females, and 19 compounds were detected in the metathorax odor gland secretions of the males.There were a total of 16 compounds shared between females and males, with 19 compounds only present in females and 3 compounds only present in males. [Conclusion] There are significant differences in the types of compounds in the metathorax odor gland secretions between females and males of Plautia stali. The chemical composition of the metathorax odor gland secretions in females was more complex than that in males, which may be related to the more complex physiological and ecological functions of the metathorax odor gland secretions in females. [ABSTRACT FROM AUTHOR]

Effective suppression of gastric acid secretion promotes healing of erosive esophagitis. Treatment guidelines recommend proton pump inhibitors (PPIs) and histamine H2–receptor antagonists (H2RAs). Emerging evidence also supports potassium‐competitive acid blockers (P‐CABs). The aim was to construct a mathematical model to examine the relationship between pH holding time ratios (HTRs) and erosive esophagitis healing rates with H2RAs, PPIs and P‐CABs. By literature search, we identified studies of H2RAs, PPIs or P‐CABs that reported mean pH >4 HTRs at steady state (days 5–8) and erosive esophagitis healing rates after 4 and/or 8 weeks. We aggregated treatments by drug class and developed a non‐linear, mixed‐effects model to explore the relationship between pH >4 HTRs and healing rates. The pH dataset included 82 studies (4297 participants; 201 dosage arms); healing rate data came from 103 studies (43,417 patients; 196 treatment arms). P‐CABs achieved the longest periods with intragastric pH >4, and the highest healing rates after 4 and 8 weeks. The predicted probabilities of achieving ≥90% healing rates at 8 weeks were 74.1% for P‐CABs, 17.3% for PPIs and 0% for H2RAs. P‐CABs provide the longest duration with intragastric pH >4 and, accordingly, the highest healing rates of erosive esophagitis. [ABSTRACT FROM AUTHOR]