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Biostimulation aims to optimize reproductive parameters as part of animal management practices by modulating animal sensory systems. Chemical signals, mostly known as pheromones, have a great potential in this regard. This study was conducted to determine the influence of short-term male rabbit exposure to different biological secretions, potentially pheromone-mediated, on reproductive parameters of males. Four groups of 18 males each were exposed to A) doe urine, B) 2-phenoxyethanol, C) doe vaginal swab, and D) distilled water (control), three times over a 2.5h exposure window, just before semen collection. Semen volume, sperm concentration and motility, as well as subpopulation analysis of the spermatozoa were assessed for each condition. Additionally, testosterone levels in blood samples were monitored at five time points over the 2.5 h exposure window. We found a higher percentage of motile, progressive, fast progressive and mid-progressive spermatozoa in any of the three experimental groups compared to the control group. In contrast, the semen volume and the percentage of immotile and non-progressive spermatozoa was generally higher in the control group. We then identified a higher proportion of a subpopulation of fast and progressive spermatozoa in groups A, B, and C compared to group D. Our data indicates that sperm motility increases when animals are exposed to specific biological fluids potentially containing pheromones, and that an increase in sperm volume does not correlate with an increase in spermatozoa concentration, progressiveness, and speed. Finally, no differences in testosterone levels were found among comparisons, although males of groups A and C (exposed to natural female biological fluids) showed a tendency towards higher testosterone levels. In conclusion, our results indicate that rabbit sperm quality increases upon exposure to the biological secretions proposed, thereby supporting further investigation into their molecular identity. This exploration could eventually pave the way for implementing the use of pheromones in rabbit husbandry to enhance reproductive and productive parameters in farmed rabbits. • Buck semen quality increases upon exposure to specific female biological fluids. • Doe urine, vaginal fluid, and 2-phenoxyethanol are potential biostimulators. • Concentration and progressiveness of spermatozoa were enhanced in exposed rabbits. • Exposure to doe urine and vaginal fluid tended to increase testosterone levels. • Pheromone biostimulation is a natural method to improve animal production. [ABSTRACT FROM AUTHOR]

Experimental studies have shown that V-type ATPase-driven H+ secretion is dependent on transepithelial voltage. On this basis, the "voltage hypothesis" of urinary acidification by the collecting duct was derived. Accordingly, it has been supposed that the lumen-negative potential created by the reabsorption of Na+ via the epithelial Na+ channel (ENaC) enhances electrogenic H+ secretion via V-type H+-ATPase. This concept continues to be widely used to explain acid/base disorders. Importantly, however, a solid proof of principle for the voltage hypothesis in physiologically relevant situations has not been reached. Rather, it has been challenged by recent in vivo functional studies. In this review, we outline the arguments and experimental observations explaining why voltage-coupled H+ secretion in the collecting duct often appears poorly applicable for rationalizing changes in H+ secretion as a function of more or less ENaC function in the collecting duct. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii, the causative agent of toxoplasmosis, infects cells and replicates inside via the secretion of factors stored in specialized organelles (rhoptries, micronemes, and dense granules) and the capture of host materials. The genesis of the secretory organelles and the processes of secretion and endocytosis depend on vesicular trafficking events whose molecular bases remain poorly known. Notably, there is no characterization of the BAR (Bin/Amphiphysin/Rvs) domaincontaining proteins expressed by T. gondii and other apicomplexans, although such proteins are known to play critical roles in vesicular trafficking in other eukaryotes. Here, by combining structural analyses with in vitro assays and cellular observations, we have characterized TgREMIND (regulators of membrane interacting domains), involved in the genesis of rhoptries and dense granules, and TgBAR2 found at the parasite cortex. We establish that TgREMIND comprises an F-BAR domain that can bind curved neutral membranes with no strict phosphoinositide requirement and exert a membrane remodeling activity. Next, we establish that TgREMIND contains a new structural domain called REMIND, which negatively regulates the membranebinding capacities of the F-BAR domain. In parallel, we report that TgBAR2 contains a BAR domain with an extremely basic membrane-binding interface able to deform anionic membranes into very narrow tubules. Our data show that T. gondii codes for two atypical BAR domain-containing proteins with very contrasting membrane-binding properties, allowing them to function in two distinct regions of the parasite trafficking system. [ABSTRACT FROM AUTHOR]

Background Essentially all individuals with multiple autoantibodies will develop clinical type 1 diabetes. Multiple autoantibodies (AABs) and normal glucose tolerance define stage 1 diabetes; abnormal glucose tolerance defines stage 2. However, the rate of progression within these stages is heterogeneous, necessitating personalized risk calculators to improve clinical implementation. Methods We developed 3 models using TrialNet's Pathway to Prevention data to accommodate the reality that not all risk variables are clinically available. The small model included AAB status, fasting glucose, hemoglobin A1c, and age, while the medium and large models added predictors of disease progression measured via oral glucose tolerance testing. Findings All models markedly improved granularity regarding personalized risk missing from current categories of stages of type 1 diabetes. Model-derived risk calculations are consistent with the expected reduction of risk with increasing age and increase in risk with higher glucose and lower insulin secretion, illustrating the suitability of the models. Adding glucose and insulin secretion data altered model predicted probabilities within stages. In those with high 2-hour glucose, a high C-peptide markedly decreased predicted risk; a lower C-peptide obviated the age-dependent risk of 2-hour glucose alone, providing a more nuanced estimate of the rate of disease progression within stage 2. Conclusion While essentially all those with multiple AABs will develop type 1 diabetes, the rate of progression is heterogeneous and not explained by any individual single risk variable. The model-based probabilities developed here provide an adaptable personalized risk calculator to better inform decisions about how and when to monitor disease progression in clinical practice. [ABSTRACT FROM AUTHOR]

The production of recombinant proteins in Escherichia coli is often challenged by cytoplasmic expression due to proteolytic degradation and inclusion body formation. Extracellular expression can overcome these problems by simplifying downstream processing and improving protein yields. This study aims to compare the efficiency of two Bacillus subtilis chitosanase signal peptides in mediating extracellular secretion in E. coli. We identified a naturally occurring mutant signal peptide (mCsn2-SP) from B. subtilis CH2 chitosanase (CH2CSN), which is characterized by a deletion of six amino acids in the N-region relative to the signal peptide (Csn1-SP) from B. subtilis CH1 chitosanase (CH1CSN). The CH1CSN and CH2CSN genes were cloned into the pET-11a vector and protein secretion was evaluated in E. coli BL21(DE3) host cells. Expression was induced with 0.1 mM and 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 30 °C for one and three days. CH2CSN showed higher secretion levels compared to CH1CSN under all experimental conditions, especially with 0.1 mM IPTG induction for 3 days, which resulted in a 2.37-fold increase in secretion. Furthermore, it was demonstrated that mCsn2-SP is capable of secreting human Cu,Zn-superoxide dismutase (hSOD) in E. coli BL21(DE3) and successfully translocating it to the periplasmic region. This study represents the inaugural investigation into the utilisation of a naturally modified signal peptide, thereby corroborating the assertion that signal peptide deletion variants can influence protein secretion efficiency. Furthermore, the findings substantiate the proposition that such variants can serve as a viable alternative for the secretion of heterologous proteins in E. coli. [ABSTRACT FROM AUTHOR]

Background and Objectives: A similar secretory pattern of prolactin (PRL) and growth hormone (GH) during the menstrual cycle has been reported in response to a high dose of ghrelin in adult healthy women. The present study aimed to assess the pattern of PRL and GH secretions in response to a submaximal dose of ghrelin during different menstrual phases in adult healthy women. Materials and Methods: Eight female subjects with normal cyclicity were enrolled. These subjects were either in the early follicular (EF), late follicular (LF), or mid-luteal (ML) phase of their cycles. Each subject received an IV dose of normal saline (2 mL each time) during the first cycle after enrollment, followed by an IV dose of ghrelin (0.30 μg/kg bw) in the second cycle. The blood samples were collected before and after the IV dosage at −15, 0, 15, 30, 45, 60, 75, 90 and 120 min, where 0 min denotes the time of IV dosage. Results: All the enrolled subjects experienced ovulatory cycles as assessed by increased serum progesterone levels. Serum estradiol levels were significantly higher in the LF than in the EF (p < 0.001) and ML phases (p < 0.01); these levels were also significantly higher in the ML than in the EF phase (p < 0.01). The administration of saline did not affect serum GH or PRL levels. Following the administration of ghrelin, plasma ghrelin levels and serum GH levels increased significantly (p < 0.001). The response amplitude of GH was similar in the three stages of cycle 2. In contrast to GH, the ghrelin injection induced a significant increase in serum PRL levels only in the LF phase (p < 0.05). Conclusions: These results show, for the first time, a different pattern of PRL and GH in response to a submaximal dose of ghrelin during the normal menstrual cycle. It is suggested that the ghrelin threshold for pituitary lactotrophs is higher than for somatotrophs and that, unlike GH, ghrelin-stimulated PRL secretion can be influenced by ovarian steroids. [ABSTRACT FROM AUTHOR]

Copyright of Retos: Nuevas Perspectivas de Educación Física, Deporte y Recreación is the property of Federacion Espanola de Asociaciones de Docentes de Educacion Fisica and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Large quantities of artificial single-stranded DNA (ssDNA) with user-defined sequences are increasingly required to exploit the potential of DNA nanotechnology. Cross-contamination-free ssDNA production can be achieved using Escherichia coli with an optimized helper plasmid in high-cell-density cultivation via the secretion of phagemid particles containing ssDNA with user-defined sequences. In our study, we aimed to reduce the number of phagemid particles for the initiation of ssDNA production. We tested different infection densities, ranging from a multiplicity of infection (MOI) of 10−6–10−2 tfu cfu−1 at the start of the initial batch phase in a 2.5 L stirred tank bioreactor. A MOI of 10−3 tfu cfu−1 was the best compromise between process time and ssDNA concentration. Early initiation of ssDNA production with low MOI reduced the number of phagemid particles by a factor of 250,000. The early infection strategy was successfully scaled up to the 25 L scale, resulting in ssDNA concentrations of >100 mg L−1 within a process time of one day. Transferring the infection strategy to a 1000 L scale gained 65 mg L−1 ssDNA because of incomplete initial infection. The versatility of the early infection strategy was further proven with a second prolonged, user-defined ssDNA sequence. [ABSTRACT FROM AUTHOR]

Background: Skin is the largest organ in the human body. Some skin parameters like moisturization and sebum secretion play a vital role in the skin's functioning. This study aims to assess the effects of topical chemical peels of different concentrations and pH, applied manually and with ultrasounds, on the level of hydration, erythema, pigmentation, and sebum secretion of the skin. Methods: The study involved 90 Caucasian females, aged 25 to 59, with dry, dehydrated skin, skin with erythema or pigmentation disorders. The patients were randomly divided into three equal groups. The subjects from Group A were applied 10% mandelic acid with 25% gluconolactone of pH 4.0 manually. In Group B, 40% mandelic acid of pH 1.5 was used. The subjects from Group C were applied 10% mandelic acid with 25% gluconolactone of pH 4.0 via sonophoresis. A series of six procedures in weekly intervals was performed. Skin functional parameters (skin hydration, erythema, and melanin indicators) were taken before the first procedure, after 14 days, 28 days, and 42 days. Results: In Group A, the level of moisturization of the skin increased statistically significantly (p = 0.0100) however, the sebum secretion and erythema did not change. In Group B, the level of moisturization improved statistically significantly, as well as erythema (p = 0.0001). Sebum secretion in the final measurement increased. The moisturization and erythema in Group C did not differ statistically significantly. On the other hand, the sebum secretion increased significantly. Conclusions: Very superficial chemical peels significantly alter selected skin parameters. AHAs and PHAs applied using the ultrasound method do not affect the level of hydration, erythema, or pigmentation of the skin. [ABSTRACT FROM AUTHOR]

Copyright of Algerian Historical Journal is the property of Algerian Historical Journal and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background/Objectives: Prematurity is a leading cause of infant mortality and mechanical ventilation increases respiratory complication risk. The effects of secretion removal techniques in premature infants remain a topic of ongoing debate. The aim of the study is to compare two secretion removal techniques in premature infants on mechanical ventilation; Methods: The participants were randomized into conventional chest physiotherapy (CPT; n = 22) or expiratory flow increase technique (EFIT; n = 21) groups. Each participant completed four sessions on consecutive days with a minimum of one and a maximum of two sessions per day. We assessed peripheral oxygen saturation (SpO2), heart (HR) and respiratory rates (RR), tidal volume (VT), and pain levels at specific time points: before the intervention, immediately after the intervention, 5 min after the intervention, and 10 min after the intervention. The partial Eta squared (ŋp2) was reported to measure the effect size; Results: HR and RR increased post-intervention in both groups (p < 0.001; HR ŋp2 = 0.51; RR ŋp2 = 0.38); post hoc comparisons showed EFIT group decreased RR from the first to last session (p = 0.045). Both groups exhibited increased VT and SpO2 in all sessions (p < 0.001; VT ŋp2 = 0.40; SpO2 ŋp2 = 0.50). The EFIT group had higher SpO2 values (p = 0.013; ŋp2 = 0.15) and lower pain scores (p < 0.001; ŋp2 = 0.46) post-intervention compared to CPT; Conclusions: CPT and EFIT resulted in similar effects on short-term respiratory parameters and heart rate; however, EFIT had advantages, including lower RR, higher SpO2, and reduced pain, suggesting it may be a more effective, comfortable neonatal respiratory treatment. [ABSTRACT FROM AUTHOR]

Background/Objectives: The respiratory system is prone to infectious diseases, especially in children below five years of age. Upper respiratory tract infections in children are often associated with Eustachian tube dysfunction and complicated by otitis media with effusion (OME), an inflammatory process within the middle ear, which can lead to hearing loss. Treatment for these infections involves a combination of medication and symptom relief, depending on the severity and cause of the infection. In recent years, natural therapeutic drugs derived from herbal medicines have been gaining popularity in treating various pathologies. Bromelain, one of the most studied natural compounds, has been investigated extensively due to its numerous pharmacological properties, offering a potential new avenue for treatment. Based on these promising findings, our study was designed to examine the efficacy of supplementation with bromelain in countering symptoms associated with OME. Methods: This study was conducted on data acquired from medical records from the Section of Audiology of the University of Palermo, focusing on the period of January 2022 to June 2023 and selecting 224 children (age range 1–8 years), namely 174 males and 50 females, who were evaluated for presumed OME at the audiology pediatric ambulatory. All patients selected before initiating pharmacological treatment underwent thorough screening regarding the functionality of the tympanic cavities, otoacoustic emissions, the auditory threshold, and the ear canal's integrity. Results: The preliminary findings of this study are significant, demonstrating that supplementation with bromelain led to notable improvements in the symptoms accompanying OME after 15 days and 60 days of therapy. Notably, patients who received the bromelain supplement reported reduced mucus secretions and improved auditory function. Conclusions: These results underscore the potential of naturally occurring compounds as adjuvants to standard therapeutic strategies in treating OME. [ABSTRACT FROM AUTHOR]

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.

Abstract 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 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 Background With a growing global population, the generation of plastic waste and the depletion of fossil resources are major concerns that need to be addressed by developing sustainable and efficient plastic recycling methods. Biocatalytic recycling is emerging as a promising ecological alternative to conventional processes, particularly in the recycling of polyethylene terephthalate (PET). However, cost-effective production of the involved biocatalyst is essential for the transition of enzymatic PET recycling to a widely used industrial technology. Extracellular enzyme production using established organisms such as Escherichia coli or Corynebacterium glutamicum offers a promising way to reduce downstream processing costs. Results In this study, we compared extracellular recombinant protein production by classical secretion in C. glutamicum and by membrane leakage in E. coli. A superior extracellular release of the cutinase ICCGDAQI was observed with E. coli in batch and fed-batch processes on a litre-scale. This phenomenon in E. coli, in the absence of a signal peptide, might be associated with membrane-destabilizing catalytic properties of the expressed cutinase. Optimisations regarding induction, expression temperature and duration as well as carbon source significantly enhanced extracellular cutinase activity. In particular, in fed-batch cultivation of E. coli at 30 °C with lactose as carbon source and inducer, a remarkable extracellular activity (137 U mL−1) and cutinase titre (660 mg L−1) were achieved after 48 h. Literature values obtained with other secretory organisms, such as Bacillus subtilis or Komagataella phaffii were clearly outperformed. The extracellular ICCGDAQI produced showed high efficacy in the hydrolysis of PET textile fibres, either chromatographically purified or unpurified as culture supernatant. In less than 18 h, 10 g L−1 substrate was hydrolysed using supernatant containing 3 mg cutinase ICCGDAQI at 70 °C, pH 9 with terephthalic acid yields of up to 97.8%. Conclusion Extracellular production can reduce the cost of recombinant proteins by simplifying downstream processing. In the case of the PET-hydrolysing cutinase ICCGDAQI, it was even possible to avoid chromatographic purification and still achieve efficient PET hydrolysis. With such production approaches and their further optimisation, enzymatic recycling of PET can contribute to a more efficient and environmentally friendly solution to the industrial recycling of plastics in the future.

Abstract 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 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 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 Background Corynebacterium glutamicum is an attractive host for secretory production of recombinant proteins, including high-value industrial enzymes and therapeutic proteins. The choice of an appropriate signaling peptide is crucial for efficient protein secretion. However, due to the limited availability of signal peptides in C. glutamicum, establishing an optimal secretion system is challenging. Result We constructed a signal peptide library for the isolation of target-specific signal peptides and developed a highly efficient secretory production system in C. glutamicum. Based on the sequence information of the signal peptides of the general secretion-dependent pathway in C. glutamicum, a synthetic signal peptide library was designed, and validated with three protein models. First, we examined endoxylanase (XynA) and one potential signal peptide (C1) was successfully isolated by library screening on xylan-containing agar plates. With this C1 signal peptide, secretory production of XynA as high as 3.2 g/L could be achieved with high purity (> 80%). Next, the signal peptide for ⍺-amylase (AmyA) was screened on a starch-containing agar plate. The production titer of the isolated signal peptide (HS06) reached 1.48 g/L which was 2-fold higher than that of the well-known Cg1514 signal peptide. Finally, we isolated the signal peptide for the M18 single-chain variable fragment (scFv). As an enzyme-independent screening tool, we developed a fluorescence-dependent screening tool using Fluorescence-Activating and Absorption-Shifting Tag (FAST) fusion, and successfully isolated the optimal signal peptide (18F11) for M18 scFv. With 18F11, secretory production as high as 228 mg/L was achieved, which was 3.4-fold higher than previous results. Conclusions By screening a fully synthetic signal peptide library, we achieved improved production of target proteins compared to previous results using well-known signal peptides. Our synthetic library provides a useful resource for the development of an optimal secretion system for various recombinant proteins in C. glutamicum, and we believe this bacterium to be a more promising workhorse for the bioindustry.

Effector secretion by different routes mediates the molecular interplay between host plant and pathogen, but mechanistic details in eukaryotes are sparse. This may limit the discovery of new effectors that could be utilized for improving host plant disease resistance. In fungi and oomycetes, apoplastic effectors are secreted via the conventional endoplasmic reticulum (ER)-Golgi pathway, while cytoplasmic effectors are packaged into vesicles that bypass Golgi in an unconventional protein secretion (UPS) pathway. In Magnaporthe oryzae, the Golgi bypass UPS pathway incorporates components of the exocyst complex and a t-SNARE, presumably to fuse Golgi bypass vesicles to the fungal plasma membrane. Upstream, cytoplasmic effector mRNA translation in M. oryzae requires the efficient decoding of AA-ending codons. This involves the modification of wobble uridines in the anticodon loop of cognate tRNAs and fine-tunes cytoplasmic effector translation and secretion rates to maintain biotrophic interfacial complex integrity and permit host infection. Thus, plant-fungal interface integrity is intimately tied to effector codon usage, which is a surprising constraint on pathogenicity. Here, we discuss these findings within the context of fungal and oomycete effector discovery, delivery, and function in host cells. We show how cracking the codon code for unconventional cytoplasmic effector secretion in M. oryzae has revealed AA-ending codon usage bias in cytoplasmic effector mRNAs across kingdoms, including within the RxLR-dEER motif-encoding sequence of a bona fide Phytophthora infestans cytoplasmic effector, suggesting its subjection to translational speed control. By focusing on recent developments in understanding unconventional effector secretion, we draw attention to this important but understudied area of host-pathogen interactions. [Figure: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Bone regeneration and repair are crucial to ambulation and quality of life. Factors such as poor general health, serious medical comorbidities, chronic inflammation, and ageing can lead to delayed healing and nonunion of fractures, and persistent bone defects. Bioengineering strategies to heal bone often involve grafting of autologous bone marrow aspirate concentrate (BMAC) or mesenchymal stem cells (MSCs) with biocompatible scaffolds. While BMAC shows promise, variability in its efficacy exists due to discrepancies in MSC concentration and robustness, and immune cell composition. Understanding the mechanisms by which macrophages and lymphocytes – the main cellular components in BMAC – interact with MSCs could suggest novel strategies to enhance bone healing. Macrophages are polarized into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, and influence cell metabolism and tissue regeneration via the secretion of cytokines and other factors. T cells, especially helper T1 (Th1) and Th17, promote inflammation and osteoclastogenesis, whereas Th2 and regulatory T (Treg) cells have anti-inflammatory pro-reconstructive effects, thereby supporting osteogenesis. Crosstalk among macrophages, T cells, and MSCs affects the bone microenvironment and regulates the local immune response. Manipulating the proportion and interactions of these cells presents an opportunity to alter the local regenerative capacity of bone, which potentially could enhance clinical outcomes. Cite this article: Bone Joint Res 2024;13(9):462–473.

Complexin determines magnitude and kinetics of synchronized secretion, but the underlying molecular mechanisms remained unclear. Here, we show that the hydrophobic face of the amphipathic helix at the C-terminus of Complexin II (CpxII, amino acids 115–134) binds to fusion-promoting SNARE proteins, prevents premature secretion, and allows vesicles to accumulate in a release-ready state in mouse chromaffin cells. Specifically, we demonstrate that an unrelated amphipathic helix functionally substitutes for the C-terminal domain (CTD) of CpxII and that amino acid substitutions on the hydrophobic side compromise the arrest of the pre-fusion intermediate. To facilitate synchronous vesicle fusion, the N-terminal domain (NTD) of CpxII (amino acids 1–27) specifically cooperates with synaptotagmin I (SytI), but not with synaptotagmin VII. Expression of CpxII rescues the slow release kinetics of the Ca2+-binding mutant Syt I R233Q, whereas the N-terminally truncated variant of CpxII further delays it. These results indicate that the CpxII NTD regulates mechanisms which are governed by the forward rate of Ca2+ binding to Syt I. Overall, our results shed new light on key molecular properties of CpxII that hinder premature exocytosis and accelerate synchronous exocytosis. [ABSTRACT FROM AUTHOR]

Background: Osteonecrosis of the femoral head (ONFH) primarily affects young individuals and is a leading cause of total hip arthroplasty in this population. Joint-preserving regenerative therapies involving core decompression (CD), enhanced with cells, growth factors, and bone substitutes, have been developed but lack extensive validation. Exosomes are emerging as a promising regenerative therapy. Human adipose stem cell (hADSC)-derived exosomes exhibit angiogenic and wound-healing effects on damaged and diseased tissues, suggesting their potential efficacy in treating early-stage ONFH. We aimed to investigate the efficacy of hADSC-derived exosomes based on CD in a medium-sized animal model (rabbit). Methods: Exosomes were extracted using the ultrafiltration filter technique from the culture supernatants of two types of hADSCs. Characterization of exosomes was performed through nanoparticle tracking analysis, transmission electron microscopy, and the detection of specific biomarkers (CD9, CD63, and CD81) by western blotting. Eighteen rabbits underwent surgical vascular occlusion and intramuscular corticosteroid injections to induce ONFH. Concurrently, CD treatment with local administration of hADSC-derived exosomes (exosome group) or saline (control group) was performed. Femoral heads were harvested at 4, 8, and 12 weeks postoperatively and evaluated using micro-computed tomography and tissue staining to assess the protective effects on osteonecrosis, angiogenesis, and osteogenesis. Results: Exosomes had average particle concentrations of 1.8 × 1012 or 1.8 × 109 particles/mL, with particle size distributions averaging 61.2 ± 14.7 or 123.1 ± 46.3 nm, and were confirmed by specific biomarkers. The exosome group exhibited a significant reduction in the severe progression of ONFH to stages 3 or 4 of the modified Ficat and Arlet classification, compared to the control group, which had four cases of stages 3 or 4. The exosome group showed significantly fewer empty lacunae in the subchondral bone area (p < 0.05) and significantly less articular cartilage injury (p < 0.05) compared to the corresponding in the control group. There were no significant differences in the microvessel number, bone trabecular structure, or volume of new bone in the medial region of the CD. Conclusions: hADSC-derived exosomes can prevent the progression of ONFH by inhibiting osteonecrosis and cartilage damage. The ultrafiltration filter technique is effective for exosome extraction, indicating that exosomes hold potential as a therapeutic agent for ONFH. [ABSTRACT FROM AUTHOR]

The use of cosmetic substances in communication is widespread in animals. Birds, for instance, use their uropygial secretion as a cosmetic in scenarios of sexual selection and parent-offspring communication. This secretion harbours symbiotic bacteria that could mediate the synthesis of pigments for cosmetic colouration. Here, we investigate the association between bacteria and the conspicuous yellow secretion used by spotless starling (Sturnus unicolor) nestlings to stain their mouths, and hypothesize a possible role of bacteria in the colour production. We also experimentally explore how nestling oxidizing condition influences the microbiota, suggesting that the possible bacterial-mediated coloured secretion acts as a reliable honest signal. An antioxidant supplementation experiment, previously known to affect secretion and mouth colouration, was conducted to assess its impact on the microbial community of secretions from control and experimental siblings. Antioxidant supplementation increased richness and phylogenetic diversity of the secretion's microbiota. Moreover, the microbiota's alpha and beta diversity, and the abundance of two bacterial genera (Parabacteroides and Pseudogracilibacillus), correlated with secretion colour. These findings demonstrate that antioxidant condition influences the gland microbiota of starling nestlings, and suggest a link between bacteria and the colouration of their cosmetic secretion. Alternative explanations for the detected links between bacteria and colouration are discussed. [ABSTRACT FROM AUTHOR]

Membrane proteins are sorted to the plasma membrane via Golgi-dependent trafficking. However, our recent studies challenged the essentiality of Golgi in the biogenesis of specific transporters. Here, we investigate the trafficking mechanisms of membrane proteins by following the localization of the polarized R-SNARE SynA versus the non-polarized transporter UapA, synchronously co-expressed in wild-type or isogenic genetic backgrounds repressible for conventional cargo secretion. In wild-type, the two cargoes dynamically label distinct secretory compartments, highlighted by the finding that, unlike SynA, UapA does not colocalize with the late-Golgi. In line with early partitioning into distinct secretory carriers, the two cargoes collapse in distinct ER-Exit Sites (ERES) in a sec31ts background. Trafficking via distinct cargo-specific carriers is further supported by showing that repression of proteins essential for conventional cargo secretion does not affect UapA trafficking, while blocking SynA secretion. Overall, this work establishes the existence of distinct, cargo-dependent, trafficking mechanisms, initiating at ERES and being differentially dependent on Golgi and SNARE interactions. [ABSTRACT FROM AUTHOR]

Background Aim Methods Results Conclusion Idiopathic granulomatous mastitis (IGM) is a rare, chronic inflammatory, and benign breast disease. Its unclear etiology may involve autoimmune reactions, secretion‐related factors, and microorganisms.To analyze data from our IGM patient series and compare potential etiological factors.We prospectively collected data using follow‐up forms for patients diagnosed with IGM at our breast clinic from September 2014 to December 2020 and analyzed it retrospectively.The study cohort included 220 patients, with a median age of 34 years (range: 20–58). A majority, 217 patients (98.6%), reported a history of breastfeeding, with a median duration of 36 months (range: 0–156). The median interval between the last breastfeeding session and disease onset was 25 (25th–75th percentiles: 15–44) months. Additionally, 116 patients (53.5%) reported breastfeeding‐related problems. In 41 surgical patients, dense milky‐brown debris in the breast ducts was noted. Although no independent seasonal fluctuations in disease onset were detected, a reduction in IGM cases during the summer months was found to correlate with a general decrease in all breast clinic visits.Most patients reported recent breastfeeding and half experienced related problems, supporting the secretion theory's relevance in IGM's etiology. The absence of seasonal fluctuations suggests that secretion‐related factors may be more central to IGM development than autoimmunity or infections. These findings offer crucial insights for future research into IGM's complex causes. [ABSTRACT FROM AUTHOR]

Simple Summary: Interactions between breast cancer (BCa) cells and carcinoma-associated fibroblasts (CAFs) in the tumor microenvironment (TME) are critical for cancer development and progression and can mitigate responses to therapies. Historically, it has been difficult to replicate in vivo long-term interactions between BCa cells and CAFs for in vitro studies and to define the precise roles of CAFs in BCa cell progression. Our novel microfluidic-capable culture devices called TAME (tissue architecture and microenvironment engineering) devices enable us to study cell-cell interactions of human breast cancer (BCa) cells and human CAFs through their secretome in 3D cultures for extended periods, up to and beyond 70 days. Using these systems, we observed that CAFs enhance BCa cell progression to an invasive phenotype. Moreover, secretome-mediated reciprocal interactions of BCa cells and CAFs promote their migration toward each other, suggesting that targeting signaling pathways to mediate interactions between BCa cells and CAFs could be a potential therapeutic approach for the prevention of BCa progression. Background/Objectives: Carcinoma-associated fibroblasts (CAFs), a prominent cell type in the tumor microenvironment (TME), significantly contributes to cancer progression through interactions with cancer cells and other TME components. Consequently, targeting signaling pathways driven by CAFs has potential to yield new therapeutic approaches to inhibit cancer progression. However, the mechanisms underlying their long-term interactions with cancer cells in vivo remains poorly understood. Methods: To address this, we developed a three-dimensional (3D) parallel coculture model of human triple-negative breast cancer (TNBC) cells and CAFs using our innovative TAME devices. This model allowed for the analysis of TNBC paracrine interactions via their secretome over extended culture periods (at least 70 days). Results: Using TNBC cell lines (MDA-MB-231, MCF10.DCIS, and HCC70), we found that TNBC spheroids in 3D parallel cocultures with CAFs exhibited more pronounced invasive finger-like outgrowths than those in cocultures of TNBC cells and normal fibroblasts (NFs) over a period of 50–70 days. We also established that the CAF-derived secretome affects TNBC migration towards the CAF secretome region. Additionally, we observed a preferential migration of CAFs, but not NFs, toward TNBC spheroids. Conclusions: Overall, our results suggest that paracrine interactions between TNBC cells and CAFs enhance TNBC invasive phenotypes and promote reciprocal migration. [ABSTRACT FROM AUTHOR]

Apolipoprotein A-I (ApoA-I), the primary component of high-density lipoprotein (HDL) cholesterol primes β-cells to increase insulin secretion, however, the mechanisms involved are not fully defined. Here, we aimed to confirm ApoA-I receptors in β-cells and delineate ApoA-I-receptor pathways in β-cell insulin output. An LRC-TriCEPS experiment was performed using the INS-1E rat β-cell model and ApoA-I for unbiased identification of ApoA-I receptors. Identified targets, alongside ATP binding cassette transporter A1 (ABCA1) (included control) were silenced in the same cells, and insulin secretion (ELISA) and mitochondrial metabolism (seahorse) were assessed with/without ApoA-I priming. Human β-cell expression data was used to investigate ApoA-I receptor pathways in type 2 diabetes (T2D). Scavenger receptor B1 (SR-BI) and regulator of microtubule dynamics 1 were identified as ApoA-I targets. SR-BI or ABCA1 silencing abolished ApoA-I induced increases in insulin secretion. ApoA-I priming increased mitochondrial OXPHOS, however this was greatly attenuated with SR-BI or ABCA1 silencing. Supporting this, human β-cell expression data investigations found SR-BI and ABCA1 to be correlated with genes associated with mitochondrial pathways. In all, SR-BI and ABCA1 correlated with 73 and 3 genes differentially expressed in T2D, respectively. We confirm that SR-BI and ABCA1 are the primary β-cell ApoA-I receptors and demonstrate that ApoA-I priming enhances β-cell insulin secretion via the upregulation of mitochondrial metabolism through ApoA-I-SR-BI and ApoA-I-ABCA1 pathways. We propose that SR-BI relies on mitochondrial and exocytotic pathways, while ABCA1 depends solely on mitochondrial pathways. Our findings uncover new targets in ApoA-I β-cell mechanism for T2D therapies. [ABSTRACT FROM AUTHOR]

Microbial communities are shaped by cell-cell interactions. Although archaea are often found in associations with other microorganisms, the mechanisms structuring these communities are poorly understood. Here, we report on the structure and function of haloarchaeal contractile injection systems (CISs). Using a combination of functional assays and time-lapse imaging, we show that Halogeometricum borinquense exhibits antagonism toward Haloferax volcanii by inducing cell lysis and inhibiting proliferation. This antagonism is contact-dependent and requires a functional CIS, which is encoded by a gene cluster that is associated with toxin-immunity pairs. Cryo-focused ion beam milling and imaging by cryo-electron tomography revealed that these CISs are bound to the cytoplasmic membrane, resembling the bacterial type six secretion systems (T6SSs). We show that related T6SS gene clusters are conserved and expressed in other haloarchaeal strains, which exhibit antagonistic behavior. Our data provide a mechanistic framework for understanding how archaea may shape microbial communities and affect the food webs they inhabit. [ABSTRACT FROM AUTHOR]

CpG ODN2006 is widely used both in vitro and in vivo to achieve B cell activation and has been previously applied in clinical trials as an adjuvant and anti-cancer agent. Recent studies have demonstrated the benefit of combining CpG ODN2006 with α-IgM antibodies to obtain optimal B cell activation in vitro. In this study, we expanded the knowledge of how both agents affect other types of peripheral blood mononuclear cells (PBMCs), thereby highlighting beneficial and potentially unfavorable properties of the combination of CpG ODN2006 and α-IgM when applied beyond isolated B cells. We elucidated the effects of both compounds on mixed PBMCs, as well as on B cell- and monocyte-depleted PBMCs, allowing us to distinguish between direct effects and indirect influences mediated by other interacting immune cells. Flow cytometry was used to measure the expression of surface markers and intracellular cytokines, while ELISA and multiplex assays were performed to determine cytokine secretion. Our results revealed that stimulation of mixed PBMCs with CpG ODN2006 and α-IgM strongly increased cytokine secretion, primarily originating from α-IgM-stimulated monocytes. Monocyte activation was confirmed by increased CD86 and HLA-DR expression and occurred independently of B cells. The high level of monocyte-derived cytokines after α-IgM exposure did not affect B cell activation. However, it represents a rather unfavorable property for clinical applications. In conclusion, α-IgM is a potent inducer of cytokine production in monocytes. Based on our findings we hypothesize that significant side effects on monocytes can occur when using α-IgM to enhance CpG ODN2006's efficacy on B cells, particularly in clinical settings. [ABSTRACT FROM AUTHOR]

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]

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]

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]

Simple Summary: Electrical stimulation-induced muscle contraction (ESMC) is a well-known rehabilitation method used to prevent muscle loss. During exercise, muscle contractions create strain on bones, which can lead to the release of undercarboxylated osteocalcin, a hormone produced by bones that is linked to improved insulin sensitivity and other health benefits. However, it is not clear whether the mechanical stress on bones caused by different frequencies of ESMC can effectively promote this hormone's release. In this study, we explored how bone strain from both low- and high-frequency ESMC impacts osteocalcin levels. Our results show that ESMC-induced bone strain does promote osteocalcin release, and that this increase is closely related to higher insulin levels. Interestingly, the frequency of stimulation plays a key role, with low-frequency ESMC being particularly effective. These findings suggest new possibilities for rehabilitation and training programs, as low-frequency electrical muscle stimulation is less painful while still producing frequent bone strain, making it a promising approach for improving health outcomes. Electrical stimulation-induced muscle contraction (ESMC) has demonstrated various physiological benefits, but its effects on the secretion of undercarboxylated osteocalcin (ucOC), a bone-derived cytokine, remain unclear. This study explored the relationship between ESMC, bone strain, and ucOC secretion through two experiments. In the first, young male Fischer 344 rats were divided into three groups: low-frequency ES (LF, 10 Hz), high-frequency ES (HF, 100 Hz), and control (CON). Acute 30-min transcutaneous ES was applied, and both bone strain and ucOC levels were measured. In the second experiment, rats underwent LF or HF long-term ES (two sessions per week for 4 weeks), with ucOC and insulin levels monitored. Results revealed a significant peak in ucOC at 6 h post-acute LF-ESMC. Despite HF-ESMC generating greater bone strain, LF-ESMC, with smaller but repetitive bone strain, proved more effective in stimulating ucOC secretion. In the long-term study, both ESMC groups exhibited early increases in ucOC, with a positive correlation to insulin levels. In conclusion, bone strain induced by ES-mediated muscle contraction promotes ucOC secretion, with both the magnitude and frequency of strain playing critical roles. [ABSTRACT FROM AUTHOR]

Filamentous plant pathogens threaten global food security and ecosystem resilience. In recent decades, significant strides have been made in deciphering the molecular basis of plant–pathogen interactions, especially the interplay between pathogens' molecular weaponry and hosts' defense machinery. Stemming from interdisciplinary investigations into the infection cell biology of filamentous plant pathogens, recent breakthrough discoveries have provided a new impetus to the field. These advances include the biophysical characterization of a novel invasion mechanism (i.e., naifu invasion) and the unraveling of novel effector secretion routes. On the plant side, progress includes the identification of components of cellular networks involved in the uptake of intracellular effectors. This exciting body of research underscores the pivotal role of logistics management by the pathogen throughout the infection cycle, encompassing the precolonization stages up to tissue invasion. More insight into these logistics opens new avenues for developing environmentally friendly crop protection strategies in an era marked by an imperative to reduce the use of agrochemicals. [ABSTRACT FROM AUTHOR]

Key Clinical Message: Syndrome of inappropriate antidiuretic hormone secretion (SIADH) induces hyponatremia accompanied by uric acid reduction. We report a case of a patient with small‐cell lung cancer who developed SIADH with hypouricemia after duloxetine treatment and discuss the potential underlying mechanisms. SIADH and hypouricemia improved after duloxetine was switched to mirogabalin. [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]

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]

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]

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]

Oral biotherapeutics hold significant promise, but their lack of controllability and targeting poses a major challenge, particularly for intestinal bacterial biotherapeutics. In response, we have developed a nanoencapsulation approach that responds to the release of enzyme activity in the organism and activates the enzyme in situ, allowing for controlled colonization of microbes in the gut. The nano-coating comprises a two-layer structure: an inner layer of polydopamine with photothermal and adhesive properties, and an outer layer of gelatin–sodium carboxymethylcellulose, which is hydrolyzed by cellulases in the gut following photothermal interaction with dopamine. We have successfully achieved controlled colonization of a wide range of microorganisms. Furthermore, in a diabetes model, this approach has had a profound impact on regulating glucagon-like peptide-1 (GLP-1) production, β-cell physiology, and promoting insulin secretion. This nanocoating is achieved by in situ activation of cellulase without the need for genetic or targeted molecular modification, representing a new paradigm and alternative strategy for microbial therapy. It not only enables precise and controlled colonization of probiotics but also demonstrates great potential for broader application in the field of oral biotherapy. We have developed a nano-encapsulation method that triggers enzyme activity in response to enzymatic activity, resulting in the controlled release and adhesion of a wide range of microorganisms in the gut. The nano coating comprises two layers: an inner layer of polydopamine with photothermal and adhesion properties, and an outer layer of a gelatin-sodium carboxymethylcellulose polymer, which can be hydrolyzed by cellulases in the intestine. Additionally, this method allows for the preparation of various microbial coatings. This approach holds significant promise for regulating GLP-1 production, the physiological function of pancreatic β-cells, and promoting insulin secretion in diabetes models. [Display omitted] [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]

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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]