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Our objective was to investigate whether the pulp and paper mill industry effluent could affect the testis and Sertoli cells in a fast exposure period. For this, the present study was carried out in immature rats at 10-day-old. Testis treated in vitro with 4% effluent for 1 h presented changes in energy metabolism in terms of a decrease in lactate content and glucose uptake. Elevation in GSH content, as an antioxidant defense mechanism, was also detected. Sertoli cells treated with 4% effluent for 1 hour showed alterations in the mitochondrial metabolism that favor the decoupling of oxidative phosphorylation and the generation of oxygen reactive species and also a time and concentration-dependent delay secretion of acidic vesicles. Our results showed that pollutants present in the pulp and paper mill effluents, in a short time of exposure, are capable of inducing alterations in important metabolic functions in the testis and in Sertoli cells that are crucial for the correct progression of spermatogenesis and fertility.

Paper wasps (Polistes dominula), parasitized by the strepsipteran Xenos vesparum, are castrated and desert the colony to gather on plants where the parasite mates and releases primary larvae, thus completing its lifecycle. One of these plants is the trumpet creeper Campsis radicans: in a previous study the majority of all wasps collected from this plant were parasitized and focused their foraging activity on C. radicans buds. The unexpected prevalence and unusual feeding strategy prompted us to investigate the influence of this plant on wasp behavior and physiology through a multidisciplinary approach. First, in a series of laboratory bioassays, we observed that parasitized wasps spent more time than non-parasitized ones on fresh C. radicans buds, rich of extra-floral nectaries (EFNs), while the same wasps ignored treated buds that lacked nectar drops. Then, we described the structure and ultra-structure of EFNs secreting cells, compatible with the synthesis of phenolic compounds. Subsequently, we analysed extracts from different bud tissues by HPLC-DAD-MS and found that verbascoside was the most abundant bioactive molecule in those tissues rich in EFNs. Finally, we tested the immune-stimulant properties of verbascoside, as the biochemical nature of this compound indicates it might function as an antibacterial and antioxidant. We measured bacterial clearance in wasps, as a proxy for overall immune competence, and observed that it was enhanced after administration of verbascoside—even more so if the wasp was parasitized. We hypothesize that the parasite manipulates wasp behavior to preferentially feed on C. radicans EFNs, since the bioactive properties of verbascoside likely increase host survival and thus the parasite own fitness.

Made available in DSpace on 2021-07-14T10:30:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-06. Added 1 bitstream(s) on 2021-07-14T11:31:52Z : No. of bitstreams: 1 S1678-91992005000200007.pdf: 240267 bytes, checksum: 7d9ed868a0d325f8cf8413feaecc45da (MD5) The venom glands are part of the most important defense weapon in Aculeata: the venom apparatus. The arrangement of these glands can vary among species, but in general they are composed of long secretory tubules connected to a muscular sac-like reservoir. Although the occurrence of these variations has been documented, many studies neglected the existence of a well-developed secretory portion in the lumen of the reservoir named convoluted gland. This study is an ultramorphological analysis of the venom glands and their histochemical relationship with the convoluted glands in the primitive social wasp Polistes versicolor. In this wasp, the venom glands are constituted by two tubular portions that penetrate individually in the venom reservoir, inside of which we can find the convoluted glands. Besides morphological differences in their cells, histochemical analysis of the venom and convoluted glands clearly show differences between them. While the venom glands indicate positive reaction only for proteins, the convoluted glands present positive reaction for proteins, neutral glycoconjugates, and lipids. The secretion of the convoluted gland cells may modify the compounds passing through the embedded tubular region. Universidade Estadual Paulista, Rio Claro Institute of Biosciences Universidade Estadual Paulista, Rio Claro Institute of Biosciences

Made available in DSpace on 2018-11-26T16:33:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-05-11 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Recent studies have reported incipient morphological caste dimorphism in the Van der Vecht organ size of some temperate Polistes paper wasps. Whether species other than the temperate ones show a similar pattern remains elusive. Here, we have studied some Neotropical Polistes species. By comparing females collected through the year, we showed caste related differences in the size of the Van der Vecht organ in P. ferreri (body size corrected Van der Vech organ size of queens = 0.45 +/- 0.06, workers = 0.38 +/- 0.07 mm(2), p = 0.0021), P. versicolor (body size corrected Van der Vech organ size of queens = 0.54 +/- 0.11, workers = 0.46 +/- 0.09 mm(2), p = 0.010), but not P. simillimus (body size corrected Van der Vech organ size of queens = 0.52 +/- 0.05, workers = 0.49 +/- 0.06 mm(2), p = 0.238). Therefore, it seems that queens and workers of some Neotropical Polistes have diverged in their ontogenic trajectory of the Van der Vecht organ size, providing clear evidence for incipient morphological caste dimorphism. As Polistes are distributed mostly in the tropics, we propose that physical caste differences may be widespread in the genus. Also, we highlight that morphological divergence in the queen-worker phenotypes may have started through differential selection of body structures, like the Van der Vecht organ. Univ Fed Vicosa, Dept Entomol, BR-36570000 Vicosa, MG, Brazil Univ Florence, Dipartimento Biol Evoluzionist Leo Pardi, Via Romana 17, I-50125 Florence, Italy Univ Fed Vicosa, Dept Biol Geral, BR-36570000 Vicosa, MG, Brazil Univ Estadual Paulista, Inst Biociencias Letras & Ciencias Exatas, Dept Zool & Bot, BR-15054000 Sao Paulo, Brazil Univ Estadual Paulista, Inst Biociencias Letras & Ciencias Exatas, Dept Zool & Bot, BR-15054000 Sao Paulo, Brazil CNPq: 143246/2011-9

YopQ in Yersinia enterocolitica (YopK in Yersinia pseudotuberculosis) is a type III secreted protein required for virulence of yersiniae. In this study YopQ expression, secretion and nucleotide sequences of the corresponding yopQ gene from different yersinia strains were analysed. The cytosolic accumulation differed significantly among serotypes of Y. enterocolitica. These differences might be attributable to variations in the nucleotide sequence and their consequences on mRNA secondary structure. An mRNA signal hypothesis has been proposed for YopQ, predicting the coupling of translation and secretion via an mRNA signal. This hypothesis claims a strictly co-translational secretion of YopQ without its intracellular accumulation. The presence of YopQ in the cytosol, even with a closed secretion apparatus, is demonstrated. Moreover, post-translational secretion of YopQ could be demonstrated. These findings do not support the mRNA signal hypothesis for co-translational secretion.

Determinants of Toxoplasma gondii virulence are still unknown, although genetic markers associated with T. gondii pathogenicity or host susceptibility to infection have been identified. To define indicator proteins of mouse virulence, type I strain parasites were attenuated by continuous passage in fibroblast culture and compared with the parental strain passaged in mice. The loss of acute virulence, evident by a 1000-fold higher pathogen dose causing 100% lethality in mice correlated with a less efficient infection of inflammatory cells at the site of inoculation, while parasite proliferation and invasiveness in vitro proved unimpaired. Infection with the attenuated parasites elicited earlier local interleukin-12 and strong interferon-gamma responses in vivo, although the activity that triggers interleukin-12 secretion in macrophages is reduced in the attenuated compared to the virulent strain variant. The interleukin-12-inducing T. gondii stimulus was identified as a protein(s) present in tachyzoite excretory products. Comparative proteome analysis combined with immunodetection and quantitation of a variety of T. gondii antigens indicated that the steady-state levels of actin, catalase, microneme protein 5, as well as dense granule proteins 1, 2, 3, 4, 5, 7, 8 and nucleoside triphosphate hydrolase 1 are decreased in the attenuated phenotype, whereas the surface antigen 1 and rhoptry protein 1 are produced at a similar level by virulent and attenuated parasites. In conclusion, these findings reveal a correlation between the efficient establishment of T. gondii infection in vivo and parasite synthesis of actin, catalase and several excretory proteins, and thus postulate a role for these molecules in acute virulence.

Two closely related pathogens, Bordetella pertussis and Bordetella bronchiseptica, share a number of virulence factors. Filamentous haemagglutinin (FHA) is widely regarded as the dominant adhesin of B. pertussis, and its multiple binding activities have been well characterized. This large protein is produced and secreted at high levels by B. pertussis and significantly lower levels by B. bronchiseptica strains. FHA secretion is mediated by a single outer-membrane accessory protein, FhaC. The genes encoding FHA and FhaC in B. bronchiseptica were characterized by sequencing and functional analyses and are highly similar to those of B. pertussis. The most distinctive feature of B. bronchiseptica FHA is additional repeats in the N-terminal portion of the predicted protein. Interestingly, a point mutation in the fhaB promoter region of the B. bronchiseptica GP1 isolate, relative to other isolates, was found to be detrimental to promoter activity and to FHA production. FhaC and the N-terminal secretion domain of FHA of B. bronchiseptica were fully functional for secretion in B. pertussis. Thus, the different levels of FHA secretion by these Bordetella species might reflect differences in physiology, composition and structure of cell envelope, or differential protein degradation. Characterization of FHA expression and function may provide clues as to the basis of host species tropism, tissue localization and receptor recognition.

The regulation of gastric acid secretion has been the subject of investigation for over a century. Inhibition of gastrin-induced acid secretion by the intestine-derived hormone secretin provides a classic physiological example of negative feedback in the gastrointestinal tract. A classic paper by Leonard R. Johnson and Morton I. Grossman clearly shows the ability of secretin to negatively regulate gastric acid secretion, providing students with an example of this feedback loop. In addition, this article demonstrates the step forward in gastrointestinal endocrinology that occurred when pure preparations of secretin and other gastrointestinal hormones first became available. The comparison of the effects of exogenous, purified secretin to the physiological stimulus of acid in the duodenum is an important example of how newly available reagents allow scientists such as Johnson and Grossman to clarify the mechanisms behind previously established processes. One or more figures from this classic paper can be used to give students insight into the role of secretin in the regulation of the function of the gastrointestinal tract and will also give students a clear example of how the careful experimentation and clear interest in gastrointestinal physiology led Johnson and Grossman to advance the field.

Phospholtptds in human liver and bile were analyzed using a neutron activation paper chromatographic technique. Phosphatidylserine, phosphoinositide, and choline plasmalogen as well as lecithin, phosphatidylethanolamine, and sphingomyelin were found to be present. Further evidence for the presence of lysolecithin in normal human bile is also presented. Similar results were obtained with rat bile using radioactive phosphorus. Lecithin comprises more than 80% of the phospholipids in human bile in contrast to liver, where it comprises 45%, indicating a selective secretion of phospholipid classes from liver into bile. The difference found in the fatty acid composition of the total phospholipids in human liver and bile by gas chromatography, coincides with the findings on the differences in phospholipid composition.

Spinal cord injury (SCI) is a traumatic disease resulting in neuronal injury. circRNAs are closely associated with human diseases. Nevertheless, the potential mechanism by which circRNAs impact SCI remains to be elucidated. The aim of this study was to investigate the regulatory roles of Circular RNAs (circRNAs) in SCI. The SCI mouse model and integrated bioinformatics analysis were used to identify the differentially expressed genes (DEGs). Functional enrichment analysis was conducted to study the related pathways. The circRNA-mediated ceRNA network and subnetwork was constructed based on circMir, TargetScan and miRanda. qRT–PCR, ELISA, flow cytometry, and luciferase reporter assays were carried out to validate the role of circ_0014637 (circ-Usp10) and microRNA(miR)-152-5p /CD84 in microglia. In all, 23 DE-circRNAs, 127 DE-miRNAs and 1327 DE-mRNAs were identified. We integrated these DEGs to construct a circRNA-miRNA-mRNA network. The circ-Usp10/miR-152-5p/CD84 axis was found to function in microglial activation. We also found that circ-Usp10 inhibited the secretion of proinflammatory factors in microglial BV2 cells. In addition, silencing circ-Usp10 significantly reduced the death of the neuronal cell line HT22. Taken together, we concluded that circ-Usp10 may function as a competing endogenous RNA (ceRNA) to promote microglial activation and induce neuronal death by targeting miR-152-5p/CD84. The circ-Usp10 may be a diagnostic biomarker and potential target for SCI therapy.

Many bacterial pathogens utilize a specialized secretion system, termed type III secretion system (T3SS), to translocate effector proteins into host cells and establish bacterial infection. The T3SS is anchored within the bacterial membranes and contains a long needle/filament that extends toward the host-cell and forms, at its distal end, a pore complex within the host membrane. The T3SS pore complex consists of two bacterial proteins, termed SctB and SctE, which have conflicting targeting indications; a signal sequence that targets to secretion to the extracellular environment via the T3SS, and transmembrane domains (TMDs) that target to membrane localization. In this study, we investigate whether the TMD sequences of SctB and SctE have special features that differentiate them from classical TMDs and allow them to escape bacterial membrane integration. For this purpose, we exchanged the SctB and SctE native TMDs for alternative hydrophobic sequences and found that the TMD sequences of SctB and SctE dictate membrane destination (bacterial versus host membrane). Moreover, we examined the role of the SctB TMD sequence in the activity of the full-length protein, post secretion, and found that the TMD does not serve only as a hydrophobic segment, but is also involved in the ability of the protein to translocate itself and other proteins into and across the host cell membrane.

Summary Plant pathogens exploit the extracellular matrix (ECM) to inhibit host immunity during their interactions with the host. The formation of ECM involves a series of continuous steps of vesicular transport events.To understand how such vesicle trafficking impacts ECM and virulence in the rice blast fungus Magnaporthe oryzae, we characterised MoSwa2, a previously identified actin‐regulating kinase MoArk1 interacting protein, as an orthologue of the auxilin‐like clathrin uncoating factor Swa2 of the budding yeast Saccharomyces cerevisiae.We found that MoSwa2 functions as an uncoating factor of the coat protein complex II (COPII) via an interaction with the COPII subunit MoSec24‐2. Loss of MoSwa2 led to a deficiency in the secretion of extracellular proteins, resulting in both restricted growth of invasive hyphae and reduced inhibition of host immunity. Additionally, extracellular fluid (ECF) proteome analysis revealed that MoSwa2‐regulated extracellular proteins include many redox proteins such as the berberine bridge enzyme‐like (BBE‐like) protein MoSef1. We further found that MoSef1 functions as an apoplastic virulent factor that inhibits the host immune response.Our studies revealed a novel function of a COPII uncoating factor in vesicular transport that is critical in the suppression of host immunity and pathogenicity of M. oryzae.

An oral antiviral against SARS-CoV-2 that also attenuates inflammatory instigators of severe COVID-19 is not available to date. Herein, we show that the apoA-I mimetic peptide 4 F inhibits Spike mediated viral entry and has antiviral activity against SARS-CoV-2 in human lung epithelial Calu3 and Vero-E6 cells. In SARS-CoV-2 infected Calu3 cells, 4 F upregulated inducers of the interferon pathway such as MX-1 and Heme oxygenase 1 (HO-1) and downregulated mitochondrial reactive oxygen species (mito-ROS) and CD147, a host protein that mediates viral entry. 4 F also reduced associated cellular apoptosis and secretion of IL-6 in both SARS-CoV-2 infected Vero-E6 and Calu3 cells. Thus, 4 F attenuates in vitro SARS-CoV-2 replication, associated apoptosis in epithelial cells and secretion of IL-6, a major cytokine related to COVID-19 morbidity. Given established safety of 4 F in humans, clinical studies are warranted to establish 4 F as therapy for COVID-19., GRAPHICAL ABSTRACT

Bone marrow mononuclear cell therapy improves cardiac repair after myocardial infarction (MI), in-part through signaling to resident cardiac cells, such as fibroblasts, which regulate scar formation. The efficacy of cell therapy declines with age, as aging of both donor and recipient cells decreases repair responses. Autophagy regulates the microenvironment by both extracellular vesicle (EV)-dependent and independent secretion pathways. We hypothesized that age-related autophagy changes in bone marrow cells (BMCs) alter paracrine signaling, contributing to lower cell therapy efficacy. Here, we demonstrate that young Sca-1+ BMCs exhibited a higher LC3II/LC3I ratio compared to old Sca-1+ BMCs, which was accentuated when BMCs were cultured under hypoxia. To examine the effect on paracrine signaling, old cardiac fibroblasts were cultured with conditioned medium (CM) from young and old Sca-1+ BMCs. Young, but not old CM, enhanced fibroblast proliferation, migration, and differentiation, plus reduced senescence. These beneficial effects were lost when autophagy or EV secretion in BMCs was blocked pharmacologically, or by siRNA knockdown of Atg7. Therefore, both EV-dependent and -independent paracrine signaling from young BMCs is responsible for paracrine stimulation of old cardiac fibroblasts. In vivo, bone marrow chimerism of old mice with young BMCs increased the number of LC3b+ cells in the heart compared to old mice reconstituted with old BMCs. These data suggest that the deterioration of autophagy with aging negatively impacts the paracrine effects of BMCs, and provide mechanistic insight into the age-related decline in cell therapy efficacy that could be targeted to improve the function of old donor cells.

Insect cells have recently proven to be an excellent platform for the high-level production of functional recombinant proteins. Autophagy is an important mechanism that promotes cell survival by eliminating damaged organelles and protein aggregates, and it also may influence recombinant protein production. In the present study, we compared the effects that autophagy inducers rapamycin, everolimus, and lithium chloride exert on recombinant lepidopteran insect cells that secrete an engineered antibody molecule. Compared with nontreatment, treatment with either rapamycin or everolimus prolonged cell growth to allow high cell density, improved viability in the declining phase, and then increased the yield of secreted antibodies. These positive effects appeared to be induced via autophagy since autophagosomes were clearly detected, particularly in cells treated with rapamycin or everolimus. Unlike rapamycin, another autophagy inducer, FK506, was ineffective in insect cells. The addition of an appropriate autophagy inducer may be effective in increasing the productivity of recombinant proteins in insect cells.

Collagen type IV (Col IV) is a basement membrane protein associated with early blood vessel morphogenesis and is essential for blood vessel stability. Defects in vascular Col IV deposition are the basis of heritable disorders, such as small vessel disease, marked by cerebral hemorrhage and drastically shorten lifespan. To date, little is known about how endothelial cells regulate the intracellular transport and selective secretion of Col IV in response to angiogenic cues, leaving a void in our understanding of this critical process. Our aim was to identify trafficking pathways that regulate Col IV deposition during angiogenic blood vessel development. We have identified the GTPase Rab10 as a major regulator of Col IV vesicular trafficking during vascular development using both in vitro imaging and biochemistry as well as in vivo models. Knockdown of Rab10 reduced de novo Col IV secretion in vivo and in vitro. Mechanistically, we determined that Rab10 is an indirect mediator of Col IV secretion, partnering with atypical Rab25 to deliver the enzyme lysyl hydroxylase 3 (LH3) to Col IV-containing vesicles staged for secretion. Loss of Rab10 or Rab25 results in depletion of LH3 from Col IV-containing vesicles and rapid lysosomal degradation of Col IV. Furthermore, we demonstrate that Rab10 is Notch responsive, indicating a novel connection between permissive Notch-based vessel maturation programs and vesicle trafficking. Our results illustrate both a new trafficking-based component in the regulated secretion of Col IV and how this vesicle trafficking program interfaces with Notch signaling to fine-tune basement membrane secretion during blood vessel development. Supplementary Information The online version contains supplementary material available at 10.1007/s10456-021-09791-9.

Mimecan encodes a secretory protein that is secreted into the human serum as two mature proteins with molecular masses of 25 and 12 kDa. We found 12-kDa mimecan to be a novel satiety hormone mediated by the upregulation of the expression of interleukin (IL)-1β and IL-6 in the hypothalamus. Mimecan was found to be expressed in human pituitary corticotroph cells and was up-regulated by glucocorticoids, while the secretion of adrenocorticotropic hormone (ACTH) in pituitary corticotroph AtT-20 cells was induced by mimecan. However, the effects of mimecan in adrenal tissue on the hypothalamic-pituitary-adrenal (HPA) axis functions remain unknown. We demonstrated that the expression of mimecan in adrenal tissues is significantly downregulated by hypoglycemia and scalded stress. It was down-regulated by ACTH, but upregulated by glucocorticoids through in vivo and in vitro studies. We further found that 12-kDa mimecan fused protein increased the corticosterone secretion of adrenal cells in vivo and in vitro. Interestingly, compared to litter-mate mice, the diurnal rhythm of corticosterone secretion was disrupted under basal conditions, and the response to restraint stress was stronger in mimecan knockout mice. These findings suggest that mimecan stimulates corticosterone secretion in the adrenal tissues under basal conditions; however, the down-regulated expression of mimecan by increased ACTH secretion after stress in adrenal tissues might play a role in maintaining the homeostasis of an organism's responses to stress.

Endoplasmic reticulum (ER) stress compromises the secretion of MUC2 from goblet cells and has been linked with inflammatory bowel disease (IBD). Although Bifidobacterium can beneficially modulate mucin production, little work has been done investigating the effects of Bifidobacterium on goblet cell ER stress. We hypothesized that secreted factors from Bifidobacterium dentium downregulate ER stress genes and modulates the unfolded protein response (UPR) to promote MUC2 secretion. We identified by mass spectrometry that B. dentium secretes the antioxidant γ-glutamylcysteine, which we speculate dampens ER stress-mediated ROS and minimizes ER stress phenotypes. B. dentium cell-free supernatant and γ-glutamylcysteine were taken up by human colonic T84 cells, increased glutathione levels, and reduced ROS generated by the ER-stressors thapsigargin and tunicamycin. Moreover, B. dentium supernatant and γ-glutamylcysteine were able to suppress NF-kB activation and IL-8 secretion. We found that B. dentium supernatant, γ-glutamylcysteine, and the positive control IL-10 attenuated the induction of UPR genes GRP78, CHOP, and sXBP1. To examine ER stress in vivo, we first examined mono-association of B. dentium in germ-free mice which increased MUC2 and IL-10 levels compared to germ-free controls. However, no changes were observed in ER stress-related genes, indicating that B. dentium can promote mucus secretion without inducing ER stress. In a TNBS-mediated ER stress model, we observed increased levels of UPR genes and pro-inflammatory cytokines in TNBS treated mice, which were reduced with addition of live B. dentium or γ-glutamylcysteine. We also observed increased colonic and serum levels of IL-10 in B. dentium- and γ-glutamylcysteine-treated mice compared to vehicle control. Immunostaining revealed retention of goblet cells and mucus secretion in both B. dentium- and γ-glutamylcysteine-treated animals. Collectively, these data demonstrate positive modulation of the UPR and MUC2 production by B. dentium-secreted compounds., Graphical abstract

Helicobacter pylori, a human pathogen that colonizes the stomach of 50% of the world's population, is associated with gastritis, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma. Diseases are characterized by severe inflammatory responses in the stomach that are induced by various chemokines and cytokines. Recently, oncostatin M (OSM), an IL-6 family cytokine, was detected in early gastric cancer biopsies. In this study, we showed that Helicobacter pylori induced secretion of OSM and overexpression of its type II receptor OSMRβ (OSM/OSMRβ) in a human gastric adenocarcinoma cell line (AGS) over 24 h of infection. Furthermore, we showed that the induction of OSM and OSMRβ was carried out by heat-sensitive Helicobacter pylori outer membrane vesicle (OMV) protein. Collectively, our results established, for the first time, a direct relation between Helicobacter pylori OMVs and the OSM/OSMRβ signaling axis.

Follicle development is a vital factor which determines the reproductive performance of poultry. Long noncoding RNAs (lncRNAs) have been reported to maintain animal reproductive function and play key roles in ovarian development and hormone secretion. But the regulatory mechanism of lncRNAs in duck follicle development has seldom been reported. In this study, to better explore the molecular mechanism of follicle development in ducks, the follicular lncRNA was sequenced and analyzed. A total of 9,551 lncRNAs were predicted in the duck follicles. Four hundred and forty-five lncRNAs were differentially expressed between the white follicles and yellow follicles. The results of our studies showed that lnc_13814 promoted cell apoptosis in duck GCs. Furthermore, the bioinformatics analysis results demonstrated that lnc_13814 was involved in a lncRNA-miRNA-mRNA coexpression network and it was observed to sponge two follicle-related miRNAs by a luciferase activity assay. Moreover, we found that overexpression of lnc_13814 significantly increased DNA damage inducible transcript 3 (DDIT3) expression and downregulated GCs apoptosis. Finally, we found that lnc_13814 directly binds to and inhibits apla-mir-145-4; then, lnc_13814 increases the expression of DDIT3 and up-regulates GCs apoptosis. Taken together, our findings demonstrate that lncRNAs have potential effects on duck ovarian follicles and lncRNAs may represent a new approach to understand follicular development.

Evidence suggests that Tripartite Motif Containing 11 (TRIM11) has pro-tumor activity in human non-small cell lung cancer (NSCLC). However, the roles and underlying mechanisms of TRIM11 in NSCLC have not yet been fully elucidated. In this work, human lung cancer cell lines (A549, H446, and H1975) were transfected with siRNA or lentiviruses to knockdown or overexpress TRIM11 and dual-specificity phosphatase 6 (DUSP6). The cell tumor response was assessed by determining the rate of proliferation, apoptosis, the uptake of 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl) amino]-2-deoxyglucose (2-NBDG), and the secretion of lactic acid (LD). Dominant-negative (dn)-MEK1 was used to block the ERK1/2 pathway. The mechanism was investigated by assessing the protein levels of pyruvate kinase isozymes M2 (PKM2) and DUSP6, as well as the activation of ERK1/2 pathway. Our data confirmed the anti-cancer effect of siTRIM11 in human lung cancer by demonstrating inhibition of cancer cell proliferation, induction of apoptosis, prevention of 2-NBDG uptake, suppression of LD production, and prevention of lung cancer cell (A549) tumorigenicity in nude mice. The underlying mechanism involved the up-regulation of DUSP6 and the inhibition of ERK1/2 activity. Overexpression of TRIM11 induced tumorigenesis of NSCLC in vitro, and the activation of ERK1/2 was significantly reversed by DUSP6 overexpression or additional dn-MEK1 treatment. Interestingly, we confirmed TRIM11 as a deubiquitinase that regulated DUSP6 accumulation, indicating that lung cancer progression is regulated via the DUSP6-ERK1/2 pathway. In conclusion, TRIM11 is an oncogene in NSCLC, likely through the DUSP6-mediated ERK1/2 signaling pathway.

Purpose Wound infections caused by Candida are life-threatening and difficult to treat. Echinocandins are highly effective against Candida species and recommended for treatment of invasive candidiasis. As penetration of echinocandins into wounds is largely unknown, we measured the concentrations of the echinocandins anidulafungin (AFG), micafungin (MFG), and caspofungin (CAS) in wound secretion (WS) and in plasma of critically ill patients. Methods We included critically ill adults with an indwelling wound drainage or undergoing vacuum-assisted closure therapy, who were treated with an echinocandin for suspected or proven invasive fungal infection. Concentrations were measured by liquid chromatography with UV (AFG and MFG) or tandem mass spectrometry detection (CAS). Results Twenty-one patients were enrolled. From eight patients, serial WS samples and simultaneous plasma samples were obtained within a dosage interval. AFG concentrations in WS amounted to Conclusion Echinocandin penetration into WS displays a high inter-individual variability. In WS of some of the patients, concentrations may be sub-therapeutic. However, the relevance of sub-therapeutic concentrations is unknown as no correlation has been established between concentration data and clinical outcome. Nevertheless, in the absence of clinical outcome studies, our data do not support the use of echinocandins at standard doses for the treatment of fungal wound infections, but underline the pivotal role of surgical debridement.

Dry age-related macular degeneration (AMD) is marked by the accumulation of extracellular and intracellular lipid-rich deposits within and around the retinal pigment epithelium (RPE). Inducing autophagy, a conserved, intracellular degradative pathway, is a potential treatment strategy to prevent disease by clearing these deposits. However, mTOR inhibition, the major mechanism for inducing autophagy, disrupts core RPE functions. Here, we screened autophagy inducers that do not directly inhibit mTOR for their potential as an AMD therapeutic in primary human RPE culture. Only two out of more than thirty autophagy inducers tested reliably increased autophagy flux in RPE, emphasizing that autophagy induction mechanistically differs across distinct tissues. In contrast to mTOR inhibitors, these compounds preserved RPE health, and one inducer, the FDA-approved compound flubendazole (FLBZ), reduced the secretion of apolipoprotein that contributes to extracellular deposits termed drusen. Simultaneously, FLBZ increased production of the lipid-degradation product β-hydroxybutyrate, which is used by photoreceptor cells as an energy source. FLBZ also reduced the accumulation of intracellular deposits, termed lipofuscin, and alleviated lipofuscin-induced cellular senescence and tight-junction disruption. FLBZ triggered compaction of lipofuscin-like granules into a potentially less toxic form. Thus, induction of RPE autophagy without direct mTOR inhibition is a promising therapeutic approach for dry AMD.

Emerging studies have noted that dysregulated lncRNAs are implicated in cancer progression and tumorigenesis. We first showed that MSC-AS1 was overexpressed in gastric cancer (GC) cells (HGC-27, MKN-45, SGC-7901 and MGC-803 cells) compared with GES cells. We observed that MSC-AS1 was upregulated in GC specimens compared with paired normal specimens. MSC-AS1 increased cell growth and cycle progression. Moreover, the overexpression of MSC-AS1 enhanced the secretion of the inflammatory mediators IL-1β, IL-6 and TNF-α. We found that the overexpression of MSC-AS1 inhibited the expression of miR-142-5p in HGC-27 cells. We noted that DDK5 was a target gene of miR-142-5p. The overexpression of miR-142-5p suppressed the luciferase activity of wild-type DDX5, but the luciferase activity of the mutant DDX5 was not changed. We showed that miR-142-5p was downregulated in GC specimens compared with paired normal specimens. MSC-AS1 expression was inversely correlated with miR-142-5p expression in GC specimens. MSC-AS1 induced cell growth, cell cycle progression and inflammatory mediator secretion by modulating DDX5. These results showed that MSC-AS1 functions as a key oncogene in the development of GC.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an emerging zoonotic pathogen of canine origin that causes an array of fatal diseases, including bacteremia and endocarditis. Despite large-scale genome sequencing projects have gained substantial insights into the genomic landscape of MRSP, current knowledge on virulence determinants that contribute to S. pseudintermedius pathogenesis during human or canine infection is very limited. Using a panel of genetically engineered MRSP variants and a mouse abscess model, we here identified the major secreted nuclease of S. pseudintermedius designated NucB and adenosine synthase A (AdsA) as two synergistically acting enzymes required for MRSP pathogenesis. Similar to Staphylococcus aureus, S. pseudintermedius requires nuclease secretion along with the activity of AdsA to degrade mammalian DNA for subsequent biosynthesis of cytotoxic deoxyadenosine. In this manner, S. pseudintermedius selectively kills macrophages during abscess formation thereby antagonizing crucial host immune cell responses. Ultimately, bioinformatics analyses revealed that NucB and AdsA are widespread in the global S. pseudintermedius population. Together, these data suggest that S. pseudintermedius deploys the canonical Nuc/AdsA pathway to persist during invasive disease and may aid in the development of new therapeutic strategies to combat infections caused by MRSP.

Accumulating evidence shows that circRNAs play critical roles in the development of human tumors. We observed that circ_0000527 was overexpressed in osteosarcoma cells (SAOS-2, HOS, MG-63 and U2OS) compared in hFOB1.19 cells. We demonstrated that the circ_0000527 level was higher in osteosarcoma specimens than in non-tumor specimens. The ectopic expression of circ_0000527 was shown to induce cell growth, cell cycle progression and the secretion of inflammatory mediators, including IL-1β, IL-6, IL-8 and TNF-α. We demonstrated that circ_0000527 sponges miR-646 in osteosarcoma cells and that ARL2 is a target gene of miR-646. MiR-646 expression was decreased and ARL2 was overexpressed in osteosarcoma cells (SAOS-2, HOS, MG-63 and U2OS) compared to hFOB1.19 cells. Overexpression of circ_0000527 was demonstrated to induce ARL2 expression in MG-63 cells. We showed that miR-646 was downregulated in osteosarcoma specimens compared to that of non-tumor specimens and that the level of circ_0000527 was negatively correlated with miR-646 expression in osteosarcoma specimens. The elevated expression of circ_0000527 was shown to promote cell growth and cell cycle progression by modulating miR-646 expression. The ectopic expression of circ_0000527 was shown to promote cell growth, cell cycle progression and the secretion of inflammatory mediators by modulating ARL2. The present study suggested that the circ_0000527/miR-646/ARL2 axis may be a potential treatment target for osteosarcoma.

Objectives We and others have previously shown that epigallocatechin gallate (EGCg) inhibits the activity of an important virulence factor, leukotoxin (LtxA), produced by the oral bacterium Aggregatibacter actinomycetemcomitans, suggesting the potential use of this molecule as an anti-virulence strategy to treat periodontal infections. Here, we sought to better understand the effects of EGCg on toxin secretion and A. actinomycetemcomitans pathogenicity in a co-culture model. Methods We used a quantitative immunoblot assay to determine the concentrations of LtxA in the bacterial supernatant and on the bacterial cell surface. Using a co-culture model, consisting of A. actinomycetemcomitans and THP-1 cells, we studied the impact of EGCg-mediated changes in LtxA secretion on the toxicity of A. actinomycetemcomitans. Key findings EGCg increased production of LtxA and changed the localization of secreted LtxA from the supernatant to the surface of the bacterial cells. In the co-culture model, a single low dose of EGCg did not protect host THP-1 cells from A. actinomycetemcomitans-mediated cytotoxicity, but a multiple dosing strategy had improved effects. Conclusions Together, these results demonstrate that EGCg has important, but complicated, effects on toxin secretion and activity; new dosing strategies and comprehensive model systems may be required to properly develop these anti-virulence activities.

The physiological functions of many vital tissues and organs continue to mature after birth, but the genetic mechanisms governing this postnatal maturation remain an unsolved mystery. Human pancreatic β-cells produce and secrete insulin in response to physiological cues like glucose, and these hallmark functions improve in the years after birth. This coincides with expression of the transcription factors SIX2 and SIX3, whose functions in native human β-cells remain unknown. Here, we show that shRNA-mediatedSIX2orSIX3suppression in human pancreatic adult islets impairs insulin secretion. However, transcriptome studies revealed thatSIX2andSIX3regulate distinct targets. Loss ofSIX2markedly impaired expression of genes governing β-cell insulin processing and output, glucose sensing, and electrophysiology, whileSIX3loss led to inappropriate expression of genes normally expressed in fetal β-cells, adult a-cells and other non-β-cells. Chromatin accessibility studies identified genes directly regulated by SIX2. Moreover, β-cells from diabetic humans with impaired insulin secretion also had reducedSIX2transcript levels. Revealing howSIX2andSIX3govern functional maturation and maintain developmental fate in native human β-cells should advance β-cell replacement and other therapeutic strategies for diabetes.

Allergic rhinitis (AR) is a symptomatic allergic disease that leads to severe inflammation. Astragaloside IV (AS-IV) is a primary active component of Astragalus membranaceus and exerts immune-regulation and anti-inflammatory effects. However, the pharmacological effect of AS-IV in the nasal epithelial cells (NECs) has not been reported. The present study aimed to assess the effect of AS-IV on inflammatory cytokines and mucin 5 subtype AC (MUC5AC) overproduction in histamine (His)-stimulated NECs and its underlying mechanism. NECs were stimulated with or without His for 24 h in the absence or presence of AS-IV. The levels of inflammatory cytokines including IL-6, IL-8, MCP-1, IL-1β, granulocyte-macrophage colony-stimulating factor (GM-CSF), eotaxin, and MUC5AC were assayed. Our findings indicated that AS-IV inhibited His-evoked release and expression of inflammatory cytokines and MUC5AC in NECs. RNA-seq analyses indicated the significant changes in expression levels involved in inflammation genes upon treatment of His-induced NECs with AS-IV. Our findings indicated that AS-IV inhibited His-evoked inflammatory cytokines secretion and MUC5AC overproduction in NECs, which were partly mediated by regulation of inflammation-related genes. Therefore, our findings provided a scientific basis for the development of AS-IV as an effective agent for clinical therapeutic strategy in the treatment of AR.

Glioblastoma is a central nervous malignancy with a very poor prognosis. This study attempted to explore the role of exosomes induced by low-dose radiation-induced (ldrEXOs) and ldrEXOs-derived circ-METRN in glioblastoma progression and radioresistance at the molecular, cellular, animal, and clinical levels. Results in the present study revealed that low-dose radiation stimulated the secretion of ldrEXOs which delivered high levels of circ-METRN. And circ-METRN-abundant ldrEXOs increased the expression of γ-H2AX, indicating an efficient DNA damage-repair process in glioblastoma cells. The ldrEXOs-derived circ-METRN enhanced the glioblastoma progression and radioresistance via miR-4709-3p/GRB14/PDGFRα pathway. Up-regulating PDGFRα can rescue the tumor-promoting function of ldrEXOs in groups previously treated with inhibition of GRB14. Additionally, in-vivo experiments revealed that treatments with ldrEXOs promoted the growth of xenografted tumors and shortened the survival period. Furthermore, clinical researches indicated that circ-METRN may be transported into the bloodstream by exosomes in the early stages of fractionated radiotherapy. It has important clinical values to detect the serum exosomal circ-METRN in the early stage of radiotherapy, which is not only conducive to predict radioresistance and prognosis but also to assist MRI diagnosis in detecting the very early recurrence of glioblastoma. In summary, this study reveals for the first time that low-dose radiation-induced exosomal circ-METRN plays an oncogenic role in glioblastoma progression and radioresistance through miR-4709-3p/GRB14/PDGFRα pathway, providing mechanistic insights into the roles of circRNAs and a valuable marker for therapeutic targets in glioblastoma.

miRNA-223 has been previously reported to play an essential role in hepatic cholesterol homeostasis. However, its role in regulation of biliary cholesterol secretion and gallstone formation remains unknown. Hence, mice with conventional knockout (KO), hepatocyte-specific knockout (ΔHepa) / knockdown (KD) or gain expression of miRNA-223 were included in the study and were subjected to lithogenic diet (LD) for various weeks. The gall bladders and liver tissues were harvested for cholesterol crystal imaging, gallstone mass measurement and molecular analysis. Levels of cholesterol, bile salt, phospholipids, and triglyceride were determined in serum, liver tissues, and bile by enzyme color reactive assays. A 3' UTR reporter gene assay was used to verify the direct target genes for miRNA-223. LD-induced gallstone formation was remarkably accelerated in miRNA-223 KO, ΔHepa, and KD mice with concurrent enhancement in total cholesterol levels in liver tissues and bile. Key biliary cholesterol transporters ABCG5 and ABCG8 were identified as direct targets of miRNA-223. Reversely, AAV-mediated hepatocyte-specific miRNA-223 overexpression prevented gallstone progression with reduced targets expression. Therefore, the present study demonstrates a novel role of miRNA-223 in the gallstone formation by targeting ABCG5 and ABCG8 and elevating miRNA-223 would be a potentially novel approach to overcome the sternness of cholesterol gallstone disease.

Extracellular vesicles derived from trophoblasts (T-EVs) play an important role in pregnancy, but the mechanism is not entirely clear. In this study, we found that HLA-E, which is mostly confined to the cytoplasm of trophoblast cells, was secreted by T-EVs. The level of HLA-E in T-EVs from unexplained recurrent spontaneous abortion (URSA) patients was lower than that in normal pregnancy (NP) and RSA patients who had an abnormal embryo karyotype (AK-RSA). T-EVs promoted secretion of IFN-γ and VEGFα by decidual NK (dNK) cells from URSA patients via HLA-E, VEGFα was necessary for angiogenesis and trophoblast growth, and IFN-γ inhibited Th17 induction. Glycolysis and oxidative phosphorylation (OxPhos) were involved in this process. Glycolysis but not OxPhos of dNK cells facilitated by T-EVs was dependent on mTORC1 activation. Inhibition of T-EV production in vivo increased the susceptibility of mice to embryo absorption, which was reversed by transferring exogenous T-EVs. T-EVs promoted secretion of IFN-γ and VEGFα by dNK cells to maintain pregnancy via Qa-1 in abortion-prone mouse models. This study reveals a new mechanism of pregnancy maintenance mediated by HLA-E via T-EVs.

Lumbar disc degeneration is a common cause of chronic low back pain and an important contributor to various degenerative lumbar spinal disorders. However, currently there is currently no effective therapeutic strategy for treating disc degeneration. The pro-inflammatory cytokine interleukin-1β (IL-1β) mediates disc degeneration by inducing apoptotic death of nucleus pulposus (NP) cells and degradation of the NP extracellular matrix. Here, we confirmed that extracellular secretion of IL-1β via secretory autophagy contributes to disc degeneration, and demonstrate that a thermosensitive reactive oxygen species (ROS)-responsive hydrogel loaded with a synthetic growth hormone-releasing hormone analog (MR409) can protect against needle puncture-induced disc degeneration in rats. Methods: The expression levels of proteins related to secretory autophagy such as tripartite motif-containing 16 (TRIM16) and microtubule-associated protein light chain 3B (LC3B) were examined in human and rat disc tissues by histology and immunofluorescence. The effects of TRIM16 expression level on IL-1β secretion were examined in THP-1 cells transfected with TRIM16 plasmid or siRNA using ELISA, immunofluorescence, and immunoblotting. The in vitro effects of MR409 on IL-1β were examined in THP-1 cells and primary rat NP cells using ELISA, immunofluorescence, immunoblotting, and qRT-PCR. Further, MR409 was subcutaneously administered to aged mice to test its efficacy against disc degeneration using immunofluorescence, X-ray, micro-CT, and histology. To achieve controllable MR409 release for intradiscal use, MR409 was encapsulated in an injectable ROS-responsive thermosensitive hydrogel. Viscosity, rheological properties, release profile, and biocompatibility were evaluated. Thereafter, therapeutic efficacy was assessed in a needle puncture-induced rat model of disc degeneration at 8 and 12 weeks post-operation using X-ray, magnetic resonance (MR) imaging, histological analysis, and immunofluorescence. Results: Secretory autophagy-related proteins TRIM16 and LC3B were robustly upregulated in degenerated discs of both human and rat. Moreover, while upregulation of TRIM16 facilitated, and knockdown of TRIM16 suppressed, secretory autophagy-mediated IL-1β secretion from THP-1 cells under oxidative stress, MR409 inhibited ROS-induced secretory autophagy and IL-1β secretion by THP-1 cells as well as IL-1β-induced pro-inflammatory and pro-catabolic effects in rat NP cells. Daily subcutaneous injection of MR409 inhibited secretory autophagy and ameliorated age-related disc degeneration in mice. The newly developed ROS-responsive MR409-encapsulated hydrogel provided a reliable delivery system for controlled MR409 release, and intradiscal application effectively suppressed secretory autophagy and needle puncture-induced disc degeneration in rats. Conclusion: Secretory autophagy and associated IL-1β secretion contribute to the pathogenesis of disc degeneration, and MR409 can effectively inhibit this pathway. The ROS-responsive thermosensitive hydrogel encapsulated with MR409 is a potentially efficacious treatment for disc degeneration.

Aim of the study The secretion rate of triglyceride from rat liver is assayed by the measurement of triglyceride accumulation in plasma when its clearance is inhibited. The aim of the study was to measure and compare the secretion rate of triglyceride from rat liver by two methods of fixed-time and continuous assays. Material and methods A single dose of 200 mg of poloxamer-407 (P-407) was injected i.p. into starved male rats. The secretion rate of triglyceride was measured by fixed-time and continuous assays. Results The time course for the changes of serum triglyceride following injection of P-407 showed three distinct phases: a lag period of about 30 minutes, a linear increase in serum triglyceride that lasted more than 4 hours, and a slight decline of triglyceride accumulation that lasted about 24 hours. The mean rate of triglyceride secretion was 234.1 ±9.6 mg/dl/h during the linear phase. The linear phase was divided into five time protocols of 240, 180, 120, 60, and 30 minutes and the secretion rate was measured at three points of time in each protocol. The mean rate of triglyceride secretion was 3.91 ±0.15, 3.83 ±0.16, 3.76 ±0.29, 3.57 ±0.43 and 3.13 ±0.34 mg/dl/min in these protocols respectively. In the kinetic assay, the change in the absorbance per three successive five minutes (ΔA/Δt) was measured and the secretion rate was calculated as 3.82 ±0.11 mg/dl/min. Conclusions The rate of triglyceride secretion can be measured by both fixed-time and kinetic assays and was about 3.82 ±0.11 mg/dl/min. The results of the two methods are more corresponded as the mean and instantaneous velocity respectively.

Pituitary neuroendocrine tumors (PitNETs) represent the neoplastic proliferation of the anterior pituitary gland. Transcription factors play a key role in the differentiation of PitNETs. However, for a substantial proportion of PitNETs, the etiology is poorly understood. According to the transcription data of 172 patients, we found the imprinting disorders of the 14q32.2 region and DLK1/MEG3 locus associated with the differentiation of PitNETs. DLK1/MEG3 locus promoted somatotroph differentiation and inhibited tumor proliferation in PIT1(+) patients, furthermore, the level of DLK1 played a critical role in the trend of somatotroph or lactotroph differentiation. Anti-DLK1 monoclonal antibody blockade or siMEG3 both indicated that the DLK1/MEG3 significantly promoted the synthesis and secretion of GH/IGF-1 and inhibited cell proliferation. In addition, loss of DLK1 activated the mTOR signaling pathway in high DLK1-expressing and PIT1(+) GH3 cell lines, a mild effect in the low DLK1-expressing and PIT1(+) MMQ cell lines and no effect in the PIT1(-) ATT20 cell line. These findings emphasize that expression at the DLK1/MEG3 locus plays a key role in the differentiation of PitNETs, especially somatotroph adenomas, and provide potential molecular target data for patient stratification and treatment in the future.

Atherosclerosis is the main cause of cardiovascular disease. Systemic inflammation is one important characteristic in atherosclerosis. Pro-inflammatory macrophages can secrete inflammatory factors and promote the inflammation of atherosclerosis. It has a great value for the treatment of atherosclerosis by inhibiting the release of inflammatory factors in macrophages. However, the detailed mechanism of this process is still unclear. In this study, we constructed an APOE-/- mice model of atherosclerosis to research the molecular mechanism of atherosclerosis. Protein tyrosine phosphatase non-receptor type 2 (PTPN2), an anti-inflammatory gene, was dramatically decreased in inflammatory mice. Deletion of PTPN2 could significantly induce monocytes toward M1 phenotype of macrophages, enhance the secretion of IL-12 and IL-1, and promote cell proliferation, invasion and metastasis. Mechanism research showed that PTPN2-mediated p65/p38/STAT3 de-phosphorylation could block the process of macrophage inflammation. In vivo experiments showed that PTPN2 may effectively inhibit the inflammatory response during atherosclerosis. In conclusion, we uncovered the negative role of PTPN2 in the occurrence of atherosclerosis, and this study provides a new potential target for atherosclerosis treatment.