AbstractArginine is an essential amino acid necessary for protein synthesis and is also a nitrogen storage compound. The genes encoding the enzymes of arginine biosynthesis in plants are not well characterized and have mainly been predicted from homologies to bacterial and fungal genes. We report the cloning and characterization of the TUMOR PRONE5 (TUP5) gene of Arabidopsis (Arabidopsis thaliana) encoding an acetylornithine aminotransferase (ACOAT), catalyzing the fourth step of arginine biosynthesis. The free arginine content was strongly reduced in the chemically induced recessive mutant tup5-1, root growth was restored by supplementation with arginine and its metabolic precursors, and a yeast (Saccharomyces cerevisiae) ACOAT mutant was complemented by TUP5. Two null alleles of TUP5 caused a reduced viability of gametes and embryo lethality, possibly caused by insufficient Arg supply from maternal tissue. TUP5 expression is positively regulated by light, and a TUP5-green fluorescent protein was localized in chloroplasts. tup5-1 has a unique light-dependent short root phenotype. Roots of light-grown tup5-1 seedlings switch from indeterminate growth to determinate growth with arresting cell production and an exhausted root apical meristem. The inhibitory activity was specific for blue light, and the inhibiting light was perceived by the root. Thus, tup5-1 reveals a novel role of amino acids and blue light in regulating root meristem function.

Abstract Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosin-mediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues.

Background. Neuron survival after ischemia-reperfusion (IR) injury is the primary determinant of motor function prognosis. MicroRNA- (miR-) based gene therapy has gained attention recently. Our previous work explored the mechanisms by which miR-137-3p modulates neuronal apoptosis in both in vivo and in vitro IR models. Methods. IR-induced motor dysfunction and spinal calpain (CAPN) subtype expression and subcellular localization were detected within 12 h post IR. Dysregulated miRs, including miR-137-3p, were identified by miR microarray analysis and confirmed by PCR. A luciferase assay confirmed CAPN-2 as a corresponding target of miR-137-3p, and their modulation of motor function was evaluated by intrathecal injection with synthetic miRs. CAPN-2 activity was measured by the intracellular Ca2+ concentration and mean fluorescence intensity in vitro. Neuronal apoptosis was detected by flow cytometry and TUNEL assay. The activities of p35, p25, Cdk5, and caspase-8 were evaluated by ELISA and Western blot after transfection with specific inhibitors and miRs. Results. The IR-induced motor dysfunction time course was closely associated with upregulated expression of the CAPN-2 protein, which was mainly localized in neurons. The miR-137-3p/CAPN-2 interaction was confirmed by luciferase assay. The miR-137-3p mimic significantly improved IR-induced motor dysfunction and decreased CAPN-2 expression, even in combination with recombinant rat calpain-2 (rr-CALP2) injection, whereas the miR-137-3p inhibitor reversed these effects. Similar changes in the intracellular Ca2+ concentration, CAPN-2 expression, and CAPN-2 activity were observed when cells were exposed to oxygen-glucose deprivation and reperfusion (OGD/R) and transfected with synthetic miRs in vitro. Moreover, double fluorescence revealed identical neuronal localization of CAPN-2, p35, p25, and caspase-8. The decrease in CAPN-2 expression and activity was accompanied by the opposite changes in p35 activity and protein expression in cells transfected with the miR-137-3p mimic, roscovitine (a Cdk5 inhibitor), or Z-IETD-FMK (a caspase-8 inhibitor). Correspondingly, the abovementioned treatments resulted in a higher neuron survival rate than that of untreated neurons, as indicated by decreases in the apoptotic cell percentage and p25, Cdk5, caspase-8, and caspase-3 protein expression. Conclusions. The miR-137-3p/CAPN-2 interaction modulates neuronal apoptosis during IR injury, possibly by inhibiting CAPN-2, which leads to p35 cleavage and inhibition of subsequent p25/Cdk5 and caspase-8 overactivation.

Silicosis is a chronic fibrotic lung disease caused by the accumulation of silica dust in the distal lung. Canonical Wnt signaling and NADPH oxidase 4 (NOX4) have been demonstrated to play a crucial role in the pathogenesis of pulmonary fibrosis including silicosis. However, the underlying mechanisms of crosstalk between these two signalings are not fully understood. In the present study, we aimed to explore the interaction of Wnt/β-catenin and NOX4 of human epithelial cells in response to an exposure of silica dust. Results demonstrated an elevated expression of key components of Wnt/β-catenin signaling and NOX4 in the lungs of silicon dioxide- (SiO2-) induced silicosis mice. Furthermore, the activated Wnt/β-catenin and NOX4 signaling are accompanied by an inhibition of cell proliferation, an increase of ROS production and cell apoptosis, and an upregulation of profibrogenic factors in BEAS-2B human lung epithelial cells exposed to SiO2. A mechanistic study further demonstrated that the Wnt3a-mediated activation of canonical Wnt signaling could augment the SiO2-induced NOX4 expression and reactive oxygen species (ROS) production but reduced glutathione (GSH), while Wnt inhibitor DKK1 exhibited an opposite effect to Wnt3a. Vice versa, an overexpression of NOX4 further activated SiO2-induced Wnt/β-catenin signaling and NFE2-related factor 2 (Nrf2) antioxidant response along with a reduction of GSH, whereas the shRNA-mediated knockdown of NOX4 showed an opposite effect to NOX4 overexpression. These results imply a positive feed forward loop between Wnt/β-catenin and NOX4 signaling that may promote epithelial-mesenchymal transition (EMT) of lung epithelial cells in response to an exposure of silica dust, which may thus provide an insight into the profibrogenic role of Wnt/β-catenin and NOX4 crosstalk in lung epithelial cell injury and pathogenesis of silicosis.

Abstract BackgroundParkinson's disease (PD) is a neurodegenerative disease characterized by the early prominent death of dopaminergic neurons and a decrease of dopamine levels. Dopamine depletion leads to several motor dysfunctions, including resting tremor, muscular rigidity, bradykinesia and postural instability. Our previous study determined that knockout of parkin, a gene of PD degrade p21, suppresses neurogenesis which is critical for a neurodegenerative disease. MethodsThus, we investigated the effect of UC2288, an inhibitor of p21, for its therapeutic effect on PD. We found that UC2288 attenuated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced behavioral impairment in Rota-rod and Pole test as well as dopamine depletion.ResultsMoreover, UC2288 recovered the number of TH positive cells, but decreased the number of GFAP and Iba-1 positive cells accompanied the decrease of BAX and cleaved caspase3 as well as iNOS and COX-2 expression. In cultured neurons, UC2288 recovered MPP+-induced neuronal cell death in a concentration dependent manner. We also found that UC2288 decreased the p21 reactive cell number, oxidative neuronal damages, cytokines product in vivo and cultured neurons. In a mechanism study, we found that UC2288 significantly decreased the activation of ERK and p38 kinase pathway in the mitogen-activated protein kinase (MAPK) pathway. In addition, 1-10 μM concentration of ERK kinase inhibitor U0126 recovered MPP+-induced neuronal cell death. However, ERK kinase inhibitor U0126 further decreased cell viability with the increase of H2O2.ConclusionThese results indicated that the administration of UC2288 exerted neuroprotective effects on the death of dopaminergic neurons through the suppression of oxidative stress and neuroinflammation via ERK pathway inhibition.

Abstract Background: The mechanism underlying the impaired cognitive function and memory loss in Minimal hepatic encephalopathy (MHE) remains unclear. Dopamine (DA) is reported to be associated with dementia. Methods: In this study, we investigated mechanism underlying DA-induced MHE pathology by immunoblotting, ELISA, FM4-64 and fluorescence staining. Results: We observed that MHE brains showed the increased content of DA, after administration of anti-DA antibody, and cognitive loss in MHE rats was recovered to the normal level, indicating the involvement of DA in the pathogenesis of MHE. Moreover, DA (10 μM) treatment obviously induced the decrease in the production of GDNF/NGF and the increase in TNFα levels in primary cultured neurons, which were blocked by addition of β-asarone (βASA). We also demonstrated that DA stimulated the activation of ASK1/JNK1 pathway. and the addition of anti-TNFα antibody reversed the inactivation of Notch signaling, the downregulation of neurotrophins and synaptic loss.Conclusions: Overall, we suggested that DA stimulated abundant production and secretion of neuronal TNFα, which elicited progressive loss of neurotrophic factors, leading to cognitive disorder of MHE.

Abstract Genome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as a strong regulatory element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. We show that genotype at the asthma-associated SNP rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a novel mechanism through which a regulatory SNP contributes to genetic risk of asthma.

Flavivirus replication is intimately associated with re-organized cellular membranes. These virus-induced changes in membrane architecture form three distinct membranous “organelles” that have specific functions during the flavivirus life cycle. One of these structures is the replication complex in which the flaviviral RNA is replicated to produce progeny genomes. We have previously observed that this process is strictly dependent on cellular cholesterol. In this study we have identified a putative cholesterol recognition/interaction amino acid consensus (CRAC) motif within the West Nile virus strain Kunjin virus (WNVKUN) NS4A protein. Site-directed mutagenesis of this motif within a WNVKUN infectious clone severely attenuated virus replication and the capacity of the mutant viruses to form the replication complex. Replication of the mutant viruses also displayed reduced co-localization with cellular markers recruited to replication sites during wild-type virus replication. In addition, we observed that the mutant viruses were significantly impaired in their ability to remodel cytoplasmic membranes. However, after extensive analysis we are unable to conclusively reveal a role for the CRAC motif in direct cholesterol binding to NS4A, suggesting additional complex lipid-protein and protein-protein interactions. We believe this study highlights the crucial role for this region within NS4A protein in recruitment of cellular and viral proteins to specialized subdomains on membrane platforms to promote efficient virus replication.

Background: Temporomandibular joint osteoarthritis (TMJOA) seriously affects the health of patients, and the current treatments are invasive and only used for advanced cases. Bone marrow mesenchymal stem cell (BMSC)-derived small extracellular vesicles (BMSC-sEVs) may represent a safer and more effective treatment, but their role in TMJOA has not been elucidated. This study attempted to analyze the cartilage reconstruction effect of BMSC-sEVs on TMJOA and the mechanism underlying this effect.Methods: BMSC-sEVs were isolated and purified by microfiltration and ultrafiltration and were subsequently characterized by nanoparticle tracking analysis, electron microscopy, and immunoblotting. TMJOA models were established in vivo and in vitro, and hematoxylin–eosin staining, immunohistochemistry, and histological scoring were performed to analyze the histological changes in TMJOA cartilage tissues treated with BMSC-sEVs. The proliferation, migratory capacity, and cell cycle distribution of TMJOA cartilage cells treated with BMSC-sEVs were detected. Furthermore, the related mechanisms were studied by bioinformatic analysis, immunoblotting, and quantitative PCR, and they were further analyzed by knockdown and inhibitor techniques.Results: The acquisition and identification of BMSC-sEVs were efficient and satisfactory. Compared with the osteoarthritis (OA) group, the condylar tissue of the OA group treated with BMSC-sEV (OAsEV) showed an increase in cartilage lacuna and hypertrophic cartilage cells in the deep area of the bone under the cartilage. Significantly upregulated expression of proliferating cell nuclear antigen and cartilage-forming factors and downregulated expression of cartilage inflammation-related factors in OAsEV were observed. In addition, we found higher rates of cell proliferation and migratory activity and alleviated G1 stagnation of the cell cycle of OAsEV. Autotaxin was found in the BMSC-sEVs, and key factors of the Hippo pathway, Yes-associated protein (YAP), phosphorylated Yes-associated protein (p-YAP), etc. were upregulated in the OAsEV group. Treatment with BMSC-sEVs after autotaxin knockdown or inhibition no longer resulted in expression changes in cartilage-forming and inflammation-related factors and key factors of the Hippo pathway.Conclusions: These results suggest that the autotaxin–YAP signaling axis plays an important role in the mechanism by which BMSC-sEVs promote cartilage reconstruction in TMJOA, which may provide guidance regarding their therapeutic applications as early and minimally invasive therapies for TMJOA, and provide insight into the internal mechanisms of TMJOA.

Herpes simplex virus type 2 (HSV2), a pathogen that causes genital herpes lesions, interferes with the host immune system via various known and unknown mechanisms. This virus has been used to study viral antigenic composition. Convalescent serum from HSV2-infected patients was used to identify viral antigens via 2-D protein electrophoresis and immunoblotting. The serum predominantly recognized several capsid scaffold proteins encoded by gene UL26.5, mainly ICP35. This protein has been primarily reported to function temporarily in viral assembly but is not expressed in mature virus particles. Further immunological studies suggested that this protein elicits specific antibody and cytotoxic T lymphocyte (CTL) responses in mice, but these responses do not result in a clinical protective effect in response to HSV2 challenge. The data suggested that immunodominance of ICP35 might be used to design an integrated antigen with other viral glycoproteins.

Sepsis-induced acute kidney injury (AKI) is a frequent complication in critically ill patients, refractory to conventional treatments. Aberrant activation of innate immune system may affect organ damage with poor prognosis for septic patients. Here, we investigated the efficacy of polymethyl methacrylate membrane (PMMA)-based continuous hemofiltration (CVVH) in modulating systemic and tissue immune activation in a swine model of LPS-induced AKI. After 3 h from LPS infusion, animals underwent to PMMA-CVVH or polysulfone (PS)-CVVH. Renal deposition of terminal complement mediator C5b-9 and of Pentraxin-3 (PTX3) deposits were evaluated on biopsies whereas systemic Complement activation was assessed by ELISA assay. Gene expression profile was performed from isolated peripheral blood mononuclear cells (PBMC) by microarrays and the results validated by Real-time PCR. Endotoxemic pigs presented oliguric AKI with increased tubulo-interstitial infiltrate, extensive collagen deposition, and glomerular thrombi; local PTX-3 and C5b-9 renal deposits and increased serum activation of classical and alternative Complement pathways were found in endotoxemic animals. PMMA-CVVH treatment significantly reduced tissue and systemic Complement activation limiting renal damage and fibrosis. By microarray analysis, we identified 711 and 913 differentially expressed genes with a fold change >2 and a false discovery rate <0.05 in endotoxemic pigs and PMMA-CVVH treated-animals, respectively. The most modulated genes were Granzyme B, Complement Factor B, Complement Component 4 Binding Protein Alpha, IL-12, and SERPINB-1 that were closely related to sepsis-induced immunological process. Our data suggest that PMMA-based CVVH can efficiently modulate immunological dysfunction in LPS-induced AKI.

Viruses have evolved many mechanisms to escape host antiviral responses. Previously, we found that classical swine fever virus (CSFV) infection induces autophagy using the autophagosome as a self-replication site, thereby evading the host immune response and promoting long-term infection. However, the underlying mechanisms used by CSFV to enter autophagosomes and the mechanism by which autophagy promotes viral replication remain unclear. We found that CSFV infection inhibited autophagy receptor nuclear dot protein 52 kDa (NDP52) expression, ubiquitination, and SUMO2-4 modification. Further analyses revealed that CSFV mediated ubiquitination and SUMOylation of NDP52 via Pten-induced kinase 1 (PINK1)-Parkin. Moreover, NDP52 inhibition also inhibited CSFV replication and the induction of mitophagy marker proteins expression. Inhibition of NDP52 reduced CD63 expression and binding to CSFV E2 protein, which has an essential role in persistent CSFV infection. As NDP52 has a close relationship with the NF-κB innate immunity pathway and plays an important role in the antiviral response, we investigated whether NDP52 inhibited CSFV replication through the release of immune factors and antivirus signals. Our results showed that inhibiting NDP52 boosted interferon and TNF release and promoted NF-κB pathway activation. In summary, we found that NDP52 inhibition not only reduces CSFV binding and entry into autophagic vesicles, but also inhibits CSFV replication by active NF-κB antiviral immune pathways. Our data reveal a novel mechanism by which NDP52, an autophagy receptor, mediates CSFV infection, and provide new avenues for the development of antiviral strategies.

Circular RNAs (circRNAs) contain microRNA (miRNA)-specific binding sites and can function as miRNA sponges to regulate gene expression by suppressing the inhibitory effect of miRNAs on their target genes. MiR-21-5p has been reported to be involved in the development of head and neck squamous cell carcinoma (HNSCC) and plays an important role in the activation of epithelial-mesenchymal transition (EMT). However, the upstream regulatory mechanism and downstream targets of miR-21-5p in tumor cells remain unknown. CircRNA_ACAP2 inhibits the function of miR-21-5p by binding to its specific binding sites in HNSCC cells. Overexpression of CircRNA_ACAP2 inhibits the proliferation and migration of HNSCC cells, while downregulation of CircRNA_ACAP2 has the opposite effect. STAT3 is a direct target gene of miR-21-5p and a transcription factor of ZEB1. We demonstrate that CircRNA_ACAP2 functions as a tumor suppressor gene in HNSCC and that its function is regulated via the miR-21-5p/STAT3 signaling axis.

Full functioning of the airway physical barrier depends on cellular integrity, which is coordinated by a series of tight junction (TJ) proteins. Due to airway spasm, edema, and mucus obstruction, positive end-expiratory alveolar pressure (also termed auto-PEEP) is a common pathophysiological phenomenon, especially in acute asthma attack. However, the influence of auto-PEEP on small airway epithelial TJs is currently unclear. We performed studies to investigate the effect of extra pressure on small airway epithelial TJs and its mechanism. The results first confirmed that a novel mechanosensitive receptor, piezo-1, was highly expressed in the airway epithelium of asthmatic mice. Extra pressure induced the degradation of occludin, ZO-1 and claudin-18 in primary human small airway epithelial cells (HSAECs), resulting in a decrease in transepithelial electrical resistance (TER) and an increase in cell layer permeability. Through in vitro investigations, we observed that exogenous pressure stimulation could elevate the intracellular calcium concentration ([Ca2+]i) in HSAECs. Downregulation of piezo-1 with siRNA and pretreatment with BAPTA-AM or ALLN reduced the degradation of TJs and attenuated the impairment of TJ function induced by exogenous pressure. These findings indicate the critical role of piezo-1/[Ca2+]i/calpain signaling in the regulation of small airway TJs under extra pressure stimulation.

Chimeric fusion proteins comprising a single domain antibody (VHH) fused to a crystallizable fragment (Fc) of an immunoglobulin are modular glycoproteins that are becoming increasingly in demand because of their value as diagnostics, research reagents and passive immunization therapeutics. Because ER-associated degradation and misfolding may potentially be limiting factors in the oxidative folding of VHH-Fc fusion proteins in the ER, we sought to explore oxidative folding in an alternative sub-compartment, the chloroplast thylakoid lumen, and determine its viability in a molecular farming context. We developed a set of in-house expression vectors for transient transformation of Nicotiana benthamiana leaves that target a VHH-Fc to the thylakoid lumen via either secretory (Sec) or twin-arginine translocation (Tat) import pathways. Compared to stromal [6.63 ± 3.41 mg/kg fresh weight (FW)], cytoplasmic (undetectable) and Tat-import pathways (5.43 ± 2.41 mg/kg FW), the Sec-targeted VHH-Fc showed superior accumulation (30.56 ± 5.19 mg/kg FW), but was less than that of the ER (51.16 ± 9.11 mg/kg FW). Additionally, the introduction of a rationally designed de novo disulfide bond enhances in planta accumulation when introduced into the Sec-targeted Fc fusion protein from 50.24 ± 4.08 mg/kg FW to 110.90 ± 6.46 mg/kg FW. In vitro immunofluorescent labeling assays on VHH-Fc purified from Sec, Tat, and stromal pathways demonstrate that the antibody still retains VHH functionality in binding Escherichia coli O157:H7 and neutralizing its intimate adherence to human epithelial type 2 cells. These results overall provide a proof of concept that the oxidative folding environment of the thylakoid lumen may be a viable compartment for stably folding disulfide-containing recombinant VHH-Fc proteins.

Background: The identification of C-repeat binding factor (CBF), and the characterization as an inducer of CBF Expression 1 (ICE1), and a major activator for C-repeat binding factor, were important breakthroughs in the cold signaling network. Methods: In the present study, the full length cDNA of ICE1 was isolated from the tea tree (Camellia sinensis). CsICE1 protein was located in the cell nucleus as revealed by subcellular localization analysis. To investigate the biological functions of CsICE1, a transgenic line fused with the CsICE1 gene in Arabidopsis thaliana (arabidopsis) was generated by the floral dip method. Results: The CsICE1 was expressed differentially in various tea tree tissues, mostly in buds and leaves, and the transcript level of CsICE1 was increased after 1 h and peaked at 2 h under cold treatment. Transcription activity assay indicated that the spermine synthase (SPMS) and arginine decarboxylase (ADC) genes were possible targets of CsICE1. In addition, the values of net photosynthetic rate, transpiration rate, stomatal conductance in transgenic lines declined by less extent than wild-type plants under low temperatures. Furthermore, transcript levels of ADC genes in the transgenic lines had no apparent alteration under normal growth conditions but substantially increased under cold conditions, consistent of changes in free polyamine levels. Taken together, these results demonstrated that CsICE1 plays a positive role in cold tolerance, which may be due to the modulation of polyamine levels through interacting with CsADC.

Cold stress can induce autophagy mediated by excess corticosterone (CORT) in the hippocampus, but the internal mechanism induced by cold stress is not clear. In vivo, male and female C57BL/6 mice were stimulated in 4 °C, 3 h per day for 1 week to build the model of cold sress. In vitro, hippocampal neuronal cell line (HT22) cells were incubated with or without mifepristone (RU486) for 1 h, then treated with 400 μM cortisol (CORT) for 3 h. In vivo, autophagy was measured by western blotting. In vitro, monodansylcadaverine staining, western blotting, flow cytometry, transmission electron microscopy, and immunofluorescence were used to characterize the mechanism of autophagy induced by excess CORT. Autophagy was shown in mouse hippocampus tissues following cold exposure, including mitochondrial damage, autophagy, and 5’ AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway activation after CORT treatment. Autophagy did not rely on the glucocorticoid receptor. In addition, autophagy in male mice was more severe. The study would provide new insight into the mechanisms and the negative effect of the cold stress response, which can inform the development of new strategies to combat the effects of hypothermia.

Recognition of the fact that bacterial biofilm may play a role in the pathogenesis of disease has led to an increased focus on identifying diseases that may be biofilm-related. Biofilm infections are typically chronic in nature, as biofilm-residing bacteria can be resilient to both the immune system, antibiotics, and other treatments. This is a comprehensive review describing biofilm diseases in the auditory, the cardiovascular, the digestive, the integumentary, the reproductive, the respiratory, and the urinary system. In most cases reviewed, the biofilms were identified through various imaging technics, in addition to other study approaches. The current knowledge on how biofilm may contribute to the pathogenesis of disease indicates a number of different mechanisms. This spans from biofilm being a mere reservoir of pathogenic bacteria, to playing a more active role, e.g., by contributing to inflammation. Observations also indicate that biofilm does not exclusively occur extracellularly, but may also be formed inside living cells. Furthermore, the presence of biofilm may contribute to development of cancer. In conclusion, this review shows that biofilm is part of many, probably most chronic infections. This is important knowledge for development of effective treatment strategies for such infections.

Bioactive natural products are typically secreted by the producer strain. Besides that, this allows the targeting of competitors, also filling a protective role, reducing the chance of self-killing. Surprisingly, DNA-degrading and membrane damaging prodiginines (PdGs) are only produced intracellularly, and are required for the onset of the second round of programmed cell death (PCD) in Streptomyces coelicolor. In this work, we investigated the influence of PdGs on the timing of the morphological differentiation of S. coelicolor. The deletion of the transcriptional activator gene redD that activates the red cluster for PdGs or nutrient-mediated reduction of PdG synthesis both resulted in the precocious appearance of mature spore chains. Transcriptional analysis revealed an accelerated expression of key developmental genes in the redD null mutant, including bldN for the developmental σ factor BldN which is essential for aerial mycelium formation. In contrast, PdG overproduction due to the enhanced copy number of redD resulted in a delay or block in sporulation. In addition, confocal fluorescence microscopy revealed that the earliest aerial hyphae do not produce PdGs. This suggests that filaments that eventually differentiate into spore chains and are hence required for survival of the colony, are excluded from the second round of PCD induced by PdGs. We propose that one of the roles of PdGs would be to delay the entrance of S. coelicolor into the dormancy state (sporulation) by inducing the leakage of the intracellular content of dying filaments thereby providing nutrients for the survivors.

Cold stress can induce neuroinflammation in the hippocampal dentate gyrus (DG), but the mechanism underlying neuronal apoptosis induced by cold stress is not well-understood. To address this issue, male and female C57BL/6 mice were exposed to a temperature of 4 °C for 3 h per day for 1 week, and glial cell activation, neuronal apoptosis, and neuroinflammation were evaluated by western blotting, immunofluorescence, terminal deoxynucleotidyl transferase 2’-deoxyuridine 5’-triphosphate (dUTP) nick end labeling, Nissl staining, and immunohistochemistry. Additionally, BV2 cells were treated with different concentrations of cortisol (CORT) for 3 h to mimic stress and molecular changes were assessed by western blotting, immunofluorescence, and co-immunoprecipitation. We found that excess CORT activated glial cells and increased neuroinflammation in the DG of mice exposed to cold temperatures, which was associated with increased acetylation and nuclear factor-κB signaling. These effects were mediated by the acetylation of lysine 9 of histone 3 and lysine 310 of p65, which resulted in increased mitogen-activated protein kinase phosphorylation, nuclear translocation of p65, microglia activation, and acetylation of high-mobility group box 1. Neuroinflammation was more severe in male compared to female mice. These findings provide new insight into the mechanisms of the cold stress response, which can inform the development of new strategies to combat the effects of hypothermia.