Intracellular Secretion Research Articles

Analysis of Microbial Community Characteristics and Function Prediction of MBBR with Magnetic Biocarriers at Low Temperature

In order to clarify effect of magnetic biocarriers on the performance of MBBR at low temperatures, the microbial diversity, community structure, functional characteristics, and nitrogen metabolism of biofilm in the reaction system were investigated. The results indicated that MBBR with magnetic biocarriers had a better pollutant removal efficiency, with the average removal rates of NH4+-N and TN being 16.2% and 12.1% higher than those in the control group (commercial biocarriers), respectively. Illumina high-throughput sequencing analysis showed that higher diversity and richness of the bacterial community was established in the biofilm of magnetic biocarriers. There were obvious differences in microbial community structure of biofilm between the two biocarrier duos to bacterial magnetic susceptibility. The relative abundances of nitrifying bacteria (e.g., Nitrosomonas and Nitrospira) and denitrifying bacteria (e.g., Sphaerotilus and Zoogloea) were increased in the magnetic biocarriers. Functional prediction analysis with PICRUSt2 showed that the microorganism of magnetic biocarriers had a better total gene function expression level, which was significantly more increased than commercial biocarriers in gene-representing signal transduction mechanisms and intracellular trafficking, secretion, and vesicular transport. Furthermore, most of the abundances of nitrogen metabolism genes were raised in the biofilm of magnetic biocarriers (e.g., genes amo and hao, were responsible for nitrification, and genes nap and nor, which were responsible for denitrification). Magnetic biocarriers increased biofilm potential for denitrification at low temperatures. Our results explained the difference in performance between the two reactors from microbiology and provided the theoretical basis for magnetic biocarrier-enhanced performances of MBBR at low temperatures.

Metabolism of NK cells during viral infections.

Cellular metabolism is essential for the correct function of immune system cells, including Natural Killer cells (NK). These cells depend on energy to carry out their effector functions, especially in the early stages of viral infection. NK cells participate in the innate immune response against viruses and tumors. Their main functions are cytotoxicity and cytokine production. Metabolic changes can impact intracellular signals, molecule production, secretion, and cell activation which is essential as the first line of immune defense. Metabolic variations in different immune cells in response to a tumor or pathogen infection have been described; however, little is known about NK cell metabolism in the context of viral infection. This review summarizes the activation-specific metabolic changes in NK cells, the immunometabolism of NK cells during early, late, and chronic antiviral responses, and the metabolic alterations in NK cells in SARS-CoV2 infection. The modulation points of these metabolic routes are also discussed to explore potential new immunotherapies against viral infections.

Extraction of Extracelluar Vesicles Derived from Mycobacterium tuberculosis and Their Effect on the Production of Reactive Oxygen Species and Expression of Inflammatory Factors in Mouse Bone Marrow-Derived Dendritic Cells

To isolate extracellular vesicles (EVs) from Mycobacterium tuberculosis ( Mtb), to examine their morphology, particle size, and distribution, to study the effect of EVs derived from Mtb ( Mtb-EVs) on intracellular reactive oxygen species (ROS) production and cytokine secretion in dendritic cells (DCs), and to make preliminary exploration of Mtb-EVs' effect on the immune regulation of DCs. Mtb-EVs were obtained by ultrafiltration concentration and the protein concentration was determined by BCA assay. The morphology of Mtb-EVs was observed through negative staining electron microscopy (EM). The particle size distribution and concentration of Mtb-EVs were determined by nanoparticle tracking analysis (NTA). Mouse bone marrow was isolated through sterile procedures and mice myeloid DCs were induced and amplified by the combined use of recombinant mouse granulocyte-macrophage colony-stimulating factor (rm GM-CSF) and recombinant mouse interleukin-4 (rm IL-4). Then, morphological and immunophenotypic characterization was performed. After that, the DCs were treated with Mtb-EVs at different concentrations and CCK-8 assay was done to measure their effect on the survival rate of DCs and to identify the appropriate stimulation concentration for subsequent experimental procedures. The intracellular ROS levels of DCs were evaluated with DCFH-DA fluorescence probe and the cytokine secretion of DCs was determined by ELISA. EM observation showed that Mtb-EVs isolated by ultrafiltration concentration were spherical vesicles of varied sizes, all being approximately 100 nm in diameter and displaying typical morphology. NTA results from NanoSight nanoparticle tracker showed that the peak particle size was 98.5 nm, that the average particle size was 110.2 nm, and that the particle size was mainly distributed between 68.4-155.7 nm. Mtb-EVs that were smaller than 250 nm accounted for 98.39% of the total. Mouse myeloid DCs directionally induced and amplified in vitro displayed typical DC phenotype and morphology, and the purity exceeded 85%. EM verified the abundance of microvilli and radial protuberance on the surface of DCs, which had uniform cytoplasm and clear nuclear membrane. Loaded with Mtb-EVs at different concentrations, including 10 2, 10 3, and 10 4 particles/cell, the DCs had significantly upregulated levels of intracellular ROS ( P<0.05). In addition, Mtb-EVs induced the release of IL-1β and IL-6 in a dose-dependent manner ( P<0.05). We established in the study a technical process for the extraction of Mtb-EVs by ultrafiltration concentration and obtained Mtb-EVs with sound morphology, high purity, and concentrated particle size distribution. Furthermore, Mtb-EVs can upregulate the intracellular ROS level in DCs and induce the release of IL-1β and IL-6 in a dose-dependent manner.

Chronic hyperadiponectinemia induced by transgenic overexpression increases plasma exosomes without significantly improving glucose and lipid metabolism.

The fat-derived factor, adiponectin, is considered a salutary circulating factor. We recently demonstrated that native adiponectin binds T-cadherin and promotes intracellular biogenesis and secretion of the exosome. Exosomes play important roles in various aspects of homeostasis, including glucose and energy metabolism. However, it remains unclear whether and how the promotion of exosome production by adiponectin in vivo is beneficial for glucose and lipid metabolism. In the present study, overexpression of human adiponectin in mice resulted in an increased number of circulating exosomes, but it did not significantly improve glucose metabolism, change body weights, or change triglyceride clearance under a high-fat diet. Multiple small doses of streptozotocin increased blood glucose and decreased triglyceride clearance similarly in both wild-type and transgenic mice. Thus, these results indicated that human adiponectin overexpression in mice increases plasma exosomes but does not significantly influence glucose and lipid metabolism.

Transcriptome Analysis Reveals Potential Mechanism in Storage Protein Trafficking within Developing Grains of Common Wheat

Gluten proteins are the major storage protein fraction in the mature wheat grain. They are restricted to the starchy endosperm, which defines the viscoelastic properties of wheat dough. The synthesis of these storage proteins is controlled by the endoplasmic reticulum (ER) and is directed into the vacuole via the Golgi apparatus. In the present study, transcriptome analysis was used to explore the potential mechanism within critical stages of grain development of wheat cultivar "Shaannong 33" and its sister line used as the control (CK). Samples were collected at 10 DPA (days after anthesis), 14 DPA, 20 DPA, and 30 DPA for transcriptomic analysis. The comparative transcriptome analysis identified that a total of 18,875 genes were differentially expressed genes (DEGs) between grains of four groups "T10 vs. CK10, T14 vs. CK14, T20 vs. CK20, and T30 vs. CK30", including 2824 up-regulated and 5423 down-regulated genes in T30 vs. CK30. Further, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment highlighted the maximum number of genes regulating protein processing in the endoplasmic reticulum (ER) during grain enlargement stages (10-20 DPA). In addition, KEGG database analysis reported 1362 and 788 DEGs involved in translation, ribosomal structure, biogenesis, flavonoid biosynthesis pathway and intracellular trafficking, secretion, and vesicular transport through protein processing within ER pathway (ko04141). Notably, consistent with the higher expression of intercellular storage protein trafficking genes at the initial 10 DPA, there was relatively low expression at later stages. Expression levels of nine randomly selected genes were verified by qRT-PCR, which were consistent with the transcriptome data. These data suggested that the initial stages of "cell division" played a significant role in protein quality control within the ER, thus maintaining the protein quality characteristics at grain maturity. Furthermore, our data suggested that the protein synthesis, folding, and trafficking pathways directed by a different number of genes during the grain enlargement stage contributed to the observed high-quality characteristics of gluten protein in Shaannong 33 (Triticum aestivum L.).

RAB27A promotes the proliferation and invasion of colorectal cancer cells

Colorectal cancer (CRC) is one of the most commonly diagnosed cancer types worldwide. Despite significant advances in prevention and diagnosis, CRC is still one of the leading causes of cancer-related mortality globally. RAB27A, the member of RAB27 family of small GTPases, is the critical protein for intracellular secretion and has been reported to promote tumor progression. However, it is controversial for the role of RAB27A in CRC progression, so we explored the exact function of RAB27A in CRC development in this study. Based on the stable colon cancer cell lines of RAB27A knockdown and ectopic expression, we found that RAB27A knockdown inhibited proliferation and clone formation of SW480 colon cancer cells, whereas ectopic expression of RAB27A in RKO colon cancer cells facilitated cell proliferation and clone formation, indicating that RAB27A is critical for colon cancer cell growth. In addition, our data demonstrated that the migration and invasion of colon cancer cells were suppressed by RAB27A knockdown, but promoted by RAB27A ectopic expression. Therefore, RAB27A is identified as an onco-protein in mediating CRC development, which may be a valuable prognostic indicator and potential therapeutic target for CRC.

In silico analysis of alternative splicing events implicated in intracellular trafficking during B-lymphocyte differentiation.

There are multiple regulatory layers that control intracellular trafficking and protein secretion, ranging from transcriptional to posttranslational mechanisms. Finely regulated trafficking and secretion is especially important for lymphocytes during activation and differentiation, as the quantity of secretory cargo increases once the activated cells start to produce and secrete large amounts of cytokines, cytotoxins, or antibodies. However, how the secretory machinery dynamically adapts its efficiency and specificity in general and specifically in lymphocytes remains incompletely understood. Here we present a systematic bioinformatics analysis to address RNA-based mechanisms that control intracellular trafficking and protein secretion during B-lymphocyte activation, and differentiation, with a focus on alternative splicing. Our in silico analyses suggest that alternative splicing has a substantial impact on the dynamic adaptation of intracellular traffic and protein secretion in different B cell subtypes, pointing to another regulatory layer to the control of lymphocyte function during activation and differentiation. Furthermore, we suggest that NERF/ELF2 controls the expression of some COPII-related genes in a cell type-specific manner. In addition, T cells and B cells appear to use different adaptive strategies to adjust their secretory machineries during the generation of effector and memory cells, with antibody secreting B cell specifically increasing the expression of components of the early secretory pathway. Together, our data provide hypotheses how cell type-specific regulation of the trafficking machinery during immune cell activation and differentiation is controlled that can now be tested in wet lab experiments.

PO087 / #896 BDNF VAL66MET POLYMORPHISM AS A POSSIBLE BIOMARKER OF EFFICACY OF VNS THERAPY IN SUBJECTS WITH DRUG-RESISTANT EPILEPSY: E-POSTER VIEWING

Vagal nerve stimulation (VNS) is safe and effective in adults and children with epilepsy according to FDA. Brain-derived Neurotrophic Factor (BDNF) is implicated in many neurophysiological processes and exerts effects on hippocampal serotonergic pathways during both acute and chronic VNS stimulation.1 BDNF gene encodes a precursor peptide (proBDNF) and non-conservative single nucleotide polymorphism (SNP) has been identified in humans producing an amino acid substitution (Val66Met). This SNP affects intracellular processing and secretion of BDNF, leading to impaired hippocampal function in humans.

Riociguat inhibits ultra-large VWF string formation on pulmonary artery endothelial cells from chronic thromboembolic pulmonary hypertension patients.

Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by elevated pulmonary arterial pressure and organized thrombi within pulmonary arteries. Riociguat is a soluble guanylate cyclase stimulator and is approved for patients with inoperable CTEPH or residual pulmonary hypertension after pulmonary endarterectomy (PEA). Previous work suggested that riociguat treatment is associated with an increased risk of bleeding, although the mechanism is unclear. The aim of this study is to assess how riociguat affects primary hemostasis by studying its effect on the interaction between platelets and endothelial cells derived from CTEPH patients. Pulmonary artery endothelial cells (PAECs) were isolated from thrombus-free regions of PEA material. Purified PAECs were cultured in flow chambers and were stimulated with 0.1 and 1 µM riociguat for 24 h before flow experiments. After stimulation with histamine, PAECs were exposed to platelets under shear stress. Platelet adhesion and expression of von Willebrand Factor (VWF) were evaluated to assess the role of riociguat in hemostasis. Under dynamic conditions, 0.1 and 1.0 µM of riociguat suppressed platelet adhesion on the surface of PAECs. Although riociguat did not affect intracellular expression and secretion of VWF, PAECs stimulated with riociguat produced fewer VWF strings than unstimulated PAECs. Flow cytometry suggested that decreased VWF string formation upon riociguat treatment may be associated with suppressed cell surface expression of P-selectin, a protein that stabilizes VWF anchoring on the endothelial surface. In conclusion, Riociguat inhibits VWF string elongation and platelet adhesion on the surface of CTEPH-PAECs, possibly by reduced P-selectin cell surface expression.

The 3D printed conductive grooved topography hydrogel combined with electrical stimulation for synergistically enhancing wound healing of dermal fibroblast cells

Patients with extensive cutaneous damage resulting from poor wound healing often have other comorbidities such as diabetes that may lead to impaired skin functions and scar formation. Many recent studies have shown that the application of electrical stimulation (ES) to cutaneous lesions significantly improves skin regeneration via activation of AKT intracellular signaling cascades and secretion of regeneration-related growth factors. In this study, we fabricated varying concentrations of gelatin-methacrylate (GelMa) hydrogels with poly(3,4-ethylenedioxythiophene) (PEDOT): polystyrene sulfonate (PSS), which is a conductive material commonly used in tissue engineering due to its efficiency among conductive thermo-elastic materials. The results showed successful modification of PEDOT:PSS with GelMa while retaining the original structural characteristics of the GelMa hydrogels. In addition, the incorporation of PEDOT:PSS increased the interactions between both the materials, thus leading to enhanced mechanical strength, improved swelling ratio, and decreased hydrophilicity of the scaffolds. Our GelMa/PEDOT:PSS scaffolds were designed to have micro-grooves on the surfaces of the scaffolds for the purpose of directional guiding. In addition, our scaffolds were shown to have excellent electrical conductivity, thus leading to enhanced cellular proliferation and directional migration and orientation of human dermal fibroblasts. In vivo studies revealed that the GelMa/PEDOT:PSS scaffolds with electrical stimulation were able to induce full skin thickness regeneration, as seen from the various stainings. These results indicate the potential of GelMa/PEDOT:PSS as an electro-conductive biomaterial for future skin regeneration applications.

Comprehensive in vitro polymer type, concentration, and size correlation analysis to microplastic toxicity and inflammation

The ubiquitous nature of microplastic particles (MP) is a growing environmental and ecological concern due to their impact on aquatic and terrestrial systems and potentially on human health. The potential impact on human health may be due to MP daily exposure by several routes, but little is known about the cellular effects. Previous in vitro and in vivo studies have described inflammation, oxidative stress, and metabolic disruption upon plastic exposure, while the effect of individual plastic parameters is not fully unraveled. To this end, we investigated plastic exposure to different polymer types, sizes, and concentrations in three human cell lines (A549, HEK293, and HeLa). Particles were polystyrene (PS) or polymethylmethacrylate (PMMA) in three sizes and concentrations, and amine-modified PS served as positive control. After MP size validation using dynamic light scattering, a high-throughput high-content imaging-based and algorithm-driven multi-z-stack analysis was established to quantify intracellular fluorescent particle accumulation in 3D objects and cell maximum intensity projections. MP uptake correlated with concentration and for PS with size (1.040 μm), while for PMMA it was maximal for 400 nm MP. Uptake increased in HEK cells independent of MP parameters. Except for positive controls, no major effect on metabolic activity, viability, and cell cycle was observed, while intracellular thiol content and cytokine secretion were affected to a considerable extent. Interestingly, particle uptake was correlated significantly with particle size and concentration, underlining the dependence of MP parameters on biological effects.

A role for N-glycosylation in active adenosine deaminase 2 production

BackgroundAdenosine deaminase 2 (ADA2) regulates extracellular levels of adenosine and the optimal expression of ADA2 is essential for modulating the immune system. However, the mechanisms regulating the production of active ADA2 enzyme are not fully understood. In this study, we examined the role of N-glycosylation in the formation of functional structures and the secretory pathway of ADA2. MethodsWe investigated the roles of N-glycosylation in the activity, homodimerization, and secretion of ADA2 via site-directed mutagenesis and the application of N-glycosylation inhibitors. Subcellular localization of ADA2 along with the endoplasmic reticulum (ER) glucosidase inhibitor was observed under confocal fluorescence microscope. ResultsInhibiting the initial N-glycosylation of ADA2 in the ER via site-directed mutagenesis or treatment with N-glycosylation inhibitors reduced the intracellular ADA2 activity and secretion. At this time, decreases in the ADA2 homodimers and ADA2 aggregation were observed in the cells. Treating the cells with castanospermine, an inhibitor of N-glycan editing in the ER, resulted in a reduction of the localization rate to the Golgi and markedly suppressed the ADA2 secretion. ConclusionsThese data suggest that the initial N-glycosylation and N-glycan editing in the ER are essential for the production of an active ADA2 enzyme and proper trafficking to the extracellular space. General significanceWith sufficient N-glycosylation in the ER, ADA2 exerts its function and is secreted extracellularly.

Effect of co-overexpression of the cargo receptor ERGIC-53/MCFD2 on antibody production and intracellular IgG secretion in recombinant Chinese hamster ovary cells

Therapeutic antibodies are attractive biopharmaceuticals because of their high therapeutic effects, fewer side effects, and prolonged half-life in the blood. Chinese hamster ovary (CHO) cells are the most widely used host cell lines to produce therapeutic antibodies in industries. High-producing recombinant CHO cells can be established via overexpression of endogenous proteins. In this study, we focused on the intracellular traffic of an antibody-producing CHO cell line, CHO-HcD6. Assembled antibodies were accumulated in the endoplasmic reticulum (ER) in the cell. We hypothesized that the accumulation was due to the insufficient number of cargo receptors in the cell and focused on a cargo receptor, the ERGIC-53-MCFD2 complex, which transports expressed proteins from the ER to the Golgi apparatus. Overexpression of the cargo receptor transport was expected to improve antibody production. Exogenous ERGIC-53 and MCFD2 were transfected into CHO-HcD6 cells, and overexpressing CHO-HcD6 cells were constructed. As a result of overexpression, antibody productivity increased in batch cultivation. However, the chase assay results and immunofluorescence microscopic observations revealed intracellular IgG accumulation in the overexpressing cells. These results suggest that overexpression of cargo receptors not only promoted extracellular secretion but also enhanced the retention of intracellular antibodies.

A yeast-based in vivo assay system for analyzing efflux of sugars mediated by glucose and xylose transporters.

Sugar transporter research focuses on the sugar uptake into cells. Under certain physiological conditions, however, the intracellular accumulation and secretion of carbohydrates (efflux) are relevant processes in many cell types. Currently, no cell-based system is available for specifically investigating glucose efflux. Therefore, we designed a system based on a hexose transporter-deficient Saccharomyces cerevisiae strain, in which the disaccharide maltose is provided as a donor of intracellular glucose. By deleting the hexokinase genes, we prevented the metabolization of glucose, and thereby achieved the accumulation of growth-inhibitory glucose levels inside the cells. When a permease mediating glucose efflux is expressed in this system, the inhibitory effect is relieved proportionally to the capacity of the introduced transporter. The assay is thereby suitable for screening of transporters and quantitative analyses of their glucose efflux capacities. Moreover, by simultaneous provision of intracellular glucose and extracellular xylose, we investigated how each sugar influences the transport of the other one from the opposite side of the membrane. Thereby, we could show that the xylose transporter variant Gal2N376F is insensitive not only to extracellular but also to intracellular glucose. Considering the importance of sugar transporters in biotechnology, the assay could facilitate new developments in a variety of applications.

Immune responses to Sinopharm/BBIBP-CorV in individuals in Sri Lanka.

As there are limited data of the immunogenicity of the Sinopharm/BBIBP-CorV in different populations, antibody responses against different SARS-CoV-2 variants of concern and T cell responses, we investigated the immunogenicity of the vaccine, in individuals in Sri Lanka. SARS-CoV-2-specific antibodies were measured in 282 individuals who were seronegative at baseline, and ACE2 receptor blocking antibodies, antibodies to the receptor-binding domain (RBD) of the wild-type (WT), alpha, beta and delta variants, ex vivo and cultured IFNγ ELISpot assays, intracellular cytokine secretion assays and B cell ELISpot assays were carried out in a sub cohort of the vaccinees at 4 and 6 weeks (2 weeks after the second dose). Ninety-five percent of the vaccinees seroconverted, although the seroconversion rates were significantly lower (p< 0.001) in individuals >60 years (93.3%) compared to those who were 20-39 years (98.9%); 81.25% had ACE2 receptor blocking antibodies at 6 weeks, and there was no difference in these antibody titres in vaccine sera compared to convalescent sera (p= 0.44). Vaccinees had significantly less (p< 0.0001) antibodies to the RBD of WT and alpha, although there was no difference in antibodies to the RBD of beta and delta compared to convalescent sera; 27.7% of 46.4% of vaccinees had ex vivo IFNγ and cultured ELISpot responses respectively, and IFNγ and CD107a responses were detected by flow cytometry. Sinopharm/BBIBP-CorV appeared to induce a similar level of antibody responses against ACE2 receptor, delta and beta as seen following natural infection.

Особливості CD3+- і CD3+CD4+-клітин периферичної крові на різних термінах вагітності в жінок із безплідністю в анамнезі

Infertile marriage is a complex socio-economic problem; the frequency of infertility in Ukraine is 17-20%, which prompts further study of its mechanisms, including immune ones, in order to develop appropriate methods of correction. Purpose - to characterize the phenotypic features of T-cells and their subpopulation of T-helpers (CD3+CD4+) with the expression of intracellular cytokines in women with a history of infertility at different stages of pregnancy. Materials and methods. Relative levels of immunocompetent cells (cl) of blood, including intracellular expression of cytokines CD3+CD4+cells, in 436 non-pregnant (the reference group n) and 514 pregnant women with infertility at the first trimester and the second trimester of pregnancy were determined using a cellular cytofluorimeter and the corresponding test systems. Characteristics of indicators in pregnant women at the different terms in groups were analyzed: a - 4-7 weeks (140 women); b - 8-9 weeks (163 women); c - 10-12 weeks (133 women); d - 13-18 weeks (63 women); e - 19-28 weeks (15 women) compared to the group n and among themselves. Results. The first 12 weeks of pregnancy in women with a history of infertility are characterized by high blood levels of T-helpers (CD3+CD4+-cl), and their activation according to the expression of markers CD25 and HLA-DR, and for 18 weeks - intracellular secretion of pro-inflammatory cytokines IFN-γ + and TNF-α, high expression of anti-inflammatory IL-4 was observed throughout the 27 weeks of follow-up. The percentage of women with high levels in the blood of CD3+CD4+IFN-γ+- and CD3+CD4+TNF-α+-L is the highest during the first 12 weeks, from 13th week these indicators for TNF-α, and with 20th - for IFN-γ do not differ from non-pregnant ones. Since the beginning of pregnancy, the relative number of women with low expression of IL-4 and IL-10 T-helpers has significantly decreased; within 13-18 weeks, the percentage of such pregnant women is significantly increased to the reference group, and such women are the most vulnerable due to the possible weak reaction of the anti-inflammatory link to the state of tolerance. Conclusions. The characteristics of peripheral blood immune system cell levels according to their CD phenotypes and intracellular cytokine expression indicate their participation in pregnancy mechanisms at different stages and individual analysis allows for negative predictions and improvement of pregnancy course in women with a history of infertility. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of the participating institution. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors. Keywords: CD-phenotype of immunocompetent cells, intracellular cytokines, pregnant women, history of infertility.

Insight Into the Molecular Mechanisms Underpinning the Mycoremediation of Multiple Metals by Proteomic Technique.

We investigated the fungus Aspergillus fumigatus PD-18 responses when subjected to the multimetal combination (Total Cr, Cd2+, Cu2+, Ni2+, Pb2+, and Zn2+) in synthetic composite media. To understand how multimetal stress impacts fungal cells at the molecular level, the cellular response of A. fumigatus PD-18 to 30 mg/L multimetal stress (5 mg/L of each heavy metal) was determined by proteomics. The comparative fungal proteomics displayed the remarkable inherent intracellular and extracellular mechanism of metal resistance and tolerance potential of A. fumigatus PD-18. This study reported 2,238 proteins of which 434 proteins were exclusively expressed in multimetal extracts. The most predominant functional class expressed was for cellular processing and signaling. The type of proteins and the number of proteins that were upregulated due to various stress tolerance mechanisms were post-translational modification, protein turnover, and chaperones (42); translation, ribosomal structure, and biogenesis (60); and intracellular trafficking, secretion, and vesicular transport (18). In addition, free radical scavenging antioxidant proteins, such as superoxide dismutase, were upregulated upto 3.45-fold and transporter systems, such as protein transport (SEC31), upto 3.31-fold to combat the oxidative stress caused by the multiple metals. Also, protein–protein interaction network analysis revealed that cytochrome c oxidase and 60S ribosomal protein played key roles to detoxify the multimetal. To the best of our knowledge, this study of A. fumigatus PD-18 provides valuable insights toward the growing research in comprehending the metal microbe interactions in the presence of multimetal. This will facilitate in development of novel molecular markers for contaminant bioremediation.

The Effects of Different Degrees of Hypoxia on Retinal Pigment Epithelium

In age-related macular degeneration (AMD), hypoxia causes abnormal changes in tissue metabolism and retinal pigment epithelium (RPE). However, the changes in cell function and intracellular molecules in RPE cells induced by hypoxia are not fully understood. In our study, cell viability, apoptosis, oxidative stress and changes in intracellular molecules at different time points were evaluated in ARPE-19 cells using a hypoxia model. A short period of mild hypoxia stimulated cell proliferation and apoptosis was induced as the period of hypoxia increased and the oxygen concentration decreased. As hypoxia worsened, the mitochondrial reactive oxygen species (ROS) accumulation was often accompanied with increased oxidative stress, increased intracellular HIF-1α accumulation and VEGF secretion to induce neovascularization. Additionally, p-ERK1 and ERK2 level was increased and p-ERK2 was decreased during hypoxia. In conclusion, our results show that the changes in the RPE is a process that depends on the degree of hypoxia and exposure time.

Broadly recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles.

Sequence homology between SARS-CoV-2 and common-cold human coronaviruses (HCoVs) raises the possibility that memory responses to prior HCoV infection can affect T cell response in COVID-19. We studied T cell responses to SARS-CoV-2 and HCoVs in convalescent COVID-19 donors and identified a highly conserved SARS-CoV-2 sequence, S811-831, with overlapping epitopes presented by common MHC class II proteins HLA-DQ5 and HLA-DP4. These epitopes are recognized by low-abundance CD4 T cells from convalescent COVID-19 donors, mRNA vaccine recipients, and uninfected donors. TCR sequencing revealed a diverse repertoire with public TCRs. T cell cross-reactivity is driven by the high conservation across human and animal coronaviruses of T cell contact residues in both HLA-DQ5 and HLA-DP4 binding frames, with distinct patterns of HCoV cross-reactivity explained by MHC class II binding preferences and substitutions at secondary TCR contact sites. These data highlight S811-831 as a highly conserved CD4 T cell epitope broadly recognized across human populations.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis.

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.