Proteins In Culture Supernatant Research Articles

Integration of line-field confocal optical coherence tomography and in situ microenvironmental mapping to investigate the living microenvironment of reconstructed human skin and melanoma models

BackgroundIn tissue engineering, real-time monitoring of tumors and of the dynamics of the microenvironment within in vitro models has traditionally been hindered by the need to harvest the cultures to obtain material to analyze. Line-field confocal optical coherence tomography (LC-OCT) has proven to be useful in evaluating in vivo skin conditions, including melanoma, by capturing dynamic, three-dimensional (3D) information without the need for invasive procedures, such as biopsies. Additionally, the M-Duo Technology® developed by IMcoMET presents a unique opportunity for continuous in situ biomarker sampling, providing insights into local cellular behavior and interactions. ObjectiveThis study aimed to validate the non-destructive mapping capabilities of two advanced methodologies (LC-OCT by DAMAE Medical and M-Duo Technology® by IMcoMET) to investigate the living microenvironment of in vitro reconstructed human skin (RhS) and melanoma-RhS (Mel-RhS). MethodsLC-OCT and M-Duo Technology® were compared to conventional analysis of the RhS and Mel-RhS microenvironments. ResultsLC-OCT successfully visualized the distinct layers of the epidermis and tumor structures within the Mel-RhS, identifying keratinocytes, melanocytes, tumor nests, and fibroblasts. The M-Duo Technology® revealed differences in in situ cytokine (IL-6) and chemokine (CCL2, CXCL10, and IL-8) secretion between Mel-RhS and the control RhS. Notably, such differences were not detected through conventional investigation of secreted proteins in culture supernatants. ConclusionThe combination of LC-OCT's high-resolution imaging and M-Duo Technology®’s in situ microenvironmental mapping has the potential to provide a synergistic platform for non-invasive, real-time analysis, allowing for prolonged observation of processes within Mel-RhS models without the need for culture disruption.

Breaking down barriers: comprehensive functional analysis of the Aspergillus niger chitin synthase repertoire

BackgroundMembers of the fungal kingdom are heterotrophic eukaryotes encased in a chitin containing cell wall. This polymer is vital for cell wall stiffness and, ultimately, cell shape. Most fungal genomes contain numerous putative chitin synthase encoding genes. However, systematic functional analysis of the full chitin synthase catalogue in a given species is rare. This greatly limits fundamental understanding and potential applications of manipulating chitin synthesis across the fungal kingdom.ResultsIn this study, we conducted in silico profiling and subsequently deleted all predicted chitin synthase encoding genes in the multipurpose cell factory Aspergillus niger. Phylogenetic analysis suggested nine chitin synthases evolved as three distinct groups. Transcript profiling and co-expression network construction revealed remarkably independent expression, strongly supporting specific role(s) for the respective chitin synthases. Deletion mutants confirmed all genes were dispensable for germination, yet impacted colony spore titres, chitin content at hyphal septa, and internal architecture of submerged fungal pellets. We were also able to assign specific roles to individual chitin synthases, including those impacting colony radial growth rates (ChsE, ChsF), lateral cell wall chitin content (CsmA), chemical genetic interactions with a secreted antifungal protein (CsmA, CsmB, ChsE, ChsF), resistance to therapeutics (ChsE), and those that modulated pellet diameter in liquid culture (ChsA, ChsB). From an applied perspective, we show chsF deletion increases total protein in culture supernatant over threefold compared to the control strain, indicating engineering filamentous fungal chitin content is a high priority yet underexplored strategy for strain optimization.ConclusionThis study has conducted extensive analysis for the full chitin synthase encoding gene repertoire of A. niger. For the first time we reveal both redundant and non-redundant functional roles of chitin synthases in this fungus. Our data shed light on the complex, multifaceted, and dynamic role of chitin in fungal growth, morphology, survival, and secretion, thus improving fundamental understanding and opening new avenues for biotechnological applications in fungi.

Protein profiling and immunoinformatic analysis of the secretome of a metal-resistant environmental isolate Pseudomonas aeruginosa S-8.

The bacterial secretome represents a comprehensive catalog of proteins released extracellularly that have multiple important roles in virulence and intercellular communication. This study aimed to characterize the secretome of an environmental isolate Pseudomonas aeruginosa S-8 by analyzing trypsin-digested culture supernatant proteins using nano-LC-MS/MS tool. Using a combined approach of bioinformatics and mass spectrometry, 1088 proteins in the secretome were analyzed by PREDLIPO, SecretomeP 2.0, SignalP 4.1, and PSORTb tool for their subcellular localization and further categorization of secretome proteins according to signal peptides. Using the gene ontology tool, secretome proteins were categorized into different functional categories. KEGG pathway analysis identified the secreted proteins into different metabolic functional pathways. Moreover, our LC-MS/MS data revealed the secretion of various CAZymes into the extracellular milieu, which suggests its strong biotechnological applications to breakdown complex carbohydrate polymers. The identified immunodominant epitopes from the secretome of P. aeruginosa showed the characteristic of being non-allergenic, highly antigenic, nontoxic, and having a low risk of triggering autoimmune responses, which highlights their potential as successful vaccine targets. Overall, the identification of secreted proteins of P. aeruginosa could be important for both diagnostic purposes and the development of an effective candidate vaccine.

Glycyrrhizae Radix suppresses lipopolysaccharide- and diazepam-induced nerve inflammation in the hippocampus, and contracts the duration of pentobarbital- induced loss of righting reflex in a mouse model

Nerve inflammation is linked to the development of various neurological disorders. This study aimed to examine whether Glycyrrhizae Radix effectively influences the duration of the pentobarbital-induced loss of righting reflex, which may increase in a mouse model of lipopolysaccharide (LPS)-induced nerve inflammation and diazepam-induced γ-aminobutyric acid receptor hypersensitivity. Furthermore, we examined the anti-inflammatory effects of Glycyrrhizae Radix extract on LPS-stimulated BV2 microglial cells, in vitro. Treatment with Glycyrrhizae Radix significantly decreased the duration of pentobarbital-induced loss of righting reflex in the mouse model. Furthermore, treatment with Glycyrrhizae Radix significantly attenuated the LPS-induced increases in interleukin-1β, interleukin-6, and tumor necrosis factor-alpha at the mRNA level, and it significantly reduced the number of ionized calcium-binding adapter molecule-1-positive cells in the hippocampal dentate gyrus 24h after LPS treatment. Treatment with Glycyrrhizae Radix also suppressed the release of nitric oxide, interleukin-1β, interleukin-6, and tumor necrosis factor protein in culture supernatants of LPS-stimulated BV2 cells. In addition, glycyrrhizic acid and liquiritin, active ingredients of Glycyrrhizae Radix extract, reduced the duration of pentobarbital-induced loss of righting reflex. These findings suggest that Glycyrrhizae Radix, as well as its active ingredients, glycyrrhizic acid and liquiritin, may be effective therapeutic agents for the treatment of nerve inflammation-induced neurological disorders.

Marine prebiotics mediate decolonization of Pseudomonas aeruginosa from gut by inhibiting secreted virulence factor interactions with mucins and enriching Bacteroides population

BackgroundPseudomonas aeruginosa intestinal carriage rates are significantly higher in immunosuppressed individuals and hospitalized patients who therefore have increased risk of infections and antibiotic-associated diarrhea. To combat intestinal dysbiosis and decolonize P. aeruginosa from gastrointestinal tract, we investigated the anti-adherence and gut microbiota modulation properties of marine prebiotic fucoidans.MethodsProteomic analysis of culture supernatant was performed by LC–MS/MS. Using lectin-based enzyme-linked immunosorbent assay, hemagglutinin domain interaction and inhibition with biomolecules were studied. We investigated the role of nutritional grade fucoidans in a mouse model and used 16S ribosomal RNA sequencing to examine fecal microbiota composition.ResultsAnalysis of culture supernatant proteins indicated the secretion of two-partner secretion (TPS) family proteins, including TpsA1/CdiA2 and TpsA2/CdiA1. Lectin like activity at the N-terminal of TpsA due to a conserved hemagglutinin domain (Pfam identifier [ID] PF05860) mediates binding to mucins that carry multiple fucosylated glycans. Fucose-rich sulfated polysaccharides (fucoidans) and sulfated dextrans were found to be potent inhibitors of the recombinant N-terminal hemagglutinin domain of TpsA (TpsA-NT-HAD) binding to mucins. In a mouse model, antibiotic-induced dysbiosis was essential for P. aeruginosa gastrointestinal colonization. After prophylactic oral fucoidans supplementation, a higher proportion (60%) of the mice were decolonized over time and resisted re-colonization, this was associated with remarkable expansion of Bacteroides (post-infection day-3 abundance, 29–50%) and consequential reductions in bloom of Enterobacteriaceae and Enterococcaceae populations. In the non-supplemented group, Parabacteroides mediated recovery from dysbiosis but failed to decolonize P. aeruginosa.ConclusionsSupplementing diet with marine prebiotic fucoidans can mediate earlier recovery from dysbiosis and decolonization of P. aeruginosa from gut by inhibiting secreted virulence factor (TpsA/CdiA) interaction with mucins and promoting the growth of beneficial Bacteroides population. We suggest the prophylactic use of nutritional grade fucoidans to decolonize P. aeruginosa from gastrointestinal tract of at-risk individuals to prevent infection and transmission of colonizing P. aeruginosa.

Secretome of Paenibacillus sp. S-12 provides an insight about its survival and possible pathogenicity.

Our aim in this study was to characterize and investigate the secretome of Paenibacillus sp. S-12 by nanoLC-MS/MS tool-based analysis of trypsin digested culture supernatant proteins. Using a bioinformatics and combined approach of mass spectrometry, we identified 657 proteins in the secretome. Bioinformatic tools such as PREDLIPO, SecretomeP 2.0, SignalP 4.1, and PSORTb were used for the subcellular localization and categorization of secretome on basis of signal peptides. Among the identified proteins, more than 25% of the secretome proteins were associated with virulence proteins including flagellar, adherence, and immune modulators. Gene ontology analysis using Blast2GO tools categorized 60 proteins of the secretome into biological processes, cellular components, and molecular functions. KEGG pathway analysis identified the enzymes or proteins involved in various biosynthesis and degradation pathways. Functional analysis of secretomes reveals a large number of proteins involved in the uptake and exchange of nutrients, colonization, and chemotaxis. A good number of proteins were involved in survival and defense mechanism against oxidative stress, the production of toxins and antimicrobial compounds. The present study is the first report of the in-depth protein profiling of Paenibacillus bacterium. In summary, the current findings of Paenibacillus sp. S-12 secretome provide basic information to understand its survival and the possible pathogenic mechanism.

Study on the Mechanism of Radix Astragali against Renal Aging Based on Network Pharmacology.

Radix Astragali is widely used in the traditional Chinese medicine with the effect of antiaging. The purpose of this study is to explore the main active ingredients and targets of Radix Astragali against renal aging by network pharmacology and further to verify the mechanism of the main active ingredients in vitro. TCMSP, ETCM, and TCMID databases were used to screen active ingredients of Radix Astragali. Targets of active ingredients were predicted using BATMAN-TCM and cross validated using kidney aging-related genes obtained from GeneCards and NCBI database. Pathways enrichment and protein-protein interaction (PPI) analysis were performed on core targets. Additionally, a pharmacological network was constructed based on the active ingredients-targets-pathways. HK-2 cell was treated with D-galactose to generate a cell model of senescence. CCK-8 and β-galactosidase were used to detect the effect of Radix Astragali active components on cell proliferation and aging. ELISA was used to detect the expression of senescence-associated secreted protein (TGF-β and IL-6) in the cell culture supernatant. Western blot was used to detect the expression of key proteins in the SIRT1/p53 pathway. Five active ingredients (Astragaloside I, II, III, IV and choline) were identified from Radix Astragali, and all these active ingredients target a total of 128 genes. Enrichment analysis showed these genes were implicated in 153 KEGG pathways, including the p53, FoxO, and AMPK pathway. 117 proteins and 572 interactions were found in PPI network. TP53 and SIRT1 were two hub genes in PPI network, which interacted with each other. The pharmacological network showed that the five main active ingredients target on some coincident genes, including TP53 and SIRT1. These targeted genes were involved in the p53, FoxO, and AMPK pathway. Proliferation of HK-2 cells was increased by Astragaloside IV treatment compared with that of the D-Gal treatment group. However, the proliferation of the SA-β-gal positive cells were inhibited. The expression of TGF-β and IL-6 in the D-Gal group was higher than that in the normal group, and the treatment of Astragaloside IV could significantly reduce the expression of TGF-β and IL-6. The expression of SIRT1 in the Astragaloside IV group was higher than that in the D-Gal group. However, the expression of p53 and p21 was less in the Astragaloside IV group than that in the D-Gal group. This study suggested that Astragaloside IV is an important active ingredient of Radix Astragali in the treatment of kidney aging via the SITR1-p53 pathway.

Effect of Dermatophagoides pteronyssinus extract on the expression of genes involved in inflammation and tissue remodeling by peripheral blood mononuclear cells of allergic asthma patients

PurposeHouse dust mite allergy constitutes a risk factor for asthma development and is associated with a faster decline of lung function in allergic asthmatics (AAs).To evaluate the effect of Dermatophagoides pteronyssinus (Dp) allergens on the expression of genes involved in inflammation and tissue remodeling by peripheral blood mononuclear cells (PBMCs) isolated from the blood of AAs. Materials and methodsThe cells from AAs, allergic rhinitis without asthma patients (ARs), and healthy controls (HCs) were cultured in the presence of Dp, lipopolysaccharide (LPS), or without any stimulation. The expression of 84 genes was evaluated using a low-density microarray whereas, the quantitative expression analysis of selected genes was performed using a real-time polymerase chain reaction. The concentration of selected proteins in the cell culture supernatants was assessed using ELISA. ResultsStimulation of PBMCs with Dp and LPS resulted in a significant upregulation of 8 and 15 among 84 studied genes, respectively. The greatest upregulation was observed for CCL2 and CCL3 using Dp and LPS, respectively. In comparison with HCs, in AAs, significantly increased upregulation of CCL2 in response to Dp was found. The secretion of CCL2 and CCL3 by PBMCs reflected the pattern of gene expression at the mRNA level. The mean Dp-stimulated secretion of CCL2 by PBMCs of ARs was less than in AAs (p ​< ​0.01), both being notably greater than in the HCs (p ​< ​0.01). ConclusionRapid and potent upregulation of CCL2 expression by PBMCs in response to Dp may constitute an important contribution to the development of allergic asthma.

Inflammatory response in human lung cells stimulated with plasma from COPD patients.

BackgroundChronic obstructive pulmonary disease (COPD) is a condition resulting from a persistent inflammatory state in the airways even after smoking cessation. Intriguingly, the reasons behind this persistence of the inflammatory influx without smoking exposure have not been fully unraveled. We aimed to explore the hypothesis that systemic inflammation in COPD patients influences lung cell inflammatory response.MethodsWe cultured human lung fibroblast and human airway epithelial cell lines with plasma from COPD patients (four emphysematous-COPD, four asthma-COPD overlap, four chronic bronchitis-COPD, and four bronchiectasis- COPD), and four smokers or ex-smokers without COPD as controls. Non-stimulated cells were used as controls. We measured Interleukine-8 (IL-8), C-reactive protein (CRP) and matrix metalloproteinase-9 (MMP-9) in plasma and culture supernatants by ELISA.ResultsCells stimulated with plasma from COPD patients and non-COPD smoker subjects produced higher CRP, IL- 8 and MMP-9 levels, an increase for COPD in CRP (p=0.029) in epithelial cells and IL-8 (p=0.039) in fibroblasts and decrease for MMP-9 (p=0.039) in fibroblasts, compared with non-stimulated cells. The response was higher in epithelial cells for IL-8 (p=0.003) and in fibroblasts for MMP-9 (p=0.063). The plasma from chronic bronchitis and bronchiectasis phenotypes induced higher IL-8 in fibroblasts.ConclusionsPlasma from COPD patients increases the inflammatory response in lung epithelial cells and lung fibroblasts, with a different response depending on the cell type and clinical phenotype.

Sildenafil Citrate Downregulates PDE5A mRNA Expression in Women with Recurrent Pregnancy Loss without Altering Angiogenic Factors-A Preliminary Study.

In our previous study, we showed that sildenafil citrate (SC), a selective PDE5A blocker, modulated NK cell activity in patients with recurrent pregnancy loss, which correlated with positive pregnancy outcomes. It was found that NK cells had a pivotal role in decidualization, angiogenesis, spiral artery remodeling, and the regulation of trophoblast invasion. Thus, in the current study, we determined the effects of SC on angiogenic factor expression and production, as well as idNK cell activity in the presence of nitric synthase blocker L-NMMA. Methods: NK cells (CD56+) were isolated from the peripheral blood of 15 patients and 15 fertile women on MACS columns and cultured in transformation media containing IL-15, TGF-β, and AZA—a methylation agent—for 7 days in hypoxia (94% N2, 1% O2, 5% CO2). Cultures were set up in four variants: (1) with SC, (2) without SC, (3) with NO, a synthase blocker, and (4) with SC and NO synthase blocker. NK cell activity was determined after 7 days of culturing as CD107a expression after an additional 4h of stimulation with K562 erythroleukemia cells. The expression of the PDE5A, VEGF-A, PIGF, IL-8, and RENBP genes was determined with quantitative real-time PCR (qRT-PCR) using TaqMan probes and ELISA was used to measure the concentrations of VEGF-A, PLGF, IL-8, Ang-I, Ang-II, IFN–γ proteins in culture supernatants after SC supplementation. Results: SC downregulated PDE5A expression and had no effect on other studied angiogenic factors. VEGF-A expression was increased in RPL patients compared with fertile women. Similarly, VEGF production was enhanced in RPL patients’ supernatants and SC increased the concentration of PIGF in culture supernatants. SC did not affect the expression or concentration of other studied factors, nor idNK cell activity, regardless of NO synthase blockade.

Bovine apo-lactoferrin affects the secretion of proteases in Mannheimia haemolytica A2.

Mannheimia haemolytica serotype A2 is the main bacterial causative agent of ovine mannheimiosis, a disease that leads to substantial economic losses for livestock farmers. Several virulence factors allow M. haemolytica to colonize the lungs and establish infection. Virulence factors can be directly secreted into the environment by bacteria but are also released through outer membrane vesicles (OMVs). In addition, due to the abuse of antibiotics in the treatment of this disease, multidrug-resistant bacterial strains of M. haemolytica have emerged. One therapeutic alternative to antibiotics or an adjuvant to be used in combination with antibiotics could be lactoferrin (Lf), a multifunctional cationic glycoprotein of the mammalian innate immune system to which no bacterial resistance has been reported. The aim of this work was to determine the effect of bovine iron-free Lf (apo-BLf) on the production and secretion of proteases into culture supernatant (CS) and on their release in OMVs. Zymography assays showed that addition of sub-MIC concentrations of apo-BLf to M. haemolytica cultures inhibited protease secretion without affecting culture growth. Biochemical characterization revealed that these proteases were mainly cysteine- and metalloproteases. The secretion of a 100 kDa metalloprotease was inhibited by sub-MIC concentrations of apo-BLf since this protease was present in the cytoplasm and OMVs but not in CS proteins, as corroborated by Western blotting. On the other hand, proteases produced by M. haemolytica caused cleavage of apo-BLf. However, when Lf is cleaved, peptides known as lactoferricins, which are more bactericidal than natural Lf, can be produced. M. haemolytica A2 protease-mediated degradation of host tissue proteins could be an important virulence factor during the infectious process of pneumonia in ovines. The mechanism of M. haemolytica protease secretion could be inhibited by treatment with apo-BLf in animals.

Infection with SARS-CoV-2 primes immunological memory in human nasal-associated lymphoid tissue

BackgroundThe coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has resulted in considerable morbidity and mortality in humans. Little is known regarding the development of immunological memory following SARS-CoV-2 infection or whether immunological memory can provide long-lasting protection against reinfection. Urgent need for vaccines is a considerable issue for all governments worldwide. MethodsA total of 39 patients were recruited in this study. Tonsillar mononuclear cells (MNCs) were co-cultured in RPMI medium and stimulated with the full-length SARS-CoV-2 spike protein in the presence and absence of a CpG-DNA adjuvant. An enzyme-linked immunosorbent assay (ELISA) was utilised to measure the specific antibody response to the spike protein in the cell culture supernatants. ResultsThe SARS-CoV-2 spike protein primed a potent memory B cell-mediated immune response in nasal-associated lymphoid tissue (NALT) from patients previously infected with the virus. Additionally, spike protein combined with the CpG-DNA adjuvant induced a significantly increased level of specific anti-spike protein IgG antibody compared with the spike protein alone (p < 0.0001, n = 24). We also showed a strong positive correlation between the specific anti-spike protein IgG antibody level in a serum samples and that produced by MNCs derived from the same COVID-19-recovered patients following stimulation (r = 0.76, p = 0.0002, n = 24). ConclusionIndividuals with serological evidence of previous SARS-CoV-2 exposure showed a significant anti-spike protein-specific memory humoral immune response to the viral spike protein upon stimulation. Additionally, our results demonstrated the functional response of NALT-derived MNCs to the viral spike protein. CpG-DNA adjuvant combined with spike protein induced significantly stronger humoral immune responses than the spike protein alone. These data indicate that the S protein antigen combined with CpG-DNA adjuvant could be used as a future vaccine candidate.

Extracellular Proteome Analysis Shows the Abundance of Histidine Kinase Sensor Protein, DNA Helicase, Putative Lipoprotein Containing Peptidase M75 Domain and Peptidase C39 Domain Protein in Leptospira interrogans Grown in EMJH Medium.

Leptospirosis is a re-emerging form of zoonosis that is caused by the spirochete pathogen Leptospira. Extracellular proteins play critical roles in the pathogenicity and survival of this pathogen in the host and environment. Extraction and analysis of extracellular proteins is a difficult task due to the abundance of enrichments like serum and bovine serum albumin in the culture medium, as is distinguishing them from the cellular proteins that may reach the analyte during extraction. In this study, extracellular proteins were separated as secretory proteins from the culture supernatant and surface proteins were separated during the washing of the cell pellet. The proteins identified were sorted based on the proportion of the cellular fractions and the extracellular fractions. The results showed the identification of 56 extracellular proteins, out of which 19 were exclusively extracellular. For those proteins, the difference in quantity with respect to their presence within the cell was found to be up to 1770-fold. Further, bioinformatics analysis elucidated characteristics and functions of the identified proteins. Orthologs of extracellular proteins in various Leptospira species were found to be closely related among different pathogenic forms. In addition to the identification of extracellular proteins, this study put forward a method for the extraction and identification of extracellular proteins.

Proteolytic Maturation of the Outer Membrane c-Type Cytochrome OmcZ by a Subtilisin-Like Serine Protease Is Essential for Optimal Current Production by Geobacter sulfurreducens.

An outer membrane c-type cytochrome (OmcZ) in Geobacter sulfurreducens is essential for optimal current production in microbial fuel cells. OmcZ exists in two forms, small and large, designated OmcZS and OmcZL, respectively. However, it is still not known how these two structures are formed. A mutant with a disruption of the GSU2075 gene encoding a subtilisin-like serine protease (designated ozpA for the OmcZprotease), which is located downstream of omcZ, produced low currents at a level similar to that of the omcZ-deficient mutant strain. Biochemical analyses revealed that the ozpA mutant accumulated OmcZL and did not produce OmcZS, which is thought to be a mature form that is essential for the extracellular electron transfer to the electrode. A heterologous expression system cell lysate from an Escherichia coli strain producing OzpA cleaved OmcZL and generated OmcZS as the proteolytic product. Among the culture supernatant, loosely bound outer surface, and intracellular protein fractions from wild-type G. sulfurreducens, only the culture supernatant protein fraction showed OmcZL cleavage activity, indicating that the mature form of OmcZ, OmcZS, can be produced outside the cells. These results indicate that OzpA is an essential protease for current production via the maturation of OmcZ, and OmcZS is the key to the extracellular electron transfer to electrodes. This proteolytic maturation of OmcZ is a unique regulation among known c-type cytochromes in G. sulfurreducens. IMPORTANCE Microbial fuel cells are a promising technology for energy generation from various waste types. However, the molecular mechanisms of microbial extracellular electron transfer to the electrode need to be elucidated. G. sulfurreducens is a common key player in electricity generation in mixed-culture microbial fuel cell systems and a model microorganism for the study of extracellular electron transfer. Outer membrane c-type cytochrome OmcZ is essential for an optimal current production by G. sulfurreducens. OmcZ proteolytic cleavage occurs during maturation, but the underlying mechanism is unknown. This study identifies a subtilisin-like protease, OzpA, which plays a role in cleaving OmcZ and generating the mature form of OmcZ (OmcZS). OzpA is essential for current production and, thus, the proteolytic maturation of OmcZ. This is a novel regulation of the c-type cytochrome for G. sulfurreducens extracellular electron transfer. This study also provides new insights into the design strategy and development of microbial extracellular electron transfer for an efficient energy conversion from chemical energy to electricity.

Velocity Gradient Separation Reveals a New Extracellular Vesicle Population Enriched in miR-155 and Mitochondrial DNA.

Extracellular vesicles (EVs) and their contents (proteins, lipids, messenger RNA, microRNA, and DNA) are viewed as intercellular signals, cell-transforming agents, and shelters for viruses that allow both diagnostic and therapeutic interventions. EVs circulating in the blood of individuals infected with human immunodeficiency virus (HIV-1) may provide insights into pathogenesis, inflammation, and disease progression. However, distinguishing plasma membrane EVs from exosomes, exomeres, apoptotic bodies, virions, and contaminating proteins remains challenging. We aimed at comparing sucrose and iodixanol density and velocity gradients along with commercial kits as a means of separating EVs from HIV particles and contaminating protein like calprotectin; and thereby evaluating the suitability of current plasma EVs analysis techniques for identifying new biomarkers of HIV-1 immune activation. Multiple analysis have been performed on HIV-1 infected cell lines, plasma from HIV-1 patients, or plasma from HIV-negative individuals spiked with HIV-1. Commercial kits, the differential centrifugation and density or velocity gradients to precipitate and separate HIV, EVs, and proteins such as calprotectin, have been used. EVs, virions, and contaminating proteins were characterized using Western blot, ELISA, RT-PCR, hydrodynamic size measurement, and enzymatic assay. Conversely to iodixanol density or velocity gradient, protein and virions co-sedimented in the same fractions of the sucrose density gradient than AChE-positive EVs. Iodixanol velocity gradient provided the optimal separation of EVs from viruses and free proteins in culture supernatants and plasma samples from a person living with HIV (PLWH) or a control and revealed a new population of large EVs enriched in microRNA miR-155 and mitochondrial DNA. Although EVs and their contents provide helpful information about several key events in HIV-1 pathogenesis, their purification and extensive characterization by velocity gradient must be investigated thoroughly before further use as biomarkers. By revealing a new population of EVs enriched in miR-155 and mitochondrial DNA, this study paves a way to increase our understanding of HIV-1 pathogenesis.

Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes.

In a disease-state-dependent manner, the histamine-resistant itch in dry skin-based skin diseases such as atopic dermatitis (AD) and xerosis is mainly due to hyperinnervation in the epidermis. Semaphorin 3A (Sema3A) is a nerve repulsion factor expressed in keratinocytes and it suppresses nerve fiber elongation in the epidermis. Our previous studies have shown that Sema3A ointment inhibits epidermal hyperinnervation and scratching behavior and improves dermatitis scores in AD model mice. Therefore, we consider Sema3A as a key therapeutic target for improving histamine-resistant itch in AD and xerosis. This study was designed to screen a library of herbal plant extracts to discover compounds with potential to induce Sema3A in normal human epidermal keratinocytes (NHEKs) using a reporter gene assay, so that positive samples were found. Among the positive samples, only the extract of S. baicalensis was found to consistently increase Sema3A levels in cultured NHEKs in assays using quantitative real-time PCR and ELISA. In evaluation of reconstituted human epidermis models, the level of Sema3A protein in culture supernatants significantly increased by application of the extract of S. baicalensis. In addition, we investigated which components in the extract of S. baicalensis contributed to Sema3A induction and found that baicalin and baicalein markedly increased the relative luciferase activity, and that baicalein had higher induction activity than baicalin. Thus, these findings suggest that S. baicalensis extract and its compounds, baicalin and baicalein, may be promising candidates for improving histamine-resistant itch via the induction of Sema3A expression in epidermal keratinocytes.

A Simple Method for In-Depth Proteome Analysis of Mammalian Cell Culture Conditioned Media Containing Fetal Bovine Serum.

A conditioned medium of a cell culture is widely used for various biological applications and frequently analyzed to characterize the functional proteins responsible for observed biological functions. However, a large number of abundant proteins in fetal bovine serum (FBS), usually included in the conditioned medium of a mammalian cell culture medium, hampers in-depth proteomic analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS). For a deep proteomic analysis of a conditioned medium by LC-MS/MS, we developed a simple albumin depletion approach coupled with data-independent acquisition (DIA)-mode LC-MS/MS for the conditioned medium of mammalian cells in this study. The results showed that this approach enabled the detection of more than 3700 cell-derived proteins in the cell culture supernatant containing FBS. We further demonstrated the potency of this approach by analyzing proteins in the conditioned media of HeLa cells with and without tumor necrosis factor (TNF) stimulation: >40 differentially accumulated proteins, including four cytokines, upon TNF stimulation were identified in the culture media, which were hardly detected by conventional proteome approaches in the literature.

Development of the high-productivity marine microalga, Picochlorum renovo, as a photosynthetic protein secretion platform

Microalgae are remarkable in their ability to convert CO2 and sunlight into renewable biomass and bioproducts. Here, we have established a novel photosynthetic protein production platform via computational prediction and genetic engineering using native microalgal protein secretory signal peptides to achieve functional secretion of the fluorescent protein mCherry. Importantly, this work was conducted in the recently characterized alga Picochlorum renovo, an alga of industrial interest due to its rapid growth rate, tolerance to both high temperature and salinity, and genetically tractable nuclear and chloroplast genomes. Genomic queries allowed the identification of native secretory signal peptides, which were N-terminally fused to mCherry allowing for secretion into the culture supernatant. Further characterization revealed no impact on fitness, a production rate of 0.19 mg/L/day, and titer of 0.37 mg/L of transgenic mCherry protein in culture supernatant. These findings lay the foundation for applied genetic engineering approaches that could enable CO2-sequestering, sustainable photoproduction of industrially relevant enzymes at low cost.

Vitamin A regulates fibroblast growth factor 23 (FGF23).

Renal phosphate and vitamin D metabolism are regulated by proteohormone fibroblast growth factor 23 (FGF23), which is secreted by bone cells. FGF23 inhibits phosphate reabsorption and the production of calcitriol, active vitamin D (1,25(OH)2D3). FGF23 generated by other cells exerts further paracrine effects in the liver, heart, and immune system. The FGF23 plasma concentration is positively associated with the onset and progression of kidney and cardiovascular diseases, disclosing FGF23 as a potential disease biomarker. The effects of vitamin A on the expression of FGF23 are controversial. Vitamin A components, retinoids, are mainly effective through nuclear retinoic acid receptors (RAR) and exert different effects on bone. The aim of this study was to clarify whether vitamin A modulates the production of FGF23. We studied the relevance of vitamin A for FGF23 production. Fgf23 transcripts were determined by real-time quantitative polymerase chain reaction in UMR106 osteoblast-like cells and IDG-SW3 osteocytes. FGF23 protein in the cell culture supernatant was measured by enzyme-linked immunosorbent assay. All-trans-retinoic acid, retinyl acetate, RAR agonist TTNPB (4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid), and 13-cis-retinoic acid downregulated the expression of the Fgf23 gene in a dose-dependent manner. This effect was significantly attenuated by RAR antagonist AGN193109 (4-[2-[5,6-Dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl]ethynyl]benzoic acid). The present study demonstrated that vitamin A is a potent suppressor of FGF23 production through RAR.

A disintegrin and metalloproteinase domain 17-epidermal growth factor receptor signaling contributes to oral cancer pain.

Cancer cells secrete pronociceptive mediators that sensitize adjacent sensory neurons and cause pain. Identification and characterization of these mediators could pinpoint novel targets for cancer pain treatment. In this study, we identified candidate genes in cancer cell lines that encode for secreted or cell surface proteins that may drive nociception. To undertake this work, we used an acute cancer pain mouse model, transcriptomic analysis of publicly available human tumor-derived cell line data, and a literature review. Cancer cell line supernatants were assigned a phenotype based on evoked nociceptive behavior in an acute cancer pain mouse model. We compared gene expression data from nociceptive and nonnociceptive cell lines. Our analyses revealed differentially expressed genes and pathways; many of the identified genes were not previously associated with cancer pain signaling. Epidermal growth factor receptor (EGFR) and disintegrin metalloprotease domain 17 (ADAM17) were identified as potential targets among the differentially expressed genes. We found that the nociceptive cell lines contained significantly more ADAM17 protein in the cell culture supernatant compared to nonnociceptive cell lines. Cytoplasmic EGFR was present in almost all (>90%) tongue primary afferent neurons in mice. Monoclonal antibody against EGFR, cetuximab, inhibited cell line supernatant-induced nociceptive behavior in an acute oral cancer pain mouse model. We infer from these data that ADAM17-EGFR signaling is involved in cancer mediator-induced nociception. The differentially expressed genes and their secreted protein products may serve as candidate therapeutic targets for oral cancer pain and warrant further evaluation.