Total Cell Protein Research Articles

Production of Reverse Transcriptase and DNA Polymerase in Bacterial Expression Systems.

DNA amplification and reverse transcription enzymes have proven to be invaluable in fast and reliable diagnostics and research applications because of their processivity, specificity, and robustness. Our study focused on the production of mutant Taq DNA polymerase and mutant M-MLV reverse transcriptase in the expression hosts Vibrio natriegens and Escherichia coli under various expression conditions. We also examined nonspecific extracellular production in V. natriegens. Intracellularly, M-MLV was produced in V. natriegens at the level of 11% of the total cell proteins (TCPs) compared with 16% of TCPs in E. coli. We obtained a soluble protein that accounted for 11% of the enzyme produced in V. natriegens and 22% of the enzyme produced in E. coli. Taq pol was produced intracellularly in V. natriegens at the level of 30% of TCPs compared with 26% of TCPs in E. coli. However, Taq pol was almost non-soluble in E. coli, whereas in V. natriegens, we obtained a soluble protein that accounted for 23% of the produced enzyme. We detected substantial extracellular production of Taq pol. Thus, V. natriegens is a suitable alternative host with the potential for production of recombinant proteins.

3-methyladenine ameliorates acute lung injury by inhibiting oxidative damage and apoptosis

BackgroundAcute lung injury (ALI) is a condition characterized by inflammation and oxidative damage. 3-methyladenine (3-MA) has great potential for regulating apoptosis, but its regulatory role in ALI is unknown. MethodsLipopolysaccharide (LPS)-treated mice and tert-butyl hydroperoxide (TBHP)-treated bronchial epithelial cells were used to simulate in vivo and in vitro ALI models, respectively. In vivo, lung injury was assessed by histopathological analysis and lung injury scoring. The total cell count, protein content, and inflammatory factors in bronchoalveolar lavage fluid (BALF) were examined. The level of apoptosis in lung tissue was assessed through TUNEL staining. In the vitro ALI model, cell viability and levels of reactive oxygen species and apoptosis were assessed. Results3-MA pretreatment ameliorated lung injury, including intra-alveolar hemorrhage and inflammatory cell accumulation, both in vitro and in vivo. 3-MA pretreatment also decreased inflammatory factor levels in the BALF. 3-MA pretreatment alleviated oxidative damage, decreased reactive oxygen species levels, and attenuated morphological changes. TUNEL and Annexin V-FITC/PI staining revealed that pretreatment with 3-MA reduced the level of apoptosis. 3-MA pretreatment significantly decreased the expression of caspase-3 and Bax but increased the expression of Bcl-2 in ALI. Mechanistically, 3-MA pretreatment also affected the PKCα/NOX4 and Nrf2 pathways, which decreased the level of apoptosis in ALI. Conclusions3-MA pretreatment inhibited inflammation and oxidative damage in ALI and inhibited apoptosis to mitigate ALI in part by inhibiting the PKCα/NOX4 pathway and activating the Nrf2 pathway. Based on these results, 3-MA might be a viable medication to treat with ALI.

Mulberry Leaf Compounds and Gut Microbiota in Alzheimer's Disease and Diabetes: A Study Using Network Pharmacology, Molecular Dynamics Simulation, and Cellular Assays.

Recently, studies have reported a correlation that individuals with diabetes show an increased risk of developing Alzheimer's disease (AD). Mulberry leaves, serving as both a traditional medicinal herb and a food source, exhibit significant hypoglycemic and antioxidative properties. The flavonoid compounds in mulberry leaf offer therapeutic effects for relieving diabetic symptoms and providing neuroprotection. However, the mechanisms of this effect have not been fully elucidated. This investigation aimed to investigate the combined effects of specific mulberry leaf flavonoids (kaempferol, quercetin, rhamnocitrin, tetramethoxyluteolin, and norartocarpetin) on both type 2 diabetes mellitus (T2DM) and AD. Additionally, the role of the gut microbiota in these two diseases' treatment was studied. Using network pharmacology, we investigated the potential mechanisms of flavonoids in mulberry leaves, combined with gut microbiota, in combating AD and T2DM. In addition, we identified protein tyrosine phosphatase 1B (PTP1B) as a key target for kaempferol in these two diseases. Molecular docking and molecular dynamics simulations showed that kaempferol has the potential to inhibit PTP1B for indirect treatment of AD, which was proven by measuring the IC50 of kaempferol (279.23 μM). The cell experiment also confirmed the dose-dependent effect of kaempferol on the phosphorylation of total cellular protein in HepG2 cells. This research supports the concept of food-medicine homology and broadens the range of medical treatments for diabetes and AD, highlighting the prospect of integrating traditional herbal remedies with modern medical research.

Construction of viral-based expression vectors for high-level production of human interferon alpha 2b in plants

Human interferon (hINF) alpha 2b is clinically important pharmaceutical product included in combinatory therapy against chronic hepatitis C and B and complex therapy against several cancer diseases. Here, we created the genetic constructions, based on genome elements of potato virus X (PVX), carrying the infα2b gene for transient expression in plant cells. The created plasmid vector constructions were tested through Agrobacterium-mediated transient gene expression method in two plant species—Nicotiana benthamiana and Ocimum basilicum (sweet basil). Production of recombinant hINF alpha 2b was more efficient in N. benthamiana than that in O. basilicum plants. The average yield of hINF alpha 2b produced in N. benthamiana plants was 0.56 mg/g of fresh leaf weight (FW) or 6% of the total soluble cell proteins (TSP). The maximal level reached up to 1.2 mg/g FW or 9% TSP. We estimated that about 0.67 mg of hINF can be obtained from one N. benthamiana plant. The yield of hINF alpha 2b obtained with the PVX-based expression cassette was about 80 times higher than the yield of hINF alpha 2b obtained with a simple expression cassette in which the infα2b gene was controlled by the 35S promoter of cauliflower mosaic virus.Key points• PVX-based expression vectors provide efficient transient expression of infα2b gene• N. benthamiana plants can produce human interferon alpha 2b at high levels• The yield of the hINF α2b reached up to 1.2 mg/g of fresh leaf weight

Anti HCV activity and expression inhibition of HCC markers by protein extract from Iberis gibraltarica.

Hepatitis C virus infection (HCV) is the foremost reason of progressive hepatic fibrosis and cirrhosis, with an elevated risk of hepatocellular carcinoma (HCC) development. Medicinal plants have been used for human health benefits for several years, but their therapeutic potential needs to be explored. The main objective of this study was to figure out the in vitro antiviral and anticancer characteristics of total crude protein of Iberis gibraltarica against HCV and HCC. Total crude protein of Iberis gibraltarica was isolated and quantified. The level of cytotoxicity was measured against the HepG2 cell line and it shows no significant cytotoxicity at the concentration of 504µg/ml. The anti-HCV effect was determined by absolute quantification via real time RT-PCR method and viral titer was reduced up to 66% in a dose dependent manner against the total protein of Iberis gibraltarica. The anticancer potential of Iberis gibraltarica was also examined through mRNA expression studies of AFP and GPC3 genes against the total protein of Iberis gibraltarica-treated HepG2 cells. The results show up to 90% of the down-regulation expression of AFP and GPC3. The obtained results indicate the therapeutic potential of total protein of Iberis gibraltarica against HCV and hepatocellular carcinoma in vitro.

Comparative assessment of beta-2 microglobulin as predictor of progression of multiple myeloma in asymptomatic and symptomatic multiple myeloma patients in a tertiary care hospital in Eastern India

Background: Multiple myeloma (MM) is a clonal late B-cell disorder in which malignant plasma cells expand and accumulate in the bone marrow and is associated with paraprotein production, bone lesions, anemia, hypercalcemia, susceptibility to infections, and renal impaired. Beta-2 microglobulin (B2M) is a serum marker of tumor burden in lymphoid malignancies, including MM. The purpose of this study was to find whether increased B2M levels can predict the progression of MM from asymptomatic to progressed symptomatic phase, as it is a less invasive and cost-effective method than the more invasive and costly ones to predict the progression of the disease. Aims and Objectives: This study was taken in a medical college, Kolkata, 50 asymptomatic Stage 1 MM patients were taken along with age and gender-matched 50 symptomatic progressed MM patients, blood samples of both groups were evaluated to estimate the difference in serum B2M (SB2M) level in both groups. Moreover, to study the difference in serum albumin, C-reactive protein, creatinine, immunoglobulin (IG), total protein, calcium, hemoglobin, and percentage of plasma cells in both groups and also their correlation with SB2M level in the progressed disease group. As well as to assess if there is increased B2M in the progressed disease group and if the B2M level correlates with the disease severity (i.e. progression of the disease). Materials and Methods: An observational descriptive non-interventional, hospital-based study conducted at central laboratory, medical college, Kolkata, for 6 months. Patients were chosen by systematic random sampling. Fifty asymptomatic MMs were chosen according to the International Myeloma Working Group criteria and out of 50 patients in the progressed disease group, 75% of patients were in Stage III and 25% of patients were in Stage II according to the Salmon and Durie system. Results: More than 80% in the progressed and symptomatic group had β-2M >3.5 mg/L. Whereas in the newly diagnosed group, all the 50 patients had their β-2M level <3.5 mg/L. Moreover, B2M value in the blood of progressed MM patients was found to be positively correlated with percentage of plasma cells from bone marrow, serum creatinine level, serum protein level, serum calcium level, and IgG level and negatively correlated with Hb level and serum albumin level. Hence, in a nutshell, increased B2M level was found in the progressed MM group, and its level positively correlated with the other predictors of the progression of MM. Conclusion: As per this study, SB2M level (which is a less invasive test than bone marrow testing and less costly) can be used as the predictor of progression of MM from asymptomatic to symptomatic. However, small sample size and cross-sectional pattern of the study were the limitations of this study. Bigger sample size and prospective cohort-type study designing method could be a better method to find a stronger correlation between MM staging and SB2M levels.

Thermotolerant PGPR consortium B3P modulates physio-biochemical and molecular machinery for enhanced heat tolerance in maize during early vegetative growth

BackgroundGlobal warming and irregular changes in temperature are a serious threat to plant growth with a significant negative impact on yield. Global maize productivity has decreased significantly due to sudden temperature fluctuations and heat waves especially in the regions severely hit by climate change.ResultsThe current study demonstrates the potential of beneficial bacteria for inducing heat tolerance in maize during early growth. Three Bacillus spp. AH-08, AH-67, SH-16, and one Pseudomonas spp. SH-29 showed the ability to grow and exhibited multiple plant-beneficial traits up to 45 ± 2 °C. At temperatures of 45 and 50 °C, Bacillus sp. SH-16 exhibited upregulation of two small heat shock proteins (HSP) of 15 and 30 kDa, while SH-16 and AH-67 showed upregulation of two large HSP of 65 and 100 kDa. Plant-inoculation with the consortium B3P (3 Bacillus + 1 Pseudomonas spp.) was carried out on six hybrid maize varieties pre-grown at 25 ± 2 ºC. Heat shock was applied to 10-day-old seedlings as: 3 h at 38ºC, 48 h recovery period, and then 48 h at 42ºC. The B3P treatment showed significant improvement in the overall plant growth (plant height, root & shoot fresh & dry weight, root and leaf area) with a higher level of CAT, POD, total chlorophyll, and carotenoids, while low concentration of MDA. A non-significant difference was observed in case of total cell protein and amino acids after B3P-treatment under stress. The expression of HSP1 and HSP18 in Malka and YH-5427 while HSP70 and HSP101 were higher in FH-1046 and Gohar as compared to non-inoculated treatment.ConclusionsThese findings indicate that heat-tolerant plant-growth promoting rhizobacteria (Ht-PGPR) exert versatile, multiphasic and differential response to improve plant growth and heat-tolerance in different maize varieties during seedling/ early vegetative growth. Subsequent research will be focused on the field evaluation of these PGPR to see the field and yield response of this consortium under natural temperature fluctuations in field.

Enhanced Mitophagy in Cholesteatoma Epithelial Cells.

Mitophagy may have a potential role in the pathogenesis of acquired cholesteatoma. Enhanced mitophagy has been proven to be involved in various cancers. However, its role in the pathogenesis of cholesteatoma, which shares some common features with cancer, is controversial. This study investigated mitophagy in cholesteatoma epithelial cells. The autophagy protein markers LC3-II and p62 and mitophagy proteins BNIP3, Parkin, and PINK1 were analyzed in cholesteatoma epithelial cells and external auditory canal epithelium cells by immunoblotting. The results were confirmed by immunohistochemistry. Adenovirus Ad-mCherry-GFP-LC3B and Ad-GFP-LC3B were used to evaluate autophagic activity. Transmission electron microscopy was used to observe and analyze autophagosomes. LC3-II expression was increased in cholesteatoma cells, whereas soluble and insoluble p62 levels were decreased. The expressions of BNIP3, Parkin, and PINK1 were higher in total protein and mitochondrial protein of cholesteatoma cells compared with normal external auditory canal epithelium cells. Autophagic activity was increased in cholesteatoma cells compared with normal external auditory canal epithelium cells. Mitophagy was enhanced in cholesteatoma epithelial cells and may have a potential role in the pathogenesis of acquired cholesteatoma.

Purification of a recombinant oncolytic virus from clarified cell culture media by anion exchange monolith chromatography.

The use of viral vectors for vaccine, gene therapy, and oncolytic virotherapy applications has received increased attention in recent years. Large-scale purification of viral vector-based biotherapeutics still presents a significant technical challenge. Chromatography is the primary tool for the purification of biomolecules in the biotechnology industry; however, the majority of chromatography resins currently available have been designed for the purification of proteins. In contrast, convective interaction media monoliths are chromatographic supports that have been designed and successfully utilized for the purification of large biomolecules, including viruses, viruslike particles, and plasmids. We present a case study on the development of a purification method for recombinant Newcastle disease virus directly from clarified cell culture media using strong anion exchange monolith technology (CIMmultus QA, BIA Separations). Resin screening studies showed at least 10 times higher dynamic binding capacity of CIMmultus QA compared to traditional anion exchange chromatography resins. Design of experiments was used to demonstrate a robust operating window for the purification of recombinant virus directly from clarified cell culture without any further pH or conductivity adjustment of the load material. The capture step was successfully scaled up from 1mL CIMmultus QA columns to the 8L column scale and achieved a greater than 30-fold reduction in process volume. Compared to the load material, total host cell proteins were reduced by more than 76%, and residual host cell DNA by more than 57% in the elution pool, respectively. Direct loading of clarified cell culture onto a high-capacity monolith stationary phase makes convective flow chromatography an attractive alternative to centrifugation or TFF-based virus purification procedures.

Computational and Experimental Investigation of the Combined Effect of Various 3D Scaffolds and Bioreactor Stimulation on Human Cells’ Feedback

Computational methods were combined with an experimental setup in order to investigate the response of human umbilical cord stem cells to 3D electrospun and printed scaffolds, when dynamically stimulated in a bioreactor. Key parameters associated to bioreactor working conditions were computationally investigated using Comsol software to use the output for the planned experimental setup. Based on the theoretical observations, the influence of the inlet velocity, cell number, and exposure time in the bioreactor were analyzed and the in vitro parameters were adjusted accordingly. MSCs were seeded in different numbers in the 3D porous scaffolds and stimulated in the bioreactor (0.5 and 2 h duration, 3 and 6 mm/s inlet velocity). Polycaprolactone 3D electrospun, and polyurethane and polylactic acid 3D-printed scaffolds were fabricated and fibronectin-coated. The computational study predicted initial events in the process of cells deposition and attachment. Total protein, osteopontin, and osteocalcin levels in cells deposited in scaffolds were investigated; SEM and confocal imaging confirmed the biomarker analysis. MSCs proliferated well in PCL. Polyurethane enabled extremely rapid proliferation followed by differentiation, while PLA induced a moderate proliferation and parallel mineralization. The scaffolds stiffness has been found as the key enabling parameter decisive for cells feedback.

Human KCC2a Protein Expression and Phosphorylation is Altered by Elimination of the Second Cryo-EM Identified Chloride Binding Site

Background: Potassium-chloride cotransporters (KCCs) are responsible for the symport of Cl- with K+ in an electroneutral manner. They play important roles in numerous crucial physiological processes, from cell volume regulation to transepithelial solute transport, intracellular ion homeostasis and regulation of GABA neurotransmission. Under isotonicity, except for KCC2, KCCs remain inhibited due to phosphorylation of residues located in cytoplasmic domains, whereas in response to hypotonic cell swelling, dephosphorylation of these residues stimulates transport activity. After several decades of studies, new breakthroughs with the cryo-EM structural determination of KCCs have deepened our understanding of their molecular basis and transport function, including architecture, ion binding sites, and coupled ion transport. In the present study, we mutated and analyzed the two chloride-binding sites in human KCC2a recently reported by cryo-EM to determine whether the phosphorylation status is modified upon their removal. Methods: Xenopus laevis oocytes and HEK-293 cells were either microinjected or transfected with human KCC2a wild type (WT) or chloride-binding site mutants, G136/V137/I138A (S1-Cl), and G436/I437/M438/Y592A (S2-Cl). KCC2a phosphorylation status in response to isotonic and hypotonic conditions was evaluated by immunoblot analysis of regulatory phosphorylation sites pThr926 and pThr1030. In parallel, phosphorylation status of SPAK/OSR1 activating regulatory site pSer373/pSer325 was analyzed under the same conditions. Results: Elimination of either chloride-binding site induced changes in total expression of KCC2a. Removal of S1-Cl slightly increased total KCC2a expression, whereas lack of S2-Cl showed a significant reduction of total protein in Xenopus laevis oocytes and HEK-293 cells. Phosphorylation of regulatory Thr1030 in S2-Cl mutant is significatively decreased on both biological models, even under isotonic inhibitory (phosphorylated) conditions. pThr1030 on S1-Cl mutant behaves similarly to WT KCC2a. Phosphorylation of regulatory SPAK kinase is not affected on both mutants. Conclusions: Our results suggest that while both sites affect KCC2a total expression, only the S2-Cl appears to be affecting KCC2a activating response. It appears S2-Cl mutation dysregulates SPAK signaling pathway, since the kinase osmotic response remains unchanged on presence of KCC2a chloride binding mutants. AM INCar 22-1345 and PdlH UNAM Papiit IN222320 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Diesel exhaust exposure impairs recovery of lung epithelial and cellular damage in murine model

Studies have investigated the relationship between diesel exhaust (DE) exposure and lung health, highlighting the potential for DE to induce pulmonary inflammation and oxidative stress. However, the resolution of inflammation upon withdrawal of DE exposure needs further investigation. Therefore, resolution of diesel exhaust-induced lung damage was studied in the murine model. Mice (6 weeks) were divided into three groups. Group 1 (control) mice were exposed to filtered air, Group 2 (DE) mice were exposed to DE (5.1 ± 0.7 mg/m3) & Group 3 (DE-FA) mice were exposed to DE followed by filtered air exposure. Airway hyper-responsiveness was recorded after 24 h of the last exposure. BALF and lung samples were collected for cytokine estimation, immunobiological assays, and western blot analysis. DE exposure showed an increase in lung resistance thereby causing alteration in lung function parameters (p < 0.05) which was restored in the DE-FA group. BALF analysis showed a significant increase in total cell count and protein content in DE with no resolution in DE-FA groups (p < 0.05). Lung histology showed no reduction in the bronchiolar thickness and damage in the DE-FA group suggesting irreversible lung damage (p < 0.05). The significant increase in inflammatory cytokine levels, and collagen deposition showed persistent inflammatory phase and lung damage in the DE-FA group(p < 0.05). ZO-1 was significantly decreased in both test groups indicating disintegrated lung epithelium where in claudin-5 expression showed increased lung permeability. A significant increase in neutrophil elastase activity and decreased expression of, Elafin, resulted in lung epithelial damage in the DE-FA group. Lung injury marker alpha1-antitrypsin was increased in DE-FA groups indicating an immune defense mechanism against neutrophil elastase. The study showed that DE exposure causes persistent lung damage via neutrophil elastase-associated disruption of the epithelial barrier integrity and membrane dysfunction.

Cyanobacterial phycobilisomes as a platform for the stable production of heterologous enzymes and other proteins

Efforts to stably over-express recombinant proteins in cyanobacteria are hindered due to cellular proteasome activity that efficiently degrades foreign proteins. Recent work from this lab showed that a variety of exogenous genes from plants, humans, and bacteria can be successfully and stably over-expressed in cyanobacteria, as fusion constructs with the abundant β-subunit of phycocyanin (the cpcB gene product) in quantities up to 10–15% of the total cell protein. The CpcB*P fusion proteins did not simply accumulate in a soluble free-floating form in the cell but, rather, they assembled as functional (α,β*P)3CpcG1 heterohexameric light-harvesting phycocyanin antenna discs, where α is the CpcA α-subunit of phycocyanin, β*P is the CpcB*P fusion protein, the asterisk denoting fusion, and CpcG1 is the 28.9 kDa phycocyanin disc linker polypeptide (Hidalgo Martinez et al., 2022). The present work showed that the CpcA α-subunit of phycocyanin and the CpcG1 28.9 kDa phycocyanin disc linker polypeptide can also successfully serve as leading sequences in functional heterohexameric (α*P,β)3CpcG1 and (α,β)3CpcG1*P fusion constructs that permit stable recombinant protein over-expression and accumulation. These were shown to form a residual light-harvesting antenna and to contribute to photosystem-II photochemistry in the cyanobacterial cells. The work suggested that cyanobacterial cells need phycocyanin for light absorption, photosynthesis, and survival and, therefore, may tolerate the presence of heterologous recombinant proteins, when the latter are in a fusion construct configuration with essential cellular proteins, e.g., phycocyanin, thus allowing their substantial and stable accumulation.

Superior protective effects of PGE2 priming mesenchymal stem cells against LPS-induced acute lung injury (ALI) through macrophage immunomodulation

BackgroundMesenchymal stem cells (MSCs) have demonstrated remarkable therapeutic promise for acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS). MSC secretomes contain various immunoregulatory mediators that modulate both innate and adaptive immune responses. Priming MSCs has been widely considered to boost their therapeutic efficacy for a variety of diseases. Prostaglandin E2 (PGE2) plays a vital role in physiological processes that mediate the regeneration of injured organs.MethodsThis work utilized PGE2 to prime MSCs and investigated their therapeutic potential in ALI models. MSCs were obtained from human placental tissue. MSCs were transduced with firefly luciferase (Fluc)/eGFP fusion protein for real-time monitoring of MSC migration. Comprehensive genomic analyses explored the therapeutic effects and molecular mechanisms of PGE2-primed MSCs in LPS-induced ALI models.ResultsOur results demonstrated that PGE2-MSCs effectively ameliorated lung injury and decreased total cell numbers, neutrophils, macrophages, and protein levels in bronchoalveolar lavage fluid (BALF). Meanwhile, treating ALI mice with PGE2-MSCs dramatically reduced histopathological changes and proinflammatory cytokines while increasing anti-inflammatory cytokines. Furthermore, our findings supported that PGE2 priming improved the therapeutic efficacy of MSCs through M2 macrophage polarization.ConclusionPGE2-MSC therapy significantly reduced the severity of LPS-induced ALI in mice by modulating macrophage polarization and cytokine production. This strategy boosts the therapeutic efficacy of MSCs in cell-based ALI therapy.

Biotoxicity comparison of Bacillus thuringiensis to control vector borne diseases against mosquito fauna

The current study was designed to evaluate the biotoxicity of screened echo-friendly Bacillus thuringiensis strains from different areas of Pakistan. Out of 50 samples, 36% Bt. isolates were quarantined from soil containing cattle waste after morphological, biochemical, and molecular characterization. The toxicity bioassays with Bt. spores and protein diet proved that 11 Bt. isolates were utmost noxious to 3rd instar larvae of mosquitoes Aedes aegypti, Anopheles stephensi, and Culex pipiens. The entopathogenic activity of first 4 Bt. toxins against A. aegypti was highly lethal as compared to the other dipteran larvae. The toxicity (LC50) of spore diet of Bt. strains GCU-DAB-NF4 (442.730 ± 0.38 μg/ml), NF6 (460.845 ± 0.29 μg/ml), NF3 (470.129 ± 0.28 μg/ml), and NF7 (493.637 ± 0.70 μg/ml) was quite high against A. aegypti as compared to the C. pipiens after 24 h of incubation. The highest toxicity of total cell protein was shown by GCU-DAB-NF4 (LC50 = 84.10 ± 50 μg/ml), NF6 (95.122 ± 0.40 μg/ml), NF3 (100.715 ± 06 μg/ml), and NF5 (103.40 ± 07 μg/ml) against A. aegypti after 24 h. So, these strains a have great potential to be used as biological control especially against A. aegypti as compared to the C. pipiens.

Maturation of pathogenic biofilms induces enhanced resistance against gaseous chlorine dioxide.

Biofilms are surface-associated microbial clusters embedded in extracellular polymeric substances. Biofilms formed on food-contact surfaces create challenges for the food industry due to their increased tolerance to antimicrobial agents and disinfectants. This study aimed to evaluate the effect of the biofilm maturation period on their resistance to gaseous ClO2. Listeria monocytogenes, Salmonellaserotype Typhimurium, and Escherichia coli O157:H7 biofilms formed on stainless steel (SS) and high-density polyethylene (HDPE) surfaces were investigated. The total cell mass and protein content significantly increased (P<.05) between the second and the fifth day of maturation, and the biofilms' resistance to gaseous ClO2 increased as they matured. Generally, the cell counts of 0-day-old L. monocytogenes, Salm. Typhimurium, and E. coli O157:H7 biofilms on SS and HDPE reduced below the detection limit (0.48 log CFU/cm2) within 5min. The cell counts of 2-day-old biofilms of the three pathogens were reduced by 6.22 to over 7.52 log, while those of 5-day-old biofilms were reduced by 3.64 to over 6.34 log after 20min of treatment with 30 ppmv of gaseous ClO2. Therefore, as resistance increases with biofilm maturation, daily gaseous ClO2 treatment would maximize the antimicrobial efficacy of the cleaning strategy against biofilms.

Molecular targeting of prodigiosin against anti-inflammatory genes cyclooxygenase-1 and -2

The prodigiosin produced by the bacteria Serratia marcescens (SM) has been isolated and optimized with the streak plate method from different natural sources, such as human urine (SM1), soil (SM2), vegetable matter (Cucurbita maxima, SM3) and water (SM4). The isolated prodigiosin was confirmed as SM by its morphological characteristics, genomic size (1.5 kb), identification, and protein purification (4 kDa) using SDS-PAGE. Further, the total cell protein was confirmed and purified through thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The prodigiosin exhibited good microbial activity against selected Gram-positive bacteria and fungi. The binding efficiency of prodigiosin against cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) genes was assessed using an in silico molecular docking method that suggested higher binding efficiency for COX-2 than COX-1. Further, prodigiosin was nontoxic to rat islet (RIN-5F) cells in an assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylteterazolium bromide) in vitro, and its morphological characteristics of cell shape, clumping, chromatin, and nuclear morphology were normal when viewed under bright light microscopy. Finally, prodigiosin was tested for anti-inflammatory properties against lipopolysaccharide-induced RIN-5F cells. The tests revealed that prodigiosin protected against elution of inflammatory markers like NO, interleukin-6 (IL6), and IL8 in a dose-dependent manner.

Diyne inactivators and activity-based fluorescent labeling of phenol hydroxylase in Pseudomonas sp. CF600.

An activity-based labeling (ABL) approach was investigated for the phenol-oxidizing bacterium, Pseudomonas sp. CF600. Phenol-grown cells were exposed to several different terminal diynes, and following cell breakage, extracts of these cells were added to copper-catalyzed alkyne/azide cycloaddition reactions containing Alexa Fluor 647 azide. Analysis of total cell proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and near-infrared scanning demonstrated covalent fluorescent labeling of a 58- and a 34-kDa polypeptide in all diyne-treated cell types. Further studies using 1,4-diethynylbenzene (DEB) demonstrated that these labeled polypeptides were consistently detected in cells grown on substrates that exhibited phenol-dependent O2 uptake activity but not observed when cells were grown on substrates such as dextrose or catechol that did not support this activity. Fluorescent labeling of the two polypeptides in DEB-treated, phenol-grown cells was time dependent and was inhibited by several known substrates for phenol hydroxylase. These results suggest that diverse diynes act as mechanism-based inactivators of phenol hydroxylase in Pseudomonas sp. CF600 and that this effect can be exploited by ABL approaches to selectively label the major 58- and 34-kDa subunits of the hydroxylase component of this complex enzyme.

Phillyrin Inhibits Isoproterenol-Induced Cardiac Hypertrophy Via P38 and NF-κB Pathways

Cardiac hypertrophy (CH) is the main compensatory response to chronic heart stress and often progresses to a decompensation state potentially leading to heart failure. Phillyrin (PHI) is a novel compound derived from Forsythia, which has shown anti-inflammatory and anti-virus activities as well as renal protective effects on diabetic nephropathy. Therefore, we investigated the effects of PHI on CH induced by isoproterenol (ISO). Cardiac hypertrophy was induced by ISO in vivo, and the H9C2 cells were treated with ISO. PHI treatment alleviated CH in isoproterenol-induced mice in 7 and 14 days. Echocardiography showed that the PHI improved ISO-induced CH heart function and structure. PHI significantly decreased heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL) ratios and improved left ventricular (LV) function in ISO-treated mice. Hematoxylin and eosin staining revealed cardiomyocyte areas of the ISO group were significantly increased, and PHI was significantly reduced at 7 and 14 days, PHI-100 groups showed significantly better improvements than PHI-50. Sirius red staining indicated PHI significantly decreased collagen deposition in heart cross-sections induced by ISO, and PHI repressed ISO-induced cTn-I and NT-proBNP expression in mouse serum. In vitro data from H9C2 cells showed that PHI decreased cell areas and total cell protein levels in cells induced by ISO, whereas ANP, BNP, IL-6, and IL-1β expression was significantly inhibited by PHI. Also, PHI simultaneously inhibited P65 and P38 phosphorylation in vivo and in vitro. In conclusion, this study demonstrated the protective effect of PHI on CH in in vivo and in vitro, and this effect was related to the suppression of inflammation through the activation of the P38/NF-κB pathway.

Activation of cannabinoid-2 receptor protects against Pseudomonas aeruginosa induced acute lung injury and inflammation

BackgroundBacterial pneumonia is a major risk factor for acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Pseudomonas aeruginosa (PA), an opportunistic pathogen with an increasing resistance acquired against multiple drugs, is one of the main causative agents of ALI and ARDS in diverse clinical settings. Given the anti-inflammatory role of the cannabinoid-2 receptor (CB2R), the effect of CB2R activation in the regulation of PA-induced ALI and inflammation was tested in a mouse model as an alternative to conventional antibiotic therapy.MethodsIn order to activate CB2R, a selective synthetic agonist, JWH133, was administered intraperitoneally (i.p.) to C57BL/6J mice. Furthermore, SR144528 (a selective CB2R antagonist) was administered in combination with JWH133 to test the specificity of the CB2R-mediated effect. PA was administered intratracheally (i.t.) for induction of pneumonia in mice. At 24 h after PA exposure, lung mechanics were measured using the FlexiVent system. The total cell number, protein content, and neutrophil population in the bronchoalveolar lavage fluid (BALF) were determined. The bacterial load in the whole lung was also measured. Lung injury was evaluated by histological examination and PA-induced inflammation was assessed by measuring the levels of BALF cytokines and chemokines. Neutrophil activation (examined by immunofluorescence and immunoblot) and PA-induced inflammatory signaling (analyzed by immunoblot) were also studied.ResultsCB2R activation by JWH133 was found to significantly reduce PA-induced ALI and the bacterial burden. CB2R activation also suppressed the PA-induced increase in immune cell infiltration, neutrophil population, and inflammatory cytokines. These effects were abrogated by a CB2R antagonist, SR144528, further confirming the specificity of the CB2R-mediated effects. CB2R-knock out (CB2RKO) mice had a significantly higher level of PA-induced inflammation as compared to that in WT mice. CB2R activation diminished the excess activation of neutrophils, whereas mice lacking CB2R had elevated neutrophil activation. Pharmacological activation of CB2R significantly reduced the PA-induced NF-κB and NLRP3 inflammasome activation, whereas CB2KO mice had elevated NLRP3 inflammasome.ConclusionOur findings indicate that CB2R activation ameliorates PA-induced lung injury and inflammation, thus paving the path for new therapeutic avenues against PA pneumonia.