Expression Pattern of Cholesterol 25-Hydroxylase and Serum Level of 25-Hydroxycholesterol and Relevant Inflammatory Cytokines in Patients with Varying Disease Severity of COVID-19.

Cholesterol 25-hydroxylase (CH25H) and its product 25-hydroxycholesterol (25HC) showed antiviral effects against various viruses in vitro. CH25H expression is regulated in mice by pro-inflammatory cytokine interferons (IFNs) in mice but data on its possible correlation with IFNs in humans are still unclear. We examined gene expression of CH25H, IFN-α, and IFN-ß and serum levels of 25HC in Iranian patients with mild and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Fifty intensive care unit (ICU) patients and outpatients with SARS-CoV-2 and 25 healthy controls were studied. Gene expression of CH25H and relevant inflammatory cytokines was quantified in peripheral blood mononuclear cells by real-time polymerase chain reaction. The expression of CH25H and serum levels of 25HC were significantly higher in ICU patients with SARS-CoV-2. Notably, IFN-α levels increased in healthy controls. However, compared to healthy controls, IFN-ß was considerably higher in outpatients. Finally, statistical analysis shows that no correlation was found between CH25H and IFN-α expression; nevertheless, a lower correlation was found with IFN-ß. The data revealed that CH25H and 25HC levels increase after SARS-CoV-2 infection. In other words, decreased levels of those factors in severe patients compared with mild patients may indicate the importance of their function in controlling the progression of the disease.

Volatilomics-Based Microbiome Evaluation of Fermented Dairy by Prototypic Headspace-Gas Chromatography-High-Temperature Ion Mobility Spectrometry (HS-GC-HTIMS) and Non-Negative Matrix Factorization (NNMF).

Fermented foods, such as yogurt and kefir, contain a versatile spectrum of volatile organic compounds (VOCs), including ethanol, acetic acid, ethyl acetate, and diacetyl. To overcome the challenge of overlapping peaks regarding these key compounds, the drift tube temperature was raised in a prototypic high-temperature ion mobility spectrometer (HTIMS). This HS-GC-HTIMS was used for the volatilomic profiling of 33 traditional kefir, 13 commercial kefir, and 15 commercial yogurt samples. Pattern recognition techniques, including principal component analysis (PCA) and NNMF, in combination with non-targeted screening, revealed distinct differences between traditional and commercial kefir while showing strong similarities between commercial kefir and yogurt. Classification of fermented dairy samples into commercial yogurt, commercial kefir, traditional mild kefir, and traditional tangy kefir was also possible for both PCA- and NNMF-based models, obtaining cross-validation (CV) error rates of 0% for PCA-LDA, PCA-kNN (k = 5), and NNMF-kNN (k = 5) and 3.3% for PCA-SVM and NNMF-LDA. Through back projection of NNMF loadings, characteristic substances were identified, indicating a mild flavor composition of commercial samples, with high concentrations of buttery-flavored diacetyl. In contrast, traditional kefir showed a diverse VOC profile with high amounts of flavorful alcohols (including ethanol and methyl-1-butanol), esters (including ethyl acetate and 3-methylbutyl acetate), and aldehydes. For validation of the results and deeper understanding, qPCR sequencing was used to evaluate the microbial consortia, confirming the microbial associations between commercial kefir and commercial yogurt and reinforcing the differences between traditional and commercial kefir. The diverse flavor profile of traditional kefir primarily results from the yeast consortium, while commercial kefir and yogurt is primarily, but not exclusively, produced through bacterial fermentation. The flavor profile of fermented dairy products may be used to directly evaluate the microbial consortium using HS-GC-HTIMS analysis.

An Updated Review of Various Medicinal Applications of p-Co umaric Acid: From Antioxidative and Anti-Inflammatory Properties to Effects on Cell Cycle and Proliferation.

P-Coumaric acid (p-CA) is a hydroxycinnamic acid, an organic compound that is a hydroxyl derivative of cinnamic acid. P-CA is the most abundant isomer in nature and can be found in a wide variety of edible plants such as fungi, peanuts, navy beans, tomatoes, carrots, basil, and garlic. Recently, the therapeutic properties of p-CA have received a great deal of attention from scientific society. Here, we described the medicinal effects of p-CA on various pathological conditions. This review was performed via evaluating PubMed reported studies from January 2010 to January 2020. Also, reference lists were checked to find additional studies. All intermediation or complementarity of animal models, case-control and cohort studies, in vitro studies, and controlled trials (CTs) on p-CA were acceptable. However plant extract studies without indication of main active substances were excluded due to the considerable diversities and heterogeneities. According to recent evidence regarding the beneficial effects of p-CA, numerous diseases such as nephropathies, cardiovascular diseases, neuroinflammatory diseases, liver diseases, cancers, and some metabolic disorders could potentially be controlled by this natural herb. Interestingly, autophagy is a novel molecular mechanism involved in the crosstalk between classic effects of p-CA and introduces alternative therapeutic pathways for this compound. Much work remains in clarifying the main therapeutic properties among the various p-CA effects; these will be the subject of forthcoming work, resulting in presenting further mechanism of action.

Quantification of Major Bacteria and Yeast Species in Kefir Consortia by Multiplex TaqMan qPCR.

Kefir grains are complex microbial systems of several groups of microorganisms. The identification and quantification of the microbial composition of milk kefirs was described in several studies, which provided an insight into the microbial consortia in this complex ecosystem. Nevertheless, the current methods for identification and quantification are not appropriate for deeper studies on kefir consortia, e.g., population dynamics and microbial interactions in kefir grains. This requires another sensitive and reliable quantitative method. Therefore, this study aims to develop multiplexed qPCR assays to specifically detect and quantify, as an example, several microorganisms of the milk kefir microbial community. Primer-probe sets, which target species-specific genes in six bacteria and five yeasts, were designed, and their sensitivity and specificity to the target species was analyzed in simplex as well as four multiplex qPCR assays. The self-designed multiplex assays were applied for the detection of target bacteria and yeast species in milk kefirs, in both, grain and beverage fractions. Detection of all target microorganisms in simplex and multiplex qPCR was achieved by good linearity, efficiency, repeatability and reproducibility in all assays. When the designed assays were applied on six kefirs, all target microorganisms were detected in different samples, but not all in one kefir sample. The two ubiquitous lactobacilli Lactobacillus kefiranofaciens and Lb. kefiri were present in all six kefirs studied, but were associated with different other yeasts and bacteria. Especially on the yeast community a significant diversity was observed. In general, multiplex TaqMan qPCR as developed here was proven to have high potential for specific identification of target microorganisms in kefir samples and for the first time, eleven target bacteria and yeasts of kefir microbiota were rapidly detected and quantified. This study, thus, provides a fast and reliable protocol for future studies on kefir and other similar microbial ecosystems.

A Big World in Small Grain: A Review of Natural Milk Kefir Starters.

Milk kefir is a traditional fermented milk product whose consumption is becoming increasingly popular. The natural starter for kefir production is kefir grain, which consists of various bacterial and yeast species. At the industrial scale, however, kefir grains are rarely used due to their slow growth, complex application, bad reproducibility and high costs. Instead, mixtures of defined lactic acid bacteria and sometimes yeasts are applied, which alter sensory and functional properties compared to natural grain-based milk kefir. In order to be able to mimic natural starter cultures for authentic kefir production, it is a prerequisite to gain deep knowledge about the nature of kefir grains, its microbial composition, morphologic structure, composition of strains on grains and the impact of environmental parameters on kefir grain characteristics. In addition, it is very important to deeply investigate the numerous multi-dimensional interactions among different species, which play important roles on the formation and the functionality of grains.

Decellularized amniotic membrane Scaffolds improve differentiation of iPSCs to functional hepatocyte-like cells.

Human-induced pluripotent stem cells-derived hepatocyte-like cells (hiPSCs-HLCs) holds considerable promise for future clinical personalized therapy of liver disease. However, the low engraftment of these cells in the damaged liver microenvironment is still an obstacle for potential application. In this study, we explored the effectiveness of decellularized amniotic membrane (dAM) matrices for culturing of iPSCs and promoting their differentiation into HLCs. The DNA content assay and histological evaluation indicated that cellular and nuclear residues were efficiently eliminated and the AM extracellular matrix component was maintained during decelluarization. DAM matrices were developed as three-dimensional scaffolds and hiPSCs were seeded into these scaffolds in defined induction media. In dAM scaffolds, hiPSCs-HLCs gradually took a typical shape of hepatocytes (polygonal morphology). HiPSCs-HLCs that were cultured into dAM scaffolds showed a higher level of hepatic markers than those cultured in tissue culture plates (TCPs). Moreover, functional activities in term of albumin and urea synthesis and CYP3A activity were significantly higher in dAM scaffolds than TCPs over the same differentiation period. Thus, based on our results, dAM scaffold might have a considerable potential in liver tissue engineering, because it can improve hepatic differentiation of hiPSCs which exhibited higher level of the hepatic marker and more stable metabolic functions.

Comparison of human-induced pluripotent stem cells and mesenchymal stem cell differentiation potential to insulin producing cells in 2D and 3D culture systems in vitro.

Diabetes is one of the most common diseases in the world that is chronic, progressive, and costly, and causes many complications. Common drug therapies are not able to cure it, and pancreas transplantation is not responsive to the high number of patients. The production of the insulin producing cells (IPCs) from the stem cells in the laboratory and their transplantation to the patient's body is one of the most promising new approaches. In this study, the differentiation potential of the induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) into IPCs was compared to each other while cultured on poly(lactic-co-glycolic) acid (PLGA)/polyethylene glycol (PEG) nanofibrous scaffold as a 3D substrate and tissue culture polystyrene (TCPS) as a 2D substrate. Although the expression level of the insulin, Glut2 and pdx-1 genes in stem cells cultured on 3D substrate was significantly higher than the stem cells cultured on 2D substrate, the highest expression level of these genes was detected in the iPSCs cultured on PLGA-PEG. Insulin and C-peptide secretions from differentiated cells were also investigated and the results showed that secretions in cultured iPSCs on the PLGA-PEG were significantly higher than cultured iPSCs on the TCPS and cultured MSCs on both PLGA-PEG and TCPS. In addition, insulin protein was also expressed in the cultured iPSCs on the PLGA-PEG significantly higher than cultured MSCs on the PLGA-PEG. It can be concluded that differentiation potential of iPSCs into IPCs is significantly higher than human MSCs at both 2D and 3D culture systems.

Platelet-rich plasma incorporated electrospun PVA-chitosan-HA nanofibers accelerates osteogenic differentiation and bone reconstruction.

Biologically active materials and polymeric materials used in tissue engineering have been one of the most attractive research areas in the past decades, especially the use of easily accessible materials from the patients that reduces or eliminates any patient's immune response. In this study, electrospun nanofibrous scaffolds were fabricated by using polyvinyl-alcohol (PVA), chitosan and hydroxyapatite (HA) polymers and platelet-rich plasma (PRP) as a bioactive substance isolated from human blood. Fabricated scaffold's structure and cytotoxicity were evaluated using scanning electron microscope and MTT assays. Scaffolds osteoinductivity was investigated by osteogenic differentiation of the mesenchymal stem cells (MSCs) at the in vitro level and then its osteoconductivity was examined by implanting at the critical-sized rat calvarial defect. The in vitro results showed that scaffolds have a good structure and good biocompatibility. Alkaline phosphatase activity, calcium content and gene expression assays were also demonstrated that their highest amount was detected in MSCs-seeded PVA-chitosan-HA(PRP) scaffold. For this reason, this scaffold alone and along with the MSCs was implanted to the animal defects. The in vivo results demonstrated that in the animals implanted with PVA-chitosan-HA(PRP), the defect was repaired to a good extent, but in those animals that received MSCs-seeded PVA-chitosan-HA(PRP), the defects was almost filled. It can be concluded that, PVA-chitosan-HA(PRP) alone or when stem cells cultured on them, has a great potential to use as an effective bone implant.

Incorporated-bFGF polycaprolactone/polyvinylidene fluoride nanocomposite scaffold promotes human induced pluripotent stem cells osteogenic differentiation.

Bioactive scaffolds that can increase transplanted cell survival time at the defect site have a great promising potential to use clinically since tissue regeneration or secretions crucially depend on the transplanted cell survival. In this study embedded basic fibroblast growth factor (bFGF)-polycaprolactone-polyvinylidene fluoride (PCL-PVDF) hybrid was designed and fabricated by electrospinning as a bio-functional nanofibrous scaffold for bone tissue engineering. After morphological characterization of the PCL-PVDF (bFGF) scaffold, nanofibers biocompatibility was investigated by culturing of the human induced pluripotent stem cells (iPSCs). Then, the bone differentiation capacity of the iPSCs was evaluated when grown on the PCL-PVDF and PCL-PVDF (bFGF) scaffolds in comparison with culture plate as a control using evaluating of the common osteogenic markers. The viability assay displayed a significant increase in iPSCs survival rate when grown on the bFGF content scaffold. The highest alkaline phosphatase activity and mineralization were detected in the iPSCs while grown on the PCL-PVDF (bFGF) scaffolds. Obtained results from gene and protein expression were also demonstrated the higher osteoinductive property of the bFGF content scaffold compared with the scaffold without it. According to the results, the release of bFGF from PCL-PVDF nanofibers increased survival and proliferation rate of the iPSCs, which followed by an increase in its osteogenic differentiation potential. Taking together, PCL-PVDF (bFGF) nanofibrous scaffold demonstrated that can be noted as a promising candidate for treating the bone lesions by tissue engineering products.

Comparison of osteogenic differentiation potential of induced pluripotent stem cells on 2D and 3D polyvinylidene fluoride scaffolds.

In recent decades, tissue engineering has been the most contributor for introducing 2D and 3D biocompatible osteoinductive scaffolds as bone implants. Polyvinylidene fluoride (PVDF), due to the unique mechanical strength and piezoelectric properties, can be a good choice for making a bone bioimplant. In the present study, PVDF nanofibers and film were fabricated as 3D and 2D scaffolds, and then, osteogenic differentiation potential of the human induced pluripotent stem cells (iPSCs) was investigated when grown on the scaffolds by evaluating the common osteogenic markers in comparison with tissue culture plate. Biocompatibility of the fabricated scaffolds was confirmed qualitatively and quantitatively by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and scanning electron microscopy assays. Human iPSCs cultured on PVDF nanofibers showed a significantly higher alkaline phosphate activity and calcium content compared with the iPSCs cultured on PVDF film. Osteogenic-related genes and proteins were also expressed in the iPSCs seeded on PVDF nanofibers significantly higher than iPSCs seeded on PVDF film, when investigated by real-time reverse transcription polymerase chain reaction and western blot analysis, respectively. According to the results, the PVDF nanofibrous scaffold showed a greater osteoinductive property compared with the PVDF film and due to the material similarity of the scaffolds, it could be concluded that the 3D structure could lead to better bone differentiation. Taken together, the obtained results demonstrated that human iPSC-seeded PVDF nanofibrous scaffold could be considered as a promising candidate for use in bone tissue engineering applications.

Population-Based Seroprevalence of Malaria in Hormozgan Province, Southeastern Iran: A Low Transmission Area.

The seroepidemiological condition of malaria in three main districts of Hormozgan Province, a low transmission area in southeast of Iran, was investigated. Methods. Sera samples (803) were collected from healthy volunteers from the three main districts (Bandar Lengeh in the west, Bandar Abbas in the center, and Bandar Jask in the east) of Hormozgan Province. A questionnaire was used to record the sociodemographic features of the participants during sample collecting. An in-house ELISA test, using crude antigens obtained from cell culture of Plasmodium falciparum, was adapted and used to detect anti-malaria antibodies in the sera. Results. The overall seroprevalence of malaria was 8.7% (70 out of 803 samples). A significant correlation was found between seropositivity and place of residence, where the highest rate of seropositivity was seen in Bandar Lengeh (west of the province). The highest seroprevalence of malaria (13.2%) was seen in the age group of 11-20 years and also in low educated individuals. Correlation between seropositivity and gender, age, and educational levels of the participants was statistically significant (P < 0.05). Conclusion. Findings of this study indicate that the rate of seropositivity to malaria in this area is not high and this might be linked to the success of malaria control programs during the last decades in the region.