P1 Cells Research Articles

Investigation of Transcription Factor and Cytokine Gene Expression Levels in Helper T Cell Subsets Among Turkish Patients Diagnosed with ICF2 (Novel ZBTB24 gene Variant) and ICF3 (CDCA7 Variant) Syndrome.

Immunodeficiency, centromeric region instability, facial anomalies syndrome (ICF), is a rare disease with autosomal recessive inheritance. ICF syndrome. It has been reported that ICF syndrome is caused by mutations in the DNMT3B (ICF1), ZBTB24 (ICF2), CDCA7 (ICF3), and HELLS (ICF4) genes. As a result of literature research, there are no studies on transcription factor and cytokine expressions of helper T cell subsets in ICF syndrome. In the study; Th1 (TBET, STAT1, STAT4), Th2 (GATA3, STAT6), Th17 (RORgt, STAT3), Treg (FoxP3, STAT5) transcription factors and the major cytokines of these cells (Th1; IFNG, Th2; IL4, Th17; IL17A-21-22, Treg; IL10, TGFβ) expressions were aimed to be evaluated by qRT-PCR. Patients (ICF3: three patients; ICF2: two patients), six heterozygous individual and five healthy controls were included in the study. All patients had hypogammaglobulinemia. Except for the CD19 cells of P2 from patients diagnosed with ICF3, the CD3, CD4, CD8, and CD19 cells in the other ICF3 patients were normal. However, the rates of these cells were low in patients with ICF2 syndrome. Factors belonging to patients' Th1, Th17 and Treg cells were significantly lower than the control. Additionally, novel mutation was detected in ZBTB24 gene (c.1121-2A > T). Our study is the first molecular study on Th cell subsets in patients with ICF syndrome and a new mutation that causes ICF2 syndrome has been identified.

The Effectiveness of Tawandakulo (Kleinhovia Hospita L.) On SGOT, SGPT Enzyme Levels, and Hepatocyte Regeneration in Paracetamol-Induced Rats

Background:Paracetamol is metabolized by cytochrome P450 enzymes into a highly toxic reactive metabolite, N-acetyl-p-benzoquinonimine (NAPQI), which can cause liver damage. Antioxidants play a crucial role in mitigating liver toxicity. Kleinhovia hospitaL. (also known as Tawandakulo) is a potent plant antioxidant due to its content of secondary metabolites, such as phenolics, flavonoids, saponins, and terpenoids. Objective:This study aimed to investigate the levels of SGOT and SGPT enzymes, as well as liver histopathology, as indicators of liver health. Methods:An experimental research design with a randomized post-test control group was used. The study involved 25 rats, randomly divided into five groups. Paracetamol was administered at a toxic dose of 2.5 g/kg body weight, dissolved in distilled water, and given orally through NGT no. 5. Results:The study found a decrease in SGOT levels in the treatment group and a significant reduction in SGPT enzyme levels between pre-intervention (P2) and post-intervention (P3). There was a significant change in enzyme levels in treatment group 2 (p = 0.00) and treatment group 3 (p = 0.03). However, no significant difference was observed in treatment group 4 (p = 0.09). Liver cells in treatment groups P1, P2, and P3 showed signs of regeneration, whereas cells in the control group did not regenerate. Group P4 exhibited slower liver cell regeneration than the other treatment groups. Conclusion:This study concludes that a decoction of Kleinhovia hospitaL. can stimulate liver cell growth and reduce SGOT and SGPT enzyme levels. Keywords: Kleinhovia hospitaL; SGOT; SGPT; Liver cell regeneration; Indonesia.

Effect of Passaging on Bovine Chondrocyte Gene Expression and Engineered Cartilage Production.

Tissue engineering strategies show great potential for repairing osteochondral defects in osteoarthritic joints; however, these approaches often rely on passaging cells multiple times to obtain enough cells to produce functional tissue. Unfortunately, monolayer expansion culture causes chondrocyte dedifferentiation, which is accompanied by a phenotypical and morphological shift in chondrocyte properties that leads to a reduction in the quality of de novo cartilage produced. Thus, the objective of this study was to evaluate transcriptional variations during in vitro expansion culture and determine how differences in cell phenotype from monolayer expansion alter development of functional engineered cartilage. We used an unbiased approach to explore genome-wide transcriptional differences in chondrocyte phenotype at passage 1 (P1), P3, and P5, and then seeded cells into hydrogel scaffolds at P3 and P5 to assess cells' abilities to produce cartilaginous extracellular matrix in three dimensional (3D). We identified distinct phenotypic differences, specifically for genes related to extracellular organization and cartilage development. Both P3 and P5 chondrocytes were able to produce chondrogenic tissue in 3D, with P3 cells producing matrix with greater compressive properties and P5 cells secreting matrix with higher glycosaminoglycan/DNA and collagen/DNA ratios. Furthermore, we identified 24 genes that were differentially expressed with passaging and enriched in human osteoarthritis (OA) genome-wide association studies, thereby prioritizing them as functionally relevant targets to improve protocols that recapitulate functional healthy cartilage with cells from adult donors. Specifically, we identified novel genes, such as TMEM190 and RAB11FIP4, which were enriched with human hip OA and may play a role in chondrocyte dedifferentiation. This work lays the foundation for several pathways and genes that could be modulated to enhance the efficacy for chondrocyte culture for tissue regeneration, which could have transformative impacts for cell-based cartilage repair strategies.

The Addition of Astaxanthin 0.5% in Sunscreen SPF 50 Inhibits the Increase of Sunburn Cells in Rats Induced By Ultraviolet Light B

Long-term exposure to ultraviolet light can cause chronic inflammation that can damage collagen and increase sunburn cells known as photoaging. There is a prevalence of up to 69% in female students in Jakarta aged 18–21 years who experience aging of the skin due to not using sunscreen. One of the signs of photoaging measured in this study is sunburn cells. This study wants to prove that the addition of astaxanthin 0.5% contained in SPF 50 sunscreen can inhibit the increase in sunburn cells in mice induced by ultraviolet B-light. Sunscreen also has protection limits, for example, SPF 30 has 97% protection, which means there is still 3% of radiation entering the skin so there is still a small portion that can cause free radicals. Additional ingredients are needed so that sunscreen can optimally protect the skin. An experimental study with a post-test-only design was used in this study comparing rats given SPF 50 sunscreen containing no astaxanthin (P1) and rats given SPF 50 sunscreen containing 0.5% astaxanthin (P2) to assess the inhibition of increased sunburn cells. 36 rats (Rattus Norvegicus) were divided into 2 groups and then shaved and exposed to UVB light with a total of 3100 mJ for 5 weeks. After the last exposure in the fifth week, skin tissue was taken fixed and stained using Hematoxylin Eosin to count sunburn cells. Sunburn Cells in group P1 had a value of 0.47 Å} 0.27 cells per high power field (hpf), and group P2 had a value of 0.16 Å} 0.18 cells per hpf. According to the Mann-Whitney test, there is a significant difference in the number of sunburn cells between groups (p < 0.05). From the results of this study, it can be concluded that astaxanthin 0.5% added to SPF 50 sunscreen products can inhibit the increase in sunburn cells.

Administration of Arabika Coffee Leaf Extract (Coffea Arabica) Reduces Tumor Necrosis Factor Alpha (TNF-α) and Sunburn Cell Levels in Male Wistar Rats (Rattus norvegicus) Exposed to Ultraviolet-B Rays

Background: Ultraviolet (UV) radiation has acute and chronic impacts on the skin. However, UVB exposure can be prevented with available natural ingredients, especially those containing antioxidants. One of the natural ingredients that contains active compounds as antioxidants is arabica coffee leaves, which contain flavonoids that have antioxidant effects. Methods: This study was an experimental research with a post-test-only control group design, using 30 male Wistar rats (Rattus norvegicus) which were divided into 5 groups; the control group (P0), treatment group 1 (P1) were given placebo, while group 2 (P2), group 3 (P3) and group 4 (P4) was given UVB rays and given ethanol extract cream from Arabica coffee leaves at a dose of 2,5%, 5%, and 10% respectively. Results: The average TNF- α levels in group P0, P1, P2, P3, and P4 were 97,88±1,19 ng/mL, 162,41±1,87 ng/mL, 138,04±1,03 g/mL, 124,71±2,65 ng/mL and 107,18±1,52 ng/mL. The average sunburn cells in P0, P1, P2, P3, and P4 were 4,0±0,13, 4,07±0,27, 2,60±0,25, 1,67±0,15 and 1,33±0,12 per field view. The administration of 10% Arabica coffee leaf extract cream was the most effective in reducing TNF-α levels by 34.01% (p<0.001) and has the lowest sunburn cells per field view (1,33±0,12 cell/field view; p < 0,001). Conclusion: Administration of ethanol extract cream from Arabica coffee leaves (Coffea arabica) can reduce TNF-α levels and the number of sunburn cells in the skin of male Wistar rats (Rattus norvegicus) exposed to UVB radiation.

Targeting of CD25+ Activated T Cells in Gvhd-Associated Marrow Suppression and Cytopenias

Introduction: Secondary cytopenias occurring after hematopoietic cell transplant (HCT) are an observed complication of graft-versus-host disease (GVHD). Causes include peripheral immune mediated destruction (hemolytic anemia or thrombocytopenia), usually associated with autoantibody production, and peripheral destruction associated with microangiopathy (TMA). However, cases have been observed where marrow production of the affected lineage is abnormal. Immune-mediated cases are treated with increased immunosuppression, e.g., steroids or calcineurin inhibitors (CNI), and TMA with complement inhibition. To better understand the mechanisms and treatment of hypoproductive cytopenias in GVHD, we combined analyses of hematopoietic stem and progenitor cells (HSPC) and peripheral blood and marrow activated T cells. We describe 3 cases of GVHD-associated cytopenias marked by HSPC depletion and presence of CD25 (IL-2R alpha subunit)-positive activated T cells in the marrow that responded to combinations of immunosuppression containing basiliximab (basil) anti-CD25 monoclonal antibody blockade. Methods/Results: We describe combined HSPC and T cell analyses in 3 patients (P) with severe GVHD-associated cytopenias. P1 was at 5 years post-HCT for acute lymphoblastic leukemia, P2 at 2 years post-HCT for severe aplastic anemia (SAA), and P3 at 1.5 months post-liver transplant for alcohol use disorder-associated cirrhosis and hepatocellular carcinoma ( Table 1). Because of refractory cytopenias, all were being considered for HCT. However, marrow analyses revealed that each had normal frequencies of phenotypic HSC, with variable decreases in cellularity and frequencies of committed progenitors ( Table 2). Chimerism analyses showed that 100% of CD34+ cells in P1 and P2 were of donor origin, and in P3 of host origin. All 3 marrows had activated CD3+ T cells per expression of CD69 and/or CD38, and frequency of CD25 on >10% of T cells. In addition, P1 had evidence of IgG bound to committed progenitor populations. The treatment strategy was to block IL-2 signaling with basil (induction of 20 mg on day 1, 4, 7, 14, 21, 28, followed by alternate week and then monthly dosing for 2-9 months), combined with suppression of IL-2 production with steroid and/or CNI. After induction, repeat analyses of the blood and marrow T-cells confirmed complete blockade of CD25. Therapy was also individualized for specific clinical conditions or as noted on marrow evaluation. P1 received prednisone, CNI, and rituximab for a presumed anti-HSPC autoantibody. At 9 months he was weaned off all therapy and remains on no immunosuppressive treatment 2 years later. P2 did not receive CNI due to chronic renal failure but received steroids and later sirolimus. At ~18 months, his cytopenias recurred, but subsequently responded to basil re-induction and steroids. P3 has received prednisone, CNI, and sirolimus indefinitely for prevention of liver rejection and/or hepatocellular carcinoma relapse. P1 had no viral reactivation; P2 had CMV pneumonia with his second course responsive to antiviral therapy, completion of basil, and tapering of steroids; and P3 had CMV reactivation responsive to antiviral therapy and EBV viremia. P3 has a recent diagnosis of EBV+ intrahepatic B cell lymphoma. In all cases, blood counts normalized except P2 who has anemia due to CKD. Discussion: HSPC analyses in these patients with GVHD and cytopenias demonstrate that peripheral changes are not due to loss of HSC, but instead to quantitative and/or qualitative defects in committed progenitor populations, likely mediated by activated CD25+ T cells. In addition, the presence of IgG on HSPCs in P1 demonstrates a novel role for antibody-mediated humoral suppression in some cases. The presence of HSC indicates that there may be no need for re-transplant. Previous studies of SAA have shown activated T cells to be drivers of aplasia and is the basis for immune suppressive therapy (anti-thymocyte globulin and CNI). Our analyses of patients with GVHD-associated cytopenias demonstrates that targeting of IL-2 signaling via combined CD25 receptor blockade and pharmacologic inhibition of IL-2 production can correct the marrow defects and cytopenias, albeit with need for attention to viral infections. Targeting CD25 in patients with evidence of CD25+ activated T cells is a rational approach to the difficult problem of GVHD-associated marrow suppression and cytopenias.

The effect of long exposure to uvb rays on histological features of wistar rats (Rattus norvegicus) in photoaging model

Introduction: Indonesia is a tropical country with exposure to UV rays from the sun throughout the year, so the Indonesian population is very vulnerable to skin aging, especially extrinsic skin aging due to prolonged exposure to UV rays. This study aimed to determine the effect of long exposure to UVB rays on the thickness of the epidermis, the thickness of the dermis, and the number of blood vessels and sunburn cells in (Rattus norvegicus) Wistar strain. Methods: This research was a post-test-only control group design using 27 rats which were divided into 3 groups, namely: control group K was untreated rats, and treatment groups P1 and P2 were rats exposed to UVB radiation for 3 weeks and 6 respectively exposure weeks. Test the effect on the study using the One-Way ANOVA test and Tukey’s Post Hoc Test. Results: The results indicated an increase in epidermal thickness of groups P1 (44.87 μm) and P2 (56.90 μm) (significant p<0.05). A decrease in dermis thickness occurred in groups P1 (726.91 μm) and P2 (559.40 μm) (significant p<0.05). In the photoaging area, there was an increased amount of sunburn cells in group P1 (6.40) and P2 (11.40) (significant p<0.05). There was a difference in the number of blood vessels between groups K and P2 and groups P1 and P2 (p<0.05). Conclusion: There was an effect of long exposure to UVB rays on histology in rats (Rattus norvegicus) of the photoaging model including the thickness of the epidermis layer and dermis layer, the number of blood vessels, and sunburn cells.

Investigating proliferation and differentiation capacities of Hanwoo steer myosatellite cells at different passages for developing cell-cultured meat

The aim of study was to investigate proliferation and differentiation capacities of Hanwoo myosatellite cells for the development of Hanwoo cell cultures. From P1 to P19, the number of live cells decreased and the cell size increased. It was confirmed that the PAX7 mRNA was higher in P3 than P6 and P9 (p < 0.05). The maximum differentiation score was measured from P1 to P12. The maximum differentiation score maintained high from P1 to P10. Immunostaining was performed for both P1 and P10 cells to investigate differentiation characteristics. And there were no significant differences in differentiation characteristics between P1 and P10 cells. MYOG mRNA was low, whereas C-FOS mRNA was high (p < 0.05) in the late passage. Myosin and Tom20 protein also showed low values in the late passage (p < 0.05). In conclusion, our results suggest that it is appropriate to use P1 to P10 for the production of cultured meat using Hanwoo muscle cells. If cell culture meat production is performed without differentiation, the passage range may increase further. These results provide basic essential data required for further development of Hanwoo cell cultures, which could provide a valuable source of protein for human populations in the future.

The Path from Nasal Tissue to Nasal Mucosa on Chip: Part 1-Establishing a Nasal In Vitro Model for Drug Delivery Testing Based on a Novel Cell Line.

In recent years, there has been a significant increase in the registration of drugs for nasal application with systemic effects. Previous preclinical in vitro test systems for transmucosal drug absorption studies have mostly been based on primary cells or on tumor cell lines such as RPMI 2650, but both approaches have disadvantages. Therefore, the aim of this study was to establish and characterize a novel immortalized nasal epithelial cell line as the basis for an improved 3D cell culture model of the nasal mucosa. First, porcine primary cells were isolated and transfected. The P1 cell line obtained from this process was characterized in terms of its expression of tissue-specific properties, namely, mucus expression, cilia formation, and epithelial barrier formation. Using air-liquid interface cultivation, it was possible to achieve both high mucus formation and the development of functional cilia. Epithelial integrity was expressed as both transepithelial electrical resistance and mucosal permeability, which was determined for sodium fluorescein, rhodamine B, and FITC-dextran 4000. We noted a high comparability of the novel cell culture model with native excised nasal mucosa in terms of these measures. Thus, this novel cell line seems to offer a promising approach for developing 3D nasal mucosa tissues that exhibit favorable characteristics to be used as an in vitro system for testing drug delivery systems.

π-Stacking among the Anthracenyl Groups of a Copper Complex Resulted in Doubling of Unit Cell Volume To Provide New Polymorphs.

Two polymorphs of the 9-N-(3-imidazolylpropylamino)methylanthracene (Hanthraimmida) containing hydrated copper(II)-2,6-pyridinedicarboxylate complex are reported. The two polymorphs have either lamellar or Herringbone arrangements of π-stacks among the anthracenyl groups of organocation. The difference between the two polymorphs originated from having face-to-face stacking arrangements between the two anthracenyl groups of the symmetry independent cations within the unit cell in one of the polymorphs. The π-stacked anthracenyl groups in consecutive layers of the polymorphs are oriented in one direction in the polymorph designated as P1, whereas the polymorph designated as P2 has such orientations in opposite directions. The unit cell volume of the polymorph P2 (Z = 4) has approximately twice the volume of the polymorph P1 (Z = 2); it happend due to coalescence of two unit cells of P1 in the ab-crystallographic plane. A mixed methanol/water solvate of the copper complex is also reported. It has a channel-like arrangement of the cations; has the anions and the solvents within the cation embraced channel-like enclosures. This complex is unstable, once taken out from the methanol solvent, it transforms in real time to P2 by replacements of the methanol molecules by water molecules.

Multiple pathways for reestablishing PAR polarity in C. elegans embryo

Asymmetric cell divisions, where cells divide with respect to a polarized axis and give rise to daughter cells with different fates, are critically important for development. In many such divisions, the conserved PAR polarity proteins accumulate in distinct cortical domains in response to a symmetry breaking cue. The one-cell C. elegans embryo is a paradigm for understanding mechanisms of PAR polarization, but much less is known about polarity in subsequent divisions. Here, we investigate the polarization of the P1 cell of the two-cell embryo. A posterior PAR-2 domain forms in the first 4 ​min, and polarization becomes stronger over time. Initial polarization depends on the PAR-1 and PKC-3 kinases, and the downstream polarity regulators MEX-5 and PLK-1. However, par-1 and plk-1 mutants exhibit delayed polarization. This late polarization correlates with the time of centrosome maturation and actomyosin flow, and loss of centrosome maturation or myosin in par-1 mutant embryos causes an even stronger polarity phenotype. Based on these and other results, we propose that PAR polarity in the P1 cell is generated by at least two redundant mechanisms: There is a novel early pathway dependent on PAR-1, PKC-3 and cytoplasmic polarity, and a late pathway that resembles symmetry breaking in the one-cell embryo and requires PKC-3, centrosome associated AIR-1 and myosin flow.

Study on in vitro differentiation of human adenoid-derived mesenchymal stem cells into olfactory sensory neurons

Objective: To investigate the feasibility of isolation and culture of human adenoid-derived mesenchymal stem cells (aMSCs) in vitro, and to observe the differentiation of aMSCs into olfactory sensory neurons. Methods: Adenoid tissues surgically removed from children with adenoid hypertrophy in the Second Xiangya Hospital of Central South University from September to November of 2020 were collected. The adenoid tissues were digested and isolated by trypsin and then cultured with adhesion method. The expressions of cell surface antigens CD45, CD73 and CD90 on aMSCs of P5 generation were tested by flow cytometry, and the ability of osteogenic and adipogenic induction were used to identify cell differentiation ability. Then, aMSCs were induced into differentiation by retinoic acid (RA), sonic hedgehog (SHH), basic fibroblast growth factor (bFGF), RA+SHH, RA+bFGF, SHH+bFGF and RA+SHH+bFGF, respectively. The morphology of differentiated cells was observed under inverted microscope. The expression of β-tubulin 3, which was the specific marker of sensory neuron, the expressions of growth associated protein-43 (GAP43) and olfactory maker protein (OMP), which were the specific markers of olfactory sensory neuron, were detected by immunofluorescence antibody assay. The expression intensities were compared by Chi-square test of four-grid table data. Results: aMSCs were successively isolated and cultured from human adenoid tissues. P0 cells generation had good adhesion and proliferation performance. P2 cells were basically purified. P5 cells expressed CD73 and CD90 with the purity of 99.3% and 99.75% respectively, without CD45 expression. P5 cells had a good ability of osteogenic differentiation and adipogenic differentiation. Neuron-like morphology and expression of β-tubulin 3 were found in differentiated cells after induced by RA, SHH, or bFGF, respectively. An induction of expression of GAP43 was found in differentiated cells of bFGF+SHH group and RA+SHH+bFGF group, without expression of OMP of each group. The intensity of GAP43 expression of RA+SHH+bFGF group was stronger than that of bFGF+SHH group (χ2=17.48, P<0.005). Conclusions: aMSCs can be cultured from human adenoid tissues, with the stably passaged and good differentiation ability. As a new population of mesenchymal stem cells, aMSCs have the neuroregenerative properties and could differentiate into immature olfactory sensory neurons under the induction of RA+SHH+bFGF in vitro.

Preclinical evaluation of teleglenastat (CB-839) in prostate cancer.

378 Background: Glutaminase (GLS) levels are upregulated in prostate cancer [PMID: 28138303, 16897757]. The androgen receptor signaling pathway enhances the expression of SLC43A1, SLC1A4 and SLC1A5 (amino acid and glutamine transporters) in prostate cancer cells [PMID: 22007000, 24052624, 28507054]. We evaluated prostate cancer data from The Cancer Genome Atlas and found that GLS expression correlates with Gleason sum. We performed in vitro and in vivo experiments to evaluate the effect CB-839, a glutaminase inhibitor (GLS1 &gt; GLS2), as monotherapy and in combination with other agents. Methods: We evaluated glutamine dependence of 3 prostate cancer cell lines [parental LNCaP (P0, hormone-sensitive), LNCaP-derived castrate resistant (CRPC) variant (P1, PMID: 34575033), and PC-3 (CRPC)] using media with and without glutamine, as well as clonogenic assays in regular media with BPTES (GLS1 inhibitor as a control) and CB-839. Combinatorial treatment and dose response studies were performed with CB-839 plus enzalutamide (ENZ), everolimus (EVE), or olaparib (OLA). We used NOD SCID female nude mice carrying P1 and PC3 prostate tumor xenografts to evaluate effects of CB-839 monotherapy, CB-839 + EVE, and EVE monotherapy. Results: P0, P1, and PC-3 all exhibited glutamine dependence. In glutamine-deplete media, all tested cell lines formed &lt; 50% of colonies from baseline. Clonogenic assays with BPTES showed IC50 of 2-6 uM. CB-839 showed single agent activity in vitro with IC50 = 1 uM (P0), IC50 = 2 (P1), and IC50 &lt; 0.1 uM (PC-3). The IC50 of CB-839 + ENZ in P1 cells was &lt; 4uM CB-839 + 2uM ENZ. CB-839 + OLA showed no additive effect as OLA alone was very active (IC50 &lt; 100 nM) in P1 cells. CB-839 + EVE showed single agent and synergistic effect (IC50 &lt; 4 uM CB-839 + 0.05 uM EVE) in both P1 and PC-3 cells. In P1 tumor xenografts, treatment response was seen in CB-839 monotherapy, EVE monotherapy, and CB-839 + EVE treatment arms. In PC-3 xenografts, treatment response was seen in all groups, with greater effect in the EVE monotherapy and CB-839 + EVE treatment groups compared with CB-839 monotherapy. Conclusions: CB-839, EVE, and CB-839 + EVE all demonstrated activity in P1 and PC-3 castrate resistant prostate cancer tumor xenografts. Additive or synergistic activity was difficult to assess with combination therapy since each drug was effective as monotherapy. Additional studies are needed to evaluate the role of EVE monotherapy or CB-839 + EVE in prostate cancer. Research Sponsors: NCI CTEP UM1 Supplement CA186688-04, NCI University of Colorado Cancer Center Support Grant 5P30CA046934-30. [Table: see text]

Detection and diversity of human enteroviruses from domestic sewage in Weishan Lake region, eastern China, 2018-2019.

Next generation sequencing (NGS) technology has been widely used in monitoring and identifying microbiomes in sewage. We aimed to evaluate the ability of NGS analysis in directly detecting enterovirus (EV) in sewage and to understand the diversity of EVs circulated in the residents in Weishan Lake region. Fourteen sewage samples were collected in Jining, Shandong Province, China from 2018 to 2019 and were parallelly investigated by the P1 amplicon-based NGS method and cell culture method. The results showed that 20 different serotypes belonging to species Enterovirus A (EV-A) (n=5), EV-B (n=13), and EV-C (n=2) were identified by NGS in the sewage concentrates, which exceeded the number of types detected by cell culture method (n=9). Echovirus 11 (E11), Coxsackievirus (CV) B5 and CVA9 were the most detected types in those sewage concentrates. Phylogenetic analysis revealed that E11 sequences obtained in this study belonged to genogroup D5 and had close genetic relationship with clinical sequences. Various EV serotypes circulated in populations near Weishan Lake. The combination of NGS technology into environmental surveillance will greatly contribute to improving our knowledge about EV circulation patterns in the population.

Comparison of the yields and properties of dedifferentiated fat cells and mesenchymal stem cells derived from infrapatellar fat pads.

Infrapatellar fat pad (IFP)-derived mesenchymal stem cells (MSCs) have high chondrogenic potential and are attractive cell sources for cartilage regeneration. During ceiling culture to acquire the characteristics of MSCs, mature adipocytes from fat tissue are known to undergo dedifferentiation, generating dedifferentiated fat (DFAT) cells. The purpose of the present study was to compare the yields and biological properties of IFP-derived MSCs and IFP-derived DFAT cells. IFPs were harvested from the knees of 8 osteoarthritis (OA) patients. DFAT cells were obtained using a ceiling culture of adipocytes isolated from the floating top layer of IFP digestion. MSCs were obtained by culturing precipitated stromal vascular fraction cells. We compared the P0 cell yields, surface antigen profile, colony formation ability, and multipotency of DFAT cells and MSCs. The P0 cell yields per flask and the estimated total cell yields from 1g of IFP were much greater for MSCs than for DFAT cells. Both MSCs and DFAT cells were positive for MSC markers. No obvious difference was observed in colony formation ability. In differentiation assays, DFAT cells produced greater amounts of lipid droplets, calcified tissue, and glycosaminoglycan than MSCs did. Adipogenic and chondrogenic gene expressions were upregulated in DFAT cells. IFP-derived DFAT cells showed higher adipogenic and chondrogenic potentials than IFP-derived MSCs, but they had a poor cell yield.

Histopathological and Gastric Histomorphometry Analysis in Mice Colibacillosis Model Induced by Ethanol 96% Oregano Vulgare Extract

Colibacillosis is a disease caused by Escherichia coli which is a normal flora bacterium of the digestive system in humans and animals. Oregano contains carvacrol and thymol which provide an antibacterial. This study was used 20 male Balb/C mice age 2-3 weeks with an average weight of 20-25 grams and were divided into 5 groups with 4 replications: negative control (KN), positive control (KP) given by streptomycin dose of 20 mg/head. E. coli dose of 3 x 109 CFU, (P1) was given by streptomycin dose of 20 mg/head, E. coli dose of 3 x 109 CFU, then given oreganoextract dose of 5 mg/head, (P2) was given by streptomycin dose of 20 mg/head, E. coli dose of 3 x 109 CFU, then given oregano extract dose of 10 mg/head, (P3) was given by streptomycin dose of 20 mg/head, E. coli dose of 3 x 109 CFU, then oregano extract was given dose of 20 mg/head. The data were analyzed by descriptive comparative. The histopathology showed epithelial cell damage or erosion caused by inflammatory cells in KP, P1, P2, and P3. In study group given oregano extract dose of 20 mg/head, P3, inflammatory cells and epithelial erosion were lower. Based on the ANOVA test of day 3 and 6 after necropsy, P1, P2, and P3 show insignificant in villi length and width of the vili (p&gt;0,05) compared to KP. The conclusion the result of the histopathology result is epithelial erosion in group with dose of 20mg/head oregano extract was able to relieve the symptoms of colibacillosis. The conclusion of histomorphometry of the length and width of the villi at P1, P2, P3 (p&gt;0.05) was not significantly different from the positive control.

Comprehensive investigations of key mitochondrial metabolic changes in senescent human fibroblasts.

There is a paucity of detailed data related to the effect of senescence on the mitochondrial antioxidant capacity and redox state of senescent human cells. Activities of TCA cycle enzymes, respiratory chain complexes, hydrogen peroxide (H2O2), superoxide anions (SA), lipid peroxides (LPO), protein carbonyl content (PCC), thioredoxin reductase 2 (TrxR2), superoxide dismutase 2 (SOD2), glutathione peroxidase 1 (GPx1), glutathione reductase (GR), reduced glutathione (GSH), and oxidized glutathione (GSSG), along with levels of nicotinamide cofactors and ATP content were measured in young and senescent human foreskin fibroblasts. Primary and senescent cultures were biochemically identified by monitoring the augmented cellular activities of key glycolytic enzymes including phosphofructokinase, lactate dehydrogenase, and glycogen phosphorylase, and accumulation of H2O2, SA, LPO, PCC, and GSSG. Citrate synthase, aconitase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and complex I-III, II-III, and IV activities were significantly diminished in P25 and P35 cells compared to P5 cells. This was accompanied by significant accumulation of mitochondrial H2O2, SA, LPO, and PCC, along with increased transcriptional and enzymatic activities of TrxR2, SOD2, GPx1, and GR. Notably, the GSH/GSSG ratio was significantly reduced whereas NAD+/NADH and NADP+/NADPH ratios were significantly elevated. Metabolic exhaustion was also evident in senescent cells underscored by the severely diminished ATP/ADP ratio. Profound oxidative stress may contribute, at least in part, to senescence pointing at a potential protective role of antioxidants in aging-associated disease.

Modeling of mitochondrial bioenergetics and autophagy impairment in MELAS-mutant iPSC-derived retinal pigment epithelial cells

BackgroundMitochondrial dysfunction and mitochondrial DNA (mtDNA) damage in the retinal pigment epithelium (RPE) have been implicated in the pathogenesis of age-related macular degeneration (AMD). However, a deeper understanding is required to determine the contribution of mitochondrial dysfunction and impaired mitochondrial autophagy (mitophagy) to RPE damage and AMD pathobiology. In this study, we model the impact of a prototypical systemic mitochondrial defect, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), in RPE health and homeostasis as an in vitro model for impaired mitochondrial bioenergetics.MethodsWe used induced pluripotent stem cells (iPSCs) derived from skin biopsies of MELAS patients (m.3243A > G tRNA leu mutation) with different levels of mtDNA heteroplasmy and differentiated them into RPE cells. Mitochondrial depletion of ARPE-19 cells (p0 cells) was also performed using 50 ng/mL ethidium bromide (EtBr) and 50 mg/ml uridine. Cell fusion of the human platelets with the p0 cells performed using polyethylene glycol (PEG)/suspension essential medium (SMEM) mixture to generate platelet/RPE “cybrids.” Confocal microscopy, FLowSight Imaging cytometry, and Seahorse XF Mito Stress test were used to analyze mitochondrial function. Western Blotting was used to analyze expression of autophagy and mitophagy proteins.ResultsWe found that MELAS iPSC-derived RPE cells exhibited key characteristics of native RPE. We observed heteroplasmy-dependent impairment of mitochondrial bioenergetics and reliance on glycolysis for generating energy in the MELAS iPSC-derived RPE. The degree of heteroplasmy was directly associated with increased activation of signal transducer and activator of transcription 3 (STAT3), reduced adenosine monophosphate-activated protein kinase α (AMPKα) activation, and decreased autophagic activity. In addition, impaired autophagy was associated with aberrant lysosomal function, and failure of mitochondrial recycling. The mitochondria-depleted p0 cells replicated the effects on autophagy impairment and aberrant STAT3/AMPKα signaling and showed reduced mitochondrial respiration, demonstrating phenotypic similarities between p0 and MELAS iPSC-derived RPE cells.ConclusionsOur studies demonstrate that the MELAS iPSC-derived disease models are powerful tools for dissecting the molecular mechanisms by which mitochondrial DNA alterations influence RPE function in aging and macular degeneration, and for testing novel therapeutics in patients harboring the MELAS genotype.

24 The CD34+ Adipose-derived Stem Cells from Fat Grafts Accelerates Keratinocyte Wound Closure

IntroductionAdipose derived stem cells (ADSCs) possess high regenerative potential and therefore are postulated to enhance re-epithelialization via recruitment of keratinocytes into the wound bed, and thusly improve healing of severe burns. Although ADSC’s are enriched in the stromal vascular fraction (SVF) of fat grafts, the ADSC frequency in these samples is very low (1%) which has hindered their further characterization. So far, the CD34+ subset of SVF cells has shown to contain up to 75% of all the ADSCs present in SVF samples but their ADSC frequency remains low (2.5%). Unfortunately, while the ADSC frequency increases in passaged SVF cells, the expression of CD34 is lost in these cells. Therefore, there is a pressing need to identify new ADSC biomarkers to study ADSC mechanisms that accelerate wound closure. In this study, we aim to identify new ADSC biomarkers and to examine the impact of ADSC-enriched cells on skin wound closure.MethodsSingle cells from SVFs were obtained from disassociated fat tissue, collected from excess lipoaspirates used in patients undergoing mastectomies. Native cells (P0), and passaged cells (P3) were placed in cell cultures and the ADSC numbers were obtained using the colony forming unit-fibroblast assays. The expression of 244 cell surface receptors was examined in P0 (low ADSCs) and P3 (high ADSCs) SVF cells to obtain new ADSC biomarkers. In another experiment, the impact of ADSC-enriched CD34+ SVF cells on wound closure was assessed. Using transwell inserts containing adult human epidermal keratinocyte (HEKa) cells in the bottom and the bulk SVF or CD34+ SVF cells in the top chamber, a scratch was induced in the HEKa cell layer and the scratch area was examined at 0h and 12h.ResultsOur cell surface antibody array experiments revealed 10 potentially new ADSC biomarkers that will be used to examine their ability to enrich for ADSCs. Both the bulk SVF and the CD34+ SVF cells showed a 2-fold increase in would closure. It is noteworthy that ADSCs frequency in the CD34+ SVF cells is still very low, and these experiments need to be conducted using the new SVF subsets with potentially higher ADSC frequencies based on our cell surface array.ConclusionsThe ADSC-enriched CD34+ SVF enhance keratinocyte wound closure, a function that is important to healing of severe burns. Further studies are needed to validate our findings using more purified SVF subsets to identify ADSC-regulated keratinocyte wound closure mechanisms.

Cultivation and characterization of primary human parathyroid cells from patients with severe secondary hyperparathyroidism

To establish an cell model of hyperparathyroidism by isolation, in vitro culture, and identification of parathyroid cells from patients with secondary hyperparathyroidism (SHPT). The parathyroid gland tissues obtained from 10 patients with SHPT were dissociated by collagenase digestion for primary culture of the parathyroid cells. Morphological changes and growth characteristics of the cells were assessed by microscopic imaging and cell counting. The mRNA and protein expression levels of parathyroid hormone (PTH), calcium-sensing receptor (CaSR), and glial cells missing 2 (GCM2) in the primary and passaged cells were determined by immunofluorescence, qRT-PCR, and Western blotting. Primary cultures of parathyroid cells were successfully obtained. The cells exhibited a high expression of PTH shown by immunofluorescence assay and had a population doubling time of approximately 71.61 h. PTH secretion in the second-passage (P2) cells was significantly lower than that in the primary (P0) and first-passage (P1) cells (P < 0.001). Despite a significant downregulation of CaSR mRNA (P=0.017) and protein (P=0.006) in P1 cells as compared with P0 cells, no significant differences were found in mRNA and protein expressions of PTH or GCM2 between the two cell generations. Primary cultures of parathyroid cells isolated from SHPT patients by collagenase digestion show similar biological properties to the cells in vivo.