Cell cycle synchronization of donor cells is an important step in mammalian somatic cell nuclear transfer (SCNT). This study was designed to compare the efficiency of serum starvation (Ss) and ...contact inhibition (cI) on cell cycle synchronization of jaguarundi, manul, and domestic cat skin fibroblasts, in the production of G0/G1 cells suitable for SCNT in felids. Ss was performed after the growing (G) cells reached 40–50% (G50+Ss), 60–70% (G70+Ss) and full confluency (Fc), i.e. in association with cI (cI+Ss). Frozen-thawed cells were cultured to the given state of confluency (d0; controls), and subjected to Ss or cI for 1, 3, and 5 days (d). In manul, the effect of Ss on arresting fibroblasts in the G0/G1 phase was noted after just 1d of culture at G70 confluence, while G50+Ss and cI+Ss were effective after 5d of treatment. In jaguarundi, 1–5d of G50+Ss and 5d of G70+Ss increased the percentage of G0/G1 cells versus d0 (P<0.01), with 5d of G70+Ss producing more (P<0.05) quiescent cells than after the same period of G50+Ss, cI+Ss and cI. In the domestic cat, Ss was efficient only after 3 and 5d of G50+Ss. In all species, cI alone failed to increase the proportion of G0/G1 cells compared to d0, however in the domestic cat, 5d of cI was more efficient than the same period of G50+Ss. In jaguarundi, >93% of cells were already in G0/G1 phase at d0 of Fc, suggesting that culture to Fc could be also a valuable method for fibroblast cell cycle synchronization in this species. In contrast to cI, prolonged Ss generated cell loss and could induce apoptosis and/or necrosis. In conclusion, Ss was the more efficient method for skin fibroblast cell cycle synchronization at the G0/G1 phase in manul, jaguarundi and the domestic cat. The response of cells to the treatments was species-specific, depending on cell confluence and duration of culture. This research may find application in preparing donor karyoplasts for SCNT in felids.
Background: Patch testing is an essential procedure in the investigation of eczema in children.
Objectives: To analyse the frequency of contact hypersensitivity and allergic contact dermatitis ...among Polish children with eczema.
Patients/methods: During an allergy screening programme involving 9320 children aged 7 and 16 years, 12.6% reported symptoms of chronic/recurrent eczema. From this group, a representative sample of 229 eczema children underwent patch testing: 96 children aged 7 years and 133 teenagers aged 16 years. Patch testing was with 10 allergens: methylchloroisothiazolinone/methylisothiazolinone (MCI/MI), nickel sulfate, mercury ammonium chloride, thimerosal, cobalt chloride, potassium dichromate, lanolin, fragrance mix I, Myroxylon pereirae (balsam of Peru), and colophonium.
Results: 49.4% tested children were found patch test (PT) positive. 43.8% of 7 year olds with eczema were PT positive, with sensitization to nickel sulfate (30.2%), thimerosal (10.4%), cobalt chloride (8.3%), fragrance mix I (7.3%), MCI/MI (6.3%), potassium dichromate (6.3%), M. pereirae (3.1%), mercury ammonium chloride (2.3%), and colophonium (1.0%). 52.6% teenagers were PT positive, with sensitization to nickel sulfate (23.3%), thimerosal (27.8%), cobalt chloride (10.5%), potassium dichromate (6.0%), mercury ammonium chloride (2.3%), M. pereirae (1.5%), and MCI/MI (0.8%). The final diagnosis of allergic contact dermatitis was confirmed in 36% of 7 year olds and 26% of 16 year olds.
Conclusions: Every second child with eczema is PT positive, whereas every third child is finally diagnosed with allergic contact dermatitis.
Along with a growing interest in mRNA-based gene therapies, efforts are increasingly focused on reaching the full translational potential of mRNA, as a major obstacle for in vivo applications is ...sufficient expression of exogenously delivered mRNA. One method to overcome this limitation is chemically modifying the 7-methylguanosine cap at the 5' end of mRNA (m
Gppp-RNA). We report a novel class of cap analogs designed as reagents for mRNA modification. The analogs carry a 1,2-dithiodiphosphate moiety at various positions along a tri- or tetraphosphate bridge, and thus are termed 2S analogs. These 2S analogs have high affinities for translation initiation factor 4E, and some exhibit remarkable resistance against the SpDcp1/2 decapping complex when introduced into RNA. mRNAs capped with 2S analogs combining these two features exhibit high translation efficiency in cultured human immature dendritic cells. These properties demonstrate that 2S analogs are potentially beneficial for mRNA-based therapies such as anti-cancer immunization.
The synthesis and biochemical properties of 17 new mRNA cap analogues are reported. Six of these nucleotides are m(7)GTP derivatives, whereas 11 are 'two headed' tetraphosphate dinucleotides based on ...a m(7)Gppppm(7)G structure. The compounds contain either a boranophosphate or phosphorothioate moiety in the nucleoside neighbouring position(s) and some of them possess an additional methylene group between β and γ phosphorus atoms. The compounds were prepared by divalent metal chloride-mediated coupling of an appropriate m(7)GMP analogue with a given P(1),P(2)-di(1-imidazolyl) derivative. The analogues were evaluated as tools for studying cap-dependent processes in a number of biochemical assays, including determination of affinity to eukaryotic initiation factor eIF4E, susceptibility to enzymatic hydrolysis, and translational efficiency in vitro. The results indicate that modification in the phosphate chain can increase binding to cap-interacting proteins and provides higher resistance to degradation. Furthermore, modified derivatives of m(7)GTP were found to be potent inhibitors of cap-dependent translation in cell free systems.
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The synthesis and biochemical properties of 17 new mRNA cap analogues are reported. Six of these nucleotides are m7GTP derivatives, whereas 11 are ‘two headed’ tetraphosphate ...dinucleotides based on a m7Gppppm7G structure. The compounds contain either a boranophosphate or phosphorothioate moiety in the nucleoside neighbouring position(s) and some of them possess an additional methylene group between β and γ phosphorus atoms. The compounds were prepared by divalent metal chloride-mediated coupling of an appropriate m7GMP analogue with a given P1,P2-di(1-imidazolyl) derivative. The analogues were evaluated as tools for studying cap-dependent processes in a number of biochemical assays, including determination of affinity to eukaryotic initiation factor eIF4E, susceptibility to enzymatic hydrolysis, and translational efficiency in vitro. The results indicate that modification in the phosphate chain can increase binding to cap-interacting proteins and provides higher resistance to degradation. Furthermore, modified derivatives of m7GTP were found to be potent inhibitors of cap-dependent translation in cell free systems.
The exact pathophysiology of severe COVID-19 is not entirely elucidated, but it has been established that hyperinflammatory responses and cytokine storms play important roles. The aim of this study ...was to examine CMV status, select chemokines, and complement components in COVID-19, and how concentrations of given molecules differ over time at both molecular and proteomic levels. A total of 210 COVID-19 patients (50 ICU and 160 non-ICU patients) and 80 healthy controls were enrolled in this study. Concentrations of select chemokines (CXCL8, CXCL10, CCL2, CCL3, CCR1) and complement factors (C2, C9, CFD, C4BPA, C5AR1, CR1) were examined at mRNA and protein levels with regard to a COVID-19 course (ICU vs. non-ICU group) and CMV status at different time intervals. We detected several significant differences in chemokines and complement profiles between ICU and non-ICU groups. Pro-inflammatory chemokines and the complement system appeared to greatly contribute to the pathogenesis and development of severe COVID-19. Higher concentrations of CXCL8 and CCL2 in the plasma, with reduced mRNA expression presumably through negative feedback mechanisms, as well as CMV-positive status, correlated with more severe courses of COVID-19. Therefore, CXCL8, CCL2, and CMV seropositivity should be considered as new prognostic factors for severe COVID-19 courses. However, more in-depth research is needed.
•A mechanism for propranolol action in infantile hemangiomas which relies on changes in MMPs-2/9 is proposed.•Evaluation of easily to be possessed patients’ serum with the use of ELISA assay is ...suggested as an valuable complement of MMPs tissue studies.•MMPs-2/9 mRNA levels appear as a potential novel biomarkers for the most individual approach in IH as well as in cancer disease.
Propranolol is a widely-known beta-blocker approved for treating infantile hemangiomas (IH). The mechanisms behind the spectacular IH involution after propranolol treatment remain unclear. Recently, there is strong evidence of overexpression of numerous angiogenic factors in IH tissues, and it is reported that propranolol influences their pathways. However, a number of MMPs studies is highly limited. Here, for the first time, we propose a comprehensive approach by analyzing the expression levels of metalloproteinases-2/9 (MMPs-2/9) and tissue metalloproteinase inhibitor-2 (TIMP-2) in vivo on both, molecular and immunohistochemical levels, and in both, IH tissues and in the serum of IH patients, and relates the obtained results to the tumor’s biology and systemic propranolol treatment.
MMPs-2/9 and TIMP-2 were analyzed in 71 IH tissue samples using immunohistochemistry and real-time PCR, and in 50 serum samples of IH patients by ELISA.
Significantly lower MMPs-2/9 and higher TIMP-2 levels were observed in IH tissues on the mRNA level as well as lower serum MMP-2 concentration among the treated individuals.
MMPs-2/9 and TIMP-2 are both involved in the biology of IH and the propranolol pathways enabling their antiangiogenic properties. The most reliable method of IH examination appears to be direct MMPs-2/9 mRNA evaluation in tumor tissue; and MMP-2 evaluation in patients’ serum is a valuable complement to it. Tissue and serum mRNA MMPs assessment may represent a suitable novel biomarker identifying tumor progression and involution processes with potential clinical impact in IH as well as in cancer disease.
A rise in the incidence of infections with severe acute respiratory syndrome coronavirus 2 has sparked the search for protective strategies against the new pathogen. It is known that individual food ...components can interact with different immune cells, modulating the immune response of the body. The aim of this study was to develop an index assessing the immunomodulatory potential of diet (POLA index) and to test its utility for the prediction of coronavirus disease 2019 (COVID-19) in a group of healthy young people following a traditional or vegetarian diet. Data on body composition, anthropometric measurements, physical activity, dietary intake, and gut microbiota were obtained from 95 adults (mean age, 34.66 ± 5.76 years). There was a strong correlation between the dietary inflammatory index and the POLA index (r = 0.90; p < 0.0001). Based on Cohen’s kappa statistic, there was a good agreement in qualitative interpretation between the two indices (kappa = 0.61; p < 0.0001). People on a diet with beneficial immunomodulatory effects had a lower risk of COVID-19 of approximately 80%, as compared with those on a diet with highly unbeneficial immunomodulatory effects. In daily practice, the POLA index might serve as a useful tool for dietitians to identify individuals whose diet is deficient in ingredients for optimal immune system function and change their dietary behavior to ensure optimal immune function that reduces the risk of infection.
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