Lactoferrin Alleviated AFM1-Induced Apoptosis in Intestinal NCM 460 Cells through the Autophagy Pathway

Aflatoxin M1 (AFM1) is the only mycotoxin with maximum residue limit in milk, which may result in serious human diseases. On the contrary, lactoferrin (Lf) is an active protein with multiple functions. Studies have confirmed that Lf has a powerful potential to protect the intestines, but the influence of Lf on mycotoxins is not clear. This study aims to explore whether Lf can protect the cytotoxicity induced by AFM1, and determine the underlying mechanisms in human normal colonic epithelial NCM460 cells. The results indicated that AFM1 decreased the cell viability, and increased the levels of apoptosis and autophagy of NCM460 cells. Lf can alleviate the cytotoxicity induced by AFM1 through enhancing cell viability, significantly down-regulated the expression of apoptotic genes and proteins (BAX, caspase3, caspase9, caspase3, and caspase9), and regulated the gene and protein expression of autophagy factors (Atg5, Atg7, Atg12, Beclin1, ULK1, ULK2, LC3, and p62). Furthermore, interference of the key gene Atg5 of autophagy can reduce AFM1-induced apoptosis, which is consistent with the role of Lf, implying that Lf may protect AFM1-induced intestinal injury by inhibiting excessive autophagy-mediated apoptosis. Taken together, our data indicated that Lf has a mitigating effect on apoptosis induced by AFM1 through the autophagy pathway.

DNA Methyltransferase HsdM Induce Drug Resistance on Mycobacterium tuberculosis via Multiple Effects

Besides the genomic variants, epigenetic mechanisms such as DNA methylation also have an effect on drug resistance. This study aimed to investigate the methylomes of totally/extensively drug-resistant M. tuberculosis clinical isolates using the PacBio single-molecule real-time technology. The results showed they were almost the same as the pan-susceptible ones. Genetics and bioinformatics analysis confirmed three DNA methyltransferases—MamA, MamB, and HsdM. Moreover, anti-tuberculosis drug treatment did not change the methylomes. In addition, the knockout of the DNA methyltransferase hsdM gene in the extensively drug-resistant clinical isolate 11826 revealed that the motifs of GTAYN4ATC modified by HsdM were completely demethylated. Furthermore, the results of the methylated DNA target analysis found that HsdM was mainly involved in redox-related pathways, especially the prodrug isoniazid active protein KatG. HsdM also targeted three drug-targeted genes, eis, embB, and gyrA, and three drug transporters (Rv0194, Rv1410, and Rv1877), which mildly affected the drug susceptibility. The overexpression of HsdM in M. smegmatis increased the basal mutation rate. Our results suggested that DNA methyltransferase HsdM affected the drug resistance of M. tuberculosis by modulating the gene expression of redox, drug targets and transporters, and gene mutation.

A novel approach for the purification of aggregation prone proteins

The protein aggregation is one of the major challenges of the biotechnological industry, especially in the areas of development and commercialization of successful protein-based drug products. The inherent high aggregation tendency of proteins during various manufacturing processes, storage, and administration has significant impact upon the product quality, safety and efficacy. We have developed an interesting protein purification approach that separates the functionally active protein from inactive aggregates using a detergent concentration gradient. The C-terminally His tagged nucleocapsid protein of Crimean Congo Hemorrhagic fever virus (CCHFV) has high aggregation tendency and rapidly precipitates upon purification by NiNTA chromatography. Using the new purification approach reported here, the freshly purified protein by NiNTA chromatography was further processed using a detergent gradient. In this new purification approach the active protein is retained in the low detergent concentration zone while the inactive aggregates are promptly removed by their rapid migration to the high detergent concentration zone. The method prevented further aggregation and retained the RNA binding activity in the native protein despite numerous freeze thaw cycles. This simple approach prevents protein aggregation by rapidly separating the preformed early aggregates and creating the appropriate microenvironment for correctly folded proteins to retain their biological activity. It will be of potential importance to the biotechnological industry and other fields of protein biochemistry that routinely face the challenges of protein aggregation.

About c-reactive protein in patients with chronic gastritis

C-c-active protein (CRP) was discovered in 1930 by Tillett and Francis. The main research results highlighting the properties and clinical and diagnostic value of C-reactive protein are summarized in reviews by A.L. Yampolsky (1958), F.L.Bukh (1958), R.V. Petrov and E.N. Kabakov (1959) and others, in which the corresponding extensive bibliography is also given.

GENDER-DEPENDENT HEMOSTASIS PROFILE IN APPARENTLY HEALTHY YOUNG PEOPLE

The objective of the study is to reveal the parameters of red blood cells, rheology, endothelin level, Willebrand factor, tPA-PAI-1 complex, antithrombin III, procoagulantive mechanisms of the hemostatic system and fibrinogen in medical students in the process of learning, taking into account gender differences. Materials and Methods. We examined 100 relatively healthy people (50 men and 50 women who did not have any complaints, were not under regular medical check-up, and did not have any proven diagnoses). The absence of diseases was confirmed by medical documentation, since all study participants underwent an annual preventive examination for admission to study. The volunteers were 18–30 years old. Results. Women had a higher endothelin level if compared with healthy men, which led to less powerful vasoconstriction and increased procoagulant blood activity. At the same time, higher levels of both procoagulant, anticoagulant and fibrinolytic blood activity were recorded. A significantly lower concentration of the tPA-PAI-1 complex and a change in the vWF index were also noted. In addition, relatively healthy women showed greater activity of VIIIa factor, higher resistance of Va factor to active protein C, and significantly higher fibrin content if compared with healthy men. Key words: sex differences, rheology, endothelin level, Willebrand factor, tPA-PAI-1 complex, AT III, procoagulantive mechanisms of the hemostatic system and fibrinogen. Цель. Выявить особенности показателей красной крови, реологии, уровня эндотелина, фактора Виллебранда, комплекса tPA-PAI-1, антитромбина III, прокоагулянтных механизмов системы гемостаза и фибриногена у студентов медицинских вузов с учетом половых различий в процессе обучения. Материалы и методы. Было обследовано 100 относительно здоровых (не предъявлявших жалоб, не состоявших на диспансерном учете и не имевших установленных диагнозов) человек (50 мужчин и 50 женщин). Отсутствие заболеваний подтверждалось медицинской документацией, так как все участники исследования проходили ежегодный профилактический осмотр для допуска к учебе. Возраст добровольцев составлял от 18 лет до 30 лет. Результаты. У женщин отмечался более высокий уровень эндотелина по сравнению со здоровыми мужчинами, что приводило к менее мощной вазоконстрикции и усилению прокоагулянтной активности крови. При этом были зарегистрированы более высокие значения как прокоагулянтной, так и противосвертывающей и фибринолитической активности крови. Одновременно с этим отмечена достоверно более низкая концентрация комплекса tPA-PAI-1, изменение показателя vWF. Кроме того, у относительно здоровых женщин была зафиксирована большая активность фактора VIIIа, более высокая резистентность фактора Vа к активному протеину С, а также достоверно более высокое содержание фибрина по сравнению со здоровыми мужчинами. Ключевые слова: половые различия, реология, уровень эндотелина, фактор Виллебранда, комплекс tPA-PAI-1, АТ III, прокоагулянтные механизмы системы гемостаза и фибриногена.

Biogenesis of Iron–Sulfur Clusters and Their Role in DNA Metabolism

Iron–sulfur (Fe/S) clusters (ISCs) are redox-active protein cofactors that their synthesis, transfer, and insertion into target proteins require many components. Mitochondrial ISC assembly is the foundation of all cellular ISCs in eukaryotic cells. The mitochondrial ISC cooperates with the cytosolic Fe/S protein assembly (CIA) systems to accomplish the cytosolic and nuclear Fe/S clusters maturation. ISCs are needed for diverse cellular functions, including nitrogen fixation, oxidative phosphorylation, mitochondrial respiratory pathways, and ribosome assembly. Recent research advances have confirmed the existence of different ISCs in enzymes that regulate DNA metabolism, including helicases, nucleases, primases, DNA polymerases, and glycosylases. Here we outline the synthesis of mitochondrial, cytosolic and nuclear ISCs and highlight their functions in DNA metabolism.

Further Findings Concerning Endothelial Damage in COVID-19 Patients

Systemic vascular damage with micro/macro-thrombosis is a typical feature of severe COVID-19. However, the pathogenesis of this damage and its predictive biomarkers remain poorly defined. For this reason, in this study, serum monocyte chemotactic protein (MCP)-2 and P- and E-selectin levels were analyzed in 204 patients with COVID-19. Serum MCP-2 and P-selectin were significantly higher in hospitalized patients compared with asymptomatic patients. Furthermore, MCP-2 increased with the WHO stage in hospitalized patients. After 1 week of hospitalization, MCP-2 levels were significantly reduced, while P-selectin increased in patients in WHO stage 3 and decreased in patients in WHO stages 5–7. Serum E-selectin was not significantly different between asymptomatic and hospitalized patients. The lower MCP-2 levels after 1 week suggest that endothelial damage triggered by monocytes occurs early in COVID-19 disease progression. MCP-2 may also predict COVID-19 severity. The increase in P-selectin levels, which further increased in mild patients and reduced in severe patients after 1 week of hospitalization, suggests that the inactive form of the protein produced by the cleavage of the active protein from the platelet membrane is present. This may be used to identify a subset of patients that would benefit from targeted therapies. The unchanged levels of E-selectin in these patients suggest that endothelial damage is less relevant.

Thermostable adenosine 5′-monophosphate phosphorylase from Thermococcus kodakarensis forms catalytically active inclusion bodies

Catalytically active inclusion bodies (CatIBs) produced in Escherichia coli are an interesting but currently underexplored strategy for enzyme immobilization. They can be purified easily and used directly as stable and reusable heterogenous catalysts. However, very few examples of CatIBs that are naturally formed during heterologous expression have been reported so far. Previous studies have revealed that the adenosine 5′-monophosphate phosphorylase of Thermococcus kodakarensis (TkAMPpase) forms large soluble multimers with high thermal stability. Herein, we show that heat treatment of soluble protein from crude extract induces aggregation of active protein which phosphorolyse all natural 5′-mononucleotides. Additionally, inclusion bodies formed during the expression in E. coli were found to be similarly active with 2–6 folds higher specific activity compared to these heat-induced aggregates. Interestingly, differences in the substrate preference were observed. These results show that the recombinant thermostable TkAMPpase is one of rare examples of naturally formed CatIBs.