Improving Homology-Directed Repair in Genome Editing Experiments by Influencing the Cell Cycle

Genome editing is currently widely used in biomedical research; however, the use of this method in the clinic is still limited because of its low efficiency and possible side effects. Moreover, the correction of mutations that cause diseases in humans seems to be extremely important and promising. Numerous attempts to improve the efficiency of homology-directed repair-mediated correction of mutations in mammalian cells have focused on influencing the cell cycle. Homology-directed repair is known to occur only in the late S and G2 phases of the cell cycle, so researchers are looking for safe ways to enrich the cell culture with cells in these phases of the cell cycle. This review surveys the main approaches to influencing the cell cycle in genome editing experiments (predominantly using Cas9), for example, the use of cell cycle synchronizers, mitogens, substances that affect cyclin-dependent kinases, hypothermia, inhibition of p53, etc. Despite the fact that all these approaches have a reversible effect on the cell cycle, it is necessary to use them with caution, since cells during the arrest of the cell cycle can accumulate mutations, which can potentially lead to their malignant transformation.     

DMT1 Protects Macrophages from Salmonella Infection by Controlling Cellular Iron Turnover and Lipocalin 2 Expression

Macrophages are at the center of innate pathogen control and iron recycling. Divalent metal transporter 1 (DMT1) is essential for the uptake of non-transferrin-bound iron (NTBI) into macrophages and for the transfer of transferrin-bound iron from the endosome to the cytoplasm. As the control of cellular iron trafficking is central for the control of infection with siderophilic pathogens such as Salmonella Typhimurium, a Gram-negative bacterium residing within the phagosome of macrophages, we examined the potential role of DMT1 for infection control. Bone marrow derived macrophages lacking DMT1 (DMT1fl/fl^LysMCre(+)) present with reduced NTBI uptake and reduced levels of the iron storage protein ferritin, the iron exporter ferroportin and, surprisingly, of the iron uptake protein transferrin receptor. Further, DMT1-deficient macrophages have an impaired control of Salmonella Typhimurium infection, paralleled by reduced levels of the peptide lipocalin-2 (LCN2). LCN2 exerts anti-bacterial activity upon binding of microbial siderophores but also facilitates systemic and cellular hypoferremia. Remarkably, nifedipine, a pharmacological DMT1 activator, stimulates LCN2 expression in RAW264.7 macrophages, confirming its DMT1-dependent regulation. In addition, the absence of DMT1 increases the availability of iron for Salmonella upon infection and leads to increased bacterial proliferation and persistence within macrophages. Accordingly, mice harboring a macrophage-selective DMT1 disruption demonstrate reduced survival following Salmonella infection. This study highlights the importance of DMT1 in nutritional immunity and the significance of iron delivery for the control of infection with siderophilic bacteria.     

Early Postoperative Immunothrombosis of Bioprosthetic Mitral Valve and Left Atrium: A Case Report

A 72-year-old female patient with mixed rheumatic mitral valve disease and persistent atrial fibrillation underwent mitral valve replacement and suffered from a combined thrombosis of the bioprosthetic valve and the left atrium as soon as 2 days post operation. The patient immediately underwent repeated valve replacement and left atrial thrombectomy. Yet, four days later the patient died due to the recurrent prosthetic valve and left atrial thrombosis which both resulted in an extremely low cardiac output. In this patient’s case, the thrombosis was notable for the resistance to anticoagulant therapy as well as for aggressive neutrophil infiltration and release of neutrophil extracellular traps (NETs) within the clot, as demonstrated by immunostaining. The reasons behind these phenomena remained unclear, as no signs of sepsis or contamination of the BHV were documented, although the patient was diagnosed with inherited thrombophilia that could impede the fibrinolysis. The described case highlights the hazard of immunothrombosis upon valve replacement and elucidates its mechanisms in this surgical setting.     

Nutritional Calcium Supply Dependent Calcium Balance,Bone Calcification and Calcium Isotope Ratios in Rats

Serum calcium isotopes (δ^44/42Ca) have been suggested as a non-invasive and sensitive Ca balance marker. Quantitative δ^44/42Ca changes associated with Ca flux across body compartment barriers relative to the dietary Ca and the correlation of δ^44/42Ca_Serum with bone histology are unknown. We analyzed Ca and δ^44/42Ca by mass-spectrometry in rats after two weeks of standard-Ca-diet (0.5%) and after four subsequent weeks of standard- and of low-Ca-diet (0.25%). In animals on a low-Ca-diet net Ca gain was 61 ± 3% and femur Ca content 68 ± 41% of standard-Ca-diet, bone mineralized area per section area was 68 ± 15% compared to standard-Ca-diet. δ^44/42Ca was similar in the diets, and decreased in feces and urine and increased in serum in animals on low-Ca-diet. δ^44/42Ca_Bone was higher in animals on low-Ca-diet, lower in the diaphysis than the metaphysis and epiphysis, and unaffected by gender. Independent of diet, δ^44/42Ca_Bone was similar in the femora and ribs. At the time of sacrifice, δ^44/42Ca_Serum inversely correlated with intestinal Ca uptake and histological bone mineralization markers, but not with Ca content and bone mineral density by µCT. In conclusion, δ^44/42Ca_Bone was bone site specific, but mechanical stress and gender independent. Low-Ca-diet induced marked changes in feces, serum and urine δ^44/42Ca in growing rats. δ^44/42Ca_Serum inversely correlated with markers of bone mineralization.     

Novel Models of Crohn’s Disease Pathogenesis Associated with the Occurrence of Mitochondrial Dysfunction in Intestinal Cells

Crohn’s disease remains one of the challenging problems of modern medicine, and the development of new and effective and safer treatments against it is a dynamic field of research. To make such developments possible, it is important to understand the pathologic processes underlying the onset and progression of Crohn’s disease at the molecular and cellular levels. During the recent years, the involvement of mitochondrial dysfunction and associated chronic inflammation in these processes became evident. In this review, we discuss the published works on pathogenetic models of Crohn’s disease. These models make studying the role of mitochondrial dysfunction in the disease pathogenesis possible and advances the development of novel therapies.     

Nutritional Management of Thyroiditis of Hashimoto

Since the thyroid gland is one of the organs most affected by autoimmune processes, many patients with thyroiditis of Hashimoto (TH) seek medical advice on lifestyle variance and dietary modifications to improve and maintain their hyroid function. In this review, we aim to present and discuss some challenges associated with the nutritional management of TH, focusing on environmental and dietary deficits, inflammatory and toxic nutrients, cyanotoxins, etc. We discuss the relationships among different diets, chronic inflammation, and microbiota, and their impact on the development and exacerbation of TH in detail. We share some novel insights into the role of vitamin D and melatonin for preserving thyroid function during chronic inflammation in autoimmune predisposed subjects. A comprehensive overview is provided on anti-inflammatory nutrients and ecological diets, including foods for cleansing and detoxification, which represent strategies to prevent relapses and achieve overall improvement of life quality. In conclusion, data from biomedical and clinical studies provide evidence that an appropriate dietary and lighting regimen could significantly improve the function of the thyroid gland and reduce the reactivity of autoantibodies in TH. Compliance with nutritional guidelines may help TH patients to reduce the need for medicines.     

The RAGE/DIAPH1 Signaling Axis & Implications for the Pathogenesis of Diabetic Complications

Increasing evidence links the RAGE (receptor for advanced glycation end products)/DIAPH1 (Diaphanous 1) signaling axis to the pathogenesis of diabetic complications. RAGE is a multi-ligand receptor and through these ligand–receptor interactions, extensive maladaptive effects are exerted on cell types and tissues targeted for dysfunction in hyperglycemia observed in both type 1 and type 2 diabetes. Recent evidence indicates that RAGE ligands, acting as damage-associated molecular patterns molecules, or DAMPs, through RAGE may impact interferon signaling pathways, specifically through upregulation of IRF7 (interferon regulatory factor 7), thereby heralding and evoking pro-inflammatory effects on vulnerable tissues. Although successful targeting of RAGE in the clinical milieu has, to date, not been met with success, recent approaches to target RAGE intracellular signaling may hold promise to fill this critical gap. This review focuses on recent examples of highlights and updates to the pathobiology of RAGE and DIAPH1 in diabetic complications.