Troglitazone, but not rosiglitazone, damages mitochondrial DNA and induces mitochondrial dysfunction and cell death in human hepatocytes

Thiazolidinediones (TZDs), such as troglitazone (TRO) and rosiglitazone (ROSI), improve insulin resistance by acting as ligands for the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ). TRO was withdrawn from the market because of reports of serious hepatotoxicity. A growing body of evidence suggests that TRO caused mitochondrial dysfunction and induction of apoptosis in human hepatocytes but its mechanisms of action remain unclear. We hypothesized that damage to mitochondrial DNA (mtDNA) is an initiating event involved in TRO-induced mitochondrial dysfunction and hepatotoxicity. Primary human hepatocytes were exposed to TRO and ROSI. The results obtained revealed that TRO, but not ROSI at equimolar concentrations, caused a substantial increase in mtDNA damage and decreased ATP production and cellular viability. The reactive oxygen species (ROS) scavenger, N-acetyl cystein (NAC), significantly diminished the TRO-induced cytotoxicity, suggesting involvement of ROS in TRO-induced hepatocyte cytotoxicity. The PPARγ antagonist (GW9662) did not block the TRO-induced decrease in cell viability, indicating that the TRO-induced hepatotoxicity is PPARγ-independent. Furthermore, TRO induced hepatocyte apoptosis, caspase-3 cleavage and cytochrome c release. Targeting of a DNA repair protein to mitochondria by protein transduction using a fusion protein containing the DNA repair enzyme Endonuclease III (EndoIII) from Escherichia coli, a mitochondrial translocation sequence (MTS) and the protein transduction domain (PTD) from HIV-1 TAT protein protected hepatocytes against TRO-induced toxicity. Overall, our results indicate that significant mtDNA damage caused by TRO is a prime initiator of the hepatoxicity caused by this drug.     

Cellular Origins of Type IV Collagen Networks in Developing Glomeruli

Laminin and type IV collagen composition of the glomerular basement membrane changes during glomerular development and maturation. Although it is known that both glomerular endothelial cells and podocytes produce different laminin isoforms at the appropriate stages of development, the cellular origins for the different type IV collagen heterotrimers that appear during development are unknown. Here, immunoelectron microscopy demonstrated that endothelial cells, mesangial cells, and podocytes of immature glomeruli synthesize collagen α1α2α1(IV). However, intracellular labeling revealed that podocytes, but not endothelial or mesangial cells, contain collagen α3α4α5(IV). To evaluate the origins of collagen IV further, we transplanted embryonic kidneys from Col4a3-null mutants (Alport mice) into kidneys of newborn, wildtype mice. Hybrid glomeruli within grafts containing numerous host-derived, wildtype endothelial cells never expressed collagen α3α4α5(IV). Finally, confocal microscopy of glomeruli from infant Alport mice that had been dually labeled with anti-collagen α5(IV) and the podocyte marker anti-GLEPP1 showed immunolabeling exclusively within podocytes. Together, these results indicate that collagen α3α4α5(IV) originates solely from podocytes; therefore, glomerular Alport disease is a genetic defect that manifests specifically within this cell type.Basement membranes are thin sheets of extracellular matrix that underlie epithelial cells, including the vascular endothelium, and surround all muscle cells, Schwann cells, and adipocytes. They are composed of polymers of laminin and type IV collagen, and also contain nidogen/entactin, and proteoglycans. During glomerulogenesis, a basement membrane beneath developing endothelial cells fuses with a separate basement membrane layer beneath differentiating podocytes, to produce the glomerular basement membrane (GBM) shared on opposing surfaces by both cell types.¹Unlike most basement membranes in the body, the laminin and collagen IV composition of the GBM changes temporally as the glomerulus develops.² The earliest GBMs of comma- and S-shaped nephrons contain laminin α1β1γ1 (laminin 111), whereas those at later developmental stages and in adulthood contain laminin α5β2γ1 (laminin 521).^2,3 Previously, we showed by postfixation immunoelectron microscopy that both endothelial cells and podocytes synthesize laminin α1 and β1 initially, and both cells then undergo a laminin isoform switch and synthesize laminin α5 and β2 as glomeruli mature.⁴ The mechanism and reason why laminin replacement occurs are unknown, but this may be necessary for achievement and maintenance of the highly differentiated states assumed by glomerular endothelial cells and podocytes. For example, mice with genetic deletions of laminin α5 fail to develop vascularized glomeruli and die before birth with incomplete neural tube closure and placental vascular defects.⁵ By transplanting embryonic day 12 (E12) metanephroi into kidney cortices of newborn wildtype mice, we partially rescued the kidney phenotype and observed well vascularized hybrid glomeruli within grafts containing wildtype endothelial cells and laminin α5 mutant podocytes.⁶ However, the GBMs that form within these glomerular hybrids are stratified and contain laminin α5 on their inner, subendothelial surfaces and laminin α1 on their outer, podocyte halves. Intriguingly, the podocytes fail to form foot processes, suggesting that an absence of the laminin α1-α5 isoform switch stunts podocyte differentiation.⁶ Unlike laminin α5, laminin β2 expression is not required for normal glomerular development. However, mice with genetic delection of the Lamb2 gene become proteinuric by 8 d after birth, display effaced foot processes, and suffer renal failure by 4 wk of age.⁷ Recently, a human mutation mapped to the LAMB2 locus has been linked to Pierson syndrome, a disease of variable phenotype that entails congenital nephrosis, diffuse mesangial sclerosis, perinatal or childhood renal failure, ocular abnormalities, and neuromuscular deficits.^8–10Six genetically distinct collagen type IV α chains form three different triple helical heterotrimers (protomers) in separate compartments of the mature glomerulus.^11,12 A network of collagen α1α2α1(IV) is found in the mesangial matrix, collagen α3α4α5(IV) is present in the GBM, and Bowman''s capsule contains networks of both α1α2α1(IV) and α5α6α5(IV).^3,13 The immature GBMs of comma, S-shaped, and early capillary loop stage glomeruli all contain collagen α1α2α1(IV).² Beginning in capillary loop stages, this network is replaced by α3α4α5(IV),² which is the only collagen IV network normally present in mature GBM. Like laminins, the mechanism for collagen IV isoform substitution in the GBM is unknown. Nevertheless, the mature α3α4α5(IV) isoform is more resistant to proteolytic degradation than α1α2α1(IV),¹⁴ which may be critical for establishment and maintenance of glomerular permselective barrier properties.Importantly, the collagen α3(IV) and α4(IV) chains associate exclusively with α5(IV), to form the α3α4α5(IV) protomer.^15,16 The carboxyl terminal noncollagenous 1 (NC1) domains of the α chain polypeptides interact specifically to select and register appropriate chains for triple helix assembly. Collagen networks then assemble through head to head interactions between NC1 domains of two protomers, to produce a NC1 hexamer structure.^11,12 Similarly, the amino termini of four protomers associate in an anti-parallel fashion, to form a three-dimensional network of polymerized collagen IV.^11,12 Mutations of the human COL4A3, COL4A4, and/or COL4A5 genes prevent the proper assembly of a stable α3α4α5(IV) network, resulting in Alport disease.^11,12,15,16 This disorder is characterized in kidney by an absence of collagen α3α4α5(IV) and persistence of collagen α1α2α1(IV) in the GBM, thickening and multilamination of the GBM, proteinuria, and in most cases eventually, renal failure. Mice homozygous for a Col4a3 mutation possess a strikingly similar phenotype, and therefore, represent an attractive experimental model of human Alport disease.^17–19As indicated earlier, we have previously shown that both glomerular endothelial cells and podocytes synthesize the different laminin isoforms at appropriate stages of glomerular development.⁴ Here, we sought to define unambiguously the cellular origins of GBM collagen α1α2α1(IV) and α3α4α5(IV) during glomerular development. Additionally, we attempted to rescue the murine Alport phenotype by grafting E12 kidneys from Col4a3 knockout mice into renal cortices of wildtype newborn hosts to generate glomerular hybrids containing wildtype endothelial cells and Alport podocytes. Our findings show that collagen α3α4α5(IV) originates only from podocytes, which indicates that Alport disease in the glomerulus is a genetic disorder of this cell type specifically.     

Chromosomal segregation in sperm of Robertsonian translocation carriers

Purpose

To study meiotic segregation patterns of Robertsonian translocations in sperm of male carriers and to assess the frequencies of unbalanced sperm formation.

Methods

FISH with combination of probes to detect all the variants of meiotic segregation was performed on decondensed sperm nuclei of 5 carriers of der(13;14), 3 carriers of der(14;21) and one carrier of a rare der(13;21) translocation.

Results

The frequency of sperm with alternate segregation and normal/balanced chromosomal complement ranged from 68 % to 94.4 % (mean 79.2 ± 8.4). Adjacent segregation was detected in 17.9 ± 7.3 % of sperm (from 5.6 % to 29 %). No significant differences in frequencies of gametes with nullisomies and disomies of chromosomes involved in translocations were observed. The mean frequency of 3:0 segregation products was 2.5 ± 1.4 %.

Conclusions

All analyzed patients showed homogenous segregation pattern with clear predominance of alternate segregation resulting in normal/balanced sperm production. Still, from 5.8–32 % (mean 20.4 ± 8.3 %) of sperm was unbalanced, which is the evidence of the increased risk of unbalanced offspring in carriers of Robertsonian translocations. Our results highlight the importance of genetic counseling of Robertsonian translocation carriers prior to ICSI or IVF.     

Cross-sectional HIV and HCV cascades of care across the regions of Ukraine between 2019 and 2020: findings from the CARE cohort

Introduction

Eastern Europe is facing major HIV and hepatitis C (HCV) epidemics, with many people living with HIV (PLHIV) and HIV/HCV coinfection living in Ukraine. Despite the previous progress towards care quality improvement, the ongoing war in Ukraine is disrupting HIV and HCV care.

Methods

We described an HIV cascade of care (CoC) in PLHIV from two clinical sites and an HCV CoC for anti-HCV-positive PLHIV from six sites in Ukraine, enrolled in the CARE cohort between 1 January 2019 and 1 June 2020. The cross-sectional HIV CoC and HCV CoC are described at study enrolment.

Results

Of 1028 PLHIV, 1014 (98.6%, 95% confidence interval [CI] 97.7–99.3) were on antiretroviral therapy (ART), and 876 (86.4% of those on ART, 95% CI 84.1–88.4) were virologically suppressed. Of 894 participants on ART >6 months, 90.8% (95% CI 88.7–92.6) were virologically suppressed (HIV-RNA <200 copies/ml). Of 2040 anti-HCV-positive PLHIV, 417 (20.4%, 95% CI 18.7–22.3) were ever tested for HCV-RNA prior to enrolment, ranging from 4.9% to 54.4% across sites, and 13.5% were currently HCV-RNA positive. One hundred and eighteen persons (7.3% of ever chronically infected) had received HCV treatment, and 25 persons (1.6% of ever chronically infected) were cured, with variations across sites (0%–7.5%). The site diagnosing 54.4% of people with chronic HCV was the only one providing free RNA testing for all anti-HCV-positive persons, while the intra-country differences in treatment coverage were driven by the number of available direct-acting antiviral (DAA) courses.

Conclusions

Over 98% of PLHIV in care in both CARE sites in Ukraine were receiving ART, and the target of 90% virally suppressed was achieved in persons >6 months on ART. Only one of six HIV/HCV study sites tested over 50% anti-HCV-positive PLHIV for HCV-RNA and treated over 25% of eligible persons. While free HCV-RNA testing and DAA treatment are paramount to achieving HCV elimination targets, they remained a challenge in Ukraine in 2019–2020. The extent of the HIV and HCV care disruption during the war will be further assessed in the CARE cohort and compared with the pre-war findings.     

COVID-Related Functional Difficulties and Concerns Among University Students During COVID-19 Pandemic: A Binational Perspective

Journal of Community Health - The COVID-19 pandemic has created a sense of threat, and stress that has surged globally at an alarming pace. University students were confronted with new challenges....     

Genetic characteristics of eighty-seven patients with the Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immune deficiency disorder characterized by thrombocytopenia, small platelet size, eczema, recurrent infections, and increased risk of autoimmune disorders and malignancies. WAS is caused by mutations in the WASP gene which encodes WASP, a 502-amino acid protein. WASP plays a critical role in actin cytoskeleton organization and signalling, and functions of immune cells. We present here the results of genetic analysis of patients with WAS from eleven Eastern and Central European (ECE) countries and Turkey. Clinical and haematological information of 87 affected males and 48 carrier females from 77 WAS families were collected. The WASP gene was sequenced from genomic DNA of patients with WAS, as well as their family members to identify carriers. In this large cohort, we identified 62 unique mutations including 17 novel sequence variants. The mutations were scattered throughout the WASP gene and included single base pair changes (17 missense and 11 nonsense mutations), 7 small insertions, 18 deletions, and 9 splice site defects. Genetic counselling and prenatal diagnosis were applied in four affected families. This study was part of the J Project aimed at identifying genetic basis of primary immunodeficiency disease in ECE countries. This report provides the first comprehensive overview of the molecular genetic and demographic features of WAS in ECE.     

Characteristics of myeloproliferative neoplasms in patients exposed to ionizing radiation following the Chernobyl nuclear accident

Myeloproliferative neoplasms (MPNs) driver mutations are usually found in JAK2, MPL, and CALR genes; however, 10%-15% of cases are triple negative (TN). A previous study showed lower rate of JAK2 V617F in primary myelofibrosis patients exposed to low doses of ionizing radiation (IR) from Chernobyl accident. To examine distinct driver mutations, we enrolled 281 Ukrainian IR-exposed and unexposed MPN patients. Genomic DNA was obtained from peripheral blood leukocytes. JAK2 V617F, MPL W515, types 1- and 2-like CALR mutations were identified by Sanger Sequencing and real time polymerase chain reaction. Chromosomal alterations were assessed by oligo-SNP microarray platform. Additional genetic variants were identified by whole exome and targeted sequencing. Statistical significance was evaluated by Fisher's exact test and Wilcoxon's rank sum test (R, version 3.4.2). IR-exposed MPN patients exhibited a different genetic profile vs unexposed: lower rate of JAK2 V617F (58.4% vs 75.4%, P = .0077), higher rate of type 1-like CALR mutation (12.2% vs 3.1%, P = .0056), higher rate of TN cases (27.8% vs 16.2%, P = .0366), higher rate of potentially pathogenic sequence variants (mean numbers: 4.8 vs 3.1, P = .0242). Furthermore, we identified several potential drivers specific to IR-exposed TN MPN patients: ATM p.S1691R with copy-neutral loss of heterozygosity at 11q; EZH2 p.D659G at 7q and SUZ12 p.V71 M at 17q with copy number loss. Thus, IR-exposed MPN patients represent a group with distinct genomic characteristics worthy of further study.