Hemorheological parameters in correlation with the risk factors for carotid atherosclerosis

The study aimed to follow the relationship between some hemorheological variables and the main risk factors (RF) for carotid atherosclerosis (CA). Carotid atherosclerosis was evaluated by color duplex sonography of the carotid arteries in 18 patients with RF for CA, 31 patients with transient ischemic attacks (TIAs), 21 patients with chronic unilateral cerebral infarctions (UCI) and 11 healthy subjects without RF for CA. The examined hemorheological variables were whole blood and plasma viscosity, hematocrit and fibrinogen. They were correlated with intima-media thickness (IMT) of the common carotid and the internal carotid arteries and with other main RF for CA: hypertension, diabetes mellitus, coronary heart disease, and hyperlipidemia. The hemorheological investigation showed an increase in blood and plasma viscosity at different shear rates and it was more expressed in the group with UCI. The neurosonographic investigation revealed an increase in the IMT and carotid artery stenoses in the patients' groups with CVD. These were also more frequent in the patients with UCI. Different correlations were established between the hemorheological parameters, the IMT of the carotid arteries and other RF for CA. In the group with UCI, the hematocrit and the whole blood viscosity correlated significantly with the IMT, arterial blood pressure and cholesterol values. These data confirm the influence of the hemorheological parameters on carotid blood vessel walls and on blood flow in patients with CVD.     

Ephrin-B2 ligand is a functional receptor for Hendra virus and Nipah virus

Hendra virus (HeV) and Nipah virus (NiV) belong to the genus Henipavirus of the family Paramyxoviridae and are unique in that they exhibit a broad species tropism and cause fatal disease in both animals and humans. They infect cells through a pH-independent membrane fusion process mediated by their fusion and attachment glycoproteins. Previously, we demonstrated identical cell fusion tropisms for HeV and NiV and the protease-sensitive nature of their unknown cell receptor and identified a human cell line (HeLa-USU) that was nonpermissive for fusion and virus infection. Here, a microarray analysis was performed on the HeLa-USU cells, permissive HeLa-CCL2 cells, and two other permissive human cell lines. From this analysis, we identified a list of genes encoding known and predicted plasma membrane surface-expressed proteins that were highly expressed in all permissive cells and absent from the HeLa-USU cells and rank-ordered them based on their relative levels. Available expression vectors containing the first 10 genes were obtained and individually transfected into HeLa-USU cells. One clone, encoding human ephrin-B2 (EFNB2), was found capable of rendering HeLa-USU cells permissive for HeV- and NiV-mediated cell fusion as well as infection by live virus. A soluble recombinant EFNB2 could potently block fusion and infection and bind soluble recombinant HeV and NiV attachment glycoproteins with high affinity. Together, these data indicate that EFNB2 serves as a functional receptor for both HeV and NiV. The highly conserved nature of EFNB2 in humans and animals is consistent with the broad tropism exhibited by these emerging zoonotic viruses.     

Single-base resolution mapping of H1–nucleosome interactions and 3D organization of the nucleosome

Despite the key role of the linker histone H1 in chromatin structure and dynamics, its location and interactions with nucleosomal DNA have not been elucidated. In this work we have used a combination of electron cryomicroscopy, hydroxyl radical footprinting, and nanoscale modeling to analyze the structure of precisely positioned mono-, di-, and trinucleosomes containing physiologically assembled full-length histone H1 or truncated mutants of this protein. Single-base resolution •OH footprinting shows that the globular domain of histone H1 (GH1) interacts with the DNA minor groove located at the center of the nucleosome and contacts a 10-bp region of DNA localized symmetrically with respect to the nucleosomal dyad. In addition, GH1 interacts with and organizes about one helical turn of DNA in each linker region of the nucleosome. We also find that a seven amino acid residue region (121–127) in the COOH terminus of histone H1 was required for the formation of the stem structure of the linker DNA. A molecular model on the basis of these data and coarse-grain DNA mechanics provides novel insights on how the different domains of H1 interact with the nucleosome and predicts a specific H1-mediated stem structure within linker DNA.     

Clinical difficulties and errors in making a diagnosis of chronic papillo-odditis

138 patients with chronic papillooditis were investigated, 15 of them in a stage of decompensation. In 83.3% of the cases, the diagnosis was not clinically determined (including 68 of 92 endoscoped before their hospitalization--82.9%). 34 patients were diagnosed as having chronic gastroduodenitis, 15--ulcer, 42--chronic cholecystitis, 11--chronic pancreatitis, 4--cholangitis, 9--postcholecystectomic status. All these diseases developed simultaneously with the papillitis. In a second endoscopic check-up with an examination of papilla Vateri, the patients were in all the cases diagnosed without difficulties and the diagnose was confirmed by biopsy. In 21 patients there was confirmed primary papillooditis and in 127--accompanying disorders: chr. gastroduodenitis--29, chr. atrophic gastritis--18, ulcer--15, chr. cholecystitis--42, postcholecystectomic status--9, choledocholithiasis--14, chr. pancreatitis--11. Most often misdiagnosis occurs if: 1) during the routine endoscopic investigation the endoscopist does not examine papilla of Vater; 2) chr. papillitis exists simultaneously with one of the already mentioned diseases that are easier of approach for diagnostics and explanation of the disorders; 3) the clinical picture of papillitis cannot be differentiated from the one of the basic or accompanying disease; 4) the bile drainage is not prevented; 5) the result of the venous biligraphy does not lead to the diagnosis and ERCP is carried out only in a case of a clinical suspicion.     

Liver dysfunction as a cytokine storm manifestation and prognostic factor for severe COVID-19

Liver damage in severe acute respiratory coronavirus 2 infection occurs in patients with or without preexisting liver disorders, posing a significant complication and mortality risk. During coronavirus disease 2019 (COVID-19), abnormal liver function is typically observed. However, liver injury may occur because of the treatment as well. Ischemia, cytokine storm, and hypoxia were identified as the three major factors contributing to liver damage during COVID-19. Indeed, raised liver enzymes during hospitalizations may be attributed to medications used, as well as sepsis and shock. As a result, the proportion of hospitalized patients afflicted with COVID-19 and pathological liver biomarkers varies from 14% to 53%. Aminotransferases and bilirubin are found most often elevated. Usually, increased gamma-glutamyltransferase, alkaline phosphatase, and decreased serum albumin levels are demonstrated. Additionally, although there is no specific treatment for COVID-19, many of the drugs used to treat the infection are hepatotoxic. In this mini-review, we focus on how liver dysfunction can be one of the features associated with the COVID-19 cytokine storm. Furthermore, data show that liver injury can be an independent predictor of severe COVID-19, the need for hospitalization, and death.     

Development of inhibitory antibodies to therapeutic factor VIII in severe hemophilia A is associated with microsatellite polymorphisms in the HMOX1 promoter

Induction of heme oxygenase-1, a stress-inducible enzyme with anti-inflammatory activity, reduces the immunogenicity of therapeutic factor VIII in experimental hemophilia A. In humans, heme oxygenase-1 expression is modulated by polymorphisms in the promoter of the heme oxygenase-1-encoding gene (HMOX1). We investigated the relationship between polymorphisms in the HMOX1 promoter and factor VIII inhibitor development in severe hemophilia A. We performed a case-control study on 99 inhibitor-positive patients and 263 patients who did not develop inhibitors within the first 150 cumulative days of exposure to therapeutic factor VIII. Direct sequencing and DNA fragment analysis were used to study (GT)_n polymorphism and single nucleotide polymorphisms located at −1135 and −413 in the promoter of HMOX1. We assessed associations between the individual allele frequencies or genotypes, and inhibitor development. Our results demonstrate that inhibitor-positive patients had a higher frequency of alleles with large (GT)_n repeats (L: n≥30), which are associated with lesser heme oxygenase-1 expression (odds ratio 2.31; 95% confidence interval 1.46–3.66; P<0.001]. Six genotypes (L/L, L/M, L/S, M/M, M/S and S/S) of (GT)_n repeats were identified (S: n<21; M: 21≤n<30). The genotype group including L alleles (L/L, L/M and L/S) was statistically more frequent among inhibitor-positive than inhibitor-negative patients, as compared to the other genotypes (33.3% versus 17.1%) (odds ratio 2.21, 95% confidence interval 1.30–3.76; P<0.01). To our knowledge, this is the first association identified between HMOX1 promoter polymorphism and development of anti-drug antibodies. Our study paves the way towards modulation of the endogenous anti-inflammatory machinery of hemophilia patients to reduce the risk of inhibitor development     

Phosphorylation of the CENP-A amino-terminus in mitotic centromeric chromatin is required for kinetochore function

The role of the mitotic phosphorylation of the amino (NH₂) terminus of Centromere Protein A (CENP-A), the histone variant epigenetic centromeric marker, remains elusive. Here, we show that the NH₂ terminus of human CENP-A is essential for mitotic progression and that localization of CENP-C, another key centromeric protein, requires only phosphorylation of the CENP-A NH₂ terminus, and is independent of the CENP-A NH₂ terminus length and amino acid sequence. Mitotic CENP-A nucleosomal complexes contain CENP-C and phosphobinding 14-3-3 proteins. In contrast, mitotic nucleosomal complexes carrying nonphosphorylatable CENP-A–S7A contained only low levels of CENP-C and no detectable 14-3-3 proteins. Direct interactions between the phosphorylated form of CENP-A and 14-3-3 proteins as well as between 14-3-3 proteins and CENP-C were demonstrated. Taken together, our results reveal that 14-3-3 proteins could act as specific mitotic “bridges,” linking phosphorylated CENP-A and CENP-C, which are necessary for the platform function of CENP-A centromeric chromatin in the assembly and maintenance of active kinetochores.Histone variants are nonallelic isoforms of conventional histones. It is widely accepted that the incorporation of histone variants generally confers novel structural and functional properties to the nucleosome (1). Centromere Protein A (CENP-A) is a histone variant, which replaces the canonical histone H3 at the centromere (2) and marks epigenetically the centromeres and the kinetochores (for reviews see refs. 3 and 4). The presence of CENP-A is required for the assembly of active kinetochores and its depletion results in numerous mitotic problems, such as chromosome misalignments and segregation defects, generation of chromosome bridges, aneuploidy, etc. (5). The resulting mitotic defects, following CENP-A depletion, were associated also with notable alterations in the composition and organization of the kinetochore, including the delocalization of the inner kinetochore proteins CENP-C, CENP-I, and CENP-H as well as the outer kinetochore components Highly Expressed in Cancer protein 1 (HEC1), Mitotic Arrest Deficient 2-like protein (Mad2), and CENP-E (5).During recent years, the studies of CENP-A were focused mainly on its histone-fold domain. The NH₂ terminus of CENP-A, which is not required for centromeric targeting (6, 7) appeared, however, to play an important role in both mitosis and meiosis. In yeast, the NH₂ tail of Chromosome Segregation Protein 4 (Cse4p) (the homolog of mammalian CENP-A) has an essential function distinct from that of the histone-fold domain in chromosome assembly and segregation (8). The reported data in Arabidopsis thaliana suggested the existence of a meiosis-specific loading pathway for CENP-A, requiring its NH₂ terminus (9). In addition, human CENP-A is phosphorylated in its NH₂ terminus at serine 7 in mitosis but the role of this phorphorylation is far from being clear (10, 11).Sequence alignments of the NH₂ termini of CENP-A from different species show very low sequence conservation in terms of amino acid composition, sequence, and length (Fig. 1A). However, expression of CENP-A with its NH₂ terminus deleted is lethal in yeast (12) and plants (13). These data create a paradox because on one hand, the NH₂ terminus of CENP-A, in certain organisms, is required for their survival and on the other hand, it appears to be nonessential because there is no evolutionary pressure to conserve at least some specific sequence elements.Open in a separate windowFig. 1.The NH₂ tail of CENP-A is required for mitosis and the H3 swapped NH₂ tail CENP-A chimera rescues the CENP-A null cell phenotype. (A) Sequence alignment of CENP-A NH₂ termini from different species. Names of species are indicated at Left. Serine residues are indicated in blue. Conservation between different species is shown (Lower). (B) Cell cycle visualization, after CENP-A suppression by siRNA treatment of naïve HeLa cells (second row) or HeLa cells stably expressing siRNA-resistant full-length GFP–CENP-A (third row) or tailless GFP–ΔN–CENP-A (fourth row) or GFP–H3–CENP-A swapped tail mutant (fifth row). First row shows naïve cells not treated with siRNA. A CREST antibody was used to visualize the centromeres in naïve cells; GFP fluorescence was used to visualize CENP-A in GFP fusion-expressing cells. An antibody against inner centromere protein (INCENP) and antilamina antibody were used to detect the midbody during cytokinesis and the nuclear envelope in interphase cells (shown in red). Blue, DNA; white arrowheads point to misaligned or lagging chromosomes and to chromatin bridges. (Scale bar, 5 µm.) (C) Detection of the GFP–CENP-A fusions and endogenous CENP-A in control (−) and siRNA treated (+) cells at 72 h posttransfection by Western blot. Cells used are indicated (Upper). Arrows indicate the positions of the GFP–CENP-A fusions or endogenous CENP-A. (D) Histograms showing the percentage of mitotic defects at 72 h posttransfection with siRNA against CENP-A in the indicated cell lines. HeLa, naïve cell line. (E) Same as D, but showing the percentage of multinucleate cells. For each experiment, at least 400 cells were counted. Data are means and SEM of five independent experiments.Here, we have analyzed the mitotic function of the NH₂ terminus of human CENP-A and its phosphorylation. We show the mere phosphorylation of CENP-A, but not its length and amino acid sequence, is required for the localization of CENP-C, a key mediator between centromeric chromatin and the outer kinetochore components. Our data reveal that in mitosis, the phosphorylated CENP-A nucleosomes are “bridged” to CENP-C via the phosphobinding 14-3-3 proteins. These 14-3-3 mitotic bridges are essential for the assembly of active kinetochores.     

Patterns of Insulin Dependence in an African Diabetic Clinic

SUMMARY Analysis of the age of onset of diabetes amongst insulin-treatedpatients in a large African diabetic clinic revealed a bimodaltype of distribution, 23 per cent having an age of onset before30 years and 77 per cent with onset at 30 years of age. All66 of the young insulin-treated group (21.7±4.8 years(mean±1 SD)), and a random selection of 50 older insulin-treatedpatients (49.7±10 years), were studied. The older groupwere better controlled (HbA₁ 8.4±1.7 per cent vs. 10.8±2.6per cent, p<0.001), on lower doses of insulin (49±23vs. 71±23 u/day, p<0.001) and had higher body massindex (26.0±5.6 vs. 21.8±3.5, p<0.001). SerumC-peptide (0.24±0.15 vs. 0.07±0.10 nmol/l, p<0.0001),and C-peptide/glucose ratio (2.57±2.65 vs. 0.56+0.98nmol/mmolx 10², p<0.001) were very significantly higher inolder patients. Patients with later onset disease thus had betterpreservation of pancreatic function, higher body mass indexand better glycaemic control on lower doses of insulin. Thesefeatures suggest that older insulin-treated patients could infact be ‘Type 2’ or non-insulin dependent patients,and the condition may be controllable with diet and/or oralhypoglycaemic agents, at least in some.