A Unified Rapid PCR Method for Detection of Normal and Expanded Trinucleotide Alleles of CAG Repeats in Huntington Chorea and CGG Repeats in Fragile X Syndrome

We report on a unified rapid betaine-based-PCR protocol for amplification of the (CAG)n region in Huntington disease (HD) and the (CGG)n region in Fragile X syndrome (FXS), followed by an electrophoretic separation on automated sequencer for precise determination of the triplet numbers. The high betaine concentration (2.5 M betaine) permits precise amplification of the CAG and CGG repeats. Ten HD affected patients and 10 healthy individuals from HD families were re-evaluated. For FXS the CGG region in normal individuals and premutations of about 100 repeats were precisely amplified by this protocol. Ten unrelated FXS premutation carriers and 24 mentally retarded non-FXS affected boys were re-examined by this method. The results totally coincided with the previous ones. This protocol is a good choice as a fast screening test. Within 24 h we can have preliminary information on the patient’s genetic status. Normal individuals, CGG premutation carriers up to 100 repeats, as well as HD patients carrying an expansion up to 50 CAG repeats can be easily clarified. This accounts for a relatively large proportion (about 90%) of the suspected HD and FXS patients, referred to our laboratory for genetic analysis. The calculation of the repeat’s number is more accurate for the correct interpretation of the results, screening tests and genetic counselling.     

Effects of the timing of applications on the incompatibility of three fungicides and one isolate of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Deuteromycotina)

Abstract: The effect of timing of application on the incompatibility of three fungicides (metalaxyl, mancozeb, copper oxide) and isolate MK2001 of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin was assessed in vitro . Isolate MK 2001 at 1 × 10⁷ conidia/ml concentration was highly pathogenous to adults of Lygus lineolaris (Hemiptera) (84% mortality at day 4 post-treatment). The fungicides at the manufacturer recommended rate (1X) were strongly fungistatic and inhibited the fungal isolate radial growth on Sabouraud dextrose agar (SDA). Mancozeb, metalaxyl and copper oxide exhibited insecticidal activity of 24, 40 and 48% respectively, on L. lineolaris adults. It is clear that application of the fungal isolate MK2001 2–4 days before applying fungicides metalaxyl, mancozeb or copper oxide synergized the insecticidal effect of the isolate. On the contrary, application of metalaxyl, mancozeb, copper oxide 2–4 days before applying isolate MK2001 antagonized the insecticidal effect. The simultaneous use of each fungicide (metalaxyl, mancozeb or copper oxide) and the isolate gave lesser insect mortality. This study revealed that although some fungicides are incompatible for use alongside fungal isolates, the proper evaluation of their time of use could be beneficial in biological control or IPM programmes. Furthermore, the application of B. bassiana isolate MK2001 followed by fungicide application could synergize insecticidal activity.     

Cytogenetic characteristics of a population of Chironomus riparius Meigen 1804 (Diptera, Chironomidae) from a polluted Po river station

A population of Chironomus riparius from a Po river station near Moncalieri (a trace-metal polluted station) was studied. In this population was established a great variability of band structure of polytene chromosomes as well as paracentric heterozygous inversions, deletions, deficiencies, partial breaks, diploid chromosome fragments, and changes in functional activity and appearance of heterochromatin. In arms A through F, some bands had an increased size compared to the standard chromosomic map. Some bands appeared in a heterozygous or normal homozygous state or were amplified. In all arms, many condensed stable bands appeared in the decondensed state when compared to the standard map. Asynaptic zones in arms E and G as well as heterozygous Balbiani rings and NORs were established. Very often the 4th chromosome was almost completely heteropycnotic and looded like a pompon chromosome. For the first time in this species, a high frequency of ectopic pairings of different arms was observed. Telomeric regions involved in ectopic pairings had a granular appearance, as did some centromeres. The hypothesis is advanced that such a high frequency of structural rearrangements could be correlated with genomic distribution of specific mobile elements.     

Co-localization of cytokinins with proteins related to cell proliferation in developing somatic embryos of Dactylis glomerata L.

The present report provides evidence for co-localization ofcytokinins with cell proliferation-associated nuclear proteins.Somatic embryos of Dactylis glomerata in two stages of developmentare used as a model system comprising both proliferating andinitially differentiated cells. Cytokinins are localized usingantibodies with marked specificity against isopentenyladenine/adenosine(2iP/2iPA) or zeatin/ riboside (Z/ZR). The proliferation-associatednuclear antigen, mitotin, is analysed using a specific monoclonalantibody. The nuclear protein BM28, required for the onset ofDNA replication and for cell division, is identified by an affinity-purifiedpolyclonal antibody. Using double immunofiuorescence labellingwith the antibodies against cytokinins and against each of thenuclear proteins, immunoreaction is observed generally in thesame nuclei of almost all cells in globular embryos and in thenuclei of cells in meristematic areas of the more developedembryos. Only small numbers of individual nuclei positive forboth type of antibodies were found in the surrounding vacuolatedparenchymatous cells. The occurrence of plant antigens homologousto BM28 and mitotin is confirmed by immunoblotting assay. InSDS-PAGE blots the anti-BM28 antibody reacts with a proteinof 58 kDa. The anti-mitotin antibody recognizes several (160,140, 125, 93, and 80 kDa) polypeptides. The data showing nuclearco-localization of cytokinins and proteins with a suggestedrole in the onset of DNA synthesis and in cell division providea new base for further study on the mode of action of cytokininsin cell cycle regulation. Key words: Immunolocalization, cytokinins, nuclear proteins, mitotin, BM28, cell proliferation, somatic embryo(s), Dactylis glomerata     

Heterogeneity of twoBeauveria bassiana strains revealed by biochemical tests,protein profiles and bio-assays ofLeptinotarsa decemlineata (Col.: Chrysomelidae) andColeomegilla macultata lengi (Col.: Coccinellidae) larvae

Biochemical profiles on API Rapid CH^* strips and protein profiles on polyacrylamide gels in the presence of sodium dodecyl sulfate were used to distinguish two strains of the entomopathogenic fungusBeauveria bassiana (Balsamo) Vuillemin, ARSEF 2991 and ATCC 44860. Next, the toxicity of these two strains was determined at concentrations of 10², 10⁴, 10⁶ and 10⁸ blastospores/ml on larvae of the Colorado potato beetleLeptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) and of its predator, the spotted ladybird beetle,Coleomegilla maculata lengi Timberlake (Coleoptera: Coccinellidae). Both strains were highly toxic toL. decemlineata larvae. However, the two strains exhibited different levels of toxicity forC. maculata larvae: ARSEF 2991 was toxic, whereas ATCC 44860 caused little coccinellid larval mortality.

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Résumé Les profils biochimiques sur galeries API Rapid CH^* et les profils protéiques sur gels de polyacrylamide ont été utilisés pour distinguer deux souches du champignon entomopathogèneBeauveria bassiana (Balsamo) Vuillemin. La toxicité de ces deux souches a été déterminée à des concentrations de 10², 10⁴, 10⁶ et 10⁸ blastospores/ml sur des larves du doryphore,Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) et de la coccinelle maculéeColeomegilla maculata lengi Timberlake (Coleoptera: Coccinellidae). Les deux souches deB. bassiana se sont avérées actives à l'égard des larves deL. decemlineata. Toutefois la souche ARSEF 2991 s'est avérée pathogène pour les larves deC. maculata, alors que la souche ATCC 44860 a provoqué une faible mortalité des larves.

     

Caffeoylquinic Acids,Cytotoxic, Antioxidant,Acetylcholinesterase and Tyrosinase Enzyme Inhibitory Activities of Six Inula Species from Bulgaria

Chlorogenic (5‐CQA), 1,5‐, 3,5‐, 4,5‐ and 3,4‐dicaffeoylquinic (DCQA) acids were identified and quantified in the methanol extracts of Inula oculus‐christi L., I. bifrons L., I. aschersoniana Janka var. aschersoniana, I. ensifolia L., I. conyza (Griess .) DC. and I. germanica L. by HPLC analysis. The amount of 5‐CQA varied from 5.48 to 28.44 mg/g DE and the highest content was detected in I. ensifolia. 1,5‐DCQA (4.05–55.25 mg/g DE) was the most abundant dicaffeoyl ester of quinic acid followed by 3,5‐DCQA, 4,5‐DCQA and 3,4‐DCQA. The extract of I. ensifolia showed the highest total phenolic content (119.92±0.95 mg GAE/g DE) and exhibited the strongest DPPH radical scavenging activity (69.41±0.55 %). I. bifrons extract was found to be the most active sample against ABTS^.+ (TEAC 0.257±0.012 mg/mL) and the best tyrosinase inhibitor. The studied extracts demonstrated a low inhibitory effect towards acetylcholinesterase and possessed low cytotoxicity in concentration range from 10 to 300 μg/mL toward non‐cancer (MDCK II) and cancer (A 549) cells.     

Absence of MMACHC in peripheral retinal cells does not lead to an ocular phenotype in mice

Combined methylmalonic aciduria with homocystinuria (cblC type) is a rare disease caused by mutations in the MMACHC gene. MMACHC encodes an enzyme crucial for intracellular vitamin B₁₂ metabolism, leading to the accumulation of toxic metabolites e.g. methylmalonic acid (MMA) and homocysteine (Hcy), and secondary disturbances in folate and one-carbon metabolism when not fully functional. Patients with cblC deficiency often present in the neonatal or early childhood period with a severe multisystem pathology, which comprises a broad spectrum of treatment-resistant ophthalmological phenotypes, including retinal degeneration, impaired vision, and vascular changes. To examine the potential function of MMACHC in the retina and how its loss may impact disease, we performed gene expression studies in human and mouse, which showed that local expression of MMACHC in the retina and retinal pigment epithelium is relatively stable over time. To study whether functional MMACHC is required for retinal function and tissue integrity, we generated a transgenic mouse lacking Mmachc expression in cells of the peripheral retina. Characterization of this mouse revealed accumulation of cblC disease related metabolites, including MMA and the folate-dependent purine synthesis intermediates AICA-riboside and SAICA-riboside in the retina. Nevertheless, fundus appearance, morphology, vasculature, and cellular composition of the retina, as well as ocular function, remained normal in mice up to 6 or 12 months of age. Our data indicates that peripheral retinal neurons do not require intrinsic expression of Mmachc for survival and function and questions whether a local MMACHC deficiency is responsible for the retinal phenotypes in patients.     

Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide

Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the “fusion peptide” (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca²⁺ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca²⁺ binding to the SARS-CoV-2-FP, the role of Ca²⁺ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca²⁺-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca²⁺-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca²⁺ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca²⁺ ions. These findings provide novel mechanistic insights regarding the role of Ca²⁺ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.     

Variability in the Essential‐Oil Composition of Sideritis scardica Griseb. from Native Bulgarian Populations

The essential‐oil composition of six native populations of Sideritis scardica from Bulgaria was studied by GC‐FID and GC/MS analyses. Altogether, 37 components, representing 73.1 to 79.2% of the total oil content were identified. Among them, α‐pinene (4.4–25.1%), β‐pinene (2.8–18.0%), oct‐1‐en‐3‐ol (2.3–8.0%), phenylacetaldehyde (0.5–9.5%), β‐bisabolene (1.3–11.0%), benzyl benzoate (1.1–14.3%), and m‐camphorene ( 1 ; 0.3–12.4%) were the main compounds. All samples were characterized by low contents of oxygenated mono‐ and sesquiterpenes (≤1.6 and 2.3%, resp.). Principal component analysis (PCA) and cluster analysis (CA) showed a significant variability in the chemical composition of the studied samples as well as a correlation between the oil profiles and the ecological conditions of the natural habitats of S. scardica.