Temperature-mediated heteroduplex analysis for detection of pncA mutations associated with pyrazinamide resistance and differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by denaturing high- performance liquid chromatography

The goal of this study was to apply temperature-mediated heteroduplex analysis using denaturing high-performance liquid chromatography to identify pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates and simultaneously differentiate between M. tuberculosis and Mycobacterium bovis. Features that contributed to an optimal assay included the use of two different reference probes for the pncA gene targets from wild-type M. tuberculosis and wild-type M. bovis, optimization of the column temperature, increasing the starting concentration of the elution buffer, and reducing the rate of elution buffer increase (slope). A total of 69 strains were studied, including 48 wild-type M. tuberculosis strains (13 were PZA-resistant strains) and 21 M. bovis strains (8 were BCG strains). In all isolates tested, wild-type M. tuberculosis generated a single-peak pattern when mixed with the M. tuberculosis probe and a double-peak pattern with the M. bovis probe. In contrast, all M. bovis isolates generated a double-peak pattern when mixed with the M. tuberculosis probe and a single-peak pattern with the M. bovis probe. PZA-resistant mutant M. tuberculosis isolates generated characteristic patterns that were easily distinguishable from both wild-type M. tuberculosis and M. bovis isolates. Chromatographic patterns generated by the two reference probes allowed the rapid detection of PZA resistance with the simultaneous ability to distinguish between M. tuberculosis and M. bovis. This approach may allow the detection of drug resistance-associated mutations, with potential application to clinical and epidemiological aspects of tuberculosis control.     

Synthetic organic hard capsule colouring agents: in vitro effect on human true and pseudo-cholinesterases

Hard capsules are made of pure gelatin and small quantities of additives, including colouring agents permitted for use in food. In this study, the effects of three colouring agents (sunset yellow, quinoline yellow and erythrosine) on true and pseudo-cholinesterases (ChE) are assessed in erythrocytes and plasma, respectively. Results indicated that the synthetic compounds affected both true and pseudo ChE activity. The concentration of sunset yellow which caused 50% inhibition (IC50) of true ChE was about 64% that of pseudo-ChE; for erythrosine, IC50 was approximately the same for both true and pseudo-ChE; and for quinoline yellow, IC50 for true ChE was 25% of pseudo-ChE, although its effect on both true and pseudo-ChE was greater than seen with the other two dyes. Inhibitions of both true and pseudo-ChE were of mixed type (competitive and non-competitive). The enzyme-inhibitor dissociation constant (Ki) indicated that quinoline yellow was most potent and erythrosine was least potent out of the three compounds. Inhibition of both true and pseudo-ChE by each of the three dyes was abolished by dialysis, indicating that the effects were reversible.     

Intestinal antilectin immunoglobulin A antibody response and immunity to Entamoeba dispar infection following cure of amebic liver abscess

We followed 93 subjects with amebic liver abscess (ALA) and 963 close associate controls at 3-month intervals for 36 months to characterize intestinal and humoral antibody responses to the amebic galactose-inhibitable lectin and to determine whether immunity developed to Entamoeba histolytica or Entamoeba dispar infection following cure of ALA. We found that ALA subjects had a higher prevalence and level of intestinal antilectin immunoglobulin A (IgA) and serum anti-LC3 (cysteine-rich recombinant lectin protein) IgA and IgG antibodies, P < 0.01 and P < 0.05, respectively, compared to controls. The intestinal antilectin IgA antibody response was sustained over a longer time period in ALA subjects (71.8% remained positive at 18 months and 52.6% at 36 months, P < 0.001 compared to 17.6% and 10.3% of controls, respectively). ALA subjects were highly immune to E. dispar infection throughout the study (0% infected at 6 and 36 months, compared to 6.5% and 4.9% of control subjects, respectively, P < 0.05). Upon entry into the study, 6.3% of ALA subjects were infected with E. histolytica; the incidence of new E. histolytica infections in controls (as determined by culture) was too low (1.4%) to determine whether ALA subjects exhibited immunity to new infections. We found that stool cultures every 3 months markedly underestimated the occurrence of new E. histolytica infections, as 15.3% of controls seroconverted after 12 months of follow-up. Unfortunately, under the field conditions present in Durban, South Africa, enzyme-linked immunosorbent assay for detection of lectin antigen in stool yielded unreliable results. In summary, subjects cured of ALA exhibited sustained mucosal IgA antibody responses to the amebic galactose-inhibitable lectin and a high level of immunity to E. dispar infection. Determination of immunity to E. histolytica following cure of ALA will require the use of more sensitive and reliable diagnostic methods.     

Median frequency of the myoelectric signal

Summary A study was performed to investigate the changes that occur in the median frequency of the myoelectric signal during local ischemia or reduction of intramuscular temperature produced by surface cooling. Data was obtained from experiments which involved the first dorsal interosseous muscle of 10 female and 16 male subjects. These subjects were asked to perform isometric constant-force abduction contractions of the index finger at 20% and 80% of maximal voluntary contraction level. The initial median frequency (IMF) of the myoelectric signal during the first 0.5 s of contraction was calculated. Results showed a significant reduction of the IMF in contractions performed under ischemic conditions; upon release, the IMF recovered quickly. At 80% maximal voluntary level of contraction, a greater decrease of the IMF was recorded. Similar results were demonstrated during reduction of intramuscular temperature with gradual recovery of the IMF after cooling. These results demonstrate that the median frequency of the myoelectric signal displays behavior similar to that reported for conduction velocity and this is consistent with the notion that accumulation of metabolic byproducts in muscle tissue causes a decrease in the conduction velocity of the muscle fibers.Dr. R. Merletti was on a leave of absence from the Institute of Electrical Engineering, Politecnico di Torino, Italy     

Thermal transition and frequency response of the dielectric behaviour of some cellulosic substances

The real part of the relative permittivity ?′_r of some cellulosic materials contaminated with commercial impurities and NaCl (determined as weight percent ash contents), and that of the purified fibres, as well as their hydrolysed residues, free of such contaminations, was measured in the frequency range 10² Hz ≤ f ≤ 10⁵ Hz and in the temperature range 283,15 K ≤ T ≤ 333,15 K. Additional measurements of the imaginary part of relative permittivity ?″_r have been carried out in the same frequency and temperature ranges for hydrocellulose samples free of inorganic impurities and contaminated with NaCl. The results obtained show that the variation with temperature of the relative permittivity of the samples examined exhibits a change in slope in the vicinity of 303,15 K except for hydrocellulose samples free of impurities. They further show that the presence of inorganic contaminations has a significant effect on the magnitude of the relative permittivity and its variation with temperature, particularly at low frequency. In the case of samples contaminated with inorganic impurities, interfacial polarization is responsible for the anomalous dielectric behaviour observed with the examined samples.     

Identification of a novel frameshift mutation in the DFNB31/WHRN gene in a Tunisian consanguineous family with hereditary non-syndromic recessive hearing loss

Approximately 80% of hereditary hearing loss is non-syndromic. Non-syndromic deafness is the most genetically heterogeneous trait. The most common and severe form of hereditary hearing impairment is autosomal recessive non-syndromic hearing loss (ARNSHL), accounting for approximately 80% of cases of genetic deafness. To date, 22 genes implicated in ARNSHL have been identified. Recently a gene, DFNB31/WHRN, which encodes a putative PDZ scaffold protein called whirlin, was found to be responsible for the ARNSHL DFNB31. We found evidence for linkage to the DFNB31locus in a consanguineous Tunisian family segregating congenital profound ARNSHL. Mutation screening of DFNB31/WHRNrevealed four nonpathogenic sequence variants and a novel frameshift mutation [c.2423delG] + [c.2423delG] that changed the reading frame and induced a novel stop codon at amino acid 818 ([p.Gly808AspfsX11] + [p.Gly808AspfsX11]). To determine the contribution of the DFNB31locus in the childhood deafness, we performed linkage analysis in 62 unrelated informative families affected with ARNSHL. No linkage was found to this locus. From this study, we concluded that DFNB31/WHRN is most likely to be a rare cause of ARNSHL in the Tunisian population.     

Presenting characteristics,comorbidities, and outcomes of patients coinfected with COVID-19 and Mycoplasma pneumoniae in the USA

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is spreading at a rapid pace, and the World Health Organization declared it as pandemic on 11 March 2020. Mycoplasma pneumoniae is an "atypical" bacterial pathogen commonly known to cause respiratory illness in humans. The coinfection from SARS-CoV-2 and mycoplasma pneumonia is rarely reported in the literature to the best of our knowledge. We present a study in which 6 of 350 patients confirmed with COVID-19 were also diagnosed with M. pneumoniae infection. In this study, we described the clinical characteristics of patients with coinfection. Common symptoms at the onset of illness included fever (six [100%] patients); five (83.3%) patients had a cough, shortness of breath, and fatigue. The other symptoms were myalgia (66.6%), gastrointestinal symptoms (33.3%-50%), and altered mental status (16.7%). The laboratory parameters include lymphopenia, elevated erythrocyte sedimentation rate, C-reactive protein, lactate dehydrogenase, interleukin-6, serum ferritin, and D-dimer in all six (100%) patients. The chest X-ray at presentation showed bilateral infiltrates in all the patients (100%). We also described electrocardiogram findings, complications, and treatment during hospitalization in detail. One patient died during the hospital course.     

Protein composition of tomato spotted wilt virus

Analysis of the protein composition of tomato spotted wilt virus (TSWV), purified by an improved procedure, by polacrylamide gel electrophoresis, revealed three major structural proteins (of MW 84,000, 50,000, and 29,000d) and a minor one of MW 220,000d. The three major proteins constitute about 98% of the total viral protein and all three were shown to be glycoproteins. One of the major proteins (MW 29,000d) and the minor protein were shown to be associated with subviral particles isolated by treatment of virus with the nonionic detergent Nonidet P-40. Only traces of the other two proteins were detected in the subviral particles.Synthesis of virus-induced proteins in TSWV-infected tobacco leaves was studied by labeling infected and healthy tissue with [³H]and [¹⁴C]valine, respectively. The labeled tissues were then fractionated into crude subcellular fractions and protein patterns of healthy and infected tissues were compared by coelectrophoresis on polyacrylamide gels. Only one virus-specific protein (of MW 49,000d) was detected in the virus-enriched fractions; this corresponded with the viral structural protein of MW 50,000d.