Kinetic properties of Na(+) -H(+) antiport in Escherichia coli membrane vesicles: Effects of imposed electrical potential, proton gradient, and internal pH

Modifications of the kinetic properties of the Escherichia coli (RA11) Na(+) - H(+) antiport system by imposed pH gradients (deltapH, interior alkaline) and membrane potential(delta(psi), interior negative) were studied by looking at the accelerating effects of deltapH and delta on downhill Na(+) efflux from membrane vesicles incubated at different external pHs. First,variations of the Na(+) efflux rate ( VNa) as a function of imposed delta pH appear to be strongly dependent on the external pH value.The individual VN, vs. deltapH relationships observed between pH 5.5 and pH 6.6 are all nonlinear and indicate the existence of a threshold deltapH above which V(Na) increases steeply as the deltapH magnitude increases; threshold deltapH values progressively decrease as the pH is raised from 5.5 to 6.6. In contrast, at or above neutrality, V(Na) acceleration is linearly related to deltapH amplitude. Strikingly, it is shown that the deltapH-dependent variations in the Na(+) efflux rate measured in vesicles incubated at different external pHs can be accounted for by variations of internal pH; the observed relationship suggests that a high internal H(+) concentration inhibits the Na(+) -H(+) antiport activity.This inhibition results from a drastic increase in the apparent K(m), of the Na(+) efflux reaction as the internal H(+) concentration increases. On the other hand, imposed Δ increases the Na(+) efflux rate linearly by a selective modification of the V(max) value of the Na(+) efflux. Together, these data indicate that the internal H(+) concentration controls the Na(+)-H(+) antiport activity and that the chemical and electrical proton gradients affect two different kinetic steps of the Na(+)-H(+) exchange reaction.     

X-linked Alport syndrome: an SSCP-based mutation survey over all 51 exons of the COL4A5 gene.

The COL4A5 gene encodes the alpha5 (type IV) collagen chain and is defective in X-linked Alport syndrome (AS). Here, we report the first systematic analysis of all 51 exons of COL4A5 gene in a series of 201 Italian AS patients. We have previously reported nine major rearrangements, as well as 18 small mutations identified in the same patient series by SSCP analysis of several exons. After systematic analysis of all 51 exons of COL4A5, we have now identified 30 different mutations: 10 glycine substitutions in the triple helical domain of the protein, 9 frameshift mutations, 4 in-frame deletions, 1 start codon, 1 nonsense, and 5 splice-site mutations. These mutations were either unique or found in two unrelated families, thus excluding the presence of a common mutation in the coding part of the gene. Overall, mutations were detected in only 45% of individuals with a certain or likely diagnosis of X-linked AS. This finding suggests that mutations in noncoding segments of COL4A5 account for a high number of X-linked AS cases. An alternative hypothesis is the presence of locus heterogeneity, even within the X-linked form of the disease. A genotype/phenotype comparison enabled us to better substantiate a significant correlation between the degree of predicted disruption of the alpha5 chain and the severity of phenotype in affected male individuals. Our study has significant implications in the diagnosis and follow-up of AS patients.     

Chromosome constitution of in vitro segregated haploid and diploid cells of the mouse

The composition in segregated haploid sets of paternal and maternal chromosomes has been studied in order to verify whether their composition is uniparental of mixed, fixed or variable. Primary cultures where prepared using kidneys from hybrids of strains of Mus musculus in which the parental chromosomes are distinguishable; the maternal set consists of 20 teleocentric chromosomes, the paternal set of 9 metacentric chromosomes, derived by Robertsonian fusion and 2 telocentrics. Applying Seabright's banding technique, an analysis of segregated haploid and diploid cells, which have originated spontaneously through polyploidisation-segregation processes was carried out. It was concluded that the haploid sets have a variable composition of paternal and maternal chromosomes.     

Hashimoto's Thyroiditis: Similar and Dissimilar Characteristics in Neighboring Areas. Possible Implications for the Epidemiology of Thyroid Cancer

Context

Medical centers worldwide report an increased frequency of Hashimoto''s thyroiditis (HT) and thyroid cancer (TC), two environmentally influenced diseases. In Sicily, data on HT are available for the province of Messina (1975–2005); data on TC are available for the whole island (2002–2004), with the volcanic province of Catania having the highest incidence.

Objective

To replicate in Catania, on comparable years, the HT data of Messina.

Design, Methods, Setting

Review of the clinical records of patients in years 1995–2005 to compare presentation and yearly changes of HT. During 1995–2005, records were computer stored in the Endocrine Divisions of the University Hospitals of Catania and Messina, two tertiary referral centers.

Results

Catania is outnumbered by Messina (742 vs. 3,409 HT patients). Similar were the linear increase in the yearly number of HT patients, rates of thyroid dysfunctions though with different proportions of subclinical and overt hypothyroidism, and rates of positiveness for TgAb or TPOAb. Different were age and its yearly trend; gender distribution and rates of the sonography variants, though yearly trends were similar.

Conclusion

The HT epidemics is smaller in Catania, with changes in presentation overlapping partially those in Messina. Whatever environmental factors might be involved, they (and/or their intensity) were not necessarily the same in these provinces. Intriguingly, the expected number of TC in HT patients with thyroid nodules in Catania is congruent with that of the general population of this province, but it is far less than in the Messina province. Thus, TC and HT incidences could be influenced by distinct environmental factors.     

Prognostic factors and predictors of outcome in patients with COVID-19 and related pneumonia: a retrospective cohort study

The aim of the present study was to simultaneously assess several potential predictors of outcome (co-morbidity, previous and in-hospital treatment, radiologic Brixia score) in patients with COVID-19.This retrospective cohort study included 258 consecutive patients with confirmed COVID-19 admitted to a medical ward at Montichiari Hospital, Brescia, Italy from February 28th to April 30rd, 2020. Patients had SARS-CoV-2 related pneumonia with respiratory failure, and were treated with hydroxychloroquine and lopinavir plus ritonavir. In some patients, additional treatment with tocilizumab, dexamethasone and enoxaparin was adopted. Outcomes (death or recovery) were assessed at the end of the discharge period or at the end of the follow-up (August 2020).During hospitalization, 59 patients died, while 6 died after discharge. The following variables were demonstrated to be associated with a worse prognosis: Radiologic Brixia score higher than 8, presence at baseline of hypertension, diabetes, chronic obstructive pulmonary disease, heart disease, cancer, previous treatment with ACE-inhibitors or anti-platelet drugs. Anticoagulant treatment during hospital admission with enoxaparin at a dose higher than 4000 U once daily was associated with a better prognosis.In conclusion, our study demonstrates that some co-morbidities and cardiovascular risk factors may affect prognosis. The radiologic Brixia score may be a useful tool to stratify the risk of death at baseline. Anticoagulant treatment with enoxaparin might be associated to a clinical benefit in terms of survival in patients with COVID-19.     

Downscaling screening cultures in a multifunctional bioreactor array-on-a-chip for speeding up optimization of yeast-based lactic acid bioproduction

A key challenge for bioprocess engineering is the identification of the optimum process conditions for the production of biochemical and biopharmaceutical compounds using prokaryotic as well as eukaryotic cell factories. Shake flasks and bench-scale bioreactor systems are still the golden standard in the early stage of bioprocess development, though they are known to be expensive, time-consuming, and labor-intensive as well as lacking the throughput for efficient production optimizations. To bridge the technological gap between bioprocess optimization and upscaling, we have developed a microfluidic bioreactor array to reduce time and costs, and to increase throughput compared with traditional lab-scale culture strategies. We present a multifunctional microfluidic device containing 12 individual bioreactors (V_t = 15 µl) in a 26 mm × 76 mm area with in-line biosensing of dissolved oxygen and biomass concentration. Following initial device characterization, the bioreactor lab-on-a-chip was used in a proof-of-principle study to identify the most productive cell line for lactic acid production out of two engineered yeast strains, evaluating whether it could reduce the time needed for collecting meaningful data compared with shake flasks cultures. Results of the study showed significant difference in the strains' productivity within 3 hr of operation exhibiting a 4- to 6-fold higher lactic acid production, thus pointing at the potential of microfluidic technology as effective screening tool for fast and parallelizable industrial bioprocess development.     

Versatile synthesis of Boc protected hydrazinoacetic acid and its application to the chemoselective ligation of TASP molecules

This paper describes a convenient synthesis of protected hydrazine derivatives, i.e. 1,2-bis-Boc-hydrazinoacetic acid, and its application for hydrazone ligation techniques in convergent template assembled synthetic protein (TASP) synthesis.