Enzymatic synthesis of chiral amino‐alcohols by coupling transketolase and transaminase‐catalyzed reactions in a cascading continuous‐flow microreactor system

Rapid biocatalytic process development and intensification continues to be challenging with currently available methods. Chiral amino‐alcohols are of particular interest as they represent key industrial synthons for the production of complex molecules and optically pure pharmaceuticals. (2S,3R)‐2‐amino‐1,3,4‐butanetriol (ABT), a building block for the synthesis of protease inhibitors and detoxifying agents, can be synthesized from simple, non‐chiral starting materials, by coupling a transketolase‐ and a transaminase‐catalyzed reaction. However, until today, full conversion has not been shown and, typically, long reaction times are reported, making process modifications and improvement challenging. In this contribution, we present a novel microreactor‐based approach based on free enzymes, and we report for the first time full conversion of ABT in a coupled enzyme cascade for both batch and continuous‐flow systems. Using the compartmentalization of the reactions afforded by the microreactor cascade, we overcame inhibitory effects, increased the activity per unit volume, and optimized individual reaction conditions. The transketolase‐catalyzed reaction was completed in under 10 min with a volumetric activity of 3.25 U ml^?1. Following optimization of the transaminase‐catalyzed reaction, a volumetric activity of 10.8 U ml^?1 was attained which led to full conversion of the coupled reaction in 2 hr. The presented approach illustrates how continuous‐flow microreactors can be applied for the design and optimization of biocatalytic processes.

     

Exercise-induced lipid mobilization in subcutaneous adipose tissue is mainly related to natriuretic peptides in overweight men

Involvement of sympathetic nervous system and natriuretic peptides in the control of exercise-induced lipid mobilization was compared in overweight and lean men. Lipid mobilization was determined using local microdialysis during exercise. Subjects performed 35-min exercise bouts at 60% of their maximal oxygen consumption under placebo or after oral tertatolol [a beta-adrenergic receptor (AR) antagonist]. Under placebo, exercise increased dialysate glycerol concentration (DGC) in both groups. Phentolamine (alpha-AR antagonist) potentiated exercise-induced lipolysis in overweight but not in lean subjects; the alpha(2)-antilipolytic effect was only functional in overweight men. After tertatolol administration, the DGC increased similarly during exercise no matter which was used probe in both groups. Compared with the control probe under placebo, lipolysis was reduced in lean but not in overweight men treated with the beta-AR blocker. Tertatolol reduced plasma nonesterified fatty acids and insulin concentration in both groups at rest. Under placebo or tertatolol, the exercise-induced changes in plasma nonesterified fatty acids, glycerol, and insulin concentrations were similar in both groups. Exercise promoted a higher increase in catecholamine and ANP plasma levels after tertatolol administration. In conclusion, the major finding of our study is that in overweight men, in addition to an increased alpha(2)-antilipolytic effect, the lipid mobilization in subcutaneous adipose tissue that persists during exercise under beta-blockade is not dependent on catecholamine action. On the basis of correlation findings, it seems to be related to a concomitant exercise-induced rise in plasma ANP when exercise is performed under tertatolol intake and a decrease in plasma insulin.     

Cytotoxic effects of metal complexes of biogenic polyamines. I. Platinum(II) spermidine compounds: prediction of their antitumour activity

Cytotoxicity and cell growth inhibition studies were performed for three distinct polynuclear platinum(II) complexes of spermidine, which showed to have significant cytotoxic and antiproliferative properties on the HeLa cancer cell line. The chemical environment of the metal centres in the drugs, as well as the coordination pattern of the ligand, were found to be strongly determinant of their cytotoxic ability. In the light of the results gathered, the most effective anticancer compound among the ones tested (IC50=5 microM) was found to be the one displaying three difunctional (PtCl2N2) moieties ((PtCl2)3(spd)2). Both the cytotoxic activity and the antiproliferative properties of the complexes studied showed to be irreversible for all the concentrations tested.     

Paracoccidioidomycosis in people living with HIV/AIDS: A historical retrospective cohort study in a national reference center for infectious diseases,Rio de Janeiro,Brazil

Paracoccidioidomycosis (PCM) is one of the main endemic systemic mycoses in Latin America, usually occurring in rural areas. When PCM occurs simultaneously with underlying immunosuppressive conditions, it can present as an opportunistic disease. Between 2000 and 2017, literature reported around 200 PCM cases in people living with HIV/AIDS (PLWHA). To address research gaps on this co-infection and to study its possible temporal changes in the last decade, we performed an active co-infection case search on the HIV/AIDS and PCM cohorts from a Brazilian reference center database from 1989 to 2019. We found 20 PLWHA among 684 PCM patients (2.92%), predominantly male (70.0%) and urban workers (80.0%). The median age of patients was higher in the 2010–2019 decade (p = 0.006). The occurrence of PCM in PLWHA was lower when compared with other fungal diseases. Although 50.0% of the patients had already been diagnosed with HIV infection and presented CD4+ T cell counts greater than 200/mm³ at the time of PCM diagnosis, the suspicion of immunosuppression in the context of atypical and more severe clinical forms of PCM revealed the diagnosis of HIV infection in 35.0% of the patients. Two (10.0%) patients had an evolution compatible with immune reconstitution inflammatory syndrome (IRIS) after starting antiretroviral therapy (ART).We highlight the importance of considering a PCM diagnosis in PLWHA to prevent a late-onset treatment and progression to severe manifestations and unfavorable outcomes. In addition, HIV investigation is recommended in PCM patients, especially those with atypical and more severe clinical presentations.     

Pepstatin-sensitive proteolytic activity of sorghum seeds

Two novel proteinases were isolated from resting sorghum seeds and purified 100-fold. The activity of the purified enzymes was completely inhibited by pepstatin A and was unaffected by PMSF, leupeptin, EDTA and E-64 (L-trans-epoxysuccinyl leucylamino 4 guanidino butane), which indicates that they belong to the class of aspartic proteinases. SDS-PAGE and native-PAGE revealed a monomeric 29-kDa enzyme and a heterodimeric 61-kDa enzyme with two S-S linked subunits of 49 and 12 kDa. The proteases have maximum activity at 45 °C and pH 3.5, with haemoglobin as substrate. Activity at 60 °C is higher than at 30 °C.     

Anindobothrium n. gen. (Eucestoda: Tetraphyllidea) inhabiting marine and freshwater potamotrygonid stingrays.

Anindobothrium n. gen. is proposed to accommodate Caulobothrium anacolum inhabiting Himantura schmardae from Colombia, and 2 new species, one inhabiting Potamotrygon orbigny in Brazil and the other inhabiting Paratrygon aereiba in Venezuela. Members of the new genus resemble members of Pararhinebothroides, Rhinebothroides, and Anthocephalum by having bothridia with poorly differentiated apical suckers and vasa deferentia expanded into external seminal vesicles. It further resembles Pararhinebothroides, Rhinebothroides, and Anthocephalum cairae by having vas deferens inserted near the poral rather than aporal end of the cirrus sac. The 3 species assigned to the new genus form an apparent monophyletic group, based on the possession of 3 putative synapomorphies: (1) genital pores in the anterior 1/4 of the proglottid, a trait that is unusual, but not unique, among phyllobothriids; (2) anteroventral ovarian lobes converging to the center of the proglottid, a character not previously reported for phyllobothriids; and (3) ovarian lobes comprising a loose network of digitiform processes.     

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction

Citrus canker is a plant disease caused by Gram‐negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm‐enriched fraction was performed for XAC cells grown in pathogenicity‐inducing (XAM‐M) and pathogenicity‐non‐inducing (nutrient broth) media using two‐dimensional electrophoresis combined with liquid chromatography‐tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up‐regulated proteins related to cellular envelope metabolism included glucose‐1‐phosphate thymidylyltransferase, dTDP‐4‐dehydrorhamnose‐3,5‐epimerase and peptidyl‐prolyl cis–trans‐isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real‐time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up‐regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60‐kDa chaperonin and glyceraldehyde‐3‐phosphate dehydrogenase were identified, suggesting the presence of post‐translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence‐related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.     

Mauriac syndrome still exists

Background/objectiveMauriac syndrome (MS) is a rare complication of type 1 diabetes mellitus (DM1). It is related to low insulin concentrations and is less common since longer-acting insulins became available. It is characterized by hepatomegaly, growth and puberty delay, and the presence of elevated transaminases and serum lipids. The aim of this study was to describe the patients from a pediatric diabetic population that fulfill the criteria of MS.Materials and methodsA retrospective analysis of the pediatric diabetic population with diagnostic criteria of MS currently followed at Hospital de Braga, was performed.ResultsFrom a population of 91 patients with DM1 18 years, 6 patients with the criteria for MS were identified: 5 girls, and 1 boy. The age at presentation was 13–17 years, with a minimum interval between DM1 diagnosis and MS criteria of 4 years. All the patients were prescribed intensive insulin therapy (median daily insulin dose: 0.88 U/kg). All had a previous history of poor glycemic control before the diagnosis of MS with glycated hemoglobin (HbA1c) between 8.8 and 12.9%. Increase of hepatic enzymes was present in all the patients; 4 of them had associated hepatomegaly. All the girls presented puberty delay and cushingoid features. None of the patients presented short stature and 5 of them presented mixed dyslipidemia.ConclusionsAlthough MS is an ancient entity described in DM1, it still exists, particularly in adolescent females. Being aware of MS is of extreme importance since most of the clinical features are reversible with better glycemic control.     

Role of Zinc in Zinc-α2-Glycoprotein Metabolism in Obesity: a Review of Literature

Biological Trace Element Research - Excessive adipose tissue promotes the manifestation of endocrine disorders such as reduction of the secretion of zinc-α2-glycoprotein (ZAG), an adipokine...     

Leprosy in a prison population: A new active search strategy and a prospective clinical analysis

BackgroundThis study evaluates an active search strategy for leprosy diagnosis based on responses to a Leprosy Suspicion Questionnaire (LSQ), and analyzing the clinical, immunoepidemiological and follow-up aspects for individuals living in a prison population.MethodsA cross-sectional study based on a questionnaire posing 14 questions about leprosy symptoms and signs that was distributed to 1,400 prisoners. This was followed by dermatoneurological examination, anti-PGL-I serology and RLEP-PCR. Those without leprosy were placed in the Non-leprosy Group (NLG, n = 1,216) and those diagnosed with clinical symptoms of leprosy were placed in the Leprosy Group (LG, n = 34).FindingsIn total, 896 LSQ were returned (64%), and 187 (20.9%) of the responses were deemed as positive for signs/symptoms, answering 2.7 questions on average. Clinically, 1,250 (89.3%) of the prisoners were evaluated resulting in the diagnosis of 34 new cases (LG), based on well-accepted clinical signs and symptoms, a new case detection rate of 2.7% within this population, while the NLG were comprised of 1,216 individuals. The confinement time medians were 39 months in the LG while it was 36 months in the NLG (p>0.05). The 31 leprosy cases who responded to the questionnaire (LSQ+) had an average of 1.5 responses. The symptoms “anesthetized skin area” and “pain in nerves” were most commonly mentioned in the LG while “tingling, numbness in the hands/feet”, “sensation of pricks and needles”, “pain in nerves” and “spots on the skin” responses were found in more than 30% of questionnaires in the NLG. Clinically, 88.2% had dysesthetic macular skin lesions and 97.1% presented some peripheral nerve impairment, 71.9% with some degree of disability. All cases were multibacillary, confirming a late diagnosis. Anti-PGL-I results in the LG were higher than in the NLG (p<0.0001), while the RLEP-PCR was positive in 11.8% of the patients.InterpretationOur findings within the penitentiary demonstrated a hidden prevalence of leprosy, although the individuals diagnosed were likely infected while living in their former communities and not as a result of exposure in the prison. The LSQ proved to be an important screening tool to help identify leprosy cases in prisons.