Enzymes that hydrolyze adenine nucleotides in chronic renal failure: relationship between hemostatic defects and renal failure severity

The activities of the enzymes NTPDase (E.C.3.6.1.5, apyrase, ATP diphosphohydrolase, ecto-CD 39) and 5'-nucleotidase (E.C.3.1.3.5, CD 73) were analyzed in platelets from patients with chronic renal failure (CRF), both undergoing hemodialysis treatment (HD) and not undergoing hemodialysis (ND), as well as from a control group. The results showed an increase in platelet NTPDase activity in CRF patients on HD treatment (52.88%) with ATP as substrate (P<0.0001). ADP hydrolysis was decreased (33.68% and 39.75%) in HD and ND patients, respectively. In addition, 5'-nucleotidase activity was elevated in the HD (160%) and ND (81.49%) groups when compared to the control (P<0.0001). Significant correlation was found among ATP, ADP and AMP hydrolysis and plasma creatinine and urea levels (P<0.0001). Patients were compared statistically according the time of hemodialysis treatment. We found enhanced NTPDase and 5'-nucleotidase activities between 49 and 72 months on HD patients. Our result suggests the existence of alterations in nucleotide hydrolysis in platelets of CRF patients. Possibly, this altered nucleotide hydrolysis could contribute to hemostasis abnormalities found in CRF.     

Anticrotalic and antitumoral activities of gel filtration fractions of aqueous extract from Tabernaemontana catharinensis (Apocynaceae)

The high mortality caused by Crotalus durissus terrificus snake venom is mainly due to crotoxin, which acts on the neuromuscular junction inhibiting the mechanism mediating acetylcholine release, thus leading to motor and respiratory paralysis and subsequently to animal death. We recently demonstrated that the aqueous extract (AE) of Tabernaemontana catharinensis can inhibit the lethal activity of C. d. terrificus venom. Eight fractions, PI to PVIII, were obtained by gel filtration of the extract on Sephadex G-10, and assayed for lethality and cytotoxicity. Fraction PVII [2.0 mg/100 g rat/50 microl saline solution (ss)] injected intramuscularly (i.m.) 20 s after the venom (240 microg) or crotoxin (200 microg/50 microl ss) neutralized the lethal activity of 2 LD50 of both. Fractions PI, PVI and PVIII (5.0 mg/100 g rat/50 microl ss) presented potent antitumoral activity in vitro against cells from human breast carcinoma (SK-BR-3) after 24 h incubation, as measured by Mosmann colorimetric method. Fraction PVII contains 12-methoxy-4-methylvoachalotine as its major component. These results demonstrate that the antivenom and antitumoral activities of the AE of T. catharinensis are exerted by different substances present in fraction PVII and fractions PI, PVI and PVIII, respectively, whose characteristics are distinct in terms of staining and Rf when analyzed by thin layer chromatography. The results also show that a preliminary fractionation by Sephadex G-10 gel filtration is a good option as a first step for isolation of biologically active substances from T. catharinensis.     

Shorter leukocyte telomere length is associated with severity of COVID-19 infection.

The infection by COVID-19 is a serious global public health problem. An efficient way to improve this disease's clinical management would be to characterize patients at higher risk of progressing to critically severe infection using prognostic biomarkers. The telomere length could be used for this purpose. Telomeres are responsible for controlling the number of maximum cell divisions. The telomere length is a biomarker of aging and several diseases. We aimed to compare leukocyte telomere length (LTL) between patients without COVID-19 and patients with different clinical severity of the infection. Were included 53 patients who underwent SARS-CoV-2 PCR divided in four groups. The first group was composed by patients with a negative diagnosis for COVID-19 (n = 12). The other three groups consisted of patients with a confirmed diagnosis of COVID-19 divided according to the severity of the disease: mild (n = 15), moderate (n = 17) and severe (n = 9). The LTL was determined by Q-PCR. The severe group had the shortest LTL, followed by the moderate group. The negative and mild groups showed no differences. There is an increase of patients with hypertension (p = 0.0099) and diabetes (p = 0.0067) in moderate and severe groups. Severe group was composed by older patients in comparison with the other three groups (p = 0.0083). Regarding sex, there was no significant difference between groups (p = 0.6279). In an ordinal regression model, only LTL and diabetes were significantly associated with disease severity. Shorter telomere length was significantly associated with the severity of COVID-19 infection, which can be useful as a biomarker or to better understand the SARS-CoV-2 pathophysiology.     

Beneficial Effects of the Activation of the Angiotensin-(1–7) Mas Receptor in a Murine Model of Adriamycin-Induced Nephropathy

Angiotensin-(1–7) [Ang-(1–7)] is a biologically active heptapeptide that may counterbalance the physiological actions of angiotensin II (Ang II) within the renin-angiotensin system (RAS). Here, we evaluated whether activation of the Mas receptor with the oral agonist, AVE 0991, would have renoprotective effects in a model of adriamycin (ADR)-induced nephropathy. We also evaluated whether the Mas receptor contributed for the protective effects of treatment with AT₁ receptor blockers. ADR (10 mg/kg) induced significant renal injury and dysfunction that was maximal at day 14 after injection. Treatment with the Mas receptor agonist AVE 0991 improved renal function parameters, reduced urinary protein loss and attenuated histological changes. Renoprotection was associated with reduction in urinary levels of TGF-β. Similar renoprotection was observed after treatment with the AT₁ receptor antagonist, Losartan. AT₁ and Mas receptor mRNA levels dropped after ADR administration and treatment with losartan reestablished the expression of Mas receptor and increased the expression of ACE2. ADR-induced nephropathy was similar in wild type (Mas^+/+) and Mas knockout (Mas ^−/−) mice, suggesting there was no endogenous role for Mas receptor activation. However, treatment with Losartan was able to reduce renal injury only in Mas^+/+, but not in Mas ^−/− mice. Therefore, these findings suggest that exogenous activation of the Mas receptor protects from ADR-induced nephropathy and contributes to the beneficial effects of AT₁ receptor blockade. Medications which target specifically the ACE2/Ang-(1–7)/Mas axis may offer new therapeutic opportunities to treat human nephropathies.     

Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (K_m) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.     

Identification of iron-regulated cellular proteins, Fe3+-reducing and -chelating compounds, in the white-rot fungus Perenniporia medulla-panis

In this paper, we present the responses of the white-rot fungus Perenniporia medulla-panis to iron availability with regard to alterations in growth, expression of cellular proteins, Fe3+-reducing activity, and Fe3+ chelators production. Iron supplementation stimulated fungal growth but did not result in a significant increase in biomass production. Catechol and hydroxamate derivatives were produced mainly under iron deficiency, and their productions were repressed under iron supplementation conditions. Perenniporia medulla-panis showed several cellular proteins in the range of 10-90 kDa. Some of them showed negative iron-regulation. Iron-supplemented medium also repressed both cell surface and extracellular Fe3+-reducing activities; however, the highest cell surface activity was detected at the initial growth phase, whereas extracellular activity increased throughout the incubation period. No significant production of chelators and extracellular Fe3+-reducing activity were observed within the initial growth phase, suggesting that the reduction of Fe3+ to Fe2+ is performed by ferrireductases.     

Molecular findings from influenza A(H1N1)pdm09 detected in patients from a Brazilian equatorial region during the pandemic period

After the World Health Organization officially declared the end of the first pandemic of the XXI century in August 2010, the influenza A(H1N1)pdm09 virus has been disseminated in the human population. In spite of its sustained circulation, very little on phylogenetic data or oseltamivir (OST) resistance is available for the virus in equatorial regions of South America. In order to shed more light on this topic, we analysed the haemagglutinin (HA) and neuraminidase (NA) genes of influenza A(H1N1)pdm09 positive samples collected during the pandemic period in the Pernambuco (PE), a northeastern Brazilian state. Complete HA sequences were compared and amino acid changes were related to clinical outcome. In addition, the H275Y substitution in NA, associated with OST resistance, was investigated by pyrosequencing. Samples from PE were grouped in phylogenetic clades 6 and 7, being clustered together with sequences from South and Southeast Brazil. The D222N/G HA gene mutation, associated with severity, was found in one deceased patient that was pregnant. Additionally, the HA mutation K308E, which appeared in Brazil in 2010 and was only detected worldwide the following year, was identified in samples from hospitalised cases. The resistance marker H275Y was not identified in samples tested. However, broader studies are needed to establish the real frequency of resistance in this Brazilian region.     

Assessment of Total and Organic Mercury Levels in Blue Sharks (Prionace glauca) from the South and Southeastern Brazilian Coast

Mercury occurrence was evaluated in samples of edible muscle tissue of 27 blue sharks (Prionace glauca) caught in the Atlantic Ocean, adjacent to the south and southeastern Brazilian coast, indicating a slight increase in comparison with previous data obtained for the same studied area and being higher than those fish caught at different sites of the Atlantic Ocean. Total Hg concentrations ranged from 0.46 to 2.40 mg kg^?1 with the organic Hg fraction ranging between 0.44 and 2.37 mg kg^?1. A negative correlation between total Hg concentration in muscle tissue and blue shark size was obtained, and 40 % of samples analyzed had Hg concentrations higher than 1.0 mg kg^?1 Hg, the maximum concentration permitted in Brazilian predator fish. Data obtained showed that total Hg can be used as a reliable predictor of organic Hg in blue shark muscle because 95 to 98 % of the total Hg measured was found to be organic mercury. The wide range of Hg concentrations obtained for our set of samples can be explained by the heterogeneity of sampled population and the large size of the studied area. Given the adverse toxicological effects of Hg on animals and humans, a regular monitoring program of Hg contamination in Brazilian marine ecosystem can be recommended.     

Trp28Arg/Ile35Thr LHB gene variants are associated with elevated testosterone levels in women with polycystic ovary syndrome

Introduction

Polycystic Ovary Syndrome (PCOS) is a complex endocrine disorder, of multifactorial etiology, which affects 6–10% of women of reproductive age. It is considered the leading cause of anovulatory infertility, menstrual disorders and hyperandrogenism in this population. The genetic basis of PCOS is still largely unknown despite significant family clustering; determining its mode of inheritance is particularly difficult given the heterogenic presentation of the disease.

Materials and methods

130 Brazilian women, aged 14–42 years, who met the 2003 Rotterdam criteria for PCOS diagnosis, were included, and 96 healthy women constituted the control group. Presence of hirsutism was classified using the modified Ferriman–Gallwey score (F–G score) as absent (≤ 7), mild (8–14), and severe (≥ 15). Blood levels of luteinizing hormone (LH), total testosterone (TT), dehydroepiandrosterone sulfate (DHEA-S) and androstenedione were determined. The coding region of the luteinizing hormone beta-subunit (LHB) gene was amplified and sequenced. Differences in allelic and genotypic frequency distribution of each polymorphism across controls and cases were estimated by the Mantel–Haenszel chi-square or Fisher's exact test (p < 0.05), and the probability of an association between the detection of a polymorphism and presence of a diagnosis of PCOS, by logistic regression.

Result(s)

Sequencing detected 8 polymorphisms in the LHB gene coding region. Two polymorphisms in linkage disequilibrium were significantly more prevalent in the presence of hyperandrogenemia: rs1800447/rs34349826 (Trp28Arg/Ile35Thr) (p = 0.02).

Conclusion(s)

In this series, a modulatory effect of LHB polymorphisms on hyperandrogenemia phenotype of PCOS was observed; however, this finding needs to be replicated in other populations.