A Non-Toxic Binuclear Vanadium(IV) Complex as Insulin Adjuvant Improves the Glycemic Control in Streptozotocin-Induced Diabetic Rats.

Diabetes mellitus (DM) complications are a burden to health care systems due to the associated consequences of poor glycemic control and the side effects of insulin therapy. Recently. adjuvant therapies, such as vanadium compounds, have gained attention due to their potential to improve glucose homeostasis in patients with diabetes. In order to determine the anti-diabetic and antioxidant effects of the oxidovanadium(IV) complex (Et3NH)2[{VO(OH}2)(ox)2(µ-ox)] or Vox2), rats with streptozotocin (STZ)-induced diabetes were treated with 30 and 100 mg/kg of Vox2, orally administered for 12 days. Vox2 at 100 mg/kg in association with insulin caused a 3.4 times decrease in blood glucose in STZ rats (424 mg/dL), reaching concentrations similar to those in the normoglycemic animals (126 mg/dL). Compared to insulin alone, the association with Vox2 caused an additional decrease in blood glucose of 39% and 65% at 30 and 100 mg/kg, respectively, and an increased pancreatic GSH levels 2.5 times. Vox2 alone did not cause gastrointestinal discomfort, diarrhea, and hepatic or renal toxicity and was not associated with changes in blood glucose level, lipid profile, or kidney or liver function. Our results highlight the potential of Vox2 in association with insulin in treating diabetes.

A magnetic bead immunoassay to detect high affinity human IgG reactive to SARS-CoV-2 Spike S1 RBD produced in Escherichia coli.

Immunological assays to detect SARS-CoV-2 Spike Receptor Binding Domain (RBD) antigen seroconversion in humans are important tools to monitor the levels of protecting antibodies in the population in response to infection and/or immunization. Here we describe a simple, low cost, and high throughput Ni2+ magnetic bead immunoassay to detect human IgG reactive to Spike S1 RBD Receptor Binding Domain produced in Escherichia coli. A 6xHis-tagged Spike S1 RBD was expressed in E. coli and purified by affinity chromatography. The protein was mobilized on the surface of Ni2+ magnetic beads and used to investigate the presence of reactive IgG in the serum obtained from pre-pandemic and COVID-19 confirmed cases. The method was validated with a cohort of 290 samples and an area under the receiver operating characteristic curve of 0.94 was obtained. The method was operated with > 82% sensitivity at 98% specificity and was also able to track human IgG raised in response to vaccination with Comirnaty at > 85% sensitivity. The IgG signal obtained with the described method was well-correlated with the signal obtained when pre fusion Spike produced in HEK cell lines was used as antigen. This novel low-cost and high throughput immunoassay may act as an important tool to investigate protecting IgG antibodies against SARS-CoV-2 in the human population.

Ultra-fast, high throughput and inexpensive detection of SARS-CoV-2 seroconversion using Ni2+ magnetic beads.

To monitor the levels of protecting antibodies raised in the population in response to infection and/or to immunization with SARS-CoV-2, we need a technique that allows high throughput and low-cost quantitative analysis of human IgG antibodies reactive against viral antigens. Here we describe an ultra-fast, high throughput and inexpensive assay to detect SARS-CoV-2 seroconversion in humans. The assay is based on Ni2+ magnetic particles coated with His tagged SARS-CoV-2 antigens. A simple and inexpensive 96 well plate magnetic extraction/homogenization process is described which allows the simultaneous analysis of 96 samples and delivers results in 7 min with high accuracy.

Antigen production and development of an indirect ELISA based on the nucleocapsid protein to detect human SARS-CoV-2 seroconversion.

Serological assays are important tools to identify previous exposure to SARS-CoV-2, helping to track COVID-19 cases and determine the level of humoral response to SARS-CoV-2 infections and/or immunization to future vaccines. Here, the SARS-CoV-2 nucleocapsid protein was expressed in Escherichia coli and purified to homogeneity and high yield using a single chromatography step. The purified SARS-CoV-2 nucleocapsid protein was used to develop an indirect enzyme-linked immunosorbent assay for the identification of human SARS-CoV-2 seroconverts. The assay sensitivity and specificity were determined analyzing sera from 140 RT-qPCR-confirmed COVID-19 cases and 210 pre-pandemic controls. The assay operated with 90% sensitivity and 98% specificity; identical accuracies were obtained in head-to-head comparison with a commercial ELISA kit. Antigen-coated plates were stable for up to 3 months at 4 °C. The ELISA method described is ready for mass production and will be an additional tool to track COVID-19 cases.

Magnetic Bead-Based Immunoassay Allows Rapid, Inexpensive, and Quantitative Detection of Human SARS-CoV-2 Antibodies.

Immunological methods to detect SARS-CoV-2 seroconversion in humans are important to track COVID-19 cases and the humoral response to SARS-CoV-2 infections and immunization to future vaccines. The aim of this work was to develop a simple chromogenic magnetic bead-based immunoassay which allows rapid, inexpensive, and quantitative detection of human antibodies against SARS-CoV-2 in serum, plasma, or blood. Recombinant 6xHis-tagged SARS-CoV-2 Nucleocapsid protein was mobilized on the surface of Ni2+ magnetic beads and challenged with serum or blood samples obtained from controls or COVID-19 cases. The beads were washed, incubated with anti-human IgG-HPR conjugate, and immersed into a solution containing a chromogenic HPR substrate. Bead transfer and homogenization between solutions was aided by a simple low-cost device. The method was validated by two independent laboratories, and the performance to detect SARS-CoV-2 seroconversion in humans was in the same range as obtained using the gold standard immunoassays ELISA and Luminex, though requiring only a fraction of consumables, instrumentation, time to deliver results, and volume of sample. Furthermore, the results obtained with the method described can be visually interpreted without compromising accuracy as demonstrated by validation at a point-of-care unit. The magnetic bead immunoassay throughput can be customized on demand and is readily adapted to be used with any other 6xHis tagged protein or peptide as antigen to track other diseases.

Serum 1,5-Anhydroglucitol Concentration as a Biomarker for Type 1 Diabetes in Adults and Children.

BACKGROUND: Type 1 diabetes (T1D) is an autoimmune disease and the leading form of diabetes among young white people. 1,5-Anhydroglucitol (1,5-AG), a nontraditional biomarker of postprandial glycemic control (after 1 - 3 days to 2 weeks), may be useful in T1D screening. We studied serum 1,5-AG concentration as a potential biomarker for T1D screening and diagnosis in adults and children. METHODS: In this case-control study, adults (n = 121; age, 19 - 61 years) and children (n = 19; age, 8 - 14 years) with T1D were matched with healthy subjects (n = 242) according to gender and age. Serum 1,5-AG levels were measured enzymatically (GlycoMark Inc., NY, USA). RESULTS: Patients showed no symptoms of overt kidney disease, assessed by serum creatinine concentrations. The median (25th - 75th percentile) 1,5-AG concentrations for the control group compared with the T1D group were 155 (128 - 183) vs. 21 (14 - 34) µmol/L in adults and 190 (158 - 237) vs. 20 (12 - 30) µmol/L in children (p < 0.001 for both). Receiver operating characteristic curves showed that 1,5-AG cutoffs of ≤ 113 and ≤ 79 µmol/L for adult men and women, respectively, and ≤ 57 µmol/L for children of both genders had > 95% sensitivity and specificity for both groups. CONCLUSIONS: Our results suggest that serum 1,5-AG concentration may be useful as an adjunct measure of hyperglycemia for diagnosing T1D and has the potential to screen for T1D in high-risk subjects.

Data for serum 1,5 anhydroglucitol concentration in different populations.

1,5 anhydroglucitol (1,5-AG), is a nonmetabolized 1-deoxy form of glucose, originate mainly from the diet. 1,5-AG is a biomarker to detect and magnify hyperglycemic excursions (postprandial hyperglycemia) in diabetic patients. Concentrations of 1,5-AG has been applied as supporting biomarker to diagnosis of the major forms of diabetes (type 1, type 2, and gestational). The serum 1,5-AG reference interval is relevant to the appropriate clinical application of this biomarker. This article contains data regards to serum concentration of the biomarker primarily for healthy subjects, capture from the literature, in different populations. Correlation analysis between 1,5-AG and markers associated with diabetes and its complication were presented. The data was complementary to the study "Reference intervals for serum 1,5-anhydroglucitol in children, adolescents, adults, and pregnant women" (Welter et al., 2018). The data present in this article improve the comparisons for 1,5-AG in different conditions and methodologies.

Reference intervals for serum 1,5-anhydroglucitol in children, adolescents, adults, and pregnant women.

BACKGROUND: 1,5-anhydroglucitol (1,5-AG) is a validated marker of short-term glycemic control. We determined the reference intervals of 1,5-AG in different age groups and during pregnancy. METHODS: Blood samples were collected from 2303 Euro-Brazilian healthy subjects: 580 children, 496 adolescents, 922 adults matched by age and sex, and 305 pregnant women in four gestational periods. Serum 1,5-AG was measured using an enzymatic reagent in an automated system. RESULTS: The calculated reference intervals (nonparametric, 2.5th-97.5th) for males and females were, respectively: children, 96-302 and 89-277 µmol/l; adolescents, 84-311 and 79-277 µmol/l; and adults, 80-260 and 62-241 µmol/l. Males consistently showed significantly higher concentrations than females. 1,5-AG reference intervals in pregnant women were 56-298 µmol/l at <23 weeks gestation (n = 110), 37-166 µmol/l at 24-28 weeks gestation (n = 106), 34-155 µmol/l at 29-32 weeks gestation (n = 52), and 33-246 µmol/l at >32 weeks gestation (n = 37). No significant differences in 1,5-AG concentration were observed between non-pregnant and pregnant women at <23 weeks of gestation. A negative correlation (r = -0.287; p < .001) between 1,5-AG concentration and age was observed. CONCLUSIONS: The reference intervals for 1,5-AG were affected by sex and age.

Association of Polymorphism rs17576 of the Metalloproteinase 9 Gene with Gestational Diabetes in Euro-Brazilian Pregnant Women.

BACKGROUND: Metalloproteinase 9 (MMP9) is involved in the degradation of extracellular matrix molecules, and its polymorphism rs17576 (Gln279Arg) has been associated with diabetes. We investigated the association of rs17576 in a case-control study with Euro-Brazilian women with gestational diabetes. METHODS: The study group consisted of a total of 262 Euro-Brazilian pregnant women classified as either healthy (n = 131, control) or with GDM (n = 131). Fluorescent probes with real time PCR (TaqMan system) were applied for genotyping. RESULTS: All groups were in Hardy-Weinberg equilibrium. The minor allele frequencies (G-allele) for rs17567 in healthy and GDM women were 27.1% [95% CI, 22 - 32] and 37.4% [95% CI, 32 - 43], p = 0.011, respectively. Genotypic comparison showed a significant difference (p < 0.05) between the groups. CONCLUSIONS: Polymorphism rs17567 was associated with GDM in the studied population and carriers of the G-allele showed an increased risk for gestational diabetes (Odds ratio 1.61; 95% CI, 1.1 - 2.3).

Characteristics of an Aeromonas trota strain isolated from cerebrospinal fluid.

Aeromonas are ubiquitous in aquatic habitats. However some species can cause infections in humans, but rarely meningitis. Here we describe the isolation and characterization of an Aeromonas strain from cerebrospinal fluid of a meningitis patient. The isolate, identified as A. trota by biochemical and molecular methods, was susceptible to ampicillin but resistant to cephalothin and cefazolin. Genome sequencing revealed virulence factor genes such as type VI secretion system, aerolysin and lateral flagella. The isolate exhibited swarming motility, hemolytic activity and adhesion and cytotoxicity on HeLa cells. This is the first report of A. trota associated with meningitis and its virulence characteristics.

The Blood Bank for Diabetes Screening: a Feasible Alternative?

BACKGROUND: Vascular complications of diabetes mellitus (DM) are associated with 5% of deaths globally every year. Early diagnosis and treatment could reduce this figure. The aim of this project was to investigate the frequency of undiagnosed DM among blood donors and the possibility of blood banks participating in DM screening. METHODS: Of the approximate 5,600 candidates for blood donation who were evaluated, 4,601 were considered suitable. Candidates with any type of DM, hypertension, thyroid disease, and/or continuous use of any drugs were excluded, resulting in the participation of 635 donors aged 18 - 69 years. Glycated hemoglobin (HbA1c) levels were used to classify the donors: HbA1c < 5.7% (low risk of DM), HbA1c 5.7 - 6.4% (pre-diabetes), and HbA1c ≥ 6.5% (diabetes). Another subsample (n = 576) that excluded donors with HbA1c levels < 5.0% or > 6.5% were classified according to the risk of developing DM in 5 years: HbA1c 5 - 5.5% (low risk, < 9%), HbA1c 5.6 - 6.0% (moderate risk, 9 - 25%), and HbA1c 6.1 - 6.5% (high risk, 26 - 50%). RESULTS: Three donors (0.5%) had HbA1c levels suggestive of DM, and 57 donors (9.0%) had levels associated with pre-DM. Regarding the risk of developing DM in 5 years, 111 donors (19.3%) were classified at moderate risk, and 10 donors (1.7%) were classified at high risk. CONCLUSIONS: DM screening in blood banks using HbA1c can identify new cases of DM and individuals at an increased risk of DM. In summary, blood banks could participate in DM screening, benefitting the general public and public health care system in Brazil.

Draft Genome Sequence of Aeromonas caviae 8LM, Isolated from Stool Culture of a Child with Diarrhea.

Aeromonas spp. are Gram-negative rods ubiquitous in aquatic environments; however, some species are able to cause a variety of infections in humans. Here, we report the draft genome sequence of Aeromonas caviae 8LM isolated from stool culture from a child with diarrhea in southern Brazil.

Biochemical characteristics, adhesion, and cytotoxicity of environmental and clinical isolates of Herbaspirillum spp.

Herbaspirillum bacteria are best known as plant growth-promoting rhizobacteria but have also been recovered from clinical samples. Here, biochemical tests, matrix-assisted laser deionization-time of flight (MALDI-TOF) mass spectrometry, adherence, and cytotoxicity to eukaryotic cells were used to compare clinical and environmental isolates of Herbaspirillum spp. Discrete biochemical differences were observed between human and environmental strains. All strains adhered to HeLa cells at low densities, and cytotoxic effects were discrete, supporting the view that Herbaspirillum bacteria are opportunists with low virulence potential.

The plasma logarithm of the triglyceride/HDL-cholesterol ratio is a predictor of low risk gestational diabetes in early pregnancy.

BACKGROUND: The plasma lipid profile changes atherogenically during normal pregnancy. Gestational diabetes mellitus (GDM) can exacerbate the changes in metabolism. The logarithm of the ratio triglycerides/HDL-cholesterol is an atherogenic index of the plasma (AIP) and can be used as a marker for plasma atherogenicity. METHODS: Serum of 576 unrelated Euro-Brazilian pregnant women was collected and the subjects were classified as healthy pregnant women (control, n=288) and gestational diabetic patients (GDM, n=288) according to the ADA 2010 criteria. Both studied groups were sub classified in 4 gestational periods: (i) 12-23, (ii) 24-28, (iii) 29-32 and (iv) >32 weeks of gestation. RESULTS: Except for the AIP, the other parameters showed low discrimination between control and GDM groups (ROC curves). When analyzed by ROC curves the AIP of subjects in the early period of gestation showed sensitivity and specificity of 82.6% and 83.4%, respectively, with a cut-off point of 0.099 (AUC 0.886, P<0.0001). CONCLUSIONS: The AIP is a valuable index to identify pregnant women with low risk of gestational diabetes before 24 weeks of gestation.

Identification and characterization of PhbF: a DNA binding protein with regulatory role in the PHB metabolism of Herbaspirillum seropedicae SmR1.

BACKGROUND: Herbaspirillum seropedicae SmR1 is a nitrogen fixing endophyte associated with important agricultural crops. It produces polyhydroxybutyrate (PHB) which is stored intracellularly as granules. However, PHB metabolism and regulatory control is not yet well studied in this organism. RESULTS: In this work we describe the characterization of the PhbF protein from H. seropedicae SmR1 which was purified and characterized after expression in E. coli. The purified PhbF protein was able to bind to eleven putative promoters of genes involved in PHB metabolism in H. seropedicae SmR1. In silico analyses indicated a probable DNA-binding sequence which was shown to be protected in DNA footprinting assays using purified PhbF. Analyses using lacZ fusions showed that PhbF can act as a repressor protein controlling the expression of PHB metabolism-related genes. CONCLUSIONS: Our results indicate that H. seropedicae SmR1 PhbF regulates expression of phb-related genes by acting as a transcriptional repressor. The knowledge of the PHB metabolism of this plant-associated bacterium may contribute to the understanding of the plant-colonizing process and the organism's resistance and survival in planta.

Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity.

The bacterial AAA+ enhancer-binding proteins (EBPs) HrpR and HrpS (HrpRS) of Pseudomonas syringae (Ps) activate σ(54)-dependent transcription at the hrpL promoter; triggering type-three secretion system-mediated pathogenicity. In contrast with singly acting EBPs, the evolution of the strictly co-operative HrpRS pair raises questions of potential benefits and mechanistic differences this transcription control system offers. Here, we show distinct properties of HrpR and HrpS variants, indicating functional specialization of these non-redundant, tandemly arranged paralogues. Activities of HrpR, HrpS and their control proteins HrpV and HrpG from Ps pv. tomato DC3000 in vitro establish that HrpRS forms a transcriptionally active hetero-hexamer, that there is a direct negative regulatory role for HrpV through specific binding to HrpS and that HrpG suppresses HrpV. The distinct HrpR and HrpS functionalities suggest how partial paralogue degeneration has potentially led to a novel control mechanism for EBPs and indicate subunit-specific roles for EBPs in σ(54)-RNA polymerase activation.

The involvement of the nif-associated ferredoxin-like genes fdxA and fdxN of Herbaspirillum seropedicae in nitrogen fixation.

The pathway of electron transport to nitrogenase in the endophytic beta-Proteobacterium Herbaspirillum seropedicae has not been characterized. We have generated mutants in two nif-associated genes encoding putative ferredoxins, fdxA and fdxN. The fdxA gene is part of the operon nifHDKENXorf1orf2fdxAnifQmodABC and is transcribed from the nifH promoter, as revealed by lacZ gene fusion. The fdxN gene is probably cotranscribed with the nifB gene. Mutational analysis suggests that the FdxA protein is essential for maximum nitrogenase activity, since the nitrogenase activity of the fdxA mutant strain was reduced to about 30% of that of the wild-type strain. In addition, the fdxA mutation had no effect on the nitrogenase switch-off in response to ammonium. Nitrogenase activity of a mutant strain lacking the fdxN gene was completely abolished. This phenotype was reverted by complementation with fdxN expressed under lacZ promoter control. The results suggest that the products of both the fdxA and fdxN genes are probably involved in electron transfer during nitrogen fixation.

Expression, purification and DNA-binding activities of two putative ModE proteins of Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae)

In prokaryotes molybdenum is taken up by a high-affinity ABC-type transporter system encoded by the modABC genes. The endophyte â-Proteobacterium Herbaspirillum seropedicae has two modABC gene clusters and two genes encoding putative Mo-dependent regulator proteins (ModE1 and ModE2). Analysis of the amino acid sequence of the ModE1 protein of H. seropedicae revealed the presence of an N-terminal domain containing a DNA-binding helix-turn-helix motif (HTH) and a C-terminal domain with a molybdate-binding motif. The second putative regulator protein, ModE2, contains only the helix-turn-helix motif, similar to that observed in some sequenced genomes. We cloned the modE1 (810 bp) and modE2 (372 bp) genes and expressed them in Escherichia coli as His-tagged fusion proteins, which we subsequently purified. The over-expressed recombinant His-ModE1 was insoluble and was purified after solubilization with urea and then on-column refolded during affinity chromatography. The His-ModE2 was expressed as a soluble protein and purified by affinity chromatography. These purified proteins were analyzed by DNA band-shift assays using the modA2 promoter region as probe. Our results indicate that His-ModE1 and His-ModE2 are able to bind to the modA2 promoter region, suggesting that both proteins may play a role in the regulation of molybdenum uptake and metabolism in H. seropedicae.

Characterization of the orf1glnKamtB operon of Herbaspirillum seropedicae.

Herbaspirillum seropedicae is an endophytic nitrogen-fixing bacterium that colonizes economically important grasses. In this organism, the amtB gene is co-transcribed with two other genes: glnK that codes for a PII-like protein and orf1 that codes for a probable periplasmatic protein of unknown function. The expression of the orf1glnKamtB operon is increased under nitrogen-limiting conditions and is dependent on NtrC. An amtB mutant failed to transport methylammonium. Post-translational control of nitrogenase was also partially impaired in this mutant, since a complete switch-off of nitrogenase after ammonium addition was not observed. This result suggests that the AmtB protein is involved in the signaling pathway for the reversible inactivation of nitrogenase in H. seropedicae.