Omentin-1 and its relationship with inflammatory factors in maternal plasma and visceral adipose tissue of women with gestational diabetes mellitus.

PURPOSE: To investigate the association of omentin-1 and inflammatory factors in serum and visceral adipose tissue (VAT) of women with gestational diabetes mellitus (GDM) compared to normal pregnant (NP) subjects. Furthermore, to examine their correlation with maternal clinical characteristics. METHODS: We compared 116 GDM women to 115 NP women, at the time of cesarean section. Circulating omentin-1 and pro-inflammatory (IL-1ß, IL-6, TNF-α), and anti-inflammatory cytokines (IL-1RA, IL-10) were examined. Moreover, their mRNA expression in VAT, along with inflammatory factors involved in the NF-κB pathway (TLR2, TLR4, NF-κB, IKκB), were examined. RESULTS: Circulating omentin-1 (p = 0.022) was lower and circulating IL-1-ß, IL-1RA, as well as IL-10 (p = 0.005, p = 0.007, and p = 0.015, respectively), were higher in GDM compared to NP women. Omentin-1 correlated negatively with pre-pregnancy and gestational BMI, and HOMA-IR in all women, but was not associated with cytokines. TLR2, TLR4, IL-1ß, IL-1RA, IL-6, IL-10 mRNA expression in VAT was lower in GDM compared with controls (p < 0.05 all). In multivariate analysis, BMI at delivery was significantly correlated to omentin-1 concentrations in all and NP subjects. In addition, omentin-1 expression was correlated to inflammatory gene expression in all, GDM and NP, women (p < 0.05 all). CONCLUSION: Serum levels and VAT gene expression of omentin-1 are not independently linked to GDM; notwithstanding, GDM women have a VAT-altered inflammatory status. In addition, no systemic association between omentin-1 and inflammatory factors was found, whereas associations between their expression in all women were observed, indicating that expression of these adipokines is linked between them regardless of GDM.

Psychosocial risk in healthcare workers after one year of COVID-19.

BACKGROUND: The COVID-19 pandemic overwhelmed the capacity of the healthcare system, affecting the volume of demands and the care tasks of healthcare workers. AIMS: To examine the health indicators and exposure to psychosocial risks of Spanish healthcare workers 1 year into the COVID-19 pandemic and compare them with the results of the first wave. METHODS: We conducted a cross-sectional study using an online questionnaire (April-May 2020 and 2021). The data stem from the COTS 1 project database, corresponding to the first wave of COVID-19 (n = 1989) and COTS 2 (n = 1716) corresponding to 1 year later. The samples were independent. The prevalence of exposure to psychosocial risks and adverse health indicators was estimated for every occupational group, segregating the data by sex. RESULTS: Professionals of all types presented worse perception of health. In general, the results were worse for women, while geriatric assistants presented the greatest exposure to psychosocial risk in COTS 2 compared to COTS 1. Sleep problems, high quantitative demands and high concern about becoming infected and spreading COVID-19 were cross-disciplinary in COTS 1, while worse perception of health, high pace of work, high work-life conflict and low development opportunities stood out in COTS 2. CONCLUSIONS: Exposure to psychosocial risks was already high during the first wave and a significant decline in working conditions was observed. The prolongation of the pandemic exacerbated these results and seems to have multiplied the pre-existing inequalities between the axes of segregation in the labour market.

Prognosis of root canal treatment in teeth with preoperative apical periodontitis: a study with cone-beam computed tomography and digital periapical radiography.

AIM: This observational cohort study aimed: (i) to determine retrospectively after a 1- to 12-year follow-up period, the strength and independence of the association of various patient-, tooth- and treatment-related prognostic variables with the outcome of root canal treatment in patients with pre-therapeutic apical periodontitis; and (ii) to establish the concordance between those predictors evaluated by both cone-beam computed tomography (CBCT) and digital periapical radiography (DPR). METHODOLOGY: The study cohort included 125 teeth in 84 individuals. The postoperative clinical signs/symptoms, plus DPR/CBCT-PAI estimations, were used to determine the healing outcome. For statistical analysis, results were dichotomized into completely healed periapical structures versus persistent disease cases. The association between candidate predictor variables with persistent disease was analysed individually and adjusted for confounders using a multivariate binary logistic regression model. RESULTS: The success rate was 53.6%. After logistic regression analysis, mandibular tooth location, periapical lesion size >10 mm, poor quality of the coronal restoration, lack of magnification/illumination, lack of disinfection of gutta-percha, time elapsed to definitive coronal restoration >1 week and poor density of root canal filling remained as robust predictors of failures. Concordance between DPR and CBCT scores varied from moderate to almost perfect agreement. CONCLUSIONS: The findings of this study suggest that several tooth- and treatment-related predictor variables, including tooth location, periapical radiolucency size, quality of the coronal restoration, magnification/illumination, disinfection of gutta-percha, time elapsed to definitive coronal restoration, as well as, the density of root canal filling may act strongly and independently for determining the root canal treatment outcome in teeth with pre-therapeutic apical periodontitis.

Sir2-Related Protein 1 from Leishmania amazonensis is a glycosylated NAD+-dependent deacetylase.

Sirtuin proteins form a family of NAD+-dependent protein deacetylases that are considered potential drug targets against parasites. Here, we present the first characterization of a sirtuin orthologue from Leishmania amazonensis, an aetiological agent of American tegumentary leishmaniasis that has been the subject of many studies focused in the development of therapeutic approaches. The protein has high sequence identity with other Kinetoplastid Silent information regulator 2 Related Protein 1 (Sir2RP1) and was named LaSir2RP1. The gene exists as a single copy, encoding a monomeric protein (LaSir2RP1) of approximately 41 kDa that has NAD+-dependent deacetylase activity. LaSir2RP1 was immunodetected in total protein extracts, in cytoplasmic granules, and in the secreted material of both promastigotes and lesion-derived amastigotes. Analysis of both lectin­affinity purified promastigote and amastigote extracts revealed the presence of a major enriched protein of approximately 66 kDa that was recognized by an anti-LaSir2RP1 serum, suggesting that a parasite sirtuin could be glycosylated in vivo.

Leishmania amazonensis: partial purification and study of the biochemical properties of the telomerase reverse transcriptase activity from promastigote-stage.

Telomeres are protein-DNA complexes that protect chromosome ends from degradation and fusion. In Leishmania spp., telomeric DNA comprises a conserved TTAGGG repeat and is maintained by telomerase. Telomerase is a multisubunit enzymatic complex that ensures the complete DNA replication by adding new telomeric repeats to the G-rich strand. In this report we aimed to purify and study the biochemical properties of Leishmania amazonensis telomerase. In a first trial we used affinity chromatography with antisense 2'-O-methyl oligonucleotide without success since the Leishmania telomerase, similarly to Trypanosoma cruzi enzyme, was not eluted by competition, but instead, it remained bound to the column. Partially purified L. amazonensis telomerase activity was achieved by fractionation of extracts on complementary ion exchange and Heparin columns. Further purification of these fractions on a G-rich telomeric DNA affinity chromatography enriched for telomerase activity. The knowledge of telomerase characteristics in Leishmania could help to develop new strategies to overcome leishmaniasis.

DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis.

Replication protein A (RPA) is a single-stranded DNA-binding protein that has been implicated in DNA metabolism and telomere maintenance. Subunit 1 of RPA from Leishmania amazonensis (LaRPA-1) has previously been affinity-purified on a column containing a G-rich telomeric DNA. LaRPA-1 binds and co-localizes with parasite telomeres in vivo. Here we describe the purification and characterization of native recombinant LaRPA-1 (rLaRPA-1). The protein was initially re-solubilized from inclusion bodies by using urea. After dialysis, rLaRPA-1 was soluble but contaminated with DNA, which was removed by an anion-exchange chromatography of the protein solubilized in urea. However, rLaRPA-1 precipitated after dialysis to remove urea. To investigate whether the contaminating DNA was involved in chaperoning the refolding of rLaRPA-1, salmon sperm DNA or heparin was added to the solution before dialysis. The addition of either of these substances prevented the precipitation of rLaRPA-1. The resulting rLaRPA-1 was soluble, correctly folded, and able to bind telomeric DNA. This is the first report showing the characterization of rLaRPA1 and of the importance of additives in chaperoning the refolding of this protein. The availability of rLaRPA-1 should be helpful in assessing the importance of this protein as a potential drug target.

Genomic organization of telomeric and subtelomeric sequences of Leishmania (Leishmania) amazonensis.

Telomeres are DNA-protein complexes that protect linear chromosomes from degradation and fusions. Telomeric DNA is repetitive and G-rich, and protrudes towards the end of the chromosomes as 3'G-overhangs. In Leishmania spp., sequences adjacent to telomeres comprise the Leishmania conserved telomere associated sequences (LCTAS) that are around 100 bp long and contain two conserved sequence elements (CSB1 and CSB2), in addition to non-conserved sequences. The aim of this work was to study the genomic organization of Leishmania (Leishmania) amazonensis telomeric/subtelomeric sequences. Leishmania amazonensis chromosomes were separated in a single Pulsed Field Gel Electrophoresis (PFGE) gel as 25 ethidium bromide-stained bands. All of the bands hybridized with the telomeric probe (5'-TTAGGG-3')3 and with probes generated from the conserved subtelomeric elements (CSB1, CSB2). Terminal restriction fragments (TRF) of L. amazonensis chromosomes were analyzed by hybridizing restriction digested genomic DNA and chromosomal DNA separated in 2D-PFGE with the telomeric probe. The L. amazonensis TRF was estimated to be approximately 3.3 kb long and the telomeres were polymorphic and ranged in size from 0.2 to 1.0 kb. Afa I restriction sites within the conserved CSB1 elements released the telomeres from the rest of the chromosome. Bal 31-sensitive analysis confirmed the presence of terminal Afa I restriction sites and served to differentiate telomeric fragments from interstitial internal sequences. The size of the L. amazonensis 3' G-overhang was estimated by non-denaturing Southern blotting to be approximately 12 nt long. Using similar approaches, the subtelomeric domains CSB1 and CSB2 were found to be present in a low copy number compared to telomeres and were organized in blocks of 0.3-1.5 kb flanked by Hinf I and Hae III restriction sites. A model for the organization of L. amazonensis chromosomal ends is provided.

Telomere biology of trypanosomatids: more questions than answers.

Trypanosomatids are severe pathogens in developing countries, where they affect both humans and domestic animals. Factors intrinsic to the host, the toxicity or subcurative effects of the available antiparasite medication and the low perspective of potential vaccines favor research on novel candidates for drug target. Telomeres are essential for the survival of most eukaryotes. In trypanosomatids, events such as antigenic variation and/or gene conversion and duplication occur at telomeric positions, possibly facilitating genome rearrangement. Understanding the role that telomere maintenance might play in the cell life span of trypanosomatids has important implications for therapeutics of parasitic diseases.

Cloning and characterization of a gene for the stage-specific 82-kDa surface antigen of metacyclic trypomastigotes of Trypanosoma cruzi.

We have cloned and sequenced a cDNA clone coding for a metacyclic trypomastigote-specific surface glycoprotein with a molecular mass of 82 kDa (MTS-gp82). By immunoblotting and immunoprecipitation, antibodies against the recombinant protein recognized an 82-kDa protein of metacyclic trypomastigotes, without any detectable reaction towards amastigotes, epimastigotes or tissue culture-derived trypomastigotes. The insert of the MTS-gp82 cDNA clone strongly hybridized with a single 2.2-kb metacyclic trypomastigote mRNA, suggesting that the steady-state levels of mRNAs for MTS-gp82 are developmentally regulated. MTS-gp82 is encoded by a multigene family whose members are distributed in several chromosomes. Sequence analysis revealed 40-56% identity at amino acid level between MTS-gp82 and members of Trypanosoma cruzi gp85/sialidase family (TSA-1, Tt34c1, SA85-1.1). MTS-gp82 showed several amino acid motifs that are characteristic of gp85/sialidase family, such as the Asp box (SxDxGxTW), the subterminal (VTVxNVFLYNR) motif and the putative GPI-anchor sequence. On the basis of its structural features, the MTS-gp82 gene could be included in the T. cruzi gp85/sialidase family, but constituting a distinct group which is preferentially expressed in metacyclic trypomastigotes.

Cloning and characterization of a gene encoding a novel immunodominant antigen of Trypanosoma cruzi.

A Trypanosoma cruzi genomic expression library was screened with a pool of sera obtained from chronic chagasic patients. The recombinant antigen (Tc40) isolated from this library reacted with a large number of serum samples of chronic chagasic patients, suggesting that the presence of anti-Tc40 antibodies may be specifically associated to Chagas' disease. The full-length sequence of the Tc40 gene was determined after isolation of genomic and cDNA clones. The Tc40 cDNA includes a large open reading frame (2745 bp-long) that encodes a polypeptide of 100 kDa without any homology with previously described T. cruzi sequences. In contrast with other T. cruzi antigens whose immunodominant B-cell epitopes are composed by amino acid repetitive motifs, Tc40 does not show any amino acid repetition. Antibodies against the Tc40 recombinant protein reacted with three native polypeptides of 100, 41 and 38 kDa which are tightly associated with membranes or cytoskeleton and expressed in all developmental stages of the parasite life cycle. A transcript of 3.9-kb was detected in Northern blot analysis which is large enough to encode a 100 kDa polypeptide. Tc40 genes were mapped on a chromosomal band of 1.1 Mbp and in a few copies per haploid genome in the G strain.

Organization and expression of the gene encoding an immunodominant repetitive antigen associated to the cytoskeleton of Trypanosoma cruzi.

We have studied the genomic organization and expression of the gene encoding a high molecular mass (300 kDa) repetitive antigen associated with the cytoskeleton of Trypanosoma cruzi. Protease digestion of the native protein, restriction analysis of genomic DNA and sequencing of genomic and cDNA clones indicated that most of the protein is built up by tandemly arranged, nearly identical repeats of 68 amino acids. The gene size was estimated to be approx. 9.4 kb based on the sizes of the transcript and the native protein. The nucleotide sequence conservation among the repeats indicates that selective sequence homogenization, presumably through gene conversion, maintained the amino-acid sequence conservation. Two duplicated allelic forms of this gene were mapped in fragments of about 20 kb. In some strains an additional allele was located in a fragment of 9.4 kb. Our results suggest that this repetitive antigen is a structural protein which could be involved in the attachment of the flagellum to the cell body.

Incidence of testicular cancer and occupation among Swedish men gainfully employed in 1970.

PURPOSE: To estimate occupation-specific risk of seminomas and nonseminoma subtypes of testicular cancer among Swedish men gainfully employed in 1970 over the period 1971-1989. METHODS: Age-period standardized incidence ratios were computed in a dataset linking cancer diagnoses from the Swedish national cancer register to occupational and demographical data obtained in the census in 1970. Log-linear Poisson models were fitted, allowing for geographical area and town size. Taking occupational sector as a proxy for socioeconomic status, occupational risks were recalculated using intra-sector analyses, where the reference group comprised other occupations in the same sector only. Risk estimators per occupation were also computed for men reporting the same occupation in 1960 and 1970, a more specifically exposed group. RESULTS: Seminomas and nonseminomas showed a substantial geographical variation. The association between germ-cell testicular tumors and high socioeconomic groups was found mainly for nonseminomas. Positive associations with particular occupations were more evident for seminomas, for which railway stationmasters, metal annealers and temperers, precision toolmakers, watchmakers, construction smiths, and typographers and lithographers exhibited a risk excess. Concrete and construction worker was the only occupation consistently associated with nonseminomas. CONCLUSIONS: Among the many occupations studied, our results corroborate the previously reported increased risk among metal workers, specifically related with seminomatous tumors in this study. Our results confirm the geographical and socioeconomical differences in the incidence of testicular tumors. These factors should be accounted for in occupational studies. The different pattern of occupations related with seminomas and nonseminomas support the need to study these tumors separately.

Trypanosoma cruzi genome project: biological characteristics and molecular typing of clone CL Brener.

Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. CL Brener was obtained by cloning procedures from bloodstream trypomastigotes isolated from mice infected with the CL strain. The doubling time of CL Brener epimastigotes cultured at 28 degrees C in liver infusion-tryptose (LIT) medium is 58 +/- 13 h. Differentiation to metacyclic forms is induced by incubation of epimastigotes in LIT-20% Grace's medium. Metacyclics give very low parasitemia in mice, contrary to what is observed for blood forms which promote 100% mortality of the animals with inocula of 5 x 10(3) parasites. CL Brener blood forms are highly susceptible to nifurtimox, benznidazole and ketoconazole. Allopurinol is inefficient in the treatment of mice experimental infection. The clone infects mammalian cultured cells and performs the complete intracellular cycle at 33 and 37 degrees C. The molecular typing of CL Brener has been done by isoenzymatic profiles; sequencing of a 24S alpha ribosomal RNA gene domain and by schizodeme, randomly amplified polymorphic DNA and DNA fingerprinting analyses. For each typing approach the patterns obtained do not change after prolonged parasite subcultivation in LIT medium (up to 100 generations). The stability of the molecular karyotype of the clone was also confirmed.

Coagglutination test (COA) for Cryptococcus neoformans circulating antigen detection in cerebral spinal fluid (CSF).

We tested 82 CSF of 24 renal transplanted patients (RT) with cerebral cryptococcosis, 8 CSF of asymptomatic RT patients, 43 CSF of proven cryptococcosis cases (positive control) and 35 CSF of patients with other diseases (histoplasmosis, paracoccidioidomycosis and bacterial infections) as negative control. The RT CSF were cultured in Sabouraud agar slant added with sunflower seeds and both control and RT CSF were qualitatively examined by cryptococcosis latex test (Crypto-LA test). The COA test was developed both qualitatively and quantitatively. The highest titre encountered was 1:2048. No false reactions appeared among the controls. The diagnostic value demonstrated by Galen and Gambino's method was: sensitivity--92.1%; specificity--92.6% and efficiency--92.3%. Besides that, the COA proved to be quick, exact and cheap, but it depends on CSF and sera pre-treatment, in order to avoid autoagglutination and increase its sensibility.

[Utilization of aminoacids in the study of the growth of Paracoccidioides brasiliensis. Influence on dimorphism]. / Utilização de aminoácidos no estudo do crescimento do Paracoccidioides brasiliensis. Influência sobre o dimorfismo.

Fifteen Paracoccidioides brasiliensis strains, in the mycelial (M) and yeast like (Y), were cultivated in minimal medium (MM) and subcultivated to be adapted to the same medium supplemented with a pool of amino acid in solution (MMS). Each of the amino acids were studied separately of the solution to provide the auxological study. The prototrophism was demonstrated by nine strains in both M and Y forms, and the auxotrophism by the remaining strains. The heterologous results has not allowed us to draw an auxological characterization of the P. brasiliensis. As far as we could observe none of the amino acid studied in this piece of research can be considered of absolute importance for to the growth and the morphogenesis maintainance of the fungus. Morphological alterations were only verified in the prototroph strains, which suggest that there could have been adaptative metabolism activity due to the absence of organic nitrogen compounds in the minimal medium (MM).

[The isolation and evaluation of Aspergillus fumigatus antigens]. / Obtenção e avaliação de antígenos de Aspergillus fumigatus.

Antigens from three strains of Aspergillus fumigatus (354, 356, and JIG) and an antiserum against the mixing of these antigens have been produced, and evaluated immunochemically. The antigens were obtained through a modified Coleman & Kaufman technique (culture filtrate concentrated by acetone). Analysis by the immunodiffusion test (ID) against homologous serum has yielded 100% sensitivity (with the studied sera). Concerning heterologous sera we found reactivity with a serum of a patient of candidiasis and another with histoplasmosis. The same result was obtained with a reference antigen in immunodiffusion, showing similar standards of response. Titration of the antiserum by ID and counterimmunoelectrophoresis showed a title of 1:32, and by complement fixation (micro-technique) a title of 1:128. Using immunoelectrophoresis (IEF), the produced antiserum yielded 8 lines of precipitation (5 in the anodic pole and 3 in the cathodic one). In SDS-PAGE at 12.5% the antigen has presented a rather complex electrophoretic profile (26 proteic subunits with a molecular weight ranging from 18 a > 100 kDa). Immunogenicity of the antigen was observed in all fractions of SDS-PAGE when the immunoblotting against the antiserum was carried out.

The sensitivity, specificity and efficiency values of some serological tests used in the diagnosis of paracoccidioidomycosis.

This work reports on the results of double immunodiffusion (ID), counterimmunoelectrophoresis (CIE), complement fixation (CF) and indirect immunofluorescence (IIF) techniques in the serodiagnosis of paracoccidioidomycosis. The study was undertaken on four groups of individuals: 46 patients with untreated paracoccidioidomycosis, 22 patients with other deep mycoses, 30 with other infectious diseases (tuberculosis and cutaneous leishmaniasis) and 47 blood donors as negative controls. Data were obtained using Paracoccidioides brasiliensis antigens, i.e., a yeast culture filtrate for ID, CIE and CF, and a yeast cell suspension for IIF. The sensitivity, specificity and efficiency values were measured according to GALEN & GAMBINO. The gel precipitation tests (ID and CIE) showed the greatest sensitivity (91.3 and 95.6%, respectively), maximum specificity (100%) and the highest efficiency values when compared to the CF and IIF tests.

RPA-1 from Leishmania amazonensis (LaRPA-1) structurally differs from other eukaryote RPA-1 and interacts with telomeric DNA via its N-terminal OB-fold domain.

Replication protein A-1 (RPA-1) is a single-stranded DNA-binding protein involved in DNA metabolism. We previously demonstrated the interaction between LaRPA-1 and telomeric DNA. Here, we expressed and purified truncated mutants of LaRPA-1 and used circular dichroism measurements and molecular dynamics simulations to demonstrate that the tertiary structure of LaRPA-1 differs from human and yeast RPA-1. LaRPA-1 interacts with telomeric ssDNA via its N-terminal OB-fold domain, whereas RPA from higher eukaryotes show different binding modes to ssDNA. Our results show that LaRPA-1 is evolutionary distinct from other RPA-1 proteins and can potentially be used for targeting trypanosomatid telomeres.

LaRbp38: a Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs.

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.