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.

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.

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.

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.

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.

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.

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA.

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres.

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.

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.

Non-Hodgkin's lymphomas and occupation in Sweden.

OBJECTIVES: To investigate whether there is a risk excess of non-Hodgkin's lymphoma among Swedish workers associated with particular occupations. METHODS: The base population was made up of Swedish men (1,779,646) and women (1,101,669) who were gainfully employed at the time of the 1970 census, had also been present in the 1960 census and were still alive and older than 24 years as of 1 January, 1971. They were followed up for 19 years until the end of 1989. Age-period standardised incidence ratios were computed in a dataset linking cancer diagnoses from the Swedish national cancer register to occupational and demographic data obtained in the census of 1970. Log-linear Poisson models were fitted, allowing for geographical area. Risk estimators per occupation were also computed for workers reporting the same occupation in 1960 and 1970, a more specifically exposed group. RESULTS: There were 7,610 non-Hodgkin's lymphomas reported in the study cohort, 5,391 cases in men and 2,219 in women. A relative risk of over 1.20 and statistically significant was observed in men among accountants and auditors, secretaries and typists, auctionists, non-specified rail and road transport workers, telecommunications traffic officers, telegraph and radio operators, photographic-laboratory workers and other production and related work. The risk excess was confirmed in men with the same occupation in both censuses. In women, only three occupations achieved statistical significance: metal platers and coaters, truck and conveyor operators and store and warehouse workers. CONCLUSIONS: The risk excess observed in telecommunication and transport workers could be explained by electromagnetic radiation exposure. We did not find a risk excess in agricultural occupations, that has been largely documented by other study groups.

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.

Telomerase in kinetoplastid parasitic protozoa.

We have identified telomerase activity in extracts of three evolutionarily diverse kinetoplastid species: Trypanosoma brucei, Leishmania major, and Leishmania tarentolae. Telomerase activity was initially detected in extracts from insect form cells of all three kinetoplastid species by using a modification of the one-tube telomere repeat amplification protocol [Kim, N., et al. (1994) Science 266, 2011-2015], although better results were subsequently achieved with the two-tube telomere repeat amplification protocol [Autexier, C., Pruzan, R., Funk, W. & Greider, C. (1996) EMBO J. 15, 5928-5935]. The activity in T. brucei extracts was sufficiently robust to enable its detection in a direct assay of telomerase; enzyme processivity was found to be relatively low. The in vitro properties of telomerase suggest a possible templating domain sequence for the telomerase RNA of T. brucei. Telomerase activity is likely to contribute to telomere maintenance in these parasitic organisms and provides a new target for chemotherapeutic intervention.

Electrophoretic karyotypes and genome sizing of the pathogenic fungus Paracoccidioides brasiliensis.

Here we present the karyotype analysis and genome sizing of Paracoccidioides brasiliensis, a pathogen refractory to conventional genetic analysis. We have established pulsed-field gel electrophoresis (PFGE) conditions to resolve the high-molecular-weight chromosomal bands of two clinical isolates of P. brasiliensis. Both isolates showed four megabase-sized bands, ranging from 2.0 to 10.0 Mbp. Significant differences in chromosome sizes and in the chromosomal location of genes for the gp43 antigen and chitin synthase were found. Different technical approaches were employed to estimate the DNA content and to define the ploidy of P. brasiliensis. An estimated genome size in the range of 45.7 to 60.9 Mbp was provided by the analysis of data generated by measuring the amplitude of fluorescence intensity of DAPI (4',6-diamidino-2-phenylindole)-stained nuclei (by confocal microscopy). The nuclear genome size estimated by confocal microscopy is twice that estimated by the average sum of the molecular weight of chromosome-sized DNA molecules by PFGE, suggesting that each separated P. brasiliensis chromosomal band is diploid.

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.

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.

[Prevalence of asthma, rhinitis and eczema in school children in the city of Cuernavaca, Mexico]. / Prevalencia de asma, rinitis y eczema en escolares de la ciudad de Cuernavaca, México.

OBJECTIVE: The measurement of asthma, rhinitis and eczema have been subject of controversy due to lack of a standardized methodology. To test the applicability of a standardized methodology for comparisons of time and space we determined the prevalence of asthma and other allergic diseases in a random sample of schoolchildren (n = 6,238) from 6 to 8 and 11 to 14 years of age living in Cuernavaca, Morelos, Mexico. MATERIAL AND METHODS: The methodology proposed by the International Study of Asthma and Allergies in Childhood (ISAAC) to determine prevalence and severity of asthma, rhinitis and eczema was applied. Current and accumulated information on prevalence was obtained by means of a standardized questionnaire answered by the children's parents. RESULTS: The accumulated prevalence of asthma by medical diagnosis and wheezing was 5.8% (5.2-6.4) and 21.8% (20.7-22.9) respectively; prevalence of wheezing in the last 12 months was 8.9% in the group of 6 to 8 years against 6.6% in the 11 to 14 year old group p < 0.001. Prevalence of the medical diagnosis of rhinitis was 4.9% (4.3-5.5). Regarding the typical symptoms of rhinitis, in the last 12 months prevalence was 9.6% (6-8 years) and 10.1% (11-14 years). Prevalence of eczema by medical diagnosis was 4.1% (3.6-4.6). Prevalence of eczema symptoms in the last 12 months was 10.1% (6-8 years) and 10.6% (11-14 years). Prevalence of severe asthma symptoms was significantly higher in the 6 to 8 year olds and in the autumn. CONCLUSIONS: Prevalence of asthma by medical diagnosis and by symptoms is relatively low with respect to other studies performed with the same methodology. The benefits of using a standardized methodology were analyzed.

The Trypanosoma cruzi genome project: nuclear karyotype and gene mapping of clone CL Brener.

By using improved pulsed field gel electrophoresis conditions, the molecular karyotype of the reference clone CL Brener selected for Trypanosoma cruzi genome project was established. A total of 20 uniform chromosomal bands ranging in size from 0.45 to 3.5 Megabase pairs (Mbp) were resolved in a single run. The weighted sum of the chromosomal bands was approximately 87 Mbp. Chromoblots were hybridized with 39 different homologous probes, 13 of which identified single chromosomes. Several markers showed linkage and four different linkage groups were identified, each comprising two markers. Densitometric analysis suggests that most of the chromosomal bands contain two or more chromosomes representing either homologous chromosomes and/or heterologous chromosomes with similar sizes.