Auto repair workers exposed to PM2.5 particulate matter in Barranquilla, Colombia: Telomere length and hematological parameters.

Exposure to 2.5 µm particulate matter (PM2.5) in automotive repair shops is associated with risks to health. We evaluated the effects of occupational exposure to PM2.5 among auto repair-shop workers. Blood and urine samples were collected from 110 volunteers from Barranquilla, Colombia: 55 active workers and 55 controls. PM2.5 concentrations were assessed at each of the sampling sites and chemical content was analyzed by SEM-EDS electron microscopy. The biological samples obtained were peripheral blood (hematological profiling, DNA extraction) and urine (malondialdehyde concentration). Telomere length was assessed by qPCR and polymorphisms in the glutathione transferase genes GSTT1 and GSTM1 by PCR-RFLP, with confirmation by allelic exclusion. White blood cell (WBC), lymphocyte (LYM%) and platelet (PLT) counts and the malondialdehyde concentration were higher (4.10 ± 0.93) in the exposed group compared to the control group (1.56 ± 0.96). TL was shorter (5071 ± 891) in the exposed individuals compared to the control group (6271 ± 805). White blood cell (WBC) and platelet counts were positively associated with exposure. Age and TBARS were correlated with TL in exposed individuals. The GSTT1 gene alleles were not in Hardy-Weinberg (H-W) equilibrium. The GSTM1 gene alleles were in H-W equilibrium and allelic exclusion analysis confirmed the presence of heterozygous GSTM1 genotypes. SEM-EDS analysis showed the presence of potentially toxic elements, including Mg, Al, Fe, Mn, Rh, Zn, and Cu. Auto repair shop workers showed effects that may be associated with exposure to mixtures of pollutants present in PM2.5. The GSTM1 and GSTT1 genes had independent modulatory effects.

Comparative Analysis of In-House RT-qPCR Detection of SARS-CoV-2 for Resource-Constrained Settings.

We developed and standardized an efficient and cost-effective in-house RT-PCR method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated sensitivity, specificity, and other statistical parameters by different RT-qPCR methods including triplex, duplex, and simplex assays adapted from the initial World Health Organization- (WHO) recommended protocol. This protocol included the identification of the E envelope gene (E gene; specific to the Sarvecovirus genus), RdRp gene of the RNA-dependent RNA polymerase (specific for SARS-CoV-2), and RNase P gene as endogenous control. The detection limit of the E and the RdRp genes were 3.8 copies and 33.8 copies per 1 µL of RNA, respectively, in both triplex and duplex reactions. The sensitivity for the RdRp gene in the triplex and duplex RT-qPCR tests were 98.3% and 83.1%, respectively. We showed a decrease in sensitivity for the RdRp gene by 60% when the E gene acquired Ct values > 31 in the diagnostic tests. This is associated with the specific detection limit of each gene and possible interferences in the protocol. Hence, developing efficient and cost-effective methodologies that can be adapted to various health emergency scenarios is important, especially in developing countries or settings where resources are limited.

Virucidal Activity of Different Mouthwashes Using a Novel Biochemical Assay.

BACKGROUND: Saliva of patients with COVID-19 has a high SARS-CoV-2 viral load. The risk of spreading the virus is not insignificant, and procedures for reducing viral loads in the oral cavity have been proposed. Little research to date has been performed on the effect of mouthwashes on the SARS-CoV-2 virus, and some of their mechanisms of action remain unknown. METHODS: SARS-CoV-2 positive nasopharyngeal swabs measured by RT-PCR were used for virucidal activity in a 1:1 ratio, with an incubation time of 1 min. The solutions used in this study were: iodopovidone (8 mg); * D-limonene, a terpene extracted from citrus peels (0.3%); † cetylpyridinium chloride (0.1%) (CPC); ‡ chlorhexidine gluconate (10%) (CHX); § a CPC (0.12%) and CHX (0.05%) containing formula; ** a formula containing essential oils; †† a CPC containing formula (0.07%); ‡‡ a D-limonene (0.2%) and CPC (0.05%) containing formula; §§ a solution containing sodium fluoride (0.05%) and CPC (0.075%); *** a solution containing CHX (0.12%) and; ††† a CHX (0.2%) containing formula. ‡‡‡ As a control reaction, saline solution or excipient solution (water, glycerin, citric acid, colorant, sodium citrate) was used. CONCLUSION: Within the limitations of this study, we can conclude that a mouthwash containing both D-limonene and CPC reduced the virucidal activity in about 6 logs (>99.999% reduction). Hence, establishing a clinical protocol for dentists is suggested, where all patients to be treated rinse pre-operatively with a mouthwash containing both D-limonene and CPC to reduce the likelihood of infection with SARS-CoV-2 for dentists. This is a relatively inexpensive way to reduce viral transmission of SARS-CoV-2 from infected individuals within the community. It is also a simple way to decrease infections from asymptomatic and pre-symptomatic patients.

Anomaly Identification during Polymerase Chain Reaction for Detecting SARS-CoV-2 Using Artificial Intelligence Trained from Simulated Data.

Real-time reverse transcription (RT) PCR is the gold standard for detecting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), owing to its sensitivity and specificity, thereby meeting the demand for the rising number of cases. The scarcity of trained molecular biologists for analyzing PCR results makes data verification a challenge. Artificial intelligence (AI) was designed to ease verification, by detecting atypical profiles in PCR curves caused by contamination or artifacts. Four classes of simulated real-time RT-PCR curves were generated, namely, positive, early, no, and abnormal amplifications. Machine learning (ML) models were generated and tested using small amounts of data from each class. The best model was used for classifying the big data obtained by the Virology Laboratory of Simon Bolivar University from real-time RT-PCR curves for SARS-CoV-2, and the model was retrained and implemented in a software that correlated patient data with test and AI diagnoses. The best strategy for AI included a binary classification model, which was generated from simulated data, where data analyzed by the first model were classified as either positive or negative and abnormal. To differentiate between negative and abnormal, the data were reevaluated using the second model. In the first model, the data required preanalysis through a combination of prepossessing. The early amplification class was eliminated from the models because the numbers of cases in big data was negligible. ML models can be created from simulated data using minimum available information. During analysis, changes or variations can be incorporated by generating simulated data, avoiding the incorporation of large amounts of experimental data encompassing all possible changes. For diagnosing SARS-CoV-2, this type of AI is critical for optimizing PCR tests because it enables rapid diagnosis and reduces false positives. Our method can also be used for other types of molecular analyses.

Nucleocapsid Protein Precursors NCp9 and NCp15 Suppress ATP-Mediated Rescue of AZT-Terminated Primers by HIV-1 Reverse Transcriptase.

In HIV-1, development of resistance to AZT (3'-azido-3'-deoxythymidine) is mediated by the acquisition of thymidine analogue resistance mutations (TAMs) (i.e., M41L, D67N, K70R, L210W, T215F/Y, and K219E/Q) in the viral reverse transcriptase (RT). Clinically relevant combinations of TAMs, such as M41L/T215Y or D67N/K70R/T215F/K219Q, enhance the ATP-mediated excision of AZT monophosphate (AZTMP) from the 3' end of the primer, allowing DNA synthesis to continue. Additionally, during HIV-1 maturation, the Gag polyprotein is cleaved to release a mature nucleocapsid protein (NCp7) and two intermediate precursors (NCp9 and NCp15). NC proteins interact with the viral genome and facilitate the reverse transcription process. Using wild-type and TAM-containing RTs, we showed that both NCp9 and NCp15 inhibited ATP-mediated rescue of AZTMP-terminated primers annealed to RNA templates but not DNA templates, while NCp7 had no effect on rescue activity. RNase H inactivation by introducing the active-site mutation E478Q led to the loss of the inhibitory effect shown by NCp9. NCp15 had a stimulatory effect on the RT's RNase H activity not observed with NCp7 and NCp9. However, analysis of RNase H cleavage patterns revealed that in the presence of NCp9, RNA/DNA complexes containing duplexes of 12 bp had reduced stability in comparison with those obtained in the absence of NC or with NCp7 or NCp15. These effects are expected to have a strong influence on the inhibitory action of NCp9 and NCp15 by affecting the efficiency of RNA-dependent DNA polymerization after unblocking DNA primers terminated with AZTMP and other nucleotide analogues.

Genome Sequence of the Siphoviridae Staphylococcus aureus Phage vB_SauS_BaqSau1.

Here, we report the genome sequence of a Siphoviridae phage named vB_SauS_BaqSau1 (BaqSau1), infecting Staphylococcus aureus Phage BaqSau1 was isolated from a sewage water treatment plant in Sahagún, Córdoba, Colombia. It has a double-stranded DNA (dsDNA) genome of 44,384 bp with 67 predicted genes, including a lysin containing a CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain.

A role of template cleavage in reduced excision of chain-terminating nucleotides by human immunodeficiency virus type 1 reverse transcriptase containing the M184V mutation.

Resistance to nucleoside reverse transcriptase (RT) inhibitors is conferred on human immunodeficiency virus type 1 through thymidine analogue resistance mutations (TAMs) that increase the ability of RT to excise chain-terminating nucleotides after they have been incorporated. The RT mutation M184V is a potent suppressor of TAMs. In RT containing TAMs, the addition of M184V suppressed the excision of 3'-deoxy-3'-azidothymidine monophosphate (AZTMP) to a greater extent on an RNA template than on a DNA template with the same sequence. The catalytically inactive RNase H mutation E478Q abolished this difference. The reduction in excision activity was similar with either ATP or pyrophosphate as the acceptor substrate. Decreased excision of AZTMP was associated with increased cleavage of the RNA template at position -7 relative to the primer terminus, which led to increased primer-template dissociation. Whether M184V was present or not, RT did not initially bind at the -7 cleavage site. Cleavage at the initial site was followed by RT dissociation and rebinding at the -7 cleavage site, and the dissociation and rebinding were enhanced when the M184V mutation was present. In contrast to the effect of M184V, the K65R mutation suppressed the excision activity of RT to the same extent on either an RNA or a DNA template and did not alter the RNase H cleavage pattern. Based on these results, we propose that enhanced RNase H cleavage near the primer terminus plays a role in M184V suppression of AZT resistance, while K65R suppression occurs through a different mechanism.

The Role of Nucleotide Excision by Reverse Transcriptase in HIV Drug Resistance.

Nucleoside reverse transcriptase (RT) inhibitors of HIV block viral replication through the ability of HIV RT to incorporate chain-terminating nucleotide analogs during viral DNA synthesis. Once incorporated, the chain-terminating residue must be removed before DNA synthesis can continue. Removal can be accomplished by the excision activity of HIV RT, which catalyzes the transfer of the 3'-terminal residue on the blocked DNA chain to an acceptor substrate, probably ATP in most infected cells. Mutations of RT that enhance excision activity are the most common cause of resistance to 3'-azido-3'-deoxythymidine (AZT) and exhibit low-level cross-resistance to most other nucleoside RT inhibitors. The resistance to AZT is suppressed by a number of additional mutations in RT, most of which were identified because they conferred resistance to other RT inhibitors. Here we review current understanding of the biochemical mechanisms responsible for increased or decreased excision activity due to these mutations.