Comprehensive study reveals phenotypic heterogeneity in Klebsiella pneumoniae species complex isolates.

Here, we conducted a comprehensive analysis of 356 Klebsiella pneumoniae species complex (KpSC) isolates that were classified as classical (cl), presumptive hypervirulent (p-hv) and hypermucoviscous-like (hmv-like). Overall, K. pneumoniae (82.3%), K. variicola (2.5%) and K. quasipneumoniae (2.5%) were identified. These isolates comprised 321 cl-KpSC, 7 p-hv-KpSC and 18 hmv-like-KpSC. A large proportion of cl-KpSC isolates were extended-spectrum-ß-lactamases (ESBLs)-producers (64.4%) and 3.4% of isolates were colistin-resistant carrying carbapenemase and ESBL genes. All p-hv-KpSC showed an antibiotic susceptible phenotype and hmv-like isolates were found to be ESBL-producers (8/18). Assays for capsule production and capsule-dependent virulence phenotypes and whole-genome sequencing (WGS) were performed in a subset of isolates. Capsule amount differed in all p-hv strains and hmv-like produced higher capsule amounts than cl strains; these variations had important implications in phagocytosis and virulence. Murine sepsis model showed that most cl strains were nonlethal and the hmv-like caused 100% mortality with 3 × 108 CFUs. Unexpectedly, 3/7 (42.9%) of p-hv strains required 108 CFUs to cause 100% mortality (atypical hypervirulent), and 4/7 (57.1%) strains were considered truly hypervirulent (hv). Genomic analyses confirmed the diverse population, including isolates belonging to hv clonal groups (CG) CG23, CG86, CG380 and CG25 (this corresponded to the ST3999 a novel hv clone) and MDR clones such as CG258 and CG147 (ST392) among others. We noted that the hmv-like and hv-ST3999 isolates showed a close phylogenetic relationship with cl-MDR K. pneumoniae. The information collected here is important to understand the evolution of clinically important phenotypes such as hypervirulent and ESBL-producing-hypermucoviscous-like amongst the KpSC in Mexican healthcare settings. Likewise, this study shows that mgrB inactivation is the main mechanism of colistin resistance in K. pneumoniae isolates from Mexico.

An experimental census of retrons for DNA production and genome editing.

Retrons are bacterial immune systems that use reverse transcribed DNA as a detector of phage infection. They are also increasingly deployed as a component of biotechnology. For genome editing, for instance, retrons are modified so that the reverse transcribed DNA (RT-DNA) encodes an editing donor. Retrons are commonly found in bacterial genomes; thousands of unique retrons have now been predicted bioinformatically. However, only a small number have been characterized experimentally. Here, we add substantially to the corpus of experimentally studied retrons. We synthesized >100 previously untested retrons to identify the natural sequence of RT-DNA they produce, quantify their RT-DNA production, and test the relative efficacy of editing using retron-derived donors to edit bacterial genomes, phage genomes, and human genomes. We add 62 new empirically determined, natural RT-DNAs, which are not predictable from the retron sequence alone. We report a large diversity in RT-DNA production and editing rates across retrons, finding that top performing editors outperform those used in previous studies, and are drawn from a subset of the retron phylogeny.

Automated EuroFlow approach for standardized in-depth dissection of human circulating B-cells and plasma cells.

Background: Multiparameter flow cytometry (FC) immunophenotyping is a key tool for detailed identification and characterization of human blood leucocytes, including B-lymphocytes and plasma cells (PC). However, currently used conventional data analysis strategies require extensive expertise, are time consuming, and show limited reproducibility. Objective: Here, we designed, constructed and validated an automated database-guided gating and identification (AGI) approach for fast and standardized in-depth dissection of B-lymphocyte and PC populations in human blood. Methods: For this purpose, 213 FC standard (FCS) datafiles corresponding to umbilical cord and peripheral blood samples from healthy and patient volunteers, stained with the 14-color 18-antibody EuroFlow BIgH-IMM panel, were used. Results: The BIgH-IMM antibody panel allowed identification of 117 different B-lymphocyte and PC subsets. Samples from 36 healthy donors were stained and 14 of the datafiles that fulfilled strict inclusion criteria were analysed by an expert flow cytometrist to build the EuroFlow BIgH-IMM database. Data contained in the datafiles was then merged into a reference database that was uploaded in the Infinicyt software (Cytognos, Salamanca, Spain). Subsequently, we compared the results of manual gating (MG) with the performance of two classification algorithms -hierarchical algorithm vs two-step algorithm- for AGI of the cell populations present in 5 randomly selected FCS datafiles. The hierarchical AGI algorithm showed higher correlation values vs conventional MG (r2 of 0.94 vs. 0.88 for the two-step AGI algorithm) and was further validated in a set of 177 FCS datafiles against conventional expert-based MG. For virtually all identifiable cell populations a highly significant correlation was observed between the two approaches (r2>0.81 for 79% of all B-cell populations identified), with a significantly lower median time of analysis per sample (6 vs. 40 min, p=0.001) for the AGI tool vs. MG, respectively and both intra-sample (median CV of 1.7% vs. 10.4% by MG, p<0.001) and inter-expert (median CV of 3.9% vs. 17.3% by MG by 2 experts, p<0.001) variability. Conclusion: Our results show that compared to conventional FC data analysis strategies, the here proposed AGI tool is a faster, more robust, reproducible, and standardized approach for in-depth analysis of B-lymphocyte and PC subsets circulating in human blood.

Genome sequence of a virulent and hypermucoviscous-like Klebsiella michiganensis clinical isolate.

OBJECTIVES: The hypermucoviscous-like phenotype has been described in Klebsiella pneumoniae species complex (KpSC) and was described as a contributor of increased virulence. This study described the characterization and whole-genome sequencing of an antibiotic susceptible and hypermucoviscous-like Klebsiella michiganensis 9273 clinical isolate. DATA DESCRIPTION: Here, we report the genome sequence of a K. michiganensis clinical isolate obtained from a urinary tract infection exhibiting the hypermucoviscous-like phenotype. The draft genome sequence consisted of 145 contigs and ~ 6.6 Mb genome size. The annotation revealed 6648 coding DNA sequences and 56 tRNA genes. The strain belongs to the sequence type (ST) 50, and the OXY-1 beta-lactam resistance gene, aph(3')-Ia gene for aminoglycoside resistance and multidrug efflux pumps were identified. The fyuA siderophore receptor of yersiniabactin siderophore was identified. Increased virulence was observed in Galleria mellonella larvae model and increased capsule production was determined by uronic acid quantification. The clinical implications of this phenotype are unknown, but the patient outcome might worsen compared to susceptible- or MDR-classical K. michiganensis isolates.

Simultaneous multi-site editing of individual genomes using retron arrays.

Our understanding of genomics is limited by the scale of our genomic technologies. While libraries of genomic manipulations scaffolded on CRISPR gRNAs have been transformative, these existing approaches are typically multiplexed across genomes. Yet much of the complexity of real genomes is encoded within a genome across sites. Unfortunately, building cells with multiple, non-adjacent precise mutations remains a laborious cycle of editing, isolating an edited cell, and editing again. Here, we describe a technology for precisely modifying multiple sites on a single genome simultaneously. This technology - termed a multitron - is built from a heavily modified retron, in which multiple donor-encoding msds are produced from a single transcript. The multitron architecture is compatible with both recombineering in prokaryotic cells and CRISPR editing in eukaryotic cells. We demonstrate applications for this approach in molecular recording, genetic element minimization, and metabolic engineering.

Objective measured physical activity and metabolic syndrome score in children and adolescents: The UP&DOWN longitudinal study.

INTRODUCTION: We aimed to analyze the cross-sectional and longitudinal association of physical activity (PA) levels and PA patterns with metabolic syndrome score (MetS) in children and adolescents. METHODS: A total of 175 children (82 females) and 188 adolescents (95 females) were included. Objective PA levels and patterns were determined by accelerometry. MetS was computed from waist circumference, systolic blood pressure, triglycerides, high-density lipoprotein cholesterol, and glucose levels. Different linear regression models were implemented to examine the associations of PA with MetS. RESULTS: Vigorous PA, moderate-vigorous PA, number of bouts per day in 10 min (N10), and total time in bouts per day in 10 min (T10) were negatively associated with MetS in male children and adolescents at cross-sectional level (ß ranging from -0.005 to -0.164, all p < 0.05). Total time in bouts per day in 20 min in male children, and vigorous PA and N10 in female children were longitudinally and negatively associated with MetS (ß ranging from -0.011 to -0.247, all p < 0.05). CONCLUSIONS: Associations of PA and MetS were observed at cross-sectional level in males and longitudinally in female children. The associations in PA patterns were found when patterns were grouped into bouts of 10 min. Therefore, for future studies of PA with health markers in the pediatric population, it would be advisable to choose bouts of shorter duration.

Temporally resolved transcriptional recording in E. coli DNA using a Retro-Cascorder.

Biological signals occur over time in living cells. Yet most current approaches to interrogate biology, particularly gene expression, use destructive techniques that quantify signals only at a single point in time. A recent technological advance, termed the Retro-Cascorder, overcomes this limitation by molecularly logging a record of gene expression events in a temporally organized genomic ledger. The Retro-Cascorder works by converting a transcriptional event into a DNA barcode using a retron reverse transcriptase and then storing that event in a unidirectionally expanding clustered regularly interspaced short palindromic repeats (CRISPR) array via acquisition by CRISPR-Cas integrases. This CRISPR array-based ledger of gene expression can be retrieved at a later point in time by sequencing. Here we describe an implementation of the Retro-Cascorder in which the relative timing of transcriptional events from multiple promoters of interest is recorded chronologically in Escherichia coli populations over multiple days. We detail the molecular components required for this technology, provide a step-by-step guide to generate the recording and retrieve the data by Illumina sequencing, and give instructions for how to use custom software to infer the relative transcriptional timing from the sequencing data. The example recording is generated in 2 d, preparation of sequencing libraries and sequencing can be accomplished in 2-3 d, and analysis of data takes up to several hours. This protocol can be implemented by someone familiar with basic bacterial culture, molecular biology and bioinformatics. Analysis can be minimally run on a personal computer.

Continuous Multiplexed Phage Genome Editing Using Recombitrons.

Bacteriophages, which naturally shape bacterial communities, can be co-opted as a biological technology to help eliminate pathogenic bacteria from our bodies and food supply1. Phage genome editing is a critical tool to engineer more effective phage technologies. However, editing phage genomes has traditionally been a low efficiency process that requires laborious screening, counter selection, or in vitro construction of modified genomes2. These requirements impose limitations on the type and throughput of phage modifications, which in turn limit our knowledge and potential for innovation. Here, we present a scalable approach for engineering phage genomes using recombitrons: modified bacterial retrons3 that generate recombineering donor DNA paired with single stranded binding and annealing proteins to integrate those donors into phage genomes. This system can efficiently create genome modifications in multiple phages without the need for counterselection. Moreover, the process is continuous, with edits accumulating in the phage genome the longer the phage is cultured with the host, and multiplexable, with different editing hosts contributing distinct mutations along the genome of a phage in a mixed culture. In lambda phage, as an example, recombitrons yield single-base substitutions at up to 99% efficiency and up to 5 distinct mutations installed on a single phage genome, all without counterselection and only a few hours of hands-on time.

Recent advances and challenges in temperature monitoring and control in microfluidic devices.

Temperature is a critical-yet sometimes overlooked-parameter in microfluidics. Microfluidic devices can experience heating inside their channels during operation due to underlying physicochemical phenomena occurring therein. Such heating, whether required or not, must be monitored to ensure adequate device operation. Therefore, different techniques have been developed to measure and control temperature in microfluidic devices. In this contribution, the operating principles and applications of these techniques are reviewed. Temperature-monitoring instruments revised herein include thermocouples, thermistors, and custom-built temperature sensors. Of these, thermocouples exhibit the widest operating range; thermistors feature the highest accuracy; and custom-built temperature sensors demonstrate the best transduction. On the other hand, temperature control methods can be classified as external- or integrated-methods. Within the external methods, microheaters are shown to be the most adequate when working with biological samples, whereas Peltier elements are most useful in applications that require the development of temperature gradients. In contrast, integrated methods are based on chemical and physical properties, structural arrangements, which are characterized by their low fabrication cost and a wide range of applications. The potential integration of these platforms with the Internet of Things technology is discussed as a potential new trend in the field.

Fitness, body composition, and metabolic risk scores in children and adolescents: the UP&DOWN study.

We aimed to analyse the longitudinal association between physical fitness (PF) and body composition (BC) with a metabolic risk score (Met4) in children and adolescents and to elucidate whether the association between PF and Met4 differs when using relativized or absolute fitness variables. A total of 188 children (86 females) and 195 adolescents (97 females) were included. Cardiorespiratory fitness (CRF) was determined by the 20-m shuttle run test, and muscular fitness (MF) was determined by hand grip and standing long jump tests. Height and weight were measured, and the body mass index (Kg/m2) was calculated. Triceps and subscapular skinfolds were assessed to compute body fat percentage. Met4 was computed from systolic blood pressure, triglycerides, high-density lipoprotein cholesterol, and glucose levels. Relative CRF was longitudinally and negatively associated with Met4 in female children (ß = -0.031, p = 0.025), while absolute CRF was positively associated with Met4 in male children and adolescents (ß = 0.000, p < 0.05). Relative upper and lower-body MF were longitudinally and negatively associated with Met4 in female adolescents (ß = -1.347, ß = -0.005, p < 0.05), while absolute lower-body MF was positively associated with Met4 in male children (ß = 0.000, p = 0.019). BC was longitudinally and positively associated with Met4 in male children (ß-ranging from 0.011 to 0.055, all p < 0.05) and male adolescents (ß-ranging from 0.011 to 0.046, all p < 0.05).  Conclusion: BC is more strongly associated with Met4 than PF in children and adolescents. An optimal body weight status should be considered the main objective of health-promoting programs at childhood and adolescence. Furthermore, the way of expressing the fitness variables determines the direction of the association with Met4. What is Known: • Physical fitness is an important health indicator in children and adolescents, with great amount of previous evidence supporting the preventive role of maintaining optimal levels of both cardiorespiratory and muscular fitness for future cardiometabolic issues. What is New: • The way of reporting physical fitness variables can affect the associations between physical fitness features and cardiometabolic outcomes. Since body composition variables have a great impact on both physical fitness and cardiometabolic health, relativizing physical fitness performance by body composition could lead to erroneous conclusions.

Breast milk preservation: thermal and non-thermal processes and their effect on microorganism inactivation and the content of bioactive and nutritional compounds.

Human Breast Milk (HBM) is widely acknowledged as the best nutritional source for neonates. Data indicates that, in 2019, 83.2% of infants in the United States received breast milk at birth, slightly reducing to 78.6% at 1 month. Despite these encouraging early figures, exclusive breastfeeding rates sharply declined, dropping to 24.9% by 6 months. This decline is particularly pronounced when direct breastfeeding is challenging, such as in Neonatal Intensive Care Units (NICU) and for working mothers. Given this, it is vital to explore alternative breast milk preservation methods. Technologies like Holder Pasteurization (HoP), High-Temperature Short-Time Pasteurization (HTST), High-Pressure Processing (HPP), UV radiation (UV), and Electric Pulses (PEF) have been introduced to conserve HBM. This review aims to enhance the understanding of preservation techniques for HBM, supporting the practice of extended exclusive breastfeeding. It explicitly addresses microbial concerns, focusing on critical pathogens like Staphylococcus aureus, Enterococcus, Escherichia coli, Listeria monocytogenes, and Cytomegalovirus, and explores how various preservation methods can mitigate these risks. Additionally, the review highlights the importance of retaining the functional elements of HBM, particularly its immunological components such as antibodies and enzymes like lysozyme and Bile Salt Stimulated Lipase (BSSL). The goal is to provide a comprehensive overview of the current state of HBM treatment, critically assess existing practices, identify areas needing improvement, and advocate for extended exclusive breastfeeding due to its vital role in ensuring optimal nutrition and overall health in infants.

Development of a standardized and validated flow cytometry approach for monitoring of innate myeloid immune cells in human blood.

Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings (11-color; 13 antibodies) and translational research (14-color; 16 antibodies). Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and in vitro functional assays, as well as for assessing the impact of sample preparation procedures and conditions. The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated (vs. manual "expert-based") gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings.

Genetic diversity and spatiotemporal dynamics of DENV-1 and DENV-2 infections during the 2012-2013 outbreak in Mexico.

Dengue fever is caused by four related dengue virus serotypes, DENV-1 to DENV-4, where each serotype comprises distinct genotypes and lineages. The last major outbreak in Mexico occurred during 2012 and 2013, when 112,698 confirmed cases were reported (DENV-1 and DENV-2 were predominant). Following partial E, NS2A and NS5 gene sequencing, based on the virus genome variability, we analyzed 396 DENV-1 and 248 DENV-2 gene sequences from serum samples from dengue acute clinical cases from 13 Mexican states, Mutations were identified, and their genetic variability estimated, along with their evolutionary relationship with DENV sequences sampled globally. DENV-1 genotype V and DENV-2 Asian-American genotype V were the only genotypes circulating during the outbreak. Mutations in NS2A and NS5 proteins were widely disseminated and suggested local emergence of new lineages. Phylogeographic analysis suggested viral spread occurred from coastal regions, and tourist destinations, such as Yucatan and Quintana Roo, which played important roles in disseminating these lineages.

UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions.

Reverse transcriptases (RTs) are enzymes capable of synthesizing DNA using RNA as a template. Within the last few years, a burst of research has led to the discovery of novel prokaryotic RTs with diverse antiviral properties, such as DRTs (Defense-associated RTs), which belong to the so-called group of unknown RTs (UG) and are closely related to the Abortive Infection system (Abi) RTs. In this work, we performed a systematic analysis of UG and Abi RTs, increasing the number of UG/Abi members up to 42 highly diverse groups, most of which are predicted to be functionally associated with other gene(s) or domain(s). Based on this information, we classified these systems into three major classes. In addition, we reveal that most of these groups are associated with defense functions and/or mobile genetic elements, and demonstrate the antiphage role of four novel groups. Besides, we highlight the presence of one of these systems in novel families of human gut viruses infecting members of the Bacteroidetes and Firmicutes phyla. This work lays the foundation for a comprehensive and unified understanding of these highly diverse RTs with enormous biotechnological potential.

CRISPR/Cas9-Mediated Allele-Specific Disruption of a Dominant COL6A1 Pathogenic Variant Improves Collagen VI Network in Patient Fibroblasts.

Collagen VI-related disorders are the second most common congenital muscular dystrophies for which no treatments are presently available. They are mostly caused by dominant-negative pathogenic variants in the genes encoding α chains of collagen VI, a heteromeric network forming collagen; for example, the c.877G>A; p.Gly293Arg COL6A1 variant, which alters the proper association of the tetramers to form microfibrils. We tested the potential of CRISPR/Cas9-based genome editing to silence or correct (using a donor template) a mutant allele in the dermal fibroblasts of four individuals bearing the c.877G>A pathogenic variant. Evaluation of gene-edited cells by next-generation sequencing revealed that correction of the mutant allele by homologous-directed repair occurred at a frequency lower than 1%. However, the presence of frameshift variants and others that provoked the silencing of the mutant allele were found in >40% of reads, with no effects on the wild-type allele. This was confirmed by droplet digital PCR with allele-specific probes, which revealed a reduction in the expression of the mutant allele. Finally, immunofluorescence analyses revealed a recovery in the collagen VI extracellular matrix. In summary, we demonstrate that CRISPR/Cas9 gene-edition can specifically reverse the pathogenic effects of a dominant negative variant in COL6A1.

A titanosaurian sauropod with Gondwanan affinities in the latest Cretaceous of Europe.

The origin of the last sauropod dinosaur communities in Europe and their evolution during the final 15 million years of the Cretaceous have become a complex phylogenetic and palaeobiogeographic puzzle characterized by the controversy on the alleged coexistence of immigrant, Gondwana-related taxa alongside relictual and insular clades. In this context, we describe a new titanosaurian sauropod dinosaur, Abditosaurus kuehnei gen. et sp. nov., from the Late Cretaceous (Maastrichtian) Tremp Group of Catalonia (Spain). Phylogenetic analyses recover Abditosaurus separately from other European titanosaurs, within a clade of otherwise South American and African saltasaurines. The affinity of the new taxon with southern landmasses is reinforced by spatiotemporal co-occurrence with Gondwanan titanosaurian oospecies in southern Europe. The large size and the lack of osteohistological features potentially related to insular dwarfism or size reduction support the idea that Abditosaurus belongs to an immigrant lineage, unequivocally distinct from some of the island dwarfs of the European archipelago. The arrival of the Abditosaurus lineage to the Ibero-Armorican Island is hypothesized to have occurred during the earliest Maastrichtian (70.6 Ma), probably as a result of a global and regional sea-level drop that reactivated ancient dispersal routes between Africa and Europe. The arrival of large-bodied titanosaurs to the European archipelago produced dramatic changes in its insular ecosystems and important evolutionary changes in its dinosaur faunas, especially with respect to the 'island rule' effect.

High expression of both desmoplastic stroma and epithelial to mesenchymal transition markers associate with shorter survival in pancreatic ductal adenocarcinoma.

Desmoplastic stroma (DS) and the epithelial-to-mesenchymal transition (EMT) play a key role in pancreatic ductal adenocarcinoma (PDAC) progression. To date, however, the combined expression of DS and EMT markers, and their association with variations in survival within each clinical stage and degree of tumor differentiation is unknown. The purpose of this study was to investigate the association between expression of DS and EMT markers and survival variability in patients diagnosed with PDAC. We examined the expression levels of DS markers alpha smooth muscle actin (α-SMA), fibronectin, and vimentin, and the EMT markers epithelial cell adhesion molecule (EPCAM), pan-cytokeratin, and vimentin, by immunohistochemistry using a tissue microarray of a retrospective cohort of 25 patients with PDAC. The results were examined for association with survival by clinical stage and by degree of tumor differentiation. High DS markers expression -α-SMA, fibronectin, and vimentin- was associated with decreased survival at intermediate and advanced clinical stages (p=0.006-0.03), as well as with both poorly and moderately differentiated tumor grades (p=0.01-0.02). Interestingly, the same pattern was observed for EMT markers, i.e., EPCAM, pan-cytokeratin, and vimentin (p=0.00008-0.03). High expression of DS and EMT markers within each clinical stage and degree of tumor differentiation was associated with lower PDAC survival. Evaluation of these markers may have a prognostic impact on survival time variation in patients with PDAC.

Contribution of APOA5, APOC3, CETP, ABCA1 and SIK3 genetic variants to hypertriglyceridemia development in Mexican HIV-patients receiving antiretroviral therapy.

OBJECTIVE: To investigate the impact of single nucleotide polymorphisms (SNPs) from APOA5, APOC3, CETP, ATP binding cassette transporter A1 and SIK3 genes in the development of hypertriglyceridemia in HIV patients under antiretroviral therapy. MATERIAL AND METHODS: A case-control study was developed. Leukocytic genomic DNA was extracted and genotyping for SNPs rs662799, rs964184, rs5128, rs2854116, rs2854117, rs3764261, rs4149310, rs4149267 and rs139961185 was performed by real time-PCR using TaqMan allelic discrimination assays, in Mexican mestizo patients with HIV infection, with hypertriglyceridemia (>1.7 mmol/L) under antiretroviral therapy. Genetic variants were also investigated in a control group of normolipidemic HIV patients (≤ 1.7 mmol/L). Haplotypes and gene interactions were analyzed. RESULTS: A total of 602 HIV patients were genotyped (316 cases and 286 controls). Age and antiretroviral regimen based on protease inhibitors were associated with hypertriglyceridemia (P = 0.0001 and P = 0.0002. respectively). SNP rs964184 GG genotype in APOA5 gene exhibited the highest association with hypertriglyceridemia risk (OR, 3.2, 95% CI, 1.7-5.8, P = 0.0001); followed by SNP rs139961185 in SIK3 gene (OR = 2.3; (95% CI, 1.1-4.8; P = 0.03 for AA vs. AG genotype; and APOC3 rs5128 GG genotype, (OR, 2.2; 95% CI, 1.1-4.9; P = 0.04) under codominant models. These associations were maintained in the adjusted analysis by age and protease inhibitors based antiretroviral regimens. CONCLUSIONS: This study reveals an association between rs964184 in APOA5; rs5128 in APOC3 and rs139961185 in SIK3 and high triglyceride concentrations in Mexican HIV-patients receiving protease inhibitors. These genetic factors may influence the adverse effects related to antiretroviral therapy.

Prokaryotic reverse transcriptases: from retroelements to specialized defense systems.

Reverse transcriptases (RTs) catalyze the polymerization of DNA from an RNA template. These enzymes were first discovered in RNA tumor viruses in 1970, but it was not until 1989 that they were found in prokaryotes as a key component of retrons. Apart from RTs encoded by the 'selfish' mobile retroelements known as group II introns, prokaryotic RTs are extraordinarily diverse, but their function has remained elusive. However, recent studies have revealed that different lineages of prokaryotic RTs, including retrons, those associated with CRISPR-Cas systems, Abi-like RTs and other yet uncharacterized RTs, are key components of different lines of defense against phages and other mobile genetic elements. Prokaryotic RTs participate in various antiviral strategies, including abortive infection (Abi), in which the infected cell is induced to commit suicide to protect the host population, adaptive immunity, in which a memory of previous infection is used to build an efficient defense, and other as yet unidentified mechanisms. These prokaryotic enzymes are attracting considerable attention, both for use in cutting-edge technologies, such as genome editing, and as an emerging research topic. In this review, we discuss what is known about prokaryotic RTs, and the exciting evidence for their domestication from retroelements to create specialized defense systems.