A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro.

In nature, bacteria usually exist as mixed-species biofilms, where they engage in a range of synergistic and antagonistic interactions that increase their resistance to environmental challenges. Biofilms are a major cause of persistent infections, and dispersal from initial foci can cause new infections at distal sites thus warranting further investigation. Studies of development and spatial interactions in mixed-species biofilms can be challenging due to difficulties in identifying the different bacterial species in situ. Here, we apply CellTrace dyes to studies of biofilm bacteria and present a novel application for multiplex labeling, allowing identification of different bacteria in mixed-species, in vitro biofilm models. Oral bacteria labeled with CellTrace dyes (far red, yellow, violet, and CFSE [green]) were used to create single- and mixed-species biofilms, which were analyzed with confocal spinning disk microscopy (CSDM). Biofilm supernatants were studied with flow cytometry (FC). Both Gram-positive and Gram-negative bacteria were well labeled and CSDM revealed biofilms with clear morphology and stable staining for up to 4 days. Analysis of CellTrace labeled cells in supernatants using FC showed differences in the biofilm dispersal between bacterial species. Multiplexing with different colored dyes allowed visualization of spatial relationships between bacteria in mixed-species biofilms and relative coverage by the different species was revealed through segmentation of the CSDM images. This novel application, thus, offers a powerful tool for studying structure and composition of mixed-species biofilms in vitro.IMPORTANCEAlthough most chronic infections are caused by mixed-species biofilms, much of our knowledge still comes from planktonic cultures of single bacterial species. Studies of formation and development of mixed-species biofilms are, therefore, required. This work describes a method applicable to labeling of bacteria for in vitro studies of biofilm structure and dispersal. Critically, labeled bacteria can be multiplexed for identification of different species in mixed-species biofilms using confocal spinning disk microscopy, facilitating investigation of biofilm development and spatial interactions under different environmental conditions. The study is an important step in increasing the tools available for such complex and challenging studies.

Efficacy and Imaging-Enabled Pharmacodynamic Profiling of KRAS G12C Inhibitors in Xenograft and Genetically Engineered Mouse Models of Cancer.

KRAS is one of the most commonly mutated oncogenes in lung, colorectal, and pancreatic cancers. Recent clinical trials directly targeting KRAS G12C presented encouraging results for a large population of non-small cell lung cancer (NSCLC), but resistance to treatment is a concern. Continued exploration of new inhibitors and preclinical models is needed to address resistance mechanisms and improve duration of patient responses. To further enable the development of KRAS G12C inhibitors, we present a preclinical framework involving translational, non-invasive imaging modalities (CT and PET) and histopathology in a conventional xenograft model and a novel KRAS G12C knock-in mouse model of NSCLC. We utilized an in-house developed KRAS G12C inhibitor (Compound A) as a tool to demonstrate the value of this framework in studying in vivo pharmacokinetic/pharmacodynamic (PK/PD) relationship and anti-tumor efficacy. We characterized the Kras G12C-driven genetically engineered mouse model (GEMM) and identify tumor growth and signaling differences compared to its Kras G12D-driven counterpart. We also find that Compound A has comparable efficacy to sotorasib in the Kras G12C-driven lung tumors arising in the GEMM, but like observations in the clinic, some tumors inevitably progress on treatment. These findings establish a foundation for evaluating future KRAS G12C inhibitors that is not limited to xenograft studies and can be applied in a translationally relevant mouse model that mirrors human disease progression and resistance.

Thickness-Dependent Interface Polarity in Infinite-Layer Nickelate Superlattices.

The interface polarity plays a vital role in the physical properties of oxide heterointerfaces because it can cause specific modifications of the electronic and atomic structure. Reconstruction due to the strong polarity of the NdNiO2/SrTiO3 interface in recently discovered superconducting nickelate films may play an important role, as no superconductivity has been observed in the bulk. By employing four-dimensional scanning transmission electron microscopy and electron energy-loss spectroscopy, we studied effects of oxygen distribution, polyhedral distortion, elemental intermixing, and dimensionality in NdNiO2/SrTiO3 superlattices grown on SrTiO3 (001) substrates. Oxygen distribution maps show a gradual variation of the oxygen content in the nickelate layer. Remarkably, we demonstrate thickness-dependent interface reconstruction due to a polar discontinuity. An average cation displacement of ∼0.025 nm at interfaces in 8NdNiO2/4SrTiO3 superlattices is twice larger than that in 4NdNiO2/2SrTiO3 superlattices. Our results provide insights into the understanding of reconstructions at NdNiO2/SrTiO3 polar interfaces.

Sustained Virologic Suppression With Dolutegravir/Lamivudine in a Test-and-Treat Setting Through 48 Weeks.

Background: We assessed the efficacy and safety of dolutegravir/lamivudine (DTG/3TC) in a US test-and-treat setting at a secondary 48-week time point of the multicenter, single-arm, phase IIIb STAT study. Methods: Participants were eligible adults newly diagnosed with human immunodeficiency virus (HIV)-1 and had started once-daily DTG/3TC within 14 days of diagnosis, before laboratory results were available. Antiretroviral therapy (ART) was modified if baseline testing indicated DTG or 3TC resistance, hepatitis B virus (HBV) coinfection, or creatinine clearance <30 mL/min per 1.73 m2, and these participants remained in the study. A proportion with HIV-1 ribonucleic acid (RNA) <50 copies/mL at Week 48 was calculated among all participants (intention-to-treat-exposed [ITT-E] missing = failure analysis) and those with available data (observed analysis). Results: At Week 48, 82% of all participants regardless of ART (107 of 131; ITT-E missing = failure) and 97% with available data (107 of 110; observed analysis) achieved HIV-1 RNA <50 copies/mL. High proportions of virologic response were seen overall, including in participants with high viral load (≥500 000 copies/mL; 89%) or low CD4+ cell count (<200 cells/mm3; 78%) at baseline. Ten participants had treatment modification (baseline HBV coinfection, n = 5; participant/proxy decision, n = 2; baseline M184V resistance mutation, adverse event [AE; rash], and pregnancy, n = 1 each) before Week 48. Two participants met confirmed virologic failure criteria. No treatment-emergent resistance was observed. Ten participants reported drug-related AEs (all grade 1-2); no serious drug-related AEs occurred. Conclusions: Results demonstrated high proportions of participants with sustained virologic suppression, no treatment-emergent resistance, and good safety over 48 weeks, supporting first-line use of DTG/3TC in a test-and-treat setting.

Population Genetics and Anastomosis Group's Geographical Distribution of Rhizoctonia solani Associated with Soybean.

Rhizoctonia solani is a species complex composed of many genetically diverse anastomosis groups (AG) and their subgroups. It causes economically important diseases of soybean worldwide. However, the global genetic diversity and distribution of R. solani AG associated with soybean are unknown to date. In this study, the global genetic diversity and distribution of AG associated with soybean were investigated based on rDNA-ITS sequences deposited in GenBank and published literature. The most prevalent AG, was AG-1 (40%), followed by AG-2 (19.13%), AG-4 (11.30%), AG-7 (10.43%), AG-11 (8.70%), AG-3 (5.22%) and AG-5 (3.48%). Most of the AG were reported from the USA and Brazil. Sequence analysis of internal transcribed spacers of ribosomal DNA separated AG associated with soybean into two distinct clades. Clade I corresponded to distinct subclades containing AG-2, AG-3, AG-5, AG-7 and AG-11. Clade II corresponded to subclades of AG-1 subgroups. Furthermore, AG and/or AG subgroups were in close proximity without corresponding to their geographical origin. Moreover, AG or AG subgroups within clade or subclades shared higher percentages of sequence similarities. The principal coordinate analysis also supported the phylogenetic and genetic diversity analyses. In conclusion, AG-1, AG-2, and AG-4 were the most prevalent AG in soybean. The clade or subclades corresponded to AG or AG subgroups and did not correspond to the AG's geographical origin. The information on global genetic diversity and distribution will be helpful if novel management measures are to be developed against soybean diseases caused by R. solani.

Effects of stabilized hypochlorous acid on oral biofilm bacteria.

BACKGROUND: Caries and periodontitis are amongst the most prevalent diseases worldwide, leading to pain and loss of oral function for those affected. Prevention relies heavily on mechanical removal of dental plaque biofilms but for populations where this is not achievable, alternative plaque control methods are required. With concerns over undesirable side-effects and potential bacterial resistance due to the use of chlorhexidine gluconate (CHX), new antimicrobial substances for oral use are greatly needed. Here we have investigated the antimicrobial effect of hypochlorous acid (HOCl), stabilized with acetic acid (HAc), on oral biofilms and compared it to that of CHX. Possible adverse effects of stabilized HOCl on hydroxyapatite surfaces were also examined. METHODS: Single- and mixed-species biofilms of six common oral bacteria (Streptococcus mutans, Streptococcus gordonii, Actinomyces odontolyticus, Veillonella parvula, Parvimonas micra and Porphyromonas gingivalis) within a flow-cell model were exposed to HOCl stabilized with 0.14% or 2% HAc, pH 4.6, as well as HOCl or HAc alone. Biofilm viability was assessed in situ using confocal laser scanning microscopy following LIVE/DEAD® BacLight™ staining. In-situ quartz crystal microbalance with dissipation (QCM-D) was used to study erosion of hydroxyapatite (HA) surfaces by stabilized HOCl. RESULTS: Low concentrations of HOCl (5 ppm), stabilized with 0.14% or 2% HAc, significantly reduced viability in multi-species biofilms representing supra- and sub-gingival oral communities, after 5 min, without causing erosion of HA surfaces. No equivalent antimicrobial effect was seen for CHX. Gram-positive and Gram-negative bacteria showed no significant differential suceptibility to stabilized HOCl. CONCLUSIONS: At low concentrations and with exposure times which could be achieved through oral rinsing, HOCl stabilized with HAc had a robust antimicrobial activity on oral biofilms, without causing erosion of HA surfaces or affecting viability of oral keratinocytes. This substance thus appears to offer potential for prevention and/or treatment of oral biofilm-mediated diseases.

Three-year durable efficacy of dolutegravir plus lamivudine in antiretroviral therapy - naive adults with HIV-1 infection.

OBJECTIVE: To assess efficacy and safety of dolutegravir (DTG) + lamivudine (3TC) vs. DTG + tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) in treatment-naive adults with HIV-1 in the prespecified 144-week secondary analyses of GEMINI-1 and GEMINI-2. DESIGN: Identical, multicenter, phase III, randomized, non-inferiority studies (double-blind through 96 weeks). METHODS: Participants with HIV-1 RNA ≤500 000 copies/ml and no major viral resistance mutations to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, or protease inhibitors were randomized 1:1 to once-daily DTG + 3TC or DTG + TDF/FTC. RESULTS: At week 144, DTG + 3TC (N = 716) was noninferior to DTG + TDF/FTC (N = 717) in proportion of participants achieving HIV-1 RNA <50 copies/ml (Snapshot algorithm) in the pooled analysis (82% vs. 84%, respectively; adjusted treatment difference [95% confidence interval (CI)], -1.8% [-5.8, 2.1]), GEMINI-1 (-3.6% [-9.4, 2.1]), and GEMINI-2 (0.0% [-5.3, 5.3]). Twelve DTG + 3TC participants and nine DTG + TDF/FTC participants met protocol-defined confirmed virologic withdrawal (CVW) criteria; none developed treatment-emergent resistance. One DTG + 3TC participant who did not meet CVW criteria developed M184V at week 132 and R263R/K at week 144, conferring a 1.8-fold change in susceptibility to DTG; non-adherence to therapy was reported. Significantly fewer drug-related adverse events occurred with DTG + 3TC vs. DTG + TDF/FTC (20% vs. 27%; relative risk [95% CI], 0.76 [0.63-0.92]). Renal and bone biomarker changes favored DTG + 3TC. CONCLUSIONS: Three-year durable efficacy, long-term tolerability, and high barrier to resistance support first-line use of DTG + 3TC for HIV-1 treatment (see Supplemental Digital Content 1, http://links.lww.com/QAD/C297; video abstract).

Mutants of the Zebrafish K+ Channel Hcn2b Exhibit Epileptic-like Behaviors.

Epilepsy is a chronic neurological disorder that affects 50 million people worldwide. The most common form of epilepsy is idiopathic, where most of the genetic defects of this type of epilepsy occur in ion channels. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization, and are mainly expressed in the heart and central and peripheral nervous systems. In humans, four HCN genes have been described, and emergent clinical data shows that dysfunctional HCN channels are involved in epilepsy. Danio rerio has become a versatile organism to model a wide variety of diseases. In this work, we used CRISPR/Cas9 to generate hcn2b mutants in zebrafish, and characterized them molecularly and behaviorally. We obtained an hcn2b mutant allele with an 89 bp deletion that produced a premature stop codon. The mutant exhibited a high mortality rate in its life span, probably due to its sudden death. We did not detect heart malformations or important heart rate alterations. Absence seizures and moderate seizures were observed in response to light. These seizures rarely caused instant death. The results show that mutations in the Hcn2b channel are involved in epilepsy and provide evidence of the advantages of zebrafish to further our understanding of the pathogenesis of epilepsy.

Dolutegravir/lamivudine as a first-line regimen in a test-and-treat setting for newly diagnosed people living with HIV.

OBJECTIVES: Dolutegravir/lamivudine (DTG/3TC) is indicated for treatment-naive and experienced people with HIV; however, questions remain about its utility in a test-and-treat setting because of potential transmitted resistance and baseline hepatitis B virus (HBV) co-infection. We present feasibility and efficacy of DTG/3TC in newly diagnosed individuals in a test-and-treat setting. DESIGN: The single-arm STAT study evaluated DTG/3TC in a US test-and-treat setting. METHODS: Eligible adults initiated DTG/3TC 14 days or less after HIV-1 diagnosis without availability of baseline laboratory results. If baseline testing indicated DTG or 3TC resistance, HBV co-infection, or creatinine clearance less than 30 ml/min per 1.73 m2, participants remained on study with treatment modification. Efficacy endpoints included proportions of participants with HIV-1 RNA less than 50 copies/ml at Week 24, regardless of antiretroviral regimen, among all participants (intention-to-treat exposed) and those with available HIV-1 RNA data (observed). RESULTS: Of 131 participants enrolled, 8% were female and 50% were non-white. Through Week 24, treatment was modified in eight participants [five with HBV co-infection, one with baseline M184V, one for adverse event (rash), one participant decision]. At Week 24, 78% (102/131) of all participants and 92% (102/111) of those with available data achieved HIV-1 RNA less than 50 copies/ml. Incidence of drug-related adverse events was low (7%); no drug-related serious adverse events occurred. CONCLUSION: These data demonstrate the feasibility, efficacy, and safety of using DTG/3TC as a first-line regimen in a test-and-treat setting, with therapy adjustments for baseline resistance or HBV co-infection occurring safely via routine clinical care as needed [ClinicalTrials.gov, NCT03945981; see Supplemental Digital Content 1, video abstract (Video abstract summarizing the STAT study design and results), http://links.lww.com/QAD/C189].

Temporal and spatial expression of zebrafish mctp genes and evaluation of frameshift alleles of mctp2b.

MCTPs (multiple C2 domain proteins with two transmembrane regions) have been proposed as novel endoplasmic reticulum calcium sensors; however, their function remains largely unknown. Here we report the structure of the four mctp genes from zebrafish (mctp1a, mctp1b, mctp2a and mctp2b), their diversity, expression pattern during embryonic development and in adult tissue and the effect of knocking down the expression of Mctp2b by CRISPR/Cas9. The four mctp genes are expressed from early development and exhibit differential expression patterns but are found mainly in the nervous and muscular systems. Mctp2b tagged with fluorescent proteins and expressed in HEK-293 cells and neurons of the fish spinal cord localized mostly in the endoplasmic reticulum but also in lysosomes and late and recycling endosomes. Knocking down mctp2b expression impaired embryonic development, suggesting that the functional participation of this gene is relevant, at least during the early stages of development.

Modeling Neuronal Diseases in Zebrafish in the Era of CRISPR.

BACKGROUND: Danio rerio is a powerful experimental model for studies in genetics and development. Recently, CRISPR technology has been applied in this species to mimic various human diseases, including those affecting the nervous system. Zebrafish offer multiple experimental advantages: external embryogenesis, rapid development, transparent embryos, short life cycle, and basic neurobiological processes shared with humans. This animal model, together with the CRISPR system, emerging imaging technologies, and novel behavioral approaches, lay the basis for a prominent future in neuropathology and will undoubtedly accelerate our understanding of brain function and its disorders. OBJECTIVE: Gather relevant findings from studies that have used CRISPR technologies in zebrafish to explore basic neuronal function and model human diseases. METHODS: We systematically reviewed the most recent literature about CRISPR technology applications for understanding brain function and neurological disorders in D. rerio. We highlighted the key role of CRISPR in driving forward our understanding of particular topics in neuroscience. RESULTS: We show specific advances in neurobiology when the CRISPR system has been applied in zebrafish and describe how CRISPR is accelerating our understanding of brain organization. CONCLUSION: Today, CRISPR is the preferred method to modify genomes of practically any living organism. Despite the rapid development of CRISPR technologies to generate disease models in zebrafish, more efforts are needed to efficiently combine different disciplines to find the etiology and treatments for many brain diseases.

Durable Efficacy of Dolutegravir Plus Lamivudine in Antiretroviral Treatment-Naive Adults With HIV-1 Infection: 96-Week Results From the GEMINI-1 and GEMINI-2 Randomized Clinical Trials.

BACKGROUND: The 2-drug regimen dolutegravir + lamivudine was noninferior to dolutegravir + tenofovir disoproxil fumarate/emtricitabine in achieving HIV-1 RNA <50 copies/mL in treatment-naive adults in the 48-week primary analysis of the GEMINI trials. We present results from the prespecified 96-week secondary analyses. SETTING: One hundred eighty-seven centers in 21 countries. METHODS: GEMINI-1 and GEMINI-2 are identical, double-blind phase III studies. Participants with screening HIV-1 RNA ≤500,000 copies/mL were randomized 1:1 to once-daily dolutegravir + lamivudine or dolutegravir + tenofovir disoproxil fumarate/emtricitabine. RESULTS: At week 96, dolutegravir + lamivudine (N = 716) was noninferior to dolutegravir + tenofovir disoproxil fumarate/emtricitabine (N = 717) in achieving HIV-1 RNA <50 copies/mL (Snapshot algorithm; -10% noninferiority margin) in the pooled analysis (proportion of responders, 86.0% vs 89.5%, respectively; adjusted treatment difference [95% CI], -3.4% [-6.7 to 0.0007]), GEMINI-1 (-4.9% [-9.8 to 0.03]), and GEMINI-2 (-1.8% [-6.4 to 2.7]). Proportions of participants in the HIV-1 RNA ≥50 copies/mL Snapshot category were largely unchanged from week 48 to 96. Eleven participants taking dolutegravir + lamivudine and 7 taking dolutegravir + tenofovir disoproxil fumarate/emtricitabine met confirmed virologic withdrawal criteria through week 96; none had treatment-emergent resistance mutations. Dolutegravir + lamivudine had a lower rate of drug-related adverse events than dolutegravir + tenofovir disoproxil fumarate/emtricitabine (19.6% vs 25.0%; relative risk ratio, 0.78; 95% CI: 0.64 to 0.95). Renal and bone biomarker changes favored dolutegravir + lamivudine. CONCLUSIONS: Consistent with 48-week data, dolutegravir + lamivudine demonstrated long-term, noninferior efficacy vs dolutegravir + tenofovir disoproxil fumarate/emtricitabine without increased risk of treatment-emergent resistance, supporting its use in treatment-naive HIV-1-infected individuals.

The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity.

KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3-5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.

IL-17A, a possible biomarker for the evaluation of treatment response in Trypanosoma cruzi infected children: A 12-months follow-up study in Bolivia.

BACKGROUND: The National Program for Chagas disease was implemented in Bolivia in 2006, and it greatly decreased the number of infections through vector control. Subsequently, a treatment regimen of benznidazole (BNZ) was started in seropositive school-age children living in certified vector control areas. METHODS AND FINDINGS: We conducted a 12-month follow-up study and seven blood samples were taken during and after the treatment. Serology, conventional diagnostic PCR (cPCR) and quantitative Real-time PCR (qPCR) were performed. Plasma Th1/Th2/Th17 cytokines levels were also determined. Approximately 73 of 103 seropositive children complied with BNZ, with three interruptions due to side effects. To evaluate each individual's treatment efficacy, the cPCR and qPCR values during the final 6 months of the follow-up period were observed. Among 57 children who completed follow-up, 6 individuals (11%) showed both cPCR(+) and qPCR(+) (non reactive), 24 (42%) cPCR(-) but qPCR(+) (ambiguous) and 27 (47%) cPCR(-) and qPCR(-) (reactive). Within 14 Th1/Th2/Th17 cytokines, IL-17A showed significantly higher levels in seropositive children before the treatment compared to age-matched seronegative children and significantly decreased to the normal level one-year after. Moreover, throughout the follow-up study, IL-17A levels were positively co-related to parasite counts detected by qPCR. At the 12 months' time point, IL-17A levels of non-reactive subjects were significantly higher than either those of reactive or ambiguous subjects suggesting that IL-17A might be useful to determine the reactivity to BNZ treatment. CONCLUSIONS: Plasma levels of IL-17A might be a bio-marker for detecting persistent infection of T. cruzi and its chronic inflammation.

A novel starch-binding laccase from the wheat pathogen Zymoseptoria tritici highlights the functional diversity of ascomycete laccases.

BACKGROUND: Laccases are multicopper oxidases, which are assigned into auxiliary activity family 1 (AA1) in the CAZy database. These enzymes, catalyzing the oxidation of phenolic and nonphenolic substrates coupled to reduction of O2 to H2O, are increasingly attractive as eco-friendly oxidation biocatalysts. Basidiomycota laccases are well characterized due to their potential in de-lignification of lignocellulose. By contrast, insight into the biochemical diversity of Ascomycota counterparts from saprophytes and plant pathogens is scarce. RESULTS: Here, we report the properties of the laccase from the major wheat pathogen Zymoseptoria tritici (ZtrLac1A), distinguished from common plant fungal pathogens by an apoplastic infection strategy. We demonstrate that ZtrLac1A is appended to a functional starch-binding module and displays an activity signature disfavoring relatively apolar phenolic redox mediators as compared to the related biochemically characterized laccases. By contrast, the redox potential of ZtrLac1A (370 mV vs. SHE) is similar to ascomycetes counterparts. The atypical specificity is consistent with distinctive sequence substitutions and insertions in loops flanking the T1 site and the enzyme C-terminus compared to characterized laccases. CONCLUSIONS: ZtrLac1A is the first reported modular laccase appended to a functional starch-specific carbohydrate binding module of family 20 (CBM20). The distinct specificity profile of ZtrLac1A correlates to structural differences in the active site region compared to previously described ascomycetes homologues. These differences are also highlighted by the clustering of the sequence of ZtrLac1A in a distinct clade populated predominantly by plant pathogens in the phylogenetic tree of AA1 laccases. The possible role of these laccases in vivo merits further investigations. These findings expand our toolbox of laccases for green oxidation and highlight the binding functionality of CBM-appended laccases as versatile immobilization tags.

Pharmaceutical intervention after evaluation of the risk of ictus in elderly patients institutionalized with atrial fibrillation.

OBJECTIVES: To evaluate the risk of having a stroke and the risk of bleeding in institutionalized patients with atrial fibrillation. Atrial fibrillation is a common cardiac arrhythmia associated with increased morbidity and mortality. It is necessary to develop pharmacotherapy plans to minimize the risk. DESIGN: A prospective study. SETTING: Institutionalized patients. PARTICIPANTS: Inclusion criteria were: patients diagnosed with atrial fibrillation, with or without treatment for the prevention of stroke. MAIN OUTCOME MEASURES: The evaluation of the CHA2DS2-VASc criteria was performed. The risk of hemorrhage was assessed using the HAS-BLED scale, based on the risk factors associated with the probability of bleeding. RESULTS: We included 53 patients (86.4 ± 6.4 years, 30.2% men). Of these, 37 (69.8%) were correctly anticoagulated. Of the remaining, 5 patients (31.2%) did not have any type of anticoagulant or antiaggregant treatment and 11 (68.7%) were treated with antiaggregant alone. The pharmaceutical intervention was performed in patients who did not meet stroke criteria. Of the untreated patients: two died before the intervention, two were recommended to be referred to cardiology and in one there was no intervention because of very advanced age. In the antiaggregant patients, it was decided not to modify the treatment. The reasons were: high risk of bleeding, very advanced age, advanced dementia or terminal illness, moderate risk of stroke, and clotting factor deficiency. CONCLUSIONS: The risk of stroke in elderly patients with atrial fibrillation is high, so it is important to control the risk factors.

Knock-Down of Specific Thyroid Hormone Receptor Isoforms Impairs Body Plan Development in Zebrafish.

The role of thyroid hormones (THs) in development has been extensively studied, however, the specific molecular mechanisms involved are far from being clear. THs act by binding to TH nuclear receptors (TR) that act as ligand-dependent transcription factors to regulate TH-dependent gene expression. Like vertebrates, zebrafish express different isoforms of functional Tr alpha and beta, some of which can bind alternative ligands like 3,5-T2. In this study, we first analyzed the effects of exogenous T3 and 3,5-T2 exposure during embryogenesis. The percentage of affected embryos was similar to those vehicle-injected, suggesting that the early exposure to low TH levels is not sufficient to elicit effects upon the phenotype of the embryo. We then generated crispants for four isoforms of thr to learn more about the role of these receptors in early development. We found that crispant larvae from thraa and a newly identified l-thrb+, but not thrab and canonical thrb1 showed profound deleterious effects upon symmetry and laterality, suggesting early novel roles for these Tr isoforms in the body plan developmental program. Since critical events that determine cell fate start in the late gastrula, we tested if some genes that are expressed during early developmental stages could indeed be TH targets. We identify early development genes, like sox10 and eve, that were specifically over-expressed in thraa and l-thrb+ crispants, suggesting that these specific thr isoforms function as transcription repressors for these genes, while transcription of zic and ets appear to be thraa and l-thrb+-mediated, respectively. Overall, present results show that TH signaling participates in early zebrafish development and identify Tr isoform-specific mediated regulation of early gene expression.

Dolutegravir plus lamivudine versus dolutegravir plus tenofovir disoproxil fumarate and emtricitabine in antiretroviral-naive adults with HIV-1 infection (GEMINI-1 and GEMINI-2): week 48 results from two multicentre, double-blind, randomised, non-inferiority, phase 3 trials.

BACKGROUND: Effective two-drug regimens could decrease long-term drug exposure and toxicity with HIV-1 antiretroviral therapy (ART). We therefore aimed to evaluate the efficacy and safety of a two-drug regimen compared with a three-drug regimen for the treatment of HIV-1 infection in ART-naive adults. METHODS: We conducted two identically designed, multicentre, double-blind, randomised, non-inferiority, phase 3 trials: GEMINI-1 and GEMINI-2. Both studies were done at 192 centres in 21 countries. We included participants (≥18 years) with HIV-1 infection and a screening HIV-1 RNA of 500 000 copies per mL or less, and who were naive to ART. We randomly assigned participants (1:1) to receive a once-daily two-drug regimen of dolutegravir (50 mg) plus lamivudine (300 mg) or a once-daily three-drug regimen of dolutegravir (50 mg) plus tenofovir disoproxil fumarate (300 mg) and emtricitabine (200 mg). Both drug regimens were administered orally. We masked participants and investigators to treatment assignment: dolutegravir was administered as single-entity tablets (similar to its commercial formulation, except with a different film colour), and lamivudine tablets and tenofovir disoproxil fumarate and emtricitabine tablets were over-encapsulated to visually match each other. Primary endpoint was the proportion of participants with HIV-1 RNA of less than 50 copies per mL at week 48 in the intention-to-treat-exposed population, using the Snapshot algorithm and a non-inferiority margin of -10%. Safety analyses were done on the safety population. GEMINI-1 and GEMINI-2 are registered with ClinicalTrials.gov, numbers NCT02831673 and NCT02831764, respectively. FINDINGS: Between July 18, 2016, and March 31, 2017, 1441 participants across both studies were randomly assigned to receive either the two-drug regimen (n=719) or three-drug regimen (n=722). At week 48 in the GEMINI-1 intention-to-treat-exposed population, 320 (90%) of 356 participants receiving the two-drug regimen and 332 (93%) of 358 receiving the three-drug regimen achieved plasma HIV-1 RNA of less than 50 copies per mL (adjusted treatment difference -2·6%, 95% CI -6·7 to 1·5); in GEMINI-2, 335 (93%) of 360 in the two-drug regimen and 337 (94%) of 359 in the three-drug regimen achieved HIV-1 RNA of less than 50 copies per mL (adjusted treatment difference -0·7%, 95% CI -4·3 to 2·9), showing non-inferiority at a -10% margin in both studies (pooled analysis: 655 [91%] of 716 in the two-drug regimen vs 669 [93%] of 717 in the three-drug regimen; adjusted treatment difference -1·7%, 95% CI -4·4 to 1·1). Numerically, more drug-related adverse events occurred with the three-drug regimen than with the two-drug regimen (169 [24%] of 717 vs 126 [18%] of 716); few participants discontinued because of adverse events (16 [2%] in the three-drug regimen and 15 [2%] in the two-drug regimen). Two deaths were reported in the two-drug regimen group of GEMINI-2, but neither was considered to be related to the study medication. INTERPRETATION: The non-inferior efficacy and similar tolerability profile of dolutegravir plus lamivudine to a guideline-recommended three-drug regimen at 48 weeks in ART-naive adults supports its use as initial therapy for patients with HIV-1 infection. FUNDING: ViiV Healthcare.

Zebrafish Got Spicy: V Course and Symposium of the Latin American Zebrafish Network.

The V Latin American Zebrafish Network Course and Symposium was held from May 4 to 12, 2018 at the Institute of Biotechnology of the National Autonomous University of Mexico. A group of 19 students from eight different countries were selected and trained during a week in a variety of topics and techniques using zebrafish as a model, followed by a 2-day symposium. In this article, we want to point out not only the outstanding activities carried out during the course but also highlight the great experience, the knowledge, and the fantastic relationships we gained from those days.