Cytosine base editing enables quadruple-edited allogeneic CART cells for T-ALL.

Allogeneic chimeric antigen receptor T-cell (CART) therapies require multiple gene edits to be clinically tractable. Most allogeneic CARTs have been created using gene editing techniques that induce DNA double-stranded breaks (DSBs), resulting in unintended on-target editing outcomes with potentially unforeseen consequences. Cytosine base editors (CBEs) install C•G to T•A point mutations in T cells, with between 90% and 99% efficiency to silence gene expression without creating DSBs, greatly reducing or eliminating undesired editing outcomes following multiplexed editing as compared with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). Using CBE, we developed 7CAR8, a CD7-directed allogeneic CART created using 4 simultaneous base edits. We show that CBE, unlike CRISPR-Cas9, does not impact T-cell proliferation, lead to aberrant DNA damage response pathway activation, or result in karyotypic abnormalities following multiplexed editing. We demonstrate 7CAR8 to be highly efficacious against T-cell acute lymphoblastic leukemia (T-ALL) using multiple in vitro and in vivo models. Thus, CBE is a promising technology for applications requiring multiplexed gene editing and can be used to manufacture quadruple-edited 7CAR8 cells, with high potential for clinical translation for relapsed and refractory T-ALL.

Overview of the participation of community health workers in primary health care in 6 Latin American countries and a proposal for their integration into the health system: a qualitative study.

BACKGROUND: According to some health programmes, implementing primary health care through community health workers (CHWs) facilitates the connection between community and health services in Latin America. However, these are isolated processes that face different obstacles and would benefit from an overview of the corresponding health policies and programmes. OBJECTIVE: To provide an overview of CHW participation in 6 Latin American countries. METHODS: This exploratory qualitative study was based on 3 sources of information: a literature review, a review of public health policy documents, and interviews with experts who have led CHW programmes in 6 Latin American countries. RESULTS: The role of CHWs in Latin America and some advances in public health policies in the region were evidenced. However, limitations arising from variable implementation of the WHO guidelines on health programmes with CHWs were also apparent. CONCLUSIONS: CHWs contribute to the primary healthcare processes in the 6 Latin American countries studied in versatile and comprehensive ways. However, they constitute an underutilized human resource because they must provide various services that are not always relevant in different work contexts. Therefore, we propose a classification of the CHW profile, using the level of access to healthcare services of the population they serve as the main differentiator. This way, CHWs will not have to provide a wide range of services but only those most relevant to the specific needs of each community.

Substance P/calcitonin gene-related peptide and their receptors expression in human periodontal ligament after root canal preparation with five different systems.

AIM: The purpose of this study was to quantify the effect of five different root canal preparation instruments on Substance P (SP), Calcitonin gene-related peptide (CGRP) and their receptors expression in healthy human periodontal ligament. METHODOLOGY: STROBE guidelines were used to design a study using 60 periodontal ligament samples obtained from healthy lower premolars where extraction was indicated for orthodontic reasons. Prior to extraction 40 of these premolars were equally divided into four groups and root canals were prepared using different systems: Mtwo, Reciproc Blue, HyFlex EDM and Plex-V. Ten premolars were prepared with hand files and served as a positive control group. The remaining 10 premolars where extracted without treatment and served as a negative control group. All periodontal ligament samples were processed to measure the expression of SP, CGRP and their receptors by radioimmunoassay. Kruskal-Wallis and Duncan tests were performed to determine statistically significant differences between the groups for each variable. RESULTS: Greater expression of all the peptides measured were found in the hand-file preparation group, followed by the Reciproc Blue, Mtwo, HyFlex EDM and Plex-V groups. The lower SP, CGRP and their receptors values were for the intact teeth control group. Kruskal-Wallis test showed statistically significant differences amongst groups (p < .001). Dunn post-hoc tests showed statistically significant differences in SP, CGRP and their receptors expression between the intact teeth and the hand-file and Reciproc Blue groups. Hand-file group showed significant differences with the other groups, except with Reciproc Blue, where no differences were observed in any of the peptides measured. Finally, no differences were observed between Plex-V and HyFlex in any of the peptides measured. CONCLUSIONS: Root canal preparation with hand files and Reciproc Blue generates the highest expression of SP, CGRP, NK1 and CGRP1R in human periodontal ligament, whilst Plex-V and HyFlex maintain the basal expression of neuropeptides and their receptors. Mtwo showed intermediate results between Reciproc Blue and HyFlex.

Neglected Tropical Diseases and Sudden Cardiac Death: The NET-Heart Project.

Sudden cardiac death (SCD) is responsible for approximately 6% of global mortality and 25% of cardiovascular (CV) deaths. SCD has been traditionally linked to coronary artery disease, valvular heart disease, cardiomyopathies, and genetic arrhythmia disorders. However, advancements in care for these diseases have not translated to a proportional reduction in SCD. This suggests an important role of underrecognized contributing pathologies. Neglected tropical diseases (NTDs) are a group of illnesses prevalent in tropical and sub-tropical regions which have been understudied partially due to their high prevalence in marginalized populations. The relationship between SCD and Chagas disease has been well-established, though emerging literature suggests that other NTDs with CV involvement may lead to fatal arrhythmias. Additionally, specific therapies for a subset of NTDs put patients at increased risk of malignant arrhythmias and other cardiac complications. This review aims to summarize the association between a group of selected NTDs and SCD.

Modulatory Impact of the sRNA Mcr11 in Two Clinical Isolates of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is a successful pathogen causing tuberculosis (TB) disease in humans. It has been shown, that some circulating strains of Mtb in TB endemic populations, are more virulent and more transmissible than others, which may be related to their evolved adaptations to modulate the host immune responses. Underlying these adaptations to the stressful conditions, different genetic regulatory networks involved sRNAs that are mostly unknown for Mtb. We have previously shown that Mcr11 is one of the main sRNAs that determine transcriptomic differences among the Colombian clinical isolates UT127 and UT205 compared to the laboratory strain H37Rv. We found that the knock-down of mcr11 using CRISPRi has a major impact on phenotypic traits, especially in the clinical isolate UT205. Through the analysis of RNA-seq during the knock-down of mcr11 in UT205, we found a downregulation of genes mainly involved in lipid synthesis, lipid metabolism, ribosomal proteins, transport systems, respiratory and energy systems, membrane and cell wall components, intermediary metabolism, lipoproteins and virulence genes. One of the most interesting genes showing transcriptomic changes is OprA (encoded by the gene rv0516c), which has been involved in the K+ regulation. Overall, our data may suggest that one of the prominent roles of the sRNA Mcr11 is to regulate genes that control Mtb growth and osmoregulation.

Dual RNA Sequencing of Mycobacterium tuberculosis-Infected Human Splenic Macrophages Reveals a Strain-Dependent Host-Pathogen Response to Infection.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), leading to pulmonary and extrapulmonary TB, whereby Mtb is disseminated to many other organs and tissues. Dissemination occurs early during the disease, and bacteria can be found first in the lymph nodes adjacent to the lungs and then later in the extrapulmonary organs, including the spleen. The early global gene expression response of human tissue macrophages and intracellular clinical isolates of Mtb has been poorly studied. Using dual RNA-seq, we have explored the mRNA profiles of two closely related clinical strains of the Latin American and Mediterranean (LAM) family of Mtb in infected human splenic macrophages (hSMs). This work shows that these pathogens mediate a distinct host response despite their genetic similarity. Using a genome-scale host-pathogen metabolic reconstruction to analyze the data further, we highlight that the infecting Mtb strain also determines the metabolic response of both the host and pathogen. Thus, macrophage ontogeny and the genetic-derived program of Mtb direct the host-pathogen interaction.

A systematic evaluation of Mycobacterium tuberculosis Genome-Scale Metabolic Networks.

Metabolism underpins the pathogenic strategy of the causative agent of TB, Mycobacterium tuberculosis (Mtb), and therefore metabolic pathways have recently re-emerged as attractive drug targets. A powerful approach to study Mtb metabolism as a whole, rather than just individual enzymatic components, is to use a systems biology framework, such as a Genome-Scale Metabolic Network (GSMN) that allows the dynamic interactions of all the components of metabolism to be interrogated together. Several GSMNs networks have been constructed for Mtb and used to study the complex relationship between the Mtb genotype and its phenotype. However, the utility of this approach is hampered by the existence of multiple models, each with varying properties and performances. Here we systematically evaluate eight recently published metabolic models of Mtb-H37Rv to facilitate model choice. The best performing models, sMtb2018 and iEK1011, were refined and improved for use in future studies by the TB research community.

Brigatinib in ALK-positive non-small cell lung cancer: real-world data in the Latin American population (Bri-world extend CLICaP).

Background: Brigatinib has demonstrated its efficacy as first-line therapy and in further lines for ALK-positive non-small cell lung cancer (NSCLC) patients; however, real-world data in Latin America are scarce. Methods: From January 2018 to March 2020, 46 patients with advanced ALK-positive NSCLC received brigatinib as second or further line of therapy in Mexico and Colombia. The primary end point was progression-free survival (PFS); secondary end point was time to treatment discontinuation (TTD). Results: At a median follow-up of 9.3 months, the median PFS was 15.2 months (95% CI: 11.6-18.8), and TTD was 18.46 months (95% CI: 9.54-27.38). The estimated overall survival at 12 months was 80%. Safety profile was consistent with previously published data. Conclusion: Brigatinib is an effective treatment for previously treated ALK-positive NSCLC patients in a real-world setting.

Acquired factor XIII deficiency in patients under therapeutic plasma exchange: A poorly explored etiology.

INTRODUCTION: Factor XIII (FXIII) deficiency may cause bleeding under certain clinical circumstances. Therapeutic plasma exchange (TPE) may lead to a transient deficiency. OBJECTIVES: To describe the clinical evolution of patients with acquired FXIII deficiency secondary to TPE. METHODS: We respectively studied a cohort of consecutive patients from 2014 to 2019 who were treated with TPE with FXIII levels <50%. The FXIII was measured after the start of the TPE course, on days between the TPE sessions, due to suspected acquired deficiency. All TPE were performed using continuous flow cell separator. In all cases, the initial replacement fluid applied was albumin. Apheresis procedures were held at 24to 48 hours intervals. RESULTS: Eighteen patients were included, 13 of them were recipients of kidney transplants. The main TPE prescription was humoral rejection. Median FXIII at diagnosis (measured on days between sessions of the TPE course) was 19%(IQR17-25). The median of apheresis procedures before measurement of FXIII was 3(IQR2-4). Among the total cohort, 10 patients suffered hemorrhages. None of the patients without history of kidney transplants had bleeding (n = 5), however, 10/13 with kidney transplants did. Five kidney transplant patients received therapy with FXIII concentrate because of life-threatening bleeding. In all cases, the bleeding stopped within the first 24 hours. All patients had their FXIII levels measured again after finishing the TPE course, with normal results. CONCLUSIONS: TPE is an under-diagnosed cause of acquired FXIII deficiency since routine coagulation tests remain unaltered. It might cause major bleeding, particularly in patients with a recent history of surgery like kidney transplants.

Expression of substance P, calcitonin gene-related peptide and vascular endothelial growth factor in human dental pulp under different clinical stimuli.

BACKGROUND: The aim of this study was to measure the dental pulp inflammatory response through neuropeptides (SP and CGRP) as a response to occlusal trauma, orthodontic movements and a combination of both, as well as the angiogenic defense mechanism through VEGF expression, which could be the initial step to mineralized tissue formation. METHODS: Forty human dental pulp samples were collected from healthy first premolars with extraction indicated due to orthodontic reasons from a sample of 20 patients. Patients were divided into four groups with 10 premolars each (1 mandibular and 1 maxillary premolar from each patient): healthy pulp control group, occlusal trauma group, moderate orthodontic forces group; and occlusal trauma plus moderate orthodontic forces group. Stimuli were applied for 24 h before tooth extraction in all experimental groups. All samples were processed, and SP, CGRP, and VEGF were measured by radioimmunoassay. The Kruskal-Wallis test was performed to assess significant differences among groups and Mann-Whitney's U post hoc pairwise comparisons were also performed. RESULTS: The highest increase in SP, CGRP, and VEGF expressions was found in the occlusal trauma plus orthodontic forces group, followed by the moderate orthodontic forces, the occlusal trauma and the control groups, with statistically significant differences between all groups for each of the 3 peptides analyzed (Kruskal-Wallis p < 0.001). All possible pairwise post-hoc comparisons were also significant for each peptide analyzed (Mann-Whitney's U p < 0.001). CONCLUSION: SP, CGRP, and VEGF expressions significantly increase in human dental pulps when stimulated by occlusal trauma combined with moderate orthodontic forces, as compared with these two stimuli applied independently. Name of the registry: Importance of Neurogenic Inflammation in the Angiogenic Response of the Dental Pulp as a Defensive Response. TRIAL REGISTRATION NUMBER: NCT03804034. Date of registration: 01/15/2019 Retrospectively registered. URL of trial registry record: https://clinicaltrials.gov/ct2/show/NCT03804034?term=NCT03804034&draw=2&rank=1 .

Design and applications of gene therapy vectors for mucopolysaccharidosis in Colombia.

The authors briefly describe their work in the construction of viral derived vectors for the use in gene therapy of muchopolysaccharide storage diseases (MPS), especially in Morquio A syndrome. The motivations to undertake that line of research about twenty years ago was the belief that gene therapy was the most plausible treatment for monogenic diseases due to the transient effect and its difficulty to reach bone tissue of the only effective treatment in use, the enzyme replacement therapy. The strategy used to increase the bone targeting was to include in the vectors an aspartic acid octapeptide that increases their affinity for the oppositely charged hydroxyapatite molecule of bone. It is also discussed the difficulties to do front line research in many developing countries, due to the extended belief that their research money should be mainly devoted to projects that render solutions in a very short time. However, the authors argue in favor of doing research in gene therapy, because it is proving to be the solution for many monogenic diseases, and therefore there is a need of people with good command of GT all over the world, in order to make good use of that therapy especially for ex-vivo treatments.

Human recombinant lysosomal ß-Hexosaminidases produced in Pichia pastoris efficiently reduced lipid accumulation in Tay-Sachs fibroblasts.

GM2 gangliosidosis, Tay-Sachs and Sandhoff diseases, are lysosomal storage disorders characterized by the lysosomal accumulation of GM2 gangliosides. This accumulation is due to deficiency in the activity of the ß-hexosaminidases Hex-A or Hex-B, which are dimeric hydrolases formed by αß or ßß subunits, respectively. These disorders show similar clinical manifestations that range from mild systemic symptoms to neurological damage and premature death. There is still no effective therapy for GM2 gangliosidoses, but some therapeutic alternatives, as enzyme replacement therapy, have being evaluated. Previously, we reported the production of active human recombinant ß-hexosaminidases (rhHex-A and rhHex-B) in the methylotrophic yeast Pichia pastoris. In this study, we evaluated in vitro the cellular uptake, intracellular delivery to lysosome, and reduction of stored substrates. Both enzymes were taken-up via endocytic pathway mediated by mannose and mannose-6-phosphate receptors and delivered to lysosomes. Noteworthy, rhHex-A diminished the levels of stored lipids and lysosome mass in fibroblasts from Tay-Sachs patients. Overall, these results confirm the potential of P. pastoris as host to produce recombinant ß-hexosaminidases intended to be used in the treatment of GM2 gangliosidosis.

Nanoparticle-templated conversion of glucose to a high surface area biocarbon for the removal of organic pollutants in water.

While extensive work has been done on the generation of adsorbents by carbonization of large polymeric structures, few works are currently available for the use of monomeric carbon molecules as precursors during carbonization. In this work we report the formation of a carbon adsorbent material from the carbonization of glucose in the presence of zinc oxide (ZnO) nanoparticle templates. Carbonization at 1,000 °C under inert atmosphere yields a product with Brunauer-Emmett-Teller (BET) surface area of 1,228.19 m2/g and 14.77 nm average pore diameter. Adsorption capacities against methylene blue, 2-naphthol and bisphenol-A at pH 7 were found to be 539 mg/g, 737 mg/g and 563 mg/g, respectively. Our material demonstrates a strong fit with the Langmuir isotherm, and adsorption kinetics show regression values near unity for the pseudo-second order kinetic model. A flow adsorption column was implemented for the remediation of tap water containing 20 mg/L methylene blue and found to quantitatively purify 11.5 L of contaminated water.

National Clinical Practice Guidelines for the management of non-small cell lung cancer in early, locally advanced and metastatic stages. Extended version. / Guía de Práctica Clínica Nacional para el manejo del cáncer de pulmón de células no pequeñas en estadios tempranos, localmente avanzados y metastásicos.

OBJECTIVE: Lung cancer is one the leading causes of mortality worldwide. Symptomatic manifestations of the disease generally occur in the advanced-stage setting, and therefore an important number of patients have advanced or metastatic disease by the time they are diagnosed. This situation contributes to a poor prognosis in the treatment of lung cancer. Evidencebased clinical recommendations are of great value to support decision-making for daily practice, and thus improving health care quality and patient outcomes. MATERIALS AND METHODS: This document was an initiative of the Mexican Society of Oncology (SMEO) in collaboration with Mexican Center of Clinical Excellence (Cenetec) according to Interna- tional Standards. Such standards included those described by the IOM, NICE, SIGN and GI-N. An interdisciplinary Guideline Development Group (GDG) was put together which included medical oncologists, surgical oncologistsc, radiation therapists, and methodologists with expertise in critical appraisal, sys- tematic reviews and clinical practice guidelines development. RESULTS: 62 clinical questions were agreed among members of the GDG. With the evidence identified from systematic reviews, the GDG developed clinical recommendations using a Modified Delphi Panel technique. Patients' representatives validated them. CONCLUSIONS: These Clinical Practice Guideline aims to support the shared decision-making process for patients with different stages of non-small cell lung cancer. Our goal is to improve health-care quality on these patients.

OBJETIVO: El cáncer de pulmón es una de las principales causas de mortalidad alrededor del mundo. Su historia natural, con la manifestación de síntomas en etapas avanzadas y el retraso en su diagnóstico hacen que una gran proporción de pacientes se diagnostiquen en estadios tardíos de la enfermedad, lo que hace muy complicado el tratamiento exitoso de la misma. De esto deriva la importancia de dar origen a recomendaciones basadas en evidencia para soportar la toma de decisiones clínicas por parte de los grupos interdisicplinarios que se encargan del manejo de este padecimiento. MATERIAL Y MÉTODOS: Este documento se desarrolló por parte de la Sociedad Mexicana de Oncología en colaboración con el Centro Nacional de Excelencia Tec- nológica de México (Cenetec) a través de la dirección de integración de Guías de Práctica Clínica en cumplimiento a estándares internacionales como los descritos por el Ins- tituto de Medicina de EUA (IOM, por sus siglas en inglés), el Instituto de Excelencia Clínica de Gran Bretaña (NICE, por sus siglas en inglés), la Red Colegiada para el Desarrollo de Guías de Escocia (SIGN, por sus siglas en inglés), la Red Internacional de Guías (G-I-N, por sus siglas en inglés); entre otros. Se integró en representación de la Sociedad Mexicana de Oncología un Grupo de Desarrollo de la Guía (GDG) de manera interdisciplinaria, considerando oncólogos médicos, cirujanos oncólogos, cirujanos de tórax, radio-oncólogos, y metodólogos con experiencia en revisiones sistemáticas de la literatura y guías de práctica clínica. RESULTADOS: Se consensuaron 62 preguntas cllínicas que abarcaron lo establecido previamente por el GDG en el documento de alcances de la Guía. Se identificó la evidencia científica que responde a cada una de estas preguntas clínicas y se evaluó críticamente la misma, antes de ser incorporada en el cuerpo de evidencia de la Guía. El GDG acordó mediante la técnica de consenso formal de expertos Panel Delphi la redacción final de las recomendaciones clínicas. C. CONCLUSIONES: Esta Guía de Práctica Clínica pretende proveer recomendaciones clínicas para el manejo de los distintos estadios de la enfermedad y que asistan en el proceso de toma de decisiones compartida. El GDG espera que esta guía contribuya a mejorar la calidad de la atención clínica en las pacientes con cáncer de pulmón de células no pequeñas.

UDiTaS™, a genome editing detection method for indels and genome rearrangements.

BACKGROUND: Understanding the diversity of repair outcomes after introducing a genomic cut is essential for realizing the therapeutic potential of genomic editing technologies. Targeted PCR amplification combined with Next Generation Sequencing (NGS) or enzymatic digestion, while broadly used in the genome editing field, has critical limitations for detecting and quantifying structural variants such as large deletions (greater than approximately 100 base pairs), inversions, and translocations. RESULTS: To overcome these limitations, we have developed a Uni-Directional Targeted Sequencing methodology, UDiTaS, that is quantitative, removes biases associated with variable-length PCR amplification, and can measure structural changes in addition to small insertion and deletion events (indels), all in a single reaction. We have applied UDiTaS to a variety of samples, including those treated with a clinically relevant pair of S. aureus Cas9 single guide RNAs (sgRNAs) targeting CEP290, and a pair of S. pyogenes Cas9 sgRNAs at T-cell relevant loci. In both cases, we have simultaneously measured small and large edits, including inversions and translocations, exemplifying UDiTaS as a valuable tool for the analysis of genome editing outcomes. CONCLUSIONS: UDiTaS is a robust and streamlined sequencing method useful for measuring small indels as well as structural rearrangements, like translocations, in a single reaction. UDiTaS is especially useful for pre-clinical and clinical application of gene editing to measure on- and off-target editing, large and small.

Tailoring the AAV2 capsid vector for bone-targeting.

BACKGROUND: Targeting specific tissues remains a major challenge to the promise of gene therapy. For example, several strategies have failed to target adeno-associated virus 2 (AAV2) vectors, to bone. We have evaluated in vitro and in vivo the affinity of an AAV2 vector to bone matrix, hydroxyapatite (HA) to treat Mucopolysacccharidosis IVA. METHODS: To increase vector affinity to HA, an aspartic acid octapeptide (D8) was inserted immediately after the N-terminal region of the VP2 capsid protein. The modified vector had physical titers and transduction efficiencies comparable to the unmodified vector. RESULTS: The bone-targeting vector had significantly higher HA affinity and vector genome copies in bone than the unmodified vector. The modified vector was also released from HA, and its enzyme activity in bone, 3 months post infusion, was 4.7-fold higher than the unmodified vector. CONCLUSION: Inserting a bone-targeting peptide into the vector capsid increases gene delivery and expression in the bone without decreasing enzyme expression. This approach could be a novel strategy to treat systemic bone diseases.

Potential of DosR and Rpf antigens from Mycobacterium tuberculosis to discriminate between latent and active tuberculosis in a tuberculosis endemic population of Medellin Colombia.

BACKGROUND: Tuberculosis (TB) remains one of the most deadly infectious diseases. One-third to one-fourth of the human population is estimated to be infected with Mycobacterium tuberculosis (Mtb) without showing clinical symptoms, a condition called latent TB infection (LTBI). Diagnosis of Mtb infection is based on the immune response to a mixture of mycobacterial antigens (PPD) or to Mtb specific ESAT-6/CFP10 antigens (IGRA), highly expressed during the initial phase of infection. However, the immune response to PPD and IGRA antigens has a low power to discriminate between LTBI and PTB. The T-cell response to a group of so-called latency (DosR-regulon-encoded) and Resuscitation Promoting (Rpf) antigens of Mtb has been proved to be significantly higher in LTBI compared to active TB across many populations, suggesting their potential use as biomarkers to differentiate latent from active TB. METHODS: PBMCs from a group LTBI (n = 20) and pulmonary TB patients (PTB, n = 21) from an endemic community for TB of the city of Medellín, Colombia, were in vitro stimulated for 7 days with DosR- (Rv1737c, Rv2029c, and Rv2628), Rpf- (Rv0867c and Rv2389c), the recombinant fusion protein ESAT-6-CFP10 (E6-C10)-, or PPD-antigen. The induced IFNγ levels detectable in the supernatants of the antigen-stimulated cells were then used to calculate specificity and sensitivity in discriminating LTBI from PTB, using different statistical approaches. RESULTS: IFNγ production in response to DosR and Rpf antigens was significantly higher in LTBI compared to PTB. ROC curve analyses of IFNγ production allowed differentiation of LTBI from PTB with areas under the curve higher than 0.70. Furthermore, Multiple Correspondence Analysis (MCA) revealed that LTBI is associated with higher levels of IFNγ in response to the different antigens compared to PTB. Analysis based on decision trees showed that the IFNγ levels produced in response to Rv2029c was the leading variable that best-classified disease status. Finally, logistic regression analysis predicted that IFNγ produced by PBMCs in response to E6-C10, Rv2029c, Rv0867c (RpfA) and Rv2389c (RpfA) antigens correlates best with the probability of being latently infected. CONCLUSIONS: The Mtb antigens E6-C10, Rv2029c (PfkB), Rv0867c (RpfA) and Rv2389c (RpfA), may be potential candidates to discriminate LTBI from PTB.

Rivaroxaban Levels in Patients' Plasmas are Comparable by Using Two Different Anti Xa Assay/Coagulometer Systems Calibrated with Two Different Calibrators.

BACKGROUND: Rivaroxaban oral anticoagulant does not need laboratory monitoring, but in some situations plasma level measurement is useful. The objective of this paper was to verify analytical performance and compare two rivaroxaban calibrated anti Xa assays/coagulometer systems with specific or other branch calibrators. METHODS: In 59 samples drawn at trough or peak from patients taking rivaroxaban, plasma levels were measured by HemosIL Liquid anti Xa in ACLTOP 300/500, and STA liquid Anti Xa in TCoag Destiny Plus. HemosIL and STA rivaroxaban calibrators and controls were used. CLSI guideline procedures EP15A3 for precision and trueness, EP6 for linearity, and EP9 for methods comparison were used. RESULTS: Coefficient of variation within run and total precision (CVR and CVWL respectively) of plasmatic rivaroxaban were < 4.2 and < 4.85% and BIAS < 7.4 and < 6.5%, for HemosIL-ACL TOP and STA-Destiny systems, respectively. Linearity verification 8 - 525 ng/mL a Deming regression for methods comparison presented R 0.963, 0.968 and 0.982, with a mean CV 13.3% when using different systems and calibrations. CONCLUSIONS: The analytical performance of plasma rivaroxaban was acceptable in both systems, and results from reagent/coagulometer systems are comparable even when calibrating with different branch material.

acal is a long non-coding RNA in JNK signaling in epithelial shape changes during drosophila dorsal closure.

Dorsal closure is an epithelial remodeling process taking place during Drosophila embryogenesis. JNK signaling coordinates dorsal closure. We identify and characterize acal as a novel negative dorsal closure regulator. acal represents a new level of JNK regulation. The acal locus codes for a conserved, long, non-coding, nuclear RNA. Long non-coding RNAs are an abundant and diverse class of gene regulators. Mutations in acal are lethal. acal mRNA expression is dynamic and is processed into a collection of 50 to 120 bp fragments. We show that acal lies downstream of raw, a pioneer protein, helping explain part of raw functions, and interacts genetically with Polycomb. acal functions in trans regulating mRNA expression of two genes involved in JNK signaling and dorsal closure: Connector of kinase to AP1 (Cka) and anterior open (aop). Cka is a conserved scaffold protein that brings together JNK and Jun, and aop is a transcription factor. Misregulation of Cka and aop can account for dorsal closure phenotypes in acal mutants.