Phylogenetic Reconstruction and Functional Characterization of the Ancestral Nef Protein of Primate Lentiviruses.

Nef is an accessory protein unique to the primate HIV-1, HIV-2, and SIV lentiviruses. During infection, Nef functions by interacting with multiple host proteins within infected cells to evade the immune response and enhance virion infectivity. Notably, Nef can counter immune regulators such as CD4 and MHC-I, as well as the SERINC5 restriction factor in infected cells. In this study, we generated a posterior sample of time-scaled phylogenies relating SIV and HIV Nef sequences, followed by reconstruction of ancestral sequences at the root and internal nodes of the sampled trees up to the HIV-1 Group M ancestor. Upon expression of the ancestral primate lentivirus Nef protein within CD4+ HeLa cells, flow cytometry analysis revealed that the primate lentivirus Nef ancestor robustly downregulated cell-surface SERINC5, yet only partially downregulated CD4 from the cell surface. Further analysis revealed that the Nef-mediated CD4 downregulation ability evolved gradually, while Nef-mediated SERINC5 downregulation was recovered abruptly in the HIV-1/M ancestor. Overall, this study provides a framework to reconstruct ancestral viral proteins and enable the functional characterization of these proteins to delineate how functions could have changed throughout evolutionary history.

HexSE: Simulating evolution in overlapping reading frames.

Gene overlap occurs when two or more genes are encoded by the same nucleotides. This phenomenon is found in all taxonomic domains, but is particularly common in viruses, where it may provide a mechanism to increase the information content of compact genomes. The presence of overlapping reading frames (OvRFs) can skew estimates of selection based on the rates of non-synonymous and synonymous substitutions, since a substitution that is synonymous in one reading frame may be non-synonymous in another and vice versa. To understand the impact of OvRFs on molecular evolution, we implemented a versatile simulation model of nucleotide sequence evolution along a phylogeny with any distribution of open reading frames in linear or circular genomes. We use a custom data structure to track the substitution rates at every nucleotide site, which is determined by the stationary nucleotide frequencies, transition bias and the distribution of selection biases (dN/dS) in the respective reading frames. Our simulation model is implemented in the Python scripting language. All source code is released under the GNU General Public License version 3 and are available at https://github.com/PoonLab/HexSE.

Clustering Highly Divergent Homologous Proteins: An Alignment-Free Method.

The comparative analysis of amino acid sequences is an important tool in molecular biology that often requires multiple sequence alignments. In comparisons between less closely related genomes, however, it becomes more difficult to accurately align protein-coding sequences, or even to identify homologous regions in different genomes. In this article, we describe an alignment-free method for the classification of homologous protein-coding regions from different genomes. This methodology was originally developed for comparing genomes within virus families, but may be adapted for other organisms. We quantify sequence homology from the overlap (intersection distance) of the k-mer (word) frequency distributions for different protein sequences. Next, we extract groups of homologous sequences from the resulting distance matrix using a combination of dimensionality reduction and hierarchical clustering methods. Finally, we demonstrate how to generate visualizations of the composition of clusters with respect to protein annotations, and by coloring protein-coding regions of genomes by cluster assignments. These provide a useful means to quickly assess the reliability of the clustering results based on the distribution of homologous genes among genomes. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Data collection and processing Basic Protocol 2: Calculating k-mer distances Basic Protocol 3: Extracting clusters of homology Support Protocol: Genome plot based on clustering results.

Restauraciones indirectas para el manejo de la Hipomineralización de Molares e Incisivos: un abordaje conservador / Restaurações indiretas para o tratamento da hipomineralização de molares e incisivos: uma abordagem conservadora / Indirect restorations for the management of hypomineralization of molars and incisors: a conservative approach

Introducción: Técnicas adhesivas contemporáneas permiten la rehabilitación conservadora y estética de dientes afectados por caries dental, trauma dentoalveolar y defectos del desarrollo del esmalte. Para el tratamiento restaurador de la hipomineralización de molares e incisivos (HMI) se ha recomendado el uso de restauraciones indirectas. Reporte de caso: Paciente sintomático de sexo femenino, 8 años, portadora de HMI severa y comportamiento levemente negativo. Los dientes 16, 36 y 46 presentaban opacidades demarcadas asociadas a dolor provocado. El tratamiento se enfocó en educar a la familia con respecto a la HMI, orientar el comportamiento, controlar la sintomatología y restaurar la función y estética de los dientes afectados por la HMI. Debido a la extensión, severidad y localización de los defectos en los dientes 16, 36 y 46, se optó por realizar restauraciones indirectas con resina compuesta. Luego de 12 meses la paciente presentaba comportamiento definitivamente positivo, no relataba sintomatología dolorosa, las restauraciones estaban sin cambio de color, bien adaptadas, con buena salud gingival y con adecuada anatomía oclusal, lisas y sin signos de lesiones de caries dental. Conclusión: En este caso de HMI severa, la restauración con resina indirecta fue una estrategia estética, conservadora, viable y efectiva durante un periodo de seguimiento de 12 meses.

Introdução: As técnicas adesivas atuais permitem a reabilitação conservadora e estética de dentes acometidos por cáries, traumas dentoalveolares e defeitos de desenvolvimento do esmalte. Para o tratamento restaurador da hipomineralização de molares e incisivos (HMI), o uso de restaurações indiretas tem sido recomendado. Relato de caso: Paciente do sexo feminino, sintomática, 8 anos, com HMI severa e comportamento levemente negativo. Os dentes 16, 36 e 46 apresentaram opacidades acentuadas associadas à dor provocada. O tratamento teve como foco a educação da família sobre a HMI, orientando o comportamento, controlando os sintomas e restaurando a função e a estética dos dentes afetados pela HMI. Devido à extensão, severidade e localização dos defeitos nos dentes 16, 36 e 46, optou-se pela realização de restaurações indiretas com resina composta. Após 12 meses, a paciente apresentou um comportamento definitivamente positivo, não relatou sintomatologia dolorosa, as restaurações estavam sem alteração de cor, bem adaptadas, com boa saúde gengival e anatomia oclusal adequada, lisas e sem sinais de lesões de cárie. Conclusão: Neste caso de MHI severa, a restauração indireta em resina foi uma estratégia estética, conservadora, viável e eficaz com um período de acompanhamento de 12 meses.

Introduction: Contemporary adhesive techniques allow the conservative and aesthetic rehabilitation of teeth affected by dental caries, dentoalveolar trauma, and enamel development defects. For the restorative treatment of hypomineralization of molars and incisors (HMI), indirect restorations have been recommended. Case report: Symptomatic female patient, 8 years old, with severe HMI and slightly negative behavior. Teeth 16, 36, and 46 presented marked opacities associated with provoked pain. The treatment focused on educating the family regarding the HMI, guiding behavior, controlling the symptoms, and restoring the function and aesthetics of the teeth affected by the HMI. Due to the extension, severity, and location of the defects in teeth 16, 36, and 46, it was decided to perform indirect restorations with composite resin. After 12 months, the patient presented definitively positive behavior, and did not report painful symptoms, the restorations were without color change, well adapted, with good gingival health and adequate occlusal anatomy, smooth and without signs of dental caries lesions. Conclusion: In this case of severe MHI, indirect resin restoration was an esthetic, conservative, viable, and effective strategy during a 12-month follow-up period

Hemodialysis vascular access flow measurements by the novel DMed NephroFlow® device: A comparative study with Transonic®.

INTRODUCTION: The current Spanish Clinical Guidelines on Vascular Access for Hemodialysis support the need for surveillance and monitoring of vascular access (VA) to avoid complications. Ultrasound dilution (UD) methods are accepted for the evaluation of VA flow and Transonic® has established the gold standard method for the measurement. The DMed NephroFlow (NIPRO®) device, based on UD method has recently been incorporated. We report a comparative study between the classic Transonic® versus the new NephroFlow® device. MATERIAL AND METHODS: For two consecutive months, measurements of VA flow using both referred systems were performed in patients with a native arteriovenous fistula (AVF) or a graft (AVG) on hemodialysis (HD) in our unit. Both studies were undertaken according to the usual recommendations: VA flow of 250 ml/min, ultrafiltration rate without modifications, both needles in the same vein, and always in the first hour of the HD session. RESULTS: Forty-five patients were included: 17 women and 28 men, mean age of 67 ± 12 years. Thirty patients were diabetic. The baseline meantime on HD was 51 ± 39 months (range: 3-163). Type of VA was: 17 patients radio-cephalic AVF, 17 brachiocephalic AVF, 7 brachiobasilic AVF, and 3 with a graft. The mean flow estimated by the Transonic® was 1222 ± 805 ml/min and the estimated flow by the NephroFlow® device was 1252 ± 975 ml/min. Good reliability between Transonic® and NephroFlow® was observed, with a reliability index of Cronbach's Alpha of 0.927 and an Intraclass Correlation Index of 0.928. CONCLUSIONS: The NephroFlow® device seems comparable with the accepted gold standard UD method for estimating VA flow. More studies must be performed to verify these results. However, they should be considered for the surveillance and monitoring of VA flow, in agreement with the Spanish Guidelines.

Using networks to analyze and visualize the distribution of overlapping genes in virus genomes.

Gene overlap occurs when two or more genes are encoded by the same nucleotides. This phenomenon is found in all taxonomic domains, but is particularly common in viruses, where it may increase the information content of compact genomes or influence the creation of new genes. Here we report a global comparative study of overlapping open reading frames (OvRFs) of 12,609 virus reference genomes in the NCBI database. We retrieved metadata associated with all annotated open reading frames (ORFs) in each genome record to calculate the number, length, and frameshift of OvRFs. Our results show that while the number of OvRFs increases with genome length, they tend to be shorter in longer genomes. The majority of overlaps involve +2 frameshifts, predominantly found in dsDNA viruses. Antisense overlaps in which one of the ORFs was encoded in the same frame on the opposite strand (-0) tend to be longer. Next, we develop a new graph-based representation of the distribution of overlaps among the ORFs of genomes in a given virus family. In the absence of an unambiguous partition of ORFs by homology at this taxonomic level, we used an alignment-free k-mer based approach to cluster protein coding sequences by similarity. We connect these clusters with two types of directed edges to indicate (1) that constituent ORFs are adjacent in one or more genomes, and (2) that these ORFs overlap. These adjacency graphs not only provide a natural visualization scheme, but also a novel statistical framework for analyzing the effects of gene- and genome-level attributes on the frequencies of overlaps.

CoVizu: Rapid analysis and visualization of the global diversity of SARS-CoV-2 genomes.

Phylogenetics has played a pivotal role in the genomic epidemiology of severe acute respiratory syndrome coronavirus 2, such as tracking the emergence and global spread of variants and scientific communication. However, the rapid accumulation of genomic data from around the world-with over two million genomes currently available in the Global Initiative on Sharing All Influenza Data database-is testing the limits of standard phylogenetic methods. Here, we describe a new approach to rapidly analyze and visualize large numbers of SARS-CoV-2 genomes. Using Python, genomes are filtered for problematic sites, incomplete coverage, and excessive divergence from a strict molecular clock. All differences from the reference genome, including indels, are extracted using minimap2 and compactly stored as a set of features for each genome. For each Pango lineage (https://cov-lineages.org), we collapse genomes with identical features into 'variants', generate 100 bootstrap samples of the feature set union to generate weights, and compute the symmetric differences between the weighted feature sets for every pair of variants. The resulting distance matrices are used to generate neighbor-joining trees in RapidNJ that are converted into a majority-rule consensus tree for each lineage. Branches with support values below 50 per cent or mean lengths below 0.5 differences are collapsed, and tip labels on affected branches are mapped to internal nodes as directly sampled ancestral variants. Currently, we process about 2 million genomes in approximately 9 h on 52 cores. The resulting trees are visualized using the JavaScript framework D3.js as 'beadplots', in which variants are represented by horizontal line segments, annotated with beads representing samples by collection date. Variants are linked by vertical edges to represent branches in the consensus tree. These visualizations are published at https://filogeneti.ca/CoVizu. All source code was released under an MIT license at https://github.com/PoonLab/covizu.

Inheritance of Rootstock Effects in Avocado (Persea americana Mill.) cv. Hass.

Grafting is typically utilized to merge adapted seedling rootstocks with highly productive clonal scions. This process implies the interaction of multiple genomes to produce a unique tree phenotype. However, the interconnection of both genotypes obscures individual contributions to phenotypic variation (rootstock-mediated heritability), hampering tree breeding. Therefore, our goal was to quantify the inheritance of seedling rootstock effects on scion traits using avocado (Persea americana Mill.) cv. Hass as a model fruit tree. We characterized 240 diverse rootstocks from 8 avocado cv. Hass orchards with similar management in three regions of the province of Antioquia, northwest Andes of Colombia, using 13 microsatellite markers simple sequence repeats (SSRs). Parallel to this, we recorded 20 phenotypic traits (including morphological, biomass/reproductive, and fruit yield and quality traits) in the scions for 3 years (2015-2017). Relatedness among rootstocks was inferred through the genetic markers and inputted in a "genetic prediction" model to calculate narrow-sense heritabilities (h 2) on scion traits. We used three different randomization tests to highlight traits with consistently significant heritability estimates. This strategy allowed us to capture five traits with significant heritability values that ranged from 0.33 to 0.45 and model fits (r) that oscillated between 0.58 and 0.73 across orchards. The results showed significance in the rootstock effects for four complex harvest and quality traits (i.e., total number of fruits, number of fruits with exportation quality, and number of fruits discarded because of low weight or thrips damage), whereas the only morphological trait that had a significant heritability value was overall trunk height (an emergent property of the rootstock-scion interaction). These findings suggest the inheritance of rootstock effects, beyond root phenotype, on a surprisingly wide spectrum of scion traits in "Hass" avocado. They also reinforce the utility of polymorphic SSRs for relatedness reconstruction and genetic prediction of complex traits. This research is, up to date, the most cohesive evidence of narrow-sense inheritance of rootstock effects in a tropical fruit tree crop. Ultimately, our work highlights the importance of considering the rootstock-scion interaction to broaden the genetic basis of fruit tree breeding programs while enhancing our understanding of the consequences of grafting.

Establishment of buttonhole technique as a puncture alternative for arteriovenous fístulas. experience of a centre over 3years. / Instauración de la técnica de buttonhole como alternativa de punción para las fístulas arteriovenosas. Experiencia de un centro en 3años.

INTRODUCTION: The buttonhole (BH) puncture technique for arteriovenous fistulas is an alternative to the classical staggered puncture. PURPOSE: We present 3years' results incorporating the BH puncture technique for arteriovenous fistulas in our dialysis unit. MATERIAL AND METHODS: Twenty-two patients were started on BH technique, 15 men and 7 women (mean age: 62 years; SD: 12), with time spent on dialysis when starting the BH technique of 34 months (SD: 34, median: 27, range: 3-136). Seven patients received acenocoumarol and 9 antiplatelet agents. The vascular access median time at the beginning of the technique was 27 months (range: 3-252). RESULTS: Between 5 and 8 consecutive dialysis sessions were necessary to achieve a proper tunnel puncture. No patient suffered major complications. The average time on BH technique until December 2015 was 12 months (SD: 10, median: 9, range: 1-45). By the end of the study, 5patients were performing self-puncture. Haemostasis times post-dialysis were reduced from 18.6min (SD: 8, prior to the BH technique), to 12.2minutes (SD: 3 after BH) (P=.0005). CONCLUSIONS: The BH technique is an alternative puncture technique for dialysis patients. Self-puncture and reduction in hemostasis time are potential beneficial aspects. A greater diffusion of this technique in the hemodialysis units would allow it to be better applied. A highly motivated nursing staff is key and a necessary condition for its implementation.

Instauración de la técnica de buttonhole como alternativa de punción para las fístulas arteriovenosas. Experiencia de un centro en 3 años / Establishment of buttonhole technique as a puncture alternative for arteriovenous fístulas. experience of a centre over 3 years

Introducción: La técnica de punción de buttonhole (BH) o de ojal para fístulas arteriovenosas es una alternativa a la punción escalonada clásica. Objetivo: Mostramos la experiencia en nuestra unidad de hemodiálisis tras la incorporación de esta técnica a la práctica clínica diaria. Material y métodos: Se ha aplicado la técnica de BH a 22 pacientes, 15 hombres y 7 mujeres, con una edad media de 62 años (DE: 12), con un tiempo en diálisis en el momento de iniciar la técnica de BH de 34 meses (DE: 34; mediana: 27; rango: 3-136). Siete pacientes recibían acenocumarol y 9 estaban antiagregados. La mediana de tiempo con el acceso vascular al inicio de la técnica de BH fue de 27 meses (rango: 3-252). Resultados: Fueron necesarias entre 5 y 8 sesiones consecutivas de diálisis para la consecución de un correcto túnel de canalización. Ningún paciente presentó complicaciones mayores. El tiempo medio en la técnica de BH fue de 12 meses (DE: 10; mediana: 9; rango: 1-45). Al final del periodo de estudio 5 pacientes realizaban autopunción. El tiempo de hemostasia posdiálisis se redujo de 18,6 min (DE: 8) previamente a la técnica de BH a 12,2 (DE: 3) posteriormente a su utilización (p=0,0005). Conclusiones: La técnica de BH es una alternativa de punción en hemodiálisis. Puede presentar aspectos beneficiosos como la autopunción o la reducción de los tiempos de hemostasia. Una mayor difusión en las unidades de hemodiálisis sería necesaria para mejorar en su aplicación adecuada. El personal de enfermería altamente motivado es clave y condición necesaria para su implantación (AU)

Introduction: The buttonhole (BH) puncture technique for arteriovenous fistulas is an alternative to the classical staggered puncture. Purpose: We present 3 years’ results incorporating the BH puncture technique for arteriovenous fistulas in our dialysis unit. Material and methods: Twenty-two patients were started on BH technique, 15 men and 7 women (mean age: 62 years; SD: 12), with time spent on dialysis when starting the BH technique of 34 months (SD: 34, median: 27, range: 3-136). Seven patients received acenocoumarol and 9 antiplatelet agents. The vascular access median time at the beginning of the technique was 27 months (range: 3-252). Results: Between 5 and 8 consecutive dialysis sessions were necessary to achieve a proper tunnel puncture. No patient suffered major complications. The average time on BH technique until December 2015 was 12 months (SD: 10, median: 9, range: 1-45). By the end of the study, 5 patients were performing self-puncture. Haemostasis times post-dialysis were reduced from 18.6min (SD: 8, prior to the BH technique), to 12.2 minutes (SD: 3 after BH) (P=.0005). Conclusions: The BH technique is an alternative puncture technique for dialysis patients. Self-puncture and reduction in hemostasis time are potential beneficial aspects. A greater diffusion of this technique in the hemodialysis units would allow it to be better applied. A highly motivated nursing staff is key and a necessary condition for its implementation (AU)

SECUENCIACIÓN DEL GENOMA COMPLETO DEL Potato yellow vein virus (PYVV) EN TOMATE (Solanum lycopersicum) EN COLOMBIA / Genome Sequencing of Potato yellow vein virus (PYVV) Strain Infecting Solanum lycopersicum in Colombia

RESUMEN Potato yellow vein virus (PYVV), es uno de los fitopatógenos más limitantes para la producción de papa en la región de Los Andes. A pesar que se le ha detectado infectando tomate en Colombia, el conocimiento de las características biológicas de las cepas presentes en este hospedante es muy limitado. En este estudio, utilizando secuenciación masiva de nueva generación (NGS), se obtuvo la secuencia completa de los tres segmentos genómicos del PYVV en plantas de tomate en Marinilla (Antioquia) y se evalúo la utilidad de tres juegos de cebadores para su detección mediante pruebas de RT-PCR convencional y en tiempo real (RT-qPCR). El genoma de la secuencia consenso presentó tamaños de 8043 nt (ARN1), 5346 nt (ARN2) y 3896 nt (ARN3) y se identificaron los diez ORF previamente reportados en este virus, aunque, en general, éstos presentaron menores niveles de identidad que los registrados entre cepas de PYVV de papa. Análisis de variación y de selección identificaron dos regiones en los ORF MET/HEL y CPm que presentan selección positiva, lo que podría estar asociado a la adaptación por hospedante. Los tres juegos de cebadores amplificaron las regiones esperadas de la cápside de PYVV, siendo posible identificar, por diferencias en valores de temperatura de fusión (Tm) y por secuenciación Sanger, la ocurrencia de al menos dos variantes principales de este virus en el Oriente Antioqueño, lo que concuerda con los niveles moderados de polimorfismos encontrados en las secuencias obtenidas por NGS.

ABSTRACT Potato yellow vein virus (PYVV) is one of the most important pathogens of potato in the Andean region. In spite of having been detected in tomato crops in Colombia, knowledge on the biological characteristics of PYVV is limited on this host. In this study, next-generation sequencing (NGS) of a PYVV strain infecting tomato in Marinilla District (Antioquia) was performed; additionally, three primer set useful in RT-PCR and RT-qPCR detection were also tested. The consensus genome consisted of three RNA segments of 8043 nt (RNA1), 5346 nt (RNA2) and 3896 nt (RNA3) encoding ten ORF with slight lower sequence identity in relation to PYVV isolates from potato. Sequence analysis suggests the presence of regions potentially undergoing positive selection in the ORFs coding for MET/HEL and CPm possibly as a result of host adaptation. Experimental validation of primers resulted in amplicon with the expected size while melting temperature analysis and sequencing suggest the presence of at least two PYVV infecting S. lycopersicum in east Antioquia in agreement with the NGS data.