Few-shot genes selection: subset of PAM50 genes for breast cancer subtypes classification.

BACKGROUND: In recent years, researchers have made significant strides in understanding the heterogeneity of breast cancer and its various subtypes. However, the wealth of genomic and proteomic data available today necessitates efficient frameworks, instruments, and computational tools for meaningful analysis. Despite its success as a prognostic tool, the PAM50 gene signature's reliance on many genes presents challenges in terms of cost and complexity. Consequently, there is a need for more efficient methods to classify breast cancer subtypes using a reduced gene set accurately. RESULTS: This study explores the potential of achieving precise breast cancer subtype categorization using a reduced gene set derived from the PAM50 gene signature. By employing a "Few-Shot Genes Selection" method, we randomly select smaller subsets from PAM50 and evaluate their performance using metrics and a linear model, specifically the Support Vector Machine (SVM) classifier. In addition, we aim to assess whether a more compact gene set can maintain performance while simplifying the classification process. Our findings demonstrate that certain reduced gene subsets can perform comparable or superior to the full PAM50 gene signature. CONCLUSIONS: The identified gene subsets, with 36 genes, have the potential to contribute to the development of more cost-effective and streamlined diagnostic tools in breast cancer research and clinical settings.

Aplicaciones clínicas de la herramienta CRISPR-Cas / Clinical Applications of the CRISPR-Cas Tool

Resumen El desarrollo de tecnologías para la edición del genoma ha abierto la posibilidad de apuntar directamente y modificar secuencias genómicas en casi todo tipo de células eucariotas. La edición del genoma ha ampliado nuestra capacidad para dilucidar la contribución de la genética a las enfermedades al promover la creación de modelos celulares y animales más precisos de procesos patológicos y ha comenzado a mostrar su potencial en una variedad de campos, que van desde la investigación básica hasta la biotecnología aplicada y biomédica. Entre estas tecnologías, el uso de las repeticiones palindrómicas cortas agrupadas regularmente espaciadas ha acelerado, en gran medida, el progreso de la edición de genes desde el concepto hasta la práctica clínica, generando, además, interés debido, no solo a su precisión y eficiencia, sino también a la rapidez y a los costos necesarios para su implementación en comparación con otras tecnologías de edición genómica. En esta revisión se presenta información recabada de publicaciones indexadas en la base de datos PubMed que se encontraron mediante el uso de palabras claves asociadas con la tecnología y que se filtraron para retener solo aquellas con evidencias de avances clínicamente relevantes y que permiten demostrar algunas de las aplicaciones que tiene esta tecnología en la investigación, pronóstico y tratamiento de enfermedades genéticas, cardiovasculares, virales, entre otras; esto con el objetivo de dar a conocer la situación actual de los avances en aplicaciones clínicas de la herramienta CRISPR-Cas y fomentar aún más la investigación en esta tecnología, la cual, tal como se evidencia a lo largo de esta revisión, posee una gran versatilidad y un amplio rango de aplicaciones, lo que ofrece una enorme oportunidad en el campo de la medicina genómica, pero que, a su vez, requiere un mayor fomento en su investigación para mejorar la tecnología y acercarla aún más a consolidar aplicaciones clínicas de uso seguro, confiable y consistente.

Abstract The development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all types of eukaryotic cells. Genome editing has expanded our ability to elucidate the contribution of genetics to disease by promoting the creation of more precise cellular and animal models of disease processes and has begun to show its potential in a variety of fields, ranging from basic research to applied and biomedical biotechnology. Among these technologies, the use of clustered regularly spaced short palindromic repeats have greatly accelerated the progress of gene editing from concept to clinical practice, further generating interest due not only to its precision and efficiency, but also to the speed and costs required for its implementation compared to other genomic editing methods. This review presents information collected from indexed publications in the PubMed database that were found by using keywords associated with the technology and filtered to retain only those with evidence of clinically relevant advances that demonstrate some of the applications that this technology has in research, prognosis, and treatment of genetic, cardiovascular, and viral diseases, among others; this with the aim of show the current situation of advances in clinical applications of the CRISPR-Cas tool and further encourage research in this technology, which, as evidenced throughout this review, has a great versatility and a wide range of applications, which offers an enormous opportunity in the field of genomic medicine but which, in turn, requires greater support in its research to improve the technology and bring it even closer to consolidating clinical applications of safe, reliable and consistent use.

Cellular and Extracellular Vesicle RNA Analysis in the Global Threat Fungus Candida auris.

Emerging and reemerging pathogens are a worldwide concern, and it is predicted that these microbes will cause severe outbreaks. Candida auris affects people with weakened immune systems, particularly those who are hospitalized or are in health care facilities. Extracellular vesicles (EVs) are lipid bilayer structures released by organisms from all domains of life. EVs can deliver functional molecules to target cells, including proteins and nucleic acids, especially RNA molecules. EVs from several pathogenic fungi species play diverse biological roles related to cell-cell communication and pathogen-host interaction. In this study, we describe a data set which we produced by sequencing the RNA content of EVs from C. auris under normal growth conditions and in the presence of the antifungal caspofungin, a first-line drug to treat this fungus. To generate a more complete data set for future comparative studies, we also sequenced the RNA cellular content of EVs under the same conditions. This data set addresses a previously unexplored area of fungal biology regarding cellular small RNA and EV RNA. Our data will provide a molecular basis for the study of the aspects associated with antifungal treatment, gene expression response, and EV composition in C. auris. These data will also allow the exploration of small RNA content in the fungal kingdom and might serve as an informative basis for studies on the mechanisms by which molecules are directed to fungal EVs. IMPORTANCE Candida auris, a relevant emerging human-pathogenic yeast, is the first fungus to be called a global public health threat by the WHO. This is because of its rapid spread on all inhabited continents, together with its extremely high frequency of drug and multidrug resistance. In our study, we generated a large data set for 3 distinct strains of C. auris and obtained cellular small RNA fraction as well as extracellular vesicle RNA (EV-RNA) during normal growth conditions and after treatment with caspofungin, the first-line drug used to treat C. auris infection.

Genome editing: propelling the next generation of crop improvement / Edição genômica: impulsionando a nova geração do melhoramento de plantas

Climate change and population size records threaten food security. Therefore, the call for a more sustainable and efficient crop production has never been more urgent. Traditional plant breeding was one of the first successful approaches to expand cultivation areas and crop yield. Later, biotechnological tools and their products, such as genetically modified organisms containing exogenous DNA, further broadened the limits of agricultural results, yet bringing huge financial, bureaucratic, and public rejection hurdles. In the 90s, scientific advances brought the opportunity to drive mutations using engineered nucleases, and since 2013 CRISPR-Cas has emerged as the most practical toolkit to edit genomes. One of the most striking possibilities is to generate edited and non-transgenic plants. In this review, we present the working mechanism behind CRISPR-induced mutations and pinpoint the latest techniques developed, as well as its myriad of applications in agriculture. The enhancing scope of CRISPR ranges from introducing traits of agronomic interest – such as herbicide resistance, resistance/tolerance to biotic and abiotic stresses, and quality and durability of products – to accelerating plant breeding processes, including haploid induction, generating male-sterile lines, fixating hybrid vigor, and overcoming self-incompatibility. We also discuss regulatory issues surrounding edited plants and derived products around the world, challenges that must be overcome, and future prospects to harness all the potential of this amazing tool to guarantee the new crop production revolution.

As mudanças climáticas e números recordes de população ameaçam a segurança alimentar. Portanto, o apelo por uma produção agrícola mais sustentável e eficiente nunca foi tão urgente. O melhoramento genético tradicional foi uma das primeiras abordagens bem-sucedidas para expandir as áreas de cultivo e o rendimento das safras. Posteriormente, ferramentas biotecnológicas e seus produtos, como organismos geneticamente modificados contendo DNA exógeno, ampliaram ainda mais os limites dos resultados agrícolas, apesar de ainda carregarem enormes obstáculos financeiros, burocráticos e de rejeição pública. Na década de 90, os avanços científicos trouxeram a oportunidade de conduzir mutações usando nucleases projetadas e, desde 2013, CRISPR-Cas surgiu como o kit de ferramentas mais prático para editar genomas. Uma das possibilidades mais marcantes é gerar plantas editadas e não transgênicas. Nesta revisão, apresentamos o mecanismo de ação por trás das mutações induzidas por CRISPR, identificando as últimas técnicas desenvolvidas, bem como sua miríade de aplicações na agricultura. O escopo de aprimoramento do CRISPR varia desde introduzir características de interesse agronômico – como resistência a herbicidas, resistência/tolerância a estresses bióticos e abióticos e qualidade e durabilidade de produtos – até acelerar processos de melhoramento genético de plantas, incluindo indução de haploidia, geração de linhagens macho-estéreis, fixação de vigor híbrido e superação da autoincompatibilidade. Também discutimos questões regulatórias em torno de plantas editadas e produtos derivados mundialmente, desafios que devem ser superados e perspectivas futuras para aproveitar todo o potencial desta ferramenta incrível para garantir a nova revolução na produção de culturas agrícolas.

Genome editing: propelling the next generation of crop improvement / Edição genômica: impulsionando a nova geração do melhoramento de plantas

Climate change and population size records threaten food security. Therefore, the call for a more sustainable and efficient crop production has never been more urgent. Traditional plant breeding was one of the first successful approaches to expand cultivation areas and crop yield. Later, biotechnological tools and their products, such as genetically modified organisms containing exogenous DNA, further broadened the limits of agricultural results, yet bringing huge financial, bureaucratic, and public rejection hurdles. In the 90s, scientific advances brought the opportunity to drive mutations using engineered nucleases, and since 2013 CRISPR-Cas has emerged as the most practical toolkit to edit genomes. One of the most striking possibilities is to generate edited and non-transgenic plants. In this review, we present the working mechanism behind CRISPR-induced mutations and pinpoint the latest techniques developed, as well as its myriad of applications in agriculture. The enhancing scope of CRISPR ranges from introducing traits of agronomic interest such as herbicide resistance, resistance/tolerance to biotic and abiotic stresses, and quality and durability of products to accelerating plant breeding processes, including haploid induction, generating male-sterile lines, fixating hybrid vigor, and overcoming self-incompatibility. We also discuss regulatory issues surrounding edited plants and derived products around the world, challenges that must be overcome, and future prospects to harness all the potential of this amazing tool to guarantee the new crop production revolution.(AU)

As mudanças climáticas e números recordes de população ameaçam a segurança alimentar. Portanto, o apelo por uma produção agrícola mais sustentável e eficiente nunca foi tão urgente. O melhoramento genético tradicional foi uma das primeiras abordagens bem-sucedidas para expandir as áreas de cultivo e o rendimento das safras. Posteriormente, ferramentas biotecnológicas e seus produtos, como organismos geneticamente modificados contendo DNA exógeno, ampliaram ainda mais os limites dos resultados agrícolas, apesar de ainda carregarem enormes obstáculos financeiros, burocráticos e de rejeição pública. Na década de 90, os avanços científicos trouxeram a oportunidade de conduzir mutações usando nucleases projetadas e, desde 2013, CRISPR-Cas surgiu como o kit de ferramentas mais prático para editar genomas. Uma das possibilidades mais marcantes é gerar plantas editadas e não transgênicas. Nesta revisão, apresentamos o mecanismo de ação por trás das mutações induzidas por CRISPR, identificando as últimas técnicas desenvolvidas, bem como sua miríade de aplicações na agricultura. O escopo de aprimoramento do CRISPR varia desde introduzir características de interesse agronômico como resistência a herbicidas, resistência/tolerância a estresses bióticos e abióticos e qualidade e durabilidade de produtos até acelerar processos de melhoramento genético de plantas, incluindo indução de haploidia, geração de linhagens macho-estéreis, fixação de vigor híbrido e superação da autoincompatibilidade. Também discutimos questões regulatórias em torno de plantas editadas e produtos derivados mundialmente, desafios que devem ser superados e perspectivas futuras para aproveitar todo o potencial desta ferramenta incrível para garantir a nova revolução na produção de culturas agrícolas.(AU)

When junk DNA turns functional: transposon-derived non-coding RNAs in plants.

Transposable elements (TEs) are major contributors to genome complexity in eukaryotes. TE mobilization may cause genome instability, although it can also drive genome diversity throughout evolution. TE transposition may influence the transcriptional activity of neighboring genes by modulating the epigenomic profile of the region or by altering the relative position of regulatory elements. Notably, TEs have emerged in the last few years as an important source of functional long and small non-coding RNAs. A plethora of small RNAs derived from TEs have been linked to the trans regulation of gene activity at the transcriptional and post-transcriptional levels. Furthermore, TE-derived long non-coding RNAs have been shown to modulate gene expression by interacting with protein partners, sequestering active small RNAs, and forming duplexes with DNA or other RNA molecules. In this review, we summarize our current knowledge of the functional and mechanistic paradigms of TE-derived long and small non-coding RNAs and discuss their role in plant development and evolution.

Assessment of Urothelial Cytotoxicity at Morphological and Molecular Levels: Urinary Bladder as Target Organ.

The urinary bladder is a target organ of several toxic agents. Exposure to those agents induces mild-to-severe changes, which can be evaluated by different methods. Among them, the scanning-electron microscopy (SEM) is the "gold standard" for characterizing urothelial damage since it provides high-definition images, making it possible to detect early lesions on the surface of the urinary bladder. In addition, molecular technologies allow detecting changes in genetic material and investigating the interaction between genes and environmental stress in disease causation. The urinary bladder epithelium is where the most common type of bladder cancer occurs in humans, that is, the transitional-cell carcinoma (TCC). In animal models, the TCC can be similar to the disease in humans. Techniques to evaluate urothelium in experimental models aid in the comprehension of risk factors for urothelial carcinogenesis.

Contrast-FEL-A Test for Differences in Selective Pressures at Individual Sites among Clades and Sets of Branches.

A number of evolutionary hypotheses can be tested by comparing selective pressures among sets of branches in a phylogenetic tree. When the question of interest is to identify specific sites within genes that may be evolving differently, a common approach is to perform separate analyses on subsets of sequences and compare parameter estimates in a post hoc fashion. This approach is statistically suboptimal and not always applicable. Here, we develop a simple extension of a popular fixed effects likelihood method in the context of codon-based evolutionary phylogenetic maximum likelihood testing, Contrast-FEL. It is suitable for identifying individual alignment sites where any among the K≥2 sets of branches in a phylogenetic tree have detectably different ω ratios, indicative of different selective regimes. Using extensive simulations, we show that Contrast-FEL delivers good power, exceeding 90% for sufficiently large differences, while maintaining tight control over false positive rates, when the model is correctly specified. We conclude by applying Contrast-FEL to data from five previously published studies spanning a diverse range of organisms and focusing on different evolutionary questions.

Partitioned Gene-Tree Analyses and Gene-Based Topology Testing Help Resolve Incongruence in a Phylogenomic Study of Host-Specialist Bees (Apidae: Eucerinae).

Incongruence among phylogenetic results has become a common occurrence in analyses of genome-scale data sets. Incongruence originates from uncertainty in underlying evolutionary processes (e.g., incomplete lineage sorting) and from difficulties in determining the best analytical approaches for each situation. To overcome these difficulties, more studies are needed that identify incongruences and demonstrate practical ways to confidently resolve them. Here, we present results of a phylogenomic study based on the analysis 197 taxa and 2,526 ultraconserved element (UCE) loci. We investigate evolutionary relationships of Eucerinae, a diverse subfamily of apid bees (relatives of honey bees and bumble bees) with >1,200 species. We sampled representatives of all tribes within the group and >80% of genera, including two mysterious South American genera, Chilimalopsis and Teratognatha. Initial analysis of the UCE data revealed two conflicting hypotheses for relationships among tribes. To resolve the incongruence, we tested concatenation and species tree approaches and used a variety of additional strategies including locus filtering, partitioned gene-trees searches, and gene-based topological tests. We show that within-locus partitioning improves gene tree and subsequent species-tree estimation, and that this approach, confidently resolves the incongruence observed in our data set. After exploring our proposed analytical strategy on eucerine bees, we validated its efficacy to resolve hard phylogenetic problems by implementing it on a published UCE data set of Adephaga (Insecta: Coleoptera). Our results provide a robust phylogenetic hypothesis for Eucerinae and demonstrate a practical strategy for resolving incongruence in other phylogenomic data sets.

Identification of patterns related to linkage groups or disequilibrium by factor analysis / Identificação de padrões relacionados a grupos de ligação ou de desequilíbrio por análise de fatores

Empirical patterns of linkage disequilibrium (LD) can be used to increase the statistical power of genetic mapping. This study was carried out with the objective of verifying the efficacy of factor analysis (AF) applied to data sets of molecular markers of the SNP type, in order to identify linkage groups and haplotypes blocks. The SNPs data set used was derived from a simulation process of an F2 population, containing 2000 marks with information of 500 individuals. The estimation of the factorial loadings of FA was made in two ways, considering the matrix of distances between the markers (A) and considering the correlation matrix (R). The number of factors (k) to be used was established based on the graph scree-plot and based on the proportion of the total variance explained. Results indicated that matrices A and R lead to similar results. Based on the scree-plot we considered k equal to 10 and the factors interpreted as being representative of the bonding groups. The second criterion led to a number of factors equal to 50, and the factors interpreted as being representative of the haplotypes blocks. This showed the potential of the technique, making it possible to obtain results applicable to any type of population, helping or corroborating the interpretation of genomic studies. The study demonstrated that AF was able to identify patterns of association between markers, identifying subgroups of markers that reflect factor binding groups and also linkage disequilibrium groups.(AU)

Padrões empíricos de desequilíbrio de ligação (LD) podem ser utilizados para aumentar o poder estatístico do mapeamento genético. Este trabalho foi realizado com o objetivo de verificar a eficácia da análise de fatores (AF) aplicada a conjuntos de dados de marcadores moleculares do tipo SNP, visando identificar grupos de ligação e blocos de haplótipos. O conjunto de dados SNPs utilizado foi oriundo de um processo de simulação de uma população F2, contendo 2000 marcas com informações de 500 indivíduos. A estimação das cargas fatoriais (loadings) da AF foi feita de duas formas, considerando a matriz de distâncias entre os marcadores (A) e considerando a matriz de correlação (R). O número de fatores (k) a ser utilizado foi estabelecido com base no gráfico scree-plot e com base na proporção da variância total explicada. Os resultados indicam que as matrizes A e R conduzem a resultados similares. Com base no scree-plot considerou-se k igual a 10 e os fatores interpretados como sendo representativos dos grupos de ligação. O segundo critério conduziu a um número de fatores igual a 50, e os fatores interpretados como sendo representativos dos blocos de haplótipos. Isto mostra o potencial da técnica que permite obter resultados aplicáveis a qualquer tipo de população, corroborando a interpretação de estudos genômicos. O trabalho demonstrou que a AF foi capaz de identificar padrões de associação entre marcadores, identificando subgrupos de marcadores que refletem grupos de ligação fatorial e também grupos de desequilíbrio de ligação.(AU)

A Likelihood Approach for Uncovering Selective Sweep Signatures from Haplotype Data.

Selective sweeps are frequent and varied signatures in the genomes of natural populations, and detecting them is consequently important in understanding mechanisms of adaptation by natural selection. Following a selective sweep, haplotypic diversity surrounding the site under selection decreases, and this deviation from the background pattern of variation can be applied to identify sweeps. Multiple methods exist to locate selective sweeps in the genome from haplotype data, but none leverages the power of a model-based approach to make their inference. Here, we propose a likelihood ratio test statistic T to probe whole-genome polymorphism data sets for selective sweep signatures. Our framework uses a simple but powerful model of haplotype frequency spectrum distortion to find sweeps and additionally make an inference on the number of presently sweeping haplotypes in a population. We found that the T statistic is suitable for detecting both hard and soft sweeps across a variety of demographic models, selection strengths, and ages of the beneficial allele. Accordingly, we applied the T statistic to variant calls from European and sub-Saharan African human populations, yielding primarily literature-supported candidates, including LCT, RSPH3, and ZNF211 in CEU, SYT1, RGS18, and NNT in YRI, and HLA genes in both populations. We also searched for sweep signatures in Drosophila melanogaster, finding expected candidates at Ace, Uhg1, and Pimet. Finally, we provide open-source software to compute the T statistic and the inferred number of presently sweeping haplotypes from whole-genome data.

Analysis of Gene Expression in Fasciola hepatica Juveniles and Adults by In Situ Hybridization.

In situ hybridization (ISH) is a technique used for the spatial localization of nucleic acids within tissues and cells. It is based on the ability of labeled nucleic acids (probes) to hybridize under the right conditions with the nucleic acids present in fixed biological specimens. In this chapter, we describe protocols for detection of RNA by ISH using digoxigenin (DIG)-labeled probes for Fasciola hepatica adults (in cryosections, given their large size) and for newly excysted juveniles (NEJs, which are ideally suited given their small size for whole-mount ISH). We describe fluorogenic and chromogenic protocols, respectively, but the detection methods can be easily interchanged by using the appropriate enzyme-conjugated antibodies and detection solutions.

Molecular Evolutionary Genetics Analysis (MEGA) for macOS.

The Molecular Evolutionary Genetics Analysis (MEGA) software enables comparative analysis of molecular sequences in phylogenetics and evolutionary medicine. Here, we introduce the macOS version of the MEGA software. This new version eliminates the need for virtualization and emulation programs previously required to use MEGA on Apple computers. MEGA for macOS utilizes memory and computing resources efficiently for conducting evolutionary analyses on macOS. It has a native Cocoa graphical user interface that is programmed to provide a consistent user experience across macOS, Windows, and Linux. MEGA for macOS is available from www.megasoftware.net free of charge.

An improved protocol for efficient transformation and regeneration of Setaria italica.

KEY MESSAGE: An efficient and improved transformation method for functional genetics studies in S. italica, being a boon for the Setaria scientific community and for crop improvement. Foxtail millet (Setaria italica) is a short life cycle C4 plant, with sequenced genome, and a potential model plant for C4 species. S. italica is also important on a global food security and healthiness context due to its importance in arid and semi-arid areas. However, despite its importance, there are just few transformation protocols directed to this species. The current protocols reached about 5.5-9% of efficiency, which do not make it a valuable model organism. Different types of explants were used in the above mentioned methods, such as immature and mature inflorescence and shoot apex. However, these techniques have many limitations, such as unavailability of explants throughout the year and a crucial, laborious and considerable time-consuming selection. Aiming a simplified and efficient methodology, we adopted dry mature seeds as explants, which are available in abundance, are constant along the year and well responsive to tissue culture, in addition to a differentiated approach that reaches on an average 19.2% transformation efficiency of S. italica. Thus, we propose a protocol that optimizes the transformation efficiency of this cereal crop allowing a high increase on transformation and regeneration rates. Our transformation protocol provides an interesting tool for Setaria community research as well as enables new strategies for breeding enhanced productivity in the species.

Step-by-Step Pipeline for the Ecological Analysis of Endophytic Fungi using ITS nrDNA Data.

The nuclear ribosomal DNA internal transcribed spacer (ITS) is accepted as the genetic marker or barcode of choice for the identification of fungal samples. Here, we present a protocol to analyze fungal ITS data, from quality preprocessing of raw sequences to identification of operational taxonomic units (OTUs), taxonomic classification, and assignment of functional traits. The pipeline relies on well-established and manually curated data collections, namely the UNITE database and the FUNGuild script. As an example, real ITS data from culturable endophytic fungi were analyzed, providing detailed descriptions for every step, parameter, and downstream analysis, and finishing with a phylogenetic analysis of the sequences and assigned ecological roles. This article constitutes a comprehensive guide for researchers that have little familiarity with bioinformatic analysis of essential steps required in further ecological studies of fungal communities. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Raw sequencing data processing Support Protocol: Building a BLAST database Basic Protocol 2: Obtaining information from databases Basic Protocol 3: Phylogenetic analysis.

An update on clinical and surgical interventions to reduce sperm DNA fragmentation in infertile men.

BACKGROUND: Sperm chromatin integrity is essential for normal embryo development and pregnancy outcome. Sperm DNA fragmentation (SDF) testing constitutes a diagnostic tool to measure the proportion of spermatozoa with damaged chromatin in the ejaculate. SDF is associated with potentially treatable conditions, including varicocele, male accessory gland infections, inadequate lifestyle, and gonadotoxin exposure, thus prompting their treatment as a means of improving sperm DNA quality and the reproductive outcomes. OBJECTIVE: To provide an up-to-date review of the role of clinical and surgical interventions on SDF values in subfertile men. MATERIALS AND METHODS: An extensive search of studies examining the relationship between male infertility conditions associated with SDF was performed using PubMed and MEDLINE, with a focus on interventional therapy. The start date for the search was not defined, whereas the end date was March 2019. Randomized and non-randomized controlled trials, observational studies, systematic and narrative reviews, and case series were evaluated. RESULTS: Treating the underlying male infertility factor seems a promising way to alleviate SDF and to increase the likelihood of achieving natural and assisted conception, but data remain limited. The best evidence relates to varicocele repair and hormonal therapy with the follicle-stimulating hormone. Antioxidant therapy and lifestyle changes might alleviate oxidative sperm markers and decrease SDF but their effects on pregnancy outcomes are still unclear. Among men with high SDF undergoing assisted reproductive technology, the use of testicular spermatozoa in preference over ejaculated spermatozoa for intracytoplasmic sperm injection (ICSI) has been shown to improve pregnancy rates possibly owing to the better sperm chromatin quality in testicular spermatozoa than in ejaculated spermatozoa. CONCLUSION: Current evidence supports interventional therapy as a means to alleviate sperm DNA damage. Identification of the conditions associated with SDF remains important to enable treatment to potentially improve pregnancy outcomes but given the limited data further research is needed to determine the exact role of specific interventional therapy for subfertile men with impaired sperm chromatin.

HyPhy 2.5-A Customizable Platform for Evolutionary Hypothesis Testing Using Phylogenies.

HYpothesis testing using PHYlogenies (HyPhy) is a scriptable, open-source package for fitting a broad range of evolutionary models to multiple sequence alignments, and for conducting subsequent parameter estimation and hypothesis testing, primarily in the maximum likelihood statistical framework. It has become a popular choice for characterizing various aspects of the evolutionary process: natural selection, evolutionary rates, recombination, and coevolution. The 2.5 release (available from www.hyphy.org) includes a completely re-engineered computational core and analysis library that introduces new classes of evolutionary models and statistical tests, delivers substantial performance and stability enhancements, improves usability, streamlines end-to-end analysis workflows, makes it easier to develop custom analyses, and is mostly backward compatible with previous HyPhy releases.

Reliable Confidence Intervals for RelTime Estimates of Evolutionary Divergence Times.

Confidence intervals (CIs) depict the statistical uncertainty surrounding evolutionary divergence time estimates. They capture variance contributed by the finite number of sequences and sites used in the alignment, deviations of evolutionary rates from a strict molecular clock in a phylogeny, and uncertainty associated with clock calibrations. Reliable tests of biological hypotheses demand reliable CIs. However, current non-Bayesian methods may produce unreliable CIs because they do not incorporate rate variation among lineages and interactions among clock calibrations properly. Here, we present a new analytical method to calculate CIs of divergence times estimated using the RelTime method, along with an approach to utilize multiple calibration uncertainty densities in dating analyses. Empirical data analyses showed that the new methods produce CIs that overlap with Bayesian highest posterior density intervals. In the analysis of computer-simulated data, we found that RelTime CIs show excellent average coverage probabilities, that is, the actual time is contained within the CIs with a 94% probability. These developments will encourage broader use of computationally efficient RelTime approaches in molecular dating analyses and biological hypothesis testing.