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Breast cancer (BC) has different molecular subgroups related to different risks and treatments. Tumor biopsies for BC detection are invasive and may not reflect tumor heterogeneity. Liquid biopsies have become relevant because they might overcome these limitations. We rationalize that liquid cfDNA biopsies through shallow whole genome sequencing (sWGS) could improve the detection of tumor alterations, complementing the genomic profiling. We evaluated the feasibility to detect somatic copy number alterations (SCNAs) in BC using shallow whole genome sequencing (sWGS) in cfDNA from archived samples from National Cancer Institute of Colombia patients. We sequenced tumor tissues from 38 BC patients with different molecular subtypes using a gene panel of 176 genes significantly mutated in cancer, and by liquid biopsies using sWGS on 20 paired samples to detect SCNAs and compare with the tumor samples. We identified an extensive intertumoral heterogeneity between the molecular subtypes of BC, with a mean tumor load of 602 mutations in the gene panel of tumor tissues. There was a 12.3% of concordance in deletions in the cfDNA-tumor pairs considering only the genes covered by the panel encompassing seven genes: BRCA1, CDK12, NF1, MAP2K4, NCOR1, TP53, and KEAP1 in three patients. This study shows the feasibility to complement the genomic analysis of tumor tissue biopsies to detect SCNA in BC using sWGS in cfDNA, providing a wider identification of potential therapeutic targets.

The epidemic potential of Chikungunya virus (CHIKV) was recently made evident by its introduction and rapid expansion in the Caribbean and the Americas. We sought to gain a detailed understanding of the dynamics of the epidemic in Mexico, the country with the highest number of confirmed CHIKV cases in the Americas, and to characterise viral evolution at the population and intra-host level. Analysis of the spatiotemporal distribution of 2,739 diagnosed cases in Mexico from December 2014 to December 2015 showed a rapid nationwide expansion of the epidemic with focalisation in the South West of the country. We sequenced the envelope glycoprotein 1 gene (E1) from 25 patients using the Illumina MiSeq platform and report synonymous and non-synonymous consensus mutations. Bayesian phylogenetic analysis using 249 Asian lineage E1 sequences gave updated estimates of nucleotide substitution rates for E1 and time to most recent common ancestor of major lineages. The analysis indicates phylogenetically-related emergent Latin American clusters in South Western Mexico, Nicaragua and Honduras and transmission of American strains in the Pacific islands. Detailed analysis showed that intra-host changes in E1 mainly occurred in two variable regions (E1:189-220 and E1:349-358) in domains II and III, respectively, in residues involved in inter and intra-envelope spike interactions. At the population level, this study sheds light on the introduction and evolutionary dynamics of CHIKV in the Americas. At the intra-host level, this study identifies mutational hotspots of the E1 protein with implications for understanding the relationship between the CHIKV quasispecies, viral fitness and pathogenesis.

Voltage-gated calcium channels contain four highly conserved transmembrane helices known as S4 segments that exhibit a positively charged residue every third position, and play the role of voltage sensing. Nonetheless, the activation range between high-voltage (HVA) and low-voltage (LVA) activated calcium channels is around 30-40 mV apart, despite the high level of amino acid similarity within their S4 segments. To investigate the contribution of S4 voltage sensors for the low-voltage activation characteristics of CaV3.3 channels we constructed chimeras by swapping S4 segments between this LVA channel and the HVA CaV1.2 channel. The substitution of S4 segment of Domain II in CaV3.3 by that of CaV1.2 (chimera IIS4C) induced a ~35 mV shift in the voltage-dependence of activation towards positive potentials, showing an I-V curve that almost overlaps with that of CaV1.2 channel. This HVA behavior induced by IIS4C chimera was accompanied by a 2-fold decrease in the voltage-dependence of channel gating. The IVS4 segment had also a strong effect in the voltage sensing of activation, while substitution of segments IS4 and IIIS4 moved the activation curve of CaV3.3 to more negative potentials. Swapping of IIS4 voltage sensor influenced additional properties of this channel such as steady-state inactivation, current decay, and deactivation. Notably, Domain I voltage sensor played a major role in preventing CaV3.3 channels to inactivate from closed states at extreme hyperpolarized potentials. Finally, site-directed mutagenesis in the CaV3.3 channel revealed a partial contribution of the S4-S5 linker of Domain II to LVA behavior, with synergic effects observed in double and triple mutations. These findings indicate that IIS4 and, to a lesser degree IVS4, voltage sensors are crucial in determining the LVA properties of CaV3.3 channels, although the accomplishment of this function involves the participation of other structural elements like S4-S5 linkers.

Behavioural phenotyping of model organisms is widely used to investigate fundamental aspects of organism biology, from the functioning of the nervous system to the effects of genetic mutations, as well as for screening new drug compounds. However, our capacity to observe and quantify the full range and complexity of behavioural responses is limited by the inability of conventional microscopy techniques to capture volumetric image information at sufficient speed. In this article we describe how combining light field microscopy with computational depth estimation provides a new method for fast, quantitative assessment of 3D posture and movement of the model organism Caenorhabditis elegans (C. elegans). We apply this technique to compare the behaviour of cuticle collagen mutants, finding significant differences in 3D posture and locomotion. We demonstrate the ability of quantitative light field microscopy to provide new fundamental insights into C. elegans locomotion by analysing the 3D postural modes of a freely swimming worm. Finally, we consider relative merits of the method and its broader application for phenotypic imaging of other organisms and for other volumetric bioimaging applications.

Acute respiratory infections are the leading cause of morbidity and mortality worldwide. Although a viral aetiological agent is estimated to be involved in up to 80% of cases, the majority of these agents have never been specifically identified. Since 2009, diagnostic and surveillance efforts for influenza virus have been applied worldwide. However, insufficient epidemiological information is available for the many other respiratory viruses that can cause Acute respiratory infections.This study evaluated the presence of 14 non-influenza respiratory viruses in 872 pharyngeal exudate samples using RT-qPCR. All samples met the operational definition of a probable case of an influenza-like illness or severe acute respiratory infection and had a previous negative result for influenza by RT-qPCR.The presence of at least one non-influenza virus was observed in 312 samples (35.8%). The most frequent viruses were rhinovirus (RV; 33.0%), human respiratory syncytial virus (HRSV; 30.8%) and human metapneumovirus (HMPV; 10.6%). A total of 56 cases of co-infection (17.9%) caused by 2, 3, or 4 viruses were identified. Approximately 62.5% of all positive cases were in children under 9 years of age.In this study, we identified 13 non-influenza respiratory viruses that could occur in any season of the year. This study provides evidence for the prevalence and seasonality of a wide range of respiratory viruses that circulate in Mexico and constitute a risk for the population. Additionally, our data suggest that including these tests more widely in the diagnostic algorithm for influenza may reduce the use of unnecessary antibiotics, reduce the hospitalisation time, and enrich national epidemiological data with respect to the infections caused by these viruses.

BACKGROUND:Common low-penetrance genetic variants have been consistently associated with colorectal cancer risk. AIM:To determine if these genetic variants are associated also with adenoma susceptibility and may improve selection of patients with increased risk for advanced adenomas and/or multiplicity (≥ 3 adenomas). METHODS:We selected 1,326 patients with increased risk for advanced adenomas and/or multiplicity and 1,252 controls with normal colonoscopy from population-based colorectal cancer screening programs. We conducted a case-control association study analyzing 30 colorectal cancer susceptibility variants in order to investigate the contribution of these variants to the development of subsequent advanced neoplasia and/or multiplicity. RESULTS:We found that 14 of the analyzed genetic variants showed a statistically significant association with advanced adenomas and/or multiplicity: the probability of developing these lesions increased with the number of risk alleles reaching a 2.3-fold risk increment in individuals with ≥ 17 risk alleles. CONCLUSIONS:Nearly half of the genetic variants associated with colorectal cancer risk are also related to advanced adenoma and/or multiplicity predisposition. Assessing the number of risk alleles in individuals within colorectal cancer screening programs may help to identify better a subgroup with increased risk for advanced neoplasia and/or multiplicity in the general population.

Avipoxvirus (APV) is a fairly common virus affecting birds that causes morbidity and mortality in wild and captive birds. We studied the prevalence of pox-like lesions and genetic diversity of APV in house sparrows (Passer domesticus) in natural, agricultural and urban areas in southern Spain in 2013 and 2014 and in central Spain for 8 months (2012-2013). Overall, 3.2% of 2,341 house sparrows visually examined in southern Spain had cutaneous lesions consistent with avian pox. A similar prevalence (3%) was found in 338 birds from central Spain. Prevalence was higher in hatch-year birds than in adults. We did not detect any clear spatial or temporal patterns of APV distribution. Molecular analyses of poxvirus-like lesions revealed that 63% of the samples were positive. Molecular and phylogenetic analyses of 29 DNA sequences from the fpv167 gene, detected two strains belonging to the canarypox clade (subclades B1 and B2) previously found in Spain. One of them appears predominant in Iberia and North Africa and shares 70% similarity to fowlpox and canarypox virus. This APV strain has been identified in a limited number of species in the Iberian Peninsula, Morocco and Hungary. The second one has a global distribution and has been found in numerous wild bird species around the world. To our knowledge, this represents the largest study of avian poxvirus disease in the broadly distributed house sparrow and strongly supports the findings that Avipox prevalence in this species in South and central Spain is moderate and the genetic diversity low.

The influenza virus is a human pathogen that causes epidemics every year, as well as potential pandemic outbreaks, as occurred in 2009. Vaccination has proven to be sufficient in the prevention and containment of viral spreading. In addition to the current egg-based vaccines, new and promising vaccine platforms, such as cell culture-derived vaccines that include virus-like particles (VLPs), have been developed. VLPs have been shown to be both safe and immunogenic against influenza infections. Although antibody persistence has been studied in traditional egg-based influenza vaccines, studies on antibody response durations induced by VLP influenza vaccines in humans are scarce. Here, we show that subjects vaccinated with an insect cell-derived VLP vaccine, in the midst of the 2009 H1N1 influenza pandemic outbreak in Mexico City, showed antibody persistence up to 24 months post-vaccination. Additionally, we found that subjects that reported being revaccinated with a subsequent inactivated influenza virus vaccine showed higher antibody titres to the pandemic influenza virus than those who were not revaccinated. These findings provide insights into the duration of the antibody responses elicited by an insect cell-derived pandemic influenza VLP vaccine and the possible effects of subsequent influenza vaccination on antibody persistence induced by this VLP vaccine in humans.

Sylvilagus floridanus Papillomavirus (SfPV) causes growth of large horn-like tumors on rabbits. SfPV was described in cottontail rabbits (probably Sylvilagus floridanus) from Kansas and Iowa by Richard Shope in 1933, and detected in S. audubonii in 2011. It is known almost exclusively from the US Midwest. We explored the University of Kansas Natural History Museum for historical museum specimens infected with SfPV, using molecular techniques, to assess if additional wild species host SfPV, and whether SfPV occurs throughout the host range, or just in the Midwest. Secondary aims were to detect distinct strains, and evidence for strain spatio-temporal specificity. We found 20 of 1395 rabbits in the KU collection SfPV symptomatic. Three of 17 lagomorph species (S. nuttallii, and the two known hosts) were symptomatic, while Brachylagus, Lepus and eight additional Sylvilagus species were not. 13 symptomatic individuals were positive by molecular testing, including the first S. nuttallii detection. Prevalence of symptomatic individuals was significantly higher in Sylvilagus (1.8%) than Lepus. Half of these specimens came from Kansas, though new molecular detections were obtained from Jalisco-Mexico's first-and Nebraska, Nevada, New Mexico, and Texas, USA. We document the oldest lab-confirmed case (Kansas, 1915), pre-dating Shope's first case. SfPV amplification was possible from 63.2% of symptomatic museum specimens. Using multiple methodologies, rolling circle amplification and, multiple isothermal displacement amplification in addition to PCR, greatly improved detection rates. Short sequences were obtained from six individuals for two genes. L1 gene sequences were identical to all previously detected sequences; E7 gene sequences, were more variable, yielding five distinct SfPV1 strains that differing by less than 2% from strains circulating in the Midwest and Mexico, between 1915 and 2005. Our results do not clarify whether strains are host species specific, though they are consistent with SfPV specificity to genus Sylvilagus.

Colorectal cancer is one of the most frequent neoplasms and an important cause of mortality in the developed world. Mendelian syndromes account for about 5% of the total burden of CRC, being Lynch syndrome and familial adenomatous polyposis the most common forms. Lynch syndrome tumors develop mainly as a consequence of defective DNA mismatch repair associated with germline mutations in MLH1, MSH2, MSH6 and PMS2. A significant proportion of variants identified by screening these genes correspond to missense or noncoding changes without a clear pathogenic consequence, and they are designated as "variants of uncertain significance", being the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene a clear example. The implication of this variant as a low-penetrance risk variant for CRC was assessed in the present study by performing a case-control study within a large cohort from the COGENT consortium-COST Action BM1206 including 18,723 individuals (8,055 colorectal cancer cases and 10,668 controls) and a case-only genotype-phenotype correlation with several clinical and pathological characteristics restricted to the Epicolon cohort. Our results showed no involvement of this variant as a low-penetrance variant for colorectal cancer genetic susceptibility and no association with any clinical and pathological characteristics including family history for this neoplasm or Lynch syndrome.