Minor Mutations Research Articles

Predictors of Virological Failure and Time to Viral Suppression of First-Line Integrase Inhibitor-Based Antiretroviral Treatment.

BackgroundIntegrase strand transfer inhibitors (InSTIs) are recommended for first-line treatment of persons with human immunodeficiency virus (HIV). We identified risk factors, including baseline minor InSTI resistance mutations, for treatment failure of InSTI-based regimens.MethodsWe studied time-to-treatment failure and time to viral suppression among 1419 drug-naive patients in the Swiss HIV Cohort Study. We performed Cox regression models adjusted for demographic factors, baseline HIV RNA/CD4 cell counts, AIDS-defining events, and the type of InSTI. In 646 patients with a baseline genotypic resistance test of the integrase, we studied the impact of minor integrase resistance mutations.ResultsWe observed 121 virological failures during 18 447 person-years of follow-up. A baseline viral load ≥100 000 copies/mL (multivariable hazard ratio [mHR], 2.2; 95% confidence interval [CI], 1.3–3.6) and an AIDS-defining event (mHR, 1.8; 95% CI. 1.1–3.0) were associated with treatment failure. CD4 counts between 200 and 500 cells/µL (mHR, 0.5; 95% CI, .3–.8) and >500 cells/µL (mHR, 0.4; 95% CI, .2–.7) were protective. Time to suppression was shorter in lower viral load strata (mHR, 0.7; 95% CI, .6–.8) and in dolutegravir-based therapy (mHR, 1.2; 95% CI, 1.0–1.4). Minor resistance mutations were found at baseline in 104 of 646 (16%) patients with no effect on treatment outcome.ConclusionsFactors associated with treatment failure on InSTI-based first-line regimen remained similar to those of older treatments, in particular high viral load and low CD4 counts.

Symmetry breaking and structural polymorphism in a bacterial microcompartment shell protein for choline utilization.

Bacterial microcompartments are protein-based organelles that carry out specialized metabolic functions in diverse bacteria. Their outer shells are built from several thousand protein subunits. Some of the architectural principles of bacterial microcompartments have been articulated, with lateral packing of flat hexameric BMC proteins providing the basic foundation for assembly. Nonetheless, a complete understanding has been elusive, partly owing to polymorphic mechanisms of assembly exhibited by most microcompartment types. An earlier study of one homologous BMC shell protein subfamily, EutS/PduU, revealed a profoundly bent, rather than flat, hexameric structure. The possibility of a specialized architectural role was hypothesized, but artifactual effects of crystallization could not be ruled out. Here we report a series of crystal structures of an orthologous protein, CutR, from a glycyl-radical type choline-utilizing microcompartment from the bacterium Streptococcus intermedius. Depending on crystal form, expression construct, and minor mutations, a range of novel quaternary architectures was observed, including two spiral hexagonal assemblies. A new graphical approach helps illuminate the variations in BMC hexameric structure, with results substantiating the idea that the EutS/PduU/CutR subfamily of BMC proteins may endow microcompartment shells with flexible modes of assembly.

The Novel Coronavirus Enigma: Phylogeny and Analyses of Coevolving Mutations Among the SARS-CoV-2 Viruses Circulating in India.

BackgroundThe RNA genome of the emerging novel coronavirus is rapidly mutating, and its human-to-human transmission rate is increasing. Hence, temporal dissection of their evolutionary dynamics, the nature of variations among different strains, and understanding the single nucleotide polymorphisms in the endemic settings are crucial. Delineating the heterogeneous genomic constellations of this novel virus will help us understand its complex behavior in a particular geographical region.ObjectiveThis is a comprehensive analysis of 95 Indian SARS-CoV-2 genome sequences available from the Global Initiative on Sharing All Influenza Data (GISAID) repository during the first 6 months of 2020 (January through June). Evolutionary dynamics, gene-specific phylogeny, and the emergence of the novel coevolving mutations in 9 structural and nonstructural genes among circulating SARS-CoV-2 strains across 12 different Indian states were analyzed.MethodsA total of 95 SARS-CoV-2 nucleotide sequences submitted from India were downloaded from the GISAID database. Molecular Evolutionary Genetics Analysis, version X software was used to construct the 9 phylogenetic dendrograms based on nucleotide sequences of the SARS-CoV-2 genes. Analyses of the coevolving mutations were done in comparison to the prototype SARS-CoV-2 from Wuhan, China. The secondary structure of the RNA-dependent RNA polymerase/nonstructural protein NSP12 was predicted with respect to the novel A97V mutation.ResultsPhylogenetic analyses revealed the evolution of “genome-type clusters” and adaptive selection of “L”-type SARS-CoV-2 strains with genetic closeness to the bat severe acute respiratory syndrome–like coronaviruses. These strains were distant to pangolin or Middle East respiratory syndrome–related coronavirus strains. With regard to the novel coevolving mutations, 2 groups have been seen circulating in India at present, the “major group” (66/95, 69.4%) and the “minor group” (21/95, 22.1%) , harboring 4 and 5 coexisting mutations, respectively. The “major group” mutations fall in the A2a clade. All the minor group mutations, except 11083G>T (L37F, NSP6 gene), were unique to the Indian isolates.ConclusionsThis study highlights the rapidly evolving SARS-CoV-2 virus and the cocirculation of multiple clades and subclades. This comprehensive study is a potential resource for monitoring the novel mutations in the viral genome, interpreting changes in viral pathogenesis, and designing vaccines or other therapeutics.

Abstract 3689: Mutation spectrum of MEF cells transformed by overexpression of novel activating KRAS mutation candidates in lung adenocarcinoma

Abstract Lung adenocarcinoma (LUAC) is a unique lung cancer subtype that is responsive to several therapeutic agents. The KRAS gene is the second most frequently mutated gene in LUAC and the majority of KRAS mutations are one of three classical activating mutations (G12, G13, and Q61). Recently, we have characterized other types of “minor” KRAS mutation (A66T, A66V, and G75E) identified among LUAC patients and found these three mutants had tumor-forming activity in mouse embryonic fibroblasts in an allograft model. RNA-Seq analysis revealed that mouse embryonic fibroblasts overexpressing these three minor KRAS mutations have distinct expression profiles compared with overexpression of the wild type but similar expression profiles compared with overexpression of the classical KRAS mutants, G12V. In this study, we have searched candidate somatic genetic alterations in the RNA-seq data and will discuss the impact of the mutations in KRAS carcinogenesis. Citation Format: Jiro Abe, Nobuhiro Tanuma, Miyuki Nomura, Shin Ito, Isao Kasugai, Ikuro Sato, Keiichi Tamai, Mai Mochizuki, Kazunori Yamaguchi, Hiroshi Shima, Yoshinori Okada, Jun Yasuda. Mutation spectrum of MEF cells transformed by overexpression of novel activating KRAS mutation candidates in lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3689.

Ultrarapid EGFR Mutation Screening Followed by Comprehensive Next-Generation Sequencing: A Feasible, Informative Approach for Lung Carcinoma Cytology Specimens With a High Success Rate

IntroductionFor patients with advanced NSCLC, cytologic samples may be the only diagnostic specimen available for molecular profiling. Although both rapid and comprehensive assessment are essential in this setting, an integrated multitest approach remains an important strategy in many laboratories, despite the risks and challenges when working with scant samples. In this study, we describe our experience and high success rate in using a multitest approach, focusing on the clinical validation and incorporation of ultrarapid EGFR testing using the Idylla system followed by comprehensive next-generation sequencing (NGS). MethodsCytology samples received for routine molecular testing were included in this study. The performance characteristics of the EGFR Idylla assay were assessed; tissue suitability parameters and interpretation criteria to supplement automated mutation calling were established. The assay performance was monitored for 1 year, comparing the results with those of concurrent NGS testing by MSK-IMPACT (primarily) or MSK-AmpliSeq and MSK-Fusion solid panel in a subset of cases. ResultsOverall, 301 samples were studied; 83 samples were included in validation (60.2% [50 of 83] were positive for EGFR mutations). Concordance with the reference method was 96.4% (80 of 83) of the samples with excellent reproducibility. The limit of detection was variable depending on the total tissue input and the specific mutation tested. Unextracted tissue inputs that maintained total EGFR cycle of quantification at less than 23 allowed all mutations to be detected if present at greater than 5% variant allele frequency. Mutations could be detected at 1% variant allele frequency with total EGFR cycle of quantification of 18. During the clinical implementation phase, 218 NSCLC samples were tested by Idylla (24.3% [53 of 218] were EGFR mutation positive). Concurrent NGS testing was requested on 165 samples and successfully performed on 96.4% (159 of 165) of the samples. The Idylla automated results were concordant with those obtained by NGS in 96.2% (153 of 159) of cases and improved to 98.7% (157 of 159) after incorporation of manual review criteria to supplement automated calling, resulting in a diagnostic sensitivity of 95.6% (95% confidence interval: 84.9%–99.5%). In general, 9% (14 of 159) of the cases tested by NGS had EGFR mutations not covered by the Idylla assay, primarily insertions in exon 19 and 20 and minor mutations co-occurring with canonical sensitizing mutations. ConclusionsComprehensive molecular testing is feasible and has a high success rate in NSCLC cytology samples when using a multitest approach. Testing with the Idylla system enables rapid and accurate determination of the EGFR status without compromising subsequent NGS testing.

Emergent Viral Infections of the CNS.

Biological evolution of the microbiome continually drives the emergence of human viral pathogens, a subset of which attack the nervous system. The sheer number of pathogens that have appeared, along with their abundance in the environment, demand our attention. For the most part, our innate and adaptive immune systems have successfully protected us from infection; however, in the past 5 decades, through pathogen mutation and ecosystem disruption, a dozen viruses emerged to cause significant neurologic disease. Most of these pathogens have come from sylvatic reservoirs having made the energetically difficult, and fortuitously rare, jump into humans. But the human microbiome is also replete with agents already adapted to the host that need only minor mutations to create neurotropic/toxic agents. While each host/virus symbiosis is unique, this review examines virologic and immunologic principles that govern the pathogenesis of different viral CNS infections that were described in the past 50 years (Influenza, West Nile Virus, Zika, Rift Valley Fever Virus, Hendra/Nipah, Enterovirus-A71/-D68, Human parechovirus, HIV, and SARS-CoV). Knowledge of these pathogens provides us the opportunity to respond and mitigate infection while at the same time prepare for inevitable arrival of unknown agents.

Non-Targeted Metabolite Profiles and Sensory Properties Elucidate Commonalities and Differences of Wines Made with the Same Variety but Different Cultivar Clones.

Grapes, one of the oldest agricultural crops, are cultivated to produce table fruits, dried fruits, juice, and wine. Grapevine variety is composed of clones that share common morphological traits. However, they can differ in minor genetic mutations which often result in not only notorious morphological changes but also in other non-visible sensorial distinctive attributes. In the present work, we identified three Vitis vinifera cv. Pinot noir clones grown under identical field conditions that showed different grape cluster types. Here, sensorial analysis together with non-targeted metabolite profiles by Ultra High performance Liquid Chromatography (UPLC) couples to Ultra High Resolution Mass Spectrometry (FT-ICR-MS) of wines elaborated from the three different grape cluster types was studied with the aim of (i) finding sensorial differences among these three types of wines, and, if there were, (ii) determining the molecular features (metabolites) associated with these sensorial attributes by a multivariate statistical approach.

Prospective study for usefulness of circulating-free DNA on prediction of third generation EGFR tyrosine kinase inhibitors.

e21510 Background: Most non-small lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations develop resistance when exposed to EGFR-tyrosine kinase inhibitors (TKIs). T790M develops in about half of patients treated with TKI and can be detected by tumor tissue and cfDNA hotspot tests. However, co-occurring mutations at other loci may impact efficacy. We conducted a prospective, multi-center, observational study to assess the detection rates and predictive values of plasma-based EGFR T790M detection methods for Japanese NSCLC patients treated with osimertinib. Methods: NSCLC patients with tumor EGFR mutations and disease progression after treatment with 1st- or 2nd-generation EGFR-TKI were enrolled. Plasma was collected at the time of clinical disease progression, before osimertinib treatment. The collected plasma was tested for EGFR T90M by in-house plasma MBP-QP and ddPCR assays and compared to clinically tested cobas (Roche) results (including tissue, plasma). The primary endpoint was to demonstrate comparability of our MBP-QP system to cobas using plasma-based EGFR T790M detection to predict the therapeuitic effect of osimertinib via objective response rate (ORR) and disease control rate (DCR). As an exploratory analysis, we used Guardant360 to retrospectively test available banked plasma samples collected describe time points. Results: From Feb 2017 to Jan 2019, 145 patients enrolled. T790M was detected by cobas in 57 cases (44 tissue, 16 plasma, 3 both). ORR and DCR in plasma cobas-positive cases were 62.5% and 81.3%, respectively. MBP-QP found T790M in 9 patients with ORR and DCR 66.7% and 77.8%. ddPCR found 17 cases with ORR and DCR 70.6% and 82.4%. ORR was not correlated to AF. In plasma samples from 54 patients, Guardant360 detected T790M in 57%. Co-occurring alterations such as amplification or minor mutations in EGFR or other genes such as TP53 did not impact ORR, but in the group with poor response to osimertinib, the number of detected gene alterations tended to be large. Two patients with small cell carcinoma transformation had RB1 mutations and MYC amplification. Conclusions: Regardless of the test system, the detection of T790M could predict a good therapeutic effect of osimertinib, but there was no difference in response to osimertinib depending on EGFR T790M AF. Compared to single-gene assessment of EGFR, NGS of cfDNA may be useful for guiding treatment decisions for patients with TKI-resistant NSCLC. Clinical trial information: UMIN000025930.

Bottleneck Size-Dependent Changes in the Genetic Diversity and Specific Growth Rate of a Rotavirus A Strain.

RNA viruses form a dynamic distribution of mutant swarms (termed "quasispecies") due to the accumulation of mutations in the viral genome. The genetic diversity of a viral population is affected by several factors, including a bottleneck effect. Human-to-human transmission exemplifies a bottleneck effect, in that only part of a viral population can reach the next susceptible hosts. In the present study, two lineages of the rhesus rotavirus (RRV) strain of rotavirus A were serially passaged five times at a multiplicity of infection (MOI) of 0.1 or 0.001, and three phenotypes (infectious titer, cell binding ability, and specific growth rate) were used to evaluate the impact of a bottleneck effect on the RRV population. The specific growth rate values of lineages passaged under the stronger bottleneck (MOI of 0.001) were higher after five passages. The nucleotide diversity also increased, which indicated that the mutant swarms of the lineages under the stronger bottleneck effect were expanded through the serial passages. The random distribution of synonymous and nonsynonymous substitutions on rotavirus genome segments indicated that almost all mutations were selectively neutral. Simple simulations revealed that the presence of minor mutants could influence the specific growth rate of a population in a mutant frequency-dependent manner. These results indicate a stronger bottleneck effect can create more sequence spaces for minor sequences.IMPORTANCE In this study, we investigated a bottleneck effect on an RRV population that may drastically affect the viral population structure. RRV populations were serially passaged under two levels of a bottleneck effect, which exemplified human-to-human transmission. As a result, the genetic diversity and specific growth rate of RRV populations increased under the stronger bottleneck effect, which implied that a bottleneck created a new space in a population for minor mutants originally existing in a hidden layer, which includes minor mutations that cannot be distinguished from a sequencing error. The results of this study suggest that the genetic drift caused by a bottleneck in human-to-human transmission explains the random appearance of new genetic lineages causing viral outbreaks, which can be expected according to molecular epidemiology using next-generation sequencing in which the viral genetic diversity within a viral population is investigated.

HIV-1 drug resistance mutations detection and HIV-1 subtype G report by using next-generation sequencing platform

BackgroundBased on world health organization (WHO) recommend, drug resistance assay should be performed in initial of treatment and after treatment for administering and monitoring of anti-retroviral regime in HIV-1 infected patients. Material and methodNGS analyses were performed on forty-one plasma samples from HIV-1 affected patients using the Sentosa SQ HIV genotyping assay (Vela-Diagnostics, Germany). This system comprises a semi-automated Ion torrent based platform and the sequencing results were analyzed based on ANRS, REGA and Stanford drug resistance algorithms. Phylogenetic analysis was analyzed based on https://comet.lih.lu database as well as MEGA5 Software. ResultsDrug resistances were identified in thirty-three samples (80%) out of forty-one samples. The Phylogenetic analysis results showed that CRF-35AD (94%) and subtypes B (2.4%) and G (2.4%) were dominant subtypes in this study. NRTI and NNRTI associated dominant mutations were M184I/V and K103 N.High-level resistance to lamivudine (3 TC) and Emtricitabine (FTC) were detected in 34.3% of patients while 53.1% were resistant to Efavirenz (EFV) and Nevirapine (NVP). The Protease inhibitor (PI) minor and major mutations were not reported but more than 95% of samples had polymorphisms mutation in K20R, M36I, H69K, L89 M positions. These mutations are subtype dependent and completely are absent in subtype B virus. The secondary mutations were reported in positions of E157Q, S230 N, and T97A of integrase gene and four samples represent low-level resistance to integrase strand transfer inhibitor (INSTI). ConclusionsThis is the first preliminary evaluation of HIV-1 drug resistance mutation (DRM) by using the Sentosa SQ HIV Genotyping Assay in Iran. The NGS represent a promising tool for the accurate detection of DRMs of CRF-35AD that is dominant subtype in Iranian HIV-1 infected population and for the first time revealed HIV-1 subtype G in Iranian population. In the present study polymorphic mutation in the position of K20R, M36I, H69K, L89 M were properly reported in CRF35AD that is dominant in Iranian HIV patients.

Comparative analysis of targeted next-generation sequencing panels for the detection of gene mutations in chronic lymphocytic leukemia: an ERIC multi-center study.

Next-generation sequencing (NGS) has transitioned from research to clinical routine, yet the comparability of different technologies for mutation profiling remains an open question. We performed a European multicenter (n=6) evaluation of three amplicon-based NGS assays targeting 11 genes recurrently mutated in chronic lymphocytic leukemia. Each assay was assessed by two centers using 48 pre-characterized chronic lymphocytic leukemia samples; libraries were sequenced on the Illumina MiSeq instrument and bioinformatics analyses were centralized. Across all centers the median percentage of target reads ≥100x ranged from 94.2-99.8%. In order to rule out assay-specific technical variability, we first assessed variant calling at the individual assay level i.e., pairwise analysis of variants detected amongst partner centers. After filtering for variants present in the paired normal sample and removal of PCR/sequencing artefacts, the panels achieved 96.2% (Multiplicom), 97.7% (TruSeq) and 90% (HaloPlex) concordance at a variant allele frequency (VAF) >0.5%. Reproducibility was assessed by looking at the inter-laboratory variation in detecting mutations and 107 of 115 (93% concordance) mutations were detected by all six centers, while the remaining eight variants (7%) were undetected by a single center. Notably, 6 of 8 of these variants concerned minor subclonal mutations (VAF <5%). We sought to investigate low-frequency mutations further by using a high-sensitivity assay containing unique molecular identifiers, which confirmed the presence of several minor subclonal mutations. Thus, while amplicon-based approaches can be adopted for somatic mutation detection with VAF >5%, after rigorous validation, the use of unique molecular identifiers may be necessary to reach a higher sensitivity and ensure consistent and accurate detection of low-frequency variants.

Polymorphisms and drug resistance analysis of HIV-1 isolates from patients on first line antiretroviral therapy (ART) in South-eastern Nigeria.

Acquisition of resistance mutations by HIV-1 isolates causes treatment failure among infected patients receiving antiretroviral therapy (ART). This study determined patterns of drug-resistance mutations (DRMs) among HIV-1 isolates from patients receiving first-line ART in South-eastern Nigeria. Blood samples were collected from HIV-1 infected patients accessing antiretroviral treatment centers at General Hospital Awo-Omamma, Imo state, State Hospital Asaba, Delta state and St Joseph's Catholic Hospital Adazi, Anambra state and used for HIV-1 DNA sequencing and phylogenetic analysis. DRMs were scored using combination of Stanford algorithm and the 2015 International Antiviral Society-USA list while drug susceptibility was predicted using Stanford algorithm. Twenty eight of the HIV-1 isolates were sequenced and identified as subtypes G (35.7%), CRF02_AG (57.1%) and unclassifiable, UG (7.1%). Major PI resistance-associated mutations were identified at two sites including M46L (16.7% of subtype G/UG) and V82L (6.3% of CRF02_AG). Minor PI resistance-associated mutations identified among subtype G/UG are L10V/I (8.3%) and K20I (100%) while L10V/I (50%), K20I (100%), L33F (6.3%) and N88D (6.3%) were identified among CRF02_AG. Other polymorphisms found include; I13V/A, E35Q, M36I/L, N37D/S/E/H, R57K/G, L63T/P/S/Q, C67E/S, H69K/R, K70R, V82I and L89M in the range of 28.6% to 100% among the different subtypes. Interpretation based on Stanford algorithm showed that Darunavir/ritonavir is the only regimen whose potency was not compromised by the circulating mutations. Identification of major and minor PI resistance mutations in this study underscores the need for drug resistance testing prior to initiation of second line antiretroviral therapy in Nigeria.

Jouer avec la page : Les Romans de Stéphane Vanderhaeghe

My paper shows how Stéphane Vanderhaeghe’s work goes against the grain of a literary mainstream whose most prominent authors (and literary prizes) favor autofiction or narratives re-exploring a more or less recent historical past. Instead, Vanderhaeghe’s radical writing seeks to challenge the formal potentialities of literature, thus inscribing his work in the experimental traditions of Oulipo and Nouveau Roman. Charøgnards, his first published novel, opens with a twenty-page prologue written in an invented, futuristic French language. The remainder of the text functions as an unpaginated journal whose chronology is constantly re-assessed in light of the numerous rewritings to which the unreliable narrator confesses. Graphemes gradually disappear from the page, figuring the slow disappearance of language as a result of the takeover of invasive crows. The color of pages veers from white to gray before turning into black, so that the book stands out in its unique materiality. Though À tous les airs presents itself as a more traditionally manufactured book, the text proves no less experimental, through the forgoing of characters and repetitions with minor mutations. These literary practices reveal the author’s singular literary identity whose ambitious nature runs the risk of alienating readers.

A novel amino acid site closely associated with the neurovirulence of live, attenuated Japanese encephalitis vaccine (SA14-14-2 strain)

Japanese encephalitis (JE) poses a serious threat to the world’s public health yet without a cure, the only way to prevent Japanese encephalitis virus (JEV) infection is vaccination. Live attenuated vaccine (SA14-14-2 strain) is the most widely used JE vaccine, and clinical data have confirmed its safety and effectiveness. Eight sitesassociated with virulence in the Envelope (E) protein are often the focus of quality control of JE vaccine. However, sequences retrieved from NCBI, as well as our previous results showed that the wild strain SA14 may harbor two different amino acids at amino acid residue 244 of the E glycoprotein (E244), and it may be related to virulence. In this study, we introduced a single mutation at nt1708 (G → A) in the full-length cDNA clone of SA14-14-2, replacing a Gly with Glu at amino acid residue 244 of the E glycoprotein, and successfully constructed the mutant virus (JEV E244). JEV E244 exhibited a similar plaque morphology and growth characteristics to JEV SA14-14-2 in cell culture. However, it had lethal neurovirulence in mice and could enter the brain following intraperitoneal inoculation. Moreover, the virulence of JEV E244 in the context of vaccine in mice is significantly different from that of the JEV E244 alone. These results suggested that E244 site should be included in the assessment of the genetic stability of the attenuated JE vaccine. The detection of minor mutations in vaccine population and influence on the safety of vaccine is discussed.

Comprehensive analysis of a novel mouse model of the 22q11.2 deletion syndrome: a model with the most common 3.0-Mb deletion at the human 22q11.2 locus

The 22q11.2 deletion syndrome (22q11.2DS) is associated with an increased risk for psychiatric disorders. Although most of the 22q11.2DS patients have a 3.0-Mb deletion, existing mouse models only mimic a minor mutation of 22q11.2DS, a 1.5-Mb deletion. The role of the genes existing outside the 1.5-Mb deletion in psychiatric symptoms of 22q11.2DS is unclear. In this study, we generated a mouse model that reproduced the 3.0-Mb deletion of the 22q11.2DS (Del(3.0 Mb)/ +) using the CRISPR/Cas9 system. Ethological and physiological phenotypes of adult male mutants were comprehensively evaluated by visual-evoked potentials, circadian behavioral rhythm, and a series of behavioral tests, such as measurement of locomotor activity, prepulse inhibition, fear-conditioning memory, and visual discrimination learning. As a result, Del(3.0 Mb)/ + mice showed reduction of auditory prepulse inhibition and attenuated cue-dependent fear memory, which is consistent with the phenotypes of existing 22q11.2DS models. In addition, Del(3.0 Mb)/ + mice displayed an impaired early visual processing that is commonly seen in patients with schizophrenia. Meanwhile, unlike the existing models, Del(3.0 Mb)/ + mice exhibited hypoactivity over several behavioral tests, possibly reflecting the fatigability of 22q11.2DS patients. Lastly, Del(3.0 Mb)/ + mice displayed a faster adaptation to experimental jet lag as compared with wild-type mice. Our results support the validity of Del(3.0 Mb)/ + mice as a schizophrenia animal model and suggest that our mouse model is a useful resource to understand pathogenic mechanisms of schizophrenia and other psychiatric disorders associated with 22q11.2DS.

Presence of V72I, G123S and R127K Integrase Inhibitor polymorphisms could reduce ART effectiveness: a retrospective longitudinal study

Objectives: Structural aspects of HIV-1 integrase complex and role of integrase minor mutations and polymorphisms in ART effectiveness is still unknown. The objective of this study was to assess the 24 and 48 weeks (W) effectiveness of ART regimens in patients with Integrase Inhibitors (InSTI) minor mutations and polymorphisms receiving InSTI-based regimens.Methods: We enrolled all ART-naïve or InSTI-naïve HIV-infected patients, with a baseline InSTI genotypic resistances test between 2011 and 2016. We analyzed integrase resistance mutations using the Stanford University HIV Drug Resistance Database (HIVdb Program, version 6.3.0). The outcome was virological response at 24 and 48 W of follow up (FU) according to snapshot analysis. We defined virological failure as two consecutive HIV-RNA > 50 copies/ml, or one >1000 copies/ml. Patients were divided in those presenting InSTI minor mutations (Group 1), and those with only polymorphisms or wild type (Group 2).Results: We enrolled 83 patients. 81 patients reached 24 W of FU: 2/20 (10%) and 4/61 (6.5%) showed virological failure in Group 1 and 2 respectively. 66 patients reached 48 W of FU: 0/17 (0%) and 2/49 (4%) showed virological failure in Group 1 and 2 respectively. Interestingly, patients with polymorphisms G123S and R127K had higher risk of failure at 24 W (respectively, relative risk - RR − 36, IQR 2.1-613, p = 0.01; RR 36, IQR 2.1-613, p = 0.01) and patients with V72I had an higher risk of failure both at 24 W (RR 6.52, IQR 1.29-32.9, p = 0.02) and 48 W (RR 21.1, IQR 1.07-414, p = 0.04).Conclusions: Our study showed that the presence of V72I, G123S and R127K polymorphisms could play a role in reducing InSTI effectiveness.

EGFR mutation genotyping and ALK status determination in liquid-based cytology samples of non-small cell lung cancer.

Personalised medicine for primary lung cancers (PLCs) requires molecular analysis of cancer tissue or cells. The primary objective of the present prospective study was to assess the concordance between epidermal growth factor receptor (EGFR) gene mutation detection and echinoderm microtubule-associated protein-like (EML) 4-anaplastic lymphoma kinase protein (ALK) expression using liquid-based cytology (LBC) samples and matched histology samples of PLC patients. A total of 117 patients who underwent surgical resection of non-small cell PLC were enrolled. Cytological specimens scratched from the resected PLC lesion were fixed in CytoRich Red. DNA extracted from LBC samples was examined for EGFR gene mutations. Anaplastic lymphoma kinase arrangement was analysed by immunostaining and fluorescence in situ hybridisation. Our patient cohort comprised 93 cases of adenocarcinoma, 16 squamous cell carcinoma, three adenosquamous carcinoma, two large cell neuroendocrine carcinoma, one pleomorphic carcinoma and two other cases. Sixty-six (58.4%) LBC samples harboured EGFR gene mutations. The overall concordance rate in EGFR gene mutation status, including minor mutations, between histologic and paired LBC specimens (N = 105) was 100%. The overall concordance rate of EGFR gene mutation status, including minor mutations and ALK status according to immunostains between histologic and paired LBC specimens, was 100% (105/105) and 100% (48/48), respectively. Genotyping and protein expression studies can be reliably performed using LBC samples prepared with CytoRich Red. Analysis of such samples may guide individual therapy in PLC patients.

Cutaneous melanoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†.

Cutaneous melanoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†.

Novel activating KRAS mutation candidates in lung adenocarcinoma

Lung adenocarcinoma (LUAC) is a unique lung cancer subtype that is responsive to several therapeutic agents. The KRAS gene is the second most frequently mutated gene in LUAC and the majority of KRAS mutations are one of three classical activating mutations (G12, G13, and Q61). Recently, other types of “minor” KRAS mutation have been identified among LUAC patients and some may have similar transforming activities to those of the classical KRAS mutations. Here we describe minor KRAS mutations in LUAC patients, some of which (A66T, A66V, and G75E) may have tumor-forming activity in mouse embryonic fibroblasts in an allograft model. RNA-Seq analysis revealed that mouse embryonic fibroblasts overexpressing these three minor KRAS mutations have distinct expression profiles compared with overexpression of the wild type but similar expression profiles compared with overexpression of the classical KRAS mutants. Our results indicate that some of the minor KRAS mutations cause varying tumor formation activity and are important targets for developing anti-RAS agents as chemotherapeutic agents.

Influenza Viruses in Mice: Deep Sequencing Analysis of Serial Passage and Effects of Sialic Acid Structural Variation.

Influenza A viruses have regularly jumped to new host species to cause epidemics or pandemics, an evolutionary process that involves variation in the viral traits necessary to overcome host barriers and facilitate transmission. Mice are not a natural host for influenza virus but are frequently used as models in studies of pathogenesis, often after multiple passages to achieve higher viral titers that result in clinical disease such as weight loss or death. Here, we examine the processes of influenza A virus infection and evolution in mice by comparing single nucleotide variations of a human H1N1 pandemic virus, a seasonal H3N2 virus, and an H3N2 canine influenza virus during experimental passage. We also compared replication and sequence variation in wild-type mice expressing N-glycolylneuraminic acid (Neu5Gc) with those seen in mice expressing only N-acetylneuraminic acid (Neu5Ac). Viruses derived from plasmids were propagated in MDCK cells and then passaged in mice up to four times. Full-genome deep sequencing of the plasmids, cultured viruses, and viruses from mice at various passages revealed only small numbers of mutational changes. The H3N2 canine influenza virus showed increases in frequency of sporadic mutations in the PB2, PA, and NA segments. The H1N1 pandemic virus grew well in mice, and while it exhibited the maintenance of some minority mutations, there was no clear evidence for adaptive evolution. The H3N2 seasonal virus did not establish in the mice. Finally, there were no clear sequence differences associated with the presence or absence of Neu5Gc.IMPORTANCE Mice are commonly used as a model to study the growth and virulence of influenza A viruses in mammals but are not a natural host and have distinct sialic acid receptor profiles compared to humans. Using experimental infections with different subtypes of influenza A virus derived from different hosts, we found that evolution of influenza A virus in mice did not necessarily proceed through the linear accumulation of host-adaptive mutations, that there was variation in the patterns of mutations detected in each repetition, and that the mutation dynamics depended on the virus examined. In addition, variation in the viral receptor, sialic acid, did not affect influenza virus evolution in this model. Overall, our results show that while mice provide a useful animal model for influenza virus pathology, host passage evolution will vary depending on the specific virus tested.