Amino Acid Substitution Patterns Research Articles

Thermosynechococcaceae as a source of thermostable C-phycocyanins: properties and molecular insights

This study describes characterisation of three thermophilic strains of cyanobacteria belonging to Thermosynechococcaceae and their utilisation as a source of thermostable C-phycocyanin. Three strains NIES 2133, NIES 2134 and PCC 6715 have been cultivated in two types of growth media and two temperatures: 45 and 50 °C and assessed for C-phycocyanin productivity. Among these strains Synechococcus lividus sp. PCC 6715, phylogenetically closer to Thermosynechococcus sp. clade than other Synechococcaceae, has been selected for subsequent study to characterise its C-phycocyanin. The strain showed faster growth rates than the other two and appeared to utilise different bicarbonate uptake mechanism based on SbtA, whilst BCT1 mechanism was found in NIES2133 and 2134. Higher growth rates translated to larger overall C-phycocyanin productivity. C-phycocyanin has been purified using chromatographic methods and its thermostability and pH stability has been assessed against that of Spirulina. Results have shown that PCC 6715 C-phycocyanin is one of the most stable proteins of this type reported to date. It exhibits 90% stability after 5-hour incubation at 50 °C and about 70% stability after 2-week incubation at the same temperature. PCC 6715 phycocyanins show high stability at acidic pH at both 4 °C and 50 °C, whilst at 50 °C maintain 100% stability during 4-hour incubation at pH 4.0 to 8.0. PCC 6715 phycocyanin shows good long-term stability characteristics and could be considered as a thermostable replacement for Spirulina phycocyanin. Analysis of molecular model of PCC 6715 C-phycocyanins and crystal structures of other phycocyanins reveals interesting pattern of amino acid substitutions that are present in thermophilic proteins that could have significant impact on their significantly higher thermostability. Phycocyanins from thermostable cyanobacteria exhibit evolutionarily conserved amino acid substitutions localised in the single structural hotspot of the protein that result in additional stabilising interactions within and between protein subunits when compared to proteins from mesophilic counterparts.

Genetic signatures of lipid metabolism evolution in Cetacea since the divergence from terrestrial ancestor.

In mammalian evolutionary history, Cetacea (whales, dolphins and porpoises) achieved astonishing success by adapting to an aquatic environment. One unique characteristic of cetaceans, contributing to this adaptive success, is efficient lipid utilization. Here, we report a comparative genetic analysis of five aquatic and five terrestrial Cetartiodactyla species using 144 genes associated with lipid metabolism. Mutation ratio (dN /dS ), amino acid substitution in functional domains and metabolic pathways were evaluated using branch-site model in PAML, Pfam and KEGG, respectively. Our tests detected 20 positively selected genes in Cetacea compared to 11 in Bovidae with little overlap between the lineages. We identified lineage-specific patterns of amino acid substitutions and functional domains that were mutually exclusive between cetaceans and bovids, supporting divergent evolution of lipid metabolism since the divergence of these taxa from a common ancestor. Moreover, a pathway analysis showed that the identified genes in cetaceans were associated with lipid digestion, lipid storage and energy-producing pathways. This study emphasizes the evolutionary context of lipid metabolism modification of cetaceans and provides a foundation for future studies of elucidating the adapted biological mechanisms of cetacean lipid metabolism and a framework for incorporating ecological context into studies aimed at investigating adaptive evolution.

Parallel evolution of site-specific changes in divergent caribou lineages.

The parallel evolution of phenotypes or traits within or between species provides important insight into the basic mechanisms of evolution. Genetic and genomic advances have allowed investigations into the genetic underpinnings of parallel evolution and the independent evolution of similar traits in sympatric species. Parallel evolution may best be exemplified among species where multiple genetic lineages, descended from a common ancestor, colonized analogous environmental niches, and converged on a genotypic or phenotypic trait. Modern North American caribou (Rangifer tarandus) originated from three ancestral sources separated during the Last Glacial Maximum (LGM): the Beringian–Eurasian lineage (BEL), the North American lineage (NAL), and the High Arctic lineage (HAL). Historical introgression between the NAL and the BEL has been found throughout Ontario and eastern Manitoba. In this study, we first characterized the functional differentiation in the cytochrome‐b (cytB) gene by identifying nonsynonymous changes. Second, the caribou lineages were used as a direct means to assess site‐specific parallel changes among lineages. There was greater functional diversity within the NAL despite the BEL having greater neutral diversity. The patterns of amino acid substitutions occurring within different lineages supported the parallel evolution of cytB amino acid substitutions suggesting different selective pressures among lineages. This study highlights the independent evolution of identical amino acid substitutions within a wide‐ranging mammal species that have diversified from different ancestral haplogroups and where ecological niches can invoke parallel evolution.

Observation of a New Pattern of Mutations in gyrA and parC within Escherichia coli Exhibiting Fluroquinolone Resistance

Therapeutic options with quinolones are severely compromised in infections caused by members of Enterobacteriaceae family. Mutations in chromosomal region are one of the major reasons for bacterial resistance towards this group of antibiotic. The aim of the study is to detect the mutations in gyrA and parC responsible for quinolone resistance among clinical isolates of Escherichia coli. A total of 96 quinolone-resistant clinical isolates of E. coli were collected from a tertiary care hospital of North-east India during March 2015 to August 2015. All the quinolone-resistant E. coli strains were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the quinolone resistance determining regions. Among the 96 E. coli isolates, 83.3% were resistant to nalidixic acid and 80.2%, 66.6%, 23.9% and 50% to ciprofloxacin, norfloxacin, levofloxacin and ofloxacin, respectively. Several alterations were detected in gyrA and parC genes. Three new patterns of amino acid substitution are reported in E. coli isolates. The findings of this study warrant a review in quinolone-based therapy in this region of the world to stop or slow down the irrational use this drug.

Crotalus durissus terrificus crotapotin naturally displays preferred positions for amino acid substitutions

BackgroundClassically, Crotalus durissus terrificus (Cdt) venom can be described, according to chromatographic criteria, as a simple venom, composed of four major toxins, namely: gyroxin, crotamine, crotoxin and convulxin. Crotoxin is a non-covalent heterodimeric neurotoxin constituted of two subunits: an active phospholipase A2 and a chaperone protein, termed crotapotin. This molecule is composed of three peptide chains connected by seven disulfide bridges. Naturally occurring variants/isoforms of either crotoxin or crotapotin itself have already been reported.MethodsThe crude Cdt venom was separated by using RP-HPLC and the toxins were identified by mass spectrometry (MS). Crotapotin was purified, reduced and alkylated in order to separate the peptide chains that were further analyzed by mass spectrometry and de novo peptide sequencing.ResultsThe RP-HPLC profile of the isolated crotapotin chains already indicated that the α chain would present isoforms, which was corroborated by the MS and tandem mass spectrometry analyses.ConclusionIt was possible to observe that the Cdt crotapotin displays a preferred amino acid substitution pattern present in the α chain, at positions 31 and 40. Moreover, substitutions could also be observed in β and γ chains (one for each). The combinations of these four different peptides, with the already described chains, would produce ten different crotapotins, which is compatible to our previous observations for the Cdt venom.

Comprehensive analysis of mutations in the MEFV gene reveal that the location and not the substitution type determines symptom severity in FMF

BackgroundFamilial Mediterranean Fever (FMF) is an autoinflammatory disorder caused by mutations in the MEFV gene. These mutations appear in different populations with different frequencies and their caused symptom severities vary from mild to moderate to severe depending on the mutation type.MethodsIn this study, we analyzed the mutations that have been reported in the MEFV gene from symptomatic FMF patients and compared their frequencies in different populations from the 1000 Genome and the Exome databases, using statistical clustering. We also analyzed the nucleotide and amino acid substitution patterns across the MEFV gene.ResultsWe found 16 (8%) nonsynonymous mutations outside exon 10 that did not cluster with known disease‐causing mutations (DCMs), due to their high frequencies in other populations. We also studied the substitution patterns for nucleotides and amino acids to determine the conserved and variable regions in the MEFV gene. In general more nonsynonymous substitutions were reported in exons 2, 3, and 10 from the FMF database (symptomatic FMF patients) compared to the 1000 Genome and the Exome databases. The same was true for amino acid (AA) substitutions where there were 1.5 times more radical (RAD) to conservative (CON) changes. However, when it came to AA substitutions exon 10 was quite conserved with a RAD/CON ratio of 0.9. In fact, we report that the most severe FMF symptoms are caused by conservative mutations in two highly conserved exon 10 regions.ConclusionWe found presumptive FMF‐causing mutations that did not cluster with DCMs based on their allele frequencies. We also observed that the type of mutation is less likely to determine the severity of the FMF symptoms; rather it was the location of the mutations that was the determining factor.

Abstract 380: Systematic identification of novel functional tumor-specific mutations in receptor tyrosine kinases based on their pan-cancer mutational profiles in Japanese patients with cancer

Abstract Background: Recent accumulation of large amounts of cancer genome information has revealed the presence of tumor-specific mutations with unknown functions; characterization of these mutations should facilitate the identification of novel therapeutic targets. Some tumor-specific mutations in receptor tyrosine kinases (RTKs) act as oncogenic drivers in multiple cancer types. Here, we aimed to identify novel, functional, tumor-specific, single nucleotide variants (SNVs) in cancer-related RTKs based on pan-cancer mutational profiles in Japanese patients with cancer. Methods: Surgically resected tissue and corresponding peripheral blood samples of 1,685 patients enrolled in Project HOPE (High-tech Omics-based Patient Evaluation), an ongoing prospective molecular profiling study at the Shizuoka Cancer Center, collected between January 2014 and December 2015, were subjected to whole-exome sequencing with an ion torrent proton platform. Newly detected tumor-specific SNVs that could represent functional mutations between the transmembrane and kinase domains of 35 cancer-related RTKs were subjected to serum response element (SRE)-reporter assay to assess effects on extracellular signal-regulated kinase (ERK) signaling relevant to cancer cell survival. Results: We identified 201,422 nonsynonymous tumor-specific SNVs in 1,685 patients. Of these SNVs, 1,049 were observed in our focused 35 cancer-related RTKs. Approximately 45% of patients with lung squamous cell carcinoma, which was the most frequent cancer type, followed by lung adenocarcinoma (39%), harbored tumor-specific SNVs in RTKs. The relative frequencies of tumor-specific SNVs in RTKs in other tumor types were 32%, 31%, 29%, 28%, and 19% in liver cancer, colorectal cancer, gastric cancer, head and neck cancer, and breast cancer, respectively. After filtering out of SNVs registered in multiple cancer-related databases, such as COSMIC, dbSNP, and DoCM, to select putative novel tumor-specific SNVs, 22 SNVs were selected as novel tumor-specific SNVs with high potential as functional mutations through an additional selection process based on amino acid substitution patterns and multiple alignment of amino acid residues coupled with information regarding functional mutations. Among these SNVs, 13 had already been assessed using an SRE reporter assay. We identified two SNVs in NTRK1 and IGF1R that were involved in activation of the ERK pathway, indicating that these SNVs may be activating mutations. Moreover, inactivating mutations were also identified. Nine SNVs in FLT3, NTRK1, EPHA5, ERBB4, FGFR3, and KDR significantly reduced ERK activity. Conclusions: A systematic evaluation of functionally unknown tumor-specific SNVs detected in cancer genome sequencing is necessary to expand the range of molecularly targeted cancer therapeutics. Citation Format: Masakuni Serizawa, Takeshi Nagashima, Yuji Shimoda, Shumpei Ohnami, Sumiko Ohnami, Keiichi Ohshima, Tohru Mochizuki, Takashi Nakajima, Kenichi Urakami, Masatoshi Kusuhara, Ken Yamaguchi. Systematic identification of novel functional tumor-specific mutations in receptor tyrosine kinases based on their pan-cancer mutational profiles in Japanese patients with cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 380. doi:10.1158/1538-7445.AM2017-380

Evolutionary and functional implications of hypervariable loci within the skin virome

Localized genomic variability is crucial for the ongoing conflicts between infectious microbes and their hosts. An understanding of evolutionary and adaptive patterns associated with genomic variability will help guide development of vaccines and antimicrobial agents. While most analyses of the human microbiome have focused on taxonomic classification and gene annotation, we investigated genomic variation of skin-associated viral communities. We evaluated patterns of viral genomic variation across 16 healthy human volunteers. Human papillomavirus (HPV) and Staphylococcus phages contained 106 and 465 regions of diversification, or hypervariable loci, respectively. Propionibacterium phage genomes were minimally divergent and contained no hypervariable loci. Genes containing hypervariable loci were involved in functions including host tropism and immune evasion. HPV and Staphylococcus phage hypervariable loci were associated with purifying selection. Amino acid substitution patterns were virus dependent, as were predictions of their phenotypic effects. We identified diversity generating retroelements as one likely mechanism driving hypervariability. We validated these findings in an independently collected skin metagenomic sequence dataset, suggesting that these features of skin virome genomic variability are widespread. Our results highlight the genomic variation landscape of the skin virome and provide a foundation for better understanding community viral evolution and the functional implications of genomic diversification of skin viruses.

Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations.

The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis. Seventy-six percent of the SeSaM-Tv P/P-generated substitutions yield chemically different amino acid substitutions compared to 64% (epPCR-low) and 69% (epPCR-high). Unique substitutions from one amino acid to others are termed distinct amino acid substitutions. In the SeSaM-Tv P/P library, 35% of all theoretical distinct amino acid substitutions were found in the 1000 mutation library compared to 25% (epPCR-low) and 26% (epPCR-high). Thirty-six percent of distinct amino acid substitutions found in SeSaM-Tv P/P were unobtainable by epPCR-low. Comparison with the SSM library showed that epPCR-low covers 15%, epPCR-high 18%, and SeSaM-Tv P/P 21% of obtainable beneficial amino acid positions. In essence, this study provides first insights on the quality of epPCR and SeSaM-Tv P/P libraries in terms of amino acid substitutions, their chemical differences, and the number of obtainable beneficial amino acid positions.

Nucleotide sequence variability analysis of Major Histocompatibility Complex Class II DQA1 gene in Nigerian goats

Major Histocompatibility Complex (MHC) molecules loaded with peptides derived from invading pathogens are recognised by the immune system to produce a highly effective and specific response against foreign pathogens. A 310-bp fragment of exon 2 of the MHC Class II DQA1 gene was amplified in 27 animals made up of three major Nigerian goat breeds [West African Dwarf (WAD), Red Sokoto (RS) and Sahel (SH)]. Twenty amino acid polymorphic sites were found in Nigerian goats. Comparison of predicted amino acid residues of DQA1 exon 2 alleles of Nigerian goats with similar alleles from other caprine species revealed considerable congruence in amino acid substitution pattern. A significant positive selection signature was detected at the DQA1 locus of Nigerian goats in that non-synonymous substitutions occurred at a faster rate compared to synonymous substitutions (dN:dS ratio = 1.28 ; Z-Statistics= 1.634; P<0.05). The evolutionary tree constructed using UPGMA, revealed that the southern WAD goat appeared to be more related to the northern RS than SH goat at the DQA1 locus. It will be interesting therefore, for future studies to investigate the association of the genetic variants in DQA1 gene of Nigerian goats with resistance/susceptiblity to diseases in order to conserve these precious animal genetic resources.

HBsAg may reappear following reactivation in individuals with spontaneous HBsAg seroclearance 8 years previously.

HBsAg reappearance may constitute not only a risk for liver disease but also an infectious source. We aimed to determine whether HBsAg may reappear after spontaneous HBsAg seroclearance. A cohort of 2999 HBsAg-positive subjects aged 30-55 years was recruited in Guangxi, China in 2004. HBsAg was tested every 6 months from July 2004 to June 2007, then, one more time in December 2013. The results showed that spontaneous HBsAg seroclearance occurred in 41 subjects in the first 3 years, giving a 0·54% annual seroclearance rate. Thirteen of the 41 subjects were randomly tested for HBsAg in 2013. Four subjects became HBsAg positive. S gene sequences of HBV were analysed from serum collected before seroclearance and after reappearance, respectively, for subject QS840 (11 and 12 clones), subject TN98 (13 and 13 clones) and subject WX227 (10 and 8 clones). Serotype, subgenotype and amino-acid substitution pattern in each sample collected after reappearance was observed in the sample collected before HBsAg seroclearance. Nucleotide similarity between the two sequences from each subject was >99% and five sequences from subject TN98 were the same. In conclusion, following reactivation, HBsAg may reappear in individuals with spontaneous HBsAg seroclearance many years previously.

Clinical Implications of TP53 Mutations in Adult Patients with Newly Diagnosed Acute Lymphoblastic Leukemia (ALL) Treated with the Hypercvad-Based Regimens

Introduction -TP53 is the most extensively studied gene in cancer and is associated with a poor outcome. The clinical implication of TP53 gene mutations in adult ALL has not been evaluated.Methods - We screened for TP53 mutations in 119 newly diagnosed patients with ALL who were treated with HCVAD-based regimens (2012-2016) and evaluated the predictive and prognostic value of TP53mutations. Bone marrow samples underwent targeted amplicon-based next-generation sequencing (NGS) based mutation analysis.Results - Among 119 patients with adult ALL, we identified 23 patients (19%, 22 B-cell; 1 T-cell) with TP53 mutations (TP53mut); 96 had wild type TP53 (TP53wt; 83 B-cell; 13 T-cell). BCR/ABL1 rearrangement was noted in 2/23 TP53mut ALL and 43/96 TP53wt ALL (p=0.001). Of the 23 patients with TP53mut, 20 were missense, 2 were nonsense mutations and 1 was an insertion/ deletion, spanning exons 2-10. The most common pattern of amino acid substitution,in 10 of the 23 patients, was a substitution of arginine to histidine or proline on different codons. The median TP53 mutant allelic frequency was 42.2% (range, 1.4 - 93.8). Seven patients (30%) with TP53mut had concurrent mutations: NRAS in 2, BRAF, NOTCH1, TET2, DNMT3A, and EZH2 - 1 each. Among patients with TP53wt, 40 patients (42%) had mutations in other genes: 9 JAK2, 7 NRAS, 5 KRAS, 5 NOTCH1, 3 IDH2, 2 each with DNMT3A, ASXL1, FLT3, PTPN11, and 1 with TET2. The clinical characteristics, pattern of mutations, response to therapy, and outcomes of patients with TP53mut ALL (n=23) vs. TP53wt ALL(n=96) were compared (Table 1 and Figure-1). Patients with TP53mutALL were significantly older at presentation (median age 60 years [24-81] versus 46 years [18-81]; p=0.01) and had significantly lower platelet counts (29 x 109 /L versus 45 x 109/L; p=0.01) and lower peripheral blood blast percentages (10% versus 43%; p=0.005). Distribution of chromosomal aberrations was significantly different among the two groups: t(9;22) was observed at a much lower rate in TP53mut ALL, while hyper and hypodiploidy were more commonly encountered in TP53mut ALL There was no significant difference in the complete remission (83% in TP53mut ALL versus 91% in TP53wt ALL, p=0.25) and minimal residual disease (66% in TP53mut ALL versus 59% in TP53wt ALL, p=0.23) rates between the 2 groups. Overall, the median follow-up was 11.3 months (range, 0.2 to 41 months). There was no statistically significant difference in outcome between patients with and without TP5mutation. (Figure -1 A-B). The 3-year event-free (EFS) and overall survival (OS) rates were 45% and 42% in patients with TP53mutand 46% and 59% in those with TP53wt. The median EFS was 18 and 26.7 months (p=0.37) respectively. The median OS was 27.5 and 37.2 months (p=0.20), respectively. Furthermore, no significant difference was observed when only patients with Philadelphia-negative B-ALL were assessed.Conclusions -TP53 mutations are seen in about 19% of adult with newly diagnosed ALL. Patients tend to be older with higher incidence of hypodiploidy. TP53 mutations had no significant negative impact on outcome in patients treated with Hyper-CVAD based regimens. Further studies are underway at our institution to expand the cohort and identify the relevance of TP53mutations in adult ALL in the era of novel chemoimmunotherapy.*3 patients in TP53 - Mutated group had 1 patient each with miscellaneous/insufficient metaphases/ not done while 6 patients had miscellaneous aberrations and 4 with insufficient metaphases TP53wild type respectively [Display omitted] [Display omitted] DisclosuresKonopleva:Calithera: Research Funding; Cellectis: Research Funding. Jain:Incyte: Research Funding; Abbvie: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Novimmune: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Infinity: Research Funding; Servier: Consultancy, Honoraria; BMS: Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; Celgene: Research Funding; Novartis: Consultancy, Honoraria. Wierda:Novartis: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Acerta: Research Funding; Abbvie: Research Funding. O'Brien:Janssen: Consultancy, Honoraria; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. Jabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy.

Polymorphism in Mitochondrial Coding Regions of Mediterranean Loggerhead Turtles: Evolutionary Relevance and Structural Effects.

We sequenced coding portions (1.6 kb) of the mtDNA in 170 loggerhead (Caretta caretta) turtles sampled in the central Mediterranean. The sequences spanned the entire ND1 and ND3 genes, the tRNAGly and tRNAArg, plus the 3' and 5' termini of COXIII and ND4L genes, respectively. Based on our sequencing results and published complete mitogenomes, we constructed a maximum parsimony phylogeny of C. caretta matrilines that sheds new light on the evolutionary relationships within the collection of lineages found in the Mediterranean and so far recognized by D-loop haplotypes only. We show that the new variants are useful to understand the ancestry of extant haplotypes, to improve genetically based studies on the philopatry and migratory behavior of the species, and for conservation purposes. In order to better understand the biological significance of the observed variation, we addressed intraspecific nonsynonymous substitutions in the context of the three-dimensional modeled structures of ND1 and ND3. The positions of variant amino acids within the folded subunits are consistent with a coadaptation with the restructuring of membrane thickness, fluidity, and lipid composition, a well-known response mechanism to thermal conditions. The pattern of amino acid substitutions departs from neutrality, suggesting local adaptation and/or polymorphism-based local selection.

TP53 mutations in newly diagnosed acute myeloid leukemia: Clinicomolecular characteristics, response to therapy, and outcomes.

Mutations in the tumor protein 53 (TP53) gene predict a poor prognosis in patients with acute myeloid leukemia (AML). Peripheral blood or bone marrow samples from 293 patients with newly diagnosed AML were analyzed with targeted, amplicon-based, next-generation sequencing-based mutation analysis. TP53 mutations were identified in 53 patients (18%; 45 were missense mutations). In 13 of the 53 patients, the most common pattern of amino acid substitution was a substitution of arginine to histidine on different codons. The clinical characteristics, pattern of mutations, response to different therapies, and outcomes of patients with AML-TP53-mutated (n = 53) versus wild-type TP53 (n = 240) were compared. TP53 mutations were significantly more likely in patients who had a complex karyotype; abnormalities of chromosome 5, 7, and 17; and therapy-related AML. Patients who had TP53-mutated AML had significantly lower incidence of mutations in Fms-like tyrosine kinase 3 (FLT3), rat sarcoma (RAS), and nucleophosmin (NPM1) and higher incidence of coexisting MPL mutations compared with those who had wild type TP53. The distribution of TP53 mutations was equal for both age groups (ages <60 years vs ≥60 years). TP53-mutated AML was associated with a lower complete remission rate (41% vs 57%; P = .04), a significantly inferior complete remission duration (at 2 years: 30% vs 55%; P = .001), and overall survival (at 2 years: 9% vs 24%; P ≤ .0001) irrespective of age or the type of treatment received (high-intensity vs low-intensity chemotherapy). The type of treatment received did not improve outcomes in younger or older patients with TP53-mutated AML. These data suggest that novel therapies are needed to improve the outcome of patients with AML who have TP53 mutations. Cancer 2016;122:3484-3491. © 2016 American Cancer Society.

Occult HBV Infection May Be Transmitted through Close Contact and Manifest as an Overt Infection.

The importance of transmission of occult HBV infection (OBI) via transfusion, organ transplantation and hemodialysis has been widely recognized. However, data regarding the transmission of OBI through close contact remain limited. In this study, serum samples were obtained from a child and his parents. The child had received the standard vaccination regimen at birth and produced protective antibody. Sera were tested for HBV serological markers. Nested PCR assays were used to detect HBV DNA and the amplicons were cloned and their sequences subjected to phylogenetic analysis. The results showed that both parents had occult infections while the child had an overt infection. Twelve, eleven and nine clones, from the father, mother and son, respectively, were sequenced. Serotypes adrq+, ayw1, ayw and ayr were found in the father and ayw1, adw2 and adwq+ in the mother; adrq+ was the only serotype in son. Genotype B, subgenotype C2 and a recombinant were identified in the father and genotype B, subgenotype C5 and three recombinants were found in the mother. Subgenotype C2 was the only genotype identified in the child. A phylogenetic tree showed that all of the child’s sequences and most of the father’s sequences clustered together. However, none of mother’s sequences clustered with those of the child. The surface gene from the child and his father had the same amino acid substitution pattern (T118K, T123N and G145A). We concluded that the father was the source of the son’s HBV infection, suggesting that occult HBV infection may be transmitted through close contact and manifest as an overt infection.

Patterns of Protein Evolution in Cytochrome c Oxidase 1 (COI) from the Class Arachnida.

Because sequence information is now available for the 648bp barcode region of cytochrome c oxidase 1 (COI) from more than 400,000 animal species, this gene segment can be used to probe patterns of mitochondrial evolution. The present study examines levels of amino acid substitution and the frequency of indels in COI from 4177 species of arachnids, including representatives from all 16 orders and 43% of its families (267/625). It examines divergences at three taxonomic levels—among members of each order to an outgroup, among families in each order and among BINs, a species proxy, in each family. Order Distances vary fourfold (0.10–0.39), while the mean of the Family Distances for the ten orders ranges fivefold (0.07–0.35). BIN Distances show great variation, ranging from 0.01 or less in 12 families to more than 0.25 in eight families. Patterns of amino acid substitution in COI are generally congruent with previously reported variation in nucleotide substitution rates in arachnids, but provide some new insights, such as clear rate acceleration in the Opiliones. By revealing a strong association between elevated rates of nucleotide and amino acid substitution, this study builds evidence for the selective importance of the rate variation among arachnid lineages. Moreover, it establishes that groups whose COI genes have elevated levels of amino acid substitution also regularly possess indels, a dramatic form of protein reconfiguration. Overall, this study suggests that the mitochondrial genome of some arachnid groups is dynamic with high rates of amino acid substitution and frequent indels, while it is ‘locked down’ in others. Dynamic genomes are most prevalent in arachnids with short generation times, but the possible impact of breeding system deserves investigation since many of the rapidly evolving lineages reproduce by haplodiploidy, a mode of reproduction absent in ‘locked down’ taxa.

Ampicillin-resistant Haemophilus influenzae isolates in Geneva: serotype, antimicrobial susceptibility, and β-lactam resistance mechanisms.

The purpose of this study was to analyze the molecular mechanisms of ampicillin-resistant Haemophilus influenzae isolated in Geneva, Switzerland. We investigated the association between specific patterns of amino acid substitutions in penicillin-binding protein 3 (with or without β-lactamase production) and β-lactam susceptibility. Another main focus for this study was to compare the accuracy of disk diffusion and Etest methods to detect resistance to ampicillin and amoxicillin/clavulanic acid. The antibiotic susceptibility to β-lactam antibiotics of 124 H. influenzae isolates was determined by disk diffusion and Etest methods, and interpreted by European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) breakpoints. Alterations in PBP3 were investigated by sequencing the ftsI gene. Of the 124 clinical isolates analyzed, ampicillin resistance was found in 36% (45 out of 124). The rate of resistance to amoxicillin/clavulanic acid was 9% and 0.8%, using EUCAST and CLSI breakpoints respectively. For the 78 β-lactamase negative ampicillin-susceptible (BLNAS) isolates for which the Etest method indicated a high degree of susceptibility (MIC ≤ 1 mg/L), the disk diffusion method revealed resistance to ampicillin and amoxicillin/clavulanic acid in 33 cases (42%). Most common amino acid substitutions were Asn526Lys and Val547Ile, followed by Asp569Ser, Ala502Val, Asp350Asn, Met377Ile, Ile449Val, and Arg517His. The patterns observed were classified into six groups (IIa, IIb, IIc, IId, III-like, and miscellaneous). Continued characterization of both invasive and respiratory H. influenzae isolates is necessary in order to observe changes in the microbiology and epidemiology of this pathogen that could lead to clinical failure when treated by empirical antibiotic therapy.

Complete genome sequence of nine isolates of canna yellow streak virus reveals its relationship to the sugarcane mosaic virus (SCMV) subgroup of potyviruses.

Complete genome sequences were obtained from nine isolates of canna yellow streak virus (CaYSV). CaYSV belongs to the sugarcane mosaic virus (SCMV) subgroup of potyviruses with johnsongrass mosaic virus (JGMV) as its closest relative. Multiple sequence alignments showed a pattern of amino acid substitutions in the CP sequences, which enabled us to relate these isolates to South East Asian or European isolates. Biological characterization of CaYSV identified Nicotiana benthamiana, Chenopodium quinoa and Phaseolus vulgaris as experimental hosts. Given the popularity and global trade of cannas, a clear picture of the genetic diversity of CaYSV is critical to disease management.

Relationship between amino acid usage and amino acid evolution in primates

Amino acid usage varies from species to species. A previous study has found a universal trend in amino acid gain and loss in many taxa and a one-way model of amino acid evolution in which the number of new amino acids increases as the number of old amino acids decreases was proposed. Later studies showed that this pattern of amino acid gain and loss is likely to be compatible with the neutral theory. The present work aimed to further study this problem by investigating the evolutionary patterns of amino acids in 8 primates (the nucleotide and protein alignments are available online http://gattaca.nju.edu.cn/pub_data.html). First, the number of amino acids gained and lost was calculated and the evolution trend of each amino acid was inferred. These values were found to be closely related to the usage of each amino acid. Then we analyzed the mutational trend of amino acid substitution in human using SNPs, this trend is highly correlated with fixation trend only with greater variance. Finally, the trends in the evolution of 20 amino acids were evaluated in human on different time scales, and the increasing rate of 5 significantly increasing amino acids was found to decrease as a function of time elapsed since divergence, and the dS/dN ratio also found to increase as a function of time elapsed since divergence. These results suggested that the observed amino acid substitution pattern is influenced by mutation and purifying selection. In conclusion, the present study shows that usage of amino acids is an important factor capable of influencing the observed pattern of amino acid evolution, and also presented evidences suggesting that the observed universal trend of amino acid gain and loss is compatible with neutral evolution.

Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication.

Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication.