Common Amino Acid Substitutions Research Articles

Molecular characterization of full-length VP2 gene of canine parvovirus type 2 strains circulating in Egypt 2019–2021

Canine parvovirus type 2 (CPV-2) is a major cause of fatal gastroenteritis and myocarditis in puppies of domestic and wild carnivores. CPV-2 has accumulated changes over time lead to the emergence of three antigenic variants CPV-2a, CPV-2b, and CPV-2c. VP2 is the major capsid protein that determines virus antigenicity, and host range. Although the three CPV-2 variants were previously identified in Egypt, most reports covered a restricted geographic region and/or time period, and only analyzed partial fragments of VP2 gene. Therefore, this study was designed to test 100 rectal swabs collected from 7 Egyptian governorates between 2019 and 2021 for CPV-2 using PCR. A total of 65 positive samples were identified, mostly in pure dog breeds of young age. The three variants co-circulated in 2019, while CPV-2b was not detected in 2020 and 2021. The frequency of CPV-2b and CPV-2c was higher in 2019 and 2021, respectively. Analysis of CPV-2 full-length VP2 gene sequence from 19/65 positive samples has identified four common amino acid substitutions F267Y, S297A, A300G, Y324I, which are characteristic for the new CPV-2 variants currently circulating worldwide. Unique substitutions including A5G, G36R, V38E, Q370R, and G392V were recognized in certain samples, and appears to have distinct effect on receptor binding, nuclear translocation, and inter-species transmission. Phylogenetic analysis showed separation of CPV-2 strains into two clades. All strains of this study were classified in clade I with Asian strains. In conclusion, this study provides updated comprehensive molecular analysis of CPV-2 variants in Egypt.

Abstract PO3-25-09: OKI-219, a PI3KαH1047R-mutant-selective inhibitor demonstrates efficacy as a single agent and drives combination responses with standard of care therapies in pre-clinical PI3KαH1047R mutant breast cancer models

Abstract Mutations in PI3Kα lead to constitutive activation of the PI3K/AKT/mTOR pathway and are highly prevalent in cancer. PI3KαH1047R is the most common amino acid substitution mutation and represents approximately one-third of all PI3Ka mutations, with 15% of breast cancers having a PI3KαH1047R mutation. In breast cancer, PI3KαH1047R mutations are associated with resistance to targeted therapies, including HER2- and estrogen receptor (ER)- targeted agents. Although targeting PI3Kα in cancer is a therapeutically proven strategy, with the currently approved drug alpelisib showing clinical efficacy alone or in combination with other therapies, treatment with non-mutant selective PI3Kα inhibitors such as alpelisib is associated with significant toxicities such as hyperglycemia, due to on-target inhibition of the wild-type enzyme. This often leads to dose modification or discontinuation. Therefore, there is a significant need to develop new PI3Kα-targeted therapies that can avoid or minimize on-target toxicity and improve the safety and clinical benefit for patients. OKI-219 is an orally bioavailable, PI3KαH1047R mutant-selective inhibitor, with ~100-fold cellular selectivity for the H1047R mutation over wild-type. OKI-219 demonstrates selectivity across a panel of 412 kinases, with no other kinases showing >30% inhibition at 1 µM. In vitro, OKI-219 drives decreased phosphorylated AKT (pAKT) and decreased proliferation specifically in PI3KαH1047R mutant cell lines across several tumor types, including breast cancer. In vivo, as a monotherapy, OKI-219 demonstrates dose dependent antitumor activity in multiple human tumor xenograft models, including the H1047R mutant breast cancer CDX models T47D and HCC1954 and several PDX models, with regression rates up to 100% at doses that show no sign of the metabolic dysfunction (increased glucose or insulin) associated with wild-type protein inhibition. The in vivo antitumor activity correlates with pathway inhibition, measured by decreased pAKT in tumors. Moreover, OKI-219 dosed in combination with the selective estrogen receptor degraders (SERDs) fulvestrant, elacestrant or camizestrant showed significant combination benefit leading to tumor regressions of up to 100% in the ER+ H1047R mutant breast cancer model xxT47D, at doses where no regressions were observed with single agent treatment. Dosing OKI-219 in combination with HER2-inhibitors, such as tucatinib, led to tumor regressions in the ER-HER2+ and H1047R mutant breast cancer CDX model HCC1954, also at doses where no regressions were observed with single agent treatment. These data indicate that OKI-219 offers improved efficacy and a wider therapeutic window compared to non-mutant selective PI3Kα inhibitors. OKI-219 is advancing into clinical trials. Citation Format: Molly A. Taylor, Qian Zhao, David Mareska, Maria Hoh, Yevgeniy Izrayelit, Kevin Litwiler, Mark L Boys, Rich Woessner, Duncan Walker, James D Winkler, Jennifer Diamond. OKI-219, a PI3KαH1047R-mutant-selective inhibitor demonstrates efficacy as a single agent and drives combination responses with standard of care therapies in pre-clinical PI3KαH1047R mutant breast cancer models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-09.

Precise genome editing underlines the distinct contributions of mutations in ERG11, ERG3, MRR1, and TAC1 genes to antifungal resistance in Candida parapsilosis.

Candida parapsilosis has recently emerged as a major threat due to the worldwide emergence of fluconazole-resistant strains causing clonal outbreaks in hospitals and poses a therapeutic challenge due to the limited antifungal armamentarium. Here, we used precise genome editing using CRISPR-Cas9 to gain further insights into the contribution of mutations in ERG11, ERG3, MRR1, and TAC1 genes and the influence of allelic dosage to antifungal resistance in C. parapsilosis. Seven of the most common amino acid substitutions previously reported in fluconazole-resistant clinical isolates (including Y132F in ERG11) were engineered in two fluconazole-susceptible C. parapsilosis lineages (ATCC 22019 and STZ5). Each mutant was then challenged in vitro against a large array of antifungals, with a focus on azoles. Any possible change in virulence was also assessed in a Galleria mellonella model. We successfully generated a total of 19 different mutants, using CRISPR-Cas9. Except for R398I (ERG11), all remaining amino acid substitutions conferred reduced susceptibility to fluconazole. However, the impact on fluconazole in vitro susceptibility varied greatly according to the engineered mutation, the stronger impact being noted for G583R acting as a gain-of-function mutation in MRR1. Cross-resistance with newer azoles, non-medical azoles, but also non-azole antifungals such as flucytosine, was occasionally noted. Posaconazole and isavuconazole remained the most active in vitro. Except for G583R, no fitness cost was associated with the acquisition of fluconazole resistance. We highlight the distinct contributions of amino acid substitutions in ERG11, ERG3, MRR1, and TAC1 genes to antifungal resistance in C. parapsilosis.

First Report of Mutations Associated With Pyrethroid (L1014F) and Organophosphate (G119S) Resistance in Belgian Culex (Diptera: Culicidae) Mosquitoes.

The emergence of West Nile virus and Usutu virus in Europe poses a significant risk to public health. In the absence of efficient antiviral therapy or vaccine candidates, the only strategy to control these arboviruses is to target the Culex (Diptera: Culicidae) mosquito vector. However, the selection pressure caused by exposure to insecticides for vector control or agricultural pest control can lead to insecticide resistance, thereby reducing the efficacy of insecticide-based vector control interventions. In Culex mosquitoes, two of the most common amino acid substitutions associated with insecticide resistance are the kdr L1014F in voltage gated sodium channels and G119S in acetylcholinesterase. In this study, Culex pipiens biotype pipiens, Culex torrentium, and Culex modestus were sampled from 2019 to 2021 in three distinct environmental habitats (urban, peri-urban, and agricultural) in and around the city of Leuven, Belgium. Individual mosquitoes were screened for two mutations resulting in L1014F and G119S amino acid substitutions. Both mutations were observed in Cx. pipiens and Cx. modestus but not in Cx. torrentium mosquitoes across the four collection sites. Furthermore, multi-resistance or cross-resistance in Cx. pipiens could be a threat in these areas, as both mutations were observed at low frequencies. These results provide the first report of kdr L1014F and ace-1 G119S resistance mutations in Cx. pipiens and Cx. modestus mosquitoes from Belgium, highlighting the importance of mosquito surveillance to design effective arbovirus outbreak control strategies.

First Description of Ceftazidime/Avibactam Resistance in a ST13 KPC-70-Producing Klebsiella pneumoniae Strain from Portugal.

The combination of ceftazidime/avibactam (CZA) is a novel β-lactam/β-lactamase inhibitor with activity against Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales. Emerging cases caused by CZA-resistant strains that produce variants of KPC genes have already been reported worldwide. However, to the best of our knowledge, no CZA-resistant strains were reported in Portugal. In September 2019, a K. pneumoniae CZA-resistant strain was collected from ascitic fluid at a surgery ward of a tertiary University Hospital Center in Lisboa, Portugal. The strain was resistant to ceftazidime/avibactam, as well as to ceftazidime, cefoxitin, gentamicin, amoxicillin/clavulanic acid, and ertapenem, being susceptible to imipenem and tigecycline. A hypermucoviscosity phenotype was confirmed by string test. Whole-genome sequencing (WGS) analysis revealed the presence of an ST13 KPC70-producing K. pneumoniae, a KPC-3 variant, differing in two amino-acid substitutions (D179Y and T263A). The D179Y mutation in the KPC Ω-loop region is the most common amino-acid substitution in KPC-2 and KPC-3, further leading to CZA resistance. The second mutation causes a KPC-70 variant in which threonine replaces alanine (T263A). The CZA-resistant strain showed the capsular locus KL3 and antigen locus O1v2. Other important virulence factors were identified: fimbrial adhesins type 1 and type 3, as well as the cluster of iron uptake systems aerobactin, enterobactin, salmochelin, and yersiniabactin included in integrative conjugative element 10 (ICEKp10) with the genotoxin colibactin cluster. Herein, we report the molecular characterization of the first hypervirulent CZA-resistant ST13 KPC-70-producing K. pneumoniae strain in Portugal. The emergence of CZA-resistant strains might pose a serious threat to public health and suggests an urgent need for enhanced clinical awareness and epidemiologic surveillance.

U2af1 is a haplo-essential gene required for hematopoietic cancer cell survival in mice.

Somatic mutations in the spliceosome gene U2AF1 are common in patients with myelodysplastic syndromes. U2AF1 mutations that code for the most common amino acid substitutions are always heterozygous, and the retained WT allele is expressed, suggesting that mutant hematopoietic cells may require the residual WT allele to be viable. We show that hematopoiesis and RNA splicing in U2af1 heterozygous knockout mice were similar to those in control mice, but that deletion of the WT allele in U2AF1(S34F) heterozygous mutant-expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of WT U2AF1 for survival in vivo and that U2AF1 is a haplo-essential cancer gene. Mutant U2AF1(S34F)-expressing cells were also more sensitive to reduced expression of WT U2AF1 than nonmutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the WT U2af1 allele was deleted compared with when it was not deleted. These results suggest that selectively targeting the WT U2AF1 allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboring U2AF1 mutations.

Molecular Epidemiology, Antifungal Susceptibility, and Virulence Evaluation of Candida Isolates Causing Invasive Infection in a Tertiary Care Teaching Hospital.

BackgroundThe incidence of invasive candidiasis is increasing worldwide. However, the epidemiology, antifungal susceptibility, and virulence of Candida spp. in most hospitals remain unclear. This study aimed to evaluate invasive candidiasis in a tertiary care hospital in Nanchang City, China.MethodsMALDI-TOF MS and 18S rDNA ITS sequencing were used to identify Candida strains. Randomly amplified polymorphic DNA analysis was used for molecular typing; biofilm production, caseinase, and hemolysin activities were used to evaluate virulence. The Sensititre™ YeastOne YO10 panel was used to examine antifungal susceptibility. Mutations in ERG11 and the hotspot regions of FKS1 of drug-resistant strains were sequenced to evaluate the possible mechanisms of antifungal resistance.ResultsWe obtained 110 Candida strains, which included 40 Candida albicans (36.36%), 37 C. parapsilosis (33.64%), 21 C. tropicalis (19.09%), 9 C. glabrata (8.18%), 2 C. rugose (1.82%), and 1 C. haemulonii (0.91%) isolates. At a limiting point of 0.80, C. albicans isolates could be grouped into five clusters, C. parapsilosis and C. tropicalis isolates into seven clusters, and C. glabrata isolates into only one cluster comprising six strains by RAPD typing. Antifungal susceptibility testing revealed that the isolates showed the greatest overall resistance against fluconazole (6.36%), followed by voriconazole (4.55%). All C. albicans and C. parapsilosis isolates exhibited 100% susceptibility to echinocandins (i.e., anidulafungin, caspofungin, and micafungin), whereas one C. glabrata strain was resistant to echinocandins. The most common amino acid substitutions noted in our study was 132aa (Y132H, Y132F) in the azole-resistant strains. No missense mutation was identified in the hotpot regions of FKS1. Comparison of the selected virulence factors detectable in a laboratory environment, such as biofilm, caseinase, and hemolysin production, revealed that most Candida isolates were caseinase and hemolysin producers with a strong activity (Pz < 0.69). Furthermore, C. parapsilosis had greater total biofilm biomass (average Abs620 = 0.712) than C. albicans (average Abs620 = 0.214, p < 0.01) or C. tropicalis (average Abs620 = 0.450, p < 0.05), although all C. glabrata strains were either low- or no-biofilm producers. The virulence level of the isolates from different specimen sources or clusters showed no obvious correlation. Interesting, 75% of the C. albicans from cluster F demonstrated azole resistance, whereas two azole-resistant C. tropicalis strains belonged to the cluster Y.ConclusionThis study provides vital information regarding the epidemiology, pathogenicity, and antifungal susceptibility of Candida spp. in patients admitted to Nanchang City Hospital.

Evolution of the primate glutamate taste sensor from a nucleotide sensor

Taste perception plays an essential role in food selection. Umami (savory) tastes are sensed by a taste receptor complex, T1R1/T1R3, that detects proteinogenic amino acids.1 High sensitivity to l-glutamate (l-Glu) is a characteristic of human T1R1/T1R3, but the T1R1/T1R3 of other vertebrates does not consistently show this l-Glu response.1,2 Here, we demonstrate that the l-Glu sensitivity of T1R1/T1R3 is a derived state that has evolved repeatedly in large primates that rely on leaves as protein sources, after their divergence from insectivorous ancestors. Receptor expression experiments show that common amino acid substitutions at ligand binding sites that render T1R1/T1R3 sensitive to l-Glu occur independently at least three times in primate evolution. Meanwhile T1R1/T1R3 senses 5'-ribonucleotides as opposed to l-Glu in several mammalian species, including insectivorous primates. Our chemical analysis reveal that l-Glu is one of the major free amino acids in primate diets and that insects, but not leaves, contain large amounts of free 5'-ribonucleotides. Altering the ligand-binding preference of T1R1/T1R3 from 5'-ribonucleotides to l-Glu might promote leaf consumption, overcoming bitter and aversive tastes. Altogether, our results provide insight into the foraging ecology of a diverse mammalian radiation and help reveal how evolution of sensory genes facilitates invasion of new ecological niches.

A thermodynamic model of protein structure evolution explains empirical amino acid substitution matrices.

Proteins evolve under a myriad of biophysical selection pressures that collectively control the patterns of amino acid substitutions. These evolutionary pressures are sufficiently consistent over time and across protein families to produce substitution patterns, summarized in global amino acid substitution matrices such as BLOSUM, JTT, WAG, and LG, which can be used to successfully detect homologs, infer phylogenies, and reconstruct ancestral sequences. Although the factors that govern the variation of amino acid substitution rates have received much attention, the influence of thermodynamic stability constraints remains unresolved. Here we develop a simple model to calculate amino acid substitution matrices from evolutionary dynamics controlled by a fitness function that reports on the thermodynamic effects of amino acid mutations in protein structures. This hybrid biophysical and evolutionary model accounts for nucleotide transition/transversion rate bias, multi‐nucleotide codon changes, the number of codons per amino acid, and thermodynamic protein stability. We find that our theoretical model accurately recapitulates the complex yet universal pattern observed in common global amino acid substitution matrices used in phylogenetics. These results suggest that selection for thermodynamically stable proteins, coupled with nucleotide mutation bias filtered by the structure of the genetic code, is the primary driver behind the global amino acid substitution patterns observed in proteins throughout the tree of life.

Molecular characteristics and phylogenetic analysis of novel goose parvovirus strains associated with short beak and dwarfism syndrome

Short beak and dwarfism syndrome (SBDS) emerged in Cherry Valley duck flocks in China in 2015, and novel goose parvovirus (NGPV) was shown to be the etiological agent of SBDS. To date, it is not known whether SBDS-related NGPV isolates possess common molecular characteristics. In this study, three new NGPV strains (namely, SDHT16, SDJN19, and SDLC19) were isolated from diseased ducks showing typical signs of SBDS and successfully passaged in embryonated goose or Cherry Valley duck eggs. The complete genome sequences of these NGPV strains were 98.9%-99.7% identical to each other but showed slightly less similarity (95.2%-96.1% identity) to classical GPV strains. A total of 16 common amino acid substitutions were present in the VP1 proteins of six NGPV strains (SDHT16, SDJN19, SDLC19, QH, JS1, and SDLC01) compared with the classical Chinese GPV strains, nine of which were identical to those found in European GPV strain B. The non-structural protein Rep1 of the six NGPV strains had 12 common amino acid substitutions compared with the classical GPV strains. Phylogenetic analysis indicated that the Chinese NGPV strains clustered with the European SBDS-related NGPV strains, forming a separate branch that was distinct from the group formed by the classical GPV strains. The present study shows the common molecular characteristics of NGPV isolates and suggests that the Chinese NGPV isolates probably share a common ancestor with European SBDS-related NGPV strains.

Genetic variability in VP1 gene of infectious bursal disease virus from the field outbreaks of Kerala, India.

Infectious bursal disease (IBD) is considered as menace as it affects poultry industry globally causing immunosuppression, high mortality and heavy economic loss. Outbreaks of IBD were reported in many states of India including Kerala. VP1 gene acts as an important factor in the process of virus encapsidation and its involvement in viral virulence and viral replication indicates its importance in infectious bursal disease virus (IBDV). The present study was conducted to carry out the molecular characterization of VP1 gene of virulent IBDV in Kerala. A total of 42 samples were processed for the detection and analysis of VP1 gene of IBDV. Out of 42 samples, 21 samples were positive for VP1 gene of IBD. The phylogenetic analysis of the partial VP1 gene sequences reveals the clustering of IBDV isolates into very virulent IBDV (vvIBDV) and non-virulent IBDV (vIBDV). Eighteen isolates (11 isolates from vaccinated flock and 7 from non-vaccinated flocks) clustered with very virulent strains. Three isolates (2 isolates were from vaccinated flock and 1 from non-vaccinated flock) clustered with non-virulent IBDV strains, showing more evolutionarily similarity to south Indian strain VCN14/ABT/MVC/India. It is observed that vvIBDV isolates from this study have common ancestor with the south Indian strain PY12 but showed 9-10% divergence from this strains. The amino acid analysis of these 21 isolates revealed that 17 isolates possessed the characteristic vvIBDV TDN amino acid triplet, while the three isolates had non-vIBDV NEG amino acid triplet at 145/146/147 position. The remaining isolate 1/CVASP/IBDV/VP1 shows unique PDN triplet instead of TDN. Two vvIBDV isolates (15/CVASP/IBDV/VP1 and 18/CVASP/IBDV/VP1) showed 100% nucleotide and amino acid similarity with intermediate plus vaccine strain. Four vvIBDV isolates showed neutral amino acid substitution K251R which was earlier reported in Indian strains but first time in south Indian isolates. The most common unique amino acid substitution observed in our study was neutral E269D amino acid substitution in 12 isolates, neutral amino acid substitution T329S in five isolates, neutral T174N and non-polar to polar amino acid substitution A178T in isolate 10/CVASP/IBDV/VP1, non-polar to polar amino acid substitution P360R in isolate 17/CVASP/IBDV/VP1 and non-polar to polar amino acid substitution P188S in isolate 1/CVASP/IBDV/VP1. These novel mutations in our study reveal the role of genetic drift in the evolution of vvIBDV strains. The isolate 2/CVASP/IBDV/VP1 from non-vaccinated flock shows VP1 gene of non-vIBDV, but possessing VP2 of vvIBDV type indicates this is evolved by genetic shift of segments A and B. This is the first genetic characterization study of field VP1 gene of IBDV isolates in Kerala, India.

Effects of a Common Eight Base Pairs Duplication at the Exon 7-Intron 7 Junction on Splicing, Expression, and Function of OCT1.

Organic cation transporter 1 (OCT1, SLC22A1) is localized in the sinusoidal membrane of human hepatocytes and mediates hepatic uptake of weakly basic or cationic drugs and endogenous compounds. Common amino acid substitutions in OCT1 were associated with altered pharmacokinetics and efficacy of drugs like sumatriptan and fenoterol. Recently, the common splice variant rs35854239 has also been suggested to affect OCT1 function. rs35854239 represents an 8 bp duplication of the donor splice site at the exon 7-intron 7 junction. Here we quantified the extent to which this duplication affects OCT1 splicing and, as a consequence, the expression and the function of OCT1. We used pyrosequencing and deep RNA-sequencing to quantify the effect of rs35854239 on splicing after minigene expression of this variant in HepG2 and Huh7 cells and directly in human liver samples. Further, we analyzed the effects of rs35854239 on OCT1 mRNA expression in total, localization and activity of the resulting OCT1 protein, and on the pharmacokinetics of sumatriptan and fenoterol. The 8 bp duplication caused alternative splicing in 38% (deep RNA-sequencing) to 52% (pyrosequencing) of the minigene transcripts when analyzed in HepG2 and Huh7 cells. The alternatively spliced transcript encodes for a truncated protein that after transient transfection in HEK293 cells was not localized in the plasma membrane and was not able to transport the OCT1 model substrate ASP+. In human liver, however, the alternatively spliced OCT1 transcript was detectable only at very low levels (0.3% in heterozygous and 0.6% in homozygous carriers of the 8 bp duplication, deep RNA-sequencing). The 8 bp duplication was associated with a significant reduction of OCT1 expression in the human liver, but explained only 9% of the general variability in OCT1 expression and was not associated with significant changes in the pharmacokinetics of sumatriptan and fenoterol. Therefore, the rs35854239 variant only partially changes splicing, causing moderate changes in OCT1 expression and may be of only limited therapeutic relevance.

The Plasmid-Borne tet(A) Gene Is an Important Factor Causing Tigecycline Resistance in ST11 Carbapenem-Resistant Klebsiella pneumoniae Under Selective Pressure

The emergence and prevalence of tigecycline-resistant Klebsiella pneumoniae have seriously compromised the effectiveness of antimicrobial agents in the treatment of infections. To explore the role of the plasmid-borne tet(A) gene in tigecycline resistance in carbapenem-resistant K. pneumoniae (CRKP), a total of 63 CRKP isolates were collected from a tertiary hospital in Hangzhou, China. The minimum inhibitory concentration (MIC) of tigecycline, mutation rate of tet(A) gene, genetic surroundings of tet(A)-carrying transmissible plasmid and the contribution of tet(A) mutation to tigecycline resistance were analyzed using antimicrobial susceptibility test, whole-genome sequencing, tigecycline resistance evolution experiment, and plasmid conjugation experiment. Our results showed that 52.4% (33 isolates) of the test isolates carried the tet(A) gene; among them, 75.8% (25 isolates) exhibited a tigecycline non-susceptible phenotype (MIC = 4 mg/L). Three clonal groups (cluster I, cluster II, and cluster III) were identified in these tet(A)-bearing isolates. All 17 isolates belonged to serotype KL21 (cluster I), which differed by only 13 SNPs, suggesting a clonal spread of tet(A)-positive ST11 K. pneumoniae with serotype KL21 occurred in the sampling hospital. The induction of tigecycline resistance experiments showed that 71.4% of strains evolved tet(A) mutations and developed a high-level tigecycline resistance. Eight amino acid substitutions were identified in these mutants. The most common amino acid substitution was A370V, followed by S251A and G300E. Twelve isolates carrying tet(A) mutants succeeded in the filter mating experiment with a conjugation efficiency of 10–3–10–8. Tigecycline MICs in E. coli EC600 transconjugants with a mutated tet(A) were 2 to 8-fold higher than those in E. coli EC600 transconjugants with a wild-type tet(A). One ColRNAI/IncFII type and two IncFII type tet(A)-bearing conjugative plasmids were identified in this study, including a class 1 integron containing multiple antibiotic resistance genes, i.e., tet(A), qnrS1, blaLAP–2, catA2, sul2, and dfrA14. Our study revealed the wide-spread situation of plasmid-borne tet(A) gene in clinical CRKP, and mutation of tet(A) is a potential driven force that lead to tigecycline resistance.

Genetic Characterization of G Protein in Respiratory Syncytial Virus ON-1 Genotype in Tehran

Aim: We investigated the genetic characterization of the respiratory syncytial virus (RSV) ON-1 genotypes and their different lineages based on the G gene among children &lt;2 years of age presenting with acute respiratory tract infections in Tehran, Iran. Materials &amp; methods: A phylogenetic tree from the Iranian samples and ON-1 strains of various parts of the world were constructed. The amino acid composition of the RSV G protein of the ON-1 genotype was mapped. Results: Human RSV ON-1 genotypes from the Iranian samples clustered in three lineages. The most common amino acid substitutions were as follows: X218Q, I240S, L289P, Y304H and L310P. Conclusion: Continuing molecular epidemiological surveys in other regions of Iran will provide deeper insight into the nature of this replacement of the dominant RSV genotype from GA2 to ON-1 in Iran.

Novel oseltamivir-resistant mutations distant from the active site of influenza B neuraminidase

We performed a neuraminidase sequence analysis of thirty-two pediatric patients with influenza B who visited Teikyo University Hospital from January 2016 to March 2017, and found oseltamivir-resistant samples belonging to the Yamagata and Victoria lineages. Comparison with the neuraminidase sequence of oseltamivir-susceptible B/Brisbane/60/2008 revealed 5 common amino acid substitutions in many of these samples. According to the binding free energy calculation, the N340D and E358K substitutions reduced the affinity of oseltamivir to neuraminidase. Unexpectedly, these substitutions were located distant from the oseltamivir-binding site in neuraminidase. According to the molecular dynamics simulations, the N340D substitution rearranged complicated hydrogen bond networks in an extensive surface region of neuraminidase. The E358K substitution extensively altered the electrostatic potential map of the overall neuraminidase structure. Through these novel mechanisms, the N340D and E358K substitutions indirectly influenced the affinity reduction. These results may be useful for designing drugs for the treatment of oseltamivir-resistant virus infections. Communicated by Ramaswamy H. Sarma

Regulation of Photosynthetic Carbohydrate Metabolism by a Raf-Like Kinase in the Liverwort Marchantia polymorpha.

To optimize growth and development, plants monitor photosynthetic activities and appropriately regulate various cellular processes. However, signaling mechanisms that coordinate plant growth with photosynthesis remain poorly understood. To identify factors that are involved in signaling related to photosynthetic stimuli, we performed a phosphoproteomic analysis with Marchantia polymorpha, an extant bryophyte species in the basal lineage of land plants. Among proteins whose phosphorylation status changed differentially between dark-treated plants and those after light irradiation but failed to do so in the presence of a photosynthesis inhibitor, we identified a B4-group Raf-like kinase, named PHOTOSYNTHESIS-RELATED RAF (MpPRAF). Biochemical analyses confirmed photosynthesis-activity-dependent changes in the phosphorylation status of MpPRAF. Mutations in the MpPRAF gene resulted in growth retardation. Measurement of carbohydrates demonstrated both hyper-accumulation of starch and reduction of sucrose in Mppraf mutants. Neither inhibition of starch synthesis nor exogenous supply of sucrose alleviated the growth defect, suggesting serious impairment of Mppraf mutants in both the synthesis of sucrose and the repression of its catabolism. As a result of the compromised photosynthate metabolism, photosynthetic electron transport was downregulated in Mppraf mutants. A mutated MpPRAF with a common amino acid substitution for inactivating kinase activity was unable to rescue the Mppraf mutant defects. Our results provide evidence that MpPRAF is a photosynthesis signaling kinase that regulates sucrose metabolism.

How good are pathogenicity predictors in detecting benign variants?

Computational tools are widely used for interpreting variants detected in sequencing projects. The choice of these tools is critical for reliable variant impact interpretation for precision medicine and should be based on systematic performance assessment. The performance of the methods varies widely in different performance assessments, for example due to the contents and sizes of test datasets. To address this issue, we obtained 63,160 common amino acid substitutions (allele frequency ≥1% and <25%) from the Exome Aggregation Consortium (ExAC) database, which contains variants from 60,706 genomes or exomes. We evaluated the specificity, the capability to detect benign variants, for 10 variant interpretation tools. In addition to overall specificity of the tools, we tested their performance for variants in six geographical populations. PON-P2 had the best performance (95.5%) followed by FATHMM (86.4%) and VEST (83.5%). While these tools had excellent performance, the poorest method predicted more than one third of the benign variants to be disease-causing. The results allow choosing reliable methods for benign variant interpretation, for both research and clinical purposes, as well as provide a benchmark for method developers.

Considerations of AOX Functionality Revealed by Critical Motifs and Unique Domains.

An understanding of the genes and mechanisms regulating environmental stress in crops is critical for boosting agricultural yield and safeguarding food security. Under adverse conditions, response pathways are activated for tolerance or resistance. In multiple species, the alternative oxidase (AOX) genes encode proteins which help in this process. Recently, this gene family has been extensively investigated in the vital crop plants, wheat, barley and rice. Cumulatively, these three species and/or their wild ancestors contain the genes for AOX1a, AOX1c, AOX1e, and AOX1d, and common patterns in the protein isoforms have been documented. Here, we add more information on these trends by emphasizing motifs that could affect expression, and by utilizing the most recent discoveries from the AOX isoform in Trypanosoma brucei to highlight clade-dependent biases. The new perspectives may have implications on how the AOX gene family has evolved and functions in monocots. The common or divergent amino acid substitutions between these grasses and the parasite are noted, and the potential effects of these changes are discussed. There is the hope that the insights gained will inform the way future AOX research is performed in monocots, in order to optimize crop production for food, feed, and fuel.

Evolution and dynamics of the pandemic H1N1 influenza hemagglutinin protein from 2009 to 2017.

The emergence of swine-origin pandemic H1N1 (pH1N1) in 2009 invigorated extensive surveillance programs worldwide which have resulted in the deposition of large numbers of H1N1 sequences to Genbank. In the present study, we report on global evolution and dynamics of the pandemic H1N1 influenza Hemagglutinin (HA) protein in viruses isolated from three different continents (North America, Europe and Asia) during the period between April 2009 until April 2017. Close to 2000 HA full protein sequences were downloaded from the Influenza Research Database of the NCBI and analyzed using DNAStar to run an alignment, the web-based NetNglyc to predict N-Glycosylation sites and finally, the BEAST software package to calculate evolution and substitution rates. Our analysis improves upon other published papers in that we report on frequencies, dynamics and impact of HA mutations in pH1N1 viruses isolated from three continents during the past decade, as well as the evolution rate and site-specific selection pressures. Sequence based analysis demonstrated substantial changes in the HA protein over the last decade. Results showed that the HA gene is under negative selection (P value; HA= -2.253). The evolution rates varied among the three continents ranging from 2.36 × 10-3 in Europe to 3.18 × 10-3 in Asia. Mutations were detected at higher frequency and faster rate at the antigenic sites surrounding the receptor-binding domain (RBD), in particular, in the Sa and Sb sites. Mutations were either gradually accumulated to become fixed in currently circulating strains (D114N, S179N, S202T, S220T, I233T, K300E and E391K) or dynamic in terms of appearance and disappearance, both spatially and temporally (A203T, N458K and E508G). Some of the reported mutations have been shown to increase infection severity (D239G/N; globular head), enhance HA binding affinity to its receptor (S200P and S202T; RBD), or have deleterious effect on HA function (N458K and E508G; stem region). The continuous accumulation of mutations at the Sa site led to the gradual acquisition of glycosylation at residue 179 starting from 2015, which became a dominant feature in all strains isolated in the following years. In addition to sharing common amino acid substitutions (e.g. S179N in HA head and E516K in HA stem) with previous seasonal strains, the pattern of glycosylation acquisition/loss at 177 and 179 positions on the globular head, which are prominent features of immune escape, implicate that pH1N1 might follow a similar evolution trend as the SC1918 pandemic virus.

Analysis of the complete genome of hepatitis B virus subgenotype C2 isolate NHB17965 from a patient with uncomplicated chronicity

The number of chronic cases of hepatitis B virus (HBV) is increasing rapidly in the world. Herein, we report a complete genome of HBV subgenotype C2 (HBV/C2) with current common amino acid substitutions from a patient with chronic HBV without liver complications. Complete genome analysis revealed that the isolated strain was a non-recombinant wild type and had several regular substitutions in the reverse transcriptase domain and small surface proteins of HBV. The isolated complete sequence could be considered as a chronic reference strain of HBV/C2 in Bangladesh. This study may help clinicians and scientists gain in-depth knowledge on common substitutions of HBV/C2 genome and to identify potential therapies against chronic HBV infections.