High Number Of Polymorphisms Research Articles

The impact of S2 mutations on Omicron SARS-CoV-2 cell surface expression and fusogenicity

ABSTRACT SARS-CoV-2 Omicron subvariants are still emerging and spreading worldwide. These variants contain a high number of polymorphisms in the spike (S) glycoprotein that could potentially impact their pathogenicity and transmission. We have previously shown that the S:655Y and P681H mutations enhance S protein cleavage and syncytia formation. Interestingly, these polymorphisms are present in Omicron S protein. Here, we characterized the cleavage efficiency and fusogenicity of the S protein of different Omicron sublineages. Our results showed that Omicron BA.1 subvariant is efficiently cleaved but it is poorly fusogenic compared to previous SARS-CoV-2 strains. To understand the basis of this phenotype, we generated chimeric S protein using combinations of the S1 and S2 domains from WA1, Delta and Omicron BA.1 variants. We found that the S2 domain of Omicron BA.1 hindered efficient cell–cell fusion. Interestingly, this domain only contains six unique polymorphisms never detected before in ancestral SARS-CoV-2 variants. WA1614G S proteins containing the six individuals S2 Omicron mutations were assessed for their fusogenicity and S surface expression after transfection in cells. Results showed that the S:N856K and N969K substitutions decreased syncytia formation and impacted S protein cell surface levels. However, we observed that “first-generation” Omicron sublineages that emerged subsequently, had convergently evolved to an enhanced fusogenic activity and S expression on the surface of infected cells while “second-generation” Omicron variants have highly diverged and showed lineage-specific fusogenic properties. Importantly, our findings could have potential implications in the improvement and redesign of COVID-19 vaccines.

Genomic insights into the glutathione metabolism of the non-conventional wine yeast <i>Starmerella bacillaris</i>

Glutathione (GSH) is an antioxidant molecule of great technological interest due to its wide range of applications in the food and beverage industry. In winemaking, although glutathione is produced during fermentation, its addition is possible for the control of oxidative spoilage of wine. Recently, to improve wine quality, mixed fermentation has been proposed by introducing a selection of non-Saccharomyces yeasts as complementary starters to the oenological species S. cerevisiae. Among them, Starmerella bacillaris, an osmophilic and high glycerol producer yeast, has been extensively studied. In the present study, the genomes of two S. bacillaris strains were compared with S. cerevisiae to identify the GSH metabolic pathway. The results showed that GSH biosynthesis includes the GSH1 and GSH2 genes in both species. The identification of a new transcription factor which binds sites in the promoter region of these genes underlined differences in the transcriptional regulation of both species. Additionally, between S. bacillaris strains, a high number of polymorphisms was found in genes involved in GSH redox balance. Preliminary laboratory scale fermentations revealed marked differences in the cell glutathione content of the two S. bacillaris strains. By comparing genomes, it was possible to gain a better understanding of the genes involved in the GSH metabolism pathway in S. bacillaris.

Persistence of Long-lived Memory B Cells specific to Duffy Binding Protein in individuals exposed to Plasmodium vivax

The major challenge in designing a protective Duffy binding protein region II (DBPII)-based vaccine against blood-stage vivax malaria is the high number of polymorphisms in critical residues targeted by binding-inhibitory antibodies. Here, longevity of antibody and memory B cell response (MBCs) to DBL-TH variants, DBL-TH2, -TH4, -TH5, -TH6 and -TH9 were analyzed in P. vivax-exposed individuals living in a low malaria transmission area of southern Thailand. Antibody to DBL-TH variants were significantly detected during P. vivax infection and it was persisted for up to 9 months post-infection. However, DBL-TH-specific MBC responses were stably maintained longer than antibody response, at least 3 years post-infection in the absence of re-infection. Phenotyping of B cell subsets showed the expansion of activated and atypical MBCs during acute and recovery phase of infection. While the persistence of DBL-TH-specific MBCs was found in individuals who had activated and atypical MBC expansion, anti-DBL-TH antibody responses was rapidly declined in plasma. The data suggested that these two MBCs were triggered by P. vivax infection, its expansion and stability may have impact on antibody responses. Our results provided evidence for ability of DBPII variant antigens in induction of long-lasting MBCs among individuals who were living in low malaria endemicity.

HIV-1 group O integrase displays lower susceptibility to raltegravir and has a different mutational pathway for resistance than HIV-1 group M.

IntroductionHIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase gene, e.g. positions L74I, S153A, G163Q and T206S. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on susceptibility to integrase inhibitors and emergence of resistance associated mutations.Viruses and MethodsWe cloned and purified integrase proteins from each of HIV-1 Group O clades A (HIV-O/A) and B (HIV-O/B), a HIV-O divergent strain (HIV-O/Div), and HIV-1 group M (subtype B, HIV-M/B) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of varying concentrations of several INSTIs. The inhibition constant (Ki) and IC50 were calculated and compared for HIV-M and HIV-O integrases. Selections for resistance-related mutations were performed using cord blood mononuclear cells and increasing concentration of INSTIs.ResultsHIV-O integrase and viruses were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. During selection, we observed different pathways of resistance depending on the drug and clade. Mutations selected in HIV-O can be classified as follows: (1) mutations described for HIV-M such as T97A, Q148R, V151A/I (RAL), T66I, E92Q, E157Q (EVG) and M50I, R263K (DTG) and (2) signature mutations for HIV-O (i.e. not described in HIV-M) F121C (HIV-O/B for RAL), V75I (HIV-O/A for RAL) and S153V (HIV-O/A for DTG). Only the HIV-O/Div selected the Q148R mutation for RAL and R263K+M50I for DTG, as previously described for HIV-M. None of the HIV-O viruses selected either N155H or Y143C. The selection of the specific S153V mutation could be explained at the nucleotide level: HIV-O at this position contains an alanine and substitution of alanine to valine (153AGGC→153VGTC) is easier than substitution of alanine to tyrosine (153AGGC→153YTAC), with only a transversion needed instead of one transition plus one transversion.ConclusionsThis is the first report of susceptibility and resistance in vitro to INSTIs for HIV-O. Our study confirmed the impact of HIV-O polymorphism, on susceptibility to INSTIs and the emergence of resistance mutations.

HIV-1 group O integrase displays lower enzymatic efficiency and higher susceptibility to raltegravir than HIV-1 group M subtype B integrase.

HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase coding region. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on enzyme function and susceptibility to integrase inhibitors. Accordingly, we cloned and purified integrase proteins from each of HIV-1 group O clades A and B, an HIV-O divergent strain, and HIV-1 group M (HIV-M, subtype B), used as a reference. To assess enzymatic function of HIV-O integrase, we carried out strand transfer and 3' processing assays with various concentrations of substrate (DNA target and long terminal repeats [LTR], respectively) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of various concentrations of several INSTIs. The inhibition constant (Ki) and 50% effective concentration (EC50) values were calculated for HIV-O integrases and HIV-O viruses, respectively, and compared with those of HIV-M. The results showed that HIV-O integrase displayed lower activity in strand transfer assays than did HIV-M enzyme, whereas 3' processing activities were similar to those of HIV-M. HIV-O integrases were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. Molecular modeling suggests that two key polymorphic residues that are close to the integrase catalytic site, 74I and 153A, may play a role in these differences.

A new series of review articles on drug metabolizing enzymes: nomenclature of isoenzyme families, genetic organization, polymorphisms, substrate specificities, clinical relevance and role in carcinogenesis

Predicting drug metabolism-dependent toxicity is of key importance in pharmacology and toxicology. The capacity for metabolic activation and inactivation strongly inXuences the toxic proWle of a substance as well as the shape of the dose–response curve (Hengstler et al. 2003; Bolt et al. 2004; Carmo et al. 2006, 2007). In recent years, metabolism has been one of the most active topics of our journal, including numerous contributions about induction or inhibition of xenobiotic metabolizing enzymes (Elovaara et al. 2007; Mitchell et al. 2007; Davila-Borja et al. 2007; Nannelli et al. 2007; Dorrenhaus et al. 2007; Ito et al. 2007; Becker et al. 2006; Bernshausen et al. 2006; Fatemi et al. 2006), metabolically competent cell systems, especially hepatocytes (Thum and Borlak 2007; Parody et al. 2007; Kurebayashi and Ohno 2006; Hewitt et al. 2007), diVerences in metabolic capacity between tissues and species (Myers and Spinnato 2007; Souma et al. 2006; Roos et al. 2006; Gultekin and Hicyilmaz 2007) and polymorphisms of drug metabolizing enzymes (Lin et al. 2007; Ulusoy et al. 2007; Kamimura 2006). A typical feature of drug metabolizing enzymes is large families of isoenzymes. An enormous diversity has evolved some 200–400 million years ago. This coincides with the period when some animals, such as initially amphibians and reptiles, moved from sea to land and started to eat plants. This led to a co-evolution of plants producing phytotoxins and animals responding with new detoxiWcation enzymes (Nebert et al. 1989). In recent years, much progress has been made in unraveling the enormous diversity of drug metabolizing enzymes. A second feature complicating research in drug metabolism is the high number of polymorphisms. Meanwhile, we know that our genome comprises approximately 3 million polymorphisms. The frequency of polymorphisms, mostly SNPs, of drug metabolizing enzymes usually is between 1 and 2 per 1000 nucleotides. Although much progress has been made, by far not all clinically relevant polymorphisms have been identiWed. Because of the dynamic progress in the Weld of drug metabolizing enzymes, the editors feel that a new series of review articles on drug metabolizing enzymes is required. Each review will focus on an individual family of isoenzymes and will focus on the following topics:

Genet size and distribution of Amanita muscaria in a suburban park, Dunedin, New Zealand

Amanita muscaria is a common ectomy‐corrhizal (ECM) fungus introduced to New Zealand. It most commonly forms associations with introduced host trees, but is also known to form ECM associations with native Nothofagus species. It may act as a “mycorrhizal weed” and could potentially have far‐reaching consequences for the diversity of indigenous fungal taxa in New Zealand. Little is known about its population biology. By analysing the banding patterns produced from randomly amplified microsatellite (RAMS) primed polymerase chain reactions, we examined the genetic structure of an established population of A. muscaria in a park in Dunedin, New Zealand. The primer BDB(ACA)5 was found to have a higher number of polymorphisms than the either of the primers DDB(CCA)5 or DHB(CGA)5, and it is possible that within any given area the number of genets detected might be greater, and the size of genets smaller, with the use of more primers. Within a 40 × 40 m area the population was found to comprise 28 genotypes, both small (< 6.0 m across) and large (> 6.0 m across). The distribution of genotypes showed no clear pattern of spatial segregation, indicating that single genotypes may comprise multiple ramets rather than contiguous genets. The presence of both small and large genets (or widely separated ramets) indicates that establishment through sexually derived basidiospores and through mycelial spread may play important roles in populations of A. muscaria such as the one studied. A. muscaria may employ a strategy combining attributes of C, S, and r‐selected ecological strategies. Frequent disturbance at the site may mean that establishment by basidiospore plays a more important role than at other sites with low levels of disturbance.

Ornithine decarboxylase: haplotype structure and trait associations in White Leghorn chickens

Sequence analysis of 4,230 bp of the 3′ end of the ornithine decarboxylase gene of 20 chickens from a noninbred White Leghorn strain revealed a total of 62 polymorphisms. Of these 61% were transitions, 24% were transversions, and 16% were deletions or insertions. Despite the high number of polymorphisms, only 3 haplotypes were present among the 40 alleles analyzed. Based on the genetic distances between haplotypes they segregated early during domestication of the chicken. Ornithine decarboxylase is a pivotal enzyme in regulating the synthesis of polyamines, cations that are important regulators of cell division, differentiation, and apoptosis. Variants of ornithine decarboxylase are, therefore, expected to affect many different traits. Association analyses between genotypes and the major egg production traits in female chickens of the same strain revealed significant effects on the onset of sexual maturity, BW at sexual maturity, eggshell thickness (a measure of calcium deposition), and residual feed consumption (a measure of the metabolic rate). Further, comparisons of the genotypes indicated that the 3 haplotypes differ in their phenotypic properties. Our results show that variations in a gene that is ubiquitously expressed in all cells of an organism may nevertheless contribute to distinct phenotypic properties of the organism as a whole.

A PCR/RFLP technique to characterize fungal species in Eucalyptus grandis Hill ex. Maiden ectomycorrhizas.

With the increasing awareness of the significance of mycorrhizas, research is focusing on studies to elucidate the contribution of the symbiosis to ecosystem dynamics. In this sense, molecular biology has acquired great significance. PCR/RFLP techniques were adapted to characterize ectomycorrhizal fungi associated with Eucalyptus grandis. The ITS region of the fungal rDNA from pure cultures and from of mycorrhizas synthesized in vitro was amplified. Primers NSA3/NLC2 were used followed by a nested reaction with primers ITS1F/NLB3. Amplicons were then digested with the enzymes MboI, HinfI and TaqI. Amplification resulted in a 1,000-bp fragment for basidiomycetes and a 1,500 bp fragment for Cenococcum geophillum (an ascomycete). There was no amplification of the plant DNA. The enzymes MboI and HinfI were more effective than TaqI, resulting in patterns of two to five fragments allowing the identification of the isolates both in culture and in mycorrhizas. HinfI allowed greater differentiation among the isolates and a higher number of polymorphisms. Restriction with TaqI resulted in too many fragments. Amplification efficiency for the fungal DNA was 64% in culture and 87% in mycorrhizas. The modified methodology represents a valuable tool to complement traditional approaches in ecosystem studies.

Analysis of mutations in the p16/CDKN2A gene in sporadic and familial melanoma in the Polish population.

Mutations in CDKN2A have been found in sporadic cutaneous malignant (CMM), in familial CMM and in other syndromes associated with melanoma. In this study DNA was obtained from 207 individuals and five cell lines. There were 157 CMM patients and 50 healthy members of melanoma patients families. The CMM group included patients with one or two melanoma cases in the family, families with dysplastic nevus syndrom (DNS) and patients with a spectrum of other types of cancers in the family. PCR-SSCP analysis and sequencing identified: six substitutions in codon 58 CGA/TGA (Arg/Stop), 16 substitutions GAC/GAT in codon 84 (Asp/Asp), six substitutions CGA/TGA in codon 148 (Arg/Thr), 14 substitutions G/C in 3'UTR and 4 double changes (two in codon 84 and 3'UTR; two in codon 148 and 3'UTR). The mutation identified in codon 58 was found in tissue only. Other substitutions were polymorphisms found in DNA from tissue and blood samples. Most of them were identified in sporadic CMM (six in codon 148 Ala/Thr, 12 in codon 84 Asp/Asp and six in 3'UTR). The frequency of the polymorphisms was also high in DNS and CMM/DNS families (four in codon 84 Asp/Asp and six in 3'UTR). No mutations or polymorphisms were found in CMM patients with one or two melanoma cases and CMM patients, with other cancers in family history. The analysis of the CDKN2A gene mutations in the Polish population demonstrated: (i) no germline mutations; (ii) a relatively high number of genetic changes in sporadic melanoma; (iii) a high number of polymorphisms in DNS and CMM/DNS families.