Search

The enzyme dihydropyrimidine dehydrogenase (DPD) is the primary catabolic pathway of fluoropyrimidines including 5 fluorouracil (5FU) and capecitabine. Cases of lethal toxicity have been reported in cancer patients with complete DPD deficiency receiving standard dose of 5FU or capecitabine. DPD is encoded by the pharmacogene DPYD in which more than 200 variants have been identified. Different approaches have been developed for screening DPD-deficiency, including DPYD genotyping and phenotyping. Plasma uracil ([U]) and dihydrouracil ([UH2]) concentrations are routinely used as surrogate markers for systemic DPD activity: [U] ≥ 16 ng/ml and < 150 ng/ml, and [U] ≥ 150 ng/mL indicate partial and complete DPD deficient phenotype, respectively, while values of 5 or 10 for [UH2]/([U] ratio are often cited. Four clinically relevant DPYD defective variants (DPYD*13, DPYD*2A, p.Asp949Val and haplotype B3), are targeted in genetic testing via PCR. In practice, pretreatment [U], alone or combined with these 4 recommended DPYD alleles guides individual dosage selection, though this approach has limitations. This is illustrated by two cases showing discrepancy between DPD deficient phenotype and normal standard genotype. In these two cases, DPYD exome sequencing with Next Generation Sequencing identified rare inactive variants, establishing concordance between phenotype and genotype. In patient 1, [U] levels of 21.1 and 25.5 ng/mL, indicated partial deficiency though the targeted genotype was normal and 5FU dose was adjusted based on the phenotype. In patient 2, [U] levels of 16.2 and 15.2 ng/mL were near the 16 ng/ml threshold. With a normal genotype, he as considered non-deficient as targeted genotype was normal and the standard dose was administered. These two cases underscore the need to pair DPD phenotyping with whole DPYD gene sequencing, due to the frequent discrepancies between these pharmacogenetic tools, the burden of rare variants and ethnic differences in variant frequencies. [ABSTRACT FROM AUTHOR]

Methane (CH4) processes and fluxes have been widely investigated in low-latitude tropical wetlands and high-latitude boreal peatlands. In the mid-latitude Mongolia Plateau, however, CH4 processes and fluxes have been less studied, particularly in riverine wetlands. In this study, in situ experiments were conducted in the riverine sandy wetlands of the Mongolia Plateau to gain a better understanding of CH4 emissions and their influencing mechanisms. Annual CH4 emissions were observed at 8.7 mg m–2 h–1 from the flowing water wetlands during November 2019 − October 2021, approximately 80% and 20% of which were emitted during the growing and non-growing seasons, respectively. In particular, CH4 emissions during the thawing period contributed < 5% to the annual total, contrary to the traditional idea that thawing plays an important role in annual CH4 emissions in boreal peatlands. CH4 emissions were significantly higher in the wetlands dominated by plant species than in that dominated by water body during the growing seasons; therefore, plant-mediated CH4 transport was explained as a favorable pathway for CH4 emissions from sandy soils to the atmosphere. Gene sequencing revealed differences in the phylogenies and taxonomies of methanogenic archaea and methanotrophs between the flowing and static water wetlands, suggesting that flowing water should bring oxygen and nutrients to microbial habitats and potentially affect the production, oxidation, and diffusion of CH4 in sandy wetlands. [ABSTRACT FROM AUTHOR]

Background: The incidence estimates for intracranial subdural empyema are 0.1 per 100,000 individuals, making it a rare clinical condition. It is a highly morbid and fatal illness that is most commonly the result of a primary infection somewhere else. Case presentation: The authors present a young male patient 15 years presented with 1 week history of headache, fever, confusion and seizure attack with initial negative CT head and nearly negative CSF analysis. With few days he developed left sided weakness. MRI brain showed right temporoparietal subdural fluid collection with midline shift. Urgent neurosurgical evacuation was made and revealed subdural empyema. The boy received combination therapy of vancomycin, ceftriaxone, and metronidazole. The culture of the pus was negative but 16S rRNA gene sequencing (bacterial) revealed streptococcus intermedius. He made a good recovery with no recollection or neurological deficit on follow up. Conclusion: This case highlights the possibility of occurrence of this rare infection in otherwise healthy individuals without obvious precipitating factor. It also indicates the superiority of MRI brain over CT head in detection of subdural collection. The rapid diagnosis and intervention improve the outcome of the patient. [ABSTRACT FROM AUTHOR]

Purpose: To describe katG and inhA mutations, clinical characteristics, treatment outcomes and clustering of drug-resistant tuberculosis (TB) in the State of São Paulo, southeast Brazil. Methods: Mycobacterium tuberculosis isolates from patients diagnosed with drug-resistant TB were screened for mutations in katG and inhA genes by line probe assay and Sanger sequencing, and typed by IS6110-restriction fragment-length polymorphism for clustering assessment. Clinical, epidemiological and demographic data were obtained from surveillance information systems for TB. Results: Among the 298 isolates studied, 127 (42.6%) were isoniazid-monoresistant, 36 (12.1%) polydrug-resistant, 93 (31.2%) MDR, 16 (5.4%) pre-extensively drug-resistant (pre-XDR), 9 (3%) extensively drug-resistant (XDR) and 17 (5.7%) susceptible after isoniazid retesting. The frequency of katG 315 mutations alone was higher in MDR isolates, while inhA promoter mutations alone were more common in isoniazid-monoresistant isolates. Twenty-six isolates phenotypically resistant to isoniazid had no mutations either in katG or inhA genes. The isolates with inhA mutations were found more frequently in clusters (75%) when compared to the isolates with katG 315 mutations (59.8%, p = 0.04). In our population, being 35–64 years old, presenting MDR-, pre-XDR- or XDR-TB and being a retreatment case were associated with unfavourable TB treatment outcomes. Conclusion: We found that katG and inhA mutations were not equally distributed between isoniazid-monoresistant and MDR isolates. In our population, clustering was higher for isolates with inhA mutations. Finally, unfavourable TB outcomes were associated with specific factors. [ABSTRACT FROM AUTHOR]

Evaluation of genetic uniformity is important in a perennial plant like coffee propagated through somatic embryogenesis. In this study, we compared the genetic homogeneity of somatic embryo-derived plants and the seed-derived plants obtained from a single mother plant of robusta coffee cultivar by employing Sequence Related Amplified Polymorphism (SRAP) and Start Codon Targeted (SCoT) molecular markers and sequencing four functional genes. The SRAP and SCoT molecular markers revealed 96% genetic similarity between somatic embryo-derived and seed-derived plants with the mother plant. The sequencing analysis has shown the minimum sequence variability in the chloroplast maturase K gene and the highest sequence variability in the zinc finger protein gene compared to NAC25 and UDP-glycosyltransferase genes. The presence of SNP and indels created polymorphism in gene sequences. The percent frequency of SNP varied from 0.0 to 0.45 in matK, 0.36 to 1.40 in NAC25, 0.81 to 2.63 in the UDP-glycosyltransferase gene and 3.30 to 8.70 in the zinc finger protein gene. In the case of NAC25 and UDP-glycosyltransferase genes, the seed-derived plants have shown a higher frequency of SNP compared to somatic embryo-derived plants. However, in the case of zinc finger protein gene, tissue culture-derived plants accumulated a two-fold higher frequency of SNP compared to the seed-derived plants. The sequence characterization of the four genes has revealed the presence of a higher frequency of non-synonymous SNPs compared to synonymous SNPs. This is the first report on the comparative genetic uniformity analysis of somatic embryo-derived and seed-derived plants in robusta coffee cultivar with significant practical implications. Key message: Molecular marker based genetic homogeneity assessment of somatic embryo and seed-derived plants of Coffea canephora revealed 96% similarity with the mother plant. The SE derived plants exhibited higher sequence variability in zinc finger protein gene. [ABSTRACT FROM AUTHOR]

We analysed mutations in katG, inhA and rpoB genes, and isoniazid phenotypic resistance levels in Mycobacterium tuberculosis isolates from drug-resistant TB patients from São Paulo state, Brazil. Isolates resistant to the critical concentration of isoniazid in MGIT (0.1 µg/mL) were screened for mutations in katG 315 codon, inhA promoter region and rpoB RRDR by MTBDRplus assay and subjected to determination of isoniazid resistance levels by MGIT 960. Discordances were resolved by Sanger sequencing. Among the 203 isolates studied, 109 (54%) were isoniazid-monoresistant, 47 (23%) MDR, 29 (14%) polydrug-resistant, 12 (6%) pre-XDR and 6 (3%) XDR. MTBDRplus detected isoniazid mutations in 75% (153/203) of the isolates. Sequencing of the entire katG and inhA genes revealed mutations in 18/50 wild-type isolates by MTBDRplus (10 with novel mutations), resulting in a total of 32/203 (16%) isolates with no mutations detected. 81/83 (98%) isolates with katG 315 mutations alone had intermediate resistance. Of the 66 isolates with inhA C-15T mutation alone, 51 (77%) showed low-level, 14 (21%) intermediate and 1 (2%) high-level resistance. 5/6 (83%) isolates with mutations in both katG and inhA had high-level resistance. Inferred mutations corresponded to 22% (16/73) of all mutations found in rpoB. Mutations detected in katG regions other than codon 315 in this study might be potential new isoniazid resistance markers and could explain phenotypic resistance in some isolates without katG and inhA classic mutations. In our setting, 16% of isoniazid-resistant isolates, some with high-level resistance, presented no mutations either in katG or inhA. [ABSTRACT FROM AUTHOR]

Root-associated compartments, including rhizosphere soil, rhizoplane soil, and the endosphere, are found to harbor distinguished bacterial populations and community composition, but how microbiome in these rhizo-compartments are affected by edaphic variables remains largely unknown. Goethite is a prevalent crystalline iron (hydr)oxide mineral of the soil matrix and strongly interact with microbial communities. The objective of our study was to determine how goethite (α-FeOOH) amendment assemble bacterial communities in the rhizo-compartments of Maize (Zea mays. L). Using sequencing of microbial 16S ribosomal RNA gene amplicons, we revealed that goethite amendment into soil enriched Actinobacteria and depleted Proteobacteria in all rhizo-compartments. Also, goethite enlarged the differences in the alpha diversity (Chao) between rhizo-compartments, with much lower mean diversity in the endosphere and rhizoplane compared with rhizosphere soil, indicating a higher selection of the microbiome assemblage. This was supported by beta Nearest Taxon Index (βNTI > + 2), indicating that changes in environmental conditions progressively increase the strength of selection. It suggests that variable selection (a deterministic process) was the dominant process influencing the microbial assembly in soil amended with goethite. According to the distance-based linear modeling (distLM), the assemblage of bacterial communities in the rhizosphere compartments was regulated by specific edaphic variables, with the major contributors being goethite (62%), total C (52%), soil pH (50%), and FeOM (25%). Stabilization of rhizosphere C in the presence of goethite would be the selective step for its accessibility and consequent microbial community. For instance, the keystone microorganisms, e.g., Pseudomonas, had more negative links within the goethite added co-occurrence network, indicating its mutual exclusions and outcompete other microbes in C/nutrients limited conditions. Thus, goethite narrows the composition of rhizosphere mainly due to "gate selection" effects on rhizodeposits, which limited microbial penetrance into inner-compartments, consequently assemble the rhizosphere bacterial community via deterministic process. [ABSTRACT FROM AUTHOR]

Primary immunodeficiencies (PI) are genetic defects of the immune system that result in chronic and often life-threatening infections and/or life-threatening autoimmunity if not diagnosed and treated. Patients with a suspected PI, but without a genetic diagnosis, commonly undergo a diagnostic odyssey that is costly, time-consuming, and arduous. This delay in diagnosis prevents appropriate disease management and treatment, contributing to prolonged suffering and decreased quality of life. Although next generation sequencing (NGS) can provide these patients with relief from such a diagnostic odyssey, it is often unavailable, mainly due to cost and inaccessibility. In January 2019, the Jeffrey Modell Foundation (JMF) launched a free genetic sequencing pilot program for Jeffrey Modell Centers Network (JMCN) patients clinically diagnosed with an underlying PI. A total of 21 sites within the JMCN were invited to participate. JMF collaborated with Invitae, and testing was comprised of Invitae's Primary Immunodeficiency Panel, which currently includes 207 genes. A questionnaire was disseminated to each participating physician to evaluate barriers to access to genetic sequencing and changes in disease management and treatment after testing. One hundred fifty-eight patients and 29 family members were tested in this pilot study. Twenty-one percent of patients with a suspected monogenic disorder received a molecular diagnosis, and others received potentially useful diagnostic leads. Based on the results of genetic sequencing, clinical diagnosis was altered in 45% of patients, disease management was altered in 40%, treatment was altered in 36%, and genetic counseling was altered in 62%. The results of this pilot program demonstrate the utility, cost-efficiency, and critical importance of NGS for PI and make the case for broad scale sequence–based diagnostics for PI patients when requested by expert immunologists. [ABSTRACT FROM AUTHOR]

Introduction: Advanced therapy-refractory biliary tract cancer (BTC) has poor prognosis and constitutes a major challenge for adequate treatment strategies. By mapping the molecular profiles of advanced BTC patients, precision cancer medicine may provide targeted therapies for these patients. Objective: In this analysis, we aimed to show the potential of PCM in metastatic BTC. Methods: In this single-center, real-world retrospective analysis of our PCM platform, we describe the molecular profiling of 30 patients diagnosed with different types of metastatic BTC. Tumor samples of the patients were examined using a 161-gene next-generation sequencing panel, immunohistochemistry (IHC), and fluorescence in situ hybridization for chromosomal translocations. Results: In total, we identified 35 molecular aberrations in 30 patients. The predominant mutations were KRAS (n = 8), TP53 (n = 7), IDH2 (n = 4), and IDH1 (n = 3) that accounted for the majority of all molecular alterations (62.86%). BRAF mutations were observed in two patients. Less frequent alterations were noted in ARID1A, CTNNB1, ESR1, FBXW7, FGFR2, MET, NOTCH2, PIK3CA, PTCH1, SMAD4, and SRC1, each in one case. FGFR fusion gene was detected in one patient. No mutations were detected in eight patients. IHC revealed EGFR and p-mTOR expression in 28 patients. Applying these results to our patients, targeted therapy was recommended for 60% of the patients (n = 18). One patient achieved stable disease. Conclusions: PCM is a feasible treatment approach and may provide molecular-guided therapy recommendations for metastatic BTC. [ABSTRACT FROM AUTHOR]

Key message: This paper shows that CH4 produced in the heartwood of living trees in upland forests is of microbial origin, and that microbial CH4 production may be suppressed in the heartwood with more phenolic compounds than carbohydrates. Methane (CH4) is a potent greenhouse gas that exerts large effects on atmospheric chemistry and the global warming. Living trees in upland forests might contribute to the global CH4 emissions but the contribution is poorly understood and the mechanisms of CH4 production in their heartwood are not completely clear. Using gene sequencing and a series of laboratory incubations, this study addressed whether CH4 produced in the heartwood of living trees in upland forests is microbial in origin and how the CH4 production is affected by secondary metabolites. Both the response of CH4 production to temperatures and the presence of methanogenic archaea indicate that the CH4 produced in heartwood was microbial in origin. Methanobacterium was the dominant methanogens in the heartwood of Populus canadensis which had high concentrations of CH4, and was absent from the heartwood of Pinus tabuliformis and Salix matsudana which produced negligible or very low CH4. Water- and ethanol-soluble extractives enhanced microbial CH4 production in heartwood incubations but acetone-soluble extractives inhibited the production. Since acetone-soluble extractives inhibited the production of microbial CH4, it is assumed that the CH4 production may be suppressed from heartwood with phenolic compounds that act as an antibiotic for methanogenic archaea in most living trees in upland forests. The inhibitory effects of secondary metabolites on microbial CH4 production in heartwood reduce the CH4 emissions from the stems of living trees in upland forests. [ABSTRACT FROM AUTHOR]

The translocation of deoxyribonucleic-acid (DNA) segments into a nanoscale channel from a bulk solution has significance to genome-related fields such as gene sequencing and genetic profiling. However, its dynamic characteristics are poorly understood because of complexities involving the size and character of the confining spaces, polymeric evolution, and driving sources. The DNA translocation was studied by mesoscale simulation using a worm-like-chain model of DNA and the dissipative particle dynamics method. Three driving sources — electric field, suction flow, and constant body force were individually applied to a λ-DNA entering a nanoscale cylindrical channel. Each source altered the translocation actions, mainly due to marked differences in conformational transformation processes. The suction flow uniquely induced strong extension of the DNA chain before channel entry. Consequently, the flow was more effective at overcoming the free-energy barrier with narrower confinements. Other driving sources permitted the DNA chain to enter in a more globular form, which was effective for larger entrances. Combining different sources is the most versatile means of controlling the trans-location of genetic materials in highly confined spaces. [ABSTRACT FROM AUTHOR]

Background: Disseminated nocardiosis is a rare disease mostly occurring in immunocompromised patients.Methods: We report a case of disseminated nocardiosis in a diabetic patient with both pulmonary and cutaneous involvement. Nocardia elegans was isolated and identified using the 16s ribosomal RNA gene sequence data.Results: Clinical improvement was observed within 3 months after initiation of antimicrobial treatment with oral doxycycline, trimethoprim-sulfamethoxazole and intravenous penicillin, but the patient died 5 months later after arbitrary discontinuation of the treatment.Conclusions: This is the first case report of disseminated nocardiosis caused by Nocardia elegans in China. [ABSTRACT FROM AUTHOR]

A novel 4.0-kb Fy was sequenced and bacterially expressed. This gene, the largest y-type HMW-GS currently reported, is 4,032-bp long and encodes a mature protein with 1,321 amino acid (AA) residues. The 4.0-kb Fy shows novel modifications in all domains. In the N-terminal, it contains only 67 AA residues, as three short peptides are absent. In the repetitive domain, the undecapeptide RYYPSVTSPQQ is completely lost and the dodecapeptide GSYYPGQTSPQQ is partially absent. A novel motif unit, PGQQ, is present in addition to the two standard motif units PGQGQQ and GYYPTSPQQ. Besides, an extra cysteine residue also occurs in the middle of this domain. The large molecular mass of the 4.0-kb Fy is mainly due to the presence of an extra-long repetitive domain with 1,279 AA residues. The novel 4.0-kb Fy gene is of interest in HMW-GS gene evolution as well as to wheat quality improvement with regard to its longest repetitive domain length and extra cysteines residues. [ABSTRACT FROM AUTHOR]

Nine novel high-molecular-weight prolamins (HMW-prolamins) were isolated from Leymus multicaulis and L. chinensis. Based on the structure of the repetitive domains, all nine genes were classified as D-hordeins but not high-molecular-weight glutenin subunits (HMW-GSs) that have been previously isolated in Leymus spp. Four genes, Lmul 1.2, 2.4, 2.7, and Lchi 2.5 were verified by bacterial expression, whereas the other five sequences (1.3 types) were classified as pseudogenes. The four Leymus D-hordein proteins had longer N-termini than those of Hordeum spp. [116/118 vs. 110 amino acid (AA) residues], whereas three (Lmul 1.2, 2.4, and 2.7) contained shorter N-termini than those of the Ps. juncea (116 vs. 118 AA residues). Furthermore, Lmul 1.2 was identified as the smallest D-hordein, and Lmul 1.2 and 2.7 had an additional cysteines. Phylogenetic analysis supported that the nine D-hordeins of Leymus formed two independent clades, with all the 1.3 types clustered with Ps. juncea Ns 1.3, whereas the others were clustered together with the D-hordeins from Hordeum and Ps. juncea and the HMW-GSs from Leymus. Within the clade of four D-hordein genes and HMW-GSs, the HMW-GSs of Leymus formed a separated branch that served as an intermediate between the D-hordeins of Ps. juncea and Leymus. These novel D-hordeins may be potentially utilized in the improvement of food processing properties particularly those relating to extra cysteine residues. The findings of the present study also provide basic information for understanding the HMW-prolamins among Triticeae species, as well as expand the sources of D-hordeins from Hordeum to Leymus. [ABSTRACT FROM AUTHOR]

Hereditary ataxias and spastic paraplegias are genetic disorders with age-dependent nearly complete penetrance. The mostly monogenetic etiology allows one to establish the diagnosis, study pathogenesis and to develop new causative therapeutic approaches for these diseases. Both the causative genes as well as the clinical presentation overlap considerably between hereditary ataxias and spastic paraplegias. This strongly argues towards a united classification for these two groups of diseases. Next generation sequencing technologies have greatly expanded the number of genes known to be causative for hereditary ataxias and spastic paraplegias and allow simultaneous time- and cost-effective diagnostic testing of > 200 genes. However, repeat expansions and large genomic deletions must be considered separately. Here, we suggest a pragmatic algorithm for genetic testing in hereditary ataxias and spastic paraplegias that we have developed in our specialized outpatient clinics. Detailed phenotyping remains crucial to interpret the multitude of genetic variants discovered by high throughput sequencing techniques. Despite recent technical advances, a substantial proportion of ataxia and spastic paraplegia families are still without a molecular diagnosis. Beside new and so far undetected ataxia and spasticity genes, unusual mutation types including noncoding variants and polygenic inheritance patterns may contribute. Because of these clinical, genetic, and technological challenges, patients with hereditary ataxias and spastic paraplegias should be referred to specialized centers offering research and clinical studies. This will also help to recruit representative patient cohorts for upcoming interventional trials. [ABSTRACT FROM AUTHOR]

Purpose: Kartagener syndrome (KS), also known as visceral inversion-nasosinusitis-bronchiectasis syndrome, or familial bronchiectasis, is an autosomal recessive inherited disease. In this study, through two cases of KS, we aimed to assess the clinical and genetic characteristics of KS caused by DNAH5 mutations. Methods: The two cases of KS from the same family underwent extensive clinical assessments, with next-generation DNA sequencing and bioinformatics analysis to identify pathogenic genes. In addition, Sanger sequencing was used to verify the pedigrees. Results: The present study employed a directional capture strategy for hereditary disease screening, which correctly identified the virulence sites in the pedigree, and facilitated the differential diagnosis among multiple genes. Two novel mutations were detected in DNAH5: c.7778C>T (missense mutation) and c.13729G>A (nonsense mutation). They were not found in dbSNP, 1000 Genomes, and ExAC. Conclusions: These findings demonstrated that new DNAH5 mutations could be used for molecular diagnosis of KS, providing families with genetic counseling and prenatal diagnosis. [ABSTRACT FROM AUTHOR]

An unknown species of rust was observed on sugarcane in Swaziland and South Africa in 2008. Infected leaves showed rust-like symptoms on the abaxial and adaxial leaf surfaces. Uredinia appeared bright orange when fresh, releasing profuse, bright orange urediniospores, which distinguished it from Puccinia melanocephala, the only rust species known to infect sugarcane in southern Africa at that time. No identical matches were obtained from the NCBI database for the combined ITS and 28S nuclear large subunit (nLSU) regions for the unknown rust. Phylogenetic analyses based on the same gene region showed that this rust was closely related to Macruropyxis fraxini and Puccinia sparganioides, which infect Fraxinus spp. (Oleaceae). Also closely related were Aecidium klugkistianum, P. mysuruensis, and P. physalidis which infect host species in the Oleaceae, Rubiaceae, and Solanaceae, respectively. This group of rusts is phylogenetically distinct from both Puccinia melanocephala and Puccinia kuehnii, which cause brown and orange rust of sugarcane, respectively. Phylogenetic reconstruction showed that the unknown rust falls outside the Puccinia I and II clades and groups with the Macruropyxis clade with strong bootstrap support. Hence the name proposed for this newly discovered rust species is Macruropyxis fulva sp. nov., causal agent of tawny rust of sugarcane. A rust observed to be infecting Miscanthus ecklonii in 2016 matched the accessioned M. fulva sp. nov. sequences and was morphologically and genetically distinct from the Miscanthus rust Puccinia miscanthidii, providing evidence of an alternate host for M. fulva sp. nov. [ABSTRACT FROM AUTHOR]

Grafted tomato plants grown in a soilless culture system in heated greenhouses in Sicily were affected by stem rot disease. Symptoms consisted of dark brown longitudinal discoloration of the basal part of the stem and petioles. Longitudinal sections of the stem revealed brown watersoaked or soft-rotted pith tissue and internal vascular discoloration. Bacterial isolates with two different colony morphologies were obtained from symptomatic tissues. Isolates were identified as Pectobacterium spp. on the basis of biochemical and molecular analyses. The analysis of the 16S rRNA gene sequence and of the concatenated sequences of the housekeeping genes rpoS and pgi revealed that the isolates of the two morphotypes belong to P. carotovorum subsp. carotovorum and P. c. subsp. brasiliensis, respectively. Isolates from both taxa reproduced disease symptoms on artificially inoculated tomato plants. This is the first report of P. carotovorum subsp. brasiliensis in Italy. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) can incur significant health care costs to the patient, their families, and society; furthermore, effective treatments are limited, as the mechanisms of AD are not fully understood. This study utilized twelve adult male tree shrews (TS), which were randomly divided into PBS and amyloidbetapeptide1-40 (Aβ1-40) groups. AD model was established via an intracerebroventricular (icv) injection of Aβ1-40 after being incubated for 4 days at 37 °C. Behavioral, pathophysiological and molecular changes were evaluated by hippocampal-dependent tasks, magnetic resonance imaging (MRI), silver staining, hematoxylin-eosin (HE) staining, TUNEL assay and gene sequencing, respectively. At 4 weeks post-injection, as compared with the PBS group, in Aβ1-40 injected animals: cognitive impairments happened, and the hippocampus had atrophied indicated by MRI findings; meanwhile, HE staining showed the cells of the CA3 and DG were significantly thinner and smaller. The average number of cells in the DG, but not the CA3, was also significantly reduced; furthermore, silver staining revealed neurotic plaques and neurofibrillary tangles (NFTs) in the hippocampi; TUNEL assay showed many cells exhibited apoptosis, which was associated with downregulated BCL-2/BCL-XL-associated death promoter (Bad), inhibitor of apoptosis protein (IAP), Cytochrome c (CytC) and upregulated tumor necrosis factor receptor 1 (TNF-R1); lastly, gene sequencing reported a total of 924 mobilized genes, among which 13 of the downregulated and 19 of the upregulated genes were common to the AD pathway. The present study not only established AD models in TS, but also reported on the underlying mechanism involved in neuronal apoptosis associated with multiple gene expression. [ABSTRACT FROM AUTHOR]

The porin gene, which is adjacent to the pigmentation region ( pgm), is usually damaged by IS100 element in highly virulent Yersinia pestis strains. In addition, the pgm region, which carries the genes responsible for virulence (high pathogenicity island) and biofilm generation ( hms-operon), is flanked by direct IS100 copies (causing its destabilization). The study of distribution of intact and truncated porin genes was conducted among 240 Y. pestis strains from 39 natural foci of Russia and countries of the near abroad and 68 Yersinia pseudotuberculosis strains from different geographical regions. Most highly virulent Y. pestis strains and some phylogenetic Y. pseudotuberculosis lines of O:1 serotype contain truncated porin genes. At the same time, deletion of the pgm region by flanked IS100 in Y. pseudotuberculosis is impossible, since IS100 is integrated in the porin gene in an orientation opposite to that of Y. pestis. The intact porin gene is carried by all Y. pestis strains with low epidemic significance and certain phylogenetic lines of highly virulent Y. pestis strains from desert foci and Caspian sandy focus, as well as most Y. pseudotuberculosis strains of O:1 serotype. A continuous deletion, which includes the porin gene and a part of the astE gene, was detected in less virulent Y. pseudotuberculosis strains of O:3 serotype. The nucleotide sequence of porin genes is identical in Y. pestis and Y. pseudotuberculosis strains from different geographical regions. Three porin gene allele only differ by IS100 integration site and orientation or absence of its integration. The nucleotide sequence of IS100 introduced in the porin gene of Yersinia has small differences only for two Y. pestis strains isolated in America. The correlation of low frequency of Hms-mutants with the intact porin gene state in Y. pestis and the absence of such a correlation in Y. pseudotuberculosis were established. [ABSTRACT FROM AUTHOR]

Nanopore-based sequencing platforms are transforming the field of genomic science. This review (containing 116 references) highlights some recent progress on nanopore-based nucleic acid analysis and sequencing. These studies are classified into three categories, biological, solid-state, and hybrid nanopores, according to their nanoporous materials. We begin with a brief description of the translocation-based detection mechanism of nanopores. Next, specific examples are given in nanopore-based nucleic acid analysis and sequencing, with an emphasis on identifying strategies that can improve the resolution of nanopores. This review concludes with a discussion of future research directions that will advance the practical applications of nanopore technology. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The most promising anti-tumor agent developed in the past three decades is Taxol. It is proven to be effective against many cancers. It is necessary to isolate pharmacologically potent endophytic microbial strains from medicinal plants with special reference to Taxol production. In the current study, endophytic fungi were isolated from the bark of the medicinal plant, Salacia oblonga. The isolated endophytes were identified morphologically, and further characterized by ITS-PCR using genomic DNA samples, later the products were sequenced for identification and phylogenetic linkage mapping. The samples were screened for the potential to produce Taxol or taxanes, employing PCR. The resulted data have been sequenced to confirm the presence of the two genes implicated in Taxol biosynthesis, 10-deacetylbaccatin III-10-O-acetyl transferase (DBAT) and C-13 phenylpropanoid side chain-CoA acyltransferase (BAPT). Seven samples showed the amplicons of DBAT gene and one showed the amplicons of BAPT gene. Sequencing of these products was carried out, of which one sample has revealed sequence homology to the original DBAT gene from Taxus . The present work confirms and substantiates the potential of genomic mining approach to discover novel Taxol-producing endophytic fungi. [ABSTRACT FROM AUTHOR]

A new species of Centrorhynchus (Centrorhynchidae) with receptacle insertion at the posterior third of the proboscis is described from the pheasant crow Centropus sinensis (Stephens) (Cuculidae) in Pakistan. Centrorhynchu sglobirostris n. sp. is similar to the 98 other known species of Centrorhynchus Lühe, 1911 in having long cylindrical trunk with anterior dilation and transverse anastomoses of the secondary lacunar vessels. However, specimens of C. globirostris differ from all other species of the genus by having a unique globular proboscis not divided into anterior proboscis with rooted hooks and posterior proboscis with rootless spines. Posterior hooks of C . globirostris emerge at the level of the receptacle insertion and are uniquely fully rooted. Sequencing and phylogenetic analysis of C . globirostris 18S and 28S ribosomal RNA genes reveals the genetic and evolutionary relationships between C. globirostris and other members of Centrorhynchidae which have representative orthologs in public databases. Comparison to known acanthocephalans confirms appropriate inclusion in the genus Centrorhynchus. [ABSTRACT FROM AUTHOR]

The precision medicine as a new emerging area and therapeutic strategy has occurred and was practiced in the individual and brought unexpected successes, and gained high attentions from professional and social aspects as a new path to improve the treatment and prognosis of patients. There will be a number of new components to appear or be discovered, of which clinical bioinformatics integrates clinical phenotypes and informatics with bioinformatics, computational science, mathematics, and systems biology. In addition to those tools, precision medicine calls more accurate and repeatable methodologies for the identification and validation of gene discovery. Precision medicine will bring more new therapeutic strategies, drug discovery and development, and gene-oriented treatment. There is an urgent need to identify and validate disease-specific, mechanism-based, or epigenetics-dependent biomarkers to monitor precision medicine, and develop 'precision' regulations to guard the application of precision medicine. [ABSTRACT FROM AUTHOR]

This study looked into a family involving a rare mother-child ABO blood type inconsistency and explored its genetic and molecular basis. In the family, the mother had type AB blood and the father was blood type B and they gave birth to a baby of blood type O. Their blood types were phenotypically identified by using different techniques, including micro-column gel test, immune inhibition test, absorption and elution tests. The sequences of all 7 exons of ABO allele from the core family members were determined by using PCR and clone-based sequencing. The loci of mutated gene were compared against normal human genes. The result showed that the mother's erythrocytes were agglutinable with monoclonal anti-A antibody (2+) and had agglutination reaction with anti-B antibody (4+). The mother's serum registered agglutination action with standard blood type A cells. The findings showed an ABO inconsistency. When domestic antibodies were used, the mother's erythrocytes yielded agglutination reaction with humanized anti-B serum (4+) and anti-B monoclonal antibody but were non-agglutinable with humanized anti-A serum and anti-A monoclonal antibody. Upon absorption and elution, the titer of anit-A antibody was 128 both before and after the absorption test, with no significant difference found between pre- and post-absorption values. Our results confirmed that the mother's allelic gene was type B and contained type A. The father's blood type was type B, and son's blood type was type O. Clone-based sequencing revealed that the mother carried a heterozygous gene of B101.01 (ntA640→G)/O01, which contained an M214→V mutation that could express a weak expression of antigen A, resulting in blood type AB. However, their son did not have the M214→V mutation, which yielded a false ABO-inconsistency between him and his mother. We were led to conclude that type B gene with a M214→V mutation can encode both antigen B and weak antigen B that can lead to false ABO-inconsistencies. [ABSTRACT FROM AUTHOR]

Vromindolines, a family of oat starch-bound proteins responsible for the extremely soft endosperm of this cereal, are characterized by a tryptophan-rich domain and ten conserved cysteine residues, as it was observed in the starch-bound proteins from other cereals, and proved to be specific of the genus Avena. Two-dimensional electrophoresis of vromindolines from different oat species, both cultivated and wild, revealed that they are present in all genomes and ploidy levels, with no major differences in their molecular weight and electrophoretic mobility. PCR amplification and sequencing of genes coding for VIN-2 and VIN-3 indicated that both proteins are strongly conserved across the species analysed. Some differences were found between diploid and polyploid accessions at specific amino acid positions along the protein. In particular the C-genome accessions ( A. clauda, A. eriantha, A. ventricosa) differed from the A-genome diploids ( A. canariensis, A. damascena, A. longiglumis, A. nuda, A. strigosa) for some peculiar sequences. Tetraploids ( A. barbata, A. magna and A. insularis) showed two sequences each for both proteins; the variability observed in them was similar to that found in A. sativa. The characteristics of these genes could help to clarify the genetic relationship among the various species of the genus Avena. Moreover, the knowledge of the genetic control of these proteins represent an important tool for the modulation of oat endosperm texture. [ABSTRACT FROM AUTHOR]

The two-line hybrid system in rice is becoming more important and employs environment-conditioned genic male sterile (EGMS) lines sensitive to photoperiod (photoperiod-sensitive genic male sterile), temperature [temperature genic male sterile (TGMS)], or a combination of the two (photoperiod temperature genic male sterile). At least 18 EGMS genes have been mapped, and two cloned, but controversies exist. For example, three different genes were reported to underlie the TGMS trait in three independently identified progenitors, Annong S-1, Zhu 1S, and Guangzhan 63S, while another study demonstrated that the TGMS genes in Annong S-1 and Zhu 1S are allelic. In the present study, we confirmed the allelism of the three TGMS genes, which means there is a common TGMS gene(s) in these lines. Knowing there is an association between the mutant allele ( RNZ) of a ribonuclease gene ( RNZ) with the TGMS trait in Guangzhuan 63S, we then sequenced RNZ for 14 commercial EGMS and 21 non-EGMS lines, and we developed two derived cleaved amplified polymorphic sequence (dCAPS) markers to detect RNZ alleles in 32 EGMS and 310 non-EGMS lines. The analyses showed that the RNZ allele existed exclusively in EGMS lines; all non-EGMS lines contained the functional RNZ or RNZ allele. Furthermore, two segregating populations that included 2,429 individuals were developed by crossing Zhu 1S ( RNZ) to two non-EGMS lines (both with RNZ); examination of the segregation of male sterile and fertile plants indicated that the TGMS trait was under the control of a single gene; analysis of the markers revealed the RNZ allele exclusively in TGMS plants and the RNZ allele only in non-TGMS plants in both populations. The dCAPS markers could therefore help select TGMS progeny in breeding programs, which will save time and labor, and improve breeding efficiency and accuracy. [ABSTRACT FROM AUTHOR]

Three y-type high-molecular-weight (HMW) glutenin gene open reading frames (ORFs), Chiy1, Chiy2, and Racy, were isolated and characterized from Leymus chinensis PI499516 and Leymus racemosus ssp. racemosus W623305. They shared an extra glutamine in the N-terminal and LAAQLPAMCRL peptides in the C-terminal with x-type HMW glutenins but had different N-terminal lengths. Like other y-type HMW glutenins, Chiy2 and Racy had 104 (or 105) amino acid (aa) residues at the N-terminal and started with EGEASR, whereas Chiy1 had 99 aa in this domain and started with QLQCER because of the deletion of EGEASR. Five other y-type glutenins, including those from Elymus ciliaris, Pseudoroegneria libanotica, and Leymus mollis, were similar to Chiy1. The ORF of Chiy2 was probably not expressed. The ORFs of both Chiy1 and Racy were expressed in bacteria. The maximum likelihood phylogenic tree based on the signal peptide and N-terminal and C-terminal aa residues revealed two clades of y-type HMW glutenins in Triticeae; the first contained Ay, By, Cy, Dy, Eey, Gy, Ky, Ry, Tay, and Uy, while the second clade contained the remaining y types, including those from Leymus. Within the second clade, HMW glutenins lacking the EGEASR peptide formed a subclade. These y-type HMW glutenins in Leymus could not be targeted to the Xm or Ns genome. [ABSTRACT FROM AUTHOR]

Summary: In this study, we used traditional morphological and molecular identification methods to preliminarily identify two strains of dermatophytes. The two strains were observed under the microscope. And then the dermatophytes were cultured on Sabouraud’s dextrose agar (SDA). The 18S rRNA regions of the two dermatophyte strains were amplified by polymerase chain reaction (PCR), and the PCR products were sequenced and compared with GenBank data. BLAST tools and DNAMAN software were used to analyze the sequences. To further determine highly homologous sequences, a phylogenetic tree was constructed using the Neighbor-Joining method. The two strains of dermatophytes were identified by traditional morphological identification as Epidermophyton floccosum and Microsporumferrugineum. The 18S rRNA sequence analyses showed high similarities to Cladosporiumcladosporioides isolate C115LM-UFPR and Ascomycete sp. LB68A1A2. Epidermophyton and Cladosporium belong to dermatophyte, while Microsporum ferrugineum and Ascomycete belong to microsporum. The two novel strains of dermatophytes were therefore identified as Cladosporiumcladosporioides isolate C115LM-UFPR (JN650537, Cladosporium) and Ascomycete sp. LB68A1A2 (AY770409, Ascomycete sp). [ABSTRACT FROM AUTHOR]

We characterized 45 LMW glutenin genes from three diploid species of Taeniatherum using 63 primer combinations, designed according to 264 genes reported in wheat and related species. The genes had 909-1,059 bp nucleotides and 301-351 amino acids. The deduced peptides shared similar structures with LMW-m proteins of wheat. The 45 genes shared 77.2-99.7% identities in peptide sequence among each other and 60.0-82.0% identities to proteins from wheat and related species. They were divided into five types according to the N-terminals, starting with METSCIP-, METSRVP-, METGRIP-, METGSIP- and VETSCIP-. The last three and some other structural domain variations were not reported previously in the Triticeae. Thirty-three genes encoded full mature proteins with intact ORFs, whereas the other 12 were pseudogenes with incomplete ORFs, in-frame stop codons or frame-shift mutations. Phylogenetic analysis showed that orthologous genes from Taeniatherum were more similar to those in the B and D genomes than in the A genome. [ABSTRACT FROM AUTHOR]

In order to clarify the phylogeny and relationships of the most confused hypotrichous ciliates, Holosticha-complex, four closely related holostichids (five populations), Holosticha bradburyae, H. diademata, Anteholosticha sp., and A. manca, were compared and analyzed using ITS2 secondary structures, ITS1-5.8S-ITS2 region and SSrRNA gene sequences. The ITS1-5.8S-ITS2 region sequences of these four species were first sequenced, and they shared sequence identities ranging from 68.0% to 90.1%, while two populations of Anteholosticha sp. differed in three nucleotides (sequence identity 99.8%). There were several minor differences among ITS2 secondary structures of these species, while two populations of Anteholosticha sp. had the identical secondary structure. Phylogenetic trees inferred from the ITS1-5.8S-ITS2 region sequences of stichotrichs using multiple algorithms (Neighbor-Joining, Maximum Parsimony and Bayesian) revealed similar topologies. The results show that: (1) Holosticha bradburyae and H. diademata firmly clustered together with strong bootstrap supports, forming a sister clade with Anteholosticha sp., (2) Anteholosticha appeared to be a paraphyletic assemblage, in which the morphotype A. manca was more closely related to Diaxonella trimarginata than to its congener Anteholosticha sp. Phylogenetic analyses based on the SSrRNA gene and the combined sequences of SSrRNA gene and ITS1-5.8S-ITS2 region revealed the similar relationships between Holosticha and Anteholosticha, nevertheless their positions within the subclass Stichotrichia differed from each other inferred from different genes. [ABSTRACT FROM AUTHOR]

Objectives:To determine the content of glutamine in major food proteins.Subjects/Methods:We used a validated 131-food item food frequency questionnaire (FFQ) to identify the foods that contributed the most to protein intake among 70 356 women in the Nurses’ Health Study (NHS, 1984). The content of glutamine and other amino acids in foods was calculated based on protein fractions generated from gene sequencing methods (Swiss Institute of Bioinformatics) and compared with data from conventional (USDA) and modified biochemical (Khun) methods. Pearson correlation coefficients were used to compare the participants’ dietary intakes of amino acids by sequencing and USDA methods.Results:The glutamine content varied from 0.01 to to 9.49 g/100 g of food and contributed from 1 to to 33% of total protein for all FFQ foods with protein. When comparing the sequencing and Kuhn's methods, the proportion of glutamine in meat was 4.8 vs 4.4%. Among NHS participants, mean glutamine intake was 6.84 (s.d.=2.19) g/day and correlation coefficients for amino acid between intakes assessed by sequencing and USDA methods ranged from 0.94 to 0.99 for absolute intake, −0.08 to 0.90 after adjusting for 100 g of protein, and 0.88 to 0.99 after adjusting for 1000 kcal. The between-person coefficient of variation of energy-adjusted intake of glutamine was 16%.Conclusions:These data suggest that (1) glutamine content can be estimated from gene sequencing methods and (2) there is a reasonably wide variation in energy-adjusted glutamine intake, allowing for exploration of glutamine consumption and disease. [ABSTRACT FROM AUTHOR]

We have evaluated the mutation profile in a sample of 127 unrelated β-thalassemia (β thal) individuals, diagnosed through A2 and fetal hemoglobin quantification by high-performance liquid chromatography (HPLC) from the Brazilian southernmost state, where a flow of Italian immigrants had occurred in the late 19th century, mainly from Northern Italy. The molecular analysis was performed by DNA sequencing of the most common mutations found in the Mediterranean region. The β0 codon 39 nonsense mutation was the most frequent alteration (50.9%), followed by β+ IVSI 110 G>A (18.1%), β0 IVSI 1 G>A (12.9%), β+ IVSI 6 T>C (9.5%), and other rare mutations (8.6%). The chosen gene sequence was able to identify 91% β-thal mutations in the population studied, showing some similarity with allele frequencies of the mainly colonizing countries of Rio Grande do Sul state. The comparison of our results to other Brazilian studies has shown significant differences. Therefore, we can conclude that the genotypic profile of β-thal shows great variability. Hence, it would be arbitrary to infer regional study results as being representative of the Brazilian whole population. Brazilian researchers of different regions should identify their most frequent genotypes to provide better understanding on this disease and state adequate public health policies. [ABSTRACT FROM AUTHOR]

In this study, we focused our investigation on two strains of Lactobacillus curvatus, L442 and LTH1174, which are able to produce bacteriocins. L. curvatus LTH1174 is widely studied for its capability to produce curvacin A, while L. curvatus L442 was isolated from traditional Greek fermented sausages and was shown to possess a strong inhibitory activity toward Listeria monocytogenes. By polymerase chain reaction, we were able to target in both strains the genes for the production of sakacin P and sakacin Q, sppA and sppQ, respectively, both encoded chromosomally. While sppA was found to be conserved when compared with other sakacin P genes, sppQ showed a deletion of about 15 nucleotides when aligned with sequences obtained from Lactobacillus sakei. This difference did not affect the activity of sakacin Q as determined by testing sensitive strains. Expression analysis highlighted that sakacin P was expressed in L. curvatus L442 but not in L. curvatus LTH1174. Curing experiments were performed on L. curvatus LTH1174 to study the effect of the megaplasmid, present in this strain. In the plasmid-cured strain, expression of the sppA gene was detected. sppQ was expressed in both plasmid-cured and wild-type L. curvatus LTH1174, although expression was higher in the plasmid-cured strain. [ABSTRACT FROM AUTHOR]

The high molecular weight (HMW) glutenin subunits, Dtx1.5 + Dty10, are special types of storage proteins found in Aegilops tauschii that are never found in common wheat ( Triticum aestivum). This study reports the characterization of the complete open reading frames (ORFs) of the HMW glutenin genes, D t x1.5 and D t y10, using a restrict-enzyme based method named the restricted deletion method’ (RDM). The D t x1.5 and D t y10 were found to have an identical structure compared with the other published HMW glutenin genes. Comparison of the deduced protein sequences also indicated that the Dty10 in Ae. tauschii differed from its counterpart Dy10 in common wheat, by having insertions and deletions in the central repetitive domain. This result confirms the two subunits with same mobility in SDS-PAGE are different types of HMW glutenin subunits. In addition, four PCR-mediated recombinants of the D t x1.5 and D t y10 genes were amplified using a PCR program with shorter extension time. The recombinants had a similar structure to their corresponding natural genes, but a significantly different central repetitive domain. Western blot analysis exhibited a normal expression of the recombinants in E. coli. In addition to its usefulness for studying structure and function of the HMW glutenin subunits, the PCR-mediated recombination may provide an efficient method to generate novel HMW glutenin genes for wheat breeding. [ABSTRACT FROM AUTHOR]

In a Japanese population study of the D1S80 locus 24 alleles ranging from allele16 to allele43 were analysed using PCR-RFLP. As two repeat units were found to contain the restriction cleavage site (CCAGG) for EcoRII, we digested the alleles with EcoRII, separated the digested fragments on polyacrylamide gels and stained with ethidium bromide. Of the 24 alleles 11 band patterns were identified and tentatively labeled E1 to E11. A total of 42 subtypes were detected in a population group of 111 unrelated individuals. All samples of allele18 were of the E3 type, while about 60% of the allele 24 samples were of the E4 type and about 40% were of the E8 type. The third most frequent allele (allele30) contained four types, E4, E8, E5 and E6. No deviations from Hardy-Weinberg equilibrium were observed. Since this method could differentiate those samples which had the same length but different sequences, it is quite useful for paternity testing and individual identification. [ABSTRACT FROM AUTHOR]

In 180 unrelated Japanese individuals 18 examples of allele 27 were detected at the locus D1S80 (MCT118). On 6% polylacrylamide gels 5 out of these 18 alleles were found to migrate between allele 26 and allele 27, but closer to allele 27, and thus were labelled variants of allele 27. All 18 examples of allele 27 were sequenced and the results were compared. Although all had the same number of base pairs (578 bp) the five variants could be subdivided into three types V1, V2 and V3. The variants and the standard allele were composed of the same kinds of repeat units, but the order of arrangement was different. We investigated whether it was possible to distinguish the standard allele 27, and the variants V1, V2, and V3 by PCR-RFLP. EcoRII and MspI which have restriction sites within the repeat units were adopted as restriction enzymes. The variants could be discriminated from each other after treatment of the PCR fragments with EcoRII or MspI, followed by PAG electrophoresis. [ABSTRACT FROM AUTHOR]

Human infections caused by Lactobacillus spp. are rarely reported in the literature. Underlying conditions are frequently reported, and identification of lactobacilli to the species level remains rare. A case of Lactobacillus casei septicaemia secondary to a vascular graft infection is reported. The 16S rRNA sequencing technique was used to definitively identify Lactobacillus casei. [ABSTRACT FROM AUTHOR]