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KEY MESSAGE: A novel locus was discovered on chromosome 7 associated with a lesion mimic in maize; this lesion mimic had a quantitative and heritable phenotype and was predicted better via subset genomic markers than whole genome markers across diverse environments. Lesion mimics are a phenotype of leaf micro-spotting in maize (Zea mays L.), which can be early signs of biotic or abiotic stresses. Dissecting its inheritance is helpful to understand how these loci behave across different genetic backgrounds. Here, 538 maize recombinant inbred lines (RILs) segregating for a novel lesion mimic were quantitatively phenotyped in Georgia, Texas, and Wisconsin. These RILs were derived from three bi-parental crosses using a tropical pollinator (Tx773) as the common parent crossed with three inbreds (LH195, LH82, and PB80). While this lesion mimic was heritable across three environments based on phenotypic ([Formula: see text] = 0.68) and genomic ([Formula: see text] = 0.91) data, transgressive segregation was observed. A genome-wide association study identified a single novel locus on chromosome 7 (at 70.6 Mb) also covered by a quantitative trait locus interval (69.3-71.0 Mb), explaining 11-15% of the variation, depending on the environment. One candidate gene identified in this region, Zm00001eb308070, is related to the abscisic acid pathway involving in cell death. Genomic predictions were applied to genome-wide markers (39,611 markers) contrasted with a marker subset (51 markers). Population structure explained more variation than environment in genomic prediction, but other substantial genetic background effects were additionally detected. Subset markers explained substantially less genetic variation (24.9%) for the lesion mimic than whole genome markers (55.4%) in the model, yet predicted the lesion mimic better (0.56-0.66 vs. 0.26-0.29). These results indicate this lesion mimic phenotype was less affected by environment than by epistasis and genetic background effects, which explain its transgressive segregation.
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Estudo de Associação Genômica Ampla , Zea mays , Zea mays/genética , Epistasia Genética , Mapeamento Cromossômico , Fenótipo , Patrimônio Genético , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The emergence of multi drug resistant clone CC320 serotype19F/19A and their capsular (cps) antigenic variants due to selective pressures such as vaccine had been reported worldwide. Hence, it is important to identify the prevalent clones, sequence types and cps variants of serotype 19F/19A in India, where PCV13 has been recently introduced. Multi-locus sequence typing (MLST) was performed for all (n = 21) invasive S. pneumoniae isolates of serotype 19A (n = 5) and 19F (n = 16) collected between the years 2012 and 2018 from children less than 5 years. The genome characterization by whole genome sequencing for the Sequence types (STs) 320 and 271(n = 7) were performed and compared with another six Indian WGSs of similar STs available from the GPS platform. The predominant STs in the serotype 19F/19A study isolates were of CC320: ST 320, 236 and 271, associated with PMEN clone Taiwan19F-14. The WGSs of CC320 study isolates showed high genomic similarity to the Taiwan19F-14 clone, and the penicillin binding protein (PBP) amino acid sequence similarity was 100% for PBP1A, 93% for PBP 2B and 2X. Whilst PBP comparison with other global MDR ST320 strains revealed that the ST320 clones in India are of low-level penicillin resistance. The presence of a few ST320/19A/19F invasive isolates with high similarity to the Taiwan clone suggests slow and gradual expansion of Taiwan19F-14 associated CC320 clones in India. Since serotype 19F/19A is covered by PCV13 vaccine, the expansion of 19F/19A cones with non-PCV13 vaccine serotype in India should be monitored.
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Resistência às Penicilinas , Infecções Pneumocócicas/microbiologia , Vacinas Pneumocócicas/uso terapêutico , Sorogrupo , Streptococcus pneumoniae/genética , Pré-Escolar , Genômica , Humanos , Índia , Tipagem de Sequências Multilocus , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/fisiologia , Sequenciamento Completo do GenomaRESUMO
An amendment to this paper has been published and can be accessed via the original article.
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BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is overexpressed in solid tumors and regarded as a putative cancer stem cell marker. Here, we report that employing EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) dual approach, for the targeted delivery of siRNA to EpCAM positive cancer cells, efficiently inhibits cancer cell proliferation. RESULTS: Targeted delivery of siRNA using polyethyleneimine is one of the efficient methods for gene delivery, and thus, we developed a novel aptamer-PEI-siRNA nanocomplex for EpCAM targeting. PEI nanocomplex synthesized with EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) showed 198 nm diameter sized particles by dynamic light scattering, spherical shaped particles, of 151 ± 11 nm size by TEM. The surface charge of the nanoparticles was -30.0 mV using zeta potential measurements. Gel retardation assay confirmed the PEI-EpApt-SiEp nanoparticles formation. The difference in size observed by DLS and TEM could be due to coating of aptamer and siRNA on PEI nanocore. Flow cytometry analysis revealed that PEI-EpApt-SiEp has superior binding to cancer cells compared to EpApt or scramble aptamer (ScrApt) or PEI-ScrApt-SiEp. PEI-EpApt-SiEp downregulated EpCAM and inhibited selectively the cell proliferation of MCF-7 and WERI-Rb1 cells. CONCLUSIONS: The PEI nanocomplex fabricated with EpApt and siEp was able to target EpCAM tumor cells, deliver the siRNA and silence the target gene. This nanocomplex exhibited decreased cell proliferation than the scrambled aptamer loaded nanocomplex in the EpCAM expressing cancer cells and may have potential for EpCAM targeting in vivo.
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Antígenos de Neoplasias/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Moléculas de Adesão Celular/metabolismo , Nanoestruturas/química , Polietilenoimina/química , RNA Interferente Pequeno/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Molécula de Adesão da Célula Epitelial , Feminino , Humanos , Células MCF-7RESUMO
Palmer amaranth (Amaranthus palmeri) is a pervasive and troublesome weed species that poses significant challenges to agriculture in the United States. Identifying the sex of Palmer amaranth plants is crucial for developing tailored control measures due to the distinct characteristics and reproductive strategies exhibited by male and female plants. Traditional methods for sex determination are expensive and time-consuming, but recent advancements in spectroscopic techniques offer new possibilities. This study explores the potential of portable Raman spectroscopy for determining the sex of mature Palmer amaranth plants in-field. Raman analysis of the plant leaves reveals spectral differences associated with nitrate salts, lipids, carotenoids, and terpenoids, allowing for high accuracy and reliable identification of the plant's sex; male plants had higher concentrations of these compounds compared to females. It was also found that male plants had higher concentrations of these compounds compared to the females. Raman spectra were analyzed using a machine learning tool, partial least squares discriminant analysis (PLS-DA), to generate accuracies of no less than 83.7% when elucidating sex from acquired spectra. These findings provide insights into the sex-specific characteristics of Palmer amaranth and suggest that Raman analysis, combined with PLS-DA, can be a promising, non-destructive, and efficient method for sex determination in field settings. This approach has implications for developing sex-specific management strategies to monitor and control this invasive weed in real-world environments, benefiting farmers, agronomists, researchers, and master gardeners.
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Weeds are attractive models for basic and applied research due to their impacts on agricultural systems and capacity to swiftly adapt in response to anthropogenic selection pressures. Currently, a lack of genomic information precludes research to elucidate the genetic basis of rapid adaptation for important traits like herbicide resistance and stress tolerance and the effect of evolutionary mechanisms on wild populations. The International Weed Genomics Consortium is a collaborative group of scientists focused on developing genomic resources to impact research into sustainable, effective weed control methods and to provide insights about stress tolerance and adaptation to assist crop breeding.
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Genômica , Plantas Daninhas , Plantas Daninhas/genética , Genômica/métodos , Controle de Plantas Daninhas/métodos , Genoma de Planta , Produtos Agrícolas/genética , Resistência a Herbicidas/genética , Melhoramento Vegetal/métodosRESUMO
The potential for gene flow between cultivated species and their weedy relatives poses agronomic and environmental concerns, particularly when there are opportunities for the transfer of adaptive or agronomic traits such as herbicide resistance into the weedy forms. Grain sorghum (Sorghum bicolor) is an important crop capable of interspecific hybridization with its weedy relative johnsongrass (Sorghum halepense). Previous findings have shown that triploid progenies resulting from S. bicolor × S. halepense crosses typically collapse with only a few developing into mature seeds, whereas tetraploids often fully develop. The objective of this experiment was to determine the impact of S. bicolor genotype and pollen competition on the frequency of hybridization between S. bicolor and S. halepense. A total of 12 different cytoplasmic male sterile S. bicolor genotypes were compared with their respective male fertile lines across 2 years, to assess the frequency of hybridization and seed set when S. halepense served as the pollinator parent. Results indicate significant differences in the frequency of interspecific hybridization among the S. bicolor genotypes, and pollen fertility in S. bicolor reduced the rate of this interspecific hybridization by up to two orders of magnitude. Further, hybridization rates greatly varied across the two study environments. Results are helpful for developing appropriate gene flow mitigation strategies and indicate that gene flow could be reduced by the selection of appropriate seed parents for sorghum hybrids.
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Steric exclusion is a key element of enzyme substrate specificity, including in polymerases. Such substrate specificity restricts the enzymatic synthesis of 2'-modified nucleic acids, which are of interest in nucleic-acid-based drug development. Here we describe the discovery of a two-residue, nascent-strand, steric control 'gate' in an archaeal DNA polymerase. We show that engineering of the gate to reduce steric bulk in the context of a previously described RNA polymerase activity unlocks the synthesis of 2'-modified RNA oligomers, specifically the efficient synthesis of both defined and random-sequence 2'-O-methyl-RNA (2'OMe-RNA) and 2'-O-(2-methoxyethyl)-RNA (MOE-RNA) oligomers up to 750 nt. This enabled the discovery of RNA endonuclease catalysts entirely composed of 2'OMe-RNA (2'OMezymes) for the allele-specific cleavage of oncogenic KRAS (G12D) and ß-catenin CTNNB1 (S33Y) mRNAs, and the elaboration of mixed 2'OMe-/MOE-RNA aptamers with high affinity for vascular endothelial growth factor. Our results open up these 2'-modified RNAs-used in several approved nucleic acid therapeutics-for enzymatic synthesis and a wider exploration in directed evolution and nanotechnology.
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RNA , Fator A de Crescimento do Endotélio Vascular , RNA/química , Oligorribonucleotídeos , RNA MensageiroRESUMO
PURPOSE: To study target-specific delivery of doxorubicin (Dox) using an RNA aptamer against epithelial cell adhesion molecule (EpCAM) in retinoblastoma (RB) cells. METHODS: The binding affinity of the EpCAM aptamer to RB primary tumor cells, Y79 and WERI-Rb1 cells, and Müller glial cell lines were evaluated with flow cytometry. Formation of physical conjugates of aptamer and Dox was monitored with spectrofluorimetry. Cellular uptake of aptamer-Dox conjugates was monitored through fluorescent microscopy. Drug efficacy was monitored with cell proliferation assay. RESULTS: The EpCAM aptamer (EpDT3) but not the scrambled aptamer (Scr-EpDT3) bound to RB tumor cells, the Y79 and WERI-Rb1 cells. However, the EpCAM aptamer and the scrambled aptamer did not bind to the noncancerous Müller glial cells. The chimeric EpCAM aptamer Dox conjugate (EpDT3-Dox) and the scrambled aptamer Dox conjugate (Scr-EpDT3-Dox) were synthesized and tested on the Y79, WERI-Rb1, and Müller glial cells. The targeted uptake of the EpDT3-Dox aptamer caused cytotoxicity in the Y79 and WERI-Rb1 cells but not in the Müller glial cells. There was no significant binding or consequent cytotoxicity by the Scr-EpDT3-Dox in either cell line. The EpCAM aptamer alone did not cause cytotoxicity in either cell line. CONCLUSIONS: The results show that the EpCAM aptamer-Dox conjugate can selectively deliver the drug to the RB cells there by inhibiting cellular proliferation and not to the noncancerous Müller glial cells. As EpCAM is a cancer stem cell marker, this aptamer-based targeted drug delivery will prevent the undesired effects of non-specific drug activity and will kill cancer stem cells precisely in RB.
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Antibióticos Antineoplásicos/farmacologia , Antígenos de Neoplasias/metabolismo , Aptâmeros de Nucleotídeos/química , Biomarcadores Tumorais/metabolismo , Moléculas de Adesão Celular/metabolismo , Doxorrubicina/farmacologia , Portadores de Fármacos/farmacologia , Antibióticos Antineoplásicos/química , Antígenos de Neoplasias/genética , Transporte Biológico , Biomarcadores Tumorais/genética , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Doxorrubicina/química , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Molécula de Adesão da Célula Epitelial , Citometria de Fluxo , Expressão Gênica , Humanos , Terapia de Alvo Molecular , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Especificidade de Órgãos , Ligação Proteica , Neoplasias da Retina/tratamento farmacológico , Neoplasias da Retina/metabolismo , Neoplasias da Retina/patologia , Retinoblastoma/tratamento farmacológico , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Espectrometria de FluorescênciaRESUMO
Crystalline hydrates of swelling clay minerals (smectites) exhibit a strong coupling between their ion exchange and hydration/dehydration reactions. The uptake or removal of water from smectite interlayers as a result of a change in the environmental conditions also leads to the partitioning of cations. Three factors, the solid ion composition, the solid basal spacing/water content, and the aqueous solution composition, are all implicated in controlling the thermodynamics of ion exchange. However, conventional approaches to measuring the exchange free energy cannot separate the influence of each of these individual factors. Here, we explore the energetics of the swelling and ion exchange reactions in montmorillonite using a potential of mean force approach and the thermodynamic integration method within molecular simulations. We investigate the influence of solution and clay composition on the spontaneity of the reactions, focusing on the 2 water-layer hydration state. The swelling simulations provide the equilibrium water content, interlayer water structure, and basal spacings, while thermodynamic integration of sodium-potassium exchange in the aqueous solution and solid phase are combined to calculate ion exchange free energies as a function of solution composition. Results confirm the tendency of the clay to collapse to lower hydration states as the concentration of the solution increases. Changes to the equilibrium water content, even at fixed hydration states, and the composition of the mixed electrolyte solution play a critical role in driving ion exchange and the selectivities of the clay to the exchanged cation, while the composition of the solid phase is shown to be insignificant. These findings underscore the extreme sensitivity of clay swelling and ion exchange thermodynamics to small (tenths of an Angstrom) deviations in layer spacing.
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Bentonita , Adsorção , Argila , Troca Iônica , TermodinâmicaRESUMO
Johnsongrass (Sorghum halepense) is a troublesome weed in row crop production in the United States. Herbicide resistance is a growing concern in this species, with resistance to ACCase-, ALS-, and EPSPS-inhibitors already reported. Pollen-mediated gene flow (PMGF) is capable of spreading herbicide resistance, but the extent of PMGF has not yet been studied in johnsongrass. Field experiments were conducted in a Nelder-wheel design to quantify the distance and frequency of PMGF from ALS-inhibitor-resistant (AR) to -susceptible (AS) johnsongrass across three environments (summer 2018, fall 2018, and fall 2019). The AR biotype (pollen donor) was established at the center of the wheel (5-m diameter), and a naturally occurring johnsongrass (AS) infestation was utilized as the pollen recipient, in eight directions and at nine distances (5, 10, 15, 20, 25, 35, 40, 45, and 50 m) within each direction. Seeds collected from the AS plants in each distance and direction were screened for survival to the ALS-inhibitor herbicide nicosulfuron (Accent Q) at 95 g ai ha-1 under greenhouse conditions. The survivors (i.e. hybrids) were further confirmed based on the presence of the Trp574Leu mutation. At the closest distance of 5 m, PMGF was 9.6-16.2% across the directions and environments, which progressively declined to 0.8-1.2% at 50 m. The exponential decay model predicted 50% reduction in PMGF at 2.2 m and 90% reduction at 5.8 m from the pollen donor block. Results demonstrate that herbicide resistance can spread between adjacent field populations of johnsongrass through PMGF, which necessitates sound monitoring and management.
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Herbicidas , Sorghum , Resistência a Herbicidas/genética , Herbicidas/farmacologia , Pólen/genética , Sorghum/genéticaRESUMO
BACKGROUND AND AIMS: The three-dimensional distributions of mineral elements in potato tubers provide insight into their mechanisms of transport and deposition. Many of these minerals are essential to a healthy human diet, and characterizing their distribution within the potato tuber will guide the effective utilization of this staple foodstuff. METHODS: The variation in mineral composition within the tuber was determined in three dimensions, after determining the orientation of the harvested tuber in the soil. The freeze-dried tuber samples were analysed for minerals using inductively coupled plasma-mass spectrometry (ICP-MS). Minerals measured included those of nutritional significance to the plant and to human consumers, such as iron, zinc, copper, calcium, magnesium, manganese, phosphorus, potassium and sulphur. KEY RESULTS: The concentrations of most minerals were higher in the skin than in the flesh of tubers. The potato skin contained about 17 % of total tuber zinc, 34 % of calcium and 55 % of iron. On a fresh weight basis, most minerals were higher in tuber flesh at the stem end than the bud end of the tuber. Potassium, however, displayed a gradient in the opposite direction. The concentrations of phosphorus, copper and calcium decreased from the periphery towards the centre of the tuber. CONCLUSIONS: The distribution of minerals varies greatly within the potato tuber. Low concentrations of some minerals relative to those in leaves may be due to their low mobility in phloem, whereas high concentrations in the skin may reflect direct uptake from the soil across the periderm. In tuber flesh, different minerals show distinct patterns of distribution in the tuber, several being consistent with phloem unloading in the tuber and limited onward movement. These findings have implications both for understanding directed transport of minerals in plants to stem-derived storage organs and for the dietary implications of different food preparation methods for potato tubers.
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Minerais/metabolismo , Tubérculos/metabolismo , Solanum tuberosum/metabolismo , Biomassa , HumanosRESUMO
Seed shattering refers to the natural shedding of seeds when they ripe, a phenomenon typically observed in wild and weedy plant species. The timing and extent of this phenomenon varies considerably among plant species. Seed shattering is primarily a genetically controlled trait; however, it is significantly influenced by environmental conditions, management practices and their interactions, especially in agro-ecosystems. This trait is undesirable in domesticated crops where consistent efforts have been made to minimize it through conventional and molecular breeding approaches. However, this evolutionary trait serves as an important fitness and survival mechanism for most weeds that utilize it to ensure efficient dispersal of their seeds, paving the way for persistent soil seedbank development and sustained future populations. Weeds have continuously evolved variations in seed shattering as an adaptation under changing management regimes. High seed retention is common in many cropping weeds where weed maturity coincides with crop harvest, facilitating seed dispersal through harvesting operations, though some weeds have notoriously high seed shattering before crop harvest. However, high seed retention in some of the most problematic agricultural weed species such as annual ryegrass (Lolium rigidum), wild radish (Raphanus raphanistrum), and weedy amaranths (Amaranthus spp.) provides an opportunity to implement innovative weed management approaches such as harvest weed seed control, which aims at capturing and destroying weed seeds retained at crop harvest. The integration of such management options with other practices is important to avoid the rapid evolution of high seed shattering in target weed species. Advances in genetics and molecular biology have shown promise for reducing seed shattering in important crops, which could be exploited for manipulating seed shattering in weed species. Future research should focus on developing a better understanding of various seed shattering mechanisms in plants in relation to changing climatic and management regimes.
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Penicillin-binding proteins are the primary targets for beta lactam drugs, which are main stay of treatment for Streptococcus pneumoniae. The emergence of increased penicillin resistance in meningeal isolates of S. pneumoniae in India is alarming. With this background, we aimed to analyze the pbp gene mutations of penicillin nonsusceptible pneumococcal (PNSP) isolates from within India and their association with international clones. A total of 32 PNSP invasive isolates with a penicillin minimal inhibitory concentrations (MIC) of ≥0.12 µg/mL were subjected to PCR and sequencing for multilocus sequence typing and the pbp genes (pbp2b, pbp2x, and pbp1a). The S. pneumoniae R6 susceptible strain was used as the reference for the comparison analyses. In the majority of the present study isolates, amino acid substitutions were only seen in one of the three active sites of one of the three pbp genes. Thus, pbp genes in the absence of the major substitutions usually associated with penicillin resistance combined with mosaicism in pbp1a resulted in a slight increase in the penicillin MIC to between 0.06 and 2.0 µg/mL, which according to meningeal break point denote resistance. Clonal analyses revealed that the emergence of PNSP in India is due to the gradual expansion of the resistant clones CC320, CC230, and CC63.
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Antibacterianos/farmacologia , Resistência às Penicilinas/genética , Proteínas de Ligação às Penicilinas/genética , Streptococcus pneumoniae/genética , Substituição de Aminoácidos , Criança , Genes Bacterianos/genética , Humanos , Índia/epidemiologia , Testes de Sensibilidade Microbiana , Tipagem de Sequências MultilocusRESUMO
Global climate change has increased the number of severe flooding events that affect agriculture, including rice production in the U.S. and internationally. Heavy rainfall can cause rice plants to be completely submerged, which can significantly affect grain yield or completely destroy the plants. Recently, a major effect submergence tolerance QTL during the vegetative stage, qSub8.1, which originated from Ciherang-Sub1, was identified in a mapping population derived from a cross between Ciherang-Sub1 and IR10F365. Ciherang-Sub1 was, in turn, derived from a cross between Ciherang and IR64-Sub1. Here, we characterize the qSub8.1 region by analyzing the sequence information of Ciherang-Sub1 and its two parents (Ciherang and IR64-Sub1) and compare the whole genome profile of these varieties with the Nipponbare and Minghui 63 (MH63) reference genomes. The three rice varieties were sequenced with 150 bp pair-end whole-genome shotgun sequencing (Illumina HiSeq4000), followed by performing the Trimmomatic-SOAPdenovo2-MUMmer3 pipeline for genome assembly, resulting in approximate genome sizes of 354.4, 343.7, and 344.7 Mb, with N50 values of 25.1, 25.4, and 26.1 kb, respectively. The results showed that the Ciherang-Sub1 genome is composed of 59-63% Ciherang, 22-24% of IR64-Sub1, and 15-17% of unknown sources. The genome profile revealed a more detailed genomic composition than previous marker-assisted breeding and showed that the qSub8.1 region is mostly from Ciherang, with some introgressed segments from IR64-Sub1 and currently unknown source(s).
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Plant height (PHT) in maize (Zea mays L.) has been scrutinized genetically and phenotypically due to relationship with other agronomically valuable traits (e.g., yield). Heritable variation of PHT is determined by many discovered quantitative trait loci; however, phenotypic effects of such loci often lack validation across environments and genetic backgrounds, especially in the hybrid state grown by farmers rather than the inbred state more often used by geneticists. A previous genome-wide association study using a topcrossed hybrid diversity panel identified two novel quantitative trait variants controlling both PHT and grain yield. Here, heterogeneous inbred families demonstrated that these two loci, characterized by two single nucleotide polymorphisms (SNPs), cause phenotypic variation in inbred lines, but that size of these effects were variable across four different genetic backgrounds, ranging from 1 to 10 cm. Weekly unoccupied aerial system flights demonstrated the two SNPs had larger effects, varying from 10 to 25 cm, in early growth while effects decreased toward the end of the season. These results show that allelic effect sizes of economically valuable loci are both dynamic in temporal growth and dynamic across genetic backgrounds, resulting in informative phenotypic variability overlooked following traditional phenotyping methods. Public genotyping data show recent favorable allele selection in elite temperate germplasm with little change across tropical backgrounds. As these loci remain rarer in tropical germplasm, with effects most visible early in growth, they are useful for breeding and selection to expand the genetic basis of maize.
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Estudo de Associação Genômica Ampla , Zea mays , Zea mays/genética , Melhoramento Vegetal , Locos de Características Quantitativas , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
A novel space-filling trialkoxysilane derivative was synthesized using a two-step strategy from commercially available starting materials to produce the precursor for the formation of low-density self-assembled monolayers. Self-assembled monolayers of the synthesized compound were prepared on three different substrates (Si/SiO(2), glass and ITO) and were characterized using contact angle, ellipsometry and sum-frequency generation spectroscopy. Removal of the space-filling protecting group, (2-chlorophenyl)diphenyl methanol, yields a carboxy-terminated surface. Correspondingly, the contact angle and film thickness decrease and the SFG spectra clearly indicate an increase in gauche defect concentration characteristic of a low-density disordered monolayer.
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The ability of reverse transcriptases (RTs) to synthesize a complementary DNA from natural RNA and a range of unnatural xeno nucleic acid (XNA) template chemistries, underpins key methods in molecular and synthetic genetics. However, RTs have proven challenging to discover and engineer, in particular for the more divergent XNA chemistries. Here we describe a general strategy for the directed evolution of RT function for any template chemistry called compartmentalized bead labelling and demonstrate it by the directed evolution of efficient RTs for 2'-O-methyl RNA and hexitol nucleic acids and the discovery of RTs for the orphan XNA chemistries D-altritol nucleic acid and 2'-methoxyethyl RNA, for which previously no RTs existed. Finally, we describe the engineering of XNA RTs with active exonucleolytic proofreading as well as the directed evolution of RNA RTs with very high complementary DNA synthesis fidelities, even in the absence of proofreading.
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Evolução Molecular , DNA Polimerase Dirigida por RNA/metabolismo , RNA/metabolismo , Biblioteca Gênica , Vírus da Leucemia Murina/enzimologia , Mutagênese Sítio-Dirigida , Técnicas de Amplificação de Ácido Nucleico , DNA Polimerase Dirigida por RNA/genéticaRESUMO
Background: Pneumococcal pneumonia is one of the major causes of mortality in children less than 5 years in Asia, especially in India. Available PCVs have less serotype coverage in India compared to western countries. Moreover, the baseline pneumococcal serotype and sequence type data is limited and available data doesn't represent the entire India. With this background we aimed to characterize invasive and carriage isolates of S. pneumoniae from a tertiary care hospital in South India. Materials and Methods: A total of 221 S. pneumoniae isolates, invasive (n=138) and carriage (n=83) between the time period of 2012-2018 were included. Isolates was identified and confirmed using standard laboratory protocols. Serotyping was performed by Customized sequential multiplex PCR and MLST as described in www.pubmlst.org. Results: The major serotypes were 19F, 6B, 14, 6A and 19A and the sequence types (ST) were ST63, 236 and 230. Predominant STs in invasive was ST 63 whereas in carriage were ST4894 and 1701. High level ST diversity in carriage was observed. Majority of the STs were SLVs or DLVs of previously reported STs or PMEN clones. Phylogenetic analyses of the STs revealed gradual expansion of three PMEN CCs CC320, 63 and 230. Conclusion: The vaccine serotypes were the predominant ones found to be associated with IPD, PMEN clones, new STs and antimicrobial resistance. Accordingly, PCV13 is expected to provide invasive serotype coverage of 75% in Indian children less than 5 years. This study provides baseline serotype and sequence type data prior to the introduction of PCV in South India.
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Sorotipagem/métodos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação , Pré-Escolar , Feminino , Humanos , Índia , Masculino , Tipagem de Sequências Multilocus , Vacinas Pneumocócicas , Streptococcus pneumoniae/imunologiaRESUMO
Diphtheria is a dreadful disease caused by Corynebacterium diphtheriae. Lysogenised bacteriophages carrying toxin gene in C. diphtheriae can make the strain toxigenic. However, such phage disseminates the toxin genes to other strains when it undergoes lytic phase. As little is known about the phage diversity in C. diphtheriae in India, the present study was undertaken to investigate the prophages integrated into the genome of 29 clinical isolates of C. diphtheriae using whole-genome shotgun sequencing. Amongst these isolates, 27 were toxigenic, while 2 were non-toxigenic strains. Of the 27 toxigenic strains, all harbored known phages carrying toxin gene and two other phages with unknown function. However, the two non-toxin strains did not harbour any of the phages in the genome. It is imperative to devise prevention strategies that hinder the dissemination of toxin by prophages, as it may increase the complications of diphtheria post-immunisation.