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1.
New Phytol ; 241(3): 1144-1160, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38072860

RESUMO

Chlorella ohadii was isolated from desert biological soil crusts, one of the harshest habitats on Earth, and is emerging as an exciting new green model for studying growth, photosynthesis and metabolism under a wide range of conditions. Here, we compared the genome of C. ohadii, the fastest growing alga on record, to that of other green algae, to reveal the genomic imprints empowering its unparalleled growth rate and resistance to various stressors, including extreme illumination. This included the genome of its close relative, but slower growing and photodamage sensitive, C. sorokiniana UTEX 1663. A larger number of ribosome-encoding genes, high intron abundance, increased codon bias and unique genes potentially involved in metabolic flexibility and resistance to photodamage are all consistent with the faster growth of C. ohadii. Some of these characteristics highlight general trends in Chlorophyta and Chlorella spp. evolution, and others open new broad avenues for mechanistic exploration of their relationship with growth. This work entails a unique case study for the genomic adaptations and costs of exceptionally fast growth and sheds light on the genomic signatures of fast growth in photosynthetic cells. It also provides an important resource for future studies leveraging the unique properties of C. ohadii for photosynthesis and stress response research alongside their utilization for synthetic biology and biotechnology aims.


Assuntos
Chlorella , Chlorella/genética , Fotossíntese , Genômica
2.
Mol Genet Genomics ; 297(4): 925-933, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35488049

RESUMO

Complex chromosomal rearrangements (CCRs), a class of structural variants (SVs) involving more than two chromosome breaks, were classically thought to be extremely rare. As advanced technologies become more available, it has become apparent that CCRs are more common than formerly thought, and are a substantial cause of genetic disorders. We attempted a novel approach for solving the mechanism of challenging CCRs, which involve repetitive sequences, by precisely identifying sequence-level changes and their order. Chromosomal microarray (CMA) and FISH analyses were used for interpretation of SVs detected by whole exome sequencing (WES). Breakpoint junctions were analyzed by Nanopore sequencing, a novel long-read whole genome sequencing tool. A large deletion identified by WES, encompassing the FOXF1 enhancer, was the cause of alveolar capillary dysplasia and respiratory insufficiency, resulting in perinatal death. CMA analysis of the newborn's mother revealed two duplications encompassing the deleted region in the proband, raising our hypothesis that the deletion resulted from the mother's CCR. Breakpoint junctions of complex SVs were determined at the nucleotide level using Nanopore long-read sequencing. According to sequencing results of breakpoint junctions, the CCR in the newborn was considered the consequence of at least one double-strand break during meiosis, and reassembly of DNA fragments by intra-chromosomal homologous recombination. Our comprehensive approach, combining cytogenetics and long-read sequencing, enabled delineation of the exact breakpoints in a challenging CCR, and proposal of a mechanism in which it arises. We suggest applying our integrative approach combining technologies for deciphering future challenging CCRs, enabling risk assessment in families.


Assuntos
Aberrações Cromossômicas , Genoma , Cromossomos , Análise Citogenética , Feminino , Genômica , Humanos , Gravidez
3.
Am J Med Genet A ; 188(5): 1420-1425, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35075769

RESUMO

Variants involving TBX4 are associated with a wide variety of disorders, including pulmonary arterial hypertension, ischiocoxopodopatellar syndrome (ICPPS)/small patella syndrome (SPS), lethal lung developmental disorders (LLDDs) in neonates, heart defects, and prenatally lethal posterior amelia with pelvic and pulmonary hypoplasia syndrome. The objective of our study was to elucidate the wide variable phenotypic expressivity and incomplete penetrance in a three-generation family with a truncating variant in TBX4. In addition to exome and genome sequencing analyses, a candidate noncoding regulatory single nucleotide variant (SNV) within the lung-specific TBX4 enhancer was functionally tested using an in vitro luciferase reporter assay. A heterozygous frameshift variant c.1112dup (p.Pro372Serfs*14) in TBX4 was identified in patients with mild interstitial lung disease (1), bronchiolitis obliterans (1), recurrent pneumothorax (1), ICPPS/SPS (1), LLDD (2), and in unaffected individuals (4). In two deceased neonates with LLDD, we identified a noncoding SNV rs62069651-C located in trans to the mutated TBX4 allele that reduced the TBX4 promoter activity by 63% in the reporter assay. Our findings provide a functional evidence for the recently reported model of complex compound inheritance in which both TBX4 coding and in trans noncoding hypomorphic variants in the lung-specific enhancer of TBX4 contribute to LLDD.


Assuntos
Pneumopatias , Anormalidades do Sistema Respiratório , Doenças do Desenvolvimento Ósseo , Quadril/anormalidades , Humanos , Recém-Nascido , Ísquio/anormalidades , Pulmão/anormalidades , Pneumopatias/genética , Patela/anormalidades , Proteínas com Domínio T/genética
4.
Plant J ; 101(6): 1269-1286, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31657869

RESUMO

Mitochondria serve as major sites of ATP production and play key roles in many other metabolic processes that are critical to the cell. As relicts of an ancient bacterial endosymbiont, mitochondria contain their own hereditary material (i.e. mtDNA, or mitogenome) and a machinery for protein biosynthesis. The expression of the mtDNA in plants is complex, particularly at the post-transcriptional level. Following transcription, the polycistronic pre-RNAs undergo extensive modifications, including trimming, splicing and editing, before being translated by organellar ribosomes. Our study focuses on N6 -methylation of adenosine ribonucleotides (m6 A-RNA) in plant mitochondria. m6 A is a prevalent modification in nuclear-encoded mRNAs. The biological significance of this dynamic modification is under investigation, but it is widely accepted that m6 A mediates structural switches that affect RNA stability and/or activity. Using m6 A-pulldown/RNA-seq (m6 A-RIP-seq) assays of Arabidopsis and cauliflower mitochondria, we provide information on the m6 A-RNA landscapes in Arabidopsis thaliana and Brassica oleracea mitochondria. The results show that m6 A targets different types of mitochondrial transcripts, including known genes, mtORFs, as well as non-coding (transcribed intergenic) RNA species. While ncRNAs undergo multiple m6 A modifications, N6 -methylation of adenosine residues with mRNAs seem preferably positioned near start codons and may modulate their translatability.


Assuntos
Adenosina/metabolismo , Expressão Gênica , Mitocôndrias/metabolismo , Organelas/metabolismo , Plantas/metabolismo , Arabidopsis/metabolismo , Brassica/metabolismo , Regulação da Expressão Gênica de Plantas , Metilação
5.
New Phytol ; 229(6): 3208-3220, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33533496

RESUMO

In multicellular organisms, Polycomb Repressive Complex2 (PRC2) is known to deposit tri-methylation of lysine 27 of histone H3 (H3K27me3) to establish and maintain gene silencing, critical for developmentally regulated processes. The PRC2 complex is absent in both widely studied model yeasts, which initially suggested that PRC2 arose with the emergence of multicellularity. However, its discovery in several unicellular species including microalgae questions its role in unicellular eukaryotes. Here, we use Phaeodactylum tricornutum enhancer of zeste E(z) knockouts and show that P. tricornutum E(z) is responsible for di- and tri-methylation of lysine 27 of histone H3. H3K27me3 depletion abolishes cell morphology in P. tricornutum providing evidence for its role in cell differentiation. Genome-wide profiling of H3K27me3 in fusiform and triradiate cells further revealed genes that may specify cell identity. These results suggest a role for PRC2 and its associated mark in cell differentiation in unicellular species, and highlight their ancestral function in a broader evolutionary context than currently is appreciated.


Assuntos
Histonas , Complexo Repressor Polycomb 2 , Diferenciação Celular/genética , Histonas/metabolismo , Metilação , Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb
6.
Nature ; 524(7565): 366-9, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26168400

RESUMO

Diatoms are one of the most ecologically successful classes of photosynthetic marine eukaryotes in the contemporary oceans. Over the past 30 million years, they have helped to moderate Earth's climate by absorbing carbon dioxide from the atmosphere, sequestering it via the biological carbon pump and ultimately burying organic carbon in the lithosphere. The proportion of planetary primary production by diatoms in the modern oceans is roughly equivalent to that of terrestrial rainforests. In photosynthesis, the efficient conversion of carbon dioxide into organic matter requires a tight control of the ATP/NADPH ratio which, in other photosynthetic organisms, relies principally on a range of plastid-localized ATP generating processes. Here we show that diatoms regulate ATP/NADPH through extensive energetic exchanges between plastids and mitochondria. This interaction comprises the re-routing of reducing power generated in the plastid towards mitochondria and the import of mitochondrial ATP into the plastid, and is mandatory for optimized carbon fixation and growth. We propose that the process may have contributed to the ecological success of diatoms in the ocean.


Assuntos
Organismos Aquáticos/metabolismo , Dióxido de Carbono/metabolismo , Diatomáceas/citologia , Diatomáceas/metabolismo , Mitocôndrias/metabolismo , Fotossíntese , Plastídeos/metabolismo , Força Próton-Motriz , Trifosfato de Adenosina/metabolismo , Organismos Aquáticos/citologia , Organismos Aquáticos/enzimologia , Organismos Aquáticos/genética , Ciclo do Carbono , Diatomáceas/enzimologia , Diatomáceas/genética , Ecossistema , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/metabolismo , NADP/metabolismo , Oceanos e Mares , Oxirredução , Oxirredutases/deficiência , Oxirredutases/metabolismo , Fenótipo , Proteínas de Plantas/metabolismo
7.
Environ Microbiol ; 21(3): 1140-1150, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30761715

RESUMO

Toxic Microcystis spp. blooms constitute a serious threat to water quality worldwide. Aeromonas veronii was isolated from Microcystis sp. colonies collected in Lake Kinneret. Spent Aeromonas media inhibits the growth of Microcystis aeruginosa MGK isolated from Lake Kinneret. The inhibition was much stronger when Aeromonas growth medium contained spent media from MGK suggesting that Aeromonas recognized its presence and produced secondary metabolites that inhibit Microcystis growth. Fractionations of the crude extract and analyses of the active fractions identified several secondary metabolites including lumichrome in Aeromonas media. Application of lumichrome at concentrations as low as 4 nM severely inhibited Microcystis growth. Inactivation of aviH in the lumichrome biosynthetic pathway altered the lumichrome level in Aeromonas and the extent of MGK growth inhibition. Conversely, the initial lag in Aeromonas growth was significantly longer when provided with Microcystis spent media but Aeromonas was able to resume normal growth. The longer was pre-exposure to Microcystis spent media the shorter was the lag phase in Aeromonas growth indicating the presence of, and acclimation to, secondary MGK metabolite(s) the nature of which was not revealed. Our study may help to control toxic Microcystis blooms taking advantage of chemical languages used in the interspecies communication.


Assuntos
Aeromonas veronii/fisiologia , Microcystis/fisiologia , Aeromonas/fisiologia , Antibiose/fisiologia , Meios de Cultura , Lagos/microbiologia , Microcystis/metabolismo
8.
New Phytol ; 221(3): 1303-1316, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30216452

RESUMO

Diatom dominance in contemporary aquatic environments indicates that they have developed unique and effective mechanisms to cope with the rapid and considerable fluctuations that characterize these environments. In view of their evolutionary history from a secondary endosymbiosis, inter-organellar regulation of biochemical activities may be of particular relevance. Diatom mitochondrial alternative oxidase (AOX) is believed to play a significant role in supplying chloroplasts with ATP produced in the mitochondria. Using the model diatom Phaeodactylum tricornutum we generated AOX knockdown lines, and followed sensitivity to stressors, photosynthesis and transcriptome and metabolome profiles of wild-type and knockdown lines. We show here that expression of the AOX gene is upregulated by various stresses including H2 O2 , heat, high light illumination, and iron or nitrogen limitation. AOX knockdown results in hypersensitivity to stress. Knockdown lines also show significantly reduced photosynthetic rates and their chloroplasts are more oxidized. Comparisons of transcriptome and metabolome profiles suggest a strong impact of AOX activity on gene expression, which is carried through to the level of the metabolome. Our data provide evidence for the involvement of mitochondrial AOX in processes central to the cell biology of diatoms, revealing that cross-talk between mitochondria and chloroplasts is crucial for maintaining sensitivity to changing environments.


Assuntos
Organismos Aquáticos/enzimologia , Cloroplastos/metabolismo , Diatomáceas/enzimologia , Diatomáceas/fisiologia , Regulação para Baixo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Estresse Fisiológico , Antioxidantes/metabolismo , Organismos Aquáticos/fisiologia , Glutationa/metabolismo , Metabolômica , Oxirredução , Fotossíntese , Transcriptoma/genética
9.
Mol Ecol ; 28(9): 2305-2320, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31025457

RESUMO

Cyanobacteria inhabiting desert biological soil crusts must prepare towards dehydration, or their revival after rewetting is severely impaired. The mechanisms involved are unknown but signalling of forthcoming dehydration by dawn illumination was demonstrated. Accurate and reproducible simulation of desert conditions enabled examination of physiological activities and transcript profiles in a model organism, Leptolyngbya ohadii, in response to specific conditions. Exposure to far red light or lack of ground warming during dawn severely reduced revival after rewetting and altered the network of gene expression. The data implicated phytochromes in light and temperature sensing. Many genes were up- or down-regulated before water content decline, while others were strongly affected by the progression of dehydration and desiccation. Transcription continues during the desiccated phase but only barely during early rewetting, although photosynthetic activity was regained. Application of rifampicin with or without a preceding dehydration phase demonstrated that RNA is stabilized/protected during desiccation, possibly by intrinsically disordered proteins. We conclude that increasing light and temperature at dawn activates a network of genes that prepare the cells towards dehydration. Quick resumption of photosynthesis upon rewetting in contrast to the slow change in the transcript profile suggested that in addition to preparing towards dehydration the cells also prepare for forthcoming rewetting, during dehydration. Unravelling the presently unknown function of many responding genes will help to clarify the networks involved.


Assuntos
Cianobactérias/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cianobactérias/efeitos dos fármacos , Desidratação , Clima Desértico , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Luz , Fotossíntese/fisiologia , Rifampina/farmacologia , Microbiologia do Solo , Temperatura , Trealose , Água
10.
Environ Microbiol ; 19(2): 535-550, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27501380

RESUMO

Filamentous cyanobacteria are the main founders and primary producers in biological desert soil crusts (BSCs) and are likely equipped to cope with one of the harshest environmental conditions on earth including daily hydration/dehydration cycles, high irradiance and extreme temperatures. Here, we resolved and report on the genome sequence of Leptolyngbya ohadii, an important constituent of the BSC. Comparative genomics identified a set of genes present in desiccation-tolerant but not in dehydration-sensitive cyanobacteria. RT qPCR analyses showed that the transcript abundance of many of them is upregulated during desiccation in L. ohadii. In addition, we identified genes where the orthologs detected in desiccation-tolerant cyanobacteria differs substantially from that found in desiccation-sensitive cells. We present two examples, treS and fbpA (encoding trehalose synthase and fructose 1,6-bisphosphate aldolase respectively) where, in addition to the orthologs present in the desiccation-sensitive strains, the resistant cyanobacteria also possess genes with different predicted structures. We show that in both cases the two orthologs are transcribed during controlled dehydration of L. ohadii and discuss the genetic basis for the acclimation of cyanobacteria to the desiccation conditions in desert BSC.


Assuntos
Cianobactérias/genética , Cianobactérias/fisiologia , Genoma Bacteriano , Microbiologia do Solo , Solo/química , Água , Aclimatação , Desidratação , Clima Desértico , Regulação Bacteriana da Expressão Gênica , Fotossíntese
11.
BMC Bioinformatics ; 17(1): 261, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-27363443

RESUMO

BACKGROUND: With the emerging interest in phytoplankton research, the need to establish genetic tools for the functional characterization of genes is indispensable. The CRISPR/Cas9 system is now well recognized as an efficient and accurate reverse genetic tool for genome editing. Several computational tools have been published allowing researchers to find candidate target sequences for the engineering of the CRISPR vectors, while searching possible off-targets for the predicted candidates. These tools provide built-in genome databases of common model organisms that are used for CRISPR target prediction. Although their predictions are highly sensitive, the applicability to non-model genomes, most notably protists, makes their design inadequate. This motivated us to design a new CRISPR target finding tool, PhytoCRISP-Ex. Our software offers CRIPSR target predictions using an extended list of phytoplankton genomes and also delivers a user-friendly standalone application that can be used for any genome. RESULTS: The software attempts to integrate, for the first time, most available phytoplankton genomes information and provide a web-based platform for Cas9 target prediction within them with high sensitivity. By offering a standalone version, PhytoCRISP-Ex maintains an independence to be used with any organism and widens its applicability in high throughput pipelines. PhytoCRISP-Ex out pars all the existing tools by computing the availability of restriction sites over the most probable Cas9 cleavage sites, which can be ideal for mutant screens. CONCLUSIONS: PhytoCRISP-Ex is a simple, fast and accurate web interface with 13 pre-indexed and presently updating phytoplankton genomes. The software was also designed as a UNIX-based standalone application that allows the user to search for target sequences in the genomes of a variety of other species.


Assuntos
Sistemas CRISPR-Cas , Genoma Humano , Internet , Humanos
12.
New Phytol ; 210(4): 1229-43, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26853530

RESUMO

Excess illumination damages the photosynthetic apparatus with severe implications with regard to plant productivity. Unlike model organisms, the growth of Chlorella ohadii, isolated from desert soil crust, remains unchanged and photosynthetic O2 evolution increases, even when exposed to irradiation twice that of maximal sunlight. Spectroscopic, biochemical and molecular approaches were applied to uncover the mechanisms involved. D1 protein in photosystem II (PSII) is barely degraded, even when exposed to antibiotics that prevent its replenishment. Measurements of various PSII parameters indicate that this complex functions differently from that in model organisms and suggest that C. ohadii activates a nonradiative electron recombination route which minimizes singlet oxygen formation and the resulting photoinhibition. The light-harvesting antenna is very small and carotene composition is hardly affected by excess illumination. Instead of succumbing to photodamage, C. ohadii activates additional means to dissipate excess light energy. It undergoes major structural, compositional and physiological changes, leading to a large rise in photosynthetic rate, lipids and carbohydrate content and inorganic carbon cycling. The ability of C. ohadii to avoid photodamage relies on a modified function of PSII and the dissipation of excess reductants downstream of the photosynthetic reaction centers. The biotechnological potential as a gene source for crop plant improvement is self-evident.


Assuntos
Chlorella/fisiologia , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Chlorella/efeitos da radiação , Clima Desértico , Solo , Estresse Fisiológico , Luz Solar
13.
New Phytol ; 202(2): 471-484, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24345283

RESUMO

Chlamydomonas reinhardtii tolerates relatively high H2 O2 levels that induce an array of antioxidant activities. However, rather than rendering the cells more resistant to oxidative stress, the cells become far more sensitive to an additional H2 O2 dose. If H2 O2 is provided 1.5-9 h after an initial dose, it induces programmed cell death (PCD) in the wild-type, but not in the dum1 mutant impaired in the mitochondrial respiratory complex III. This mutant does not exhibit a secondary oxidative burst 4-5 h after the inducing H2 O2 , nor does it activate metacaspase-1 after the second H2 O2 treatment. The intracellular dehydroascorbate level, a product of ascorbate peroxidase, increases under conditions leading to PCD. The addition of dehydroascorbate induces PCD in the wild-type and dum1 cultures, but higher levels are required in dum1 cells, where it is metabolized faster. The application of dehydroascorbate induces the expression of metacaspase-2, which is much stronger than the expression of metacaspase-1. The presence or absence of oxidative stress, in addition to the rise in internal dehydroascorbate, may determine which metacaspase is activated during Chlamydomonas PCD. Cell death is strongly affected by the timing of H2 O2 or dehydroascorbate admission to synchronously grown cultures, suggesting that the cell cycle phase may distinguish cells that perish from those that do not.


Assuntos
Apoptose , Chlamydomonas reinhardtii/metabolismo , Ácido Desidroascórbico/metabolismo , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Ascorbato Peroxidases/metabolismo , Caspases/metabolismo , Ciclo Celular , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/fisiologia , Ácido Desidroascórbico/farmacologia , Peróxido de Hidrogênio/farmacologia , Mutação , Oxirredução
14.
Microbiol Spectr ; 11(3): e0389522, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37195188

RESUMO

As a result of the increasing use of sensitive nucleic acid amplification tests, Kingella kingae is being recognized as a common pathogen of early childhood, causing medical conditions ranging from asymptomatic oropharyngeal colonization to bacteremia, osteoarthritis, and life-threatening endocarditis. However, the genomic determinants associated with the different clinical outcomes are unknown. Employing whole-genome sequencing, we studied 125 international K. kingae isolates derived from 23 healthy carriers and 102 patients with invasive infections, including bacteremia (n = 23), osteoarthritis (n = 61), and endocarditis (n = 18). We compared their genomic structures and contents to identify genomic determinants associated with the different clinical conditions. The mean genome size of the strains was 2,024,228 bp, and the pangenome comprised 4,026 predicted genes, of which 1,460 (36.3%) were core genes shared by >99% of the isolates. No single gene discriminated between carried and invasive strains; however, 43 genes were significantly more frequent in invasive isolates, compared to asymptomatically carried organisms, and a few showed a significant differential distribution among isolates from skeletal system infections, bacteremia, and endocarditis. The gene encoding the iron-regulated protein FrpC was uniformly absent in all 18 endocarditis-associated strains but was present in one-third of other invasive isolates. Similar to other members of the Neisseriaceae family, the K. kingae differences in invasiveness and tropism for specific body tissues appear to depend on combinations of multiple virulence-associated determinants that are widely distributed throughout the genome. The potential role of the absence of the FrpC protein in the pathogenesis of endocardial invasion deserves further investigation. IMPORTANCE The wide range of clinical severities exhibited by invasive Kingella kingae infections strongly suggests that isolates differ in their genomic contents, and strains associated with life-threatening endocarditis may harbor distinct genomic determinants that result in cardiac tropism and severe tissue damage. The results of the present study show that no single gene discriminated between asymptomatically carried isolates and invasive strains. However, 43 putative genes were significantly more frequent among invasive isolates than among pharyngeal colonizers. In addition, several genes displayed a significant differential distribution among isolates from bacteremia, skeletal system infections, and endocarditis, suggesting that the virulence and tissue tropism of K. kingae are multifactorial and polygenic, depending on changes in the allele content and genomic organization. Further analysis of these putative genes may identify genomic determinants of the invasiveness of K. kingae and its affinity for specific body tissues and potential targets for a future protective vaccine.


Assuntos
Bacteriemia , Endocardite , Kingella kingae , Humanos , Pré-Escolar , Kingella kingae/genética , Virulência/genética , Fatores de Virulência/genética , Bacteriemia/patologia
15.
Sci Rep ; 13(1): 18036, 2023 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-37865712

RESUMO

Recent advances in genomic technologies expand the scope and efficiency of preimplantation genetic testing (PGT). We previously developed Haploseek, a clinically-validated, variant-agnostic comprehensive PGT solution. Haploseek is based on microarray genotyping of the embryo's parents and relatives, combined with low-pass sequencing of the embryos. Here, to increase throughput and versatility, we aimed to develop a sequencing-only implementation of Haploseek. Accordingly, we developed SHaploseek, a universal PGT method to determine genome-wide haplotypes of each embryo based on low-pass (≤ 5x) sequencing of the parents and relative(s) along with ultra-low-pass (0.2-0.4x) sequencing of the embryos. We used SHaploseek to analyze five single lymphoblast cells and 31 embryos. We validated the genome-wide haplotype predictions against either bulk DNA, Haploseek, or, at focal genomic sites, PCR-based PGT results. SHaploseek achieved > 99% concordance with bulk DNA in two families from which single cells were derived from grown-up children. In embryos from 12 PGT families, all of SHaploseek's focal site haplotype predictions were concordant with clinical PCR-based PGT results. Genome-wide, there was > 99% median concordance between Haploseek and SHaploseek's haplotype predictions. Concordance remained high at all assayed sequencing depths ≥ 2x, as well as with only 1ng of parental DNA input. In subtelomeric regions, significantly more haplotype predictions were high-confidence in SHaploseek compared to Haploseek. In summary, SHaploseek constitutes a single-platform, accurate, and cost-effective comprehensive PGT solution.


Assuntos
Diagnóstico Pré-Implantação , Gravidez , Feminino , Criança , Humanos , Diagnóstico Pré-Implantação/métodos , Testes Genéticos/métodos , Haplótipos , Embrião de Mamíferos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA , Aneuploidia , Blastocisto
16.
Front Med (Lausanne) ; 10: 1292665, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38020140

RESUMO

Coinfection of HIV and multidrug-resistant tuberculosis (MDR-TB) presents significant challenges in terms of the treatment and prognosis of tuberculosis, leading to complexities in managing the disease and impacting the overall outcome for TB patients. This study presents a remarkable case of a patient with MDR-TB and HIV coinfection who survived for over 8 years, despite poor treatment adherence and comorbidities. Whole genome sequencing (WGS) of the infecting Mycobacterium tuberculosis (Mtb) strain revealed a unique genomic deletion, spanning 18 genes, including key genes involved in hypoxia response, intracellular survival, immunodominant antigens, and dormancy. This deletion, that we have called "Del-X," potentially exerts a profound influence on the bacterial physiology and its virulence. Only few similar deletions were detected in other non-related Mtb genomes worldwide. In vivo evolution analysis identified drug resistance and metabolic adaptation mutations and their temporal dynamics during the patient's treatment course.

17.
Sci Rep ; 11(1): 22372, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34785703

RESUMO

More than 900 variants have been described in the GLA gene. Some intronic variants and copy number variants in GLA can cause Fabry disease but will not be detected by classical Sanger sequence. We aimed to design and validate a method for sequencing the GLA gene using long-read Oxford Nanopore sequencing technology. Twelve Fabry patients were blindly analyzed, both by conventional Sanger sequence and by long-read sequencing of a 13 kb PCR amplicon. We used minimap2 to align the long-read data and Nanopolish and Sniffles to call variants. All the variants detected by Sanger (including a deep intronic variant) were also detected by long-read sequencing. One patient had a deletion that was not detected by Sanger sequencing but was detected by the new technology. Our long-read sequencing-based method was able to detect missense variants and an exonic deletion, with the added advantage of intronic analysis. It can be used as an efficient and cost-effective tool for screening and diagnosing Fabry disease.


Assuntos
Sequência de Bases , Doença de Fabry/genética , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Deleção de Sequência , alfa-Galactosidase/genética , Adulto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade
18.
Nat Commun ; 12(1): 4802, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376665

RESUMO

During meiosis, gene expression is silenced in aberrantly unsynapsed chromatin and in heterogametic sex chromosomes. Initiation of sex chromosome silencing is disrupted in meiocytes with sex chromosome-autosome translocations. To determine whether this is due to aberrant synapsis or loss of continuity of sex chromosomes, we engineered Caenorhabditis elegans nematodes with non-translocated, bisected X chromosomes. In early meiocytes of mutant males and hermaphrodites, X segments are enriched with euchromatin assembly markers and active RNA polymerase II staining, indicating active transcription. Analysis of RNA-seq data showed that genes from the X chromosome are upregulated in gonads of mutant worms. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, our data indicate that unsynapsed X segments are transcribed. Therefore, our results suggest that sex chromosome chromatin has a unique character that facilitates its meiotic expression when its continuity is lost, regardless of whether or not it is synapsed.


Assuntos
Caenorhabditis elegans/genética , Inativação Gênica , Meiose/genética , Cromossomo X/genética , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Pareamento Cromossômico/genética , Feminino , Células Germinativas/citologia , Células Germinativas/metabolismo , Histonas/metabolismo , Hibridização in Situ Fluorescente , Masculino , Microscopia de Fluorescência , Transcrição Gênica
19.
Sci Adv ; 7(34)2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34407941

RESUMO

Desiccation-tolerant cyanobacteria can survive frequent hydration/dehydration cycles likely affecting inorganic carbon (Ci) levels. It was recently shown that red/far-red light serves as signal-preparing cells toward dehydration. Here, the effects of desiccation on Ci assimilation by Leptolyngbya ohadii isolated from Israel's Negev desert were investigated. Metabolomic investigations indicated a decline in ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation activity, and this was accelerated by far-red light. Far-red light negatively affected the Ci affinity of L. ohadii during desiccation and in liquid cultures. Similar effects were evident in the non-desiccation-tolerant cyanobacterium Synechocystis The Synechocystis Δcph1 mutant lacking the major phytochrome exhibited reduced photosynthetic Ci affinity when exposed to far-red light, whereas the mutant ΔsbtB lacking a Ci uptake inhibitory protein lost the far-red light inhibition. Collectively, these results suggest that red/far-red light perception likely via phytochromes regulates Ci uptake by cyanobacteria and that this mechanism contributes to desiccation tolerance in strains such as L. ohadii.

20.
FEMS Microbiol Rev ; 45(6)2021 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-34165541

RESUMO

Biological soil crusts (BSCs) are found in drylands, cover ∼12% of the Earth's surface in arid and semi-arid lands and their destruction is considered an important promoter of desertification. These crusts are formed by the adhesion of soil particles to polysaccharides excreted mostly by filamentous cyanobacteria, which are the pioneers and main primary producers in BSCs. Desert BSCs survive in one of the harshest environments on Earth, and are exposed to daily fluctuations of extreme conditions. The cyanobacteria inhabiting these habitats must precisely read the changing conditions and predict, for example, the forthcoming desiccation. Moreover, they evolved a comprehensive regulation of multiple adaptation strategies to enhance their stress tolerance. Here, we focus on what distinguishes cyanobacteria able to revive after dehydration from those that cannot. While important progress has been made in our understanding of physiological, biochemical and omics aspects, clarification of the sensing, signal transduction and responses enabling desiccation tolerance are just emerging. We plot the trajectory of current research and open questions ranging from general strategies and regulatory adaptations in the hydration/desiccation cycle, to recent advances in our understanding of photosynthetic adaptation. The acquired knowledge provides new insights to mitigate desertification and improve plant productivity under drought conditions.


Assuntos
Cianobactérias , Solo , Clima Desértico , Ecossistema , Leitura , Microbiologia do Solo
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