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1.
Braz J Biol ; 83: e246520, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34468518

RESUMO

Chitin and its derived products have immense economic value due to their vital role in various biological activities as well as biomedical and industrial application. Insects, microorganism and crustaceans are the main supply of chitin but the crustaceans shell like shrimp, krill, lobsters and crabs are the main commercial sources. Chitin content of an individual varies depending on the structures possessing the polymer and the species. In this study edible crabs' shells (Callinectes sapidus) were demineralized and deproteinized resulting in 13.8% (dry weight) chitin recovery from chitin wastes. FTIR and XRD analyses of the experimental crude as well as purified chitins revealed that both were much comparable to the commercially purchased controls. The acid pretreatment ceded 54g of colloidal chitin that resulted in 1080% of the crude chitin. The colloidal chitin was exploited for isolation of eighty five chitinolytic bacterial isolates from different sources. Zone of clearance was displayed by the thirty five isolates (41.17%) succeeding their growth at pH 7 on colloidal chitin agar medium. Maximum chitinolytic activity i.e. 301.55 U/ml was exhibited by isolate JF70 when cultivated in extracted chitin containing both carbon and nitrogen. The study showed wastes of blue crabs can be utilized for extraction of chitin and isolation of chitinolytic bacteria that can be used to degrade chitin waste, resolve environmental pollution as well as industrial purpose.


Assuntos
Braquiúros , Decápodes , Exoesqueleto , Animais , Bactérias , Quitina , Meios de Cultura
2.
J Med Microbiol ; 70(9)2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34486972

RESUMO

Introduction. Non-invasive sample collection and viral sterilizing buffers have independently enabled workflows for more widespread COVID-19 testing by reverse-transcriptase polymerase chain reaction (RT-PCR).Gap statement. The combined use of sterilizing buffers across non-invasive sample types to optimize sensitive, accessible, and biosafe sampling methods has not been directly and systematically compared.Aim. We aimed to evaluate diagnostic yield across different non-invasive samples with standard viral transport media (VTM) versus a sterilizing buffer eNAT- (Copan diagnostics Murrieta, CA) in a point-of-care diagnostic assay system.Methods. We prospectively collected 84 sets of nasal swabs, oral swabs, and saliva, from 52 COVID-19 RT-PCR-confirmed patients, and nasopharyngeal (NP) swabs from 37 patients. Nasal swabs, oral swabs, and saliva were placed in either VTM or eNAT, prior to testing with the Xpert Xpress SARS-CoV-2 (Xpert). The sensitivity of each sampling strategy was compared using a composite positive standard.Results. Swab specimens collected in eNAT showed an overall superior sensitivity compared to swabs in VTM (70 % vs 57 %, P=0.0022). Direct saliva 90.5 %, (95 % CI: 82 %, 95 %), followed by NP swabs in VTM and saliva in eNAT, was significantly more sensitive than nasal swabs in VTM (50 %, P<0.001) or eNAT (67.8 %, P=0.0012) and oral swabs in VTM (50 %, P<0.0001) or eNAT (58 %, P<0.0001). Saliva and use of eNAT buffer each increased detection of SARS-CoV-2 with the Xpert; however, no single sample matrix identified all positive cases.Conclusion. Saliva and eNAT sterilizing buffer can enhance safe and sensitive detection of COVID-19 using point-of-care GeneXpert instruments.


Assuntos
Teste de Ácido Nucleico para COVID-19 , Manejo de Espécimes/métodos , Adulto , Idoso , COVID-19/diagnóstico , Contenção de Riscos Biológicos , Meios de Cultura , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Boca/virologia , Nasofaringe/virologia , Nariz/virologia , Testes Imediatos , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Sensibilidade e Especificidade , Manejo de Espécimes/normas
3.
Appl Microbiol Biotechnol ; 105(14-15): 6047-6057, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34342709

RESUMO

The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. KEY POINTS: • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality.


Assuntos
Reatores Biológicos , Animais , Diferenciação Celular , Meios de Cultura , Netrina-1 , Netrinas
4.
Klin Lab Diagn ; 66(8): 509-512, 2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34388323

RESUMO

The results of comparative experimental studies of identification of nontoxigenic C. diphtheriae strain by three different commercial laboratories are presented. A typical nontoxigenic strain of C. diphtheriae biovar mitis was used. For the studies, three lines of ten-fold dilutions of bacterial culture were prepared, followed by control planting on the medium and counting CFU/ml. In the experiment, tampons were pooled with a 24-hour bacterial culture of a nontoxigenic C. diphtheriae strain. Tampons were provided from three different laboratories - ∑-Transwab® with Ames liquid medium (from the first and second laboratories) and a viscose tampon with coal medium (from the third laboratory). After pooled, tampons were delivered to commercial laboratories. And as a result of the experiment, Corynebacterium spp. was identified in first laboratory (103 CFU/tamp), S. epidermidis (102 CFU/ml) - in second laboratory and nontoxigenic C. diphtheriae biovar gravis - in third laboratory. The study indicates that there is a need to the supervision of bacteriological investigations conducted in various laboratories. This will improve the quality of investigations on diphtheria infection and identify of diphtheria carrier, which is a reservoir of the causative agent of diphtheria, and will contribute to the maintenance of sanitary and epidemiological well-being in our country.


Assuntos
Corynebacterium diphtheriae , Difteria , Corynebacterium diphtheriae/genética , Meios de Cultura , Difteria/diagnóstico , Humanos
5.
Artigo em Inglês | MEDLINE | ID: mdl-34444305

RESUMO

The collection and storage of water-related matrices such as biofilm from collection to processing are critical for the detection of Legionella pneumophila by cultural and molecular tests. SRK™ is a liquid medium that acts both as an antimicrobial neutralizing agent and a transport medium for bacterial culture enumeration and is useful to maintain the stability of the sample from collection to analysis. The aims of this study were to evaluate Legionella pneumophila viability and bacterial nucleic acids' stability in SRK™ medium over time at different storage conditions. Artificial bacterial inoculates with an approximate concentration of 104, 103 and 102 CFU/mL were made using Legionella pneumophila certified reference material suspended in SRK™ medium. Bacteria recovery was analyzed by cultural and molecular methods at time 0, 24 and 48 h at room temperature and at 0, 24, 48 and 72 h at 2-8 °C, respectively. SRK™ medium supported Legionella pneumophila culture viability with CFU counts within the expected range. The recovery after 72 h at 2-8 °C was 83-100% and 75-95% after 48 h at room temperature. Real-time PCR appropriately detected Legionella pneumophila DNA at each temperature condition, dilution and time point. Results demonstrated a good performance of SRK™ medium for the reliable recovery of environmental Legionella.


Assuntos
Legionella pneumophila , Legionella , Meios de Cultura , Legionella/genética , Legionella pneumophila/genética , Reação em Cadeia da Polimerase em Tempo Real , Microbiologia da Água
6.
Nat Commun ; 12(1): 4730, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354063

RESUMO

Brain organoids derived from human pluripotent stem cells provide a highly valuable in vitro model to recapitulate human brain development and neurological diseases. However, the current systems for brain organoid culture require further improvement for the reliable production of high-quality organoids. Here, we demonstrate two engineering elements to improve human brain organoid culture, (1) a human brain extracellular matrix to provide brain-specific cues and (2) a microfluidic device with periodic flow to improve the survival and reduce the variability of organoids. A three-dimensional culture modified with brain extracellular matrix significantly enhanced neurogenesis in developing brain organoids from human induced pluripotent stem cells. Cortical layer development, volumetric augmentation, and electrophysiological function of human brain organoids were further improved in a reproducible manner by dynamic culture in microfluidic chamber devices. Our engineering concept of reconstituting brain-mimetic microenvironments facilitates the development of a reliable culture platform for brain organoids, enabling effective modeling and drug development for human brain diseases.


Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Dispositivos Lab-On-A-Chip , Neurogênese/fisiologia , Organoides/crescimento & desenvolvimento , Organoides/fisiologia , Animais , Encéfalo/citologia , Meios de Cultura , Fenômenos Eletrofisiológicos , Matriz Extracelular/fisiologia , Estudos de Viabilidade , Perfilação da Expressão Gênica , Humanos , Hidrogéis , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Modelos Anatômicos , Modelos Neurológicos , Neurogênese/genética , Neuroglia/citologia , Neuroglia/fisiologia , Técnicas de Cultura de Órgãos/instrumentação , Técnicas de Cultura de Órgãos/métodos , Organoides/citologia , Suínos
7.
In Vivo ; 35(5): 2669-2673, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34410955

RESUMO

BACKGROUND/AIM: Erythropoietin and its receptor are expressed in the male reproductive system. Initial studies have shown that erythropoietin affects the motility of spermatozoa. The aim of the present study was to investigate the in vitro effect of erythropoietin in the motility and vitality of human spermatozoa. MATERIALS AND METHODS: Forty-three semen samples, obtained after 2-4 days of abstinence from sex, were analyzed and processed using density gradient centrifugation. Aliquots containing one million of spermatozoa were treated with either erythropoietin, at concentrations of 10 and 100 mIU/µl or standard culture medium for one hour. RESULTS: Progressive motility and vitality of spermatozoa significantly increased following treatment with erythropoietin. The effect was not dose-dependent. CONCLUSION: The supplementation of culture medium with erythropoietin improves sperm processing in terms of vitality and motility. Future research should focus on the effects of erythropoietin on sperm capacitation as well as on the signal transduction pathways activated by erythropoietin and its receptor in spermatozoa.


Assuntos
Eritropoetina , Motilidade Espermática , Meios de Cultura , Eritropoetina/farmacologia , Humanos , Masculino , Espermatozoides
8.
Braz J Med Biol Res ; 54(11): e11191, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34431872

RESUMO

The present study focused on the scenario of confirmed cases of SARS-CoV-2 infection in the state of Minas Gerais (MG), Brazil, from March 2020 to March 2021. We evaluated the evolution of COVID-19 prevalence and death in one municipality from each of the 14 health macro-regions of MG state. Socio-demographic characteristics and variables related to the municipalities were analyzed. The raw dataset used in this study was freely sourced from the website Brasil.io. From the raw dataset, two time series were extracted: the cumulative confirmed cases of COVID-19 and cumulative death counts, and they were compared to the state data using a nowcasting approach. In order to make time series comparisons possible, all data was normalized per 100,000 inhabitants. When analyzing in light of colored wave code interventions initiated in August 2020 in MG, for the majority of the municipalities, there was an absence of clear influence on prevalence and deaths. The national holidays in the first semester of 2020 had a small impact on the COVID-19 prevalence of the municipalities, but the holidays in the second semester of 2020 and beginning of 2021 caused important impacts on COVID-19 prevalence. The low number of ICU beds in some municipalities contributed to the higher number of deaths. The analysis showed here is expected to contribute to the improvement of decision making of the MG government, as it opened a huge possibility to have the total macro-regions and state data analyzed.


Assuntos
COVID-19 , Brasil/epidemiologia , Cidades/epidemiologia , Meios de Cultura , Humanos , SARS-CoV-2
9.
Nat Biomed Eng ; 5(8): 926-940, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34373601

RESUMO

Current protocols for the differentiation of human pluripotent stem cells (hPSCs) into chondrocytes do not allow for the expansion of intermediate progenitors so as to prospectively assess their chondrogenic potential. Here we report a protocol that leverages PRRX1-tdTomato reporter hPSCs for the selective induction of expandable and ontogenetically defined PRRX1+ limb-bud-like mesenchymal cells under defined xeno-free conditions, and the prospective assessment of the cells' chondrogenic potential via the cell-surface markers CD90, CD140B and CD82. The cells, which proliferated stably and exhibited the potential to undergo chondrogenic differentiation, formed hyaline cartilaginous-like tissue commensurate to their PRRX1-expression levels. Moreover, we show that limb-bud-like mesenchymal cells derived from patient-derived induced hPSCs can be used to identify therapeutic candidates for type II collagenopathy and we developed a method to generate uniformly sized hyaline cartilaginous-like particles by plating the cells on culture dishes coated with spots of a zwitterionic polymer. PRRX1+ limb-bud-like mesenchymal cells could facilitate the mass production of chondrocytes and cartilaginous tissues for applications in drug screening and tissue engineering.


Assuntos
Proteínas de Homeodomínio/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Condrócitos/citologia , Condrócitos/metabolismo , Condrócitos/transplante , Condrogênese , Doenças do Colágeno/terapia , Meios de Cultura/química , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Pluripotentes/metabolismo , Polímeros/química , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Antígenos Thy-1/metabolismo , Engenharia Tecidual
12.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360992

RESUMO

Several protocols exist for generating megakaryocytes (MKs) and platelets from human induced pluripotent stem cells (hiPSCs) with limited efficiency. We observed previously that mesoderm induction improved endothelial and stromal differentiation. We, therefore, hypothesized that a protocol modification prior to hemogenic endothelial cell (HEC) differentiation will improve MK progenitor (MKP) production and increase platelet output. We further asked if basic media composition affects MK maturation. In an iterative process, we first compared two HEC induction protocols. We found significantly more HECs using the modified protocol including activin A and CHIR99021, resulting in significantly increased MKs. MKs released comparable platelet amounts irrespective of media conditions. In a final validation phase, we obtained five-fold more platelets per hiPSC with the modified protocol (235 ± 84) compared to standard conditions (51 ± 15; p < 0.0001). The regenerative potency of hiPSC-derived platelets was compared to adult donor-derived platelets by profiling angiogenesis-related protein expression. Nineteen of 24 angiogenesis-related proteins were expressed equally, lower or higher in hiPSC-derived compared to adult platelets. The hiPSC-platelet's coagulation hyporeactivity compared to adult platelets was confirmed by thromboelastometry. Further stepwise improvement of hiPSC-platelet production will, thus, permit better identification of platelet-mediated regenerative mechanisms and facilitate manufacture of sufficient amounts of functional platelets for clinical application.


Assuntos
Plaquetas/citologia , Diferenciação Celular , Técnicas de Reprogramação Celular/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Megacariócitos/citologia , Células Cultivadas , Meios de Cultura/química , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo
13.
Appl Microbiol Biotechnol ; 105(18): 6679-6689, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34459953

RESUMO

A series of culture media for haloarchaea were evaluated to optimize the production of ultrahigh-molecular-weight (UHMW) poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Haloferax mediterranei. Cells of H. mediterranei grew (> 1 g/L of dry cell weight) and accumulated PHBV upon flask cultivation in 10 medium types with neutral pH and NaCl concentration > 100 g/L. Molecular weight and compositional analysis revealed that the number-average molecular weight (Mn) of PHBV produced with six selected types of media ranged from 0.8 to 3.5 × 106 g/mol and the 3-hydroxyvalerate (3HV) composition ranged from 8 to 36 mol%. Cultivation in two NBRC media, 1214 and 1380, resulted in the production of PHBV with an Mn of more than 3.0 × 106 g/mol and a weight-average molecular weight of more than 5.0 × 106 g/mol, indicating the production of UHMW-PHBV. These culture media contained small amount of complex nutrients like yeast extract and casamino acids, suggesting that H. mediterranei likely produced UHMW-PHBV on poor nutrient condition. Haloferax mediterranei grown in NBRC medium 1380 produced PHBV with the highest 3HV composition. A solvent-cast film of UHMW-PHBV with 26.4 mol% 3HV produced from 1-L flask cultivation with NBRC medium 1380 was found to be flexible and semi-transparent. Thermal analysis of the UHMW-PHBV cast film revealed melting and glass-transition temperatures of 90.5 °C and - 2.7 °C, respectively. KEY POINTS: • Haloarchaeal culture media were evaluated to produce UHMW-PHBV by H. mediterranei. • UHMW-PHBV with varied molecular weight was produced dependent on culture media. • Semi-transparent film could be made from UHMW-PHBV with 26.4 mol% 3HV.


Assuntos
Haloferax mediterranei , Poli-Hidroxialcanoatos , Meios de Cultura , Peso Molecular , Poliésteres
14.
Bioresour Technol ; 340: 125729, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34391189

RESUMO

The mechanism and nitrogen removal performance of anammox process under different concentrations of Ca2+ and Mg2+ were explored from the perspective of molecular biology analysis based on the metabolic changes of the second messenger cyclic diguanylate (c-di-GMP). After 100-day operation, reactor with 98 mg/L Ca2+ and 30 mg/L Mg2+ achieved a higher anammox performance with an average total nitrogen (TN) removal efficiency of 85.8%. Under the Mg2+concentration of 30 mg/L, a higher Ca2+ could accelerate anammox process by promoting the amplification of Candidatus Brocadia (0.62%) and production of Diguanylate cyclase (DGC-s: 6.54 × 108 copies/µL DNA) which function was to synthesize c-di-GMP. While under the Ca2+concentration of 49 mg/L, Mg2+ concentration at appropriate rang could promote the degradation process of c-di-GMP. Since Ca2+ had positive linear relationship with TN removal (R2 = 0.96), a higher Ca2+ concentration is recommended in the culture medium. This study provided a potential method for optimization of anammox process.


Assuntos
Bactérias , Nitrogênio , Anaerobiose , Reatores Biológicos , Meios de Cultura , Desnitrificação , Oxirredução
15.
Curr Microbiol ; 78(10): 3686-3695, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34406433

RESUMO

Aflatoxin B1 (AFB1) contamination in feed and food seriously threatens the healthy growth of animals and humans, and it may lead to huge economic losses in livestock and poultry production. Therefore, screening of high-efficient AFB1-degrading bacteria is necessary to ensure the safety of feed and food. The study aims to isolate and characterize bacteria from various sources to explore its AFB1 degradation potential. Fifteen bacterial were obtained using a medium containing coumarin as the sole carbon source; only one strain showed a good-degrading ability in culture media by adding AFB1 and it was selected for further studies. A gram-negative and spore-forming, designated E1, was identified as Paenibacillus pabuli, with the highest sequence similarity to P. pabuli NBRC13638T (98.97%). The growth of the strain E1 was observed under 22-47 °C, pH 5.5-9.5 and NaCl concentration 0-6% (w/v), with optimum growth at 37 °C, pH 7.5 and 1% NaCl. The biodegradation characteristics of object strain were detected by high performance liquid chromatography (HPLC). The degradation ratio of AFB1 reached 55% at 24 h and 70.2% at 48 h. After 96 h, the degradation rate of AFB1 reached 85.9%. The active degradation components were present in the cell-free supernatant of strain E1, and the degradation ratio of AFB1 reached 80.0% after 96 h. It is the first report that genus Paenibacillus could degrade AFB1. Moreover, E1 has highly adaptable to diverse environmental conditions. It will be a potential candidate for biodegradation of mycotoxins in feed and food.


Assuntos
Aflatoxina B1 , Paenibacillus , Animais , Biodegradação Ambiental , Meios de Cultura , Humanos , Paenibacillus/genética
16.
Methods Mol Biol ; 2287: 187-197, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270030

RESUMO

The production of doubled haploids (DHs) has proved to be a highly valuable tool to obtain new cultivars. Among the cereals, barley (Hordeum vulgare L.) is the most successful species in large-scale haploid production. Techniques employed for this purpose are based on either the gynogenetic or the androgenetic pathway. Interspecific cross with Hordeum bulbosum L., haploid gene inducer (the hap gene), ovary culture, anther culture (AC), and isolated microspore culture (IMC) are the most used methods. Among all of them, IMC is regarded as a particularly effective system owing to the great increase in green plant numbers per spike and also the higher induction of chromosome doubling when compared with other methods. Thus, IMC provides the best way to mass scale production of new varieties.


Assuntos
Cromossomos de Plantas , Gametogênese Vegetal , Hordeum/crescimento & desenvolvimento , Técnicas de Cultura de Tecidos/métodos , Meios de Cultura , Haploidia , Hordeum/genética , Pólen/genética , Pólen/crescimento & desenvolvimento
17.
Methods Mol Biol ; 2287: 199-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270031

RESUMO

In plant research and breeding, haploid technology is employed upon crossing, induced mutagenesis or genetic engineering to generate populations of meiotic recombinants that are themselves genetically fixed. Thanks to the speed and efficiency in producing true-breeding lines, haploid technology has become a major driver of modern crop improvement. In the present study, we used embryogenic pollen cultures of winter barley ( Hordeum vulgare ) for Cas9 endonuclease-mediated targeted mutagenesis in haploid cells, which facilitates the generation of homozygous primary mutant plants. To this end, microspores were extracted from immature anthers, induced to undergo cell proliferation and embryogenic development in vitro, and were then inoculated with Agrobacterium for the delivery of T-DNAs comprising expression units for Cas9 endonuclease and target gene-specific guide RNAs (gRNAs). Amongst the regenerated plantlets, mutants were identified by PCR amplification of the target regions followed by sequencing of the amplicons. This approach also enabled us to discriminate between homozygous and heterozygous or chimeric mutants. The heritability of induced mutations and their homozygous state were experimentally confirmed by progeny analyses. The major advantage of the method lies in the preferential production of genetically fixed primary mutants, which facilitates immediate phenotypic analyses and, relying on that, a particularly efficient preselection of valuable lines for detailed investigations using their progenies.


Assuntos
Endonucleases/metabolismo , Haploidia , Hordeum/crescimento & desenvolvimento , Hordeum/genética , Mutagênese Sítio-Dirigida/métodos , Melhoramento Vegetal/métodos , RNA Guia/genética , Sistemas CRISPR-Cas , Meios de Cultura , Endonucleases/genética , Edição de Genes , Engenharia Genética , Genoma de Planta , Homozigoto , Hordeum/embriologia , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/crescimento & desenvolvimento
18.
Methods Mol Biol ; 2287: 281-293, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270037

RESUMO

Isolated microspore culture systems have been designed in maize by several groups, mainly from the late 1980s to early 2000s. However, even with optimized protocols, microspore embryogenesis induction has remained very dependent on the genotype in maize, with elite germplasm generally displaying no response or very low response. Yet, these last few years, significant progress has been accomplished in understanding and controlling microspore embryogenesis induction in model dicot and monocot species. This knowledge may be transferred to maize, and isolated microspore culture may gain new interest in this crop, at least for embryogenesis research. The methods we hereby present in detail permit the purification of 3-12 × 105 viable microspores per maize tassel, at the favorable stage for microspore embryogenesis. When cultured in appropriate liquid media, microspores from responsive genotypes give rise to androgenic embryos, which can then be regenerated into fertile doubled haploid plants.


Assuntos
Gametogênese Vegetal , Técnicas de Cultura de Tecidos/métodos , Zea mays/crescimento & desenvolvimento , Meios de Cultura , Haploidia , Pólen/embriologia , Pólen/genética , Pólen/crescimento & desenvolvimento , Zea mays/embriologia , Zea mays/genética
19.
Methods Mol Biol ; 2287: 295-312, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270038

RESUMO

Here, we describe a method of triticale isolated microspore culture for production of doubled haploid plants via androgenesis. We use this method routinely because it is highly efficient and works well on different triticale genotypes. To force microspores into becoming embryogenic, we apply a 21-day cold pretreatment. The shock of cold facilitates redirecting microspores from their predestined pollen developmental program into the androgenesis pathway. Ovaries are included in our culture methods to help with embryogenesis, and the histone deacytelase inhibitor Trichostatin A (TSA) is added to further improve androgenesis and increase our ability to recover green doubled haploid plants.


Assuntos
Gametogênese Vegetal , Técnicas de Cultura de Tecidos/métodos , Triticale/crescimento & desenvolvimento , Meios de Cultura , Haploidia , Pólen/embriologia , Pólen/genética , Pólen/crescimento & desenvolvimento , Triticale/embriologia , Triticale/genética
20.
Appl Microbiol Biotechnol ; 105(14-15): 6059-6072, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34328537

RESUMO

The QuantaMatrix Microfluidic Agarose Channel (QMAC) system was used for rapid drug susceptibility testing (DST). Here, we performed DST using QMAC integrated with the mycobacteria growth indicator tube (MGIT) liquid culture employing a specially designed cross agarose channel for the tuberculosis chip. MGIT-, QMAC-, and Löwenstein-Jensen (LJ)-DSTs were performed using 13 drugs. The protocol for QMAC-DST was optimized using the inoculum obtained after the disaggregation of Mycobacterium tuberculosis clumps in MGIT culture. The completion times of QMAC-DST and MGIT-DST were analyzed, and the results of all three DSTs were compared. Discrepant results were analyzed using line probe assays and DNA sequencing. Nontuberculous mycobacteria were distinguished using the ρ-nitrobenzoic acid inhibition test. The overall agreement rate of QMAT-DST and LJ-DST was 97.0% and that of QMAT-DST and MGIT-DST was 86.3%. An average turnaround time for DST was 5.4 days, which was considerably less than the time required for MGIT-DST. The overall time required to obtain DST results using QMAC-DST integrated with MGIT culture was an average of 18.6 days: 13.2 days for culture and identification and 5.4 days for DST. Hence, QMAC-DST integrated with liquid culture can be used to perform DSTs with short turnaround times and effective detection. KEY POINTS: • QMAC system can simultaneously perform phenotypic DST with 13 anti-TB drugs and PNB. • An optimized DST protocol led to a marked decrease in clumping in MGIT culture. • QMAC system integrated with MGIT liquid culture system reduced the turnaround time.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Técnicas Bacteriológicas , Meios de Cultura , Humanos , Testes de Sensibilidade Microbiana , Microfluídica , Mycobacterium tuberculosis/genética , Sefarose
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