Drought stress enhances the efficiency of floral dip method of Agrobacterium-mediated transformation in Arabidopsis thaliana / Estresse hídrico aumenta a eficiência do método de imersão floral de transformação mediado por Agrobacterium em Arabidopsis thaliana

The Agrobacterium-mediated floral dip protocol is the most extensively used transformation method for a model plant Arabidopsis thaliana. Several useful methods for Agrobacterium tumefaciens-mediated transformations of Arabidopsis are existing, but they are time consuming and with low transformation efficiency. Here, we developed a transgenic Arabidopsis lines TET12p::TET12-RFP in a short period of time and enhanced transformation efficiency by using a modified transformation method by applying drought stress after floral dip. In this protocol, Agrobacterium cells carrying TET12p::TET12-RFP recombinant vectors were resuspended in a solution of 5% sucrose, 0.05% (v/v) silwet L-77 to transform female gametes of developing Arabidopsis inflorescences. Treated Arabidopsis were then applied with different levels of drought stresses to stimulate plants for the utilization of maximum plant energy in seed maturation process. The applied stresses achieved the fast maturation of already treated inflorescences while stopped the growing of newly arising untreated inflorescence, thus decreased the chances of wrong collection of untransformed seeds. Consequently, the collected seeds were mostly transgenic with a transformation frequency of at least 10%, thus the screening for positive transformants selection was more advantageous on a selective medium as compared to a classical floral dip method. Within 2-3 months, two hundred of individual transgenic plants were produced from just 10 infiltrated plants. This study concludes that application of drought stresses in a specific stage of plant is a beneficial strategy for achieving the transgenic Arabidopsis in a short period of time with high transformation efficiency.

O protocolo de imersão floral mediado por Agrobacterium é o método de transformação mais amplamente utilizado para uma planta-modelo Arabidopsis thaliana. Existem vários métodos úteis para transformações de Arabidopsis mediados por Agrobacterium tumefaciens, mas são demorados e com baixa eficiência de transformação. Aqui, desenvolvemos uma linha transgênica de Arabidopsis TET12p::TET12-RFP em um curto período de tempo e eficiência de transformação aprimorada usando um método de transformação modificado por meio da aplicação de estresse hídrico após o mergulho floral. Neste protocolo, células de Agrobacterium transportando vetores recombinantes TET12p::TET12-RFP foram ressuspensas em uma solução de 5% de sacarose, 0,05% (v/v) silwet L-77 para transformar gametas femininos de inflorescências de Arabidopsis em desenvolvimento. Arabidopsis tratadas foram então aplicadas com diferentes níveis de estresse hídrico para estimular as plantas a utilizar o máximo de energia da planta no processo de maturação das sementes. Os estresses aplicados alcançaram a rápida maturação das inflorescências já tratadas enquanto paravam o crescimento de inflorescências não tratadas recém-surgidas, diminuindo, assim, as chances de coleta errada de sementes não transformadas. Consequentemente, as sementes coletadas eram principalmente transgênicas, com uma frequência de transformação de pelo menos 10%; portanto, a triagem para seleção dos transformantes positivos foi mais vantajosa em um meio seletivo em comparação com um método clássico de imersão floral. Dentro de 2-3 meses, 200 plantas transgênicas individuais foram produzidas a partir de apenas 10 plantas infiltradas. Este estudo conclui que a aplicação de estresse hídrico em um estágio específico da planta é uma estratégia benéfica para alcançar a Arabidopsis transgênica em um curto período de tempo com alta eficiência de transformação.

Cadmium (Cd) influences calcium (Ca) levels in the skeleton of a freshwater fish Channa gachua / Influência do cádmio (Cd) nos níveis de Ca no esqueleto de peixes de água doce Channa gachua

Abstract Environmental contamination with heavy metals is a threat to the organisms due to their toxicity, persistence and bioaccumulation in food chains. The study was aimed to assess cadmium (Cd) effect on calcium (Ca) level in bones of a freshwater fish Channa gachua. 42 fish individuals were kept into six (6) aquaria; labelled aq.0, 1, 2, 3,4 and 5 in the laboratory for treatment. Aq.0 was control group and aq.1, 2,3,4,5 were experimental with treatment solution of Cd 0, 0.1ppm, 0.5ppm, 1ppm, 2.5ppm and 5ppm respectively for three months. After exposure, bones tissue were examined for Cd accumulation and Ca concentration. Highest accumulation of Cd were recorded in aq.5 mean 46.86 ± 0.46 mgkg-1 .and lowest in the control group with mean 0.61 ± 0.06 mgkg-1. The order of Cd bioaccumulation in bones were aq.5 > aq.4 >aq.3 > aq.2 > aq.1 > aq.0. Highest concentration of Ca were noted in aq.0 (Control group) mean 7888.06 ± 4827.22 mgkg-1 and lowest were 1132.36 ± 203.73 mgkg-1 in aq.5 (at 5.0 ppm). Generally a pattern of decreasing Ca level were observed with each rise of Cd bioaccumulation aq.0 > aq.1 > aq.2 > aq.3 > aq.4 > aq.5. Current study indicated that Cd accumulation have substantial effect on Ca level in bones and hence on skeleton system. Strict rules must be implemented by government to control metals pollution and exploitations of biota.

A contaminação ambiental com metais pesados ​​é uma ameaça aos organismos devido à sua toxicidade, persistência e bioacumulação nas cadeias alimentares. O estudo teve como objetivo avaliar o efeito do Cd sobre o nível de Ca em ossos de peixes de água doce Channa gachua. Quarenta e dois indivíduos de peixes foram mantidos em seis aquários; marcado aq. 0, 1, 2, 3, 4 e 5 no laboratório para tratamento. Aq. 0 foi o grupo controle e aq. 1, 2, 3, 4, 5 foram experimentais com solução de tratamento de Cd 0, 0,1 ppm, 0,5 ppm, 1 ppm, 2,5 ppm e 5 ppm respectivamente por três meses. Após a exposição, o tecido ósseo foi examinado quanto ao acúmulo de Cd e concentração de Ca. O maior acúmulo de Cd foi registrado em aq. 5 com média de 46,86 ± 0,46 mgkg-1 e o menor no grupo controle com média de 0,61 ± 0,06 mgkg-1. A ordem de bioacumulação de Cd nos ossos foi aq. 5 > aq. 4 > aq. 3 > aq. 2 > aq. 1 > aq. 0. A concentração mais alta de Ca foi observada em aq. 0 (grupo controle) média de 7.888,06 ± 4.827,22 mgkg-1 e a mais baixa foi de 1.132,36 ± 203,73 mgkg-1 em aq. 5 (a 5,0 ppm). Geralmente um padrão de diminuição do nível de Ca foi observado com cada aumento da bioacumulação de Cd aq. 0 > aq. 1 > aq. 2 > aq. 3 > aq. 4 > aq. 5. O estudo atual indicou que o acúmulo de Cd tem efeito substancial no nível de Ca nos ossos e, portanto, no sistema esquelético. Regras estritas devem ser implementadas pelo governo para controlar a poluição por metais e a exploração da biota.

Organoleptic characteristics and compositional profile of meat of growing Japanese quail fed different levels of poultry byproducts compost.

The poultry industry generates a lot of waste, including dead birds, manure, and poultry litter. Poultry waste should never be disposed of improperly because it can seriously harm the environment. The waste can be recycled as a feedstock for use in poultry feed by composting the litter and deceased birds. The compositional profile and organoleptic properties of the meat of growing Japanese quail were examined over the course of a 4-week trial to ascertain the effect of adding compost to the diet. In a completely randomized design (CRD), 1200 newly hatched quail chicks (Coturnix coturnix japonica) were divided into five treatment groups (diets with 0, 2.5, 5, 7.5, and 10% compost), each consisting of 40 birds with six replicates. The addition of compost to the diet had no noticeable effects on the organoleptic qualities of appearance, color, aroma, taste, texture, juiciness, tenderness, and acceptability (P>0.05). The compositional profile characteristics for chicks given compost at any level compared to chicks fed the control diet showed no differences (P>0.05). These findings suggest that the sensory characteristics and compositional profile of growing meat quails can be maintained when fed diets including up to 10% compost.

The developing role of extracellular vesicles in autoimmune diseases: special attention to mesenchymal stem cell-derived extracellular vesicles.

Autoimmune diseases are complex, chronic inflammatory conditions initiated by the loss of immunological tolerance to self-antigens. Nowadays, there is no effective and useful therapy for autoimmune diseases, and the existing medications have some limitations due to their nonspecific targets and side effects. During the last few decades, it has been established that mesenchymal stem cells (MSCs) have immunomodulatory functions. It is proposed that MSCs can exert an important therapeutic effect on autoimmune disorders. In parallel with these findings, several investigations have shown that MSCs alleviate autoimmune diseases. Intriguingly, the results of studies have demonstrated that the effective roles of MSCs in autoimmune diseases do not depend on direct intercellular communication but on their ability to release a wide spectrum of paracrine mediators such as growth factors, cytokines and extracellular vehicles (EVs). EVs that range from 50 to 5,000 nm were produced by almost any cell type, and these nanoparticles participate in homeostasis and intercellular communication via the transfer of a broad range of biomolecules such as modulatory proteins, nucleic acids (DNA and RNA), lipids, cytokines, and metabolites. EVs derived from MSCs display the exact properties of MSCs and can be safer and more beneficial than their parent cells. In this review, we will discuss the features of MSCs and their EVs, EVs biogenesis, and their cargos, and then we will highlight the existing discoveries on the impacts of EVs from MSCs on autoimmune diseases such as multiple sclerosis, arthritis rheumatic, inflammatory bowel disease, Type 1 diabetes mellitus, systemic lupus erythematosus, autoimmune liver diseases, Sjögren syndrome, and osteoarthritis, suggesting a potential alternative for autoimmune conditions therapy.

Designing Effective Multi-Target Drugs and Identifying Biomarkers in Recurrent Pregnancy Loss (RPL) Using In Vivo, In Vitro, and In Silico Approaches.

Recurrent pregnancy loss (RPL) occurs in approximately 5% of women. Despite an abundance of evidence, the molecular mechanism of RPL's pathology remains unclear. Here, we report the protective role of polo-like kinase 1 (PLK1) during RPL. We aimed to construct an RPL network utilizing GEO datasets and identified hub high-traffic genes. We also investigated whether the expressions of PLK1 were altered in the chorionic villi collected from women with RPL compared to those from healthy early pregnant women. Gene expression differences were evaluated using both pathway and gene ontology (GO) analyses. The identified genes were validated using in vivo and in vitro models. Mice with PLK1-overexpression and PLK1-knockdown in vitro models were produced by transfecting certain plasmids and si-RNA, respectively. The apoptosis in the chorionic villi, mitochondrial function, and NF-κB signaling activity was evaluated. To suppress the activation of PLK1, the PLK1 inhibitor BI2536 was administered. The HTR-8/SVneo and JEG-3 cell lines were chosen to establish an RPL model in vitro. The NF-κB signaling, Foxo signaling, PI3K/AKT, and endometrial cancer signaling pathways were identified via the RPL regulatory network. The following genes were identified: PLK1 as hub high-traffic gene and MMP2, MMP9, BAX, MFN1, MFN2, FOXO1, OPA1, COX15, BCL2, DRP1, FIS1, TRAF2, and TOP2A. Clinical samples were examined, and the results demonstrated that RPL patients had tissues with decreased PLK1 expression in comparison to women with normal pregnancies (p < 0.01). In vitro, PLK1 knockdown induced the NF-κB signaling pathway and apoptosis activation while decreasing cell invasion, migration, and proliferation (p < 0.05). Furthermore, the in vivo model proved that cell mitochondrial function and chorionic villi development are both hampered by PLK1 suppression. Our findings revealed that the PLK1/TRAF2/NF-κB axis plays a crucial role in RPL-induced chorionic villi dysfunction by regulating mitochondrial dynamics and apoptosis and might be a potential therapeutic target in the clinic.

Honey as a bioindicator of environmental organochlorine insecticides contamination / Mel como bioindicador de contaminação ambiental de inseticidas organoclorados

Honey is a suitable matrix for the evaluation of environmental contaminants including organochlorine insecticides. The present study was conducted to evaluate residues of fifteen organochlorine insecticides in honey samples of unifloral and multifloral origins from Dir, Pakistan. Honey samples (5 g each) were extracted with GC grade organic solvents and then subjected to Rotary Evaporator till dryness. The extracts were then mixed with n-Hexane (5 ml) and purified through Column Chromatography. Purified extracts (1μl each) were processed through Gas Chromatograph coupled with Electron Capture Detector (GC-ECD) for identification and quantification of the insecticides. Of the 15 insecticides tested, 46.7% were detected while 53.3% were not detected in the honey samples. Heptachlor was the most prevalent insecticide with a mean level of 0.0018 mg/kg detected in 80% of the samples followed by β-HCH with a mean level of 0.0016 mg/kg detected in 71.4% of the honey samples. Honey samples from Acacia modesta Wall. were 100% positive for Heptachlor with a mean level of 0.0048 mg/kg followed by β-HCH with a mean level of 0.003 mg/kg and frequency of 83.3%. Minimum levels of the tested insecticides were detected in the unifloral honey from Ziziphus jujuba Mill. Methoxychlor, Endosulfan, Endrin and metabolites of DDT were not detected in the studied honey samples. Some of the tested insecticides are banned in Pakistan but are still detected in honey samples indicating their use in the study area. The detected levels of all insecticides were below the Maximum Residue Levels (MRLs) and safe for consumers. However, the levels detected can cause mortality in insect fauna. The use of banned insecticides is one of the main factors responsible for the declining populations of important insect pollinators including honeybees.

O mel é uma matriz adequada para a avaliação de contaminantes ambientais, incluindo inseticidas organoclorados. O presente estudo foi conduzido para avaliar resíduos de 15 inseticidas organoclorados em amostras de mel de origem unifloral e multifloral de Dir, Paquistão. Amostras de mel (5 g cada) foram extraídas com solventes orgânicos de grau GC e, em seguida, submetidas ao evaporador rotativo até a secura. Os extratos foram então misturados com n-hexano (5 ml) e purificados por cromatografia em coluna. Os extratos purificados (1μl cada) foram processados através de cromatógrafo gasoso acoplado a detector de captura de elétrons (GC-ECD) para identificação e quantificação dos inseticidas. Dos 15 inseticidas testados, 46,7% foram detectados enquanto 53,3% não foram detectados nas amostras de mel. O heptacloro foi o inseticida mais prevalente com um nível médio de 0,0018 mg / kg detectado em 80% das amostras, seguido por β-HCH com um nível médio de 0,0016 mg / kg detectado em 71,4% das amostras de mel. Amostras de mel da parede de Acacia modesta foram 100% positivos para heptacloro com um nível médio de 0,0048 mg / kg seguido por β-HCH com um nível médio de 0,003 mg / kg e frequência de 83,3%. Níveis mínimos dos inseticidas testados foram detectados no mel unifloral de Ziziphus jujuba da usina. Metoxicloro, Endosulfan, Endrin e metabólitos do DDT não foram detectados nas amostras de mel estudadas. Alguns dos inseticidas testados são proibidos no Paquistão, mas ainda são detectados em amostras de mel, indicando seu uso na área de estudo. Os níveis detectados de todos os inseticidas estavam abaixo dos Níveis Máximos de Resíduos (MRLs) e seguros para os consumidores. No entanto, os níveis detectados podem causar mortalidade na fauna de insetos. O uso de inseticidas proibidos é um dos principais fatores responsáveis pelo declínio das populações de importantes insetos polinizadores, incluindo as abelhas.

Honey as a bioindicator of environmental organochlorine insecticides contamination

Abstract Honey is a suitable matrix for the evaluation of environmental contaminants including organochlorine insecticides. The present study was conducted to evaluate residues of fifteen organochlorine insecticides in honey samples of unifloral and multifloral origins from Dir, Pakistan. Honey samples (5 g each) were extracted with GC grade organic solvents and then subjected to Rotary Evaporator till dryness. The extracts were then mixed with n-Hexane (5 ml) and purified through Column Chromatography. Purified extracts (1l each) were processed through Gas Chromatograph coupled with Electron Capture Detector (GC-ECD) for identification and quantification of the insecticides. Of the 15 insecticides tested, 46.7% were detected while 53.3% were not detected in the honey samples. Heptachlor was the most prevalent insecticide with a mean level of 0.0018 mg/kg detected in 80% of the samples followed by -HCH with a mean level of 0.0016 mg/kg detected in 71.4% of the honey samples. Honey samples from Acacia modesta Wall. were 100% positive for Heptachlor with a mean level of 0.0048 mg/kg followed by -HCH with a mean level of 0.003 mg/kg and frequency of 83.3%. Minimum levels of the tested insecticides were detected in the unifloral honey from Ziziphus jujuba Mill. Methoxychlor, Endosulfan, Endrin and metabolites of DDT were not detected in the studied honey samples. Some of the tested insecticides are banned in Pakistan but are still detected in honey samples indicating their use in the study area. The detected levels of all insecticides were below the Maximum Residue Levels (MRLs) and safe for consumers. However, the levels detected can cause mortality in insect fauna. The use of banned insecticides is one of the main factors responsible for the declining populations of important insect pollinators including honeybees.

Resumo O mel é uma matriz adequada para a avaliação de contaminantes ambientais, incluindo inseticidas organoclorados. O presente estudo foi conduzido para avaliar resíduos de 15 inseticidas organoclorados em amostras de mel de origem unifloral e multifloral de Dir, Paquistão. Amostras de mel (5 g cada) foram extraídas com solventes orgânicos de grau GC e, em seguida, submetidas ao evaporador rotativo até a secura. Os extratos foram então misturados com n-hexano (5 ml) e purificados por cromatografia em coluna. Os extratos purificados (1l cada) foram processados através de cromatógrafo gasoso acoplado a detector de captura de elétrons (GC-ECD) para identificação e quantificação dos inseticidas. Dos 15 inseticidas testados, 46,7% foram detectados enquanto 53,3% não foram detectados nas amostras de mel. O heptacloro foi o inseticida mais prevalente com um nível médio de 0,0018 mg / kg detectado em 80% das amostras, seguido por -HCH com um nível médio de 0,0016 mg / kg detectado em 71,4% das amostras de mel. Amostras de mel da parede de Acacia modesta foram 100% positivos para heptacloro com um nível médio de 0,0048 mg / kg seguido por -HCH com um nível médio de 0,003 mg / kg e frequência de 83,3%. Níveis mínimos dos inseticidas testados foram detectados no mel unifloral de Ziziphus jujuba da usina. Metoxicloro, Endosulfan, Endrin e metabólitos do DDT não foram detectados nas amostras de mel estudadas. Alguns dos inseticidas testados são proibidos no Paquistão, mas ainda são detectados em amostras de mel, indicando seu uso na área de estudo. Os níveis detectados de todos os inseticidas estavam abaixo dos Níveis Máximos de Resíduos (MRLs) e seguros para os consumidores. No entanto, os níveis detectados podem causar mortalidade na fauna de insetos. O uso de inseticidas proibidos é um dos principais fatores responsáveis pelo declínio das populações de importantes insetos polinizadores, incluindo as abelhas.

Honey as a bioindicator of environmental organochlorine insecticides contamination / Mel como bioindicador de contaminação ambiental de inseticidas organoclorados

Abstract Honey is a suitable matrix for the evaluation of environmental contaminants including organochlorine insecticides. The present study was conducted to evaluate residues of fifteen organochlorine insecticides in honey samples of unifloral and multifloral origins from Dir, Pakistan. Honey samples (5 g each) were extracted with GC grade organic solvents and then subjected to Rotary Evaporator till dryness. The extracts were then mixed with n-Hexane (5 ml) and purified through Column Chromatography. Purified extracts (1μl each) were processed through Gas Chromatograph coupled with Electron Capture Detector (GC-ECD) for identification and quantification of the insecticides. Of the 15 insecticides tested, 46.7% were detected while 53.3% were not detected in the honey samples. Heptachlor was the most prevalent insecticide with a mean level of 0.0018 mg/kg detected in 80% of the samples followed by β-HCH with a mean level of 0.0016 mg/kg detected in 71.4% of the honey samples. Honey samples from Acacia modesta Wall. were 100% positive for Heptachlor with a mean level of 0.0048 mg/kg followed by β-HCH with a mean level of 0.003 mg/kg and frequency of 83.3%. Minimum levels of the tested insecticides were detected in the unifloral honey from Ziziphus jujuba Mill. Methoxychlor, Endosulfan, Endrin and metabolites of DDT were not detected in the studied honey samples. Some of the tested insecticides are banned in Pakistan but are still detected in honey samples indicating their use in the study area. The detected levels of all insecticides were below the Maximum Residue Levels (MRLs) and safe for consumers. However, the levels detected can cause mortality in insect fauna. The use of banned insecticides is one of the main factors responsible for the declining populations of important insect pollinators including honeybees.

Resumo O mel é uma matriz adequada para a avaliação de contaminantes ambientais, incluindo inseticidas organoclorados. O presente estudo foi conduzido para avaliar resíduos de 15 inseticidas organoclorados em amostras de mel de origem unifloral e multifloral de Dir, Paquistão. Amostras de mel (5 g cada) foram extraídas com solventes orgânicos de grau GC e, em seguida, submetidas ao evaporador rotativo até a secura. Os extratos foram então misturados com n-hexano (5 ml) e purificados por cromatografia em coluna. Os extratos purificados (1μl cada) foram processados através de cromatógrafo gasoso acoplado a detector de captura de elétrons (GC-ECD) para identificação e quantificação dos inseticidas. Dos 15 inseticidas testados, 46,7% foram detectados enquanto 53,3% não foram detectados nas amostras de mel. O heptacloro foi o inseticida mais prevalente com um nível médio de 0,0018 mg / kg detectado em 80% das amostras, seguido por β-HCH com um nível médio de 0,0016 mg / kg detectado em 71,4% das amostras de mel. Amostras de mel da parede de Acacia modesta foram 100% positivos para heptacloro com um nível médio de 0,0048 mg / kg seguido por β-HCH com um nível médio de 0,003 mg / kg e frequência de 83,3%. Níveis mínimos dos inseticidas testados foram detectados no mel unifloral de Ziziphus jujuba da usina. Metoxicloro, Endosulfan, Endrin e metabólitos do DDT não foram detectados nas amostras de mel estudadas. Alguns dos inseticidas testados são proibidos no Paquistão, mas ainda são detectados em amostras de mel, indicando seu uso na área de estudo. Os níveis detectados de todos os inseticidas estavam abaixo dos Níveis Máximos de Resíduos (MRLs) e seguros para os consumidores. No entanto, os níveis detectados podem causar mortalidade na fauna de insetos. O uso de inseticidas proibidos é um dos principais fatores responsáveis pelo declínio das populações de importantes insetos polinizadores, incluindo as abelhas.

Honey as a bioindicator of environmental organochlorine insecticides contamination / Mel como bioindicador de contaminação ambiental de inseticidas organoclorados

Honey is a suitable matrix for the evaluation of environmental contaminants including organochlorine insecticides. The present study was conducted to evaluate residues of fifteen organochlorine insecticides in honey samples of unifloral and multifloral origins from Dir, Pakistan. Honey samples (5 g each) were extracted with GC grade organic solvents and then subjected to Rotary Evaporator till dryness. The extracts were then mixed with n-Hexane (5 ml) and purified through Column Chromatography. Purified extracts (1μl each) were processed through Gas Chromatograph coupled with Electron Capture Detector (GC-ECD) for identification and quantification of the insecticides. Of the 15 insecticides tested, 46.7% were detected while 53.3% were not detected in the honey samples. Heptachlor was the most prevalent insecticide with a mean level of 0.0018 mg/kg detected in 80% of the samples followed by β-HCH with a mean level of 0.0016 mg/kg detected in 71.4% of the honey samples. Honey samples from Acacia modesta Wall. were 100% positive for Heptachlor with a mean level of 0.0048 mg/kg followed by β-HCH with a mean level of 0.003 mg/kg and frequency of 83.3%. Minimum levels of the tested insecticides were detected in the unifloral honey from Ziziphus jujuba Mill. Methoxychlor, Endosulfan, Endrin and metabolites of DDT were not detected in the studied honey samples. Some of the tested insecticides are banned in Pakistan but are still detected in honey samples indicating their use in the study area. The detected levels of all insecticides were below the Maximum Residue Levels (MRLs) and safe for consumers. However, the levels detected can cause mortality in insect fauna. The use of banned insecticides is one of the main factors responsible for the declining populations of important insect pollinators including honeybees.(AU)

O mel é uma matriz adequada para a avaliação de contaminantes ambientais, incluindo inseticidas organoclorados. O presente estudo foi conduzido para avaliar resíduos de 15 inseticidas organoclorados em amostras de mel de origem unifloral e multifloral de Dir, Paquistão. Amostras de mel (5 g cada) foram extraídas com solventes orgânicos de grau GC e, em seguida, submetidas ao evaporador rotativo até a secura. Os extratos foram então misturados com n-hexano (5 ml) e purificados por cromatografia em coluna. Os extratos purificados (1μl cada) foram processados através de cromatógrafo gasoso acoplado a detector de captura de elétrons (GC-ECD) para identificação e quantificação dos inseticidas. Dos 15 inseticidas testados, 46,7% foram detectados enquanto 53,3% não foram detectados nas amostras de mel. O heptacloro foi o inseticida mais prevalente com um nível médio de 0,0018 mg / kg detectado em 80% das amostras, seguido por β-HCH com um nível médio de 0,0016 mg / kg detectado em 71,4% das amostras de mel. Amostras de mel da parede de Acacia modesta foram 100% positivos para heptacloro com um nível médio de 0,0048 mg / kg seguido por β-HCH com um nível médio de 0,003 mg / kg e frequência de 83,3%. Níveis mínimos dos inseticidas testados foram detectados no mel unifloral de Ziziphus jujuba da usina. Metoxicloro, Endosulfan, Endrin e metabólitos do DDT não foram detectados nas amostras de mel estudadas. Alguns dos inseticidas testados são proibidos no Paquistão, mas ainda são detectados em amostras de mel, indicando seu uso na área de estudo. Os níveis detectados de todos os inseticidas estavam abaixo dos Níveis Máximos de Resíduos (MRLs) e seguros para os consumidores. No entanto, os níveis detectados podem causar mortalidade na fauna de insetos. O uso de inseticidas proibidos é um dos principais fatores responsáveis pelo declínio das populações de importantes insetos polinizadores, incluindo as abelhas.(AU)

Cadmium (Cd) influences calcium (Ca) levels in the skeleton of a freshwater fish Channa gachua.

Environmental contamination with heavy metals is a threat to the organisms due to their toxicity, persistence and bioaccumulation in food chains. The study was aimed to assess cadmium (Cd) effect on calcium (Ca) level in bones of a freshwater fish Channa gachua. 42 fish individuals were kept into six (6) aquaria; labelled aq.0, 1, 2, 3,4 and 5 in the laboratory for treatment. Aq.0 was control group and aq.1, 2,3,4,5 were experimental with treatment solution of Cd 0, 0.1ppm, 0.5ppm, 1ppm, 2.5ppm and 5ppm respectively for three months. After exposure, bones tissue were examined for Cd accumulation and Ca concentration. Highest accumulation of Cd were recorded in aq.5 mean 46.86 ± 0.46 mgkg-1 .and lowest in the control group with mean 0.61 ± 0.06 mgkg-1. The order of Cd bioaccumulation in bones were aq.5 > aq.4 >aq.3 > aq.2 > aq.1 > aq.0. Highest concentration of Ca were noted in aq.0 (Control group) mean 7888.06 ± 4827.22 mgkg-1 and lowest were 1132.36 ± 203.73 mgkg-1 in aq.5 (at 5.0 ppm). Generally a pattern of decreasing Ca level were observed with each rise of Cd bioaccumulation aq.0 > aq.1 > aq.2 > aq.3 > aq.4 > aq.5. Current study indicated that Cd accumulation have substantial effect on Ca level in bones and hence on skeleton system. Strict rules must be implemented by government to control metals pollution and exploitations of biota.

Drought stress enhances the efficiency of floral dip method of Agrobacterium-mediated transformation in Arabidopsis thaliana.

The Agrobacterium-mediated floral dip protocol is the most extensively used transformation method for a model plant Arabidopsis thaliana. Several useful methods for Agrobacterium tumefaciens-mediated transformations of Arabidopsis are existing, but they are time consuming and with low transformation efficiency. Here, we developed a transgenic Arabidopsis lines TET12p::TET12-RFP in a short period of time and enhanced transformation efficiency by using a modified transformation method by applying drought stress after floral dip. In this protocol, Agrobacterium cells carrying TET12p::TET12-RFP recombinant vectors were resuspended in a solution of 5% sucrose, 0.05% (v/v) silwet L-77 to transform female gametes of developing Arabidopsis inflorescences. Treated Arabidopsis were then applied with different levels of drought stresses to stimulate plants for the utilization of maximum plant energy in seed maturation process. The applied stresses achieved the fast maturation of already treated inflorescences while stopped the growing of newly arising untreated inflorescence, thus decreased the chances of wrong collection of untransformed seeds. Consequently, the collected seeds were mostly transgenic with a transformation frequency of at least 10%, thus the screening for positive transformants selection was more advantageous on a selective medium as compared to a classical floral dip method. Within 2-3 months, two hundred of individual transgenic plants were produced from just 10 infiltrated plants. This study concludes that application of drought stresses in a specific stage of plant is a beneficial strategy for achieving the transgenic Arabidopsis in a short period of time with high transformation efficiency.

Impact of the Salud Mesoamerica Initiative on delivery care choices in Guatemala, Honduras, and Nicaragua.

BACKGROUND: The Salud Mesoamérica Initiative (SMI) is a public-private collaboration aimed to improve maternal and child health conditions in the poorest populations of Mesoamerica through a results-based aid mechanism. We assess the impact of SMI on the staffing and availability of equipment and supplies for delivery care, the proportion of institutional deliveries, and the proportion of women who choose a facility other than the one closest to their locality of residence for delivery. METHODS: We used a quasi-experimental design, including baseline and follow-up measurements between 2013 and 2018 in intervention and comparison areas of Guatemala, Nicaragua, and Honduras. We collected information on 8754 births linked to the health facility closest to the mother's locality of residence and the facility where the delivery took place (if attended in a health facility). We fit difference-in-difference models, adjusting for women's characteristics (age, parity, education), household characteristics, exposure to health promotion interventions, health facility level, and country. RESULTS: Equipment, inputs, and staffing of facilities improved after the Initiative in both intervention and comparison areas. After adjustment for covariates, institutional delivery increased between baseline and follow-up by 3.1 percentage points (ß = 0.031, 95% CI -0.03, 0.09) more in intervention areas than in comparison areas. The proportion of women in intervention areas who chose a facility other than their closest one to attend the delivery decreased between baseline and follow-up by 13 percentage points (ß = - 0.130, 95% CI -0.23, - 0.03) more than in the comparison group. CONCLUSIONS: Results indicate that women in intervention areas of SMI are more likely to go to their closest facility to attend delivery after the Initiative has improved facilities' capacity, suggesting that results-based aid initiatives targeting poor populations, like SMI, can increase the use of facilities closest to the place of residence for delivery care services. This should be considered in the design of interventions after the COVID-19 pandemic may have changed health and social conditions.

Honey as a bioindicator of environmental organochlorine insecticides contamination.

Honey is a suitable matrix for the evaluation of environmental contaminants including organochlorine insecticides. The present study was conducted to evaluate residues of fifteen organochlorine insecticides in honey samples of unifloral and multifloral origins from Dir, Pakistan. Honey samples (5 g each) were extracted with GC grade organic solvents and then subjected to Rotary Evaporator till dryness. The extracts were then mixed with n-Hexane (5 ml) and purified through Column Chromatography. Purified extracts (1µl each) were processed through Gas Chromatograph coupled with Electron Capture Detector (GC-ECD) for identification and quantification of the insecticides. Of the 15 insecticides tested, 46.7% were detected while 53.3% were not detected in the honey samples. Heptachlor was the most prevalent insecticide with a mean level of 0.0018 mg/kg detected in 80% of the samples followed by ß-HCH with a mean level of 0.0016 mg/kg detected in 71.4% of the honey samples. Honey samples from Acacia modesta Wall. were 100% positive for Heptachlor with a mean level of 0.0048 mg/kg followed by ß-HCH with a mean level of 0.003 mg/kg and frequency of 83.3%. Minimum levels of the tested insecticides were detected in the unifloral honey from Ziziphus jujuba Mill. Methoxychlor, Endosulfan, Endrin and metabolites of DDT were not detected in the studied honey samples. Some of the tested insecticides are banned in Pakistan but are still detected in honey samples indicating their use in the study area. The detected levels of all insecticides were below the Maximum Residue Levels (MRLs) and safe for consumers. However, the levels detected can cause mortality in insect fauna. The use of banned insecticides is one of the main factors responsible for the declining populations of important insect pollinators including honeybees.

ENTPD3 Marks Mature Stem Cell-Derived ß-Cells Formed by Self-Aggregation In Vitro.

Stem cell-derived ß-like cells (sBC) carry the promise of providing an abundant source of insulin-producing cells for use in cell replacement therapy for patients with diabetes, potentially allowing widespread implementation of a practical cure. To achieve their clinical promise, sBC need to function comparably with mature adult ß-cells, but as yet they display varying degrees of maturity. Indeed, detailed knowledge of the events resulting in human ß-cell maturation remains obscure. Here we show that sBC spontaneously self-enrich into discreet islet-like cap structures within in vitro cultures, independent of exogenous maturation conditions. Multiple complementary assays demonstrate that this process is accompanied by functional maturation of the self-enriched sBC (seBC); however, the seBC still contain distinct subpopulations displaying different maturation levels. Interestingly, the surface protein ENTPD3 (also known as nucleoside triphosphate diphosphohydrolase-3 [NDPTase3]) is a specific marker of the most mature seBC population and can be used for mature seBC identification and sorting. Our results illuminate critical aspects of in vitro sBC maturation and provide important insights toward developing functionally mature sBC for diabetes cell replacement therapy.

Performance of passive case detection for malaria surveillance: results from nine countries in Mesoamerica and the Dominican Republic.

BACKGROUND: In malaria elimination settings, available metrics for malaria surveillance have been insufficient to measure the performance of passive case detection adequately. An indicator for malaria suspected cases with malaria test (MSCT) is proposed to measure the rate of testing on persons presenting to health facilities who satisfy the definition of a suspected malaria case. This metric does not rely on prior knowledge of fever prevalence, seasonality, or external denominators, and can be used to compare detection rates in suspected cases within and between countries, including across settings with different levels of transmission. METHODS: To compute the MSCT, an operational definition for suspected malaria cases was established, including clinical and epidemiological criteria. In general, suspected cases included: (1) persons with fever detected in areas with active malaria transmission; (2) persons with fever identified in areas with no active transmission and travel history to, or residence in areas with active transmission (either national or international); and (3) persons presenting with fever, chills and sweating from any area. Data was collected from 9 countries: Belize, Colombia (in areas with active transmission), Costa Rica, Dominican Republic, El Salvador, Guatemala, Honduras, Nicaragua, and Panama (September-March 2020). A sample of eligible medical records for 2018 was selected from a sample of health facilities in each country. An algorithm was constructed to assess if a malaria test was ordered or performed for cases that met the suspected case definition. RESULTS: A sample of 5873 suspected malaria cases was obtained from 239 health facilities. Except for Nicaragua and Colombia, malaria tests were requested in less than 10% of all cases. More cases were tested in areas with active transmission than areas without cases. Travel history was not systematically recorded in any country. CONCLUSIONS: A statistically comparable, replicable, and standardized metric was proposed to measure suspected malaria cases with a test (microscopy or rapid diagnostic test) that enables assessing the performance of passive case detection. Cross-country findings have important implications for malaria and infectious disease surveillance, which should be promptly addressed as countries progress towards malaria elimination. Local and easy-to-implement tools could be implemented to assess and improve passive case detection.

Agronomic efficiency and profitability of cotton on integrated use of phosphorus and plant microbes / Eficiência e rentabilidade agronômica do algodão no uso integrado de fósforo e micróbios vegetais

Cotton crop, plays a significant role in Pakistans economy by ruling a prominent place in edible oil and local textile industry. Phosphorus (P) inaccessibility and deficiency of soil organic matter are the key restraints for low crop productivity in cotton. Therefore, a two years field study was designed during 2014-15, to explore the influence of phosphate solubilizing bacteria (PSB), farmyard manure (FYM), poultry manure (PM) and inanimate sources of P on various physiological, growth, yield and quality parameters of cotton crop at CCRI Multan. Field responses of seeds inoculated with two distinctive phosphate solubilizing bacteria (PSB) strains viz. S0 = control, S1 =strain-1, S2 = strain-2 and eight organic, inorganic P sources viz., P0= control, P1 = 80 kg ha-1 P from inorganic source, P2 = 80 kg ha-1 P from FYM, P3 = 80 kg ha-1 P from PM, P4 = 40 kg ha-1 P from FYM + 40 kg ha-1 P from inorganic source, P5 = 40 kg ha-1 P from PM + 40 kg ha-1 P from inorganic source, P6 = 80 kg ha-1 P from FYM + 40 kg ha-1 P from inorganic source, P7 = 80 kg ha-1 P from PM + 40 kg ha-1 P from inorganic source and P8 = 40 kg ha-1 P from FYM + 40 kg ha-1 P from PM were evaluated. Results revealed that inoculation of seeds with PSB and collective use of inorganic and organic sources of P had considerably increased the yield contributing attributes in cotton. However, the treatment P7 (80 kg P ha-1 from PM + 40 kg P ha-1 from inorganic source) in coincidence with seeds inoculated with PSB (S1) produced taller plant, maximum boll weight, significantly higher LAI and CGR. Significantly higher seed cotton yield, lint yield, fiber length and maximum BCR of 1.95 and 1.81 was also obtained from the P7 treatment during both crop-growing seasons. In conclusion, combined use of 80 kg P ha-1 from PM + 40 kg P ha-1 from inorganic source and cotton seeds inoculated with strain-1 improved phosphorus uptake ensuing in greater consumption of photo-assimilates for maximum growth and yield.(AU)

A safra de algodão, desempenha um papel significativo na economia do Paquistão, ao ocupar um lugar de destaque no óleo comestível e na indústria têxtil local. A inacessibilidade e a deficiência de fósforo (P) da matéria orgânica do solo são as principais restrições para a baixa produtividade das culturas em algodão. Portanto, um estudo de campo de dois anos foi desenvolvido durante 2014-15, para explorar a influência de bactérias solubilizantes de fosfato (PSB), esterco de capoeira (FYM), esterco de aves (PM) e fontes inanimadas de P sobre vários fatores fisiológicos, crescimento, rendimento e parâmetros de qualidade da cultura do algodão no CCRI Multan. Respostas de campo de sementes inoculadas com duas cepas distintas de bactérias solubilizantes de fosfato (PSB) viz. S0 = controle, S1 = cepa-1, S2 = cepa-2 e oito fontes orgânicas de P inorgânicas viz., P0 = controle, P1 = 80 kg ha-1 P de fonte inorgânica, P2 = 80 kg ha-1 P de FYM, P3 = 80 kg ha-1 P de PM, P4 = 40 kg ha-1 P de FYM + 40 kg ha-1 P de fonte inorgânica, P5 = 40 kg ha-1 P de PM + 40 kg ha-1 P de fonte inorgânica, P6 = 80 kg ha-1 P de FYM + 40 kg ha-1 P de fonte inorgânica, P7 = 80 kg ha-1 P de PM + 40 kg ha-1 P de fonte inorgânica e P8 = 40 Foram avaliados kg ha-1 P da FYM + 40 kg ha-1 P da PM. Os resultados revelaram que a inoculação de sementes com PSB e o uso coletivo de fontes inorgânicas e orgânicas de P aumentaram consideravelmente os atributos que contribuem para a produtividade no algodão. No entanto, o tratamento P7 (80 kg P ha-1 da PM + 40 kg P ha-1 da fonte inorgânica) em coincidência com sementes inoculadas com PSB (S1) produziu planta mais alta, peso máximo de cápsula, IAF e CGR significativamente maiores. Produtividade significativamente maior do algodão, rendimento de fiapos, comprimento da fibra e BCR máximo de 1,95 e 1,81 também foi obtida a partir do tratamento P7 durante as duas épocas de cultivo. Em conclusão, o uso combinado de 80 kg P ha-1 de PM + 40 kg P ha-1 de fonte inorgânica e sementes de algodão inoculadas com a cepa-1 melhoraram a captação de fósforo, resultando em maior consumo de foto assimilados para obter crescimento e produtividade máximos.(AU)

Agronomic efficiency and profitability of cotton on integrated use of phosphorus and plant microbes.

Cotton crop, plays a significant role in Pakistan's economy by ruling a prominent place in edible oil and local textile industry. Phosphorus (P) inaccessibility and deficiency of soil organic matter are the key restraints for low crop productivity in cotton. Therefore, a two years field study was designed during 2014-15, to explore the influence of phosphate solubilizing bacteria (PSB), farmyard manure (FYM), poultry manure (PM) and inanimate sources of P on various physiological, growth, yield and quality parameters of cotton crop at CCRI Multan. Field responses of seeds inoculated with two distinctive phosphate solubilizing bacteria (PSB) strains viz. S0 = control, S1 =strain-1, S2 = strain-2 and eight organic, inorganic P sources viz., P0= control, P1 = 80 kg ha-1 P from inorganic source, P2 = 80 kg ha-1 P from FYM, P3 = 80 kg ha-1 P from PM, P4 = 40 kg ha-1 P from FYM + 40 kg ha-1 P from inorganic source, P5 = 40 kg ha-1 P from PM + 40 kg ha-1 P from inorganic source, P6 = 80 kg ha-1 P from FYM + 40 kg ha-1 P from inorganic source, P7 = 80 kg ha-1 P from PM + 40 kg ha-1 P from inorganic source and P8 = 40 kg ha-1 P from FYM + 40 kg ha-1 P from PM were evaluated. Results revealed that inoculation of seeds with PSB and collective use of inorganic and organic sources of P had considerably increased the yield contributing attributes in cotton. However, the treatment P7 (80 kg P ha-1 from PM + 40 kg P ha-1 from inorganic source) in coincidence with seeds inoculated with PSB (S1) produced taller plant, maximum boll weight, significantly higher LAI and CGR. Significantly higher seed cotton yield, lint yield, fiber length and maximum BCR of 1.95 and 1.81 was also obtained from the P7 treatment during both crop-growing seasons. In conclusion, combined use of 80 kg P ha-1 from PM + 40 kg P ha-1 from inorganic source and cotton seeds inoculated with strain-1 improved phosphorus uptake ensuing in greater consumption of photo-assimilates for maximum growth and yield.

Agronomic efficiency and profitability of cotton on integrated use of phosphorus and plant microbes / Eficiência e rentabilidade agronômica do algodão no uso integrado de fósforo e micróbios vegetais

Cotton crop, plays a significant role in Pakistan's economy by ruling a prominent place in edible oil and local textile industry. Phosphorus (P) inaccessibility and deficiency of soil organic matter are the key restraints for low crop productivity in cotton. Therefore, a two years field study was designed during 2014-15, to explore the influence of phosphate solubilizing bacteria (PSB), farmyard manure (FYM), poultry manure (PM) and inanimate sources of P on various physiological, growth, yield and quality parameters of cotton crop at CCRI Multan. Field responses of seeds inoculated with two distinctive phosphate solubilizing bacteria (PSB) strains viz. S0 = control, S1 =strain-1, S2 = strain-2 and eight organic, inorganic P sources viz., P0= control, P1 = 80 kg ha-1 P from inorganic source, P2 = 80 kg ha-1 P from FYM, P3 = 80 kg ha-1 P from PM, P4 = 40 kg ha-1 P from FYM + 40 kg ha-1 P from inorganic source, P5 = 40 kg ha-1 P from PM + 40 kg ha-1 P from inorganic source, P6 = 80 kg ha-1 P from FYM + 40 kg ha-1 P from inorganic source, P7 = 80 kg ha-1 P from PM + 40 kg ha-1 P from inorganic source and P8 = 40 kg ha-1 P from FYM + 40 kg ha-1 P from PM were evaluated. Results revealed that inoculation of seeds with PSB and collective use of inorganic and organic sources of P had considerably increased the yield contributing attributes in cotton. However, the treatment P7 (80 kg P ha-1 from PM + 40 kg P ha-1 from inorganic source) in coincidence with seeds inoculated with PSB (S1) produced taller plant, maximum boll weight, significantly higher LAI and CGR. Significantly higher seed cotton yield, lint yield, fiber length and maximum BCR of 1.95 and 1.81 was also obtained from the P7 treatment during both crop-growing seasons. In conclusion, combined use of 80 kg P ha-1 from PM + 40 kg P ha-1 from inorganic source and cotton seeds inoculated with strain-1 improved phosphorus uptake ensuing in greater consumption of photo-assimilates for maximum growth and yield.

A safra de algodão, desempenha um papel significativo na economia do Paquistão, ao ocupar um lugar de destaque no óleo comestível e na indústria têxtil local. A inacessibilidade e a deficiência de fósforo (P) da matéria orgânica do solo são as principais restrições para a baixa produtividade das culturas em algodão. Portanto, um estudo de campo de dois anos foi desenvolvido durante 2014-15, para explorar a influência de bactérias solubilizantes de fosfato (PSB), esterco de capoeira (FYM), esterco de aves (PM) e fontes inanimadas de P sobre vários fatores fisiológicos, crescimento, rendimento e parâmetros de qualidade da cultura do algodão no CCRI Multan. Respostas de campo de sementes inoculadas com duas cepas distintas de bactérias solubilizantes de fosfato (PSB) viz. S0 = controle, S1 = cepa-1, S2 = cepa-2 e oito fontes orgânicas de P inorgânicas viz., P0 = controle, P1 = 80 kg ha-1 P de fonte inorgânica, P2 = 80 kg ha-1 P de FYM, P3 = 80 kg ha-1 P de PM, P4 = 40 kg ha-1 P de FYM + 40 kg ha-1 P de fonte inorgânica, P5 = 40 kg ha-1 P de PM + 40 kg ha-1 P de fonte inorgânica, P6 = 80 kg ha-1 P de FYM + 40 kg ha-1 P de fonte inorgânica, P7 = 80 kg ha-1 P de PM + 40 kg ha-1 P de fonte inorgânica e P8 = 40 Foram avaliados kg ha-1 P da FYM + 40 kg ha-1 P da PM. Os resultados revelaram que a inoculação de sementes com PSB e o uso coletivo de fontes inorgânicas e orgânicas de P aumentaram consideravelmente os atributos que contribuem para a produtividade no algodão. No entanto, o tratamento P7 (80 kg P ha-1 da PM + 40 kg P ha-1 da fonte inorgânica) em coincidência com sementes inoculadas com PSB (S1) produziu planta mais alta, peso máximo de cápsula, IAF e CGR significativamente maiores. Produtividade significativamente maior do algodão, rendimento de fiapos, comprimento da fibra e BCR máximo de 1,95 e 1,81 também foi obtida a partir do tratamento P7 durante as duas épocas de cultivo. Em conclusão, o uso combinado de 80 kg P ha-1 de PM + 40 kg P ha-1 de fonte inorgânica e sementes de algodão inoculadas com a cepa-1 melhoraram a captação de fósforo, resultando em maior consumo de foto assimilados para obter crescimento e produtividade máximos.