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Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance.
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BACKGROUND: The coronavirus disease of 2019 (COVID-19) pandemic was associated with a high mortality rate. It posed a formidable challenge to healthcare systems worldwide. In this study, we evaluated the predictive value of the blood urea nitrogen (BUN)/albumin ratio as a mortality marker in patients with moderate to severe COVID-19 infection in the emergency department (ED). METHODOLOGY: A retrospective evaluation of 352 patients with moderate to severe COVID-19 infections was conducted. Out of the 352 patients, 183 (51.99%) were discharged and 169 (48.01%) succumbed. Comprehensive demographic, clinical, biochemical, and haematological data was compiled for each patient. BUN to albumin ratios were determined for all patients, and all data were compared between survivors and non-survivors. RESULTS: This study included 352 patients. The average length of stay in the hospital was 13 days. In the survivor group, the median BAR value was 0.012, but in the non-survivor group, it was 0.022 (p > 0.001). Also, it was determined that the differences in creatinine, BUN, and albumin between the two groups were statistically significant. The median BAR value was significantly higher in the non-survivor group (0.022 [0.014-0.033]) as opposed to the survivor group. Also, the median values of creatinine were higher and albumin was lower in the non-survivor group. This difference was statistically significant. CONCLUSION: The BUN/albumin ratio can be utilized as a marker of mortality in patients with COVID-19 infection presenting to the emergency department.
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Metal-organic framework (MOF) based proton conductors are synthesized by the Avrami model (time-temperature modalities). Our objective here is to obtain a material with high proton conductivity in anhydrous conditions, improved catalytic behaviour and morphology control of conductivity, band gap and catalysis. For this purpose, we try to understand the role of morphology on mass transportation using computational fluid dynamics and the experimental realisation using the synthesis of MOF membranes with high protonic conductivity. In order to alter the morphology, the membranes are synthesized from protic ionic liquid (dimethyl ethyl amine H2PO4) and metal ion (Co3O4) at different temperatures and duration. A high protonic conductivity of 0.0286 S cm-1 with a high transference number >0.99 is observed in anhydrous conditions with the change in morphology. Furthermore, catalyst properties along with high activity (Tafel slope = 39 mV decade-1) with the alteration in morphology are also investigated in detail and observed adsorption governed conduction. This adsorption governed conduction is verified using computational fluid dynamics simulations with the alteration in morphology. This study suggests that morphology not only plays a pivotal role in obtaining a robust proton exchange membrane, it also improves the catalytic functionality and stability of the membrane.
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BACKGROUND: Urinary tract infections (UTIs) are more common in females than males and predominantly based on their unhealthy practices in their day to day lives. This study is trying to assess the occurrence of UTI and strategies used by female student to prevent UTI. MATERIALS AND METHODS: A cross-sectional descriptive study carried out in a tertiary care teaching institute. A total of 338 conveniently selected B. Sc. Nursing and MBBS Medical students were surveyed. The data were collected by using a pretested, a self-structured questionnaire related to occurrence and practices to prevent UTI along with necessary demographic details. Descriptive (frequency, percentage, mean and standard deviation) and inferential (Chi-square test) statistics was used to compute the data. RESULTS: The result of this study shows that 10.05% female students reported of having UTI. The proportion was higher in nursing students (67.6%) than medical (32.4%) but did not show a significance difference (P = 0.61). Age, duration of hostel life, and marital status were significantly associated with the status of UTI (P = 0.001). To prevent UTI, students were using commercial product with Tea Tree Oil, soap, drinking 3-4 l water/day, increased frequency of peri-wash during menstruation period, and use of Citrus Fruits. The use of western toilet was also significantly associated with status of UTI (P = 0.04). CONCLUSION: The occurrence of UTI was more among nursing students. Most of the perineal hygienic practices by the female students were associated with occurrence of UTI; hence, it is necessary to emphasize on preventive practices to minimize re-occurrence.
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BACKGROUND: Coronavirus disease 2019 (COVID19) has evolved as a global pandemic. The patients with COVID-19 infection can present as mild, moderate, and severe disease forms. The reported mortality of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) infection is around 6.6%, which is lower than that of SARS-CoV and (middle east respiratory syndrome CoV). However, the fatality rate of COVID-19 infection is higher in the geriatric age group and in patients with multiple co-morbidities. The study aimed to evaluate the utility of early warning scores (EWS) to predict mortality in patients with moderate to severe COVID-19 infection. METHODS: This retrospective study was carried out in a tertiary care institute of Uttarakhand. Demographic and clinical data of the admitted patients with moderate-to-severe COVID-19 infection were collected from the hospital record section and utilized to calculate the EWS-National early warning score (NEWS), modified early warning score (MEWS), Rapid Acute Physiology Score (RAPS), rapid emergency medicine score (REMS), and worthing physiological scoring system (WPS). RESULTS: The area under the curve for NEWS, MEWS, RAPS, REMS, and WPS was 0.813 (95% confidence interval [CI]; 0.769-0.858), 0.770 (95% CI; 0.717-0.822), 0.755 (95% CI; 0.705-0.805), 0.892 (95% CI; 0.859-0.924), and 0.892 (95% CI; 0.86-0.924), respectively. CONCLUSION: The EWS at triage can be used for early assessment of severity as well as predict mortality in patients with COVID-19 patients.
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BACKGROUND: Coronavirus disease 2019 (COVID-19) was first reported in Wuhan, China in December 2019. It is caused by SARS-CoV-2, a beta coronavirus. In this study, we assessed the association of biomarkers such as neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR) and lymphocyte monocyte ratio (LMR) with the severity of COVID-19 in patients. METHODS: This retrospective observational study was carried out at a tertiary care hospital of the sub-Himalayan region of Uttarakhand over a period of six months from May to October 2020. A total of 350 patients with confirmed RT-PCR COVID-19 infection were included in the study. Detailed clinical, demographic and biochemical data of each patient was obtained from the hospital record section after permission from the Institute Ethical Committee. NLR, PLR and LMR ratios were calculated and compared with the outcomes in each patient. The patients were subdivided into two sub-groups: those with saturation less than 94% and those with saturation more than 94%. The patients were categorised as mild (with SpO2 of > 94%) and moderate-severe (with SpO2 of ≤94%) based on oxygen saturation. RESULTS: A total of 350 patients with Covid-19 pneumonia were enrolled in the study. The mean age of the patients with oxygen saturation of ≤94% was 54.91 ± 13.29 years, which was comparable to the other group. Absolute neutrophil count (ANC) and NLR were significantly higher in patients with a saturation of < 94%. However, LMR and PLR were significantly lower in the group with saturation of <94%. Thus, a significant association was found between haematological inflammatory ratios and the severity of COVID-19 infection. CONCLUSION: NLR, LMR and PLR ratios can be utilised as point of care markers to assess severity in patients with COVID-19 pneumonia.
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In India telemedicine is ideal because of its diverse landmass, difficult terrain and huge population. India, a developing country is witnessing an increase in economic growth but growing health expenditure is still posing a challenge. Telemedicine offers a solution to bridge the gap between resources available and growing needs in the health care set up. It offers an opportunity for effective collaboration of primary and secondary health care setups and to reach population staying in underserved areas. The purpose of this article is to discuss current scenario, future possibilities and applicability of telemedicine in hilly and remote areas in India.
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Breast regression protein 39 (BRP39) is a 39 kDa protein that is a member of chitolectin class of glycosyl hydrolase family 18 (GH18). High expression levels of BRP39 have been detected in breast carcinoma. It helps in proliferation of cells during the progression of this disease and may act as a signaling factor. BRP39 may act as a potential candidate for rational structure-based drug design against breast carcinoma. In this study, we report the crystal structure of mouse recombinant BRP39 expressed in E. coli. The structure was solved by molecular replacement and refined to 2.6 Å resolution. The overall structure of BRP39 consisted of two globular domains: a large (ß/α)8 triosephosphate isomerase (TIM) barrel domain and a small (α + ß) domain. Three non-proline cis-peptides were detected in the sugar-binding cleft of BRP39, including Ser57-Phe58, Leu141-Tyr142, and Trp353-Ala354. The latter residues were conserved in other GH18 family members. It was notable that the conformation of critical Trp100 residue within the sugar-binding cleft was oriented away from the barrel. The side-chain conformation was found to be similar to that observed in chitinases, however, it was oriented into the barrel in other chitinase-like proteins (CLPs). The conformation of this critical residue may have significant implications in sugar binding. Further, two amino acid substitutions were observed in the sugar-binding groove of BRP39. The conserved Asn100 and Arg263 in Hcgp39 and other CLPs proteins (SPX-40 structures) were substituted by Lys101 and Lys264 in BRP39 which may have a significant impact on the sugar-binding properties.
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Proteína 1 Semelhante à Quitinase-3/química , Proteína 1 Semelhante à Quitinase-3/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Proteína 1 Semelhante à Quitinase-3/genética , Proteína 1 Semelhante à Quitinase-3/isolamento & purificação , Quitinases/química , Cristalização , Cristalografia por Raios X , Escherichia coli/genética , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Açúcares/metabolismo , Triptofano/químicaRESUMO
Karan Fries is an Indian composite breed of cattle with superior milk production with a greater disease resistance than many other cattle. Understanding of the basic sperm and semen characteristics is necessary to develop the artificial insemination and sperm sexing methods. This study was conducted to evaluate the morphological, rheological and biochemical characteristics of Karan Fries (Indian composite breed) bull semen. Semen analysis was conducted using Computer Assisted Sperm Analysis (CASA) and rheological methods. Results from correlation assessments indicated there was a positive correlation among values for sperm velocity variables. Motility of bull sperm movements in a linear path were the important variables evaluated in this study. There was also characterization of the individual spermatozoa using scanning electron microscopy (SEM) and Raman spectroscopy. The sperm head length and width were 8.8⯱â¯0.8 and 3.9⯱â¯0.6 µm, respectively, and the approximate flagellum length was 59.4⯱â¯4.5 µm. There was a difference in the Raman intensity among the individual spermatozoa. Results of this study could be important in designing and developing methods of artificial insemination, cryopreservation of semen and semen sexing.
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Bovinos/genética , Bovinos/fisiologia , Análise do Sêmen/veterinária , Espermatozoides/fisiologia , Animais , Criopreservação/veterinária , Masculino , Preservação do Sêmen/veterinária , Motilidade dos EspermatozoidesRESUMO
Oviduct-specific glycoprotein (OVGP1) is a high molecular weight chitinase-like protein belonging to GH18 family. It is secreted by non-ciliated epithelial cells of oviduct during estrous cycle providing an essential milieu for fertilization and embryo development. The present study reports the characterization of buffalo OVGP1 through structural modeling, carbohydrate-binding properties and evolutionary analysis. Structural model displayed the typical fold of GH18 family members till the boundary of chitinase-like domain further consisting of a large (ß/α)8 TIM barrel sub-domain and a small (α+ß) sub-domain. Two critical catalytic residues were found substituted in the catalytic centre (Asp to Phe118, Glu to Leu120) compared with the active chitinase. The carbohydrate-binding groove in TIM barrel was lined with various conserved aromatic residues. Molecular docking with different sugars revealed the involvement of various residues in hydrogen-bonding and non-bonded contacts. Most of the substrate-binding residues were conserved except for a few replacements (Ser13, Lys48, Asp49, Pro50, Asp167, Glu199, Gln272 and Phe275) in comparison with other GH18 members. The residues Trp10, Trp79, Asn80, Gln272, Phe275 and Trp334 were involved in recognition of all six ligands. The α+ß sub-domain participated in sugar-binding through Thr270, Gln272, Tyr242 and Phe275. The binding assays revealed significant sugar-binding with purified native and recombinant OVGP1. Phylogenetic analysis revealed that OVGP1 was closely related to AMCases followed by other CLPs and evolution of OVGP1 occurred through several gene duplications. This is the first study describing the structural characteristics of OVGP1 that will further help to understand its interaction with gametes to perform crucial reproductive functions.
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Búfalos/genética , Glicoproteínas/ultraestrutura , Conformação Proteica , Relação Estrutura-Atividade , Animais , Domínio Catalítico/genética , Feminino , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Simulação de Acoplamento MolecularRESUMO
MGP-40 is a mammary gland-specific glycoprotein which is expressed during involution and is an important marker for mammary gland apoptosis. It is an inactive chitinase-like protein belonging to Glycosyl Hydrolase family 18. The present study reports sequence characterization, tissue-specific expression analysis, production of recombinant MGP-40 and its mutant (A117D and L119E) in both E. coli and COS1 cells for their chitin-binding and chitinase activity analysis. The cDNA of buffalo MGP-40 was cloned and sequenced which corresponded to 1803 bp with an open reading frame of 1152 bp (361 aa), signal sequence of 63 bp (21 aa), 5' and 3' UTR of 144 bp and 507 bp, respectively. The 3' UTR analysis revealed potential sites for high level expression and stability during involution. The half-life of buffalo MGP-40 was found to be 11.7 h. MGP-40 was highly expressed in mammary gland followed by small intestine, spleen and mammary epithelial cells. The purified recombinant MGP-40 and its mutant expressed in E.coli were observed to bind chitin efficiently, however, no chitinase activity was observed. Further, chitinase activity was also not observed by expressing mutant recombinant MGP-40 in COS1 cells ruling out the possible role of post-translational modifications. Structure-based in-silico mutagenesis by FoldX algorithm showed a drastic decrease in overall fold stability which might be a possible reason for inability to recover its activity. Therefore, chitinase activity could not be restored in MGP-40 even after reverting back two critical residues in active site which may be due to detrimental effect of mutations on structural stability.
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Búfalos/metabolismo , Proteína 1 Semelhante à Quitinase-3/metabolismo , Proteína 1 Semelhante à Quitinase-3/fisiologia , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Búfalos/genética , Búfalos/fisiologia , Células COS , Proteína 1 Semelhante à Quitinase-3/genética , Quitinases/genética , Quitinases/metabolismo , Chlorocebus aethiops , Clonagem Molecular/métodos , DNA Complementar/genética , Escherichia coli/genética , Feminino , Glicoproteínas/genética , Glândulas Mamárias Animais/enzimologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/fisiologia , Fases de Leitura Aberta , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/genéticaRESUMO
Crimean-Congo haemorrhagic fever (CCHF) is a tick borne viral disease reported from different parts of the world. The distribution of the CCHF cases are linked with the distribution of the principal vector, Hyalomma anatolicum in the ecosystem. Presently, vector control is mainly dependent on repeated application of acaricides, results in partial efficacy and generated acaricide resistant tick strains. Amongst the different components of integrated management programme, immunization of hosts is considered as one of the sustainable component. To restrict CCHF virus spreading, use of anti-Hyalomma vaccines appears as a viable solution. Accordingly, present study was under taken to characterize and evaluate vaccine potential of two conserved molecules, ferritin2 (FER2) and tropomyosin (TPM). Silencing of the genes conferred a cumulative reduction (rejection + unable to engorge) of 61.3% in FER2 and 70.2% in TPM respectively. Furthermore, 44.2% and 72.7% reduction in engorgement weight, 63.6% and 94.9% reduction in egg masses in FER2 and TPM silenced ticks in comparison to LUC-control group was recorded. The recombinant protein, rHaFER2 was characterized as 35 kDa protein with pI of 5.84 and possesses iron binding domains. While rHaTPM is a 51kDa protein with pI of 4.94 having calcium binding domains. Immunization of cross-bred calves by rHaFER2 conferred 51.7% and 51.2% protection against larvae and adults of H. anatolicum challenge infestations. While rHaTPM conferred 63.7% and 66.4% protection against larvae and adults infestations, respectively. The results were comparable with the data generated by RNAi and it clearly showed the possibility for the development of anti-hyalomma vaccine to manage CCHF virus and Theileria annulata infection in human and animals.
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Vetores Aracnídeos/imunologia , Doenças dos Bovinos/prevenção & controle , Ferritinas/imunologia , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Febre Hemorrágica da Crimeia/veterinária , Vacinas Protozoárias/administração & dosagem , Carrapatos/virologia , Tropomiosina/imunologia , Animais , Vetores Aracnídeos/virologia , Bovinos , Doenças dos Bovinos/virologia , Vetores de Doenças , Febre Hemorrágica da Crimeia/prevenção & controle , Febre Hemorrágica da Crimeia/virologia , Humanos , Imunização/veterinária , Masculino , Contagem de Ovos de Parasitas , Vacinas Protozoárias/imunologia , Proteínas Recombinantes/imunologiaRESUMO
Pregnancy-associated glycoproteins (PAGs) are expressed during pregnancy by the trophoectodermal cells of fetus. Presence of PAGs in dam's circulation has been widely used in pregnancy diagnosis. The present study reports the identification and characterization of different PAG isoforms in buffalo during early stages of pregnancy. The PAG mRNAs isolated from fetal cotyledons (Pregnancy stages: 45, 75 and 90 days) were successfully cloned in pJET1.2 vector and transformed in E. coli. A total of 360 random clones were sequenced and correlated with their stages of expression. A total of 12 isoforms namely, BuPAG 1, 2, 4, 6, 7, 8, 9, 13, 15, 16, 18 and one new isoform were identified. BuPAG 7 was found as the most abundant isoform in all three stages followed by BuPAG 18. Further, a large number of variants were found for most of these isoforms. Phylogenetic relationship of identified BuPAGs showed that BuPAG 2 belonged to an ancient group while other members clustered with modern group. Three-dimensional (3D) structure of BuPAG 7 was determined by homology modeling and molecular dynamic (MD) simulations which displayed a typical fold represented by other aspartic proteinase (AP) family members. Molecular docking of Pepstatin inhibitor with BuPAG 7 revealed to interact through various hydrogen bonding and hydrophobic interactions. Various amino acid substitutions were observed in peptide-binding cleft of BuPAG 7. Superimposition of BuPAG 7 with homologous structures revealed the presence of a 35-41 amino acid long insertion (alpha helix connected by two loops) near the N- terminus which seems to be a unique feature of BuPAG 7 in AP family. This is the first report on identification and sequence characterization of PAG isoforms in buffalo with unique finding that these isoforms represent many transcript variants. We also report 3D structure of the most abundant isoform BuPAG 7 for the first time.
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Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Búfalos/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Evolução Molecular , Feminino , Humanos , Modelos Moleculares , Pepstatinas/metabolismo , Filogenia , Gravidez , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Análise de Sequência de Proteína , Homologia Estrutural de Proteína , Sus scrofaRESUMO
The crosstalk between fetus and mother starts with the onset of placental attachment to the uterus. The cotyledons and caruncles are the two anatomically distinct structures that play a crucial role in this physiological communication. Using Agilent Gene Chip Genome microarray, we measured the expression profile of pregnancy cotyledons in comparison to caruncular reminiscence of the uteri in non-pregnant buffalo (Bubalus bubalis) for the detection of the early post-pregnancy rapid changes in cellular expression of mRNA transcripts. We identified a total of 497 up- and 578 down-regulated genes with <0.05 the FDR corrected p-values using 4 replicates in each group (cotyledons and caruncles) and their role in pregnancy. Deep bioinformatics analysis of data revealed the cluster of genes involved at the placentome level for various functions such as fetus attachment, transport of nutrition, and immune response. Importantly, the pathways like Hedgehog/Calcium/Wnt signalling, cell cycle regulation and immune responses regulatory functions were highly enriched by the differentially identified genes. A very highly up-regulated IL-2 specific gene showed the role of interleukin-2 signalling in the attachment of the embryo. It was observed that the genes responsible for immune response were downregulated, suggesting an immune suppressive environment which is required to adopt the semiallogeneic fetus for a successful pregnancy. To further evaluate and validate the data, we have performed qRT-PCR analysis of twenty-one genes. The present study highlights the repertoire of active transcripts in the junction of cotyledons and caruncles, which are essential for a successful onset and completion of pregnancy.
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Búfalos/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Placenta/fisiologia , Animais , Adesão Celular , Ciclo Celular , Análise por Conglomerados , Biologia Computacional , Feminino , Genômica , Sistema Imunitário , Interleucina-2/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Prenhez , Análise de Sequência de RNA , Transdução de Sinais , TranscriptomaRESUMO
BACKGROUND: An oviduct- specific glycoprotein, OVGP1, is synthesized and secreted by non-ciliated epithelial cells of the mammalian oviduct which provides an essential milieu for reproductive functions. The present study reports the effects of recombinant buffalo OVGP1 that lacks post-translational modifications, and native Buffalo OVGP1 isolated from oviductal tissue, on frozen- thawed sperm functions and in vitro embryo development. RESULTS: The proportion of viable sperms was greater (P < 0.05) in the recombinant OVGP1-treated group compared to the native OVGP1-treated group at 2 h, 3 h, and 4 h of incubation. The proportion of motile sperms at 3 h and 4 h of incubation; and membrane- intact sperms at 4 h was greater (P < 0.05) in the native OVGP1-treated group compared to the control and recombinant OVGP1-treated groups. The proportion of capacitated and acrosome- reacted sperms was greater (P < 0.05) in the native OVGP1-treated group compared to the recombinant OVGP1 group at 4 h. The rates of cleavage of embryos and their development to the blastocyst stage were greater (P < 0.05) in the presence of either native or recombinant OVGP1 in comparison to control at 10 µg/mL concentration as compared to 5 or 20 µg/mL. CONCLUSIONS: The study suggests that both native and recombinant OVGP1 impart a positive effect on various sperm features and in vitro embryo development. However, native OVGP1 was found to have a more pronounced effect in comparison to recombinant non-glycosylated OVGP1 on various sperm functions except viability. Hence, our current findings infer that glycosylation of OVGP1 might be essential in sustaining the sperm functions but not the in vitro embryo development.
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BACKGROUND: Escherichia coli have become the enterobacteriaceae species most affected by extended-spectrum ß-lactamases (ESBLs) in view of the emergence of CTX-M-type ESBLs. These CTX-M-positive E. coli have been reported in numerous regions worldwide. Virulence determinants of already reported CTX-M-positive E. coli were investigated. METHODOLOGY: To gain insights into the mechanism underlying this phenomenon, we assessed serogroup, susceptibility pattern and diversity of virulence profiles within a collection of nine bla CTX-M-positive E. coli strains and their virulent determinant using miniaturized DNA microarray techniques. The nine ESBL-positive E. coli isolates were from eight male and one female patient(s) selected for study based on previous work. Virulence potential was inferred by detection of 63 virulence factor (VF) genes. RESULTS: Four (44.4%) of the 9 E. coli isolates exhibited the same set of core characteristics: serotype O8:Hnt, while all were positive for OXA-1, ciprofloxacin resistance. Five of the isolates exhibited highly similar (91% to 100%) VF profiles. CONCLUSION: The findings describe a broadly disseminated, bla CTX-M-positive and virulent E. coli serogroup with highly homogeneous virulence genotypes, suggesting recent emergence in this zone. Understanding how this clone has emerged and successfully disseminated within the hospital and community, including across national boundaries, should be a public health priority.
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Oviductin is a high molecular weight oviduct-specific glycoprotein secreted by the non-ciliated epithelial cells of oviduct during estrous cycle and early pregnancy. It plays an important role during fertilization and early embryonic development. The oviductin gene from oviductal tissues of buffalo was successfully cloned and sequenced. The sequence analysis revealed that buffalo and cattle oviductin share very high homology between their cDNA sequences. The predicted amino acid sequences of the buffalo oviductin exhibited the highest percent of identity of 97 % with bovine followed by 94 % with goat, 93 % with sheep, 78 % with porcine, 72 % with human, 67 % with hamster and rabbit and 65 % with mouse. Oviductin was also observed to share high similarity with the mammalian chitinase, however oviductins do not show chitinase activity due to GluâIle mutation in the active site responsible for chitinase activity. The phylogenetic tree based on amino acid sequences of oviductin indicated that buffalo oviductin was closely related to its cattle counterpart, and this clustering is in accordance with the classic taxonomic relationship. Tissue specific expression of the transcripts for buffalo oviductin revealed a high level expression in oviduct and ovary followed by testis, mammary gland, kidney, while in mammary epithelial cells and liver its expression was very low. The full length matured oviductin and its domains constituting chitinase-like domain and mucin-like domain were cloned into pET and pGEX series of expression vectors and over expressed in E. coli. The soluble recombinant oviductin was successfully purified to homogeneity. Full length recombinant oviductin was expressed partially in soluble form, where as the chitinase-like and mucin-like domains of oviductin were expressed in insoluble form and aggregating to form inclusion bodies at both 37 and 16 °C induction temperatures.
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Expressão Gênica , Serina Endopeptidases/genética , Acrossomo/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Búfalos , Clonagem Molecular , Sequência Conservada , DNA Complementar/genética , Escherichia coli , Tubas Uterinas/enzimologia , Feminino , Glicoproteínas/genética , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Glicoproteínas/fisiologia , Rim/enzimologia , Masculino , Glândulas Mamárias Animais/enzimologia , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Serina Endopeptidases/fisiologia , Espermatozoides/fisiologia , Testículo/enzimologiaRESUMO
OBJECTIVES: To investigate the occurrence of antimicrobial resistance genes of human clinical relevance in Salmonella isolated from livestock in Great Britain. METHODS: Two hundred and twenty-five Salmonella enterica isolates were characterized using an antimicrobial resistance gene chip and disc diffusion assays. Plasmid profiling, conjugation experiments and identification of Salmonella genomic island 1 (SGI1) were performed for selected isolates. RESULTS: Approximately 43% of Salmonella harboured single or multiple antimicrobial resistance genes with pig isolates showing the highest numbers where 96% of Salmonella Typhimurium harboured one or more resistance genes. Isolates harbouring multiple resistances divided into three groups. Group 1 isolates harboured ampicillin/streptomycin/sulphonamide/tetracycline resistance and similar phenotypes. This group contained isolates from pigs, cattle and poultry that were from several serovars including Typhimurium, 4,[5],12:i:-, Derby, Ohio and Indiana. All Group 2 isolates were from pigs and were Salmonella Typhimurium. They contained a non-sul-type class 1 integron and up to 13 transferrable resistances. All Group 3 isolates harboured a class 1 integron and were isolated from all animal species included in the study. Most isolates were Salmonella Typhimurium and harboured SGI1. CONCLUSIONS: Salmonella isolated from livestock was shown to harbour antimicrobial resistance genes although no or little resistance to third-generation cephalosporins or ciprofloxacin, respectively, was detected. The preponderance in pigs of multidrug-resistant Salmonella Typhimurium makes it important to introduce control measures such as improved biosecurity to ensure that they do not pass through the food chain and limit human therapeutic options.