RESUMO
Brucellosis is a zoonotic disease caused by various species of the genus Brucella. The control of this disease mainly depends on its accurate and early diagnosis. Culture methods employed for diagnosis are time consuming and require well equipped biosafety level 3 laboratories and hence serological tests are favored alternative for brucellosis diagnosis. At present serological diagnosis is based on LPS (lipopolysaccharide) which is less specific as it shows cross reactivity with other gram-negative bacteria. There is a need to develop serological diagnostic assay based on purified recombinant antigen of Brucella. T4SS (Type IV Secretion System) is an important virulent factor of Brucella and required for infection suggesting their expression in vivo and can be targeted as serological marker for infection. To test this concept, the present study is designed to clone, express and purify virB10 gene of Brucella T4SS under denaturing conditions and to evaluate its use as serological marker of Brucella infection. The immunoreactivity of this recombinant antigen was checked with antisera collected after experimental infection in Balb/C mice with B. melitensis 16M, BR31 (human clinical isolate) and Y. enterocolitica O:9. The recombinant protein was also tested against a panel of 46 bovine sera samples collected from Leh, India. Antibody response against VirB10 was detected in experimental and natural host suggesting that it can be explored as potential target for serodiagnosis of Brucella infection.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Brucella melitensis/metabolismo , Brucelose/diagnóstico , Testes Sorológicos/métodos , Animais , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , Brucella melitensis/imunologia , Brucelose/imunologia , Brucelose/metabolismo , Bovinos , Expressão Gênica , Humanos , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Brucellosis is an important zoonotic disease caused by different Brucella species and human brucellosis is commonly prevalent in different states of India. Among various Brucella species, B. melitensis is most pathogenic to human and included as category B biothreat which can cause infection through aerosol, cut, wounds in skin and contact with infected animals. The diagnosis of human brucellosis is very important for proper treatment and management of disease as there is no vaccine available for human use. The present study was designed to clone, express and purify immunodominant recombinant omp2a (rOmp2a) porin protein of B. melitensis and to evaluate this new antigen candidate for specific serodiagnosis of human brucellosis by highly sensitive iELISA (indirect enzyme linked immunosorbent assay). METHOD: Omp2a gene of B. melitensis 16 M strain was cloned and expressed in pET-SUMO expression system. The recombinant protein was purified under denaturing conditions using 8 M urea. The purified recombinant protein was confirmed by western blotting by reacting with anti-HIS antibody. The sero-reactivity of the recombinant protein was also checked by reacting with antisera of experimentally infected mice with B. melitensis 16 M at different time points. Serodiagnostic potential of recombinant porin antigen was tested against 185 clinical serum samples collected from regions endemic to brucellosis in southern part of India by iELISA. The samples were grouped into five groups. Group 1 contained cultured confirmed positive serum samples of brucellosis (n = 15), group 2 contained sera samples from positive cases of brucellosis previously tested by conventional methods of RBPT (n = 28) and STAT (n = 26), group 3 contained sera samples negative by RBPT(n = 36) and STAT (n = 32), group 4 contained sera samples of other febrile illness and PUO case (n = 35) and group 5 contained confirmed negative sera samples from healthy donors (n = 23). RESULT: The rOmp2a was found to be immunoreactive by iELISA and western blotting. The test showed a sensitivity of 93.75% and specificity of 95.83% when tested against 185 serum samples. For determination of statistical significance between experimental groups and control groups, Student's t test was performed on the data. CONCLUSION: Omp2a emerges as a potential antigen candidate for serodiagnosis of human brucellosis.
Assuntos
Proteínas de Bactérias/imunologia , Brucelose/diagnóstico , Porinas/imunologia , Testes Sorológicos/métodos , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Brucella/imunologia , Brucella/patogenicidade , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Índia , Camundongos , Porinas/genética , Porinas/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
Laboratory-based evolution and whole-genome sequencing can link genotype and phenotype. We used evolution of acid resistance in exponential phase Escherichia coli to study resistance to a lethal stress. Iterative selection at pH 2.5 generated five populations that were resistant to low pH in early exponential phase. Genome sequencing revealed multiple mutations, but the only gene mutated in all strains was evgS, part of a two-component system that has already been implicated in acid resistance. All these mutations were in the cytoplasmic PAS domain of EvgS, and were shown to be solely responsible for the resistant phenotype, causing strong upregulation at neutral pH of genes normally induced by low pH. Resistance to pH 2.5 in these strains did not require the transporter GadC, or the sigma factor RpoS. We found that EvgS-dependent constitutive acid resistance to pH 2.5 was retained in the absence of the regulators GadE or YdeO, but was lost if the oxidoreductase YdeP was also absent. A deletion in the periplasmic domain of EvgS abolished the response to low pH, but not the activity of the constitutive mutants. On the basis of these results we propose a model for how EvgS may become activated by low pH.
Assuntos
Ácidos/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Evolução Molecular , Proteínas Quinases/genética , Sequência de Aminoácidos , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Estrutura Terciária de ProteínaRESUMO
Aim: To compare the effect of different analgesic regimens on the time to initiate breastfeeding (BF) and ambulation after cesarean section (CS). Methods: This prospective, double-blinded, placebo-controlled randomized study included 300 women (20-40 years of age) of the American Society of Anesthesiologists status 1 or 2 with singleton term pregnancies scheduled for CS under spinal anesthesia. Women were allocated to three groups of 100 each by computer-generated randomization. As an adjunct to 1,000 mg intravenous acetaminophen, Group 1 received 100 mg rectal diclofenac, Group 2 received 100 mg rectal tramadol, and Group 3 received rectal glycerin suppository. The time to initiate BF and ambulation was compared between different analgesic regimens and corelated with pain score. Results: BF (both with and without support) was initiated significantly earlier in Groups 1 and 2 as compared with control Group 3 (p < 0.001). A significantly shorter time was taken to initiate BF without support in Group 1 as compared with Group 2 (p = 0.028). The time to start ambulation (both with and without assistance) was significantly lower in Groups 1 and 2 as compared with Group 3 and in Group 1 versus Group 2 (p < 0.001). A significant positive correlation was found between the time to initiate BF with support and ambulation without assistance and postoperative pain score at 0, 1, and 6 hours. Conclusion: Effective post-CS analgesia affects early initiation of BF and ambulation in the immediate postnatal period. The inclusion of rectal diclofenac suppository in post-CS analgesic regimens is a promising approach to postoperative delivery care.
Assuntos
Analgesia Obstétrica , Diclofenaco , Feminino , Gravidez , Humanos , Diclofenaco/uso terapêutico , Analgésicos Opioides , Cesárea/efeitos adversos , Estudos Prospectivos , Aleitamento Materno , Dor Pós-Operatória/tratamento farmacológico , Dor Pós-Operatória/prevenção & controle , Método Duplo-Cego , CaminhadaRESUMO
The detrimental effects of high concentrations of colonic iron have been linked to intestinal inflammation and microbial dysbiosis. Exploiting chelation against this luminal pool of iron may restore intestinal health and have beneficial impacts on microbial communities. This study aimed to explore whether lignin, a heterogenous polyphenolic dietary component, has iron-binding affinity and can sequester iron within the intestine and thus, potentially modulate the microbiome. Within in vitro cell-culture models, the treatment of RKO and Caco-2 cells with lignin almost abolished intracellular iron import (96% and 99% reduction of iron acquisition respectively) with corresponding changes in iron metabolism proteins (ferritin and transferrin receptor-1) and reductions in the labile-iron pool. In a Fe-59 supplemented murine model, intestinal iron absorption was significantly inhibited by 30% when lignin was co-administered compared to the control group with the residual iron lost in the faeces. The supplementation of lignin into a microbial bioreactor colonic model increased the solubilisation and bio-accessibility of iron present by 4.5-fold despite lignin-iron chelation previously restricting intracellular iron absorption in vitro and in vivo. The supplementation of lignin in the model increased the relative abundance of Bacteroides whilst levels of Proteobacteria decreased which could be attributed to the changes in iron bio-accessibility due to iron chelation. In summary, we demonstrate that lignin is an effective luminal iron chelator. Iron chelation leads to the limitation of intracellular iron import whilst, despite increasing iron solubility, favouring the growth of beneficial bacteria.
Assuntos
Microbioma Gastrointestinal , Ferro , Humanos , Animais , Camundongos , Ferro/metabolismo , Lignina , Radioisótopos de Ferro/farmacologia , Células CACO-2 , Intestinos/microbiologia , Quelantes de Ferro/farmacologiaRESUMO
The coexistence of different mating strategies, whereby a species can reproduce both by selfing and outcrossing, is an evolutionary enigma. Theory predicts two predominant stable mating states: outcrossing with strong inbreeding depression or selfing with weak inbreeding depression. As these two mating strategies are subject to opposing selective forces, mixed breeding systems are thought to be a rare transitory state yet can persist even after multiple speciation events. We hypothesise that if each mating strategy plays a distinctive role during some part of the species life history, opposing selective pressures could be balanced, permitting the stable co-existence of selfing and outcrossing sexual morphs. In this scenario, we would expect each morph to be specialised in their respective roles. Here we show, using behavioural, physiological and gene expression studies, that the selfing (hermaphrodite) and outcrossing (female) sexual morphs of the trioecious nematode Auanema freiburgensis have distinct adaptations optimised for their different roles during the life cycle. A. freiburgensis hermaphrodites are known to be produced under stressful conditions and are specialised for dispersal to new habitat patches. Here we show that they exhibit metabolic and intestinal changes enabling them to meet the cost of dispersal and reproduction. In contrast, A. freiburgensis females are produced in favourable conditions and facilitate rapid population growth. We found that females compensate for the lack of reproductive assurance by reallocating resources from intestinal development to mate-finding behaviour. The specialisation of each mating system for its role in the life cycle could balance opposing selective forces allowing the stable maintenance of both mating systems in A. freiburgensis.
Assuntos
Evolução Biológica , Depressão por Endogamia , Animais , Feminino , Estágios do Ciclo de Vida , Reprodução/fisiologiaRESUMO
The toxic effects of excess dietary iron within the colonic lumen are well documented, particularly in the context of Inflammatory Bowel Disease (IBD) and Colorectal Cancer (CRC). Proposed mechanisms that underpin iron-associated intestinal disease include: (1) the pro-inflammatory and ROS-promoting nature of iron, (2) gene-expression alterations, and (3) intestinal microbial dysbiosis. However, to date no studies have examined the effect of iron on the colonic epigenome. Here we demonstrate that chronic iron exposure of colonocytes leads to significant hypomethylation of the epigenome. Bioinformatic analysis highlights a significant epigenetic effect on NRF2 (nuclear factor erythroid 2-related factor 2) pathway targets (including NAD(P)H Quinone Dehydrogenase 1 [NQO1] and Glutathione peroxidase 2 [GPX2]); this demethylating effect was validated and subsequent gene and protein expression quantified. These epigenetic modifications were not observed upon the diminishment of cellular lipid peroxidation with endogenous glutathione and the subsequent removal of iron. Additionally, the induction of TET1 expression was found post-iron treatment, highlighting the possibility of an oxidative-stress induction of TET1 and subsequent hypomethylation of NRF2 targets. In addition, a strong time dependence on the establishment of iron-orchestrated hypomethylation was found which was concurrent with the increase in the intracellular labile iron pool (LIP) and lipid peroxidation levels. These epigenetic changes were further validated in murine intestinal mucosa in models administered a chronic iron diet, providing evidence for the likelihood of dietary-iron mediated epigenetic alterations in vivo. Furthermore, significant correlations were found between NQO1 and GPX2 demethylation and human intestinal tissue iron-status, thus suggesting that these iron-mediated epigenetic modifications are likely in iron-replete enterocytes. Together, these data describe a novel mechanism by which excess dietary iron is able to alter the intestinal phenotype, which could have implications in iron-mediated intestinal disease and the regulation of ferroptosis.
Assuntos
Enterócitos/metabolismo , Epigênese Genética , Glutationa Peroxidase/genética , Mucosa Intestinal/metabolismo , Ferro da Dieta , Ferro/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , Animais , Células CACO-2 , Colo/metabolismo , Metilação de DNA , Epigenoma , Ferritinas/genética , Ferritinas/metabolismo , Compostos Ferrosos/farmacologia , Glutationa Peroxidase/metabolismo , Humanos , Camundongos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Nematodes belong to one of the most diverse animal phyla. However, functional genomic studies in nematodes, other than in a few species, have often been limited in their reliability and success. Here we report that by combining liposome-based technology with microinjection, we were able to establish a wide range of genomic techniques in the newly described nematode genus Auanema. The method also allowed heritable changes in dauer larvae of Auanema, despite the immaturity of the gonad at the time of the microinjection. As proof of concept for potential functional studies in other nematode species, we also induced RNAi in the free-living nematode Pristionchus pacificus and targeted the human parasite Strongyloides stercoralis.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Lipossomos , Mutagênese , Interferência de RNA , Transfecção , Animais , Sistemas CRISPR-Cas , Caenorhabditis elegans/genética , Expressão Gênica , Nematoides/genética , RNA de Cadeia Dupla/genéticaRESUMO
No ideal vaccine exists to control plague, a deadly dangerous disease caused by Yersinia pestis. In this context, we cloned, expressed and purified recombinant F1, LcrV antigens of Y. pestis and heat shock protein70 (HSP70) domain II of M. tuberculosis in E. coli. To evaluate the protective potential of each purified protein alone or in combination, Balb/C mice were immunized. Humoral and cell mediated immune responses were evaluated. Immunized animals were challenged with 100 LD50 of Y. pestis via intra-peritoneal route. Vaccine candidates i.e., F1 and LcrV generated highly significant titres of anti-F1 and anti-LcrV IgG antibodies. A significant difference was noticed in the expression level of IL-2, IFN-γ and TNF-α in splenocytes of immunized animals. Significantly increased percentages of CD4+ and CD8+ T cells producing IFN-γ in spleen of vaccinated animals were observed in comparison to control group by flow cytometric analysis. We investigated whether the F1, LcrV and HSP70(II) antigens alone or in combination can effectively protect immunized animals from any histopathological changes. Signs of histopathological lesions noticed in lung, liver, kidney and spleen of immunized animals on 3rd day post challenge whereas no lesions in animals that survived to day 20 post-infection were observed. Immunohistochemistry showed bacteria in lung, liver, spleen and kidney on 3rd day post-infection whereas no bacteria was observed on day 20 post-infection in surviving animals in LcrV, LcrV+HSP70(II), F1+LcrV, and F1+LcrV+HSP70(II) vaccinated groups. A significant difference was observed in the expression of IL-2, IFN-γ, TNF-α, and CD4+/CD8+ T cells secreting IFN-γ in the F1+LcrV+HSP70(II) vaccinated group in comparison to the F1+LcrV vaccinated group. Three combinations that included LcrV+HSP70(II), F1+LcrV or F1+LcrV+HSP70(II) provided 100% protection, whereas LcrV alone provided only 75% protection. These findings suggest that HSP70(II) of M. tuberculosis can be a potent immunomodulator for F1 and LcrV containing vaccine candidates against plague.