RESUMEN
Nano-based drug delivery systems are increasingly used for diagnosis, prevention and treatment of several diseases, thanks to several beneficial properties, including the ability to target specific cells or organs, allowing to reduce treatment costs and side effects frequently associated with chemotherapeutic medications, thereby improving treatment compliance of patients. In the field of communicable diseases, especially those caused by intracellular bacteria, the delivery of antibiotics targeting specific cells is of critical importance to maximize their treatment efficacy. Brucella melitensis, an intracellular obligate bacterium surviving and replicating inside macrophages is hard to be eradicated, mainly because of the low ability of antibiotics to enter these phagocityc cells . Although different antibiotics regimens including gentamicin, doxycycline and rifampicin are in fact used against the Brucellosis, no efficient treatment has been attained yet, due to the intracellular life of the respective pathogen. Nano-medicines responding to environmental stimuli allow to maximize drug delivery targeting macropages, thereby boosting treatment efficacy. Several drug delivery nano-technologies, including solid lipid nanoparticles, liposomes, chitosan, niosomes, and their combinations with chitosan sodium alginate can be employed in combination of antibiotics to successfully eradicate Brucellosis infection from patients.
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Brucella melitensis , Brucelosis , Quitosano , Humanos , Quitosano/farmacología , Brucelosis/tratamiento farmacológico , Brucelosis/microbiología , Brucelosis/prevención & control , Antibacterianos/uso terapéutico , Antibacterianos/farmacología , Sistemas de Liberación de MedicamentosRESUMEN
CRISPR/Cas9 technology is one of the common methods of genome editing and targeted gene mutation, which has recently been used for manipulating microalgae such as Chlamydomonas reinhardtii. Besides, this technology can play a role in the fight against greenhouse gases (e.g., carbon dioxide) production by studying genetic pathways to improve algal strains. Among several genes in algae that respond to CO2 and regulators control the expression of each; Cia5 is one of the most critical transcriptional regulators. In this research, we knocked out the Cia5 gene using the CRISPR/Cas9 technique and analysed the ability of C. reinhardtii to perform CO2 sequestration. Our results showed that C. reinhardtii has better performance (i.e., response to CO2 treatment) in both control and mutant species at 0.5% CO2 concentration than other concentrations. However, the difference between the control microalgae species and the mutant species was in the CO2 removal efficiency. Additionally, our findings revealed that the control type isolate in CO2 concentrations of 0.04%, 0.5% and 1% had removal efficiencies of 27%, 37% and 21%, respectively. Nevertheless, for mutant species in the same concentrations, the observed removal efficiencies were 16%, 23% and 9%.
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Chlamydomonas reinhardtii , Sistemas CRISPR-Cas , Dióxido de Carbono/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Factores de Transcripción/genéticaRESUMEN
PURPOSE: Chitosan is a natural polymer that has excellent properties include biocompatibility, biodegradability, no cytotoxicity, high charge density, low cost, mucoadhesive, permeation enhancing (ability to cross tight junction), and immunomodulating ability that makes the spectrum of its applicability much broader. This study was conducted to investigate the stabilizing, preservative and immunogenicity properties of N-trimethyl chitosan nanospheres (N-TMCNS). MATERIALS AND METHODS: The tetanus toxoid (TT) was encapsulated into N-TMCNS and then characterized by scanning electron microscope, atomic force microscope, and dynamic light scattering. For stabilizer assay of N-TMCNS after storage of TT-N-TMCNS at different temperatures for 3 weeks, they were used for immunization of mice and different temperatures groups' anti-TT-N-TMCNS production compared with other groups. Finally, the immunized mice were challenged with tetanus toxin. The preservation activity of TT-N-TMCNS against Escherichia coli was compared with thimerosal formulated TT. RESULTS: Our results revealed that heat-treated TT-N-TMCNS could induce higher titer of neutralizing immunoglobulin G in compared to TT vaccine and was able to protect the mice better than TT vaccine in challenge test. Furthermore, N-TMCNS as a preservative inhibited the growth of E. coli more effective than thimerosal. CONCLUSION: Overall, the obtained results indicated that the N-TMCNS is one of the best stabilizer and preservative agent that can be used in the formulation of TT vaccine.
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BACKGROUND: Shigella flexneri is a pathogen responsible for shigellosis around the world, especially in developing countries. Many immunogenic antigens have been introduced as candidate vaccines against Shigella, including N-terminal region of IpaD antigen (NIpaD). OBJECTIVE: To evaluate the efficiency of O-metylated free trimethyl chitosan nanoparticles (TMC NPs) in the oral delivery of NIpaD. METHODS: TMC was synthesized by a two-step method from high molecular weight chitosan. The recombinant NIpaD protein was used as the immunogen. The protein was overexpressed in E. coli BL21 (DE3) and characterized by gel electrophoresis. The NIpaD-loaded TMC NPs were synthesized by ionic gelation method and were characterized by electron microscopy. NPs were orally administered to guinea pigs and specific humoral and mucosal immune responses were assessed by serum IgG and secretory IgA, respectively. The protectivity of the formulation was assessed by keratoconjunctivitis (Sereny) test. RESULTS: The immunized guinea pigs showed a significant raise in rNIpaD-specific serum IgG and faecal IgA titers. Specific secretory IgA was detected in eye-washes. Sereny test results showed that immunized animals vaccinated with IpaD-loaded TMC NPS tolerated the wild type of Shigella flexneri 2a in Sereny test. However, in the group immunized with NIpaD antigen and non-immunized group, no increase was observed in antibody titer against NIpaD. These animals were infected following the challenge with Shigella flexneri 2a (p<0.0152). CONCLUSION: The recombinant rNIpaD formulated with TMC obtained from high molecular weight chitosan, can be considered as a mucosal vaccine against Shigella flexneri through oral route.
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Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Disentería Bacilar/inmunología , Nanopartículas/metabolismo , Vacunas contra la Shigella/metabolismo , Shigella flexneri/fisiología , Administración Oral , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Quitosano/química , Modelos Animales de Enfermedad , Cobayas , Humanos , Inmunidad Mucosa , Inmunización , Inmunoglobulina A/metabolismo , Inmunoglobulina G/sangre , Nanopartículas/química , Dominios Proteicos/genética , Vacunas contra la Shigella/inmunología , VacunaciónRESUMEN
Along with robust immunogenicity, an ideal vaccine candidate should be able to produce a long lasting protection. In this regard, the frequency of memory B-cells is possibly an important factor in memory B-cell persistency and duration of immunological memory. On this basis, binding domains of tetanus toxin (HcT), botulinum type A1 toxin (HcA), and heat-labile toxin (LTB) were selected as antigen models that induced long-term, midterm and short-term immune memory, respectively. In the present study, the frequency of total memory B-cells after immunization with HcT, HcA and LTB antigens after 90 and 180 days, and also after one booster, in 190 days, was evaluated. The results showed a significant correlation between frequency of total memory B-cells and duration of humoral immunity. Compared to other antigens, the HcT antibody titers and HcT total memory B-cell populations were greater and persistent even after 6 months. At 6 months after the final immunization, all HcT- and HcA-immunized mice survived against tetanus and botulinum toxins, and also LT toxin binding to GM1 ganglioside was blocked in LTB-immunized mice. We conclude the frequency of memory B-cells and their duration are likely a key factor for vaccine memory duration.
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Antígenos Bacterianos/inmunología , Subgrupos de Linfocitos B/inmunología , Toxinas Bacterianas/inmunología , Toxinas Botulínicas/inmunología , Enterotoxinas/inmunología , Proteínas de Escherichia coli/inmunología , Memoria Inmunológica , Toxina Tetánica/inmunología , Animales , Antígenos Bacterianos/administración & dosificación , Toxinas Bacterianas/administración & dosificación , Toxinas Botulínicas/administración & dosificación , Enterotoxinas/administración & dosificación , Proteínas de Escherichia coli/administración & dosificación , Ratones , Toxina Tetánica/administración & dosificación , Factores de TiempoRESUMEN
BACKGROUND: Final elimination of some intracellular bacterial agents, such as Brucella, is often a complex issue and impossible to achieve, primarily due to the presence and survival of the bacteria within phagocytic cells. By penetrating into the cell membrane, drug delivery nanosystems can reduce the number of intracellular bacteria. The aim of this study was to assess the efficacy of chitosan nanoparticles on the delivery of gentamicin into Brucella infected J774A.1 murine cells in vitro. MATERIALS AND METHODS: Chitosan nanoparticles (NPs) were synthesized using ionic gelation technique. The shape, size and charge of NPs, loading rate and release of the drug were investigated. Finally, the effects of gentamicin-loaded chitosan NPs (Gen-Cs) and free gentamicin on J774A.1 murine cells infected with these bacteria were examined. RESULTS: The mean size and charge of NPs were computed as 100 nm and +28mV, respectively. The loading capacity of NPs was 22%. About 70% of the drug was released from NPs during the first 8 hours. Antimicrobial activity of the two formulations showed that MIC (minimum inhibitory concentration) of the Gen-Cs and free drug was 3.1 and 6.25 µg, respectively. The minimum bactericidal concentration of the NPs-loaded drug and free drug was 6.25 and 12.5 µg, respectively. Cell culture analysis revealed that there was a significant reduction in the load of the intercellular bacteria in J774A.1 murine cells in both formulations. CONCLUSION: Our results showed the Gen-Cs have a proper potential for optimal treatment of intracellular bacterial agents.
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Lung cancer, the leading cause of cancer mortality, needs urgent development of newly qualified diagnostic and therapeutic biomarkers. Recently, Human Endogenous Retroviruses (HERVs) have been introduced for cancer diagnosis. In this case-control study, we have collected blood samples from 60 lung cancer patients and 20 healthy controls. Quantitative gene expression analysis of various HERV env genes, including HERV-R, HERV-H, HERV-K, and HERV-P was performed by real-time PCR. Results indicate that expression of all four HERV env mRNAs is significantly increased in the blood of lung cancer patients than healthy controls (P-values<0.01). Furthermore, we have observed a positive and significant pairwise correlation between the expressions of four HERV env genes. The level of HERV env transcript in the blood of adenocarcinoma patients was generally much higher than squamous cell carcinoma (SCC) and small-cell lung cancer (SCLC) patients. Also, the expression of three HERV P, HERV H, and HERV K in the blood of lung cancer patients could significantly differentiate between adenocarcinoma and other types of lung cancer. In conclusion, these four HERV families could be considered as promising non-invasive blood-based biomarkers for prognosis, early detection, and monitoring of lung cancer.
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Adenocarcinoma/sangre , Retrovirus Endógenos/genética , Productos del Gen env/sangre , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/virología , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Productos del Gen env/genética , Humanos , Neoplasias Pulmonares/virología , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Recombinant vaccine technology is one of the most developed means in controlling infectious diseases. However, an effective vaccine against Shigella is still missing. OBJECTIVE: To evaluate recombinant IpaC protein of Shigella as a vaccine candidate. METHODS: In this study we cloned IpaC gene into an expression vector in prokaryotic system. The protein expression was evaluated by SDS-PAGE and Western-Blotting analysis. The recombinant protein was purified using Ni-NTA affinity chromatography. Guinea pigs were immunized with the recombinant protein and the level of immunogenicity was examined by ELISA and Western blotting of IpaC. Challenge test was done through the intraoculary injection of Shigella dysenteriae (6×108 CFU/eye) and after 48 hours was scored for keratoconjunctivitis. RESULTS: The results showed a remarkable level of immunogenicity in terms of antibody response and protection against keratoconjunctivitis in tested animals. The recombinant IpaC protein provided a protective system against Shigella dysenteriae type I during the challenge test. CONCLUSION: The results showed the potential of using recombinant IpaC in preparation of vaccine in perspective studies.
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Antígenos Bacterianos/inmunología , Disentería Bacilar/inmunología , Vacunas contra la Shigella/inmunología , Shigella dysenteriae/inmunología , Animales , Formación de Anticuerpos , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/genética , Disentería Bacilar/complicaciones , Ingeniería Genética , Cobayas , Humanos , Inmunización , Queratoconjuntivitis/prevención & control , Masculino , Vacunas contra la Shigella/administración & dosificación , Vacunas contra la Shigella/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Shigella is known as pathogenic intestinal bacteria in high dispersion and pathogenic bacteria due to invasive plasmid antigen (Ipa). So far, a number of Ipa proteins have been studied to introduce a new candidate vaccine. Here, for the first time, we examined whether the N-terminal region of IpaD(72-162) could be a proper candidate for Shigella vaccine. Initially, the DNA sequence coding N-terminal region was isolated by PCR from Shigella dysenteriae type I and cloned into pET-28a expression vector. Then, the heterologous protein was expressed, optimized and purified by affinity Ni-NTA column. Western blot analysis using, His-tag and IpaD(72-162) polyclonal antibodies, confirmed the purity and specificity of the recombinant protein, respectively. Subsequently, the high immunogenicity of the antigen was shown by ELISA. The results of the sereny test in Guinea pigs showed that IpaD(72-162) provides a protective system against Shigella flexneri 5a and S. dysenteriae type I.
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Antígenos Bacterianos , Clonación Molecular , Disentería Bacilar/prevención & control , Vacunas contra la Shigella , Shigella dysenteriae , Shigella flexneri , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/farmacología , Disentería Bacilar/genética , Disentería Bacilar/inmunología , Expresión Génica , Cobayas , Masculino , Reacción en Cadena de la Polimerasa , Estructura Terciaria de Proteína , Vacunas contra la Shigella/biosíntesis , Vacunas contra la Shigella/química , Vacunas contra la Shigella/inmunología , Vacunas contra la Shigella/farmacologíaRESUMEN
Recombinant Escherichia coli displaying organophosphorus hydrolase (OPH) was used to overcome the diffusion barrier limitation of organophosphorus pesticides. A new anchor system derived from the N-terminal domain of ice-nucleation protein from Pseudomonas syringae InaV (InaV-N) was used to display OPH onto the surface. The designed sequence was cloned in the vector pET-28a(+) and then was expressed in E. coli. Tracing of the expression location of the recombinant protein using SDS-PAGE showed the presentation of OPH by InaV-N on the outer membrane, and the ability of recombinant E. coli to utilize diazinon as the sole source of energy, without growth inhibition, indicated its significant activity. The location of OPH was detected by comparing the activity of the outer membrane fraction with the inner membrane and cytoplasm fractions. Studies revealed that recombinant E. coli can degrade 50% of 2 mM chlorpyrifos in 2 min. It can be concluded that InaV-N can be used efficiently to display foreign functional protein, and these results highlight the high potential of an engineered bacterium to be used in bioremediation of pesticide-contaminated sources in the environment.
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Arildialquilfosfatasa/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Arildialquilfosfatasa/genética , Proteínas de la Membrana Bacteriana Externa/genética , Cloropirifos/metabolismo , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos , Organismos Modificados Genéticamente , Plásmidos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Cancer is now the main cause of increasing mortality throughout the world. Minor alterations in the cell cycle which are inherited and not removed by apoptosis are important risk factors. Blood cancers are asmong the types which most readily cause death. Here in this study, usual but important factors such as age, gender, Rh and ABO blood typing, weight, and platelet counts are analyzed for impact on blood cancers. Frequencies and distributions, correlations and chi-square test were utilized in order to clarify the perspective of important factors. Our statistical results show males and females to have same risk in blood cancer but A blood type (40%) along with positive Rh (73%) had the highest risk. Low platelet counts are related to more than 80% of cases. Obesity has a statistically ignorable role in blood cancer prevalence. The fact that blood cancer cases increase during the second decade of life (45.7%) which might be because of involvement of maturation processes.
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Neoplasias Hematológicas , Neoplasias , Humanos , Irán/epidemiología , Prevalencia , Factores de RiesgoRESUMEN
BACKGROUND: The combustion of sulfur-rich fossil fuels leads to release of sulfur oxide pollution in the environment. In biodesulfurization process, an organism is able to remove sulfur from fossil fuels without decreasing the caloric value of those substrates. The main aim of this research was to design a recombinant microorganism to remove the highest amount of sulfur compounds in fossil fuels. METHODS: Three genes (dszA,B,C) from dsz operon are responsible for the 4S pathway (biodesulfurization pathway) in Rhodococcus erythropolis IGTS8 were inserted into the chromosome of a novel indigenous Pseudomonas putida. The reaction catalyzed by products of dszA,B,C genes require FMNH2 supplied by dszD enzyme. Thus, pVLT31 vector harboring dszD gene was transferred into this recombinant strain. RESULTS: The results demonstrated a higher biodesulfurization activity when the flavin reductase gene was transferred into recombinant P. putida harboring dszA,B,C. These results were approved by the Gibbs test and HPLC analysis. CONCLUSION: These analyses showed that this novel indigenous engineered P. putida could be a promising candidate for an industrial and environmental application for Biodesulfurization process.