Draft Genome Sequence of Bacillus albus Strain IB84, Isolated from Mexican Soil.

Bacillus albus is a new species, but it lies on the borderline with Bacillus thuringiensis In this work, we report a strain previously identified as Bacillus thuringiensis IB84, which now, based on average nucleotide identity and rRNA 16S, gyrB, groEL, and xre gene sequences, must be identified as Bacillus albus.

Draft Genome Sequence of Bacillus toyonensis Strain GM18, Isolated from Agricultural Soil.

Bacillus toyonensis is a recently described species related to Bacillus cereus and Bacillus thuringiensis The GM18 strain previously identified as B. thuringiensis is now classified as B. toyonensis based on the RNA 16S sequence and whole-genome average nucleotide identity. The genome analysis revealed the presence of insecticide, nematicide, and antitumoral proteins.

Electrospun Polyvinylpyrrolidone-Gelatin and Cellulose Acetate Bi-Layer Scaffold Loaded with Gentamicin as Possible Wound Dressing.

Acceleration of wound healing can be achieved with the use of wound dressings. Through the electrospinning technique, a polymeric scaffold composed of two layers was processed: a gelatin and polyvinylpyrrolidone layer with gentamicin, and a second layer of cellulose acetate. The conditions for the electrospinning process were standardized for voltage parameters, feed flow and the distance from the injector to the collector. Once the values of the main variables for the electrospinning were optimized, a three-hour processing time was established to allow the separation of the material from the collector. The obtained material was characterized by observations on scanning electron microscopy, Fourier transform infrared spectroscopy and thermal analysis; contact angle measurement was performed to evaluate wettability properties, and antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus were evaluated using the Kirby-Bauer test. The obtained fibers that form the bi-layer scaffold present diameters from 100 to 300 nm. The scaffold presents chemical composition, thermal stability, wettability characteristics and antibacterial activity that fulfill the proposal from this study, based on obtaining a scaffold that could be used as a drug delivery vehicle and a wound dressing material.

In vitro anticancer activity of methanolic extract of Granulocystopsis sp., a microalgae from an oligotrophic oasis in the Chihuahuan desert.

With the purpose of discovering new anticancer molecules that might have fewer side effects or reduce resistance to current antitumor drugs, a bioprospecting study of the microalgae of the Cuatro Cienegas Basin (CCB), an oasis in the Chihuahuan desert in Mexico was conducted. A microalgae was identified as Granulocystopsis sp. through sequencing the rbcL gene and reconstruction of a phylogenetic tree, and its anticancer activities were assessed using various in vitro assays and different cell lines of human cancers, including lung, skin melanoma, colorectal, breast and prostatic cancers, as well as a normal cell line. The values of IC50 of the microalgae methanolic extract using the MTT assay were lower than 20 µg/ml, except that in the lung cancer line and the normal cell line. In vitro, the microalgae extract caused the loss of membrane integrity, monitored by the trypan blue exclusion test and exhibited marked inhibition of adhesion and cell proliferation in cancer cell lines, through the evaluation of the clonogenic assay. Also, typical nuclear changes of apoptotic processes were observed under the microscope, using the dual acridine orange/ethidium bromide fluorescent staining. Finally, the microalgae extract increased the activity of caspases 3 and 7 in skin melanoma, colon, breast and prostate cancer cells, in the same way as the apoptotic inductor and powerful antitumoral drug, doxorubicin. This study shows the anticancer activity from Granulocystopsis sp., a microalgae isolated from the CCB.

Anticancer potential of Thevetia peruviana fruit methanolic extract.

BACKGROUND: Thevetia peruviana (Pers.) K. Schum or Cascabela peruviana (L.) Lippold (commonly known as ayoyote, codo de fraile, lucky nut, or yellow oleander), native to Mexico and Central America, is a medicinal plant used traditionally to cure diseases like ulcers, scabies, hemorrhoids and dissolve tumors. The purpose of this study was to evaluate the cytotoxic, antiproliferative and apoptotic activity of methanolic extract of T. peruviana fruits on human cancer cell lines. METHODS: The cytotoxic activity of T. peruviana methanolic extract was carried out on human breast, colorectal, prostate and lung cancer cell lines and non-tumorigenic control cells (fibroblast and Vero), using the MTT assay. For proliferation and motility, clonogenic and wound-healing assays were performed. Morphological alterations were monitored by trypan blue exclusion, as well as DNA fragmentation and AO/EB double staining was performed to evaluate apoptosis. The extract was separated using flash chromatography, and the resulting fractions were evaluated on colorectal cancer cells for their cytotoxic activity. The active fractions were further analyzed through mass spectrometry. RESULTS: The T. peruviana methanolic extract exhibited cytotoxic activity on four human cancer cell lines: prostate, breast, colorectal and lung, with values of IC50 1.91 ± 0.76, 5.78 ± 2.12, 6.30 ± 4.45 and 12.04 ± 3.43 µg/mL, respectively. The extract caused a significant reduction of cell motility and colony formation on all evaluated cancer cell lines. In addition, morphological examination displayed cell size reduction, membrane blebbing and detachment of cells, compared to non-treated cancer cell lines. The T. peruviana extract induced apoptotic cell death, which was confirmed by DNA fragmentation and AO/EB double staining. Fractions 4 and 5 showed the most effective cytotoxic activity and their MS analysis revealed the presence of the secondary metabolites: thevetiaflavone and cardiac glycosides. CONCLUSION: T. peruviana extract has potential as natural anti-cancer product with critical effects in the proliferation, motility, and adhesion of human breast and colorectal cancer cells, and apoptosis induction in human prostate and lung cancer cell lines, with minimal effects on non-tumorigenic cell lines.

Antibody profile to Borrelia burgdorferi in veterinarians from Nuevo León, Mexico, a non-endemic area of this zoonosis.

OBJECTIVES: Lyme disease is a tick-borne disease caused by infections with Borrelia. Persons infected with Borrelia can be asymptomatic or can develop disseminated disease. Diagnosis and recognition of groups at risk of infection with Borrelia burgdorferi is of great interest to contemporary rheumatology. There are a few reports about Borrelia infection in Mexico, including lymphocytoma cases positive to B. burgdorferi sensu stricto by PCR and a patient with acrodermatitis chronica atrophicans. Veterinarians have an occupational risk due to high rates of tick contact. The aim of this work was to investigate antibodies to Borrelia in students at the Faculty of Veterinary Medicine and Zootechnics, at Nuevo León, Mexico, and determine the antibody profile to B. burgdorferi antigens. MATERIAL AND METHODS: Sera were screened using a C6 ELISA, IgG and IgM ELISA using recombinant proteins from B. burgdorferi, B. garinii and B. afzelii. Sera with positive or grey-zone values were tested by IgG Western blot to B. burgdorferi sensu stricto. RESULTS: All volunteers reported tick exposures and 72.5% remembered tick bites. Only nine persons described mild Lyme disease related symptoms, including headaches, paresthesias, myalgias and arthralgias. None of the volunteers reported erythema migrans. Nine samples were confirmed by IgG Western blot. The profile showed 89% reactivity to OspA, 67% to p83, and 45% to BmpA. CONCLUSIONS: Positive sera samples shared antibody reactivity to the markers of late immune response p83 and BmpA, even if individuals did not present symptoms of Lyme arthritis or post-Lyme disease. The best criterion to diagnose Lyme disease in our country remains to be established, because it is probable that different strains coexist in Mexico. This is the first report of antibodies to B. burgdorferi in Latin American veterinarians. Veterinarians and high-risk people should be alert to take precautionary measures to prevent tick-borne diseases.

Bacteriocins synthesized by Bacillus thuringiensis: generalities and potential applications.

The members of the Bacillus thuringiensis group, commonly known as Bt, produce a huge number of metabolites, which show biocidal and antagonistic activity. B. thuringiensis is widely known for synthesizing Cry, Vip and Cyt proteins, active against insects and other parasporins with biocidal activity against certain types of cancerous cells. Nevertheless, B. thuringiensis also synthesizes compounds with antimicrobial activity, especially bacteriocins. Some B. thuringiensis bacteriocins resemble lantibiotics and other small linear peptides (class IIa) from the lactic acid bacteria bacteriocins classification system. Although many bacteriocins produced by Bt have been reported, there is no proper classification for them. In this work, we have grouped these based on molecular weight and functionality. Bacteriocins are small peptides synthesized by bacteria, presenting inhibitory activity against Gram-positive and Gram-negative bacteria and to a lesser extent against fungi. These molecules represent a good study model in the search for microbial control alternatives. Lactic acid bacteria produces a huge number of these types of molecules with great potential. Nonetheless, members of the Bacillus, cereus group, especially B. thuringiensis, emerge as an attractive alternative for obtaining bacteriocins showing novel activities. This review describes the potential applications of B. thuringiensis bacteriocins in the control of foodborne pathogens, environment and medical area.

A Pycnoporus sanguineus laccase for denim bleaching and its comparison with an enzymatic commercial formulation.

A laccase from the basidiomycete Pycnoporus sanguineus strain RVAN5 was evaluated for its ability to decolorize synthetic dyes and denim bleaching. Dye color reduction and denim bleaching were monitored at different dye concentrations and incubation times. Dye decolorization by Pycnoporus sanguineus fungal crude extract (FCE) ranged from 80 to 96% within 2-4 h at 25-65 °C. Comparable results were obtained when violuric acid (VA) was added as mediator to the FCE, however, the number of decolorized dyes increased significantly. Dye decolorization rates with VA varied of initial and final optical density (595 nm) values of 2.5-3.0 and 0.2-0.02, respectively. P. sanguineus FCE had no substantial effect on denim bleaching when used alone, notwithstanding, the mixture of FCE with VA (10 mM) showed significant denim color reduction values and considerably higher than those obtained with a bleaching enzyme from a commercial formulation; CIElab values obtained with FCE/VA mixture were of ΔL = 6.4, versus a ΔL 1.4 value obtained with an enzyme from commercial formulation.

Shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris bioreactor cultures.

Recently, we engineered a Pichia pastoris Mut(+) strain to produce and secrete recombinant Litopenaeus vannamei trypsinogen. Despite the observed toxicity of the recombinant shrimp trypsinogen to the P. pastoris cell host, when high density cell cultures in shake flasks with alanine in the induction medium were used recombinant shrimp trypsinogen could be produced. To further improve the product yield, in this work, we evaluated L. vannamei trypsinogen production in P. pastoris using a bioreactor and two recombinant P. pastoris strains with different methanol utilization (Mut) phenotypes. The effect of pH and temperature during the induction step on the trypsinogen production was also evaluated. The results indicate that temperature, pH, and Mut phenotypes influence the production of the recombinant protein, with almost no observed effect on cell growth. All cultures with the Mut(+) strain had significant operational difficulties, such as in lowering the induction temperature, maintaining dissolved oxygen (DO) above 20%, and maintaining the methanol concentration at a constant value, and showed a decrease in metabolic activity due to trypsinogen toxicity to the cell host. In the culture with the Mut(s) strain, however, the temperature, methanol concentration, and DO could be more easily controlled, the temperature could be easily decreased, and the trypsinogen caused the lowest toxicity to the host cells. After 96 h of Mut(s) strain induction (pH 6 and 25°C), about 250 mg/L recombinant trypsinogen was detected in the culture medium.

Development and in vitro evaluation of biopolymers as a delivery system against periodontopathogen microorganisms.

Periodontal disease is the major cause of tooth loss in adults. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are considered key pathogens in periodontitis. The treatment consists of oral hygiene education, instrumentation for removal of calculus (scaling), chemotherapy and periodontal surgery. Several agents are commercially available; these chemicals can alter oral microbiota and have undesirable side-effects such as vomiting, diarrhea and tooth staining. Hence, the search for alternative products continues and natural phytochemicals isolated from plants used as traditional medicine and the use of biomaterials are considered good alternatives. Chitosan and pullulan are polymers that have been proposed due to their favorable properties such as biocompatibility, biodegradability, and adhesion ability. They can be used as local delivery systems of active principles of plant extracts. Thymus vulgaris, Matricaria chamomilla, Croton lechleri, Calendula officinalis L. and Juliana adstringens Schl. are known to have medicinal activity, and they are used in Mexican traditional medicine. Their extracts were tested in vitro for antimicrobial activity against P. gingivalis and A. actinomycetemcomitans, using agar diffusion and microdilution methods. The antimicrobial activity of films from biopolymers with plant extracts was evaluated by measuring the zones of inhibition against the tested organisms. The aim of this study was to develop bioadhesive films from chitosan and pullulan with added plant extracts and determine the antimicrobial activity of films against periodontal pathogens.

Development and in vitro evaluation of biopolymers as a delivery system against periodontopathogen microorganisms

Periodontal disease is the major cause of tooth loss in adults. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are considered key pathogens in periodontitis. The treatment consists of oral hygiene education, instrumentation for removal of calculus (scaling), chemotherapy and periodontal surgery. Several agents are commercially available; these chemicals can alter oral microbiota and have undesirable sideeffects such as vomiting, diarrhea and tooth staining. Hence, the search for alternative products continues and natural phytochemicals isolated from plants used as traditional medicine and the use of biomaterials are considered good alternatives. Chitosan and pullulan are polymers that have been proposed due to their favorable properties such as biocompatibility, biodegradability, and adhesion ability. They can be used as local delivery systems of active principles of plant extracts. Thymus vulgaris, Matricaria chamomilla, Croton lechleri, Calendula officinalis L. and Juliana adstringens Schl. are known to have medicinal activity, and they are used in Mexican traditional medicine. Their extracts were tested in vitro for antimicrobial activity against P. gingivalis and A. actinomycetemcomitans, using agar diffusion and microdilution methods. The antimicrobial activity of films from biopolymers with plant extracts was evaluated by measuring the zones of inhibition against the tested organisms. The aim of this study was to develop bioadhesive films from chitosan and pullulan with added plant extracts and determine the antimicrobial activity of films against periodontal pathogens.

La enfermedad periodontal es la principal causa de perdida de dientes en los adultos. Los agentes causales comunmente identificados con la enfermedad son Porphyromonas gingivalis y Aggregatibacter actinomycetemcomitans. El tratamiento de la enfermedad consiste en educacion sobre higiene oral, remocion de calculos por medio de instrumentacion (raspado y alisado de la raiz), la administracion de medicamentos y cirugia. Hay multiples agentes quimicos disponibles comercialmente; estos pueden alterar la microflora oral y tener efectos secundarios indeseables como vomito, diarrea y pigmentacion dental. Por lo tanto, los productos naturales como los fitoquimicos aislados de plantas que son usadas como medicinas tradicionales y los biomateriales, son considerados buenas alternativas. El quitosan y el pululan son polimeros que han sido propuestos debido a sus propiedades de biocompatibilidad, biodegradabilidad, habilidad de adhesion y que pueden ser usados como sistemas de liberacion de los principios activos de extractos de plantas. Los extractos de Thymus vulgaris, Matricaria chamomilla, Croton lechleri, Calendula officinalis L. y Juliana adstringens Schl. son conocidos por tener actividad medicinal y se usan en la medicina tradicional Mexicana. La actividad antimicrobiana de sus extractos fue probada in vitro contra P. gingivalis y A. actinomycetemcomitans usando los metodos de difusion en agar y de microdilucion. La actividad antimicrobiana de peliculas a base de biopolimeros con extractos de plantas fue evaluada midiendo las zonas de inhibicion de crecimiento de los organismos probados. El proposito de este estudio fue desarrollar peliculas bioadhesivas de quitosan y pululan adicionadas con extractos de plantas y evaluar su actividad antimicrobiana contra periodontopatogenos.

Detection of Porphyromonas gingivalis and Streptococcus intermedius in chronic periodontitis patients by multiplex PCR.

A Multiplex PCR assay for the detection of Porphyromonas gingivalis and Streptococcus intermedius in chronic periodontitis is presented. A total of 180 samples from 65 adults with untreated periodontitis and 17 healthy volunteers were taken and processed in a simple boiling step. Cell lysates were used as DNA source for multiplex PCR assays. Primers were designed from 16S rRNA gene sequences from the GenBank-EMBL database showing specificity for target pathogens. This multiplex PCR system could detect 8.2 P gingivalis and S. intermedius cells. In untreated periodontitis patients, only 78.5% were positive for one or both bacteria; 37% were positive for P gingivalis only, 17% for S. intermedius and 24.5% for both. P. gingivalis was detected in 23.5% of healthy volunteers, while S. intermedius was not detected in the same patients. The distribution of these bacteria was related to the periodontal probing depth, while 95.23% of patients with pockets wih 6 to 7 mm deep were positive for either or both, only 70.45% of of them with 4 to 5 mm pockets were positive.

Transcriptional upregulation of genes related to virulence activation in Entamoeba histolytica.

BACKGROUND: To understand the molecular basis of virulence variability in Entamoeba histolytica, this study presents results about differential gene expression induced by E. histolytica trophozoites in liver of hamsters in order to produce experimental amebic liver abscess (ALA) and consequently reactivate its virulence. METHODS: Amebic cultures were studied before (BALA) and after (AALA) inoculation in hamster peritoneal cavity. Markers of pathogenicity such as the rate of erythrophagocytosis, hemolytic activity, and cytotoxic effects on MDCK cell monolayers were evaluated in order to correlate these phenotypic characteristics to differential gene expression between virulent and non-virulent strains. Genotypic variability was determined by genetic polymorphism using the random-amplified polymorphic DNA (RAPD) technique, which defines the parasite genomic plasticity. mRNA differential display was used in order to identify variable transcripts levels. RESULTS: The rate of erythrophagocytosis and hemolytic activity were notably increased in AALA in comparison with BALA E. histolytica cultures, as well as the cytotoxic effect on MDCK cells. An increment in the transcription level of several mRNA was shown. CONCLUSIONS: The RAPD technique allowed us to confirm differences in number and size of polymorphic markers bands between virulent and non-virulent stages, suggesting genomic adaptability in E. histolytica. Eight different genes (membrane-bound acid phosphatase, cysteine proteinase, two different ribosomal proteins, heat shock transcription factor, ribosomal RNA, aldehyde dehydrogenase-1 and patatin-like phospholipase) were sequenced and may be associated with a biological function related to the virulence of E. histolytica. Together these findings show genomic variability between virulent and non-virulent cultures of E. histolytica.

Genotyping of recombinant Pichia pastoris strains.

A simplified amplified-fragment length polymorphism (AFLP) method was used to genotype Pichia pastoris strains obtained by transformation of P. pastoris strain GS115 with a single integration vector. A total of 14 transformants and 3 control strains were analyzed, which generated 16 different band patterns. A clonal variation was obtained after the transformation process due to genetic differences generated during the transformation event of the host strain. Furthermore, the cluster analysis showed that the transformants with lesser genetic differences with respect to the P. pastoris host strain are the recombinant strains with the highest level of recombinant protein production.

Characterization of Mexican Bacillus thuringiensis strains toxic for lepidopteran and coleopteran larvae.

Bacillus thuringiensis strains C-4, C-9, GM-7, and GM-10, isolated from northeast Mexico and selected for their high toxicity against lepidopteran and coleopteran pests, were characterized following United States Environmental Protection Agency (EPA)'s guidelines. Flagellar serotyping revealed that GM-7 and GM-10 belonged to serotype aizawai, whereas C-4, C-9 corresponded to the kumamotoensis serotype. GM-10 and C-9 were also shown to be the most effective against lepidoptera and coleoptera larvae, respectively. None of the tested strains produced beta-exotoxin or showed activity against mosquitoes. GM-7 and GM-10 were sensitive to R-41 and CP-51 phages. All strains synthesized crystal proteins of 130-140 kDa. PCR analysis showed that C-4, GM-7, and GM-10 strains expressed cry1 genes, and C-9 expressed cry3 and cry7/8 genes, but not cry1. However, the C-9 strain had no cross-reaction with antisera raised against Cry3A and Cry7A proteins. GM-7 and GM-10 were sensitive to R-41 and CP-51 phages. When the delta-endotoxin (crystal) from the four strains was subcutaneously injected to Balb/c mice, alone or in combination with spores, only C-4 and C-9 provoked tissue necrosis similar to that caused by the beta-exotoxin producer HD-41. Tissue necrosis was prevented with the injection of pentoxifylline, an inhibitor of tumor necrosis factor alpha (TNF-alpha) production, suggesting a role of this cytokine in the observed effect. Our results demonstrated that GM-7 and GM-10 strains are effective and suitable for control of lepidopteran pests and safe for mammals under EPA regulations. The potential of the C-9 strain for the control of several coleopteran pests, and the induction of tissue necrosis in mice by C-4 and C-9 strains, are discussed.