L-Lactate Electrochemical Biosensor Based on an Integrated Supramolecular Architecture of Multiwalled Carbon Nanotubes Functionalized with Avidin and a Recombinant Biotinylated Lactate Oxidase.

L-Lactate is an important bioanalyte in the food industry, biotechnology, and human healthcare. In this work, we report the development of a new L-lactate electrochemical biosensor based on the use of multiwalled carbon nanotubes non-covalently functionalized with avidin (MWCNT-Av) deposited at glassy carbon electrodes (GCEs) as anchoring sites for the bioaffinity-based immobilization of a new recombinant biotinylated lactate oxidase (bLOx) produced in Escherichia coli through in vivo biotinylation. The specific binding of MWCNT-Av to bLOx was characterized by amperometry, surface plasmon resonance (SPR), and electrochemical impedance spectroscopy (EIS). The amperometric detection of L-lactate was performed at -0.100 V, with a linear range between 100 and 700 µM, a detection limit of 33 µM, and a quantification limit of 100 µM. The proposed biosensor (GCE/MWCNT-Av/bLOx) showed a reproducibility of 6.0% and it was successfully used for determining L-lactate in food and enriched serum samples.

Antibody detection against Kunitz-type protein in Fasciola hepatica experimentally infected sheep using enzyme-linked immunosorbent assay (ELISA).

Fasciolosis is a parasitic disease considered as emerging and neglected by the WHO. Sheep are highly susceptible to this disease, and affected flocks experience decreased productivity due to increased mortality, and the reduced quality of their products, such as wool and meat. To effectively control this disease, reliable and early diagnosis is essential for making decisions regarding antiparasitic application and/or the removal of affected animals. Currently, the diagnosis of F. hepatica in sheep relies on the detection of parasite eggs in faeces, a method that becomes reliable from week 10 post-infection. Consequently, there is a need for earlier diagnostic tools based on immune response. However, obtaining antigens for antibody detection has proven to be difficult and expensive. The aim of this study was to evaluate members of the Kunitz protein family of F. hepatica expressed in the form of a fusion protein in the serological diagnosis of F. hepatica in sheep. The performance of three recombinant F. hepatica Kunitz-type inhibitors (FhKT1.1, FhKT1.3, and FhKT4) was compared with a synthetic Kunitz-type peptide (sFhKT) in sera from sheep experimentally infected with F. hepatica, using an ELISA. Of these, FhKT1.1 showed the most promising diagnostic indicators, exhibiting high precision and low cross-reactivity, and thus potential for standardized production. The results of our study demonstrated that the application of FhKT1.1 is a valuable tool for early-stage diagnosis of F. hepatica in sheep. Such an early diagnosis can aid in implementing timely interventions and effectively managing the disease in sheep populations.

Biotecnología y salud bucal: Terapia probiótica para la prevención de caries dentales / Biotechnology and oral health: Probiotic therapy to prevent dental caries

Los probióticos son microorganismos vivos que cuando son administrados adecuadamente proveen beneficios para la salud del huésped. La terapia con probióticos ha sido usada con éxito para el control de enfermedades intestinales y actualmente se plantea comouna nueva estrategia para la prevención de enfermedades de la cavidad oral. Con esta terapia se propone la utilización de microorganismos benéficos que poseen la capacidad de desplazar a los microorganismos cariogénicos y colonizar la cavidad oral. En esta editorial se comentará, con un enfoque biotecnológico, sobre la aplicación de la terapia probiótica para la prevención de caries dentales.

Biophysical characterization of the recombinant chitinase chi18-5 with potential biotechnological interest.

Chitinase chi18-5 is an enzyme able to hydrolyze chitin and chitosan producing chitooligosaccharides (COS) of potential technological interest. chi18-5 is produced naturally by the fungus Trichoderma atroviride. It belongs to the glycosyl hydrolase (GH) family 18 of the Carbohydrate Active Enzyme (CAZy) database and it has 83% identity compared to the well-characterized chi42 of Trichoderma harzianum. Several efforts have been made to characterize the biochemical activity of the enzyme and its structure. Here, we studied the biophysical properties of recombinant chi18-5. In order to gain insight into its structure and stability, we studied thermal denaturation by Circular Dichroism (CD), Intrinsic Fluorescence (FL), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT-IR) at several pH between 3 and 8. We observed that the conformation of chi18-5 changes near its pI, and the transitions as a function of the temperature involved an increment in ß-sheet secondary structure at the expenses of ⍺-helix. We also performed amide hydrogen exchange dynamics in selected conditions. At pH ≤ 6, the proportion of fast exchanging residues are larger than at pH ≥ 6. Our results suggest that at pH below pI, chi18-5 is in a less compact structure which may have influence in the interaction with substrate and enzyme activity. KEY POINTS: • Characterization of enzyme behavior is critical for their wide applications • We produced and characterized biophysically a chitinase as a function of pH • The pH of optimum activity correlates with a less compact structure of chi18-5.

Human α-Galactosidase A Mutants: Priceless Tools to Develop Novel Therapies for Fabry Disease.

Fabry disease (FD) is a lysosomal storage disease caused by mutations in the gene for the α-galactosidase A (GLA) enzyme. The absence of the enzyme or its activity results in the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different tissues, leading to a wide range of clinical manifestations. More than 1000 natural variants have been described in the GLA gene, most of them affecting proper protein folding and enzymatic activity. Currently, FD is treated by enzyme replacement therapy (ERT) or pharmacological chaperone therapy (PCT). However, as both approaches show specific drawbacks, new strategies (such as new forms of ERT, organ/cell transplant, substrate reduction therapy, or gene therapy) are under extensive study. In this review, we summarize GLA mutants described so far and discuss their putative application for the development of novel drugs for the treatment of FD. Unfavorable mutants with lower activities and stabilities than wild-type enzymes could serve as tools for the development of new pharmacological chaperones. On the other hand, GLA mutants showing improved enzymatic activity have been identified and produced in vitro. Such mutants could overcome several complications associated with current ERT, as lower-dose infusions of these mutants could achieve a therapeutic effect equivalent to that of the wild-type enzyme.

DNA repair, NFKß, and TP53 polymorphisms associated with potentially malignant disorders and oral squamous cell carcinoma in Argentine patients.

OBJECTIVE: An important strategy in cancer prevention is to identify individual susceptibilities for cancer development through the genomic profile. Developing countries such as Argentina have no data on genetic composition. The aim of this study was to evaluate the single nucleotide polymorphisms of genes related to DNA repair (XCCR3, XPD), cell cycle arrest/apoptosis (TP53), and inflammation (NFKß) of patients with precancer and oral cancer and to contribute to recognizing potential risk of developing these pathologies, and incorporate the risk patients into a clinical follow-up program in Córdoba, Argentina. STUDY DESIGN: A cross-sectional study was performed on 140 patients with oral squamous cell carcinoma (OSCC), oral potentially malignant disorders (OPMDs), and controls. Genotyping of single nucleotide polymorphisms was performed using allele-specific polymerase chain reaction or restriction fragment length polymorphism techniques. The variables were evaluated by bivariate and multivariate statistical methods, with P < .05 statistically significant. RESULTS: The multiple correspondence analyses showed that patients with OSCC are clustered with the T allele of XRCC3 T241 M and the C allele of TP53 R72 P, and patients with OPMDs are clustered with the T allele of NFKß-519. CONCLUSION: Our preliminary results showed that the C allele of the Pro72 variant of TP53 was related to OSSC and OPMD, and the T allele of NFKß-519 is related to OPMDs in Argentine patients.

Combining Modules for Versatile and Optimal Labeling of Lactic Acid Bacteria: Two pMV158-Family Promiscuous Replicons, a Pneumococcal System for Constitutive or Inducible Gene Expression, and Two Fluorescent Proteins.

Labeling of bacterial cells with fluorescent proteins allows tracking the bacteria in competition and interactomic in vivo and in vitro studies. During the last years, a few plasmid vectors have been developed aimed at the fluorescent labeling of specific members of the lactic acid bacteria (LAB), a heterogeneous group that includes microorganisms used in the food industry, as probiotics, or as live vectors for mucosal vaccines. Successful and versatile labeling of a broad range of LAB not only requires a vector containing a promiscuous replicon and a widely recognized expression system for the constitutive or regulated expression of the fluorescence determinant, but also the knowledge of the main features of the entire plasmid/host/fluorescent protein ensemble. By using the LAB model species Lactococcus lactis, we have compared the utility properties of a set of labeling vectors constructed by combining a promiscuous replicon (pMV158 or pSH71) of the pMV158 plasmid family with the gene encoding either the EGFP or the mCherry fluorescent protein placed under control of promoter PX or PM from the pneumococcal mal gene cluster for maltosaccharide uptake and utilization, respectively. Some vectors carrying PM also harbor the malR gene, whose product represses transcription from this promoter, thus enabling maltose-inducible synthesis of the fluorescent proteins. We have determined the plasmid copy number (PCN) and segregational stability of the different constructs, as well as the effect of these features on the fitness and fluorescence intensity of the lactococcal host. Constructs based on the pSH71 replicon had a high copy number (∼115) and were segregationally stable. The copy number of vectors based on the pMV158 replicon was lower (∼8-45) and varied substantially depending on the genetic context of the plasmid and on the bacterial growth conditions; as a consequence, inheritance of these vectors was less stable. Synthesis of the fluorescent proteins encoded by these plasmids did not significantly decrease the host fitness. By employing inducible expression vectors, the fluorescent proteins were shown to be very stable in this bacterium. Importantly, conditions for accurate quantification of the emitted fluorescence were established based on the maturation times of the fluorescent proteins.

Study of the TP53 codon 72 polymorphism in oral cancer and oral potentially malignant disorders in Argentine patients.

The aim of this work was to evaluate the prevalence of TP53Arg72Pro mutations and their possible relationship with oral carcinoma and oral potentially malignant disorders in Argentine patients. A cross-sectional study was performed on 111 exfoliated cytologies from patients with oral cancer (OC), oral potentially malignant disorders (OPMD) and controls. The TP53Arg72Pro mutations were determined using conventional PCR. We evaluated univariate and multivariate study variables, setting p < 0.05. We found: (a) a low frequency of Pro72 variant in control group and a high frequency in OC and OPMD, as well in OC and oral leukoplakia (OL) diagnosis; (b) multivariate association among the TP53CC genotype and females over 45 years with no tobacco nor alcohol habits with oral lichen planus pathology; (c) multivariate association between the TP53GC genotype and males with alcohol and tobacco habits and OC and OL pathologies. Our results showed that the wild-type Arg72variant was related to control patients and Pro72variant was related to OC and OPMD, in Argentine patients.

Malignancy risk models for oral lesions

OBJECTIVES: The aim of this work was to assess risk habits, clinical and cellular phenotypes and TP53 DNA changes in oral mucosa samples from patients with Oral Potentially Malignant Disorders (OPMD), in order to create models that enable genotypic and phenotypic patterns to be obtained that determine the risk of lesions becoming malignant. STUDY DESIGN: Clinical phenotypes, family history of cancer and risk habits were collected in clinical histories. TP53 gene mutation and morphometric-morphological features were studied, and multivariate models were applied. Three groups were estabished: a) oral cancer (OC) group (n=10), b) oral potentially malignant disorders group (n=10), and c) control group (n=8).RESULTS: An average of 50% of patients with malignancy were found to have smoking and drinking habits. A high percentage of TP53 mutations were observed in OC (30%) and OPMD (average 20%) lesions (p=0.000). The majority of these mutations were GC TA transversion mutations (60%). However, patients with OC presented mutations in all the exons and introns studied. Highest diagnostic accuracy (p=0.0001) was observed when incorporating alcohol and tobacco habits variables with TP3 mutations. CONCLUSIONS: Our results prove to be statistically reliable, with parameter estimates that are nearly unbiased even for small sample sizes. Models 2 and 3 were the most accurate for assessing the risk of an OPMD becoming cancerous. However, in a public health context, model 3 is the most recommended because the characteristics considered are easier and less costly to evaluate (AU)

Malignancy risk models for oral lesions.

OBJECTIVES: The aim of this work was to assess risk habits, clinical and cellular phenotypes and TP53 DNA changes in oral mucosa samples from patients with Oral Potentially Malignant Disorders (OPMD), in order to create models that enable genotypic and phenotypic patterns to be obtained that determine the risk of lesions becoming malignant. STUDY DESIGN: Clinical phenotypes, family history of cancer and risk habits were collected in clinical histories. TP53 gene mutation and morphometric-morphological features were studied, and multivariate models were applied. Three groups were estabished: a) oral cancer (OC) group (n=10), b) oral potentially malignant disorders group (n=10), and c) control group (n=8). RESULTS: An average of 50% of patients with malignancy were found to have smoking and drinking habits. A high percentage of TP53 mutations were observed in OC (30%) and OPMD (average 20%) lesions (p=0.000). The majority of these mutations were GC TA transversion mutations (60%). However, patients with OC presented mutations in all the exons and introns studied. Highest diagnostic accuracy (p=0.0001) was observed when incorporating alcohol and tobacco habits variables with TP3 mutations. CONCLUSIONS: Our results prove to be statistically reliable, with parameter estimates that are nearly unbiased even for small sample sizes. Models 2 and 3 were the most accurate for assessing the risk of an OPMD becoming cancerous. However, in a public health context, model 3 is the most recommended because the characteristics considered are easier and less costly to evaluate.

Cloning and functional expression of secreted phospholipases A(2) from Bothrops diporus (Yarará Chica).

Bothrops diporus is a very common viper in Argentina. At present, no complete sequence of secreted phospholipase A(2) (sPLA(2)) from this snake has been reported. We have cloned two sPLA(2) isoenzymes as well as a putative sPLA(2)-like myotoxin from venom gland. The two sPLA(2) were expressed as inclusion bodies in Escherichia coli with an N-terminal tag of ubiquitin. After in vitro renaturation and cleavage step, using an ubiquitin specific peptidase, the recombinants exhibited sPLA(2) activity when analyzed by means of Langmuir dilauroylphosphatidylcholine monolayers as substrate. Both enzymes have a similar surface pressure-activity profile when compared with non-recombinant purified isoforms. To our knowledge, this is the first time that analysis of optimal lateral pressure of substrate monolayers by using the surface barostat technique is performed on recombinant sPLA(2)s.

Graph models for phenotype and genotype association between oral mucosa and submandibular gland tumorigenesis in rat.

BACKGROUND: In recent years, success of statistics in field of genetics has been the identification of genes that affect the process of disease. Experimental models using animals enable early stages of tumor development to be studied. The aim of this study was to apply graph models to assess the association between the observed phenotypic changes in rat oral mucosa and induced tumorigenesis in the submandibular gland (SMG). MATERIALS AND METHODS: We studied changes in oncogenes TP53 and bcl-2, histopathological and immunomarker variables in samples of oral mucosa and SMG of Wistar male rats, 60 days old and 180 g in weight, in which tumorigenesis was induced in their SMG by a 0.5% solution of 9,10-dimethyl-1,2-benzanthracene in acetone. A set of linear structural equations were defined, with each formula indicating the response variables and the direct influences. In graph models, saliva was considered as a latent variable. The association was analyzed using Graphical Gaussian Markov models and odd ratios. RESULTS: About 40% of animals treated with 9, 10-dimethyl-1, 2-benzanthracene showed histological alterations in the epithelial basal strata of their oral mucosa only at 150 days. Statistical models indicated a relationship between gene alteration in gene bcl-2 in the SMG and histological changes observed in the oral mucosa (P = 0.04). CONCLUSION: Graph statistical model with one latent variable allows to conclude that these results associated with other clinical parameters may be useful in detecting early changes in SMG tumorigenesis. Furthermore, the design of randomized sampling of oral mucosa allows to validate these results and establish a reliable methodology for presumptive diagnosis or screening in the future.

Early phenotypic and genotypic alterations in submandibular gland oncogenesis in rats.

The present study evaluates the phenotypic and genotypic changes that take place during early oncogenesis. The submandibular glands of male rats were injected with a 0.5% solution of 9,10-dimethyl-1,2-benzanthracene (DMBA) in acetone. Gland samples were taken at 0, 7, 30 and 150 days post-injection and submitted to histological, biochemical, immunocytochemical and PCR evaluation. Histopathological analysis was performed on hematoxylin-eosin stained slides. Total protein content was assessed by Lowry's method and the protein profile was analyzed by 12% SDS-PAGE. Bcl-2 was demonstrated by silver-enhanced gold immunolabeling. p53 immunolabeling was performed using the streptavidin-biotin system. All the treated animals developed carcinoma-like lesions at 30 and 150 days. Total protein concentration rose significantly (p < 0.05) above control values at 7, 30 and 150 days. The treated glands exhibited positive immunolabeling for p53 in the nuclei of neoplastic cells at 30 and 150 days. Treated glands also showed positive cytoplasmic immunolabeling for Bcl-2, exhibiting statistically significant differences between 7, 30 and 150 days (p = 0.0015), and with controls (p < 0.0001). No p53 mutations were observed whereas a point mutation, C-to-A, of the Bcl-2 gene was detected at 7, 30 and 150 days by PCR amplification. This mutation led to a single aminoacid change (thre --> asn) in the protein molecule. Our results suggest that the early histopathological changes correspond to quantitative and qualitative protein changes. The histopathological, biochemical, immunocytochemical and genetic alterations observed during the course of experimental carcinogenesis in the submandibular gland of the rat could constitute reproducible indices of malignant transformation applicable to human oncogenesis, given the high degree of homology between the oncogenes of mice, rats and human beings.

Early phenotypic and genotypic alterations in submandibular gland oncogenesis in rats

Se evaluaron modificaciones del fenotipo y genotipo en glándulas submandibulares durante el desarrollo temprano de la tumorogénesis. Glándulas submandibulares de ratas macho fueron inyectadas con una solución de 0.5 por ciento de 9,10 dimetil 1,2-benzathracene (DMBA) diluida en acetona. Muestras de glándulas fueron analizadas mediante técnicas histológicas, bioquímicas, inmunocitoquímicas y por PCR a los 0, 7, 30 y 150 días postinyección. Para los estudios histopatológicos se utilizó la técnica de hematoxilina-eosina. Se determinó la concentración de proteínas totales por el método de Lowry y se realizaron corridas electroforéticas en gel de poliacrilamida SDS-PAGE al 12 por ciento para determinar el perfil proteico. Se realizó inmunomarcación para Bcl-2 con oro coloidal-plata y para p53 por streptavidina-biotina. Todos los animales tratados desarrollaron cambios similares a carcinomas a los 30 y 150 días. La concentracióan de proteínas totales aumentó significativamente (p<0,05) a los 7, 30 y 150 días en relación a los controles. En glándulas inducidas la inmunomarcación fue positiva para la proteína p53 en núcleos de células neoplásicas a los 30 y 150 días. En las mismas glándulas, la marcación citoplasmática de Bcl-2 fue positiva a los 7, 30 y 150 días(p=0,0015) y en relación a los controles (p<0,0001). No se observaron mutaciones de p53, mientras que se observó una mutación puntual, C A del gen bcl-2 a los 7, 30 y 150 días que generó un cambio de aminoácidos en la proteína (thre asn). Nuestros resultados sugieren que los cambios histopatológicos tempranos corresponden a modificaciones cuantitativas y cualitativas de las proteínas. Las modificaciones observadas a nivel histopatológico, bioquímico, inmunocitoquímico y genético en la carcinomgénesis experimental de glándula submandibular de rata podrían representar parámetros reproducibles de transformaciones malignas transferibles al ser humano, dada la alta homología de estos oncogenes entre rata, ratones...

Early phenotypic and genotypic alterations in submandibular gland oncogenesis in rats

Se evaluaron modificaciones del fenotipo y genotipo en glándulas submandibulares durante el desarrollo temprano de la tumorogénesis. Glándulas submandibulares de ratas macho fueron inyectadas con una solución de 0.5 por ciento de 9,10 dimetil 1,2-benzathracene (DMBA) diluida en acetona. Muestras de glándulas fueron analizadas mediante técnicas histológicas, bioquímicas, inmunocitoquímicas y por PCR a los 0, 7, 30 y 150 días postinyección. Para los estudios histopatológicos se utilizó la técnica de hematoxilina-eosina. Se determinó la concentración de proteínas totales por el método de Lowry y se realizaron corridas electroforéticas en gel de poliacrilamida SDS-PAGE al 12 por ciento para determinar el perfil proteico. Se realizó inmunomarcación para Bcl-2 con oro coloidal-plata y para p53 por streptavidina-biotina. Todos los animales tratados desarrollaron cambios similares a carcinomas a los 30 y 150 días. La concentracióan de proteínas totales aumentó significativamente (p<0,05) a los 7, 30 y 150 días en relación a los controles. En glándulas inducidas la inmunomarcación fue positiva para la proteína p53 en núcleos de células neoplásicas a los 30 y 150 días. En las mismas glándulas, la marcación citoplasmática de Bcl-2 fue positiva a los 7, 30 y 150 días(p=0,0015) y en relación a los controles (p<0,0001). No se observaron mutaciones de p53, mientras que se observó una mutación puntual, C A del gen bcl-2 a los 7, 30 y 150 días que generó un cambio de aminoácidos en la proteína (thre asn). Nuestros resultados sugieren que los cambios histopatológicos tempranos corresponden a modificaciones cuantitativas y cualitativas de las proteínas. Las modificaciones observadas a nivel histopatológico, bioquímico, inmunocitoquímico y genético en la carcinomgénesis experimental de glándula submandibular de rata podrían representar parámetros reproducibles de transformaciones malignas transferibles al ser humano, dada la alta homología de estos oncogenes entre rata, ratones...(AU)

Early phenotypic and genotypic alterations in submandibular gland oncogenesis in rats

Se evaluaron modificaciones del fenotipo y genotipo en glándulas submandibulares durante el desarrollo temprano de la tumorogénesis. Glándulas submandibulares de ratas macho fueron inyectadas con una solución de 0.5 por ciento de 9,10 dimetil 1,2-benzathracene (DMBA) diluida en acetona. Muestras de glándulas fueron analizadas mediante técnicas histológicas, bioquímicas, inmunocitoquímicas y por PCR a los 0, 7, 30 y 150 días postinyección. Para los estudios histopatológicos se utilizó la técnica de hematoxilina-eosina. Se determinó la concentración de proteínas totales por el método de Lowry y se realizaron corridas electroforéticas en gel de poliacrilamida SDS-PAGE al 12 por ciento para determinar el perfil proteico. Se realizó inmunomarcación para Bcl-2 con oro coloidal-plata y para p53 por streptavidina-biotina. Todos los animales tratados desarrollaron cambios similares a carcinomas a los 30 y 150 días. La concentracióan de proteínas totales aumentó significativamente (p<0,05) a los 7, 30 y 150 días en relación a los controles. En glándulas inducidas la inmunomarcación fue positiva para la proteína p53 en núcleos de células neoplásicas a los 30 y 150 días. En las mismas glándulas, la marcación citoplasmática de Bcl-2 fue positiva a los 7, 30 y 150 días(p=0,0015) y en relación a los controles (p<0,0001). No se observaron mutaciones de p53, mientras que se observó una mutación puntual, C A del gen bcl-2 a los 7, 30 y 150 días que generó un cambio de aminoácidos en la proteína (thre asn). Nuestros resultados sugieren que los cambios histopatológicos tempranos corresponden a modificaciones cuantitativas y cualitativas de las proteínas. Las modificaciones observadas a nivel histopatológico, bioquímico, inmunocitoquímico y genético en la carcinomgénesis experimental de glándula submandibular de rata podrían representar parámetros reproducibles de transformaciones malignas transferibles al ser humano, dada la alta homología de estos oncogenes entre rata, ratones...(AU)