Dr. Ashutosh Sharma | Tecnológico de Monterrey (original) (raw)

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Papers by Dr. Ashutosh Sharma

Agronomy

MicroRNAs (miRNAs) are a key gene regulator and play essential roles in several biological and pa... more MicroRNAs (miRNAs) are a key gene regulator and play essential roles in several biological and pathological mechanisms in the human system. In recent years, plenty of miRNAs have been identified to be involved in the development of neurodegenerative disorders (NDDs), thus making them an attractive option for therapeutic approaches. Hence, in this review, we provide an overview of the current research of miRNA-based therapeutics for a selected set of NDDs, either for their high prevalence or lethality, such as Alzheimer's, Parkinson's, Huntington's, Amyotrophic Lateral Sclerosis, Friedreich's Ataxia, Spinal Muscular Atrophy, and Frontotemporal Dementia. We also discuss the relevant delivery techniques, pertinent outcomes, their limitations, and their potential to become a new generation of human therapeutic drugs in the near future.

Superfruit guava (Psidium guajava L.) is one of the healthiest fruits due to its high antioxidant... more Superfruit guava (Psidium guajava L.) is one of the healthiest fruits due to its high antioxidant dietary fiber and vitamin content. However, the growth and development of this plant are severely affected by salinity stress, mostly at the seedling stage. MicroRNAs (miRNAs) are small, noncoding, endogenous, highly conserved RNA molecules that play key regulatory roles in plant development, organ morphogenesis, and stress response signaling. In this study, applying computational approaches and following high stringent filtering criteria, a total of 40 potential microRNAs belonging to 19 families were characterized from guava. The identified miRNA precursors formed stable stem-loop structures and exhibited high sequence conservation among diverse and evolutionarily distant plant species. Differential expression pattern of seven selected guava miRNAs (pgu-miR156f-5p, pgu-miR160c-5p, pgu-miR162-3p, pgu-miR164b-5p, pgu-miR166t, pgu-miR167a-5p, and pgu-miR390b-5p) were recorded under salinity stress and pgu-miR162-3p, pgu-miR164b-5p as well as pgu-miR166t were found to be the most affected ones. Using the psRNATarget tool, a total of 49 potential target transcripts of the characterized guava miRNAs were identified in this study which are mostly involved in metabolic pathways, cellular development, and stress response signaling. A biological network has also been constructed to understand the miRNA mediated gene regulation using the minimum free energy (MFE) values of the miRNA-target interaction. To the best of our knowledge, this is the first report of guava miRNAs and their targets.

Chikungunya (CHIKV) and Dengue (DENV) viruses cause an acute febrile illness which is hard to cli... more Chikungunya (CHIKV) and Dengue (DENV) viruses cause an acute febrile illness which is hard to clinically differentiate and treat since both exhibit similar symptoms. Hence, this study was aimed at identifying the expression profiles of cytokines on co-infected samples and compare with CHIKV and DENV mono-infected samples. Serum samples of 292 suspected patients during 2009-2011 were analyzed. The presence of primary (IgM)/secondary (IgG) antibodies and early NS1 Dengue antigens were confirmed by capture ELISA. Molecular diagnosis and serotypes were discriminated by RT-PCR, confirmed by sequencing. All the plasma samples were assayed for cytokine expression by BDTM cytometry bead array (CBA) and compared with independent mono-infection viral load. Among the tested samples, 82 were confirmed as Dengue positive; 52 through IgM (17.8%), and 30 through IgG (10.2%). Additionally, 186 samples were confirmed as Chikungunya, 96 through IgM (32.6%) and 92 through IgG (31.5%) ELISA, respectively. Interestingly , 19 patients were co-infection positive in which, only 6 were confirmed for CHIKV and 7 for DENV by RT-PCR. Among 8 cytokines, IL-2, IL-8, IFNα, IFN γ, and IL-12 were found to be significantly different between co-infected and CHIKV mono-infected patients and correlated with viral load. DENV viral load was correlated with cytokine expression and a significant difference in IL-2 and IL-12 was observed between DENV mono-infected and DENV and CHIKV co-infected patients. Results indicated that apart from serological and molecular confirmation, cytokines could be used as a specific biomarker for the diagnosis of DENV and CHIKV. In the future, the role of independent cytokines can be determined to understand the pathogenesis and etiology of these dreadful diseases.

Background: The banana (Musa sp., AAA) genome is constantly increasing due to high-frequency of s... more Background: The banana (Musa sp., AAA) genome is constantly increasing due to high-frequency of somaclonal variations. Due to its large diversity, a conventional numerical and morphological based taxonomic identification of banana cultivars is laborious, difficult and often leads to subject of disagreements. Results: Hence, in the present study, we used universal DNA barcode ITS2 region to identify and to find the genetic relationship between the cultivars and varieties of banana. Herein, a total of 16 banana cultivars were PCR amplified using ITS2 primer pair. In addition, 321 sequences which were retrieved from GenBank, USA, were used in this study. The sequences were then aligned using Clustal W and genetic distances were computed using MEGA V5.1. The study showed significant divergence between the intra-and inter-specific genetic distances in ITS2 region. BLAST1 and Distance methods proved that ITS2 DNA barcode region successfully identified and distinguished the cultivar and varieties of banana.

Endophytic fungi represent a promising biotechnological tool to identify and produce in large sca... more Endophytic fungi represent a promising biotechnological tool to identify and produce in large scale novel anti-inflammatory bioactive compounds. In this study, Crescentia alata Kunth was selected following the eth-nomedical criteria and a total of 219 isolates grouped in 86 morphotypes were obtained. From these, 44 isolates that presented a pigment-producing morphotype were selected as the screening panel. The ITS2 ribotypes of the selected endophytic fungi were annotated and classified phylogenetically based on a sequence-structure analysis. The isolates belonged to 17 genera: Colletotrichum, Fusarium, Lophotrichus, Podo-spora, Xylaria, Diaporthe, Aspergillus, Periconia, Didymella, Prosthemium, Trematophoma, Cladosporium, Cerco-spora, Pseudocercosporella, Aureobasidium, Bjerkandera, and Trametes. From the anti-inflammatory screening with the Griess assay only 14.77% were highly active with no significant difference compared to indometha-cin, and showed promising in vitro anti-inflammatory effect tested in murine macrophages induced with bacterial lipopolysaccharides (LPS). Interestingly, 11.36% of the extracts increased the production of nitrite on LPS-induced macrophages. None of the extracts at the tested concentrations presented a pro-inflammatory effect on non-induced macrophages, nor a cytotoxic effect (cell viability >85%) in the resazurin bioassay. Metabolic profiling of the endo-metabolome and exo-metabolome extracts using Thin Layer Chromatography (TLC), revealed that the exo-metanolome extracts had a relative higher number and diversity of chemical groups. The 1 H NMR metabolomic analysis showed characteristic signals that differentiate the fungal genera with high anti-inflammatory activity from those with the least activity. These signals could be associated with the group of terpenes. This is the first report on the isolation of endophytes from C. alata, from which 13 isolates exhibit pharmacological value as sources of potential anti-inflammatory and immuno-modulatory compounds. These bioactive metabolites are likely to belong to the groups of terpenes.

MDPI, 2020

Triple-negative breast cancer (TNBC) cells are deficient in estrogen, progesterone and ERBB2 rece... more Triple-negative breast cancer (TNBC) cells are deficient in estrogen, progesterone and ERBB2 receptor expression, presenting a particularly challenging therapeutic target due to their highly invasive nature and relatively low response to therapeutics. There is an absence of specific treatment strategies for this tumor subgroup, and hence TNBC is managed with conventional therapeutics, often leading to systemic relapse. In terms of histology and transcription profile these cancers have similarities to BRCA-1-linked breast cancers, and it is hypothesized that BRCA1 pathway is non-functional in this type of breast cancer. In this review article, we discuss the different receptors expressed by TNBC as well as the diversity of different signaling pathways targeted by TNBC therapeutics, for example, Notch, Hedgehog, Wnt/b-Catenin as well as TGF-beta signaling pathways. Additionally, many epidermal growth factor receptor (EGFR), poly (ADP-ribose) polymerase (PARP) and mammalian target of rapamycin (mTOR) inhibitors effectively inhibit the TNBCs, but they face challenges of either resistance to drugs or relapse. The resistance of TNBC to conventional therapeutic agents has helped in the advancement of advanced TNBC therapeutic approaches including hyperthermia, photodynamic therapy, as well as nanomedicine-based targeted therapeutics of drugs, miRNA, siRNA, and aptamers, which will also be discussed. Artificial intelligence is another tool that is presented to enhance the diagnosis of TNBC.

Molecules, 2018

Galphimia glauca (Cav.) Kuntze is an important endemic plant species, which possesses many medici... more Galphimia glauca (Cav.) Kuntze is an important endemic plant species, which possesses many medicinal properties and has been used in the Mexican traditional medicine for its sedative, anxiolytic, anticonvulsant, antiasthmatic and antiallergic properties. The therapeutic properties of this plant are mainly due to the presence of diverse bioactive compounds such as flavonoids, triterpenoids, and phenolics. Several triterpenoids and flavonoids compounds have been isolated and identified. Modern studies have demonstrated many biological activities such as anti-inflammatory, antidiarrheal, gastroenteritis, antimalarial and cytotoxic activities. Nevertheless, many studies are restricted to the crude extract, and many bioactive compounds are yet to be identified and validated according to its traditional use. However, its commercial exploitation and use are highly limited due to the non-availability of enough plant material and lack of knowledge about its agronomical practices. Moreover, the misinterpretation and mislabeling of closely related species of the genus Galphimia Cav. as G. glauca or G. gracilis is a common problem for its rigorous scientific study and commercial exploitation. The present review provides comprehensive knowledge based on the available scientific literature. To the best of our knowledge, this is the first review on G. glauca. This comprehensive information will certainly provide a guide for the better understanding and utilization of G. glauca for its scientific and industrial exploitation.

Journal of Natural products, 2019

Two new prenylated acylphloroglucinols, paleacenins A (1) and B (2), were isolated from the rhizo... more Two new prenylated acylphloroglucinols, paleacenins A (1) and B (2), were isolated from the rhizome n-hexane and chloroform extracts of the fern Elaphoglossum paleaceum. Both compounds were found to possess the same geranylated filicinic acid moiety but have a different phloroglucinol ring substituent. Their structures were determined using 1 H and 13 C NMR spectroscopic, HRMS, and ECD analysis. The plant extracts and purified compounds were assayed for inhibition of monoamine oxidase (MAO) activity, and the n-hexane and chloroform extracts displayed 25.0% and 26.5% inhibition of MAO-A, respectively, as well as 42.5% and 23.7% inhibition of MAO-B, respectively. Compounds 1 and 2 exhibited IC 50 values of 31.0 (1.3) μM for MAO-A and 4.7 (4.4) μM for MAO-B. Paleacenin A (1) showed a higher selective index (SI) toward MAO-B (SI MAO-B/MAO-A 0.1), and paleacenin B (2) exhibited selectivity to MAO-A (SI MAO-B/MAO-A , 3.5). The extracts showed cytotoxicity against a panel of prostate, cervix, breast, and colon cancer cell lines (IC 50 values between 1.7 and 10.6 μg/mL); the pure compounds were more active against the prostate, cervix, and colon cancer cell lines. Paleacenins A (1) and B (2), with IC 50 values of 46 and 41 μM, respectively, inhibited nitric oxide production by the RAW264.7 murine macrophage model.

Molecular Biology Report, 2019

Ovalbumin is considered a protein of high nutritional value because it contains essential amino a... more Ovalbumin is considered a protein of high nutritional value because it contains essential amino acids and is highly digestible. Therefore, it has a high biological value. Currently, the high food demand requires worldwide attention because food production is insufficient. Therefore, other alternatives are necessary to satisfy food demands, such as protein engineering. In this work, a protein with a high essential amino acid content similar to ovalbumin was synthesized by protein engineering , expressed, and digested in vitro. The assembly and sequential overlap extension PCR strategy was used to synthesize a 345-bp gene that encodes a high essential amino acid content protein (HEAAP). The 345-bp product was cloned into the vector pBAD TOPO®, and expressed in Escherichia coli BL21. PCR reactions and sequencing demonstrated the presence, orientation, and correct sequence of the insert. HEAAP expression was induced by l-arabinose and then purified using Ni-NTA affinity chromatography. The expression in E. coli was low and barely detected by Western blot assay. The in vitro multienzyme digestibility of HEAAP was around 79%, which suggests that the protein is potentially nutritious. Virtual analysis classifies the protein as unstable and hydrophilic, with a half-life in E. coli of 10 h. The recombinant HEAAP was successfully synthesized, but it is necessary to improve the digestibility and to optimize expression including selecting other expression models.

Journal of the Institute of Tropical Medicine of São Paulo, 2019

Multidrug resistance (MDR), virulence and transferable elements potentiate Pseudomonas aeruginosa... more Multidrug resistance (MDR), virulence and transferable elements potentiate Pseudomonas aeruginosa's role as an opportunistic pathogen creating a high risk for public health. In this study, we evaluated the possible association of multidrug resistance, virulence factors and integrons with intrahospital P. aeruginosa strains isolated from patients at Cumana hospital, Venezuela. Relevant clinical-epidemiological data were collected to study 176 strains (2009-2016) isolated from different hospital units. Bacterial resistance was classified as susceptible, low-level resistant (LDR), multidrug resistant (MDR) and extensively drug-resistant (XDR). Most strains produced pyoverdine, DNase, gelatinase and hemolysin. Around 73% of the strains showed some type of movement. MDR and XDR strains increased from 2009 (24.2% and 4.8%, respectively) to 2016 (53.1% and 18.8%); while LDR decreased from 64.5% to 6.3%. The exoU and exoS genes were found in a significant number of strains (38.1 and 7.4%, respectively). Class I integrons were detected in 35.8% of the strains and the frequency was associated with resistance (42.9, 22.4, 41.4 and 61.9%, for susceptible, LDR, MDR and XDR, respectively). The MDR/XDR strains were positively associated with hemolysins and exoU, but negatively associated with bacterial twitching. MDR/XDR phenotypes were also associated with the Intensive Care Unit (ICU), septicemia, bronchial infection and diabetic foot ulcers, as well as long hospital stay (≥10 days) and previous antimicrobial treatment. High frequency of MDR/XDR strains and their association with class I integrons and virulence factors can increase the infection potential, as well as morbidity and mortality of patients attending this hospital and could spread infection to the community, creating a health risk for the region.

3Biotech, 2019

In the present study, a novel transformation protocol for Opuntia ficus-indica was generated by m... more In the present study, a novel transformation protocol for Opuntia ficus-indica was generated by means of particle bombardment. The best conditions obtained were: 900 psi rupture disk pressure, 8 cm microprojectile travel distance, and 4 h of exposition to 0.2 M mannitol. For all experiments, gold particles coated with 1.0 µg/µL of pBI426 plasmid DNA were used. With all these conditions, a 23% of transformation efficiency in terms of regeneration in selection media (100 mg/L kanamy-cin) was obtained. Interestingly, the presence of both transgenes: nptII and uidA, by means of PCR and RT-PCR assays was detected. The regeneration percentage achieved in terms of stable integration for both genes was 10%. In addition, we also detected adequate amounts of β-glucuronidase activity by means of the GUS fluorometric assay. The procedure described in the present investigation reveals the feasibility of using nopal for the introduction, expression, and possible production of heterologous proteins.

Life, 2019

In recent years, metabolic engineering of microorganisms has attained much research interest to p... more In recent years, metabolic engineering of microorganisms has attained much research interest to produce biofuels and industrially pertinent chemicals. Owing to the relatively fast growth rate, genetic malleability, and carbon neutral production process, cyanobacteria has been recognized as a specialized microorganism with a significant biotechnological perspective. Metabolically engineering cyanobacterial strains have shown great potential for the photosynthetic production of an array of valuable native or non-native chemicals and metabolites with profound agricultural and pharmaceutical significance using CO 2 as a building block. In recent years, substantial improvements in developing and introducing novel and efficient genetic tools such as genome-scale modeling, high throughput omics analyses, synthetic/system biology tools, metabolic flux analysis and clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (CRISPR/cas) systems have been made for engineering cyanobacterial strains. Use of these tools and technologies has led to a greater understanding of the host metabolism, as well as endogenous and heterologous carbon regulation mechanisms which consequently results in the expansion of maximum productive ability and biochemical diversity. This review summarizes recent advances in engineering cyanobacteria to produce biofuel and industrially relevant fine chemicals of high interest. Moreover, the development and applications of cutting-edge toolboxes such as the CRISPR-cas9 system, synthetic biology, high-throughput "omics", and metabolic flux analysis to engineer cyanobacteria for large-scale cultivation are also discussed.

Phyton, 2019

Torin 1 is an ATP-competitive TOR inhibitor which inhibits the signaling of TOR and S6K kinase in... more Torin 1 is an ATP-competitive TOR inhibitor which inhibits the signaling of TOR and S6K kinase in mammals and plants. The objective of this research is to determine the effect of Torin 1 in a relatively simple and homogeneous plant system such as the NT-1 tobacco suspension cell cultures. Cultures of NT-1 cells were tested with 5, 50, 150 and 250 nM of Torin 1. During kinetics growth of NT-1 tobacco suspension cell cultures, 150 and 250 nM Torin 1 inhibits the early growth and later enhanced the cellular proliferation during exponential growth by means of an increased expression of E2F1 and cyclin B. Furthermore, Torin 1 stimulates the growth of NT-1 cells during log phase with small shaped cell, characteristic of tobacco suspension cell cultures with high mitotic activity.

Canadian Journal of Plant Sciences, 2019

MicroRNAs (miRNAs) are highly conserved, endogenous, short (21-24 nucleotides), non-coding RNA mo... more MicroRNAs (miRNAs) are highly conserved, endogenous, short (21-24 nucleotides), non-coding RNA molecules that play significant roles in post-transcriptional gene silencing by directing target mRNA cleavage or translational inhibition. Nonetheless, highly nutritious "super grain" quinoa (Chenopodium quinoa) is an extreme abiotic stress tolerant Andean seed crop of many potential uses, with outstanding protein quality and a load of vitamins, minerals, as well as flavonoid antioxidants. In this study, applying genome-wide in silico approaches (referring to the recently published quinoa genome) and following a set of stringent filtering measures, a total of 22 potentially conserved microRNAs belonging to 18 families were characterized from quinoa and 11 randomly selected putative microRNAs cqu-miR398b) were validated successfully by RT-PCR. Using the psRNATarget tool, a sum of 59 potential miRNA targets, mostly transcription factors, were identified that are involved in biosynthesis, metabolic processes, and signal transduction. Among the detected targets, six target transcripts (F-Box proteins, TCP, MYB, WD protein, NAC, and CSD) were reported to have specific roles in both flavonoids biosynthesis and stress response signaling in some plants. To the best of our knowledge, this is the first report of quinoa microRNAs and their targets. Résumé : Les microARN (miARN) sont de courtes molécules d'ARN (21 à 24 nucléotides) endogènes, fort bien conservées, qui ne codent rien, mais jouent un rôle important dans le silençage de l'expression génique après la transcription par le clivage de certains ARNm ou l'inhibition de leur traduction. Le quinoa (Chenopodium quinoa), une super céréale, est une culture grainière des Andes qui tolère extrêmement bien les stress abiotiques. Elle possède de nombreux usages potentiels et sa protéine, de qualité exceptionnelle, renferme une foule de vitamines, d'oligoéléments et de flavonoïdes antioxydants. Les auteurs ont appliqué une approche in silico au génome complet (en recourant au génome du quinoa récemment publié) et recouru à un jeu de mesures de filtrage rigoureuses pour recueillir 22 microARN éventuellement conservés appartenant à 18 familles du quinoa. Onze microARN pos-cqu-miR398b), ont été validés par RT-PCR. Grâce à l'outil psRNATarget, les chercheurs ont identifié 59 cibles potentielles intervenant dans la photosynthèse, divers proces-sus métaboliques et la transduction des signaux pour ces miRNA, essentiellement des facteurs de transcription. Parmi elles figurent six transcrits (protéines F-Box, TCP, MYB, WD, NAC, et CSD) qui jouent un rôle spécifique dans la biosynthèse des flavonoïdes et le signalement de la réaction au stress chez quelques plantes. Pour autant qu'on le sache, il s'agit de la première fois où l'on rapporte l'existence de microARN et de leurs cibles chez le quinoa. [Traduit par la Rédaction]

Applied Nanoscience, 2020

Copper oxide nanoparticles possess a high absorption coefficient and are non-toxic to animal cell... more Copper oxide nanoparticles possess a high absorption coefficient and are non-toxic to animal cells. Biological agents inhabit many compounds that can be explored for the synthesis of monodispersed and non-toxic copper oxide nanoparticles. This is the most important advantage of biological syntheses over physical and chemical methods. In the present work, copper oxide nanoparticles have been synthesized with a simple and green technique by using an aqueous extract of mixed leaf and flowers of Galphimia glauca. The nanoparticles were synthesized at 80 °C, with a 15 mM Copper sulfate solution and at varying pH range (2, 4, inherent, i.e. 5.3, 8, 10, 12). The particle size of the nanoparticles was confirmed to be 5-10 nm using TEM and XRD at pH 12. The functional groups on the surface of the nanoparticles were characterized using FTIR. Moreover, GCMS analysis of the aqueous extracts showed that different flavonoids were responsible for the biosynthesis of copper oxide nanoparticles. The cytotoxicity assays were also determined using MRC-5 and HeLa cell lines for copper oxide nanoparticles and was found that the nanoparticles are non-toxic to the normal cells and are relatively toxic to cancer cells.

Journal of Material Science, 2019

There are enormous methods such as physical, chemical, and biological, for the synthesis of metal... more There are enormous methods such as physical, chemical, and biological, for the synthesis of metallic nanoparticles (MNPs), which has become a matter of focus among material scientists. Green chemistry-based MNP synthesis is an area, which has gained much importance presently due to their non-toxicity and monodispersed nanoparticle preparation methodologies. Among green synthesis methods, plants are considered as efficient candidates for nanoparticle synthesis. The meticulous formation of different sizes and shapes of the nanoparticles using plants has spurred encouraging interest. The rate kinetics and stability of nanoparticle synthesis are well studied as well as appreciated in the arena of materials. Their capability to sequester metal ions and fastidiously define the dimensions using a plethora of capping proteins such as glutathione and phytochelatins is intriguing giving it a monodispersed size. This review is a comprehensive understanding of the metal nanoparticles synthesized by plants and apprehends the mechanism of nanoparticle synthesis exhaustively.

Agronomy

MDPI, 2020

Molecules, 2018

Journal of Natural products, 2019

Molecular Biology Report, 2019

Journal of the Institute of Tropical Medicine of São Paulo, 2019

3Biotech, 2019

Life, 2019

Phyton, 2019

Canadian Journal of Plant Sciences, 2019

Applied Nanoscience, 2020

Journal of Material Science, 2019

ELSEVIER, 2019

The surface area of all teeth, gums, dental implants, and periodontal pockets offers potential co... more The surface area of all teeth, gums, dental implants, and periodontal pockets offers potential colonization sites for a wide
range of both early and late colonizers. Organism communities making up biofilms arrange themselves nonrandomly into
colonies of bacterial cells, yeasts, and other fungi, a matrix known as glycocalyx. Aerobic species occupy the biofilm
surface, whereas anaerobic species arrange themselves in deeper layers, which creates actual gas diffusion gradients. Typically,
biofilms begin their formation by propagating laterally, and after these first developmental stages, biofilm begins to
grow vertically (Fig. 5.1) (Socransky and Haffajee, 2002, 2005).
Thus, differential geographical locations are produced during the first growth phases, while dense accumulations of
microorganisms attached to organic or inorganic polysaccharide matrices take place in lower biofilm layers, where stationary
groups can be differentiated from free groups. Biofilms are dynamic communities where nutritional components
and gases are transported by molecular diffusion (Lang and Tonetti, 2003). Microbial colonies are often composed of different
species within a matrix surrounded by water channels that allow for nutrients and other agents to pass, resembling a
primitive circulatory system. These channels create an enormous maze consisting mostly of exopolysaccharides, whose
role is essential in safeguarding the integrity of a constantly-growing biofilm (Socransky and Haffajee, 2002). Within
the biofilm, at the microbial species level, cell physiology is quite heterogeneous, which means that cells can be at different
physiological stages even when they are close to one another. When environmental factors affect one species, cells of the
same species in deeper layers of the biofilm can remain unharmed (Marsh, 2005). The human host contributes directly, due
to lack of dental hygiene, or indirectly, by ingestion, to the promotion of biofilm growth and stability, making the oral cavity
a haven for microbial resistance (Marsh and Devine, 2011) (Fig. 5.2). The complexity of the cell-to-cell interactions
required for the biofilm formation and stability is worth the advantages of physical protection obtained by colonizers from
hostile factors in the oral environment. Most often, these factors are variations in pH, temperature, redox reactions, and
nutrient concentration, as well as potentially toxic substances such as antibiotics and the host’s immunological response
mechanisms (Socransky and Haffajee, 2002).

SPringer, 2019

The lung is a specialized organ that facilitates the gas exchange between an organism and the env... more The lung is a specialized organ that facilitates the gas exchange between an organism and the environment. Its function is to supply the blood with oxygen that the body uses and eliminate the carbon dioxide produced by metabolism. Chronic inhalation of environmental contaminants can result in the overwhelming production of reactive oxygen species (ROS). Oxidative stress in tissues is a process of cellular deterioration dependent on the production of free radicals by an imbalance between ROS and antioxidant agents of endogenous. In the patho-genesis and evolution of numerous pulmonary diseases of high prevalence, inflammation and oxidative stress seem to coexist with an important degree of interaction between both. During the inflammatory process, increased production of ROS may induce damage to lipid structures, proteins, and DNA, the inhibition of apoptosis, and activation of proto-oncogenes when initiating signal transduction pathways. Diet and nutrition are becoming recognized as modifi-able contributors to the development and progression of pulmonary diseases.

SPRINGER, 2019

Current knowledge in the use of hairy root cell cultures is growing. The use of this technology i... more Current knowledge in the use of hairy root cell cultures is growing. The use of this technology in the production of secondary metabolites is also increasing with the general purpose of generating medicinal alternatives from ethnophar-maceutical origins. Strategies include either genetically modified hairy roots that overexpress genes that codify for key enzymes in complex metabolic pathways or the use of certain elicitors for the natural production of certain compounds in hairy roots. The importance of understanding basic root technology and the complex genetic regulation of root development helps in establishing well-designed strategies in order to obtain higher yields in terms of metabolites or recombinant protein production. Also, bioprocess factors such as the use of bioreactors, elicitors, nutrients, and phytohormones for the induction of compounds of economic importance are also growing. Thus, the aim of the present chapter is to summarize the most important aspects regarding the technology to generate these hairy roots and their main applications in novel fields such as phytoremediation.

Factores, mecanismos y bioprocesos involucrados en la formación y destrucción de la biopelícula e... more Factores, mecanismos y bioprocesos involucrados en la formación y destrucción de la biopelícula en la cavidad oral. La gran mayoría de los microorganismos en la naturaleza están adheridos a superficies sobre las cuales crecen y forman biopelículas o biofilms. Las biopelículas generadas por el microbioma oral son comunidades metabólicas dinámicas embebidas en una matriz de exo-polisacáridos. La matriz extracelular prove de andamio, estabilidad mecánica y adherencia a la población de la microbiota y está compuesta por agua, bio-polímeros o glicoproteínas, enzimas y macromoléculas secretadas al medio extracelular. En conjunto todos estos diferentes componentes y actividades interaccionan creando a nivel local una serie de condiciones microambientales que promueven el desarrollo funcional del consorcio microbiano. La arquitectura de la matriz está sujeta a diversos factores extrínsecos e intrínsecos. La acción combinada de dichos factores produce un dinámico y heterogéneo microambiente adecuado para la retención, adhesión, coagregación y multiplicación de microorganismos. La coagregación microbiana es un fenómeno que involucra una gran variedad de especies en la comunidad de la biopelícula oral, las cuales pueden estar sujetas a selección natural, un medio ambiente bajo condiciones fluctuantes, baja concentración de nutrientes, estrés y presencia de agentes antimicrobianos. Al coagregarse, los microorganismos de vida planctónica forman comunidades que les permiten nutrirse, multiplicarse y alcanzar una mayor tasa de sobrevida. En general, las biopelículas presentan una estructura muy organizada donde cada especie interacciona, compite y se reproduce bajo el control de la comunidad. La expresión génica en cada miembro del consorcio es significativamente diferente a su vida planctónica, debido a que se producen controles regulatorios dentro de la biopelícula que garantizan comunicación metabólica, balance ecológico, resistencia y sucesión de especies. Los microorganismos que conforman una biopelícula son menos susceptibles a los agentes antimicrobianos, depredación y estrés medioambiental. En general, las biopelículas están compuestas por una mezcla de comunidades microbianas y por los productos del metabolismo que se generan y liberan al medio oral.

Field of the Invention; The invention relates to a novel process for purifying and/or isolating n... more Field of the Invention; The invention relates to a novel process for purifying and/or isolating nucleic acids from fecal/epithelial tissue in cattle and buffalo. Background of the Invention; Genomic DNA acts as a major starting material for any kind of molecular genetic work like, identification, paternity control, genetic testing, QTL analysis, etc. Common genomic DNA isolation is based on a procedure in which the source material is either blood or any other soft tissue. In diary animals, different types of source materials are used like, the traditional blood sample, ear tag, hair follicles, semen, milk (Lipkin et al., 1993) etc. For forensic purposes, DNA is isolated from traces of tissue samples present in blood stains, fingerprints, dental remains, skeletal evidence, specimens from fires, explosions, airplane crashes, and other traumatic events. For most mammalian species, DNA isolation from blood is a universal and widely used method for any type of molecular biology work, as this method gives a good quality and quantity of DNA and a little amount of blood is enough to get the required amount of DNA. Blood is easily available and can be collected without harming the animal. Mammalian tissue exhibit a considerable variation in their gross and microscopic structures; hence a single DNA extraction technique is not suitable for all tissue types. Many techniques are current used for genomic DNA isolation depending upon the type, nature and amount of tissue sample available (. DNA extraction methods from such a variety of of biological samples involves three basic steps. The first step is the lysis of the animal cell, nuclear membrane and mitochondrial membrane to release genomic DNA mitochondrial DNA and cytoplasmic RNA. This is achieved by a strong anionic detergent such as sodium dodecyl sulfate (SDS) by the disruption of cell membranes. Since DNA is very robust, it is not adversely affected by SDS treatment. The lysis step is assisted by Proteinase K, a serine protease which digests the protein and frees the DNA from any proteins associated with it (euchromatin structures/histones, etc.). The second step is the removal of (separation of) complex macromolecules (protein, lipid and polysaccharides) from aqueous solutions containing nucleic acids, upon treatment with organic solvents such as phenol/guanidine hydrochloride, followed by centrifugation. Buffer saturated phenol at a neutral pH brings all macromolecules together at the buffer-phenol interface. Finally, the DNA » is precipitated out of the aqueous solution by alcohols in the presence of mild cationic salts. Some commercial methods/kits are available which avoid or reduce the last two steps and the DNA is allowed to bind to a solid matrix or silica by favoring hydrophobic or ionic interactions. The bound DNA is selectively eluted after extensive washing to remove the impurities. The development of non-invasive genetic sampling is increasingly popular/important to field biologists as the methods offer new ways to obtain genomic DNA for molecular analysis. Non-invasive sources of DNA include hair (in primates, bears, and dairy animals), milk, semen, urine and faces. Hair and feces are the most common non invasive sources of mammalian DNA. The epithelial cells shed from the intestinal lining and deposited on the surface of the feces could be considered another important non invasive source of mammalian DNA, which is retrievable from exfoliated intestinal epithelial cells in feces. Fecal DNA isolation was reported from a variety of animals such as primates (Utami etal., 2002), lions (Ernest et al., 2000), coyotes (Kohn et ah, 1999), bears (Tablerlet et al 1997), ungulates (Flagstad et. al, 1999), dolphins, bats (Veg. and Mccracken 2001 and rhinos with varying success rates. Fecal samples are the only resource for genetic and ecological studies of wild and captive animals (Constable et ah, 1995; H'oss et ah, 1992; Kohn and Wayne, 1997, Zhang et al., 2006). The quality and quantity of the available DNA depends on the type of sample, their collection methods, preservation conditions and procedures applied to isolate the DNA. Commercial kits are available (QIAamp DNA stool Mini Kit, Qiagen) for DNA extraction. However, these kits are expensive and sometimes reported to be ineffective in herbivores (Zhang et ah, 2006). There are no reports available for DNA isolation methods from fecal tissue in dairy animals. Normal DNA isolation procedure from farm animals: Presently, genomic DNA is isolated by invasive methods like blood (venipuncture), ear tag, semen (in case of bull), milk (rare, in case of milking female). The remaining DNA isolation methodology is standard. We used a novel approach/protocol to process the fecal mucus tissue/dead tissue to obtain the genomic DNA. Use of Fecal materials for the isolation of DNA and PCR: The use of fecal material from humans as a means to obtain genomic DNA for use in genetic analysis, identification of infectious microorganisms and the diagnosis of cancerous and pre-cancerous colorectal lesions has been reported by various groups. Non-invasive sources of DNA include hair (in primates, bears, and dairy animals), milk, semen, urine and faces. Hair and feces are the most common non invasive sources of mammalian DNA. The epithelial cells shed from the intestinal lining and deposited on the surface of the feces could be considered another important non invasive source of mammalian DNA, which is retrievable from exfoliated intestinal epithelial cells in feces. Fecal DNA isolation was reported from a variety of animals such as primates, dolphins, bats (Veg and Mccracken 2001 and rhinos with varying success rates. Fecal samples are the only resources for genetic and ecological studies of wild and captive animals (Constable et al., 1995; H"oss et al., 1992; Kohn and Wayne, 1997, Zhang et al., 2006) The quality and quantity of the available DNA depends on the type of sample, their collection methods, preservation conditions and procedures applied to isolate the DNA. Commercial kits are available (QIAamp DNA Stool Mini Kit, Qiagen) for DNA extraction. However, these kits are expensive and sometimes reported to be ineffective in herbivores (Zhang et al., 2006). There are no reports available for DNA isolation methods from fecal tissue in diary animals. Drawbacks in invasive procedures: The existing approaches require invasive means of collecting tissue for genomic DNA isolation. For the collection of sample for genomic DNA isolation from ear tags/peripheral blood, the animal has to be restrained in a special crate. Moreover, manpower is required to handle the animal while collecting the tissue sample invasively. It is a big problem to collect sample by invasive means in aggressive animals and bulls. Drawbacks in isolating DNA from feces; The presence of inhibitors in fecal materials is a major obstacle limiting the usefulness of the PCR for detecting microorganisms in feces. A number of inhibitors are present in human feces which include bile salts, hemoglobin degradation products, and complex polysaccharides (Monteiro, L., D. Bonnemaison, A. Vekris, K.G. Retry, J. Bonnett, R. Vidal, J. Cabrita, and F. Megraud. 1997. Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model. J. Clin. Microbiol. 35:995-998.). in addition, polyphenolic substances from plant tissues are inhibitory to PCR (Koonjul, P.K., W.F. Brandt, J.M. Farrant, and G.G. Lindsey. 1999. Inclusion of polyvinylpyrrolidone in the polymerase chain reaction reverses the inhibitory effects of polyphenolic contamination of RNA. Nucleic Acids Res. 27:915-916). It is a completely noninvasive method with enough sources for DNA; This method does not require invasive tissues like blood and ear tags used routinely for DNA isolation (in cattle and buffalo). At present no alternative noninvasive method exists for genomic DNA isolation in Dairy animals. The amount of tissue available from the surface of the any fecal matter is a sufficient source for DNA. Objects of the Invention: The object of this invention is to develop a simplified DNA isolation method in cattle and buffalo. Other object is to use a non-invasive DNA isolation methodology in Dairy animals. Another object is to use Genomic DNA from fecal/epithelial tissue in cattle and buffalo. Other object is to isolate nucleic acid which are found to be considerably intact and not sheared. Another object is to avoid use of strong organic reagents such as phenol which is a inhibitor of PCR. Yet another object is to isolate DNA from sufficient amount of available tissue. Other object is to use fecal epithelial dead tissue for DNA isolation wherein it is resistant to any common enzymatic treatment because of its outer protective mucus layer. Another object is to remove any probable inhibitors of DNA modifying enzymes for further processing of the genomic DNA. Yet another object is to develop a process that can be used for DNA isolation from any tissue. Brief description of accompanying Figs; Fig 1. Shows gel electrophoresis of genomic DNA isolated from cattle fecal epithelial tissue. Fig 2. Shows gel electrophoresis of genomic DNA isolated from buffalo fecal tissue. Fig 3. Shows Restriction Enzymatic digestion of isolated genomic DNA of cattle. Fig 4. Shows PCR amplification of mitochondrial DNA from isolated buffalo and cattle.