Comparative genomic analysis of Pseudomonas aeruginosa strains susceptible and resistant to carbapenems and aztreonam isolated from patients with healthcare-associated infections in a Mexican hospital.

Pseudomonas aeruginosa (PA) is an important opportunistic pathogen that causes different infections on immunocompromised patients. Within PA accessory genome, differences in virulence, antibiotic resistance and biofilm formation have been described between strains, leading to the emergence of multidrug-resistant strains. The genome sequences of 17 strains isolated from patients with healthcare-associated infections in a Mexican hospital were genomically and phylogenetically analyzed and antibiotic resistance genes, virulence genes, and biofilm formation genes were detected. Fifteen of the 17 strains were resistant to at least two of the carbapenems meropenem, imipenem, and the monobactam aztreonam. The antibiotic resistance (mexA, mexB, and oprM) and the biofilm formation (pslA and pslD) genes were detected in all strains. Differences were found between strains in accessory genome size. The strains had different sequence types, and seven strains had sequence types associated with global high risk epidemic PA clones. All strains were represented in two groups among PA global strains. In the 17 strains, horizontally acquired resistance genes to aminoglycosides and beta-lactams were found, mainly, and between 230 and 240 genes that encode virulence factors. The strains under study were variable in terms of their accessory genome, antibiotic resistance, and virulence genes. With these characteristics, we provide information about the genomic diversity of clinically relevant PA strains.

Transcriptomic Analysis Reveals the Response Mechanisms of Bell Pepper (Capsicum annuum) to Phosphorus Deficiency.

Phosphorus (P) is an important nutritional element needed by plants. Roots obtain P as inorganic phosphate (Pi), mostly in H2PO-4 form. It is vital for plants to have a sufficient supply of Pi since it participates in important processes like photosynthesis, energy transfer, and protein activation, among others. The physicochemical properties and the organic material usually make Pi bioavailability in soil low, causing crops and undomesticated plants to experience variations in accessibility or even a persistent phosphate limitation. In this study, transcriptome data from pepper roots under low-Pi stress was analyzed in order to identify Pi starvation-responsive genes and their relationship with metabolic pathways and functions. Transcriptome data were obtained from pepper roots with Pi deficiency by RNASeq and analyzed with bioinformatic tools. A total of 97 differentially expressed genes (DEGs) were identified; Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment revealed that metabolic pathways, such as porphyrin and chlorophyll metabolism, were down-regulated, and galactose and fatty acid metabolism were up-regulated. The results indicate that bell pepper follows diverse processes related to low Pi tolerance regulation, such as the remobilization of internal Pi, alternative metabolic pathways to generate energy, and regulators of root development.

Transcriptomic analysis of bell pepper (Capsicum annuum L.) revealing key mechanisms in response to low temperature stress.

BACKGROUND: Bell pepper (Capsicum annuum L.) is one of the most economically and nutritionally important vegetables worldwide. However, its production can be affected by various abiotic stresses, such as low temperature. This causes various biochemical, morphological and molecular changes affecting membrane lipid composition, photosynthetic pigments, accumulation of free sugars and proline, secondary metabolism, as well as a change in gene expression. However, the mechanism of molecular response to this type of stress has not yet been elucidated. METHODS AND RESULTS: To further investigate the response mechanism to this abiotic stress, we performed an RNA-Seq transcriptomic analysis to obtain the transcriptomic profile of Capsicum annuum exposed to low temperature stress, where libraries were constructed from reads of control and low temperature stress samples, varying on average per treatment from 22,952,190.5-27,305,327 paired reads ranging in size from 30 to 150 bp. The number of differentially expressed genes (DEGs) for each treatment was 388, 417 and 664 at T-17 h, T-22 h and T-41 h, respectively, identifying 58 up-regulated genes and 169 down-regulated genes shared among the three exposure times. Likewise, 23 DEGs encoding TFs were identified at T-17 h, 30 DEGs at T-22 h and 47 DEGs at T-42 h, respectively. GO analysis revealed that DEGs were involved in catalytic activity, response to temperature stimulus, oxidoreductase activity, stress response, phosphate ion transport and response to abscisic acid. KEGG pathway analysis identified that DEGs were related to flavonoid biosynthesis, alkaloid biosynthesis and plant circadian rhythm pathways in the case of up-regulated genes, while in the case of down-regulated genes, they pertained to MAPK signaling and plant hormone signal transduction pathways, present at all the three time points of low temperature exposure. Validation of the transcriptomic method was performed by evaluation of five DEGs by quantitative polymerase chain reaction (q-PCR). CONCLUSIONS: The data obtained in the present study provide new insights into the transcriptome profiles of Capsicum annuum stem in response to low temperature stress. The data generated may be useful for the identification of key candidate genes and molecular mechanisms involved in response to this type of stress.

Response Surface Methodology for Optimization of Multiplex-PCR Protocols for Detection of TYLCV, TSWV and Fol Molecular Markers: Analytical Performance Evaluation.

Molecular markers linked to disease resistance genes which affect economically important crops are of great interest. In the case of tomato, a major focus on resistance breeding to multiple fungal and viral pathogens such as Tomato yellow leaf curl virus (TYLCV), Tomato spotted wilt virus (TSWV) and Fusarium oxysporum f. sp. lycopersici (Fol), have led to the introgression of several resistance genes; therefore, molecular markers have become important in molecular-assisted selection (MAS) of tomato varieties resistant to those pathogens. However, assays that allow simultaneous evaluation of resistant genotypes, such as multiplex PCR, need to be optimized and evaluated to demonstrate their analytical performance, as many factors can affect them. This work aimed to generate multiplex PCR protocols for the joint detection of the molecular markers associated with pathogen resistance genes in tomato plants that are sensitive, specific and repeatable. For the optimization a central composite design of a response surface methodology (RSM-CCD) was used. For analytical performance evaluation, specificity/selectivity and sensibility (limit of detection and dynamic range) were analyzed. Two protocols were optimized: the first one with a desirability of 1.00, contained two markers (At-2 and P7-43) linked to I- and I-3-resistant genes. The second one with a desirability of 0.99, contained markers (SSR-67, SW5 and P6-25) linked to I-, Sw-5-, and Ty-3-resistant genes. For protocol 1, all the commercial hybrids (7/7) were resistant to Fol, and for protocol 2, two hybrids were resistant to Fol, one to TSWV and one to TYLCV with good analytical performance. In both protocols, the varieties considered susceptible to the pathogens, no-amplicon or susceptible amplicons, were observed. The optimized multiplex PCR protocols showed dynamic ranges from 5.97 up to 161.3 ng DNA. The limit of detection was 17.92 ng and 53.76 ng DNA for protocols 1 and 2, respectively, giving 100% positive results in the test replicates. This method allowed to develop optimized multiplex PCR protocols with few assays which translates into less time and resources, without sacrificing method performance.

Current knowledge in the use of bacteriophages to combat infections caused by Pseudomonas aeruginosa in cystic fibrosis.

Pseudomonas aeruginosa (PA) is considered the first causal agent of morbidity and mortality in people with cystic fibrosis (CF) disease. Multi-resistant strains have emerged due to prolonged treatment with specific antibiotics, so new alternatives have been sought for their control. In this context, there is a renewed interest in therapies based on bacteriophages (phages) supported by several studies suggesting that therapy based on lytic phages and biofilm degraders may be promising for the treatment of lung infections in CF patients. However, there is little clinical data about phage studies in CF and the effectiveness and safety in patients with this disease has not been clear. Therefore, studies regarding on phage characterization, selection, and evaluation in vitro and in vivo models will provide reliable information for designing effective cocktails, either using mixed phages or in combination with antibiotics, making a great progress in clinical research. Hence, this review focuses on the most relevant and recent findings on the activity of lytic phages against PA strains isolated from CF patients and hospital environments, and discusses perspectives on the use of phage therapy on the treatment of PA in CF patients.

Cytotoxic Activity of Polyphenol Extracts from Three Oregano Species: Hedeoma patens, Lippia graveolens and Lippia palmeri, and Antiproliferative Potential of Lippia graveolens against Two Types of Breast Cancer Cell Lines (MDA-MB-231 and MCF-7).

Oregano infusions have traditionally been used to treat some diseases related to inflammation and cancer; also, some species have shown antiproliferative activity on cancer cell lines, for example, colon and liver, and this has been attributed to its phytochemical profile, mainly its phenolic compounds. This study aimed to evaluate the cytotoxicity and antiproliferative potential of the polyphenols-rich extracts (PRE) of the oregano species H. patens, L. graveolens, and L. palmeri on breast cancer cell lines. The PRE of the three oregano species were obtained from dried leaves. The extract was characterized by determining antioxidant activity, total phenols content, and identifying the profile of phenolic acids and flavonoids by chromatography UPLC-MS/MS. Furthermore, the cytotoxicity of the extracts was evaluated in vitro on a non-cancer cell line of fibroblast NIH3T3 and the antiproliferative potential on the breast cancer cell lines MDA-MB-231 and MCF-7. L. graveolens showed the highest antioxidant capacity and significantly inhibited the proliferation of MCF-7 and MDA-MB-231 cells at non-cytotoxic concentrations in normal cells, with a similar effect to that cisplatin in MDA-MB-231 cells. Therefore, the polyphenol-rich extract from L. graveolens showed the greatest potential to guide future research on the antiproliferative mechanism of action.

Optimization of PCR-based TYLCV molecular markers by response surface methodology.

Tomato (Solanum lycopersicum L.) is one of the most economically important vegetables worldwide. However, its production is affected by the tomato yellow leaf curl virus (TYLCV), causing the greatest devastation in the crop. One strategy to cope with TYLCV implies the use of resistant varieties, whose development can be accelerated by molecular markers. The aim of this study was to optimize endpoint PCR protocols for the detection of the molecular markers TG178, TG105A and P6-25, linked to Ty-1, Ty-2 and Ty-3 resistance genes, respectively, through a response surface methodology (RSM) using a central composite design (CCD) for four factors (temperature of annealing (Ta), DNA amount, MgCl2 and primer concentrations). Applicability, the limit of detection and dynamic range were also analyzed. The optimized PCR conditions were: for TG178: Ta = 60 °C, 90 ng DNA, 3.36 mM MgCl2 and 0.13 µM primers; for TG105A: Ta = 54.4 °C, 10 ng DNA, 1.5 mM MgCl2 and 0.9 µM primers; for P6-25, Ta = 52.5 °C, 50 ng DNA, 2.5 mM MgCl2 and 0.5 µM primers. Dynamic ranges varied from 0.42 to 103.3 ng of DNA, while the limit of detection was 3.82, 0.42 and 11.47 ng of DNA for the TG178, TG105A and P6-25 molecular makers respectively and was 100% positive in replicates. CCD allowed the optimization of PCR protocols for molecular markers, which may further apply in identifying TYLCV resistant tomato lines.

The complete genome and comparative analysis of the phage phiC120 infecting multidrug-resistant Escherichia coli and Salmonella strains.

Phages infecting Salmonella and Escherichia coli are promising agents for therapeutics and biological control of these foodborne pathogens, in particular those strains with resistance to several antibiotics. In an effort to assess the potential of the phage phiC120, a virulent phage isolated from horse feces in Mexico, we characterized its morphology, host range and complete genome. Herein, we showed that phiC120 possesses strong lytic activity against several multidrug-resistant E. coli O157: H7 and Salmonella strains, and its morphology indicated that is a member of Myoviridae family. The phiC120 genome is double-stranded DNA and consists of 186,570 bp in length with a 37.6% G + C content. A total of 281 putative open reading frames (ORFs) and two tRNAs were found, where 150 ORFs encoded hypothetical proteins with unknown function. Comparative analysis showed that phiC120 shared high similarity at nucleotide and protein levels with coliphages RB69 and phiE142. Detailed phiC120 analysis revealed that ORF 94 encodes a putative depolymerase, meanwhile genes encoding factors associated with lysogeny, toxins, and antibiotic resistance were absent; however, ORF 95 encodes a putative protein with potential allergenic and pro-inflammatory properties, making needed further studies to guarantee the safety of phiC120 for human use. The characterization of phiC120 expands our knowledge about the biology of coliphages and provides novel insights supporting its potential for the development of phage-based applications to control unwanted bacteria.

The impact of quorum sensing on the modulation of phage-host interactions.

Bacteriophages are the most diverse and abundant biological entities on the Earth and require host bacteria to replicate. Because of this obligate relationship, in addition to the challenging conditions of surrounding environments, phages must integrate information about extrinsic and intrinsic factors when infecting their host. This integration helps to determine whether the infection becomes lytic or lysogenic, which likely influences phage spreading and long-term survival. Although a variety of environmental and physiological clues are known to modulate lysis-lysogeny decisions, the social interplay among phages and host populations has been overlooked until recently. A growing body of evidence indicates that cell-cell communication in bacteria and, more recently, peptide-based communication among phage-phage populations, affect phage-host interactions by controlling phage lysis-lysogeny decisions and phage counter-defensive strategies in bacteria. Here, we explore and discuss the role of signal molecules as well as quorum sensing and quenching factors that mediate phage-host interactions. Our aim is to provide an overview of population-dependent mechanisms that influence phage replication, and how social communication may affect the dynamics and evolution of microbial communities, including their implications in phage therapy.

Occurrence and Abundance of Pathogenic Vibrio Species in Raw Oysters at Retail Seafood Markets in Northwestern Mexico.

Seafood has frequently been associated with foodborne illness because pathogens are easily introduced during seafood cultivation, handling, and processing. Vibrio parahaemolyticus and Vibrio cholerae are human pathogens that cause gastroenteritis and cholera, respectively, and Vibrio vulnificus can cause fatal wound infections and septicemia. However, information about the occurrence of these pathogens in oysters from the Pacific coast of Mexico is limited to V. parahaemolyticus. In the present study, we evaluated the presence and abundance of these three Vibrio species in 68 raw oysters (Crassostrea corteziensis) obtained from retail seafood markets in Sinaloa, Mexico. The most probable number (MPN)-PCR assay was used for amplification of the tlh (thermolabile hemolysin), ompW (outer membrane protein), and vvhA (hemolytic cytolysin) genes that are specific to V. parahaemolyticus, V. cholerae, and V. vulnificus, respectively. All oyster samples were positive for at least one Vibrio species. V. parahaemolyticus, V. cholerae, and V. vulnificus prevalences were 77.9, 8.8, and 32.3% overall, respectively, and most species were present in all sample periods with increased prevalence in period 3. The tdh (thermostable direct hemolysin) gene was detected in 30.1%, trh (TDH-related hemolysin) was detected in 3.7%, and tdh/trh was detected in 7.5% of the total tlh-positive samples (53 of 68), whereas the pandemic serotype O3:K6 (orf8 positive) was detected in only 1 sample (1.8%). The total prevalence of tdh and/or trh was 41.5%. In none of the samples positive for V. cholerae were the cholera toxin (ctxA) and cholix (chxA) toxigenic genes or the rfb gene encoding the O1 and O139 antigens amplified, suggesting the presence of non-O1 non-O139 V. cholerae strains. Our results clearly indicated a high prevalence of pathogenic Vibrio species in raw oysters from retail seafood markets in Mexico. Consumption of these raw oysters carries the potential risk of foodborne illness, which can be limited by cooking.

Involvement of OpsLTP1 from Opuntia streptacantha in abiotic stress adaptation and lipid metabolism.

Plant lipid transfer proteins (LTPs) exhibit the ability to transfer lipids between membranes in vitro, and have been implicated in diverse physiological processes associated to plant growth, reproduction, development, biotic and abiotic stress responses. However, their mode of action is not yet fully understood. To explore the functions of the OpsLTP1 gene encoding a LTP from cactus pear Opuntia streptacantha Lem., we generated transgenic Arabidopsis thaliana (L.) Heynh. plants to overexpress OpsLTP1 and contrasted our results with the loss-of-function mutant ltp3 from A. thaliana under abiotic stress conditions. The ltp3 mutant seeds showed impaired germination under salt and osmotic treatments, in contrast to OpsLTP1 overexpressing lines that displayed significant increases in germination rate. Moreover, stress recovery assays showed that ltp3 mutant seedlings were more sensitive to salt and osmotic treatments than wild-type plants suggesting that AtLTP3 is required for stress-induced responses, while the OpsLTP1 overexpressing line showed no significant differences. In addition, OpsLTP1 overexpressing and ltp3 mutant seeds stored lower amount of total lipids compared with wild-type seeds, showing changes primarily on 16C and 18C fatty acids. However, ltp3 mutant also lead changes in lipid profile and no over concrete lipids which may suggest a compensatory activation of other LTPs. Interestingly, linoleic acid (18:2ω6) was consistently increased in neutral, galactoglycerolipids and phosphoglycerolipids of OpsLTP1 overexpressing line indicating a role of OpsLTP1 in the modulation of lipid composition in A. thaliana.

Dental Pulp Stem Cells: Current Advances in Isolation, Expansion and Preservation.

Dental pulp stem cells (DPSCs) are mesenchymal stem cells with high self-renewal potential that have the ability to differentiate into several cell types. Thus, DPSCs have become a promising source of cells for several applications in regenerative medicine, tissue engineering, and stem cell therapy. Numerous methods have been reported for the isolation, expansion, and preservation of DPSCs. However, methods are diverse and do not follow specific rules or parameters, which can affect stem cell properties, adding more variation to experimental results. In this review, we compare and analyze current experimental evidence to propose some factors that can be useful to establish better methods or improved protocols to prolong the quality of DPSCs. In addition, we highlight other factors related to biological aspects of dental tissue source (e.g., age, genetic background) that should be considered before tooth selection. Although current methods have reached significant advances, optimization is still required to improve culture stability and its maintenance for an extended period without losing stem cell properties. In addition, there is still much that needs to be done toward clinical application due to the fact that most of DPSCs procedures are not currently following good manufacturing practices. The establishment of optimized general or tailored protocols will allow obtaining well-defined DPSCs cultures with specific properties, which enable more reproducible results that will be the basis to develop effective and safe therapies.

Síndrome cardiorrenal: Nuevas perspectivas / Cardiorenal syndrome: New perspectives

La disfunción cardiaca y la renal coexisten con alta prevalencia. Se ha definido a esta entidad clínica como síndrome cardiorrenal y una de sus características principales es la resistencia al tratamiento. Se han descrito múltiples hipótesis para explicar su fisiopatología, como la desregulación hemodinámica y, recientemente, mecanismos neurohumorales e inmunológicos que intervienen en su desarrollo y perpetuación. Se clasifica de acuerdo con su forma de presentación y componentes fisiopatológicos. Existen distintos enfoques terapéuticos para controlar o limitar el progreso de la enfermedad. Esta revisión discute y analiza la información actual sobre la fisiopatología, la clasificación y el tratamiento de esta entidad.

There is a high prevalence of heart failure associated to kidney failure or viceversa. This association has been defined as a clinical entity: cardiorenal syndrome and one of its main characteristics is the resistance to treatment. Multiple hypotheses have been proposed to explain the pathophysiology of this syndrome, such as hemodynamic deregulation and recently, other neurohormonal and immunological mechanisms involved in the development and perpetuation of this pathology. Classifications have been based on the form of presentation or physiopathological manifestations. Different therapeutic approaches have been proposed to control or limit the progress of this disease. This review discusses and analyzes the current information on pathophysiology, different classifications and treatment.

The basal transcription machinery as a target for cancer therapy.

General transcription is required for the growth and survival of all living cells. However, tumor cells require extraordinary levels of transcription, including the transcription of ribosomal RNA genes by RNA polymerase I (RNPI) and mRNA by RNA polymerase II (RNPII). In fact, cancer cells have mutations that directly enhance transcription and are frequently required for cancer transformation. For example, the recent discovery that MYC enhances the transcription of the majority genes in the genome correlates with the fact that several transcription interfering drugs preferentially kill cancer cells. In recent years, advances in the mechanistic studies of the basal transcription machinery and the discovery of drugs that interfere with multiple components of transcription are being used to combat cancer. For example, drugs such as triptolide that targets the general transcription factors TFIIH and JQ1 to inhibit BRD4 are administered to target the high proliferative rate of cancer cells. Given the importance of finding new strategies to preferentially sensitize tumor cells, this review primarily focuses on several transcription inhibitory drugs to demonstrate that the basal transcription machinery constitutes a potential target for the design of novel cancer drugs. We highlight the drugs' mechanisms for interfering with tumor cell survival, their importance in cancer treatment and the challenges of clinical application.

The genetic depletion or the triptolide inhibition of TFIIH in p53-deficient cells induces a JNK-dependent cell death in Drosophila.

Transcription factor IIH (TFIIH) participates in transcription, nucleotide excision repair and the control of the cell cycle. In the present study, we demonstrate that the Dmp52 subunit of TFIIH in Drosophila physically interacts with the fly p53 homologue, Dp53. The depletion of Dmp52 in the wing disc generates chromosome fragility, increases apoptosis and produces wings with a reduced number of cells; cellular proliferation, however, is not affected. Interestingly, instead of suppressing the apoptotic phenotype, the depletion of Dp53 in Dmp52-depleted wing disc cells increases apoptosis and the number of cells that suffer from chromosome fragility. The apoptosis induced by the depletion of Dmp52 alone is partially dependent on the JNK pathway. In contrast, the enhanced apoptosis caused by the simultaneous depletion of Dp53 and Dmp52 is absolutely JNK-dependent. In this study, we also show that the anti-proliferative drug triptolide, which inhibits the ATPase activity of the XPB subunit of TFIIH, phenocopies the JNK-dependent massive apoptotic phenotype of Dp53-depleted wing disc cells; this observation suggests that the mechanism by which triptolide induces apoptosis in p53-deficient cancer cells involves the activation of the JNK death pathway.

Physical and functional interactions between Drosophila homologue of Swc6/p18Hamlet subunit of the SWR1/SRCAP chromatin-remodeling complex with the DNA repair/transcription factor TFIIH.

The multisubunit DNA repair and transcription factor TFIIH maintains an intricate cross-talk with different factors to achieve its functions. The p8 subunit of TFIIH maintains the basal levels of the complex by interacting with the p52 subunit. Here, we report that in Drosophila, the homolog of the p8 subunit (Dmp8) is encoded in a bicistronic transcript with the homolog of the Swc6/p18(Hamlet) subunit (Dmp18) of the SWR1/SRCAP chromatin remodeling complex. The SWR1 and SRCAP complexes catalyze the exchange of the canonical histone H2A with the H2AZ histone variant. In eukaryotic cells, bicistronic transcripts are not common, and in some cases, the two encoded proteins are functionally related. We found that Dmp18 physically interacts with the Dmp52 subunit of TFIIH and co-localizes with TFIIH in the chromatin. We also demonstrated that Dmp18 genetically interacts with Dmp8, suggesting that a cross-talk might exist between TFIIH and a component of a chromatin remodeler complex involved in histone exchange. Interestingly, our results also show that when the level of one of the two proteins is decreased and the other maintained, a specific defect in the fly is observed, suggesting that the organization of these two genes in a bicistronic locus has been selected during evolution to allow co-regulation of both genes.