Atherosclerosis is the leading cause of cardiovascular diseases in Mexico and worldwide. The membrane transporters ABCA1 and ABCG1 are involved in the reverse transport of cholesterol and stimulate ...the HDL synthesis in hepatocytes, therefore the deficiency of these transporters promotes the acceleration of atherosclerosis. MicroRNA-33 (miR-33) plays an important role in lipid metabolism and exerts a negative regulation on the transporters ABCA1 and ABCG1. It is known that by inhibiting the function of miR-33 with antisense RNA, HDL levels increase and atherogenic risk decreases. Therefore, in this work, a genetic construct, pPEPCK-antimiR-33-IRES2-EGFP, containing a specific antimiR-33 sponge with two binding sites for miR-33 governed under the PEPCK promoter was designed, constructed, and characterized, the identity of which was confirmed by enzymatic restriction, PCR, and sequencing. Hep G2 and Hek 293 FT cell lines, as well as a mouse hepatocyte primary cell culture were transfected with this plasmid construction showing expression specificity of the PEPCK promoter in hepatic cells. An analysis of the relative expression of miR-33 target messengers showed that the antimiR-33 sponge indirectly induces the expression of its target messengers (ABCA1 and ABCG1). This strategy could open new specific therapeutic options for hypercholesterolemia and atherosclerosis, by blocking the miR-33 specifically in hepatocytes.
Helicobacter pylori is a major aetiologic agent associated with gastritis. H. pylori infections increase the expression of the Toll-like receptor (TLR), which in turn modulates the expression of ...microRNA (miRNA)-146a and miRNA-155. The objective of this study was to compare the expression of miRNA-146a and miRNA-155 in gastric lesions of paediatric and adult patients with different pathologies and in Mongolian gerbils (Meriones unguiculatus) infected with H. pylori 26,695.
Quantification of miRNA expression was performed by quantitative real-time polymerase chain reaction (qRT-PCR) of paraffin-embedded gastric lesions of children with or without an infection (n = 25), adults with follicular gastritis and metaplasia (n = 32) and eight-week-old M. unguiculatus males (Hsd:MON) infected with H. pylori 26,695 for 0, 3, 6, 12 and 18 months (n = 25). The genes RNU48 and RNU6 were used as endogenous controls for data normalization. Statistical analyses were performed using Kruskal-Wallis, Mann-Whitney, ANOVA and Student's t-test.
The expression of miRNA-146a and miRNA-155 in infected children increased by 247.6- and 79.4-fold (on average), respectively, compared to that observed in the control group. However, these results were not significant (p = 0.12 and p = 0.07 respectively). In some children a gradual increase in expression was observed, while in others, expression was very high. Additionally, the expression levels of miRNA-146a and miRNA-155 increased by an average of 21.7- and 62-fold, respectively, in adult patients with follicular gastritis when compared to those of the controls. In M. unguiculatus infected with H. pylori 26,695, the expression of both miRNAs increased as the infection progressed.
This is the first report to show differences in the expression of miRNA-146a and miRNA-155 in paediatric and adult patients with gastritis who were infected with H. pylori. In addition, in M. unguiculatus infected with H. pylori, miRNA expression was associated with the progression of infection and the ability of the bacteria to adapt to the host.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
is a resident of the human gut. However, certain
toxigenic strains exist that secrete the nonribosomal peptide tilivalline (TV) cytotoxin. TV is a pyrrolobenzodiazepine that causes ...antibiotic-associated hemorrhagic colitis (AAHC). The biosynthesis of TV is driven by enzymes encoded by the
and NRPS operons. In this study, we determined the effect of environmental signals such as carbon sources, osmolarity, and divalent cations on the transcription of both TV biosynthetic operons. Gene expression was enhanced when bacteria were cultivated in tryptone lactose broth. Glucose, high osmolarity, and depletion of calcium and magnesium diminished gene expression, whereas glycerol increased transcription of both TV biosynthetic operons. The cAMP receptor protein (CRP) is a major transcriptional regulator in bacteria that plays a key role in metabolic regulation. To investigate the role of CRP on the cytotoxicity of
, we compared levels of expression of TV biosynthetic operons and synthesis of TV in wild-type strain MIT 09-7231 and a Δ
isogenic mutant. In summary, we found that CRP directly activates the transcription of the
and NRPS operons and that the absence of CRP reduced cytotoxicity of
on HeLa cells, due to a significant reduction in TV production. This study highlights the importance of the CRP protein in the regulation of virulence genes in enteric bacteria and broadens our knowledge on the regulatory mechanisms of the TV cytotoxin.
Mycobacteria, like other microorganisms, survive under different environmental variations by expressing an efficient adaptive response, oriented by regulatory elements, such as transcriptional ...repressors of the TetR family. These repressors in mycobacteria also appear to be related to cholesterol metabolism. In this study, we have evaluated the effect of a fatty acid (oleic–palmitic–stearic)/cholesterol mixture on some phenotypic and genotypic characteristics of a tetR-mutant strain (
BCG_2177c
mutated gene) of
M. bovis
BCG, a homologous of
Rv2160A
of
M. tuberculosis
. In order to accomplish this, we have analyzed the global gene expression of this strain by RNA-seq and evaluated its neutral-lipid storage capacity and potential to infect macrophages. We have also determined the macrophage response by measuring some pro- and anti-inflammatory cytokine expressions. In comparison with wild-type microorganisms, we showed that the mutation in the
BCG_2177c
gene did not affect the growth of
M. bovis
BCG in the presence of lipids but it probably modified the structure/composition of its cell envelope. Compared to with dextrose, an overexpression of the transcriptome of the wild-type and mutant strains was observed when these mycobacteria were cultured in lipids, mainly at the exponential phase. Twelve putative intracellular redox balance maintenance genes and four others coding for putative transcriptional factors (including WhiB6 and three TetR-like) were the main elements repeatedly overexpressed when cultured in the presence of lipids. These genes belonged to the central part of what we called the “genetic lipid signature” for
M. bovis
BCG. We have also found that all these mycobacteria genotypic changes affected the outcome of BCG-infected macrophages, being the mutant strain most adapted to persist longer inside the host. This high persistence result was also confirmed when mutant-infected macrophages showed overexpression of the anti-inflammatory cytokine TGF-β versus pro-inflammatory cytokines. In summary, the lack of this TetR-like repressor expression, within a lipid environment, may help mycobacteria overcome intracellular redox stress and survive longer inside their host.
The slow-growing, nontuberculous mycobacterium
possesses two rRNA operons,
and
, located downstream from the
and
genes, respectively. Here, we report the sequence and organization of the promoter ...regions of these two
operons. In the
operon, transcription can be initiated from the two promoters, named P1
and PCL1, while in
, transcription can only start from one, called P1
. Both
operons show a similar organization to the one described in
and
. Furthermore, by qRT-PCR analyses of the products generated from each promoter, we report that stress conditions such as starvation, hypoxia, and cellular infection affect the contribution of each operon to the synthesis of pre-rRNA. It was found that the products from the PCL1 promoter of
play a pivotal role in rRNA synthesis during all stress conditions. Interestingly, the main participation of the products of transcription from the P1 promoter of
was found during hypoxic conditions at the NRP1 phase. These results provide novel insights into pre-rRNA synthesis in mycobacteria, as well as the potential ability of
to produce latent infections.
The acquisition of Salmonella pathogenicity island 2 (SPI-2) conferred on Salmonella the ability to survive and replicate within host cells. The
bicistronic operon, located in SPI-2, encodes the ...SsrAB two-component system (TCS), which is the central positive regulator that induces the expression of SPI-2 genes as well as other genes located outside this island. On the other hand, CpxRA is a two-component system that regulates expression of virulence genes in many bacteria in response to different stimuli that perturb the cell envelope. We previously reported that the CpxRA system represses the expression of SPI-1 and SPI-2 genes under SPI-1-inducing conditions by decreasing the stability of the SPI-1 regulator HilD. Here, we show that under SPI-2-inducing conditions, which mimic the intracellular environment, CpxRA represses the expression of SPI-2 genes by the direct action of phosphorylated CpxR (CpxR-P) on the
regulatory operon. CpxR-P recognized two sites located proximal and distal from the promoter located upstream of
. Consistently, we found that CpxRA reduces the replication of Salmonella enterica serovar Typhimurium inside murine macrophages. Therefore, our results reveal CpxRA as an additional regulator involved in the intracellular lifestyle of Salmonella, which in turn adds a new layer to the intricate regulatory network controlling the expression of Salmonella virulence genes.
SPI-2 encodes a type III secretion system (T3SS) that is a hallmark for the species Salmonella enterica, which is essential for the survival and replication within macrophages. Expression of SPI-2 genes is positively controlled by the two-component system SsrAB. Here, we determined a regulatory mechanism involved in controlling the overgrowth of Salmonella inside macrophages. In this mechanism, CpxRA, a two-component system that is activated by extracytoplasmic stress, directly represses expression of the
regulatory operon; as a consequence, expression of SsrAB target genes is decreased. Our findings reveal a novel mechanism involved in the intracellular lifestyle of Salmonella, which is expected to sense perturbations in the bacterial envelope that Salmonella faces inside host cells, as the synthesis of the T3SS-2 itself.
There has been very limited investigation regarding the genetic diversity of
(MTb) strains isolated from human immunodeficiency virus (HIV)-infected patients in Mexico. In this study, we isolated 93 ...MTb strains from pulmonary and extrapulmonary samples of HIV-infected patients treated in a public hospital in Mexico City to evaluate the genetic diversity using spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) typing (based on 24 loci). The cohort comprised 80 male and 13 female individuals. There was a positive correlation between a high HIV viral load (>100,000 copies) and extrapulmonary tuberculosis (TB) (
= 0.306,
= 0.008). Lineage 4 was the most frequent lineage (79 strains). In this lineage, we found the H clade (
= 24), including the Haarlem, H3, and H1 families; the T clade (
= 22), including T1 and T2; the X clade (
= 15), including X1 and X3; the LAM clade (
= 14), including LAM1, LAM2, LAM3, LAM6, and LAM9; the S clade (
= 2); Uganda (
= 1); and Ghana (
= 1). We also found 12 strains in the EAI clade belonging to lineage 1, including the EAI2-Manila and EAI5 families. Interestingly, we identified one strain belonging to the Beijing family, which is part of lineage 2. One strain could not be identified. This study reports high genetic diversity among MTb strains, highlighting the need for a molecular epidemiological surveillance system that can help to monitor the spread of these strains, leading to more appropriate measures for TB control in HIV-infected patients.
Drug-resistant tuberculosis (TB) is associated with higher mortality rates in patients with human immunodeficiency virus (HIV). In Mexico, the number of deaths due to TB among the HIV-positive ...population has tripled in recent years.
Ninety-three
strains isolated from the same number of HIV-infected patients treated in a public hospital in Mexico City were studied to determine the drug resistance to first- and second-line anti-TB drugs and to identify the mutations associated with the resistance.
Of the 93 patients, 82.7% were new TB cases, 86% were male, and 73% had extrapulmonary TB. Most patients (94%) with a CD4 T-lymphocyte count <350 cells/mm
were associated with extrapulmonary TB (
<0.0001), whilst most patients (78%) with a CD4 T-lymphocyte count >350 cells/mm
were associated with pulmonary TB (
= 0.0011). Eighty-two strains were pan-susceptible, four mono-resistant, four poly-resistant, two multidrug-resistant, and one was extensively drug-resistant. In the rifampicin-resistant strains,
S531L was the mutation most frequently identified, whereas the
C15T and
S315T1 mutations were present in isoniazid-resistant strains. The extensively drug-resistant strain also contained the mutation
D94A.
These data highlight the need to promptly diagnose the drug resistance of
among all HIV-infected patients by systematically offering access to first- and second-line drug susceptibility testing and to tailor the treatment regimen based on the resistance patterns to reduce the number of deaths in HIV-infected patients.
•The microbiological quality of packaged ice was evaluated.•The presence of E. coli and diarrheagenic E. coli pathotypes in ice was assessed.•The occurrence of nontuberculous mycobacteria (NTM) in ...ice samples was determined.•Thirty-eight (44.7%) ice samples were outside of Mexico's official guidelines.•NTM species associated with illness were identified in some ice samples.
This study aimed to determine the bacteriological quality and presence of diarrheagenic Escherichia coli pathotypes (DEP) and nontuberculous mycobacteria (NTM) species in 85 packaged ice samples from 12 different states of central Mexico. Three samples had a pH of 9.8 and therefore fell outside of the acceptable range for pH. All samples were positive for aerobic-mesophilic bacteria, with limits ranging from 1 to 3.47 log CFU/mL. In total, 35, 11, and 3 ice samples were positive for total coliforms (TC), fecal coliforms (FC), and E. coli, respectively. In the samples, the TC concentration ranged from <1.1 to >23 MPN/100 mL and from <1.1 to 23 MPN/100 mL for FC and E. coli. In total, 38 (44.7%) ice samples were outside of Mexico's official guidelines. None of the 12 E. coli strains isolated from the three ice samples belonged to DEP. NTM were recovered from 20 ice samples and included M. neoaurum (n = 7), M. porcinum (n = 2), M. flavescens (n = 2), M. fortuitum (n = 1), M. abscessus (n = 1), M. senegalense (n = 1), M. conceptionense (n = 1), and M. sp. (n = 1). In the remaining four samples, two NTM were isolated simultaneously. Thus, we recommend that producers should evaluate the microbiological quality of purified water used as a raw material as well as that of the final product, the ice should be packed in thick bags to avoid stretching and tearing during transportation or storage to prevent environmental contamination of ice, personnel involved in the production, and handling of ice should be trained in relative hygiene matters and how ice-machines should be cleaned and disinfected and the implementation of hazard analysis and critical control points must be applied throughout the chain of production. Finally, regular inspection by the authorities is also of great importance. These recommendations can be applied in different countries with low microbiological quality packaged ice.
Gliomas are heterogeneous, solid, and intracranial tumors that originate from glial cells. Malignant cells from the tumor undergo metabolic alterations to obtain the energy required for proliferation ...and the invasion of the cerebral parenchyma. The alterations in the expression of the genes related to the metabolic pathways can be detected in biopsies of gliomas of different CNS WHO grades. In this study, we evaluated the expression of 16 candidate reference genes in the HMC3 microglia cell line. Then, statistical algorithms such as BestKeeper, the comparative ΔC
method, geNorm, NormFinder, and RefFinder were applied to obtain the genes most suitable to be considered as references for measuring the levels of expression in glioma samples. The results show that
and
are two novel genes suitable for genic expression studies on gliomas. Finally, we analyzed the expression of genes involved in metabolic pathways in clinical samples of brain gliomas of different CNS WHO grades. RT-qPCR analysis showed that in CNS WHO grade 3 and 4 gliomas, the expression levels of
,
,
,
,
,
,
,
, and
were higher than those of CNS WHO grade 1 and 2 glioma biopsies. Hence, our results suggest that reference genes from metabolic pathways have different expression profiles depending on the stratification of gliomas and constitute a potential model for studying the development of this type of tumor and the search for molecular targets to treat gliomas.
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