The mammary gland reaches a fully differentiated phenotype at lactation, a stage characterized by the abundant expression of beta-casein. We have investigated the expression and regulation of gap junction proteins (connexins, Cx) during the various developmental stages of mouse mammary gland. Immunohistochemical analysis, with specific antibodies, reveals that Cx26 and Cx32 are expressed and confined to the cell borders of luminal epithelial cells in all developmental stages of the gland. Cx26 and Cx32 expression, at the mRNA and protein levels, increases in pregnancy and peaks in lactation. Whereas Cx43 mRNA decreases in pregnancy and lactation, the functional activity of Cx43 protein, which has been localized to myoepithelial cells, is regulated (through phosphorylation) during pregnancy and peaks during lactation. Cx30 mRNA and proteins have, for the first time, been detected in mammary gland epithelia. Using reverse transcription/polymerase chain reaction and sequencing techniques, we show that Cx30 is abundant in pregnant and lactating mammary gland. Cx30 protein levels have not been detected in the mammary gland prior to day 15 of pregnancy, whereas maximum expression occurs at the onset of lactation. In mouse mammary cells in culture, Cx30 is epithelial-cell-specific and is induced by lactogenic hormones. These data identify a novel player in mammary differentiation and suggest a potential role for Cx30 in the fully differentiated gland.

Cardiovascular diseases (CVDs) are significantly high in the Lebanese population with the two most predominant forms being atherosclerosis and venous thrombosis. The purpose of our study was to assess the association of a spectrum of CVD related genes and combined state of hypertension hypercholesterolemia (HH) in unrelated Lebanese. Twelve polymorphisms were studied by multiplex PCR and reverse hybridization of DNA from 171 healthy individuals and 144 HH subjects. Two genes were significantly associated with HH: ACE (OR: 9.20, P<0.0001) and PAI-1 (OR: 2.29, P = 0.007), respectively with the occurrence of the risky alleles “Del” and “4G”. The frequencies of the Del and 4G alleles were found to be 0.98 and 0.90 in the HH group versus 0.84 and 0.79 in the healthy group, respectively. Serum ACE activity and PAI-I increased significantly with Del/Del and 4G/5G genotypes. The co-expression of Del/4G(+/+) was detected in 113 out of 171 (66.0%) controls and 125 out of 144 (86.8%) HH subjects. Del/4G(-/-) was detected in only 6 (3.5%) controls and undetected in the HH group. Three venous thrombosis related genes [FV(Leiden), MTHFR(A1298C) and FXIII(V34L)] were significantly related to the prominence of the co-expression of Del/4G(+/+). A range of 2 to 8 combined polymorphisms co-expressed per subject where 5 mutations were the most detected. In Del/4G(+/+) subjects, peripheral blood mononuclear cells (PBMCs) produced significant elevated levels of IFN-γ and TNF-α contrary to IL-10, and no variations occurred for IL-4. ACE inhibitor (ramipril) in combination with statin (atorvastatin) and not alone reversed significantly the situation. This first report from Lebanon sheds light on an additional genetic predisposition of a complex spectrum of genes involved in CVD and suggests that the most requested gene FVL by physicians may not be sufficient to diagnose eventual future problems that can occur in the cardiovascular system. Subjects expressing the double mutations (Del/4G) are at high risk for the onset of CVDs.

PCR is commonly used for mRNA quantitation. Previously described procedures are applied to one or a few specific mRNA sequences. We show here that methods used for amplifying heterogeneous eDNA populations can be applied to the quantitation of many mRNA species. This quantitation is achieved by dot blotting and hybridization with the corresponding probes after amplifying a bulk mRNA population. Only a single, two-round-amplification assay is required for quantitation of a whole set of mRNA species. The proportionality of input molecules to output signal was shown by performing a series of control experiments. We applied this technique to measure the relative variations of the MBP, Po, and MAG mRNA sequences in the normal trembler mouse model. The results were consistent with previously described Northern blot data. This quantitative PCR method provides a rapid and reliable way to quantify relative amounts of mRNA species in small amounts of total RNA by using internal controls. Estimation of the amount of one or several mRNA species is a common way to monitor responses of biological systems to environmental stimuli or changes in develomental programs. The classic RNA hybridization technique has the disadvantage of requiring a considerable amount of mRNA (at least 10s-106 specific molecules per sample). With reverse transcription polymerase chain reaction (RT-PCR) the detection threshold has been lowered at least 1000-fold. Various protocols using PCR for quantitating specific RNA species have been developed in the last few years. ~ Reasonably good results have been obtained when an RNA molecule of known concentration is added to the sample and used as a standard and when the reaction is monitored during its exponential phase. (1'2'12) Alternative quantitative procedures involving reactions that have been driven to the plateau have been also developed. (9'1~ However, these methods have some drawbacks. First, preliminary calibration analysis has to be carried out for each individual template. Second, such procedures allow one analysis and therefore give only one answer per PCR assay for the sample under study. Alternatively, amplification of complex DNA or cDNA mixtures have already been described ~ for the constitution of DNA and cDNA libraries. Here, we show that such amplification techniques can be adapted to a quantitation end. Our strategy is outlined in Figure 1. First, a double-stranded cDNA population is synthesized from the RNA population to be studied. Second, a specially designed linker-adapter is bluntend ligated to this cDNA; third, the population is amplified in two successive rounds of 20-cycle PCR; fourth, aliquots of the amplification products are taken during the exponential phase of the second PCR round, dot-blotted, and hybridized with different specific probes. Finally, hybridization signals are plotted on a semi-log scale against the number of cycles, and quantitation of a given mRNA species is obtained by extrapolating to zero cycle. This provides an easy, accurate, and reproducible relative...

Introduction: The increasing incidence of infections caused by multidrug-resistant bacteria is considered a global health problem. This study aimed to investigate this resistance in Gram-negative bacteria isolated from patients hospitalized in North-Lebanon. Methodology: All isolates were identified using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antibiotic susceptibility testing was achieved using disk diffusion, E-test and Broth microdilution methods. Phenotypic detection of carbapenemase was carried out using the CarbaNP test. RT-PCR, standard-PCR and sequencing were performed to detect resistance genes and oprD gene. Conjugal transfer was carried out between our isolates and Escherichia coli J53 to detect the genetic localization of resistance genes. MLST was conducted to determine the genotype of each isolate. Results: Twenty-three carbapenem-resistant Enterobacterales of which eight colistin-resistant Escherichia coli, and Twenty carbapenem-resistant Pseudomonas aeruginosa were isolated. All isolates showed an imipenem MIC greater than 32 mg/mL with MICs for colistin greater than 2 mg/L for E. coli isolates. All the Enterobacterales isolates had at least one carbapenemase-encoding gene, with E. coli isolates coharboring blaNDM-4 and mcr-1 genes. Moreover, 16/20 Pseudomonas aeruginosa harbored the blaVIM-2 gene and 18/20 had mutations in the oprD gene. MLST revealed that the isolates belonged to several clones. Conclusions: We report here the first description in the world of clinical E. coli isolates coharboring blaNDM-4 and mcr-1 genes, and K. pneumoniae isolates producing NDM-6 and OXA-48 carbapenemases. Also, we describe the emergence of NDM-1-producing E. cloacae in Lebanon. Screening for these isolates is necessary to limit the spread of resistant microorganisms in hospitals.

No abstract