Structural changes affecting cardiomyocyte function may contribute to the pathophysiological

remodeling underlying cardiac function impairment. Recent reports have shown that

endogenous nitric oxide (NO) plays an important role in this process. In order to examine the role

of NO in cardiomyocyte remodeling, male rats were acclimated to room temperature (22± 1
C) or

cold (4 ± 1
C) and treated with 2.25% L-arginine
HCl or 0.01% L-NAME (Nx-nitro-L-arginine

methyl ester)
HCl for 45 days. Untreated groups served as controls. Right heart ventricles were routinely

prepared for light microscopic examination. Stereological estimations of volume densities of

cardiomyocytes, surrounding blood vessels and connective tissue, as well as the morphometric measurements

of cardiomyocyte diameters were performed. Tissue sections were also analyzed for

structural alterations. We observed that both L-arginine and L-NAME supplementation induced

cardiomyocyte hypertrophy, regardless of ambient temperature. However, cardiomyocyte hypertrophy

was associated with fibrosis and extra collagen deposition only in the L-NAME treated group.

Taken together, our results suggest that NO has a modulatory role in right heart ventricle remodeling

by coordinating hypertrophy of cardiomyocytes and fibrous tissue preventing cardiac fibrosis.

Purpose: The first aim of this study was to analyse the presence of circulating Claudin 4 (CLDN4) mRNA in serum and to evaluate whether the rate of CLDN4 mRNA can be used as a tumor marker to discriminate breast cancer patients and healthy women. The second aim was to compare CLDN4 mRNA measured using real-time quantitative RT-PCR with CA 15-3 measured using immunoassay as biomarker for the diagnosis of breast cancer.

Experimental Design: The present study collected serum samples

from 118 patients with recently diagnosed breast cancer and 30 matched healthy women. The number of copies of CLDN4 mRNA and the CA-15-3 rate were analysed and the percentage of patients with increased marker level were compared.

Results: The quality and the stability of mRNA which were evaluated

using the housekeeping RPLPO gene in all studied serum samples were confirmed (p>0.05). The expression level of CLDN4 mRNA was found significantly increased in serum of 64% patients with breast cancer from the threshold calculated in healthy women (p<0.001) versus 6% for CA 15-3. The expression of claudin4 was not correlated with histological type, tumour size, nodal status, tumor grade, and status of ER, PR and Her2 but only with age.

Conclusions:

Our results showed that the expression of CLDN4 mRNA by real-time quantitative PCR give the opportunity to predict breast cancer before pathological diagnosis in a large majority of patients.

Leishmania pyrimidine salvage is replete with opportunities for therapeutic intervention with enzyme inhibitors or antimetabolites. Their uptake into cells depends upon specific transporters; therefore it is essential to establish whether various Leishmania species possess similar pyrimidine transporters capable of drug uptake. Here, we report a comprehensive characterization of pyrimidine transport in L. major and L. mexicana. In both species, two transporters for uridine/adenosine were detected, one of which also transported uracil and the antimetabolites 5-fluoruracil (5-FU) and 5F,2'deoxyuridine (5F,2'dUrd), and was designated uridine-uracil transporter 1 (UUT1); the other transporter mediated uptake of adenosine, uridine, 5F,2'dUrd and thymidine and was designated Nucleoside Transporter 1 (NT1). To verify the reported L. donovani model of two NT1-like genes encoding uridine/adenosine transporters, and an NT2 gene encoding an inosine transporter, we cloned the corresponding L. major and L. mexicana genes, expressing each in T. brucei. Consistent with the L. donovani reports, the NT1-like genes of either species mediated the adenosine-sensitive uptake of [3H]-uridine but not of [3H]-inosine. Conversely, the NT2-like genes mediated uptake of [3H]-inosine but not [3H]-uridine. Among pyrimidine antimetabolites tested, 5-FU and 5F,2'dUrd were the most effective antileishmanials; resistance to both analogs was induced in L. major and L. mexicana. In each case it was found that the resistant cells had lost the transport capacity for the inducing drug. Metabolomics analysis found that the mechanism of action of 5-FU and 5F-2'dUrd was similar in both Leishmania species, with major changes in deoxynucleotide metabolism. We conclude that the pyrimidine salvage system is highly conserved in Leishmania species - essential information for the development of pyrimidine-based chemotherapy.

 The tetracycline promoter (tet) has been manipulated in several systems as an inducible regulator of gene expression. In this study the effect of doxycycline on C. glabrata viability has been examined in water and according to CLSI standard method. In addition, flow cytometry analysis was performed to detect SLT2 protein activation by sub-inhibitory concentrations of doxycycline. Our findings show that 100 µg/ml of doxycycline is the maximum tolerated dosage in media; however, C. glabrata was sensitive to 40-50µg/ml in water. 50 µg/ml of doxycycline induced SLT2p phosphorylation and our results support the idea of involvement of doxycycline in alteration of C. glabrata oxidative status. These results should be considered when the tet promoter system is used in yeast molecular studies

Abstract:

Germplasm (Oocyte and embryo) and ovarian tissue cryopreservation have become routine procedure in human in vitro fertilization and embryo transfer. Human oocyte and embryo cryopreservation field has been much progress during its fourth decade history. Two basic strategies have ruled the mammalian germplasm cryopreservation field: the traditional slow freezing technique also referred as conventional slow freezing or controlled slow freezing which started in the 1970s, and followed by vitrification technique developed in the late 1980s, In the slow freezing protocol, the embryo is placed in a hypertonic solution and slowly cooled using a programmable freezer to sub-zero temperatures, and then plunged into liquid nitrogen, whereas vitrification technique can be performed by using high cooling rates and high concentration of cryoprotectants. In recent years vitrification technique is a common method for cryopreservation of oocytes and embryos in comparable to slow freezing method. Vitrification appears to be associated with a significantly higher post-thawing survival rate, higher implantation rate and more pregnancies than slow freezing. Vitrification protocol simplifies the cryopreservation process without physical damage to embryos and oocytes and decreases the chilling injuries of germplasm as it passes through a very low temperature very rapidly. Although germplasm cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the in vitro fertilization cycle, further prospective trials are necessary to confirm that vitrification is a superior technique to slow freezing method. The purpose of the present mini-review is to highlight oocyte and embryo cryopreservation technique in general, with comparing the different laboratory methods in particular slow freezing (vs) vitrification and successful implantation after transfer. 

The antifungal effect of thymoquinone, a component of black seed essential oil, has been studied on different types of fungi. Its mechanism of action as an antifungal has not been described yet. This study demonstrates the fungicidal effect of thymoquinone on different Candida species with particular emphasis on C. glabrata planktonic cells and biofilms. Since cell death was induced via the generation of oxidative stress as evidenced by the abrogation of thymoquinone toxicity in cells incubated with antioxidants, a part of thymoquinone’s mechanism of action includes a direct involvement as a pro-oxidant. This was further confirmed by measuring the generation of reactive oxygen species, glutathione level reduction and decrease in mitochondrial membrane potential. The oxidative stress caused by thymoquinone was confirmed to be the cause of death and not a result of cell death.

The Calgary Biofilm Device (CBD) has been described as a technology for the rapid and reproducible assay of biofilm susceptibilities to antibiotics. In this study a simple and inexpensive alternative to the CBD was developed from polypropylene (PP) microcentrifuge tubes and pipette tip boxes. The utility of the device was demonstrated using Candida glabrata, a yeast that can develop antimicrobial-resistant biofilm communities. Biofilms of C. glabrata were formed on the outside surface of microcentrifuge tubes and examined by quantitative analysis and scanning electron microscopy. Growth of three C. glabrata strains, including a clinical isolate, demonstrated that biofilms could be formed on the microcentrifuge tubes. After 24 h incubation the three C. glabrata strains produced biofilms that were recovered into cell suspension and quantified. The method was found to produce uniform and reproducible results with no significant differences between biofilms formed on PP tubes incubated in various compartments of the device. In addition, the difference between maximum and minimum counts for each strain was comparable to those which have been reported for the CBD device.

One of the most promising new targets for trypanocidal drugs to emerge in recent years is the cyclic AMP (cAMP) phosphodiesterase (PDE) activity encoded by TbrPDEB1 and TbrPDEB2. These genes were genetically confirmed as essential, and a highaffinity inhibitor, CpdA, displays potent antitrypanosomal activity. To identify effectors of the elevated cAMP levels resulting from CpdA action and, consequently, potential sites for adaptations giving resistance to PDE inhibitors, resistance to the drug was induced. Selection of mutagenized trypanosomes resulted in resistance to CpdA as well as cross-resistance to membranepermeable cAMP analogues but not to currently used trypanocidal drugs. Resistance was not due to changes in cAMP levels or in PDEB genes. A second approach, a genome-wide RNA interference (RNAi) library screen, returned four genes giving resistance to CpdA upon knockdown. Validation by independent RNAi strategies confirmed resistance to CpdA and suggested a role for the identified cAMP Response Proteins (CARPs) in cAMP action. CARP1 is unique to kinetoplastid parasites and has predicted cyclic nucleotide binding-like domains, and RNAi repression resulted in >100-fold resistance. CARP2 and CARP4 are hypothetical conserved proteins associated with the eukaryotic flagellar proteome or with flagellar function, with an orthologue of CARP4 implicated in human disease. CARP3 is a hypothetical protein, unique to Trypanosoma. CARP1 to CARP4 likely represent components of a novel cAMP signaling pathway in the parasite. As cAMP metabolism is validated as a drug target in Trypanosoma brucei, cAMP effectors highly divergent from the mammalian host, such as CARP1, lend themselves to further pharmacological development