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

 Candida glabrata cells suspended in water are under hypo-osmotic stress and undergo cell death in 1–2 days, unless they are at a density of more than 105 CFU mL 1. The dying cells exhibit FITC-annexin V staining, indicative of programmed cell death (apoptosis). In a higher cell density, cells are protected and survive at least for 4 days. Filtrates from cells at high density can protect those at lower density, indicating that cells release substances, amounting to c. 5mgL 1 of cell suspension, that protect each other against hypo-osmotic stress. In a concentrated form, the released materials can support growth, indi cating that the protective material includes carbon and nitrogen sources, as well as vitamins that are required by C. glabrata for growth. We conclude that cell death from hypo-osmotic stress can be alleviated by small amounts of nutrients.

Background: African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host, but it is unknown whether either process is essential to the parasite. Methodology/Principal Findings: Pyrimidine requirements for growth were investigated using strictly pyrimidine-free media, with or without single added pyrimidine sources. Growth rates of wild-type bloodstream form Trypanosoma brucei brucei were unchanged in pyrimidine-free medium. The essentiality of the de novo pyrimidine biosynthesis pathway was studied by knocking out the PYR6-5 locus that produces a fusion product of orotate phosphoribosyltransferase (OPRT) and Orotidine Monophosphate Decarboxylase (OMPDCase). The pyrimidine auxotroph was dependent on a suitable extracellular pyrimidine source. Pyrimidine starvation was rapidly lethal and non-reversible, causing incomplete DNA content in new cells. The phenotype could be rescued by addition of uracil; supplementation with uridine, 29deoxyuridine, and cytidine allowed a diminished growth rate and density. PYR6-52/ 2 trypanosomes were more sensitive to pyrimidine antimetabolites and displayed increased uracil transport rates and uridine phosphorylase activity. Pyrimidine auxotrophs were able to infect mice although the infection developed much more slowly than infection with the parental, prototrophic trypanosome line. Conclusions/Significance: Pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the py

African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host. However, uptake of pyrimidines in bloodstream form trypanosomes has not been investigated, making it difficult to judge the relative importance of salvage and synthesis or to design a pyrimidine-based chemotherapy. Detailed characterization of pyrimidine transport activities in bloodstream form Trypanosoma brucei brucei found that these cells express a high-affinity uracil transporter (designated TbU3) that is clearly distinct from the procyclic pyrimidine transporters. This transporter had low affinity for uridine and 2′deoxyuridine and was the sole pyrimidine transporter expressed in these cells. In addition, thymidine was taken up inefficiently through a P1-type nucleoside transporter. Of importance, the anticancer drug 5-fluorouracil was an excellent substrate for TbU3, and several 5-fluoropyrimidine analogs were investigated for uptake and trypanocidal activity; 5F-orotic acid, 5F-2′deoxyuridine displayed activity in the low micromolar range. The metabolism and mode of action of these analogs was determined using metabolomic assessments of T. brucei clonal lines adapted to high levels of these pyrimidine analogs, and of the sensitive parental strains. The analysis showed that 5-fluorouracil is incorporated into a large number of metabolites but likely exerts toxicity through incorporation into RNA. 5F-2′dUrd and 5F-2′dCtd are not incorporated into nucleic acids but act as prodrugs by inhibiting thymidylate synthase as 5F-dUMP. We present the most complete model of pyrimidine salvage in T. brucei to date, supported by genome-wide profiling of the predicted pyrimidine biosynthesis and conversion enzymes.

ABSTRACT

The objective of the study was to evaluate the effect of Diabetes in elderly females.

Oxidative stress is regarded as a main causal factor for natural ageing. This study tested

the hypothesis that healthy elderly people show higher oxidative DNA damage levels and

lower antioxidative enzymatic defense capacities. In type II diabetic patients, persistence

of hyperglycemia is one of the causes of increased in oxygen free radicals. This increase

oxidative stress and makes the life threatening cardiovascular complications. Blood

samples were drawn from selective 100 elderly females (with and without diabetes). In

elderly females having menopause, there occur hypercholesterolemia,

hypertriglyceridemia, and hyperlipoproteinemia, these on the body, escort an increase in

free radicals, with mounting oxidative stress. In diabetes the body suffers with a poor or

negligible glycemic control. All diabetic post menopausal females with CVD have been

reported a significant high levels of Fasting Blood Sugar, Total Cholesterol, Triglycerides,

LDL-C, VLDL-C, CAT, NT-proBNP and MDA and significantly lower levels of HDL-C, GPx,

SOD as compared to control subjects. In type-II diabetes mellitus elderly female patients

during the postmenopause, the probability of secondary complications of cardiovascular

diseases increases.

Given the pressing need for new antiprotozoal drugs without cross-resistance with current (failing) chemotherapy, we have explored 3-tridecylpyridinium alkaloids (3TPAs), derivatives of viscosamine, as antiparasitic agents. We have developed a simple synthetic route toward viscosamine and related cyclic and linear monomers and oligomers. Evaluation for cytotoxicity on the protozoan parasites Trypanosoma brucei, Leishmania spp., and Plasmodium falciparum revealed several 3TPAs with antiprotozoal activity in the nanomolar range. Their promising selectivity index in vitro prompted us to study the dynamics of cytotoxicity on trypanosomes in more detail. Parasites were killed relatively slowly at therapeutically safe concentrations, in a process that did not target the cell cycle. Clearance of T. brucei cultures was observed at drug concentrations of 1–10 μM.