Follicle size-dependent effects of sow follicular fluid on in vitro cumulus expansion, nuclear maturation and blastocyst formation of sow cumulus oocytes complexes

Follicular fluid from 2 to 4 and 5 to 8 mm diameter non-atretic follicles (SFF and LFF, respectively) of sows was added during IVM of cumulus oocytes complexes (COCs) to study its effects on cumulus expansion, nuclear maturation, and subsequent fertilization and embryo development in presence or absence of recombinant human FSH. COCs aspirated from 2 to 5 mm follicles of sow ovaries, were cultured for the first 22 h in TCM-199 and 100 microM cysteamine, with or without 10% pFF and/or 0.05 IU/ml recombinant hFSH. For the next 22 h, the COCs were cultured in the same medium, but without pFF and FSH. After culture, cumulus cells were removed and the oocytes were either fixed and stained to evaluate nuclear stages or co-incubated with fresh sperm. Twenty-four hours after fertilization, presumptive zygotes were fixed to examine fertilization or cultured for 6 days to allow blastocyst formation. Subsequently, embryos were evaluated and the blastocysts were fixed and stained to determine cell numbers. When LFF was added to maturation medium, cumulus expansion and percentage of nuclear maturation (277 +/- 61 microm and 72%, respectively) of COCs were significantly higher (P < 0.05) than those in SFF (238 +/- 33 microm and 55%, respectively). However, in the presence of FSH both FF stimulated cumulus expansion and nuclear maturation to a similar degree. No differences were observed with regards to sperm penetration, male pronucleus formation, and to polyspermia between fertilized oocytes matured either in SFF or LFF. Fertilized oocytes matured in the presence of LFF without or with FSH showed a higher cleavage (45 +/- 7% and 51 +/- 7%, respectively) and blastocyst (14 +/- 4% and 22 +/- 6%, respectively) formation rate compared to SFF (cleavage, 35 +/- 8% and 41 +/- 4%, blastocyst: 8 +/- 3 and 13 +/-3, respectively; P < 0.05). The mean number of cells per blastocyst did not differ significantly between treatments. These findings indicate that factor(s) within follicles at later stages of development play an important role during oocyte maturation and thereby enhance developmental competence to occur.     

ARMC4 Mutations Cause Primary Ciliary Dyskinesia with Randomization of Left/Right Body Asymmetry

The motive forces for ciliary movement are generated by large multiprotein complexes referred to as outer dynein arms (ODAs), which are preassembled in the cytoplasm prior to transport to the ciliary axonemal compartment. In humans, defects in structural components, docking complexes, or cytoplasmic assembly factors can cause primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease and defects in laterality. By using combined high resolution copy-number variant and mutation analysis, we identified ARMC4 mutations in twelve PCD individuals whose cells showed reduced numbers of ODAs and severely impaired ciliary beating. Transient suppression in zebrafish and analysis of an ENU mouse mutant confirmed in both model organisms that ARMC4 is critical for left-right patterning. We demonstrate that ARMC4 is an axonemal protein that is necessary for proper targeting and anchoring of ODAs.     

Approved spectrofluorimetric strategies for assurance of three modern antineoplastic drugs: tepotinib,sotorasib, and darolutamide in their dose forms and biological liquids utilizing mercurochrome

An approved, straightforward, fast, and delicate spectrofluorimetric strategy was developed for the estimation of tepotinib (TEPO), sotorasib (SOTO), and darolutamide (DARO) as new antineoplastic drugs. The spectrofluorimetric strategy was based on quantitative fluorescence quenching of MER at 538 nm after being excited at 350 nm by the addition of the cited drugs in the presence of acetate buffer (pH 3.5). The degree of fluorescence quenching was directly proportional to the concentrations of the cited drugs within the concentration range of 0.5–10.0, 0.2–10, and 0.4–10.0 μg ml⁻¹ for TEPO, SOTO, and DARO, respectively. Mean ± standard deviation (SD) were calculated for the studied drugs as follows; 99.9 ± 0.87, 99.72 ± 1.08, and 100.21 ± 1.44, for TEPO, SOTO, and DARO, respectively. Limit of detection (LOD) values were 0.16, 0.05, and 0.11 μg ml⁻¹, whereas limit of quantitation (LOQ) values were 0.5, 0.15, and 0.36 μg ml⁻¹ for TEPO, SOTO, and DARO, respectively. Statistical comparison through detailed strategies produced greater understanding and found that there were no noteworthy contrasts in exactness and exactness between strategies. The proposed strategy was used effectively to analyze the measurement of different forms of the examined drugs. Moreover, the recommended fluorimetric strategy was used for examination of TEPO, SOTO, and DARO in human plasma and urine tests.     

Synthesis of new bisaryl cyclopentathiophene and thieno-cyclopentoxazolidine derivatives as potential cytotoxic agents

The synthesis of new bisaryl thienocyclopentoxazolidine derivatives was achieved through a Suzuki cross-coupling procedure with the aim to enhance the previously reported cytotoxicity of the series. The biological activity, evaluated in the NCI's in vitro human disease-oriented tumor cell line screening panel, was however partially conserved by the pharmacomodulations.     

PEGylated derivatives of cystamine as enhanced treatments for nephropathic cystinosis

The genetic disease, nephropathic cystinosis is characterized by lysosomal accumulation of the amino acid cystine. Crystallization of cystine in affected organs, if untreated, results in mortality of the affected individuals by their middle to late teens. The only approved treatment for cystinosis is administration of cysteamine. However, cysteamine is associated with an offending odor and taste and this, coupled to a rapid first pass metabolism and a 6 h dosing regimen, suggest a clear need to improve the therapy. A number of PEGylated derivatives of cystamine, the disulfide counterpart of cysteamine, have been synthesised and evaluated in cultured cystinotic fibroblasts for toxicity and efficacy. All of the tested compounds were non-cytotoxic and displayed a remarkable depletion of intralysosomal cystine.     

Synthesis and in vitro evaluation of novel pro-drugs for the treatment of nephropathic cystinosis

As part of our continuing work to obtain new pro-drugs for the treatment of nephropathic cystinosis, a number of glutaric and succinic acid derivatives of cystamine have been designed, synthesised and biologically evaluated in vitro. These compounds have been designed as odourless and tasteless pro-drugs which will release multiple molecules of cysteamine upon administration. All of the synthesised compounds evaluated in this study were non-cytotoxic and displayed a greater ability than cysteamine to deplete the levels of cystine in cultured fibroblasts.     

Binding and distribution of three prototype calcium channel blockers in perfused rat liver

This work represents a study of the binding and distribution of three different calcium channel blockers in the Sprague-Dawley rat liver, using an in situ perfusion technique. For this purpose, [3H] desmethoxyverapamil, [3H] PN200-110 (isradipine) and [3H] azidopine were used as binding probes interacting with calcium channels. The perfusion steps of the liver involved both portal vein and thoracic inferior vena cava cannulations as inlet and outlet respectively. The subhepatic inferior vena cava was ligated to prevent leakage of the perfusate. Buffer, containing the tracer drug, was administered via the portal vein at a rate of l mL/min and perfusate collected at the same rate within specified time intervals during 50 min. The concentration of the tracer solutes in the perfusate's outlet increased with time, and steady state was observed for all tracers at 40 min. The effect of adding cold isradipine to tracer desmethoxyverapamil, or cold verapamil to tracer PN200-110 were also assessed. First order rate constants for hepatocellular influx, efflux and calcium channel binding of the tracer substances were obtained using a simplified model from Goresky et al. [25]. These constants were mathematically manipulated and changed into permeability constants, second order binding constants, and residency times.Tracer solute influx across hepatocellular membranes is solubility-diffusion controlled, is inversely related to the molecular weights and is different in value from the efflux constants. Cold isradipine reduced the binding constant of desmethoxyverapamil by 36%, while cold verapamil reduced the binding constant of PN200-110 by 23%. Azidopine cellular distribution was low, however, binding to its receptor was analogous to desmethoxyverapamil and PN200-110. Moreover, PN200-110 had the highest residency time with no effect of cold verapamil on its receptor binding, while desmethoxyverapamil had the lowest residency time which significantly increased in the presence of cold isradipine.     

Testosterone,gonadotropins and androgen receptor during spermatogenesis of Biomphalaria alexandrina snails (Pulmonata: Basommatophora)

Endocrine regulation of reproductive processes of the snail Biomphalaria alexandrina is poorly recognized. Thus, the aims of the study were: (1) to acquire histological images of the ovotestis; (2) to determine the hemolymph concentrations of testosterone (T) and gonadotropic hormones (luteinizing hormone: LH and follicle stimulating hormone: FSH), (3) to demonstrate androgen receptor (AR) immunolocalization in the ovotestis, and (4) to show LH and FSH protein expression in cerebral ganglia of small (diameter shell: 4–6 mm), medium (7–11 mm) and large (12–16 mm) B. alexandrina snails. These three groups represented different reproductive stages of the snail. The AR immunoexpression was found in the periphery and inside the acini of small (immature) snails as well as in spermatocytes, spermatids, Sertoli cells, the interstitial cells and the acinus lining epithelium of medium (mature) snails. Low AR immunoexpression was demonstrated in the interstitial cells of large (aged) snails. The neurons at the periphery of the cerebral ganglia and connective sheath of the ganglia showed a positive FSH and LH immunostaining. T concentration in the hemolymph was higher in medium snails than in small and large snails. In contrast, LH concentration was higher in medium snails than in small and large snails. These data suggests that gonadotropins and T play a role in the gonadal development in B. alexandrina.