Cláudia Alves

Unique (Y;13) translocation in a male with oligozoospermia: cytogenetic and molecular studies.

The incidence of Y/autosome translocations is low. Whereas involvement of non-acrocentric chromosomes often leads to infertility, cases related with acrocentric chromosomes are usually familial with no or minimal effect on fertility. A de novo (Yp/13p) translocation was found in a 32-year-old male referred for severe oligozoospermia. Conventional cytogenetic procedures (GTG, CBG and NOR banding) and molecular cytogenetic techniques (Fluorescence In Situ Hybridization, FISH) were performed on high-resolution chromosomes obtained after peripheral blood lymphocyte culture as also on interphase nuclei of spermatogenic cells from semen samples. Screening of AZF microdeletions in the Yq11.2 region known to be involved with spermatogenesis defects was also performed. GTG banding showed a (Yp/13p) translocation in all scored metaphases. CBG and NOR staining of the derivative chromosome revealed the maintenance of Yq heterochromatin and of the 13p NOR region. FISH with centromeric Y and 13/21 probes, SRY specific probe and X/Y (p and q arms) sub-telomeric probes gave the expected number/location of fluorescent signals. Hybridisation with a pan-telomeric repeat (TTAGGG) probe showed an absence of the telomeric sequences at the fusion point of the rearranged chromosome. FISH analysis with probes to chromosomes X, Y, 13 and 18 showed an abnormal segregation of the translocated chromosome during meiosis I, which explains that only 13.6% of the secondary spermatocytes were normal. Most of these became arrested, as after meiosis II the large majority of the round spermatids were normal (70%), as were in consequence most of the sperm (85.1%). Multiplex-PCR confirmed the intactness of the SRY region and showed absence of AZF microdeletions. We report a novel de novo (Yp;13p) translocation characterised by loss of the 13p and Yp telomeres. Meiotic studies using FISH demonstrated meiosis I chromosome unpairing and mal segregation that justifies the severe oligozoospermia. Although most sperm have a normal chromosomal constitution, preimplantation genetic diagnosis should be considered an option for this patient.

Cytological and Expression Studies and Quantitative Analysis of the Temporal and Stage-Specific Effects of Follicle-Stimulating Hormone and Testosterone During Cocultures of the Normal Human Seminiferous Epithelium

Abstract In vitro culturing of normal human seminiferous epithelium remains largely unexplored. To study normal human spermatogenesis in vitro, we used a micromethod for the purification and culture of Sertoli cells, spermatogonia A, spermatocytes, and early round spermatids. Cytological quantitative data for Sertoli and premeiotic germ cell cocultures isolated from normal testicular biopsies demonstrated that cells were able to proliferate (4%), complete meiosis (6.7%), and differentiate into late round (54%), elongating (49%), and elongated (17%) spermatids at similar in vivo time delays (up to 16 days) in response to FSH + testosterone stimulation. Cells maintained normal meiotic segregation, chromosome complements, and specific gene expression profiles. Follicle-stimulating hormone + testosterone stimulated spermatogonia proliferation and Sertoli cell survival. Follicle-stimulating hormone and especially FSH + testosterone increased diploid germ cell survival during the first week, whereas only FSH + testosterone was able to inhibit cell death during the second week of culture. Follicle-stimulating hormone and especially FSH + testosterone also stimulated meiosis resumption, although this was restricted to late pachytene and secondary spermatocytes. In contrast, spermiogenesis was only stimulated by FSH + testosterone. Expression studies showed that apoptosis was induced in the nucleus of diploid cells, and in nuclear and cytoplasmic compartments of spermatids, mainly triggered by the Fas pathway. Although junctional complexes between Sertoli and premeiotic germ cells were partially reacquired, the same did not apply to spermatids, suggesting that FSH potentiated by testosterone was unable to render Sertoli cells competent to bind round spermatids.

Gemcitabine anti-proliferative activity significantly enhanced upon conjugation with cell-penetrating peptides

Gemcitabine proven efficiency against a wide range of solid tumors and undergoes deamination to its inactive uridine metabolite, which underlies its low bioavailability, and tumour resistance was also associated with nucleoside transporter alterations. Hence, we have conjugated gemcitabine to cell-penetrating peptides (CPP), in an effort to both mask its aniline moiety and facilitate its delivery into cancer cells. Two CPP-drug conjugates have been synthesized and studied regarding both the time-dependent kinetics of gemcitabine release and their anti-proliferative activity on three different human cancer cell lines. Results obtained reveal a dramatic increase in the anti-proliferative activity of gemcitabine in vitro, upon conjugation with the CPPs. As such, CPP-gemcitabine conjugates emerge as promising leads for cancer therapy.

Contamination of public transports by Staphylococcus aureus and its carriage by biomedical students: point-prevalence, related risk factors and molecular characterization of methicillin-resistant strains.

OBJECTIVES To analyse the contamination of public transports by Staphylococcus aureus and assess its carriage by biomedical students, focussing on the point-prevalence, related risk factors and molecular characterization of methicillin-resistant strains. STUDY DESIGN Cross-sectional survey. METHODS Methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolated from handrails of buses (n = 112) and trains (n = 79) circulating in Porto and from nasal swabs of local university students (n = 475) were quantified, characterized by molecular typing methods and related to possible risk factors. RESULTS The MRSA prevalence in buses (16.1%) was not significantly different from trains (8.9%). There was also no identifiable association between the counts of MSSA and MRSA in buses and trains and the number of travellers in each sampling day, specific routes (including those passing by main hospitals) or other risk factors. Of the students, 37.1% carried S. aureus, and having a part-time job or smoking were found to be risk factors for carriage. EMRSA-15 (ST22-SCCmecIVh) was the prevalent MRSA clonal lineage, found not only in the buses (n = 14) and trains (n = 2) but also in the single MRSA-carrier among the students. The characteristics of the community-associated Southwest Pacific MRSA clone were found in a single ST30-IVa isolate, which may suggest a recent SCCmec acquisition by an MSSA background in the community. CONCLUSIONS The spread of EMRSA-15, a common hospital-associated lineage, among different public transports and as a nasal coloniser is of concern and warrants adequate public health control measures.

Structure and function of a novel antioxidant peptide from the skin of tropical frogs

ABSTRACT The amphibian skin plays an important role protecting the organism from external harmful factors such as microorganisms or UV radiation. Based on biorational strategies, many studies have investigated the cutaneous secretion of anurans as a source of bioactive molecules. By a peptidomic approach, a novel antioxidant peptide (AOP) with in vitro free radical scavenging ability was isolated from Physalaemus nattereri. The AOP, named antioxidin‐I, has a molecular weight [M+H]+ = 1543.69 Da and a TWYFITPYIPDK primary amino acid sequence. The gene encoding the antioxidin‐I precursor was expressed in the skin tissue of three other Tropical frog species: Phyllomedusa tarsius, P. distincta and Pithecopus rohdei. cDNA sequencing revealed highly homologous regions (signal peptide and acidic region). Mature antioxidin‐I has a novel primary sequence with low similarity compared with previously described amphibians AOPs. Antioxidin‐I adopts a random structure even at high concentrations of hydrophobic solvent, it has poor antimicrobial activity and poor performance in free radical scavenging assays in vitro, with the exception of the ORAC assay. However, antioxidin‐I presented a low cytotoxicity and suppressed menadione‐induced redox imbalance when tested with fibroblast in culture. In addition, it had the capacity to substantially attenuate the hypoxia‐induced production of reactive oxygen species when tested in hypoxia exposed living microglial cells, suggesting a potential neuroprotective role for this peptide. Graphical abstract Figure. No caption available. HighlightsAntioxidin‐I is a new antioxidant peptide isolated from the skin tropical frogs.The bioactive peptide presented very low cytotoxicity against mammalian cells.It was able to avoid redox imbalance in oxidative challenged cells.Antioxidin‐I had the capacity to suppress ROS levels in hypoxia‐exposed microglia.Results support the application of the peptide for neuroprotection.

Caracterización citogenética molecular de las células germinales masculinas en la azoospermia secretora: parada de la maduración

Resumen Cerca de una tercera parte de los pacientes con azoospermia no obstructive presentan parada de la maduracion (MA, maturation arrest). Queda por evaluar por que algunos casos exhiben unas pocas celulas germinales que escapan al bloqueo meiotico y forman espermatozoides (MA incompleta) mientras otros presentan una MA completa en la meiosis (MA completa). En este estudio comparamos ambas situaciones por hibridacion in situ fluorescente (FISH) de los cromosomas sexuales (X, Y) y autosomicos (7, 18) en poblaciones puras de los distintos estadios de celulas germinales. Se aislaron por micromanipulacion celulas de Sertoli, espermatocitos primarios, espermatocitos secundarios, espermatidas redondas y elongadas de biopsias en fresco de 7 pacientes con azoospermia obstructiva (controles) y 20 pacientes con MA (9 MA completa, 11 MA incompleta). En todos los casos, los pacientes presentaron cariotipo y desarrollo de los caracteres sexuales secundarios normales, ausencia de endocrinopatias y microdeleciones en Yq11.2. La MA incompleta se caracterizo por cifras incrementadas de aneuploidias en las celulas de Sertoli, disminucion en el emparejamiento de los cromosomas homologos en la meiosis I y una incidencia incrementada de aneuploidias en los espermatocitos secundarios, sugiriendo que puede ser debido a deficiencias en el ensamblaje del complejo sinaptonemico. Por el contrario, la MA completa se asocio con cifras incrementadas de aneuploidias en los espermatocitos primarios, originadas en las divisiones mitoticas de la espermatogonia, y emparejamiento normal, sugiriendo que la MA meiotica puede ser debida a deficiencias en el sistema de reparacion del ADN.