In situ electroporation of mammalian cells through SiO2 thin film capacitive microelectrodes.

Electroporation is a widely used non-viral technique for the delivery of molecules, including nucleic acids, into cells. Recently, electronic microsystems that miniaturize the electroporation machinery have been developed as a new tool for genetic manipulation of cells in vitro, by integrating metal microelectrodes in the culture substrate and enabling electroporation in-situ. We report that non-faradic SiO2 thin film-insulated microelectrodes can be used for reliable and spatially selective in-situ electroporation of mammalian cells. CHO-K1 and SH-SY5Y cell lines and primary neuronal cultures were electroporated by application of short and low amplitude voltage transients leading to cell electroporation by capacitive currents. We demonstrate reliable delivery of DNA plasmids and exogenous gene expression, accompanied by high spatial selectivity and cell viability, even with differentiated neurons. Finally, we show that SiO2 thin film-insulated microelectrodes support a double and serial transfection of the targeted cells.

Stimulation of Ca²+ signals in neurons by electrically coupled electrolyte-oxide-semiconductor capacitors.

Electrolyte-oxide-semiconductor capacitors (EOSCs) are a class of microtransducers for extracellular electrical stimulation that have been successfully employed to activate voltage-dependent sodium channels at the neuronal soma to generate action potentials in vitro. In the present work, we report on their use to control Ca²+ signalling in cultured mammalian cells, including neurons. Evidence is provided that EOSC stimulation with voltage waveforms in the microsecond or nanosecond range activates two distinct Ca²+ pathways, either by triggering Ca²+ entry through the plasma membrane or its release from intracellular stores. Ca²+ signals were activated in non-neuronal and neuronal cell lines, CHO-K1 and SH-SY5Y. On this basis, stimulation was tailored to rat and bovine neurons to mimic physiological somatic Ca²+ transients evoked by glutamate. Being minimally invasive and easy to use, the new method represents a versatile complement to standard electrophysiology and imaging techniques for the investigation of Ca²+ signalling in dissociated primary neurons and cell lines.

Space and time-resolved gene expression experiments on cultured mammalian cells by a single-cell electroporation microarray.

Single-cell experiments represent the next frontier for biochemical and gene expression research. Although bulk-scale methods averaging populations of cells have been traditionally used to investigate cellular behavior, they mask individual cell features and can lead to misleading or insufficient biological results. We report on a single-cell electroporation microarray enabling the transfection of pre-selected individual cells at different sites within the same culture (space-resolved), at arbitrarily chosen time points and even sequentially to the same cells (time-resolved). Delivery of impermeant molecules by single-cell electroporation was first proven to be finely tunable by acting on the electroporation protocol and then optimized for transfection of nucleic acids into Chinese Hamster Ovary (CHO-K1) cells. We focused on DNA oligonucleotides (ODNs), short interfering RNAs (siRNAs), and DNA plasmid vectors, thus providing a versatile and easy-to-use platform for time-resolved gene expression experiments in single mammalian cells.

[Remuneration and productivity: the case of the Minas Gerais Hospital Foundation, Brazil, 1992-1995]. / Remuneración y productividad: el caso de la Fundación hospitalaria del Estado de Minas Gerais, Brasil, 1992-1995.

This article presents an analysis of the Minas Gerais State Hospital Foundation immediately after the introduction in its health services units in early 1993 of a new system of rewards for good performance and productivity, as a complement to salaried remuneration. Analysis was based on a cross-sectional study of changes in indicators of production and productivity in the Foundation during the 1992-1995 period. Data were obtained from hospitalization authorization forms, payment authorization guides, and bulletins from the Human Resource Administration. The strategy of conditional remuneration and incentives was adopted not only to step up production and productivity, but also to increase the employees' commitment to the institution. Analysis of the selected indicators appears to confirm other study results in that remuneration based on results (conditional incentives) leads to a positive change in the production level of services and productivity, even if it does not last for the long term. Study results also support the notion that such alternatives may be deliberately used as part of a more general strategy of organizational development and not only as an isolated element for concrete improvements in productivity.

Germ cell apoptosis and endothelial nitric oxide synthase (eNOS) expression following ischemia-reperfusion injury to testis.

There is evidence to suggest that reactive oxygen species (ROS) are involved in ischemia-reperfusion injury to the testis. Nitric oxide (NO), a ubiquitous free radical produced by the nitric oxide synthases (NOS), has been implicated in physiologic and pathologic interactions with ROS. We examined the effect of testicular ischemia on germ cell apoptosis and endothelial NOS (eNOS) expression. Adult rats were subjected to unilateral 720 degrees testicular torsion for 1 or 3 hours and 24 hours later, testes were harvested for immunohistochemical studies. Apoptosis was detected by in situ 3' end-labeling of DNA with digoxigenin-ddUTP and eNOS protein was detected using an eNOS monoclonal antibody. Tests subjected to 3 hours of torsion had a threefold increase in apoptotic germ cells per cross-sectional area compared to sham testes (P < .05). In addition to its known expression in Leydig, Sertoli, and vascular endothelial cells, eNOS was detected in the cytoplasm of degenerating germ cells. Consecutive testis sections stained for eNOS and cellular DNA fragmentation demonstrated co-localization of eNOS protein and germ cell apoptosis. The detection of strong immunostaining in apoptotic germ cells supports a role of eNOS in germ cell degeneration after testicular ischemia-reperfusion and suggests that NO is associated with germ cell apoptosis.

The acid-fast stain is a superior stain for use in the mean mature spermatid count for testicular biopsies.

The mean mature spermatid count (MMSC) provides a useful, simplified quantitative evaluation of human spermatogenesis that is based on the number of mature spermatids in histological sections of testicular biopsies. Here, the activity of the acid-fast (AF) stain was compared to that of the usual hematoxylin and eosin (H&E) stain in performing the MMSC. Thirty bilateral testicular biopsies showing normal spermatogenesis were chosen retrospectively from 15 subfertile patients with obstructive azoospermia or severe oligospermia. The MMSC was determined on each biopsy by utilizing both H&E and AF stains. The AF stain proved to be specific for the mature spermatids normally counted for the MMSC. It simplified recognition of mature spermatids, thereby shortening the overall time required for the procedure. The mean AF MMSC was lower than the mean H&E MMSC, and the mean interobserver differences were decreased. The AF stain is a superior stain for the MMSC when used in conjunction with the H&E stain for descriptive histology.

[Splenic epidermoid cyst. Clinical case and review of the literature]. / Cisti epidermoide della milza. Caso clinico e revisione della letteratura.

The occasional discovery of an epidermoid cyst of the spleen, rare kind of lesion, in a 25 years old man who underwent a US for urethral colic, permit to the authors a review of the literature. Treatment is individualized in a partial or total splenectomy.

Submicroscopic deletions in the Y chromosome of infertile men.

Recent investigations have suggested a high prevalence of Y chromosome submicroscopic deletions in men with severely impaired spermatogenesis. We evaluated the frequency of Y chromosome deletions in 160 infertile men using a series of 36 sequence-tagged-sites, emphasizing intervals 5 and 6 of the long arm of the Y chromosome. Peripheral leukocyte DNA was extracted and amplified with two parallel techniques to minimize potential overestimation of the frequency of deletions. The presence of deletions was evaluated relative to patient's sperm concentration, testis volume, and hormonal parameters. Men with sperm concentration <5 x 10(6)/ml had a 7% prevalence of submicroscopic Y chromosome deletions. Deletions were detected in 7% of azoospermic men, 10% of men with <1 x 10(6) spermatozoa/ml, and 8% of men with >1 x 10(6) but <5 x 10(6) spermatozoa/ml. Other clinical parameters did not identify men with Y chromosome deletions prior to polymerase chain reaction (PCR)-based testing for the presence of sequence-tagged-sites. Two distinct regions of Y chromosome deletions were detected, approximately 3.6 Mb and 1.4 Mb in length respectively. These deleted regions are present in AZFb and AZFc respectively. No deletions were detected in AZFa. The loss of these two distinct areas is supported by the finding of highly repetitive sequences along the Y chromosome, predisposing to deletion of specific intervals on the Y chromosome during meiosis. Men with severe male infertility are at high risk for Y chromosome deletions. Testing of men for these genetic abnormalities is indicated prior to treatment with assisted reproduction.

Clinical review 87: In vitro fertilization for male factor infertility.

Since the first U.S. report of a successful delivery from in vitro fertilization in 1982 (65), progress in the field of assisted reproduction and micromanipulation has been truly dramatic. Perhaps the most exciting advances have been in the area of male factor infertility. Couples who previously would have been offered donor insemination or adoption are now achieving pregnancies despite severe impairments in semen quality, the presence of only single numbers of sperm in the ejaculate, or unreconstructable reproductive tract obstruction. Techniques of micromanipulation that were revolutionary less than five yr ago are now obsolete, replaced by even more successful methods. Even nonobstructive azoospermia resulting from maturation arrest or other impairments in germ cell development have been added to the list of treatable factors in male infertility, as sperm can frequently be extracted directly from testicular parenchyma that is aspirated or surgically biopsied. For patients without sperm in the testicular parenchyma, round spermatid or secondary spermatocyte injections are at least theoretically possible. Several important questions remain with regard to IVF-ICSI. 1) What should be the specific indications for IVF and IVF-ICSI? Should IVF alone ever be used for male factor infertility? 2) What are the reasons for failure to achieve pregnancy after ICSI, which still represent over half of our attempts at achieving ongoing pregnancies? 3) Can we be certain that using severely impaired or less mature sperm will not result in significant birth defects or in genetic abnormalities that could affect the offspring in adolescence or adulthood? 4) What is the most successful and cost effective approach for the infertile couple with impaired semen parameters? For couples with male factor infertility, careful evaluation and treatment of the man should be considered before assisted reproduction, including ICSI. Contemporary application of ICSI for severe male factor infertility can allow pregnancy rates up to 52% (33), with ongoing pregnancy and live delivery rates as high as 37% per IVF cycle attempt (27). As long as viable sperm are present in the ejaculate or retrievable from the reproductive tract, then ICSI procedures can be applied.

Vasectomy and vasectomy reversal.

PIP: Each year in the US, nearly half a million men undergo vasectomy and at least 15 million rely on this form of contraception. This article presents state-of-the-art information on vasectomy and vasectomy reversal. First, it describes the conventional, no-scalpel, percutaneous, and open-ended vasectomy techniques and presents research evidence on short-term complications, time required to achieve azoospermia, and vasectomy failure. The article then reviews findings on the local effects of vasectomy on the testis, rete testis and epididymis, vas deferens, and seminal vesicles and prostate and the development of sperm granulomas. It reports the available evidence on long-term systemic consequences of vasectomy and the risk of genitourinary cancer. Finally, the article considers sterilization reversal, including preoperative evaluation, surgical technique, anesthesia, factors that influence the success of reversal surgery, and the recovery of fertility.^ieng

[Anorectal manometry in hemorrhoidal disease]. / La manometria anorettale nella malattia emorroidaria.

Manometry is an important approach to anorectal function. In the haemorrhoids the evaluation of the anal resting tone, of the squeeze and of the sphincterial length is very useful to realize the best surgical treatment. Anorectal manometry is neither invasive nor expensive; it can be ready repeated and the results are reproducible. This examination must be considerated as essential part of the diagnostic routine of the anorectal diseases.

[Submucosal reconstructive hemorrhoidectomy (A.G. Parks operation)]. / L'emorroidectomia sottomucosa ricostruttiva (operazione di A.G. Parks).

Indications and procedure of submucosal reconstructive hemorrhoidectomy (A.G. Parks hemorrhoidectomy) are described and discussed. This procedure is more difficult and time consuming than other techniques, but results are better in high degree hemorrhoids. Authors consider complications and sequelae on 278 patients operated in their Department.

[When to combine internal sphincterotomy with hemorrhoidectomy]. / Quando associare la sfinterotomia interna all'emorroidectomia.

396 patients, treated with hemorrhoidectomy (278 Parks, 118 Milligan-Morgan) were submitted to preoperative manometry, which, in 158 patients (39.9%), revealed high anal resting pressure (TBS), with or without an associated anal fissure; this group was submitted to a regulated lateral sphincterotomy (SILR) in association with the haemorroidectomy. The manometric follow-up, two mounts after the operations showed similar TBS values in both groups of patients. The authors conclude that normal values of TBS after Hemorroidectomy can be reached only by associating sphincterotomy in manometrically selected cases.

[Continence in low resections]. / La continenza nelle resezioni basse.

Anorectal function is an important problem after low anterior resection procedure. This paper reports the results from 14 patients undergoing to low resection at Surgical Pathology Institute of the University of Verona. In 12 cases the restoration of the bowel continuity has been obtained by colon-anal anastomosis (Parks Operation) and in 3 cases by colo-rectal anastomosis at the anorectal ring level. The patients have been examined in the preoperative and in the postoperative period by clinical and manometric study. The results confirm that low resection does not involve faecal continence.