Insights into differentiation and function of the transition region between the seminiferous tubule and rete testis.

Seminiferous tubules physically connect to the rete testis through short segments called the transition region (TR). During fetal development, this specialized junction is considered the initial site where testis cords begin to form and to grow in length well beyond birth and into adulthood and form convoluted tubular cores. Mitotic activity of the Sertoli cell, the somatic cell of the epithelium, ceases before puberty, but modified Sertoli cells in the TR remain immature and capable of proliferation. This review presents what is known about this specialized region of the testis, with an emphasis on the morphological, molecular and physiological features, which support the hypothesis that this short region of epithelial transition serves as a specialized niche for undifferentiated Sertoli cells and spermatogonial stem cells. Also, the region is populated by an elevated number of immune cells, suggesting an important activity in monitoring and responding to any leakage of autoantigens, as sperm enter the rete testis. Several structure/function characteristics of the transition region are discussed and compared across species.

Zymosan enhances in vitro phagocyte function and the immune response of mice infected with Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is the major etiologic agent of Paracoccidioidomycosis (PCM), the most frequent human deep mycosis in Latin America. It is proposed that masking of ß-glucan in P. brasiliensis cell wall is a critical virulence factor that contributes to the development of a chronic disease characterized by a long period of treatment, which is usually toxic. In this context, the search for immunomodulatory agents for therapeutic purposes is highly desirable. One strategy is to use pattern recognition receptors (PRRs) ligands to stimulate the immune response mediated by phagocytes. Here, we sought to evaluate if Zymosan, a ß-glucan-containing ligand of the PRRs Dectin-1/TLR-2, would enhance phagocyte function and the immune response of mice challenged with P. brasiliensis. Dendritic cells (DCs) infected with P. brasiliensis and treated with Zymosan showed improved secretion of several proinflammatory cytokines and expression of maturation markers. In addition, when cocultured with splenic lymphocytes, these cells induced the production of a potential protective type 1 and 17 cytokine patterns. In macrophages, Zymosan ensued a significant fungicidal activity associated with nitric oxide production and phagolysosome acidification. Importantly, we observed a protective effect of Zymosan-primed DCs delivered intranasally in experimental pulmonary PCM. Overall, our findings support the potential use of ß-glucan-containing compounds such as Zymosan as an alternative or complementary antifungal therapy. LAY SUMMARY: We report for the first time that Paracoccidioides brasiliensis-infected phagocytes treated with Zymosan (cell wall extract from bakers' yeast) show enhanced cytokine production, maturation, and fungal killing. Also, Zymosan-primed phagocytes induce a protective immune response in infected mice.

Dimethylacetamide in Rooster Semen Cryopreservation.

BACKGROUND: Sperm cryopreservation of cockerels is a major challenge, and so far there is no adequate information to enable commercial use of frozen semen. OBJECTIVE: To test the toxicity of dimethylacetamide (DMA). MATERIALS AND METHODS: DMA was added at 3%, 6%, 9% and 12% to the freezing diluent, and maintained for equilibration with the semen sample for 1 min, 3 min, 5 min, 7 min and 9 min prior to freezing. Thawed semen was evaluated for kinetic characteristics by computer-assisted semen analysis (CASA) and for structural and functional properties by flow cytometry (plasma membrane rupture, mitochondrial functionality and plasma membrane functionality). RESULTS AND CONCLUSION: The addition of 6% DMA for 3-min equilibration resulted in the highest total and progressive motility, 42.0% and 36.9%, respectively. The point of intersection between a good protection and low plasma membrane rupture was obtained with the addition of 6% of DMA for 3-min equilibration with the rooster semen.

Occult Tumour Cells in Lymph Nodes from Gastric Cancer Patients: Should Isolated Tumour Cells Also Be Considered?

INTRODUCTION: Regional lymph node metastasis is an important prognostic factor for patients with gastric cancer. Occult tumour cells (OTCs), including either micrometastases (MMs) or isolated tumour cells (ITCs), may be a key factor in the development of cancer recurrence in pN0 patients. AIMS: We aimed to determine the frequency and prognostic significance for disease recurrence of OTCs. MATERIALS AND METHODS: This retrospective cohort study included all consecutive patients with pN0 gastric adenocarcinoma between January 2000 and December 2011 (n = 73). Immunohistochemistry using the pan-cytokeratin antibody AE1/AE3 was used to detect OTCs in 1257 isolated lymph nodes. RESULTS: OTCs were identified in 30 patients (41%), including 20 cases with MMs (27%) and 10 cases with ITCs (14%). Disease recurrence and cancer-related death were observed in 24 (33%) and 20 patients (27%), respectively, and both were significantly associated with the detection of OTCs. A significant difference was also observed for the mean survival time between patients with OTCs and those without OTCs [100 vs 158 months (p = 0.015)]. The presence of OTCs was statistically significantly associated with the Lauren classification, tumour size and lymphatic permeation. Multivariate analyses revealed that only age, T stage and the presence of ITCs in lymph nodes were independent factors for recurrence. The presence of ITCs increased the risk for recurrence by 11.1-fold. CONCLUSIONS: In a significant proportion of patients diagnosed as stage pN0, OTCs may be identified in lymph nodes if carefully searched for, which can negatively affect their prognosis. The presence of ITCs was found to be an independent factor for recurrence and after proper validation should be considered during lymph node assessment for prognosis definition.

Immunomodulatory activity of ß-glucan-containing exopolysaccharides from Auricularia auricular in phagocytes and mice infected with Cryptococcus neoformans.

Current antifungal drugs present poor effectiveness and there is no available vaccine for fungal infections. Thus, novel strategies to treat or prevent invasive mycosis, such as cryptococcosis, are highly desirable. One strategy is the use of immunomodulators of polysaccharide nature isolated from mushrooms. The purpose of the present work was to evaluate the immunostimulatory activity of ß-(1,3)-glucan-containing exopolysaccharides (EPS) from the edible mushrooms Auricularia auricula in phagocytes and mice infected with Cryptococcus neoformans. EPS triggered macrophages and dendritic cell activation upon binding to Dectin-1, a pattern recognition receptor of the C-type lectin receptor family. Engagement of Dectin-1 culminated in pro-inflammatory cytokine production and cell maturation via its canonical Syk-dependent pathway signaling. Furthermore, upon EPS treatment, M2-like phenotype macrophages, known to support intracellular survival and replication of C. neoformans, repolarize to M1 macrophage pattern associated with enhanced production of the microbicidal molecule nitric oxide that results in efficient killing of C. neoformans. Treatment with EPS also upregulated transcript levels of genes encoding products associated with host protection against C. neoformans and Dectin-1 mediated signaling in macrophages. Finally, orally administrated ß-glucan-containing EPS from A. auricular enhanced the survival of mice infected with C. neoformans. In conclusion, the results demonstrate that EPS from A. auricula exert immunostimulatory activity in phagocytes and induce host protection against C. neoformans, suggesting that polysaccharides from this mushroom may be promising as an adjuvant for vaccines or antifungal therapy.

Effects of SSRI medication on heart rate and blood pressure in individuals with hypertension and depression.

OBJECTIVE: To test the role of escitalopram on blood pressure and heart rate of individuals with hypertension and depression. METHODS: A total of 30 individuals participated in this study who were being treated for hypertension and were diagnosed with major depression. Escitalopram (10-20 mg) was administered to 15 individuals, while the other 15 received placebo. These individuals were followed for 8 weeks with regular monitoring of blood pressure and heart rate. Scores on the Hamilton Depression Rating Scale were evaluated within the first, second, fourth, and eighth weeks of the study onset. RESULTS: Comparing with placebo, heart rate was lower in the escitalopram group (66.79 ± 9.85 vs. 74.10 ± 9.52 bpm, p = 0.044). There was not a significant decrease of systolic blood pressure (140.80 ± 16.48 vs 139.61 ± 18.92 mmHg, p = 0.85) and diastolic blood pressure (80.55 ± 12.64 vs 80.18 ± 16.36 mmHg, p = 0.94). CONCLUSION: Escitalopram decreases HR, but not BP, in individuals with hypertension and depression. Abbreviation: SH: systemic hypertension; BP: blood pressure; DSM: Diagnostic and Statistical Manual of Mental Disorders; SRQ 20: Self-Report Questionnaire; SCID: Structured Clinical Interview for DSM-IV; HR: heart rate; SNS: Sympathetic nervous system; HPA: hypothalamus-pituitary-adrenal axis; RAA: renin, angiotensin, aldosterone system; NE: norepinephrine; CSF: cerebrospinal fluid; HAM-D: Hamilton Depression Rating Scale; CRF: corticotropin releasing factor; ACTH: adrenocorticotropic hormone; BMI: Body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; t: time.

Population structure of methicillin-susceptible Staphylococcus aureus (MSSA) in Portugal over a 19-year period (1992-2011).

Despite their clinical relevance, few studies have addressed the epidemiology of methicillin-susceptible S. aureus (MSSA). In particular, it is not clear how MSSA population structure has evolved over time and how it might have been shaped by the emergence of MRSA in the community (CA-MRSA). In the present study we have evaluated the MSSA population structure over time, its geographical distribution and relatedness with MRSA in Portugal. A total of 465 MSSA from infection and colonization, collected over a 19-year period (1992-2011) in the northern, central and southern regions of Portugal were analyzed. Isolates were characterized by spa typing and multilocus-sequence typing (MLST). Isolates with predominant spa types were characterized by pulsed-field gel electrophoresis (PFGE). Isolates relatedness was analyzed by eBURST and BURP. The 172 spa types found among the 465 MSSA were grouped into 18 spa-CC (clonal complexes). Ten clonal types were more prevalent (40 %): one major clone (ST30-t012) was present in the entire study period and all over the country and the other nine were intermittently detected over time (ST5-t002, ST8-t008, ST15-t084, ST34-t166, ST72-t148, ST1-t127, ST7-t091, ST398-t571 and ST34-t136). Interestingly, three MSSA clonal types observed only after 1996 were closely related with CA-MRSA epidemic strains (ST8-t008, ST72-t148 and ST1-t127) found currently in Portugal. The MSSA population in Portugal is genetically diverse; however, some dominant clonal types have been established and widely disseminated for almost two decades. We identified MSSA isolates that were related with emergent CA-MRSA clones found in Portugal.

Oral L-arginine modulates blood lactate and interleukin-6 after exercise in HIV-infected men.

The acute administration of L-arginine (L-arg), a nitric oxide (NO) precursor, reduces lactate (LAC) concentration after exercise in healthy individuals. Lower concentration of L-arg may enhance the action of some inflammatory cytokines in HIV-1 infected patients. We tested the hypothesis that acute L-arg administration may reduce post-exercise blood LAC and inflammatory cytokines levels in HIV-infected patients. 10 HIV-infected men performed 2 maximal incremental cardiopulmonary exercise tests, separated by one week. 30 min before each test, patients received oral placebo or 20 g of L-arg, in random order. Blood LAC, tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-10 (IL-10) were measured before and up to 60 min after exercise. L-arg administration had no significant effect on exercise performance. Compared to placebo, L-arg administration reduced maximal post-exercise blood LAC from 8.7±0.6 to 6.9±0.4 mmol.L-1 (p<0.05). L-arg administration had no significant effect on TNF-alpha or IL-10 concentrations, but increased post-exercise IL-6 (placebo=19±3pg.mL-1; L-arg=63±8 pg.mL-1; p<0.05). In HIV-1 infected men, acute administration of L-arg reduces post-exercise blood LAC and increases IL-6 levels, suggesting the activation of the L-arg-NO pathway, with possible anti-inflammatory consequences.

Response of high-risk of recurrence/progression bladder tumours expressing sialyl-Tn and sialyl-6-T to BCG immunotherapy.

BACKGROUND: High risk of recurrence/progression bladder tumours is treated with Bacillus Calmette-Guérin (BCG) immunotherapy after complete resection of the tumour. Approximately 75% of these tumours express the uncommon carbohydrate antigen sialyl-Tn (Tn), a surrogate biomarker of tumour aggressiveness. Such changes in the glycosylation of cell-surface proteins influence tumour microenvironment and immune responses that may modulate treatment outcome and the course of disease. The aim of this work is to determine the efficiency of BCG immunotherapy against tumours expressing sTn and sTn-related antigen sialyl-6-T (s6T). METHODS: In a retrospective design, 94 tumours from patients treated with BCG were screened for sTn and s6T expression. In vitro studies were conducted to determine the interaction of BCG with high-grade bladder cancer cell line overexpressing sTn. RESULTS: From the 94 cases evaluated, 36 had recurrence after BCG treatment (38.3%). Treatment outcome was influenced by age over 65 years (HR=2.668; (1.344-5.254); P=0.005), maintenance schedule (HR=0.480; (0.246-0.936); P=0.031) and multifocality (HR=2.065; (1.033-4.126); P=0.040). sTn or s6T expression was associated with BCG response (P=0.024; P<0.0001) and with increased recurrence-free survival (P=0.001). Multivariate analyses showed that sTn and/or s6T were independent predictive markers of recurrence after BCG immunotherapy (HR=0.296; (0.148-0.594); P=0.001). In vitro studies demonstrated higher adhesion and internalisation of the bacillus to cells expressing sTn, promoting cell death. CONCLUSION: s6T is described for the first time in bladder tumours. Our data strongly suggest that BCG immunotherapy is efficient against sTn- and s6T-positive tumours. Furthermore, sTn and s6T expression are independent predictive markers of BCG treatment response and may be useful in the identification of patients who could benefit more from this immunotherapy.

MLL2 mosaic mutations and intragenic deletion-duplications in patients with Kabuki syndrome.

Kabuki syndrome (KS) is a rare multi-system disorder that can result in a variety of congenital malformations, typical dysmorphism and variable learning disability. It is caused by MLL2 point mutations in the majority of the cases and, rarely by deletions involving KDM6A. Nearly one third of cases remain unsolved. Here, we expand the known genetic basis of KS by presenting five typical patients with the condition, all of whom have novel MLL2 mutation types- two patients with mosaic small deletions, one with a mosaic whole-gene deletion, one with a multi-exon deletion and one with an intragenic multi-exon duplication. We recommend MLL2 dosage studies for all patients with typical KS, where traditional Sanger sequencing fails to identify mutations. The prevalence of such MLL2 mutations in KS may be comparable with deletions involving KDM6A. These findings may be helpful in understanding the mutational mechanism of MLL2 and the disease mechanism of KS.

High prevalence of hospital-associated methicillin-resistant Staphylococcus aureus in the community in Portugal: evidence for the blurring of community-hospital boundaries.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of infection in the community (CA-MRSA), but in spite of its relevance, no data exist concerning its epidemiology in Portugal. In this study, we aimed to evaluate the prevalence, population structure, and origin of MRSA in the Portuguese community. A total of 527 isolates, both methicillin-susceptible S. aureus (MSSA) and MRSA, were collected from individuals with no healthcare-related risk factors attending 16 healthcare institutions in Portugal. Isolates were characterized for the presence of mecA, Panton-Valentine leukocidin (PVL), and arginine catabolic mobile element (ACME), and by staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), spa, and multilocus sequence typing (MLST). Susceptibility to a panel of 13 antibiotics was tested. Isolates relatedness was analyzed by goeBURST and BURP. We found a high frequency (21.6%) of MRSA in the community. However, only 11.4% of the isolates belonged to typical CA-MRSA epidemic clones (USA300, USA400, USA700, Southwest Pacific, European, and ST398). The remaining isolates, which constituted the great majority (88.6%), belonged to hospital-associated MRSA (HA-MRSA) epidemic clones, namely, to the EMRSA-15 clone (77.2%). PVL was rare and carried by 17 isolates only (five MRSA and 12 MSSA). In the whole collection, some MRSA and MSSA were highly related. The high frequency of MRSA in the community in Portugal seems to result mainly from dissemination from the hospital. They might also have emerged from an extant MSSA population, by SCCmec acquisition, or MRSA clonal introduction from abroad.

Serum TSH as a predictor of malignancy in indeterminate thyroid nodules.

INTRODUCTION: Thyroid nodules are lesions that are radiologically distinct from the thyroid parenchyma. Cervical ultrasound diagnoses 19-67% of nodules and is crucial in identifying those that lack cytological characterisation. Approximately 25% of biopsies reveal an indeterminate cytological result (Bethesda III), in which the risk of malignancy is variable (5-15%). The clinical importance of the diagnostic strategy used for thyroid nodules results from the need to exclude malignancy. The aim of this study was to evaluate the usefulness of serum thyroid-stimulating hormone (TSH) levels as a predictor of malignancy in cytologically indeterminate thyroid nodules. METHODS: Our retrospective study included 40 patients with cytologically indeterminate thyroid nodules seen in our hospital between January 2013 and December 2017. Clinical parameters were reviewed, including age, gender, serum TSH levels, family history of thyroid carcinoma, radiation exposure and some sonographic features of the nodules. Statistical analysis was performed using SPSS. Statistical significance was defined as p<0.05. RESULTS: Female gender was predominant (85%) and the mean (SD) age was 53.3 (15) years. Thyroid carcinoma was confirmed in 28% of patients. Median TSH levels were higher in patients with malignant (2.73µIU/ml) compared with benign (1.56µIU/ml) nodules (p<0.05). We demonstrated an increased risk of malignancy in patients with TSH levels of 2.68µIU/ml or above (p<0.05). CONCLUSION: Higher serum TSH levels are associated with an increased risk of thyroid carcinoma in cytologically indeterminate nodules. TSH can become a fundamental diagnostic tool in stratifying the risk of malignancy and assist in diagnostic and therapeutic approaches to these nodules.

Polo kinase and Asp are needed to promote the mitotic organizing activity of centrosomes.

Interfering with the activity of polo-like kinases can lead to the formation of monopolar spindles. Polo-like kinases also regulate mitotic entry, activation of the anaphase-promoting complex and the necessary preconditions for cytokinesis. Similarities between the phenotypes of the Drosophila mutants asp and polo point towards a common role in spindle pole function. The abnormal spindles of asp mutants are bipolar but have disorganized broad poles at which gamma-tubulin has an abnormal distribution. Moreover, the synergism or of polo1 aspE3 double mutants indicates a possible involvement of these genes in a common process. Asp is a microtubule-associated protein of relative molecular mass 220,000 (Mr 220K) found at the face of the centrosome that contacts spindle microtubules. In partially purified centrosomes, it is required with gamma-tubulin to organize microtubule asters. Here, we show that Asp is the previously identified Mr 220K substrate of Polo kinase. Polo phosphorylates Asp in vitro, converting it into an MPM2 epitope. Polo and Asp proteins immunoprecipitate together and exist as part of a 25-38S complex. Extracts of polo-derived embryos are unable to restore the ability of salt-stripped centrosomes to nucleate microtubule asters. This can be rescued by addition of phosphorylated Asp or active Polo kinase.

Hepatitis C treatment outcome in former or current intravenous drug users coinfected with HIV, with or without directly observed therapy.

BACKGROUND: Intravenous drug users (IDUs) with hepatitis C virus (HCV)/human immunodeficiency virus (HIV)-coinfection are recognised as a high-risk, vulnerable group. METHODS: Between February 2015 and April 2018, a single-centre, non-interventional cohort study was conducted in an outpatient setting, to evaluate the sustained virologic response (SVR12) and assess treatment uptake models. The study included 385 former or recent IDUs divided into two groups: A-without use of opioid substitution treatment (OST) and B-patients taking opioid substitution; patients in group B received OST and self-administered therapy (B1) or OST and therapy under DOT (B2). Patients were characterised by demographic and clinical features and compared for treatment response. Correlations between SVR12 and independent variables were determined by logistic regression. RESULTS: Patients were mostly males (88.3%) with a mean age of 46 ± 5 years and HCV genotype 1a (63.7%). Approximately 28% were treatment-experienced and 84.9% received sofosbuvir/ledipasvir. The mean CD4+T count was 649 cells/mm3, and most individuals were on antiretroviral therapy with undetectable viral loads (97.4%). SVR12 was achieved in 94.8%, and only eight patients relapsed. No significant differences were found in treatment effect between individuals taking opioid substitutes under different treatment models. Correlations were found between HCV viral response and both HIV suppression and albumin levels. CONCLUSIONS: IDU with HCV/HIV coinfection, including individuals on self-administration of HCV therapy and opioid substitution treatments or in DOT programmes, are no longer considered a difficult-to-treat group, as they achieve high rates of SVR12.

Prevalence and clonality of methicillin-resistant Staphylococcus aureus (MRSA) in the Atlantic Azores islands: predominance of SCCmec types IV, V and VI.

In order to obtain insights into the methicillin-resistant Staphylococcus aureus (MRSA) population structure in the Azores archipelago, 106 MRSA isolates were collected from patients attending an Azorean central hospital between January 2007 and February 2008. Antimicrobial resistance was determined for all isolates. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing (MLST), staphylococcal chromosome cassette mec (SCCmec) typing and the presence of Panton-Valentine leukocidin (PVL). The majority of the isolates (87%, n = 92) belonged to the EMRSA-15 clone (ST22, SCCmec-IVh), followed by the Pediatric clone (ST5-VI/IVc) (11%, n = 12). The Berlin clone (ST45-IVa) and a new clone (spa type t1839, ST1339 and SCCmec V variant) were represented by single isolates. All of the isolates carried SCCmec types IV, V or VI and a non-multiresistant antibiotic profile, resembling the currently emerging community MRSA. Moreover, PVL was described for the first time to be associated with the Pediatric clone carrying SCCmec type VI. We provided the first description of the population structure of MRSA in the Azores islands, which seems to be shaped by genetic events occurring locally, as well as by the regular population exchange between the islands, continental Portugal, the United Kingdom and the United States.

Combined cytotoxic and genotoxic effects of ochratoxin A and fumonisin B1 in human kidney and liver cell models.

Food products can be contaminated by several fungi species and each species may produce different mycotoxins, leading to human combined exposure. Although predictions about the joint toxic effects of mycotoxins can be made from their individual toxicities, experimental data is still limited to allow a reliable hazard assessment. Thus, this study aimed to characterize the combined cytotoxic and genotoxic effects of ochratoxin A (OTA) and fumonisin B1 (FB1) in cell lines representative of their target organs, kidney and liver. Interactions were ascertained using mathematical extensions to the reference models of concentration addition and independent action. Cytotoxicity (MTT assay) data modeling revealed a synergistic pattern for low doses of both FB1 and OTA shifting to antagonism at higher concentration levels, irrespectively of the reference model applied. Concerning genotoxicity assessment, neither OTA nor FB1, individually or in combination, induced a prominent increase in DNA damage (comet assay) or oxidative DNA damage (FPG-comet assay). In conclusion, this study revealed a synergistic cytotoxic effect for OTA and FB1 at low concentration levels. Given that human co-exposure to these two mycotoxins is probable to occur at low doses, these results raise concerns regarding their potential health outcomes that seem to differ from those predicted by an additive model.

A landslide risk index for municipal land use planning in Portugal.

In Portugal landslides caused 237 fatalities and >1600 displaced people in the period 1865-2015. Spatial distribution and temporal patterns of slope instability can be related with a complex set of natural and human factors responsible for generating damages. It is essential to develop new methodologies to synthetize risk dimensions to contribute to the landslide risk management at the municipal level. This work proposed a municipal landslide risk index (LRI) considering three risk dimensions: hazard, exposure and physical vulnerability of buildings. The hazard dimension includes the landslide susceptibility performed at the national scale, the probability of weather types associated with landslides and an extreme precipitation susceptibility index. The exposure dimension considered the population density and the number of buildings, whereas the average physical vulnerability of the buildings was computed using four statistical variables from the official census: (i) construction technique and construction materials; (ii) reinforced structure; (iii) number of floors; and (iv) conservation status. Each variable includes different classes that were empirically weighted. After evaluating the three risk dimensions and the LRI, a cluster analysis was performed in order to identify the most important landslide risk drivers in each municipality. Exposure is the main driving force of LRI in the metropolitan areas of Lisbon and Porto, while the hazard is more relevant in the NW municipalities and the physical vulnerability is the major driving force in the south of the country. This methodological approach contributes to a comprehensive and synthetized knowledge about the landslide risk driving forces within the 278 Portuguese municipalities. In addition, it contributes to the diversification and context-oriented strategies of landslide risk management that still lacks in most of the national-level risk governance processes. Finally, this methodology can be generalized to other geographical contexts, improving the risk management, land use planning and the disaster risk reduction.

Metaphase arrest with centromere separation in polo mutants of Drosophila.

The Drosophila gene polo encodes a conserved protein kinase known to be required to organize spindle poles and for cytokinesis. Here we report two strongly hypomorphic mutations of polo that arrest cells of the larval brain at a point in metaphase when the majority of sister kinetochores have separated by between 20-50% of the total spindle length in intact cells. In contrast, analysis of sister chromatid separation in squashed preparations of cells indicates that some 83% of sisters remain attached. This suggests the separation seen in intact cells requires the tension produced by a functional spindle. The point of arrest corresponds to the spindle integrity checkpoint; Bub1 protein and the 3F3/2 epitope are present on the separated kinetochores and the arrest is suppressed by a bub1 mutation. The mutant mitotic spindles are anastral and have assembled upon centrosomes that are associated with Centrosomin and the abnormal spindle protein (Asp), but neither with gamma-tubulin nor CP190. We discuss roles for Polo kinase in recruiting centrosomal proteins and in regulating progression through the metaphase-anaphase checkpoint.

Drosophila polo kinase is required for cytokinesis.

A number of lines of evidence point to a predominance of cytokinesis defects in spermatogenesis in hypomorphic alleles of the Drosophila polo gene. In the pre-meiotic mitoses, cytokinesis defects result in cysts of primary spermatocytes with reduced numbers of cells that can contain multiple centrosomes. These are connected by a correspondingly reduced number of ring canals, structures formed by the stabilization of the cleavage furrow. The earliest defects during the meiotic divisions are a failure to form the correct mid-zone and mid-body structures at telophase. This is accompanied by a failure to correctly localize the Pavarotti kinesin- like protein that functions in cytokinesis, and of the septin Peanut and of actin to be incorporated into a contractile ring. In spite of these defects, cyclin B is degraded and the cells exit M phase. The resulting spermatids are frequently binuclear or tetranuclear, in which case they develop either two or four axonemes, respectively. A significant proportion of spermatids in which cytokinesis has failed may also show the segregation defects previously ascribed to polo1 mutants. We discuss these findings in respect to conserved functions for the Polo-like kinases in regulating progression through M phase, including the earliest events of cytokinesis.

Genetic characterization of the UCS and Kex1 loci of Pneumocystis jirovecii.

Nucleotide variation in the Pneumocystis jirovecii upstream conserved sequence (UCS) and kexin-like serine protease (Kex1) loci was studied in pulmonary specimens from Portuguese HIV-positive patients. DNA was extracted and used for specific molecular sequence analysis. The number of UCS tandem repeats detected in 13 successfully sequenced isolates ranged from three (9 isolates, 69%) to four (4 isolates, 31%). A novel tandem repeat pattern and two novel polymorphisms were detected in the UCS region. For the Kex1 gene, the wild-type (24 isolates, 86%) was the most frequent sequence detected among the 28 sequenced isolates. Nevertheless, a nonsynonymous (1 isolate, 3%) and three synonymous (3 isolates, 11%) polymorphisms were detected and are described here for the first time.