OnePot PURE Cell-Free System.

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic biology. Unfortunately, commercially available systems are costly, and their tunability is limited. In comparison, a home-made approach can be customized based on user needs. However, the preparation of home-made systems is time-consuming and arduous due to the need for ribosomes as well as 36 medium scale protein purifications. Streamlining protein purification by coculturing and co-purification allows for minimizing time and labor requirements. Here, we present an easy, adjustable, time- and cost-effective method to produce all PURE system components within 1 week, using standard laboratory equipment. Moreover, the performance of the OnePot PURE is comparable to commercially available systems. The OnePot PURE preparation method expands the accessibility of the PURE system to more laboratories due to its simplicity and cost-effectiveness.

Automated method for the determination of the band gap energy of pure and mixed powder samples using diffuse reflectance spectroscopy.

An automated method to determine the band gap energy (E g ) of pure and mixed powder compounds using diffuse reflectance spectroscopy is presented. This method is based on a five-step algorithm that mimics the judgment made by an expert analyst in identifying the linear segments in Tauc plots and subsequent estimation of the E g value. It is demonstrated that the method to estimate E g by intersecting the straight-line fit of the Tauc segment with the photon energy axis is not appropriate for those samples containing more than one optical absorbing phase because systematic underestimation of the E g value results. The automated method accounts for such cases by introducing a base line function. The robustness of the implemented algorithm was tested using three model systems, ZnO-Al2O3, ZnO-CoO and ZnO-CdO. The estimated E g 's using the automated method differ in less than 1% than those obtained by its manual counterpart.

Mutación en el gen supresor tumoral PTCH1 en el síndrome de Gorlin. Presentación de un caso / Mutation in the PTCH1 tumor suppressor gene in Gorlin Syndrome. A case report

El síndrome de Gorlin, también conocido como síndrome del carcinoma basocelular nevoide (SCBN), es una enfermedad hereditaria, autosómica dominante, con penetrancia alta y expresividad clínica variable. El SCBN se caracteriza por la presencia de múltiples carcinomas basocelulares, fibromas de ovario y una variedad de características clínicas, clasificadas según criterios mayores y menores que permiten orientar el diagnóstico. El SCBN corresponde a una enfermedad genética con baja incidencia y poca prevalencia en México. Está asociado a mutaciones en el gen supresor de tumores PTCH1. Presentamos el caso de una niña de 13 años, producto del primer embarazo de padres sanos y sin antecedentes heredofamiliares de importancia. Los signos clínicos en esta paciente incluían los siguientes: macrocefalia, frontal amplio, puente nasal ancho, telecanto y paladar alto y ojival. En la piel se observaron 8 nevos y hoyuelos palmares o plantares. Mediante un estudio radiológico se observó la presencia de quistes odontogénicos, que eran recurrentes. El estudio molecular demostró una mutación heterocigota en el gen supresor de tumores PTCH1. Los hallazgos mostraron una mutación novel, no descrita en la bibliografía o en bases de datos públicas; sin embargo, la mutación expresa las manifestaciones fenotípicas características del SCBN. Actualmente, no existe un tratamiento definitivo para esta afección, por lo que es necesario un abordaje preventivo multidisciplinario y el asesoramiento genético (AU)

Gorlin syndrome is a hereditary disease, and it is also known as nevoid basal cell carcinoma (NBCC). NBCC follows an autosomal dominant inheritance pattern, with high penetrance and variable clinical expression. NBCC is characterized by multiple basal cell carcinomas, ovarian fibroma and a variety of clinical manifestation known as minor or mayor criteria. NBCC is a genetic disease with low incidence in México and it is associated with mutated PTCH1 suppressor gen. We present the case of a 13 years old feminine patient was a healthy product of the first gestation of parents with no history of disease. Her clinical characteristics include macrocephaly, broad forehead, broad nasal bridge, telecanthus, high-arched palate, with 8 palmar and plantar pits. The radiology dental studies showed chists odontogenic with a recurrent pattern. Molecular studies showed a heterocigotic mutation in the suppressor gene PTCH1. Molecular analysis showed a novel mutation and clinical manifestation of the NBCC, not described before. For the NBCC there is no definitive treatment, and a multidisciplinary medical team is necessary for prevention and genetic counseling (AU)

Conservative management of early-stage epithelial ovarian cancer: results of a large retrospective series.

BACKGROUND: To assess the long-term oncological outcome and the fertility of young women with early-stage epithelial ovarian cancer (ES/EOC) treated with fertility-sparing surgery (FSS). PATIENTS AND METHODS: All patients treated with FSS for ES/EOC in two Italian centers were considered for this analysis. Univariate and multivariate analyses were used to test demographic characteristics and clinical features for the association with overall survival (OS), recurrence-free survival (RFS) and fertility. RESULTS: From 1982 to 2010, 240 patients with malignant ES/EOC were treated with FSS in two tertiary centers in Italy. At a median follow-up of 9 years, 27 patients had relapsed (11%) and 11 (5%) had died of progressive disease. Multivariate analysis found only grade 3 negatively affected the prognosis of patients [hazard ratio (HR) for recurrence: 4.2, 95% confidence interval (CI): 1.5-11.7, P=0.0067; HR for death: 7.6, 95% CI: 2.0-29.3, P=0.0032]. Grade 3 was also significantly associated with extra-ovarian relapse (P=0.006). Of the 105 patients (45%) who tried to become pregnant, 84 (80%) were successful. CONCLUSIONS: Conservative treatment can be proposed to all young patients when tumor is limited to the ovaries, as ovarian recurrences can always be managed successfully. Patients with G3 tumors are more likely to have distant recurrences and should be closely monitored.

Number and tumor size are not sufficient criteria to select patients for liver transplantation for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is an indication for liver resection or transplantation (LT). In most centers, patients whose HCC meets the Milan criteria are considered for LT. The first objective of this study was to analyze whether there is a correlation between the pathologic characteristics of the tumor, survival and recurrence rate. Second, we focused our attention on vascular invasion (VI). METHODS: From January 1997 to December 2007, a total of 196 patients who had a preoperative diagnosis of HCC were included. The selection criteria for LT satisfied both the Milan and the San Francisco criteria (UCSF). Demographic, clinical, and pathologic information were recorded. RESULTS: HCC was confirmed in 168 patients (85.7%). The median follow-up was 74 months. The pathologic findings showed that 106 patients (54.1%) satisfied the Milan criteria, 134 (68.4%) the UCSF criteria of whom 28 (14.3%) were beyond the Milan criteria but within the UCSF criteria, and 34 (17.3%) beyond the UCSF criteria. VI was detected in 41 patients (24%). The 1-, 3-, and 5-year overall survival rates were 90%, 85%, and 77%, respectively, according to the Milan criteria and 90%, 83%, and 76%, respectively, according to the UCSF criteria (P = NS). In univariate and multivariate analyses, tumor size and VI were significant prognostic factors affecting survival (P < 0.001). Two factors were significantly associated with VI: alfa-fetoprotein level of >400 ng/ml and tumor grade G3. CONCLUSIONS: Tumor size and VI were the only significant prognostic factors affecting survival of HCC patients. Primary liver resection could be a potential selection treatment before LT.

Continuous infusion of local anesthesia after living donor nephrectomy: a comparative analysis.

INTRODUCTION: Today local anesthetic wound infiltration is widely recognized as a useful adjunct in a multimodality approach to postoperative pain management. The effectiveness of continuous wound infusion of ropivacaine for postoperative pain relief after laparoscopic living donor nephrectomy was analyzed in this retrospective, comparative analysis. METHODS: Twenty patients undergoing living donor nephrectomy were divided into two groups: standard analgesic therapy (n=10) and ropivacaine continuous infusion group (n = 10). RESULTS: We observed a significant difference in term of visual analogue scale scores, use of morphine, hospital stay, and bowel recovery in favor of the ropivacaine group. The cost analysis demonstrated an overall savings of 985 Euros/patient. DISCUSSION: Surgical wound infusion with ropivacaine was safe and seemed to improve pain relief and accelerate recovery and discharge, reducing the overall costs of care. Postoperative pain control in the donor is of primary importance for better patient compliance and greater perceived quality of health care service.

Proteomic analysis reveals suppression of bark chitinases and proteinase inhibitors in citrus plants affected by the citrus sudden death disease.

Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges grafted on Rangpur lime rootstock, the most important rootstock in Brazilian citriculture. We performed a proteomic analysis to generate information related to this plant pathogen interaction. Protein profiles from healthy, CSD-affected and CSD-tolerant stem barks, were generated using two-dimensional gel electrophoresis. The protein spots were well distributed over a pI range of 3.26 to 9.97 and a molecular weight (MW) range from 7.1 to 120 kDa. The patterns of expressed proteins on 2-DE gels made it possible to distinguish healthy barks from CSD-affected barks. Protein spots with MW around 30 kDa and pI values ranging from 4.5 to 5.2 were down-regulated in the CSD-affected root-stock bark. This set of protein spots was identified as chitinases. Another set of proteins, ranging in pI from 6.1 to 9.6 with an MW of about 20 kDa, were also suppressed in CSD-affected rootstock bark; these were identified as miraculin-like proteins, potential trypsin inhibitors. Down-regulation of chitinases and proteinase inhibitors in CSD-affected plants is relevant since chitinases are well-known pathogenesis-related protein, and their activity against plant pathogens is largely accepted.

Diagnostic markers for diseases: SELDI-TOF profiling of pig sera for PRRS.

Porcine reproductive and respiratory syndrome (PRRS) is a highly infectious viral disease causing severe losses to the pig industry. Most weaning piglets are likely to be exposed to the infection and show at least asymptomatic PRRS viremia strongly related to productive performance. The aims of this study were to set up experimental conditions for pig sera proteomic profiling and to identify biomarkers that differentiate weaning asymptomatic piglets positive to PRRS viremia from negative controls (PCR tested) with potential predictive value for the subsequent occurrence of clinical PRRS. Protein profiles were generated by SELDI-TOF MS using the Bio-Rad Chips WCX, IMAC30 and H50. The discovery phase revealed that a consistent number of highly significant protein peaks can be detected by the WCX and IMAC30 surfaces; however none of these peaks were statistically confirmed by the subsequent validation phase, highlighting that serum concentration of the contaminant and most abundant proteins is a crucial parameterfor SELDI-TOF MS studies. Current protocols are being furtheroptimized and adapted to pig sera to reduce the unfavourable effects of the most abundant proteins and to increase the number of potential detectable biomarkers. Furthermore, proteomic fingerprint profiling has been shown to be a promising diagnostic tool that, in the future, may be useful to provide also insights into the mechanisms of early viral infection in vivo.

RET oncoproteins induce tyrosine phosphorylation changes of proteins involved in RNA metabolism.

We report the identification of proteins induced in response to RET/PTC2, an oncogene implicated in thyroid cancers. Anti-phosphotyrosine antibody affinity resin was used to purify Tyr(P)-containing and interacting proteins from 293T and NIH3T3 cells which were transfected with kinase active or inactive RET/PTC and RETMEN2 oncogenes. Proteins were separated by one-dimensional SDS-PAGE, extracted by in-gel digestion, and identified by MALDI-TOF peptide mass fingerprinting. The expression and tyrosine phosphorylation of Sam68, a protein implicated in mRNA nucleocytoplasmic translocation and splicing, were further examined in RET-transfected cells and thyroid tumors. Of relevance, cells transfected with RETMEN2B examined for anti-phosphotyrosine bound proteins, showed other proteins implicated in splicing: DEAD-box p68 RNA helicase, SYNCRIP, and hnRNP K. Western blotting analysis suggested that these proteins are singularly tyrosine phosphorylated in RETMEN2B-transfected cells, and that they constitutively bind with Sam68. The study concludes that regulation of splicing factors is likely to be important in RET-mediated thyroid carcinogenesis.

Main diagnosis and cause of death in a neonatal intensive care unit: do clinicians and pathologists agree?

AIM: To determine the agreement rates between clinical and autopsy diagnoses in a neonatal intensive care unit (NICU), distinguishing between the main diagnosis and cause of death. METHODS: Clinical and autopsy records of 75 infants who died in two consecutive years in the NICU (autopsy rate 42.6%) of a pediatric hospital in Mexico City were reviewed. RESULTS: Ninety-two percent of main clinical diagnoses were confirmed by autopsy. Four conditions (congenital cardiopathy, prematurity, specific congenital syndromes and hyaline membrane disease) accounted for more than two-thirds of diagnoses. However, for cause of death, the global agreement was only 50%. The most common conditions considered by clinicians (77%) and pathologists (56%) to be the causes of death were cardiogenic, septic or mixed shocks. Additionally, clinicians omitted 34 relevant conditions in 30 (40.0%) patients, and 21 of these conditions possibly played a role in the deaths of 17 (22.7%) patients. The most frequently omitted diagnosis was pneumonia, in 9 (26.5%) patients. Omissions were not related to gestational age, age at death, days as an inpatient, or gender. CONCLUSION: Despite a high agreement rate in the main diagnoses, notable imprecisions were present regarding cause of death and antemortem overlooking of potentially fatal conditions, confirming the useful role of autopsy to verify clinical diagnoses and suggesting that differentiation between the main diagnosis and cause of death should be carried out in future studies.

Randomized controlled trial of single-agent paclitaxel versus cyclophosphamide, doxorubicin, and cisplatin in patients with recurrent ovarian cancer who responded to first-line platinum-based regimens.

PURPOSE: To assess the activity, efficacy, and tolerability of single-agent paclitaxel and a platinum-containing regimen in previously treated patients with recurrent ovarian cancer. PATIENTS AND METHODS: Patients who achieved complete remission with platinum-based regimens and whose disease recurred after a progression-free interval of more than 12 months were included in the study. Every 21 days, patients received paclitaxel 175 mg/m(2) intravenously (IV) over 3 hours or cyclophosphamide 500 mg/m(2), doxorubicin 50 mg/m(2), and cisplatin 50 mg/m(2) (CAP) IV. RESULTS: Between June 1992 and May 1995, 97 consecutive patients with assessable or measurable disease were randomized to paclitaxel (n = 50) or CAP (n = 47). The median number of cycles on each arm was six. Toxicities included grade 3/4 leukopenia (4% for paclitaxel v 34% for CAP), grade 3/4 neutropenia (13% v 36%), grade 1/2 myalgia (19% v 4%), allergic reactions (15% v 2%), and grade 2/3 nausea and vomiting (17% v 51%). Complete responses were achieved in 17% and 30% of patients receiving paclitaxel and CAP, respectively, and partial responses were achieved in 28% and 25%, respectively (P =.062). At a median follow-up time of 49 months, median progression-free intervals were 9 months for paclitaxel and 15.7 months for CAP (Cox analysis: hazards ratio [HR], 0.60; 95% confidence interval [CI], 0.37 to 0.97; P =.038); median overall survival times were 25.8 months for paclitaxel and 34.7 months for CAP (Cox analysis: HR, 0.58; 95% CI, 0.34 to 0.98; P =.043). CONCLUSION: Rechallenge with either single-agent paclitaxel or platinum-based chemotherapy is effective in this patient population. Preliminary results suggest that single-agent paclitaxel may not be as active as platinum-based chemotherapy in recurrent ovarian cancer. Larger randomized trials are needed.

Survival and reproductive function after treatment of malignant germ cell ovarian tumors.

PURPOSE: Germ cell ovarian tumors are curable. The possible sequelae of chemotherapy on long-term survivors are still unknown, but these patients may expect normal lives. The aim of this study was to evaluate the outcome and reproductive function in a population of women treated since 1982. MATERIALS AND METHODS: Between 1982 and 1996, 169 women with malignant germ cell ovarian tumors were seen (70 dysgerminomas, 28 endodermal sinus tumors, 24 mixed tumors, and 47 immature teratomas). Seventy-one had advanced or recurrent disease. Fertility-sparing surgery was performed in 138 (81%) women, 81 of whom received postoperative chemotherapy. RESULTS: With a median follow-up of 67 months, the survival rate was 94% for dysgerminoma, 89% for endodermal sinus tumors, 100% for mixed types, and 98% for immature teratoma. For women who were treated conservatively, the survival rate was 98%, 90%, 100%, and 100%, respectively. Two women had adnexal recurrences, and both received salvage treatment. After treatment, all but one postpubertal woman had recovery of menses within 9 months. During follow-up, 12 untreated and 20 treated patients had 55 conceptions. We recorded 40 pregnancies at term, six terminations, and nine miscarriages. Four malformations were observed: one in 14 conceptions of patients who had not received chemotherapy and three in 41 conceptions of treated patients. CONCLUSION: Irrespective of subtype and stage, conservative surgery should become the standard approach to treating most patients with malignant ovarian germ cell tumors. Fertility seems to be only marginally affected by treatments. Miscarriages are in the expected range for the general population. The malformation rate is slightly higher than in the general population, but no significant difference was seen between patients who did and did not receive chemotherapy.

Neuro- and ototoxicity of high-dose carboplatin treatment in poor prognosis ovarian cancer patients.

BACKGROUND: Hematopoietic toxicity of high-dose carboplatin (HD-CBDCA) chemotherapy can be managed effectively with autologous blood cell support, but no conclusive data are available on its neuro- and ototoxicity. PATIENTS AND METHODS: We determined the neuro- and ototoxicity of HD-CBDCA in 10 patients affected by advanced ovarian cancer. HD-CBDCA was delivered as 24-hour continuous infusion or as 5-day schedules. Each patient underwent an extended clinical and instrumental neurological and otological evaluation before, during and after treatment. RESULTS: After HD-CBDCA only 1 patient had a clinically-evident peripheral neuropathy, while 3 additional patients had only distal paresthesias. Neurophysiological examination evidenced mild, although diffuse, sensory nerve impairment. Motor nerve impairment was also occasionally observed. All the sensory and motor pathological changes had a favorable course during the follow-up period. Ototoxicity was more severe than neurotoxicity and, in one case it was dose-limiting and audiologic impairment tended to remain constant also in the follow-up period. CONCLUSIONS: HD-CBDCA treatment can be tolerated by most of the patients, but careful monitoring of neuro- and, especially, ototoxicity should be planned.

Diffusion of HCV through peritoneal membrane in HCV positive patients treated with continuous ambulatory peritoneal dialysis.

PURPOSE OF THE STUDY: We evaluated the presence of HCV in the peritoneal effluents of viraemic patients treated with continuous ambulatory peritoneal dialysis (CAPD) to evaluate the risk of transmitting the infection with this procedure. PROCEDURE: Fifteen of 81 CAPD patients (18.5%) had anti-HCV antibodies and eight were viraemic. At the beginning of CAPD two of the viraemic patients had ascites with a clinical picture of chronic active hepatitis and cirrhosis. Peritoneal dialysates were collected after an overnight exchange with 1.36% glucose and after a 4-h exchange with 3.86% glucose. Fluids from the overnight exchange were spun to obtain a cellular pellet and the supernatant 100-fold concentrated. RESULTS: No viral genome could be detected in unconcentrated samples and in cellular pellets, while HCV-RNA at low titre was detected in concentrated dialysates from the two patients with active liver disease. CONCLUSIONS: Our findings confirm that HCV may be present in the CAPD effluent of some patients; however, the titre of virus in the effluent was extremely low, at the limit of detection of the PCR assay. Peritoneal fluids originating from patients with HCV associated severe liver disease may be a potential source of infection.

Expression of human placental lactogen and variant growth hormone genes in placentas.

Previous studies comparing the expression levels of human placental lactogen (hPL) genes have shown varying results, due to, perhaps, the fact that in all of them only one placenta was being analyzed. Here, the expression of hPL and growth hormone variant (hGH-V) genes in fifteen term placentas was comparatively analyzed at the RNA level, using reverse transcription coupled to polymerase chain reaction (RT-PCR). The abundance of the combined RNA transcripts derived from these genes varied from one placenta to another. The authors found that hPL-4 transcripts were more abundant than those of hPL-3 in most samples (ratios from 1:1 to 6:1), transcripts from the putative hPL-1 pseudogene were more abundant at the unprocessed stage while those of the hGH-V gene were mostly processed. Again, the authors of this study observed wide variation from placenta to placenta in the abundance of both of these types of transcripts. The same was observed when a group of six placentas from abortuses and nine from pregnancies complicated by preclampsia, diabetes and hypertension was studied. The authors conclude that the disagreeing results reported in the literature which are not in agreement concerning the expression levels of hPL genes could be explained by normal variations of their expression levels among the different placentas analyzed.

[Brain metastases in patients with malignant ovarian epithelial tumors]. / Metastasi cerebrali in pazienti affette da tumore maligno epiteliale ovarico.

From September 1983 to September 1994, 23 patients with Central Nervous System (CNS) metastases from ovarian carcinoma were observed in our institution. The mean age at the time of CNS metastases diagnosis was 59 years, the mean interval between diagnosis of ovarian carcinoma and documentation of the CNS involvement was 35 months. All the patients presented neurological symptoms. One patient had meningeal carcinomatosis; 22 presented parenchymal lesions. Nine patients had a single CNS lesion and 13 had multiple metastatic sites. CNS was the only site of disease in 9 patients, while 8 had concomitant extraperitoneal dissemination. Four patients received hormonal treatment with a mean survival (MS) of 3 months; 14 received radiotherapy alone (MS 5.5 months), 5 underwent surgical resection of solitary lesion followed by radiotherapy (MS 17 months). Number of CNS lesions, extent of the disease at time of CNS metastases and the treatment were the factors which significantly affected survival. The prognosis of these patients appears poor, however, early diagnosis followed by multimodal treatment may result in significant palliation an improve overall survival in a selected group of patients.

Pure ovarian immature teratoma, a unique and curable disease: 10 years' experience of 32 prospectively treated patients.

OBJECTIVE: To report and evaluate a conservative and individualized treatment policy in a homogeneously selected series of patients affected by pure ovarian immature teratoma. METHODS: This prospective trial, with specific treatment policies according to stage and grade, was planned and started in 1982. The study population consisted of 32 patients affected by pure immature teratoma, with the exclusion of mixed germ cell tumors. Fertility-sparing surgery was performed whenever possible. Surgery alone, with careful follow-up, was adopted for stage I or II according to the International Federation of Gynecology and Obstetrics (FIGO) and grade 1 or 2 tumors. The other patients, with stage III or with grade 3 stage I or II tumors, or those referred at relapse, were treated with platinum-based chemotherapy regimens. RESULTS: Thirty of 32 patients underwent fertility-sparing surgery. Ten of 32 patients received chemotherapy after surgery, either as adjuvant treatment or in the presence of visible tumor. All 32 patients are alive and disease-free, with a median follow-up from surgery of 47 months (range 11-138). In six patients, regardless of the administration of chemotherapy, the tumor either spontaneously differentiated toward mature glia or increased in volume, mimicking progression but still remaining completely mature. Five of six patients wishing to procreate had a total of seven normal pregnancies. CONCLUSIONS: Pure ovarian immature teratoma is a potentially curable disease with a unique natural history. Our data substantiate the hypothesis that low-grade and low-stage tumors do not require chemotherapy, and that a fertility-sparing surgical approach is warranted in all cases.

Single-center comparison of results of 1000 prenatal diagnoses with chorionic villus sampling and 1000 diagnoses with amniocentesis.

Large multicenter studies have confirmed the safety and accuracy of chorionic villus sampling as a prenatal genetic diagnostic procedure, but there have been few single-center evaluations. We report our experience with 1000 consecutive chorionic villus sampling procedures compared with 1000 consecutive amniocentesis procedures during the same period. The procedures were performed by the same genetic counselors, sonographers, obstetricians, and laboratory personnel. Indications for referral, demographic characteristics of patients, numbers of attempts per patient, fetal loss rates, laboratory results, and evaluation of accuracy are included. Analysis of all data suggests that chorionic villus sampling is a safe and accurate alternative to amniocentesis in our community-based teaching hospital.