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Recombinant DNA is a fundamental tool in biotechnology and medicine. These DNA sequences are often built, replicated, and delivered in the form of plasmids. Validation of these plasmid sequences is a critical and time-consuming step, which has been dominated for the last 35 years by Sanger sequencing. As plasmid sequences grow more complex with new DNA synthesis and cloning techniques, we need new approaches that address the corresponding validation challenges at scale. Here we prototype a high-throughput plasmid sequencing approach using DNA transposition and Oxford Nanopore sequencing. Our method, Circuit-seq, creates robust, full-length, and accurate plasmid assemblies without prior knowledge of the underlying sequence. We demonstrate the power of Circuit-seq across a wide range of plasmid sizes and complexities, generating full-length, contiguous plasmid maps. We then leverage our long-read data to characterize epigenetic marks and estimate plasmid contamination levels. Circuit-seq scales to large numbers of samples at a lower per-sample cost than commercial Sanger sequencing, accelerating a key step in synthetic biology, while low equipment costs make it practical for individual laboratories.

Recombinant DNA is a fundamental tool in biotechnology and medicine. Validation of the resulting plasmid sequence is a critical and time-consuming step, which has been dominated for the last 35 years by Sanger sequencing. As plasmid sequences grow more complex with new DNA synthesis and cloning techniques, we need new approaches that address the corresponding validation challenges at scale. Here we prototype a high-throughput plasmid sequencing approach using DNA transposition and Oxford Nanopore sequencing. Our method, Circuit-seq, creates robust, full-length, and accurate plasmid assemblies without prior knowledge of the underlying sequence for approximately $1.50 per plasmid. We demonstrate the power of Circuit-seq across a wide range of plasmid sizes and complexities, generating accurate and contiguous plasmid maps. We then leverage our long read-data to characterize epigenetic marks and estimate plasmid contamination levels. Circuit-seq scales to large numbers of samples at a lower cost than commercial Sanger sequencing, accelerating a key step in synthetic biology, with low startup costs make it practical for individual laboratories.

Ureteric stents are one of the most used tools in urology. As they are often necessary it is imperative to correctly indicate a stent placement, following conscious and evidence-based criteria. However, they present a significant drawback, since up to 80% of patients present bothersome symptoms that negatively affect quality of life. There are many strategies to reduce stented patient’s morbidity: First a proper stenting indication is needed to avoid unnecessary stenting; Minimizing stent indwelling time is also crucial; Currently, numerous materials are available including relatively stiff (polyurethane) to relatively soft (silicone), materials that’s seems to influence patient stent-related symptoms; Finally, choosing a proper stent length according to the patients anatomy and a correct placement may reduce further stent bothersome. All these different approaches may be implemented in daily practice to help reduce adverse effects and complications in stented patients.

B cells are a rare subset in the thymus, an environment that supports T cell development and inhibits commitment to other immune cell lineages. Yet thymic B cells play an important role in the negative selection of autoreactive T cells. The origin of thymic B cells from either committed B cells or early thymocyte progenitors remains uncertain. We hypothesize that early thymic progenitor cells are a heterogenous population that can give rise to both T cells and B cells. In our study we combined the CRISPR/Cas9-dependent lineage recorder GESTALT with single cell RNAseq to capture cell lineage and type information to follow progressive cell fate decisions and to identify thymic B cell progenitors and transcriptional regulators permitting B cell differentiation in the thymus. Additionally, to evaluate the functional importance of thymic B cells in shaping the T cell repertoire, we performed TCR-seq on conventional CD4 T cells and Tregs. In the absence of B cells, amino acid usage within either TCR alpha or beta CDR3 regions remained unchanged. However, the dominant TCR alpha CDR3 length from the most abundant thymic Treg clones was reduced compared to wild type. Whether this repertoire shift impacts self-tolerance to B cell or AIRE-dependent antigens, both of which are expressed by thymic B cells, remains to be determined. Together, our data directly assesses lineage relationships leading to thymic T and B cell fate commitment and deepen our understanding of intra-thymic development and function of B cells that are required for central tolerance. Supported by AI089805 Burroughs-Wellcome Fellowship

To evaluate the learning curve of the simplified fluoroscopic biplanar (0-90º) puncture technique for percutaneous nephrolithotomy.We prospectively evaluated patients with renal stones treated with percutaneous nephrolithotomy by a single institution's fellows employing the simplified bi-planar (0-90º) fluoroscopic puncture technique for renal access. The learning curve was assessed with the fluoroscopic screening time and the percutaneous renal puncture time. Data obtained were compared to a subset of patients operated by a senior surgeon.Eighty-nine patients were included in the study. Forty patients were operated by fellow-1, 39 by fellow-2, and 10 patients by the senior surgeon. Demographic data of all patients between groups were homogeneous, with no difference in gender (p = 0.432), age (p = 0.92), stone volume (p = 0.78), puncture laterality (p = 0.755), and body mass index (p = 0.365). The mean puncture time was 7.5, 4, and 3.1 min for fellow-1, fellow-2, and expert, respectively. The mean fluoroscopic screening time for the puncture was 10, 11, and 5.1 s for fellow-1, fellow-2, and the expert, respectively. Stone cases, both fellows needed to complete 10 procedures to match the senior surgeon in the mean puncture time (p = 0.046); meanwhile, the fluoroscopic screening time was equal even before to complete 10 procedures.This study suggests that with the simplified biplanar (0-90º) puncture technique, the fluoroscopic screening time used in the learning process is brief. A novice fellow could require to complete ten cases to flatten the learning curve treating complex stone cases, and a flat learning curve is seen since the beginning when treating simple renal stones.

To compare the safety and efficacy of RIRS in patients ≥ 80 years to a younger population.We retrospectively compared the data from patients ≥ 80 years of age undergoing RIRS with the data of a group of patients from 18 and 80 years. Perioperative outcomes, complications and emergency department visits were compared between two groups.A total of 173 patients were included in the study. Mean age was 44 (27-79) and 81 years-old (80-94), for younger and elderly group, respectively. Elderly patients had higher ASA scores (≥ 3) (28.6% vs 75.8%; p = 0.0001) and Charlson comorbidity index (1.99 vs 7.86; p = 0.0001), more diabetes (p = 0.006) and respiratory comorbidities (p = 0.002). No statistical difference was found between two groups in stone size (p = 0.614) and number (p = 0.152). Operative time (74.48 vs 102.96 min; p = 0.0001) and duration of hospitalisation (1.7 vs 2.9 days; p = 0.001) were longer for the elderly. Intraoperative complication rate did not show differences between the two groups (p = 0.166). Postoperative complications rates were similar between the cohorts (7.7% vs 9.5%; p = 0.682). The success rates were 67.5% in the younger group and 71.4% in the elderly group (p = 0.584). No difference was seen in stone recurrence (p = 0.73). A higher rate of visits to the emergency department was found in younger cohort (23.6% vs 11.6%; p = 0.046), mostly duo to stent-related symptoms.Despite the higher rate of comorbidity in the elderly group, RIRS was a safe procedure with similar complication rate and outcomes at an expense of higher operative time and hospital stay.

Background: Clinical and molecular factors are essential to define the prognosis in patients with glioblastoma (GBM). O6-methylguanine-DNA methyltransferase (MGMT) methylation status, age, Karnofsky Performance Status (KPS), and extent of surgical resection are the most relevant prognostic factors. Our investigation of the role of gender in predicting prognosis shows a slight survival advantage for female patients. Methods: We performed a prospective evaluation of the Project of Emilia Romagna on Neuro-Oncology (PERNO) registry to identify prognostic factors in patients with GBM who received standard treatment. Results: A total of 169 patients (99 males [58.6%] and 70 females [41.4%]) were evaluated prospectively. MGMT methylation was evaluable in 140 patients. Among the male patients, 36 were MGMT methylated (25.7%) and 47 were unmethylated (33.6%); among the female patients, 32 were methylated (22.9%) and 25 were unmethylated (17.9%). Survival was longer in the methylated females compared with the methylated males (P = 0.028) but was not significantly different between the unmethylated females and the unmethylated males (P = 0.395). In multivariate analysis, gender and MGMT methylation status considered together (methylated females vs. methylated males; hazard ratio [HR], 0.459; 95% confidence interval [CI], 0.242-0.827; P = 0.017), age (HR, 1.025; 95% CI, 1.002-1.049; P = 0.032), and KPS (HR, 0.965; 95% CI, 0.948-0.982; P < 0.001) were significantly correlated with survival. Conclusions: Survival was consistently longer among MGMT methylated females compared with males. Gender can be considered as a further prognostic factor.

There are currently 3holmium laser, YAG (Ho:YAG) endolithotripsy procedures that are considered basic (fragmentation, pulverisation, "pop-corn" technique). We present the technique of fragmentation targeted at preferred discontinuities (FTPD), a new concept of endolithotripsy by Ho:YAG laser.The FTPD technique is based on the selective application of energy (targeting a specific preselected point) to an area that is visually prone to the formation of a fracture line or preferred discontinuity (conditioned by the anisotropy of the urolithiasis). The ideal energy regimen (setting) is a high range of working energy (2-3J) with a very low frequency range (5-8Hz) and short pulse width. Between January 2015 to February 2017, the FTPD technique was used in 37 procedures (7 NLP, 16 RIRS, 12 URS, 2 cystolithotomies), with a Ho:YAG laser (Lumenis Pulse 120HStrategic improvement was achieved in all cases using the TFPD technique to continue the endolithotripsy or remove fragments. No complications were recorded after the use of this method.FTPD can be considered a complementary option in combination with the basic methods of fragmentation and pulverisation. In our experience, it constitutes significant progress in optimising the performance of Ho:YAG laser endolithotripsy.

L’ostilità è uno dei fattori psicosociali che influiscono sulla salute ed è predittore dello sviluppo di malattie, comportamenti e abitudini poco salutari. Il presente lavoro ha come obiettivo la presentazione della validazione italiana della Cynical Distrust Scale (CynDis; Julkunen et al. , 1994) per la misurazione della sfiducia cinica, componente cognitiva dell’ostilità. Nonostante l’ampia letteratura dedicata allo studio di tale costrutto, in Italia non sono ancora disponibili strumenti validi e attendibili in grado di misurare in maniera veloce e precisa la sfiducia cinica. La CynDis è stata somministrata, insieme alle PANAS, alla MCSD-8 e a due domande sulla percezione del proprio stato di salute, a 311 partecipanti (169 femmine e 142 maschi, età media 39.07 anni, DS = 14.98). Le caratteristiche psicometriche della scala, analizzate tramite analisi fattoriale esplorativa, studio della consistenza interna e della stabilità nel tempo, e correlazioni con criteri esterni, appaiono soddisfacenti. La scala appare pertanto adatta come strumento di screening valido e attendibile, da utilizzare nei contesti di prevenzione e promozione della salute.

Background As yet, no population-based prospective studies have been conducted to investigate the incidence and clinical outcome of glioblastoma (GBM) or the diffusion and impact of the current standard therapeutic approach in newly diagnosed patients younger than aged 70 years. Methods Data on all new cases of primary brain tumors observed from January 1, 2009, to December 31, 2010, in adults residing within the Emilia-Romagna region were recorded in a prospective registry in the Project of Emilia Romagna on Neuro-Oncology (PERNO). Based on the data from this registry, a prospective evaluation was made of the treatment efficacy and outcome in GBM patients. Results Two hundred sixty-seven GBM patients (median age, 64 y; range, 29–84 y) were enrolled. The median overall survival (OS) was 10.7 months (95% CI, 9.2–12.4). The 139 patients ≤aged 70 years who were given standard temozolomide treatment concomitant with and adjuvant to radiotherapy had a median OS of 16.4 months (95% CI, 14.0–18.5). With multivariate analysis, OS correlated significantly with KPS (HR = 0.458; 95% CI, 0.248–0.847; P = .0127), MGMT methylation status (HR = 0.612; 95% CI, 0.388–0.966; P = .0350), and treatment received in a high versus low-volume center (HR = 0.56; 95% CI, 0.328–0.986; P = .0446). Conclusions The median OS following standard temozolomide treatment concurrent with and adjuvant to radiotherapy given to (72.8% of) patients aged ≤70 years is consistent with findings reported from randomized phase III trials. The volume and expertise of the treatment center should be further investigated as a prognostic factor.

Fluconazole is recommended in the prophylaxis of oropharyngeal candidiasis (OPC) in patients undergoing radiotherapy for head-neck tumours; however, the actual effectiveness of fluconazole in this setting remains unclear. Adult patients with cervico-cephalic carcinoma submitted to radical or adjuvant radiotherapy were randomized to 100 mg fluconazole (n = 138) or matched placebo (n = 132) oral suspension once daily from the sixth session of radiotherapy up to the end of treatment. The final analysis of the investigation showed a higher rate of the OPC outbreak-free survival in the fluconazole compared with placebo (P = 0.008 in the log-rank test). The mean time (95% CI) to OPC outbreak was 56 (53-59) days in the fluconazole group and 47 (43-51) days with placebo. The mean duration of radiotherapy was 43.5 and 39.9 days, respectively in the two groups (P = 0.027). Adverse effects were reported in 70.3% of patients in the fluconazole group and in 67.4% with placebo. The results showed prophylaxis with fluconazole given in irradiated patients with head-neck tumours significantly reduces the rate and the time to development of OPC compared with placebo.

Adenoid cystic carcinoma is a malignant tumour involving the salivary glands, rarely developing in the nasopharynx. The biological behaviour of adenoid cystic carcinoma is characterized by slow growth rate, high tendency to local recurrence and metastatic spread. Its histological features are particularly important for prognostic prediction: solid pattern has the worst outcome. Initial presentation of nasopharyngeal adenoid cystic carcinoma with paresis of cranial nerves and Horner’ s syndrome is infrequent. The Authors present a case of a rare adenoid cystic carcinoma of the nasopharynx, at admission with Horner’ s syndrome, in a 66-year-old male. Magnetic resonance imaging showed an expansive submucosal lesion of the nasopharynx involving the para-pharyngeal space, tensor and levator veli palati muscles and the apex of petrous bone. Positron emission tomography excluded distant metastasis. Definitive histopathological examination revealed an adenoid cystic carcinoma with mixed cribriform and solid pattern. The patient was treated exclusively with radiotherapy (70 Gy) in 35 fractions with partial reduction of the neoplastic mass.

The research methodology in the "Associazione Italiana di Radioterapia (AIRO)" (The Italian Association of Radiation Oncology) is presented according to two interconnected aspects: the consensus research on similar projects with other Scientific Associations and the clinical research carried out in the ten years of life of the Association. The mechanism of the "Federazione Intersocietaria" (Federation of Associations) with the Radiology, Oncology and Physics Associations can be a stimulant to broaden the horizons and feasibility of cancer research which is one of the main causes of mortality and which is increasing due to the population aging. The results of three national multicentric trials on the clinical research on cancer of the rectum, oropharynx and in the elderly, are reported. The study methodology, based on the clinical evidence and multidisciplinarity, shows that the time is ripe for a development of research and, if coordinated by the Association, it would contribute to the prestige and importance of the Italian Radiotherapy.

Summary Pelvic osteosarcoma is a very rare disease, with poor prognosis affecting young people. Since June 1987 we have observed 26 patients, F/M: 12/14, median age 23 years (range 14–55), 7 with lung metastases at diagnosis. Four patients underwent surgery as first line treatment; 4 patients received intravenous (i.v.) chemotherapy; eighteen out of 26 received 1–3 cycles of neoadjuvant chemotherapy consisting of high dose i.v. methotrexate plus leucovorin rescue, intrarterial cisplatin and i.v. epidoxorubicin. We obtained 8 PR, 9 SD, 1 PD. Hemipelvectomy has been performed in 4 cases (0%, 0%, 50%, 70% necrosis). The other 14 patients, judged not operable, received radiotherapy plus daily low doses of cisplatin. All patients have been treated with i.v. epidoxorubicin, ifosfamide, high dose methotrexate, cisplatin and etoposide. Two out of four patients who underwent surgery died at 17 and 18 months, 2 are alive and free of disease at 28+ and 17+ months from surgery. Among the 14 patients treated with radiotherapy, 9 died with a median survival of 18 months (5–22); 5 are alive with a median survival of 20 months (14–31). Although pelvic osteosarcoma has a limited cure rate at present, results could be improved in the future by means of earlier diagnosis and better combined treatment.

The major role of radiotherapy and its outcomes in the treatment of cancer are now supported by a 100-year experience. To cover the cost of the equipment needed for an effective application of RT in Italy, the allocation decided according to the Census of 1995 is definitely inadequate. The Italian radiotherapists to cope with this situation should acquire a sound professional preparation as well as be familiar with the organizational management. The knowledge of the National and European regulations and the programs of Quality Control and Quality Assurance, the use of guidelines agreed on with other specialists, the application of standardized procedures controllable on inspection and audit, the plan of the budget, the appraisal of costs are all indispensable to safeguard the personal professional dignity and to improve the quality of life of the patients.

The optimal sequence of a paclitaxel-radiation combination was investigated in vitro in two human colon adenocarcinoma cell lines, HT29 and LoVo. Three schedules of combined treatment were tested by clonogenic and flow cytometric assays. Paclitaxel was given 24 h prior to a single radiation shot (first schedule) or 24 h (second schedule) or 48 h (third schedule) before 3 days of concomitant radiation. Dose-response data were fit to a linear quadratic model, and mean inactivation dose and sensitizer enhanced ratio were calculated. In HT29 cells, the first and second schedule resulted in an additive effect, whereas a supraadditive interaction was observed with the third combination schedule. This effect was obtained with amounts of paclitaxel lower than IC50, which did not result in cell cycle perturbation, and with low radiation dose (2 Gy) that may be given in a clinical setting. LoVo cells were less sensitive to combined treatment than HT29 cells, switching from infraadditive (first and second schedule) to additive interaction (third schedule). Posttreatment recovery studies of third schedule showed a loss of cell survival in HT29 cells but not in LoVo cells. In contrast to LoVo cells, the third schedule in HT29 cells was able to induce perturbation of cell cycle kinetics, an effective impairment of DNA repair, and apoptotic cell death. HT29 and LoVo cells showed constitutional different characteristics: HT29 cells were more sensitive to paclitaxel exposure, less radiosensitive, and had a different cell cycle redistribution after radiation exposure than LoVo cells; moreover, HT29 cells showed a major propensity to undergo apoptosis. These results suggest that the radiosensitizing effect of paclitaxel was strictly schedule dependent, and the inhibition of DNA repair, cell cycle redistribution, and apoptosis could be the mechanisms for the induction of radiosensitization by paclitaxel.

Sixteen patients affected by previously untreated non-small cell lung cancer stage IIIB or IV received radiotherapy and paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) as radiation sensitizer in an open, nonrandomized pilot study to find the maximum tolerated dose of the drug concomitantly combined with radiation. Paclitaxel was given as a 3-hour infusion once weekly at a dose escalating by 10 mg/m2/wk for every patient cohort, starting at 40 mg/m2/wk and continuing to 80 mg/m2/wk. Conventionally fractionated (2 Gy/d for 5 d/wk for 5 weeks) radiotherapy up to 50 Gy was delivered to the primary tumor and mediastinum with a 6-mv linear accelerator. Hematologic toxicity has been very low; grade 3 World Health Organization nonhematalogic toxicities have been registered only at the 80 mg/m2/wk dose level. Seven patients achieved a major response, three patients had stable disease, and five patients progressed; three patients are still responding, whereas the others are relapsed and five of them died of disease. The median duration of response was 5 months. Paclitaxel may be safely combined with radiation at the maximum tolerated dose of 70 mg/m2/wk. Our data seem to confirm the radiosensitizing effect of the drug, independent of the dose level. Low doses of paclitaxel given as a single agent are unable to control metastatic disease.

To establish the maximum tolerated dose of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) as a radiosensitizing agent and to determine its optimal therapeutic dose when combined with conventionally fractionated radiotherapy in the treatment of non-small cell lung cancer, a phase I/II study was undertaken in 16 treatment-naive patients. Beginning at 40 mg/m2/wk with doses escalated in 10 mg/m2 increments until dose-limiting toxicity was encountered, paclitaxel was administered over 3 hours to successive three-patient cohorts. Radiotherapy (2 Gy/d x 5 d/wk; maximum total dose, 50 Gy) was delivered after the paclitaxel infusion. Treatment continued for 5 successive weeks. All 16 patients are evaluable for response and toxicity. Hematologic toxicity was low, with red blood cells and platelets remaining stable and leukocyte decreases (mean, 60%) attributed to radiotherapy. Nonhematologic toxicity included grade 2/3 esophagitis and neurologic sequelae. Responses were noted at all paclitaxel dose levels, including two complete and five partial responses, but the median time to progression was only 5 months. Paclitaxel may be combined safely with radiotherapy without major toxicity, and the radiosensitizing effect of paclitaxel was evident at all doses.

Osteosarcoma is a very rare disease affecting young people : it is calculated not to exceed 0.2 % of all the tumors. Pelvic osteosarcoma (p.o.) is even more uncommon, representing about 5 % of all osteosarcomas reported.

Fourteen acute non-lymphoid leukaemias (ANLL) were identified among 947 consecutive patients with Hodgkin's disease (HD) treated in five collaborating centres in Italy between January 1969 and December 1979. Leukaemia developed 12-118 months after the diagnosis of HD and was always preceded by a pre-leukaemic phase. The karyotype of the leukaemic cells was studied in nine of the 14 patients and was abnormal in seven cases. Bone marrow agar cultures were performed in five of the 14 patients and were abnormal in every case. The actuarial risk of developing ANLL at 10 years was 6.2% for combined therapy, 4.8% for salvage therapy and 2.2% for chemotherapy alone. No case of ANLL was documented among 136 patients treated with radiotherapy alone. One patient developed acute promyelocytic leukaemia following treatment with ABVD and radiotherapy. Both univariate and multivariate analyses seem to demonstrate that the induction therapy modality is a significant variable in development of secondary acute leukaemia. The recognition of a significant risk of ANLL in patients treated for HD should create changes in treatment policy.