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Many modern applications of online changepoint detection require the ability to process high-frequency observations, sometimes with limited available computational resources. Online algorithms for detecting a change in mean often involve using a moving window, or specifying the expected size of change. Such choices affect which changes the algorithms have most power to detect. We introduce an algorithm, Functional Online CuSUM (FOCuS), which is equivalent to running these earlier methods simultaneously for all sizes of windows, or all possible values for the size of change. Our theoretical results give tight bounds on the expected computational cost per iteration of FOCuS, with this being logarithmic in the number of observations. We show how FOCuS can be applied to a number of different changes in mean scenarios, and demonstrate its practical utility through its state-of-the-art performance at detecting anomalous behaviour in computer server data.

Background: There is a paucity of robust clinical data on major postoperative complications following robotic-assisted resection for primary lung cancer. This study assessed the incidence and outcomes of patients who experienced major complications after robotic anatomic pulmonary resection. Methods: This was a multicenter, retrospective review of patients who underwent robotic anatomic pulmonary resection between 2002 and 2018. Major complications were defined as grade III or higher complications according to the Clavien–Dindo classification. Statistical analysis was performed based on patient-, surgeon-, and treatment-related factors. Results: During the study period, 1264 patients underwent robotic anatomic pulmonary resections, and 64 major complications occurred in 54 patients (4.3%). Univariate analysis identified male sex, forced expiratory volume in 1 second, diffusion capacity of the lung for carbon monoxide, neoadjuvant therapy, and extent of resection as associated with increased likelihood of a major postoperative complication. Patient age, performance status, body mass index, reoperation status, and surgeon experience did not have a significant impact on major complications. Patients who experienced at least 1 major complication were at higher risk for an intensive care unit stay of >24 hours (17.0% vs 1.4%; P < .001) and prolonged hospitalization (8.5 days vs 4 days; P < .001). Patients who experienced a major postoperative complication had a 14.8% risk of postoperative death. Conclusions: In this series, the major complication rate during the postoperative period was 4.3%. A number of identified patient- and treatment-related factors were associated with an increased risk of major complications. Major complications had a significant impact on mortality and duration of stay.

Applying digestate, the residue from anaerobic digestion, to soil as a replacement for inorganic fertiliser is of growing interest in agriculture. However, the impacts of different fractions of digestate on the soil carbon (C) cycle remain unclear and provide the focus for the research reported here. We examined the effects of applying whole digestate (WD) and solid digestate (SD) on carbon dioxide (CO2C) efflux, the concentrations of dissolved organic carbon (DOC), microbial biomass C (Cmicro) and phospholipid fatty acids, alongside carbon use efficiency (CUE). A 21‐day laboratory microcosm incubation was used to investigate the impacts of digestate when applied to two grassland soils of high versus low initial nutrient content. Application rates for SD and WD were based on recommended nitrogen (N) inputs to grassland soils for these organic materials. Compared to control treatments, cumulative CO2C efflux and the concentration of DOC increased significantly after WD and SD application, although only within the low nutrient soil. Both Cmicro and the fungal‐to‐bacterial ratio increased significantly following SD application, regardless of the initial soil nutrient content. These observations are likely to reflect the larger input of C, alongside the dominance of more strongly lignified compounds, associated with SD compared to WD to achieve a constant N application rate. Our results also indicate that the two digestate fractions generated significantly different CUE. The application of SD led to increases in Cmicro and positive values of CUE, whereas decreases in Cmicro and negative values of CUE were observed following WD application. These findings emphasize the need to carefully plan the management of digestate in agricultural production systems, to minimize negative impacts on C storage within soils whilst maximizing the agronomic value derived from digestate.

Robotic-assisted pulmonary resection has greatly increased over the last few years, yet data on the application of robotic surgery in high-risk patients are still lacking. The objective of this study is to evaluate the perioperative outcomes in ASA III-IV patients who underwent robotic-assisted lung resection for NSCLC. Between January 2010 and December 2017, we retrospectively collected the data of 148 high-risk patients who underwent lung resection for NSCLC via a robotic approach at our institution. For this study, the prediction of operative risk was based on the ASA-PS score, considering patients in ASA III and IV classes as high-risk patients: of the 148 high-risk patients identified, 146 patients were classified as ASA III (44.8%) and two as ASA IV (0.2%). Possible prognostic factors were also analysed. The average hospital stay was 6 days (8–30). Post-operative complications were observed in 87 (58.8%) patients. Patients with moderate/severe COPD developed in 33 (80.5%) cases post-operative complications, while elderly patients in 25 (55%) cases, with a greater incidence of high-grade complications. No difference was observed when comparing the data of obese and non-obese patients. Robotic surgery appears to be associated with satisfying post-operative results in ASA III-IV patients. Both marginal respiratory function and advanced age represent negative prognostic factors. Due to its safety and efficacy, robotic surgery can be considered the treatment of choice in high-risk patients.

Background Robotic-assisted thoracic surgery has been shown as a safe and feasible surgical procedure to treat a broad range of thoracic malignancies with a shorten hospital stay, a quicker return to normal daily activities and superior quality of life compared to open approach. Nonetheless, its utilization has predominantly been restricted to the average surgical intervention. In the latest years, given the technological improvements and the advanced skills of the “robotic” surgeons, this approach has been applied to more complex and challenging procedures (e.g., advanced stages/ after neoadjuvant therapies). The aim of this study is to show the results and the advantages of redo thoracic procedures performed with a robot-assisted approach. Methods Retrospective analysis of a prospectively collected database (2017–2019). We analysed the first case series undergoing robotic approach (totally endoscopic) in patients who underwent previous surgery and/or radiotherapy for thoracic malignancies. Results Nine patients (M/F =5/4) with a median age of 73 years underwent robotic procedures after previous radical surgery (n=6) or radiation therapy treatment (n=3). The mean time interval between the two interventions was 96.12 months (range, 7–444 months). The conversion rate was 0%. No postoperative death occurred. Only one complication arose (air leaking) treated conservatively. The median length of stay and chest tube duration were 5 days (range, 4–10 days) and 2 days (range, 2–5 days). The 30-, 60- and 90-day mortality was 0%. With a median follow-up of 10 months (range, 2–12 months), all patients are currently alive, disease free, and with an overall survival and a disease-free survival of 10 months. Conclusions The robotic surgery is a safe and feasible approach in case of redo-robotic surgery, showing good outcomes, permitting a fast return to full daily activity, and enabling the patients to rapidly undergo adjuvant treatments, when required.

Open pulmonary resection is considered the gold standard treatment of early-stage non-small cell lung cancer (NSCLC). However, in the last decades, the use of minimal-invasive techniques has given promising results. Survival in lung cancer, after surgery, depends on the number of pathological nodes (pN), thus lymph nodal upstaging can be considered a surrogate for surgical quality of the procedure. Several studies have demonstrated a lower rate of upstaging in video-assisted thoracic surgery than in open surgery, suggesting an approach-related difference in lymphadenectomy. Features of robotic technique could consent a lymph nodal dissection similar to open surgery. The aim of the study is to compare nodal upstaging between thoracotomy and robotic approaches to evaluate the oncologic radicality. Between January 2013 and December 2016, 212 consecutive cN0 NSCLC patients underwent lobectomy and lymphadenectomy (N1 + N2 stations) by either thoracotomy (Open Group) or robotic surgery (Robotic Group). Lobectomy and lymphadenectomy were performed in 106 cN0–cN1 NSCLC patients by robotic surgery and in 106 cN0–cN1 NSCLC patients by open surgery. A mean of 14.42 ± 6.99 lymph nodes was removed in the Robotic Group (RG) and a mean of 14.32 ± 7.34 nodes in the Open Group (OG). Nodal upstaging was observed in 22 (20.75%) RG patients and in 19 OG (17.92%) patients. Robotic lobectomy for clinical N0–N1 NSCLC appears to be equivalent to thoracotomy in terms of efficacy of lymph node dissection and nodal upstaging. Given that the nodal upstaging is a surrogate of quality of surgery, we can consider robotic lobectomy an appropriate procedure which ensures similar result to the open approach.

International audience; Whilst there are a plethora of algorithms for detecting changes in mean in univariate time-series, almost all struggle in real applications where there is autocorrelated noise or where the mean fluctuates locally between the abrupt changes that one wishes to detect. In these cases, default implementations, which are often based on assumptions of a constant mean between changes and independent noise, can lead to substantial over-estimation of the number of changes. We propose a principled approach to detect such abrupt changes that models local fluctuations as a random walk process and autocorrelated noise via an AR(1) process. We then estimate the number and location of changepoints by minimising a penalised cost based on this model. We develop a novel and efficient dynamic programming algorithm, DeCAFS, that can solve this minimisation problem; despite the additional challenge of dependence across segments, due to the autocorrelated noise, which makes existing algorithms inapplicable. Theory and empirical results show that our approach has greater power at detecting abrupt changes than existing approaches. We apply our method to measuring gene expression levels in bacteria.

Patient Reported Outcome and Experience Measures (PROMs and PREMs) play an increasingly important role in monitoring the quality of the oncological pathway. The aim of this study is to describe the case of five hospitals a year after the adoption of PROMs and PREMs for robotic oncological colorectal surgery in Tuscany and to investigate how the clinicians can impact the process of implementation and the efficacy of such measures. We used 14 months of data from the five robotic centers in Tuscany. Above all, the physician's personal motivation to improve the treatment of patients, the teamwork, and the possibility to use data for research purposes proved to be the essential factors for their engagement and the successful implementation of patient reported measures. Physicians play a key role in the adoption of systematic PROMs and PREMs. The higher their level of engagement, the higher the collection success, both in terms of number of patients enrolled and response rates. Moreover, the collection of patient reported measures may become part of physicians' daily practice and may lead to a change in their relationship and communication with patients, as clinicians accept to have their job reviewed and are not afraid to be evaluated by their patients.

Objective(s) Robotic lobectomy is becoming a widespread surgical procedure in NSCLC treatment, but data on oncologic outcomes is still lacking. The aim of this study was to analyze long term oncologic results of robotic lobectomy for non small lung cancer. Methods Clinical records of consecutive NSCLC patients underwent robotic major surgery, between January 2010 and December 2015, were collected and analyzed. Results We analyzed data of 212 patients (127 male and 85 female), with a median age of 66.3 years. The median follow-up time was 40.3 months (range 4–83). The median disease free survival was 66.3 months. Free disease survival stage-correlated was 75.6 months for stage I, 42.3 months for stage II, 51.2 months for stage III and 10.3 months for stage IV. The median overall survival was 78.6 months. Overall survival stage-correlated was 82 months for stage I, 73.5 months for stage II, 61.4 months for stage III and 41.3 months for stage IV. Conclusions This study suggests high safety level, positive post-operative and oncologic outcomes for patients NSCLC underwent robotic major surgery, also in advanced stages.

The original version of this article, unfortunately, contained an error. In Fig. 2 - panel d, incorrect image was published and this is now presented correctly in this article.

The human P2X7 receptor is significant and exhibits several functions in neoplasia. At present, little is known with regard to its regulation. P2X7 expression may be regulated post-transcriptionally and putative microRNA (miRNA) binding sites are considered to be involved. The aim of this study was to determine whether miRNAs (miR-21, let-7 g and miR-205) regulate P2X7 mRNA stability. In addition, the impact of P2X7 expression in patients with non-small cell lung cancer (NSCLC) was investigated. P2X7 mRNA and mature Let-7 g, miR-21, and miR-205 expression levels were quantified in 96 NSCLC cases using quantitative reverse transcription polymerase chain reaction. In all samples, epidermal growth factor receptor and K-Ras mutational analysis was also performed. Samples with low P2X7 expression were found to exhibit a higher fold change in miR-21 expression when compared with samples exhibiting high P2X7 expression. Significantly higher miR-21 expression was observed in the tumors of NSCLC patients with a K-Ras mutation when compared with patients who had K-Ras wild-type tumors (P=0.003). Additionally, to evaluate the association between P2X7 expression and prognosis in NSCLC patients, survival analysis was performed using the Kaplan-Meier method. A significant difference in the progression-free survival and overall survival in the NSCLC patients with high P2X7 expression was identified, when compared with that of patients with low expression (P=0.03 and P=0.02, respetively). Therefore, we hypothesized that high levels of miR-21 expression in NSCLC patients with K-Ras mutations may be regulated by a complex circuit, including P2X7 downregulation and together these processes may promote tumor progression.

The CDK9-related pathway is an important regulator of mammalian cell biology and is also involved in the replication cycle of several viruses, including the human immunodeficiency virus type 1. CDK9 is present in two isoforms termed CDK9-42 and CDK9-55 that bind noncovalently type T cyclins and cyclin K. This association forms a heterodimer, where CDK9 carries the enzymatic site and the cyclin partner functions as a regulatory subunit. This heterodimer is the main component of the positive transcription elongation factor b, which stabilizes RNA elongation via phosphorylation of the RNA pol II carboxyl terminal domain. Abnormal activities in the CDK9-related pathway were observed in human malignancies and cardiac hypertrophies. Thus, the elucidation of the CDK9 pathway deregulations may provide useful insights into the pathogenesis and progression of human malignancies, cardiac hypertrophy, AIDS and other viral-related maladies. These studies may lead to the improvement of kinase inhibitors for the treatment of the previously mentioned pathological conditions. This review describes the CDK9-related pathway deregulations in malignancies and the development of kinase inhibitors in cancer therapy, which can be classified into three categories: antagonists that block the ATP binding site of the catalytic domain, allosteric inhibitors, and small molecules that disrupt protein-protein interactions.

According to National Comprehensive Cancer Network guidelines concerning non–small cell lung cancer, robotic lobectomy is considered an advisable oncologic procedure for patients with lung cancer, with no anatomic or surgical contraindications. At present, da Vinci is the only available robotic system (Intuitive Surgical, Inc., Sunnyvale, CA), with 2 platforms (da Vinci SI and da Vinci XI) requiring different port-mapping and cart positioning. Robotic left lower lobectomy can be considered one of the most technically simple of all lobectomies. We describe our endoscopic technique, with 4 surgical ports, using CO2 insufflation.

Integrating gene delivery systems allow for a more stable transgene expression in mammalian cells than the episomal ones. However, the integration of the shuttle vector within the cellular chromosomal DNA is associated with the risk of insertional mutagenesis, which, in turn, may cause malignant cell transformation. The use of a retroviral-derived vector system was responsible for the development of leukemia in five children, who participated in various clinical trials for the treatment of severe combined immunodeficiency (SCID-X1) in France and in the United Kingdom. Unfortunately, the hematological malignancy claimed the life of one patient in 2004, who was enrolled in the French clinical trial. In addition, adeno-associated-viral-(AAV-) mediated gene transfer induced tumors in animal models, whereas the Sleeping Beauty (SB) DNA transposon system was associated with insertional mutagenesis events in cell culture systems. On these grounds, it is necessary to develop safer gene delivery systems for the genetic manipulation of mammalian cells. This paper discusses the latest achievements that have been reported in the field of vector design.

Background: The incidental detection of solitary pulmonary nodule (SPN) is currently increasing due to the widespread use of computed tomography (CT) during the follow up in oncological patients or in screening trials. A quick and definitive histological diagnosis of these nodes is mandatory as, in case of primitive lung cancer, an early detection could improve both surgical results and prognosis. The minimally invasive pulmonary resection (MIPR) is the gold standard procedure for diagnosis and treatment of small lung nodules, but it can be difficult to localize deep nonpalpable nodes that lie in the lung parenchyma. Hence, throughout the years several techniques have been developed to better localize deep or sub solid nodes. We describe our experience with radio-guided technique. Methods: Patients with SPN smaller than 10 mm and/or with a distance from the visceral pleura equal or larger than 10 mm underwent MIPR after CT injection of a solution containing 0.2 mL 99 Tc-labelled human serum albumin microspheres and 0.1 mL of non-ionic contrast. During surgical procedures, a collimated probe, connected to a gamma-ray detector, was used to scan the lung and detect the target area. The area of major radioactivity was then resected. Results: Between 2010 and 2015, 175 patients (M/F:97/78) underwent minimally invasive resection (thoracoscopic or robotic) with radio-guided technique. The mean node diameter was 13 mm (range, 5–20 mm), and the mean distance from the visceral pleura was 15 mm (range, 6–39 mm). No significant CT-guided-injection-related complications occurred, except for 13 patients (7.4%) who developed a pneumothorax (PNX) not requiring chest tube insertion. This technique guaranteed a successfully intra-operative node localization in 100% of cases. The mean duration of the surgical procedure was 44 min (range, 25–130 min). The mean length of pleural drainage and mean hospital stay was 2.6 days (range, 1–5 days) and 3.9 days (range, 1–7 days) respectively. No mortality or perioperative complications occurred. Pathology reports showed 105 metastases, 55 primitive lung cancers and 15 benign lesions. In case of suspicion of primitive lung tumour an intraoperative frozen exam was conducted, and all patients underwent lobectomy or segmentectomy with lymphadenectomy [41 patients with thoracotomy, 11 with robotic surgery and 3 with video-assisted thoracoscopic surgery (VATS)]. Conclusions: Our experience confirms that radio-guided thoracoscopic surgery (RGTS) is a feasible and safe procedure, thanks to its high success rate the radio-guided technique is helpful to overcome the lack of tactile feedback during MIPR [VATS and robotic-assisted thoracoscopic surgery (RATS)] and to limit conversion to open surgery.

Ochratoxin A (OTA) is a natural mycotoxin, involved in the development of important human and animal diseases. In this work we have studied the role of oxidative stress in the development of OTA nephrotoxicity and the effect of a new recombinant mitochondrial manganese containing superoxide dismutase (rMnSOD) to prevent kidney damage induced by OTA. Blood pressure, glomerular filtration rate and renal histology were analyzed in control rats and in OTA treated rats. In addition, lipid peroxidation, catalase and superoxide dismutase productions were measured. Our data showed that animals treated with OTA presented hypertension and reduction of glomerular filtration rate (GFR). These effects are most probably related to an increase in the reactive oxygen species (ROS) productions. In fact, we have shown that treatment with rMnSOD restored the levels of blood pressure and GFR simultaneously. Moreover, we have noted that OTA induced alteration on glomerular and tubular degeneration and interstitial infiltrates and that use of rMnSOD combined with OTA prevent this renal histological damage confirming the potential therapeutic role in the treatment of rMnSOD OTA nephrotoxicity. Ochratoxin A (OTA) is a natural mycotoxin, involved in the development of important human and animal diseases. In this work we have studied the role of oxidative stress in the development of OTA nephrotoxicity and the effect of a new recombinant mitochondrial manganese containing superoxide dismutase (rMnSOD) to prevent kidney damage induced by OTA. Blood pressure, glomerular filtration rate and renal histology were analyzed in control rats and in OTA treated rats. In addition, lipid peroxidation, catalase and superoxide dismutase productions were measured. Our data showed that animals treated with OTA presented hypertension and reduction of glomerular filtration rate (GFR). These effects are most probably related to an increase in the reactive oxygen species (ROS) productions. In fact, we have shown that treatment with rMnSOD restored the levels of blood pressure and GFR simultaneously. Moreover, we have noted that OTA induced alteration on glomerular and tubular degeneration and interstitial infiltrates and that use of rMnSOD combined with OTA prevent this renal histological damage confirming the potential therapeutic role in the treatment of rMnSOD OTA nephrotoxicity.

The Id family of helix-loop-helix proteins is known to be involved in the proliferation and differentiation of several types of cells. The type 1 IGF receptor (IGF-IR) induces either proliferation or differentiation in 32D cells, a murine hemopoietic cell line, depending on the availability of the appropriate substrates for the receptor. We have previously reported that the IGF-IR regulates the expression of the Id2 gene in 32D cells. We now show that the IGF-IR controls the increase in Id2 gene expression through at least three pathways. These three pathways originate from the tyrosine residue at 950, a domain in the C-terminus, and the activation of the insulin receptor substrate-1 (IRS-1) by the receptor. IRS-1 is the preponderant signal, and its effect on Id2 gene expression requires a functional phosphotyrosine binding domain. With wild-type IRS-1, Id2 gene expression is increased, even in those cells that express IGF-I receptors defective in Id2 signaling. Rapamycin, an inhibitor of p70(S6K), a downstream effector of IRS-1 signaling, partially inhibits (but does not completely abrogate) the increase in Id2 gene expression. A mutant IRS-1 with a deletion of the Pleckstrin domain is as effective as wild-type IRS-1 in up-regulating Id2 gene expression. In addition, it seems to increase the stability of p70(S6K). Our results indicate that the IGF-IR regulates Id2 gene expression through different pathways. At least in 32D cells, increased Id2 gene expression seems to correlate more with inhibition of differentiation than with proliferation.

H19-7/IGF-IR cells are rat hippocampal cells expressing a human IGF-I receptor, which differentiate to a neuronal phenotype when stimulated by IGF-I at 39 degrees C. H19-7/IGF-IR cells have low levels of expression of insulin receptor substrate-l (IRS-1), a major substrate of the IGF-IR. IGF-I induces serine-phosphorylation and down-regulation of the endogenous IRS-1 upon differentiation of H19-7/IGF-IR cells. The profound influence of IRS-1 on differentiation of H19-7/IGF-IR cells was confirmed by transfecting these cells with a plasmid expressing mouse IRS-1. Over-expression of wild type IRS-1 in H19-7/IGF-IR cells abolishes IGF-I-induced differentiation at 39 degrees C. A mutant of IRS-1 lacking the PTB domain loses the ability to inhibit the differentiation program. H19-7/IGF-IR/IRS-1 cells at 39 degrees C show a stronger and prolonged activation of Akt, when compared to H19-7/IGF-IR cells. The role of Akt in the inhibition of the differentiation program was confirmed by using the inhibitor of Class I PI3 kinases LY29400, which restores IGF-I-induced differentiation of H19-7/IGF-IR/IRS-1 cells. H19-7/IGF-IR/IRS-1 cells show a strong reduction in MAP kinases signaling, which is related to the superactivation of Akt. This was confirmed by expressing in H19-7/IGF-IR cells a constitutively active Akt, which inhibited MAP kinases activation in these cells. These experiments confirm the importance of MAPK in the mechanism of IGF-I-mediated differentiation of H19-7/IGF-IR cells

LNCaP cells are human prostatic cancer cells that have a frame-shift mutation of the tumor suppressor gene PTEN and do not express the insulin receptor substrate1 (IRS-1), a major substrate of the type 1 insulin-like growth factor receptor (IGF-IR). Ectopic expression of IRS-1 in LNCaP cells increases cell adhesion and decreases cell motility by an IGF-I-independent mechanism. We show now that these eAects of IRS-1 are accompanied by serine phosphorylation of IRS-1 and are inhibited by inhibitors of phosphatidylinositol 3-kinase (PI3K). We have confirmed the requirement for PI3K activity and serine phosphorylation by the use of IRS-1 mutants, expressed in LNCaP cells. Serine phosphorylation inhibits IGF-I-induced tyrosyl phosphorylation of IRS-1, which is restored by the expression of wild-type PTEN or by inhibition of PI3K activity. Finally, IRS-1 in LNCaP cells co-immunoprecipitates with integrin a 5 b 1, and the association is again IGF-I-independent. We conclude that in LNCaP cells, IRS-1 is serine phosphorylated by PI3K, generating eAects that are diAerent, and even opposite, from those generated by IGF-I. Oncogene (2001) 20, 490‐500.

We present a new inflatable self-locking intramedullary nailing system for the treatment of long bone fractures. The features of this system and the advantages of the technique include: fixation along the entire length of the nail, lack of proximal or distal interlocking screws, optional reaming, reduced exposure of the surgeon to X-ray and reduced operating room time. First preliminary clinical and radiographic results are reported. Out of 41 patients who received implants for closed fractures, 29 were available for follow-up. We observed fracture consolidation in all 29 cases (100%), with absence of nonunions, infections or rotational malalignments. Only in 2 (6.9%) cases were complication reported, consisting in the opening of the fracture after inflation of the nail. Good stability of the nail, however, assured a complete callus formation. We conclude that the use of this intramedullary nail is an innovative, effective, simple and minimally invasive treatment for long bone shaft fractures.

LNCaP prostatic cancer cells are characterized by having a PTEN mutation, low levels of type 1 insulin-like growth factor receptor (IGF-IR) and no IRS-1, one of the major substrates of the IGF-IR. The absence of IRS-1, an activator of PI3-kinase, is compensated in these cells by the mutation in PTEN, an inhibitor of PI3-kinase. However, IGF-IR signaling in the absence of IRS-1 can cause cell differentiation and growth arrest. We hypothesized that these three characteristics may not be unrelated, specifically that, together, they may favor the metastatic spread of prostatic cancer cells without decreasing their growth potential. In support of this hypothesis, we report here that: (1) IRS-1 expression increases cell adhesion and decreases cell motility; (2) over-expression of the IGF-IR, in the absence of IRS-1, causes growth arrest and (3) a combination of IGF-IR and IRS-1 restores the transformed phenotype of LNCaP cells. These findings suggest a mechanism by which prostatic cancer cells can achieve metastatic potential without interfering with their growth potential. Oncogene (2000).

Over the last decade, more than 300 phase I and phase II gene-based clinical trials have been conducted worldwide for the treatment of cancer and monogenic disorders. Lately, these trials have been extended to the treatment of AIDS and, to a lesser extent, cardiovascular diseases. There are 27 currently active gene therapy protocols for the treatment of HIV-1 infection in the USA. Preclinical studies are currently in progress to evaluate the possibility of increasing the number of gene therapy clinical trials for cardiopathies, and of beginning new gene therapy programs for neurologic illnesses, autoimmuno diseases, allergies, regeneration of tissues, and to implement procedures of allogeneic tissues or cell transplantation. In addition, gene transfer technology has allowed for the development of innovative vaccine design, known as genetic immunization. This technique has already been applied in the AIDS vaccine programs in the USA. These programs aim to confer protective immunity against HIV-1 transmission to individuals who are at risk of infection. Research programs have also been considered to develop therapeutic vaccines for patients with AIDS and generate either preventive or therapeutic vaccines against malaria, tuberculosis, hepatitis A, B and C viruses, influenza virus, La Crosse virus, and Ebola virus. The potential therapeutic applications of gene transfer technology are enormous. However, the effectiveness of gene therapy programs is still questioned. Furthermore, there is growing concern over the matter of safety of gene delivery and controversy has arisen over the proposal to begin in utero gene therapy clinical trials for the treatment of inherited genetic disorders. From this standpoint, despite the latest significant achievements reported in vector design, it is not possible to predict to what extent gene therapeutic interventions will be effective in patients, and in what time frame.

The clinical efficacy of therapeutic complement (C)-activating monoclonal antibodies (mAb) to melanoma-associated antigens can be impaired by the levels of expression of C-inhibitory molecules on neoplastic cells. Protectin (CD59) is a glycosylphosphatidylinositol (GPI)-anchored cell membrane glycoprotein, acting as terminal regulator of C cascade, which is heterogeneously expressed in melanomas and represents the main restriction factor of C-mediated lysis of melanoma cells. Thus, we investigated whether the overexpression of CD59 could influence the constitutive susceptibility of distinct melanoma cells to homologous C. Infection of CD59-positive Mel 100 and 70-W melanoma cells by a retroviral vector carrying the CD59 cDNA, significantly (P < 0.05) upregulated their constitutive expression of CD59, whereas it did not affect that of additional C-regulatory molecules. Transduced CD59 was entirely GPI-anchored and showed a molecular weight identical to native CD59. Additionally, higher amounts of soluble CD59 were detected in the conditioned media of CD59-transduced melanoma cells compared with parental cells. CD59-transduced melanoma cells, sensitized by the anti-GD3 disialoganglioside mAb R24, were significantly (P < 0.05) less susceptible to homologous C-lysis than were parental cells; this effect was fully reverted by the masking of CD59 with F(ab′)2 fragments of the anti-CD59 mAb YTH53.1. These results provide conclusive evidence demonstrating that absolute levels of CD59 expression regulate the susceptibility to homologous C of specific melanoma cells, and suggest an additional explanation for the poor clinical results obtained with C-activating mAb in the clinical setting. J. Cell. Physiol. 185:317–323, 2000. © 2000 Wiley-Liss, Inc.

We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium butyrate to further increase expression of each component, we achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components. This system can be used both for the generation of high titer retroviral stocks for transduction and for the rapid screening of a large number of MLV gag-pol or env mutants.

Cdk9 and Cdk7 are cdc2-like serine/threonine kinases that stabilize RNA transcript elongation through RNA polII carboxyl terminal domain (CTD) phosphorylation and are considered suitable targets for cancer therapy. The effects of flavopiridol and of siRNA-mediated inhibition of Cdk9 and/or Cdk7 were analyzed in human glioblastoma and human prostate cancer cell lines. One finding revealed that Cdk9 and Cdk7 could substitute each other in RNA polII CTD phosphorylation in contrast to the in vitro system. Thus, a simultaneous inhibition of Cdk9 and Cdk7 might be required both for targeting malignant cells and developing a platform for microarray analysis. However, these two pathways are not redundant, as indicated by differential effects observed in cell cycle regulation following siRNA-mediated inhibition of Cdk9 and/or Cdk7 in human PC3 prostate cancer cell line. Specifically, siRNA-mediated inhibition of Cdk9 caused a shift from G 0/G 1 to G 2/M phase in human PC3 prostate cancer cell line. Another finding showed that flavopiridol treatment induced a substantial AKT-Ser473 phosphorylation in human glioblastoma T98G cell line in contrast to siRNA-mediated inhibition of Cdk9 and Cdk9 combined with Cdk7, whereas siRNA-mediated silencing of Cdk7 caused a minor increase in AKT-Ser473 phosphorylation. AKT-Ser473 is a hallmark of AKT pathway activation and may protect cells from apoptosis. This finding also shows that Cdk9 and Cdk7 pathways are not redundant and may have important implications in drug development and for studying the mechanism of chemoresistance in malignant cells.

Cyclin-dependent kinase 9 (Cdk9) is a cdc2-like serine/threonine kinase. The so-called Cdk9-related pathway comprises two Cdk9 isoforms (Cdk9-42 and Cdk9-55), cyclin T1, cyclin T2a, cyclin T2b and cyclin K. The association between Cdk9 and one of its cyclin partners forms a heterodimer, which is the main component of the positive transcription elongation factor (P-TEFb). The latter stabilizes the elongation process of RNA polymerase II (polII) transcripts. Through the control of RNA polII-mediated gene expression, the Cdk9-related pathway performs an important role in several biological processes, such as cell growth, proliferation, protection from apoptosis and differentiation. Incidentally, the P-TEFb that contains the heterodimer Cdk9-cyclin T1 is also critical for HIV-1 and HIV-2 replication in human cells. A deregulation in the Cdk9-related pathway is associated with various types of human malignancies and cardiomyocytes hypertrophy. On these grounds, the characterization of Cdk9-related pathway deregulation might have a two-fold purpose: (1) the development of novel kinase inhibitors for the treatment of cancer, AIDS and cardiac hypertrophy and (2) a better understanding of the pathogenesis and progression of these maladies.

The hematopoietic system consists of: (1) a network of stem and progenitor cells of varying degrees of maturity interacting with other cells that possess supportive and regulatory capacities and (2) vascular stem cell niches with supporting stem cells in their self-renewal, proliferation, differentiation, and mobilization to the circulation. Recent data suggest that selective expression of organ-specific chemokines promotes the mobilization of bone-marrow-derived pluripotent cells, a process that is essential for tissue vascularization and organ regeneration. Despite intensive investigation, the pathways by which mechanical signals are converted to biochemical responses are not completely understood. Recent studies have suggested that chromatin shifts and cell cycle effects stem cell gene expression, and thus results in changes of its surface receptor expression at different points of the cell cycle machinery, therefore changing cell cycle transit. This review will attempt to discuss new approaches to determine the regulation of stem cell growth and differentiation by underlying the significance of the chaotic dynamics of transcriptional networks within a cell, with a combination of chemokines and cytokines in the environment, and mechanical forces, such as; stretch, strain and laminar flow, all involving both cooperation and competition. J. Cell. Physiol. 213:672–678. © 2007 Wiley-Liss, Inc.

The transcriptional and chromatin profile of the promoter, first exon and first intron of the human TH gene were analyzed in human neuroblastoma BE(2)-C-16 and human renal carcinoma 293FT cell lines. The latter is a cell culture system that is not permissive for TH gene expression, whereas the former has a 50% cell fraction that tests positive for TH. The engineering of a 6.3 kb recombinant human TH promoter revealed the presence of repressors of transcription between positions (-6,244/-194). The addition of a 1.2 kb fragment of the first intron of the human TH gene (+730/+1,653) enhanced transcriptional activity of the recombinant promoter. However, both constructs were not specific for TH-positive BE(2)-C-16 cells. Chromatin immunoprecipitation (Chip) analysis was carried out on BE(2)-C-16 and 293FT cells to probe sequences of promoter, first exon and first intron of the human TH gene from position (-448/+1,204). The presence of nucleosomes was observed approximately from position (-20/+473) in both cell lines. Chip analysis was then conducted to determine the acetylation of various lysine residues of H3 and H4 in both cell lines. All analyzed lysine residues of H3 and H4 were acetylated in BE(2)-C-16 cells, whereas 293FT cells tested positive for acetylation only in the external lysine residues of the histone tail. Our data are compatible with an active TH gene expression in a 50% cell fraction of BE(2)-C-16 cells. Further analysis of epigenetic programming might lead to the identification of the factors that determine TH gene expression specifically in dopaminergic neurons.

Stem cell research has greatly contributed to the field of oncology with the identification and isolation of cancer stem cells from a variety of tumors. The discovery of rare subpopulations of cancer stem cells has indeed entirely changed the focus of cancer research. Normal adult stem cells and cancer stem cells can both self-renew and undergo a differentiation program that, in turn, gives rise to a high number of differentiated cells. Adult stem cells and their malignant counterparts share almost all of the same intrinsic and extrinsic factors to regulate self-renewal, differentiation and proliferation pathways. Fractions of normal and cancer stem cells are naturally more resistant to toxic injuries than any other cell type. Overall, these observations lead to the conclusion that adult stem or progenitor cells can eventually become malignant by generating cancer stem cells, which are responsible for the development and maintenance of the tumor mass. In addition, chemo-resistant cancer stem cells may cause the relapse of the disease following an apparent beneficial treatment. Indeed, the study of the biology of cancer stem cells might lead to the improvement of preventive cancer diagnosis and to the development of novel therapeutics, which must be designed to selectively target malignant stem cells without affecting normal adult stem cells.

A previous study on the human tyrosine hydroxylase (TH) promoter revealed remarkable differences in the mechanism of TH gene regulation between the human and murine models. Indeed, a low degree of homology was observed in the sequence of TH promoters among human, mouse, and rat systems. Only five short conserved regions (CRs) could be identified among the three species. A human TH minimal promoter was engineered and assembled into a self-inactivating lentiviral vector system. This human TH minimal promoter contained the five CRs plus the first -194 bp from the transcription start of the human TH promoter and the first 35 bp of the untranslated messenger RNA leader of the human TH gene. A significant degree of specificity for this human TH minimal promoter was observed only for human neuronal progenitor cells (hNPCs), but not for TH-positive differentiated mouse primary striatal and substantia nigra cells, indicating a significant difference in TH gene regulation between the human and mouse systems. Not only is the degree of homology between the human and mouse promoters in the range of only 46%, but also those few elements that share a high degree of homology display totally different functions in human and mouse brain-derived cells. In the rodent system, NR4A2 (Nurr1) is required for the transactivation of TH minimal promoters. Intriguingly, neither the dimeric nor the heterodimeric binding sites for Nurr1 are present in the 13 kb DNA sequence that contains the human TH promoter. Instead, the CRs termed one and four of the human TH promoter encode only for a half palindromic binding site sequence for Nurr1, which failed to bind Nurr1 in an in vitro electrophoretic mobility shift assay (EMSA). Additionally, of the three monomeric NGFI-B response element (NBRE) core sites (AGGTCA) and two NBRE-related sites present in the human TH promoter, only one core and two NBRE-related sites formed protein binding complexes. Interestingly, there was no increase of protein binding complex formation upon TH induction and in no case could antibodies supershift Nurr1 from the complex. These findings, taken together, demonstrate that NBRE-related binding sites for Nurr1 do not play a direct role in mediating an interaction between Nurr1 and the human TH promoter. Likewise, immunohistochemical and Western blot analysis have also confirmed that both endogenous and exogenous Nurr1 expression does not positively correlate with TH gene expression in hNPCs, in contrast to the mouse model. In addition, real-time PCR analysis revealed that the downregulation of human Nurr1 gene expression mediated by silencing RNA molecules did not affect human TH gene expression in differentiated hNPCs. A better understanding of human TH gene regulation may have important implications both for the development of novel therapeutic approaches and the study of the pathogenesis of a variety of neurological illnesses, including Parkinson's disease, bipolar disorder, and schizophrenia.