A Contemporary Review of the Clubhouse Model of Psychosocial Rehabilitation: Past, Present, and Emerging Directions.

Since the 1940s, the Clubhouse model of psychosocial rehabilitation has evolved towards a comprehensive practice of social theory and intervention. Despite the model's cost effectiveness and observational evidence of its efficacy, empirical research remains lacking. The current narrative review examines studies from recent years (2015-2021), not to assess study rigor, but to identify trends in research aims, findings, and methodology, as well as specify future research directions. A narrative review was conducted using PRISMA guidelines. Using the search term "Clubhouse," 194 articles were identified in online databases. 38 met criteria for inclusion. Most studies were qualitative (60.5%) and few utilized experimental or quasi-experimental designs (7.9%). Narrative synthesis revealed research aims and outcome variables falling into six key areas: social integration and connectedness, quality of life (QOL), recovery outcomes, relational dynamics, policy, and virtual adaptations of the model. Findings indicate that recent Clubhouse-related research trends have primarily involved studies of social connectedness, QOL, recovery, relationships, and policy, as well as studies examining the value of the virtual Clubhouse in maintaining well-being. However, heterogeneity of methodologies and measures present a critical limitation to assessing results across studies. Options for increasing experimental methodologies in this area are reviewed. Recommended future directions involve moving towards a biopsychosocial approach to clarifying the mechanisms through which the model promotes recovery-aims that may yield implications beyond the realm of serious mental illness.

Gene transfection of primary mouse hepatocytes in 384-well plates.

We report the development of an improved in vitro transfection assay to test the efficiency of non-viral vector DNA nanoparticle transfection of primary hepatocytes. The protocol describes the isolation of viable hepatocytes from a mouse by collagenous perfusion. Primary mouse hepatocytes are plated in 384-well plates and cultured for 24 h prior to transfection with polyethylenimine (PEI) or peptide DNA nanoparticles. Luciferase expression is measured after 24 h following the addition of ONE-Glo substrate. The gene transfer assay for primary hepatocytes was optimized for cell plating number, DNA dose, and PEI to DNA ratio. The assay was applied to compare the expression mediated by mRNA relative to two plasmids possessing different promoters. The reported assay provides reliable in vitro expression results that allow direct comparison of the efficiency of different non-viral gene delivery vectors.

Fluorescent labeling of plasmid DNA for gene delivery: Implications of dye hydrophobicity on labeling efficiencies and nanoparticle size.

Covalent fluorescent labels are important tools for monitoring the in vitro and in vivo localization of plasmid DNA nanoparticles, but must meet several criteria including high DNA labeling efficiencies and minimal impact on nanoparticle size. We developed a novel fluorescent labeling strategy utilizing an aryl azide photolabel conjugated to a short cationic peptide to label plasmid DNA with Cyanine 5 and sulfo-Cyanine 5. Using a simple camera flash apparatus, photolabel-peptide-dyes can be conjugated to DNA in minutes with preservation of DNA structure and minimal dye photobleaching. The addition of two anionic sulfonates to the Cyanine 5 core greatly improved labeling efficiencies from ~13 to ~53% and mitigated PEGylated polyacridine peptide-DNA nanoparticle size increases over a range of labeling densities. Comparison of our sulfo-Cyanine 5 peptide label to the Mirus Bio Label IT-Cy5 kit revealed that while both did not affect nanoparticle sizes appreciably, labeling efficiencies with our conjugate were higher, possibly due to the higher positive charge density on the peptide linker. The results from this work provide important considerations for choosing fluorophore tags to track DNA nanoparticles.

The reduced-charge melittin analogue MelP5 improves the transfection of non-viral DNA nanoparticles.

Melittin is a 26-amino acid amphiphilic alpha-helical peptide derived from honeybee venom. Prior studies have incorporated melittin into non-viral delivery systems to effect endosomal escape of DNA nanoparticles and improve transfection efficiency. Recent advances have led to the development of two newer melittin analogues, MelP5 and Macrolittin 70, with improved pore formation in lipid bilayers while possessing fewer positive charges relative to natural melittin. Consequently, MelP5 and Macrolittin 70 were conjugated through a disulfide bond to a DNA binding polyacridine peptide. The resulting peptide conjugates were used to prepare DNA nanoparticles to compare their relative endosomolytic potency by transfection of HepG2 cells. Melittin and MelP5 conjugates were equally potent at mediating in vitro gene transfer, whereas PEGylation of DNA nanoparticles revealed improved transfection with MelP5 relative to melittin. The results demonstrate the ability to substitute a potent, reduced-charge analogue of melittin to improve overall DNA nanoparticle biocompatibility needed for in vivo testing.

Jail-based competency treatment comes of age: Multi-site outcomes and challenges to the implementation of an evidence-based forensic continuum.

The jail-based competency treatment (JBCT) model has become an established forensic practice across the country. From the perspective of implementation science and the three core elements of the Promoting Action on Research Implementation in Health Service (PARiHS) framework, the JBCT model is a remarkable example of how context (an unrelenting and overwhelmingly strong demand for forensic beds) has driven multiple state governments to facilitate implementation of a methodology in the absence of empirical evidence supporting its efficacy. This 7-year study of outcomes from four JBCT program sites provides this much-needed evidence by showing that JBCT restored 56% of 1553 male and 336 female patients over an average of 48.7 days. At the same time, the study highlights how variations in JBCT models, methods, and preadmission stabilization time present challenges to planned and effective implementation of evidence-based practice at the statewide system level. By identifying differential responsiveness to JBCT treatment by diagnosis and other factors, the study suggests preliminary implementation ideas for what types of patients are well served by the JBCT model as part of a continuum of restoration options that includes inpatient, outpatient and diversion. Significant findings showed that JBCT patients were restored at a higher rate and in a shorter time if they were female, < 20 years old (highest restoration rate; those < 60 years old also significantly better rates), free of co-occurring intellectual and cognitive deficits, and malingering. Of the major diagnoses, schizoaffective disorder required a significantly longer length of JBCT treatment for restoration, and lower restoration rates than schizophrenia and bipolar disorder, although this was moderated by a significant interaction with abuse of amphetamines.

Why We Come: Clubhouse Members Seek Connection, Purpose and Meaning.

Approximately 4.5% of U.S. adults live with a serious mental illness (SMI) (Substance Abuse and Mental Health Services Administration, 2018). Creating a place for people seeking structure, connection, and purpose following psychiatric hospitalization or for mental health recovery is difficult given several factors associated with illness trajectories, lack of community-based support programs, and reliance on traditional models of care. Using semi-structured interviews, the current study examined the reasons people attend community programs referred to as psychosocial "clubhouses." Interviews with 140 people across 10 clubhouse programs in one state were conducted. Qualitative analyses revealed that social connections and the need to reduce social isolation were driving forces for attending. Further, individuals noted that the "structure" of engaging in meaningful activities and roles was a main reason for participating. Authors discuss the continued need for community models that provide a "place" for all people to successfully engage and recover from psychiatric illnesses.

Heat-shrinking DNA nanoparticles for in vivo gene delivery.

The particle size of a PEG-peptide DNA nanoparticle is a key determinant of biodistribution following i.v. dosing. DNA nanoparticles of <100 nm in diameter are sufficiently small to cross through fenestrated endothelial cells to target hepatocytes in the liver. In addition, DNA nanoparticles must be close to charge-neutral to avoid recognition and binding to scavenger receptors found on Kupffer cells and endothelial cells in the liver. In the present study, we demonstrate an approach to heat shrink DNA nanoparticles to reduce their size to <100 nm to target hepatocytes. An optimized protocol heated plasmid DNA at 100 °C for 10 min resulting in partial denaturation. The immediate addition of a polyacridine PEG-peptide followed by cooling to room temperature resulted in heat-shrunken DNA nanoparticles that were ~70 nm in diameter compared with 170 nm when heating was omitted. Heat shrinking resulted in the conversion of supercoiled DNA into open circular to remove strain during compaction. Heat-shrunken DNA nanoparticles were stable to freeze-drying and reconstitution in saline. Hydrodynamic dosing established that 70 nm heat-shrunken DNA nanoparticles efficiently expressed luciferase in mouse liver. Biodistribution studies revealed that 70 nm DNA nanoparticles are rapidly and transiently taken up by liver whereas 170 nm DNA nanoparticles avoid liver uptake due to their larger size. The results provide a new approach to decrease the size of polyacridine PEG-peptide DNA nanoparticles to allow penetration of the fenestrated endothelium of the liver for the purpose of transfecting hepatocytes in vivo.

A review of the antimicrobial potential of precious metal derived nanoparticle constructs.

The field of nanotechnology is rapidly growing. The promise of pharmacotherapeutics emerging from this vast field has drawn the attention of many researchers. However, with the increase in the prevalence of antibiotic resistant microorganisms, the manifestations of these promises are needed now more than ever. Many have postulated the antimicrobial potential of nanoparticle constructs derived from precious metals/noble metals nanoparticles (NMNPs), such as silver nanoparticles that show activity against multidrug resistant bacteria. In this review we will evaluate the current studies and explore the data to obtain a clear picture of the potential of these particles and the validity of the claims of drug resistant treatments with NMNPs.

A prospective study of health-related quality of life outcomes among men treated for intermediate- and high-risk prostate cancer: the impact of primary and secondary therapies.

INTRODUCTION: To assess the impact of primary and secondary therapies for high- and intermediate-risk prostate cancer on health-related quality of life (HRQoL). MATERIALS AND METHODS: A prospective study was initiated in 2007 at Center for Prostate Disease Research Multicenter National Database sites. Longitudinal patterns in HRQoL from baseline (pre-treatment) to 5 years post-diagnosis were examined for patients with high- and intermediate-risk prostate cancer, treated by radical prostatectomy (RP) or external beam radiation therapy (EBRT). Change in HRQoL was modeled using linear regression models fit with generalized estimating equations. The probability of maintaining HRQoL was compared between patients receiving RP only versus RP with secondary treatment. RESULTS: Of 445 men with high- and intermediate-risk prostate cancer, 228 underwent RP and 143 had EBRT± androgen deprivation therapy (ADT). Fifty received secondary therapy (EBRT and/or ADT or chemotherapy) after RP. RP patients showed a greater decline over time in sexual function and bother and urinary function compared to EBRT±ADT patients. Patients who had secondary therapy after RP were less likely to maintain their HRQoL compared to those who had RP alone. These differences were most pronounced for sexual and hormonal function. CONCLUSIONS: Prostate cancer patients experience significant declines in HRQoL after primary therapy. Additional secondary therapy after RP, in the form of EBRT and/or ADT, appears to be responsible for further deterioration in HRQoL outcomes.

Metabolically stabilized double-stranded mRNA polyplexes.

The metabolic instability of mRNA currently limits its utility for gene therapy. Compared to plasmid DNA, mRNA is significantly more susceptible to digestion by RNase in the circulation following systemic dosing. To increase mRNA metabolic stability, we hybridized a complementary reverse mRNA with forward mRNA to generate double-stranded mRNA (dsmRNA). RNase A digestion of dsmRNA established a 3000-fold improved metabolic stability compared to single-stranded mRNA (ssmRNA). Formulation of a dsmRNA polyplex using a PEG-peptide further improved the stability by 3000-fold. Hydrodynamic dosing and quantitative bioluminescence imaging of luciferase expression in the liver of mice established the potent transfection efficiency of dsmRNA and dsmRNA polyplexes. However, hybridization of the reverse mRNA against the 5' and 3' UTR of forward mRNA resulted in UTR denaturation and a tenfold loss in expression. Repeat dosing of dsmRNA polyplexes produced an equivalent transient expression, suggesting the lack of an immune response in mice. Co-administration of excess uncapped dsmRNA with a dsmRNA polyplex failed to knock down expression, suggesting that dsmRNA is not a Dicer substrate. Maximal circulatory stability was achieved using a fully complementary dsmRNA polyplex. The results established dsmRNA as a novel metabolically stable and transfection-competent form of mRNA.

PEG-Peptide Inhibition of Scavenger Receptor Uptake of Nanoparticles by the Liver.

PEGylated polylysine peptides represent a new class of scavenger receptor inhibitors that may find utility at inhibiting DNA nanoparticle uptake by Kupffer cells in the liver. PEG-peptides inhibit scavenger receptors in the liver by a novel mechanism involving in situ formation of albumin nanoparticles. The present study developed a new in vivo assay used to explore the structure-activity-relationships of PEG-peptides to find potent scavenger receptor inhibitors. Radio-iodinated PEG-peptides were dosed i.v. in mice and shown to saturate liver uptake in a dose-dependent fashion. The inhibition potency (IC50) was dependent on both the length of a polylysine repeat and PEG molecular weight. PEG30kda-Cys-Tyr-Lys25 was confirmed to be a high molecular weight (33.5 kDa) scavenger receptor inhibitor with an IC50 of 18 µM. Incorporation of multiple Leu residues improved potency, allowing a decrease in PEG MW and Lys repeat, resulting in PEG5kda-Cys-Tyr-Lys-(Leu-Lys4)3-Leu-Lys that inhibited scavenger receptors with an IC50 = 20 µM. A further decrease in PEG MW to 2 kDa increased potency further, resulting in a low molecular weight (4403 g/mol) PEG-peptide with an IC50 of 3 µM. Optimized low molecular weight PEG-peptides also demonstrated potency when inhibiting the uptake of radio-iodinated DNA nanoparticles by the liver. This study demonstrates an approach to discover low molecular weight PEG-peptides that serve as potent scavenger receptor inhibitors to block nanoparticle uptake by the liver.

Longitudinal regret after treatment for low- and intermediate-risk prostate cancer.

BACKGROUND: Prostate cancer patients diagnosed with low- and intermediate-risk disease have several treatment options. Decisional regret after treatment is a concern, especially when poor oncologic outcomes or declines in health-related quality of life (HRQoL) occur. This study assessed determinants of longitudinal decisional regret in prostate cancer patients attending a multidisciplinary clinic and treated with radical prostatectomy (RP), external beam radiation therapy (EBRT), brachytherapy (BT), or active surveillance (AS). METHODS: Patients newly diagnosed with prostate cancer at the Walter Reed National Military Medical Center who attended a multidisciplinary clinic were enrolled into a prospective study from 2006 to 2014. The Decision Regret Scale was administered at 6, 12, 24, and 36 months posttreatment. HRQoL was also assessed at regular intervals using the Expanded Prostate Cancer Index Composite and 36-item RAND Medical Outcomes Study Short Form questionnaires. Adjusted probabilities of reporting regret were estimated via multivariable logistic regression fitted with generalized estimating equations. RESULTS: A total of 652 patients met the inclusion criteria (395 RP, 141 EBRT, 41 BT, 75 AS). Decisional regret was consistently low after all of these treatments. In multivariable models, only African American race (odds ratio, 1.67; 95% confidence interval, 1.12-2.47) was associated with greater regret across time. Age and control preference were marginally associated with regret. Regret scores were similar between RP patients who did and did not experience biochemical recurrence. Declines in HRQoL were weakly correlated with greater decisional regret. CONCLUSION: In the context of a multidisciplinary clinic, decisional regret did not differ significantly between treatment groups but was greater in African Americans and those reporting poorer HRQoL. Cancer 2017;123:4252-4258. © 2017 American Cancer Society.

Prophylactic Treatment with Cerium Oxide Nanoparticles Attenuate Hepatic Ischemia Reperfusion Injury in Sprague Dawley Rats.

BACKGROUND: Hepatic ischemia reperfusion is one the main causes for graft failure following transplantation. Although, the molecular events that lead to hepatic failure following ischemia reperfusion (IR) are diverse and complex, previous studies have shown that excessive formation of reactive oxygen species (ROS) are responsible for hepatic IR injury. Cerium oxide (CeO2) nanoparticles have been previously shown to act as an anti-oxidant and anti-inflammatory agent. Here, we evaluated the protective effects of CeO2 nanoparticles on hepatic ischemia reperfusion injury. METHODS: Male Sprague Dawley rats were randomly assigned to one of the four groups: Control, CeO2 nanoparticle only, hepatic ischemia reperfusion (IR) group and hepatic ischemia reperfusion (IR) plus CeO2 nanoparticle group (IR+ CeO2). Partial warm hepatic ischemia was induced in left lateral and median lobes for 1h, followed by 6h of reperfusion. Animals were sacrificed after 6h of reperfusion and blood and tissue samples were collected and processed for various biochemical experiments. RESULTS: Prophylactic treatment with CeO2 nanoparticles (0.5mg/kg i.v (IR+CeO2 group)) 1 hour prior to hepatic ischemia and subsequent reperfusion injury lead to a decrease in serum levels of alanine aminotransaminase and lactate dehydrogenase at 6 hours after reperfusion. These changes were accompanied by significant decrease in hepatocyte necrosis along with reduction in several serum inflammatory markers such as macrophage derived chemokine, macrophage inflammatory protein-2, KC/GRO, myoglobin and plasminogen activator inhibitor-1. However, immunoblotting demonstrated no significant changes in the levels of apoptosis related protein markers such as bax, bcl2 and caspase 3 in IR and IR+ CeO2 groups at 6 hours suggesting necrosis as the main pathway for hepatocyte death. CONCLUSION: Taken together, these data suggest that CeO2 nanoparticles attenuate IR induced cell death and can be used as a prophylactic agent to prevent hepatic injury associated with graft failure.

Treatment and Clinical Outcomes of Patients with Teratoma with Somatic-Type Malignant Transformation: An International Collaboration.

PURPOSE: We assessed prognostic factors, treatments and outcomes in patients with teratoma with malignant transformation, a rare occurrence among germ cell tumors. MATERIALS AND METHODS: Data on patients diagnosed with teratoma with malignant transformation between June 1981 and August 2014 were collected across 5 referral centers. Chemotherapy was dichotomized as based on germ cell tumor or teratoma with malignant transformation. Cox analyses were done to evaluate prognostic factors of overall survival, the primary end point. Each factor was evaluated in a univariable model. Forward stepwise selection was used to construct an optimal model. RESULTS: Among 320 patients the tumor primary site was gonadal in 287 (89.7%), retroperitoneal in 17 (5.3%) and mediastinal in 16 (5%). Teratoma with malignant transformation and germ cell tumor were diagnosed concurrently in 130 patients (40.6%). A total of 49 patients (16.8%) initially presented with clinical stage I. The remaining patients were at good (123 or 42.3%), intermediate (42 or 14.4%) and poor (77 or 26.5%) risk for metastasis according to IGCCCG (International Germ Cell Cancer Collaborative Group). First line chemotherapy was given for germ cell tumor in 159 patients (49.7%), chemotherapy for teratoma with malignant transformation was performed in 14 (4.4%) and only surgery was done in 147 (45.9%). Median followup was 25.1 months (IQR 5.4-63.8). Five-year overall survival was 83.4% (95% CI 61.3 to 93.5) in patients with clinical stage I and it was also worse than expected in those with metastasis. On multivariable analyses nonprimitive neuroectodermal tumor histology (overall p = 0.004), gonadal primary tumor (p = 0.005) and fewer prior chemotherapy regimens (p <0.001) were independent predictors of better overall survival. Chemotherapy was not independently prognostic. CONCLUSIONS: Less heavily pretreated teratoma with malignant transformation with a gonadal primary tumor and nonprimitive neuroectodermal tumor histology appears to be associated with longer overall survival. Generally, teratoma with malignant transformation had a worse prognosis than germ cell tumor. Uncertainties persist regarding optimal chemotherapy.

Climate structures genetic variation across a species' elevation range: a test of range limits hypotheses.

Gene flow may influence the formation of species range limits, and yet little is known about the patterns of gene flow with respect to environmental gradients or proximity to range limits. With rapid environmental change, it is especially important to understand patterns of gene flow to inform conservation efforts. Here we investigate the species range of the selfing, annual plant, Mimulus laciniatus, in the California Sierra Nevada. We assessed genetic variation, gene flow, and population abundance across the entire elevation-based climate range. Contrary to expectations, within-population plant density increased towards both climate limits. Mean genetic diversity of edge populations was equivalent to central populations; however, all edge populations exhibited less genetic diversity than neighbouring interior populations. Genetic differentiation was fairly consistent and moderate among all populations, and no directional signals of contemporary gene flow were detected between central and peripheral elevations. Elevation-driven gene flow (isolation by environment), but not isolation by distance, was found across the species range. These findings were the same towards high- and low-elevation range limits and were inconsistent with two common centre-edge hypotheses invoked for the formation of species range limits: (i) decreasing habitat quality and population size; (ii) swamping gene flow from large, central populations. This pattern demonstrates that climate, but not centre-edge dynamics, is an important range-wide factor structuring M. laciniatus populations. To our knowledge, this is the first empirical study to relate environmental patterns of gene flow to range limits hypotheses. Similar investigations across a wide variety of taxa and life histories are needed.

Outcomes of postchemotherapy retroperitoneal lymph node dissection following high-dose chemotherapy with stem cell transplantation.

BACKGROUND: Characterizing the role of postchemotherapy retroperitoneal lymph node dissection (PC-RPLND) after high-dose chemotherapy (HDCT) has been limited by small sample sizes. This study reports on survival after HDCT with stem cell support and PC-RPLND as well as histologic findings in the retroperitoneum. METHODS: The prospectively maintained testicular cancer database of Indiana University was queried for patients receiving HDCT with stem cell transplantation before PC-RPLND. The cause and date of death were obtained through patient chart review and contact with referring physicians. The Kaplan-Meier method was used to evaluate overall survival (OS). The log-rank test was used for tests of significance. A multivariate, backward, stepwise Cox regression model was built to evaluate predictors of overall mortality. RESULTS: A total of 92 patients were included in the study. In the entire cohort, the retroperitoneal (RP) histology findings at the time of PC-RPLND were necrosis (26%), teratoma (34%), and cancer (38%). Sixty-six patients (72%) harbored either a teratoma or active cancer in the RP specimen at PC-RPLND. The median follow-up for the entire cohort was 80.6 months. A total of 28 patients (30%) died during follow-up. The 5-year OS rate of the entire cohort was 70%. The most significant predictor of death was PC-RPLND performed in the desperation setting with elevated markers. CONCLUSIONS: Despite these patients being heavily pretreated with multiple cycles of chemotherapy, including HDCT, approximately three-fourths were found to have a teratoma and/or active cancer in the retroperitoneum. This underscores the importance of PC-RPLND for rendering patients free of disease and providing a potential for cure.

Therapeutic Potential of Cerium Oxide Nanoparticles for the Treatment of Peritonitis Induced by Polymicrobial Insult in Sprague-Dawley Rats.

OBJECTIVES: Peritonitis is a life-threatening disease that is associated with high mortality. The purpose of this study was to determine if cerium oxide nanoparticles can be used to diminish intra-abdominal infection-induced mortality and systemic inflammatory response syndrome in the laboratory rat. DESIGN: Randomized, controlled animal study and cell culture study. SETTING: University research laboratory. SUBJECTS: Male Sprague-Dawley rats aged 12 weeks, RAW 246.7 macrophage cell line. INTERVENTIONS: Intra-abdominal infection or peritonitis was induced by intraperitoneal injection of cecal material (600 mg/kg in 5% sterile dextrose water at a dosage of 5 mL/kg) obtained from healthy donors. Rats in control and peritonitis groups received 200 µL of sterile deionized water IV via the tail vein, whereas rats in cerium oxide-only group and peritonitis+cerium oxide group received cerium oxide nanoparticles (0.5 mg/kg) IV at the time of polymicrobial injection. Survival rate was monitored for 14 days, while in other experiments, animals were killed at 3 and 18 hours after induction of peritonitis for biochemical analysis. MEASUREMENTS AND MAIN RESULTS: Administration of a single dose (0.5 mg/kg) of cerium oxide nanoparticles IV to rats in the peritonitis group significantly improved survival rates and functioned to restore core body temperature toward baseline. Treatment-induced increases in animal survivability were associated with reduced systemic and hepatic oxidative stress, diminished serum cytokines, and chemokine levels. Changes in serum inflammatory markers with treatment were accompanied by decreased monocyte and lymphocyte extravasation into the peritoneal cavity along with decreased infiltration of macrophages into liver. In the heart, treatment diminished extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase-Stat-3 signaling and attenuated endothelial expression of P-selectin and vascular cell adhesion molecule-1. CONCLUSIONS: Cerium oxide nanoparticles attenuate the systemic inflammatory response associated with peritonitis, suggesting potential use as a novel therapeutic agent for the treatment of severe intra-abdominal infection.

Miniaturization of gene transfection assays in 384- and 1536-well microplates.

The miniaturization of gene transfer assays to either 384- or 1536-well plates greatly economizes the expense and allows much higher throughput when transfecting immortalized and primary cells compared with more conventional 96-well assays. To validate the approach, luciferase and green fluorescent protein (GFP) reporter gene transfer assays were developed to determine the influence of cell seeding number, transfection reagent to DNA ratios, transfection time, DNA dose, and luciferin dose on linearity and sensitivity. HepG2, CHO, and NIH 3T3 cells were transfected with polyethylenimine (PEI)-DNA in both 384- and 1536-well plates. The results established optimal transfection parameters in 384-well plates in a total assay volume of 35µl and in 1536-well plates in a total assay volume of 8µl. A luciferase assay performed in 384-well plates produced a Z' score of 0.53, making it acceptable for high-throughput screening. Primary hepatocytes were harvested from mouse liver and transfected with PEI DNA and calcium phosphate DNA nanoparticles in 384-well plates. Optimal transfection of primary hepatocytes was achieved on as few as 250cellsperwell in 384-well plates, with CaPO4 proving to be 10-fold more potent than PEI.

Structure-Activity Relationship of PEGylated Polylysine Peptides as Scavenger Receptor Inhibitors for Non-Viral Gene Delivery.

PEGylated polylysine peptides of the general structure PEG30 kDa-Cys-Trp-LysN (N = 10 to 30) were used to form fully condensed plasmid DNA (pGL3) polyplexes at a ratio of 1 nmol of peptide per µg of DNA (ranging from N:P 3:1 to 10:1 depending on Lys repeat). Co-administration of 5 to 80 nmols of excess PEG-peptide with fully formed polyplexes inhibited the liver uptake of (125)I-pGL3-polyplexes. The percent inhibition was dependent on the PEG-peptide dose and was saturable, consistent with inhibition of scavenger receptors. The scavenger receptor inhibition potency of PEG-peptides was dependent on the length of the Lys repeat, which increased 10-fold when comparing PEG30 kDa-Cys-Trp-Lys10 (IC50 of 20.2 µM) with PEG30 kDa-Cys-Trp-Lys25 (IC50 of 2.1 µM). We hypothesize that PEG-peptides inhibit scavenger receptors by spontaneously forming small 40 to 60 nm albumin nanoparticles that bind to and saturate the receptor. Scavenger receptor inhibition delayed the metabolism of pGL3-polyplexes, resulting in efficient gene expression in liver hepatocytes following delayed hydrodynamic dosing. PEG-peptides represent a new class of scavenger inhibitors that will likely have broad utility in blocking unwanted liver uptake and metabolism of a variety of nanoparticles.

Three-tiered nodal classification system for bladder cancer: a new proposal.

AIM: To evaluate a three-tiered prognostic stratification using one, two to five and >five positive lymph nodes (LNs) and this nodal staging system performs across different pelvic LN dissection (PLND) templates and adjuvant chemotherapy status. METHODS: We evaluated 244 patients with positive LN urothelial cancer who underwent radical cystectomy and PLND between 2000 and 2011. Survival analyses utilizing the Kaplan-Meier method and log rank test were performed. Median follow-up was 55.3 months (range: 0.4-141). Multivariable Cox proportional hazards models were built to evaluate the prognostic stratification. RESULTS: Extended PLND template was performed on 152 (62.3%) patients and standard on 92 (37.7%). The median number of LNs resected was 14 in the standard group vs 22 in the extended group (p < 0.01) and positive LNs was 2 vs 3 (p = 0.09), respectively. Stratification in patients with: one positive LN, two to five positive LNs or >five positive LNs lead to 5-year recurrence-free survival of: 48.6, 34.5 and 15.9% for each group, while the 5-year overall survival was: 43.0, 22.1 and 11.3%, respectively. Stratification in the three groups was also verified irrespective of PLND template and adjuvant chemotherapy. Two multivariable models confirmed the findings when controlling for demographic features and known pathologic risk factors. CONCLUSION: Three-tiered nodal classification system using the number of metastatic LNs (one, two to five and >five) stratifies patients with lymphatic disease into distinct prognostic groups.