Novel drug resistance mechanisms and drug targets in BRAF-mutated peritoneal metastasis from colorectal cancer.

BACKGROUND: Patients with peritoneal metastasis from colorectal cancer (PM-CRC) have inferior prognosis and respond particularly poorly to chemotherapy. This study aims to identify the molecular explanation for the observed clinical behavior and suggest novel treatment strategies in PM-CRC. METHODS: Tumor samples (230) from a Norwegian national cohort undergoing surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) with mitomycin C (MMC) for PM-CRC were subjected to targeted DNA sequencing, and associations with clinical data were analyzed. mRNA sequencing was conducted on a subset of 30 samples to compare gene expression in tumors harboring BRAF or KRAS mutations and wild-type tumors. RESULTS: BRAF mutations were detected in 27% of the patients, and the BRAF-mutated subgroup had inferior overall survival compared to wild-type cases (median 16 vs 36 months, respectively, p < 0.001). BRAF mutations were associated with RNF43/RSPO aberrations and low expression of negative Wnt regulators (ligand-dependent Wnt activation). Furthermore, BRAF mutations were associated with gene expression changes in transport solute carrier proteins (specifically SLC7A6) and drug metabolism enzymes (CES1 and CYP3A4) that could influence the efficacy of MMC and irinotecan, respectively. BRAF-mutated tumors additionally exhibited increased expression of members of the novel butyrophilin subfamily of immune checkpoint molecules (BTN1A1 and BTNL9). CONCLUSIONS: BRAF mutations were frequently detected and were associated with particularly poor survival in this cohort, possibly related to ligand-dependent Wnt activation and altered drug transport and metabolism that could confer resistance to MMC and irinotecan. Drugs that target ligand-dependent Wnt activation or the BTN immune checkpoints could represent two novel therapy approaches.

Added value of EUS-B-FNA to bronchoscopy and EBUS-TBNA in diagnosing and staging of lung cancer.

Background: Bronchoscopy and EBUS are standard procedures in lung cancer work-up but have low diagnostic yield in lesions outside the central airways and hilar/mediastinal lymph nodes. Growing evidence on introducing the EBUS endoscope into the oesophagus (EUS-B) in the same session as bronchoscopy/EBUS gives access to new anatomical areas that can be safely biopsied. Objective: To summarize the current evidence of the added value of EUS-B-FNA to bronchoscopy and EBUS-TBNA in lung cancer work-up. Methods: A narrative review. Results: Few randomized trials or prospective studies are available. Prospective studies show that add-on EUS-B-FNA increases diagnostic yield when sampling abnormal mediastinal lymph nodes, para-oesophageal lung and left adrenal gland. A large retrospective series on EUS-B-FNA from retroperitoneal lymph nodes suggests high diagnostic yield without safety concerns, as do casuistic reports on EUS-B-FNA from mediastinal pleural thickening, pancreatic lesions, ascites fluid and pericardial effusions. No study has systematically assessed both diagnostic yield, safety, patient reported outcomes, adverse events and costs. Conclusion: The diagnostic value of add-on EUS-B to standard bronchoscopy and EBUS in lung cancer work-up appears very promising without safety concerns, giving the pulmonologist access to a variety of sites out of reach with other minimally invasive techniques. Little is known on patient-reported outcomes and costs. Future and prospective research should focus on effectiveness aspects to clarify whether overall benefits of add-on EUS-B sufficiently exceed overall downsides.

The criteria for chronic rhinosinusitis in children with cystic fibrosis are rarely fulfilled after initiation of CFTR modulator treatment.

The vast majority of people with cystic fibrosis (pwCF) have untreated secondary chronic rhinosinusitis (CRS). Whereas the introduction of the cystic fibrosis transmembrane conductance regulator modulator (CFTRm) treatment regime has improved the lung function of pwCF, few studies have been published examining the effect on sinonasal symptoms in children. Our aim was to explore the effect of double CFTRm treatment on CRS and olfaction in children with CF. pwCF were included in this non-randomized cross-sectional study, where an otolaryngologist performed a complete ENT examination before initiating treatment with elaxacaftor/tezacaftor/ivacaftor (ETI). Twenty-three pwCF aged 6-12 years were included. Eighteen of 23 patients were on a double CFTRm treatment, and 5 patients were CFTRm naive, respectively. Altogether, 19 had normal olfaction, 20 had none or mild CRS symptoms according to SNOT-22, and 14 had a normal endoscopy. None of the patients had symptoms of chronic rhinosinusitis lasting for more than 12 weeks, thus none of the patients fulfilled the criteria for CRS. Children with CF treated with double CFTRm have few to no symptoms of CRS and normal olfaction, which is an improvement compared with children following treatment modalities prior to CFTRm.

Using the Endoscope for Endobronchial Ultrasound in the Esophagus.

EUS-B is a procedure using the echoendobronchoscope in the esophagus and stomach. The procedure is a minimally invasive, safe, and feasible approach that pulmonologists can use to visualize and biopsy structures adjacent to the esophagus and stomach. EUS-B gives access to many structures of which some may also be reached by EBUS (mediastinal lymph nodes, lung or pleural tumors, pericardial fluid) while others cannot be reached such as retroperitoneal lymph nodes, ascites, and lesions in the liver, pancreas or left adrenal gland. The procedure is a pulmonologist- and patient- friendly version of the gastroenterologists' EUS using the thin EBUS endoscope that the pulmonologist already masters. Thus EUS-B training should be easy and a natural continuation of EBUS. With the patient under conscious sedation and in the supine position, the echoendoscope is introduced either through the nostril or mouth into the oropharynx. Then the patient is encouraged to swallow while the endoscope is slowly bent posteriorly and introduced into the esophagus and stomach. Using the ultrasonic image, the operator identifies the six landmarks by EUS-B and EUS: the left liver lobe, abdominal aorta (with the celiac trunk and superior mesenteric artery), left adrenal gland, and mediastinal lymph node stations 7, 4L, and 4R. Biopsies can be taken from suspected lesions under real-time ultrasonographic guidance- fine needle aspiration (EUS-B-FNA) using a technique similar to that used with EBUS-TBNA. The biopsy order is M1b-M1a-N3-N2-N1-T (M = metastasis, N = lymph node, T = tumor) to avoid iatrogenic upstaging. Pre- and post-procedural observation is similar to that of bronchoscopy. EUS-B is safe and feasible in the hands of experienced interventional pulmonologists and provides a significant expansion of the diagnostic possibilities in providing safe, fast, and thorough diagnosis and staging of lung cancer.

High-resolution visualization and assessment of basal and OXPHOS-induced mitophagy in H9c2 cardiomyoblasts.

Mitochondria are susceptible to damage resulting from their activity as energy providers. Damaged mitochondria can cause harm to the cell and thus mitochondria are subjected to elaborate quality-control mechanisms including elimination via lysosomal degradation in a process termed mitophagy. Basal mitophagy is a house-keeping mechanism fine-tuning the number of mitochondria according to the metabolic state of the cell. However, the molecular mechanisms underlying basal mitophagy remain largely elusive. In this study, we visualized and assessed the level of mitophagy in H9c2 cardiomyoblasts at basal conditions and after OXPHOS induction by galactose adaptation. We used cells with a stable expression of a pH-sensitive fluorescent mitochondrial reporter and applied state-of-the-art imaging techniques and image analysis. Our data showed a significant increase in acidic mitochondria after galactose adaptation. Using a machine-learning approach we also demonstrated increased mitochondrial fragmentation by OXPHOS induction. Furthermore, super-resolution microscopy of live cells enabled capturing of mitochondrial fragments within lysosomes as well as dynamic transfer of mitochondrial contents to lysosomes. Applying correlative light and electron microscopy we revealed the ultrastructure of the acidic mitochondria confirming their proximity to the mitochondrial network, ER and lysosomes. Finally, exploiting siRNA knockdown strategy combined with flux perturbation with lysosomal inhibitors, we demonstrated the importance of both canonical as well as non-canonical autophagy mediators in lysosomal degradation of mitochondria after OXPHOS induction. Taken together, our high-resolution imaging approaches applied on H9c2 cells provide novel insights on mitophagy during physiologically relevant conditions. The implication of redundant underlying mechanisms highlights the fundamental importance of mitophagy.Abbreviations: ATG: autophagy related; ATG7: autophagy related 7; ATP: adenosine triphosphate; BafA1: bafilomycin A1; CLEM: correlative light and electron microscopy; EGFP: enhanced green fluorescent protein; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; OXPHOS: oxidative phosphorylation; PepA: pepstatin A; PLA: proximity ligation assay; PRKN: parkin RBR E3 ubiquitin protein ligase; RAB5A: RAB5A, member RAS oncogene family; RAB7A: RAB7A, member RAS oncogene family; RAB9A: RAB9A, member RAS oncogene family; ROS: reactive oxygen species; SIM: structured illumination microscopy; siRNA: short interfering RNA; SYNJ2BP: synaptojanin 2 binding protein; TEM: transmission electron microscopy; TOMM20: translocase of outer mitochondrial membrane 20; ULK1: unc-51 like kinase 1.

ImmunoPeCa trial: Long-term outcome following intraperitoneal MOC31PE immunotoxin treatment in colorectal peritoneal metastasis.

BACKGROUND: The ImmunoPeCa trial investigated the use of intraperitoneal MOC31PE immunotoxin as a novel therapeutic principle for the treatment of peritoneal metastasis from colorectal cancer (PM-CRC). We here report long-term outcome from the trial. METHODS: This was a dose-finding trial aiming to evaluate safety and toxicity (primary endpoint) upon a single dose of intraperitoneal MOC31PE in patients with PM-CRC undergoing CRS-HIPEC with mitomycin C. Overall survival (OS) and disease-free survival (DFS) were secondary endpoints. Twenty-one patients received the study drug at four dose levels on the first postoperative day, including six patients constituting an expansion cohort. RESULTS: With a 34-month follow-up, the median OS was not reached and the estimated 3-year OS was 78%. Median DFS for all patients was 21 months and the 3-year DFS was 33%, with a median follow-up of 31 months. When excluding patients with potential favorable characteristics from the analysis (n = 4), the median DFS was 13 months and the 3-year OS 72%. CONCLUSIONS: The promising long-term outcome combined with low systemic absorbance, high drug concentration and cytotoxic activity in peritoneal fluid support further investigations of clinical efficacy.

Perirenal fat thickness as a predictor of postoperative complications after laparoscopic distal gastrectomy for gastric cancer.

BACKGROUND: Laparoscopic distal gastrectomy is used widely in surgery for gastric cancer. Excess visceral fat can limit the ability to dissect the suprapancreatic region, potentially increasing the risk of local complications, particularly pancreatic fistula. This study evaluated perirenal fat thickness as a surrogate for visceral fat to see whether this was related to complications after laparoscopic distal gastrectomy. METHODS: Perirenal fat thickness was measured dorsal to the left kidney as an indicator of visceral fat in patients with gastric cancer who underwent laparoscopic distal gastrectomy. Patients were divided into two groups: those with and those without complications. The relationship between perirenal fat thickness and postoperative complications was evaluated. RESULTS: The optimal cut-off value for predicting morbidity using adipose tissue thickness was 10·7 mm; a distance equal to or greater than this was considered a positive perirenal fat thickness sign (PTS). A positive PTS showed a significant correlation with visceral fat area. Multivariable analysis found that a positive PTS was an independent risk factor for complications (hazard ratio 4·42, 95 per cent c.i. 2·31 to 8·86; P < 0·001). CONCLUSION: Perirenal fat thickness as an indicator of visceral fat was an independent predictor of postoperative complications after laparoscopic distal gastrectomy for gastric cancer.

ANTECEDENTES: La gastrectomía distal laparoscópica se utiliza ampliamente en la cirugía del cáncer gástrico. El exceso de grasa visceral puede limitar la capacidad para disecar la región suprapancreática, aumentando potencialmente riesgo de complicaciones locales, especialmente de fistula pancreática. El propósito de este estudio fue evaluar el grosor de la grasa perirrenal como marcador subrogado de grasa visceral para determinar si se relacionaba con complicaciones tras gastrectomía distal laparoscópica. MÉTODOS: El grosor de la grasa perirrenal se midió a nivel dorsal del riñón izquierdo como indicador de grasa visceral en pacientes con cáncer gástrico sometidos a gastrectomía distal laparoscópica. Los pacientes fueron divididos en dos grupos: aquellos con y sin complicaciones. Se evaluó la relación entre grosor de la grasa perirrenal y las complicaciones postoperatorias. RESULTADOS: El punto de corte óptimo para predecir la morbilidad utilizando el grosor del tejido adiposo fue de 10,7 mm, por lo que una distancia igual o mayor a este nivel fue considerado como signo positivo de engrosamiento de la grasa perirrenal (peri-renal fat thickness sign, PTS). Un PTS positivo mostró una correlación significativa con el área de grasa visceral. Los análisis multivariables demostraban que un PTS positivo era un factor de riesgo independiente para complicaciones (razón de oportunidades, odds ratio 4,418; i.c. del 95% 2,307-8,855; P < 0,001). CONCLUSIÓN: El grosor de grasa perirrenal como indicador de la grasa visceral fue un predictor independiente de complicaciones postoperatorias tras una gastrectomía distal laparoscópica por cáncer gástrico.

Laparoscopic proximal gastrectomy with double-flap technique versus laparoscopic subtotal gastrectomy for proximal early gastric cancer.

BACKGROUND: Laparoscopic proximal gastrectomy with double-flap technique (LPG-DFT) and laparoscopic subtotal gastrectomy (LSTG) may replace laparoscopic total gastrectomy (LTG) for proximal early gastric cancer. The aim of this study was to evaluate short- and long-term outcomes after LPG-DFT and LSTG. METHODS: Patients who underwent LPG-DFT or LSTG at the Cancer Institute Hospital in Tokyo between January 2006 and April 2015 were included in this retrospective study. Operative procedures were selected based on the distance from the cardia to the proximal boundary of the tumour, tumour location and predicted remnant stomach volume. Patient characteristics, surgical data, markers of postoperative nutritional status, such as blood chemistry and bodyweight loss, and endoscopic findings were compared between procedures. The main study outcome was nutritional status. RESULTS: A total of 161 patients (LPG-DFT 51, LSTG 110) were included. Types of postoperative complication occurring more than 30 days after surgery differed between the two procedures. Remnant stomach ulcers, including anastomotic ulcers, were observed only after LPG-DFT, whereas complications involving the small intestine, such as internal hernia or small bowel obstruction, occurred more frequently after LSTG. Values for total protein, albumin, prealbumin and bodyweight loss were comparable between the two procedures at 36 months after surgery. Haemoglobin concentrations were higher after LPG-DFT than after LSTG at 24 months (13·4 versus 12·8 g/dl respectively; P = 0·045) and 36 months (13·5 versus 12·8 g/dl; P = 0·007) after surgery. The rate of Los Angeles grade B or more severe reflux oesophagitis was comparable. CONCLUSION: LPG-DFT and LSTG for proximal early gastric cancer have similar outcomes, but different types of complication.

ANTECEDENTES: La gastrectomía proximal laparoscópica con técnica de doble derivación (double flap technique, LPG-DFT) y la gastrectomía subtotal laparoscópica (laparoscopic subtotal gastrectomy, LsTG) pueden sustituir a la gastrectomía total laparoscópica (laparoscopic total gastrectomy, LTG) para el cáncer gástrico precoz (early gastric cancer, EGC) proximal. El objetivo de este estudio fue evaluar los resultados a corto y a largo plazo tras LPG-DFT y LsTG. MÉTODOS: En este estudio retrospectivo se incluyeron pacientes que fueron sometidos a LPG-DFT o LsTG en el Hospital del Instituto del Cáncer de Tokio entre enero 2006 y abril 2015. Las técnicas quirúrgicas se seleccionaron en base a la distancia entre el cardias y el borde proximal del tumor, localización del tumor, y el volumen previsto del remanente gástrico. Las características de los pacientes, datos quirúrgicos, marcadores del estado nutricional postoperatorio, tales como la bioquímica sanguínea y la pérdida de peso corporal (body weight loss, BWL), y los hallazgos endoscópicos se compararon entre las técnicas. El resultado principal del estudio fue el estado nutricional. RESULTADOS: Se incluyeron un total de 161 pacientes (LPG-DFT 51, LsTG 110). Los tipos de complicaciones postoperatorias que aparecieron a más de 30 días después de la cirugía variaron entre ambas técnicas. Las úlceras en el remanente gástrico, incluyendo úlceras anastomóticas, solo se observaron tras una LPG-DFT, mientras las complicaciones relacionadas con el intestino delgado, como la hernia interna o la obstrucción de intestino delgado, sucedió con más frecuencia tras una LsTG. Los valores de proteínas totales, albúmina, prealbúmina, y BWL fueron comparables entre ambas técnicas a los 36 meses después de la cirugía. Las concentraciones de hemoglobina fueron más altas tras una LPG-DFT que tras una LsTG a los 24 (13,4 versus 12,8 mg/dL, P = 0,045) y 36 meses (13,5 versus 12,8 mg/mL, P = 0,007) después de la cirugía. Las incidencias de esofagitis por reflujo grado B Los Angeles o más grave fueron comparables. CONCLUSIÓN: La LPG-DFT y la LsTG para el EGC proximal presentan resultados bastante similares, pero difieren en el tipo de complicaciones.

The 3F Library: Fluorinated Fsp3 -Rich Fragments for Expeditious 19 F†NMR Based Screening.

Fragment-based drug discovery (FBDD) is a popular method in academia and the pharmaceutical industry for the discovery of early lead candidates. Despite its wide-spread use, the approach still suffers from laborious screening workflows and a limited diversity in the fragments applied. Presented here is the design, synthesis, and biological evaluation of the first fragment library specifically tailored to tackle both these challenges. The 3F library of 115 fluorinated, Fsp3 -rich fragments is shape diverse and natural-product-like with desirable physicochemical properties. The library is perfectly suited for rapid and efficient screening by NMR spectroscopy in a two-stage workflow of 19 F NMR and subsequent 1 H NMR methods. Hits against four diverse protein targets are widely distributed among the fragment scaffolds in the 3F library and a 67 % validation rate was achieved using secondary assays. This collection is the first synthetic fragment library tailor-made for 19 F NMR screening and the results demonstrate that the approach should find broad application in the FBDD community.

The FlpTRAP system for purification of specific, endogenous chromatin regions.

The repressor element 1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) binds to repressor element 1/neuron-restrictive silencer element (RE1/NRSE) sites in the genome and recruits effector proteins to repress its target genes. Here, we developed the FlpTRAP system to isolate endogenously assembled DNA-protein complexes such as the REST/NRSF complex. In the FlpTRAP system, we take advantage of the step-arrest variant of the Flp recombinase, FlpH305L, which, in the presence of Flp recognition target (FRT) DNA, accumulates as FRT DNA-protein adduct. The FlpTRAP system consists of three elements: (i) FlpH305L-containing cell extracts or isolates, (ii) a cell line engineered to harbor the DNA motif of interest flanked by FRT sites, and (iii) affinity selection steps to isolate the target chromatin. Specifically, 3×FLAG-tagged FlpH305L was expressed in insect cell cultures infected with baculovirus, and cell lysates were prepared. The lysate was used to capture the FRT-SNAP25 RE1/NRSE-FRT chromatin from a human medulloblastoma cell line, and the target RE1/NRSE chromatin was isolated by anti-FLAG immunoaffinity chromatography. Using electrophoretic mobility shift assays (EMSAs) and chromatin immunopurification (ChIP), we show that FlpH305L recognized and bound to the FRT sites. Overall, we suggest the FlpTRAP system as a tool to purify endogenous, specific chromatin loci from eukaryotic cells.

Life-threatening arrhythmic presentation in patients with arrhythmogenic cardiomyopathy before and after entering the genomic era; a two-decade experience from a large volume center.

BACKGROUND: Arrhythmogenic cardiomyopathy (AC) is an inheritable progressive heart disease with high risk of life-threatening ventricular arrhythmia (VA). We aimed to explore the prevalence of VA as presenting event in patients with AC over two decades, symptoms preceding VA and compare the clinical presentations and rate of AC-diagnosis over time. METHODS: We included consecutive AC-patients from our tertiary referral center. We recorded clinical history, VA (aborted cardiac arrest, sustained ventricular tachycardia or appropriate implantable cardioverter-defibrillator therapy), cardiac symptoms preceding VA in AC, and compared the history of patients diagnosed before and after implementation of genetic testing. RESULTS: We included 179 consecutive AC-patients and mutation-positive family members (95 [53%] probands, 84 [45%] female, 49 ±â€¯17 years), 33 (18%) diagnosed before and 146 (82%) after genetic testing became available. VA led to the AC-diagnosis in 46 (26%), and was less prevalent after implementation of genetic testing (17[52%] vs. 29[20%], p < 0.001), also when adjusted for proband status (Adjusted OR 2.7, 95% CI 1.1-6.7, p = 0.03). Yearly rate of AC-diagnosis increased after implementation of genetic testing in probands (2.7 ±â€¯1.3 vs. 6.8 ±â€¯4.3, p = 0.01) and family members (0.7 ±â€¯1.1 vs. 7.7 ±â€¯5.9, p = 0.002). Most patients with VA (92%) reported cardiac symptoms prior to event, and exercise-induced syncope was the strongest marker of subsequent VA (Adjusted OR 5.3, 95% CI 1.7-16.4, p = 0.004). CONCLUSION: VA led to AC-diagnosis in 46% of probands and was preceded by cardiac symptoms in the majority of cases. Yearly rate of AC-diagnoses increased after the implementation of genetic testing and life-threatening presentation of AC-disease seemed to decrease.

Number of pregnancies and subsequent phenotype in a cross-sectional cohort of women with arrhythmogenic cardiomyopathy.

Aims: We aimed to assess the relation between number of pregnancies and cardiac structure, function, and arrhythmic events in women with arrhythmogenic cardiomyopathy (AC). Methods and results: We included female AC patients in a cross-sectional study. Number of pregnancies and pregnancy related symptoms were recorded. Ventricular arrhythmias were defined as aborted cardiac arrest, sustained ventricular tachycardia, or appropriate implantable cardioverter-defibrillator therapy. Right and left ventricular dimensions and function, including strain analyses, were assessed by echocardiography and magnetic resonance imaging. We created a new AC severity score to grade the severity of AC disease. We included 77 women (age 47 ± 16, 43 probands and 34 AC mutation positive female relatives), 19 ± 14 years after last pregnancy. Median number of pregnancies was 2 (0-4); 19 had no previous pregnancies, 16 had 1 pregnancy, 30 had 2, and 12 had ≥3 pregnancies. Presence of a definite AC diagnosis (P = 0.36), severity of AC disease (P = 0.53), and arrhythmic events (P = 0.25) did not differ between groups of pregnancies. Number of pregnancies was related to increased right ventricular outflow tract diameter in single variable analyses [odds ratio (OR) 1.76, 95% confidence interval (CI) 1.08-2.87; P = 0.02], but not when adjusted for body surface area and age (OR 1.56, 95% CI 0.91-2.66; P = 0.11). The number of pregnancies was not associated with any other measures of cardiac structure and function. Conclusion: Higher number of pregnancies did not seem to relate to a worse phenotype in women with AC.

Three-Year Prospective Follow-up of Potential Pediatric Candidate for Intestinal Transplantation.

Intestinal transplantation (ITx) is a treatment for refractory intestinal failure (IF). However, the indications for and timing of ITx are still controversial because the course of IF is unknown. We performed a prospective multi-institutional cohort study to identify the prognostic factors for referral to an ITx facility. Patients under 18 years of age in Japan who suffered from IF and had received parenteral nutrition for longer than 6 months were enrolled in this study. They were followed up for 3 years. Seventy-two patients were followed. The mean age at the beginning of the study was 7.0 years. Diagnoses were short gut syndrome (n = 25), motility disorder (n = 45), and other (n = 2). The overall 3-year survival rate was 95%. The 3-year survival rate was 86% in patients with intestinal-failure-associated liver disease (IFALD) (n = 6) compared to 97% in those without IFALD (n = 66) (P = .0003). Furthermore, the 3-year survival rates of patients who did and did not meet the criteria for ITx were 82% (n = 11) and 97% (n = 62), respectively (P = .034). Six (44%) of 14 patients whose performance status (PS) was ≥3 at enrollment were dead or still had a PS ≥ 3 at 3 years. This study indicates that IFALD is a poor prognostic factor in pediatric patients with IF. Our indication for ITx, namely the presence of IFALD or loss of more than 2 parenteral nutrition access sites, seems to be applicable.

Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5.

The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins. Here, we demonstrate that human full-length SNF5 tagged with a C-terminal 3 × FLAG can be expressed and purified from insect cell extracts in monomeric and dimeric forms. To this end, we constructed a set of donor and acceptor vectors for the expression of individual proteins and protein complexes in the baculovirus/insect cell expression system under the control of a polyhedrin (polh), p10, or a minimal Drosophila melanogaster Hsp70 promoter. We show that the SNF5 expression level could be modulated by the selection of the promoter used to control expression. The vector set also comprises vectors that encode a 3 × FLAG tag, Twin-Strep tag, or CBP-3 × FLAG-TEV-ProteinA triple tag to facilitate affinity selection and detection. By gel filtration and split-ubiquitin assays, we show that human full-length SNF5 has the ability to self-interact. Overall, the toolbox developed herein offers the possibility to flexibly select the promoter strength as well as the affinity tag and is suggested to advance the recombinant expression of chromatin remodeling factors and other challenging proteins.

Intensified training increases salivary free light chains in trained cyclists: Indication that training volume increases oral inflammation.

Periods of short-term intensified training (IT) are often used by athletes during training cycles over the season and undergoing phases of increased physical stress may impact upon the immune system. This study investigated the effects of a period of IT on free light chains (FLCs) in saliva - an emerging immune biomarker of oral inflammation - and matched serum samples in well-trained athletes. It also examined if IT influences basal FLC levels and FLC flux during acute exercise. Highly trained male cyclists (n = 10) underwent a 9-day period of IT; before and after IT participants performed a 1 h time trial (TT) on a cycle ergometer, with blood and saliva samples collected pre- and post-exercise. FLCs were assessed in serum and saliva, and IgG, IgA, IgM and creatinine were also measured in serum. Weekly training volume increased by 143% (95% CI 114-172%), p < 0.001, during IT compared with pre-trial baseline training. Following IT, the cyclists demonstrated higher salivary FLC levels. Both salivary lambda FLC concentrations (p < 0.05, η2 = 0.384) and secretion rates, and kappa FLC concentrations and secretion rates increased after IT. Salivary FLCs concentration and secretion rates decreased in response to the TT following IT (p < 0.05, η2 = 0.387-0.428), but not in response to the TT prior to IT. No significant effects of IT on serum FLCs were observed. There were no significant changes in serum FLCs in response to the TT, before or after the IT period, nor did IT impact upon other serological responses to the TT. In conclusion, IT increased basal salivary FLC parameters and amplified decreases in salivary FLCs in response to acute exercise. Increases in salivary FLC concentration likely reflects alterations to oral inflammation during times of heavy training, and we show for the first time that FLCs may have utility as a marker of exercise stress and oral health status.

The effect of Mg-Ca-Sr alloy degradation products on human mesenchymal stem cells.

Biodegradable Mg alloys have the potential to replace currently used metallic medical implant devices, likely eliminating toxicity concerns and the need for secondary surgeries, while also providing a potentially stimulating environment for tissue growth. A recently developed Mg-Ca-Sr alloy possesses advantageous characteristics over other Mg alloys, having a good combination of strength and degradation behavior, while also displaying potentially osteogenic properties. To better understand the effect of alloy degradation products on cellular mechanisms, in vitro studies using human bone marrow-derived mesenchymal stem cells were conducted. Ionic products of alloy dissolution were found to be nontoxic but changed the proliferation profile of stem cells. Furthermore, their presence changed the progress of osteogenic development, while concentrations of Mg in particular appeared to induce stem cell differentiation. The work presented herein provides a foundation for future alloy design where structures can be tailored to obtain specific implant performance. These potentially bioactive implants would reduce the risks for patients by shortening their healing time, minimizing discomfort and toxicity concerns, while reducing hospital costs. © 2017 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 697-704, 2018.

Novel Treatment with Intraperitoneal MOC31PE Immunotoxin in Colorectal Peritoneal Metastasis: Results From the ImmunoPeCa Phase 1 Trial.

BACKGROUND: MOC31PE immunotoxin was developed to rapidly kill cells expressing the tumor-associated epithelial cell adhesion molecule, which is highly expressed in colorectal cancer. Although cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) may offer long-term survival to patients with peritoneal metastasis from colorectal cancer (PM-CRC), most patients experience disease relapse and novel therapeutic options are needed. On this basis, MOC31PE is being developed as a novel therapeutic principle to target PM-CRC. METHODS: This was a dose-escalating phase I trial to evaluate the safety and toxicity (primary endpoint), pharmacokinetic profile, and neutralizing antibody response (secondary endpoints) upon intraperitoneal administration of MOC31PE in patients with PM-CRC undergoing CRS-HIPEC with Mitomycin C. Fifteen patients received the study drug at four dose levels (3+3+3+6), administered intraperitoneally as a single dose the day after CRS-HIPEC. RESULTS: No dose-limiting toxicity was observed, and the maximum tolerated dose was not reached. There was negligible systemic absorption of the study drug. Drug concentrations in peritoneal fluid samples were in the cytotoxic range and increased in a dose-dependent manner. MOC31PE recovered from peritoneal cavity retained its cytotoxic activity in cell-based assays. All patients developed neutralizing antibodies. CONCLUSIONS: Intraperitoneal administration of MOC31PE was safe and well tolerated, and combined with low systemic uptake, MOC31PE seems ideal for local intraperitoneal treatment. The drug will be further evaluated in an ongoing phase II expansion cohort.

Randomized clinical trial comparing standard diet with perioperative oral immunonutrition in total gastrectomy for gastric cancer.

BACKGROUND: Total gastrectomy for gastric cancer is associated with excessive weight loss and decreased calorie intake. Nutritional support using eicosapentaenoic acid modulates immune function and limits catabolism in patients with advanced cancer, but its impact in the perioperative period is unclear. METHODS: This was a randomized phase III clinical trial of addition of eicosapentaenoic acid-rich nutrition to a standard diet in patients having total gastrectomy for gastric cancer. Patients were randomized to either a standard diet or standard diet with oral supplementation of an eicosapentaenoic acid (ProSure®), comprising 600 kcal with 2·2 g eicosapentaenoic acid, for 7 days before and 21 days after surgery. The primary endpoint was percentage bodyweight loss at 1 and 3 months after surgery. RESULTS: Of 127 eligible patients, 126 were randomized; 124 patients (61 standard diet, 63 supplemented diet) were analysed for safety and 123 (60 standard diet, 63 supplemented diet) for efficacy. Across both groups, all but three patients underwent total gastrectomy with Roux-en-Y reconstruction. Background factors were well balanced between the groups. Median compliance with the supplement in the immunonutrition group was 100 per cent before and 54 per cent after surgery. The surgical morbidity rate was 13 per cent in patients who received a standard diet and 14 per cent among those with a supplemented diet. Median bodyweight loss at 1 month after gastrectomy was 8·7 per cent without dietary supplementation and 8·5 per cent with eicosapentaenoic acid enrichment (P = 0·818, adjusted P = 1·000). Similarly, there was no difference between groups in percentage bodyweight loss at 3 months (P = 0·529, adjusted P = 1·000). CONCLUSION: Immunonutrition based on an eicosapentaenoic acid-enriched oral diet did not reduce bodyweight loss after total gastrectomy for gastric cancer compared with a standard diet. Registration number: UMIN000006380 ( http://www.umin.ac.jp/).

Soluble ST2 is associated with disease severity in arrhythmogenic right ventricular cardiomyopathy.

PURPOSE: Diagnostic and prognostic evaluation remains challenging in arrhythmogenic right ventricular cardiomyopathy (ARVC). We measured plasma concentration of soluble ST2 (sST2) and assessed its association with right ventricular (RV) function and ventricular arrhythmias in patients with ARVC. METHODS: We included patients with ARVC and genotype positive relatives. Soluble ST2 was determined by ELISA. We assessed myocardial function by echocardiography including strain by speckle tracking technique. RESULTS: We included 44 subjects (age 41 ± 15 years, 21 (48%) female). Soluble ST2 was associated with RV global strain (r = 0.44; p = 0.008), as well as with left ventricular (LV) function. Plasma levels of sST2 were higher in patients with ventricular arrhythmias than in patients without ventricular arrhythmias (35 ± 13 ng/mL vs. 26 ± 7 ng/mL, p = 0.009). The association between sST2 and ventricular arrhythmias remained significant even after adjusting for RV function (Wald = 5.2; p = 0.02). CONCLUSIONS: Soluble ST2 is associated with RV and LV function in patients with ARVC. Soluble ST2 may aid in the determination of disease severity in ARVC.