Effects of sheared chromatin length on ChIP-seq quality and sensitivity.

Chromatin immunoprecipitation followed by massively parallel, high throughput sequencing (ChIP-seq) is the method of choice for genome-wide identification of DNA segments bound by specific transcription factors or in chromatin with particular histone modifications. However, the quality of ChIP-seq datasets varies widely, with a substantial fraction being of intermediate to poor quality. Thus, it is important to discern and control the factors that contribute to variation in ChIP-seq. In this study, we focused on sonication, a user-controlled variable, to produce sheared chromatin. We systematically varied the amount of shearing of fixed chromatin from a mouse erythroid cell line, carefully measuring the distribution of resultant fragment lengths prior to ChIP-seq. This systematic study was complemented with a retrospective analysis of additional experiments. We found that the level of sonication had a pronounced impact on the quality of ChIP-seq signals. Over-sonication consistently reduced quality, while the impact of under-sonication differed among transcription factors, with no impact on sites bound by CTCF but frequently leading to the loss of sites occupied by TAL1 or bound by POL2. The bound sites not observed in low-quality datasets were inferred to be a mix of both direct and indirect binding. We leveraged these findings to produce a set of CTCF ChIP-seq datasets in rare, primary hematopoietic progenitor cells. Our observation that the amount of chromatin sonication is a key variable in success of ChIP-seq experiments indicates that monitoring the level of sonication can improve ChIP-seq quality and reproducibility and facilitate ChIP-seq in rare cell types.

Ventricular Assist Device Implantation and Bariatric Surgery: A Route to Transplantation in Morbidly Obese Patients with End-Stage Heart Failure.

We reviewed our experience of morbidly obese patients with end-stage heart failure that underwent left ventricular assist device (LVAD) implantation. From January 1, 2008 to January 1, 2018, 240 adult LVADs were implanted at our center. We reviewed the cases of patients presenting with end-stage heart failure and morbid obesity (preoperative body mass index [BMI] ≥ 35 kg/m2) who underwent LVAD-alone, and compared that to a group that underwent LVAD and bariatric surgery (laparoscopic sleeve gastrectomy [LSG]) as a means for weight reduction. Demographic characteristics, perioperative details, BMI, and status of transplant candidacy were recorded. Statistical analysis was performed (SPSS version 25) with χ2 analysis, Kaplan-Meier survival analysis, regression analysis, and Student's t-test. Twenty-nine patients met criteria and underwent LVAD implantation. Fifteen patients underwent LVAD-alone. Fourteen patients underwent LVAD + LSG. Both groups showed good survival outcomes, LVAD-alone (88.9 ± 5.9 months) versus LVAD +LSG (96.1 ± 12.4 months) but were not significantly different. However, we did note that more patients in the LVAD + LSG group were bridged to heart transplantation (p < 0.001). LVAD-alone and/or LVAD + LSG are both technically feasible and effective treatment options for the long-term survival of morbidly obese patients with end-stage heart failure. Combining LVAD + LSG can help bridge patients to heart transplantation.

Oncolytic measles virus therapy enhances tumor antigen-specific T-cell responses in patients with multiple myeloma.

Oncolytic virus therapy leads to immunogenic death of virus-infected tumor cells and this has been shown in preclinical models to enhance the cytotoxic T-lymphocyte response against tumor-associated antigens (TAAs), leading to killing of uninfected tumor cells. To investigate whether oncolytic virotherapy can increase immune responses to tumor antigens in human subjects, we studied T-cell responses against a panel of known myeloma TAAs using PBMC samples obtained from ten myeloma patients before and after systemic administration of an oncolytic measles virus encoding sodium iodide symporter (MV-NIS). Despite their prior exposures to multiple immunosuppressive antimyeloma treatment regimens, T-cell responses to some of the TAAs were detectable even before measles virotherapy. Measurable baseline T-cell responses against MAGE-C1 and hTERT were present. Furthermore, MV-NIS treatment significantly (P < 0.05) increased T-cell responses against MAGE-C1 and MAGE-A3. Interestingly, one patient who achieved complete remission after MV-NIS therapy had strong baseline T-cell responses both to measles virus proteins and to eight of the ten tested TAAs. Our data demonstrate that oncolytic virotherapy can function as an antigen agnostic vaccine, increasing cytotoxic T-lymphocyte responses against TAAs in patients with multiple myeloma, providing a basis for continued exploration of this modality in combination with immune checkpoint blockade.

An integrative view of the regulatory and transcriptional landscapes in mouse hematopoiesis.

Thousands of epigenomic data sets have been generated in the past decade, but it is difficult for researchers to effectively use all the data relevant to their projects. Systematic integrative analysis can help meet this need, and the VISION project was established for validated systematic integration of epigenomic data in hematopoiesis. Here, we systematically integrated extensive data recording epigenetic features and transcriptomes from many sources, including individual laboratories and consortia, to produce a comprehensive view of the regulatory landscape of differentiating hematopoietic cell types in mouse. By using IDEAS as our integrative and discriminative epigenome annotation system, we identified and assigned epigenetic states simultaneously along chromosomes and across cell types, precisely and comprehensively. Combining nuclease accessibility and epigenetic states produced a set of more than 200,000 candidate cis-regulatory elements (cCREs) that efficiently capture enhancers and promoters. The transitions in epigenetic states of these cCREs across cell types provided insights into mechanisms of regulation, including decreases in numbers of active cCREs during differentiation of most lineages, transitions from poised to active or inactive states, and shifts in nuclease accessibility of CTCF-bound elements. Regression modeling of epigenetic states at cCREs and gene expression produced a versatile resource to improve selection of cCREs potentially regulating target genes. These resources are available from our VISION website to aid research in genomics and hematopoiesis.

APOBEC3B-mediated corruption of the tumor cell immunopeptidome induces heteroclitic neoepitopes for cancer immunotherapy.

APOBEC3B, an anti-viral cytidine deaminase which induces DNA mutations, has been implicated as a mediator of cancer evolution and therapeutic resistance. Mutational plasticity also drives generation of neoepitopes, which prime anti-tumor T cells. Here, we show that overexpression of APOBEC3B in tumors increases resistance to chemotherapy, but simultaneously heightens sensitivity to immune checkpoint blockade in a murine model of melanoma. However, in the vaccine setting, APOBEC3B-mediated mutations reproducibly generate heteroclitic neoepitopes in vaccine cells which activate de novo T cell responses. These cross react against parental, unmodified tumors and lead to a high rate of cures in both subcutaneous and intra-cranial tumor models. Heteroclitic Epitope Activated Therapy (HEAT) dispenses with the need to identify patient specific neoepitopes and tumor reactive T cells ex vivo. Thus, actively driving a high mutational load in tumor cell vaccines increases their immunogenicity to drive anti-tumor therapy in combination with immune checkpoint blockade.

Prepping for Prevention: An Intervention to Optimize Skin Antisepsis and Peri-Operative Antibiotic Prophylaxis.

Background: The U.S. Centers for Disease Control and Prevention recommend bathing prior to surgery, surgical skin antisepsis, peri-operative antibiotic administration, normothermia throughout the procedure, serum glucose concentration <200 mg/dL throughout the procedure, and hyperoxygenation in the immediate post-operative period to prevent surgical site infection (SSI). We developed interventions to standardize skin antisepsis and peri-operative antibiotic administration at our institution. Methods: This is a cross-sectional evaluation of surgical skin antisepsis and antibiotic administration before and after a series of interventions designed to standardize the processes. Results: One hundred twenty-four surgical skin antisepsis opportunities were observed; significant improvement was seen in hand hygiene prior to performing skin antisepsis (compliance changing from 1% to 48%; p < 0.001), sleeves being worn during skin antisepsis (1% versus 67%; p < 0.001), use of the correct cleansing time (47% versus 85%; p < 0.001), allowance for adequate drying time (67% versus 87%; p = 0.02), and use of a cleansing motion from the incision to the periphery (78% versus 95%; p = 0.004). Pre-operative antibiotic order placement, correct antibiotic selection, and optimal antibiotic dose were evaluated in 466 surgical procedures. Significant improvement was seen in both peri-operative order placement (59% versus 70%; p = 0.02) and correct antibiotic selection (52% versus 95%; p < 0.001). Conclusion: An intervention to standardize skin antisepsis and to encourage early ordering of peri-operative antibiotics was successful.

Comparative Oncology Evaluation of Intravenous Recombinant Oncolytic Vesicular Stomatitis Virus Therapy in Spontaneous Canine Cancer.

Clinical translation of intravenous therapies to treat disseminated or metastatic cancer is imperative. Comparative oncology, the evaluation of novel cancer therapies in animals with spontaneous cancer, can be utilized to inform and accelerate clinical translation. Preclinical murine studies demonstrate that single-shot systemic therapy with a vesicular stomatitis virus (VSV)-IFNß-NIS, a novel recombinant oncolytic VSV, can induce curative remission in tumor-bearing mice. Clinical translation of VSV-IFNß-NIS therapy is dependent on comprehensive assessment of clinical toxicities, virus shedding, pharmacokinetics, and efficacy in clinically relevant models. Dogs spontaneously develop cancer with comparable etiology, clinical progression, and response to therapy as human malignancies. A comparative oncology study was carried out to investigate feasibility and tolerability of intravenous oncolytic VSV-IFNß-NIS therapy in pet dogs with spontaneous cancer. Nine dogs with various malignancies were treated with a single intravenous dose of VSV-IFNß-NIS. Two dogs with high-grade peripheral T-cell lymphoma had rapid but transient remission of disseminated disease and transient hepatotoxicity that resolved spontaneously. There was no shedding of infectious virus. Correlative pharmacokinetic studies revealed elevated levels of VSV RNA in blood in dogs with measurable disease remission. This is the first evaluation of intravenous oncolytic virus therapy for spontaneous canine cancer, demonstrating that VSV-IFNß-NIS is well-tolerated and safe in dogs with advanced or metastatic disease. This approach has informed clinical translation, including dose and target indication selection, leading to a clinical investigation of intravenous VSV-IFNß-NIS therapy, and provided preliminary evidence of clinical efficacy and potential biomarkers that correlate with therapeutic response. Mol Cancer Ther; 17(1); 316-26. ©2017 AACR.

Alu-dependent RNA editing of GLI1 promotes malignant regeneration in multiple myeloma.

Despite novel therapies, relapse of multiple myeloma (MM) is virtually inevitable. Amplification of chromosome 1q, which harbors the inflammation-responsive RNA editase adenosine deaminase acting on RNA (ADAR)1 gene, occurs in 30-50% of MM patients and portends a poor prognosis. Since adenosine-to-inosine RNA editing has recently emerged as a driver of cancer progression, genomic amplification combined with inflammatory cytokine activation of ADAR1 could stimulate MM progression and therapeutic resistance. Here, we report that high ADAR1 RNA expression correlates with reduced patient survival rates in the MMRF CoMMpass data set. Expression of wild-type, but not mutant, ADAR1 enhances Alu-dependent editing and transcriptional activity of GLI1, a Hedgehog (Hh) pathway transcriptional activator and self-renewal agonist, and promotes immunomodulatory drug resistance in vitro. Finally, ADAR1 knockdown reduces regeneration of high-risk MM in serially transplantable patient-derived xenografts. These data demonstrate that ADAR1 promotes malignant regeneration of MM and if selectively inhibited may obviate progression and relapse.

The use of the NIS reporter gene for optimizing oncolytic virotherapy.

INTRODUCTION: Oncolytic viruses are experimental cancer therapies being translated to the clinic. They are unique in their ability to amplify within the body, therefore requiring careful monitoring of viral replication and biodistribution. Traditional monitoring strategies fail to recapitulate the dynamic nature of oncolytic virotherapy. Consequently, clinically relevant, noninvasive, high resolution strategies are needed to effectively track virotherapy in real time. AREAS COVERED: The expression of the sodium iodide symporter (NIS) reporter gene is tightly coupled to viral genome replication and mediates radioisotope concentration, allowing noninvasive molecular nuclear imaging of active viral infection with high resolution. This provides insight into replication kinetics, biodistribution, the impact of vector design, administration, and dosing on therapeutic outcomes, and highlights the heterogeneity of spatial distribution and temporal evolution of infection. NIS-mediated imaging in clinical trials confirms the feasibility of this technology to noninvasively and longitudinally observe oncolytic virus infection, replication, and distribution. EXPERT OPINION: NIS-mediated imaging provides detailed functional and molecular information on the evolution of oncolytic virus infection in living animals. The use of NIS reporter gene imaging has rapidly advanced to provide unparalleled insight into the spatial and temporal context of oncolytic infection which will be integral to optimization of oncolytic treatment strategies.

Perfusion Pressure Is a Critical Determinant of the Intratumoral Extravasation of Oncolytic Viruses.

Antitumor efficacy of oncolytic virotherapy is determined by the density and distribution of infectious centers within the tumor, which may be heavily influenced by the permeability and blood flow in tumor microvessels. Here, we investigated whether systemic perfusion pressure, a key driver of tumor blood flow, could influence the intratumoral extravasation of systemically administered oncolytic vesicular stomatitis virus (VSV) in myeloma tumor-bearing mice. Exercise was used to increase mean arterial pressure, and general anesthesia to decrease it. A recombinant VSV expressing the sodium iodide symporter (NIS), which concentrates radiotracers at sites of infection, was administered intravenously to exercising or anesthetized mice, and nuclear NIS reporter gene imaging was used to noninvasively track the density and spatial distribution of intratumoral infectious centers. Anesthesia resulted in decreased intratumoral infection density, while exercise increased the density and uniformity of infectious centers. Perfusion state also had a significant impact on the antitumor efficacy of the VSV therapy. In conclusion, quantitative dynamic radiohistologic imaging was used to noninvasively interrogate delivery of oncolytic virotherapy, highlighting the critical importance of perfusion pressure as a driver of intratumoral delivery and efficacy of oncolytic viruses.

Noninvasive 3-dimensional imaging of liver regeneration in a mouse model of hereditary tyrosinemia type 1 using the sodium iodide symporter gene.

Cell transplantation is a potential treatment for the many liver disorders that are currently only curable by organ transplantation. However, one of the major limitations of hepatocyte (HC) transplantation is an inability to monitor cells longitudinally after injection. We hypothesized that the thyroidal sodium iodide symporter (NIS) gene could be used to visualize transplanted HCs in a rodent model of inherited liver disease: hereditary tyrosinemia type 1. Wild-type C57Bl/6J mouse HCs were transduced ex vivo with a lentiviral vector containing the mouse Slc5a5 (NIS) gene controlled by the thyroxine-binding globulin promoter. NIS-transduced cells could robustly concentrate radiolabeled iodine in vitro, with lentiviral transduction efficiencies greater than 80% achieved in the presence of dexamethasone. Next, NIS-transduced HCs were transplanted into congenic fumarylacetoacetate hydrolase knockout mice, and this resulted in the prevention of liver failure. NIS-transduced HCs were readily imaged in vivo by single-photon emission computed tomography, and this demonstrated for the first time noninvasive 3-dimensional imaging of regenerating tissue in individual animals over time. We also tested the efficacy of primary HC spheroids engrafted in the liver. With the NIS reporter, robust spheroid engraftment and survival could be detected longitudinally after direct parenchymal injection, and this thereby demonstrated a novel strategy for HC transplantation. This work is the first to demonstrate the efficacy of NIS imaging in the field of HC transplantation. We anticipate that NIS labeling will allow noninvasive and longitudinal identification of HCs and stem cells in future studies related to liver regeneration in small and large preclinical animal models.

The effect of preoperative skin preparation products on surgical site infection.

Skin preparation products contribute to surgical site infection (SSI) prevention. In a case-control study, diabetes was associated with increased SSI (adjusted odds ratio [OR], 5.74 [95% confidence interval (CI), 1.22-27.0]), while the use of chlorhexidine gluconate (CHG) plus isopropyl alcohol versus CHG alone was found to be protective (adjusted OR, 2.64 [95% CI, 1.12-6.20]).

Maternal embryonic leucine zipper kinase regulates pancreatic ductal, but not ß-cell, regeneration.

The maternal embryonic leucine zipper kinase (MELK) is expressed in stem/progenitor cells in some adult tissues, where it has been implicated in diverse biological processes, including the control of cell proliferation. Here, we described studies on its role in adult pancreatic regeneration in response to injury induced by duct ligation and ß-cell ablation. MELK expression was studied using transgenic mice expressing GFP under the control of the MELK promoter, and the role of MELK was studied using transgenic mice deleted in the MELK kinase domain. Pancreatic damage was initiated using duct ligation and chemical beta-cell ablation. By tracing MELK expression using a MELK promoter-GFP transgene, we determined that expression was extremely low in the normal pancreas. However, following duct ligation and ß-cell ablation, it became highly expressed in pancreatic ductal cells while remaining weakly expressed in α-cells and ß- cells. In a mutant mouse in which the MELK kinase domain was deleted, there was no effect on pancreatic development. There was no apparent effect on islet regeneration, either. However, following duct ligation there was a dramatic increase in the number of small ducts, but no change in the total number of duct cells or duct cell proliferation. In vitro studies indicated that this was likely due to a defect in cell migration. These results implicate MELK in the control of the response of the pancreas to injury, specifically controlling cell migration in normal and transformed pancreatic duct cells.

Reporter gene imaging identifies intratumoral infection voids as a critical barrier to systemic oncolytic virus efficacy.

Systemically administered oncolytic viruses have the ability to cause tumor destruction through the expansion and coalescence of intratumoral infectious centers. Efficacy is therefore dependent upon both the density and intratumoral distribution of virus-infected cells achieved after initial virus infusion, and delivery methods are being developed to enhance these critical parameters. However, the three-dimensional (3D) mapping of intratumoral infectious centers is difficult using conventional immunohistochemical methodology, requiring painstaking 3D reconstruction of numerous sequential stained tumor sections, with no ability to study the temporal evolution of spreading infection in a single animal. We therefore developed a system using very high-resolution noninvasive in vivo micro single-photon emitted computed tomography/computed tomography (microSPECT/CT) imaging to determine the intratumoral distribution of thyroid radiotracers in tumors infected with an oncolytic virus encoding the thyroidal sodium-iodide symporter (NIS). This imaging system was used for longitudinal analysis of the density, distribution, and evolution of intratumoral infectious centers after systemic administration of oncolytic vesicular stomatitis virus in tumor-bearing mice and revealed heterogeneous delivery of virus particles both within and between tumors in animals receiving identical therapy. This study provides compelling validation of high resolution in vivo reporter gene mapping as a convenient method for serial monitoring of intratumoral virus spread that will be necessary to address critical barriers to systemic oncolytic virus efficacy such as intratumoral delivery.

Sustained reduction in surgical site infection after abdominal hysterectomy.

BACKGROUND: During a period of five years, the rate of surgical site infection (SSI) after abdominal hysterectomy at our institution was >10%. With the implementation of a multifaceted intervention designed to reduce this, the rate of SSI fell to <2% in the post-intervention period. The pre- and post-intervention periods were compared to determine which of the interventions in the multifaceted array of interventions was most valuable in decreasing SSI. METHODS: A retrospective chart review was done to identify: (1) Parameters associated with SSI, and (2) parameters that differed in the pre- and post-intervention periods. The intervention included providing departmental SSI rates to the gynecology faculty, re-educating operating room (OR) staff personnel about appropriate perioperative antibiotic choice and timing, and changing the preferred sterile preparation for abdominal surgery from 10% povidone-iodine (PI) to 4% chlorhexidine gluconate (CHG). The preliminary results of our review also led to the suggestion that surgeons use blood products sparingly, although an absolute threshold for transfusion was not specified. RESULTS: Twenty-one of 192 patients (10.7%) developed an SSI in the pre-intervention period, whereas 1 of 84 patients (1.2%) developed an SSI in the post-intervention period (p=0.006). Surgical site infection was associated with obesity (a body mass index [BMI] ≥30) (11.5% vs. 4.8%, p=0.04), receipt of a blood transfusion (18.2% vs. 6.6%, p=0.03), and abdominal skin preparation with PI as opposed to CHG (10.1% vs. 2.0%, p=0.07). Chlorhexidine gluconate was used more commonly for abdominal skin preparation in the post- than in the pre-intervention period (6.6% pre-intervention vs. 50.7% post-intervention, p <0.0001). CONCLUSIONS: A multifaceted intervention decreased dramatically the rate of SSI after abdominal hysterectomy at our institution. No single component of the intervention could be identified as most responsible for the improvement.

Hydrogel nanoparticles with covalently linked coomassie blue for brain tumor delineation visible to the surgeon.

Delineation of tumor margins is a critical and challenging objective during brain cancer surgery. A tumor-targeting deep-blue nanoparticle-based visible contrast agent is described, which, for the first time, offers in vivo tumor-specific visible color staining. This technology thus enables color-guided tumor resection in real time, with no need for extra equipment or special lighting conditions. The visual contrast agent consists of polyacrylamide nanoparticles covalently linked to Coomassie Blue molecules (for nonleachable blue color contrast), which are surface-conjugated with polyethylene glycol and F3 peptides for efficient in vivo circulation and tumor targeting, respectively.

Sustained Medication Reduction Following Unilateral VIM Thalamic Stimulation for Essential Tremor.

BACKGROUND: Deep brain stimulation (DBS) is an increasingly utilized therapeutic modality for the management of medication refractory essential tremor (ET). The aim of this study was to determine whether DBS allowed for anti-tremor medication reduction within the year after the procedure was performed. METHODS: We conducted a retrospective chart review and telephone interviews on 34 consecutive patients who had been diagnosed with ET, and who had undergone unilateral DBS surgery. RESULTS: Of the 34 patients in our cohort, 31 patients (91%) completely stopped all anti-tremor medications either before surgery (21 patients, 62%) or in the year following DBS surgery (10 patients, 29%). Patients who discontinued tremor medications before DBS surgery did so because their tremors either became refractory to anti-tremor medication, or they developed adverse events to tremor medications. Patients who stopped tremor medications after DBS surgery did so due to sufficient tremor control. Only three patients (9%) who were taking tremor medications at the time of surgery continued the use of a beta-blocker post-operatively for the purpose of hypertension management in all cases. DISCUSSION: The data from this study indicate that medication cessation is common following unilateral DBS for ET.

Maternal embryonic leucine zipper kinase is upregulated and required in mammary tumor-initiating cells in vivo.

Maternal embryonic leucine zipper kinase (MELK) is expressed in several developing tissues, in the adult germ line, and in adult neural progenitors. MELK expression is elevated in aggressive undifferentiated tumors, correlating with poor patient outcome in human breast cancer. To investigate the role of MELK in mammary tumorigenesis in vivo, we used a MELK-green fluorescent protein (GFP) reporter mouse, which allows prospective isolation of MELK-expressing cells based on GFP fluorescence. We found that in the normal mammary gland, cells expressing high levels of MELK were enriched in proliferating cells that express markers of mammary progenitors. The isolation of cells with high levels of MELK in mammary tumors from MMTV-Wnt1/MELK-GFP bitransgenic mice resulted in a significant enrichment of tumorsphere formation in culture and tumor initiation after transplantation into mammary fat pads of syngeneic mice. Furthermore, using lentiviral delivery of MELK-specific shRNA and limiting dilution cell transplantations, we showed that MELK function is required for mammary tumorigenesis in vivo. Our findings identify MELK as a potential target in breast tumor-initiating cells.

Reduction of circulating endothelial cells in peripheral blood of ALS patients.

BACKGROUND: Amyotrophic Lateral Sclerosis (ALS) treatment is complicated by the various mechanisms underlying motor neuron degeneration. Recent studies showed that the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) are compromised in an animal model of ALS due to endothelial cell degeneration. A later study demonstrated a loss of endothelium integrity in the spinal cords of ALS patients. Since circulating endothelial cells (CECs) in the peripheral blood are associated with endothelium damage, being detached dysfunctional endothelial cells, we hypothesized that CEC levels may reflect endothelium condition in ALS patients. METHODOLOGY/PRINCIPAL FINDINGS: CEC levels were estimated in whole blood smears from ALS patients with moderate stage ((M)ALS), severe stage ((S)ALS), and healthy controls by CD146 expression using immunocytochemistry. A significant reduction of CECs was detected in (M)ALS and (S)ALS patients. CONCLUSIONS/SIGNIFICANCE: CECs did not predict endothelium state in ALS patients; however, endothelial damage and/or impaired endothelium repair may occur in ALS leading to BBB/BSCB dysfunction. Reduced CECs in peripheral blood of ALS patients may indicate different mechanisms of endothelial damage and repair, rather than only detachment of dysfunctional endothelial cells. Although a potential mechanism of CEC reduction is discussed, establishing a reliable indicator of endothelial dysfunction/damage is important for evaluation of BBB/BSCB status in ALS patients during disease progression.