Integrase-deficient lentiviral vectors mediate efficient gene transfer to human vascular smooth muscle cells with minimal genotoxic risk

We have previously shown that injury-induced neointima formation was rescued by adenoviral-Nogo-B gene delivery. Integrase-competent lentiviral vectors (ICLV) are efficient at gene delivery to vascular cells but present a risk of insertional mutagenesis. Conversely, integrase-deficient lentiviral vectors (IDLV) offer additional benefits through reduced mutagenesis risk, but this has not been evaluated in the context of vascular gene transfer. Here, we have investigated the performance and genetic safety of both counterparts in primary human vascular smooth muscle cells (VSMC) and compared gene transfer efficiency and assessed the genotoxic potential of ICLVs and IDLVs based on their integration frequency and insertional profile in the human genome. Expression of enhanced green fluorescent protein (eGFP) mediated by IDLVs (IDLV-eGFP) demonstrated efficient transgene expression in VSMCs. IDLV gene transfer of Nogo-B mediated efficient overexpression of Nogo-B in VSMCs, leading to phenotypic effects on VSMC migration and proliferation, similar to its ICLV version and unlike its eGFP control and uninfected VSMCs. Large-scale integration site analyses in VSMCs indicated that IDLV-mediated gene transfer gave rise to a very low frequency of genomic integration compared to ICLVs, revealing a close-to-random genomic distribution in VSMCs. This study demonstrates for the first time the potential of IDLVs for safe and efficient vascular gene transfer

A Multi-Proxy Approach to Archaeobotanical Research: Archaic and Fremont Diets, Utah

New analytical techniques in archaeobotany allow researchers to examine human plant use by developing interrelated, yet independent lines of evidence. Here we outline the results of a two-method archaeobotanical approach to investigate Archaic and Fremont Great Basin diets. We conducted both macro- and microbotanical (starch granule) analyses at nine archaeological sites located in central and southwestern Utah. Our results show that in contexts where macrobotanical remains are poorly preserved, the application of microbotanical methods can produce additional sets of information, thus improving interpretations about past human diets. In this study, macrobotanical remains represented seed-based dietary contributions, while microbotanical remains came primarily from geophytes. Results suggest largely overlapping diets for Archaic and Fremont residents of Utah

Impact of gastric per‐oral endoscopic myotomy on static and dynamic pyloric function in gastroparesis patients

BackgroundFunctional Lumen Imaging Probe (EndoFLIP) tests typically measure static pyloric parameters, but the pylorus exhibits phasic variations on manometry. Dynamic changes in pyloric function have not been quantified using EndoFLIP, and the impact of Gastric Per‐Oral Endoscopic Myotomy (G‐POEM) on static and dynamic pyloric activity in gastroparesis is unknown.MethodsEndoFLIP balloon inflation to 30, 40, and 50 mL was performed to measure mean, maximum, and minimum values and variability in pyloric diameter and distensibility before and after G‐POEM in 20 patients with refractory gastroparesis. The impact of phasic contractions on these pyloric measures was compared.Key ResultsG‐POEM increased mean (P < .0001) and maximum (P = .0002) pyloric diameters and mean (P = .02) and maximum (P = .02) pyloric distensibility on 50 mL EndoFLIP inflation but not intraballoon pressures or minimum diameters or distensibility. Temporal variability of pyloric diameter (P = .02) and distensibility (P = .02) also increased after G‐POEM. Phasic coupled contractions propagating from the antrum through the pylorus were observed in 37.5% of recordings; other phasic activity including isolated pyloric contractions were seen in 23.3%. Variability of pyloric diameter and distensibility tended to be higher during recordings with phasic activity. Some pyloric responses to G‐POEM were influenced by age, gastroparesis etiology, gastric emptying, and prior botulinum toxin injection.Conclusions & InferencesPyloric activity exhibits dynamic changes on EndoFLIP testing in gastroparesis. G‐POEM increases maximal but not minimal diameter and distensibility with increased variations, suggesting this therapy enhances pyloric opening but may not impair pyloric closure. Phasic pyloric contractions contribute to variations in pyloric activity.We employed Functional Lumen Imaging Probe (EndoFLIP)tests toshowincreases in pyloric diameter and variability of diameter after gastricperoralendoscopicmyotomy(G‐POEM ingastroparesis patients (left graphs). Variability of pyloric activity was noted before and after G‐POEM which was partly due to propagated antropyloriccontractions (3‐D plot on right) detected by EndoFLIP.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163489/2/nmo13892_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163489/1/nmo13892.pd

Convergent Validity of a Single Question with Multiple Classification Options for Depression Screening in Medical Settings

The purpose of this study was to assess the convergent validity of a single depression question with multiple classification options for depression screening. Participants were 40 medical inpatients. The age range of our sample was 18 to 85 years (M = 56.15, SD = 17.66). A clinical interview and the BDI-II were administered. The correlation between patients’ self-rating classification of depression and their BDI-II classification was significant, rs(38) = .90, p < .01. Follow-up repeated-measures chi-square revealed a statistically significant association between BDI-II classification and patients’ self-rating classification, χ2(9, N = 40) = 47.79, p < .005. Significant positive standardized residuals revealed a clear linear relationship between BDI-II and patient self-rating classifications. Our data support the use of a single depression question with multiple classification options as a useful and valid means of quickly screening for the presence of depression by frontline health care professionals

Profiling of transcriptional and epigenetic changes during directed endothelial differentiation of human embryonic stem cells identifies FOXA2 as a marker of early mesoderm commitment

Introduction: Differentiation of vascular endothelial cells (ECs) in clinically relevant numbers for injection into ischaemic areas could offer therapeutic potential in the treatment of cardiovascular conditions, including myocardial infarction, peripheral vascular disease and stroke. While we and others have demonstrated successful generation of functional endothelial-like cells from human embryonic stem cells (hESCs), little is understood regarding the complex transcriptional and epigenetic changes that occur during differentiation, in particular during early commitment to a mesodermal lineage. Methods: We performed the first gene expression microarray study of hESCs undergoing directed differentiation to ECs using a monolayer-based, feeder-free and serum-free protocol. Microarray results were confirmed by quantitative RT-PCR and immunocytochemistry, and chromatin immunoprecipitation (ChIP)-PCR analysis was utilised to determine the bivalent status of differentially expressed genes. Results: We identified 22 transcription factors specific to early mesoderm commitment. Among these factors, FOXA2 was observed to be the most significantly differentially expressed at the hESC–EC day 2 timepoint. ChIP-PCR analysis revealed that the FOXA2 transcription start site is bivalently marked with histone modifications for both gene activation (H3K4me3) and repression (H3K27me3) in hESCs, suggesting the transcription factor may be a key regulator of hESC differentiation. Conclusion: This enhanced knowledge of the lineage commitment process will help improve the design of directed differentiation protocols, increasing the yield of endothelial-like cells for regenerative medicine therapies in cardiovascular disease

The CSF in neurosarcoidosis contains consistent clonal expansion of CD8 T cells, but not CD4 T cells

The tissue-specific drivers of neurosarcoidosis remain poorly defined. To identify cerebrospinal fluid (CSF) specific, antigen-driven T and B cell responses, we performed single-cell RNA sequencing of CSF and blood cells from neurosarcoid participants coupled to T and B cell receptor sequencing. In contrast to pulmonary sarcoidosis, which is driven by CD4 T cells, we found CD8 T cell clonal expansion enriched in the neurosarcoid CSF. These CSF-enriched CD8 T cells were composed of two subsets with differential expression of EBI2, CXCR3, and CXCR4. Lastly, our data suggest that IFNγ signaling may distinguish neurosarcoidosis from other neurological disorders

The Grizzly, September 25, 2003

The State of Iraq: Critical, but not Hopeless says Trudy Rubin • Family Day Celebrates Students\u27 Second Home • The Journey of a Lifetime • Aftermath of Isabel: A Photo Essay • SERV Serves Students During Campus Emergencies • Opinions: Common Sentiments About the Common Experience; UC Fashion: Mood-Based; People Want to Know the Real Truth; WTO Talks Collapse: Possibly a Good Thing; Out of the Middle East: Part 3 • Collegeville\u27s Hot Spots • Playwriting Debut • Meet the Star Among Us • London Living: A Warm City • The Outhouse Revisited: A Review • Field Hockey: Young Sets School Record in 5-0 win • Men\u27s Soccer: Continuing to Battle Against Tough Competition • UC Football: Albright Stomps the Bearshttps://digitalcommons.ursinus.edu/grizzlynews/1542/thumbnail.jp

The Grizzly, October 2, 2003

Sexual Assault Reported on Campus • Water Worries Burst in Reimert • Senior in the Market for Success • Tough Crowd: Bush Speaks to the United Nations • Opinions: Out of the Middle East, Part 4; Pharming: The Scariest New Drug Fad; To Tan or Not to Tan?; Maples-palooza! • Is Collegeville Boring? • Smart Shopping • CAB Events • The Glory of Ursinus: Bomberger Memorial Hall • Bears Obliterate Mt. Ida, 47-0 • Women\u27s Soccer Take Two • Volleyball Ends Losing Streak • Bears Overtake Quakers in Hockey Season Opener • Japanese Women Wrestlers • UC Field Hockey Team Continues Domination in C.C. Play • Ursinus XC Invades Celtic Fest 2003https://digitalcommons.ursinus.edu/grizzlynews/1543/thumbnail.jp

Discovery and targeting of a noncanonical mechanism of sarcoma resistance to ADI-PEG20 mediated by the microenvironment

PURPOSE: Many cancers lack argininosuccinate synthetase 1 (ASS1), the rate-limiting enzyme of arginine biosynthesis. This deficiency causes arginine auxotrophy, targetable by extracellular arginine-degrading enzymes such as ADI-PEG20. Long-term tumor resistance has thus far been attributed solely to ASS1 reexpression. This study examines the role of ASS1 silencing on tumor growth and initiation and identifies a noncanonical mechanism of resistance, aiming to improve clinical responses to ADI-PEG20. EXPERIMENTAL DESIGN: Tumor initiation and growth rates were measured for a spontaneous Ass1 knockout (KO) murine sarcoma model. Tumor cell lines were generated, and resistance to arginine deprivation therapy was studied in vitro and in vivo. RESULTS: Conditional Ass1 KO affected neither tumor initiation nor growth rates in a sarcoma model, contradicting the prevalent idea that ASS1 silencing confers a proliferative advantage. Ass1 KO cells grew robustly through arginine starvation in vivo, while ADI-PEG20 remained completely lethal in vitro, evidence that pointed toward a novel mechanism of resistance mediated by the microenvironment. Coculture with Ass1-competent fibroblasts rescued growth through macropinocytosis of vesicles and/or cell fragments, followed by recycling of protein-bound arginine through autophagy/lysosomal degradation. Inhibition of either macropinocytosis or autophagy/lysosomal degradation abrogated this growth support effect in vitro and in vivo. CONCLUSIONS: Noncanonical, ASS1-independent tumor resistance to ADI-PEG20 is driven by the microenvironment. This mechanism can be targeted by either the macropinocytosis inhibitor imipramine or the autophagy inhibitor chloroquine. These safe, widely available drugs should be added to current clinical trials to overcome microenvironmental arginine support of tumors and improve patient outcomes

Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis.

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43-positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the approximately 25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis