The safety of a 2'-O-methoxyethyl antisense oligonucleotide (ASO) was investigated in Mauritius cynomolgus monkeys in a 41-week Good Laboratory Practice (GLP) toxicity study after multiple intrathecal (IT) administrations. Histopathological examination revealed ectopic formation of lymphoid follicles in the spinal cord (SC) at the injection site at all doses and the presence of granular material in neurons of the SC in high-dose animals. The granular material was seen in all the segments of the SC, but mainly in the lumbar segment and persisted at the end of the 26-week recovery period, while the lymphoid follicles showed a reversibility trend. Findings associated with repeated IT administration of ASOs have been described in nonhuman primate (NHP) toxicity studies, specifically in the brain, but findings in the SC are rarely reported. In the present study, we report a high incidence of findings in the SC compared to brain, especially in the lumbar segment in proximity to IT injection sites. An extensive panel of immunohistochemistry markers showed that the ectopic lymphoid follicle formation (LFF) had a cellular composition and organization consistent with tertiary lymphoid structure (TLS) without associated axonal damage in the adjacent nervous tissue. In situ hybridization with an miRNA probe complementary to the ASO revealed that the granular material represented a dose-dependent ASO accumulation in the cytoplasm of neurons without inducing cell death or apoptosis. Glial and ependymal cells in the SC also showed dose-dependent accumulation of the ASO preceding detection of granular material by hematoxylin and eosin (H&E). Based on these molecular localization data, the presence of LFF in SC suggests a chronic local immune activation. Considering the absence of neuronal dysfunction or injury and transient clinical signs previously reported with other 2'-MOE ASOs, the presence of TLS and ASO was considered non-adverse.

Peripheral nervous system (PNS) toxicity assessment in non-clinical safety studies is challenging and relies mostly on histopathological assessment. The present work aims to identify blood-based biomarkers that could detect peripheral neuropathy in rats upon exposure to neurotoxic compounds. Three anticancer agents (oxaliplatin, cisplatin, paclitaxel) and a developmental compound (NVS-1) were assessed in male rats (Wistar Han). Clinical and/or functional endpoints (i.e., electronic Von Frey, Cold Plate, and Paw Pressure tests) and blood biomarkers (i.e., neurofilament light chain (NfL), neurofilament heavy chain (NF-H), microtubule-associated protein Tau (Tau), neuron specific enolase (NSE), vascular endothelial growth factor A (VEGFA), and glial fibrillary acidic protein (GFAP)) were assessed. Drug exposure and histopathological evaluations were conducted on selected nervous tissues. Oxaliplatin, cisplatin and paclitaxel treatment resulted in a significant decrease of nociceptive thresholds. Clinical signs suggestive of PNS toxicity were observed with NVS-1. NfL was consistently increased in the NVS-1 study and correlated with moderate microscopic findings in dorsal root ganglia (DRG). Only minimal microscopic findings were observed in oxaliplatin-treated animals, whereas no treatment-related microscopic findings were observed in animals treated with cisplatin and paclitaxel. For all compounds, exposure was confirmed in the PNS tissues. Clinical and functional changes were observed with all the compounds evaluated. NfL levels in plasma proved to be the most sensitive indicator of PNS toxicities, capturing moderate nervous degeneration in DRG. A combined approach that includes both functional assessments and biomarker measurements offers a more comprehensive evaluation than histopathological analysis alone when monitoring drug-induced neurotoxicity in rat models.

In nonhuman primates (NHPs), adeno-associated virus serotype 9 (AAV9) vectorized gene therapy can cause asymptomatic microscopic injury to dorsal root ganglia (DRG) and trigemiment light chain (NfL) to diffuse into cerebrospinal fluid (CSF) and blood. Data from 260 cynomolgus macaques administered vehicle or AAV9 vectors (intrathecally or intravenously) were analyzed to investigate NfL as a soluble biomarker for monitoring DRG/TG microscopic findings. The incidence of key DRG/TG findings with AAV9 vectors was 78% (maximum histopathology severity, moderate) at 2-12 weeks after the dose. When examined up to 52 weeks after the dose, the incidence was 42% (maximum histopathology severity, minimal). Terminal NfL concentrations in plasma, serum, and CSF correlated with microscopic severity. After 52 weeks, NfL returned to pre-dose baseline concentrations, correlating with microscopic findings of lesser incidence and/or severity compared with interim time points. Blood and CSF NfL concentrations correlated with asymptomatic DRG/TG injury, suggesting that monitoring serum and plasma concentrations is as useful for assessment as more invasive CSF sampling. Longitudinal assessment of NfL concentrations related to microscopic findings associated with AAV9 administration in NHPs indicates NfL could be a useful biomarker in nonclinical toxicity testing. Caution should be applied for any translation to humans.

To better understand the pathogenetics of pseudoxanthoma elasticum (PXE), we performed a mutational analysis of ATP-binding cassette subfamily C member 6 (ABCC6) in 122 unrelated patients with PXE, the largest cohort of patients yet studied. Thirty-six mutations were characterized, and, among these, 28 were novel variants (for a total of 43 PXE mutations known to date). Twenty-one alleles were missense variants, six were small insertions or deletions, five were nonsense, two were alleles likely to result in aberrant mRNA splicing, and two were large deletions involving ABCC6. Although most mutations appeared to be unique variants, two disease-causing alleles occurred frequently in apparently unrelated individuals. R1141X was found in our patient cohort at a frequency of 18.8% and was preponderant in European patients. ABCC6del23-29 occurred at a frequency of 12.9% and was prevalent in patients from the United States. These results suggested that R1141X and ABCC6del23-29 might have been derived regionally from founder alleles. Putative disease-causing mutations were identified in approximately 64% of the 244 chromosomes studied, and 85.2% of the 122 patients were found to have at least one disease-causing allele. Our results suggest that a fraction of the undetected mutant alleles could be either genomic rearrangements or mutations occurring in noncoding regions of the ABCC6 gene. The distribution pattern of ABCC6 mutations revealed a cluster of disease-causing variants within exons encoding a large C-terminal cytoplasmic loop and in the C-terminal nucleotide-binding domain (NBD2). We discuss the potential structural and functional significance of this mutation pattern within the context of the complex relationship between the PXE phenotype and the function of ABCC6.

SESSION TITLE: Critical Care Posters II SESSION TYPE: Original Investigation Poster PRESENTED ON: Wednesday, October 28, 2015 at 01:30 PM - 02:30 PM PURPOSE: Airway pressures are often elevated in morbidly obese patients when they are mechanically ventilated. The current standard of practice is to limit ventilator plateau pressure (PPLAT) to less than 30 cmH20 to prevent ventilation induced lung injury (VILI) and mechanical complications such as pneumothorax. In obese patients PPLAT may not be an accurate predictor of transpulmonary pressure as their pleural pressure is variable. EPM provides a more accurate estimate of transpulmonary pressure and more accurately informs the titration of Positive Pressure Ventilation. METHODS: In this retrospective study, we reviewed the charts of all mechanically ventilated adult patients in whom esophageal pressure monitoring was performed from April 2010 to February 2014. 26 patients had esophageal pressure monitoring. Full data sets were recorded for 17 patients and were included in the study. Their age, body mass index (BMI), as well as the tidal volume, plateau pressure, PEEP, esophageal pressure, intra-abdominal pressure and chest compliance were recorded. A linear regression analysis was performed. RESULTS: The mean and standard deviation of BMI, chest wall compliance, PPLAT, PEEP and tidal volumes were 44.5+ 15, 130+97 ml/cmH2O, 40+9.9 cmH2O, 22+6 cmH2O, 322+114 ml respectively. There was no correlation between intra-abdominal pressures and transpulmonary pressure or between chest wall compliance and body mass index. The P values on both sides were 0.47 and 0.67 respectively. The P value for transpulmonary pressure and plateau pressure was 0.0051 and it was significant. CONCLUSIONS: Morbidly obese patients with elevated plateau pressure demonstrated variable chest wall compliance. BMI and intra-abdominal pressure had no correlation with transpulmonary pressure (as estimated via EPM), Pplat significantly overestimated transpulmonary pressure, and therefore overestimates the risk of VILI. CLINICAL IMPLICATIONS: In morbidly obese patients with elevated Pplat, low dynamic and static lung compliance and/or difficulty oxygenating, EPM has value in more accurately assessing transpulmonary pressures, assessing compliance curves, and in titrating pressure-dependent ventilator settings. DISCLOSURE: The following authors have nothing to disclose: Arul Chandran, Stevan Whitt, Troy Whitacre, Yuji Oba, Eric Johnson, Joe Devasahayam, Fahad Omar No Product/Research Disclosure Information

The authors describe a geometric layout of quantum-dot cellular automata (QCA) cells and an associated set of clock signals that can be used to implement a programmable array of logic (PAL). PALs are an important category of programmable logic that can be programmed (typically once) to perform a particular sum-of-products Boolean operation. The particular device described has six inputs, four product terms, and one output. The connections between the inputs and the product terms are fully reprogrammable, while the connections between the product terms and the output are hardwired. This device takes 22 clock cycles to load the connection data and then completes the calculation in 57 cycles. The connection data are preserved in memory, so additional calculations with the same set of connections can be performed in just 57 cycles each.

This paper outlines the use of a Model-Eliciting Activity (MEA) in a logic design course that introduces students to design through an authentic client-driven problem. The MEA involved creating a model of a traffic signal controller for an intersection of a U.S. highway and a county road near the university. Students were given background information on traffic signal controllers and actual traffic flow data for the intersection and were then asked to analyze the data, transform the problem to the digital design space, and create a state diagram for a traffic signal controller that would be effective for the given intersection. The results using the MEA in the course were very encouraging. All seven groups in the class created valid traffic signal controller state diagrams and six of the seven groups were able to create working implementations in a programmable logic device. In addition, on the course's final exam, a similar modeling problem was included and the students who participated in the MEA scored higher on the problem than last year's students that did not have the MEA experience.

Cerebral cavernous malformations (CCMs) are vascular anomalies of the central nervous system, comprising dilated blood-filled capillaries lacking structural support. The lesions are prone to rupture, resulting in seizures or hemorrhagic stroke. CCM can occur sporadically, manifesting as solitary lesions, but also in families, where multiple lesions generally occur. Familial cases follow autosomal-dominant inheritance due to mutations in one of three genes, CCM1/KRIT1, CCM2/malcavernin or CCM3/PDCD10. The difference in lesion burden between familial and sporadic CCM, combined with limited molecular data, suggests that CCM pathogenesis may follow a two-hit molecular mechanism, similar to that seen for tumor suppressor genes. In this study, we investigate the two-hit hypothesis for CCM pathogenesis. Through repeated cycles of amplification, subcloning and sequencing of multiple clones per amplicon, we identify somatic mutations that are otherwise invisible by direct sequencing of the bulk amplicon. Biallelic germline and somatic mutations were identified in CCM lesions from all three forms of inherited CCMs. The somatic mutations are found only in a subset of the endothelial cells lining the cavernous vessels and not in interstitial lesion cells. These data suggest that CCM lesion genesis requires complete loss of function for one of the CCM genes. Although widely expressed in the different cell types of the brain, these data also suggest a unique role for the CCM proteins in endothelial cell biology.

We introduce a vertical carbon nanotube capacitor with high capacitance per unit area. Using an electrical model of single-walled, metallic carbon nanotubes and the extracted capacitance values of a carbon nanotube bundle network, we develop an electrical model for the capacitor. The device can exhibit a capacitance greater than 175fF/mum 2 .

Retaining first-year engineering students is an area of concern for many colleges and universities and has recently received even more attention. While time can be invested in implementing strong first-year courses and general support programs, the fact still remains that some students will leave engineering during the first year without truly experiencing the possibilities that engineering has to offer them. While retaining traditional students is challenging, the rapidly changing demographics of the students pursuing engineering degrees adds a whole new dimension to the problem. The additional needs of these international and minority student are difficult to predict and are often different than those that have been commonly observed. The cultural differences alone can often make traditional support programs ineffective. Increasing student persistence in the field of engineering for all students is a complex task that has no obvious solution. One approach is to find the factors that have the greatest impact on student success and to support all students in these areas. Some factors can include academic preparation, interest in engineering topics, additional activities, study habits, and sense of community. The purpose of this study was to determine these primary factors that affected students leaving the field of engineering to pursue other careers. A student survey of first-year students at midterm of the first semester was implemented to collect information on the 2008 cohort. The survey concentrated on student confidence in various areas, study habits, campus activities, and sense of community. This paper will describe the study of how academic preparation and survey results correlate with the retention of these students.

Cerebral cavernous malformations (CCMs) are vascular abnormalities of the brain that can result in hemorrhagic stroke and seizures. Familial forms of CCM are inherited in an autosomal-dominant fashion, and three CCM genes have been identified. We recently determined that large genomic deletions in the CCM2 gene represent 22% of mutations in a large CCM cohort from the USA. In particular, a 77.6 kb deletion spanning CCM2 exons 2-10 displays an identical recombination event in eight CCM probands/families and appears to be common in the US population. In the current study, we report the identification of six additional probands/families from the USA with this same large deletion. Haplotype analysis strongly suggests that this common deletion derives from an ancestral founder. We also examined an Italian CCM cohort consisting of 24 probands/families who tested negative for mutations in the CCM1, CCM2, and CCM3 genes by DNA sequence analysis. Surprisingly, the common CCM2 deletion spanning exons 2-10 is not present in this population. Further analysis of the Italian cohort by multiplex ligation-dependent probe analysis identified a total of ten deletions and one duplication. The overall spectrum of genomic rearrangements in the Italian cohort is thus quite different than that seen in a US cohort. These results suggest that there are elements within all three of the CCM genes that predispose them to large deletion/duplication events but that the common deletion spanning CCM2 exons 2-10 appears to be specific to the US population due to a founder effect.

Teaching probability and statistics to undergraduate engineering students is a challenge because of the theoretical nature of the topic. There are many advantages to introducing this topic to first-year students. Students who have seen and worked with topics in probability and statistics are more likely to understand these principles when they are used in later courses, and they more clearly understand the relationship of statistics to their disciplinary coursework. In this paper we describe how probability and statistics can be introduced within a first-semester engineering course. A modular approach was used, which included a lecture with a number of active learning exercises, a homework assignment, and a hands-on experiment to help them better understand the theoretical background. Assessment results illustrate that after the module, the students believed they could meet all of the modulepsilas learning objectives and that each of the learning opportunities contributed to their achievement of the objectives.

Cerebral cavernous malformations (CCMs) are vascular abnormalities of the brain that can result in a variety of neurological disabilities, including hemorrhagic stroke and seizures. Mutations in the gene KRIT1 are responsible for CCM1, mutations in the gene MGC4607 are responsible for CCM2, and mutations in the gene PDCD10 are responsible for CCM3. DNA sequence analysis of the known CCM genes in a cohort of 63 CCM-affected families showed that a high proportion (40%) of these lacked any identifiable mutation. We used multiplex ligation-dependent probe analysis to screen 25 CCM1, -2, and -3 mutation-negative probands for potential deletions or duplications within all three CCM genes. We identified a total of 15 deletions: 1 in the CCM1 gene, 0 in the CCM3 gene, and 14 in the CCM2 gene. In our cohort, mutation screening that included sequence and deletion analyses gave disease-gene frequencies of 40% for CCM1, 38% for CCM2, 6% for CCM3, and 16% with no mutation detected. These data indicate that the prevalence of CCM2 is much higher than previously predicted, nearly equal to CCM1, and that large genomic deletions in the CCM2 gene represent a major component of this disease. A common 77.6-kb deletion spanning CCM2 exons 2-10 was identified, which is present in 13% of our entire CCM cohort. Eight probands exhibit an apparently identical recombination event in the CCM2 gene, involving an AluSx in intron 1 and an AluSg distal to exon 10. Haplotype analysis revealed that this CCM2 deletion occurred independently at least twice in our families. We hypothesize that these deletions occur in a hypermutable region because of surrounding repetitive sequence elements that may catalyze the formation of intragenic deletions.

Cerebral cavernous malformations (CCMs) are vascular abnormalities of the brain that can result in a variety of neurological disabilities, including stroke and seizures. Linkage analyses using autosomal dominant families manifesting CCMs have identified three different causative loci on chromosomes 7q21.2 (CCM1), 7p13 (CCM2), and 3q25.2-q27 (CCM3). Mutations in the gene Krit1 are responsible for CCM1, mutations in the gene MGC4607 are responsible for CCM2, and mutations in the gene PDCD10 were recently reported to be responsible for CCM3. We report here that sequence analysis of PDCD10 in a panel of 29 probands lacking Krit1 and MGC4607 mutations revealed only three mutations. The frequency of identified mutations in the PDCD10 gene was surprisingly low, especially given that this panel was heavily biased towards non-CCM1, non-CCM2 probands. These data are in stark contrast with the linkage data, which suggests that 40% of inherited cases would be due to mutations in this gene. Interestingly, when examining the haplotypes of previously published CCM3 families, we found a distinct recombination event in one of the largest CCM3 families that excludes the PDCD10 gene. Although there are many potential explanations for this observation, when combined with the apparent under-representation of causative CCM mutations in PDCD10, this recombination event in a CCM3-linked family suggests that there may be an additional CCM gene in the same chromosomal region.

In the mammalian testis, the germ line stem cells are a small subpopulation of type A spermatogonia that pro liferate and ultimately differentiate into sperm under the control of both endocrine and paracrine factors. To study the early phases of spermatogenesis at the molecular level, an in vitro system must be devised whereby germ line stem cells can be either cultured for a prolonged period of time or expanded as cell lines. In the study reported here, we chose to immortalize type A spermatogonia using the Simian virus large T-antigen gene (LTAg) under the control of an ecdysone-inducible promoter. While the cells escaped the hormonal control after a finite number of generations and expressed the LTAg constitutively, their growth remained slow and the cells exhibited morphological features typical of spermatogonia at the light microscopic level. Moreover, the cells expressed detectable levels of protein markers specific for germ cells such as Dazi, and specific for germ line stem cells such as Oct-4, a transcription factor, and GFRalpha-1, the receptor for glial cell line-derived neurotrophic factor (GDNF). Further analysis confirmed the spermatogonial phenotype and also revealed the expression of markers expressed in stem cells such as Piwi12 and Prame11. Since the cells respond to GDNF by a marked increase in their rate of proliferation, this cell line represents a good in vitro model for studying aspects of mouse germ line stem cell biology.

Most applications of distance learning involve an instructor at a central, technology-rich location delivering a virtual classroom experience to students at one or more remote sites. However, there are many occasions when instructors need to be away from campus to attend conferences, perform on-site research projects, or administer off-campus academic programs, and they may need to remotely teach from a technology-poor environment (such as a hotel room or an apartment) back to a centralized location such as a campus classroom. During the spring semester of 2005, the electrical and computer engineering department at Valparaiso University conducted just such a course, which was taught by a member of the department who was spending the semester in Reutlingen, Germany. The authors describe the technology used to make this connection, appropriate course policies related to examinations, homework, and design projects, and unique challenges and opportunities presented by the nature of such a course