Solution Cathode Glow Discharge Coupled to Atmospheric Pressure Chemical Ionization for Elemental Detection of S and P in Organic Compounds.

We report a post-plasma chemical ionization approach to evaluate solution cathode glow discharge (SCGD) for S and P elemental analysis. Here, the SCGD serves as a reactor to produce chemical vapors for S and P from organic compounds containing these elements, while a corona discharge operated in negative mode is used to ionize the products. The approach creates long-lived ions in atmospheric pressure, enabling direct investigation of chemical vapor products via mass spectrometric and ion mobility separations. The investigations indicate that SCGD converts S and P to H2SO4 and H3PO4, respectively. These species are then ionized as HSO4HNO3 - and H3PO4NO3HNO3- via reactions with NO3HNO3- produced by corona discharge. The response factors for P among several small molecules varies within 10% of the average response from the compounds, suggesting a reasonable species-independent characteristic. The response factors for S show larger variations among compounds, indicating a higher dependence of chemical vapor generation efficiency on analytes' chemical structures. Detection limits of 15 and 29 ng/mL are achieved for P and S detection, respectively. These figures are limited by background equivalent concentrations and low ion flux in the utilized ion mobility-time of flight mass spectrometer, indicating potential for significant improvements. In particular, the specificity of clustering for S and P-containing ions produced in this approach suggest facile analysis of S and P using quadrupole-based mass spectrometers for improved analytical performance.

Mechanism of Insoluble Aggregate Formation in a Reconstituted Solution of Spray-Dried Protein Powder.

BACKGROUND: Spray-drying is considered a promising alternative drying method to lyophilization (freeze-drying) for therapeutic proteins. Particle counts in reconstituted solutions of dried solid dosage forms of biologic drug products are closely monitored to ensure product quality. We found that high levels of particles formed after reconstitution of protein powders that had been spray-dried under suboptimal conditions. METHODS: Visible and subvisible particles were evaluated. Soluble proteins in solution before spray-drying and in the reconstituted solution of spray-dried powder were analyzed for their monomer content levels and melting temperatures. Insoluble particles were collected and analyzed by Fourier transform infrared microscopy (FTIR), and further analyzed with hydrogen-deuterium exchange (HDX). RESULTS: Particles observed after reconstitution were shown not to be undissolved excipients. FTIR confirmed their identity as proteinaceous in nature. These particles were therefore considered to be insoluble protein aggregates, and HDX was applied to investigate the mechanism underlying aggregate formation. Heavy-chain complementarity-determining region 1 (CDR-1) in the aggregates showed significant protection by HDX, suggesting CDR-1 was critical for aggregate formation. In contrast, various regions became more conformationally dynamic globally, suggesting the aggregates have lost protein structural integrity and partially unfolded after spray-drying. DISCUSSION: The spray-drying process could have disrupted the higher-order structure of proteins and exposed the hydrophobic residues in CDR-1 of the heavy chain, contributing to the formation of aggregate through hydrophobic interactions upon reconstitution of spray-dried powder. These results can contribute to efforts to design spray-dry resilient protein constructs and improve the robustness of the spray-drying process.

High-Resolution Elemental Mass Spectrometry Using LC-ICP-Nanospray-Orbitrap for Simultaneous and Species-Independent Quantitation of Fluorinated and Chlorinated Compounds.

Simultaneous elemental detection of F and Cl offers quantitation of fluorinated and chlorinated compounds and their transformation products without compound-specific standards. Despite wide-ranging applications, this capability has been hindered by fundamental and technical shortcomings of current inductively coupled plasma (ICP)-MS methods in ion formation and isobaric interference elimination. These hurdles are alleviated here via a chemical ionization method. Fluorine and chlorine in analytes are first converted to HF and HCl by an ICP with post-plasma recombination and subsequently react with barium-containing ions supplied by a nanospray, yielding BaF+ and BaCl+ as elemental reporter ions. Notably, the method is readily interfaced to an Orbitrap MS which eliminates isobaric interferences at resolving powers as low as 35,000, far greater than that of current ICP-MS instruments. Moreover, the instrument is easily reverted to the ESI-MS mode for complementary molecular characterization. To demonstrate analytical capabilities, a workflow for rapid quantitation of compounds separated by reversed-phase liquid chromatography is developed using a species-independent calibration. The independent F and Cl measurements agree with each other, providing recoveries of >90% and LODs of 8-12 pmol Cl and 5-12 pmol F on the column. The workflow along with LC-ESI-MS on the same instrument is then applied to identify and quantify in-vitro drug metabolites, yielding total drug-related material recoveries of >80% and quantitation of minor metabolites summing to 8% of the total drug-related compounds. These results highlight the strengths of simultaneous F and Cl speciation for rapid quantitation with applications in early drug development.

The impact of the wake maintenance zone on attentional capacity, physiological drowsiness, and subjective task demands during sleep deprivation.

We aimed to investigate the impact of the Wake Maintenance Zone (WMZ) on measures of drowsiness, attention, and subjective performance under rested and sleep deprived conditions. We studied 23 healthy young adults (18 males; mean age = 25.41 ± 5.73 years) during 40 hr of total sleep deprivation under constant routine conditions. Participants completed assessments of physiological drowsiness (EEG-scored slow eye movements and microsleeps), sustained attention (PVT), and subjective task demands every two hours, and four-hourly ocular motor assessment of inhibitory control (inhibition of reflexive saccades on an anti-saccade task). Tests were analyzed relative to dim light melatonin onset (DLMO); the WMZ was defined as the 3 hr prior to DLMO, and the preceding 3 hr window was deemed the pre-WMZ. The WMZ did not mitigate the adverse impact of ~37 hr sleep deprivation on drowsiness, sustained attention, response inhibition, and self-rated concentration and difficulty, relative to rested WMZ performance (~13 hr of wakefulness). Compared to the pre-WMZ, though, the WMZ improved measures of sustained attention, and subjective concentration and task difficulty, during sleep deprivation. Cumulatively, these results expand on previous work by characterizing the beneficial effects of the WMZ on operationally-relevant indices of drowsiness, inhibitory attention control, and self-rated concentration and task difficulty relative to the pre-WMZ during sleep deprivation. These results may inform scheduling safety-critical tasks at more optimal circadian times to improve workplace performance and safety.

Perturbation-induced high-frequency pulsing of nano-ESI with facile ion selection at atmospheric pressure.

Nano-ESI is a commonly used ionization technique with continually expanding analytical advantages. Here, we report a facile way for high-frequency (500-3800 Hz) pulsing of nano-ESI, providing a high flux of mobility-selected ions. The pulsing is accomplished using a relatively low-voltage modulation (80 V peak-to-peak) of an electrode placed <1 cm downstream of a nano-ESI emitter biased to a constant potential. Configuring the electrode as an ion gate enables mobility-based ion selection by scanning the modulation frequency. Our investigations indicate that the electrode modulation perturbs continuous nano-ESI, resulting in solution accumulation at the emitter tip between spray pulses. Selective transmission of ions occurs at frequencies corresponding to harmonics of a fundamental frequency determined by the travel time of each ion from the emitter to the ion gate (pulsing electrode). Remarkably, the intensities of ions selected in this fashion are similar across the harmonics, suggesting that the ionization efficiencies of analytes have minimal dependence on the accumulated volume at the emitter tip. Moreover, intensities of ion-mobility-selected analytes using this technique reach >50% of those in continuous nano-ESI without ion selection, underscoring efficient ion generation via high-frequency pulsing. These findings indicate the potential of the pulsed nano-ESI for enhanced analytical utility, such as a high-flux selected-reagent-ion supplier at atmospheric pressure, and chart new avenues to further enhance the analytical performance of nano-ESI.

Performance of a prototype stationary catadioptric concentrating photovoltaic module.

A stationary catadioptric concentrating photovoltaic module with aperture area over 100 cm2, geometric concentration of 180×, and collection within 60° of polar incidence was designed, prototyped, and characterized. The module performance followed modeling closely with a peak power conversion efficiency of 26% for direct irradiance. Tracking of the sun is accomplished via translational micro-tracking completely internal to the module, avoiding the cost and complexity of mechanical two-axis trackers that point towards the sun. This demonstrates the potential for concentrating photovoltaic modules with significantly higher efficiency than industry standard silicon photovoltaic modules that could be installed in stationary configurations on rooftops.

Pulsed Nano-ESI Atmospheric-Pressure Ion Mobility Mass Spectrometry with Enhanced Ion Sampling.

Ion mobility-mass spectrometry (IM-MS) has gained considerable attention for detection of clusters and weakly bound species created by electrospray ionization (ESI). Atmospheric-pressure (AP) IM-MS offers an advantage in these studies compared to its low-pressure counterpart, owing to soft introduction of ions into the mobility cell with minimal ion activation. Here, we report new approaches to improve the sensitivity and soft ion introduction in AP-IM-MS. For the former, we demonstrate enhanced aerodynamic sampling of ions from the mobility cell into the MS using pulsed-field sampling. In this approach, ions are driven toward the MS, and the field is shut down once the ions reach the vicinity of the MS inlet orifice. The pulsed-field operation provides arrival times without the need for an exit ion gate in the mobility cell and leads to improvements in sensitivity of up to 1 order of magnitude. For soft ion generation, we report a pulsed nano-ESI source to introduce a packet of ions into the room-temperature mobility cell without induced desolvation. Further, we demonstrate the application of the pulsed nano-ESI AP-IM-MS with enhanced ion sampling for detection of solvent clusters of amines and peptide aggregates.

Acute air pollution exposure and risk of suicide completion.

Research into environmental factors associated with suicide has historically focused on meteorological variables. Recently, a heightened risk of suicide related to short-term exposure to airborne particulate matter was reported. Here, we examined the associations between short-term exposure to nitrogen dioxide, particulate matter, and sulfur dioxide and completed suicide in Salt Lake County, Utah (n = 1,546) from 2000 to 2010. We used a time-stratified case-crossover design to estimate adjusted odds ratios for the relationship between suicide and exposure to air pollutants on the day of the suicide and during the days preceding the suicide. We observed maximum heightened odds of suicide associated with interquartile-range increases in nitrogen dioxide during cumulative lag 3 (average of the 3 days preceding suicide; odds ratio (OR) = 1.20, 95% confidence interval (CI): 1.04, 1.39) and fine particulate matter (diameter ≤2.5 µm) on lag day 2 (day 2 before suicide; OR = 1.05, 95% CI: 1.01, 1.10). Following stratification by season, an increased suicide risk was associated with exposure to nitrogen dioxide during the spring/fall transition period (OR = 1.35, 95% CI: 1.09, 1.66) and fine particulate matter in the spring (OR = 1.28, 95% CI: 1.01, 1.61) during cumulative lag 3. Findings of positive associations between air pollution and suicide appear to be consistent across study locations with vastly different meteorological, geographical, and cultural characteristics.

Common genetic variants on 5p14.1 associate with autism spectrum disorders.

Autism spectrum disorders (ASDs) represent a group of childhood neurodevelopmental and neuropsychiatric disorders characterized by deficits in verbal communication, impairment of social interaction, and restricted and repetitive patterns of interests and behaviour. To identify common genetic risk factors underlying ASDs, here we present the results of genome-wide association studies on a cohort of 780 families (3,101 subjects) with affected children, and a second cohort of 1,204 affected subjects and 6,491 control subjects, all of whom were of European ancestry. Six single nucleotide polymorphisms between cadherin 10 (CDH10) and cadherin 9 (CDH9)-two genes encoding neuronal cell-adhesion molecules-revealed strong association signals, with the most significant SNP being rs4307059 (P = 3.4 x 10(-8), odds ratio = 1.19). These signals were replicated in two independent cohorts, with combined P values ranging from 7.4 x 10(-8) to 2.1 x 10(-10). Our results implicate neuronal cell-adhesion molecules in the pathogenesis of ASDs, and represent, to our knowledge, the first demonstration of genome-wide significant association of common variants with susceptibility to ASDs.

Autism genome-wide copy number variation reveals ubiquitin and neuronal genes.

Autism spectrum disorders (ASDs) are childhood neurodevelopmental disorders with complex genetic origins. Previous studies focusing on candidate genes or genomic regions have identified several copy number variations (CNVs) that are associated with an increased risk of ASDs. Here we present the results from a whole-genome CNV study on a cohort of 859 ASD cases and 1,409 healthy children of European ancestry who were genotyped with approximately 550,000 single nucleotide polymorphism markers, in an attempt to comprehensively identify CNVs conferring susceptibility to ASDs. Positive findings were evaluated in an independent cohort of 1,336 ASD cases and 1,110 controls of European ancestry. Besides previously reported ASD candidate genes, such as NRXN1 (ref. 10) and CNTN4 (refs 11, 12), several new susceptibility genes encoding neuronal cell-adhesion molecules, including NLGN1 and ASTN2, were enriched with CNVs in ASD cases compared to controls (P = 9.5 x 10(-3)). Furthermore, CNVs within or surrounding genes involved in the ubiquitin pathways, including UBE3A, PARK2, RFWD2 and FBXO40, were affected by CNVs not observed in controls (P = 3.3 x 10(-3)). We also identified duplications 55 kilobases upstream of complementary DNA AK123120 (P = 3.6 x 10(-6)). Although these variants may be individually rare, they target genes involved in neuronal cell-adhesion or ubiquitin degradation, indicating that these two important gene networks expressed within the central nervous system may contribute to the genetic susceptibility of ASD.

Surface structured optical coatings with near-perfect broadband and wide-angle antireflective properties.

Optical thin-film coatings are typically limited to designs where the refractive index varies in only a single dimension. However, additional control over the propagation of incoming light is possible by structuring the other two dimensions. In this work, we demonstrate a three-dimensional surface structured optical coating that combines the principles of thin-film optical design with bio-inspired nanostructures to yield near-perfect antireflection. Using this hybrid approach, we attain average reflection losses of 0.2% on sapphire and 0.6% on gallium nitride for 300-1800 nm light. This performance is maintained to very wide incidence angles, achieving less than 1% reflection at all measured wavelengths out to 45° for sapphire. This hybrid design has the potential to significantly enhance the broadband and wide-angle properties for a number of optical systems that require high transparency.

Design of antireflective nanostructures and optical coatings for next-generation multijunction photovoltaic devices.

The successful development of multijunction photovoltaic devices with four or more subcells has placed additional importance on the design of high-quality broadband antireflection coatings. Antireflective nanostructures have shown promise for reducing reflection loss compared to the best thin-film interference coatings. However, material constraints make nanostructures difficult to integrate without introducing additional absorption or electrical losses. In this work, we compare the performance of various nanostructure configurations with that of an optimized multilayer antireflection coating. Transmission into a four-junction solar cell is computed for each antireflective design, and the corresponding cell efficiency is calculated. We find that the best performance is achieved with a hybrid configuration that combines nanostructures with a multilayer thin-film optical coating. This approach increases transmitted power into the top subcell by 1.3% over an optimal thin-film coating, corresponding to an increase of approximately 0.8% in the modeled cell efficiency.

Effect of neuronal nicotinic acetylcholine receptor genes (CHRN) on longitudinal cigarettes per day in adolescents and young adults.

INTRODUCTION: Few studies have sought to identify specific genetic markers associated with cigarettes per day (CPD) during adolescence and young adulthood, the period of greatest vulnerability for the development of nicotine dependence. METHODS: We used a longitudinal design to investigate the effect of neuronal nicotinic acetylcholine receptor (CHRN) subunit genes on CPD from 15 to 21 years of age in young smokers of European descent (N = 439, 59% female). The number of CPD typically smoked during the previous 30 days was self-reported. Single nucleotide polymorphisms (SNPs) from CHRN genes were genotyped using DNA extracted from saliva samples collected at the 5-year assessment. Mixed-model analyses of SNP effects were computed across age at the time of assessment using log-transformed CPD as the phenotype. Data from the 1000 Genomes Project were used to clarify the architecture of CHRN genes to inform SNP selection and interpretation of results. RESULTS: CPD was associated with a CHRNB3A6 region tagged by rs2304297, with CHRNA5A3B4 haplotype C (tagged by rs569207), and with the CHRNA2 SNP rs2271920, ps < .004. The reliability of single-SNP associations was supported by the correspondence between a more extensive set of SNP signals and the underlying genetic architecture. The 3 signals identified in this study appear to make independent contributions to CPD, and their combined effect accounts for 5.5% of the variance in log-transformed CPD. CONCLUSIONS: Level of CPD during adolescence and young adulthood is associated with CHRNB3A6, CHRNA5A3B4, and CHRNA2.

Utility of live/real time three-dimensional transesophageal echocardiography in the assessment and percutaneous intervention of bioprosthetic pulmonary valve stenosis.

Accurate echocardiographic evaluation of the pulmonary valve is technically difficult because of its close proximity to the left lung, which often limits decision making. Pulmonary valvotomy is the intervention of choice for symptomatic pulmonary valve stenosis, but fluoroscopy lacks appropriate real time anatomic detail. In this report, we present a case where direct imaging of the pulmonary valve with live/real time three-dimensional transesophageal echocardiography (3DTEE) aided in accurate evaluation and was then used to help guide and monitor successful valvuloplasty of a stenotic pulmonary valve bioprosthesis. We demonstrate that even in cases where two-dimensional (2D) evaluation of the pulmonary valve is difficult, the use of live/real time 3DTEE allows for accurate evaluation of bioprosthetic pulmonary valve structure and function, and enhances the precision and monitoring of percutaneous valvuloplasty.

Live/real time three-dimensional transesophageal echocardiographic assessment of the spinal cord.

We present an adult patient in whom live/real time three-dimensional transesophageal echocardiography (3DTEE) provided incremental value in the assessment of the spinal cord as compared to two-dimensional transesophageal echocardiographic (2DTEE) findings published in the literature. It improved accurate identification and assessment of the anterior radiculomedullary spinal arteries which may have an important clinical application in monitoring for spinal cord ischemia during thoracic aortic surgery. Because the spinal cord and spinal canal could be examined using not only transverse but also coronal (frontal), sagittal, and oblique planes, 3DTEE further allowed for three-dimensional measurements of the dimensions and volumetric analysis of the visualized spinal cord and spinal canal. These may have implications in the assessment of spinal cord edema due to trauma and other conditions which result in increase in the size and volume of the spinal cord.

Ultrasound- versus landmark-guided femoral catheterization in the pediatric catheterization laboratory: a randomized-controlled trial.

Ultrasound (US) is the standard of care for vascular access in many clinical scenarios. Limited data exist regarding the benefits of US- versus landmark (LM)-guided femoral vascular access in the pediatric catheterization laboratory. This study aimed to compare US- and LM-guided vascular access in the pediatric catheterization laboratory. A single operator randomized 95 patients (201 vessels) to undergo either LM- or US-guided vascular access. The primary end point was the access success rate. Number of attempts, inadvertent access, time to sheath placement, and complications also were compared between the two groups. No difference was seen in the overall access success rate: 98 % with US versus 93 % with LM (p = 0.17). The success rate for the targeted vessel was higher with US (89 %) than with LM (67 %) (p = 0.012). US facilitated fewer attempts (1.1 ± 0.4 vs 1.4 ± 0.9; p = 0.048) and improved the first-attempt success rate (87 vs 77 %; p = 0.049). The time to access did not differ significantly between the two groups (US 2:55 ± 4:03 vs LM 3:37 ± 2:54; p = 0.28). No differences in complication rates were noted. The benefits of US were accentuated in the subgroup weighing less than 10 kg. In this study, US access in the pediatric catheterization laboratory did not improve overall success. However, US improved accuracy and reduced the number attempts necessary for access without prolonging the access time of the procedure. Small children realized the greatest benefit of US-guided access.

In Situ Smoothing of Facets on Spalled GaAs(100) Substrates during OMVPE Growth of III-V Epilayers, Solar Cells, and Other Devices: The Impact of Surface Impurities/Dopants.

One possible pathway toward reducing the cost of III-V solar cells is to remove them from their growth substrate by spalling fracture, and then reuse the substrate for the growth of multiple cells. Here we consider the growth of III-V cells on spalled GaAs(100) substrates, which typically have faceted surfaces after spalling. To facilitate the growth of high-quality cells, these faceted surfaces should be smoothed prior to cell growth. In this study, we show that these surfaces can be smoothed during organometallic vapor-phase epitaxy growth, but the choice of epilayer material and modification of the various surfaces by impurities/dopants greatly impacts whether or not the surface becomes smooth, and how rapidly the smoothing occurs. Representative examples are presented along with a discussion of the underlying growth processes. Although this work was motivated by solar cell growth, the methods are generally applicable to the growth of any III-V device on a nonplanar substrate.

Terrestrial surface stabilisation by modern analogues of the earliest land plants: A multi-dimensional imaging study.

The evolution of the first plant-based terrestrial ecosystems in the early Palaeozoic had a profound effect on the development of soils, the architecture of sedimentary systems, and shifts in global biogeochemical cycles. In part, this was due to the evolution of complex below-ground (root-like) anchorage systems in plants, which expanded and promoted plant-mineral interactions, weathering, and resulting surface sediment stabilisation. However, little is understood about how these micro-scale processes occurred, because of a lack of in situ plant fossils in sedimentary rocks/palaeosols that exhibit these interactions. Some modern plants (e.g., liverworts, mosses, lycophytes) share key features with the earliest land plants; these include uni- or multicellular rhizoid-like anchorage systems or simple roots, and the ability to develop below-ground networks through prostrate axes, and intimate associations with fungi, making them suitable analogues. Here, we investigated cryptogamic ground covers in Iceland and New Zealand to better understand these interactions, and how they initiate the sediment stabilisation process. We employed multi-dimensional and multi-scale imaging, including scanning electron microscopy (SEM) and X-ray Computed Tomography (µCT) of non-vascular liverworts (Haplomitriopsida and complex thalloids) and mosses, with additional imaging of vascular lycopods. We find that plants interact with their substrate in multiple ways, including: (1) through the development of extensive surface coverings as mats; (2) entrapment of sediment grains within and between networks of rhizoids; (3) grain entwining and adherence by rhizoids, through mucilage secretions, biofilm-like envelopment of thalli on surface grains; and (4) through grain entrapment within upright 'leafy' structures. Significantly, µCT imaging allows us to ascertain that rhizoids are the main method for entrapment and stabilisation of soil grains in the thalloid liverworts. This information provides us with details of how the earliest land plants may have significantly influenced early Palaeozoic sedimentary system architectures, promoted in situ weathering and proto-soil development, and how these interactions diversified over time with the evolution of new plant organ systems. Further, this study highlights the importance of cryptogamic organisms in the early stages of sediment stabilisation and soil formation today.

T-cell responses induced by ChAdOx1 nCoV-19 (AZD1222) vaccine to wild-type severe acute respiratory syndrome coronavirus 2 among people with and without HIV in South Africa.

OBJECTIVES: This study aimed to investigate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T-cell responses 14 days after single-dose ChAdOx1 nCoV-19 (AZD1222) vaccination in black Africans with and without HIV in South Africa, as well as determine the effect of AZD1222 vaccination on cell-mediated immune responses in people with HIV (PWH) with prior SARS-CoV-2 infection. METHODS: A total of 70 HIV-uninfected people and 104 PWH were prospectively enrolled in the multicentre, randomized, double-blinded, placebo-controlled, phase Ib/IIa trial (COV005). Peripheral blood mononuclear cells (PBMCs) were collected from trial participants 14 days after receipt of first dose of study treatment (placebo or AZD1222 vaccine). T-cell responses against the full-length spike (FLS) glycoprotein of wild-type SARS-CoV-2 and mutated S-protein regions found in the Alpha, Beta and Delta variants were assessed using an ex-vivo ELISpot assay. RESULTS: Among AZD1222 recipients without preceding SARS-CoV-2 infection, T-cell responses to FLS of wild-type SARS-CoV-2 were similarly common in PWH and HIV-uninfected people (30/33, 90.9% vs. 16/21, 76.2%; P = 0.138); and magnitude of response was similar among responders (78 vs. 56 SFCs/106 PBMCs; P = 0.255). Among PWH, AZD1222 vaccinees with prior SARS-CoV-2 infection, displayed a heightened T-cell response magnitude compared with those without prior infection (186 vs. 78 SFCs/106 PBMCs; P = 0.001); and similar response rate (14/14, 100% vs. 30/33, 90.9%; P = 0.244). CONCLUSION: Our results indicate comparable T-cell responses following AZD1222 vaccination in HIV-uninfected people and PWH on stable antiretroviral therapy. Our results additionally show that hybrid immunity acquired through SARS-CoV-2 infection and AZD1222 vaccination, induce a heightened T-cell response.

Genome-wide analyses of exonic copy number variants in a family-based study point to novel autism susceptibility genes.

The genetics underlying the autism spectrum disorders (ASDs) is complex and remains poorly understood. Previous work has demonstrated an important role for structural variation in a subset of cases, but has lacked the resolution necessary to move beyond detection of large regions of potential interest to identification of individual genes. To pinpoint genes likely to contribute to ASD etiology, we performed high density genotyping in 912 multiplex families from the Autism Genetics Resource Exchange (AGRE) collection and contrasted results to those obtained for 1,488 healthy controls. Through prioritization of exonic deletions (eDels), exonic duplications (eDups), and whole gene duplication events (gDups), we identified more than 150 loci harboring rare variants in multiple unrelated probands, but no controls. Importantly, 27 of these were confirmed on examination of an independent replication cohort comprised of 859 cases and an additional 1,051 controls. Rare variants at known loci, including exonic deletions at NRXN1 and whole gene duplications encompassing UBE3A and several other genes in the 15q11-q13 region, were observed in the course of these analyses. Strong support was likewise observed for previously unreported genes such as BZRAP1, an adaptor molecule known to regulate synaptic transmission, with eDels or eDups observed in twelve unrelated cases but no controls (p = 2.3x10(-5)). Less is known about MDGA2, likewise observed to be case-specific (p = 1.3x10(-4)). But, it is notable that the encoded protein shows an unexpectedly high similarity to Contactin 4 (BLAST E-value = 3x10(-39)), which has also been linked to disease. That hundreds of distinct rare variants were each seen only once further highlights complexity in the ASDs and points to the continued need for larger cohorts.