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Wastewater-based epidemiology has gained attention throughout the world for detection of SARS-CoV-2 RNA in wastewater to supplement clinical testing. Raw wastewater consists of small particles, or solids, suspended in liquid. Methods have been developed to measure SARS-CoV-2 RNA in the liquid and the solid fraction of wastewater, with some studies reporting higher concentrations in the solid fraction. To investigate this relationship further, six laboratories collaborated to conduct a study across five publicly owned treatment works (POTWs) where both primary settled solids obtained from primary clarifiers and raw wastewater influent samples were collected and quantified for SARS-CoV-2 RNA. Settled solids and influent samples were processed by participating laboratories using their respective methods and retrospectively paired based on date of collection. SARS-CoV-2 RNA concentrations, on a mass equivalent basis, were higher in settled solids than in influent by approximately three orders of magnitude. Concentrations in matched settled solids and influent were positively and significantly correlated at all five POTWs. RNA concentrations in both settled solids and influent were correlated to COVID-19 incidence rates in the sewersheds and thus representative of disease occurrence; the settled solids methods appeared to produce a comparable relationship between SARS-CoV-2 RNA concentration measurements and incidence rates across all POTWs. Settled solids and influent methods showed comparable sensitivity, N gene detection frequency, and calculated empirical incidence rate lower limits. Analysis of settled solids for SARS-CoV-2 RNA has the advantage of using less sample volume to achieve similar sensitivity to influent methods.

v3 updates: changes to the S gene primers and probes for detection of all SARS-CoV-2 variants This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and ORF1a and a duplex assay targeting Bovine Coronavirus Vaccine (BCoV) and Pepper Mild mottle virus (PMMoV) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. This protocol describes 2 separate PCR reactions, one containing primer/probe mixtures targeting the three SARS-CoV-2 targets and one containing primer/probe mixtures targeting BCoV and PMMoV. BCoV is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition. PMMoV is an enveloped virus which is abundant in human fecal waste and serves as an endogenous control for data normalization. PMMoV RNA is abundant at such high levels in wastewater samples that the samples must be diluted by a factor of 100 before quantification. The readout of this assay is a concentration of each target in the extracted RNA samples (copies/uL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

Background: The effective reproductive number, Re, is a critical indicator to monitor disease dynamics, inform regional and national policies, and estimate the effectiveness of interventions. It describes the average number of new infections caused by a single infectious person through time. To date, Re estimates are based on clinical data such as observed cases, hospitalizations, and/or deaths. These estimates are temporarily biased when clinical testing or reporting strategies change. Objectives: We show that the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater can be used to estimate Re in near real time, independent of clinical data and without the associated biases. Methods: We collected longitudinal measurements of SARS-CoV-2 RNA in wastewater in Zurich, Switzerland, and San Jose, California, USA. We combined this data with information on the temporal dynamics of shedding (the shedding load distribution) to estimate a time series proportional to the daily COVID-19 infection incidence. We estimated a wastewater-based Re from this incidence. Results: The method to estimate Re from wastewater worked robustly on data from two different countries and two wastewater matrices. The resulting estimates were as similar to the Re estimates from case report data as Re estimates based on observed cases, hospitalizations, and deaths are among each other. We further provide details on the effect of sampling frequency and the shedding load distribution on the ability to infer Re Discussion: To our knowledge, this is the first time Re has been estimated from wastewater. This method provides a low-cost, rapid, and independent way to inform SARS-CoV-2 monitoring during the ongoing pandemic and is applicable to future wastewater-based epidemiology targeting other pathogens. https://doi.org/10.1289/EHP10050 ISSN:1552-9924 ISSN:0091-6765

Please note that while this protocol is for TNA extraction using the Perkin Elmer Chemagic 360, RNA extraction with resuspended solids from this protocol has been verified to perform well using the Kingfisher MagMax kit as another high throughput, automated option and two manual Qiagen kits - the All Prep Powerviral DNA/RNA Kit and the Qiamp Viral RNA Mini Kit. This process instruction describes the steps for purification of nucleic acids from wastewater solids and preparation for downstream quantitative analysis with Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR). Due to the large quantities of substances that have inhibitory effects on PCR in wastewater samples, a subsequent PCR inhibitor removal step is required after nucleic acid purification. Both steps of the process are carried out in a 96-well plate format. This method uses the resuspended solids generated using this protocol: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. RNA purification is carried out using a kit optimized for the purification of viral on for the Perkin Elmer Chemagic 360. Although only RNA is used in the downstream applications from this protocol, DNA is also eluted in this process. A crucial component of the purification kit are the magnetic particles coated with poly vinyl alcohol (M-PVA Magnetic Beads) which have a hydrophilic surface giving them an affinity for nucleic acids but not many other biological molecules. The workflow involves binding nucleic acids in a sample to the beads which are then transferred through a series of wash buffers to remove debris with a robotic head with magnetic rods. The OneStep PCR Inhibitor Removal Kits are PCR inhibitor clean up kits that contain all the components needed for efficient removal of contaminants that can inhibit downstream enzymatic reactions (e.g. PCR and RT) from DNA and RNA preparations. The column matrices in these PCR inhibitor clean up kits have been specifically designed for the efficient removal of polyphenolic compounds, humic/fulvic acids, tannins, melanin, etc. from the most impure DNA and RNA preparations. This process instruction applies to extraction of RNA from wastewater samples using the Chemagic™ Viral DNA/RNA 300 Kit H96 for the Perkin Elmer Chemagic 360 followed by PCR Inhibitor Removal with the Zymo OneStep-96 PCR Inhibitor Removal Kit.

This process instruction describes the steps for pre-analytical processing of primary settled solids from wastewater treatment plants for downstream nucleic acid purification and quantification. Previous research has demonstrated that enveloped viral particles, such as SARS-CoV-2, associate with the solids in wastewater. Therefore, concentrating the solids in the sample and removing the water concentrates the viral particles as well and increases the sensitivity of the assay. Bovine coronavirus vaccine (BCoV) is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition. The dry weight of the sample is determined to account for sample variability between different treatment plants and between samples collected on different days and allows for the final quantification to be normalized to the precise quantity of solids in the sample. A dry weight conversion factor to adjust for the final amount of solids in the quantified sample is determined by measuring the difference in mass between the “wet” dewatered solid and after the sample is dried at 110°C overnight. For long term storage, up to 50mL of the original primary settled solid sample is stored at 4°C and small aliquot of the dewatered solids is stored at -80°C. This process instruction applies to sample dewatering, BCoV control spike in, homogenization, dry weight measurement, aliquoting and storage.

Wastewater-based epidemiology has gained attention throughout the world for detection of SARS-CoV-2 RNA in wastewater to supplement clinical testing. Methods have been developed using both the liquid and the solid fraction of wastewater, with some studies reporting higher concentrations in solids. To investigate this relationship further, we collaborated with six other laboratories to conduct a study across five publicly owned treatment works (POTWs) where both primary solids and raw wastewater influent samples were collected and quantified for SARS-CoV-2 RNA. Solids and influent samples were processed by participating laboratories using their respective methods and retrospectively paired based on date of collection. SARS-CoV-2 RNA concentrations by mass (gene copies per gram) were higher in solids than in influent by approximately three orders of magnitude. Concentrations in matched solids and influent were positively and significantly correlated at all five POTWs. RNA concentrations in both solids and influent were correlated to COVID-19 incidence rates in the sewershed and thus representative of disease burden; the solids methods appeared to produce a comparable relationship between SARS-CoV-2 RNA concentration measurements and incidence rates across all POTWs. Solids and influent methods showed comparable sensitivity, N gene detection frequency, and calculated empirical incidence rate lower limits. Analysis of solids has the advantage of using less sample volume to achieve similar sensitivity to influent methods.

A number of recent retrospective studies have demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in wastewater are associated with coronavirus disease 2019 (COVID-19) cases in the corresponding sewersheds. Implementing high-resolution, prospective efforts across multiple plants depends on sensitive measurements that are representative of COVID-19 cases, scalable for high-throughput analysis, and comparable across laboratories. We conducted a prospective study across eight publicly owned treatment works (POTWs). A focus on SARS-CoV-2 RNA in solids enabled us to scale up our measurements with a commercial lab partner. Samples were collected daily, and results were posted to a website within 24 h. SARS-CoV-2 RNA in daily samples correlated with the incidence of COVID-19 cases in the sewersheds; a 1 log10 increase in SARS-CoV-2 RNA in settled solids corresponds to a 0.58 log10 (4×) increase in sewershed incidence rate. SARS-CoV-2 RNA signals measured with the commercial laboratory partner were comparable across plants and comparable to measurements conducted in a university laboratory when normalized by pepper mild mottle virus (PMMoV) RNA. Results suggest that SARS-CoV-2 RNA should be detectable in settled solids for COVID-19 incidence rates of >1/100,000 (range, 0.8 to 2.3 cases per 100,000). These sensitive, representative, scalable, and comparable methods will be valuable for future efforts to scale up wastewater-based epidemiology. IMPORTANCE Access to reliable, rapid monitoring data is critical to guide response to an infectious disease outbreak. For pathogens that are shed in feces or urine, monitoring wastewater can provide a cost-effective snapshot of transmission in an entire community via a single sample. In order for a method to be useful for ongoing COVID-19 monitoring, it should be sensitive for detection of low concentrations of SARS-CoV-2, representative of incidence rates in the community, scalable to generate data quickly, and comparable across laboratories. This paper presents a method utilizing wastewater solids to meet these goals, producing measurements of SARS-CoV-2 RNA strongly associated with COVID-19 cases in the sewershed of a publicly owned treatment work. Results, provided within 24 h, can be used to detect incidence rates as low as approximately 1/100,000 cases and can be normalized for comparison across locations generating data using different methods.

A number of recent retrospective studies have demonstrated that SARS-CoV-2 RNA concentrations in wastewater are associated with COVID-19 cases in the corresponding sewersheds. Implementing high-resolution, prospective efforts across multiple plants depends on sensitive measurements that are representative of COVID-19 cases, scalable for high throughput analysis, and comparable across laboratories. We conducted a prospective study across eight publicly owned treatment works (POTWs). A focus on SARS-CoV-2 RNA in solids enabled us to scale-up our measurements with a commercial lab partner. Samples were collected daily and results were posted to a website within 24-hours. SARS-CoV-2 RNA in daily samples correlated to incidence COVID-19 cases in the sewersheds; a 1 log10 increase in SARS-CoV-2 RNA in settled solids corresponds to a 0.58 log10 (4X) increase in sewershed incidence rate. SARS-CoV-2 RNA signals measured with the commercial laboratory partner were comparable across plants and to measurements conducted in a university laboratory when normalized by pepper mild mottle virus PMMoV RNA. Results suggest that SARS-CoV-2 RNA should be detectable in settled solids for COVID-19 incidence rates > 1/100,000 (range 0.8 - 2.3 cases per 100,000). These sensitive, representative, scalable, and comparable methods will be valuable for future efforts to scale-up wastewater-based epidemiology.ImportanceAccess to reliable, rapid monitoring data is critical to guide response to an infectious disease outbreak. For pathogens that are shed in feces or urine, monitoring wastewater can provide a cost-effective snapshot of transmission in an entire community via a single sample. In order for a method to be useful for ongoing COVID-19 monitoring, it should be sensitive for detection of low concentrations of SARS-CoV-2, representative of incidence rates in the community, scalable to generate data quickly, and comparable across laboratories. This paper presents a method utilizing wastewater solids to meet these goals, producing measurements of SARS-CoV-2 RNA strongly associated with COVID-19 cases in the sewershed of a publicly owned treatment work. Results, provided within 24 hrs, can be used to detect incidence rates as low as approximately 1/100,000 cases and can be normalized for comparison across locations generating data using different methods.

The mechanisms by which common risk variants of small effect interact to contribute to complex genetic disorders remain unclear. Here, we apply a genetic approach, using isogenic human induced pluripotent stem cells (hiPSCs), to evaluate the effects of schizophrenia-associated common variants predicted to function as brain expression quantitative trait loci (SZ-eQTLs). By integrating CRISPR-mediated gene editing, activation and repression technologies to study one putative SZ-eQTL (FURIN rs4702) and four top-ranked SZ-eQTL genes (FURIN, SNAP91, TSNARE1, CLCN3), our platform resolves pre- and post-synaptic neuronal deficits, recapitulates genotype-dependent gene expression differences, and identifies convergence downstream of SZ-eQTL gene perturbations. Our observations highlight the cell-type-specific effects of common variants and demonstrate a synergistic effect between SZ-eQTL genes that converges on synaptic function. We propose that the links between rare and common variants implicated in psychiatric disease risk constitute a potentially generalizable phenomenon occurring more widely in complex genetic disorders.

The effective reproductive number, Re, is a critical indicator to monitor disease dynamics, inform regional and national policies, and estimate the effectiveness of interventions. It describes the average number of new infections caused by a single infectious person through time. To date, Re estimates are based on clinical data such as observed cases, hospitalizations, and/or deaths. Here we show that the dynamics of SARS-CoV-2 RNA in wastewater can be used to estimate Re in near real-time, independent of clinical data and without associated biases stemming from clinical testing and reporting strategies. The method to estimate Re from wastewater is robust and applicable to data from different countries and wastewater matrices. The resulting estimates are as similar to the Re estimates from case report data as Re estimates based on observed cases, hospitalizations, and deaths are among each other. We further provide details on the effect of sampling frequency and the shedding load distribution on the ability to infer Re. To our knowledge, this is the first time Re has been estimated from wastewater. This method provides a low cost, rapid, and independent way to inform SARS-CoV-2 monitoring during the ongoing pandemic and is applicable to future wastewater-based epidemiology targeting other pathogens. Significance statementThe effective reproductive number, Re, is widely used during the COVID-19 pandemic to track disease dynamics, inform regional and national policies, and estimate the effectiveness of interventions. Re is typically estimated from clinical case data, and can be biased by e.g. changes in testing and reporting. We show longitudinal measurements of SARS-CoV-2 RNA in wastewater can be used to estimate Re, across different regions, and provide an independent assessment of the dynamics of COVID-19. Given widespread wastewater sampling during this pandemic, these Re estimates are directly applicable as a rapid, low-cost method to inform public health policy. The method can be adapted to other pathogens, including those for which clinical data is not available.

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and ORF1a and a duplex assay targeting Bovine Coronavirus Vaccine (BCoV) and Pepper Mild mottle virus (PMMoV) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. This protocol describes 2 separate PCR reactions, one containing primer/probe mixtures targeting the three SARS-CoV-2 targets and one containing primer/probe mixtures targeting BCoV and PMMoV. BCoV is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition. PMMoV is an enveloped virus which is abundant in human fecal waste and serves as an endogenous control for data normalization. PMMoV RNA is abundant at such high levels in wastewater samples that the samples must be diluted by a factor of 100 before quantification. The readout of this assay is a concentration of each target in the extracted RNA samples (copies/uL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

Schizophrenia (SZ) is a psychiatric disorder with complex genetic risk dictated by interactions between hundreds of risk variants. Epigenetic factors, such as histone posttranslational modifications (PTMs), have been shown to play critical roles in many neurodevelopmental processes, and when perturbed may also contribute to the precipitation of disease. Here, we apply an unbiased proteomics approach to evaluate combinatorial histone PTMs in human induced pluripotent stem cell (hiPSC)-derived forebrain neurons from individuals with SZ. We observe hyperacetylation of H2A.Z and H4 in neurons derived from SZ cases, results that were confirmed in postmortem human brain. We demonstrate that the bromodomain and extraterminal (BET) protein, BRD4, is a bona fide ‘reader’ of H2A.Z acetylation, and further provide evidence that BET family protein inhibition ameliorates transcriptional abnormalities in patient-derived neurons. Thus, treatments aimed at alleviating BET protein interactions with hyperacetylated histones may aid in the prevention or treatment of SZ.

Causal genes and variants within genome-wide association study (GWAS) loci can be identified by integrating GWAS statistics with expression quantitative trait loci (eQTL) and determining which variants underlie both GWAS and eQTL signals. Most analyses, however, consider only the marginal eQTL signal, rather than dissect this signal into multiple conditionally independent signals for each gene. Here we show that analyzing conditional eQTL signatures, which could be important under specific cellular or temporal contexts, leads to improved fine mapping of GWAS associations. Using genotypes and gene expression levels from post-mortem human brain samples (n = 467) reported by the CommonMind Consortium (CMC), we find that conditional eQTL are widespread; 63% of genes with primary eQTL also have conditional eQTL. In addition, genomic features associated with conditional eQTL are consistent with context-specific (e.g., tissue-, cell type-, or developmental time point-specific) regulation of gene expression. Integrating the 2014 Psychiatric Genomics Consortium schizophrenia (SCZ) GWAS and CMC primary and conditional eQTL data reveals 40 loci with strong evidence for co-localization (posterior probability > 0.8), including six loci with co-localization of conditional eQTL. Our co-localization analyses support previously reported genes, identify novel genes associated with schizophrenia risk, and provide specific hypotheses for their functional follow-up.

SARS-CoV-2 RNA in wastewater settled solids is associated with COVID-19 incidence in sewersheds and therefore, there is a strong interest in using these measurements to augment traditional disease surveillance methods. A wastewater surveillance program should provide rapid turn around for sample measurements (ideally within 24 hours), but storage of samples is necessary for a variety of reasons including biobanking. Here we investigate how storage of wastewater solids at 4°C, -20°C, and -80°C affects measured concentrations of SARS-CoV-2 RNA. We find that short term (7-8 d) storage of raw solids at 4°C has little effect on measured concentrations of SARS-CoV-2 RNA, whereas longer term storage at 4°C (35-122 d) or freezing reduces measurements by 60%, on average. We show that normalizing SARS-CoV-2 RNA concentrations by concentrations of pepper mild mottle virus (PMMoV) RNA, an endogenous wastewater virus, can correct for changes during storage as storage can have a similar effect on PMMoV RNA as on SARS-CoV-2 RNA. The reductions in SARS-CoV-2 RNA in solids during freeze thaws is less than those reported for the same target in liquid influent by several authors.

The brain-specific tyrosine phosphatase, STEP (STriatal-Enriched protein tyrosine Phosphatase) is an important regulator of synaptic function. STEP normally opposes synaptic strengthening by increasing N-methyl D-aspartate glutamate receptor (NMDAR) internalization through dephosphorylation of GluN2B and inactivation of the kinases extracellular signal-regulated kinase 1/2 and Fyn. Here we show that STEP61 is elevated in the cortex in the Nrg1+/- knockout mouse model of schizophrenia (SZ). Genetic reduction or pharmacological inhibition of STEP prevents the loss of NMDARs from synaptic membranes and reverses behavioral deficits in Nrg1+/- mice. STEP61 protein is also increased in cortical lysates from the central nervous system-specific ErbB2/4 mouse model of SZ, as well as in human induced pluripotent stem cell (hiPSC)-derived forebrain neurons and Ngn2-induced excitatory neurons, from two independent SZ patient cohorts. In these selected SZ models, increased STEP61 protein levels likely reflect reduced ubiquitination and degradation. These convergent findings from mouse and hiPSC SZ models provide evidence for STEP61 dysfunction in SZ.

Background Schizophrenia (SCZ) is a severe psychiatric disorder affecting ~1% of the world’s population. It is largely heritable with genetic risk reflected by a combination of common variants of small effect and highly penetrant rare mutations. Chromatin modifications are known to play critical roles in the mediation of many neurodevelopmental processes, and, when disturbed, may also contribute to the precipitation of psychiatric disorders, such as SCZ. While a handful of candidate-based studies have measured changes in promoter-bound histone modifications, few mechanistic studies have been carried out to explore how these modifications may affect chromatin to precipitate behavioral phenotypes associated with the disease. Methods We applied an unbiased proteomics approach to evaluate the epigenetic landscape of SCZ in human induced pluripotent stem cells (hiPSC), neural progenitor cells (NPCs) and neurons from SCZ patients vs. matched controls. We utilized proteomics-based, label free liquid chromatography mass spectrometry (LC-MS/MS) on purified histones from these cells and confirmed our results by western blotting in postmortem SCZ cortical brain tissues. Furthermore we validated our findings with the application of histone interaction assays and structural and biophysical assessments to identify and confirm novel chromatin ‘readers’. To relate our findings to a SCZ phenotype we used a SCZ rodent model of prepulse inhibition (PPI) to perform pharmacological manipulations and behavioral assessments. Results Using label free mass spectrometry we performed PTM screening of hiPSCs, NPCs and matured neurons derived from SCZ patients and matched controls. We identified, amongst others, altered patterns of hyperacetylation in SCZ neurons. Additionally we identified enhanced binding of particular acetylation ‘reader’ proteins. Pharmacological inhibition of such proteins in an animal model of amphetamine sensitization ameliorated PPI deficits further validating this epigenetic signature in SCZ. Discussion Recent evidence indicates that relevance and patterns of acetylation in epigenetics advances beyond its role in transcription and small molecule inhibitors of these aberrant interactions hold promise as useful therapeutics. This study identifies a role for modulating gene expression changes associated with a SCZ epigenetic signature and warrants further investigation in terms of how this early gene expression pattern perhaps determines susceptibility or severity of the SCZ disease trajectory.

The identification of therapeutic nanoparticles formulation with preferential distribution to the tumor microenvironment remains an unsolved challenge. We have developed a high-throughput synthesis and screening platform to discover novel tumor-targeting liposomal nanoparticles. Over 1,000 unique liposomal formulations were synthesized using a combinatorial approach. The characterization of physicochemical properties (e.g., size, charge and stability) allowed the prioritization of ~500 formulations further profiled with in vitro cell-based assays (e.g., cell binding, cytotoxicity and cell barrier crossing). A subset of formulations with diverse properties was selected to create a library of 47 DNA-barcoded nanoparticles. The pharmacokinetics and biodistribution of the formulations library were measured in tumor xenografts mouse models in a highly multiplexed fashion by sequencing the DNA barcodes. The established synthesis and screening platform provides an effective approach to identify novel liposomal formulations with improved tumor and tissue distribution profile. Note: This abstract was not presented at the conference. Citation Format: Mark Audeh, Manali Dwarakanath, Michael Hopkins, Alisha Knudson, Sally Kwok, Stephen Morton, Nikki Peck, Tim Ruckh, Sarah Sanowar, Zach Scott, Peter Thana, Gary Tong, Aaron Topol, Alberto C Vitari, Eugeni Vaisberg, Jane Wang, Stanley Wong. High-throughput synthesis and screening for tumor-targeting liposomal nanoparticles [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B66.

Consistent with recent reports indicating that neurons differentiated in vitro from human-induced pluripotent stem cells (hiPSCs) are immature relative to those in the human brain, gene expression comparisons of our hiPSC-derived neurons to the Allen BrainSpan Atlas indicate that they most resemble fetal brain tissue. This finding suggests that, rather than modeling the late features of schizophrenia (SZ), hiPSC-based models may be better suited for the study of disease predisposition. We now report that a significant fraction of the gene signature of SZ hiPSC-derived neurons is conserved in SZ hiPSC neural progenitor cells (NPCs). We used two independent discovery-based approaches—microarray gene expression and stable isotope labeling by amino acids in cell culture (SILAC) quantitative proteomic mass spectrometry analyses—to identify cellular phenotypes in SZ hiPSC NPCs from four SZ patients. From our findings that SZ hiPSC NPCs show abnormal gene expression and protein levels related to cytoskeletal remodeling and oxidative stress, we predicted, and subsequently observed, aberrant migration and increased oxidative stress in SZ hiPSC NPCs. These reproducible NPC phenotypes were identified through scalable assays that can be applied to expanded cohorts of SZ patients, making them a potentially valuable tool with which to study the developmental mechanisms contributing to SZ.

Infectious diseases disproportionately affect indigent regions and are the greatest cause of childhood mortality in developing countries. Practical, low-cost vaccines for use in these countries are paramount to reducing disease burdens and concomitant poverty. Algae are a promising low-cost system for producing vaccines that can be orally delivered, thereby avoiding expensive purification and injectable delivery. We engineered the chloroplast of the eukaryotic alga Chlamydomonas reinhardtii to produce a chimeric protein consisting of the 25-kDa Plasmodium falciparum surface protein (Pfs25) fused to the β subunit of the cholera toxin (CtxB) to investigate an alga-based whole-cell oral vaccine. Pfs25 is a promising malaria transmission-blocking vaccine candidate that has been difficult to produce in traditional recombinant systems due to its structurally complex tandem repeats of epidermal growth factor-like domains. The noncatalytic CtxB domain of the cholera holotoxin assembles into a pentameric structure and acts as a mucosal adjuvant by binding GM1 ganglioside receptors on gut epithelial cells. We demonstrate that CtxB-Pfs25 accumulates as a soluble, properly folded and functional protein within algal chloroplasts, and it is stable in freeze-dried alga cells at ambient temperatures. In mice, oral vaccination using freeze-dried algae that produce CtxB-Pfs25 elicited CtxB-specific serum IgG antibodies and both CtxB- and Pfs25-specific secretory IgA antibodies. These data suggest that algae are a promising system for production and oral delivery of vaccine antigens, but as an orally delivered adjuvant, CtxB is best suited for eliciting secretory IgA antibodies for vaccine antigens against pathogens that invade mucosal surfaces using this strategy.

Over 100 genetic loci harbor schizophrenia associated variants, yet how these common variants confer risk is uncertain. The CommonMind Consortium has sequenced dorsolateral prefrontal cortex RNA from schizophrenia cases (n=258) and control subjects (n=279), creating the largest publicly available resource to date of gene expression and its genetic regulation; ∼5 times larger than the latest release of GTEx. Using this resource, we find that ∼20% of the schizophrenia risk loci have common variants that could explain regulation of brain gene expression. In five loci, these variants modulate expression of a single gene: FURIN, TSNARE1, CNTN4, CLCN3 or SNAP91. Experimentally altered expression of three of them, FURIN, TSNARE1, and CNTN4, perturbs the proliferation and apoptotic index of neural progenitors and leads to neuroanatomical deficits in zebrafish. Furthermore, shRNA mediated knock-down of FURIN1 in neural progenitor cells derived from human induced pluripotent stem cells produces abnormal neural migration. Although 4.2% of genes (N = 693) display significant differential expression between cases and controls, 44% show some evidence for differential expression. All fold changes are ≤ 1.33, and an independent cohort yields similar differential expression for these 693 genes (r = 0.58). These findings are consistent with schizophrenia being highly polygenic, as has been reported in investigations of common and rare genetic variation. Co-expression analyses identify a gene module that shows enrichment for genetic associations and is thus relevant for schizophrenia. Taken together, these results pave the way for mechanistic interpretations of genetic liability for schizophrenia and other brain diseases.

Converging evidence indicates that microRNAs (miRNAs) may contribute to disease risk for schizophrenia (SZ). We show that microRNA-9 (miR-9) is abundantly expressed in control neural progenitor cells (NPCs) but also significantly downregulated in a subset of SZ NPCs. We observed a strong correlation between miR-9 expression and miR-9 regulatory activity in NPCs as well as between miR-9 levels/activity, neural migration, and diagnosis. Overexpression of miR-9 was sufficient to ameliorate a previously reported neural migration deficit in SZ NPCs, whereas knockdown partially phenocopied aberrant migration in control NPCs. Unexpectedly, proteomic- and RNA sequencing (RNA-seq)-based analysis revealed that these effects were mediated primarily by small changes in expression of indirect miR-9 targets rather than large changes in direct miR-9 targets; these indirect targets are enriched for migration-associated genes. Together, these data indicate that aberrant levels and activity of miR-9 may be one of the many factors that contribute to SZ risk, at least in a subset of patients.

Over 100 genetic loci harbor schizophrenia-associated variants, yet how these variants confer liability is uncertain. The CommonMind Consortium sequenced RNA from dorsolateral prefrontal cortex of people with schizophrenia (N = 258) and control subjects (N = 279), creating a resource of gene expression and its genetic regulation. Using this resource, ∼20% of schizophrenia loci have variants that could contribute to altered gene expression and liability. In five loci, only a single gene was involved: FURIN, TSNARE1, CNTN4, CLCN3 or SNAP91. Altering expression of FURIN, TSNARE1 or CNTN4 changed neurodevelopment in zebrafish; knockdown of FURIN in human neural progenitor cells yielded abnormal migration. Of 693 genes showing significant case-versus-control differential expression, their fold changes were ≤ 1.33, and an independent cohort yielded similar results. Gene co-expression implicates a network relevant for schizophrenia. Our findings show that schizophrenia is polygenic and highlight the utility of this resource for mechanistic interpretations of genetic liability for brain diseases.

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.

Neural stem/progenitor cells (NSPCs) have the potential to differentiate into neurons, astrocytes, and/or oligodendrocytes. Because these cells can be expanded in culture, they represent a vast source of neural cells. With the recent discovery that patient fibroblasts can be reprogrammed directly into induced NSPCs, the regulation of NSPC fate and function, in the context of cell-based disease models and patient-specific cell-replacement therapies, warrants review.

Subunit vaccines are significantly more expensive to produce than traditional vaccines because they are based primarily on recombinant proteins that must be purified from the expression system. Despite the increased cost, subunit vaccines are being developed because they are safe, effective, and can elicit antibodies that confer protection against diseases that are not currently vaccine-preventable. Algae are an attractive platform for producing subunit vaccines because they are relatively inexpensive to grow, genetically tractable, easily scaled to large volumes, have a short generation time, and are devoid of inflammatory, viral, or prion contaminants often present in other systems. We tested whether algal chloroplasts can produce malaria transmission blocking vaccine candidates, Plasmodium falciparum surface protein 25 (Pfs25) and 28 (Pfs28). Antibodies that recognize Pfs25 and Pfs28 disrupt the sexual development of parasites within the mosquito midgut, thus preventing transmission of malaria from one human host to the next. These proteins have been difficult to produce in traditional recombinant systems because they contain tandem repeats of structurally complex epidermal growth factor-like domains, which cannot be produced in bacterial systems, and because they are not glycosylated, so they must be modified for production in eukaryotic systems. Production in algal chloroplasts avoids these issues because chloroplasts can fold complex eukaryotic proteins and do not glycosylate proteins. Here we demonstrate that algae are the first recombinant system to successfully produce an unmodified and aglycosylated version of Pfs25 or Pfs28. These antigens are structurally similar to the native proteins and antibodies raised to these recombinant proteins recognize Pfs25 and Pfs28 from P. falciparum. Furthermore, antibodies to algae-produced Pfs25 bind the surface of in-vitro cultured P. falciparum sexual stage parasites and exhibit transmission blocking activity. Thus, algae are promising organisms for producing cysteine-disulfide-containing malaria transmission blocking vaccine candidate proteins.

The genetic and epigenetic factors contributing to risk for schizophrenia (SZ) remain unresolved. Here we demonstrate, for the first time, perturbed global protein translation in human-induced pluripotent stem cell (hiPSC)-derived forebrain neural progenitor cells (NPCs) from four SZ patients relative to six unaffected controls. We report increased total protein levels and protein synthesis, together with two independent sets of quantitative mass spectrometry evidence indicating markedly increased levels of ribosomal and translation initiation and elongation factor proteins, in SZ hiPSC NPCs. We posit that perturbed levels of global protein synthesis in SZ hiPSC NPCs represent a novel post-transcriptional mechanism that might contribute to disease progression.

Aberrant behavior and function of neurons are believed to be the primary causes of most neurological diseases and psychiatric disorders. Human postmortem samples have limited availability and, while they provide clues to the state of the brain after a prolonged illness, they offer limited insight into the factors contributing to disease onset. Conversely, animal models cannot recapitulate the polygenic origins of neuropsychiatric disease. Novel methods, such as somatic cell reprogramming, deliver nearly limitless numbers of pathogenic human neurons for the study of the mechanism of neuropsychiatric disease initiation and progression. First, this article reviews the advent of human induced pluripotent stem cell (hiPSC) technology and introduces two major methods, “directed differentiation” and “neuronal induction,” by which it is now possible to generate neurons for modeling neuropsychiatric disease. Second, it discusses the recent applications, and the limitations, of these technologies to in vitro studies of psychiatric disorders.