Transmission Dynamics and Rare Clustered Transmission Within an Urban University Population Before Widespread Vaccination.

BACKGROUND: Universities returned to in-person learning in 2021 while SARS-CoV-2 spread remained high. At the time, it was not clear whether in-person learning would be a source of disease spread. METHODS: We combined surveillance testing, universal contact tracing, and viral genome sequencing to quantify introductions and identify likely on-campus spread. RESULTS: Ninety-one percent of viral genotypes occurred once, indicating no follow-on transmission. Less than 5% of introductions resulted in >3 cases, with 2 notable exceptions of 40 and 47 cases. Both partially overlapped with outbreaks defined by contact tracing. In both cases, viral genomics eliminated over half the epidemiologically linked cases but added an equivalent or greater number of individuals to the transmission cluster. CONCLUSIONS: Public health interventions prevented within-university transmission for most SARS-CoV-2 introductions, with only 2 major outbreaks being identified January to May 2021. The genetically linked cases overlap with outbreaks identified by contact tracing; however, they persisted in the university population for fewer days and rounds of transmission than estimated via contact tracing. This underscores the effectiveness of test-trace-isolate strategies in controlling undetected spread of emerging respiratory infectious diseases. These approaches limit follow-on transmission in both outside-in and internal transmission conditions.

Viral Dynamics of Omicron and Delta Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants With Implications for Timing of Release from Isolation: A Longitudinal Cohort Study.

BACKGROUND: In January 2022, US guidelines shifted to recommend isolation for 5 days from symptom onset, followed by 5 days of mask-wearing. However, viral dynamics and variant and vaccination impact on culture conversion are largely unknown. METHODS: We conducted a longitudinal study on a university campus, collecting daily anterior nasal swabs for at least 10 days for reverse-transcription polymerase chain reaction (RT-PCR) testing and culture, with antigen rapid diagnostic testing (RDT) on a subset. We compared culture positivity beyond day 5, time to culture conversion, and cycle threshold trend when calculated from diagnostic test, from symptom onset, by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, and by vaccination status. We evaluated sensitivity and specificity of RDT on days 4-6 compared with culture. RESULTS: Among 92 SARS-CoV-2 RT-PCR-positive participants, all completed the initial vaccine series; 17 (18.5%) were infected with Delta and 75 (81.5%) with Omicron. Seventeen percent of participants had positive cultures beyond day 5 from symptom onset, with the latest on day 12. There was no difference in time to culture conversion by variant or vaccination status. For 14 substudy participants, sensitivity and specificity of day 4-6 RDT were 100% and 86%, respectively. CONCLUSIONS: The majority of our Delta- and Omicron-infected cohort culture-converted by day 6, with no further impact of booster vaccination on sterilization or cycle threshold decay. We found that rapid antigen testing may provide reassurance of lack of infectiousness, though guidance to mask for days 6-10 is supported by our finding that 17% of participants remained culture-positive after isolation.

Early Introduction and Rise of the Omicron Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variant in Highly Vaccinated University Populations.

BACKGROUND: The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible in vaccinated and unvaccinated populations. The dynamics that govern its establishment and propensity toward fixation (reaching 100% frequency in the SARS-CoV-2 population) in communities remain unknown. Here, we describe the dynamics of Omicron at 3 institutions of higher education (IHEs) in the greater Boston area. METHODS: We use diagnostic and variant-specifying molecular assays and epidemiological analytical approaches to describe the rapid dominance of Omicron following its introduction into 3 IHEs with asymptomatic surveillance programs. RESULTS: We show that the establishment of Omicron at IHEs precedes that of the state and region and that the time to fixation is shorter at IHEs (9.5-12.5 days) than in the state (14.8 days) or region. We show that the trajectory of Omicron fixation among university employees resembles that of students, with a 2- to 3-day delay. Finally, we compare cycle threshold values in Omicron vs Delta variant cases on college campuses and identify lower viral loads among college affiliates who harbor Omicron infections. CONCLUSIONS: We document the rapid takeover of the Omicron variant at IHEs, reaching near-fixation within the span of 9.5-12.5 days despite lower viral loads, on average, than the previously dominant Delta variant. These findings highlight the transmissibility of Omicron, its propensity to rapidly dominate small populations, and the ability of robust asymptomatic surveillance programs to offer early insights into the dynamics of pathogen arrival and spread.

Comparison of BinaxNOW and SARS-CoV-2 qRT-PCR Detection of the Omicron Variant from Matched Anterior Nares Swabs.

The COVID-19 pandemic has increased use of rapid diagnostic tests (RDTs). In winter 2021 to 2022, the Omicron variant surge made it apparent that although RDTs are less sensitive than quantitative reverse transcription-PCR (qRT-PCR), the accessibility, ease of use, and rapid readouts made them a sought after and often sold-out item at local suppliers. Here, we sought to qualify the Abbott BinaxNOW RDT for use in our university testing program as a method to rule in positive or rule out negative individuals quickly at our priority qRT-PCR testing site. To perform this qualification study, we collected additional swabs from individuals attending this site. All swabs were tested using BinaxNOW. Initially as part of a feasibility study, test period 1 (n = 110) samples were stored cold before testing. In test period 2 (n = 209), samples were tested immediately. Combined, 102/319 samples tested severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive via qRT-PCR. All sequenced samples were Omicron (n = 92). We calculated 53.9% sensitivity, 100% specificity, a 100% positive predictive value, and an 82.2% negative predictive value for BinaxNOW (n = 319). Sensitivity would be improved (75.3%) by changing the qRT-PCR positivity threshold from a threshold cycle (CT) value of 40 to a CT value of 30. The receiver operating characteristic (ROC) curve shows that for qRT-PCR-positive CT values of between 24 and 40, the BinaxNOW test is of limited value diagnostically. Results suggest BinaxNOW could be used in our setting to confirm SARS-CoV-2 infection in individuals with substantial viral load, but a significant fraction of infected individuals would be missed if we used RDTs exclusively to rule out infection. IMPORTANCE Our results suggest BinaxNOW can rule in SARS-CoV-2 infection but would miss infections if RDTs were exclusively used.

Linking contact tracing with genomic surveillance to deconvolute SARS-CoV-2 transmission on a university campus.

Contact tracing and genomic data, approaches often used separately, have both been important tools in understanding the nature of SARS-CoV-2 transmission. Linked analysis of contact tracing and sequence relatedness of SARS-CoV-2 genomes from a regularly sampled university environment were used to build a multilevel transmission tracing and confirmation system to monitor and understand transmission on campus. Our investigation of an 18-person cluster stemming from an athletic team highlighted the importance of linking contact tracing and genomic analysis. Through these findings, it is suggestive that certain safety protocols in the athletic practice setting reduced transmission. The linking of traditional contact tracing with rapid-return genomic information is an effective approach for differentiating between multiple plausible transmission scenarios and informing subsequent public health protocols to limit disease spread in a university environment.

Examination of SARS-CoV-2 In-Class Transmission at a Large Urban University With Public Health Mandates Using Epidemiological and Genomic Methodology.

Importance: SARS-CoV-2, the causative agent of COVID-19, has displayed person-to-person transmission in a variety of indoor situations. This potential for robust transmission has posed significant challenges and concerns for day-to-day activities of colleges and universities where indoor learning is a focus for students, faculty, and staff. Objective: To assess whether in-class instruction without any physical distancing, but with other public health mitigation strategies, is a risk for driving SARS-CoV-2 transmission. Design, Setting, and Participants: This cohort study examined the evidence for SARS-CoV-2 transmission on a large urban US university campus using contact tracing, class attendance, and whole genome sequencing during the 2021 fall semester. Eligible participants were on-campus and off-campus individuals involved in campus activities. Data were analyzed between September and December 2021. Exposures: Participation in class and work activities on a campus with mandated vaccination and indoor masking but that was otherwise fully open without physical distancing during a time of ongoing transmission of SARS-CoV-2, both at the university and in the surrounding counties. Main Outcomes and Measures: Likelihood of in-class infection was assessed by measuring the genetic distance between all potential in-class transmission pairings using polymerase chain reaction testing. Results: More than 600 000 polymerase chain reaction tests were conducted throughout the semester, with 896 tests (0.1%) showing detectable SARS-CoV-2; there were over 850 cases of SARS-CoV-2 infection identified through weekly surveillance testing of all students and faculty on campus during the fall 2021 semester. The rolling mean average of positive tests ranged between 4 and 27 daily cases. Of more than 140 000 in-person class events and a total student population of 33 000 between graduate and undergraduate students, only 9 instances of potential in-class transmission were identified, accounting for 0.0045% of all classroom meetings. Conclusions and Relevance: In this cohort study, the data suggested that under robust transmission abatement strategies, in-class instruction was not an appreciable source of disease transmission.

Comparison of anterior nares CT values in asymptomatic and symptomatic individuals diagnosed with SARS-CoV-2 in a university screening program.

At our university based high throughput screening program, we test all members of our community weekly using RT-qPCR. RT-qPCR cycle threshold (CT) values are inversely proportional to the amount of viral RNA in a sample and are a proxy for viral load. We hypothesized that CT values would be higher, and thus the viral loads at the time of diagnosis would be lower, in individuals who were infected with the virus but remained asymptomatic throughout the course of the infection. We collected the N1 and N2 target gene CT values from 1633 SARS-CoV-2 positive RT-qPCR tests of individuals sampled between August 7, 2020, and March 18, 2021, at the BU Clinical Testing Laboratory. We matched this data with symptom reporting data from our clinical team. We found that asymptomatic patients had CT values significantly higher than symptomatic individuals on the day of diagnosis. Symptoms were followed by the clinical team for 10 days post the first positive test. Within the entire population, 78.1% experienced at least one symptom during surveillance by the clinical team (n = 1276/1633). Of those experiencing symptoms, the most common symptoms were nasal congestion (73%, n = 932/1276), cough (60.0%, n = 761/1276), fatigue (59.0%, n = 753/1276), and sore throat (53.1%, n = 678/1276). The least common symptoms were diarrhea (12.5%, n = 160/1276), dyspnea on exertion (DOE) (6.9%, n = 88/1276), foot or skin changes (including rash) (4.2%, n = 53/1276), and vomiting (2.1%, n = 27/1276). Presymptomatic individuals, those who were not symptomatic on the day of diagnosis but became symptomatic over the following 10 days, had CT values higher for both N1 (median = 27.1, IQR 20.2-32.9) and N2 (median = 26.6, IQR 20.1-32.8) than the symptomatic group N1 (median = 21.8, IQR 17.2-29.4) and N2 (median = 21.4, IQR 17.3-28.9) but lower than the asymptomatic group N1 (median = 29.9, IQR 23.6-35.5) and N2 (median = 30.0, IQR 23.1-35.7). This study supports the hypothesis that viral load in the anterior nares on the day of diagnosis is a measure of disease intensity at that time.

Buildout and integration of an automated high-throughput CLIA laboratory for SARS-CoV-2 testing on a large urban campus.

In 2019, the first cases of SARS-CoV-2 were detected in Wuhan, China, and by early 2020 the first cases were identified in the United States. SARS-CoV-2 infections increased in the US causing many states to implement stay-at-home orders and additional safety precautions to mitigate potential outbreaks. As policies changed throughout the pandemic and restrictions lifted, there was an increase in demand for COVID-19 testing which was costly, difficult to obtain, or had long turn-around times. Some academic institutions, including Boston University (BU), created an on-campus COVID-19 screening protocol as part of a plan for the safe return of students, faculty, and staff to campus with the option for in-person classes. At BU, we put together an automated high-throughput clinical testing laboratory with the capacity to run 45,000 individual tests weekly by Fall of 2020, with a purpose-built clinical testing laboratory, a multiplexed reverse transcription PCR (RT-qPCR) test, robotic instrumentation, and trained staff. There were many challenges including supply chain issues for personal protective equipment and testing materials in addition to equipment that were in high demand. The BU Clinical Testing Laboratory (CTL) was operational at the start of Fall 2020 and performed over 1 million SARS-CoV-2 PCR tests during the 2020-2021 academic year.

Viral dynamics of Omicron and Delta SARS-CoV-2 variants with implications for timing of release from isolation: a longitudinal cohort study.

Background: In January 2022, United States guidelines shifted to recommend isolation for 5 days from symptom onset, followed by 5 days of mask wearing. However, viral dynamics and variant and vaccination impact on culture conversion are largely unknown. Methods: We conducted a longitudinal study on a university campus, collecting daily anterior nasal swabs for at least 10 days for RT-PCR and culture, with antigen rapid diagnostic testing (RDT) on a subset. We compared culture positivity beyond day 5, time to culture conversion, and cycle threshold trend when calculated from diagnostic test, from symptom onset, by SARS-CoV-2 variant, and by vaccination status. We evaluated sensitivity and specificity of RDT on days 4-6 compared to culture. Results: Among 92 SARS-CoV-2 RT-PCR positive participants, all completed the initial vaccine series, 17 (18.5%) were infected with Delta and 75 (81.5%) with Omicron. Seventeen percent of participants had positive cultures beyond day 5 from symptom onset with the latest on day 12. There was no difference in time to culture conversion by variant or vaccination status. For the 14 sub-study participants, sensitivity and specificity of RDT were 100% and 86% respectively. Conclusions: The majority of our Delta- and Omicron-infected cohort culture-converted by day 6, with no further impact of booster vaccination on sterilization or cycle threshold decay. We found that rapid antigen testing may provide reassurance of lack of infectiousness, though masking for a full 10 days is necessary to prevent transmission from the 17% of individuals who remain culture positive after isolation. Main Point: Beyond day 5, 17% of our Delta and Omicron-infected cohort were culture positive. We saw no significant impact of booster vaccination on within-host Omicron viral dynamics. Additionally, we found that rapid antigen testing may provide reassurance of lack of infectiousness.

Minimal SARS-CoV-2 classroom transmission at a large urban university experiencing repeated into campus introduction.

SARS-CoV-2, the causative agent of COVID-19, has displayed person to person transmission in a variety of indoor situations. This potential for robust transmission has posed significant challenges to day-to-day activities of colleges and universities where indoor learning is a focus. Concerns about transmission in the classroom setting have been of concern for students, faculty and staff. With the simultaneous implementation of both non-pharmaceutical and pharmaceutical control measures meant to curb the spread of the disease, defining whether in-class instruction without any physical distancing is a risk for driving transmission is important. We examined the evidence for SARS-CoV-2 transmission on a large urban university campus that mandated vaccination and masking but was otherwise fully open without physical distancing during a time of ongoing transmission of SARS-CoV-2 both at the university and in the surrounding counties. Using weekly surveillance testing of all on-campus individuals and rapid contact tracing of individuals testing positive for the virus we found little evidence of in-class transmission. Of more than 140,000 in-person class events, only nine instances of potential in-class transmission were identified. When each of these events were further interrogated by whole-genome sequencing of all positive cases significant genetic distance was identified between all potential in-class transmission pairings, providing evidence that all individuals were infected outside of the classroom. These data suggest that under robust transmission abatement strategies, in-class instruction is not an appreciable source of disease transmission.

Assessment of a COVID-19 Control Plan on an Urban University Campus During a Second Wave of the Pandemic.

Importance: The COVID-19 pandemic has severely disrupted US educational institutions. Given potential adverse financial and psychosocial effects of campus closures, many institutions developed strategies to reopen campuses in the fall 2020 semester despite the ongoing threat of COVID-19. However, many institutions opted to have limited campus reopening to minimize potential risk of spread of SARS-CoV-2. Objective: To analyze how Boston University (BU) fully reopened its campus in the fall of 2020 and controlled COVID-19 transmission despite worsening transmission in Boston, Massachusetts. Design, Setting, and Participants: This multifaceted intervention case series was conducted at a large urban university campus in Boston, Massachusetts, during the fall 2020 semester. The BU response included a high-throughput SARS-CoV-2 polymerase chain reaction testing facility with capacity to deliver results in less than 24 hours; routine asymptomatic screening for COVID-19; daily health attestations; adherence monitoring and feedback; robust contact tracing, quarantine, and isolation in on-campus facilities; face mask use; enhanced hand hygiene; social distancing recommendations; dedensification of classrooms and public places; and enhancement of all building air systems. Data were analyzed from December 20, 2020, to January 31, 2021. Main Outcomes and Measures: SARS-CoV-2 diagnosis confirmed by reverse transcription-polymerase chain reaction of anterior nares specimens and sources of transmission, as determined through contact tracing. Results: Between August and December 2020, BU conducted more than 500 000 COVID-19 tests and identified 719 individuals with COVID-19, including 496 students (69.0%), 11 faculty (1.5%), and 212 staff (29.5%). Overall, 718 individuals, or 1.8% of the BU community, had test results positive for SARS-CoV-2. Of 837 close contacts traced, 86 individuals (10.3%) had test results positive for COVID-19. BU contact tracers identified a source of transmission for 370 individuals (51.5%), with 206 individuals (55.7%) identifying a non-BU source. Among 5 faculty and 84 staff with SARS-CoV-2 with a known source of infection, most reported a transmission source outside of BU (all 5 faculty members [100%] and 67 staff members [79.8%]). A BU source was identified by 108 of 183 undergraduate students with SARS-CoV-2 (59.0%) and 39 of 98 graduate students with SARS-CoV-2 (39.8%); notably, no transmission was traced to a classroom setting. Conclusions and Relevance: In this case series of COVID-19 transmission, BU used a coordinated strategy of testing, contact tracing, isolation, and quarantine, with robust management and oversight, to control COVID-19 transmission in an urban university setting.

Interleukin-1 genotypes modulate the long-term effect of lipoprotein(a) on cardiovascular events: The Ioannina Study.

BACKGROUND: Lipoprotein(a) [Lp(a)] is a genetic risk factor for cardiovascular disease (CVD), and proinflammatory interleukin-1 (IL-1) genotypes may influence Lp(a)-mediated CVD events. The genotype IL-1(+) is associated with higher rates of inflammation than IL-1(-) genotype. Targeting IL-1ß was recently shown to decrease CVD events independent of low-density lipoprotein-cholesterol levels. OBJECTIVE: The objective of the study is to assess the modulatory effect of IL-1 genotypes on risk mediated by Lp(a) METHODS: We assessed whether IL-1 genotypes modulate the effect of Lp(a) on major adverse cardiovascular events (cardiovascular death, myocardial infarction, and stroke/transient ischemic attack) and angiographically determined coronary artery disease (CAD). IL-1 genotypes and Lp(a) were measured in 603 patients without diabetes mellitus undergoing angiography. Major adverse cardiovascular events and CAD were assessed over a median of 45 months. RESULTS: In multivariable-adjusted analysis, Lp(a) was associated with major adverse cardiovascular events (hazard ratio [HR] [95% confidence interval {CI}]: 2.95 [1.16-7.54], P = .023) and CAD (odds ratio [OR] [95% CI]: 1.84 [1.12-3.03], P = .016) comparing quartile 4 vs quartile 1. In Cox regression analysis, IL-1(+) patients with Lp(a) above the median (>9.2 mg/dL) had a worse event-free cumulative survival (HR [95% CI]: 3.59 [1.07-12.03], P = .039) compared to IL-1(-) patients with Lp(a) below the median. In IL-1(+) patients aged ≤60 years, Lp(a) was also associated with angiographically determined CAD (OR [95% CI]: 2.90 [1.07-7.86], P = .036) comparing quartile 4 vs quartile 1 but not IL-1(-) patients. CONCLUSION: Proinflammatory IL-1(+) genotypes modulate the risk of Lp(a) long-term CVD events and CAD. These data suggest that the dual genetic contributions of elevated Lp(a) levels and IL-1(+) genotypes may identify younger subjects at particularly high risk for CVD events.

Influence of Obesity on Periodontitis Progression Is Conditional on Interleukin-1 Inflammatory Genetic Variation.

BACKGROUND: This study evaluates whether specific patterns of interleukin (IL)-1 gene variants, known to affect periodontitis severity, influence the previously reported association between obesity and subsequent periodontitis progression in a longitudinal database. The study population included 292 men (aged 29 to 64 years at entry) from the Veterans Affairs Dental Longitudinal Study from whom DNA and dental and anthropometric endpoints were collected during multiple examinations (approximately every 3 years for up to 27 years). METHODS: Key variables assessed included: 1) periodontitis; 2) body mass index; 3) waist circumference to height (WHTR) ratio for central adiposity; 4) age; 5) smoking; 6) glucose tolerance; and 7) two previously reported versions of IL-1 genetic patterns associated with periodontitis severity and progression. Disease progression was determined using predefined criteria that used a combination of change in classification of disease severity based on alveolar bone loss and tooth loss during follow-up. Extended Cox regression analyses were used to estimate hazards of experiencing periodontal disease progression with or without adjustments for appropriate covariates. RESULTS: In hazard ratio analyses, men with WHTR >50% at baseline and positive for either IL-1 genotype version were at significantly higher risk (two-fold) for disease progression (P for interaction = 0.04). Participants positive for IL-1 genotype version 2 exhibited earlier progression (fewer years from baseline to first incidence of progression) than those who were negative (P = 0.02, adjusted for age and smoking). CONCLUSION: In this longitudinally monitored male population, observed effect of baseline central adiposity on future periodontitis progression is conditional on proinflammatory IL-1 genetic variations.

Counterpoint: Risk factors, including genetic information, add value in stratifying patients for optimal preventive dental care.

BACKGROUND: There is disagreement as to whether patient stratification by a combination of diabetes, smoking, and genetic test results is useful for informing the frequency of dental prophylaxes. METHODS: The authors appeal to basic tenets of clinical study design and statistical analysis of clinical investigations, and highlight how secondary ad hoc analyses, such as those of Diehl and colleagues, are frequently underpowered and inconclusive. They also provide evidence from numerous studies supporting the use of genetics to identify risk. RESULTS: The authors believe the conclusions reached from their original analyses are valid and the analyses of Diehl and colleagues serve to simply reinforce the authors' specific intent of avoiding such underpowered analyses altogether with the Michigan Personalized Prevention Study. CONCLUSIONS: Until full genome sequencing in many people with highly specified disease phenotypes is feasible, experimental approaches based on biological findings and hypothesis testing should not be summarily discounted. PRACTICAL IMPLICATIONS: Stratification of patients to provide "personalized" treatment remains an important, yet elusive, goal. The current debate serves to highlight the need for large, clinical utility studies that can adequately determine how phenotypic and genotypic data can be best used to improve oral health in the US population.

Functional modularity of nuclear hormone receptors in a Caenorhabditis elegans metabolic gene regulatory network.

Gene regulatory networks (GRNs) provide insights into the mechanisms of differential gene expression at a systems level. GRNs that relate to metazoan development have been studied extensively. However, little is still known about the design principles, organization and functionality of GRNs that control physiological processes such as metabolism, homeostasis and responses to environmental cues. In this study, we report the first experimentally mapped metazoan GRN of Caenorhabditis elegans metabolic genes. This network is enriched for nuclear hormone receptors (NHRs). The NHR family has greatly expanded in nematodes: humans have 48 NHRs, but C. elegans has 284, most of which are uncharacterized. We find that the C. elegans metabolic GRN is highly modular and that two GRN modules predominantly consist of NHRs. Network modularity has been proposed to facilitate a rapid response to different cues. As NHRs are metabolic sensors that are poised to respond to ligands, this suggests that C. elegans GRNs evolved to enable rapid and adaptive responses to different cues by a concurrence of NHR family expansion and modular GRN wiring.

A C. elegans genome-scale microRNA network contains composite feedback motifs with high flux capacity.

MicroRNAs (miRNAs) and transcription factors (TFs) are primary metazoan gene regulators. Whereas much attention has focused on finding the targets of both miRNAs and TFs, the transcriptional networks that regulate miRNA expression remain largely unexplored. Here, we present the first genome-scale Caenorhabditis elegans miRNA regulatory network that contains experimentally mapped transcriptional TF --> miRNA interactions, as well as computationally predicted post-transcriptional miRNA --> TF interactions. We find that this integrated miRNA network contains 23 miRNA <--> TF composite feedback loops in which a TF that controls a miRNA is itself regulated by that same miRNA. By rigorous network randomizations, we show that such loops occur more frequently than expected by chance and, hence, constitute a genuine network motif. Interestingly, miRNAs and TFs in such loops are heavily regulated and regulate many targets. This "high flux capacity" suggests that loops provide a mechanism of high information flow for the coordinate and adaptable control of miRNA and TF target regulons.

Matrix and Steiner-triple-system smart pooling assays for high-performance transcription regulatory network mapping.

Yeast one-hybrid (Y1H) assays provide a gene-centered method for the identification of interactions between gene promoters and regulatory transcription factors (TFs). To date, Y1H assays have involved library screens that are relatively expensive and laborious. We present two Y1H strategies that allow immediate prey identification: matrix assays that use an array of 755 individual Caenorhabditis elegans TFs, and smart-pool assays that use TF multiplexing. Both strategies simplify the Y1H pipeline and reduce the cost of protein-DNA interaction identification. We used a Steiner triple system (STS) to create smart pools of 4-25 TFs. Notably, we uniplexed a small number of highly connected TFs to allow efficient assay deconvolution. Both strategies outperform library screens in terms of coverage, confidence and throughput. These versatile strategies can be adapted both to TFs in other systems and, likely, to other biomolecules and assays as well.

Transcription factor modularity in a gene-centered C. elegans core neuronal protein-DNA interaction network.

Transcription regulatory networks play a pivotal role in the development, function, and pathology of metazoan organisms. Such networks are comprised of protein-DNA interactions between transcription factors (TFs) and their target genes. An important question pertains to how the architecture of such networks relates to network functionality. Here, we show that a Caenorhabditis elegans core neuronal protein-DNA interaction network is organized into two TF modules. These modules contain TFs that bind to a relatively small number of target genes and are more systems specific than the TF hubs that connect the modules. Each module relates to different functional aspects of the network. One module contains TFs involved in reproduction and target genes that are expressed in neurons as well as in other tissues. The second module is enriched for paired homeodomain TFs and connects to target genes that are often exclusively neuronal. We find that paired homeodomain TFs are specifically expressed in C. elegans and mouse neurons, indicating that the neuronal function of paired homeodomains is evolutionarily conserved. Taken together, we show that a core neuronal C. elegans protein-DNA interaction network possesses TF modules that relate to different functional aspects of the complete network.

A gene-centered C. elegans protein-DNA interaction network.

Transcription regulatory networks consist of physical and functional interactions between transcription factors (TFs) and their target genes. The systematic mapping of TF-target gene interactions has been pioneered in unicellular systems, using "TF-centered" methods (e.g., chromatin immunoprecipitation). However, metazoan systems are less amenable to such methods. Here, we used "gene-centered" high-throughput yeast one-hybrid (Y1H) assays to identify 283 interactions between 72 C. elegans digestive tract gene promoters and 117 proteins. The resulting protein-DNA interaction (PDI) network is highly connected and enriched for TFs that are expressed in the digestive tract. We provide functional annotations for approximately 10% of all worm TFs, many of which were previously uncharacterized, and find ten novel putative TFs, illustrating the power of a gene-centered approach. We provide additional in vivo evidence for multiple PDIs and illustrate how the PDI network provides insights into metazoan differential gene expression at a systems level.

Towards a proteome-scale map of the human protein-protein interaction network.

Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.