Characterization of the distribution and dynamics of chromatin states in the C. elegans germline reveals substantial H3K4me3 remodeling during oogenesis.

Chromatin organization in the C. elegans germline is tightly regulated and critical for germ cell differentiation. Although certain germline epigenetic regulatory mechanisms have been identified, how they influence chromatin structure and ultimately gene expression remains unclear, in part because most genomic studies have focused on data collected from intact worms comprising both somatic and germline tissues. We therefore analyzed histone modification and chromatin accessibility data from isolated germ nuclei representing undifferentiated proliferating and meiosis I populations to define chromatin states. We correlated these states with overall transcript abundance, spatiotemporal expression patterns, and the function of small RNA pathways. Because the essential role of the germline is to transmit genetic information and establish gene expression in the early embryo, we compared epigenetic and transcriptomic profiles from undifferentiated germ cells to those of embryos to define the epigenetic changes during this developmental transition. The active histone modification H3K4me3 shows particularly dynamic remodeling as germ cells differentiate into oocytes, which suggests a mechanism for establishing early transcription of essential genes during zygotic genome activation. This analysis highlights the dynamism of the chromatin landscape across developmental transitions and provides a resource for future investigation into epigenetic regulatory mechanisms in germ cells.

The art of observation: bridging science and art to see the unexpected.

Observation is the heart of research, but it can be challenging to observe deeply and go beyond expected observations. Here, we describe activities designed for scientists to enhance their observational skills by engaging with art. In collaboration with an art gallery at our university, our lab practiced observing representational paintings in a systematic way, separating the act of observation from interpretation. Applying this skill to our microscopy images allowed us to access information in the data that may otherwise have been overlooked. In addition, these activities highlighted the power of collecting observations from multiple observers before generating interpretations, as well as the value of discussing the creative and emotional aspects of data collection and interpretation. We provide concrete examples of how we will incorporate these skills into our research processes, as well as details that other groups can use to engage in similar art-based training activities to enhance their own observational skills.

The Upstream Sequence Transcription Complex dictates nucleosome positioning and promoter accessibility at piRNA genes in the C. elegans germ line.

The piRNA pathway is a conserved germline-specific small RNA pathway that ensures genomic integrity and continued fertility. In C. elegans and other nematodes, Type-I piRNAs are expressed from >10,000 independently transcribed genes clustered within two discrete domains of 1.5 and 3.5 MB on Chromosome IV. Clustering of piRNA genes contributes to their germline-specific expression, but the underlying mechanisms are unclear. We analyze isolated germ nuclei to demonstrate that the piRNA genomic domains are located in a heterochromatin-like environment. USTC (Upstream Sequence Transcription Complex) promotes strong association of nucleosomes throughout piRNA clusters, yet organizes the local nucleosome environment to direct the exposure of individual piRNA genes. Localization of USTC to the piRNA domains depends upon the ATPase chromatin remodeler ISW-1, which maintains high nucleosome density across piRNA clusters and ongoing production of piRNA precursors. Overall, this work provides insight into how chromatin states coordinate transcriptional regulation over large genomic domains, with implications for global genome organization.

Nop9 recognizes structured and single-stranded RNA elements of preribosomal RNA.

Nop9 is an essential factor in the processing of preribosomal RNA. Its absence in yeast is lethal, and defects in the human ortholog are associated with breast cancer, autoimmunity, and learning/language impairment. PUF family RNA-binding proteins are best known for sequence-specific RNA recognition, and most contain eight α-helical repeats that bind to the RNA bases of single-stranded RNA. Nop9 is an unusual member of this family in that it contains eleven repeats and recognizes both RNA structure and sequence. Here we report a crystal structure of Saccharomyces cerevisiae Nop9 in complex with its target RNA within the 20S preribosomal RNA. This structure reveals that Nop9 brings together a carboxy-terminal module recognizing the 5' single-stranded region of the RNA and a bifunctional amino-terminal module recognizing the central double-stranded stem region. We further show that the 3' single-stranded region of the 20S target RNA adds sequence-independent binding energy to the RNA-Nop9 interaction. Both the amino- and carboxy-terminal modules retain the characteristic sequence-specific recognition of PUF proteins, but the amino-terminal module has also evolved a distinct interface, which allows Nop9 to recognize either single-stranded RNA sequences or RNAs with a combination of single-stranded and structured elements.

E-cigarette environmental and fire/life safety risks in schools reported by secondary school teachers.

BACKGROUND: To identify if e-cigarette usage is an on-campus problem for secondary schools and evaluate initial school survey responses. More specifically, this survey can aid in identifying where students are seen using e-cigarettes, if smoke alarms have been newly inserted on school property, if smoke alarms have been tampered with to allow for vaping without detection; and, if any e-cigarette fires or explosions have occurred on school property. METHODS: This survey, disseminated to New Jersey secondary school teachers across seven sessions January-July 2019, resulted in 104 complete responses of 109 respondents. The survey was conducted after fire prevention, exit/egress, and life safety portions of "OSHA 10 Plus for General Industry" courses. Survey questions included number of times teachers observed students vaping and location where vaping in last 12 months, fire alarm installation and tampering, and fires or explosions and injuries from vaping/e-cigarette devices. RESULTS: Many (63%) respondents reported very or moderately common rates of vaping within their schools; however, three of four questions regarding school fire and smoke alarm use specifically to detect vaping had a majority of unsure/I don't know responses. CONCLUSION: Results suggested concerns regarding student vaping and e-cigarette use inside and outside secondary schools. Improved school detection and response are warranted. TRIAL REGISTRATION: Not applicable.

Structural analysis reveals the flexible C-terminus of Nop15 undergoes rearrangement to recognize a pre-ribosomal RNA folding intermediate.

The RNA recognition motif (RRM) is the most abundant RNA-binding domain in eukaryotes, and it plays versatile roles in RNA metabolism. Despite its abundance, diversity of RRM structure and function is generated by variations on a conserved core. Yeast Nop15 is an RRM protein that is essential for large ribosomal subunit biogenesis. We determined a 2.0 Å crystal structure of Nop15 that reveals a C-terminal α-helical region obscures its canonical RNA-binding surface. Small-angle X-ray scattering, NMR and RNA-binding analyses further reveal that the C-terminal residues of Nop15 are highly flexible, but essential for tight RNA binding. Moreover, comparison with a recently reported cryo-electron microscopy structure indicates that dramatic rearrangement of the C-terminal region of Nop15 in the pre-ribosome exposes the RNA-binding surface to recognize the base of its stem-loop target RNA and extends a newly-formed α helix to the distal loop where it forms protein interactions.

Exploring the utility of "next-generation" sequence data on inferring the phylogeny of the South American Valeriana (Valerianaceae).

This study aimed to investigate the phylogenetic utility of genotyping-by-sequencing (GBS) data in the southern South American subclade of Valerianaceae (Dipsacales). The variety of forms that has arisen in this clade, presumably over the past 5-10 million years, has all the signatures of an adaptive and rapid radiation. While the phylogeny of Valerianaceae has received a great deal of attention in the last decade, species relationships have been hard to resolve using traditional phylogenetic markers. Here, we collected high-throughput genomic sequence data from reduced-representation libraries obtained through GBS protocols. Putative orthologs were identified using within- and among-sample clustering using the computer software pyRAD. We recovered over 3000 loci for 14 species of southern South AmericanValeriana,with 140 loci present across all samples.We analyzed a set of phylogenetic trees generated from each locus using maximum likelihood methods, as well as multispecies coalescent (∗BEAST) methods. For comparative purposes, we also used a supermatrix approach to infer the phylogeny for these taxa. Across different methods and data sets, we recovered consistent relationships for the southern South American valerians that we sampled with varying degrees of support.

Transcriptome analysis uncovers Arabidopsis F-BOX STRESS INDUCED 1 as a regulator of jasmonic acid and abscisic acid stress gene expression.

BACKGROUND: The ubiquitin 26S proteasome system (UPS) selectively degrades cellular proteins, which results in physiological changes to eukaryotic cells. F-box proteins are substrate adaptors within the UPS and are responsible for the diversity of potential protein targets. Plant genomes are enriched in F-box genes, but the vast majority of these have unknown roles. This work investigated the Arabidopsis F-box gene F-BOX STRESS INDUCED 1 (FBS1) for its effects on gene expression in order elucidate its previously unknown biological function. RESULTS: Using publically available Affymetrix ATH1 microarray data, we show that FBS1 is significantly co-expressed in abiotic stresses with other well-characterized stress response genes, including important stress-related transcriptional regulators. This gene suite is most highly expressed in roots under cold and salt stresses. Transcriptome analysis of fbs1-1 knock-out plants grown at a chilling temperature shows that hundreds of genes require FBS1 for appropriate expression, and that these genes are enriched in those having roles in both abiotic and biotic stress responses. Based on both this genome-wide expression data set and quantitative real-time PCR (qPCR) analysis, it is apparent that FBS1 is required for elevated expression of many jasmonic acid (JA) genes that have established roles in combatting environmental stresses, and that it also controls a subset of JA biosynthesis genes. FBS1 also significantly impacts abscisic acid (ABA) regulated genes, but this interaction is more complex, as FBS1 has both positive and negative effects on ABA-inducible and ABA-repressible gene modules. One noteworthy effect of FBS1 on ABA-related stress processes, however, is the restraint it imposes on the expression of multiple class I LIPID TRANSFER PROTEIN (LTP) gene family members that have demonstrated protective effects in water deficit-related stresses. CONCLUSION: FBS1 impacts plant stress responses by regulating hundreds of genes that respond to the plant stress hormones JA and ABA. The positive effect that FBS1 has on JA processes and the negative effect it has on at least some ABA processes indicates that it in part regulates cellular responses balanced between these two important stress hormones. More broadly then, FBS1 may aid plant cells in switching between certain biotic (JA) and abiotic (ABA) stress responses. Finally, because FBS1 regulates a subset of JA biosynthesis and response genes, we conclude that it might have a role in tuning hormone responses to particular circumstances at the transcriptional level.

Oncogenic Kras induces spatiotemporally specific tissue deformation through converting pulsatile into sustained ERK activation.

Tissue regeneration and maintenance rely on coordinated stem cell behaviours. This orchestration can be impaired by oncogenic mutations leading to cancer. However, it is largely unclear how oncogenes perturb stem cells' orchestration to disrupt tissue. Here we used intravital imaging to investigate the mechanisms by which oncogenic Kras mutation causes tissue disruption in the hair follicle. Through longitudinally tracking hair follicles in live mice, we found that KrasG12D, a mutation that can lead to squamous cell carcinoma, induces epithelial tissue deformation in a spatiotemporally specific manner, linked with abnormal cell division and migration. Using a reporter mouse capture real-time ERK signal dynamics at the single-cell level, we discovered that KrasG12D, but not a closely related mutation HrasG12V, converts ERK signal in stem cells from pulsatile to sustained. Finally, we demonstrated that interrupting sustained ERK signal reverts KrasG12D-induced tissue deformation through modulating specific features of cell migration and division.

The Upstream Sequence Transcription Complex Dictates Nucleosome Positioning and Promoter Accessibility at piRNA Genes in the C. elegans Germ Line.

The piRNA pathway is a conserved germline-specific small RNA pathway that ensures genomic integrity and continued fertility. In C. elegans and other nematodes, Type-I piRNA precursor transcripts are expressed from over 10,000 small, independently regulated genes clustered within two discrete domains of 1.5 and 3.5 MB on Chromosome IV. These large clusters likely play a significant role in promoting germline-specific expression of piRNAs, but the underlying mechanisms are unclear. By examining the chromatin environment specifically in isolated germ nuclei, we demonstrate that piRNA clusters are located in closed chromatin, and confirm the enrichment for the inactive histone modification H3K27me3. We further show that the piRNA biogenesis factor USTC (Upstream Sequence Transcription Complex) plays two roles - it promotes a strong association of nucleosomes throughout the piRNA clusters, and it organizes the local nucleosome environment to direct the exposure of individual piRNA genes. Overall, this work reveals new insight into how chromatin state coordinates transcriptional regulation over large genomic domains, which has implications for understanding global genome organization in the germ line.

Organ function is preserved despite reorganization of niche architecture in the hair follicle.

The ability of stem cells to build and replenish tissues depends on support from their niche. Although niche architecture varies across organs, its functional importance is unclear. During hair follicle growth, multipotent epithelial progenitors build hair via crosstalk with their remodeling fibroblast niche, the dermal papilla, providing a powerful model to functionally interrogate niche architecture. Through mouse intravital imaging, we show that dermal papilla fibroblasts remodel individually and collectively to form a morphologically polarized, structurally robust niche. Asymmetric TGF-ß signaling precedes morphological niche polarity, and loss of TGF-ß signaling in dermal papilla fibroblasts leads them to progressively lose their stereotypic architecture, instead surrounding the epithelium. The reorganized niche induces the redistribution of multipotent progenitors but nevertheless supports their proliferation and differentiation. However, the differentiated lineages and hairs produced by progenitors are shorter. Overall, our results reveal that niche architecture optimizes organ efficiency but is not absolutely essential for organ function.

Live imaging reveals chromatin compaction transitions and dynamic transcriptional bursting during stem cell differentiation in vivo.

Stem cell differentiation requires dramatic changes in gene expression and global remodeling of chromatin architecture. How and when chromatin remodels relative to the transcriptional, behavioral, and morphological changes during differentiation remain unclear, particularly in an intact tissue context. Here, we develop a quantitative pipeline which leverages fluorescently-tagged histones and longitudinal imaging to track large-scale chromatin compaction changes within individual cells in a live mouse. Applying this pipeline to epidermal stem cells, we reveal that cell-to-cell chromatin compaction heterogeneity within the stem cell compartment emerges independent of cell cycle status, and instead is reflective of differentiation status. Chromatin compaction state gradually transitions over days as differentiating cells exit the stem cell compartment. Moreover, establishing live imaging of Keratin-10 (K10) nascent RNA, which marks the onset of stem cell differentiation, we find that Keratin-10 transcription is highly dynamic and largely precedes the global chromatin compaction changes associated with differentiation. Together, these analyses reveal that stem cell differentiation involves dynamic transcriptional states and gradual chromatin rearrangement.

Maternal Piwi regulates primordial germ cell development to ensure the fertility of female progeny in Drosophila.

In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the Drosophila early embryo. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.

Time-Resolved Characterization of Indoor Air Quality due to Human Activity and Likely Outdoor Sources during Early Evening Secondary School Wrestling Matches.

Despite positive health outcomes associated with physical activity as well as individual and team sports, poor indoor air and environmental quality can adversely affect human health, performance, and comfort. We conducted a 14-month field case study incorporating two winter sports regular seasons (12/2017-2/2019) including analyses of particulate matter (PMx) in air and in dust, carbon dioxide (CO2), temperature, and relative humidity (RH%) during secondary or high school wrestling activities in southern New Jersey, USA. After planning and piloting methods during the first winter sports regular season (12/2017-2/2018), during the second winter sports regular season (1-2/2019), we conducted a purposeful simultaneous real-time sampling midgymnasium adjacent to the wrestling mats. Gymnasium occupancy ranged 100-500 people. Data collected included inhalable PM10 resuspended from floor mats, fine respirable PM2.5, and indoor CO2, temperature, and RH%. Short-term real-time elevated PM10 levels were directly compared with simultaneously documented wrestling match bouts, e.g., student-athlete takedowns and pins. PM10 and PM2.5 levels were compared with other known documented activities indoors (e.g., snack bar) and sources outdoors (e.g., adjacent parking lot and major freeway). To understand CO2, temperature, and RH% data, we characterized the HS gym mechanical ventilation system-no doors or windows outdoors-and recorded occupancy during match hours. Indoor CO2 levels ranged ∼700-1000 ppm during match #1 on 1/23/2019 but ranged from ∼900 to 1900 ppm during match #2 on 1/30/2019, with >1000 ppm for the majority of the time (and throughout the entire varsity match when occupancy was at maximum). Future research should further characterize PM10 constituents in mat dust and indoor air with larger samples of schools and matches.

Fall 2020 COVID-19 Needs Assessment among New Jersey Secondary School Educational Professionals.

Secondary or high school (HS) educational professionals expressed concerns about dealing with environmental and occupational health and safety protocols due to COVID-19. Concerns related to fall 2020 school re-opening and getting back into in-person teaching-whether full-time, part-time or some other approved hybrid model-plus ongoing uncertainty with how the state and federal government will be handling matters about mandates for virtual learning, rapid testing, vaccine distribution, etc. These concerns were related to both their experience as educational professionals and genuine interest in personal and student well-being. This study was a cross-sectional online survey in early fall from mid-September-early October 2020. Of a possible maximum participation of 740 New Jersey (NJ) supervisory-level HS teachers and administrators (e.g., department chairs, district and school principals), 100 confirmed unique respondents (13.5%) consented and completed the survey. Of 100 experienced (mean 18 years teaching) participants, 70% responded to the gender identity question (overall, 61% female, 39% male; by NJ region, gender ratios were similar). There were statistically significant differences (using Fischer's exact test) between NJ regions regarding provision of online counseling and support services for teachers (p < 0.001); for resources and equipment for teachers to mediate online learning (p = 0.02); for assistive video technology tools (p = 0.03) and accessibility to structured online learning and professional development (p = 0.002); concerning learning aids to engage students in online instruction, online counseling, and support services for students and their families (p = 0.006); appropriate protocol is clean and disinfect areas used by a person with COVID-19 (p = 0.002); and, immediately separate staff and students who screen positive for COVID-19 (p = 0.03). There were few statistical differences by gender. This study reported what participants wanted regarding the development of future policies then implemented as reopening practices. Data can inform recommendations in NJ and elsewhere at federal, state, and local levels. Data provide new insights and valuable information to inform the consideration of acceptability of various policy measures among HS education professionals.

Online and in-Person Violence, Harassment, Intimidation and Bullying in New Jersey: 2011-2016.

BACKGROUND: New Jersey (NJ) Safe Schools Program, primarily funded by the NJ Department of Education, has aims concerning safety and health including helping to alleviate harassment, intimidation, and bullying (HIB) in secondary school because HIB can affect learning. We evaluated officially reported adolescent HIB total incidents and estimated rates in NJ public city and county school districts for the 2011-2016 school years. METHODS: A cross-sectional observational study using population-based state-reported public data. We calculated point prevalence and incidence rates for K-12 students in 22 public city school districts, students in 21 career-technical-vocational education (CTE) school districts, and 8 special services school districts during 2011-2016. RESULTS: HIB is prevalent in NJ school classrooms. HIB comprised ≥75% of total officially reported in-school violence-related incidents in a given school year. Rates per 100 enrollees of total officially reported incidents for the 5-year period of 2011-2016 was highest in special services school districts (5.00; 95% confidence interval [CI]: 4.67, 5.34) followed by CTE districts (1.94; 95% CI: 1.86, 2.01), and lowest in city districts (1.46; 95% CI: 1.44, 1.48). The special services school district also had the highest rates per year and the 5-year period. CONCLUSIONS: Data analyses suggested ideas for further research and improvements for school HIB incident reports. Policies and programs could remedy issues observed in state secondary school classrooms. Best practices within districts and schools can help protect students from HIB and promote safety, health, learning, and maturation.

Incidence reporting via online high school concussion surveillance by certified athletic trainers and school nurses, 2015-2018.

BACKGROUND: There is an increasing concern over adolescent concussions in sports due to risks of long-term negative effects. This study analyzed data over three school years on reported concussion incidence rates by season, high school grade levels and gender, and reported symptoms by school nurses versus athletic trainers, from New Jersey student-athlete concussion data available from an online school-based surveillance system. METHODS: School nurses and athletic trainers reported 300 concussions within five days from when each occurred over three school years, 2015-2018, in team sports and physical education in New Jersey high schools. Analysis was further conducted on symptoms and number of symptoms reported by school nurses versus school athletic trainers for each documented student-athlete concussion. Estimated concussion incidence rates were calculated using state agency verified school enrollment data. FINDINGS: Concussions most commonly occurred during fall, followed by spring, then winter. Concussion incidence rates ranged from 6.3/1000 (4.99, 7.55) - 9.1/1000 (7.27, 10.98) students over the three school years of the study. Athletic trainers completed 86% of the reports while nurses completed 11% (position or title of 3%, or n = 7, were not disclosed); the values were similar when considering only fall pre-season and regular season sports (88, 10, 2%, respectively). On average, across the three school years, athletic trainers reported about 3.5 symptoms per report while nurses reported 2.7 (values in fall seasons only were 3.7 and 3.1, respectively.) CONCLUSIONS: Certified athletic trainers, compared to school nurses, more often completed concussion report forms and reported more symptoms per injured student, perhaps due partly to closer contact and immediate care provided after injury. Additionally, this study had a higher concussion incidence rate during fall sports seasons compared to winter and spring. Future research can further improve our understanding of concussions among adolescent student-athletes to better inform concussion identification, management and recovery protocols.

Injury surveillance trends in career-technical education: New Jersey 1999-2018.

INTRODUCTION: Adolescents engaging in school-sponsored work experiences may be at risk of injury due to factors such as inexperience. This article examines trends in 20 years of reported injuries among New Jersey (NJ) adolescents engaging in school-sponsored work experiences, and compares a transition from paper to online reporting format. METHODS: New Jersey requires reporting of injuries occurring during school-sponsored work experiences to the NJ Department of Education. Injuries reported by NJ schools from 1999 to 2018 (n = 2,119) were examined; incidence rates for 2008 to 2018 (n = 743) were calculated using publicly available NJ Department of Education enrollment data for the denominator, including for specific groups of students: career and technical education; special healthcare needs. RESULTS: A downward trend in reported injuries in NJ schools was observed. However, the year online reporting became required by code (2013), an increase of nearly 50% was seen from 2012 (59-89), followed by a decline in reported incidents 2014 to 2018 (mean = 65, range 76 down to 47). Injury rate trends over time paralleled those of reported incidents. CONCLUSIONS: This study suggested worker safety and public health benefits of improvements from State of NJ code required training programs and online injury surveillance report form. Practical Applications: One potential method to address the safety and health of adolescents engaging in work experiences is the use of online reporting forms, to aid in surveillance efforts, coupled with occupational safety and health training specifically geared toward teachers and administrators who both supervise young, relatively inexperienced and vulnerable workers and who make relatively frequent worksite visits. Incorporation of specific details of the instructions on the use of a reporting form into required trainings, in addition to providing a clear, accessible guidance manual online, could further help improve youth worker safety surveillance efforts.

Developing and Piloting a School-Based Online Adolescent Student-Athlete Concussion Surveillance System.

BACKGROUND: Despite increased awareness of concussions, epidemiologic surveillance efforts have been scarce, especially among adolescents. This project, which was developed with school stakeholders (certified athletic trainers [ATCs], nurses, athletic directors), piloted a public secondary school-based online surveillance tool for interscholastic and intramural sports and physical education-related concussions in New Jersey during 2014-2017 school years (SY). METHODS: Participating public high schools (5 within 4 districts) and career-technical-vocational education districts (2 with 5 campuses) completed forms anonymously online via PsychData within 5 days. RESULTS: There were 208 concussions reported, 115 in 2015-2016 SY and 93 in 2016-2017 SY. In fall 2015, 86 concussions were reported, including 16 from summer preseason. In fall 2016, 56 concussions were reported; 3 occurred during preseason. There were 7 concussions reported in winter 2016 and 16 in winter 2017. Twenty-two concussions were reported in spring of both 2016 and 2017. Most online forms were completed in <10 minutes, usually using either desktop computers or tablets/iPads. School nurses followed by ATCs were primary sources of data entered online, usually by ATCs. CONCLUSIONS: Cooperation of nurses and ATCs at participating schools suggested online surveillance was valued and viable. Data inform future concussion prevention education and ongoing injury surveillance.

Contrasting Asymptomatic and Drug Resistance Gene Prevalence of Plasmodium falciparum in Ghana: Implications on Seasonal Malaria Chemoprevention.

Malaria is a significant public health problem in Ghana. Seasonal Malaria Chemoprevention (SMC) using a combination of sulfadoxine-pyrimethamine and amodiaquine has been implemented since 2015 in northern Ghana where malaria transmission is intense and seasonal. In this study, we estimated the prevalence of asymptomatic P. falciparum carriers in three ecological zones of Ghana, and compared the sensitivity and specificity of different molecular methods in identifying asymptomatic infections. Moreover, we examined the frequency of mutations in pfcrt, pfmdr1,pfdhfr, and pfdhps that relate to the ongoing SMC. A total of 535 asymptomatic schoolchildren were screened by microscopy and PCR (18s rRNA and TARE-2) methods. Among all samples, 28.6% were detected as positive by 18S nested PCR, whereas 19.6% were detected by microscopy. A high PCR-based asymptomatic prevalence was observed in the north (51%) compared to in the central (27.8%) and south (16.9%). The prevalence of pfdhfr-N51I/C59R/S108N/pfdhps-A437G quadruple mutant associated with sulfadoxine-pyrimethamine resistance was significantly higher in the north where SMC was implemented. Compared to 18S rRNA, TARE-2 serves as a more sensitive molecular marker for detecting submicroscopic asymptomatic infections in high and low transmission settings. These findings establish a baseline for monitoring P. falciparum prevalence and resistance in response to SMC over time.