An epigenetic switch controls an alternative NR2F2 isoform that unleashes a metastatic program in melanoma.

Metastatic melanoma develops once transformed melanocytic cells begin to de-differentiate into migratory and invasive melanoma cells with neural crest cell (NCC)-like and epithelial-to-mesenchymal transition (EMT)-like features. However, it is still unclear how transformed melanocytes assume a metastatic melanoma cell state. Here, we define DNA methylation changes that accompany metastatic progression in melanoma patients and discover Nuclear Receptor Subfamily 2 Group F, Member 2 - isoform 2 (NR2F2-Iso2) as an epigenetically regulated metastasis driver. NR2F2-Iso2 is transcribed from an alternative transcriptional start site (TSS) and it is truncated at the N-terminal end which encodes the NR2F2 DNA-binding domain. We find that NR2F2-Iso2 expression is turned off by DNA methylation when NCCs differentiate into melanocytes. Conversely, this process is reversed during metastatic melanoma progression, when NR2F2-Iso2 becomes increasingly hypomethylated and re-expressed. Our functional and molecular studies suggest that NR2F2-Iso2 drives metastatic melanoma progression by modulating the activity of full-length NR2F2 (Isoform 1) over EMT- and NCC-associated target genes. Our findings indicate that DNA methylation changes play a crucial role during metastatic melanoma progression, and their control of NR2F2 activity allows transformed melanocytes to acquire NCC-like and EMT-like features. This epigenetically regulated transcriptional plasticity facilitates cell state transitions and metastatic spread.

Pittsburgh Air Pollution Changes During the COVID-19 Lockdown.

The rapid spread of COVID-19 resulted in various public lockdowns across the globe. Previous studies showed that resultant travel restrictions improved air quality. The novel results presented here focus on source-specific changes and compare air quality for multiple years controlled for precipitation. This study sought to analyze air pollution changes in Pittsburgh, a city where an industrial past and present has led to elevated levels of particulate matter with representative diameter of ≤ 2.5µm (PM2.5). Data from the Allegheny County Health Department, from monitors located near a variety of site types, were analyzed with generalized linear models that used a gamma distribution with a log link to determine the magnitude and significance of changes in air pollution during the COVID-19 lockdown. The hypothesis was that nitrogen dioxide (NO2), which is primarily linked to vehicular traffic, would decrease significantly while potential decreases in particulate matter (PM2.5 and PM10) would be less apparent. Results of the regression models showed that NO2 was significantly reduced during lockdown at both monitoring sites and that PM10 was also significantly reduced at the majority of monitoring sites. However, decreases in PM2.5 pollution were only observed at half of the monitoring locations, and the location which observed the greatest decreases is located adjacent to an industrial source. Decreases in PM2.5 at this monitoring site were likely a result of reduced industrial processes both dependent and independent of the COVID-19 lockdown. This study suggests that industrial sources are a larger contributor of particulate matter than vehicular transportation in the city of Pittsburgh and that future air pollution reduction efforts should focus attention on emission reduction at these industrial facilities.

Whole-genome screen identifies diverse pathways that negatively regulate ciliogenesis.

We performed a high-throughput whole-genome RNAi screen to identify novel inhibitors of ciliogenesis in normal and basal breast cancer cells. Our screen uncovered a previously undisclosed, extensive network of genes linking integrin signaling and cellular adhesion to the extracellular matrix (ECM) with inhibition of ciliation in both normal and cancer cells. Surprisingly, a cohort of genes encoding ECM proteins was also identified. We characterized several ciliation inhibitory genes and showed that their silencing was accompanied by altered cytoskeletal organization and induction of ciliation, which restricts cell growth and migration in normal and breast cancer cells. Conversely, supplying an integrin ligand, vitronectin, to the ECM rescued the enhanced ciliation observed on silencing this gene. Aberrant ciliation could also be suppressed through hyperactivation of the YAP/TAZ pathway, indicating a potential mechanistic basis for our findings. Our findings suggest an unanticipated reciprocal relationship between ciliation and cellular adhesion to the ECM and provide a resource that could vastly expand our understanding of controls involving "outside-in" and "inside-out" signaling that restrain cilium assembly.

Impact of Low-Cost Point-of-Use Water Treatment Technologies on Enteric Infections and Growth among Children in Limpopo, South Africa.

Enteric infections early in life have been associated with poor linear growth among children in low-resource settings. Point-of-use water treatment technologies provide effective and low-cost solutions to reduce exposure to enteropathogens from drinking water, but it is unknown whether the use of these technologies translates to improvements in child growth. We conducted a community-based randomized controlled trial of two water treatment technologies to estimate their effects on child growth in Limpopo, South Africa. We randomized 404 households with a child younger than 3 years to receive a silver-impregnated ceramic water filter, a silver-impregnated ceramic tablet, a safe-storage water container alone, or no intervention, and these households were followed up quarterly for 2 years. We estimated the effects of the interventions on linear and ponderal growth, enteric infections assessed by quantitative molecular diagnostics, and diarrhea prevalence. The silver-impregnated ceramic water filters and tablets consistently achieved approximately 1.2 and 3 log reductions, respectively, in total coliform bacteria in drinking water samples. However, the filters and tablets were not associated with differences in height (height-for-age z-score differences compared with no intervention: 0.06, 95% CI: -0.29, 0.40, and 0.00, 95% CI: -0.35, 0.35, respectively). There were also no effects of the interventions on weight, diarrhea prevalence, or enteric infections. Despite their effectiveness in treating drinking water, the use of the silver-impregnated ceramic water filters and tablets did not reduce enteric infections or improve child growth. More transformative water, sanitation, and hygiene interventions that better prevent enteric infections are likely needed to improve long-term child growth outcomes.

BRCA1 and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells.

Long interspersed element-1 (LINE-1, or L1) is the only autonomous retrotransposon that is active in human cells. Different host factors have been shown to influence L1 mobility; however, systematic analyses of these factors are limited. Here, we developed a high-throughput microscopy-based retrotransposition assay that identified the double-stranded break (DSB) repair and Fanconi anemia (FA) factors active in the S/G2 phase as potent inhibitors and regulators of L1 activity. In particular, BRCA1, an E3 ubiquitin ligase with a key role in several DNA repair pathways, directly affects L1 retrotransposition frequency and structure and plays a distinct role in controlling L1 ORF2 protein translation through L1 mRNA binding. These results suggest the existence of a 'battleground' at the DNA replication fork between homologous recombination (HR) factors and L1 retrotransposons and reveal a potential role for L1 in the genotypic evolution of tumors characterized by BRCA1 and HR repair deficiencies.

Comprehensive Scanning Mutagenesis of Human Retrotransposon LINE-1 Identifies Motifs Essential for Function.

Long Interspersed Nuclear Element-1 (LINE-1, L1) is the only autonomous active transposable element in the human genome. The L1-encoded proteins ORF1p and ORF2p enable the element to jump from one locus to another via a "copy-and-paste" mechanism. ORF1p is an RNA-binding protein, and ORF2p has endonuclease and reverse transcriptase activities. The huge number of truncated L1 remnants in the human genome suggests that the host has likely evolved mechanisms to prevent full L1 replication, and thereby decrease the proliferation of active elements and reduce the mutagenic potential of L1. In turn, L1 appears to have a minimized length to increase the probability of successful full-length replication. This streamlining would be expected to lead to high information density. Here, we describe the construction and initial characterization of a library of 538 consecutive trialanine substitutions that scan along ORF1p and ORF2p to identify functionally important regions. In accordance with the streamlining hypothesis, retrotransposition was overall very sensitive to mutations in ORF1p and ORF2p; only 16% of trialanine mutants retained near-wild-type (WT) activity. All ORF1p mutants formed near-WT levels of mRNA transcripts and 75% formed near-WT levels of protein. Two ORF1p mutants presented a unique nucleolar-relocalization phenotype. Regions of ORF2p that are sensitive to mutagenesis but lack phylogenetic conservation were also identified. We provide comprehensive information on the regions most critical to retrotransposition. This resource will guide future studies of intermolecular interactions that form with RNA, proteins, and target DNA throughout the L1 life cycle.

KLF4 as a rheostat of osteolysis and osteogenesis in prostate tumors in the bone.

We previously showed that KLF4, a gene highly expressed in murine prostate stem cells, blocks the progression of indolent intraepithelial prostatic lesions into aggressive and rapidly growing tumors. Here, we show that the anti-tumorigenic effect of KLF4 extends to PC3 human prostate cancer cells growing in the bone. We compared KLF4 null cells with cells transduced with a DOX-inducible KLF4 expression system, and find KLF4 function inhibits PC3 growth in monolayer and soft agar cultures. Furthermore, KLF4 null cells proliferate rapidly, forming large, invasive, and osteolytic tumors when injected into mouse femurs, whereas KLF4 re-expression immediately after their intra-femoral inoculation blocks tumor development and preserves a normal bone architecture. KLF4 re-expression in established KLF4 null bone tumors inhibits their osteolytic effects, preventing bone fractures and inducing an osteogenic response with new bone formation. In addition to these profound biological changes, KLF4 also induces a transcriptional shift from an osteolytic program in KLF4 null cells to an osteogenic program. Importantly, bioinformatic analysis shows that genes regulated by KLF4 overlap significantly with those expressed in metastatic prostate cancer patients and in three individual cohorts with bone metastases, strengthening the clinical relevance of the findings in our xenograft model.

Understanding and Perception of Overlapping Surgery in an Orthopaedic Trauma Population: A Survey Study.

INTRODUCTION: Recently, overlapping surgery has received attention on the national scale. This study quantifies orthopaedic trauma patients' familiarity and concern with overlapping surgery as it relates to their care. METHODS: A 15-question survey was voluntarily completed by 200 orthopaedic trauma patients in the outpatient setting of a level I trauma center. Three domains were evaluated in the survey: demographic data, familiarity with overlapping surgery, and the degree of concern with overlapping surgery. Patients read a position statement explaining the practice of overlapping surgery, and their changes in level of concern were evaluated. Descriptive statistics were used to evaluate the data. RESULTS: A total of 200 patients completed the survey, of which 98 (49%) were male. The age range was broadly distributed. After surgery, 124 patients (62%) were seen for follow up. The remaining 76 patients (38%) did not undergo surgery. Regarding the practice of overlapping surgery, 116 respondents (58%) had no knowledge. There were 127 patients (63%) who reported their concern level as a 1 on an ordinal scale from 1 to 5, corresponding to the lowest possible level. Overall, 182 patients (91%) reported a level of concern of 3 (the median) or less with an average score of 1.7, indicating a low average level of concern. Six patients (3%) reported the maximum level of concern. On the whole, 160 patients (80%) reported either a decreased level of concern or no change after reading our department's position statement on overlapping surgery. Of the 124 patients, 81 (65%) postoperatively reported that they perceived no effect by overlapping surgery. The most common factors cited as areas of concern by patients were the absence of attending physician in the operating room (26%), risk of error by the resident (34%), and risk of a missed step in the surgical procedure (31%). CONCLUSION: These data indicate that most respondents had no previous knowledge of overlapping surgery and had a generally low level of concern with its use as practiced at our institution. Disclosing the use of overlapping surgery and its purpose to patients is an important component of preoperative counseling. LEVEL OF EVIDENCE: Level V.

Whole genome screen reveals a novel relationship between Wolbachia levels and Drosophila host translation.

Wolbachia is an intracellular bacterium that infects a remarkable range of insect hosts. Insects such as mosquitos act as vectors for many devastating human viruses such as Dengue, West Nile, and Zika. Remarkably, Wolbachia infection provides insect hosts with resistance to many arboviruses thereby rendering the insects ineffective as vectors. To utilize Wolbachia effectively as a tool against vector-borne viruses a better understanding of the host-Wolbachia relationship is needed. To investigate Wolbachia-insect interactions we used the Wolbachia/Drosophila model that provides a genetically tractable system for studying host-pathogen interactions. We coupled genome-wide RNAi screening with a novel high-throughput fluorescence in situ hybridization (FISH) assay to detect changes in Wolbachia levels in a Wolbachia-infected Drosophila cell line JW18. 1117 genes altered Wolbachia levels when knocked down by RNAi of which 329 genes increased and 788 genes decreased the level of Wolbachia. Validation of hits included in depth secondary screening using in vitro RNAi, Drosophila mutants, and Wolbachia-detection by DNA qPCR. A diverse set of host gene networks was identified to regulate Wolbachia levels and unexpectedly revealed that perturbations of host translation components such as the ribosome and translation initiation factors results in increased Wolbachia levels both in vitro using RNAi and in vivo using mutants and a chemical-based translation inhibition assay. This work provides evidence for Wolbachia-host translation interaction and strengthens our general understanding of the Wolbachia-host intracellular relationship.

Functional Interactions Between rsks-1/S6K, glp-1/Notch, and Regulators of Caenorhabditis elegans Fertility and Germline Stem Cell Maintenance.

The proper accumulation and maintenance of stem cells is critical for organ development and homeostasis. The Notch signaling pathway maintains stem cells in diverse organisms and organ systems. In Caenorhabditis elegans, GLP-1/Notch activity prevents germline stem cell (GSC) differentiation. Other signaling mechanisms also influence the maintenance of GSCs, including the highly-conserved TOR substrate ribosomal protein S6 kinase (S6K). Although C. elegans bearing either a null mutation in rsks-1/S6K or a reduction-of-function (rf) mutation in glp-1/Notch produce half the normal number of adult germline progenitors, virtually all these single mutant animals are fertile. However, glp-1(rf) rsks-1(null) double mutant animals are all sterile, and in about half of their gonads, all GSCs differentiate, a distinctive phenotype associated with a significant reduction or loss of GLP-1 signaling. How rsks-1/S6K promotes GSC fate is unknown. Here, we determine that rsks-1/S6K acts germline-autonomously to maintain GSCs, and that it does not act through Cyclin-E or MAP kinase in this role. We found that interfering with translation also enhances glp-1(rf), but that regulation through rsks-1 cannot fully account for this effect. In a genome-scale RNAi screen for genes that act similarly to rsks-1/S6K, we identified 56 RNAi enhancers of glp-1(rf) sterility, many of which were previously not known to interact functionally with Notch. Further investigation revealed at least six candidates that, by genetic criteria, act linearly with rsks-1/S6K. These include genes encoding translation-related proteins, cacn-1/Cactin, an RNA exosome component, and a Hedgehog-related ligand. We found that additional Hedgehog-related ligands may share functional relationships with glp-1/Notch and rsks-1/S6K in maintaining germline progenitors.

Transcription factor profiling reveals molecular choreography and key regulators of human retrotransposon expression.

Transposable elements (TEs) represent a substantial fraction of many eukaryotic genomes, and transcriptional regulation of these factors is important to determine TE activities in human cells. However, due to the repetitive nature of TEs, identifying transcription factor (TF)-binding sites from ChIP-sequencing (ChIP-seq) datasets is challenging. Current algorithms are focused on subtle differences between TE copies and thus bias the analysis to relatively old and inactive TEs. Here we describe an approach termed "MapRRCon" (mapping repeat reads to a consensus) which allows us to identify proteins binding to TE DNA sequences by mapping ChIP-seq reads to the TE consensus sequence after whole-genome alignment. Although this method does not assign binding sites to individual insertions in the genome, it provides a landscape of interacting TFs by capturing factors that bind to TEs under various conditions. We applied this method to screen TFs' interaction with L1 in human cells/tissues using ENCODE ChIP-seq datasets and identified 178 of the 512 TFs tested as bound to L1 in at least one biological condition with most of them (138) localized to the promoter. Among these L1-binding factors, we focused on Myc and CTCF, as they play important roles in cancer progression and 3D chromatin structure formation. Furthermore, we explored the transcriptomes of The Cancer Genome Atlas breast and ovarian tumor samples in which a consistent anti-/correlation between L1 and Myc/CTCF expression was observed, suggesting that these two factors may play roles in regulating L1 transcription during the development of such tumors.

LINE-1 protein localization and functional dynamics during the cell cycle.

LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology. However, a clear understanding of L1's lifecycle and the processes involved in restricting its insertion and intragenomic spread remains elusive. Here we identify modes of L1 proteins' entrance into the nucleus, a necessary step for L1 proliferation. Using functional, biochemical, and imaging approaches, we also show a clear cell cycle bias for L1 retrotransposition that peaks during the S phase. Our observations provide a basis for novel interpretations about the nature of nuclear and cytoplasmic L1 ribonucleoproteins (RNPs) and the potential role of DNA replication in L1 retrotransposition.

Challenges to Sustainable Safe Drinking Water: A Case Study of Water Quality and Use across Seasons in Rural Communities in Limpopo Province, South Africa.

Consumption of microbial-contaminated water can result in diarrheal illnesses and enteropathy with the heaviest impact upon children below the age of five. We aimed to provide a comprehensive analysis of water quality in a low-resource setting in Limpopo province, South Africa. Surveys were conducted in 405 households in rural communities of Limpopo province to determine their water-use practices, perceptions of water quality, and household water-treatment methods. Drinking water samples were tested from households for microbiological contamination. Water from potential natural sources were tested for physicochemical and microbiological quality in the dry and wet seasons. Most households had their primary water source piped into their yard or used an intermittent public tap. Approximately one third of caregivers perceived that they could get sick from drinking water. All natural water sources tested positive for fecal contamination at some point during each season. The treated municipal supply never tested positive for fecal contamination; however, the treated system does not reach all residents in the valley; furthermore, frequent shutdowns of the treatment systems and intermittent distribution make the treated water unreliable. The increased water quantity in the wet season correlates with increased treated water from municipal taps and a decrease in the average contaminant levels in household water. This research suggests that wet season increases in water quantity result in more treated water in the region and that is reflected in residents' water-use practices.

Computer-assisted surgery: The use of stored intraoperative images for accurate restoration of femoral length and rotational alignment after fracture.

Most femoral fractures are now managed with minimally invasive internal fixation. In the absence of formal exposure of the fracture lines, these procedures make heavy use of C-arm fluoroscopy to allow both fracture reduction and placement of implants, at the expense of measurable radiation exposure to both patient and surgeon. Although this technology has been commercially available for over a decade, it has not yet been widely accepted by the Orthopaedic community.

Evaluation of Orthopedic Trauma Surgery Follow-up and Impact of a Routine Callback Program.

A high rate of patients lost to follow-up is a common problem in orthopedic trauma surgery. This adversely affects the ability to produce accurate clinical outcomes research. The purpose of this project was to (1) evaluate the rate of loss to follow-up at an academic level I trauma center; (2) identify the patient-reported reasons for loss to follow-up; and (3) evaluate the efficacy of a routine patient callback program. All patients who underwent surgery in the orthopedic trauma division of the University of Virginia Medical Center from April 1, 2014, to September 30, 2014, and did not complete their postoperative clinic follow-up were analyzed. The characteristics of these patients were evaluated, and the primary reason for not completing the recommended follow-up was identified. All patients were then offered additional orthopedic follow-up at the time of contact. Of the 480 patients who met the inclusion criteria, 41 (8.5%) failed to complete the recommended postoperative follow-up course. The most common reason for being lost to follow-up was feeling well and not having the need to be seen (46.3%). Only 6 (14.6%) of the 41 patients requested follow-up care at the time of contact. The lost to follow-up rate in this study, 8.5%, was considerably lower than that previously reported, but patient characteristics were consistent with those of prior studies on this subject. The low lost to follow-up rate may reflect a difference in geographic location or patient population. The patient callback program had a low yield of patients requesting additional follow-up after being contacted. [Orthopedics. 2017; 40(2):e312-e316.].

Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are an essential tool for modeling how causal genetic variants impact cellular function in disease, as well as an emerging source of tissue for regenerative medicine. The preparation of somatic cells, their reprogramming and the subsequent verification of iPSC pluripotency are laborious, manual processes limiting the scale and reproducibility of this technology. Here we describe a modular, robotic platform for iPSC reprogramming enabling automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. We demonstrate that automated reprogramming and the pooled selection of polyclonal pluripotent cells results in high-quality, stable iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single-colony subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines.

Three-dimensional navigation is more accurate than two-dimensional navigation or conventional fluoroscopy for percutaneous sacroiliac screw fixation in the dysmorphic sacrum: a randomized multicenter study.

OBJECTIVES: To evaluate the accuracy of computer-assisted sacral screw fixation compared with conventional techniques in the dysmorphic versus normal sacrum. DESIGN: Review of a previous study database. SETTING: Database of a multinational study with 9 participating trauma centers. PATIENTS: The reviewed group included 130 patients, 72 from the navigated group and 58 from the conventional group. Of these, 109 were in the nondysmorphic group and 21 in the dysmorphic group. INTERVENTION: Placement of sacroiliac (SI) screws was performed using standard fluoroscopy for the conventional group and BrainLAB navigation software with either 2-dimensional or 3-dimensional (3D) navigation for the navigated group. MAIN OUTCOME MEASUREMENTS: Accuracy of SI screw placement by 2-dimensional and 3D navigation versus conventional fluoroscopy in dysmorphic and nondysmorphic patients, as evaluated by 6 observers using postoperative computerized tomography imaging at least 1 year after initial surgery. Intraobserver agreement was also evaluated. RESULTS: There were 11.9% (13/109) of patients with misplaced screws in the nondysmorphic group and 28.6% (6/21) of patients with misplaced screws in the dysmorphic group, none of which were in the 3D navigation group. Raw agreement between the 6 observers regarding misplaced screws was 32%. However, the percent overall agreement was 69.0% (kappa = 0.38, P < 0.05). CONCLUSIONS: The use of 3D navigation to improve intraoperative imaging for accurate insertion of SI screws is magnified in the dysmorphic proximal sacral segment. We recommend the use of 3D navigation, where available, for insertion of SI screws in patients with normal and dysmorphic proximal sacral segments. LEVEL OF EVIDENCE: Therapeutic level I.

Characterization and molecular profiling of PSEN1 familial Alzheimer's disease iPSC-derived neural progenitors.

Presenilin 1 (PSEN1) encodes the catalytic subunit of γ-secretase, and PSEN1 mutations are the most common cause of early onset familial Alzheimer's disease (FAD). In order to elucidate pathways downstream of PSEN1, we characterized neural progenitor cells (NPCs) derived from FAD mutant PSEN1 subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying PSEN1 mutations. PSEN1 mutant fibroblasts, and NPCs produced greater ratios of Aß42 to Aß40 relative to their control counterparts, with the elevated ratio even more apparent in PSEN1 NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in PSEN1 NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our PSEN1 iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aß42/40, and have characterized novel expression changes.

ß-cell dysfunction due to increased ER stress in a stem cell model of Wolfram syndrome.

Wolfram syndrome is an autosomal recessive disorder caused by mutations in WFS1 and is characterized by insulin-dependent diabetes mellitus, optic atrophy, and deafness. To investigate the cause of ß-cell failure, we used induced pluripotent stem cells to create insulin-producing cells from individuals with Wolfram syndrome. WFS1-deficient ß-cells showed increased levels of endoplasmic reticulum (ER) stress molecules and decreased insulin content. Upon exposure to experimental ER stress, Wolfram ß-cells showed impaired insulin processing and failed to increase insulin secretion in response to glucose and other secretagogues. Importantly, 4-phenyl butyric acid, a chemical protein folding and trafficking chaperone, restored normal insulin synthesis and the ability to upregulate insulin secretion. These studies show that ER stress plays a central role in ß-cell failure in Wolfram syndrome and indicate that chemical chaperones might have therapeutic relevance under conditions of ER stress in Wolfram syndrome and other forms of diabetes.