Decryption of sequence, structure, and functional features of SINE repeat elements in SINEUP non-coding RNA-mediated post-transcriptional gene regulation.

RNA structure folding largely influences RNA regulation by providing flexibility and functional diversity. In silico and in vitro analyses are limited in their ability to capture the intricate relationships between dynamic RNA structure and RNA functional diversity present in the cell. Here, we investigate sequence, structure and functional features of mouse and human SINE-transcribed retrotransposons embedded in SINEUPs long non-coding RNAs, which positively regulate target gene expression post-transcriptionally. In-cell secondary structure probing reveals that functional SINEs-derived RNAs contain conserved short structure motifs essential for SINEUP-induced translation enhancement. We show that SINE RNA structure dynamically changes between the nucleus and cytoplasm and is associated with compartment-specific binding to RBP and related functions. Moreover, RNA-RNA interaction analysis shows that the SINE-derived RNAs interact directly with ribosomal RNAs, suggesting a mechanism of translation regulation. We further predict the architecture of 18 SINE RNAs in three dimensions guided by experimental secondary structure data. Overall, we demonstrate that the conservation of short key features involved in interactions with RBPs and ribosomal RNA drives the convergent function of evolutionarily distant SINE-transcribed RNAs.

Impact of high- and low-flow nebulised saline on airway hydration and mucociliary transport.

Background: Nebulised drugs, including osmotic agents and saline, are increasingly used during noninvasive respiratory support, including nasal high-flow therapy. The authors conducted an in vitro study to compare the hydration effect of nebulised isotonic 0.9% and hypertonic 7.0% saline on mucociliary transport. Methods: In a perfused organ bath, 10 sheep tracheas were exposed to 7.5 mL nebulised 0.9% and 7.0% saline entrained into heated (38°C) and humidified air delivered at high and low flow (20 and 7 L·min-1, respectively). Simultaneous measurements of the airway surface liquid height, mucus transport velocity, cilia beat frequency and surface temperature were made over time. The data are presented as mean±sd. Results: The airway surface liquid height increased significantly with both 0.9% and 7.0% saline: at low-flow by 37.2±10.0 µm and 152.7±10.9 µm, respectively, and at high-flow by 62.3±5.6 µm and 163.4±25.4 µm, respectively (p<0.001). Mucus velocity was increased by both 0.9% and 7.0% saline from a baseline of 8.2±0.8 mm·min-1 to 8.8±0.7 mm·min-1 and 17.1±0.5 mm·min-1, respectively, with low-flow and at high-flow to 9.8±0.02 mm·min-1 (p=0.04) and 16.9±0.5 mm·min-1 (p<0.05), respectively. Ciliary beating did not change with 0.9% saline, but declined from 13.1±0.6 Hz to 10.2±0.6 Hz and 11.1±0.6 Hz (p<0.05) with 7.0% saline at low- and high-flow, respectively. Conclusions: The findings demonstrate that nebulised isotonic 0.9% saline, like hypertonic 7.0% saline, significantly stimulates basal mucociliary transport, and the use of high-flow delivery had no significantly different hydration effects compared with low-flow delivery. Hypertonic 7.0% saline suppressed ciliary beating, indicating an increase in airway surface liquid osmolarity, which may have negative effects on the airway surface with frequent use.

Understanding online ratings of joint arthroplasty surgeons: A national cross-sectional analysis.

Background: Patient reviews provide an important referral source for physicians and an opportunity to improve practice performance. This study's objective was to characterize the online reviews of hip and knee arthroplasty surgeons published by three of the industry's leading platforms. Methods: A random sample of 1000 hip and knee arthroplasty surgeons across all 50 US states (10 hip and 10 knee surgeons per state) was generated using Google Search. A total of 7842 online reviews posted for those surgeons on Healthgrades, Vitals, and Google were analyzed. A range of surgeons, affiliated hospitals, and reviewer attributes was compared to identify significant predictors of patient satisfaction. Results: The study cohort had 98.1% male surgeons with a mean age of 53.55 ± 8.94 years and mean experience of 26.43 ± 9.21 years. Younger age (p < 0.001), shorter years of experience (p < 0.001), and arthroplasty fellowship training (p < 0.001) were associated with more positive ratings. Reviewer anonymity, observed in 30.93% of all reviews, tended to correlate with more negative ratings (p < 0.001). Overall, 86.93% of patient remarks were positive, and only 74.81% of remarks centered on physician attributes. The five leading components of patient satisfaction were perceptions of physician competence (34.81%, p < 0.001), bedside manner (23.83%, p = 0.002), and communication (16.17%, p = 0.94); interactions with physician extenders (14.75%, p < 0.001); and wait time (2.73%, p < 0.001). Conclusion: While most ratings of hip and knee arthroplasty surgeons were positive, more than a quarter of reviews were either not directly related to the individual surgeons or were submitted anonymously. Caution is advised regarding overreliance on patient experience surveys as predictors of physician performance.

Modification of the Folmer primers for the cytochrome c oxidase gene facilitates identification of mosquitoes.

BACKGROUND: Accurate identification of mosquito species is essential for the development and optimization of strategies to control mosquitoes and mosquito-borne diseases. Problems with the morphological identification of mosquito species have led to the use of molecular identification techniques, in particular the Folmer cytochrome c oxidase subunit I (COI) PCR system (FCOS), originally designed to identify a range of other invertebrates. METHODS: As there can be difficulties identifying mosquitoes using FCOS, we re-evaluated the FCOS primers and developed a new COI-based SYBR PCR (the Auburn COI system-AUCOS) to improve the molecular identification of mosquitoes. Sequence data in GenBank for 33 species from 10 genera of mosquitoes were used to develop our AUCOS primers. Two molecular assays (AUCOS, FCOS) and morphological identification were carried out on mosquitoes collected from the field in Auburn, Alabama (USA) and on Saint Kitts. RESULTS: With a convenience sample of individual mosquitoes comprising 19 species from six genera in Saint Kitts (n = 77) and Auburn (n = 48), our AUCOS provided higher-quality sequence data than FCOS. It also proved more sensitive than FCOS, successfully amplifying 67.5% (85/126) as opposed to 16.7% (21/126) of the samples. The species determined by morphology, or genus with damaged samples, matched that as determined by AUCOS for 84.9% (62/73) of the samples. Morphological classification was confirmed by FCOS with 81.0% (17/21) of samples producing utilizable sequences. While both FCOS and AUCOS correctly identified all the Aedes, Anopheles, Deinocerites, and Uranotaenia species in the study, identification of Culex species was less successful with both methods: 50.0% (3/6) by FCOS and 35.7% (5/14) by AUCOS. CONCLUSIONS: The AUCOS DNA barcoding system for mosquito species described in this study is superior to the existing FCOS for the identification of mosquito species. As AUCOS and FCOS amplify the same variable region of the COI, the large amount of existing data on GenBank can be used to identify mosquito species with sequences produced by either PCR.

Early and Direct Rehab Transfer Leads to Significant Cost Savings and Decreased Hospital Length of Stay for Total Joint Arthroplasty in a Veteran Population.

BACKGROUND: Total joint arthroplasty is the most common elective orthopedic procedure in the Veterans Affairs hospital system. In 2019, physical medicine and rehabilitation began screening patients before surgery to select candidates for direct transfer to acute rehab after surgery. The primary outcome of this study was to demonstrate that the accelerated program was successful in decreasing inpatient costs and length of stay (LOS). The secondary outcome was to show that there was no increase in complication, reoperation, and readmission rates. METHODS: A retrospective review of total joint arthroplasty patients was conducted with three cohorts: 1) control (n = 193), 2) transfer to rehab orders on postop day #1 (n = 178), and 3) direct transfers to rehab (n = 173). To assess for demographic disparities between cohorts, multiple analysis of variance tests followed by a Bonferroni P-value correction were used. Differences between test groups regarding primary outcomes were assessed with analysis of variance tests followed by pairwise t-tests with Bonferroni P-value corrections. RESULTS: There were no significant differences between the cohort demographics or comorbidities. The mean total LOS decreased from 7.0 days in the first cohort, to 6.9 in the second, and 6.0 in the third (P = .00034). The mean decrease in cost per patient was $14,006 between cohorts 1 and 3, equating to over $5.6 million in savings annually. There was no significant change in preintervention and postintervention short-term complications (P = .295). CONCLUSIONS: Significant cost savings and decrease in total LOS was observed. In the current health care climate focused on value-based care, a similar intervention could be applied nationwide to improve Veterans Affair services.

Effects of seasonality and land use on the diversity, relative abundance, and distribution of mosquitoes on St. Kitts, West Indies.

BACKGROUND: Mosquito surveys that collect local data on mosquito species' abundances provide baseline data to help understand potential host-pathogen-mosquito relationships, predict disease transmission, and target mosquito control efforts. METHODS: We conducted an adult mosquito survey from November 2017 to March 2019 on St. Kitts, using Biogents Sentinel 2 traps, set monthly and run for 48-h intervals. We collected mosquitoes from a total of 30 sites distributed across agricultural, mangrove, rainforest, scrub and urban land covers. We investigated spatial variation in mosquito species richness across the island using a hierarchical Bayesian multi-species occupancy model. We developed a mixed effects negative binomial regression model to predict the effects of spatial variation in land cover, and seasonal variation in precipitation on observed counts of the most abundant mosquito species observed. RESULTS: There was high variation among sites in mosquito community structure, and variation in site level richness that correlated with scrub forest, agricultural, and urban land covers. The four most abundant species were Aedes taeniorhynchus, Culex quinquefasciatus, Aedes aegpyti and Deinocerites magnus, and their relative abundance varied with season and land cover. Aedes aegypti was the most commonly occurring mosquito on the island, with a 90% probability of occurring at between 24 and 30 (median = 26) sites. Mangroves yielded the most mosquitoes, with Ae. taeniorhynchus, Cx. quinquefasciatus and De. magnus predominating. Psorophora pygmaea and Toxorhynchites guadeloupensis were only captured in scrub habitat. Capture rates in rainforests were low. Our count models also suggested the extent to which monthly average precipitation influenced counts varied according to species. CONCLUSIONS: There is high seasonality in mosquito abundances, and land cover influences the diversity, distribution, and relative abundance of species on St. Kitts. Further, human-adapted mosquito species (e.g. Ae. aegypti and Cx. quinquefasciatus) that are known vectors for many human relevant pathogens (e.g. chikungunya, dengue and Zika viruses in the case of Ae. aegypti; West Nile, Spondweni, Oropouche virus, and equine encephalitic viruses in the case of Cx. quinqefasciatus) are the most wide-spread (across land covers) and the least responsive to seasonal variation in precipitation.

SINEUP long non-coding RNA acts via PTBP1 and HNRNPK to promote translational initiation assemblies.

SINEUPs are long non-coding RNAs (lncRNAs) that contain a SINE element, and which up-regulate the translation of target mRNA. They have been studied in a wide range of applications, as both biological and therapeutic tools, although the underpinning molecular mechanism is unclear. Here, we focused on the sub-cellular distribution of target mRNAs and SINEUP RNAs, performing co-transfection of expression vectors for these transcripts into human embryonic kidney cells (HEK293T/17), to investigate the network of translational regulation. The results showed that co-localization of target mRNAs and SINEUP RNAs in the cytoplasm was a key phenomenon. We identified PTBP1 and HNRNPK as essential RNA binding proteins. These proteins contributed to SINEUP RNA sub-cellular distribution and to assembly of translational initiation complexes, leading to enhanced target mRNA translation. These findings will promote a better understanding of the mechanisms employed by regulatory RNAs implicated in efficient protein translation.

No evidence for sylvatic cycles of chikungunya, dengue and Zika viruses in African green monkeys (Chlorocebus aethiops sabaeus) on St. Kitts, West Indies.

BACKGROUND: Dengue, chikungunya and Zika viruses (DENV, CHIKV and ZIKV) are transmitted in sylvatic transmission cycles between non-human primates and forest (sylvan) mosquitoes in Africa and Asia. It remains unclear if sylvatic cycles exist or could establish themselves elsewhere and contribute to the epidemiology of these diseases. The Caribbean island of St. Kitts has a large African green monkey (AGM) (Chlorocebus aethiops sabaeus) population and is therefore ideally suited to investigate sylvatic cycles. METHODS: We tested 858 AGM sera by ELISA and PRNT for virus-specific antibodies and collected and identified 9704 potential arbovirus vector mosquitoes. Mosquitoes were homogenized in 513 pools for testing by viral isolation in cell culture and by multiplex RT-qPCR after RNA extraction to detect the presence of DENV, CHIKV and ZIKVs. DNA was extracted from 122 visibly blood-fed individual mosquitoes and a polymorphic region of the hydroxymethylbilane synthase gene (HMBS) was amplified by PCR to determine if mosquitoes had fed on AGMs or humans. RESULTS: All of the AGMs were negative for DENV, CHIKV or ZIKV antibodies. However, one AGM did have evidence of an undifferentiated Flavivirus infection. Similarly, DENV, CHIKV and ZIKV were not detected in any of the mosquito pools by PCR or culture. AGMs were not the source of any of the mosquito blood meals. CONCLUSION: Sylvatic cycles involving AGMs and DENV, CHIKV and ZIKV do not currently exist on St. Kitts.

RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions.

Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure.

Single-cell transcriptomics reveals expansion of cytotoxic CD4 T cells in supercentenarians.

Supercentenarians, people who have reached 110 y of age, are a great model of healthy aging. Their characteristics of delayed onset of age-related diseases and compression of morbidity imply that their immune system remains functional. Here we performed single-cell transcriptome analysis of 61,202 peripheral blood mononuclear cells (PBMCs), derived from 7 supercentenarians and 5 younger controls. We identified a marked increase of cytotoxic CD4 T cells (CD4 cytotoxic T lymphocytes [CTLs]) as a signature of supercentenarians. Furthermore, single-cell T cell receptor sequencing of 2 supercentenarians revealed that CD4 CTLs had accumulated through massive clonal expansion, with the most frequent clonotypes accounting for 15 to 35% of the entire CD4 T cell population. The CD4 CTLs exhibited substantial heterogeneity in their degree of cytotoxicity as well as a nearly identical transcriptome to that of CD8 CTLs. This indicates that CD4 CTLs utilize the transcriptional program of the CD8 lineage while retaining CD4 expression. Indeed, CD4 CTLs extracted from supercentenarians produced IFN-γ and TNF-α upon ex vivo stimulation. Our study reveals that supercentenarians have unique characteristics in their circulating lymphocytes, which may represent an essential adaptation to achieve exceptional longevity by sustaining immune responses to infections and diseases.

Sylvatic cycles of arboviruses in non-human primates.

Arboviruses infecting people primarily exist in urban transmission cycles involving urban mosquitoes in densely populated tropical regions. For dengue, chikungunya, Zika and yellow fever viruses, sylvatic (forest) transmission cycles also exist in some regions and involve non-human primates and forest-dwelling mosquitoes. Here we review the investigation methods and available data on sylvatic cycles involving non-human primates and dengue, chikungunya, Zika and yellow fever viruses in Africa, dengue viruses in Asia and yellow fever virus in the Americas. We also present current putative data that Mayaro, o'nyong'nyong, Oropouche, Spondweni and Lumbo viruses exist in sylvatic cycles.

Multi-year whole-blood transcriptome data for the study of onset and progression of Parkinson's Disease.

Parkinson's disease (PD) is an age-related, chronic and progressive neurodegenerative disorder characterized by a loss of multifocal neurons, resulting in both non-motor and motor symptoms. While several genetic and environmental contributory risk factors have been identified, more exact methods for diagnosing and assessing prognosis of PD have yet to be established. Here we describe the generation and validation of a dataset comprising whole-blood transcriptomes originally intended for use in detection of blood biomarkers and transcriptomic network changes indicative of PD. Whole-blood samples extracted from both early-stage PD patients and healthy controls were sequenced using no-amplification non-tagging cap analysis of gene expression (nAnT-iCAGE) to analyse differences in global RNA expression patterns across the conditions. Subsequent sampling of a subset of PD patients one-year later provides the opportunity to study changes in transcriptomes arising due to disease progression.

Computer-Aided Recognition of Facial Attributes for Fetal Alcohol Spectrum Disorders.

OBJECTIVES: To compare the detection of facial attributes by computer-based facial recognition software of 2-D images against standard, manual examination in fetal alcohol spectrum disorders (FASD). METHODS: Participants were gathered from the Fetal Alcohol Syndrome Epidemiology Research database. Standard frontal and oblique photographs of children were obtained during a manual, in-person dysmorphology assessment. Images were submitted for facial analysis conducted by the facial dysmorphology novel analysis technology (an automated system), which assesses ratios of measurements between various facial landmarks to determine the presence of dysmorphic features. Manual blinded dysmorphology assessments were compared with those obtained via the computer-aided system. RESULTS: Areas under the curve values for individual receiver-operating characteristic curves revealed the computer-aided system (0.88 ± 0.02) to be comparable to the manual method (0.86 ± 0.03) in detecting patients with FASD. Interestingly, cases of alcohol-related neurodevelopmental disorder (ARND) were identified more efficiently by the computer-aided system (0.84 ± 0.07) in comparison to the manual method (0.74 ± 0.04). A facial gestalt analysis of patients with ARND also identified more generalized facial findings compared to the cardinal facial features seen in more severe forms of FASD. CONCLUSIONS: We found there was an increased diagnostic accuracy for ARND via our computer-aided method. As this category has been historically difficult to diagnose, we believe our experiment demonstrates that facial dysmorphology novel analysis technology can potentially improve ARND diagnosis by introducing a standardized metric for recognizing FASD-associated facial anomalies. Earlier recognition of these patients will lead to earlier intervention with improved patient outcomes.

Uterine angioleiomyoma in an African green monkey (Chlorocebus aethiops sabaeus).

BACKGROUND: A uterine neoplasm was observed, as an incidental finding, during post-mortem examination of a 26-year-old female multiparous African green monkey (Chlorocebus aethiops sabaeus). The intramural, expansile, 2 to 3 cm well-demarcated, dark-red, nodular neoplasm was located on the anterior uterine body (corpus) wall. METHODS: The mass was examined by light microscopy and by immunohistochemistry. RESULTS: The mass was confirmed as a cavernous uterine angioleiomyoma (syn. vascular leiomyoma) characterized by abundant intratumoural vasculature lined by Factor VIII-positive endothelial cells and surrounded by smooth muscle actin-positive cell proliferations. CONCLUSION: Angioleiomyoma sharing the characteristics of intramural human cavernous uterine angioleiomyoma should be considered in the differential diagnosis of uterine tumours in non-human primates.

Uterine prolapse with endometrial eversion in association with an unusual diffuse, polypoid, fibrosing perimetritis and parametritis in a cat.

CASE SUMMARY: This case describes a young non-pregnant cat that presented with uterine prolapse in association with an unusual diffuse, polypoid, fibrosing perimetritis and parametritis. Following ovariohysterectomy the cat recovered fully. No intra-abdominal complications were seen on ultrasound examination 3 months postsurgery. At the time of writing, the cat remains healthy. RELEVANCE AND NOVEL INFORMATION: Uterine prolapse in the cat is relatively rare and usually associated with the periparturient period. Inflammatory polypoid perimetritis and parametritis have not previously been documented in cats, and in dogs have only been reported in association with the administration of oestrogenic compounds. The polypoid inflammation affecting the uterus and parametrium may have contributed to increased laxity of the uterine ligaments and predisposed to the development of uterine prolapse.