Establishment and Characterization of Three Human Ocular Adnexal Sebaceous Carcinoma Cell Lines.

Ocular adnexal sebaceous carcinoma (SebCA) represents one of the most clinically problematic periocular tumors, often requiring aggressive surgical resection. The pathobiology of this tumor remains poorly understood, and few models exist that are suitable for preclinical testing. The aim of this study was to establish new cell lines to serve as models for pathobiological and drug testing. With patient consent, freshly resected tumor tissue was cultured using conditional reprogramming cell conditions. Standard techniques were used to characterize the cell lines in terms of overall growth, clonogenicity, apoptosis, and differentiation in vitro. Additional analyses including Western blotting, short tandem repeat (STR) profiling, and next-generation sequencing (NGS) were performed. Drug screening using mitomycin-C (MMC), 5-fluorouricil (5-FU), and 6-Diazo-5-oxo-L-norleucine (DON) were performed. JHH-SebCA01, JHH-SebCA02, and JHH-SebCA03 cell lines were established from two women and one man undergoing surgical resection of eyelid tumors. At passage 15, they each showed a doubling time of two to three days, and all could form colonies in anchorage-dependent conditions, but not in soft agar. The cells contained cytoplasmic vacuoles consistent with sebaceous differentiation, and adipophilin protein was present in all three lines. STR profiling confirmed that all lines were derived from their respective patients. NGS of the primary tumors and their matched cell lines identified numerous shared mutations, including alterations similar to those previously described in SebCA. Treatment with MMC or 5-FU resulted in dose-dependent growth inhibition and the induction of both apoptosis and differentiation. MYC protein was abundant in all three lines, and the glutamine metabolism inhibitor DON, previously shown to target high MYC tumors, slowed the growth of all our SebCA models. Ocular adnexal SebCA cell lines can be established using conditional reprogramming cell conditions, and our three new models are useful for testing therapies and interrogating the functional role of MYC and other possible molecular drivers. Current topical chemotherapies promote both apoptosis and differentiation in SebCA cells, and these tumors appear sensitive to inhibition or MYC-associated metabolic changes.

Trends in orbital cellulitis severity and management: 2016 through 2022.

PURPOSE: To characterize trends in orbital cellulitis (OC) severity and management from 2016 through 2022, including assessment of effects from the COVID-19 pandemic. METHODS: A retrospective chart review was conducted at a tertiary care center using an electronic medical record search of OC from January 1 2016 to January 1 2023. Radiography was used to categorize patients into three cohorts - OC without abscess, OC with subperiosteal abscess (SPA), and OC with orbital abscess (OA). Infection cohort and age group [pediatric (<9 years old), adolescent (9-18 years), adult (>18 years)] were compared across years. Subgroup analysis was performed in the SPA cohort. Comparisons were made between the time periods prior to and after the start of the COVID-19 pandemic. RESULTS: From 2016-2022, there were 247 (67.3%) cases of OC, 103 (28.1%) SPA, and 17 (4.6%) OA. The year 2022 incurred higher rates of SPA and OA (p = 0.035), pediatric infections (p = 0.008), and co-morbid sinusitis (p = 0.005) compared to all preceding years. Rates were similar from 2016 through 2021. Within the SPA cohort, rates of surgical intervention and outcomes were similar across all years. Rate of abscess formation was significantly higher during the COVID-19 pandemic compared to the pre-pandemic era (p = 0.033). CONCLUSION: The severity of OC leading up to the COVID-19 pandemic appeared steady. After pandemic onset, a delayed increase in abscess formation, predominantly affecting pediatric patients and correlating with a greater rate of sinusitis, was observed in 2022.

Ocular adnexal sebaceous carcinoma in a patient with Li-Fraumeni syndrome.

Li-Fraumeni syndrome (LFS) is caused by a pathogenic germline variant at the TP53 locus and is associated with an increased predisposition to a variety of cancers. The neoplasms most frequently associated with LFS are sarcomas, breast cancer, brain tumors, and adrenocortical carcinomas. In this case report, we present a 43-year-old male diagnosed with an ocular adnexal sebaceous carcinoma of the right upper eyelid who was confirmed to have LFS with subsequent genetic testing. The mutational profile of both the patient's genetic screen and tumor sequencing were congruent, demonstrating the same pathogenic loss-of-function TP53 variant. This case report highlights the importance of pursuing genetic testing in patients with a history of multiple tumor types, particularly those with uncommon diagnoses. In this case, confirmation of LFS had important implications for personalized patient care, including identification of contraindicated treatment interventions and the imaging modalities necessary for vigilant follow-up screening.

Rapidly Growing Eyelid Lesion in a 73-Year-Old Man.

A man aged 73 years presented with a 5-day history of a fast-growing right eyebrow nodule with raised borders and central ulceration. There was no discomfort, and the patient denied vision changes. The lesion was refractory to treatment with erythromycin, clindamycin, and trimethoprim/sulfamethoxazole. What would you do next?

Psychometric properties of 3-meter backward walk test (3MBWT) in people with Parkinson disease.

BACKGROUND AND PURPOSE: The purpose of this study was to investigate the 3-meter backward walk test (3MBWT) in individuals with Parkinson Disease (PD) to determine the following: (1) concurrent validity with other gait velocity measures and (2) interrater and intrarater reliability of in-person and video assessment. METHODS: A convenience sample of 25 people with PD participated. Forward gait velocity was measured using a computerized walkway (Zeno Walkway System), the 10-meter walk test (10MWT), and 4-meter walk test (4MWT). Backward gait velocity was measured using the 3MBWT. Concurrent validity was assessed using Pearson's correlations. Reliability was assessed using intra-class correlation coefficients (ICC(2,1)). RESULTS: All relationships between the 3MBWT and gait outcome measures were significant. The 3MBWT demonstrated strong correlations with the 4MWT dual task (r = .795, p=<.001) and moderate correlations with 4MWT comfortable walking speed (r = .658, p < .001), 4MWT fast walking speed (r = .601,p=.002), 10MWT comfortable walking speed (r = .512, p = .009), and 10MWT dual task (r = .535, p = .006). A low yet significant correlation was noted with the 10MWT fast walking speed (r = .398, p = .049). Association between the 3MBWT and the Zeno Walkway System revealed moderate correlations. All reliability tests were significant at p < .001. Interrater reliability ICC(2,1) values were very high for 3MBWT (ICC(2,1) = 0.93, [0.83-0.91]). Intrarater reliability was also very high (ICC(2,1) = 0.96 [0.90-0.98]). CONCLUSION: The 3MBWT demonstrates validity and reliability as a tool for assessing gait speed in the posterior direction in people with PD.

Bilateral Dilated Superior Ophthalmic Veins in a Patient With an Arteriovenous Dialysis Fistula.

A 64-year-old man presented with 4 months of diplopia. He had end-stage renal disease requiring a cephalic transposition brachiocephalic fistula that was no longer in use following successful renal transplantation. On presentation, he had bilateral proptosis, extraocular movement restriction, chemosis, tortuous episcleral vessels, and caruncular injection. Non-contrast CT of the orbits demonstrated dilation of both superior ophthalmic veins, and CT angiography showed asymmetric enlargement of both cavernous sinuses and superior ophthalmic veins. A carotid-cavernous fistula was suspected, but cerebral angiography revealed shunting from the old fistula with intracranial drainage and cerebral venous hypertension. Aberrant retrograde drainage resulted from anatomical compression of the left brachiocephalic vein. The fistula was ligated, and at 1-week follow-up, the patient had marked improvement in extraocular movements and orbital congestion with near complete resolution of diplopia. Postoperative CT angiography obtained 2 months later demonstrated decreased size of both superior ophthalmic veins, consistent with improvement of venous hypertension.

Impact of a Virtual Game, 'Name That Band,' on Older People's and PharmD Students' Feelings of Social Isolation During COVID-19.

Background Prior to the COVID-19 pandemic, PharmD students at the University of Arizona (UArizona) had a long-standing relationship with the older people at St. Luke's Home, a local Eden Alternative assisted-living community. Hosting community engagement programs for assisted-living residents was challenging with COVID-19 precautions and older individuals suffering from social isolation and loneliness. Objective To determine the impact of playing a virtual game, 'Name That Band,' on older people's and PharmD students' feelings of social isolation during the COVID-19 pandemic. Methods Questionnaires were administered before and after a virtual game to residents at St. Luke's Home and PharmD students at UArizona. Participants were asked about their mood before COVID-19 and pre-and postgame, as well as their social interactions and feelings of loneliness using the Modified UCLA Three-Item Loneliness Scale, which assesses a composite score of lack of companionship, feelings of being left out, and isolation. Results Fifteen older people and 11 students participated in the game (N = 26). All participants completed the pregame survey and 25 completed the postgame survey. The older people reported fewer feelings of isolation and loneliness (measured by a reduction in UCLA Loneliness Scale score) postgame compared with pregame. The students reported a higher total UCLA Loneliness Scale score during the pandemic than prepandemic, but there was no difference in their scores postgame compared with pregame. More older people and students reported feeling 'happy' after playing the virtual game together compared with before the pandemic and before playing. The aspect of the activity that helped older people and students feel more socially engaged was playing a game. Conclusion A social intervention using a virtual game may be a tool that can be used to decrease feelings of isolation and increase engagement for older people residing in an assisted-living community.

Evaluating Functional Performance Tests in those with Non-arthritic Intra-articular Hip Pain: An International Consensus Statement.

Background: Non-arthritic intra-articular hip pain, caused by various pathologies, leads to impairments in range of motion, strength, balance, and neuromuscular control. Although functional performance tests offer valuable insights in evaluating these patients, no clear consensus exists regarding the optimal tests for this patient population. Purpose: This study aimed to establish expert consensus on the application and selection of functional performance tests in individuals presenting with non-arthritic intra-articular hip pain. Study Design: A modified Delphi technique was used with fourteen physical therapy experts, all members of the International Society for Hip Arthroscopy (ISHA). The panelists participated in three rounds of questions and related discussions to reach full consensus on the application and selection of functional performance tests. Results: The panel agreed that functional performance tests should be utilized at initial evaluation, re-evaluations, and discharge, as well as criterion for assessing readiness for returning to sports. Tests should be as part of a multimodal assessment of neuromuscular control, strength, range of motion, and balance, applied in a graded fashion depending on the patient's characteristics. Clinicians should select functional performance tests with objective scoring criteria and prioritize the use of tests with supporting psychometric evidence. A list of recommended functional performance tests with varying intensity levels is provided. Low-intensity functional performance tests encompass controlled speed in a single plane with no impact. Medium-intensity functional performance tests involve controlled speed in multiple planes with low impact. High-intensity functional performance tests include higher speeds in multiple planes with higher impact and agility requirements. Sport-specific movement tests should mimic the patient's particular activity or sport. Conclusion: This international consensus statement provides recommendations for clinicians regarding selection and utilization of functional performance tests for those with non-arthritic intra-articular hip pain. These recommendations will encourage greater consistency and standardization among clinicians during a physical therapy assessment.

Crystal Structure of Inhibitor-Bound GII.4 Sydney 2012 Norovirus 3C-Like Protease.

Norovirus is the leading cause of viral gastroenteritis worldwide, and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections. The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI.1 noroviruses, despite most global outbreaks being caused by GII.4 noroviruses. Here, we determined the crystal structures of the GII.4 Sydney 2012 ligand-free norovirus protease at 2.79 Å and at 1.83 Å with a covalently bound high-affinity (IC50 = 0.37 µM) protease inhibitor (NV-004). We show that the active sites of the ligand-free protease structure are present in both open and closed conformations, as determined by their Arg112 side chain orientation. A comparative analysis of the ligand-free and ligand-bound protease structures reveals significant structural differences in the active site cleft and substrate-binding pockets when an inhibitor is covalently bound. We also report a second molecule of NV-004 non-covalently bound within the S4 substrate binding pocket via hydrophobic contacts and a water-mediated hydrogen bond. These new insights can guide structure-aided drug design against the GII.4 genogroup of noroviruses.

Off-Label Reduced Dose Apixaban in Older Adults With Atrial Fibrillation and Associated Outcomes.

BACKGROUND: Apixaban is commonly used to prevent stroke in older adults with nonvalvular atrial fibrillation (AF). Although its package insert has specific dose reduction criteria, providers may dose reduce outside of these parameters based on clinical scenarios. OBJECTIVE: The primary objective was to determine the incidence of apixaban off-label reduced dosing, while secondarily determining the safety and efficacy outcomes associated with such dosing. METHODS: A retrospective analysis of patients aged 65 and older with orders for apixaban for AF was institutional review board (IRB)-approved and conducted across 3 academic medical centers. Patients receiving off-label reduced-dose apixaban (ie, "underdosed") were matched to a cohort of patients dosed according to the package insert at the standard dosing (5 mg twice daily) using stratified random sampling. Secondary outcomes included 1-year incidence of major bleeding, clinically relevant non-major bleeding (CRNMB), stroke or transient ischemic attack (TIA), and mortality. The Fisher exact tests were used to compare between-group differences. RESULTS: Of the 1172 patients meeting initial inclusion criteria, 201 (17%) were dosed off-label, with 175 (15%) "underdosed." The 147 "underdosed" patients with documented follow-up were matched with 139 patients receiving standard Food and Drug Administration (FDA)-labeled dosing. There were no significant differences in incidence of stroke (2.7% vs 2.2%), major bleeding (0% vs 0.7%), and CRNMB (2.7% vs 1.4%) in the off-label reduced dosing versus standard dosing groups. All-cause mortality was higher in the off-label reduced-dose group (16 [10.9%] vs 2 [1.4%], P < 0.05). CONCLUSION AND RELEVANCE: Older adults with nonvalvular AF are commonly prescribed lower-than-recommended doses of apixaban. However, no significant association was found between empiric off-label reduced dosing and stroke or bleeding outcomes.

Progressive muscle relaxation, meditation, and mental practice-based interventions for the treatment of tremor after traumatic brain injury.

BACKGROUND AND PURPOSE: Individuals post traumatic brain injury (TBI) are likely to experience cognitive, sensory, and motor impairments. Tremor is a common movement disorder that can affect this patient population. The purpose of this case report was to describe the effects that progressive muscle relaxation, meditation, and mental practice-based interventions have on the treatment of chronic right upper extremity tremor in a patient post-TBI. CASE DESCRIPTION: A 47-year-old male with a traumatic brain injury presented with a resting and postural tremor of the right wrist flexors and deficits in cognition, sensation, proprioception, balance, and motor function. The patient's primary goal for therapy was to decrease the tremor to improve his quality of life and ability to perform activities of daily living. METHODS: Progressive muscle relaxation, breath-focused meditation, and mental practice-based intervention were administered to the patient. Active range of motion activities occurred during weekly in-person sessions and daily practice occurred at home. RESULTS: The patient demonstrated improvements in quality of life, self-ratings of tremor severity, ratings of tremor impact on ADL's, gross motor object manipulation, frequency of tremor via surface electromyography, and visual inspection of a spirograph. CONCLUSION: A combination of progressive muscle relaxation, meditation, and mental practice-based interventions appeared to positively impact tremor across physical and psychosocial domains in a patient with a chronic tremor. Further investigation is needed to verify these findings.

The 2022 International Society for Hip Preservation (ISHA) physiotherapy agreement on assessment and treatment of greater trochanteric pain syndrome (GTPS): an international consensus statement.

The 2022 International Society of Hip Preservation (ISHA) physiotherapy agreement on assessment and treatment of greater trochanteric pain syndrome (GTPS) was intended to present a physiotherapy consensus on the assessment and surgical and non-surgical physiotherapy management of patients with GTPS. The panel consisted of 15 physiotherapists and eight orthopaedic surgeons. Currently, there is a lack of high-quality literature supporting non-operative and operative physiotherapy management. Therefore, a group of physiotherapists who specialize in the treatment of non-arthritic hip pathology created this consensus statement regarding physiotherapy management of GTPS. The consensus was conducted using a modified Delphi technique to guide physiotherapy-related decisions according to the current knowledge and expertise regarding the following: (i) evaluation of GTPS, (ii) non-surgical physiotherapy management, (iii) use of corticosteroids and orthobiologics and (iv) surgical indications and post-operative physiotherapy management.

Terabase-Scale Coassembly of a Tropical Soil Microbiome.

Petabases of environmental metagenomic data are publicly available, presenting an opportunity to characterize complex environments and discover novel lineages of life. Metagenome coassembly, in which many metagenomic samples from an environment are simultaneously analyzed to infer the underlying genomes' sequences, is an essential tool for achieving this goal. We applied MetaHipMer2, a distributed metagenome assembler that runs on supercomputing clusters, to coassemble 3.4 terabases (Tbp) of metagenome data from a tropical soil in the Luquillo Experimental Forest (LEF), Puerto Rico. The resulting coassembly yielded 39 high-quality (>90% complete, <5% contaminated, with predicted 23S, 16S, and 5S rRNA genes and ≥18 tRNAs) metagenome-assembled genomes (MAGs), including two from the candidate phylum Eremiobacterota. Another 268 medium-quality (≥50% complete, <10% contaminated) MAGs were extracted, including the candidate phyla Dependentiae, Dormibacterota, and Methylomirabilota. In total, 307 medium- or higher-quality MAGs were assigned to 23 phyla, compared to 294 MAGs assigned to nine phyla in the same samples individually assembled. The low-quality (<50% complete, <10% contaminated) MAGs from the coassembly revealed a 49% complete rare biosphere microbe from the candidate phylum FCPU426 among other low-abundance microbes, an 81% complete fungal genome from the phylum Ascomycota, and 30 partial eukaryotic MAGs with ≥10% completeness, possibly representing protist lineages. A total of 22,254 viruses, many of them low abundance, were identified. Estimation of metagenome coverage and diversity indicates that we may have characterized ≥87.5% of the sequence diversity in this humid tropical soil and indicates the value of future terabase-scale sequencing and coassembly of complex environments. IMPORTANCE Petabases of reads are being produced by environmental metagenome sequencing. An essential step in analyzing these data is metagenome assembly, the computational reconstruction of genome sequences from microbial communities. "Coassembly" of metagenomic sequence data, in which multiple samples are assembled together, enables more complete detection of microbial genomes in an environment than "multiassembly," in which samples are assembled individually. To demonstrate the potential for coassembling terabases of metagenome data to drive biological discovery, we applied MetaHipMer2, a distributed metagenome assembler that runs on supercomputing clusters, to coassemble 3.4 Tbp of reads from a humid tropical soil environment. The resulting coassembly, its functional annotation, and analysis are presented here. The coassembly yielded more, and phylogenetically more diverse, microbial, eukaryotic, and viral genomes than the multiassembly of the same data. Our resource may facilitate the discovery of novel microbial biology in tropical soils and demonstrates the value of terabase-scale metagenome sequencing.

Artificial Intelligence Automation of Proptosis Measurement: An Indicator for Pediatric Orbital Abscess Surgery.

INTRODUCTION: To evaluate the ability of artificial intelligence (AI) software to quantify proptosis for identifying patients who need surgical drainage. METHODS: We pursued a retrospective study including 56 subjects with a clinical diagnosis of subperiosteal orbital abscess (SPOA) secondary to sinusitis at a tertiary pediatric hospital from 2002 to 2016. AI computer software was developed to perform 3D visualization and quantitative assessment of proptosis from computed tomography (CT) images acquired at the time of hospital admission. The AI software automatically computed linear and volume metrics of proptosis to provide more practice-consistent and informative measures. Two experienced physicians independently measured proptosis using the interzygomatic line method on axial CT images. The AI software and physician proptosis assessments were evaluated for association with eventual treatment procedures as standalone markers and in combination with the standard predictors. RESULTS: To treat the SPOA, 31 of 56 (55%) children underwent surgical intervention, including 18 early surgeries (performed within 24 h of admission), and 25 (45%) were managed medically. The physician measurements of proptosis were strongly correlated (Spearman r = 0.89, 95% CI 0.82-0.93) with 95% limits of agreement of ± 1.8 mm. The AI linear measurement was on average 1.2 mm larger (p = 0.007) and only moderately correlated with the average physicians' measurements (r = 0.53, 95% CI 0.31-0.69). Increased proptosis of both AI volumetric and linear measurements were moderately predictive of surgery (AUCs of 0.79, 95% CI 0.68-0.91, and 0.78, 95% CI 0.65-0.90, respectively) with the average physician measurement being poorly to fairly predictive (AUC of 0.70, 95% CI 0.56-0.84). The AI proptosis measures were also significantly greater in the early as compared to the late surgery groups (p = 0.02, and p = 0.04, respectively). The surgical and medical groups showed a substantial difference in the abscess volume (p < 0.001). CONCLUSION: AI proptosis measures significantly differed from physician assessments and showed a good overall ability to predict the eventual treatment. The volumetric AI proptosis measurement significantly improved the ability to predict the likelihood of surgery compared to abscess volume alone. Further studies are needed to better characterize and incorporate the AI proptosis measurements for assisting in clinical decision-making.

Maternity and family leave experiences among female ophthalmologists in the United States.

OBJECTIVE: To evaluate family and maternity leave policies and examine the social and professional impacts on female ophthalmologists. PARTICIPANTS: Participants were recruited through the Women in Ophthalmology online list-serv to complete a survey evaluating maternity leave policies and their impacts. Survey questions were repeated for each birth event after medical school for up to five birth events. RESULTS: The survey was accessed 198 times, and 169 responses were unique. Most participants were practicing ophthalmologists (92%), with a minority in residency (5%), in fellowship (1.2%), on disability/leave (0.6%), or retired (0.6%). Most participants (78%) were within their first ten years of practice. Experiences were recorded for each leave event, with 169 responses for the first leave, 120 for the second, 28 for the third, and 2 for the fourth. Nearly half of participants reported the information they received about maternity leave to be somewhat or extremely inadequate (first: 50%; second: 42%; third: 41%). Many reported a greater sense of burnout after returning to work (first: 61%, second: 58%, third: 46%). A minority of participants received full pay during the first through third maternity leave events, 39%, 27%, and 33%, respectively. About a third of participants reported being somewhat or very dissatisfied with their maternity leave experience (first: 42%, second: 35%; third: 27%). CONCLUSIONS: Female ophthalmologists have varying experiences with maternity leave, but many encounter similar challenges. This study demonstrates that many women receive inadequate information about family leave, desire more weeks of leave, experience a wide variation in pay practices, and lack support for breastfeeding. Understanding the shared experiences of women in ophthalmology identifies areas where improvements are needed in maternity leave practices within the field to create a more supportive environment for physician mothers.

Utility of diffusion-weighted imaging to differentiate benign and malignant solid orbital tumours.

OBJECTIVE: We assessed the utility of apparent diffusion coefficients (ADCs) derived from diffusion-weighted imaging to differentiate benign and malignant orbital tumours by oculoplastic surgeons in the clinical setting and sought to validate observed ADC cut-off values. DESIGN AND PARTICIPANTS: Retrospective review of patients with benign or malignant biopsy-confirmed orbital tumours. METHODS: Blinded graders including 2 oculoplastic surgeons, 1 neuroradiologist, and 1 medical student located and measured orbital tumour ADCs (10-6 mm2/s) using the Region of Interest tool. OUTCOME MEASURES: Nonradiologist measurements were compared with each other to assess reliability and with an expert neuroradiologist measurement and final pathology to assess accuracy. RESULTS: Twenty-nine orbital tumours met inclusion criteria, consisting of 6 benign tumours and 23 malignant tumours. Mean ADC values for benign orbital tumours were 1430.59 ± 254.81 and 798.68 ± 309.12 mm2/s for malignant tumours. Our calculated optimized ADC cut-off to differentiate benign from malignant orbital tumours was 1120.84 × 10-6 mm2/s (sensitivity 1, specificity 0.9). Inter-rater reliability was excellent (intraclass correlation coefficient = 0.92; 95% CI, 0.86-0.96). Our 3 graders had a combined accuracy of 84.5% (92.3%, 92.3%, and 65.4%). CONCLUSIONS: Our ADC cut-off of 1120.84 × 10-6 mm2/s for benign and malignant orbital tumours agrees with previously established values in literature. Without priming with instructions, training, or access to patient characteristics, most tumours were correctly classified using rapid ADC measurements. Surgeons without radiologic expertise can use the ADC tool to quickly risk stratify orbital tumours during clinic visits to guide patient expectations and further work-up.

HT-SIP: a semi-automated stable isotope probing pipeline identifies cross-kingdom interactions in the hyphosphere of arbuscular mycorrhizal fungi.

BACKGROUND: Linking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive for this goal, Stable-isotope probing-SIP-remains among the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, actively growing microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated, high-throughput SIP (HT-SIP) pipeline to support well-replicated, temporally resolved amplicon and metagenomics experiments. We applied this pipeline to a soil microhabitat with significant ecological importance-the hyphosphere zone surrounding arbuscular mycorrhizal fungal (AMF) hyphae. AMF form symbiotic relationships with most plant species and play key roles in terrestrial nutrient and carbon cycling. RESULTS: Our HT-SIP pipeline for fractionation, cleanup, and nucleic acid quantification of density gradients requires one-sixth of the hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We applied HT-SIP to 13C-AMF hyphosphere DNA from a 13CO2 plant labeling study and created metagenome-assembled genomes (MAGs) using high-resolution SIP metagenomics (14 metagenomes per gradient). SIP confirmed the AMF Rhizophagus intraradices and associated MAGs were highly enriched (10-33 atom% 13C), even though the soils' overall enrichment was low (1.8 atom% 13C). We assembled 212 13C-hyphosphere MAGs; the hyphosphere taxa that assimilated the most AMF-derived 13C were from the phyla Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia-oxidizing archaeon genus Nitrososphaera. CONCLUSIONS: Our semi-automated HT-SIP approach decreases operator time and improves reproducibility by targeting the most labor-intensive steps of SIP-fraction collection and cleanup. We illustrate this approach in a unique and understudied soil microhabitat-generating MAGs of actively growing microbes living in the AMF hyphosphere (without plant roots). The MAGs' phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling. Video Abstract.

Use of Melatonin and/on Ramelteon for the Treatment of Insomnia in Older Adults: A Systematic Review and Meta-Analysis.

To investigate the efficacy of melatonin and/or ramelteon reporting sleep outcomes for older adults with chronic insomnia, a systematic review and a meta-analysis of PubMed, EMBASE, Cochrane library, International Pharmaceutical Abstracts, PsycINFO, science citation index, center for reviews and dissemination, CINAHL, grey literature and relevant sleep journal searches were conducted from 1 January 1990 to 20 June 2021. Randomized controlled trials and other comparative studies with melatonin and/or ramelteon use among older patients with chronic insomnia were included. Funnel plot and Egger's test was used to determine publication bias. A forest plot was constructed to obtain a pooled standardized mean difference using either a fixed or random effects model for each of the two broad categories of sleep outcomes: objective and subjective. Of 5247 studies identified, 17 studies met the inclusion criteria for MA. Study sample size ranged from 10 to 829 with the mean age ≥55 years. There were significant improvements in total sleep time (objective), sleep latency and sleep quality (objective and subjective) for melatonin and/or ramelteon users compared with placebo. Sleep efficiency was not significantly different. The effects of these agents are modest but with limited safe treatment options for insomnia in older adults, these could be the drugs of choice.

Detection of Human Papillomavirus in Squamous Lesions of the Conjunctiva Using RNA and DNA In-Situ Hybridization.

In-situ hybridization provides a convenient and reliable method to detect human papillomavirus (HPV) infection in formalin-fixed paraffin-embedded tissue. Cases of conjunctival papillomas, conjunctival intraepithelial neoplasia (CIN), conjunctival carcinoma in situ (cCIS), and invasive squamous cell carcinoma (SCC), in which low-risk (LR) and/or high-risk (HR) HPV types were evaluated by RNA or DNA in-situ hybridization, were retrospectively identified. LR HPV types were frequently detected in conjunctival papillomas (25/30, 83%), including 17/18 (94%) with RNA probes, compared to 8/12 (75%) with DNA probes. None of the CIN/cCIS or SCC cases were positive for LR HPV by either method. HR HPV was detected by RNA in-situ hybridization in 1/16 (6%) of CIN/cCIS cases and 2/4 (50%) of SCC cases, while DNA in-situ hybridization failed to detect HPV infection in any of the CIN/cCIS lesions. Reactive atypia and dysplasia observed in papillomas was generally associated with the detection of LR HPV types. Collectively, our findings indicate RNA in-situ hybridization may provide a high-sensitivity approach for identifying HPV infection in squamous lesions of the conjunctiva and facilitate the distinction between reactive atypia and true dysplasia. There was no clear association between HPV infection and atopy in papillomas or dysplastic lesions.