The Prevalence of Incontinence and Its Association With Urinary Tract Infections, Dermatitis, Slips and Falls, and Behavioral Disturbances Among Older Adults in Medicare Fee-for-Service.

PURPOSE: The purpose of this study was to examine the prevalence of urinary (UI), fecal (FI), and dual incontinence (DI) in older adults and their association with urinary tract infections, dermatitis, slips and falls, and behavioral disturbances based on Medicare fee-for-service (FFS) claims data. DESIGN: Retrospective analysis. SUBJECTS AND SETTINGS: Data from administrative claims from the CMS Medicare Limited Data Set (5% sample) for all months in 2018 were reviewed. The analysis was limited to FFS Medicare beneficiaries, with minimum of 3-month enrollment in Parts A and B who were at least 65 years old. This cohort included 1.2 million beneficiaries in the United States. METHODS: We used diagnosis codes to identify members with incontinence and grouped these members into 3 categories (UI only, FI only, and DI). We also divided claims based on 4 sites of care (nursing home, skilled nursing facility, home health, and self- or family care). We then determined the prevalence of (1) urinary tract infections (UTIs), (2) dermatitis, (3) slips and falls, and (4) behavioral disturbances for each type of incontinence. RESULTS: We found that 11.2% of Medicare members had a claims-based diagnosis of incontinence in 2018. On average, those diagnosed with incontinence experienced 5 times more UTIs, 2 times as many dermatitis events, more than twice as many slips and falls, and 2.8 times more behavior disturbances compared to those without an incontinence diagnosis. For those with DI, the prevalence of the 4 outcomes was significantly higher (between 22% and 185%) compared to those with UI only. CONCLUSIONS: Findings show that Medicare beneficiaries diagnosed as incontinent experience a much higher prevalence of UTIs, dermatitis, slips and falls, and behavioral disturbances compared to those without a diagnosis of incontinence. Our results suggest that incontinence may be an important indicator diagnosis for multiple other conditions and, if not well-managed, may challenge the desire for those who are incontinent to age at home.

Developmental basis of SHH medulloblastoma heterogeneity.

Many genes that drive normal cellular development also contribute to oncogenesis. Medulloblastoma (MB) tumors likely arise from neuronal progenitors in the cerebellum, and we hypothesized that the heterogeneity observed in MBs with sonic hedgehog (SHH) activation could be due to differences in developmental pathways. To investigate this question, here we perform single-nucleus RNA sequencing on highly differentiated SHH MBs with extensively nodular histology and observed malignant cells resembling each stage of canonical granule neuron development. Through innovative computational approaches, we connect these results to published datasets and find that some established molecular subtypes of SHH MB appear arrested at different developmental stages. Additionally, using multiplexed proteomic imaging and MALDI imaging mass spectrometry, we identify distinct histological and metabolic profiles for highly differentiated tumors. Our approaches are applicable to understanding the interplay between heterogeneity and differentiation in other cancers and can provide important insights for the design of targeted therapies.

Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude.

Answer ALS, a large-scale resource for sporadic and familial ALS combining clinical and multi-omics data from induced pluripotent cell lines.

Answer ALS is a biological and clinical resource of patient-derived, induced pluripotent stem (iPS) cell lines, multi-omic data derived from iPS neurons and longitudinal clinical and smartphone data from over 1,000 patients with ALS. This resource provides population-level biological and clinical data that may be employed to identify clinical-molecular-biochemical subtypes of amyotrophic lateral sclerosis (ALS). A unique smartphone-based system was employed to collect deep clinical data, including fine motor activity, speech, breathing and linguistics/cognition. The iPS spinal neurons were blood derived from each patient and these cells underwent multi-omic analytics including whole-genome sequencing, RNA transcriptomics, ATAC-sequencing and proteomics. The intent of these data is for the generation of integrated clinical and biological signatures using bioinformatics, statistics and computational biology to establish patterns that may lead to a better understanding of the underlying mechanisms of disease, including subgroup identification. A web portal for open-source sharing of all data was developed for widespread community-based data analytics.

Openness, economic uncertainty, government responses, and international financial market performance during the coronavirus pandemic.

This study explores the impact of COVID-19 on financial markets controlled by macroeconomic and administrative factors. As natural experimentation, we employ panel data analysis to test 50 stock market indices from January 01 to August 20, 2020. The findings suggest daily growth of COVID-19 confirmed cases have considerable adverse effects on stock returns and positive impacts on investment risks across markets. The government prompt interventions offset adverse impacts of the pandemic. Market reactions to the outbreak and authority responses information are more momentous in more developed economies due to their better information efficiency. The country-specific features of globalisation, uncertainty, healthcare system readiness, and economic development levels appear to have substantial impacts on equity market reactions. Financial markets in countries with higher levels of globalisation, economic policy and financial uncertainty experience more chaos during the pandemic. While those hostile effects are less significant in countries with robust healthcare systems.

A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila.

Advances in CRISPR technology have immensely improved our ability to manipulate nucleic acids, and the recent discovery of the RNA-targeting endonuclease Cas13 adds even further functionality. Here, we show that Cas13 works efficiently in Drosophila, both ex vivo and in vivo. We test 44 different Cas13 variants to identify enzymes with the best overall performance and show that Cas13 could target endogenous Drosophila transcripts in vivo with high efficiency and specificity. We also develop Cas13 applications to edit mRNAs and target mitochondrial transcripts. Our vector collection represents a versatile tool collection to manipulate gene expression at the post-transcriptional level.

Polypharmacy, Medication Possession, and Deprescribing of Potentially Non-Beneficial Drugs in Hospice Patients.

Patients frequently have comorbidities that when combined with their primary diagnosis qualifies the patient for hospice. Consequently, patients are at risk for polypharmacy due to the number of medications prescribed to treat both the underlying conditions and the related symptoms. Polypharmacy is associated with negative consequences, including increased risk for adverse drug events, drug-drug and drug-disease interactions, reduced functional status and falls, multiple geriatric syndromes, medication nonadherence, and increased mortality. Polypharmacy also increases the complexity of medication management for caregivers and contributes to the cost of prescription drugs for hospices and patients. Deprescribing or removing nonbeneficial or ineffective medications can reduce polypharmacy in hospice. We study medication possession ratios and rates of deprescribing of commonly prescribed but potentially nonbeneficial classes of medication using a large hospice pharmacy database. Prevalence of some classes of potentially inappropriate medications is high. We report possession ratios for 10 frequently prescribed classes, and, because death and prescription termination are competing events, we calculate prescription termination rates using Cumulative Incidence Functions. Median duration of antifungal and antiviral medications is brief (5 and 7 days, respectively), while statins and diabetes medications have slow discontinuance rates (median termination durations of 93 and 197 days). Almost all patients with a proton pump inhibitor prescription have the drug for their entire hospice stay. Data from this study identify those drug classes that are commonly deprescribed slowly, suggesting drug classes and diagnoses that hospices may wish to focus on more closely, as they act to limit polypharmacy and reduce prescription costs.

Spatial and temporal control of gene manipulation in Drosophila via drug-activated Cas9 nucleases.

Advances in CRISPR/Cas9 have revolutionized molecular biology and greatly facilitated the ability to manipulate gene function through the creation of precisely engineered mutants. We recently reported a collection of modular gateway-compatible Cas9/gRNA Drosophila lines to interfere with gene expression in a tissue-specific manner, including polytene tissues. However, most current in vivo CRISPR/Cas9 tools cannot temporally control the induction of Cas9 or gRNAs via external stimuli such as RU486. A drug-inducible CRISPR/Cas9 system would allow studying genes at later stages where early lethality is an issue. This would be especially useful when combined with tissue-specific expression of Cas9 or gRNAs, allowing for full spatiotemporal control. Here, we present a RU486-inducible version of Cas9 and also show that a Rapamycin-inducible Cas9, previously used in mammalian cell culture, works in Drosophila as well. Both RU486 and rapamycin-inducible Cas9 work in vivo and in Drosophila cell culture. We also present split Cas9 constructs for rapamycin-dependent gene disruption and activation. These approaches establish drug-inducible and thus temporally controlled CRISPR/Cas9 tools for gene disruption and expression in a living model organism. Our CRISPR/Cas9 vector collection can be easily adapted for any tissue and provides higher fidelity compared to RNAi approaches.

Tumescent Injections in Subcutaneous Pig Tissue Disperse Fluids Volumetrically and Maintain Elevated Local Concentrations of Additives for Several Hours, Suggesting a Treatment for Drug Resistant Wounds.

PURPOSE: Bolus injection of fluid into subcutaneous tissue results in accumulation of fluid at the injection site. The fluid does not form a pool. Rather, the injection pressure forces the interstitial matrix to expand to accommodate the excess fluid in its volume, and the fluid becomes bound similar to that in a hydrogel. We seek to understand the properties and dynamics of externally tumesced (swollen) subcutaneous tissue as a first step in assessing whether tumescent antibiotic injections into wounds may provide a novel method of treatment. METHODS: Subcutaneous injections of saline are performed in live and dead pigs and the physical properties (volume, expansion ratio, residence time, apparent diffusion constant) of the resulting fluid deposits are observed with diffusion-weighted magnetic resonance imaging, computed tomography, and 3D scanning. RESULTS: Subcutaneous tissue can expand to a few times its initial volume to accommodate the injected fluid, which is dispersed thoroughly throughout the tumescent volume. The fluid spreads to peripheral unexpanded regions over the course of a few minutes, after which it remains in place for several hours. Eventually the circulation absorbs the excess fluid and the tissue returns to its original state. CONCLUSIONS: Given the evidence for dense fluid dispersal and several-hour residence time, a procedure is proposed whereby tumescent antibiotic injections are used to treat drug-resistant skin infections and chronic wounds that extend into the subcutaneous tissue. The procedure has the potential to effectively treat otherwise untreatable wounds by keeping drug concentrations above minimum inhibitory levels for extended lengths of time.

Snail synchronizes endocycling in a TOR-dependent manner to coordinate entry and escape from endoreplication pausing during the Drosophila critical weight checkpoint.

The final body size of any given individual underlies both genetic and environmental constraints. Both mammals and insects use target of rapamycin (TOR) and insulin signaling pathways to coordinate growth with nutrition. In holometabolous insects, the growth period is terminated through a cascade of peptide and steroid hormones that end larval feeding behavior and trigger metamorphosis, a nonfeeding stage during which the larval body plan is remodeled to produce an adult. This irreversible decision, termed the critical weight (CW) checkpoint, ensures that larvae have acquired sufficient nutrients to complete and survive development to adulthood. How insects assess body size via the CW checkpoint is still poorly understood on the molecular level. We show here that the Drosophila transcription factor Snail plays a key role in this process. Before and during the CW checkpoint, snail is highly expressed in the larval prothoracic gland (PG), an endocrine tissue undergoing endoreplication and primarily dedicated to the production of the steroid hormone ecdysone. We observed two Snail peaks in the PG, one before and one after the molt from the second to the third instar. Remarkably, these Snail peaks coincide with two peaks of PG cells entering S phase and a slowing of DNA synthesis between the peaks. Interestingly, the second Snail peak occurs at the exit of the CW checkpoint. Snail levels then decline continuously, and endoreplication becomes nonsynchronized in the PG after the CW checkpoint. This suggests that the synchronization of PG cells into S phase via Snail represents the mechanistic link used to terminate the CW checkpoint. Indeed, PG-specific loss of snail function prior to the CW checkpoint causes larval arrest due to a cessation of endoreplication in PG cells, whereas impairing snail after the CW checkpoint no longer affected endoreplication and further development. During the CW window, starvation or loss of TOR signaling disrupted the formation of Snail peaks and endocycle synchronization, whereas later starvation had no effect on snail expression. Taken together, our data demonstrate that insects use the TOR pathway to assess nutrient status during larval development to regulate Snail in ecdysone-producing cells as an effector protein to coordinate endoreplication and CW attainment.

A Multi-Omics Interpretable Machine Learning Model Reveals Modes of Action of Small Molecules.

High-throughput screening and gene signature analyses frequently identify lead therapeutic compounds with unknown modes of action (MoAs), and the resulting uncertainties can lead to the failure of clinical trials. We developed an approach for uncovering MoAs through an interpretable machine learning model of transcriptomics, epigenomics, metabolomics, and proteomics. Examining compounds with beneficial effects in models of Huntington's Disease, we found common MoAs for compounds with unrelated structures, connectivity scores, and binding targets. The approach also predicted highly divergent MoAs for two FDA-approved antihistamines. We experimentally validated these effects, demonstrating that one antihistamine activates autophagy, while the other targets bioenergetics. The use of multiple omics was essential, as some MoAs were virtually undetectable in specific assays. Our approach does not require reference compounds or large databases of experimental data in related systems and thus can be applied to the study of agents with uncharacterized MoAs and to rare or understudied diseases.

Autologous Transplantation of Skin-Derived Precursor Cells in a Porcine Model.

BACKGROUND: Hirschprung's disease is characterized by aganglionic bowel and often requires surgical resection. Cell-based therapies have been investigated as potential alternatives to restore functioning neurons. Skin-derived precursor cells (SKPs) differentiate into neural and glial cells in vitro and generate ganglion-like structures in rodents. In this report, we aimed to translate this approach into a large animal model of aganglionosis using autologous transplantation of SKPs. METHODS: Juvenile pigs underwent skin procurement from the shoulder and simultaneous chemical denervation of an isolated colonic segment. Skin cells were cultured in neuroglial-selective medium and labeled with fluorescent dye for later identification. The cultured SKPs were then injected into the aganglionic segments of colon, and the specimens were retrieved within seven days after transplantation. SKPs in vitro and in vivo were assessed with histologic samples for various immunofluorescent markers of multipotency and differentiation. SKPs from the time of harvest were compared to those at the time of injection using PCR. RESULTS: Prior to transplantation, 72% of SKPs stained positive for nestin and S100b, markers of neural and glial precursor cells of neural crest origin, respectively. Markers of differentiated neurons and gliocytes, TUJ1 and GFAP, were detected in 47% of cultured SKPs. After transplantation, SKPs were identified in both myenteric and submucosal plexuses of the treated colon. Nestin co-expression was detected in the SKPs within the aganglionic colon in vivo. Injected SKPs appeared to migrate and express early neuroglial differentiation markers. CONCLUSIONS: Autologous SKPs implanted into aganglionic bowel demonstrated immunophenotypes of neuroglial progenitors. Our results suggest that autologous SKPs may be potentially useful for cell-based therapy for patients with enteric nervous system disorders. TYPE OF STUDY: Basic science.

Conversion of D-fructose to 5-acetoxymethyl-2-furfural Using Immobilized Lipase and Cation Exchange Resin.

5-Acetoxymethyl-2-furfural (AMF) was prepared from D-fructose via 1,6-diacetylfructose (DAF) through a simple two-step reaction pathway. Immobilized enzyme (Novozym 435) was found to be the best enzymatic catalyst for the trans-esterification step (yielding 94.6% DAF). In the dehydration step, while soluble H2SO4 was found to be the best acidic catalyst (yielding 86.6% AMF), we opted to utilize heterogeneous cation exchange resin (Amberlyst 15) together with recyclable industrial solvents (1,4-dioxane) for a more sustainable AMF synthesis procedure. Although the total yield of AMF was a little lower, both the enzyme and the solid acid catalyst could be recycled for five cycles without a significant loss of activity, which has a major contribution to the cost-efficient aspect of the entire process.

Glycogen branching enzyme controls cellular iron homeostasis via Iron Regulatory Protein 1 and mitoNEET.

Iron Regulatory Protein 1 (IRP1) is a bifunctional cytosolic iron sensor. When iron levels are normal, IRP1 harbours an iron-sulphur cluster (holo-IRP1), an enzyme with aconitase activity. When iron levels fall, IRP1 loses the cluster (apo-IRP1) and binds to iron-responsive elements (IREs) in messenger RNAs (mRNAs) encoding proteins involved in cellular iron uptake, distribution, and storage. Here we show that mutations in the Drosophila 1,4-Alpha-Glucan Branching Enzyme (AGBE) gene cause porphyria. AGBE was hitherto only linked to glycogen metabolism and a fatal human disorder known as glycogen storage disease type IV. AGBE binds specifically to holo-IRP1 and to mitoNEET, a protein capable of repairing IRP1 iron-sulphur clusters. This interaction ensures nuclear translocation of holo-IRP1 and downregulation of iron-dependent processes, demonstrating that holo-IRP1 functions not just as an aconitase, but throttles target gene expression in anticipation of declining iron requirements.

Intestinal Electrical Stimulation to Increase the Rate of Peristalsis.

BACKGROUND: Pediatric gastrointestinal motility disorders are a large and broad group. Some of these disorders have been effectively treated with electrical stimulation. The goal of our present study is to determine whether the rate of intestinal peristalsis can be increased with electrical stimulation. METHODS: Juvenile mini-Yucatan pigs were placed under general anesthesia and a short segment of the jejunum was transected. Ultrasound gel was placed inside the segment. The segment of the jejunum was first monitored for 20 min under no stimulation, followed by direct electrical stimulation using a planar electrode. The gel extruded out of the intestine via peristalsis was collected and weighed for each 20-min time interval. RESULTS: Effective delivery of the current to the intestine was confirmed via direct measurements. When there was no direct intestinal electrical stimulation, an average of 0.40 g of gel was expelled in 20 min, compared to 1.57 g of gel expelled during direct electrical stimulation (P < 0.01). CONCLUSIONS: Direct intestinal electrical stimulation accelerates the transit of gastrointestinal contents. This approach may be useful in the treatment of a range of pediatric motility disorders.

Double plication for spring-mediated in-continuity intestinal lengthening in a porcine model.

BACKGROUND: Short bowel syndrome is a condition with substantial morbidity and mortality, yet definitive therapies are lacking. Distraction enterogenesis uses mechanical force to "grow" new intestine. In this study, we examined whether intestinal plication can be used to safely achieve spring-mediated intestinal lengthening in a functioning segment of jejunum in its native position. METHODS: A total of 12 juvenile, miniature Yucatan pigs underwent laparotomy to place either compressed springs or expanded springs within a segment of jejunum (n = 6 per group). The springs were secured within the jejunum by performing intestinal plication to narrow the intestinal lumen around the spring. After 3 weeks, the jejunum was retrieved and examined for lengthening and for histologic changes. RESULTS: There were no intraoperative or postoperative complications, and the pigs tolerated their diets and gained weight. Segments of jejunum containing expanded springs showed no significant change in length over the 3 weeks. In contrast, jejunum containing compressed springs showed nearly a 3-fold increase in length (P < .001). Histology of the retrieved jejunum showed a significant increase in thickness of the muscularis propria and in crypt depth relative to normal jejunum. CONCLUSION: Intestinal plication is effective in securing endoluminal springs to lengthen the jejunum. This approach is a clinically relevant model because it allows for normal GI function and growth of animals during intestinal lengthening, which may be useful in lengthening intestine in patients with short bowel syndrome.

Intestinal lengthening via multiple in-continuity springs.

BACKGROUND: Short bowel syndrome is a debilitating condition with few effective treatments. Spring-mediated distraction enterogenesis can be used to lengthen intestine. The purpose of this study is to determine whether multiple springs in series can safely increase the total amount of lengthening. METHODS: Juvenile mini-Yucatan pigs each received three nitinol springs placed within their jejunum. Plication was used to narrow the intestine around each spring to secure them. Compressed springs were used in the experimental group, while uncompressed springs were used in the control group. The intestine was examined 3 weeks later for lengthening and histologic changes. RESULTS: All pigs tolerated diets postoperatively with continued weight gain, and no dilation or obstruction of the intestine was observed. Segments of intestine that contained compressed springs had a significant increase in length from 2.5 cm to 3.9 ±â€¯0.2 cm per spring, compared to segments containing control springs that showed no change (p < 0.001). CONCLUSIONS: Intestinal plication can be safely used to secure multiple springs in series to achieve intestinal lengthening without compromising intestinal function. Using several springs at once allows for a greater amount of total lengthening. This is a promising model that has potential in the treatment of short bowel syndrome.

A Drosophila CRISPR/Cas9 Toolkit for Conditionally Manipulating Gene Expression in the Prothoracic Gland as a Test Case for Polytene Tissues.

Targeting gene function with spatial or temporal specificity is a key goal in molecular genetics. CRISPR-Cas9 has greatly facilitated this strategy, but some standard approaches are problematic. For instance, simple tissue-specific or global overexpression of Cas9 can cause significant lethality or developmental delays even in the absence of gRNAs. In particular, we found that Gal4-mediated expression of UAS-Cas9 in the Drosophila prothoracic gland (PG) was not a suitable strategy to disrupt gene expression, since Cas9 alone caused widespread lethality. The PG is widely used for studying endocrine gland function during animal development, but tools validating PG-specific RNAi phenotypes are lacking. Here, we present a collection of modular gateway-compatible CRISPR-Cas9 tools that allow precise modulation of target gene activity with temporal and spatial specificity. We also demonstrate that Cas9 fused to the progesterone ligand-binding domain can be used to activate gene expression via RU486. Using these approaches, we were able to avoid the lethality associated with simple GAL4-mediated overexpression of Cas9 in the PG. Given that the PG is a polytene tissue, we conclude that these tools work effectively in endoreplicating cells where Cas9 has to target multiple copies of the same locus. Our toolkit can be easily adapted for other tissues and can be used both for gain- and loss-of-function studies.