Interventional analytics in skilled nursing facilities associated with reduced readmissions.

OBJECTIVE: To assess differences in longitudinal profiles for 30-day risk-adjusted readmission rates in skilled nursing facilities (SNFs) associated with Penn Medicine's Lancaster General Hospital (LGH) that implemented an interventional analytics (IA) platform vs other LGH facilities lacking IA vs other SNFs in Pennsylvania vs facilities in all other states. STUDY DESIGN: Retrospective longitudinal analysis of CMS readmissions data from 2017 through 2022, and cross-sectional analysis using CMS quality metrics data. METHODS: CMS SNF quality performance data were aggregated and compared with risk-adjusted readmissions by facility and time period. Each SNF was assigned to a cohort based on location, referral relationship with LGH, and whether it had implemented IA. Multivariable mixed effects modeling was used to compare readmissions by cohort, whereas quality measures from the fourth quarter of 2022 were compared descriptively. RESULTS: LGH profiles differed significantly from both state and national profiles, with LGH facilities leveraging IA demonstrating an even greater divergence. In the most recent 12 months ending in the fourth quarter of 2022, LGH SNFs with IA had estimated readmission rates that were 15.24, 12.30, and 13.06 percentage points lower than the LGH SNFs without IA, Pennsylvania, and national cohorts, respectively (all pairwise P < .0001). SNFs with IA also demonstrated superior CMS claims-based quality metric outcomes for the 12 months ending in the fourth quarter of 2022. CONCLUSIONS: SNFs implementing the studied IA platform demonstrated statistically and clinically significant superior risk-adjusted readmission rate profiles compared with peers nationally, statewide, and within the same SNF referral network (P < .0001). A more detailed study on the use of IA in this setting is warranted.

Dysplastic Hips That Are Too Late for Periacetabular Osteotomy Are Not Too Early for Total Hip Arthroplasty.

BACKGROUND: Total hip arthroplasty (THA) is often performed in symptomatic patients who have hip dysplasia and do not qualify for periacetabular osteotomy. The impact of osteoarthritis (OA) severity on postoperative outcomes in dysplasia patients who undergo THA is not well described. We hypothesized that dysplasia patients who have mild OA have slower initial recovery postoperatively but similar one-year patient-reported outcome measures (PROMs) compared to dysplasia patients who have severe OA. METHODS: We performed a retrospective review at a single academic institution over a 6-year period of patients who have dysplasia who underwent THA compared to patients who have primary OA who underwent THA. There were 263 patients who had dysplasia, compared to 1,225 THA patients who did not have dysplasia. Within the dysplasia cohort, we compared PROMs stratified by dysplasia and OA severity. The diagnosis of dysplasia was verified using the radiographic lateral center edge angle. A minimum one-year follow-up was required. The PROMs were collected through one year postoperatively. Logistic and linear regression models were used, adjusting for age, sex, body mass index, and Charlson comorbidity index. RESULTS: No significant differences were found in postoperative PROMs or revision rates (P = .58). When stratified by dysplasia severity, patients who had lower lateral center edge angle had more improvement in physical function scores from preoperative to 2 weeks (P < .01) and higher physical function scores at 2 weeks (P = .03). When stratified by OA severity, patients who had a worse Tönnis score had more improvement in physical function scores from preoperative to 2 weeks (P < .01). Recovery curves in dysplasia patients based on dysplasia and OA severity were not significantly different at 6 weeks, 1 year, and 2 years postoperative. CONCLUSIONS: Patients who had hip dysplasia and mild OA had similar recovery curves compared to those who had severe OA or who did not have dysplasia. We believe that THA is a reasonable surgical intervention for symptomatic dysplasia patients who have mild arthritis and do not qualify for periacetabular osteotomy.

RNA sequence to structure analysis from comprehensive pairwise mutagenesis of multiple self-cleaving ribozymes.

Self-cleaving ribozymes are RNA molecules that catalyze the cleavage of their own phosphodiester backbones. These ribozymes are found in all domains of life and are also a tool for biotechnical and synthetic biology applications. Self-cleaving ribozymes are also an important model of sequence-to-function relationships for RNA because their small size simplifies synthesis of genetic variants and self-cleaving activity is an accessible readout of the functional consequence of the mutation. Here, we used a high-throughput experimental approach to determine the relative activity for every possible single and double mutant of five self-cleaving ribozymes. From this data, we comprehensively identified non-additive effects between pairs of mutations (epistasis) for all five ribozymes. We analyzed how changes in activity and trends in epistasis map to the ribozyme structures. The variety of structures studied provided opportunities to observe several examples of common structural elements, and the data was collected under identical experimental conditions to enable direct comparison. Heatmap-based visualization of the data revealed patterns indicating structural features of the ribozymes including paired regions, unpaired loops, non-canonical structures, and tertiary structural contacts. The data also revealed signatures of functionally critical nucleotides involved in catalysis. The results demonstrate that the data sets provide structural information similar to chemical or enzymatic probing experiments, but with additional quantitative functional information. The large-scale data sets can be used for models predicting structure and function and for efforts to engineer self-cleaving ribozymes.

Multiple tissue-specific epigenetic alterations regulate persistent gene expression changes following developmental DES exposure in mouse reproductive tissues.

Clinically, developmental exposure to the endocrine disrupting chemical, diethylstilboestrol (DES), results in long-term male and female infertility. Experimentally, developmental exposure to DES results in abnormal reproductive tract phenotypes in male and female mice. Previously, we reported that neonatal DES exposure causes ERα-mediated aberrations in the transcriptome and in DNA methylation in seminal vesicles (SVs) of adult mice. However, only a subset of DES-altered genes could be explained by changes in DNA methylation. We hypothesized that alterations in histone modification may also contribute to the altered transcriptome during SV development. To test this idea, we performed a series of genome-wide analyses of mouse SVs at pubertal and adult developmental stages in control and DES-exposed wild-type and ERα knockout mice. Neonatal DES exposure altered ERα-mediated mRNA and lncRNA expression in adult SV, including genes encoding chromatin-modifying proteins that can impact histone H3K27ac modification. H3K27ac patterns, particularly at enhancers, and DNA methylation were reprogrammed over time during normal SV development and after DES exposure. Some of these reprogramming changes were ERα-dependent, but others were ERα-independent. A substantial number of DES-altered genes had differential H3K27ac peaks at nearby enhancers. Comparison of gene expression changes, H3K27ac marks and DNA methylation marks between adult SV and adult uterine tissue from ovariectomized mice neonatally exposed to DES revealed that most of the epigenetic changes and altered genes were distinct in the two tissues. These findings indicate that the effects of developmental DES exposure cause reprogramming of reproductive tract tissue differentiation through multiple epigenetic mechanisms.

Bacterial Metabolism and Transport Genes Are Associated with the Preference of Drosophila melanogaster for Dietary Yeast.

Many animal traits are influenced by their associated microorganisms ("microbiota"). To expand our understanding of the relationship between microbial genotype and host phenotype, we report an analysis of the influence of the microbiota on the dietary preference of the fruit fly Drosophila melanogaster. First, we confirmed through experiments on flies reared bacteria-free ("axenic") or in monoassociation with two different strains of bacteria that the microbiota significantly influences fruit fly dietary preference across a range of ratios of dietary yeast:dietary glucose. Then, focusing on microbiota-dependent changes in fly dietary preference for yeast (DPY), we performed a metagenome-wide association (MGWA) study to define microbial species specificity for this trait and to predict bacterial genes that influence it. In a subsequent mutant analysis, we confirmed that disrupting a subset of the MGWA-predicted genes influences fly DPY, including for genes involved in thiamine biosynthesis and glucose transport. Follow-up tests revealed that the bacterial influence on fly DPY did not depend on bacterial modification of the glucose or protein content of the fly diet, suggesting that the bacteria mediate their effects independent of the fly diet or through more specific dietary changes than broad ratios of protein and glucose. Together, these findings provide additional insight into bacterial determinants of host nutrition and behavior by revealing specific genetic disruptions that influence D. melanogaster DPY. IMPORTANCE Associated microorganisms ("microbiota") impact the physiology and behavior of their hosts, and defining the mechanisms underlying these interactions is a major gap in the field of host-microbe interactions. This study expands our understanding of how the microbiota can influence dietary preference for yeast (DPY) of a model host, Drosophila melanogaster. First, we show that fly preferences for a range of different dietary yeast:dietary glucose ratios vary significantly with the identity of the microbes that colonize the fruit flies. We then performed a metagenome-wide association study to identify candidate bacterial genes that contributed to some of these bacterial influences. We confirmed that disrupting some of the predicted genes, including genes involved in glucose transport and thiamine biosynthesis, resulted in changes to fly DPY and show that the influence of two of these genes is not through changes in dietary ratios of protein to glucose. Together, these efforts expand our understanding of the bacterial genetic influences on a feeding behavior of a model animal host.

An altered microbiome in a Parkinson's disease model Drosophila melanogaster has a negative effect on development.

Parkinson's disease (PD) is the second most common neurodegenerative disease, besides Alzheimer's Disease, characterized by multiple symptoms, including the well-known motor dysfunctions. It is well-established that there are differences in the fecal microbiota composition between Parkinson's disease (PD) patients and control populations, but the mechanisms underlying these differences are not yet fully understood. To begin to close the gap between description and mechanism we studied the relationship between the microbiota and PD in a model organism, Drosophila melanogaster. First, fecal transfers were performed with a D. melanogaster model of PD that had a mutation in the parkin (park25) gene. Results indicate that the PD model feces had a negative effect on both pupation and eclosion in both control and park25 flies, with a greater effect in PD model flies. Analysis of the microbiota composition revealed differences between the control and park25 flies, consistent with many human studies. Conversely, gnotobiotic treatment of axenic embryos with feces-derived bacterial cultures did not affect eclosure. We speculate this result might be due to similarities in bacterial prevalence between mutant and control feces. Further, we confirmed a bacteria-potentiated impact on mutant and control fly phenotypes by measuring eclosure rate in park25 flies that were mono-associated with members of the fly microbiota. Both the fecal transfer and the mono-association results indicate a host genotype-microbiota interaction. Overall, this study concludes functional effects of the fly microbiota on PD model flies, providing support to the developing body of knowledge regarding the influence of the microbiota on PD.

Fragmentation of Apolipoprotein E4 is Required for Differential Expression of Inflammation and Activation Related Genes in Microglia Cells.

The apolipoprotein E4 (APOE4) allele represents the single greatest risk factor for late-onset Alzheimer's disease (AD) and accumulating evidence suggests that fragmentation with a toxic-gain of function may be a key molecular step associated with this risk. Recently, we demonstrated strong immunoreactivity of a 151 amino-terminal fragment of apoE4 (E4-fragment) within the nucleus of microglia in the human AD brain. In vitro, this fragment led to toxicity and activation of inflammatory processes in BV2 microglia cells. Additionally, a transcriptome analysis following exogenous treatment of BV2 microglia cells with this E4 fragment led to a > 2-fold up regulation of 1,608 genes, with many genes playing a role in inflammation and microglia activation. To extend these findings, we here report a similar transcriptome analysis in BV2 microglia cells following treatment with full-length ApoE4 (FL-ApoE4). The results indicated that full-length ApoE4 had a very small effect on gene expression compared to the fragment. Only 48 differentially expressed genes (DEGs) were identified (p < 0.05, and greater than 2-fold change). A gene ontology analysis of these DEGs indicated that they are not involved in inflammatory and activation processes, in contrast to the genes up regulated by the E4-fragment. In addition, genes that showed a negative fold-change upon FL-E4 treatment typically showed a strong positive fold-change upon treatment with the fragment (Pearson's r = -0.7). Taken together, these results support the hypothesis that a key step in the conversion of microglia to an activated phenotype is proteolytic cleavage of FL-ApoE4. Therefore, the neutralization of this amino-terminal fragment of ApoE4, specifically, may serve as an important therapeutic strategy in the treatment of AD.

Experimental Resurrection of Ancestral Mammalian CPEB3 Ribozymes Reveals Deep Functional Conservation.

Self-cleaving ribozymes are genetic elements found in all domains of life, but their evolution remains poorly understood. A ribozyme located in the second intron of the cytoplasmic polyadenylation binding protein 3 gene (CPEB3) shows high sequence conservation in mammals, but little is known about the functional conservation of self-cleaving ribozyme activity across the mammalian tree of life or during the course of mammalian evolution. Here, we use a phylogenetic approach to design a mutational library and a deep sequencing assay to evaluate the in vitro self-cleavage activity of numerous extant and resurrected CPEB3 ribozymes that span over 100 My of mammalian evolution. We found that the predicted sequence at the divergence of placentals and marsupials is highly active, and this activity has been conserved in most lineages. A reduction in ribozyme activity appears to have occurred multiple different times throughout the mammalian tree of life. The in vitro activity data allow an evaluation of the predicted mutational pathways leading to extant ribozyme as well as the mutational landscape surrounding these ribozymes. The results demonstrate that in addition to sequence conservation, the self-cleavage activity of the CPEB3 ribozyme has persisted over millions of years of mammalian evolution.

A mutant form of ERα associated with estrogen insensitivity affects the coupling between ligand binding and coactivator recruitment.

A homozygous missense mutation in the gene encoding the estrogen receptor α (ERα) was previously identified in a female patient with estrogen insensitivity syndrome. We investigated the molecular features underlying the impaired transcriptional response of this mutant (ERα-Q375H) and four other missense mutations at this position designed to query alternative mechanisms. The identity of residue 375 greatly affected the sensitivity of the receptor to agonists without changing the ligand binding affinity. Instead, the mutations caused changes in the affinity of coactivator binding and alterations in the balance of coactivator and corepressor recruitment. Comparisons among the transcriptional regulatory responses of these six ERα genotypes to a set of ER agonists showed that both steric and electrostatic factors contributed to the functional deficits in gene regulatory activity of the mutant ERα proteins. ERα-coregulator peptide binding in vitro and RIME (rapid immunoprecipitation mass spectrometry of endogenous) analysis in cells showed that the degree of functional impairment paralleled changes in receptor-coregulator binding interactions. These findings uncover coupling between ligand binding and coregulator recruitment that affects the potency rather than the efficacy of the receptor response without substantially altering ligand binding affinity. This highlights a molecular mechanism for estrogen insensitivity syndrome involving mutations that perturb a bidirectional allosteric coupling between ligand binding and coregulator binding that determines receptor transcriptional output.

Transcriptome Analyses in BV2 Microglial Cells Following Treatment With Amino-Terminal Fragments of Apolipoprotein E.

Despite the fact that harboring the apolipoprotein E4 (APOE4) allele represents the single greatest risk factor for late-onset Alzheimer's disease (AD), the exact mechanism by which ApoE4 contributes to disease progression remains unknown. Recently, we demonstrated that a 151 amino-terminal fragment of ApoE4 (nApoE41-151) localizes within the nucleus of microglia in the human AD brain and traffics to the nucleus causing toxicity in BV2 microglia cells. In the present study, we examined in detail what genes may be affected following treatment by nApoE41-151. Transcriptome analyses in BV2 microglial cells following sublethal treatment with nApoE41-151 revealed the upregulation of almost 4,000 genes, with 20 of these genes upregulated 182- to 715-fold compared to untreated control cells. The majority of these 20 genes play a role in the immune response and polarization toward microglial M1 activation. As a control, an identical nApoE31-151 fragment that differed by a single amino acid at position 112 (Cys→Arg) was tested and produced a similar albeit lower level of upregulation of an identical set of genes. In this manner, enriched pathways upregulated by nApoE31-151 and nApoE41-151 following exogenous treatment included Toll receptor signaling, chemokine/cytokine signaling and apoptosis signaling. There were unique genes differentially expressed by at least two-fold for either fragment. For nApoE31-151, these included 16 times as many genes, many of which are involved in physiological functions within microglia. For nApoE41-151, on the other hand the number genes uniquely upregulated was significantly lower, with many of the top upregulated genes having unknown functions. Taken together, our results suggest that while nApoE31-151 may serve a more physiological role in microglia, nApoE41-151 may activate genes that contribute to disease inflammation associated with AD. These data support the hypothesis that the link between harboring the APOE4 allele and dementia risk could be enhanced inflammation through activation of microglia.

ESR1 Mutations Associated With Estrogen Insensitivity Syndrome Change Conformation of Ligand-Receptor Complex and Altered Transcriptome Profile.

Estrogen insensitivity syndrome (EIS) arises from rare mutations in estrogen receptor-α (ERα, encoded by ESR1 gene) resulting in the inability of estrogen to exert its biological effects. Due to its rarity, mutations in ESR1 gene and the underlying molecular mechanisms of EIS have not been thoroughly studied. Here, we investigate known ESR1 mutants, Q375H and R394H, associated with EIS patients using in vitro and in vivo systems. Comparison of the transcriptome and deoxyribonucleic acid methylome from stable cell lines of both Q375H and R394H clinical mutants shows a differential profile compared with wild-type ERα, resulting in loss of estrogen responsiveness. Molecular dynamic simulation shows that both ESR1 mutations change the ERα conformation of the ligand-receptor complexes. Furthermore, we generated a mouse model Esr1-Q harboring the human mutation using CRISPR/Cas9 genome editing. Female and male Esr1-Q mice are infertile and have similar phenotypes to αERKO mice. Overall phenotypes of the Esr1-Q mice correspond to those observed in the patient with Q375H. Finally, we explore the effects of a synthetic progestogen and a gonadotropin-releasing hormone inhibitor in the Esr1-Q mice for potentially reversing the impaired female reproductive tract function. These findings provide an important basis for understanding the molecular mechanistic consequences associated with EIS.

The microbiota influences the Drosophila melanogaster life history strategy.

Organisms are locally adapted when members of a population have a fitness advantage in one location relative to conspecifics in other geographies. For example, across latitudinal gradients, some organisms may trade off between traits that maximize fitness components in one, but not both, of somatic maintenance or reproductive output. Latitudinal gradients in life history strategies are traditionally attributed to environmental selection on an animal's genotype, without any consideration of the possible impact of associated microorganisms ("microbiota") on life history traits. Here, we show in Drosophila melanogaster, a key model for studying local adaptation and life history strategy, that excluding the microbiota from definitions of local adaptation is a major shortfall. First, we reveal that an isogenic fly line reared with different bacteria varies the investment in early reproduction versus somatic maintenance. Next, we show that in wild fruit flies, the abundance of these same bacteria was correlated with the latitude and life history strategy of the flies, suggesting geographic specificity of the microbiota composition. Variation in microbiota composition of locally adapted D. melanogaster could be attributed to both the wild environment and host genetic selection. Finally, by eliminating or manipulating the microbiota of fly lines collected across a latitudinal gradient, we reveal that host genotype contributes to latitude-specific life history traits independent of the microbiota and that variation in the microbiota can suppress or reverse the differences between locally adapted fly lines. Together, these findings establish the microbiota composition of a model animal as an essential consideration in local adaptation.

A Novel, Highly Related Jumbo Family of Bacteriophages That Were Isolated Against Erwinia.

Erwinia amylovora is a plant pathogen from the Erwiniaceae family and a causative agent of the devastating agricultural disease fire blight. Here we characterize eight lytic bacteriophages of E. amylovora that we isolated from the Wasatch front (Utah, United States) that are highly similar to vB_EamM_Ea35-70 which was isolated in Ontario, Canada. With the genome size ranging from 271 to 275 kb, this is a novel jumbo family of bacteriophages. These jumbo bacteriophages were further characterized through genomic and proteomic comparison, mass spectrometry, host range and burst size. Their proteomes are highly unstudied, with over 200 putative proteins with no known homologs. The production of 27 of these putative proteins was confirmed by mass spectrometry analysis. These bacteriophages appear to be most similar to bacteriophages that infect Pseudomonas and Ralstonia rather than Enterobacteriales bacteria by protein similarity, however, we were only able to detect infection of Erwinia and the closely related strains of Pantoea.

Connecting Solution-Phase to Single-Molecule Properties of Ni(Salophen).

We present a strong correlation of the Ni(salophen) structure and properties measured in single-molecule vs bulk quantities and in ultra high vacuum vs solution phase. Under a scanning tunneling microscope (STM), Ni(salophen) forms a self-assembled monolayer (SAM) on Au(111) at 23 °C with molecular structure identical to that of the X-ray crystallographic measurement. The HOMO and LUMO levels are determined using elastic tunneling spectroscopy at the single-molecule level with confirmation by monolayer-quantity ultraviolet photoelectron spectroscopy (UPS) and by cyclic voltammetry (CV) measurements. The STM-determined HOMO-LUMO gap of 3.28 eV and (HOMO-1)-HOMO gap of 0.36 eV form a new foundation for the selection of hybrid functionals with a simple basis set to be effective in accurately calculating single-molecule Ni(salophen) frontier MO levels. Our results suggest that microscopy-based experiments on a surface, along with free-molecule gas-phase calculations, can provide useful insights into the physical properties of metal(salen) complexes, especially when such direct measurements are not available in solution.

A Fragment of Apolipoprotein E4 Leads to the Downregulation of a CXorf56 Homologue, a Novel ER-Associated Protein, and Activation of BV2 Microglial Cells.

Despite the fact that harboring the apolipoprotein E4 (APOE4) allele represents the single greatest risk factor for late-onset Alzheimer's disease (AD), the exact mechanism by which apoE4 contributes to disease progression remains unknown. Recently, we demonstrated that a 151 amino-terminal fragment of apoE4 (nApoE41-151) localizes within the nucleus of microglia in the human AD brain, suggesting a potential role in gene expression. In the present study, we investigated this possibility utilizing BV2 microglia cells treated exogenously with nApoE41-151. The results indicated that nApoE41-151 leads to morphological activation of microglia cells through, at least in part, the downregulation of a novel ER-associated protein, CXorf56. Moreover, treatment of BV2 cells with nApoE41-151 resulted in a 68-fold increase in the expression of the inflammatory cytokine, TNFα, a key trigger of microglia activation. In this regard, we also observed a specific binding interaction of nApoE41-151 with the TNFα promoter region. Collectively, these data identify a novel gene-regulatory pathway involving CXorf56 that may link apoE4 to microglia activation and inflammation associated with AD.

Synthesis and Characterization of Hydrazine-Appended BODIPY Dyes and the Related Aminomethyl Complexes.

The synthesis and characterization of three new organic hydrazines containing BODIPY dyes is described. The respective aminomethyl complexes were also synthesized to aid in the assignment of the physical properties that were hydrazine-based vs. BODIPY-based. Incorporation of a BODIPY dye into an organic hydrazine introduced a reduction event (average value of -1.70 V vs. Cp2Fe/Cp2Fe+). Although two irreversible oxidation events were observed, it was unclear whether the oxidation events arose from BODIPY-based or amine/hydrazine-based oxidations. The respective BODIPY-appended hydrazine complexes exhibited excited state lifetimes on the order of 2-6 ns, suggesting the presence of a singlet excited state. The excited state lifetimes of the BODIPY-appended hydrazine complexes were about a factor of ten greater than the respective aminomethyl complexes. Computational analysis showed that by appending a BODIPY dye to a hydrazine fragment the hydrazine fragment becomes more susceptible to transfer H2 equivalents as protons and hydrides as opposed to H-atoms, which occurs with common organic hydrazines. Computational analysis also revealed that the BODIPY-based redox events can be used to manipulate the mechanism for H2 transfer from the BODIPY-appended hydrazine, where a BODIPY-based reduction favors H-atom transfer and a BODIPY-based oxidation favors proton transfer followed by hydride transfer.

Nondestructive X-ray diffraction measurement of warpage in silicon dies embedded in integrated circuit packages.

Transmission X-ray diffraction imaging in both monochromatic and white beam section mode has been used to measure quantitatively the displacement and warpage stress in encapsulated silicon devices. The displacement dependence with position on the die was found to agree well with that predicted from a simple model of warpage stress. For uQFN microcontrollers, glued only at the corners, the measured misorientation contours are consistent with those predicted using finite element analysis. The absolute displacement, measured along a line through the die centre, was comparable to that reported independently by high-resolution X-ray diffraction and optical interferometry of similar samples. It is demonstrated that the precision is greater than the spread of values found in randomly selected batches of commercial devices, making the techniques viable for industrial inspection purposes.

Clinician's Guide to Prevention and Treatment of Osteoporosis.

The Clinician's Guide to Prevention and Treatment of Osteoporosis was developed by an expert committee of the National Osteoporosis Foundation (NOF) in collaboration with a multispecialty council of medical experts in the field of bone health convened by NOF. Readers are urged to consult current prescribing information on any drug, device, or procedure discussed in this publication.