Allele-specific transcription factor binding across human brain regions offers mechanistic insight into eQTLs.

Transcription factors (TFs) regulate gene expression by facilitating or disrupting the formation of transcription initiation machinery at particular genomic loci. Because TF occupancy is driven in part by recognition of DNA sequence, genetic variation can influence TF-DNA associations and gene regulation. To identify variants that impact TF binding in human brain tissues, we assessed allele-specific binding (ASB) at heterozygous variants for 94 TFs in nine brain regions from two donors. Leveraging graph genomes constructed from phased genomic sequence data, we compared ChIP-seq signals between alleles at heterozygous variants within each brain region and identified thousands of variants exhibiting ASB for at least one TF. ASB reproducibility was measured by comparisons between independent experiments both within and between donors. We found that rare alleles in the general population more frequently led to reduced TF binding, whereas common alleles had an equal likelihood of increasing or decreasing binding. Further, for ASB variants in predicted binding motifs, the favored allele tended to be the one with the stronger expected motif match, but this concordance was not observed within highly occupied sites. We also found that neuron-specific cis-regulatory elements (cCREs), in contrast with oligodendrocyte-specific cCREs, showed depletion of ASB variants. We identified 2670 ASB variants associated with evidence for allele-specific gene expression in the brain from GTEx data and observed increasing eQTL effect direction concordance as ASB significance increases. These results provide a valuable and unique resource for mechanistic analysis of cis-regulatory variation in human brain tissue.

Minimal Clinically Important Difference, Substantial Clinical Benefit, and Patient Acceptable Symptom State Values After Hip Arthroscopy for Femoroacetabular Impingement Are Highly Dependent on Their Study Population and Calculation Methods: A Systematic Review.

PURPOSE: To provide a summary of available literature on the minimal clinically important difference (MCID), substantial clinical benefit (SCB), and patient acceptable symptom state (PASS) after hip arthroscopy for femoroacetabular impingement (FAI). METHODS: A systematic review was conducted via the Cochrane Library, PubMed, Ovid MEDLINE, and Embase to identify studies that calculated MCID, SCB, or PASS for patient-reported outcome measures after hip arthroscopy for FAI. The electronic search strategy used was as follows: hip AND arthroscopy AND (MCID OR "minimal clinically important difference" OR SCB OR "substantial clinical benefit" OR PASS OR "patient acceptable symptom state"). Inclusion criteria were English-language studies published from 1980 to 2023 reporting clinical outcome scores and calculated values of MCID, PASS, or SCB for patients undergoing hip arthroscopy for FAI. RESULTS: Forty-two studies (5 Level II, 19 Level III, and 18 Level IV) met inclusion and exclusion criteria. The most commonly used outcome measures across MCID, SCB, and PASS were the Hip Outcome Score sports-specific subscale and the activities of daily living subscale, the modified Harris Hip Score, and the 12-item international Hip Outcome Tool. The range of MCID values for Hip Outcome Score sports-specific subscale, Hip Outcome Score activities of daily living subscale, modified Harris Hip Score, and 12-item international Hip Outcome Tool were 7.2-15.7, 7.3-15.4, 7.2-16.8, and 8.8-16.2 respectively. Similarly, for SCB the values ranged from 77.9-96.9, 90.4-98.5, 20.0-98.4, and 66.7-87.5, respectively. Lastly, the PASS values ranged from 63.9-80.9, 85.9-99.2, 74.0-97.0, and 59.5-86.0, respectively. CONCLUSIONS: MCID, SCB, and PASS values for patient-reported outcome measures after hip arthroscopy for the management of FAI are highly dependent on their associated study including study population and calculation methods. LEVEL OF EVIDENCE: IV, systematic review of Level II-IV studies.

Female Orthopaedic Surgeons Are Less Represented as Head Team Physicians in Collegiate Athletic Programs.

PURPOSE: The purpose of this study was to evaluate National Collegiate Athletic Association (NCAA) head team physicians (HTP), focusing on gender as it relates to divisional variability, medical specialty, and research productivity. METHODS: In December 2022, the NCAA member directory was utilized to obtain HTP information from the top 5 conferences within Divisions I, II, and III (DI, DII, DIII, respectively). Division I schools were selected from the previously established "Power 5" conferences. Divisions II and III used NCAA rankings. HTP data was collected from publicly available verifiable data sources. Data for gender, Scopus H-index, residency programs, and fellowship programs were collected. Mean, median, skewness, p-values, and odds ratio were calculated for analysis. RESULTS: One hundred and eighty six NCAA institutions were evaluated: 69 DI (37%), 65 DII (35%), and 52 DIII (28%). DIII had the highest female representation (8; 16%). Out of the 67 orthopaedic surgery HTPs, 5 (7.5%) were female and 62 male (92.5%). There is a statistically significant difference in female orthopedic surgeon representation in the HTP field compared to males (p-value = 0.038, alpha = 0.05). Female orthopaedic surgeons have 38% lower odds of being represented as HTPs compared to males (p-value = 0.046, alpha = 0.05). Female HTPs in Divisions I and III had higher H-indexes than the overall average and median values for their respective divisons. CONCLUSION: Female HTPs are significantly less represented compared to males in the NCAA. Furthermore, female orthopaedic surgeons were found to have lower odds of being represented as HTPs compared to their male counterparts. For research productivity, female HTPs impacted the distribution as outliers in Divisions I and III.

The START domain mediates Arabidopsis GLABRA2 dimerization and turnover independently of homeodomain DNA binding.

Class IV homeodomain leucine-zipper transcription factors (HD-Zip IV TFs) are key regulators of epidermal differentiation that are characterized by a DNA-binding HD in conjunction with a lipid-binding domain termed steroidogenic acute regulatory-related lipid transfer (START). Previous work established that the START domain of GLABRA2 (GL2), a HD-Zip IV member from Arabidopsis (Arabidopsis thaliana), is required for TF activity. Here, we addressed the functions and possible interactions of START and the HD in DNA binding, dimerization, and protein turnover. Deletion analysis of the HD and missense mutations of a conserved lysine (K146) resulted in phenotypic defects in leaf trichomes, root hairs, and seed mucilage, similar to those observed for START domain mutants, despite nuclear localization of the respective proteins. In vitro and in vivo experiments demonstrated that while HD mutations impair binding to target DNA, the START domain is dispensable for DNA binding. Vice versa, protein interaction assays revealed impaired GL2 dimerization for multiple alleles of START mutants, but not HD mutants. Using in vivo cycloheximide chase experiments, we provided evidence for the role of START, but not HD, in maintaining protein stability. This work advances our mechanistic understanding of HD-Zip TFs as multidomain regulators of epidermal development in plants.

Continuous flow strategies for using fluorinated greenhouse gases in fluoroalkylations.

Large quantities of fluorinated gases are generated as intermediates or byproducts from fluorinated polymer production annually, and they are effective ozone depleting substances or greenhouse gases. On the other hand, the incorporation of fluoroalkyl groups into drug molecules or bioactive compounds has been shown to enhance biological properties such as the bioavailability, binding selectivity, and metabolic stability. Extraction of fluoroalkyl sources, including trifluoromethyl and difluoromethyl groups, from the fluorinated gases is highly desirable, yet challenging under regular batch reaction conditions. Flow chemistry is an emerging and promising technique to address long-standing challenges in gas-liquid batch reactions such as insufficient interfacial contact and scalability issues. In this review, we highlight recent advances in continuous flow strategies toward enabling the use of fluorinated greenhouse gases in organic synthesis.

Mouse models of two missense mutations in actin-binding domain 1 of dystrophin associated with Duchenne or Becker muscular dystrophy.

Missense mutations in the dystrophin protein can cause Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) through an undefined pathomechanism. In vitro studies suggest that missense mutations in the N-terminal actin-binding domain (ABD1) cause protein instability, and cultured myoblast studies reveal decreased expression levels that can be restored to wild-type with proteasome inhibitors. To further elucidate the pathophysiology of missense dystrophin in vivo, we generated two transgenic mdx mouse lines expressing L54R or L172H mutant dystrophin, which correspond to missense mutations identified in human patients with DMD or BMD, respectively. Our biochemical, histologic and physiologic analysis of the L54R and L172H mice show decreased levels of dystrophin which are proportional to the phenotypic severity. Proteasome inhibitors were ineffective in both the L54R and L172H mice, yet mice homozygous for the L172H transgene were able to express even higher levels of dystrophin which caused further improvements in muscle histology and physiology. Given that missense dystrophin is likely being degraded by the proteasome but whole body proteasome inhibition was not possible, we screened for ubiquitin-conjugating enzymes involved in targeting dystrophin to the proteasome. A myoblast cell line expressing L54R mutant dystrophin was screened with an siRNA library targeting E1, E2 and E3 ligases which identified Amn1, FBXO33, Zfand5 and Trim75. Our study establishes new mouse models of dystrophinopathy and identifies candidate E3 ligases that may specifically regulate dystrophin protein turnover in vivo.

Endogenous endophthalmitis and other ocular manifestations of injection drug use.

PURPOSE OF REVIEW: The United States has experienced a dramatic rise in opioid and injection drug use over the past 2 decades. A public health emergency was declared in 2017 and subsequently, there have been several new reports on the rise of endogenous endophthalmitis specifically associated with injection drug use. The purpose of this review is to provide a current perspective of the ocular harms posed by injection drug use. RECENT FINDINGS: The opioid epidemic has prompted several new studies from New England, one of the US regions most heavily affected, that examine the trends and characteristics of injection drug use-associated endogenous endophthalmitis. Patients may delay seeking care and may be infected with a variety of rare and atypical microbes, and as a result clinical appearance may vary widely. Injection drug use also leads to embolic phenomena such as talc retinopathy and septic emboli from endocarditis. HIV is highly associated with injection drug use and although HAART has drastically reduced the morbidity and mortality of HIV-associated infections, a variety of ocular disease may accompany an immunocompromised patient. SUMMARY: Healthcare providers must remain vigilant in the recognition of injection drug use patients with vision loss and ocular inflammation to ensure prompt medical and/or surgical treatment.

PhPAd-DalPhos: Ligand-Enabled, Nickel-Catalyzed Cross-Coupling of (Hetero)aryl Electrophiles with Bulky Primary Alkylamines.

The base metal-catalyzed C-N cross-coupling of bulky α,α,α-trisubstituted primary alkylamines with (hetero)aryl electrophiles represents a challenging and under-developed class of transformations that is of significant potential utility, including in the synthesis of lipophilic active pharmaceutical ingredients. Herein, we report that a new, air-stable Ni(II) pre-catalyst incorporating the optimized ancillary ligand PhPAd-DalPhos enables such transformations of (hetero)aryl chloride, bromide, and tosylate electrophiles to be carried out for the first time with substrate scope rivalling that achieved using state-of-the-art Pd catalysts, including room temperature cross-couplings of (hetero)aryl chlorides that are unprecedented for any catalyst (Pd, Ni, or other).

Exploiting Ancillary Ligation To Enable Nickel-Catalyzed C-O Cross-Couplings of Aryl Electrophiles with Aliphatic Alcohols.

The use of (L)Ni( o-tolyl)Cl precatalysts (L = PAd-DalPhos or CyPAd-DalPhos) enables the C( sp2)-O cross-coupling of primary, secondary, or tertiary aliphatic alcohols with (hetero)aryl electrophiles, including unprecedented examples of such nickel-catalyzed transformations employing (hetero)aryl chlorides, sulfonates, and pivalates. In addition to offering a competitive alternative to palladium catalysis, this work establishes the feasibility of utilizing ancillary ligation as a complementary means of promoting challenging nickel-catalyzed C( sp2)-O cross-couplings, without recourse to precious-metal photoredox catalytic methods.

Independent variability of microtubule perturbations associated with dystrophinopathy.

Absence of the protein dystrophin causes Duchenne muscular dystrophy. Dystrophin directly binds to microtubules in vitro, and its absence in vivo correlates with disorganization of the subsarcolemmal microtubule lattice, increased detyrosination of α-tubulin, and altered redox signaling. We previously demonstrated that the dystrophin homologue utrophin neither binds microtubules in vitro nor rescues microtubule lattice organization when overexpressed in muscles of dystrophin-deficient mdx mice. Here, we fine-mapped the dystrophin domain necessary for microtubule binding to spectrin-like repeats 20­22. We show that transgenic mdx mice expressing a full-length dystrophin/utrophin chimera completely lacking microtubule binding activity are surprisingly rescued for all measured dystrophic phenotypes, including full restoration of microtubule lattice organization. Conversely, despite the presence of dystrophin at the sarcolemma, ß-sarcoglycan-deficient skeletal muscle presents with a disorganized and densified microtubule lattice. Finally, we show that the levels of α-tubulin detyrosination remain significantly elevated to that of mdx levels in transgenic mdx mice expressing nearly full-length dystrophin. Our results demonstrate that the microtubule-associated perturbations of mdx muscle are distinct, separable, and can vary independently from other parameters previously ascribed to dystrophin deficiency.

An anatomically comprehensive atlas of the adult human brain transcriptome.

Neuroanatomically precise, genome-wide maps of transcript distributions are critical resources to complement genomic sequence data and to correlate functional and genetic brain architecture. Here we describe the generation and analysis of a transcriptional atlas of the adult human brain, comprising extensive histological analysis and comprehensive microarray profiling of ∼900 neuroanatomically precise subdivisions in two individuals. Transcriptional regulation varies enormously by anatomical location, with different regions and their constituent cell types displaying robust molecular signatures that are highly conserved between individuals. Analysis of differential gene expression and gene co-expression relationships demonstrates that brain-wide variation strongly reflects the distributions of major cell classes such as neurons, oligodendrocytes, astrocytes and microglia. Local neighbourhood relationships between fine anatomical subdivisions are associated with discrete neuronal subtypes and genes involved with synaptic transmission. The neocortex displays a relatively homogeneous transcriptional pattern, but with distinct features associated selectively with primary sensorimotor cortices and with enriched frontal lobe expression. Notably, the spatial topography of the neocortex is strongly reflected in its molecular topography-the closer two cortical regions, the more similar their transcriptomes. This freely accessible online data resource forms a high-resolution transcriptional baseline for neurogenetic studies of normal and abnormal human brain function.

Comparative Genome Analysis and Global Phylogeny of the Toxin Variant Clostridium difficile PCR Ribotype 017 Reveals the Evolution of Two Independent Sublineages.

The diarrheal pathogen Clostridium difficile consists of at least six distinct evolutionary lineages. The RT017 lineage is anomalous, as strains only express toxin B, compared to strains from other lineages that produce toxins A and B and, occasionally, binary toxin. Historically, RT017 initially was reported in Asia but now has been reported worldwide. We used whole-genome sequencing and phylogenetic analysis to investigate the patterns of global spread and population structure of 277 RT017 isolates from animal and human origins from six continents, isolated between 1990 and 2013. We reveal two distinct evenly split sublineages (SL1 and SL2) of C. difficile RT017 that contain multiple independent clonal expansions. All 24 animal isolates were contained within SL1 along with human isolates, suggesting potential transmission between animals and humans. Genetic analyses revealed an overrepresentation of antibiotic resistance genes. Phylogeographic analyses show a North American origin for RT017, as has been found for the recently emerged epidemic RT027 lineage. Despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents.

The Quality and Utility of Surgical and Anesthetic Data at a Ugandan Regional Referral Hospital.

BACKGROUND: There are little primary data available on the delivery or quality of surgical treatment in rural sub-Saharan African hospitals. To initiate a quality improvement system, we characterized the existing data capture at a Ugandan Regional Referral Hospital. METHODS: We examined the surgical ward admission (January 2008-December/2011) and operating theater logbooks (January 2010-July 2011) at Mbarara Regional Referral Hospital. RESULTS: There were 6346 admissions recorded over three years. The mean patient age was 31.4 ± 22.3 years; 29.8 % (n = 1888) of admissions were children. Leading causes of admission were general surgical problems (n = 3050, 48.1 %), trauma (n = 2041, 32.2 %), oncology (n = 718, 11.3 %) and congenital condition (n = 193, 3.0 %). Laparotomy (n = 468, 35.3 %), incision and drainage (n = 188, 14.2 %) and hernia repair (n = 90, 6.8 %) were the most common surgical procedures. Of 1325 operative patients, 994 (75 %) had an ASA I-II score. Of patients undergoing 810 procedures booked as non-elective, 583 (72 %) had an ASA "E" rating. Records of 41.3 % (n-403/975) of patients age 5 years or older undergoing non-obstetric operations were missing from the ward logbook. Missing patients were younger (25 [13,40] versus 30 [18,46] years, p = 0.002) and had higher ASA scores (ASA III-V 29.0 % versus 18.9 %, p < 0.001) than patients recorded in the logbbook; there was no diffence in gender (male 62.8 % versus 67.0 %, p = 0.20). CONCLUSIONS: The hospital records system measures surgical care, but improved data capture is needed to determine outcomes with sufficient accuracy to guide and record expansion of surgical capacity.

Nickel-Catalyzed N-Arylation of Primary Amides and Lactams with Activated (Hetero)aryl Electrophiles.

The first nickel-catalyzed N-arylation of amides with (hetero)aryl (pseudo)halides is reported, enabled by use of the air-stable pre-catalyst (PAd-DalPhos)Ni(o-tolyl)Cl (C1). A range of structurally diverse primary amides and lactams were cross-coupled successfully with activated (hetero)aryl chloride, bromide, triflate, tosylate, mesylate, and sulfamate electrophiles.

Genomic Epidemiology of a Protracted Hospital Outbreak Caused by a Toxin A-Negative Clostridium difficile Sublineage PCR Ribotype 017 Strain in London, England.

Clostridium difficile remains the leading cause of nosocomial diarrhea worldwide, which is largely considered to be due to the production of two potent toxins: TcdA and TcdB. However, PCR ribotype (RT) 017, one of five clonal lineages of human virulent C. difficile, lacks TcdA expression but causes widespread disease. Whole-genome sequencing was applied to 35 isolates from hospitalized patients with C. difficile infection (CDI) and two environmental ward isolates in London, England. The phylogenetic analysis of single nucleotide polymorphisms (SNPs) revealed a clonal cluster of temporally variable isolates from a single hospital ward at University Hospital Lewisham (UHL) that were distinct from other London hospital isolates. De novo assembled genomes revealed a 49-kbp putative conjugative transposon exclusive to this hospital clonal cluster which would not be revealed by current typing methodologies. This study identified three sublineages of C. difficile RT017 that are circulating in London. Similar to the notorious RT027 lineage, which has caused global outbreaks of CDI since 2001, the lineage of toxin-defective RT017 strains appears to be continually evolving. By utilization of WGS technologies to identify SNPs and the evolution of clonal strains, the transmission of outbreaks caused by near-identical isolates can be retraced and identified.

Site selection for heart failure clinical trials in the USA.

There are more than 1 million primary hospitalizations for heart failure (HF) annually in the USA alone, and post-discharge outcomes remain persistently poor despite available therapies and quality improvement initiatives. Recent international randomized clinical trials in hospitalized HF have repeatedly failed to improve this post-discharge event rate. A potential reason for this persistent lack of clinical trial success that has not previously received significant attention relates to site selection and the generally low level of patient enrollment from the USA. Only ~5 % of US hospitals participate in clinical trials, and in four recent randomized trials of hospitalized HF, only one-third of patients were enrolled in North America. This poor participation among US centers has necessitated disproportionate enrollment from non-US sites. Regional variations in HF patient characteristics and clinical outcomes are well documented, and a lack of US patient representation in clinical trials limits the generalizability of results and presents obstacles for US regulatory agency approval. There are multiple impediments to successful US enrollment including a lack of incentive for investigators and institutions, the relative value unit-based compensation system, poor institutional framework for identification of appropriate patients, and increasing liability to conduct trials. In this manuscript, we specifically identify barriers to successful hospitalized HF clinical trial participation in the USA and suggest possible solutions.

Palladium-catalyzed mono-α-arylation of acetone at room temperature.

The first examples of acetone mono-α-arylation at room temperature are described, enabled by use of a [Pd(cinnamyl)Cl]2 /JosiPhos catalyst system. (Hetero)aryl chloride, bromide, and iodide electrophiles featuring or lacking ortho-substitution, and comprising a range of functionalities (e.g., alkoxy, cyano, fluoro, trifluoromethyl, or alkenyl) and heteroaryl motifs (e.g., pyrrole, pyridine, isoquinoline, quinoline, quinaldine, (benzo)thiophene, benzothiazole, or benzodioxole) were successfully accommodated. Proof-of-principle experiments confirm that other (hetero)aryl methyl ketones can also be employed in such room temperature mono-α-arylations. The established substrate scope is the most extensive reported to date for acetone mono-α-arylation under any conditions, and more generally represents the first room temperature ketone mono-α-arylations employing a structurally diverse set of (hetero)aryl chlorides.

Nickel-catalyzed monoarylation of ammonia.

Structurally diverse (hetero)aryl chloride, bromide, and tosylate electrophiles were employed in the Ni-catalyzed monoarylation of ammonia, including chemoselective transformations. The employed JosiPhos/[Ni(cod)2] catalyst system enables the use of commercially available stock solutions of ammonia, or the use of ammonia gas in these reactions, thereby demonstrating the versatility and potential scalability of the reported protocol. Proof-of-principle experiments established that air-stable [(JosiPhos)NiCl2] precatalysts can be employed successfully in such transformations.

Emergence of the P2 phenotype in Pseudomonas aeruginosa PAO1 strains involves various mutations in mexT or mexF.

We recently demonstrated that Pseudomonas aeruginosa PAO1 undergoes a pronounced phenotypic change when introduced into the intestines of rats during surgical injury. Recovered strains displayed a specific phenotype (termed the P2 phenotype) characterized by altered pyocyanin production, high collagenase activity, high swarming motility, low resistance to chloramphenicol, and increased killing of Caenorhabditis elegans compared to the inoculating strain (termed the P1 phenotype). The aims of this study were to characterize the differences between the P. aeruginosa P1 and P2 phenotypes in quorum sensing and competitiveness. We then determined the presence of the P2 phenotype among PAO1 strains from various laboratories. Results demonstrated that P2 cells display accelerated growth during early exponential phase and early activation of quorum-sensing systems and overcome the growth of P1 cells in a mixed population. Among eight PAO1 strains obtained from different laboratories, four exhibited the P2 phenotype. Of 27 mutants analyzed from the P. aeruginosa MPAO1 transposon library, 25 displayed P2 phenotypes. The P2 phenotype in both cases correlated with a lack of expression of mexE or mexF due to mutations in mexT and mexF genes. In summary, strains possessing the P2 phenotype are distributed among PAO1 strains commonly used across a variety of research laboratories. Genetically, they are characterized by various mutations in mexT or mexF.

Adherence to dabigatran therapy and longitudinal patient outcomes: insights from the veterans health administration.

BACKGROUND: Dabigatran is a novel oral anti-coagulant (NOAC) that reduces risk of stroke in patients with non-valvular atrial fibrillation (NVAF). It does not require routine monitoring with laboratory testing which may have an adverse impact on adherence. We aimed to describe adherence to dabigatran in the first year after initiation and assess the association between non-adherence to dabigatran and clinical outcomes in a large integrated healthcare system. METHODS: We studied a national cohort of 5,376 patients with NVAF, initiated on dabigatran between October-2010 and September-2012 at all Veterans Affairs hospitals. Adherence to dabigatran was calculated as proportion of days covered (PDC) and association between PDC and outcomes was assessed using standard regression techniques. RESULTS: Mean age of the study cohort was 71.3 ± 9.7 years; 98.3% were men and mean CHADS2 score was 2.4 ± 1.2 (mean CHA2DS2VASc score 3.2 ± 1.4). Median PDC was 94% (IQR 76%-100%; mean PDC 84% ± 22%) over a median follow-up of 244 days (IQR 140-351). A total of 1,494 (27.8%) patients had a PDC <80% and were classified as non-adherent. After multivariable adjustment, lower adherence was associated with increased risk for combined all-cause mortality and stroke (HR 1.13, 95% CI 1.07-1.19 per 10% decrease in PDC). Adherence to dabigatran was not associated with non-fatal bleeding or myocardial infarction. CONCLUSIONS: In the year after initiation, adherence to dabigatran for a majority of patients is very good. However, 28% of patients in our cohort had poor adherence. Furthermore, lower adherence to dabigatran was associated with increased adverse outcomes. Concerted efforts are needed to optimize adherence to NOACs.