Dynamic Changes in Lymphocyte Populations Establish Zebrafish as a Thymic Involution Model.

The thymus is the site of T lymphocyte development and T cell education to recognize foreign, but not self, Ags. B cells also reside and develop in the thymus, although their functions are less clear. During "thymic involution," a process of lymphoid atrophy and adipose replacement linked to sexual maturation, thymocytes decline. However, thymic B cells decrease far less than T cells, such that B cells comprise ∼1% of human neonatal thymocytes but up to ∼10% in adults. All jawed vertebrates possess a thymus, and we and others have shown zebrafish (Danio rerio) also have thymic B cells. In this article, we investigated the precise identities of zebrafish thymic T and B cells and how they change with involution. We assessed the timing and specific details of zebrafish thymic involution using multiple lymphocyte-specific, fluorophore-labeled transgenic lines, quantifying the changes in thymic T- and B-lymphocytes pre- versus postinvolution. Our results prove that, as in humans, zebrafish thymic B cells increase relative to T cells postinvolution. We also performed RNA sequencing on D. rerio thymic and marrow lymphocytes of four novel double-transgenic lines, identifying distinct populations of immature T and B cells. Collectively, this is, to our knowledge, the first comprehensive analysis of zebrafish thymic involution, demonstrating its similarity to human involution and establishing the highly genetically manipulatable zebrafish model as a template for involution studies.

Clustering of multi-tissue transcriptomes in gilts with normal cyclicity or delayed puberty reveals genes related to pubertal development†.

In gilts, puberty is marked by standing estrus in the presence of a boar. Delayed puberty (DP; failure to display pubertal estrus) is a major reason for gilt removal. To investigate the physiological determinants underlying DP in gilts, transcriptomic data from tissues relevant to estrus and puberty, such as mediobasal hypothalamus, anterior pituitary gland, ovarian cortex, olfactory bulb, amygdala, and hippocampus, were obtained from age-matched DP (n = 8) and cyclic control gilts at follicular phase (n = 8) and luteal phase (n = 8) of the estrous cycle. A gene expression module analysis via three-way gene × individual × tissue clustering using tensor decomposition identified pituitary and ovary gene modules contributing to regulation of pubertal development. Analysis of gene expression in the hypothalamic-pituitary-ovary axis identified reduced expression of hypothalamic genes critical for stimulating gonadotropin secretion (KISS1 and TAC3) and reduced expression of LHB in the anterior pituitary of DP gilts compared with their cyclic counterparts. Consequently, luteinizing hormone-induced genes in the ovary important for folliculogenesis (OXTR, RUNX2, and PTX3) were less expressed in DP gilts. Other intrafollicular genes (AHR, PTGS2, PTGFR, and IGFBP7) and genes in the steroidogenesis pathways (STAR and CYP11A1) necessary to complete the ovulatory cascade were also less expressed in DP gilts. This is the first clustering of multi-tissue expression data from DP and cyclic gilts to identify genes differentially expressed in gilts of similar ages but at different levels of sexual development. A critical lack of gonadotropin support and reduced ovarian responsiveness underlie DP in gilts.

Does pes anserinus tenotomy affect surgical site infection rate in operative tibial plateau fractures?

PURPOSE: To assess if pes anserinus tenotomy (PAT) during definitive open reduction and internal fixation (ORIF) of tibial plateau fractures is associated with a decreased risk of surgical site infection (SSI) and other postoperative complications. METHODS: A retrospective review of all adults who underwent ORIF for tibial plateau fractures from April 2005 to February 2022 at single level 1 trauma center was performed. Patients who had a medial approach to the plateau with minimum three-month follow-up were required for inclusion. All patients with fasciotomy for compartment syndrome or with traumatically avulsed or damaged pes anserinus prior to ORIF were excluded. Two groups were created: those who received a pes anserinus tenotomy with repair (PAT group) and those whose pes anserinus were spared and left intact (control group). Patient demographics, injury and operative characteristics, and surgical outcomes were compared. The primary outcomes were rates of deep and superficial SSI. RESULTS: The PAT group had significantly lower rates of deep SSI (9.2% vs. 19.7%, P = 0.009), superficial SSI (14.2% vs. 26.5%), P = 0.007), and any SSI (15.8% vs. 28.9%, P = 0.005). Multiple logistic regression showed that heart failure (aOR = 7.215, 95% CI 2.291-22.719, P < 0.001), and presence of open fracture (aOR = 4.046, 95% CI 2.074-7.895, P < 0.001) were independently associated with increased odds of deep SSI, while PAT was associated with a decreased odds of deep SSI (aOR = 0.481, 95% CI 0.231-0.992, P = 0.048). PAT had significantly lower rates of unplanned return to the operating room (20.8% vs. 33.7%, P = 0.010) and implant removal (10.0% vs. 18.0%, P = 0.042). CONCLUSION: While these data do not allow for discussion of functional recovery or strength, pes anserinus tenotomy was independently associated with significantly lower rates of infection, unplanned operation, and implant removal. LEVEL OF EVIDENCE: Level III.

Analysis of CheW-like domains provides insights into organization of prokaryotic chemotaxis systems.

The ability to control locomotion in a dynamic environment provides a competitive advantage for microorganisms, thus driving the evolution of sophisticated regulatory systems. In total, 19 known categories of chemotaxis systems control motility mediated by flagella or Type IV pili, plus other cellular functions. A key feature that distinguishes chemotaxis systems from generic two-component regulatory systems is separation of receptor and kinase functions into distinct proteins, linked by CheW scaffold proteins. This arrangement allows for formation of varied arrays with remarkable signaling properties. We recently analyzed sequences of CheW-like domains found in CheA kinases and CheW and CheV scaffold proteins. In total, 16 Architectures of CheA, CheW, and CheV proteins contain ~94% of all CheW-like domains and form six Classes with likely functional specializations. We surveyed chemotaxis system categories and proteins containing CheW-like domains in ~1900 prokaryotic species, the most comprehensive analysis to date, revealing new insights. Co-occurrence analyses suggested that many chemotaxis systems occur in non-random combinations within species, implying synergy or antagonism. Furthermore, many Architectures of proteins containing CheW-like domains occurred predominantly with specific categories of chemotaxis systems, suggesting specialized functional interactions. We propose Class 1 (~80%) and Class 6 (~20%) CheW proteins exhibit preferences for distinct chemoreceptor structures. Furthermore, rare (~1%) Class 2 CheW proteins frequently co-occurred with methyl-accepting coiled coil proteins, which contain both receptor and kinase functions and so do not require connection via a CheW scaffold but may benefit from arrays. Last, rare multidomain CheW proteins may interact with different receptors than single-domain CheW proteins.

Functional genomics of reproduction in pigs: Are we there yet?

Reproductive failure is the main reason for culling females in swine herds and is both a financial and sustainability issue. Because reproductive traits are complex and lowly to moderately heritable, genomic selection within populations can achieve substantial genetic gain in reproductive efficiency. A better understanding of the physiological components affecting the expression of these traits will facilitate greater understanding of the genes affecting reproductive traits and is necessary to improve and optimize management strategies to maximize reproductive success of gilts and sows. Large-scale genotyping with single-nucleotide polymorphism (SNP) arrays are used for genome-wide association studies (GWAS) and have facilitated identification of positional candidate genes. Transcriptomic data can be used to weight SNP for GWAS and could lead to previously unidentified candidate genes. Resequencing and fine mapping of candidate genes are necessary to identify putative functional variants and some of these have been incorporated into new genotyping arrays. Sequence imputation and genotype by sequence are newer strategies that could reveal novel functional mutations. In this study, these approaches are discussed. Advantages and limitations are highlighted where additional research is needed.

Role of gonadotropin-releasing hormone-II and its receptor in swine reproduction.

The pig represents the only livestock mammal capable of producing a functional protein for the second mammalian form of gonadotropin-releasing hormone (GnRH-II) and its receptor (GnRHR-II). To examine the role of GnRH-II and its receptor in pig reproduction, we produced a unique swine line with ubiquitous knockdown of endogenous GnRHR-II levels (GnRHR-II knockdown [KD]), which is largely the focus of this review. In mature GnRHR-II KD males, circulating testosterone concentrations were 82% lower than littermate control boars, despite similar luteinizing hormone (LH) levels. In addition, nine other gonadal steroids were reduced in the serum of GnRHR-II KD boars, whereas adrenal steroids (except 11-deoxycortisol) did not differ between lines. Interestingly, testes from GnRHR-II KD males had fewer, hypertrophic Leydig cells and fewer, enlarged seminiferous tubules than control testes. As expected, downstream reproductive traits such as androgen-dependent organ weights and semen characteristics were also significantly reduced in GnRHR-II KD versus control boars. Next, we explored the importance of this novel ligand/receptor complex in female reproduction. Transgenic gilts had fewer, but heavier, corpora lutea with smaller luteal cells than littermate control females. Although the number of antral follicles were similar between lines, the diameter of antral follicles tended to be larger in GnRHR-II KD females. In regard to steroidogenesis, circulating concentrations of progesterone and 17ß-estradiol were lower in transgenic compared to control gilts, even though serum levels of follicle-stimulating hormone and LH were similar. Thus, GnRH-II and GnRHR-II represent a potential avenue to enhance fertility and promote the profitability of pork producers.

How do surgeons decide when to treat proximal humerus fractures with operative versus nonoperative management?

PURPOSE: The objective of this study was to determine the underlying factors that drive the decision for surgeons to pursue operative versus nonoperative management for proximal humerus fractures (PHF) and if fellowship training had an impact on these decisions. METHODS: An electronic survey was distributed to members of the Orthopaedic Trauma Association and the American Shoulder and Elbow Surgeons Society to assess differences in patient selection for operative versus nonoperative management of PHF. Descriptive statistics were reported for all respondents. RESULTS: A total of 250 fellowship trained Orthopaedic Surgeons responded to the online survey. A greater proportion of trauma surgeons preferred nonoperative management for displaced PHF fractures in patients over the age of 70. Operative management was preferred for older patients with fracture dislocations (98%), limited humeral head bone subchondral bone (78%), and intraarticular head split (79%). Similar proportions of trauma surgeons and shoulder surgeons cited that acquiring a CT was crucial to distinguish between operative and nonoperative management. CONCLUSION: We found that surgeons base their decisions on when to operate primarily on patient's comorbidities, age, and the amount of fracture displacement when treating younger patients. Further, we found a greater proportion of trauma surgeons elected to proceed with nonoperative management in patients older than the age of 70 years old as compared to shoulder surgeons.

Generalizable strategy to analyze domains in the context of parent protein architecture: A CheW case study.

Domains are the three-dimensional building blocks of proteins. An individual domain can occur in a variety of domain architectures that perform unique functions and are subject to different evolutionary selective pressures. We describe an approach to evaluate the variability in amino acid sequences of a single domain across architectural contexts. The ability to distinguish different evolutionary outcomes of one protein domain can help determine whether existing knowledge about a specific domain will apply to an uncharacterized protein, lead to insights and hypotheses about function, and guide experimental priorities. We developed and tested our approach on CheW-like domains (PF01584), which mediate protein/protein interactions and are difficult to compare experimentally. CheW-like domains occur in CheW scaffolding proteins, CheA kinases, and CheV proteins that regulate bacterial chemotaxis. We analyzed 16 domain architectures that included 94% of all CheW-like domains found in nature. We identified six Classes of CheW-like domains with presumed functional differences. CheV and most CheW proteins contained Class 1 domains, whereas some CheW proteins contained Class 6 (~20%) or Class 2 (~1%) domains instead. Most CheA proteins contained Class 3 domains. CheA proteins with multiple Hpt domains contained Class 4 domains. CheA proteins with two CheW-like domains contained one Class 3 and one Class 5. We also created SimpLogo, an innovative method for visualizing amino acid composition across large sets of multiple sequence alignments of arbitrary length. SimpLogo offers substantial advantages over standard sequence logos for comparison and analysis of related protein sequences. The R package for SimpLogo is freely available.

General strategies for using amino acid sequence data to guide biochemical investigation of protein function.

The rapid increase of '-omics' data warrants the reconsideration of experimental strategies to investigate general protein function. Studying individual members of a protein family is likely insufficient to provide a complete mechanistic understanding of family functions, especially for diverse families with thousands of known members. Strategies that exploit large amounts of available amino acid sequence data can inspire and guide biochemical experiments, generating broadly applicable insights into a given family. Here we review several methods that utilize abundant sequence data to focus experimental efforts and identify features truly representative of a protein family or domain. First, coevolutionary relationships between residues within primary sequences can be successfully exploited to identify structurally and/or functionally important positions for experimental investigation. Second, functionally important variable residue positions typically occupy a limited sequence space, a property useful for guiding biochemical characterization of the effects of the most physiologically and evolutionarily relevant amino acids. Third, amino acid sequence variation within domains shared between different protein families can be used to sort a particular domain into multiple subtypes, inspiring further experimental designs. Although generally applicable to any kind of protein domain because they depend solely on amino acid sequences, the second and third approaches are reviewed in detail because they appear to have been used infrequently and offer immediate opportunities for new advances. Finally, we speculate that future technologies capable of analyzing and manipulating conserved and variable aspects of the three-dimensional structures of a protein family could lead to broad insights not attainable by current methods.

The Prevalence of Metabolic and Endocrine Disturbances on Fracture Nonunion.

OBJECTIVE: This study aims to determine the prevalence of metabolic disturbance in all fracture nonunion cases and identify the most common endocrine abnormalities seen using a simple screening algorithm. METHODS: A retrospective review study was performed evaluating patients who underwent operative intervention for nonunion from January 2010 to December 2018 at 2 level-1 trauma centers. Preoperative laboratory values were recorded for a 9-test "nonunion panel." A metabolic or endocrine abnormality, specifically an abnormality in the thyroid or parathyroid axis, was evaluated. RESULTS: 42% of patients had an undiagnosed metabolic laboratory abnormality. When multiple tests were used, the rate of metabolic dysfunction was between 60% and 75%, depending on the definition of vitamin D insufficiency vs deficiency used. CONCLUSION: Results indicate a relatively high prevalence of metabolic disturbance in patients with nonunion and suggest metabolic screening for all nonunion patients not only those without a mechanical or infectious cause. LEVEL OF EVIDENCE: IV, retrospective case series.

Isothermal Microcalorimetry Improves the Time to Diagnosis of Fracture-related Infection Compared With Conventional Tissue Cultures.

BACKGROUND: A consensus definition recently was formulated for fracture-related infection, which centered on confirmatory criteria including conventional cultures that take time to finalize and have a 10% to 20% false-negative rate. During this time, patients are often on broad-spectrum antibiotics and may remain hospitalized until cultures are finalized to adjust antibiotic regimens. QUESTIONS/PURPOSES: (1) What is the diagnostic accuracy of isothermal microcalorimetry, and how does its accuracy compare with that of conventional cultures? (2) Does isothermal microcalorimetry decrease time to detection (or diagnosis) of fracture-related infection compared with conventional cultures? (3) Does isothermal microcalorimetry have a diagnostic accuracy or time advantage over conventional cultures in patients on chronic suppressive antibiotics? METHODS: Between July 2020 and August 2021, we treated 310 patients with concerns for infection after prior fracture repair surgery. Of those, we considered all patients older than 18 years of age with fixation hardware in place at the time of presentation as potentially eligible. All included patients returned to the operating room with cultures obtained and assessed by both isothermal microcalorimetry and conventional cultures, and all were diagnosed using the consensus criteria for fracture-related infection. Based on that, 81% (250 of 310) of patients were eligible; a further 51% (157 of 310) were excluded because of the following reasons: the capacity of the isothermal microcalorimetry instrument limited the throughput on that day (34% [106 of 310]), they had only swab cultures obtained in surgery (15% [46 of 310]), or they had less than 3 months follow-up after surgery for infectious concerns (2% [5 of 310]), leaving 30% (93 of 310) of the originally identified patients for analysis. We obtained two to five cultures from each patient during surgery, which were sent to our clinical microbiology laboratory for standard processing (conventional cultures). This included homogenization of each tissue sample individually and culturing for aerobic, anaerobic, acid-fast bacilli, and fungal culturing. The remaining homogenate from each sample was then taken to our orthopaedic research laboratory, resuspended in growth media, and analyzed by isothermal microcalorimetry for a minimum of 24 hours. Aerobic and anaerobic cultures were maintained for 5 days and 14 days, respectively. Overall, there were 93 patients (59 males), with a mean age of 43 ± 14 years and a mean BMI of 28 ± 8 kg/m 2 , and 305 tissue samples (mean 3 ± 1 samples per patient) were obtained and assessed by conventional culturing and isothermal microcalorimetry. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of isothermal microcalorimetry to diagnose fracture-related infection were compared with conventional cultures using a McNemar test based on the consensus definition of fracture-related infection. This consensus criteria is comprised of two levels of certainty for the diagnostic variables. The first is confirmatory criteria, where infection is considered definitely present and includes the presence of fistula/sinus tract/wound breakdown, purulent drainage or the presence of pus, presence of microorganisms in deep tissue specimens on histopathologic examination, presence of more than five neutrophils/high-powered field by histopathologic examination (only for chronic/late onset cases), and identification of phenotypically indistinguishable pathogens by conventional culture from at least two separate deep tissue/implant specimens. The second is suggestive criteria in which further investigation is required to achieve confirmatory status. Fracture-related infection was diagnosed for this study to minimize subjectivity based on the presence of at least one of the confirmatory criteria as documented by the managing surgeon. When suggestive criteria were present without confirmatory criteria, patients were considered negative for fracture-related infection and followed further in clinic after surgical exploration (n = 25 patients). All 25 patients deemed not to have fracture-related infection were considered infection-free at latest follow-up (range 3 to 12 months). The time to detection or diagnosis was recorded and compared via the Mann-Whitney U test. RESULTS: Using the consensus criteria for fracture-related infection, there were no differences with the numbers available between isothermal microcalorimetry and conventional cultures in terms of sensitivity (87% [95% confidence interval 77% to 94%] versus 81% [95% CI 69% to 89%]), specificity (100% [95% CI 87% to 100%] versus 96% [95% CI 79% to 99%]), PPV (100% [95% CI 90% to 100%] versus 98% [95% CI 89% to 99%]), NPV (74% [95% CI 60% to 84%] versus 65% [95% CI 52% to 75%]), or accuracy (90% [95% CI 83% to 96%] versus 85% [95% CI 76% to 91%]; p = 0.13). The concordance by sample between conventional cultures and isothermal microcalorimetry was 85%. Isothermal microcalorimetry had a shorter median (range) time to detection or diagnosis compared with conventional cultures (2 hours [0.5 to 66] versus 51 hours [18 to 147], difference of medians 49 hours; p < 0.001). Additionally, 32 patients used antibiotics for a median (range) duration of 28 days (7 to 1095) before presentation. In these unique patients, there were no differences with the numbers available between isothermal microcalorimetry and conventional cultures in terms of sensitivity (89% [95% CI 71% to 98%] versus 74% [95% CI 53% to 88%]), specificity (100% [95% CI 48% to 100%] versus 83% [95% CI 36% to 99%]), PPV (100% [95% CI 85% to 100%] versus 95% [95% CI 77% to 99%]), NPV (63% [95% CI 37% to 83%] versus 42% [95% CI 26% to 60%]), or accuracy (91% [95% CI 75% to 98%] versus 78% [95% CI 57% to 89%]; p = 0.17). Isothermal microcalorimetry again had a shorter median (range) time to detection or diagnosis compared with conventional cultures (1.5 hours [0.5 to 48] versus 51.5 hours [18 to 125], difference of medians 50 hours; p < 0.001). CONCLUSION: Given that isothermal microcalorimetry considerably decreases the time to the diagnosis of a fracture-related infection without compromising the accuracy of the diagnosis, managing teams may eventually use isothermal microcalorimetry-pending developmental improvements and regulatory approval-to rapidly detect infection and begin antibiotic management while awaiting speciation and susceptibility testing to modify the antibiotic regimen. Given the unique thermograms generated, further studies are already underway focusing on speciation based on heat curves alone. Additionally, increased study sizes are necessary for both overall fracture-related infection diagnostic accuracy and test performance on patients using long-term antibiotics given the promising results with regard to time to detection for this groups as well. LEVEL OF EVIDENCE: Level II, diagnostic study.

Understanding postoperative rehabilitation preferences in operatively managed proximal humerus fractures: do trauma and shoulder surgeons differ?

BACKGROUND: Proximal humerus fractures (PHFs) are common, and their incidence is increasing as the population ages. Despite this, postoperative rehabilitation remains unstandardized and little is known about surgeon preferences. The aim of this study was to assess differences in postoperative rehabilitation preferences and patient education between orthopedic trauma and shoulder surgeons. METHODS: An electronic survey was distributed to members of the Orthopaedic Trauma Association and the American Shoulder and Elbow Surgeons to assess differences in postoperative rehabilitation preferences and patient counseling. Descriptive statistics were reported for all respondents, trauma surgeons, and shoulder surgeons. Chi-square and unpaired 2-sample t tests were used to compare responses. Multinomial regression was used to further elucidate the influence of fellowship training independent of confounding characteristics. RESULTS: A total of 293 surgeons completed the survey, including 172 shoulder and 78 trauma surgeons. A greater proportion of trauma surgeons preferred an immediate weightbearing status after arthroplasty compared to shoulder surgeons (45% vs. 19%, P = .003), but not after open reduction and internal fixation (ORIF) (62% vs. 75%, P = .412). A greater proportion of shoulder surgeons preferred home exercise therapy taught by the physician or using a handout following reverse shoulder arthroplasty (RSA) (21% vs. 2%, P = .009). A greater proportion of trauma surgeons began passive range of motion (ROM) <2 weeks after 2-part fractures (70% vs. 41%, P < .001). Conversely, a greater proportion of shoulder surgeons began passive ROM between 2 and 6 weeks for 2-part (57% vs. 24%, P < .001) and 4-part fractures (65% vs. 43%, P = .020). On multinomial regression analysis, fellowship training in shoulder surgery was associated with preference for a nonweightbearing duration of >12 weeks vs. 6-12 weeks after ORIF. Similarly, fellowship training in shoulder surgery was associated with increased odds of preferring a nonweightbearing duration of <6 weeks vs. no restrictions and >12 weeks vs. 6-12 weeks after arthroplasty. Training in shoulder surgery was associated with greater odds of preferring a nonweightbearing duration prior to beginning passive ROM of 2-6 weeks vs. <2 weeks or >6 weeks for 2-part fractures, but not 4-part fractures. CONCLUSION: Trauma surgeons have a more aggressive approach to rehabilitation following operative PHF repair compared to shoulder surgeons regarding time to weightbearing status and passive ROM. Given the increasing incidence of PHFs and substantial variations in reported treatment outcomes, differences in rehabilitation after PHF treatment should be further evaluated to determine the role it may play in the outcomes of treatment studies.

Factors influencing surgical management of proximal humerus fractures: do shoulder and trauma surgeons differ?

BACKGROUND: Proximal humerus fractures (PHFs) are managed with open reduction and internal fixation (ORIF), hemiarthroplasty (HA), reverse shoulder arthroplasty (RSA), or nonoperatively. Given the mixed results in the literature, the optimal treatment is unclear to surgeons. The purpose of this study was to survey orthopedic shoulder and trauma surgeons to identify the patient- and fracture-related characteristics that influence surgical decision-making. METHODS: We distributed a 23-question closed-response email survey to members of the American Shoulder and Elbow Surgeons and Orthopaedic Trauma Association. Questions posed to respondents included demographics, surgical planning, indications for ORIF and arthroplasty, and the use of surgical augmentation with ORIF. Numerical and multiple-choice responses were compared between shoulder and trauma surgeons using unpaired t-tests and χ2 tests, respectively. RESULTS: Respondents included 172 shoulder and 78 trauma surgeons. When surgery is indicated, most shoulder and trauma surgeons treat 2-part (69%) and 3-part (53%) PHFs with ORIF. Indications for managing PHFs with arthroplasty instead of ORIF include an intra-articular fracture (82%), bone quality (76%), age (72%), and previous rotator cuff dysfunction (70%). In patients older than 50 years, 90% of respondents cited a head-split fracture as an indication for arthroplasty. Both shoulder and trauma surgeons preferred RSA for treating PHFs presenting with a head-split fracture in an elderly patient (94%), pre-existing rotator cuff tear (84%), and pre-existing glenohumeral arthritis with an intact cuff (75%). Similarly, both groups preferred ORIF for PHFs in young patients with a fracture dislocation (94%). In contrast, although most trauma surgeons preferred to manage PHFs in low functioning patients with a significantly displaced fracture or nonreconstructable injury nonoperatively (84% and 86%, respectively), shoulder surgeons preferred either RSA (44% and 46%, respectively) or nonoperative treatment (54% and 49%, respectively) (P < .001). Similarly, although trauma surgeons preferred to manage PHFs in young patients with a head-split fracture or limited humeral head subchondral bone with ORIF (98% and 87%, respectively), shoulder surgeons preferred either ORIF (54% and 62%, respectively) or HA (43% and 34%, respectively) (P < .001). CONCLUSIONS: ORIF and HA are preferred for treating simple PHFs in young patients with good bone quality or fracture dislocations, whereas RSA and nonoperative management are preferred for complex fractures in elderly patients with poor bone quality, rotator cuff dysfunction, or osteoarthritis. The preferred management differed between shoulder and trauma surgeons for half of the common PHF presentations, highlighting the need for future research.

Role of Position K+4 in the Phosphorylation and Dephosphorylation Reaction Kinetics of the CheY Response Regulator.

Two-component signaling is a primary method by which microorganisms interact with their environments. A kinase detects stimuli and modulates autophosphorylation activity. The signal propagates by phosphotransfer from the kinase to a response regulator, eliciting a response. Response regulators operate over a range of time scales, corresponding to their related biological processes. Response regulator active site chemistry is highly conserved, but certain variable residues can influence phosphorylation kinetics. An Ala-to-Pro substitution (K+4, residue 113) in the Escherichia coli response regulator CheY triggers a constitutively active phenotype; however, the A113P substitution is too far from the active site to directly affect phosphochemistry. To better understand the activating mechanism(s) of the substitution, we analyzed receiver domain sequences to characterize the evolutionary role of the K+4 position. Although most featured Pro, Leu, Ile, and Val residues, chemotaxis-related proteins exhibited atypical Ala, Gly, Asp, and Glu residues at K+4. Structural and in silico analyses revealed that CheY A113P adopted a partially active configuration. Biochemical data showed that A113P shifted CheY toward a more activated state, enhancing autophosphorylation. By characterizing CheY variants, we determined that this functionality was transmitted through a hydrophobic network bounded by the ß5α5 loop and the α1 helix of CheY. This region also interacts with the phosphodonor CheAP1, suggesting that binding generates an activating perturbation similar to the A113P substitution. Atypical residues like Ala at the K+4 position likely serve two purposes. First, restricting autophosphorylation may minimize background noise generated by intracellular phosphodonors such as acetyl phosphate. Second, optimizing interactions with upstream partners may help prime the receiver domain for phosphorylation.

Evidence that pubertal status impacts kisspeptin/neurokinin B/dynorphin neurons in the gilt†.

Puberty onset is a complex physiological process, which enables the capacity for reproduction through increased gonadotropin-releasing hormone and subsequently luteinizing hormone secretion. While cells that coexpress kisspeptin, neurokinin B (NKB), and dynorphin in the hypothalamic arcuate nucleus are believed to govern the timing of puberty, the degree to which kisspeptin/NKB/dynorphin (KNDy) neurons exist and are regulated by pubertal status remains to be determined in the gilt. Hypothalamic tissue from prepubertal and postpubertal, early follicular phase gilts was used to determine the expression of kisspeptin, NKB, and dynorphin within the arcuate nucleus. Fluorescent in situ hybridization revealed that the majority (>74%) of arcuate nucleus neurons that express mRNA for kisspeptin coexpressed mRNA for NKB and dynorphin. There were fewer arcuate nucleus cells that expressed mRNA for dynorphin in postpubertal gilts compared to prepubertal gilts (P < 0.05), but the number of arcuate nucleus cells expressing mRNA for kisspeptin or NKB was not different between groups. Within KNDy neurons, mRNA abundance for kisspeptin, NKB, and dynorphin of postpubertal gilts was the same as, less than, and greater than, respectively, prepubertal gilts. Immunostaining for kisspeptin did not differ between prepubertal and postpubertal gilts, but there were fewer NKB immunoreactive fibers in postpubertal gilts compared to prepubertal gilts (P < 0.05). Together, these data reveal novel information about KNDy neurons in gilts and support the idea that NKB and dynorphin play a role in puberty onset in the female pig.

Localization of kisspeptin, NKB, and NK3R in the hypothalamus of gilts treated with the progestin altrenogest.

Mechanisms in the brain controlling secretion of gonadotropin hormones in pigs, particularly luteinizing hormone (LH), are poorly understood. Kisspeptin is a potent LH stimulant that is essential for fertility in many species, including pigs. Neurokinin B (NKB) acting through neurokinin 3 receptor (NK3R) is involved in kisspeptin-stimulated LH release, but organization of NKB and NK3R within the porcine hypothalamus is unknown. Hypothalamic tissue from ovariectomized (OVX) gilts was used to determine the distribution of immunoreactive kisspeptin, NKB, and NK3R cells in the arcuate nucleus (ARC). Almost all kisspeptin neurons coexpressed NKB in the porcine ARC. Immunostaining for NK3R was distributed throughout the preoptic area (POA) and in several hypothalamic areas including the periventricular and retrochiasmatic areas but was not detected within the ARC. There was no colocalization of NK3R with gonadotropin-releasing hormone (GnRH), but NK3R-positive fibers in the POA were in close apposition to GnRH neurons. Treating OVX gilts with the progestin altrenogest decreased LH pulse frequency and reduced mean circulating concentrations of LH compared with OVX control gilts (P < 0.01), but the number of kisspeptin and NKB cells in the ARC did not differ between treatments. The neuroanatomical arrangement of kisspeptin, NKB, and NK3R within the porcine hypothalamus confirms they are positioned to stimulate GnRH and LH secretion in gilts, though differences with other species exist. Altrenogest suppression of LH secretion in the OVX gilt does not appear to involve decreased peptide expression of kisspeptin or NKB.

Potential functional variants in AHR signaling pathways are associated with age at puberty in swine.

Puberty in female pigs is defined as age at first estrus and gilts that have an earlier age at puberty are more likely to have greater lifetime productivity. Because age at puberty is predictive for sow longevity and lifetime productivity, but not routinely measured in commercial herds, it would be beneficial to use genomic or marker-assisted selection to improve these traits. A GWAS at the US Meat Animal Research Center (USMARC) identified several loci associated with age at puberty in pigs. Candidate genes in these regions were scanned for potential functional variants using sequence information from the USMARC swine population founder animals and public databases. In total, 135 variants (SNP and insertion/deletions) in 39 genes were genotyped in 1284 phenotyped animals from a validation population sired by Landrace and Yorkshire industry semen using the Agena MassArray system. Twelve variants in eight genes were associated with age at puberty (P < 0.005) with estimated additive SNP effects ranging from 1.6 to 5.3 days. Nine of these variants were non-synonymous coding changes in AHR, CYP1A2, OR2M4, SDCCAG8, TBC1D1 and ZNF608, two variants were deletions of one and four codons in aryl hydrocarbon receptor, AHR, and the most significant SNP was near an acceptor splice site in the acetyl-CoA carboxylase alpha, ACACA. Several of the loci identified have a physiological and a genetic role in sexual maturation in humans and other animals and are involved in AHR-mediated pathways. Further functional validation of these variants could identify causative mutations that influence age at puberty in gilts and possibly sow lifetime productivity.

Modulation of Response Regulator CheY Reaction Kinetics by Two Variable Residues That Affect Conformation.

Microorganisms and plants utilize two-component systems to regulate adaptive responses to changing environmental conditions. Sensor kinases detect stimuli and alter their autophosphorylation activity accordingly. Signal propagation occurs via the transfer of phosphoryl groups from upstream kinases to downstream response regulator proteins. Removal of phosphoryl groups from the response regulator typically resets the system. Members of the same protein family may catalyze phosphorylation and dephosphorylation reactions with different efficiencies, exhibiting rate constants spanning many orders of magnitude to accommodate response time scales from milliseconds to days. We previously found that variable positions one or two residues to the C-terminal side of the conserved Asp phosphorylation site (D+2) or Thr/Ser (T+1/T+2) in response regulators alter reaction kinetics by direct interaction with phosphodonor or phosphoacceptor molecules. Here, we explore the kinetic effects of amino acid substitutions at the two positions immediately C-terminal to the conserved Lys (K+1/K+2) in the model Escherichia coli response regulator CheY. We measured CheY autophosphorylation and autodephosphorylation rate constants for 27 pairs of K+1/K+2 residues that represent 84% of naturally occurring response regulators. Effects on autodephosphorylation were modest, but autophosphorylation rate constants varied by 2 orders of magnitude, suggesting that the K+1/K+2 positions influence reaction kinetics by altering the conformational spectrum sampled by CheY at equilibrium. Additional evidence supporting this indirect mechanism includes the following: the effect on autophosphorylation rate constants is independent of the phosphodonor, the autophosphorylation rate constants and dissociation constants for the phosphoryl group analog BeF3- are inversely correlated, and the K+1/K+2 positions are distant from the phosphorylation site.IMPORTANCE We have identified five variable positions in response regulators that allow the rate constants of autophosphorylation and autodephosporylation reactions each to be altered over 3 orders of magnitude in CheY. The distributions of variable residue combinations across response regulator subfamilies suggest that distinct mechanisms associated with different variable positions allow reaction rates to be tuned independently during evolution for diverse biological purposes. This knowledge could be used in synthetic-biology applications to adjust the properties (e.g., background noise and response duration) of biosensors and may allow prediction of response regulator reaction kinetics from the primary amino acid sequence.

Risk Factors for Surgical Site Infection after Operative Fixation of Acetabular Fractures: Is Psoas Density a Useful Metric?

BACKGROUND: Surgical site infection (SSI) occurs in 5% to 7% of patients undergoing operative fixation of acetabular fractures, with reported risk factors including longer operative duration, increased blood loss, pelvic artery embolization, and concurrent abdominal organ injury, among others. Psoas muscle density is a measure of muscle quality and, as a metric for sarcopenia and/or nutrition status, has been associated with poor outcomes such as mortality across multiple surgical specialties. To date, psoas muscle density has not been explored for its associations with SSI in acetabular fracture patients.Questions/purposes (1) Is decreased psoas muscle density, as measured by Hounsfield units, associated with an increased SSI risk after acetabular fracture fixation? (2) What patient, operative, and hospital variables are associated with an increased SSI risk after acetabular fracture fixation? METHODS: Between 2012 to 2017, surgeons performed 684 acetabular ORIF procedures at one level I trauma center. Of those, 8% (56 of 684) did not meet inclusion criteria, leaving 92% (n = 628) for analysis in this study. The median (range) follow-up duration was 12 months (0.5-77). Patient demographics, comorbidities, operative and in-hospital variables, and psoas muscle density measured using preoperative pelvic CT images-acquired for all operative acetabular fracture patients-were analyzed. SSI was defined by positive culture results obtained during irrigation and débridement. Overall, 7% (42 of 628) of patients had an SSI. A multivariable regression analysis was performed to identify independent risk factors. Sensitivity analysis was performed with minimum follow-up set at 3 months and 6 months. RESULTS: There was no difference in the mean psoas muscle density between patients with SSI (50.9 ± 10.2 Hounsfield units [HUs]) and those who did not have an SSI within 1 year of open reduction and internal fixation (51.4 ± 8.1 HUs) (mean difference: 0.5 [95% confidence interval -2.34 to 3.32]; p = 0.69). Four variables were independently associated with an increased risk of SSI: increased operative time (1.04 [95% CI 1.00 to 1.07]; p = 0.03), estimated blood loss (1.08 [95% CI 1.02 to 1.14]; p = 0.01), female sex (2.34 [95% CI 1.19 to 4.60]; p = 0.01), and intravenous drug use (3.95 [95% CI 1.51 to 10.33]; p = 0.01). Sensitivity analysis showed no change in results using either 3-month or 6-month minimum follow-up. CONCLUSIONS: Risk factors for SSI after acetabular fixation include female sex, intravenous drug use, prolonged operative times, and increased intraoperative blood loss. Although the density of the psoas muscle may be a surrogate for nutritional markers, it was not associated with SSI in our patients with acetabular fractures. Thus, it is not useful for risk assessment of SSI in the general population with acetabular fracture; however, future studies with larger sample sizes of patients older than 60 years may re-investigate this marker for SSI risk. Contrary to the results of previous studies, pelvic artery embolization, intraoperative blood transfusion, and intensive care unit stay did not increase the risk of SSI; however, we may have been underpowered to detect differences in these secondary endpoints. Future large, multisite studies may be needed to address these conflicting results more definitively. LEVEL OF EVIDENCE: Level III, therapeutic study.

Role of the highly conserved G68 residue in the yeast phosphorelay protein Ypd1: implications for interactions between histidine phosphotransfer (HPt) and response regulator proteins.

BACKGROUND: Many bacteria and certain eukaryotes utilize multi-step His-to-Asp phosphorelays for adaptive responses to their extracellular environments. Histidine phosphotransfer (HPt) proteins function as key components of these pathways. HPt proteins are genetically diverse, but share a common tertiary fold with conserved residues near the active site. A surface-exposed glycine at the H + 4 position relative to the phosphorylatable histidine is found in a significant number of annotated HPt protein sequences. Previous reports demonstrated that substitutions at this position result in diminished phosphotransfer activity between HPt proteins and their cognate signaling partners. RESULTS: We report the analysis of partner binding interactions and phosphotransfer activity of the prototypical HPt protein Ypd1 from Saccharomyces cerevisiae using a set of H + 4 (G68) substituted proteins. Substitutions at this position with large, hydrophobic, or charged amino acids nearly abolished phospho-acceptance from the receiver domain of its upstream signaling partner, Sln1 (Sln1-R1). An in vitro binding assay indicated that G68 substitutions caused only modest decreases in affinity between Ypd1 and Sln1-R1, and these differences did not appear to be large enough to account for the observed decrease in phosphotransfer activity. The crystal structure of one of these H + 4 mutants, Ypd1-G68Q, which exhibited a diminished ability to participate in phosphotransfer, shows a similar overall structure to that of wild-type. Molecular modelling suggests that the highly conserved active site residues within the receiver domain of Sln1 must undergo rearrangement to accommodate larger H + 4 substitutions in Ypd1. CONCLUSIONS: Phosphotransfer reactions require precise arrangement of active site elements to align the donor-acceptor atoms and stabilize the transition state during the reaction. Any changes likely result in an inability to form a viable transition state during phosphotransfer. Our data suggest that the high degree of evolutionary conservation of residues with small side chains at the H + 4 position in HPt proteins is required for optimal activity and that the presence of larger residues at the H + 4 position would cause alterations in the positioning of active site residues in the partner response regulator.