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Background: Race and insurance status are independent predictors of healthcare outcomes following lower-extremity trauma. Level 1 trauma centers show better outcomes overall, but it is has not been extensively studied as to whether they specifically lower complication rates and shorten length of stay in those with Black race, with low socioeconomic status, and/or a lack of private health insurance. We performed a study with the objective of determining whether Level I trauma centers can improve the complication rate of those shown to be at high risk of experiencing adverse outcomes due to socioeconomic differences., Hypothesis: Level 1 trauma centers will be successful in mitigating the disparity in complication rates and length of stay associated with racial and socioeconomic differences among trauma patients experiencing an open tibia fracture., Patients and Methods: The National Trauma Databank was reviewed from 2008 to 2015, identifying 81,855 encounters with an open tibia fracture, and 33,047 at a Level I trauma center. Regression models determined effects of race and insurance status on outcomes by trauma center while controlling for confounders., Results: Black race [OR 1.36, 95% CI, 1.17-1.58; p < 0.05] and "other" race [OR 1.28, 95% CI, 1.07-1.52; p < 0.05] were associated with higher odds of injury-specific complications. Patients without private insurance and of non-White or Black race in comparison to White patients had a significantly longer length of stay [coefficient 1.66, 95% CI, 1.37-1.94; p < 0.001]. These differences persisted in patients treated at an American College of Surgeons (ACS) Level I trauma center., Discussion: Treatment at an ACS Level I trauma center did not reduce the independent effects of race and insurance status on outcomes after open tibia fracture, emphasizing the need to recognize this disparity and improve care for at-risk populations., (© 2023 Delhi Orthopedic Association. All rights reserved.)

The vulnerabilities of cancer cells constitute a promising strategy for drug therapeutics. This paper integrates proteomics, bioinformatics, and cell genotype together with in vitro cell proliferation assays to identify key biological processes and potential novel kinases that could account, at least in part, for the clinical differences observed in colorectal cancer (CRC) patients. This study started by focusing on CRC cell lines stratified by their microsatellite ( MS ) state and p53 genotype. It shows that cell-cycle checkpoint, metabolism of proteins and RNA, signal transduction, and WNT signaling processes are significantly more active in MSI-High p53-WT cell lines. Conversely, MSI-High cell lines with a mutant (Mut) p53 gene showed hyperactivation of cell signaling, DNA repair, and immune-system processes. Several kinases were linked to these phenotypes, from which RIOK1 was selected for additional exploration. We also included the KRAS genotype in our analysis. Our results showed that RIOK1's inhibition in CRC MSI-High cell lines was dependent on both the p53 and KRAS genotypes. Explicitly, Nintedanib showed relatively low cytotoxicity in MSI-High with both mutant p53 and KRAS (HCT-15) but no inhibition in p53 and KRAS WT (SW48) MSI-High cells. This trend was flipped in CRC MSI-High bearing opposite p53-KRAS genotypes (e.g., p53-Mut KRAS-WT or p53-WT KRAS-Mut), where observed cytotoxicity was more extensive compared to the p53-KRAS WT-WT or Mut-Mut cells, with HCT 116 (KRAS-Mut and p53-WT) being the most sensitive to RIOK1 inhibition. These results highlight the potential of our in silico computational approach to identify novel kinases in CRC sub-MSI-High populations as well as the importance of clinical genomics in determining drug potency.

Purpose: Regional anesthesia (RA) is commonly used in distal radius fracture surgery to reduce pain and opioid consumption. The purpose of this study was to evaluate the real-world impact of RA on inpatient and outpatient opioid consumption and demand in patients undergoing distal radius fracture surgery., Methods: All patients ages 18 and older undergoing distal radius fracture surgery between 7/2013 and 7/2018 at a single institution ( n  = 969) were identified. Inpatient opioid consumption and outpatient opioid prescribing in oxycodone 5-mg equivalents (OE's) up to 90-d post-operative were recorded for patients with and without RA. Adjusted models were used to evaluate the impact of RA on opioid outcomes., Results: Adjusted models demonstrated decreases in inpatient opioid consumption in patients with RA (10.7 estimated OE's without RA vs. 7.6 OE's with RA from 0 to 24 h post-op, 10.2 vs. 5.3 from 24 to 48 h post-op and 7.5 vs. 5.0 from 48 to 72 h post-op, p <.05). Estimated cumulative outpatient opioid demand was significantly higher in patients with RA (65.3 OE's without RA vs. 81.0 with RA from 1-month pre-op to 2-week post-discharge, 76.1 vs. 87.7 OE's to 6-weeks, and 80.8 vs. 93.5 OE's to 90-d, all p values for RA <.05) though rates of refill were significantly lower in patients with RA from 2-week to 6-week post-op compared to patients without RA., Conclusions: Patients undergoing RA in distal radius fracture surgery had decreased inpatient opioid consumption but increased outpatient demand after adjustment for patient and operative characteristics., Level of Evidence: Level III, retrospective, therapeutic cohort study.

Introduction: Regional anesthesia (RA) is used reduce pain in proximal humerus and humeral shaft fracture surgery. The study hypothesis was that RA would decrease opioid demand in patients undergoing fracture surgery., Materials and Methods: Opioid demand was recorded in all patients ages 18 and older undergoing proximal humerus or humeral shaft fracture surgery at a single, Level I trauma center from 7/2013 - 7/2018 (n = 380 patients). Inpatient opioid consumption from 0-24, 24-48, and 48-72 h and outpatient opioid demand from 1-month pre-operative to 90-days post-operative were converted to oxycodone 5-mg equivalents (OE's). Unadjusted and adjusted models were constructed to evaluate the impact of RA and other factors on opioid utilization., Results: Adjusted models demonstrated increases in inpatient opioid consumption in patients with RA (6.8 estimated OE's without RA vs 8.8 estimated OE's with RA from 0-24 h post-op; 10 vs 13.7 from 24-48 h post-op; and 8.7 vs 11.6 from 48-72 h post-op; all p  < 0.05). Estimated cumulative outpatient opioid demand was significantly higher in patients with RA at all timepoints., Discussion: In proximal humerus and humeral shaft fracture surgery, RA was associated with increased inpatient and outpatient opioid demand after adjusting for baseline patient and treatment characteristics., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2021.)

Purpose: The purpose of this study was to examine the differences in functional outcomes between direct and indirect surgical fixation methods of the posterior malleolus in the setting of trimalleolar fractures and identify any variables affecting patient outcomes., Methods: Primary outcomes were evaluated by PROMIS scores for short-term outcomes regarding total pain (TP) and total function (TF) comparing 40 patients with direct fixation with 77 with indirect fixation. Continuous variables were analyzed using t tests for parametric variables and the Mann-Whitney U test for nonparametric variables. Categorical variables were analyzed using a χ 2 test. Univariate and multivariate linear regression models were performed to analyze factors that affect outcomes of TP and TF., Results: There was no difference in TP or TF between groups ( P = 0.65 vs. P = 0.19). On univariate linear regression for TP, BMI, incidence of complication, tobacco use, and open injury showed significance in increasing pain levels with open injuries providing the greatest effect (coef = 11.8). On multivariate analysis, BMI, incidence of complication, open injury, and tourniquet time all significantly increased pain. For TF, univariate analysis showed age, BMI, incidence of complication, and diabetes to decrease function, and use of external fixator and tourniquet time increased function. In the multivariate model, increased BMI, open injuries, and increasing tourniquet time all decreased TF while use of an external fixator increased TF., Conclusion: This study showed no difference in TP and TF using the PROMIS outcome scores when comparing direct fixation versus indirect fixation under univariate and multivariate models., Level of Evidence: Therapeutic III., Competing Interests: The authors report no conflict of interest., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Association for the Study of Pain.)

Background: Titinopathies are inherited muscular diseases triggered by genetic mutations in the titin gene. Muscular dystrophy with myositis (mdm) is one such disease caused by a LINE repeat insertion, leading to exon skipping and an 83-amino acid residue deletion in the N2A-PEVK region of mouse titin. This region has been implicated in a number of titin-titin ligand interactions, hence are important for myocyte signaling and health. Mice with this mdm mutation develop a severe and progressive muscle degeneration. The range of phenotypic differences observed in mdm mice shows that the deletion of this region induces a cascade of transcriptional changes extending to numerous signaling pathways affected by the titin filament. Previous research has focused on correlating phenotypic differences with muscle function in mdm mice. These studies have provided understanding of the downstream physiological effects resulting from the mdm mutation but only provide insights on processes that can be physiologically observed and measured. We used differential gene expression (DGE) to compare the transcriptomes of extensor digitorum longus (EDL), psoas and soleus muscles from wild-type and mdm mice to develop a deeper understand of these tissue-specific responses., Results: The overall expression pattern observed shows a well-differentiated transcriptional signature in mdm muscles compared to wild type. Muscle-specific clusters observed within the mdm transcriptome highlight the level of variability of each muscle to the deletion. Differential gene expression and weighted gene co-expression network analysis showed a strong directional response in oxidative respiration-associated mitochondrial genes, which aligns with the poor shivering and non-shivering thermogenesis previously observed. Sln, which is a marker associated with shivering and non-shivering thermogenesis, showed the strongest expression change in fast-fibered muscles. No drastic changes in MYH expression levels were reported, which indicated an absence of major fiber-type switching events. Overall expression shifts in MYH isoforms, MARPs, and extracellular matrix associated genes demonstrated the transcriptional complexity associated with mdm mutation. The expression alterations in mitochondrial respiration and metabolism related genes in the mdm muscle dominated over other transcriptomic changes, and likely account for the late stage cellular responses in the mdm muscles., Conclusions: We were able to demonstrate that the complex nature of mdm mutation extends beyond a simple rearrangement in titin gene. EDL, psoas and soleus exemplify unique response modes observed in skeletal muscles with mdm mutation. Our data also raises the possibility that failure to maintain proper energy homeostasis in mdm muscles may contribute to the pathogenesis of the degenerative phenotype in mdm mice. Understanding the full disease-causing molecular cascade is difficult using bulk RNA sequencing techniques due to intricate nature of the disease. The development of the mdm phenotype is temporally and spatially regulated, hence future studies should focus on single fiber level investigations., (© 2022. The Author(s).)

The conformational sensitivity of intrinsically disordered proteins to shifts in pH due to their high degree of charged residues has been recognized for well over a decade. However, the role of the non-ionizable residues in this pH sensitivity remains poorly understood. Our lab has been investigating the pH sensitivity of the poly-E motifs of the PEVK region of the muscle protein titin, which provides an ideal model system to explore this question. Using a series of 15-amino acid peptides derived from one of the poly-E motif sequences, we have investigated the role of side-chain chemistry in the conformational flexibility of this region. Our results demonstrate that aromatic side chains and proline content are the two variables that most influence pH sensitivity. The introduction of aromatic side chains resulted in a more collapsed structure, even at pH 7, while the removal of prolines resulted in a higher degree of pH sensitivity. These results highlight the importance of considering the impact of non-ionizable residues on IDP function, especially when considering the impact of pH on conformational flexibility.

Muscular dystrophy with myositis ( mdm ) is a naturally occurring mutation in the mouse Ttn gene that results in higher passive stress in muscle fibers and intact muscles compared to wild-type (WT). The goal of this study was to test whether alternative splicing of titin exons occurs in mdm muscles, which contain a small deletion in the N2A-PEVK regions of titin, and to test whether splicing changes are associated with an increase in titin-based passive tension. Although higher levels of collagen have been reported previously in mdm muscles, here we demonstrate alternative splicing of titin in mdm skeletal muscle fibers. We identified Z-band, PEVK, and C-terminus Mex5 exons as splicing hotspots in mdm titin using RNA sequencing data and further reported upregulation in ECM-associated genes. We also treated skinned mdm soleus fiber bundles with trypsin, trypsin + KCl, and trypsin + KCL + KI to degrade titin. The results showed that passive stress dropped significantly more after trypsin treatment in mdm fibers (11 ± 1.6 mN/mm 2 ) than in WT fibers (4.8 ± 1 mN/mm 2 ; p = 0.0004). The finding that treatment with trypsin reduces titin-based passive tension more in mdm than in WT fibers supports the hypothesis that exon splicing leads to the expression of a stiffer and shorter titin isoform in mdm fibers. After titin extraction by trypsin + KCl + KI, mdm fibers (6.7 ± 1.27 mN/mm 2 ) had significantly higher collagen-based passive stress remaining than WT fibers (2.6 ± 1.3 mN/mm 2 ; p = 0.0014). We conclude that both titin and collagen contribute to higher passive tension of mdm muscles., Competing Interests: The authors declare no conflict of interest.

Background: Regional anesthesia (RA) has been used to reduce pain and opioid usage in elective orthopedic surgery. The hypothesis of this study was that RA would be associated with decreased opioid demand in tibial plateau fracture surgery., Methods: Inpatient opioid consumption and 90-day outpatient opioid prescribing in all patients ≥18 years of age undergoing tibial plateau fracture surgery from July 2013 to July 2018 (n = 264) at a single, level I trauma center were recorded. The presence or absence of perioperative RA was noted. Of 60 patients receiving RA, 52 underwent peripheral nerve blockade (PNB) with single-shot sciatic-popliteal (40.0%; n = 24), femoral (26.7%; n = 16), adductor canal (18.3%; n = 11), or fascia iliaca (1.7%; n = 1) block with ropivacaine. Ten patients received epidural analgesia (EA) with either single-shot spinal (11.7%; n = 7) blocks or continuous epidural (5.0%; n = 3). Additional baseline and treatment characteristics were recorded, including age, sex, race, body mass index (BMI), smoking, chronic opioid use, American Society of Anesthesiologists (ASA) score, injury mechanism, additional injuries, open injury, and additional inpatient surgery. Statistical models, including multivariable generalized linear models with propensity score weighting to adjust for baseline patient and treatment characteristics, were used to assess perioperative opioid demand with and without RA., Results: RA was associated with reduced inpatient opioid usage from 0 to 24 hours postoperatively of approximately 5.2 oxycodone 5-mg equivalents (0.74 incident rate ratio [IRR]; 0.63-0.86 CI; P < .001) and from 24 to 48 hours postoperatively of approximately 2.9 oxycodone 5-mg equivalents (0.78 IRR; 0.64-0.95 CI; P = .014) but not at 48 to 72 hours postoperatively. From 1 month preoperatively to 2 weeks postoperatively, RA was associated with reduced outpatient opioid prescribing of approximately 24.0 oxycodone 5-mg equivalents (0.87; 0.75-0.99; P = .044) and from 1 month preoperatively to 90 days postoperatively of approximately 44.0 oxycodone 5-mg equivalents (0.83; 0.71-0.96; P = .011), although there was no significant difference from 1 month preoperatively to 6 weeks postoperatively. There were no cases of acute compartment syndrome in this cohort., Conclusions: In tibial plateau fracture surgery, RA was associated with reduced inpatient opioid consumption up to 48 hours postoperatively and reduced outpatient opioid demand up to 90 days postoperatively without an associated risk of acute compartment syndrome. RA should be considered for patients undergoing tibial plateau fracture fixation., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 International Anesthesia Research Society.)

The disordered PEVK region of titin contains two main structural motifs: PPAK and poly-E. The distribution of these motifs in the PEVK region contributes to the elastic properties of this region, but the specific mechanism of how these motifs work together remains unclear. Previous work from our lab has demonstrated that 28-amino acid peptides of the poly-E motif are sensitive to shifts in pH, becoming more flexible as the pH decreases. We extend this work to longer poly-E constructs, including constructs containing PPAK motifs. Our results demonstrate that longer poly-E motifs have a much larger range of pH sensitivity and that the inclusion of the PPAK motif reduces this sensitivity. We also demonstrate that binding calcium can increase the conformational flexibility of the poly-E motif, though the PPAK motif can block this calcium-dependent change. The data presented here suggest a model where PPAK and calcium can alter the stiffness of the poly-E motif by modulating the degree of charge repulsion in the glutamate clusters.

While protein refolding has been studied for over 50 years since the pioneering work of Christian Anfinsen, there have been a limited number of studies correlating results between chemical, thermal, and mechanical unfolding. The limited knowledge of the relationship between these processes makes it challenging to compare results between studies if different refolding methods were applied. Our current work compares the energetic barriers and folding rates derived from chemical, thermal, and mechanical experiments using an immunoglobulin-like domain from the muscle protein titin as a model system. This domain, I83, has high solubility and low stability relative to other Ig domains in titin, though its stability can be modulated by calcium. Our experiments demonstrated that the free energy of refolding was equivalent with all three techniques, but the refolding rates exhibited differences, with mechanical refolding having slightly faster rates. This suggests that results from equilibrium-based measurements can be compared directly but care should be given comparing refolding kinetics derived from refolding experiments that used different unfolding methods.

Introduction: We hypothesized that the modified Fragility Index (mFI), which predicts surgical complications, would be applicable to surgical complications in patients older than 50 years with distal humerus fractures (DHF)., Methods: We retrospectively reviewed the American College of Surgeons National Surgery Quality Improvement Program database, including patients older than 50 years who underwent open reduction and internal fixation of a DHF. A 5-item mFI score was calculated. Postoperative complications, readmission and reoperation rates, and length of stay were recorded. Univariate as well as a multivariable statistical analysis was performed, controlling for age, sex, body mass index, length of stay, and operative time., Results: We identified 864 patients (mean age, 68.6 years ± 10.4), and 74.1% were female. As the mFI increased from 0 to 2 or greater, 30-day readmission rate increased from 3% to 10% ( P value = .01), rate of discharge to rehabilitation facility increased from 12% to 32% ( P value = .0), and any complication rate increased from 4% to 19% ( P value = .0). Rates of pulmonary complications increased significantly in patients with the mFI of 2 or greater ( P value = .047). Patients with the mFI of 2 or greater were nearly 4 times more likely to be readmitted within 30 days (odds ratio [OR] = 3.5, P value = .007) and had an increased OR of 30-day reoperation and any complication (OR = 3.7, P value = .02; OR = 4.5, P  value = .00, respectively) on multivariate analysis., Conclusion: A fragility state is predictive of postoperative complications, readmission, and reoperation after surgical management of DHF. Our data suggest that a fragility evaluation can help inform surgical decision-making in patients older than 50 years with DHF.

After-hours surgery represents a novel solution that can effectively combat surgical fatigue of care teams in addition to addressing the high volume of surgical backlog associated with the repercussions of the COVID-19 pandemic. This commentary seeks to rationalize how successful employment of a dedicated after-hours surgical team and protocol has tremendous potential for increased efficiency while maintaining good surgical outcomes in patients., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2021.)

Open tibia fractures are often associated with considerable soft tissue injuries. Management of open tibia fractures can be challenging, and some patients require amputation. The patient and treatment factors have not been described on a population level in the United States. A retrospective analysis was completed using the 2000 to 2011 Nationwide Inpatient Sample. Amputation rates during the index hospitalization after open tibia fracture were computed based on injury, patient, and hospital characteristics in patients 18 years or older. The overall amputation rate in open tibia fractures during the index hospitalization was 2.2% (n=3769). Patients with midshaft tibia fractures comprised the largest portion of patients undergoing amputation (46.8% of total amputations) compared with distal tibia (34.0%) and proximal tibia (19.3%) fractures. Patients with no neurovascular injury comprised the largest portion of patients undergoing amputation (85.9%), followed by isolated arterial injury (11.1%), combined neurovascular injury (1.9%), and isolated nerve injury (1.1%). Amputation rates were significantly increased for midshaft tibia fractures with neurovascular injury (odds ratio, 12.39; 95% CI, 5.52-27.83) and distal tibia fractures with neurovascular injury (odds ratio, 5.45; 95% CI, 1.73-17.19) compared with tibia fractures with no neurovascular injury while controlling for confounders. On the basis of a review of the Nationwide In-patient Sample during the past decade, the authors have shown that the early amputation rate in open tibia fractures for all-comers is 2.2%. Rates of amputation varied based on fracture site, associated neurovascular injury, medical comorbidities, and hospital location. [Orthopedics. 2021;44(1):48-53.]., (Copyright 2020, SLACK Incorporated.)

Hypothesis: We hypothesized that the modified Fragility Index (mFI) would predict complications in patients older than 50 years who underwent operative intervention for a proximal humerus fracture., Methods: We retrospectively reviewed the American College of Surgeons National Surgery Quality Improvement Program database, including patients older than 50 years who underwent open reduction and internal fixation of a proximal humerus fracture. A 5-item mFI score was then calculated for each patient. Postoperative complications, readmission and reoperation rates as well as length of stay (LOS) were recorded. Univariate as well as multivariable statistical analyses were performed, controlling for age, sex, body mass index, LOS, and operative time., Results: We identified 2,004 patients (median age, 66 years; interquartile range: 59-74), of which 76.2% were female. As mFI increased from 0 to 2 or greater, 30-day readmission rate increased from 2.8% to 6.7% ( P -value = .005), rate of discharge to rehabilitation facility increased from 7.1% to 25.3% ( P -value < .001), and rates of any complication increased from 6.5% to 13.9% ( P -value < .001). Specifically, the rates of renal and hematologic complications increased significantly in patients with mFI of 2 or greater ( P -value = .042 and  P -value < .001, respectively). Compared with patients with mFI of 0, patients with mFI of 2 or greater were 2 times more likely to be readmitted within 30 days (odds ratio = 2.2, P -value .026). In addition, patients with mFI of 2 or greater had an increased odds of discharge to a rehabilitation center (odds ratio = 2.3, P -value < .001). However, increased fragility was not significantly associated with an increased odds of 30-day reoperation or any complication after controlling for demographic data, LOS, and operative time., Conclusion: An increasing level of fragility is predictive of readmission and discharge to a rehabilitation center after open reduction and internal fixation of proximal humerus fractures. Our data suggest that a simple fragility evaluation can help inform surgical decision-making and counseling in patients older than 50 years with proximal humerus fractures., (© 2021 The Authors.)

The muscle protein titin plays a crucial role in passive elasticity and the disordered PEVK region within titin is central to that function. The PEVK region is so named due to its high proline, glutamate, valine and lysine content and the high charge density in this region results in a lack of organized structure within this domain. The PEVK region is highly extensible but the molecular interactions that contribute to the elastic nature of the PEVK still remain poorly described. The PEVK region is formed by two unique sequence motifs. The PPAK motif is a 26 to 28 amino acid sequence that contains a mixture of charged and hydrophobic residues and is the primary building block for the PEVK region. Poly-E sequence motifs vary in length and contain clusters of 3-4 glutamic acids distributed throughout the motif. In this study, we derived two 28-residue peptides from the human titin protein sequence and measured their structural characteristics over a range of pHs. Our results demonstrate that the poly-E peptide undergoes a shift from a more rigid and elongated state to a more collapsed state as pH decreases with the midpoint of this transition being at pH ~5.5. Interestingly, a similar conformational shift is not observed in the PPAK peptide. These results suggest that the poly-E motif might provide a nucleating site for the PEVK when the muscle is not in an extended state., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

Background: Individual skeletal muscles have evolved to perform specific tasks based on their molecular composition. In general, muscle fibers are characterized as either fast-twitch or slow-twitch based on their myosin heavy chain isoform profiles. This approach made sense in the early days of muscle studies when SDS-PAGE was the primary tool for mapping fiber type. However, Next Generation Sequencing tools permit analysis of the entire muscle transcriptome in a single sample, which allows for more precise characterization of differences among fiber types, including distinguishing between different isoforms of specific proteins. We demonstrate the power of this approach by comparing the differential gene expression patterns of extensor digitorum longus (EDL), psoas, and soleus from mice using high throughput RNA sequencing., Results: EDL and psoas are typically classified as fast-twitch muscles based on their myosin expression pattern, while soleus is considered a slow-twitch muscle. The majority of the transcriptomic variability aligns with the fast-twitch and slow-twitch characterization. However, psoas and EDL exhibit unique expression patterns associated with the genes coding for extracellular matrix, myofibril, transcription, translation, striated muscle adaptation, mitochondrion distribution, and metabolism. Furthermore, significant expression differences between psoas and EDL were observed in genes coding for myosin light chain, troponin, tropomyosin isoforms, and several genes encoding the constituents of the Z-disk., Conclusions: The observations highlight the intricate molecular nature of skeletal muscles and demonstrate the importance of utilizing transcriptomic information as a tool for skeletal muscle characterization.

As bundled reimbursement models continue to evolve, there is a continued effort to increase the value of care for patients undergoing arthroplasty. The authors sought to evaluate the effect of surgeon specialization (arthroplasty vs non-arthroplasty) on acute outcomes for patients with hip fracture who underwent total hip arthroplasty (THA), in an effort to determine whether the value of care can be improved by surgeons specializing in these procedures. They performed a multicenter retrospective cohort study of patients who had hip fracture and were treated with THA between June 2013 and February 2018 at 2 academic institutions that were involved in bundled reimbursement initiatives. Patients were stratified based on the subspecialty training of the operative surgeon (fellowship-trained adult reconstruction vs other orthopedic sub-specialty), and 90-day readmissions, length of stay, and discharge disposition were compared between groups. A total of 291 patients were included in the final cohort, with 120 (41.2%) undergoing surgery performed by a fellowship-trained adult reconstruction surgeon. No significant difference was found in age, sex, race, or American Society of Anesthesiologists score between the 2 groups. In addition, no significant difference was found in length of stay, discharge to a facility, or 90-day readmissions on univariable or multivariable analysis when adjusted for age, sex, body mass index, and American Society of Anesthesiologists score. This study showed that the acute outcomes used to assess the value of care for patients undergoing THA were not significantly different when the surgery was performed by an adult reconstruction specialist compared with other orthopedic surgeons at 2 high-volume academic centers with perioperative care pathways. Alternative modalities to significantly improve acute postoperative outcomes in a bundled reimbursement model must be investigated. [Orthopedics. 2020;43(5):e442-e446.]., (Copyright 2020, SLACK Incorporated.)

Titin is a large filamentous protein that spans half a sarcomere, from Z-disk to M-line. The N2A region within the titin molecule exists between the proximal immunoglobulin (Ig) region and the PEVK region and protein-protein interactions involving this region are required for normal muscle function. The N2A region consists of four Ig domains (I80-I83) with a 105 amino acid linker region between I80 and I81 that has a helical nature. Using chemical stability measurements, we show that predicted differences between the adjacent Ig domains (I81-I83) correlate with experimentally determined differences in chemical stability and refolding kinetics. Our work further shows that I83 has the lowest ΔG unfolding , which is increased in the presence of calcium (pCa 4.3), indicating that Ca 2+ plays a role in stabilizing this immunoglobulin domain. The characteristics of N2A's three Ig domains provide insight into the stability of the binding sites for proteins that interact with the N2A region. This work also provides insights into how Ca 2+ might influence binding events involving N2A., (© 2020 The Protein Society.)

This study sought to determine (1) whether surgeons can accurately predict functional outcomes of operative fixation of pilon fractures based on injury and initial postoperative radiographs, (2) whether the surgeon's level of experience is associated with the ability to successfully predict outcome, and (3) the association between patients' demographic and clinical characteristics and surgeons' prediction scores. A blinded, randomized provider survey was conducted at a level I trauma center. Seven fellowship-trained orthopedic traumatologists and 4 orthopedic trauma fellows who were blinded to outcome reviewed data regarding 95 pilon fractures in random order. Injury ankle radiographs, initial postoperative fixation radiographs, and brief patient histories were assessed. Midterm follow-up functional outcome scores obtained a mean 4.9 years after surgery were available for all patients. Main outcome measures were Pearson correlation coefficient-assessed functional outcomes and surgeon-predicted outcomes. A mixed-effect model determined the association between patients' characteristics and surgeons' prediction scores. Minimal positive correlation was observed between functional outcomes and prediction scores. No difference was noted between the attending and fellow groups in prediction ability. When surgeons' prediction confidence level was greater than 1 SD above the mean confidence level, correlation between functional outcome and prediction improved, although poor correlation was still observed. AO/OTA type 43C fractures, high-energy mechanisms, and older patient age were characteristics associated with lower prediction scores. Surgeons had poor ability to predict functional outcomes of patients with pilon fractures based on injury and initial postoperative radiographs, and level of experience was not associated with ability to predict outcome. [Orthopedics. 2020; 43(1): e43-e46.]., (Copyright 2019, SLACK Incorporated.)

Background: A current limitation in the care of patients with severe lower extremity traumatic injuries is the lack of a rigorously developed patient-reported outcome (PRO) instrument specific to lower extremity trauma patients., Objective: This mixed methods protocol aims to describe phases I and II of the development of a PRO instrument for lower extremity trauma patients, following international PRO development guidelines., Methods: The phase I study follows an interpretive description approach. Development of the PRO instrument begins with identifying the concepts that are important to patients, after which a preliminary conceptual framework is devised from a systematic literature review and used to generate an interview guide. Patients aged 18 years or above with limb-threatening lower extremity traumatic injuries resulting in reconstruction, amputation, or amputation after failed reconstruction will be recruited. The subjects will participate in semistructured, in-depth qualitative interviews to identify all important concepts of interest. The qualitative interview data will be coded with top-level domains, themes, and subthemes. The codes will then be utilized to refine the conceptual framework and generate preliminary items and a set of scales. The preliminary scales will be further refined via a process of conducting cognitive debriefing interviews with lower extremity trauma patients and soliciting expert opinions. Phase III will include a large-scale field test, using Rasch measurement theory to analyze the psychometric properties of the instrument; shortening and finalizing the scales; and determining the reliability, validity, and responsiveness of the instrument., Results: Phases I and II of this study have been funded. Phase I of this study has been completed, and phase II began in January 2019 and is expected to be completed in November 2019. Phase III will begin following the completion of phase II., Conclusions: This protocol describes the initial phases of development of a novel PRO instrument for use in lower extremity trauma patients., International Registered Report Identifier (irrid): DERR1-10.2196/14397., (©Lily R Mundy, Anne Klassen, Jordan Grier, Matthew J Carty, Andrea L Pusic, Scott T Hollenbeck, Mark J Gage. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 17.10.2019.)

Rising and more variable global temperatures pose a challenge for biodiversity, with reproduction and fertility being especially sensitive to heat. Here, we assessed the potential for thermal adaptation in sperm and egg function using Tribolium flour beetles, a warm-temperate-tropical insect model. Following temperature increases through adult development, we found opposing gamete responses, with males producing shorter sperm and females laying larger eggs. Importantly, this gamete phenotypic plasticity was adaptive: thermal translocation experiments showed that both sperm and eggs produced in warmer conditions had superior reproductive performance in warmer environments, and vice versa for cooler production conditions and reproductive environments. In warmer environments, gamete plasticity enabled males to double their reproductive success, and females could increase offspring production by one-third. Our results reveal exciting potential for sensitive but vital traits within reproduction to handle increasing and more variable thermal regimes in the natural environment., Competing Interests: RV, AS, KS, MD, AL, MG No competing interests declared, (© 2019, Vasudeva et al.)

Background: Advances in fracture fixation and soft tissue coverage continue to improve the care of patients after limb-threatening lower extremity (LE) trauma. However, debate continues regarding which treatment option-reconstruction or amputation-is most appropriate. Many authors have attempted to quantify the patient experience in this treatment paradigm; however, they have not used patient-reported outcome (PRO) instruments specific to this population. Our aim was to identify and evaluate PRO instruments developed specifically for LE trauma, applicable to reconstruction and amputation, using established PRO instrument development and validation guidelines., Methods: A multidisciplinary team used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses method to query PubMed, Medline Ovid, EMBASE, Cochrane, Medline Web of Science, and Psych Info databases from inception to November 2016. Publications were included that described the development and/or validation of a PRO instrument assessing satisfaction and/or quality of life in LE trauma, applicable to both amputation and reconstruction. Two authors independently reviewed each full-text citation., Results: After removing duplicates, 6,290 abstracts were identified via the database query. Following a preliminary title and abstract screen, 657 full-text citations were reviewed. Of these references, none satisfied the previously established inclusion criteria., Conclusions: No studies were identified that described a PRO instrument developed to assess outcomes in LE trauma patients applicable to both reconstruction and amputation. There is thus a need for a PRO instrument designed specifically for patients who have sustained limb-threatening LE trauma to guide treatment decisions.

Mutations in titin are responsible for many cardiac and muscle diseases, yet the underlying mechanisms remain largely unexplained. Numerous studies have established roles for titin in muscle function, and Ca 2+ -dependent interactions between titin and actin have been suggested to play a role in muscle contraction. The present study used co-sedimentation assays, dynamic force spectroscopy (DFS), and in vitro motility (IVM) assays to determine whether the N2A region of titin, overlooked in previous studies, interacts with actin in the presence of Ca 2+ . Co-sedimentation demonstrated that N2A - F-actin binding increases with increasing protein and Ca 2+ concentration, DFS demonstrated increased rupture forces and decreased k off in the presence of Ca 2+ , and IVM demonstrated a Ca 2+ -dependent reduction in motility of F-actin and reconstituted thin filaments in the presence of N2A. These results indicate that Ca 2+ increases the strength and stability of N2A - actin interactions, supporting the hypothesis that titin plays a regulatory role in muscle contraction. The results further support a model in which N2A - actin binding in active muscle increases titin stiffness, and that impairment of this mechanism contributes to the phenotype in muscular dystrophy with myositis. Future studies are required to determine whether titin - actin binding occurs in skeletal muscle sarcomeres in vivo.

Background: The need for reoperation or wound infection treatments between pulsatile and gravity irrigation are statistically equivalent, however, it is unclear which method maximizes operative efficiency and expeditious irrigation. In this study we set out to determine the differences in irrigation rate between these various treatment methods., Methods: This was an ex-vivo experimental laboratory study not involving human subjects. Irrigation rates were tested based on the time in seconds required to empty a three-liter bag of normal saline hanging at either 6 or 9 ft. Three forms of irrigation were tested: gravity irrigation (GI6, GI9), low-pressure pulsatile irrigation (LP6, LP9) and high-pressure pulsatile irrigation. One-way ANOVA and Student's t -test were used to compare rates based on height and form of irrigation., Results: Significant differences in irrigation rates were noted at 6 ft between all three forms of irrigation with gravity irrigation the fastest followed by high-pressure and low-pressure pulsatile irrigation (GI6, mean 142 s ± 3.2; HP6, mean 189 s ± 10.2; LP6, mean 323 s ± 22.5; p  < 0.001). This difference was also found at 9 ft (GI9, mean 114 s ± 1.5; HP9, mean 186 s ± 10.5; LP9, mean 347 s ± 3.5; p  < 0.001). Gravity irrigation was significantly faster ( p  < 0.001) at an increased height, whereas the high and low-pressure irrigation rates were unaffected by height. List price comparison found pulsatile irrigation to cost approximately 3.3 times more than gravity lavage., Conclusions: Gravity irrigation provided the most rapid rate of irrigation tested, regardless of the height. With existing literature demonstrating equivalent clinical outcomes between methods, gravity lavage offers a faster and potentially more cost-effective form of irrigation.

In the majority of insects, sperm fertilize the egg via a narrow canal through the outer chorion called the micropyle. Despite having this one primary function, there is considerable unexplained variation in the location, arrangement and number of micropyles within and between species. Here, we examined the relationship between micropyle number and female mating pattern through a comparative analysis across Lepidoptera. Three functional hypotheses could explain profound micropylar variation: (i) increasing micropyle number reduces the risk of infertility through sperm limitation in species that mate infrequently; (ii) decreasing micropyle number reduces the risk of pathological polyspermy in species that mate more frequently; and (iii) increasing micropyle number allows females to exert greater control over fertilization within the context of post-copulatory sexual selection, which will be more intense in promiscuous species. Micropyle number was positively related to the degree of female promiscuity as measured by spermatophore count, regardless of phylogenetic signal, supporting the hypothesis that micropyle number is shaped by post-copulatory sexual selection. We discuss this finding in the context of cryptic female choice, sperm limitation and physiological polyspermy., (© 2016 The Author(s).)

There is increasing evidence that females can somehow improve their offspring fitness by mating with multiple males, but we understand little about the exact stage(s) at which such benefits are gained. Here, we measure whether offspring fitness is influenced by mechanisms operating solely between sperm and egg. Using externally fertilizing and polyandrous Atlantic salmon (Salmo salar), we employed split-clutch and split-ejaculate in vitro fertilization experiments to generate offspring using designs that either denied or applied opportunities for sperm competition and cryptic female choice. Following fertilizations, we measured 140 days of offspring fitness after hatch, through growth and survival in hatchery and near-natural conditions. Despite an average composite mortality of 61%, offspring fitness at every life stage was near-identical between groups fertilized under the absence versus presence of opportunities for sperm competition and cryptic female choice. Of the 21 551 and 21 771 eggs from 24 females fertilized under monandrous versus polyandrous conditions, 68% versus 67.8% survived to the 100-day juvenile stage; sub-samples showed similar hatching success (73.1% versus 74.3%), had similar survival over 40 days in near-natural streams (57.3% versus 56.2%) and grew at similar rates throughout. We therefore found no evidence that gamete-specific interactions allow offspring fitness benefits when polyandrous fertilization conditions provide opportunities for sperm competition and cryptic female choice.

Reproduction through sex carries substantial costs, mainly because only half of sexual adults produce offspring. It has been theorized that these costs could be countered if sex allows sexual selection to clear the universal fitness constraint of mutation load. Under sexual selection, competition between (usually) males and mate choice by (usually) females create important intraspecific filters for reproductive success, so that only a subset of males gains paternity. If reproductive success under sexual selection is dependent on individual condition, which is contingent to mutation load, then sexually selected filtering through 'genic capture' could offset the costs of sex because it provides genetic benefits to populations. Here we test this theory experimentally by comparing whether populations with histories of strong versus weak sexual selection purge mutation load and resist extinction differently. After evolving replicate populations of the flour beetle Tribolium castaneum for 6 to 7 years under conditions that differed solely in the strengths of sexual selection, we revealed mutation load using inbreeding. Lineages from populations that had previously experienced strong sexual selection were resilient to extinction and maintained fitness under inbreeding, with some families continuing to survive after 20 generations of sib × sib mating. By contrast, lineages derived from populations that experienced weak or non-existent sexual selection showed rapid fitness declines under inbreeding, and all were extinct after generation 10. Multiple mutations across the genome with individually small effects can be difficult to clear, yet sum to a significant fitness load; our findings reveal that sexual selection reduces this load, improving population viability in the face of genetic stress.

Context: During team athletic events, athletic trainers commonly provide fluids with water bottles. When a limited number of water bottles exist, various techniques are used to deliver fluids., Objective: To determine whether fluid delivered via water-bottle administration influenced fluid consumption and hydration status., Design: Crossover study., Setting: Outdoor field (22.2°C ± 3.5°C)., Patients or Other Participants: Nineteen participants (14 men, 5 women, age = 30 ± 10 years, height = 176 ± 8 cm, mass = 72.5 ± 10 kg) were recruited from the university and local running clubs., Intervention(s): The independent variable was fluid delivery with 3 levels: self-administration with mouth-to-bottle direct contact (SA-DC), self-administration with no contact between mouth and bottle (SA-NC), and external administration with no contact between the mouth and the bottle (EA-NC). Participants warmed up for 10 minutes before completing 5 exercise stations, after which an ad libitum fluid break was given, for a total of 6 breaks., Main Outcome Measure(s): We measured the fluid variables of total volume consumed, total number of squirts, and average volume per squirt. Hydration status via urine osmolality and body-mass loss, and perceptual variables for thirst and fullness were recorded. We calculated repeated-measures analyses of variance to assess hydration status, fluid variables, and perceptual measures to analyze conditions across time., Results: The total volume consumed for EA-NC was lower than for SA-DC (P = .001) and SA-NC (P = .001). The total number of squirts for SA-DC was lower than for SA-NC (P = .009). The average volume per squirt for EA-NC was lower than for SA-DC (P = .020) and SA-NC (P = .009). Participants arrived (601.0 ± 21.3 mOsm/L) and remained (622.3 ± 38.3 mOsm/L) hydrated, with no difference between conditions (P = .544); however, the EA-NC condition lost more body mass than did the SA-DC condition (P = .001). There was no main effect for condition on thirst (P = .147) or fullness (P = .475)., Conclusions: External administration of fluid decreased total volume consumed via a decreased average volume per squirt. The SA-DC method requires fewer squirts within a specific time frame. Fluid breaks every 15 minutes resulted in maintenance of euhydration; however, loss of body mass was influenced by fluid administration. Athletic trainers should avoid external administration to promote positive hydration behaviors. When fluid is self-administered, individual bottles may be the best clinical practice because more volume can be consumed per squirt.

Adaptations at the gamete level (a) evolve quickly, (b) appear sensitive to inbreeding and outbreeding and (c) have important influences on potential to reproduce. We apply this understanding to problems posed by escaped farm salmon and measure their potential to reproduce in the wild. Farm Atlantic salmon (Salmo salar) are a threat to biodiversity, because they escape in large numbers and can introgress, dilute or disrupt locally adapted wild gene pools. Experiments at the whole fish level have found farm reproductive potential to be significant, but inferior compared to wild adults, especially for males. Here, we assess reproductive performance at the gamete level through detailed in vitro comparisons of the form, function, fertility, compatibility and competitiveness of farm versus wild Atlantic salmon sperm and eggs, in conditions mimicking the natural gametic microenvironment, using fish raised under similar environmental conditions. Despite selective domestication and reduced genetic diversity, we find functional equivalence in all farm fish gamete traits compared with their wild ancestral strain. Our results identify a clear threat of farm salmon reproduction with wild fish and therefore encourage further consideration of using triploid farm strains with optimized traits for aquaculture and fish welfare, as triploid fish remain reproductively sterile following escape.

Background: Currently many habitats suffer from quality loss due to environmental change. As a consequence, evolutionary trajectories might shift due to environmental effects and potentially increase extinction risk of resident populations. Nevertheless, environmental variation has rarely been incorporated in studies of sexual selection and sexual conflict, although local environments and individuals' condition undoubtedly influence costs and benefits. Here, we utilise polyandrous and monogamous selection lines of flour beetles, which evolved in presence or absence of sexual selection for 39 generations. We specifically investigated effects of low vs. standard food quality (i.e. stressful vs. benign environments) on reproductive success of cross pairs between beetles from the contrasting female and male selection histories to assess gender effects driving fitness., Results: We found a clear interaction of food quality, male selection history and female selection history. Monogamous females generally performed more poorly than polyandrous counterparts, but reproductive success was shaped by selection history of their mates and environmental quality. When monogamous females were paired with polyandrous males in the standard benign environment, females seemed to incur costs, possibly due to sexual conflict. In contrast, in the novel stressful environment, monogamous females profited from mating with polyandrous males, indicating benefits of sexual selection outweigh costs., Conclusions: Our findings suggest that costs and benefits of sexually selected adaptations in both sexes can be profoundly altered by environmental quality. With regard to understanding possible impacts of environmental change, our results further show that the ecology of mating systems and associated selection pressures should be considered in greater detail.

Despite evidence that variation in male-female reproductive compatibility exists in many fertilization systems, identifying mechanisms of cryptic female choice at the gamete level has been a challenge. Here, under risks of genetic incompatibility through hybridization, we show how salmon and trout eggs promote fertilization by conspecific sperm. Using in vitro fertilization experiments that replicate the gametic microenvironment, we find complete interfertility between both species. However, if either species' ova were presented with equivalent numbers of both sperm types, conspecific sperm gained fertilization precedence. Surprisingly, the species' identity of the eggs did not explain this cryptic female choice, which instead was primarily controlled by conspecific ovarian fluid, a semiviscous, protein-rich solution that bathes the eggs and is released at spawning. Video analyses revealed that ovarian fluid doubled sperm motile life span and straightened swimming trajectory, behaviors allowing chemoattraction up a concentration gradient. To confirm chemoattraction, cell migration tests through membranes containing pores that approximated to the egg micropyle showed that conspecific ovarian fluid attracted many more spermatozoa through the membrane, compared with heterospecific fluid or water. These combined findings together identify how cryptic female choice can evolve at the gamete level and promote reproductive isolation, mediated by a specific chemoattractive influence of ovarian fluid on sperm swimming behavior., (© 2013 The Authors. Evolution published by Wiley Periodicals, Inc. on behalf of The Society for the Study of Evolution.)

Theory predicts that males should evolve mechanisms to assess competition and allocate resources accordingly. This requires phenotypic plasticity, to accurately match responses to the environment. Plastic responses in males to sexual competition are diverse and widespread. However, our ability to understand and predict how they evolve is limited because their benefits are rarely measured, and costs are, as yet, entirely unquantified. In the fruit fly Drosophila melanogaster, males that anticipate strong competition for matings or fertilizations subsequently mate for longer and transfer more of two key seminal fluid proteins. This results in significantly elevated reproductive output. In this study, we examined the fitness effects of male responses to rivals across the entire male life span. Males were exposed to rivals or not throughout life while controlling mating opportunities. Males showed significant responses to rivals throughout their lifetimes, associated with significant early-life fitness benefits. However, these disappeared after the third mating. There were also significant costs--males exposed to rivals took significantly fewer mating opportunities in later life and had significantly shorter life spans than controls. The data suggest that there are substantial costs for males of mounting plastic responses to the threat of sexual competition., (© 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.)

Plasticity in behaviour is of fundamental significance when environments are variable. Such plasticity is particularly important in the context of rapid changes in the socio-sexual environment. Males can exhibit adaptive plastic responses to variation in the overall level of reproductive competition. However, the extent of behavioural flexibility within individuals, and the degree to which rapidly changing plastic responses map onto fitness are unknown. We addressed this by determining the behaviour and fitness profiles of individual Drosophila melanogaster males subjected to up to three episodes of exposure to rivals or no rivals, in all combinations. Behaviour (mating duration) was remarkably sensitive to the level of competition and fully reversible, suggesting that substantial costs arise from the incorrect expression of even highly flexible behaviour. However, changes in mating duration matched fitness outcomes (offspring number) only in scenarios in which males experienced zero then high competition. Following the removal of competition, mating duration, but not offspring production, decreased to below control levels. This indicates that the benefit of increasing reproductive investment when encountering rivals may exceed that of decreasing investment when rivals disappear. Such asymmetric fitness benefits and mismatches with behavioural responses are expected to exert strong selection on the evolution of plasticity.

Leiomyosarcomas are smooth muscle-derived tumors generally found intra-abdominally in the retoperitoneum, mesentery, or omentum. Only approximately 5% of these tumors originate from vessel wall smooth muscle. Those derived from the splenic vein are exceedingly rare, with only one previously published case in the literature. We present a second case of leiomyosarcoma of the splenic vein in a 58-year-old woman with 2 months of epigastric pain. A distal pancreatectomy was performed to include the tumor found centered in the splenic vein at the splenic and portal vein confluence and growing into the pancreas in the body on the posterior aspect. A saphenous vein patch was used for reconstruction., (Copyright © 2012 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

The widespread phenomenon of polyandry (mating by females with multiple males) is an evolutionary puzzle, because females can sustain costs from promiscuity, whereas full fertility can be provided by a single male. Using the red flour beetle, Tribolium castaneum, we identify major fitness benefits of polyandry to females under inbreeding, when the risks of fertilization by incompatible male haplotypes are especially high. Fifteen generations after inbred populations had passed through genetic bottlenecks, we recorded increased levels of female promiscuity compared with noninbred controls, most likely due to selection from prospective fitness gains through polyandry. These data illustrate how this common mating pattern can evolve if population genetic bottlenecks increase the risks of fitness depression due to fertilization by sperm carrying genetically incompatible haplotypes.

Across many species, males exhibit plastic responses when they encounter mating rivals. The ability to tailor responses to the presence of rivals allows males to increase investment in reproduction only when necessary. This is important given that reproduction imposes costs that limit male reproductive capacity, particularly when sperm competition occurs. Fruitfly (Drosophila melanogaster) males exposed to rivals subsequently mate for longer and thus accrue fitness benefits under increased competition, in line with theory. Here, we show that male D. melanogaster detect rivals by using a suite of cues and that the resulting responses lead directly to significant fitness benefits. We used multiple techniques to systematically remove auditory, olfactory, tactile, and visual cues, first singly and then in all possible combinations. No single cue alone was sufficient to allow males to detect rivals. However, the perception of any two cues from sound, smell, or touch permitted males to detect and respond adaptively to rivals through increased offspring production. Vision was only of marginal importance in this context. The findings indicate adaptive redundancy through the use of multiple, but interchangeable, cues. We reveal the robust mechanisms by which males assess their socio-sexual environment to precisely attune responses via the expression of plastic behavior., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Between-individual variance in potential reproductive rate theoretically creates a load in reproducing populations by driving sexual selection of male traits for winning competitions, and female traits for resisting the costs of multiple mating. Here, using replicated experimental evolution under divergent operational sex ratios (OSR, 9:1 or 1:6 ♀:♂) we empirically identified the parallel reproductive fitness consequences for females and males in the promiscuous flour beetle Tribolium castaneum. Our results revealed clear evidence that sexual conflict resides within the T. castaneum mating system. After 20 generations of selection, females from female-biased OSRs became vulnerable to multiple mating, and showed a steep decrease in reproductive fitness with an increasing number of control males. In contrast, females from male-biased OSRs showed no change in reproductive fitness, irrespective of male numbers. The divergence in reproductive output was not explained by variation in female mortality. Parallel assays revealed that males also responded to experimental evolution: individuals from male-biased OSRs obtained 27% greater reproductive success across 7-day competition for females with a control male rival, compared to males from the female-biased lines. Subsequent assays suggest that these differences were not due to postcopulatory sperm competitiveness, but to precopulatory/copulatory competitive male mating behavior., (© 2010 The Author(s). Evolution© 2010 The Society for the Study of Evolution.)

As populations decline to levels where reproduction among close genetic relatives becomes more probable, subsequent increases in homozygous recessive deleterious expression and/or loss of heterozygote advantage can lead to inbreeding depression. Here, we measure how inbreeding across replicate lines of the flour beetle Tribolium castaneum impacts on male reproductive fitness in the absence or presence of male-male competition. Effects on male evolution from mating pattern were removed by enforcing monogamous mating throughout. After inbreeding across eight generations, we found that male fertility in the absence of competition was unaffected. However, we found significant inbreeding depression of sperm competitiveness: non-inbred males won 57 per cent of fertilizations in competition, while inbred equivalents only sired 42 per cent. We also found that the P(2) 'offence' role in sperm competition was significantly more depressed under inbreeding than sperm 'defence' (P(1)). Mating behaviour did not explain these differences, and there was no difference in the viability of offspring sired by inbred or non-inbred males. Sperm length variation was significantly greater in the ejaculates of inbred males. Our results show that male ability to achieve normal fertilization success was not depressed under strong inbreeding, but that inbreeding depression in these traits occurred when conditions of sperm competition were generated.

Spermatozoal haplotypic DNA is prone to damage, leading to male fertility problems. So far, the assessment of sperm DNA breakage has been challenging because protamines render the nuclear chromatin highly compacted. Here, we report the application of a new test to quantify DNA fragmentation in spermatozoa of an externally fertilizing teleost fish. The sperm chromatin dispersion (SCD) test uses a species-specific lysing solution to generate controlled protein depletion that, followed by DNA-specific fluorescent labelling, allows an easy morphological discrimination between nuclei affected by DNA damage. Using tench (Tinca tinca) as our model, we first trialled the test against established, but more technically demanding, assays employing in situ nick translation (ISNT) and the comet assay. The SCD test showed high concordance with ISNT, comet assay measures and a chromatin-swelling test, confirming the application of this straightforward SCD technique to various aspects of reproductive biology. Second, we examined between-male variation in DNA damage, and measured changes through time following spermatozoal activation. Between-male variation in the basal levels of average DNA damage ranged from 0 to 20% of sperm showing damage, and all showed increases in DNA fragmentation through time (0-60 min). The rates of DNA damage increase are the fastest so far recorded in sperm for a living organism, and may relate to the external fertilization mode. Our findings have relevance for broodstock selection and optimizing IVF protocols routinely used in modern aquaculture.

Although theory and widespread evidence show that the evolution of egg size is driven primarily by offspring and maternal fitness demands, an additional explanation invokes sperm limitation as a selective force that could also influence egg size optima. Levitan proposed that constraints from gamete encounter in external fertilization environments could select for enlargement of ova to increase the physical size of the fertilization target. We test this theory using in vitro fertilization experiments in an externally fertilizing fish. Sockeye salmon (Onchorhyncus nerka) females show considerable between-individual variation in ovum size, and we explored the consequences of this natural variation for the fertilization success of individual eggs under conditions of sperm limitation. By engineering consistent conditions where in vitro fertilization rate was always intermediate, we were able to compare the sizes of fertilized and unfertilized eggs across 20 fertilization replicates. After controlling for any changes in volume through incubation, results showed that successfully fertilized eggs were significantly larger than the eggs that failed to achieve fertilization. Under conditions without sperm limitation, fertility was unaffected by egg size. Our findings therefore support Levitan's theory, demonstrating empirically that some element of egg size variation could be selected by fertilization demands under sperm limitation. However, further research on sperm limitation in natural spawnings is required to assess the selective importance of these results.

Polyandry and post-copulatory sexual selection provide opportunities for the evolution of female differential sperm selection. Here, we examined the influence of variation in major histocompatibility (MH) class I allelic composition upon sperm competition dynamics in Atlantic salmon. We ran in vitro fertilization competitions that mimicked the gametic microenvironment, and replicated a paired-male experimental design that allowed us to compare differences in sperm competition success among males when their sperm compete for eggs from females that were genetically either similar or dissimilar at the MH class I locus. Concurrently, we measured variation in spermatozoal traits that are known to influence relative fertilization success under these conditions. Contrary to the findings demonstrating mechanisms that promote MH complex heterozygosity, our results showed that males won significantly greater relative fertilization success when competing for eggs from genetically similar females at the MH class I. This result also showed covariation with the known influences of sperm velocity on relative fertilization success. We discuss these unexpected findings in relation to sperm-egg recognition and hybridization avoidance mechanisms based upon immunogenetic variation.

Conflicts between and within species can drive fast evolutionary change. A recent study has documented remarkably rapid counter-adaptations in the wild to an extreme sex-ratio distortion caused by a bacterial symbiont.

Current theory to explain the adaptive significance of sex change over gonochorism predicts that female-first sex change could be adaptive when relative reproductive success increases at a faster rate with body size for males than for females. A faster rate of reproductive gain with body size can occur if larger males are more effective in controlling females and excluding competitors from fertilizations. The most simple consequence of this theoretical scenario, based on sexual allocation theory, is that natural breeding sex ratios are expected to be female biased in female-first sex changers, because average male fecundity will exceed that of females. A second prediction is that the intensity of sperm competition is expected to be lower in female-first sex-changing species because larger males should be able to more completely monopolize females and therefore reduce male-male competition during spawning. Relative testis size has been shown to be an indicator of the level of sperm competition, so we use this metric to examine evolutionary responses to selection from postcopulatory male-male competition. We used data from 116 comparable female-first sex-changing and nonhermaphroditic (gonochoristic) fish species to test these two predictions. In addition to cross-species analyses we also controlled for potential phylogenetic nonindependence by analyzing independent contrasts. As expected, breeding sex ratios were significantly more female biased in female-first sex-changing than nonhermaphroditic taxa. In addition, males in female-first sex changers had significantly smaller relative testis sizes that were one-fifth the size of those of nonhermaphroditic species, revealing a new evolutionary correlate of female-first sex change. These results, which are based on data from a wide range of taxa and across the same body-size range for either mode of reproduction, provide direct empirical support for current evolutionary theories regarding the benefits of female-first sex change.

The mangrove killifish is the only vertebrate known to have a mixed-mating strategy, where hermaphrodites reproduce by either self-fertilisation or cross-breeding. New molecular evidence from this species reveals that occasional cross-breeding between common hermaphroditic individuals and rare pure males results in an injection of genetic variation into otherwise highly homozygous 'clonal' lineages.

When close relatives are forced to reproduce, the resulting offspring inherit above average homozygosity and reduced fitness. Biologists now recognize inbreeding depression in the wild, a phenomenon that will probably increase as natural populations become depleted and fragmented. Inbreeding depression is most commonly expressed as compromised fertility and embryogenesis, but actual mechanisms remain poorly understood, especially for wild populations. Here, we examine how reduced heterozygosity influences spermatozoal and gonadal traits in wild rabbits (Oryctolagus cuniculus) sampled across the United Kingdom. By using a suite of 29 microsatellite markers (analyzed to confirm representation of individual heterozygosity across our sample), we found a significant negative relationship between heterozygosity and the production of normal sperm; the relationship was significant both between (n = 12) and within (n = 91 [total males], 42 [island], 49 [mainland]) populations. Reduced heterozygosity was also associated with decreased testis size across males (n = 112), but no relationship was seen at the population level, suggesting environmental confounds. Our results show, for a wild mammal, that inbreeding is associated with decreased sperm quality, confirming suggestions of links between inbreeding and elevated sperm abnormalities in rare felids . These findings could explain why inbreeding depression so frequently arises via compromised fertility and embryogenesis .

The P22 tailspike protein folds by forming a folding competent monomer species that forms a dimeric, then a non-native trimeric (protrimer) species by addition of folding competent monomers. We have found three residues, R549, R563, and D572, which play a critical role in both the stability of the native tailspike protein and assembly and maturation of the protrimer. King and colleagues reported previously that substitution of R563 to glutamine inhibited protrimer formation. We now show that the R549Q and R563K variants significantly delay the protrimer-to-trimer transition both in vivo and in vitro. Previously, variants that destabilize intermediates have shown wild-type chemical stability. Interestingly, both the R549Q and R563K variants destabilize the tailspike trimer in guanidine denaturation studies, indicating that they represent a new class of tailspike folding variants. R549Q has a midpoint of unfolding at 3.2M guanidine, compared to 5.6M for the wild-type tailspike protein, while R563K has a midpoint of unfolding of 1.8 M. R549Q and R563K also denature over a broader pH range than the wild-type tailspike protein and both proteins have increased sensitivity to pH during refolding, suggesting that both residues are involved in ionic interactions. Our model is that R563 and D572 interact to stabilize the adjacent turn, aiding the assembly of the dimer and protrimer species. We believe that the interaction between R563 and D572 is also critical following assembly of the protrimer to properly orient D572 in order to form a salt bridge with R549 during protrimer maturation.

Female redback spiders cannibalise mating males. New research has shown they have evolved two separate sperm storage organs, allowing them to make post-copulatory paternity choices. Counter-adaptation by males, to maximise paternity after cannibalism, has led to the evolution of an abdominal constricting mechanism, which enhances short-term survival and mating success after attack by the female and maximises the chances of inseminating both sperm storage sites.

Research on seminal fluid proteins is providing fundamental insights into the interactive evolution of male and female reproductive strategies. Two new studies demonstrate, first, how an influential male sex peptide in Drosophila is delivered to the female bound directly onto sperm cells, and second, that its subsequent release has significant reproductive costs for females.