Biologics in rotator cuff repair.

Rotator cuff tears are common in middle-aged and elderly patients. Despite advances in the surgical repair of rotator cuff tears, the rates of recurrent tear remain high. This may be due to the complexity of the tendons of the rotator cuff, which contributes to an inherently hostile healing environment. During the past 20 years, there has been an increased interest in the use of biologics to complement the healing environment in the shoulder, in order to improve rotator cuff healing and reduce the rate of recurrent tears. The aim of this review is to provide a summary of the current evidence for the use of forms of biological augmentation when repairing rotator cuff tears.

Lower Extremity Trauma is Associated With an Increased Rate of New Mental Disorder Diagnosis and Suicide Attempt.

OBJECTIVES: To evaluate the risk of developing a new mental disorder diagnosis within 2 years of lower extremity fracture. DESIGN: Retrospective cohort study. SETTING: National insurance claims database. PATIENT SELECTION CRITERIA: Patients between 18 and 65 years with lower extremity, pelvis, and acetabular fractures without prior mental disorders as defined using International Classification of Diseases, 9th and 10th revision diagnosis codes were included. Mental disorders evaluated included alcohol use disorder, generalized anxiety disorder, bipolar disorder, major depressive disorder, drug use disorder, panic disorder, posttraumatic stress disorder, and suicide attempt. OUTCOME MEASURES AND COMPARISONS: The individual lower extremity fracture cohorts were matched 1:4 with nonfracture controls. The specific groups of interest were pelvis fractures, acetabulum fractures, proximal femur fractures, femoral shaft fractures, distal femur fractures, patella fractures, tibia plateau fractures, tibia shaft fractures, ankle fractures, pilon fractures, calcaneus fractures, and Lisfranc fractures. Rates of mental disorders after primary lower extremity fractures within 2 years were compared using multivariable logistic regression. RESULTS: Overall, the 263,988 patient-fracture group was 57.2% female with an average age of 46.6 years. Compared with controls with no fracture, patients who sustained pelvis, acetabulum, proximal femur, femoral shaft, distal femur, patella, tibia plateau, tibia shaft, pilon, calcaneus, or Lisfranc fracture had a statistically significantly increased risk of being diagnosed with a queried mental disorder within 2 years of fracture. When comparing all fracture patients by location, those suffering from fractures proximal to the knee joint, including pelvis fractures [OR: 1.51, 95% confidence interval (CI): 1.39-1.64] and proximal femur fractures [odds ratio (OR): 1.36, 95% CI: 1.26-1.47], demonstrated greater risk of developing any of the queried mental disorders compared with fractures distal to the knee, including ankle fractures (OR: 0.99, 95% CI: 0.95-1.03) and pilon fractures (OR: 1.05, 95% CI: 0.81-1.36). When comparing specific fracture patients with patients without fracture by mental disorder, patients demonstrated an increased risk of suicide attempt following fracture of the pelvis, acetabulum, femoral shaft, distal femur, and calcaneus, as well as patients sustaining a Lisfranc fracture. CONCLUSIONS: There is an increased risk of being diagnosed with a new mental disorder following lower extremity trauma in patients without prior mental disorder diagnosis compared with matched individuals without a lower extremity fracture. Among the fractures studied, those that were more proximal, such as pelvis and proximal femur fractures, carried greater risk compared with more distal fracture sites, including ankle and pilon fractures. Patients who experienced certain lower extremity fractures had a significantly higher rate of suicide attempt compared with patients without fracture. Physicians should consider increased mental health screening and potential referral for mental health evaluation for patients following lower extremity trauma. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Smokeless Tobacco Use is Associated with Worse Outcomes Following Open Tibia Fracture.

Background: Smoking is a known risk factor for complications after open tibia fractures, but it is unclear if smokeless tobacco confers a similar risk. Objective: The purpose of this study was to evaluate the rates of postoperative complications after surgical fixation of open tibia fractures in smokeless tobacco users as compared to matched tobacco naïve controls. Methods: A retrospective cohort study was conducted using the national PearlDiver database. For patients who had a unilateral open tibia fracture, smokeless tobacco users (n=121) and tobacco-naïve controls (n=242) were matched at a 1:2 ratio. Rates of bone complications within 1 year were compared using multivariable logistic regression. Results: Compared to controls, the smokeless tobacco user cohort demonstrated significantly higher rates of nonunion (OR: 3.42, 95% CI: 1.05 - 11.12), infection / inflammatory reaction of implant (OR: 12.45, 95% CI: 2.67 - 58.93), and osteomyelitis of the leg (OR: 6.15, 95% CI: 1.90 - 19.93) within 1 year of open tibia fracture. Compared to the most recent population figures, smokeless tobacco use was significantly under-reported in both males and females. Conclusions: Smokeless tobacco use confers an increased risk of fracture nonunion, infection or inflammatory response to implant, and osteomyelitis of the leg in patients with open tibia fractures. Surgeons should consider this risk when counseling patients and deciding on treatment plans for patients with tibia fractures.

The Jarring Reality: Adolescent and Pediatric Gunshot Trauma on the Rise in a Major Urban Institution.

INTRODUCTION: Firearms are the leading cause of death in the pediatric population, and the incidence of firearm injury is on the rise in the United States. The purpose of this study was to examine the incidence of pediatric gunshot wounds (GSWs) in New Orleans from 2012 to 2023 and evaluate factors that contribute to mortality. METHODS: A retrospective analysis of pediatric GSWs was conducted using the trauma database at a Level I trauma center in New Orleans, Louisiana. All patients aged 0 to 18 years with a GSW between January 1, 2012, and January 1, 2023, were evaluated. Demographic data, Injury Severity Score (ISS), hospital length of stay (LOS), discharge disposition, intent of injury, mortality, firearm type, orthopaedic injuries, and related surgical procedures were extracted from the database. Linear regression was used to assess the trend of GSWs over time, and logistic regression was used to identify variables that predicted mortality. Statistical significance was defined as α = 0.05. Geographic information system (GIS) mapping was conducted using the ZIP code location of injury to identify geographic areas with the greatest number of GSWs. RESULTS: A significant increasing trend was observed in the number of pediatric GSWs when adjusted for changes in population ( P = 0.014), and the number of GSWs increased 43.2% over the duration of the study. Higher ISS, shorter length of stay, and self-inflicted intent of injury were notable predictors of mortality. Three hundred fifty-four patients (31.4%) had at least one orthopaedic injury, and of the survivors, 365 patients (35.6%) required at least one surgical procedure in the operating room during their admission. DISCUSSION: The number of GSWs in the pediatric population in New Orleans presenting at a large level 1 trauma center demonstrated a statistically significant increasing trend over the past 11 years. ISS and self-inflicted intent were predictive of mortality within this pediatric patient population.

Association of Smokeless Tobacco and Complications Following Ankle and Hindfoot Arthrodesis.

BACKGROUND: The purpose of this study was to (1) evaluate the rates of postoperative complications following ankle or hindfoot arthrodesis among current smokeless tobacco users and people who smoke compared to matched controls, and (2) compare rates of postoperative complications in current smokeless tobacco users vs people who smoke tobacco cigarettes. METHODS: A retrospective cohort study was conducted using a large national database. For patients who underwent ankle or hindfoot arthrodesis, smokeless tobacco users (n = 131) and people who smoke (n = 1948) were matched 1:4 with controls, and smokeless tobacco users (n = 131) were matched 1:4 with people who smoke tobacco cigarettes (n = 524). Orthopaedic complications within 90 days, 1 year, and 2 years were compared using multivariable logistic regressions. RESULTS: Within 90 days of ankle or hindfoot arthrodesis, smokeless tobacco users demonstrated significantly higher rates of hardware removal (odds ratio [OR] 5.01, 95% CI 1.65-15.20), wound disruption or dehiscence (OR 3.00, 95% CI 1.21-7.44), and pooled complications (16.0% vs 5.9%, OR 2.84, 95% CI 1.50-5.38) compared with tobacco-naïve controls. The rates of hardware removal, wound disruption and dehiscence, and pooled complications remained significant in the smokeless tobacco cohort at 1 and 2 years. At 2 years following ankle or hindfoot arthrodesis, smokeless tobacco users additionally demonstrated significantly higher rates of infection (OR 6.08, 95% CI 1.15-32.05) and nonunion (OR 2.35, 95% CI 1.31-4.20) compared with tobacco-naïve controls. Within 90 days of ankle or hindfoot arthrodesis, smokeless tobacco users demonstrated significantly lower rates of malunion or nonunion than patients who smoke tobacco cigarettes (OR 0.34, 95% CI 0.12-0.97), whereas all other complications were not significantly different. CONCLUSION: Smokeless tobacco use is associated with higher rates of complications following ankle or hindfoot arthrodesis compared with tobacco-naïve controls, and physicians should screen for smokeless tobacco use specifically and encourage cessation before operating electively. LEVEL OF EVIDENCE: Level III, retrospective cohort study.

The push-pull intercrop Desmodium does not repel, but intercepts and kills pests.

Over two decades ago, an intercropping strategy was developed that received critical acclaim for synergizing food security with ecosystem resilience in smallholder farming. The push-pull strategy reportedly suppresses lepidopteran pests in maize through a combination of a repellent intercrop (push), commonly Desmodium spp., and an attractive, border crop (pull). Key in the system is the intercrop's constitutive release of volatile terpenoids that repel herbivores. However, the earlier described volatile terpenoids were not detectable in the headspace of Desmodium, and only minimally upon herbivory. This was independent of soil type, microbiome composition, and whether collections were made in the laboratory or in the field. Furthermore, in oviposition choice tests in a wind tunnel, maize with or without an odor background of Desmodium was equally attractive for the invasive pest Spodoptera frugiperda. In search of an alternative mechanism, we found that neonate larvae strongly preferred Desmodium over maize. However, their development stagnated and no larva survived. In addition, older larvae were frequently seen impaled and immobilized by the dense network of silica-fortified, non-glandular trichomes. Thus, our data suggest that Desmodium may act through intercepting and decimating dispersing larval offspring rather than adult deterrence. As a hallmark of sustainable pest control, maize-Desmodium push-pull intercropping has inspired countless efforts to emulate stimulo-deterrent diversion in other cropping systems. However, detailed knowledge of the actual mechanisms is required to rationally improve the strategy, and translate the concept to other cropping systems.

Incontinence Is an Independent Risk Factor for Total Hip and Knee Arthroplasty.

Background: Urinary incontinence has been linked to worse postoperative pain, decreased physical function, and reduced quality of life in patients following total joint arthroplasty. The purpose of this study was to analyze whether incontinence is associated with increased postoperative medical and joint complications following primary total hip arthroplasty (THA) and total knee arthroplasty (TKA). Methods: A retrospective cohort study was conducted using a national insurance database. Thirty-two thousand eight hundred eleven patients with incontinence who underwent primary THA were identified and matched 1:4 with 129,073 patients without incontinence. Ninety-one thousand nine hundred thirty-five patients with incontinence who underwent primary TKA were matched 1:4 with 367,285 patients without incontinence. Medical and joint complication rates at 90 days and 2 years, respectively, were then compared for patient cohorts using multivariable logistic regressions. Results: Patients who underwent primary THA with incontinence had statistically higher rates of dislocation, periprosthetic fracture, aseptic revisions, and overall joint complications compared to controls. Patients who underwent primary TKA with incontinence had higher rates of mechanical failure, aseptic revision, and all-cause revision compared to controls. Conclusions: This study demonstrated an association between patients with incontinence and higher rates of dislocation, periprosthetic fractures, aseptic revisions, and overall joint complications following primary THA compared to controls. Patients with incontinence experience higher rates of mechanical failure, aseptic revision, and all-cause revision following TKA compared to controls. As such, perioperative management of urinary incontinence may help mitigate the risk of postoperative complications.

Machine learning-aided design and screening of an emergent protein function in synthetic cells.

Recently, utilization of Machine Learning (ML) has led to astonishing progress in computational protein design, bringing into reach the targeted engineering of proteins for industrial and biomedical applications. However, the design of proteins for emergent functions of core relevance to cells, such as the ability to spatiotemporally self-organize and thereby structure the cellular space, is still extremely challenging. While on the generative side conditional generative models and multi-state design are on the rise, for emergent functions there is a lack of tailored screening methods as typically needed in a protein design project, both computational and experimental. Here we describe a proof-of-principle of how such screening, in silico and in vitro, can be achieved for ML-generated variants of a protein that forms intracellular spatiotemporal patterns. For computational screening we use a structure-based divide-and-conquer approach to find the most promising candidates, while for the subsequent in vitro screening we use synthetic cell-mimics as established by Bottom-Up Synthetic Biology. We then show that the best screened candidate can indeed completely substitute the wildtype gene in Escherichia coli. These results raise great hopes for the next level of synthetic biology, where ML-designed synthetic proteins will be used to engineer cellular functions.

Adult Reconstruction Surgeons Manage Patients With Higher Medical Complexities and Still Achieve Comparable Outcomes to Sports Medicine Surgeons Following Total Knee Arthroplasty.

Background: Orthopaedic surgeons who are fellowship-trained in adult reconstruction (AR) specialize specifically in total joint arthroplasty, including total knee arthroplasty (TKA). However, TKA procedures are not only performed by AR surgeons. The purpose of this study was to compare the patient demographics and postoperative outcomes of patients who had a TKA procedure performed by an AR surgeon vs a sports medicine (SM) surgeon. Methods: A retrospective cohort study was conducted using a national insurance database. Patients who underwent a primary elective TKA procedure by an AR surgeon (n = 56,570) and an SM surgeon (n = 72,888) were identified. Patient demographics, rates of joint complications within 2 years, and medical complications within 90 days postoperatively were compared using multivariable logistic regression. Results: Compared to the cohort of patients undergoing TKA by SM surgeons, the patient cohort of AR surgeons had a higher mean Elixhauser comorbidity index (4.2 vs 4.0, P < .001), and had significantly higher rates of several comorbidities. Within 90 days, patients of AR surgeons demonstrated significantly lower rates of acute kidney injury and transfusions. When compared to patients of SM surgeons, patients of AR surgeons demonstrated significantly lower rate of manipulation under anesthesia or lysis of adhesions within 2 years. Rates of all other joint-related complications were statistically comparable between the 2 cohorts. Conclusions: As a cohort, AR surgeons perform TKA on a higher-risk cohort of patients compared to sports medicine surgeons. Despite the higher-risk patient population, outcomes of TKA by AR surgeons appear equivalent compared to their SM colleagues.

Similar Outcomes Are Found Between Quadriceps Tendon Repair With Transosseous Tunnels and Suture Anchors: A Systematic Review and Meta-Analysis.

Purpose: To evaluate the clinical outcomes and biomechanical performance of transosseous tunnels compared with suture anchors for quadriceps tendon repair. Methods: In accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, a systematic search was performed in April 2021 in the following databases: Cochrane Database of Systematic Reviews, PubMed (1980-2021), MEDLINE (1980-2021), Embase (1980-2021), and CINAHL (1980-2021). Level I-IV studies were included if they provided outcome data for surgical repair of the quadriceps tendon using transosseous tunnels or suture anchors with minimum 1-year follow-up. Biomechanical studies comparing transosseous tunnels and suture anchors were separately analyzed. Results: The systematic search yielded 1,837 citations, 23 of which met inclusion criteria (18 clinical, 5 biomechanical). In total, 13 studies reported results for transosseous repair and 7 studies reported results for repair with suture anchors. There were results for 508 patients from clinical studies. The average postoperative Lysholm score ranged from 88 to 92 for suture anchor repairs and 72.8 to 94 for transosseous repairs with range of motion ranging from 117° to 138° and 116° to 135°, respectively. Synthesis of the biomechanical data revealed the mean difference in load to failure was not significant between constructs (137.21; 95% confidence interval -10.14 to 284.57 N; P = .068). Conclusions: Transosseous and suture anchor techniques for quadriceps tendon repair result in similar biomechanical and postoperative outcomes. No difference between techniques in regard to ultimate load to failure among comparative biomechanical studies were observed. Level of Evidence: Level IV, systematic review level III-IV studies.

Efficacy and Safety of an Extravascular Implantable Cardioverter-Defibrillator.

BACKGROUND: The extravascular implantable cardioverter-defibrillator (ICD) has a single lead implanted substernally to enable pause-prevention pacing, antitachycardia pacing, and defibrillation energy similar to that of transvenous ICDs. The safety and efficacy of extravascular ICDs are not yet known. METHODS: We conducted a prospective, single-group, nonrandomized, premarket global clinical study involving patients with a class I or IIa indication for an ICD, all of whom received an extravascular ICD system. The primary efficacy end point was successful defibrillation at implantation. The efficacy objective would be met if the lower boundary of the one-sided 97.5% confidence interval for the percentage of patients with successful defibrillation was greater than 88%. The primary safety end point was freedom from major system- or procedure-related complications at 6 months. The safety objective would be met if the lower boundary of the one-sided 97.5% confidence interval for the percentage of patients free from such complications was greater than 79%. RESULTS: A total of 356 patients were enrolled, 316 of whom had an implantation attempt. Among the 302 patients in whom ventricular arrhythmia could be induced and who completed the defibrillation testing protocol, the percentage of patients with successful defibrillation was 98.7% (lower boundary of the one-sided 97.5% confidence interval [CI], 96.6%; P<0.001 for the comparison with the performance goal of 88%); 299 of 316 patients (94.6%) were discharged with a working ICD system. The Kaplan-Meier estimate of the percentage of patients free from major system- or procedure-related complications at 6 months was 92.6% (lower boundary of the one-sided 97.5% CI, 89.0%; P<0.001 for the comparison with the performance goal of 79%). No major intraprocedural complications were reported. At 6 months, 25 major complications were observed, in 23 of 316 patients (7.3%). The success rate of antitachycardia pacing, as assessed with generalized estimating equations, was 50.8% (95% CI, 23.3 to 77.8). A total of 29 patients received 118 inappropriate shocks for 81 arrhythmic episodes. Eight systems were explanted without extravascular ICD replacement over the 10.6-month mean follow-up period. CONCLUSIONS: In this prospective global study, we found that extravascular ICDs were implanted safely and were able to detect and terminate induced ventricular arrhythmias at the time of implantation. (Funded by Medtronic; ClinicalTrials.gov number, NCT04060680.).

Tracing back variations in archaeal ESCRT-based cell division to protein domain architectures.

The Endosomal Sorting Complex Required for Transport (ESCRT) system is a multi-protein machinery that is involved in cell division of both Eukaryotes and Archaea. This spread across domains of life suggests that a precursor ESCRT machinery existed already at an evolutionary early stage of life, making it a promising candidate for the (re)construction of a minimal cell division machinery. There are, however, only few experimental data about ESCRT machineries in Archaea, due to high technical challenges in cultivation and microscopy. Here, we analyse the proteins of ESCRT machineries in archaea bioinformatically on a protein domain level, to enable mechanistical comparison without such challenging experiments. First, we infer that there are at least three different cell division mechanisms utilizing ESCRT proteins in archaea, probably similar in their constriction mechanisms but different in membrane tethering. Second, we show that ESCRT proteins in the archaeal super-phylum Asgard are highly similar to eukaryotic ESCRT proteins, strengthening the recently developed idea that all Eukaryotes descended from archaea. Third, we reconstruct a plausible evolutionary development of ESCRT machineries and suggest that a simple ESCRT-based constriction machinery existed in the last archaeal common ancestor. These findings not only give very interesting insights into the likely evolution of cell division in Archaea and Eukaryotes, but also offer new research avenues by suggesting hypothesis-driven experiments for both, cell biology and bottom-up synthetic biology.

Hidden protein functions and what they may teach us.

Bottom-up synthetic biology is a new research field with the goal of constructing living systems from a minimal number of functional components. The key challenges are, first, to identify a necessary canon of functions for a system to be considered alive, and second, to reconstitute these respective modules in vitro. When using proteins as obvious candidates, it appears that not only some of their described physiological functions fail to unfold outside the cellular context, but that completely new and unexpected functions are being observed. We put these insights in the context of other recent findings on protein functionality and discuss their potential role in the emergence and evolution of life.

Developmentally regulated volatiles geosmin and 2-methylisoborneol attract a soil arthropod to Streptomyces bacteria promoting spore dispersal.

Volatile compounds emitted by bacteria are often sensed by other organisms as odours, but their ecological roles are poorly understood1,2. Well-known examples are the soil-smelling terpenoids geosmin and 2-methylisoborneol (2-MIB)3,4, which humans and various animals sense at extremely low concentrations5,6. The conservation of geosmin biosynthesis genes among virtually all species of Streptomyces bacteria (and genes for the biosynthesis of 2-MIB in about 50%)7,8, suggests that the volatiles provide a selective advantage for these soil microbes. We show, in the present study, that these volatiles mediate interactions of apparent mutual benefit between streptomycetes and springtails (Collembola). In field experiments, springtails were attracted to odours emitted by Streptomyces colonies. Geosmin and 2-MIB in these odours induce electrophysiological responses in the antennae of the model springtail Folsomia candida, which is also attracted to both compounds. Moreover, the genes for geosmin and 2-MIB synthases are under the direct control of sporulation-specific transcription factors, constraining emission of the odorants to sporulating colonies. F. candida feeds on the Streptomyces colonies and disseminates spores both via faecal pellets and through adherence to its hydrophobic cuticle. The results indicate that geosmin and 2-MIB production is an integral part of the sporulation process, completing the Streptomyces life cycle by facilitating dispersal of spores by soil arthropods.

Ecological and Phylogenetic Relationships Shape the Peripheral Olfactory Systems of Highly Specialized Gall Midges (Cecidomiiydae).

Insects use sensitive olfactory systems to detect relevant host volatiles and avoid unsuitable hosts in a complex environmental odor landscape. Insects with short lifespans, such as gall midges (Diptera: Cecidomyiidae), are under strong selection pressure to detect and locate suitable hosts for their offspring in a short period of time. Ephemeral gall midges constitute excellent models for investigating the role of olfaction in host choice, host shift, and speciation. Midges mate near their site of emergence and females migrate in order to locate hosts for oviposition, thus females are expected to be more responsive to olfactory cues emitted by the host compared to males. In this study, we explored the correlation between host choice and the function of the peripheral olfactory system in 12 species of gall midges, including species with close phylogenetic relationships that use widely different host plants and more distantly related gall midge species that use similar hosts. We tested the antennal responses of males and females of the 12 species to a blend of 45 known insect attractants using coupled gas chromatographic-electroantennographic detection. When the species-specific response profiles of the gall midges were compared to a newly generated molecular-based phylogeny, we found they responded to the compounds in a sex- and species-specific manner. We found the physiological response profiles of species that use annual host plants, and thus have to locate their host every season, are similar for species with similar hosts despite large phylogenetic distances. In addition, we found closely related species with perennial hosts demonstrated odor response profiles that were consistent with their phylogenetic history. The ecology of the gall midges affects the tuning of the peripheral olfactory system, which in turn demonstrates a correlation between olfaction and speciation in the context of host use.

Complete Genome Sequence of Cluster J Mycobacteriophage Superphikiman.

Mycobacteriophage Superphikiman is a cluster J bacteriophage which was isolated from soil collected in Philadelphia, PA. Superphikiman has a 109,799-bp genome with 239 predicted genes, including 2 tRNA genes.

Sonographic pictorial review of benign palpable paediatric lumps: head to foot.

No abstract available.

A novel interaction linking the FAS-II and phthiocerol dimycocerosate (PDIM) biosynthetic pathways.

The fatty acid biosynthesis (FAS-II) pathway in Mycobacterium tuberculosis generates long chain fatty acids that serve as the precursors to mycolic acids, essential components of the mycobacterial cell wall. Enzymes in the FAS-II pathway are thought to form one or more noncovalent multi-enzyme complexes within the cell, and a bacterial two-hybrid screen was used to search for missing components of the pathway and to furnish additional data on interactions involving these enzymes in vivo. Using the FAS-II beta-ketoacyl synthase, KasA, as bait, an extensive bacterial two-hybrid screen of a M. tuberculosis genome fragment library unexpectedly revealed a novel interaction between KasA and PpsB as well as PpsD, two polyketide modules involved in the biosynthesis of the virulence lipid phthiocerol dimycocerosate (PDIM). Sequence analysis revealed that KasA interacts with PpsB and PpsD in the region of the acyl carrier domain of each protein, raising the possibility that lipids could be transferred between the FAS-II and PDIM biosynthetic pathways. Subsequent studies utilizing purified proteins and radiolabeled lipids revealed that fatty acids loaded onto PpsB were transferred to KasA and also incorporated into long chain fatty acids synthesized using a Mycobacterium smegmatis lysate. These data suggest that in addition to producing PDIMs, the growing phthiocerol product can also be shuttled into the FAS-II pathway via KasA as an entry point for further elongation. Interactions between these biosynthetic pathways may exist as a simple means to increase mycobacterial lipid diversity, enhancing functionality and the overall complexity of the cell wall.

Probing mechanisms of resistance to the tuberculosis drug isoniazid: Conformational changes caused by inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis.

The frontline tuberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis (FAS-II) enoyl reductase from Mycobacterium tuberculosis (MTB), via formation of a covalent adduct with NAD(+) (the INH-NAD adduct). Resistance to INH can be correlated with many mutations in MTB, some of which are localized in the InhA cofactor binding site. While the InhA mutations cause a substantial decrease in the affinity of InhA for NADH, surprisingly the same mutations result in only a small impact on binding of the INH-NAD adduct. Based on the knowledge that InhA interacts in vivo with other components of the FAS-II pathway, we have initiated experiments to determine whether enzyme inhibition results in structural changes that could affect protein-protein interactions involving InhA and how these ligand-induced conformational changes are modulated in the InhA mutants. Significantly, while NADH binding to wild-type InhA is hyperbolic, the InhA mutants bind the cofactor with positive cooperativity, suggesting that the mutations permit access to a second conformational state of the protein. While cross-linking studies indicate that enzyme inhibition causes dissociation of the InhA tetramer into dimers, analytical ultracentrifugation and size exclusion chromatography reveal that ligand binding causes a conformational change in the protein that prevents cross-linking across one of the dimer-dimer interfaces in the InhA tetramer. Interestingly, a similar ligand-induced conformational change is also observed for the InhA mutants, indicating that the mutations modulate communication between the subunits without affecting the two conformational states of the protein that are present.

Activation of the JAK/STAT pathway in vascular smooth muscle by serotonin.

Serotonin (5-hydroxytryptamine, 5-HT) is a vasoconstrictor and mitogen whose levels are elevated in diabetes. Previous studies have shown the presence of 5-HT2A, 5-HT2B, and 5-HT1B receptors in vascular smooth muscle cells (VSMCs). There are currently no data regarding 5-HT2B and 5-HT1B receptor activation of the JAK/STAT pathway in VSMCs and resultant potential alterations in 5-HT signaling in diabetes. Therefore, we tested the hypothesis that 5-HT differentially activates the JAK/STAT pathway in VSMCs under conditions of normal (5 mM) and high (25 mM) glucose. Treatment of rat VSMCs with 5-HT (10(-6) M) resulted in time-dependent activation ( approximately 2-fold) of JAK2, JAK1, and STAT1, but not STAT3 (maximal at 5 min, returned to baseline by 30 min). The 5-HT2B receptor agonist BW723C86 and the 5-HT1B receptor agonist CGS12066A (10(-9)-10(-5) M, 5-min stimulation) did not activate the JAK/STAT pathway. Treatment with the 5-HT2A receptor antagonist ketanserin (10 nM) inhibited JAK2 activation by 5-HT. Treatment of streptozotocin-induced diabetic rats with ketanserin (5 mg.kg-1.day-1) reduced activation of JAK2 and STAT1 but not STAT3 in endothelium-denuded thoracic aorta in vivo. 5-HT (10(-6) M) treatment resulted in increased cell proliferation and increased DNA synthesis, which were inhibited by the JAK2 inhibitor AG490. Further studies with apocynin, diphenyleneiodonium chloride, catalase, and virally transfected superoxide dismutase had no effect at either glucose concentration on activation of the JAK/STAT pathway by 5-HT. Therefore, we conclude that 5-HT activates JAK2, JAK1, and STAT1 via the 5-HT2A receptors in a reactive oxygen species-independent manner under both normal and high glucose conditions.