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Abstract Purpose To identify the proportion of Emergency Department (ED) visits in cancer patients associated with a mental and substance use disorder (MSUD) and the subsequent healthcare costs. Methods Nationally representative data on ED visits from 2009 to 2018 was obtained from the Nationwide Emergency Department Sample (NEDS). We identified cancer‐related visits with or without a MSUD using the Clinical Classifications Software diagnoses documented during the ED visit. Survey‐adjusted frequencies and proportions of ED visits among adult cancer patients with or without a MSUD was evaluated. Survey‐adjusted multivariable logistic regression models were used to examine demographic and clinical predictors of the presence of an MSUD and the likelihood of hospital admission for patients with a primary MSUD. Results Among 54,004,462 ED visits with a cancer diagnosis between 2009 and 2018, 11,803,966 (22%) were associated with a MSUD. Compared to a primary diagnosis of cancer, patients who presented to the ED with a chief complaint of MSUD were more likely to be female (54% vs. 49%), younger (median: 58 vs. 66), more likely to have Medicaid insurance, and more likely to be discharged home. The three most common MSUD diagnoses among cancer patients were alcohol‐related disorders, anxiety disorders, and depressive disorders. The total costs associated with a primary MSUD from 2009 to 2018 was $3,133,432,103, of which alcohol‐related disorders claimed the largest majority. Younger age (OR per 10‐year increase: 0.86, 95% CI: 0.85, 0.86) and female sex (OR: 1.34, 95% CI: 1.33–1.35) were associated with higher odds of having an MSUD. Conclusions Our findings demonstrate a high burden of psychiatric and substance use illness in the cancer population and provide the rationale for early psychosocial intervention to support these patients.

Introduction: Neonatal sepsis could be defined as a clinical entity because of generalized bacterial infection within 28 days of life and showing a positive blood culture. It is probably responsible for 30- 50% of the total neonatal deaths each year. Timely diagnosis of neonatal sepsis is critical because in neonates the illness can progress more rapidly than adults. The blood culture report takes at least 72 hours. Therefore, a simple test with quick availability of results can be helpful to reduce neonatal morbidity and mortality. Aim: To evaluate the utility of sepsis screen in early diagnosis of neonatal septicemia and to study various hematological parameters, changes in peripheral blood smear, evaluate the performance of microerythrocyte sedimentation rate, serum C-reactive protein and serum direct bilirubin in neonates with clinical suspicion of sepsis. Materials and Methods: The present, study was done in our institute from October 2013 to October 2015. CBC was done on 191 clinically suspected cases of neonatal sepsis along with Micro ESR, Serum CRP and direct bilirubin. Differential leukocyte count, absolute neutrophil count, immature neutrophils: total neutrophils ratio was done from Field stained peripheral smears. Blood culture was done in Microbiology Department. Exclusion criteria were neonates with major congenital anomalies and those who have already received antibiotics. Statistical Analysis: Statistical analysis was done using SPSS software, version 20th and unpaired ‘t’- test. Result: Out of 191 cases studied, 91 were culture positive. CRP (84.6%) and immature : total neutrophils ratio(75.8%) showed highest sensitivity, Whereas absolute neutrophil count(99.0%) along with serum direct bilirubin (93.0%) and corrected total leucocyte count (93.0%)showed highest specificity. Positive predictive value was highest for absolute neutrophil count (97.5%) and CRP (84.8%). Conclusion: Serum CRP is the most sensitive marker of sepsis. Use of peripheral smear study, serum direct bilirubin and micro ESR together with CRP can be used effectively as a sepsis screen for early diagnosis of neonatal sepsis.

Introduction: The humeral shaft gap non-union treatment surgically after unsuccessful fixation is difficult. In this study, the functional outcome of unsuccessful fixation of humeral shaft gap non-unions using a locking compression plate (LCP), fibular strut graft, and iliac bone graft was evaluated., Case Report: After taking ethical approval, we studied 10 patients with humeral shaft gap non-unions with unsuccessful surgical fixation treated with open reduction and internal fixation using LCP with fibular strut graft and iliac cancellous bone graft. The study duration was from January 2022-January 2024. The mean time from non-union was 7 months. Disabilities of the arm, shoulder, and hand (DASH) scores improved significantly from a pre-operative average of 59.2 ± 7.3 to a mean final score of 24.6 ± 4.8 reflecting a mean improvement of 34.6 with no complications., Conclusion: This study shows that LCP with fibular strut graft and iliac bone grafts is a good alternative for diaphyseal humerus gap non-union along with statistically significant improvement in functional outcome, with union seen in all cases. This construct augments biomechanical stability and good biological healing in these gap non-unions., Competing Interests: Conflict of Interest: Nil, (Copyright: © Indian Orthopaedic Research Group.)

Introduction: There is a paucity of literature regarding a neglected shoulder dislocation, as it is unusual to miss it clinically due to the apparent deformity. Nevertheless, in some cases, particularly those who receive primary treatment from a local bonesetter, they may present with a neglected shoulder dislocation. In the absence of comprehensive studies, health-care professionals have to resort to lower-tier evidence and practical experience to guide their treatment decisions. Therefore, most of the treatment recommendations are based on level four studies and the literature for recurrent dislocation of the shoulder., Case Report: We have described three cases of neglected anterior dislocation of the shoulders in two patients, which were managed by open reduction, Latarjet procedure, remplissage, and rotator cuff repair. Both of our patients after 1-year follow-up had a painless joint with an improved functional range of motion. This case discussion contributes to understanding the approach to treating these patients., Conclusion: Recurrent shoulder joint instability with bone loss in the younger and older age groups has to be managed differently. Based on this case report involving individuals older than 50 years, it can be inferred that the approach to managing neglected locked shoulder dislocations with off-track lesions is with open reduction and fixation with Latarjet procedure, coupled with RCR and remplissage, has yielded adequate joint stability and favorable post-operative outcomes., Competing Interests: Conflict of Interest: Nil, (Copyright: © Indian Orthopaedic Research Group.)

Introduction: Securing stable internal fixation for fractures in osteoporotic intra-articular distal femur proves to be a demanding task due to thin cortices, a wide medullary canal, diminished bone stock, and fracture comminution. No singular therapeutic approach has successfully tackled all facets of this injury. Consequently, we now introduce a pioneering fixation method in our report, aiming to offer a holistic solution to the intricate challenges associated with this scenario., Case Report: A 60-year-old female presented with an intra-articular distal femur fracture, and underwent a combination fixation of distal femur plate and intramedullary interlocking nailing. The patient was rehabilitated with active knee range of motion on post-operative day 7 and has now attained full knee range of motion., Conclusion: The utilization of anatomical plates with locking mechanisms, in tandem with intramedullary interlocking nailing, holds promise for the secure stabilization and fixation of osteoporotic distal femur fractures, potentially leading to an expedited recovery process., Competing Interests: Conflict of Interest: Nil, (Copyright: © Indian Orthopaedic Research Group.)

Competing Interests: Conflict of Interest: Nil

Background: Surgical resection is a necessary component of curative-intent treatment for most solid-organ cancers but is occasionally aborted, most often due to occult metastatic disease or unanticipated unresectability. Despite its frequency, little research has been performed on the experiences, care needs, and treatment preferences of patients who experience an aborted cancer surgery., Methods: Semistructured interviews of patients who had previously experienced an aborted cancer surgery were conducted, focusing on their recalled experiences and stated preferences. All interviews were audio recorded, transcribed, and coded by two independent researchers by using NVivo 12. An integrative approach to qualitative analysis was used-both inductive and deductive methods-and iteratively identifying themes until saturation was reached., Results: Fifteen patients with an aborted cancer surgery participated in the interviews. Cancer types included pancreatic (n = 9), cholangiocarcinoma (n = 3), hepatocellular carcinoma (n = 1), gallbladder (n = 1), and neuroendocrine (n = 1). The most common reasons for aborting surgery included local tumor unresectability (n = 8) and occult metastatic disease (n = 7). Five subthemes that characterized the patient experience following an aborted cancer surgery emerged, including physical symptoms, emotional responses, impact on social and life factors, coping mechanisms, and support received., Conclusions: This qualitative study characterizes the impact of aborted cancer surgery on multiple domains of quality of life: physical, emotional, social, and existential. These results highlight the importance of developing patient-centered interventions that focus on enhancing quality of life after aborted cancer surgery., (© 2023. Society of Surgical Oncology.)

Purpose: Image-based Robotic Total knee Arthroplasty (RA-TKA)was developed with the purpose of enhancing the accuracy in determining the component sizes preoperatively and helping surgeons in minimizing errors and improve patient outcomes. The research aims to find the reliability of robotic-assisted TKR based on images in determining the correct component sizes using preoperative three-dimensional (3D) computer tomography., Method: After ethical approval, we conducted a prospective study from March 2022 to December 2022. A total of 100 knees underwent image-based RA-TKA having grade 4 Osteoarthritis knee (Kellegren Lawrence classification). A single senior surgeon performed on all patients. Postoperative implant sizes and fit were assessed by five radiographic markers by an independent observer., Results: In our study, we found the mean age was (64.96 ± 7.3) years, with female to male ratio of 43:22. The preoperative 3D CT accuracy is 100% for femoral component sizing and 97% for the tibial component. There was a statistically significant improvement in varus deformity from preoperative 7.370 ± 3.70° to 1.24 0 ± 0.910° after surgery., p = 0.001. Improvement in flexion deformity correction was from preoperative 6.50 ± 6.30 to postoperative 1.640 ± 1.770, p = 0.001., Conclusion: Our study concludes that the use of pre-operative 3D CT helps in predicting the component sizes, minimizes surgical time, and enhances implant position accuracy, as well as improves postoperative limb alignment in the coronal and sagittal planes., (© 2023 Professor P K Surendran Memorial Education Foundation. Published by Elsevier B.V. All rights reserved.)

Background and Objectives: Pathology is a broad subject consisting of various branches, such as hematology, clinical pathology, biochemistry, histopathology, cytology, blood banking, and others. All the subspecialties are difficult to cover in depth in a 3-year MD Pathology course and curriculum. Hence, it was decided to investigate whether the MD Pathology curriculum is able to meet the challenges of today's laboratory medicine practice. So, a survey was conducted among newly qualified pathologists who have passed their exam within last 5 years. Purpose was to know their views about MD Pathology curriculum with special emphasis on challenges they face during their day-to-day practice., Materials and Methods: Study is designed as cross-sectional survey conducted via web-based Google forms questionnaire. Study population is pathologists qualified within last 5 years. A convenience sample of eligible pathologists was taken by sending a web-based Google form to various pathology groups on social media apps. Data were collected in the Google spreadsheet software and various responses were summarized as percentages, graphs, and pie-charts as necessary. Chi square test was used to find the significance of difference in responses from government, private, and deemed university respondents., Results: Different numbers of responses were received to different survey questions. Out of 187 respondents, 65 (34.8%) worked in a hospital-based laboratory, 61 (32.6%) did jobs in a private laboratory, 49 (26.2%) worked in a medical college, and 37 (19.8%) worked in their own private laboratories. Out of 193 respondents, 158 (81.9%) were working in hematology, 149 (77.2%) in clinical pathology, 139 (72%) in cytopathology, 118 (61.7%) in histopathology, whereas 103 (53.4%) worked in clinical biochemistry, and least number of respondents, i.e., 38 (19.7%) were working in blood bank. Almost all the respondents had overlapping work in hematology and other areas. The level of confidence rated by the respondents was best in hematology, followed by clinical pathology; it was least for clinical biochemistry and blood bank. Out of 192 respondents, 64.1% (123) felt that the curriculum does not equip them for lab management and administration, 21.9% (42) felt that the curriculum somewhat equipped them; whereas only 14.1% (27) felt that curriculum equips them for this task. There were 191 responses to the question regarding satisfaction with MD Pathology curriculum; out of which, 51.8% (99) were not satisfied, 24.6% (47) said they may be satisfied, whereas 23.6% (45) were satisfied with the curriculum. There was no significant difference in responses from government, private, and deemed university respondents., Conclusions: The graduating MD Pathology students expressed confidence in reporting routine cases of hematology, clinical pathology, routine cytology, and histopathology. However, there is difficulty in clinical biochemistry, blood banking, and reporting of malignancies. The laboratory management and administration, communication skills, and quality assurance are also other areas of weakness. MD Pathology program needs more focus on these weaknesses, so that newly qualified graduate would feel confident in day-to-day laboratory working., Competing Interests: None

Introduction: This study was proposed to evaluate the efficacy of fibrin clot augmentation in meniscal tear using inside-out meniscal repair., Methods: A total of 35 patients with meniscus tears were operated on with inside-out meniscus repair and fibrin clot augmentation. Patients were evaluated preoperatively and postoperatively with clinical criteria, Lysholm knee scoring system, and MRI., Results: Out of the total 35 cases, 5 cases were lost to follow-up. Clinical improvement was observed in 29 out of 30 patients (96.6%). The mean Lysholm score improved significantly from 67.63 ± 6.55 points preoperatively to 92.0 ± 2.9 points postoperatively (P < 0.05) in 3 years follow-up. Follow-up MRI in all patients revealed complete healing except in 1 case where the patient presented with recurrence of symptoms such as pain and locking which resolved with partial meniscectomy. Paraesthesia in the anterior part of the knee was observed in 2 cases. (6.6%)., Conclusion: We conclude that fibrin clot augmentation is a good cost-effective modality of treatment for repairable meniscus tears to preserve the meniscus and decrease the point contact pressure on the condyles which may prevent the early occurrence of osteoarthritis., Competing Interests: On behalf of all authors, the corresponding author states that there is no conflict of interest., (© 2022 Delhi Orthopedic Association. All rights reserved.)

Background: Infection around the ankle joint after fracture fixation, or septic arthritis with active discharging sinuses is often challenging to manage with conventional procedures of arthrodesis. The Ilizarov method of arthrodesis gives a better alternative for salvage in such cases., Methods: This was a retrospective study including 20 patients who were subjected to tibiotalar arthrodesis with Ilizarov method. The major pathologies included internal fixation of ankle fractures complicated by infection, posttraumatic infected ankle arthritis, and osteomyelitis. The patients were evaluated on the basis of Association for the Study and Application of the Method of Ilizarov (ASAMI) criteria. The aim of the surgery was to achieve plantigrade, stable, and painless foot with no signs of infection., Results: A total of 20 patients were operated and reviewed at our institute. The study comprised of 15 male and 5 female patients with a mean age of 43.9 years (range 33-55 years). Out of 20 patients, 4 patients had complications of pin-tract infection and one had wire breakage of the forefoot ring. According to the ASAMI criteria, 17 patients had excellent bone scores and 18 patients had good functional scores. Union was achieved in all patients with resolution of infection and the mean limb length discrepancy was 1.92 cm (range 1-2.5 cm)., Conclusions: The Ilizarov fixator for ankle arthrodesis provides an excellent way for strong bone fusion, infection eradication, early weight-bearing, and the added benefits of compression at the arthrodesis site post-operatively., Competing Interests: Conflict of InterestAjit Chalak, Sushmit Singh, Ashok Ghodke, Sachin Kale, Javed Hussain, and Ronak Mishra declare that they have no conflict of interest., (© Indian Orthopaedics Association 2022.)

Background and Objectives: Infertility is a sensitive subject carrying with it economic, social, and psychological implications. Work up of male infertility is often hampered by a lack of infrastructure and facilities, as well as inadequate training of pathology residents. The purpose of this research survey was to evaluate the current status of semen analysis practices and compare them to the standards laid down by the World Health Organization (WHO)., Materials and Methods: A web-based questionnaire was designed consisting of questions related to semen analysis practices and procedures being followed currently by pathologists in India. A total of 194 pathologists responded. Questions regarding the procedures followed for semen collection, sperm count, volume, normal range, lower normal limit of sperm count, morphology, etc., were included in the survey. These data were recorded. The differences and gaps in the practice with respect to the WHO standards were analyzed., Results: The survey revealed that the printed instructions for semen analysis were available with 38.7% of the respondents; 58.8% of the respondents had a separate room for semen collection; 95.9% performed the analysis manually, and; only 4.1% used automated analyzers. Only 53.6 and 52.6% of the respondents were correctly reporting the normal range of semen volume and sperm counts, respectively. Only 19.6% stated as having read the WHO manual and were also practicing its guidelines, while 14.4% had not read the WHO manual even once., Conclusion: The present study showed a large gap between the practice of semen analysis by respondents from various parts of India and standard procedures as laid down by the WHO. Many laboratories do not follow the standard instructions. There is a need to improve the quality of practice related to semen analysis in this country through appropriate teaching and training in medical institutions as well as through Continuing Medical Education (CMEs) or regular update programs., Competing Interests: None

Introduction: Gap non-union of tibia occurring mostly after trauma and many times complicated by infection, is a difficult problem to treat. The study aimed to assess the outcome of the three-ring construct of the Ilizarov fixator frame in the management of gap non-union of the tibia., Methods: This retrospective study included 30 patients of gap non-union of tibia operated from April 2016 to March 2019 with a three-ring Ilizarov fixator frame and follow-up done till March 2021. The mean age was 39.27 (range 10-66) years. The results were assessed by the Association for the Study and Application of the Method of Ilizarov (ASAMI) criteria. MPTA, PPTA, and LDTA after removal of the frame were also measured., Results: Out of the total 30 cases, all the patients showed complete union. The Ilizarov fixator was kept for an average period of 11.43 months and the mean defect size was 7.17 (range 2-12) cm. All patients were followed up for an average period of 39.36 (range 24-54) months. According to the ASAMI score bone/radiological results, 27 were classified as excellent, 2 as good, and 1 as poor. Functionally 28 were graded as excellent and the remainder as good. The normal ranges of MPTA, LDTA & PPTA were also achieved in a majority (80%) of patients., Conclusion: Our results after using only a three-ring Ilizarov fixator frame are almost equivalent to earlier studies and have advantages such as less weight, better patient compliance, superior radiographic visualization, easy mobilization, and reduced costs. Ilizarov ring fixator remains an excellent treatment modality for tibial non-union with a defect, regarding bone union, deformity correction, infection eradication, limb-length achievement, and limb function., Competing Interests: On behalf of all authors, the corresponding author states that there is no conflict of interest., (© 2021 Delhi Orthopedic Association. All rights reserved.)

Background: A tetravalent live attenuated dengue vaccine, Dengvaxia, sensitised naïve recipients to severe dengue illness upon a subsequent natural dengue infection and is suspected to be due to antibody-dependent enhancement (ADE). ADE has also been implicated in the severe neurological outcomes of Zika virus (ZIKV) infection. It has become evident that cross-reactive antibodies targeting the viral pre-membrane protein and fusion-loop epitope are ADE-competent. A pre-clinical tetravalent dengue sub-unit vaccine candidate, DSV4, eliminates these ADE-competent epitopes., Methods: We compared protective efficacy and ADE-competence of murine polyclonal antibodies induced by DSV4, Dengvaxia and an 'in house' tetravalent mixture of all four laboratory DENV strains, TV DENV, using established mouse models., Findings: DSV4-induced antibodies, known to be predominantly type-specific, provided significant protection against lethal DENV challenge, but did not promote ADE of either DENV or ZIKV infection in vivo. Antibodies elicited by Dengvaxia and TV DENV, which are predominantly cross-reactive, not only failed to offer protection against lethal DENV challenge, but also promoted ADE of both DENV and ZIKV infection in vivo., Interpretation: Protective efficacy against DENV infection may be linked to the induction of neutralising antibodies which are type-specific rather than cross-reactive. Whole virus-based dengue vaccines may be associated with ADE risk, despite their potent virus-neutralising capacity. Vaccines designed to eliminate ADE-competent epitopes may help eliminate/minimise ADE risk., Funding: This study was supported partly by ICGEB, India, the National Biopharma Mission, DBT, Government of India, Sun Pharmaceutical Industries Limited, India, and NIAID, NIH, USA., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives : To assess the safety and reactogenicity of single oral dose of heat-stable rotavirus vaccine (HSRV) in healthy adults aged 18-45 years followed by assessment of safety, reactogenicity, and immunogenicity of three doses of HSRV in healthy infants aged 6-8 weeks at enrollment. Trial Design : Single-center randomized controlled, sequential, blinded (adults) and open-label (infants). Setting : Single site at International Center for Diarrheal Disease Research, Bangladesh (icddr,b). Participants : Fifty eligible adults randomized in 1:1 ratio (HSRV: Placebo) followed by 50 eligible infants randomized in 1:1 ratio (HSRV: Comparator (RotaTeq®, pentavalent human-bovine (WC3) reassortant live-attenuated, rotavirus vaccine)). Intervention : Adults received either a single dose of HSRV or placebo and followed for 14 days. Infants received three doses of either HSRV or comparator with a follow-up for 28 days after each dose. Main Outcome Measures : Solicited and unsolicited adverse events (AEs) along with any serious adverse events (SAEs) were part of the safety and reactogenicity assessment in adults and infants whereas serum anti-rotavirus IgA response rates were part of immunogenicity assessment in infants only. Post-vaccination fecal shedding of vaccine-virus rotavirus strains was also determined in adults and infants. Results : In this study, HSRV, when compared with placebo, did not result in increase in solicited adverse events (solicited AEs) in adults. In infants, HSRV had a safety profile similar to comparator vis-à-vis solicited AEs. In infants, fecal shedding of vaccine-virus strains was not detected in HSRV recipients but was observed in two comparator recipients. Percentage of infants exhibiting threefold rise in serum anti-rotavirus IgA titers from baseline to 1-month post-dose 3 in HSRV group was 88% (22/25) and 84% (21/25) in comparator group. Conclusion : HSRV was found to be generally well-tolerated in both adults and infants and immunogenic in infants.

To develop a safe and efficacious heat-stable rotavirus vaccine, new lyophilized formulations were developed using rotavirus serotypes constituting RotaTeq®. A series of formulation compositions, differing in buffering agents, bulking agents, cryoprotectants, amino acids and divalent cations, were screened for their ability to provide stability to rotavirus serotypes during lyophilization and when stored under elevated temperatures for extended periods. Lead formulations and lyophilization cycles were further optimized. Stability profiles of thus optimized formulations showed their ability to retain the potency of rotavirus for > 36 months at 5°C, 20 months at 37°C, and 7 months at 45°C. The heat-stable lyophilized rotavirus formulations developed met the all critical quality attributes for appearance, heat-stability during storage, moisture content as well as pH, viability and stability after reconstitution and has great potential to be used as vaccine candidates for improving access in low-income countries.

Objective: To determine whether inflammatory and hemostasis response in patients hospitalized for pneumonia varies by age and whether these differences explain higher mortality in the elderly., Methods: In an observational cohort of subjects with community-acquired pneumonia (CAP) recruited from emergency departments (ED) in 28 hospitals, we divided subjects into 5 age groups (<50, 51-64, 65-74, 75-84, and ≥85). We measured circulating levels of inflammatory (TNF, IL-6, and IL-10), hemostasis (D-dimer, Factor IX, thrombin-antithrombin complex, antithrombin and plasminogen-activator inhibitor-1), and cell-surface markers (TLR-2, TLR-4, and HLA-DR) during the first week of hospitalization and at discharge and compared 90-day mortality. We used logistic regression to compare odds ratios (OR) for 90-day mortality between age groups, adjusting for differences in pre-infection factors alone and then additionally adjusting for immune markers., Results: Of 2,183 subjects, 495, 444, 403, 583, and 258 subjects were <50, 51-64, 65-74, 75-84, and ≥85 years of age, respectively. Large age-related differences were observed in 90-day mortality (0.82% vs. 3.2% vs. 6.4% vs. 12.8% vs. 13.6%, p<0.01). No age-related differences in inflammatory and cell surface markers occurred during the first week. Older subjects had higher pro-coagulant markers on ED presentation and over first week (p ≤ 0.03), but these differences were modest (1.0-1.7-fold differences). Odds of death for older adults changed minimally in models incorporating differences in hemostasis and inflammatory markers (for subjects ≥ 85 compared to those <50, OR = 4.36, when adjusted for pre-infection factors and OR = 3.49 when additionally adjusted for hemostasis markers). At discharge, despite clinical recovery as evidenced by normal vital signs in >85% subjects, older subjects had modestly increased hemostasis markers and IL-6 levels (p<0.01)., Conclusions: Modest age-related increases in coagulation response occur during hospitalization for CAP; however these differences do not explain the large differences in mortality. Despite clinical recovery, immune resolution may be delayed in older adults at discharge.

Most bacterial pyruvate dehydrogenase complexes from either gram-positive or gram-negative bacteria have E1 components with an alpha(2) homodimeric quaternary structure. In a sequel to our previous publications, we present the first NMR study on the flexible regions of the E1 component from Escherichia coli and its biological relevance. We report sequence-specific NMR assignments for 6 residues in the N-terminal 1-55 region and for a glycine in each of the two mobile active center loops of the E1 component, a 200-kDa homodimer. This was accomplished by using site-specific substitutions and appropriate labeling patterns along with a peptide with the sequence corresponding to the N-terminal 1-35 amino acids of the E1 component. To study the functions of these mobile regions, we also examined the spectra in the presence of (a) a reaction intermediate analog known to affect the mobility of the active center loops, (b) an E2 component construct consisting of a lipoyl domain and peripheral subunit binding domain, and (c) a peptide corresponding to the amino acid sequence of the E2 peripheral subunit binding domain. Deductions from the NMR studies are in excellent agreement with our functional finding, providing a clear indication that the N-terminal region of the E1 interacts with the E2 peripheral subunit binding domain and that this interaction precedes reductive acetylation. The results provide the first structural support to the notion that the N-terminal region of the E1 component of this entire class of bacterial pyruvate dehydrogenase complexes is responsible for binding the E2 component.

Cooperativity is extensively used by enzymes, particularly those acting at key metabolic branch points, to "fine tune" catalysis. Thus, cooperativity and enzyme catalysis are intimately linked, yet their linkage is poorly understood. Here we show that negative cooperativity in the rate-determining step in the E1 component of the Escherichia coli pyruvate dehydrogenase multienzyme complex is an outcome of redistribution of a "rate-promoting" conformational pre-equilibrium. An array of biophysical and biochemical studies indicates that non-catalytic but conserved residues directly regulate the redistribution. Furthermore, factors such as ligands and temperature, individually or in concert, also strongly influence the redistribution. As a consequence, these factors also exert their influence on catalysis by profoundly influencing the pre-equilibrium facilitated dynamics of communication between multienzyme components. Our observations suggest a mode of cooperativity in the E1 component that is consistent with the dynamical hypothesis shown to satisfactorily explain cooperativity in many well studied enzymes. The results point to the likely existence of multiple modes of communication between subunits when the entire class of thiamin diphosphate-dependent enzymes is considered.

The region encompassing residues 401-413 on the E1 component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli comprises a loop (the inner loop) which was not seen in the X-ray structure in the presence of thiamin diphosphate, the required cofactor for the enzyme. This loop is seen in the presence of a stable analogue of the pre-decarboxylation intermediate, the covalent adduct between the substrate analogue methyl acetylphosphonate and thiamin diphosphate, C2α-phosphonolactylthiamin diphosphate. It has been shown that the residue H407 and several other residues on this loop are required to reduce the mobility of the loop so electron density corresponding to it can be seen once the pre-decarboxylation intermediate is formed. Concomitantly, the loop encompassing residues 541-557 (the outer loop) appears to work in tandem with the inner loop and there is a hydrogen bond between the two loops ensuring their correlated motion. The inner loop was shown to: a) sequester the active center from carboligase side reactions; b) assist the interaction between the E1 and the E2 components, thereby affecting the overall reaction rate of the entire multienzyme complex; c) control substrate access to the active center. Using viscosity effects on kinetics it was shown that formation of the pre-decarboxylation intermediate is specifically affected by loop movement. A cysteine-less variant was created for the E1 component, onto which cysteines were substituted at selected loop positions. Introducing an electron spin resonance spin label and an (19)F NMR label onto these engineered cysteines, the loop mobility was examined: a) both methods suggested that in the absence of ligand, the loop exists in two conformations; b) line-shape analysis of the NMR signal at different temperatures, enabled estimation of the rate constant for loop movement, and this rate constant was found to be of the same order of magnitude as the turnover number for the enzyme under the same conditions. Furthermore, this analysis gave important insights into rate-limiting thermal loop dynamics. Overall, the results suggest that the dynamic properties correlate with catalytic events on the E1 component of the pyruvate dehydrogenase complex.

Protein motions are ubiquitous and are intrinsically coupled to catalysis. Their specific roles, however, remain largely elusive. Dynamic loops at the active center of the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex are essential for several catalytic functions starting from a predecarboxylation event and culminating in transfer of the acetyl moiety to the E2 component. Monitoring the kinetics of E1 and its loop variants at various solution viscosities, we show that the rate of a chemical step is modulated by loop dynamics. A cysteine-free E1 construct was site-specifically labeled on the inner loop (residues 401-413), and the EPR nitroxide label revealed ligand-induced conformational dynamics of the loop and a slow "open <--> close" conformational equilibrium in the unliganded state. An (19)F NMR label placed at the same residue revealed motion on the millisecond-second time scale and suggested a quantitative correlation of E1 catalysis and loop dynamics for the 200,000-Da protein. Thermodynamic studies revealed that these motions may promote covalent addition of substrate to the enzyme-bound thiamin diphosphate by reducing the free energy of activation. Furthermore, the global dynamics of E1 presumably regulate and streamline the catalytic steps of the overall complex by inducing an entirely entropic (nonmechanical) negative cooperativity with respect to substrate binding at higher temperatures. Our results are consistent with, and reinforce the hypothesis of, coupling of catalysis and regulation with enzyme dynamics and suggest the mechanism by which it is achieved in a key branchpoint enzyme in sugar metabolism.

Our crystallographic studies have shown that two active center loops (an inner loop formed by residues 401-413 and outer loop formed by residues 541-557) of the E1 component of the Escherichia coli pyruvate dehydrogenase complex become organized only on binding a substrate analog that is capable of forming a stable thiamin diphosphate-bound covalent intermediate. We showed that residue His-407 on the inner loop has a key role in the mechanism, especially in the reductive acetylation of the E. coli dihydrolipoamide transacetylase component, whereas crystallographic results showed a role of this residue in a disorder-order transformation of these two loops, and the ordered conformation gives rise to numerous new contacts between the inner loop and the active center. We present mapping of the conserved residues on the inner loop. Kinetic, spectroscopic, and crystallographic studies on some inner loop variants led us to conclude that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for substrate entry to the active site, for sequestering active site chemistry from undesirable side reactions, as well as for communication between the E1 and E2 components of the E. coli pyruvate dehydrogenase multienzyme complex.