Accuracy of intraoperative approximation of pelvic tilt using preoperative standing radiographs.

Background: Anterior approach surgeons who utilize intraoperative fluoroscopy often try to match a preoperative radiograph as a reference for intraoperative cup position. Every degree of inaccuracy in tilt leads to a roughly 0.7° change in anteversion. This study aimed to determine how closely pelvic tilt (PT) is approximated intraoperatively when compared to preoperative anteroposterior (AP) radiographs. Methods: This was a retrospective review of 193 primary THA's done by 2 surgeons at an academic tertiary referral center between September 2021-January 2023. There were 24 patients excluded for distorted anatomy, post-traumatic arthritis, insufficient x-rays, or a sacroiliac joint that could not be visualized on film. Data collected included age and BMI. PT was calculated using the formula, Tilt = -(ln((B/A) x (1/0.483)))/0.051. Value A is the distance from the base of the SI joint to the superior margin of the obturator foramen; value B is the height of the obturator foramen. Results: Mean preoperative PT was 0.2° versus intraoperative PT was 3.4° (p < 0.001). Mean absolute difference was 6.5°. 48% of patients (n = 81) had an absolute difference less than 5°, 31% (n = 52) between 5° and 10°, 14% (n = 24) between 10° and 15°, and 7% (n = 12) greater than 15°. There was no correlation between BMI or age and PT discrepancy. Conclusion: Of the patients, 21% had a discrepancy of 10° or greater between their preoperative radiographs and intraoperative fluoroscopic images. Surgeons should be aware of potential errors in cup positioning and be particularly diligent in high-risk cases.

Incidence and Risk Factors for Acid-fast Bacillus/Fungal Culture Positivity in Primary, Conversion, and Revision Hip and Knee Arthroplasty.

INTRODUCTION: Previous literature has reported minimal incidences of positive fungal/AFB cultures, questioning the routine use of these tests. With growing concern for excessive use, predictive factors for patients at higher risk for intraoperative AFB/fungal infections would help surgeons limit unnecessary testing. This study evaluates the positivity rate and predictive factors of positive fungal and/or acid-fast bacillus (AFB) cultures after primary, conversion, or revision hip and knee arthroplasty. METHOD: Two hundred thirty-eight knee and hip procedures were done between January 2007 and 2022 where intraoperative AFB/fungal cultures were obtained. Procedures included primary total knee arthroplasty, primary total hip arthroplasty, conversion, first of two-stage, second of two-stage, irrigation and débridement polyexchange, and aseptic revision. Positivity rates of intraoperative AFB/fungal cultures were calculated as binomial exact proportions with 95% confidence intervals and are displayed as percentages. Univariable generalized linear mixed models estimated the unadjusted effects of demographics, individual comorbid conditions, and procedural characteristics on the logit of positive AFB/fungal cultures. RESULTS: Two hundred thirty-eight knee and hip procedures recorded an overall positivity rate of 5.8% for intraoperative AFB/fungal cultures. Aseptic revisions showed the lowest rates of positivity at 3.6%, while conversions showed the highest rates of positivity at 14.3%. The positivity rates are highest among patients who are male (9.0%), of Hispanic origin (12.0%), with body mass index <30 (6.4%), and a Charlson Comorbidity Index <5 (6.1%). History of a prior infection in the same surgical joint showed statistically significant influence of odds of culture positivity with an odds ratio of 3.47 (P-value: 0.039). Other demographic factors that we investigated including age, sex, race, ethnicity, body mass index, and Charlson Comorbidity Index did not show any notable influence on AFB/fungal positivity rates. CONCLUSION: These results suggest utility in obtaining routine intraoperative AFB/fungal cultures, given the relatively high positivity and poor predictive factors.

A survey of experts to identify methods to detect problematic studies: Stage 1 of the INSPECT-SR Project.

Background: Randomised controlled trials (RCTs) inform healthcare decisions. Unfortunately, some published RCTs contain false data, and some appear to have been entirely fabricated. Systematic reviews are performed to identify and synthesise all RCTs which have been conducted on a given topic. This means that any of these 'problematic studies' are likely to be included, but there are no agreed methods for identifying them. The INSPECT-SR project is developing a tool to identify problematic RCTs in systematic reviews of healthcare-related interventions. The tool will guide the user through a series of 'checks' to determine a study's authenticity. The first objective in the development process is to assemble a comprehensive list of checks to consider for inclusion. Methods: We assembled an initial list of checks for assessing the authenticity of research studies, with no restriction to RCTs, and categorised these into five domains: Inspecting results in the paper; Inspecting the research team; Inspecting conduct, governance, and transparency; Inspecting text and publication details; Inspecting the individual participant data. We implemented this list as an online survey, and invited people with expertise and experience of assessing potentially problematic studies to participate through professional networks and online forums. Participants were invited to provide feedback on the checks on the list, and were asked to describe any additional checks they knew of, which were not featured in the list. Results: Extensive feedback on an initial list of 102 checks was provided by 71 participants based in 16 countries across five continents. Fourteen new checks were proposed across the five domains, and suggestions were made to reword checks on the initial list. An updated list of checks was constructed, comprising 116 checks. Many participants expressed a lack of familiarity with statistical checks, and emphasized the importance of feasibility of the tool. Conclusions: A comprehensive list of trustworthiness checks has been produced. The checks will be evaluated to determine which should be included in the INSPECT-SR tool.

Evaluating the impact of genomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) on hospital infection prevention and control decisions.

Genomic epidemiology enhances the ability to detect and refute methicillin-resistant Staphylococcus aureus (MRSA) outbreaks in healthcare settings, but its routine introduction requires further evidence of benefits for patients and resource utilization. We performed a 12 month prospective study at Cambridge University Hospitals NHS Foundation Trust in the UK to capture its impact on hospital infection prevention and control (IPC) decisions. MRSA-positive samples were identified via the hospital microbiology laboratory between November 2018 and November 2019. We included samples from in-patients, clinic out-patients, people reviewed in the Emergency Department and healthcare workers screened by Occupational Health. We sequenced the first MRSA isolate from 823 consecutive individuals, defined their pairwise genetic relatedness, and sought epidemiological links in the hospital and community. Genomic analysis of 823 MRSA isolates identified 72 genetic clusters of two or more isolates containing 339/823 (41 %) of the cases. Epidemiological links were identified between two or more cases for 190 (23 %) individuals in 34/72 clusters. Weekly genomic epidemiology updates were shared with the IPC team, culminating in 49 face-to-face meetings and 21 written communications. Seventeen clusters were identified that were consistent with hospital MRSA transmission, discussion of which led to additional IPC actions in 14 of these. Two outbreaks were also identified where transmission had occurred in the community prior to hospital presentation; these were escalated to relevant IPC teams. We identified 38 instances where two or more in-patients shared a ward location on overlapping dates but carried unrelated MRSA isolates (pseudo-outbreaks); research data led to de-escalation of investigations in six of these. Our findings provide further support for the routine use of genomic epidemiology to enhance and target IPC resources.

Inpatient Hospital Costs, Emergency Department Visits, and Readmissions for Revision Hip and Knee Arthroplasty.

BACKGROUND: Revision total hip arthroplasty (THA) and total knee arthroplasty (TKA) tremendously burden hospital resources. This study evaluated factors influencing perioperative costs, including emergency department (ED) visits, readmissions, and total costs-of-care within 90 days following revision surgery. METHODS: A retrospective analysis of 772 revision TKAs and THAs performed on 630 subjects at a single center between January 2007 and December 2019 was conducted. Cost data was available from January 2015 to December 2019 for 277 patients. Factors examined included comorbidities, demographic information, pre-operative Anesthesia Society of Anesthesiologists score, implant selection, and operative indication using mixed-effects linear regression models. RESULTS: Among 772 revisions (425 THA and 347 TKA), 213 patients required an ED visit, and 90 required hospital readmission within 90 days. There were 22.6% of patients who underwent a second procedure after their initial revision. Liver disease was a significant predictor of ED readmission for THA patients (multivariable OR [odds ratio]: 3.473, P = 0.001), while aseptic loosening, osteolysis, or instability significantly reduced the odds of readmission for TKA patients (OR: 0.368, P = 0.014). In terms of ED visits, liver disease increased the odds for THA patients (OR: 1.845, P = 0.100), and aseptic loosening, osteolysis, or instability decreased the odds for TKA patients (OR: 0.223, P < 0.001). Increased age was associated with increased costs in both THA and TKA patients, with significant cost factors including congestive heart failure for TKA patients (OR: $7,308.17, P = 0.004) and kidney disease for THA patients. Revision surgeries took longer than primary ones, with TKA averaging 3.0 hours (1.6 times longer) and THA 2.8 hours (1.5 times longer). CONCLUSION: Liver disease increases ED readmission risk in revision THA, while aseptic loosening, osteolysis, or instability decreases it in revision TKA. Increased age and CHF are associated with increased costs. These findings inform postoperative care and resource allocation in revision arthroplasty.

A rare case of taper junction corrosion in semi-constrained total knee arthroplasty.

Metallosis is a known yet rare late complication of unicompartmental and total knee arthroplasty (TKA), usually secondary to either metal-backed patellar component failure, mobile-bearing polyethylene dislocation, or catastrophic polyethylene failure and wear through. The majority of literature surrounding metallosis has been published in relation to total hip arthroplasty (THA) metal on metal bearing wear or mechanically assisted crevice corrosion.This case report describes the development of metallosis in a 77-year-old male patient with advanced (Kellgren-Lawrence Grade 4) osteoarthritis with associated valgus deformity, who underwent index TKA with a semiconstrained revision knee system due to intraoperative medial collateral ligament laxity. The taper junction between the titanium alloy stem and cobalt chromium femoral component was the source of diffuse intra-articular metallosis.

A structure-based designed small molecule depletes hRpn13Pru and a select group of KEN box proteins.

Proteasome subunit hRpn13 is partially proteolyzed in certain cancer cell types to generate hRpn13Pru by degradation of its UCHL5/Uch37-binding DEUBAD domain and retention of an intact proteasome- and ubiquitin-binding Pru domain. By using structure-guided virtual screening, we identify an hRpn13 binder (XL44) and solve its structure ligated to hRpn13 Pru by integrated X-ray crystallography and NMR to reveal its targeting mechanism. Surprisingly, hRpn13Pru is depleted in myeloma cells following treatment with XL44. TMT-MS experiments reveal a select group of off-targets, including PCNA clamp-associated factor PCLAF and ribonucleoside-diphosphate reductase subunit M2 (RRM2), that are similarly depleted by XL44 treatment. XL44 induces hRpn13-dependent apoptosis and also restricts cell viability by a PCLAF-dependent mechanism. A KEN box, but not ubiquitination, is required for XL44-induced depletion of PCLAF. Here, we show that XL44 induces ubiquitin-dependent loss of hRpn13Pru and ubiquitin-independent loss of select KEN box containing proteins.

Protocol for the development of a tool (INSPECT-SR) to identify problematic randomised controlled trials in systematic reviews of health interventions.

INTRODUCTION: Randomised controlled trials (RCTs) inform healthcare decisions. It is now apparent that some published RCTs contain false data and some appear to have been entirely fabricated. Systematic reviews are performed to identify and synthesise all RCTs that have been conducted on a given topic. While it is usual to assess methodological features of the RCTs in the process of undertaking a systematic review, it is not usual to consider whether the RCTs contain false data. Studies containing false data therefore go unnoticed and contribute to systematic review conclusions. The INveStigating ProblEmatic Clinical Trials in Systematic Reviews (INSPECT-SR) project will develop a tool to assess the trustworthiness of RCTs in systematic reviews of healthcare-related interventions. METHODS AND ANALYSIS: The INSPECT-SR tool will be developed using expert consensus in combination with empirical evidence, over five stages: (1) a survey of experts to assemble a comprehensive list of checks for detecting problematic RCTs, (2) an evaluation of the feasibility and impact of applying the checks to systematic reviews, (3) a Delphi survey to determine which of the checks are supported by expert consensus, culminating in, (4) a consensus meeting to select checks to be included in a draft tool and to determine its format and (5) prospective testing of the draft tool in the production of new health systematic reviews, to allow refinement based on user feedback. We anticipate that the INSPECT-SR tool will help researchers to identify problematic studies and will help patients by protecting them from the influence of false data on their healthcare. ETHICS AND DISSEMINATION: The University of Manchester ethics decision tool was used, and this returned the result that ethical approval was not required for this project (30 September 2022), which incorporates secondary research and surveys of professionals about subjects relating to their expertise. Informed consent will be obtained from all survey participants. All results will be published as open-access articles. The final tool will be made freely available.

Integrin-αvß6 targeted peptide-toxin therapy in a novel αvß6-expressing immunocompetent model of pancreatic cancer.

Previously we reported that a novel αvß6-specific peptide-drug conjugate (SG3299) could eliminate established human pancreatic ductal adenocarcinoma (PDAC) xenografts. However the development of effective therapies for PDAC, which is an essential need, must show efficacy in relevant immunocompetent animals. Previously we reported that the KPC mouse transgenic PDAC model that closely recapitulates most stages of development of human PDAC, unlike in humans, failed to express αvß6 on their tumours or metastases. In this study we have taken the KPC-derived PDAC line TB32043 and engineered a variant line (TB32043mb6S2) that expresses mouse integrin αvß6. We report that orthotopic implantation of the αvß6 over-expressing TB32043mb6S2 cells promotes shorter overall survival and increase in metastases. Moreover, systemic treatment of mice with established TB32043mb6S2 tumours in the pancreas with SG2399 lived significantly longer (p < 0.001; mean OS 48d) compared with PBS or control SG3511 (mean OS 25.5d and 26d, respectively). Thus SG3299 is confirmed as a promising candidate therapeutic for the therapy of PDAC.

Biofilm and How It Relates to Prosthetic Joint Infection.

Prosthetic joint infection following total joint arthroplasty is a devastating complication, resulting in increased morbidity and mortality for the patient. The formation of a biofilm on implanted hardware contributes to the difficulty in successful identification and eradication of the infection. Antibiotic therapy and surgical intervention are necessary for addressing this condition; we present a discussion on different treatment options, including those that are not yet routinely utilized in the clinical setting or are under investigation, to highlight the present and future of PJI management.

Percutaneous thrombolysis via cholecystostomy catheter to dissolve biliary clots causing obstructive jaundice.

Haemobilia, or bleeding within the biliary tree, is rare. It can cause biliary obstruction secondary to blood clots. A comorbid 87-year-old was admitted to hospital with acute cholecystitis, choledocholithiasis, and an Escherichia coli bacteremia. He had a partial pancreatectomy and gastrojejunostomy 35 years prior for severe pancreatitis. He was treated with antibiotics and a percutaneous cholecystostomy. He developed atrial fibrillation and was subsequently commenced on warfarin. He re-presented 5 days after discharge with abdominal pain and fevers. Liver function tests revealed cholestasis and a supratherapeutic international normalised ratio. Imaging showed cholecystitis, biliary obstruction, and extensive biliary blood clots. He improved with antibiotics, vitamin K, and alteplase flushes through the percutaneous cholecystostomy. Repeat cholangiogram demonstrated dissolution of the biliary clots. Due to altered anatomy and comorbidities, alteplase flushes were utilized to relieve this patient's biliary obstruction. Thrombolytics may assist in treating biliary clots when first-line options are not possible or favourable.

Friend or foe? Reciprocal regulation between E3 ubiquitin ligases and deubiquitinases.

Protein ubiquitination is a post-translational modification that entails the covalent attachment of the small protein ubiquitin (Ub), which acts as a signal to direct protein stability, localization, or interactions. The Ub code is written by a family of enzymes called E3 Ub ligases (∼600 members in humans), which can catalyze the transfer of either a single ubiquitin or the formation of a diverse array of polyubiquitin chains. This code can be edited or erased by a different set of enzymes termed deubiquitinases (DUBs; ∼100 members in humans). While enzymes from these distinct families have seemingly opposing activities, certain E3-DUB pairings can also synergize to regulate vital cellular processes like gene expression, autophagy, innate immunity, and cell proliferation. In this review, we highlight recent studies describing Ub ligase-DUB interactions and focus on their relationships.

Next-generation sequencing identifies two novel HLA-DPB1 alleles: HLA-DPB1*1069:01 and DPB1*1072:01.

Characterization of two novel HLA-DPB1 alleles: HLA-DPB1*1069:01, and DPB1*1072:01 containing non-synonymous nucleotide substitutions.

Antibiotic resistance determination using Enterococcus faecium whole-genome sequences: a diagnostic accuracy study using genotypic and phenotypic data.

BACKGROUND: DNA sequencing could become an alternative to in vitro antibiotic susceptibility testing (AST) methods for determining antibiotic resistance by detecting genetic determinants associated with decreased antibiotic susceptibility. Here, we aimed to assess and improve the accuracy of antibiotic resistance determination from Enterococcus faecium genomes for diagnosis and surveillance purposes. METHODS: In this retrospective diagnostic accuracy study, we first conducted a literature search in PubMed on Jan 14, 2021, to compile a catalogue of genes and mutations predictive of antibiotic resistance in E faecium. We then evaluated the diagnostic accuracy of this database to determine susceptibility to 12 different, clinically relevant antibiotics using a diverse population of 4382 E faecium isolates with available whole-genome sequences and in vitro culture-based AST phenotypes. Isolates were obtained from various sources in 11 countries worldwide between 2000 and 2018. We included isolates tested with broth microdilution, Vitek 2, and disc diffusion, and antibiotics with at least 50 susceptible and 50 resistant isolates. Phenotypic resistance was derived from raw minimum inhibitory concentrations and measured inhibition diameters, and harmonised primarily using the breakpoints set by the European Committee on Antimicrobial Susceptibility Testing. A bioinformatics pipeline was developed to process raw sequencing reads, identify antibiotic resistance genetic determinants, and report genotypic resistance. We used our curated database, as well as ResFinder, AMRFinderPlus, and LRE-Finder, to assess the accuracy of genotypic predictions against phenotypic resistance. FINDINGS: We curated a catalogue of 228 genetic markers involved in resistance to 12 antibiotics in E faecium. Very accurate genotypic predictions were obtained for ampicillin (sensitivity 99·7% [95% CI 99·5-99·9] and specificity 97·9% [95·8-99·0]), ciprofloxacin (98·0% [96·4-98·9] and 98·8% [95·9-99·7]), vancomycin (98·8% [98·3-99·2] and 98·8% [98·0-99·3]), and linezolid resistance (after re-testing false negatives: 100·0% [90·8-100·0] and 98·3% [97·8-98·7]). High sensitivity was obtained for tetracycline (99·5% [99·1-99·7]), teicoplanin (98·9% [98·4-99·3]), and high-level resistance to aminoglycosides (97·7% [96·6-98·4] for streptomycin and 96·8% [95·8-97·5] for gentamicin), although at lower specificity (60-90%). Sensitivity was expectedly low for daptomycin (73·6% [65·1-80·6]) and tigecycline (38·3% [27·1-51·0]), for which the genetic basis of resistance is not fully characterised. Compared with other antibiotic resistance databases and bioinformatic tools, our curated database was similarly accurate at detecting resistance to ciprofloxacin and linezolid and high-level resistance to streptomycin and gentamicin, but had better sensitivity for detecting resistance to ampicillin, tigecycline, daptomycin, and quinupristin-dalfopristin, and better specificity for ampicillin, vancomycin, teicoplanin, and tetracycline resistance. In a validation dataset of 382 isolates, similar or improved diagnostic accuracies were also achieved. INTERPRETATION: To our knowledge, this work represents the largest published evaluation to date of the accuracy of antibiotic susceptibility predictions from E faecium genomes. The results and resources will facilitate the adoption of whole-genome sequencing as a tool for the diagnosis and surveillance of antimicrobial resistance in E faecium. A complete characterisation of the genetic basis of resistance to last-line antibiotics, and the mechanisms mediating antibiotic resistance silencing, are needed to close the remaining sensitivity and specificity gaps in genotypic predictions. FUNDING: Wellcome Trust, UK Department of Health, British Society for Antimicrobial Chemotherapy, Academy of Medical Sciences and the Health Foundation, Medical Research Council Newton Fund, Vietnamese Ministry of Science and Technology, and European Society of Clinical Microbiology and Infectious Disease.

Multi-monoubiquitylation controls VASP-mediated actin dynamics.

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitylation regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitylation impacts VASP activity was unknown. Here, we show that mimicking multi-monoubiquitylation of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitylated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitylated VASP maintained the ability to bind and protect barbed ends from capping protein. Finally, we demonstrate the electroporation of recombinant multi-monoubiquitylated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitylation controls VASP-mediated actin dynamics.

Total Joint Arthroplasty in Patients With Lymphedema as Compared to a Propensity-Matched Control Cohort.

Background: Lymphedema is rare in arthroplasty patients but has been associated with a higher complication rate. This study sought to determine the outcomes of total joint arthroplasty in patients with lymphedema as compared to a matched control cohort. Methods: Treatment-control propensity matching was implemented on 335 patients following total knee or hip arthroplasty generating 5-patient sets of one patient with presurgery lymphedema (67 total) to 4 patients without presurgery lymphedema (268 total) and matched on age, sex, and surgery year. Body mass index and presence of diabetes were controlled using multivariable generalized estimating equations. Results: In the lymphedema cohort, 1 patient (1.5%) had a deep vein thrombosis within 90 days of their surgery, 36 (53.7%) were discharged to a rehabilitation center, 16 (23.9%) had a readmission, 14 (20.9%) were admitted to the emergency room within 90 days, 6 (9.0%) experienced infection, and 6 (9.0%) had a revision/reoperation. Lymphedema significantly increased emergency room admission within 90 days (odds ratio [OR] 4.56, P = .01) and non-home discharge (OR 4.14, P < .01), affected readmission within 90 days (OR 2.21, P = .09), revision/reoperation (OR 2.82, P = .09), and no effect on deep vein thrombosis within 90 days (OR 0.57, P = .45), postsurgical infection (OR 1.47, P = .45), length of stay (OR 0.00, P = .99), operative time (OR 0.04, P = .38), or estimated blood loss (OR 0.09, P = .47), after adjusting for various factors. Conclusions: Preoperative lymphedema is a significant risk factor for patients who are undergoing total joint arthroplasty. Preoperative and postoperative modalities should be utilized to help control lymphedema and mitigate these increased risks.

Infection and Prophylaxis During Normothermic Liver Perfusion: Audit of Incidence and Pharmacokinetics of Antimicrobial Therapy.

BACKGROUND: Ex situ normothermic liver perfusion (NMP) in a blood-based perfusate is associated with a risk of microbe growth, resulting in life-threatening posttransplant sepsis. Antibiotics are widely used, but the pharmacokinetics of these agents are unknown as is their efficacy. We wished to assess the perfusate concentrations of the meropenem and fluconazole that we use and to audit the incidence of infection with this antimicrobial therapy. METHODS: Fluconazole and meropenem (100 mg each) were added to the perfusate before NMP began, and serial samples were taken and assayed for drug concentrations. Perfusate cultures were available from 210 of the 242 perfusions performed between February 1, 2018, and April 6, 2023; these were reviewed. RESULTS: Following administration of 100 mg fluconazole, levels fell slightly from a median of 24.9 mg/L at 1 h to 22.6 mg/L at 10 h. In contrast, meropenem concentrations fell over time, from a median of 21.8 mg/L at 1 h to 9.4 mg/L at 10 h. There were 4 significant microorganisms grown in the perfusions, including 3 Candida species and an Enterococcus faecium. All the Candida-infected livers were transplanted with no adverse consequences, the recipients being treated with anidulafungin upon identification of the infecting organism; the Enterococcus-infected liver was not transplanted. CONCLUSIONS: Serious infection is a risk with NMP but appears to be mitigated with a protocol combining fluconazole and meropenem. This combination may not be appropriate in areas where resistance is prevalent. Routine culture of NMP perfusate is essential to identify breakthrough organisms early and enable recipient treatment.

Using NMR to Monitor TET-Dependent Methylcytosine Dioxygenase Activity and Regulation.

The active removal of DNA methylation marks is governed by the ten-eleven translocation (TET) family of enzymes (TET1-3), which iteratively oxidize 5-methycytosine (5mC) into 5-hydroxymethycytosine (5hmC), and then 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TET proteins are frequently mutated in myeloid malignancies or inactivated in solid tumors. These methylcytosine dioxygenases are α-ketoglutarate (αKG)-dependent and are, therefore, sensitive to metabolic homeostasis. For example, TET2 is activated by vitamin C (VC) and inhibited by specific oncometabolites. However, understanding the regulation of the TET2 enzyme by different metabolites and its activity remains challenging because of limitations in the methods used to simultaneously monitor TET2 substrates, products, and cofactors during catalysis. Here, we measure TET2-dependent activity in real time using NMR. Additionally, we demonstrate that in vitro activity of TET2 is highly dependent on the presence of VC in our system and is potently inhibited by an intermediate metabolite of the TCA cycle, oxaloacetate (OAA). Despite these opposing effects on TET2 activity, the binding sites of VC and OAA on TET2 are shared with αKG. Overall, our work suggests that NMR can be effectively used to monitor TET2 catalysis and illustrates how TET activity is regulated by metabolic and cellular conditions at each oxidation step.

Sublethal effects of imidacloprid-contaminated honey stores on colony performance, queens, and worker activities in fall and early winter colonies.

Neonicotinoid-contaminated sugar stores can have both near term and long term effects on honey bees due to their persistence in honey stores. Effects of imidacloprid food stores contaminants were examined in subtropical colonies that experience reduced brood rearing and foraging during overwintering. Colonies were given treatment sugar syrup containing 0 ppb (control), 20 ppb (field relevant), or 100 ppb (above field relevant) imidacloprid over six weeks to simulate contaminated fall nectar. Colonies were evaluated immediately (post-treatment) and 10 weeks (mid-winter) after treatment to compare proximal and latent effects. Post-treatment 0 ppb and 20 ppb colonies had more workers than 100 ppb colonies while 0 ppb colonies more brood than 20 ppb or 100 ppb colonies. Mid-winter 0 ppb and 20 ppb colonies had more workers than 100 ppb colonies and 0 ppb colonies more brood than 100 ppb colonies. Colonies experienced seasonal declines in stored pollen but no treatment effects. Lower 100 ppb colony performance was associated with reduced effort rather than lifespan. RFID (Radio Frequency Identification) tracking revealed that workers had similar adult lifespans across treatments; however, 100 ppb workers engaged in activities outside the colony for less time than 0 ppb workers. Imidacloprid exposure affected queen but not worker nutritional physiology. Nurses retained well-developed hypopharyngeal glands (as indicated by head protein) across treatments. Mid-winter queens from 0 ppb colonies had marginally higher ovary protein than queens from 100 ppb colonies and more ovary lipids than queens from 20 ppb colonies. However, queen nutrient stores in non-reproductive tissues (fat bodies) did not differ across treatments. Queens from different treatments were attended by comparable numbers of retinue workers and had similar gland contents of four QMP (Queen Mandibular Pheromone) components essential to queen care. High levels of imidacloprid in sugar stores can negatively affect colony performance months after initial storage.

A new experimental evidence-weighted signaling pathway resource in FlyBase.

Research in model organisms is central to the characterization of signaling pathways in multicellular organisms. Here, we present the comprehensive and systematic curation of 17 Drosophila signaling pathways using the Gene Ontology framework to establish a dynamic resource that has been incorporated into FlyBase, providing visualization and data integration tools to aid research projects. By restricting to experimental evidence reported in the research literature and quantifying the amount of such evidence for each gene in a pathway, we captured the landscape of empirical knowledge of signaling pathways in Drosophila.