Profiling Complex RAS-Effector Interactions Using NMR Spectroscopy.

Knowledge of how effectors interact with RAS GTPases is key to understanding how these switch-like proteins function in cells. Effectors bind specifically to GTP-loaded RAS using RAS association (RA) or RAS binding domains (RBDs) that show wide-ranging affinities and thermodynamic characteristics. Both normal development and RAS-induced tumorigenesis depend on multiple distinct effector proteins that are frequently co-expressed and co-localized, suggesting an antagonistic nature to signaling whereby multiple proteins compete for a limited pool of activated GTPase. NMR spectroscopy offers a powerful approach to multiplex effectors and/or regulatory enzymes and quantifies their interaction with RAS, expanding our biophysical and systems-level understanding of RAS signaling in a more integrated and physiologically relevant setting. Here we describe a method to directly quantitate GTPase binding to competing effectors, using wild-type KRAS complex with ARAF and PLCε1 as a model. Unlabeled RBD/RA domains are added simultaneously to isotopically labeled RAS, and peak intensities at chemical shifts characteristic of individually bound domains provide quantitation. Similar competition-based assays can be run with small molecule interactors, GEF/GAP domains, or regulatory enzymes that drive posttranslational modifications. Such efforts bring in vitro interaction experiments in line with more complex cellular environments.

Interactions between zinc and NRF2 in vascular redox signalling.

Recent evidence highlights the importance of trace metal micronutrients such as zinc (Zn) in coronary and vascular diseases. Zn2+ plays a signalling role in modulating endothelial nitric oxide synthase and protects the endothelium against oxidative stress by up-regulation of glutathione synthesis. Excessive accumulation of Zn2+ in endothelial cells leads to apoptotic cell death resulting from dysregulation of glutathione and mitochondrial ATP synthesis, whereas zinc deficiency induces an inflammatory phenotype, associated with increased monocyte adhesion. Nuclear factor-E2-related factor 2 (NRF2) is a transcription factor known to target hundreds of different genes. Activation of NRF2 affects redox metabolism, autophagy, cell proliferation, remodelling of the extracellular matrix and wound healing. As a redox-inert metal ion, Zn has emerged as a biomarker in diagnosis and as a therapeutic approach for oxidative-related diseases due to its close link to NRF2 signalling. In non-vascular cell types, Zn has been shown to modify conformations of the NRF2 negative regulators Kelch-like ECH-associated Protein 1 (KEAP1) and glycogen synthase kinase 3ß (GSK3ß) and to promote degradation of BACH1, a transcriptional suppressor of select NRF2 genes. Zn can affect phosphorylation signalling, including mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinases and protein kinase C, which facilitate NRF2 phosphorylation and nuclear translocation. Notably, several NRF2-targeted proteins have been suggested to modify cellular Zn concentration via Zn exporters (ZnTs) and importers (ZIPs) and the Zn buffering protein metallothionein. This review summarises the cross-talk between reactive oxygen species, Zn and NRF2 in antioxidant responses of vascular cells against oxidative stress and hypoxia/reoxygenation.

Effect of vitamin E-enhanced highly cross-linked polyethylene on wear rate and particle debris in anatomic total shoulder arthroplasty: a biomechanical comparison to ultrahigh-molecular-weight polyethylene.

BACKGROUND: Particle-induced osteolysis resulting from polyethylene wear remains a source of implant failure in anatomic total shoulder designs. Modern polyethylene components are irradiated in an oxygen-free environment to induce cross-linking, but reducing the resulting free radicals with melting or heat annealing can compromise the component's mechanical properties. Vitamin E has been introduced as an adjuvant to thermal treatments. Anatomic shoulder arthroplasty models with a ceramic head component have demonstrated that vitamin E-enhanced polyethylene show improved wear compared with highly cross-linked polyethylene (HXLPE). This study aimed to assess the biomechanical wear properties and particle size characteristics of a novel vitamin E-enhanced highly cross-linked polyethylene (VEXPE) glenoid compared to a conventional ultrahigh-molecular-weight polyethylene (UHMWPE) glenoid against a cobalt chromium molybdenum (CoCrMo) head component. METHODS: Biomechanical wear testing was performed to compare the VEXPE glenoid to UHMWPE glenoid with regard to pristine polyethylene wear and abrasive endurance against a polished CoCrMo alloy humeral head in an anatomic shoulder wear-simulation model. Cumulative mass loss (milligrams) was recorded, and wear rate calculated (milligrams per megacycle [Mc]). Under pristine wear conditions, particle analysis was performed, and functional biologic activity (FBA) was calculated to estimate particle debris osteolytic potential. In addition, 95% confidence intervals for all testing conditions were calculated. RESULTS: The average pristine wear rate was statistically significantly lower for the VEXPE glenoid compared with the HXLPE glenoid (0.81 ± 0.64 mg/Mc vs. 7.00 ± 0.45 mg/Mc) (P < .05). Under abrasive wear conditions, the VEXPE glenoid had a statistically significant lower average wear rate compared with the UHMWPE glenoid comparator device (18.93 ± 5.80 mg/Mc vs. 40.47 ± 2.63 mg/Mc) (P < .05). The VEXPE glenoid demonstrated a statistically significant improvement in FBA compared with the HXLPE glenoid (0.21 ± 0.21 vs. 1.54 ± 0.49 (P < .05). CONCLUSIONS: A new anatomic glenoid component with VEXPE demonstrated significantly improved pristine and abrasive wear properties with lower osteolytic particle debris potential compared with a conventional UHMWPE glenoid component. Vitamin E-enhanced polyethylene shows early promise in shoulder arthroplasty components. Long-term clinical and radiographic investigation needs to be performed to verify if these biomechanical wear properties translate to diminished long-term wear, osteolysis, and loosening.

Effect of making skin incision with electrocautery on positive Cutibacterium acnes culture rates in shoulder arthroplasty: a prospective randomized clinical trial.

BACKGROUND: Cutibacterium acnes remains the most commonly detected organism in shoulder arthroplasty. C acnes infection is thought to occur during shoulder arthroplasty through contamination of the surgical field with C acnes from the incised dermis. The purpose of this study was to examine whether using electrocautery for making skin incisions would decrease C acnes culture rates at the incised dermis compared to using scalpels during shoulder arthroplasty. METHODS: Patients undergoing primary shoulder arthroplasty were randomized into 2 groups, electrocautery vs. scalpel incision group. All patients received a standard preoperative antiseptic preparation including chlorhexidine gluconate showers, intravenous antibiotic administration, and topical application of hydrogen peroxide, povidone iodine, isopropyl alcohol, and DuraPrep. Cultures were obtained from the incised dermal edge immediately after skin incision and later from surgeon's gloves and forceps immediately prior to humeral component implantation. The primary outcome was positive C acnes culture rates compared between the groups. RESULTS: A total of 64 patients (32 in each group) were enrolled. There were 24 males in each group. Regarding dermis cultures, 10 patients (31%) in the scalpel group were positive with 8 of them positive for C acnes, whereas no patients in the electrocautery group were positive (P < .001). Regarding glove cultures, the electrocautery group had 8 patients positive C acnes, while the scalpel group had 8 (P = .777). Regarding forceps cultures, the electrocautery group had 4 patients positive for C acnes, and the scalpel group had 6 (P = .491). All positive cultures were exclusively from male patients. There were no wound complications or infection in the electrocautery group while the scalpel group had 1 acute postoperative infection. CONCLUSIONS: Making skin incisions using electrocautery resulted in 0 C acnes culture at the incised dermis, suggesting its potential effect against C acnes. However, despite this initial antibacterial effect, C acnes still appeared on surgeon's gloves and forceps during surgery of male patients. All positive cultures were from male patients, suggesting that the source of C acnes was specifically related to the male body. While the study hypothesis was supported by the results, the present study also raises new questions and calls for further research.

Defining bone fide effectors of RAS GTPases.

RAS GTPases play essential roles in normal development and are direct drivers of human cancers. Three decades of study have failed to wholly characterize pathways stimulated by activated RAS, driven by engagement with 'effector' proteins that have RAS binding domains (RBDs). Bone fide effectors must bind directly to RAS GTPases in a nucleotide-dependent manner, and this interaction must impart a clear change in effector activity. Despite this, for most proteins currently deemed effectors there is little mechanistic understanding of how binding to the GTPase alters protein function. There has also been limited effort to comprehensively resolve the specificity of effector binding to the full array of RAS superfamily GTPase proteins. This review will summarize what is known about RAS-driven activation for an array of potential effector proteins, focusing on structural and mechanistic effects and highlighting how little is still known regarding this key paradigm of cellular signal transduction.

Vascular protection afforded by zinc supplementation in human coronary artery smooth muscle cells mediated by NRF2 signaling under hypoxia/reoxygenation.

Zinc (Zn) has antioxidant, anti-inflammatory and anti-proliferative actions, with Zn dysregulation associated with coronary ischemia/reperfusion injury and smooth muscle cell dysfunction. As the majority of studies concerning Zn have been conducted under non-physiological hyperoxic conditions, we compare the effects of Zn chelation or supplementation on total intracellular Zn content, antioxidant NRF2 targeted gene transcription and hypoxia/reoxygenation-induced reactive oxygen species generation in human coronary artery smooth muscle cells (HCASMC) pre-adapted to hyperoxia (18 kPa O2) or normoxia (5 kPa O2). Expression of the smooth muscle marker SM22-α was unaffected by lowering pericellular O2, whereas calponin-1 was significantly upregulated in cells under 5 kPa O2, indicating a more physiological contractile phenotype under 5 kPa O2. Inductively coupled plasma mass spectrometry established that Zn supplementation (10 µM ZnCl2 + 0.5 µM pyrithione) significantly increased total Zn content in HCASMC under 18 but not 5 kPa O2. Zn supplementation increased metallothionein mRNA expression and NRF2 nuclear accumulation in cells under 18 or 5 kPa O2. Notably, NRF2 regulated HO-1 and NQO1 mRNA expression in response to Zn supplementation was only upregulated in cells under 18 but not 5 kPa. Furthermore, whilst hypoxia increased intracellular glutathione (GSH) in cells pre-adapted to 18 but not 5 kPa O2, reoxygenation had negligible effects on GSH or total Zn content. Reoxygenation-induced superoxide generation in cells under 18 kPa O2 was abrogated by PEG-superoxide dismutase but not by PEG-catalase, and Zn supplementation, but not Zn chelation, attenuated reoxygenation-induced superoxide generation in cells under 18 but not 5kPaO2, consistent with a lower redox stress under physiological normoxia. Our findings highlight that culture of HCASMC under physiological normoxia recapitulates an in vivo contractile phenotype and that effects of Zn on NRF2 signaling are altered by oxygen tension.

Prospective Randomized Controlled Clinical Trial Comparing Hyperosmolar Saline to Standard Isotonic Irrigation Fluid for Arthroscopic Knee Surgery: Initial Clinical Outcomes.

Numerous in vitro studies suggest higher osmolarity irrigation fluids potentiate a chondroprotective environment, and a recent clinical study using hyperosmolar saline for shoulder arthroscopy reported potential clinical advantages. This prospective randomized double-blind controlled clinical trial was designed to assess initial clinical outcomes associated with use of a hyperosmolar irrigation solution in patients undergoing arthroscopic knee surgery. With institutional review board approval and informed consent, patients scheduled for arthroscopic knee surgery were randomized to surgery with either isotonic lactated Ringer's (273 mOsm/L) or hyperosmolar saline (593 mOsm/L) irrigation solution. Outcomes included perioperative blood pressure, knee girth, visual analogue scale (VAS) pain scores, and narcotic pain medication consumption. Forty-six patients underwent arthroscopic knee surgery with isotonic (n = 23) or hyperosmolar (n = 23) irrigation fluids. There were 11 males and 12 females (mean age = 44.0 years) in the isotonic cohort and 8 males and 15 females (mean age = 40.2 years) in the hyperosmolar cohort. There were no significant differences with respect to surgical duration (pump time) or amount of irrigation fluid used between the two cohorts. There were no significant differences with respect to change in knee girth, blood pressure, or VAS pain scores. However, patients treated with hyperosmolar saline consumed less narcotic medication on postoperative day 3 (4.0 ± 7.6 vs. 15.5 ± 17.4 mg, p = 0.01). The results of this randomized clinical trial suggest that a hyperosmolar irrigation solution is safe and relatively inexpensive for use in patients undergoing arthroscopic knee surgery and contributes to a reduction in initial postoperative narcotic pain medication consumption. A hyperosmolar saline irrigation fluid was not associated with any detrimental effects on the execution of the surgical procedure, postoperative pain, or periarticular fluid extravasation. Taken together with previous basic science, translational, and clinical studies, hyperosmolar saline irrigation fluid is promising alternative to traditional isotonic irrigation fluids for knee arthroscopy. This study is a prospective trial and reflects level of evidence I.

Functional biomechanical comparison of Latarjet vs. distal tibial osteochondral allograft for anterior glenoid defect reconstruction.

INTRODUCTION: Glenoid reconstruction is indicated for recurrent glenohumeral instability with significant glenoid bone deficiency. Coracoid autograft (Latarjet) and distal tibial osteochondral allograft (DTA) reconstructions have been used to successfully restore glenohumeral stability. Relative advantages and disadvantages associated with each reconstruction technique have been described. However, direct comparisons of functional glenohumeral biomechanics associated with Latarjet vs. DTA reconstruction are lacking. This study was designed to compare these 2 glenoid reconstruction techniques with respect to joint kinematics and cartilage pressure mapping using a robotic testing system. METHODS: In accordance with institutional review board policies, human cadaveric shoulders (n = 8) were cyclically tested in the neutral position and 90° of external rotation with 60° and 90° of abduction under a 45-N joint-compression load to measure clinically relevant translations, loads, and torques. Joint contact pressure maps were obtained under a 120-N joint-compression load using pressure mapping sensors. After confirming that a 25% anterior glenoid defect resulted in glenohumeral dislocation, testing was performed to compare 3 conditions: native intact glenoid, 25% anterior glenoid defect with Latarjet reconstruction, and 25% anterior glenoid defect with DTA reconstruction. Analyses of variance and t tests were used to analyze data with statistical significance set at P < .05. RESULTS: Significant differences in anterior translation, inferior drawer, anterior drawer, compression loads, horizontal abduction, negative elevation (adduction), and external rotation torques during cyclical testing in 90° of external rotation with 60° and/or 90° of abduction were noted when comparing the 2 different glenoid bone reconstruction techniques to native, intact shoulders. The only significant difference between Latarjet and DTA reconstructions for measured translations, loads, and torques was a significantly higher absolute maximum compressive load for Latarjet compared to DTA at 60° of abduction. CONCLUSION: Latarjet coracoid osseous autograft and distal tibial osteochondral allograft reconstructions of large (25%) glenoid bone defects prevent failure (dislocation) and are associated with significant glenohumeral kinematic differences that largely confer less translation, load, and torque on the joint in abduction when compared to the native state. These findings suggest that these 2 surgical techniques exhibit similar glenohumeral kinematics such that each provides adequate functional stability following anterior glenoid bone reconstruction. Joint compression load and articular contact pressure distribution may favor distal tibial osteochondral allograft reconstruction for treatment of large (25%) anterior glenoid bone defects associated with shoulder instability.

Mediating Effects of Diagnostic Route on the Comorbidity Gap in Survival of Patients with Diffuse Large B-Cell or Follicular Lymphoma in England.

Background: Socioeconomic inequalities in survival from non-Hodgkin lymphoma persist. Comorbidities are more prevalent amongst those in more deprived areas and are associated with diagnostic delay (emergency diagnostic route), which is also associated with poorer survival probability. We aimed to describe the effect of comorbidity on the probability of death mediated by diagnostic route (emergency vs. elective route) amongst patients with diffuse large B-cell (DLBCL) or follicular lymphoma (FL). Methods: We linked the English population-based cancer registry and hospital admission records (2005-2013) of patients aged 45-99 years. We decomposed the effect of comorbidity on survival into an indirect effect acting through diagnostic route and a direct effect not mediated by diagnostic route. Furthermore, we estimated the proportion of the comorbidity effect on survival mediated by diagnostic route. Results: For both DLBCL (n = 27,379) and FL (n = 14,043), those with any comorbidity, or living in more deprived areas, were more likely to experience diagnostic delay and poorer survival. The indirect effect of comorbidity on mortality through diagnostic route was highest at 12 months since diagnosis (DLBCL: Odds Ratio 1.10 [95% CI 1.07-1.13], FL: OR 1.09 [95% CI 1.04-1.14]). Within the first 12 months since diagnosis, emergency diagnostic route accounted for 24% (95% CI 17.5-29.5) and 16% (95% CI 6.0-25.6) of the comorbidity effect on mortality, for DLBCL and FL, respectively. Conclusion: Efforts to reduce diagnostic delay (emergency diagnosis) amongst patients with comorbidity would reduce inequalities in DLBCL and FL survival by 24% and 16%, respectively. Further public health programs and interventions are needed to reduce diagnostic delay amongst lymphoma patients with comorbidities.

Local Infiltration Analgesia Versus Interscalene Block for Pain Management Following Shoulder Arthroplasty: A Prospective Randomized Clinical Trial.

BACKGROUND: While providing effective analgesia following shoulder arthroplasty, an interscalene block has known complications. Local infiltration analgesia (LIA) using ropivacaine has been successfully employed in other joint arthroplasties, but its efficacy in shoulder arthroplasty has not been studied extensively. The purpose of this study was to compare pain and opioid consumption between LIA and an interscalene block following shoulder arthroplasty. METHODS: Patients undergoing primary shoulder arthroplasty were prospectively randomized into 2 groups: the block group received an interscalene block using liposomal bupivacaine, and the injection group received an LIA injection intraoperatively. The LIA injection included ropivacaine, epinephrine, ketorolac, and normal saline solution. Postoperative visual analog scale pain scores, opioid consumption in morphine milligram equivalents, and complications were compared between the groups. The mean pain scores during the first 24 hours postoperatively were used to test noninferiority of LIA compared with an interscalene block. RESULTS: The study included 74 patients (52 men and 22 women with a mean age of 69 years; 37 were in the injection group and 37 in the block group). There was no significant difference between the groups with respect to pain scores at any postoperative time points (p > 0.05), except for the 8-hour time point, when the injection group had a significantly higher pain score than the block group (p = 0.01). There was no significant difference in opioid consumption between the groups at any time points postoperatively (p > 0.05). The amount of intraoperative opioid consumption was significantly higher in the injection group (p < 0.001). In noninferiority testing for the mean pain scores during the first 24 hours, the injection group was found to be noninferior to the block group. One patient in the block group developed transient phrenic nerve palsy. One patient in the injection group developed dislocation after reverse arthroplasty related to noncompliance. The mean procedure hospital charge was $1,718 for an interscalene block and $157 for LIA. CONCLUSIONS: LIA and an interscalene block provided similar analgesia during the first 24 hours after primary shoulder arthroplasty. LIA was associated with worse pain at 8 hours postoperatively and more intraoperative opioid consumption but was also substantially less costly. LEVEL OF EVIDENCE: Therapeutic Level I . See Instructions for Authors for a complete description of levels of evidence.

Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels.

Iron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study exploits the metalloprotein geNOps in cultured HEK293T epithelial and EA.hy926 endothelial cells to test the iron-dependency in cells adapted to standard room air (18 kPa O2) or physiological normoxia (5 kPa O2). We show that cells in culture require iron supplementation to activate the metalloprotein geNOps and demonstrate for the first time that cells adapted to physiological normoxia require significantly lower iron compared to cells adapted to hyperoxia. This study establishes an essential role for recapitulating oxygen levels in vivo and uncovers a previously unrecognized requirement for ferrous iron supplementation under standard cell culture conditions to achieve geNOps functionality.

Tertiary syphilis mimicking metastatic rectal cancer.

Syphilis is a sexually transmitted infection caused by the bacterium Treponema pallidum. Tertiary syphilis, a late-stage multi-visceral complication of the disease is characterized by its diversity of clinical manifestations. Here, we present the first documented case of tertiary syphilis that clinically and radiologically mimicked primary rectal cancer with hepatic metastasis.

Functional diversity in the RAS subfamily of small GTPases.

RAS small GTPases regulate important signalling pathways and are notorious drivers of cancer development and progression. While most research to date has focused on understanding and addressing the oncogenic potential of three RAS oncogenes: HRAS, KRAS, and NRAS; the full RAS subfamily is composed of 35 related GTPases with diverse cellular functions. Most remain deeply understudied despite strong evolutionary conservation. Here, we highlight a group of 17 poorly characterized RAS GTPases that are frequently down-regulated in cancer and evidence suggests may function not as oncogenes, but as tumour suppressors. These GTPases remain largely enigmatic in terms of their cellular function, regulation, and interaction with effector proteins. They cluster within two families we designate as 'distal-RAS' (D-RAS; comprised of DIRAS, RASD, and RASL10) and 'CaaX-Less RAS' (CL-RAS; comprised of RGK, NKIRAS, RERG, and RASL11/12 GTPases). Evidence of a tumour suppressive role for many of these GTPases supports the premise that RAS subfamily proteins may collectively regulate cellular proliferation.

Interpretation of Dihydrorhodamine-1,2,3 Flow Cytometry in Chronic Granulomatous Disease: an Atypical Exemplar.

PURPOSE: This is a functional characterization of a novel CYBA variant associated with normal DHR flow cytometry. Chronic granulomatous disease (CGD) is an inborn error of immunity characterized by recurrent bacterial and fungal infections and dysregulated inflammatory responses due to defective phagocytic cell function leading to the formation of granulomas. CGD patients have pathogenic variants in any of the five components of the phagocytic NADPH oxidase, which transfers electrons through the phagosomal membrane and produces superoxide upon bacterial uptake. Here, we report a pediatric female patient with a novel homozygous missense variant (c.293C > T, p.(Ser98Leu)) in CYBA, encoding the p22phox protein, associated with autosomal recessive CGD. METHODS AND RESULTS: The patient presented with severe recurrent pneumonia. Specific pathogens identified included Burkholderia and Serratia species suggesting neutrophil functional abnormalities; however, the dihydrorhodamine-1,2,3 (DHR) flow cytometric and cytochrome c reduction assays for neutrophil respiratory burst fell within the low side of the normal range. Western blot and flow cytometric analysis of individual NADPH oxidase components revealed reduced levels of p22phox and gp91phoxphox proteins. The pathological consequence of the p.Ser98Leu variant was further evaluated in heterologous expression systems, which confirmed reduced p22phox protein stability and oxidase activity. CONCLUSIONS: Although this patient did not exhibit all the classic features of CGD, such as granulomas and skin infections, she had recurrent pneumonias with oxidant-sensitive pathognomonic organisms, resulting in appropriate targeted CGD testing. This case emphasizes the need to contextually interpret laboratory data, especially using clinical findings to direct additional assessments including genetic analysis.

Effectiveness of caregiver interventions for people with cancer and non-cancer-related chronic pain: a systematic review and meta-analysis.

BACKGROUND: People with chronic pain frequently have difficulties in completing everyday tasks to maintain independence and quality of life. Informal caregivers may provide support to these individuals. However, the effectiveness of interventions to train and support these individuals in caregiving remains unclear. The purpose of this study was to systematically review the evidence to determine the effectiveness of caregiver interventions to support informal caregivers of people with chronic pain. METHODS: A systematic review of published and unpublished literature databases was undertaken (9 April 2021). Trials reporting clinical outcomes of caregiver interventions to train informal caregivers to support community-dwelling people with chronic pain were included. Meta-analysis was undertaken and each outcome was assessed using Grading of Recommendations, Assessment, Development and Evaluation. RESULTS: Twenty-seven studies were eligible (N = 3427 patients). Twenty-four studies assessed patients with cancer pain and three with musculoskeletal pain. No other patient groups were identified. There was very low-quality evidence that caregiver interventions were beneficial for caregiver health-related quality of life (standardised mean difference = 0.26, 95% confidence interval = 0.01 to 0.52; N = 231). There was moderate-quality evidence that caregiving interventions were effective in reducing pain in the short-term (standardised mean difference = 0.16, 95% confidence interval = -0.29 to -0.03). There was low-quality evidence that caregiving interventions had no beneficial effect over usual care for psychological outcomes, fatigue, coping or physical function in the long-term. CONCLUSION: Caregiving interventions may be effective for patients and caregivers but only in the shorter-term and for a limited number of outcomes. There is insufficient evidence examining the effectiveness of caregiver interventions for people with non-cancer-related pain.

Investigating the inequalities in route to diagnosis amongst patients with diffuse large B-cell or follicular lymphoma in England.

INTRODUCTION: Diagnostic delay is associated with lower chances of cancer survival. Underlying comorbidities are known to affect the timely diagnosis of cancer. Diffuse large B-cell (DLBCL) and follicular lymphomas (FL) are primarily diagnosed amongst older patients, who are more likely to have comorbidities. Characteristics of clinical commissioning groups (CCG) are also known to impact diagnostic delay. We assess the association between comorbidities and diagnostic delay amongst patients with DLBCL or FL in England during 2005-2013. METHODS: Multivariable generalised linear mixed-effect models were used to assess the main association. Empirical Bayes estimates of the random effects were used to explore between-cluster variation. The latent normal joint modelling multiple imputation approach was used to account for partially observed variables. RESULTS: We included 30,078 and 15,551 patients diagnosed with DLBCL or FL, respectively. Amongst patients from the same CCG, having multimorbidity was strongly associated with the emergency route to diagnosis (DLBCL: odds ratio 1.56, CI 1.40-1.73; FL: odds ratio 1.80, CI 1.45-2.23). Amongst DLBCL patients, the diagnostic delay was possibly correlated with CCGs that had higher population densities. CONCLUSIONS: Underlying comorbidity is associated with diagnostic delay amongst patients with DLBCL or FL. Results suggest a possible correlation between CCGs with higher population densities and diagnostic delay of aggressive lymphomas.

What Are the Risk Factors for an Upper Extremity Deep Venous Thrombosis After Orthopaedic Irrigation and Debridement and Peripherally Inserted Central Catheter Placement?

BACKGROUND: Peripherally inserted central catheter (PICC) placement is necessary for delivery of intravenous (IV) antibiotics to treat bone and soft tissue infections. Upper extremity deep venous thrombosis (DVT) after PICC placement is a complication with unknown incidence in the orthopaedic literature. The major objectives of this study are Identifying the rate of upper extremity PICC-associated DVTs after orthopaedic procedures;Which orthopaedic subspecialties are most likely to encounter an upper extremity PICC-associated DVT?What surgeries or medical comorbidities are risk factors for upper extremity PICC-associated DVTs?Does type of DVT chemoprophylaxis decrease the risk of an upper extremity PICC-associated DVT? METHODS: A retrospective review of electronic medical records (EMR) was performed to include all patients undergoing irrigation and debridement (I&D) for treatment of orthopaedic surgery-related infections over a 10-year period. All patients with PICC placement were included for analyses. Age, sex, and medical comorbidities were extracted from the EMR. Mann-Whitney non-parametric tests, Fisher's exact tests, Chi-square tests, and Cochran-Mantel-Haenszel (CMH) tests were used to determine associations with DVT events for those with PICCs based on medical comorbidities, PICC lumen size, team placing the PICC, impact of implant removal, and protective effect of DVT chemoprophylaxis. Significance was set at p<0.05. RESULTS: Twenty-one of 660 patients (3.18% rate) were found to have an upper extremity PICC-associated DVT. A history of DVT (OR=8.99 [95% CI: 3.39, 49.42]) was significantly associated with an upper extremity PICC-associated DVT. The greatest risk for an upper extremity PICC-associated DVT was intramedullary implant removal (OR=12.43 [95% CI: 3.13, 49.52]). The type of DVT chemoprophylaxis did not significantly affect the likelihood of an upper extremity PICC-associated DVT. CONCLUSION: Intramedullary implant removal and a history of DVT are risk factors for an upper extremity PICC-associated DVT. The results of this study should be of particular interest to surgeons who do not typically give DVT prophylaxis and plan to perform surgery on patients with CHF, a history of a DVT, or plan to manipulate the intramedullary canal.

Orthopedic Manifestations of Cystic Fibrosis.

Cystic fibrosis (CF) is a relatively common disease seen in Whites of northern European descent. Classically, it was a lethal disease and uncommon for the orthopedic practitioner to interact with CF patients. Recent pharmaceutical breakthroughs targeting the CF transmembrane conductance regulator (CFTR) gene have significantly prolonged patient life expectancy. This makes it increasingly likely that orthopedic surgeons will encounter CF patients in their clinic. In this article, the authors discuss pertinent musculoskeletal manifestations of the CF population, including the increased risk of decreased bone mineral density and bone mineral content, muscle deconditioning, spinal kyphosis, fractures, and elevated systemic inflammation predisposing these individuals to CF-related arthralgia. The diagnoses are grouped into subspecialties (arthroplasty, pediatrics, spine, sports, and trauma) most likely to evaluate the patient. Additionally, the authors review treatment options for these conditions and discuss the need for these patients to be seen in the perioperative period by their CF care team for patient optimization due to their diminished pulmonary function. Interspersed with this literature review, the authors present 2 unique cases. The first case details a patient with pain over her spine due to multilevel spinous process bursitis caused by a high-frequency chest wall oscillation system, which masquerades as an infection. The second case is a non-contact midsubstance rectus femoris tear in an athlete. These cases highlight the need for increased vigilance for uncommon diagnoses in the CF patient population. [Orthopedics. 2021;44(3):e440-e445.].

NMR Detection Methods for Profiling RAS Nucleotide Cycling.

RAS oncoproteins exhibit a switch-like behavior to drive diverse signaling cascades. In the active GTP-bound state, a conformational change occurs in these enzymes that enables interaction with downstream effectors. Nucleotide-dependent conformational exchange is easily detected with real-time NMR (RT-NMR) spectroscopy. RT-NMR has been firmly established as an effective assay to measure RAS oncoprotein nucleotide exchange and GTP hydrolysis kinetics and can further determine the regulatory activity of guanine exchange factors (GEFs) and GTPase activating proteins (GAPs). It is now possible to multiplex these assays, allowing for the precise monitoring of activation states for mixtures of RAS oncoproteins or other RAS superfamily GTPases. Here, we describe the protocols necessary to express and purify isotopically labeled RAS and detail how to carry out an RT-NMR assay on a singular RAS protein or on a mixture of small GTPases.