Efficient and economic protein labeling for NMR in mammalian expression systems: Application to a preT-cell and T-cell receptor protein.

Protein nuclear magnetic resonance (NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with 15N and 13C, respectively. Moreover, E. coli can grow and express proteins in uniformly deuterium-substituted water (D2O), a strategy useful for experiments targeting high molecular weight proteins. Unfortunately, many proteins, particularly those requiring specific posttranslational modifications like disulfide bonding or glycosylation for proper folding and/or function, cannot be readily expressed in their functional forms using E. coli-based expression systems. One such class of proteins includes T-cell receptors and their related preT-cell receptors. In this study, we present an expression system for isotopic labeling of proteins using a nonadherent human embryonic kidney cell line, Expi293F, and a specially designed media. We demonstrate the application of this platform to the ß subunit common to both receptors. In addition, we show that this expression system and media can be used to specifically label amino acids Phe, Ile, Val, and Leu in this system, utilizing an amino acid-specific labeling protocol that allows targeted incorporation at high efficiency without significant isotopic scrambling. We demonstrate that this system can also be used to express proteins with fluorinated amino acids. We were routinely able to obtain an NMR sample with a concentration of 200 µM from 30 mL of culture media, utilizing less than 20 mg of the labeled amino acids.

Parsing digital or analogue TCR performance through piconewton forces.

αß T-cell receptors (TCRs) recognize aberrant peptides bound to major histocompatibility complex molecules (pMHCs) on unhealthy cells, amplifying specificity and sensitivity through physical load placed on the TCR-pMHC bond during immunosurveillance. To understand this mechanobiology, TCRs stimulated by abundantly and sparsely arrayed epitopes (NP 366-374 /D b and PA 224-233 /D b , respectively) following in vivo influenza A virus infection were studied with optical tweezers. While certain NP repertoire CD8 T lymphocytes require many ligands for activation, others are digital, needing just few. Conversely, all PA TCRs perform digitally, exhibiting pronounced bond lifetime increases through sustained, energizing volleys of structural transitioning. Optimal digital performance is superior in vivo, correlating with ERK phosphorylation, CD3 loss, and activation marker upregulation in vitro . Given neoantigen array paucity, digital TCRs are likely critical for immunotherapies. One Sentence Summary: Quality of ligand recognition in a T-cell repertoire is revealed through application of physical load on clonal T-cell receptor (TCR)-pMHC bonds.

Primary Total Hip Arthroplasty in Juvenile Idiopathic Arthritis: Survivorship After a Median Follow-Up of 12 Years.

BACKGROUND: Juvenile idiopathic arthritis (JIA) is a chronic inflammatory condition of childhood that frequently affects the hip. Total hip arthroplasty (THA) in JIA can be challenging due to the patient's young age, small proportion, complex anatomy, and bone loss. Outcome data are limited. METHODS: We reviewed prospectively collected data in 57 JIA patients (83 hips) who underwent THA between 1986 and 2020 by a single surgeon. The median patient age at surgery was 26 years (range, 14 to 62). Reoperation-free survival was assessed via the cumulative incidence function, accounting for the competing risk of death. Relationships between patient and implant factors and survivorship were evaluated by stratification of the cumulative incidence function and Gray's tests. Wilcoxon signed rank tests were used to assess the preoperative to latest postoperative change in patient-reported outcome measures. RESULTS: At a median (interquartile range) of 12 (4, 20) years of follow-up, 13 (16%) patients underwent reoperation, most commonly for polyethylene wear and osteolysis (7 hips). The estimated incidence of 10-year, 20-year, and 30-year revision (95% confidence interval) were 11.3% (4.5, 21.6%), 18.5% (8.9, 30.9%), and 40.6% (19.4, 60.9%), respectively. There were no differences in survival based on patient age, sex, implant fixation method, polyethylene type, or thickness. All patient-reported outcome measures improved from preoperative to latest follow-up. CONCLUSIONS: Primary THA is a durable and effective treatment for JIA patients with severe hip involvement and results in major improvements in pain and function. We did not identify any factors predictive of failure.

Primary thoracic intramedullary spinal cord tumor with likely metastases of glial origin to the lumbosacral vertebrae: Illustrative case.

Background: Metastasis of systemic neoplasms to the spine is common; however, the metastasis of primary spinal cord tumors to other regions in the body is an infrequent occurrence. A few case reports have described the metastasis of primary spinal cord tumors, and in most cases, patients were younger than 30 years of age. Case Description: We present an illustrative case of a 47-year-old female with metastatic lesions to the lumbosacral vertebrae years after the initial diagnosis of an intradural, intramedullary spinal cord tumor (IMSCT). Although the surgical biopsy of the IMSCT was nondiagnostic, the patient was not found to have a separate primary neoplastic source, and the specimens of the metastatic lesions from the lumbar vertebral body were of glial origin. Conclusion: Metastasis from primary IMSCTs is extremely rare. Distant vertebral body and intracranial metastasis are even rarer yet possible. The clinical course is highly aggressive and responds poorly to current standard treatment.

An Open-Source Curriculum to Teach Practical Academic Research Skills.

Academic productivity is important for career advancement, yet not all trainees have access to structured research programs. Without formal teaching, acquiring practical skills for research can be challenging. A comprehensive research course that teaches practical skills to translate ideas into publications could accelerate trainees' productivity and liberate faculty mentors' time. We share our experience designing and teaching "A Practical Introduction to Academic Research", a course that teaches practical skills including building productive habits, recognizing common statistical pitfalls, writing cover letters, succinct manuscripts, responding to reviewers, and delivering effective presentations. We share open-source educational material used during the Winter 2022 iteration to facilitate curriculum adoption at peer institutions.

Why the Treatment Sequence Matters: Interplay Between Chemotherapy Cycles Received, Cumulative Dose Intensity, and Survival in Resected Early-stage Pancreas Cancer.

OBJECTIVE: To define the optimal threshold of perioperative chemotherapy completion and relative dose intensity (RDI) for patients with resected pancreatic ductal adenocarcinoma (PDAC). BACKGROUND: Many patients who undergo pancreatectomy for PDAC fail to initiate or complete recommended perioperative chemotherapy. The association between the amount of perioperative chemotherapy received and overall survival (OS) is not well-defined. METHODS: Single-institution analysis of 225 patients who underwent pancreatectomy for stage I/II PDAC (2010-2021). Associations between OS, chemotherapy cycles completed, and RDI were analyzed. RESULTS: Regardless of treatment sequence, completion of ≥67% of recommended cycles was associated with improved OS compared with no chemotherapy [median OS: 34.5 vs 18.1 months; hazard ratio (HR): 0.43; 95% CI: 0.25-0.74] and <67% of cycles (median OS: 17.9 months; HR: 0.39; 95% CI: 0.24-0.64). A near-linear relationship existed between cycles completed and the RDI received (ß = 0.82). A median RDI of 56% corresponded to the completion of 67% of cycles. Receipt of ≥56% RDI was associated with improved OS compared with no chemotherapy (median OS: 35.5 vs 18.1 months; HR: 0.44; 95% CI: 0.23-0.84) and <56% RDI (median OS: 27.2 months; HR: 0.44; 95% CI: 0.20-0.96). Neoadjuvant chemotherapy is associated with increased odds of receiving ≥67% of recommended cycles (odds ratio: 2.94; 95% CI: 1.45-6.26) and ≥56% RDI (odds ratio: 4.47; 95% CI: 1.72-12.50). CONCLUSIONS: Patients with PDAC who received ≥67% of recommended chemotherapy cycles or ≥56% cumulative RDI had improved OS. Neoadjuvant therapy was associated with increased odds of receiving ≥67% of cycles and ≥56% cumulative RDI and should be considered in all patients with resectable PDAC.

Single, Recurrent, Synchronous, and Metachronous Periprosthetic Joint Infections in Patients With Multiple Hip and Knee Arthroplasties.

BACKGROUND: The rate for periprosthetic joint infection (PJI) exceeds 1% for primary arthroplasties. Over 30% of patients who have a primary arthroplasty require an additional arthroplasty, and the impact of PJI on this population is understudied. Our objective was to assess the prevalence of recurrent, synchronous, and metachronous PJI in patients who had multiple arthroplasties and to identify risk factors for a subsequent PJI. METHODS: We identified 337 patients who had multiple arthroplasties and at least 1 PJI that presented between 2003 and 2021. The mean follow-up after revision arthroplasty was 3 years (range, 0 to 17.2). Patients who had multiple infected prostheses were categorized as synchronous (ie, presenting at the same time as the initial infection) or metachronous (ie, presenting at a different time as the initial infection). The PJI diagnosis was made using the MusculoSkeletal Infection Society (MSIS) criteria. RESULTS: There were 39 (12%) patients who experienced recurrent PJI in the same joint, while 31 (9%) patients developed PJI in another joint. Positive blood cultures were more likely in the second joint PJI (48%) compared to recurrent PJI (23%) or a single PJI (15%, P < .001). Synchronous PJI represented 42% of the second joint PJI cases (n = 13), while metachronous PJI represented 58% (n = 18). Tobacco users had 75% higher odds of metachronous PJI (odds ratio 1.75, 95% confidence interval: 1.1-2.9, P = .041). CONCLUSION: Over 20% of the patients with multiple arthroplasties and a single PJI will develop a subsequent PJI in another arthroplasty with 12% recurring in the initial arthroplasty and nearly 10% ocurring in another arthroplasty. Particular caution should be taken in patients who use tobacco, have bacteremia, or have Staphylococcus aureus isolation at time of their initial PJI. Optimizing the management of this high-risk patient population is necessary to reduce the additional burden of subsequent PJI. LEVEL OF EVIDENCE: Prognostic Level IV.

Perceptions of Community Service in Dermatology Residency Training Programs: A Survey-Based Study of Program Directors, Residents, and Recent Dermatology Residency Graduates.

Integrating community service (CS) into the dermatology residency program curriculum creates a rewarding training environment that promotes an interest in health disparities, increases skin health equity, and augments the cultural sensitivity of its trainees. To better understand the importance of CS activities in dermatology residency programs, program directors, residents, and recent dermatology residency graduates were surveyed about their perceptions and participation in CS-defined as participation in activities to increase dermatologic access, education, and resources in under-served communities.

The First Pilot Study of an Interactive, 360° Augmented Reality Visualization Platform for Neurosurgical Patient Education: A Case Series.

BACKGROUND: Interactive technologies are increasingly being used for patient education. Augmented reality (AR) is the technology of superimposing digital content on the reality that the user observes. OBJECTIVE: To evaluate a brand new, commercial prototype of a 360° AR visualization platform (360 ARVP) to enhance patient education for neurosurgical patients. METHODS: This pilot study was a single-center, single-provider study that took place in the clinic setting of the senior author. Patients were given the opportunity to use the 360 ARVP (Surgical Theater) concurrently with the neurosurgeon. All patients completed a survey on their experience with the 360 ARVP immediately after use. RESULTS: A total of 24 patients participated in the study. All patients (19 [79.2%] strongly agreed and 5 [20.8%] agreed) reported that using the 360 ARVP system helped improve understanding of their medical condition. The total percentage of patients who either agreed or strongly agreed that experiencing the 360 ARVP helped improve their comfort levels and that they felt included in decisions about their treatment was 95.8%. When asked to rate their level of understanding of their treatment plans on a 0 to 10 scale, the difference in means for the patients' ratings before and after the use of 360 ARVP was statistically significant (P < .0017). CONCLUSION: This pilot study demonstrates that AR can be used as an adjunctive tool for patient education. Patients found that the 360 ARVP increased their understanding of their medical conditions and improved their comfort level with the proposed treatments.

Dracoleuctra, a new wingless stonefly genus from southern Oregon, U.S.A. (Plecoptera: Leuctridae).

Dracoleuctra siskiyou Lee Baumann gen. nov. sp. nov. a new apterous genus and species in the stonefly family Leuctridae (Insecta, Plecoptera), is described from males and females collected in southern Oregon, U.S.A. Habitus and terminalia are illustrated using both light photography images and scanning electron micrographs. Diagnostic characters, habitat, biological notes, and stonefly apterism in the Klamath Mountains region are discussed.

Miliary pattern in secondary central nervous system T-cell lymphoma.

BACKGROUND: Secondary central nervous system lymphoma may manifest in a variety of ways on imaging, but most commonly presents with leptomeningeal disease, isolated parenchymal lesions, or both. We present a case of secondary central nervous system T-cell lymphoma with miliary pattern of spread noted on imaging. CASE DESCRIPTION: Our patient had known systemic T-cell lymphoma involving the gastrointestinal and respiratory tracts and underwent stereotactic biopsy confirming secondary cerebral metastasis. This spread pattern is an uncommon manifestation of disease and in our experience carries a very poor prognosis. CONCLUSION: We highlight the need to maintain a broad differential diagnosis that includes other metastatic disease and infectious etiologies, including toxoplasmosis and tuberculosis. High clinical suspicion and timely confirmatory testing including biopsy or cerebrospinal fluid flow cytometry are critical to treatment.

The Role of Instrumentation in the Surgical Treatment of Spondylodiscitis and Spinal Epidural Abscess: A Single-Center Retrospective Cohort Study.

BACKGROUND: Despite the high incidence of spinal infections that require an operation, there is no consensus on the most appropriate initial surgical management for these patients regarding decompression with vs without instrumented fusion. In this study, we investigated the differences in clinical outcomes, complication rates, and reoperation rates between patients with spinal epidural abscess who underwent decompression alone vs decompression with instrumented fusion. METHODS: Records of patients undergoing operative intervention for spondylodiscitis with spinal epidural abscess at the authors' institution between 2011 and 2018 were reviewed. Two cohorts were observed: patients who underwent decompression alone and patients who underwent decompression with instrumented fusion as the initial operation. Patient demographics and primary outcomes were analyzed and compared. RESULTS: Medical records of 74 patients with spinal infection were reviewed, and 47 patients met the inclusion criteria. There were 27 (57.4%) patients who underwent decompression alone and 20 (42.6%) patients who underwent decompression and fusion. There were no significant differences in the comorbidities, level, and/or extent of infectious involvement between the decompression alone cohort and the decompression with fusion cohort. Although no significant differences were seen between groups with regard to complication rates and neurological outcomes, the reoperation rate was significantly higher in the patients who underwent decompression alone (51.9% vs 10%, P = 0.004). CONCLUSIONS: Decompression with instrumented fusion delivers neurological outcomes and complication rates similar to those seen with decompression alone in patients with spondylodiscitis. However, there was a significantly higher reoperation rate in the decompression only cohort compared to the decompression and fusion cohort.

BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1-ubiquitin clusters.

ATG9A, the only multi-pass transmembrane protein among core ATG proteins, is an essential regulator of autophagy, yet its regulatory mechanisms and network of interactions are poorly understood. Through quantitative BioID proteomics, we identify a network of ATG9A interactions that includes members of the ULK1 complex and regulators of membrane fusion and vesicle trafficking, including the TRAPP, EARP, GARP, exocyst, AP-1, and AP-4 complexes. These interactions mark pathways of ATG9A trafficking through ER, Golgi, and endosomal systems. In exploring these data, we find that ATG9A interacts with components of the ULK1 complex, particularly ATG13 and ATG101. Using knockout/reconstitution and split-mVenus approaches to capture the ATG13-ATG101 dimer, we find that ATG9A interacts with ATG13-ATG101 independently of ULK1. Deletion of ATG13 or ATG101 causes a shift in ATG9A distribution, resulting in an aberrant accumulation of ATG9A at stalled clusters of p62/SQSTM1 and ubiquitin, which can be rescued by an ULK1 binding-deficient mutant of ATG13. Together, these data reveal ATG9A interactions in vesicle-trafficking and autophagy pathways, including a role for an ULK1-independent ATG13 complex in regulating ATG9A.

MRI-guided laser interstitial thermal therapy using the Visualase system and Navigus frameless stereotaxy in an infant: technical case report.

Laser interstitial thermal therapy (LITT) is increasingly used as a surgical option for the treatment of epilepsy. Placement of the laser fibers relies on stereotactic navigation with cranial fixation pins. In addition, the laser fiber is stabilized in the cranium during the ablation using a cranial bolt that assumes maturity of the skull. Therefore, younger infants (< 2 years of age) have traditionally not been considered as candidates for LITT. However, LITT is an appealing option for patients with familial epilepsy syndromes, such as tuberous sclerosis complex (TSC), due to the multiplicity of lesions and the likely need for multiple procedures. A 4-month-old infant with TSC presented with refractory focal seizures despite receiving escalating doses of 5 antiepileptic medications. Electrographic and clinical seizures occurred numerous times daily. Noninvasive evaluations, including MRI, magnetoencephalography, scalp EEG, and SPECT, localized the ictal onset to a left frontal cortical tuber in the premotor area. In this paper, the authors report a novel technique to overcome the challenges of performing LITT in an infant with an immature skull by repurposing the Navigus biopsy skull mount for stereotactic placement of a laser fiber using electromagnetic-based navigation. The patient underwent successful ablation of the tuber and remained seizure free 4 months postoperatively. To the authors' knowledge, this is the youngest reported patient to undergo LITT. A safe method is described to perform LITT in an infant using commonly available tools without dedicated instrumentation beyond standard stereotactic navigation, a biopsy platform, and the Visualase system.

Chordoma-Current Understanding and Modern Treatment Paradigms.

Chordoma is a low-grade notochordal tumor of the skull base, mobile spine and sacrum which behaves malignantly and confers a poor prognosis despite indolent growth patterns. These tumors often present late in the disease course, tend to encapsulate adjacent neurovascular anatomy, seed resection cavities, recur locally and respond poorly to radiotherapy and conventional chemotherapy, all of which make chordomas challenging to treat. Extent of surgical resection and adequacy of surgical margins are the most important prognostic factors and thus patients with chordoma should be cared for by a highly experienced, multi-disciplinary surgical team in a quaternary center. Ongoing research into the molecular pathophysiology of chordoma has led to the discovery of several pathways that may serve as potential targets for molecular therapy, including a multitude of receptor tyrosine kinases (e.g., platelet-derived growth factor receptor [PDGFR], epidermal growth factor receptor [EGFR]), downstream cascades (e.g., phosphoinositide 3-kinase [PI3K]/protein kinase B [Akt]/mechanistic target of rapamycin [mTOR]), brachyury-a transcription factor expressed ubiquitously in chordoma but not in other tissues-and the fibroblast growth factor [FGF]/mitogen-activated protein kinase kinase [MEK]/extracellular signal-regulated kinase [ERK] pathway. In this review article, the pathophysiology, diagnosis and modern treatment paradigms of chordoma will be discussed with an emphasis on the ongoing research and advances in the field that may lead to improved outcomes for patients with this challenging disease.

Hybrid Anterior Column Realignment-Pedicle Subtraction Osteotomy for Severe Rigid Sagittal Deformity.

OBJECTIVE: Recently, a hybrid anterior column realignment-pedicle subtraction osteotomy (ACR-PSO) approach has been conceived for patients with severe rigid sagittal deformity, the clinical and radiographic outcomes of which require further investigation compared with ACR only. METHODS: A single-center, retrospective chart review identified patients undergoing a combination of hyperlordotic lateral lumbar interbody grafting (ACR) and concurrent Schwab grade 3 three-column osteotomy and propensity-matched patients undergoing ACR only in the same time frame. Anterior longitudinal ligament was directly released or partially sectioned in all patients. Chart data included demographics, Oswestry Disability Index scores, ACR and osteotomy locations, cage dimensions, fusion length, and complications. Radiographic measurements included lumbar lordosis, sagittal vertical axis, pelvic tilt (PT), and proximal junctional kyphosis. RESULTS: Fourteen patients were enrolled in the ACR + PSO group and 36 in the ACR-only group. Mean ages were 68.5 and 63.9 years, 64% and 67% were female, average body mass index was 27.9 and 29.2, and cardiopulmonary comorbidities were 21% and 17%, respectively. There was no difference in complications (P = 0.347). The average follow-up for the ACR + PSO and ACR-only groups were 22 and 18 months, respectively. Excluding 2 mortalities, fusion occurred in all patients. Average change in lumbar lordosis measured -40.8 ± 9.2 degrees and -19.1 ± 15.7 degrees (P = 0.0006), and PT correction measured 10.5 ± 3.4 degrees and 27.3 ± 1.6 degrees (P < 0.0001), respectively. CONCLUSIONS: For patients with severe rigid sagittal deformity, the hybrid ACR-PSO approach offers significant restoration of lumbar lordosis compared with ACR only, with similar complications but reduced PT correction.

Body Composition, Natriuretic Peptides, and Adverse Outcomes in Heart Failure With Preserved and Reduced Ejection Fraction.

OBJECTIVES: The purpose of this study was to determine the relationship between body composition, N-terminal B-type natriuretic peptide (NT-proBNP) levels, and heart failure (HF) phenotypes and outcomes. BACKGROUND: Abnormalities in body composition can influence metabolic dysfunction and HF severity; however, data assessing fat distribution and skeletal muscle (SM) size in HF with reduced (HFrEF) and preserved EF (HFpEF) are limited. Further, whether NPs relate more closely to axial muscle mass than measures of adiposity is not well studied. METHODS: We studied 572 adults without HF (n = 367), with HFrEF (n = 113), or with HFpEF (n = 92). Cardiac magnetic resonance was used to assess subcutaneous and visceral abdominal fat, paracardial fat, and axial SM size. We measured NT-proBNP in 334 participants. We used Cox regression to analyze the relationship between body composition and mortality. RESULTS: Compared with controls, pericardial and subcutaneous fat thickness were significantly increased in HFpEF, whereas patients with HFrEF had reduced axial SM size after adjusting for age, sex, race, and body height (p < 0.05 for comparisons). Lower axial SM size, but not fat, was significantly predictive of death in unadjusted (standardized hazard ratio: 0.63; p < 0.0001) and multivariable-adjusted analyses (standardized hazard ratio = 0.72; p = 0.0007). NT-proBNP levels more closely related to lower axial SM rather than fat distribution or body mass index (BMI) in network analysis, and when simultaneously assessed, only SM (p = 0.0002) but not BMI (p = 0.18) was associated with NT-proBNP. However, both NT-proBNP and axial SM mass were independently predictive of death (p < 0.05). CONCLUSIONS: HFpEF and HFrEF have distinct abnormalities in body composition. Reduced axial SM, but not fat, independently predicts mortality. Greater axial SM more closely associates with lower NT-proBNP rather than adiposity. Lower NT-proBNP levels in HFpEF compared with HFrEF relate more closely to muscle mass rather than obesity.

Riga-Fede Disease: A Case of Sublingual Trauma Not Associated With Abuse.

OBJECTIVES: The following case presents a pediatric patient with Riga-Fede disease (RFD), a rare disorder in children under 2 years of age characterized by sublingual ulceration of the tongue due to trauma from repeated rubbing against the primary teeth. Riga-Fede disease is well reported in dental literature but is relatively unknown to the general pediatric community. It can be confused with nonaccidental trauma (NAT) from forced feeding or other abusive trauma because it presents with injury of oral structures including the sublingual frenulum and often results in problems with growth. This case highlights the importance for physicians to recognize RFD as a source of isolated oral injury that is distinct from NAT. Although approximately 35 cases of RFD have been reported in the literature, this is the first case to describe the process of distinguishing this disease from child abuse. METHODS: We present a case of an 8-month-old female infant who presented with extensive injury of her ventral tongue from RFD who underwent a full workup for NAT. We review the literature for guidance on diagnosing RFD and discuss how to distinguish it from abuse. RESULTS: Infants with characteristic ulceration of the ventral tongue opposing new teeth and with no other medical, social, or developmental concerns do not need to undergo further workup. Clinical examination and thorough history are sufficient to make the diagnosis. CONCLUSIONS: Traumatic intraoral injury in nonmobile children is highly concerning for child abuse. However, RFD is a well described cause of sublingual ulceration in infants that is not associated with abuse.