Ultrafast His-Tagged Protein Purification.

This article details how to use a vortex fluidic device (VFD) to accelerate protein purification via immobilized metal affinity chromatography (IMAC). Building upon a previous report of VFD-based purification, we introduce a membrane insert to simplify the purification protocol and the resin recovery step. This new platform can be adapted to different types of IMAC resins and purification membranes. Proteins can be purified directly from clarified lysate, non-clarified lysate, and even non-lysed cultures without concerns of system clogging. Strong binding between the Ni2+ and the target protein's His6-tag effectively captures the target protein on IMAC resins or membranes placed in the VFD. Continuous flow of different solutions through the VFD allows dynamic binding, washing, and elution of the target protein. Furthermore, the system dramatically accelerates protein purification; a typical purification from cell lysate requires approximately 4 min. Herein, we demonstrate the single-step purification of two His6-tagged proteins from both clarified and non-clarified cell lysates without requiring batch binding. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of the resin-loaded membrane insert and the vortex fluidic device (VFD) setup prior to purification Basic Protocol 2: Purification of His6-tagged proteins using the VFD Alternate Protocol: VFD-mediated His6-tagged protein purification from non-clarified lysate Support Protocol: Preparation of chemically modified glass fiber membrane for VFD-mediated immobilized metal affinity chromatography purification.

Laser Ablation of Periventricular Nodular Heterotopia for Medically Refractory Epilepsy.

OBJECTIVE: Periventricular nodular heterotopia (PVNH) is the most common neuronal heterotopia, frequently resulting in pharmaco-resistant epilepsy. Here, we characterize variables that predict good epilepsy outcomes following surgical intervention using stereo-electroencephalography (SEEG) -informed magnetic resonance-guided laser interstitial thermal therapy (MRgLITT). METHODS: A retrospective review of consecutive cases from a single high-volume epilepsy referral center identified patients who underwent SEEG evaluation for PVNH to characterize the intervention and outcomes. RESULTS: Thirty-nine patients underwent SEEG-guided MRgLITT of the seizure onset zone (SoZ) in PVNH and associated epileptic tissue. PVNH and polymicrogyria (PMG) were densely sampled with a mean of 16.5 (SD = 2)/209.4 (SD = 36.9) SEEG probes/recording contacts per patient. Ablation principally targeted just the PVNH and cortex that was abnormal on imaging was ablated (5 patients) only if implicated in the SoZ. Volumetric analyses revealed a high percentage of PVNH SoZ ablation (96.6%, SD = 5.3%) in unilateral and bilateral (92.9%, SD = 7.2%) cases. Mean follow-up duration was 31.4 months (SD = 20.9). Seizure freedom (ILAE 1) was excellent: unilateral PVNH without other imaging abnormalities, 80%; PVNH with mesial temporal sclerosis (MTS) or PMG, 63%; bilateral PVNH, 50%. SoZ ablation percentage significantly impacted surgical outcomes (p < 0.001). INTERPRETATION: PVNH plays a central role in seizure genesis as revealed by dense recordings and selective targeting by LITT. MRgLITT represents a transformative technological advance in PVNH-associated epilepsy with seizure control outcomes consistent with those seen in focal lesional epilepsies. In localized unilateral cases and otherwise normal imaging, PVNH ablation without invasive recordings may be considered, and this approach deserves to be explored further. ANN NEUROL 2024.

Multiscale mapping of transcriptomic signatures for cardiotoxic drugs.

Drug-induced gene expression profiles can identify potential mechanisms of toxicity. We focus on obtaining signatures for cardiotoxicity of FDA-approved tyrosine kinase inhibitors (TKIs) in human induced-pluripotent-stem-cell-derived cardiomyocytes, using bulk transcriptomic profiles. We use singular value decomposition to identify drug-selective patterns across cell lines obtained from multiple healthy human subjects. Cellular pathways affected by cardiotoxic TKIs include energy metabolism, contractile, and extracellular matrix dynamics. Projecting these pathways to published single cell expression profiles indicates that TKI responses can be evoked in both cardiomyocytes and fibroblasts. Integration of transcriptomic outlier analysis with whole genomic sequencing of our six cell lines enables us to correctly reidentify a genomic variant causally linked to anthracycline-induced cardiotoxicity and predict genomic variants potentially associated with TKI-induced cardiotoxicity. We conclude that mRNA expression profiles when integrated with publicly available genomic, pathway, and single cell transcriptomic datasets, provide multiscale signatures for cardiotoxicity that could be used for drug development and patient stratification.

Comparative Analysis of Dorsal Onlay Buccal Mucosal Graft and Vaginal Wall Graft Urethroplasty for Female Urethral Stricture at a Tertiary Care Centre.

OBJECTIVE: To compare dorsal onlay Buccal Mucosal Graft (BMG) and Vaginal Wall Graft (VWG) urethroplasty in the management of Female Urethral Stricture (FUS). METHODS: A retrospective analysis was conducted on 33 women undergoing dorsal onlay urethroplasty using BMG (n = 17) and VWG (n = 16) for urethral stricture at a tertiary care centre. Data including patient demographics, comorbidities, stricture characteristics, preoperative, and postoperative Female Sexual Function Index (FSFI) scores and operative outcomes were analyzed. Their preoperative diagnosis was confirmed with uroflowmetry, micturating cystourethrogram, urethral calibration, and urethrocystoscopy. Per urethral catheter was removed after 3 weeks and patients were followed up at regular intervals with urine analysis, uroflowmetry and post void residual urine assessment. RESULTS: The overall mean age was 47 years (range: 21-75) and follow-up was 16 months (range: 6-26). The overall change in American Urological Association (AUA) symptom score was from 18 to 4, maximum flow rate (Q max) from 5 mL/s to 24 mL/s, Post Void Residual Urine (PVRU) from 110 mL to 10 mL, average operative time of 97 minutes (range: 80 to 118 minutes) with no statistical difference between the 2 groups. Other parameters showed no difference. The overall urethral patency rate was 93.9% with no statistical difference in these groups (0.862). None of the patients developed urinary incontinence. CONCLUSION: Dorsal onlay substitution with BMG and VWG shows equivalent outcomes with low complication rate. Substitution urethroplasty elicits a robust and enduring therapeutic response and should be offered to patients presenting with FUS to avoid the adversities of repeated urethral dilatations.

Contemporary Practice Patterns in the Treatment of Cervical Stenosis and Central Cord Syndrome: A Survey of the Cervical Spine Research Society.

STUDY DESIGN: Cross-sectional study. OBJECTIVE: To evaluate for areas of consensus and divergence of opinion within the spine community regarding the management of cervical spondylotic conditions and acute traumatic central cord syndrome (ATCCS) and the influence of the patient's age, disease severity, and myelomalacia. SUMMARY OF BACKGROUND DATA: There is ongoing disagreement regarding the indications for, and urgency of, operative intervention in patients with mild degenerative myelopathy, moderate to severe radiculopathy, isolated axial symptomatology with evidence of spinal cord compression, and ATCCS without myelomalacia. METHODS: A survey request was sent to 330 attendees of the Cervical Spine Research Society (CSRS) 2021 Annual Meeting to assess practice patterns regarding the treatment of cervical stenosis, myelopathy, radiculopathy, and ATCCS in 16 unique clinical vignettes with associated MRIs. Operative versus nonoperative treatment consensus was defined by a management option selected by >80% of survey participants. RESULTS: Overall, 116 meeting attendees completed the survey. Consensus supported nonoperative management for elderly patients with axial neck pain and adults with axial neck pain without myelomalacia. Operative management was indicated for adult patients with mild myelopathy and myelomalacia, adult patients with severe radiculopathy, elderly patients with severe radiculopathy and myelomalacia, and elderly ATCCS patients with pre-existing myelopathic symptoms. Treatment discrepancy in favor of nonoperative management was found for adult patients with isolated axial symptomatology and myelomalacia. Treatment discrepancy favored operative management for elderly patients with mild myelopathy, adult patients with mild myelopathy without myelomalacia, elderly patients with severe radiculopathy without myelomalacia, and elderly ATCCS patients without preceding symptoms. CONCLUSIONS: Although there is uncertainty regarding the treatment of mild myelopathy, operative intervention was favored for nonelderly patients with evidence of myelomalacia or radiculopathy and for elderly patients with ATCCS, especially if pre-injury myelopathic symptoms were present. LEVEL OF EVIDENCE: Level V.

Paraspinal Sarcopenia and Lower Hounsfield Units are Independent Predictors of Increased Risk for Proximal Junctional Complications Following Thoracolumbar Fusions Terminating in the Upper Thoracic Spine.

STUDY DESIGN: Retrospective cohort study. OBJECTIVE: The impact of paraspinal sarcopenia following fusions that extend to the upper thoracic spine remain unknown. The purpose of the present study was to assess the impact of sarcopenia on the development of PJK and PJF following spine fusion surgery from the upper thoracic spine to the pelvis. METHODS: We performed a retrospective review of patients who underwent spine fusion surgery that extended caudally to the pelvis and terminated cranially between T1-6. The cohort was divided into 2 groups: (1) patients without PJK or PJF and (2) patients with PJK and/or PJF. Univariate and multivariate analyses were performed to determine risk factors for the development of proximal junctional complications. RESULTS: We identified 81 patients for inclusion in this study. Mean HU at the UIV was 186.1 ± 47.5 in the cohort of patients without PJK or PJF, which was substantially higher than values recorded in the PJK/PJF subgroup (142.4 ± 40.2) (P < 0.001). Severe multifidus sarcopenia was identified at a higher rate in the subgroup of patients who developed proximal junction pathology (66.7%) than in the subgroup of patients who developed neither PJK nor PJF (7.4%; P < 0.001). Multivariate analysis demonstrated both low HU at the UIV and moderate-severe multifidus sarcopenia to be risk factors for the development of PJK and PJF. CONCLUSIONS: Severe paraspinal sarcopenia and diminished bone density at the UIV impart an increased risk of developing PJK and PJF in following thoracolumbar fusions from the upper thoracic spine to the pelvis.

Unilateral versus bilateral pedicle screw fixation with anterior lumbar interbody fusion: a comparison of postoperative outcomes.

PURPOSE: To determine of the impact of ALIF with minimally invasive unilateral pedicle screw fixation (UPSF) versus bilateral pedicle screw fixation (BPSF) on perioperative outcomes, radiographic outcomes, and the rates of fusion, subsidence, and adjacent segment stenosis. METHODS: All adult patients who underwent one-level ALIF with UPSF or BPSF at an academic institution between 2015 and 2022 were retrospectively identified. Postoperative outcomes including length of hospital stay (LOS), wound complications, readmissions, and revisions were determined. The rates of fusion, screw loosening, adjacent segment stenosis, and subsidence were assessed on one-year postoperative CT. Lumbar alignment including lumbar lordosis, L4-S1 lordosis, regional lordosis, pelvic tilt, pelvic incidence, and sacral slope were assessed on standing x-rays at preoperative, immediate postoperative, and final postoperative follow-up. Univariate and multivariate analysis compared outcomes across posterior fixation groups. RESULTS: A total of 60 patients were included (27 UPSF, 33 BPSF). Patients with UPSF were significantly younger (p = 0.011). Operative time was significantly greater in the BPSF group in univariate (p < 0.001) and multivariate analysis (ß=104.1, p < 0.001). Intraoperative blood loss, LOS, lordosis, pelvic parameters, fusion rate, subsidence, screw loosening, adjacent segment stenosis, and revision rate did not differ significantly between fixation groups. Though sacral slope (p = 0.037) was significantly greater in the BPSF group, fixation type was not a significant predictor on regression. CONCLUSIONS: ALIF with UPSF relative to BPSF predicted decreased operative time but was not a significant predictor of postoperative outcomes. ALIF with UPSF can be considered to increase operative efficiency without compromising construct stability.

Transforaminal lumbar interbody fusion subsidence: computed tomography analysis of incidence, associated risk factors, and impact on outcomes.

OBJECTIVE: The aims of this study were to 1) define the incidence of transforaminal lumbar interbody fusion (TLIF) interbody subsidence; 2) determine the relative importance of preoperative and intraoperative patient- and instrumentation-specific risk factors predictive of postoperative subsidence using CT-based assessment; and 3) determine the impact of TLIF subsidence on postoperative complications and fusion rates. METHODS: All adult patients who underwent one- or two-level TLIF for lumbar degenerative conditions at a multi-institutional academic center between 2017 and 2019 were retrospectively identified. Patients with traumatic injury, infection, malignancy, previous fusion at the index level, combined anterior-posterior procedures, surgery with greater than two TLIF levels, or incomplete follow-up were excluded. Interbody subsidence at the superior and inferior endplates of each TLIF level was directly measured on the endplate-facing surface of both coronal and sagittal CT scans obtained greater than 6 months postoperatively. Patients were grouped based on the maximum subsidence at each operative level classified as mild, moderate, or severe based on previously documented < 2-mm, 2- to 4-mm, and ≥ 4-mm thresholds, respectively. Univariate and regression analyses compared patient demographics, medical comorbidities, preoperative bone quality, surgical factors including interbody cage parameters, and fusion and complication rates across subsidence groups. RESULTS: A total of 67 patients with 85 unique fusion levels met the inclusion and exclusion criteria. Overall, 28% of levels exhibited moderate subsidence and 35% showed severe subsidence after TLIF with no significant difference in the superior and inferior endplate subsidence. Moderate (≥ 2-mm) and severe (≥ 4-mm) subsidence were significantly associated with decreases in cage surface area and Taillard index as well as interbody cages with polyetheretherketone (PEEK) material and sawtooth surface geometry. Severe subsidence was also significantly associated with taller preoperative disc spaces, decreased vertebral Hounsfield units (HU), the absence of bone morphogenetic protein (BMP) use, and smooth cage surfaces. Regression analysis revealed decreases in Taillard index, cage surface area, and HU, and the absence of BMP use predicted subsidence. Severe subsidence was found to be a predictor of pseudarthrosis but was not significantly associated with revision surgery. CONCLUSIONS: Patient-level risk factors for TLIF subsidence included decreased HU and increased preoperative disc height. Intraoperative risk factors for TLIF subsidence were decreased cage surface area, PEEK cage material, bullet cages, posterior cage positioning, smooth cage surfaces, and sawtooth surface designs. Severe subsidence predicted TLIF pseudarthrosis; however, the causality of this relationship remains unclear.

Sisal Fiber Reinforced and Cenosphere Hybridized Polypropylene-SEBS Composite: An Insight into Crystallographic, Dynamic Mechanical and Rheological Behavior.

The current study attempts to explore the crystallographic, rheological and, dynamic mechanical properties of the submicron-treated cenosphere (t- CSF) particles and sisal fiber (SF) reinforced Styrene-(Ethylene-Butylene)-Styrene (SEBS) toughened PP hybrid composites. Moreover, the composites reinforced with 25 wt.% of SF and 5 wt.% of CSF (Treated 6 wt.% cetrimonium bromide (CTAB)) demonstrated the most significant storage modulus (E'), loss modulus (E"), and lowest damping (tan δ) factor throughout the temperature range. Likewise, X-ray diffraction techniques were used to assess the samples' crystallographic properties. The composites reported an enhanced ß phase (responsible for high impact strength and reduced α phase of the base matrix compared to pristine PP. Likewise, all the composites' rheological properties showed an improved complex viscosity (η*) compared to the BM but lower than that of the pristine PP. Overall processing parameters of the BM and composites were improved due to the decrement in the η* of all the composites. The rheological properties confirmed the easy processing of the fabricated composites due to the improved flowability. The storage (G') and loss (G") modulus of all the composites were desirably higher than that of the BM.

Factors Related to Clinical Performance in Spine Surgery Fellowship: Can We Predict Success.

INTRODUCTION: The factors most important in the spine fellowship match may not ultimately correlate with quality of performance during fellowship. This study examined the spine fellow applicant metrics correlated with high application rank compared with the metrics associated with the strongest clinical performance during fellowship. METHODS: Spine fellow applications at three academic institutions were retrieved from the San Francisco Match database (first available to 2021) and deidentified for application review. Application metrics pertaining to research, academics, education, extracurriculars, leadership, examinations, career interests, and letter of recommendations were extracted. Attending spine surgeons involved in spine fellow selection at their institutions were sent a survey to rank (1) fellow applicants based on their perceived candidacy and (2) the strength of performance of their previous fellows. Pearson correlation assessed the associations of application metrics with theoretical fellow rank and actual performance. RESULTS: A total of 37 spine fellow applications were included (Institution A: 15, Institution B: 12, Institution C: 10), rated by 14 spine surgeons (Institution A: 6, Institution B: 4, Institution C: 4). Theoretical fellow rank demonstrated a moderate positive association with overall research, residency program rank, recommendation writer H-index, US Medical Licensing Examination (USMLE) scores, and journal reviewer positions. Actual fellow performance demonstrated a moderate positive association with residency program rank, recommendation writer H-index, USMLE scores, and journal reviewer positions. Linear regressions identified journal reviewer positions (ß = 1.73, P = 0.002), Step 1 (ß = 0.09, P = 0.010) and Step 3 (ß = 0.10, P = 0.002) scores, recommendation writer H-index (ß = 0.06, P = 0.029, and ß = 0.07, P = 0.006), and overall research (ß = 0.01, P = 0.005) as predictors of theoretical rank. Recommendation writer H-index (ß = 0.21, P = 0.030) and Alpha Omega Alpha achievement (ß = 6.88, P = 0.021) predicted actual performance. CONCLUSION: Residency program reputation, USMLE scores, and a recommendation from an established spine surgeon were important in application review and performance during fellowship. Research productivity, although important during application review, was not predictive of fellow performance. LEVEL OF EVIDENCE: III. STUDY DESIGN: Cohort Study.

Comparison of Twin Screw Derotation Type Versus Single Helical Blade Type Cephalomedullary Nail in the Management of Unstable Intertrochanteric Fractures.

BACKGROUND: The intertrochanteric fracture is a frequently occurring fracture, often attributed to osteoporosis in older populations. Recently, there has been a proposal to perform early surgical fixation on elderly patients to facilitate early rehabilitation. This approach has been shown to have a beneficial effect in lowering comorbidities. The study aims to compare the efficacy of the twin screw derotation type cephalomedullary nail with that of the single helical blade type cephalomedullary nail in the management of unstable intertrochanteric fractures. METHODOLOGY:  The research sample included patients from the orthopedic outpatient and emergency departments of Adesh Medical College and Hospital, Ambala Cantt, India, who were scheduled for surgery for unstable intertrochanteric femur fractures. The patients were categorized into two groups according to the kind of implant they were given: either a twin screw derotation cephalomedullary nail or a single helical blade cephalomedullary nail. The functional result was evaluated by comparing the modified Harris hip score (HHS). Patients with unstable intertrochanteric fractures, including reverse oblique fractures and fractures with posteromedial comminution, as well as patients who provided consent, were included in this study. RESULTS:  Thirteen individuals received treatment with proximal femoral nail antirotation (PFNA2), whereas 19 individuals received treatment with proximal femoral nail (PFN). The mean age in the PFNA2 group was 69.51, whereas the mean age in the PFN group was 70.804. There were three patients in the PFNA2 group and five patients in the PFN group who had a tip apex distance of more than 25 mm. According to the Cleveland index, nine patients in the PFNA2 group and eight patients in the PFN group had an implant location that was not optimum. Four patients in the PFNA2 group and seven patients in the PFN group had a neck shaft angle difference of more than 10° between their undamaged and operated sides. The mean HHS was 74.55 for the PFNA2 group and 69.88 for the PFN group. The PFNA2 group exhibited four problems, whereas the PFN group had five issues. CONCLUSION:  The study found that both implants offer similar functional outcomes, with adherence to specific radiological parameters optimizing results. While both face similar challenges with osteoporosis, there was no notable distinction between them. Notably, the PFNA2 group showed superior outcomes in perioperative morbidity.

Treatment of Unresectable BRAF V600E, TERT-Mutated Differentiated Papillary Thyroid Cancer With Dabrafenib and Trametinib.

Complete surgical resection of differentiated papillary thyroid cancer (PTC) is associated with an excellent prognosis. However, for locally invasive PTC, disease-specific morbidity and mortality increases when microscopic margin negative resection (R0) or complete macroscopic resection (R1) is not feasible. Neoadjuvant dabrafenib and trametinib (DT) used in BRAF V600E-positive, unresectable anaplastic thyroid cancer has allowed for R0 or R1 resection and improved survival rates. We demonstrate feasibility of using neoadjuvant DT in a patient with BRAF V600E and TERT-mutated PTC for whom R0/R1 resection was initially aborted due to predicted unacceptable morbidity. The patient was treated with neoadjuvant DT for 5 months, at which time disease was undetectable on imaging with near resolution on final pathology; however, subsequent rapid recurrence after discontinuation of neoadjuvant DT occurred. Neoadjuvant DT offers promise in future cohorts of patients with locally invasive BRAF V600E and TERT-mutated PTC for whom neoadjuvant therapy can reduce surgical morbidity while still allowing for R0/R1 resection.

Change in spinal bone mineral density as estimated by Hounsfield units following osteoporosis treatment with romosozumab, teriparatide, denosumab, and alendronate: an analysis of 318 patients.

OBJECTIVE: The purpose of this study was to determine the effect of osteoporosis medications on opportunistic CT-based Hounsfield units (HU). METHODS: Spine and nonspine surgery patients were retrospectively identified who had been treated with romosozumab for 3 to 12 months, teriparatide for 3 to 12 months, teriparatide for > 12 months, denosumab for > 12 months, or alendronate for > 12 months. HU were measured in the L1-4 vertebral bodies. One-way ANOVA was used to compare the mean change in HU among the five treatment regimens. RESULTS: In total, 318 patients (70% women) were included, with a mean age of 69 years and mean BMI of 27 kg/m2. There was a significant difference in mean HU improvement (p < 0.001) following treatment with romosozumab for 3 to 12 months (n = 32), teriparatide for 3 to 12 months (n = 30), teriparatide for > 12 months (n = 44), denosumab for > 12 months (n = 123), and alendronate for > 12 months (n = 100). Treatment with romosozumab for a mean of 10.5 months significantly increased the mean HU by 26%, from a baseline of 85 to 107 (p = 0.012). Patients treated with teriparatide for > 12 months (mean 23 months) experienced a mean HU improvement of 25%, from 106 to 132 (p = 0.039). Compared with the mean baseline HU, there was no significant difference after treatment with teriparatide for 3 to 12 months (110 to 119, p = 0.48), denosumab for > 12 months (105 to 107, p = 0.68), or alendronate for > 12 months (111 to 113, p = 0.80). CONCLUSIONS: Patients treated with romosozumab for a mean of 10.5 months and teriparatide for a mean of 23 months experienced improved spinal bone mineral density as estimated by CT-based opportunistic HU. Given the shorter duration of effective treatment, romosozumab may be the preferred medication for optimization of osteoporotic patients in preparation for elective spine fusion surgery.