Sequence-independent activity of a predicted long disordered segment of the human papillomavirus type 16 L2 capsid protein during virus entry.

The activity of proteins is thought to be invariably determined by their amino acid sequence or composition, but we show that a long segment of a viral protein can support infection independent of its sequence or composition. During virus entry, the papillomavirus L2 capsid protein protrudes through the endosome membrane into the cytoplasm to bind cellular factors such as retromer required for intracellular virus trafficking. Here, we show that an ~110 amino acid segment of L2 is predicted to be disordered and that large deletions in this segment abolish infectivity of HPV16 pseudoviruses by inhibiting cytoplasmic protrusion of L2, association with retromer, and proper virus trafficking. The activity of these mutants can be restored by insertion of protein segments with diverse sequences, compositions, and chemical properties, including scrambled amino acid sequences, a tandem array of a short sequence, and the intrinsically disordered region of an unrelated cellular protein. The infectivity of mutants with small in-frame deletions in this segment directly correlates with the size of the segment. These results indicate that the length of the disordered segment, not its sequence or composition, determines its activity during HPV16 pseudovirus infection. We propose that a minimal length of L2 is required for it to protrude far enough into the cytoplasm to bind cytoplasmic trafficking factors, but the sequence of this segment is largely irrelevant. Thus, protein segments can carry out complex biological functions such as Human papillomavirus pseudovirus infection in a sequence-independent manner. This finding has important implications for protein function and evolution.

TRM integrins CD103 and CD49a differentially support adherence and motility after resolution of influenza virus infection.

Tissue-resident memory CD8 T (TRM) cells are a unique immune memory subset that develops and remains in peripheral tissues at the site of infection, providing future host resistance upon reexposure to that pathogen. In the pulmonary system, TRM are identified through S1P antagonist CD69 and expression of integrins CD103/ß7 and CD49a/CD29(ß1). Contrary to the established role of CD69 on CD8 T cells, the functions of CD103 and CD49a on this population are not well defined. This study examines the expression patterns and functions of CD103 and CD49a with a specific focus on their impact on T cell motility during influenza virus infection. We show that the TRM cell surface phenotype develops by 2 wk postinfection, with the majority of the population expressing CD49a and a subset that is also positive for CD103. Despite a previously established role in retaining TRM in peripheral tissues, CD49a facilitates locomotion of virus-specific CD8 T cells, both in vitro and in vivo. These results demonstrate that CD49a may contribute to local surveillance mechanisms of the TRM population.

Availability of Consumer Prices for Arthroscopic Meniscus Surgery.

The purpose is to examine the availability of consumer pricing information for arthroscopic meniscal surgery in the United States. Secondary objectives were comparing the price of meniscal repair to meniscectomy and regional pricing differences. Orthopaedic sports medicine clinics were sorted by state and randomly selected from American Orthopaedic Society for Sports Medicine's online directory. Following standardized script, each clinic was called a maximum of three times to obtain pricing information for meniscal surgery. A total of 1,008 distinct orthopaedic sport medicine practices were contacted. Six (6%) clinics were able to provide complete bundle pricing, and 183 (18.2%) clinics were able to provide physician-only fees for either meniscectomy or meniscal repair. Physician-only fees and bundle pricing were significantly less for meniscal repairs as compared to meniscectomies. There were no geographic regional differences in pricing for physician-only fees. There is a paucity of information regarding price transparency for arthroscopic meniscal surgery. (Journal of Surgical Orthopaedic Advances 32(2):083-087, 2023).

Minimal Clinically Important Difference in Robotic-Assisted Total Knee Arthroplasty Versus Standard Manual Total Knee Arthroplasty.

BACKGROUND: The purpose of this study was to determine whether robotic total knee arthroplasty (R-TKA) demonstrated evidence of improvement in minimal clinically important difference (MCID) in early (<4 weeks) and intermittent (4-8 month) patient-reported outcomes compared with manual total knee arthroplasty (M-TKA). METHODS: A prospectively collected database was reviewed of 1160 consecutive patients undergoing R-TKA or M-TKA from December 2017 to October 2019. Primary outcomes consisted of Knee Injury and Osteoarthritis Outcome Score for Joint Replacement (KOOS-JR) and Patient-Reported Outcomes Measurement Information System Global Health Measures of Physical Health (PH) and Mental Health (MH). Statistical analysis included MCID via the distribution method. RESULTS: Univariate analysis demonstrated conflicting results for early MCID achievement favoring M-TKA (4-week KOOS-JR, P = .03) for the multisurgeon cohort, but favored R-TKA (4-week Patient-Reported Outcomes Measurement Information System-PH, P = .04) in the single-surgeon analysis, and the remaining outcome scores were similar. Ultimately, multivariate analysis demonstrated similar 4-week and 6-month MCID achievement in all measures. Lower preoperative scores consistently achieved MCID at a higher rate in M-TKA, although in R-TKA, the higher baseline scores improved at a rate comparable with those with lower scores in all but the short-term postoperative KOOS-JR. CONCLUSION: R-TKA demonstrated comparable MCID achievement to M-TKA across the larger cohort. Single-surgeon comparison did show some early benefit. Confounding variables such as surgical technique, implant fixation, and responsiveness of an outcome measure may be as important as simply what tools are used during surgery. Such granular data should be sought out in future studies.

Machine learning: a useful radiological adjunct in determination of a newly diagnosed glioma's grade and IDH status.

INTRODUCTION: Machine learning methods have been introduced as a computer aided diagnostic tool, with applications to glioma characterisation on MRI. Such an algorithmic approach may provide a useful adjunct for a rapid and accurate diagnosis of a glioma. The aim of this study is to devise a machine learning algorithm that may be used by radiologists in routine practice to aid diagnosis of both: WHO grade and IDH mutation status in de novo gliomas. METHODS: To evaluate the status quo, we interrogated the accuracy of neuroradiology reports in relation to WHO grade: grade II 96.49% (95% confidence intervals [CI] 0.88, 0.99); III 36.51% (95% CI 0.24, 0.50); IV 72.9% (95% CI 0.67, 0.78). We derived five MRI parameters from the same diagnostic brain scans, in under two minutes per case, and then supplied these data to a random forest algorithm. RESULTS: Machine learning resulted in a high level of accuracy in prediction of tumour grade: grade II/III; area under the receiver operating characteristic curve (AUC) = 98%, sensitivity = 0.82, specificity = 0.94; grade II/IV; AUC = 100%, sensitivity = 1.0, specificity = 1.0; grade III/IV; AUC = 97%, sensitivity = 0.83, specificity = 0.97. Furthermore, machine learning also facilitated the discrimination of IDH status: AUC of 88%, sensitivity = 0.81, specificity = 0.77. CONCLUSIONS: These data demonstrate the ability of machine learning to accurately classify diffuse gliomas by both WHO grade and IDH status from routine MRI alone-without significant image processing, which may facilitate usage as a diagnostic adjunct in clinical practice.

The DNA copy number landscape of a collision tumor.

Intracranial collision tumors are composed of two histologically distinct but merging components, and are rare. Their genetic profile has rarely been described. Comparative genome hybridization of a combined meningioma and oligodendroglioma demonstrated deletion of chromosome 22q and of 19q in both tumors. Somatic deletion of chromosome 22q and 19q is associated with development of an intracranial collision tumor.
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Absence of 1p/19q codeletion in oligodendroglioma-like areas of pilocytic astrocytomas.

The presence of oligodendroglioma-like areas in pilocytic astrocytoma may give rise to pathologic diagnostic uncertainty. This study aims to determine if the oligodendroglioma-like areas present in some pilocytic astrocytomas (PA) possess the signature 1p/19q codeletion that is characteristic of classical oligodendroglioma. Array comparative genomic hybridization was carried out on 12 PA samples, from which oligodendroglioma-like areas were microdissected and used as the template DNA source. 1p/19q codeletions were not found in any of the oligodendroglioma areas in PAs. We conclude that PAs with oligodendroglioma-like areas do not share the same molecular genetics as classic oligodendroglioma.
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Editorial Commentary: Arthroscopic Subscapularis Repair: Bridging Successful Tendon Healing and Improved Patient Outcomes.

Open repair has been regarded as the gold standard for the treatment of subscapularis tendon tears. However, recent studies on newer arthroscopic techniques and published results of arthroscopic repair have shown promising results. As such, there exists a growing interest in understanding the best fixation technique to obtain successful functional and patient-reported clinical outcomes. While the literature shows superior outcomes after double-row repair for posterosuperior rotator cuff tears compared with single-row repair, the evidence is inconclusive for subscapularis tears. We believe that arthroscopic double-row transosseous equivalent repair of full-thickness subscapularis tears leads to superior clinical outcomes with higher healing rates and will become the standard of care in the future.

Patients With Infected Total Hip Arthroplasty Undergoing 2-Stage Exchange Arthroplasty Experience Massive Blood Loss.

BACKGROUND: Two-stage exchange arthroplasty is the preferred treatment for chronic periprosthetic joint infection following total hip arthroplasty (THA). These patients are at high risk of substantial blood loss and perioperative blood transfusion. Our study aimed at determining risk factors for blood transfusion during a 2-stage exchange for infected THA. METHODS: Medical records of 297 patients with infected THA who underwent 2-stage exchange arthroplasty from 1997 to 2016 were reviewed. Blood loss was calculated using a validated formula. Transfusion data, clinical information, and operative data were gathered to determine predictors of blood loss and risk factors for perioperative allogeneic blood transfusion. RESULTS: Calculated blood loss was significantly higher during reimplantation than resection arthroplasty (5156.0 ± 3402 mL vs 3706.9 ± 2148 mL; P < .0001). Blood transfusion was needed in 81% after resection and 81.1% after reimplantation. Allogeneic blood transfusion averaged 3.6 ± 1.8 units for stage 1 and 4.2 ± 2.9 units for stage 2 (P = .0066). Patient characteristics that increased the likelihood for perioperative blood transfusions were increasing preoperative international normalized ratio, type 2 diabetes, current smoking, age, and transfusion requirement in the first stage. Tranexamic acid usage was associated with decreased blood loss. CONCLUSION: Patients with periprosthetic joint infection following THA have significant blood loss during both stages of exchange arthroplasty, especially reimplantation. Hematological optimization should be considered in all patients requiring a transfusion after the first stage, as these patients are at greater risk of requiring transfusion after the second stage. The use of tranexamic acid dramatically decreases the risk of requiring a transfusion in both stages and should be more ubiquitously incorporated into blood management protocols.

Concurrent DNA Copy-Number Alterations and Mutations in Genes Related to Maintenance of Genome Stability in Uninvolved Mammary Glandular Tissue from Breast Cancer Patients.

Somatic mosaicism for DNA copy-number alterations (SMC-CNAs) is defined as gain or loss of chromosomal segments in somatic cells within a single organism. As cells harboring SMC-CNAs can undergo clonal expansion, it has been proposed that SMC-CNAs may contribute to the predisposition of these cells to genetic disease including cancer. Herein, the gross genomic alterations (>500 kbp) were characterized in uninvolved mammary glandular tissue from 59 breast cancer patients and matched samples of primary tumors and lymph node metastases. Array-based comparative genomic hybridization showed 10% (6/59) of patients harbored one to 359 large SMC-CNAs (mean: 1,328 kbp; median: 961 kbp) in a substantial portion of glandular tissue cells, distal from the primary tumor site. SMC-CNAs were partially recurrent in tumors, albeit with considerable contribution of stochastic SMC-CNAs indicating genomic destabilization. Targeted resequencing of 301 known predisposition and somatic driver loci revealed mutations and rare variants in genes related to maintenance of genomic integrity: BRCA1 (p.Gln1756Profs*74, p.Arg504Cys), BRCA2 (p.Asn3124Ile), NCOR1 (p.Pro1570Glnfs*45), PALB2 (p.Ser500Pro), and TP53 (p.Arg306*). Co-occurrence of gross SMC-CNAs along with point mutations or rare variants in genes responsible for safeguarding genomic integrity highlights the temporal and spatial neoplastic potential of uninvolved glandular tissue in breast cancer patients.

Modulation of chemotherapeutic drug resistance in neuroblastoma SK-N-AS cells by the neural apoptosis inhibitory protein and miR-520f.

The acquisition of multidrug resistance is a major impediment to the successful treatment of neuroblastoma, a clinically heterogeneous cancer accounting for ∼15% of all pediatric cancer deaths. The MYCN transcription factor, whose gene is amplified in ∼30% of high-risk neuroblastoma cases, influences drug resistance by regulating a cadre of genes, including those involved with drug efflux, however, other high-risk subtypes of neuroblastoma lacking MYCN amplification, such as those with chromosome 11q deletions, also acquire multidrug resistance. To elucidate additional mechanisms involved with drug resistance in non-MYCN amplified tumour cells, an SK-N-AS subline (SK-N-AsCis24) that is significantly resistant to cisplatin and cross resistant to etoposide was developed through a pulse-selection process. High resolution aCGH analysis of SK-N-AsCis24 revealed a focal gain on chromosome 5 containing the coding sequence for the neural apoptosis inhibitory protein (NAIP). Significant overexpression of NAIP mRNA and protein was documented, while experimental modulation of NAIP levels in both SK-N-AsCis24 and in parental SK-N-AS cells confirmed that NAIP was responsible for the drug resistant phenotype by apoptosis inhibition. Furthermore, a decrease in the NAIP targeting microRNA, miR-520f, was also demonstrated to be partially responsible for increased NAIP levels in SK-N-AsCis24. Interestingly, miR-520f levels were determined to be significantly lower in postchemotherapy treatment tumours relative to matched prechemotherapy samples, consistent with a role for this miRNA in the acquisition of drug resistance in vivo, potentially through decreased NAIP targeting. Our findings provide biological novel insight into neuroblastoma drug-resistance and have implications for future therapeutic research.

Extractive FTIR spectroscopy with cryogen-free low-temperature inert preconcentration for autonomous measurements of atmospheric organics: 1: Instrument development and preliminary performance.

In collaboration with the Jefferson County Department of Health and the Environmental Protection Agency (EPA), the University of Alabama in Huntsville developed a novel sensor for detecting very low levels of volatile organic compounds (VOCs). This sensor uses a commercial Fourier-transform infrared (FTIR) spectrometer, a commercial long-path IR gas cell, a commercial acoustic Stirling cyrocooler, and a custom cryogen-free cryotrap to improve sensitivity in an autonomous system with on-board quality control and quality assurance. Laboratory and initial field results show this methodology is sensitive to and well-suited for a wide variety of VOC atmospheric research and monitoring applications, including EPA National Air Toxics Trends Stations and the National Core monitoring network.

miR-199a-5p silencing regulates the unfolded protein response in chronic obstructive pulmonary disease and α1-antitrypsin deficiency.

RATIONALE: Retention of abnormal α1-antitrypsin (AAT) activates the unfolded protein response in AAT-deficient monocytes. The regulatory role of microRNAs (miRNAs) in unfolded protein responses and chronic obstructive pulmonary disease pathogenesis has not been investigated. OBJECTIVES: To investigate miRNA expression and function in MM and ZZ monocytes and identify miRNA(s) regulating the unfolded protein response. METHODS: Peripheral blood monocytes were isolated from asymptomatic and symptomatic MM and ZZ individuals for miRNA expression profiling and pyrosequencing analysis. miRNA/gene and protein expression was measured with quantitative polymerase chain reaction and Western blotting. Overexpression and inhibition studies were performed with pre-miR or anti-miR, respectively. Luciferase reporter genes were used to elucidate direct miRNA-target interactions. Inflammatory cytokines were detected using the Meso Scale Discovery Plex assays. MEASUREMENTS AND MAIN RESULTS: Forty-three miRNAs were differentially expressed, with miR-199a-5p most highly up-regulated in asymptomatic ZZ versus MM monocytes. miR-199a-2 promoter hypermethylation inhibits miR-199a-5p expression and was increased in symptomatic MM and ZZ monocytes compared with asymptomatic counterparts. GRP78, activating transcription factor 6, p50, and p65 were increased in symptomatic versus asymptomatic ZZ monocytes. Reciprocal down- or up-regulation of these markers was observed after miRNA modulation. Direct miR-199a-5p targeting of activating transcription factor 6, p50, and p65 by miR-199a-5p was demonstrated using luciferase reporter systems. Overexpression of miR-199a-5p also decreased other arms of the UPR and expression of cytokines that are not putative targets. CONCLUSIONS: miR-199a-5p is a key regulator of the unfolded protein response in AAT-deficient monocytes, and epigenetic silencing of its expression regulates this process in chronic obstructive pulmonary disease.

Administration of Aspirin as a Prophylaxis Agent Against Venous Thromboembolism Results in Lower Incidence of Periprosthetic Joint Infection.

The efficacy and safety of aspirin (ASA) for prevention of venous thromboembolism (VTE) following total joint arthroplasty (TJA) have been demonstrated. Our hypothesis was that postoperative ASA compared to warfarin lowers the incidence of periprosthetic joint infection (PJI). Between January 2006 and December 2012, 1456 patients received ASA and 1700 patients received warfarin following primary TJA as standard VTE prophylaxis. Logistic regression was utilized to identify independent risk factors of PJI. Incidence of PJI was significantly lower at 0.4% in patients receiving ASA vs. 1.5% in patients receiving warfarin (P<0.001). Warfarin and elevated BMI were independent risk factors for PJI following TJA (P<0.05). Our research suggests that the use of ASA compared to warfarin for VTE prophylaxis reduces the risk of PJI following TJA.

Genetic features of oligodendrogliomas and presence of seizures. The relationship of seizures and genetics in LGOs.

Low grade oligodendrogliomas (LGO) are diffusely infiltrating World Health Organization (WHO) grade II gliomas, 20 - 30% of which show contrast enhancement. Seizures are a common presenting feature. It has been suggested that 1p19q co-deletion is associated with occurrence of seizures in adults, however, to date, the relationship of tumor genetics and seizure activity has not been extensively investigated. We sought to assess the influence of 1p19q co-deletion, IDH1-R132H positivity, and radiological variables on seizure activity in LGO patients. Specifically, we examined whether these characteristics were associated with seizure at initial presentation, or if they could predict outcome in terms of seizure free survival. In 62 LGOs, neither tumor location nor tumor enhancement were associated with seizures. 1p19q co-deletion status did not predict seizures when controlled for mutant IDH1-R132H expression, tumor location, or enhancement status (odds ratio (OR) 0.9, 95% confidence interval (CI) 0.1 - 4.3). This study, although of limited statistical power, did not demonstrate an association between 1p19q status and seizure occurrence in LGO's. Replication in a larger cohort would further support our hypothesis that 1p19q status alone cannot be used as a reliable predictor of seizure occurrence in LGO's.

Surgical Management of a Torn ACL and Bucket-Handle Meniscal Tear in the Pregnant Patient: A Case Report.

CASE: A 36-year-old, 7-month pregnant woman presented to the office with a locked knee and a displaced bucket-handle medial meniscus tear, in the setting of chronic anterior cruciate ligament (ACL) insufficiency. After thorough discussion with the patient and her husband, the obstetrician, and the anesthesiologist, the patient was treated with left knee ACL reconstruction and medial meniscus repair. CONCLUSION: With sufficient preoperative planning and coordinated multidisciplinary care among orthopaedic, anesthesiologist, and obstetric specialists, elective knee surgery can be performed safely in time-sensitive situations during pregnancy.

Proteogenomic characterization of primary colorectal cancer and metastatic progression identifies proteome-based subtypes and signatures.

Metastatic progression of colorectal adenocarcinoma (CRC) remains poorly understood and poses significant challenges for treatment. To overcome these challenges, we performed multiomics analyses of primary CRC and liver metastases. Genomic alterations, such as structural variants or copy number alterations, were enriched in oncogenes and tumor suppressor genes and increased in metastases. Unsupervised mass spectrometry-based proteomics of 135 primary and 123 metastatic CRCs uncovered distinct proteomic subtypes, three each for primary and metastatic CRCs, respectively. Integrated analyses revealed that hypoxia, stemness, and immune signatures characterize these 6 subtypes. Hypoxic CRC harbors high epithelial-to-mesenchymal transition features and metabolic adaptation. CRC with a stemness signature shows high oncogenic pathway activation and alternative telomere lengthening (ALT) phenotype, especially in metastatic lesions. Tumor microenvironment analysis shows immune evasion via modulation of major histocompatibility complex (MHC) class I/II and antigen processing pathways. This study characterizes both primary and metastatic CRCs and provides a large proteogenomics dataset of metastatic progression.

ATM-deficiency-induced microglial activation promotes neurodegeneration in ataxia-telangiectasia.

While ATM loss of function has long been identified as the genetic cause of ataxia-telangiectasia (A-T), how it leads to selective and progressive degeneration of cerebellar Purkinje and granule neurons remains unclear. ATM expression is enriched in microglia throughout cerebellar development and adulthood. Here, we find evidence of microglial inflammation in the cerebellum of patients with A-T using single-nucleus RNA sequencing. Pseudotime analysis revealed that activation of A-T microglia preceded upregulation of apoptosis-related genes in granule and Purkinje neurons and that microglia exhibited increased neurotoxic cytokine signaling to granule and Purkinje neurons in A-T. To confirm these findings experimentally, we performed transcriptomic profiling of A-T induced pluripotent stem cell (iPSC)-derived microglia, which revealed cell-intrinsic microglial activation of cytokine production and innate immune response pathways compared to controls. Furthermore, A-T microglia co-culture with either control or A-T iPSC-derived neurons was sufficient to induce cytotoxicity. Taken together, these studies reveal that cell-intrinsic microglial activation may promote neurodegeneration in A-T.

Differential DNA methylation patterns define status epilepticus and epileptic tolerance.

Prolonged seizures (status epilepticus) produce pathophysiological changes in the hippocampus that are associated with large-scale, wide-ranging changes in gene expression. Epileptic tolerance is an endogenous program of cell protection that can be activated in the brain by previous exposure to a non-harmful seizure episode before status epilepticus. A major transcriptional feature of tolerance is gene downregulation. Here, through methylation analysis of 34,143 discrete loci representing all annotated CpG islands and promoter regions in the mouse genome, we report the genome-wide DNA methylation changes in the hippocampus after status epilepticus and epileptic tolerance in adult mice. A total of 321 genes showed altered DNA methylation after status epilepticus alone or status epilepticus that followed seizure preconditioning, with >90% of the promoters of these genes undergoing hypomethylation. These profiles included genes not previously associated with epilepsy, such as the polycomb gene Phc2. Differential methylation events generally occurred throughout the genome without bias for a particular chromosomal region, with the exception of a small region of chromosome 4, which was significantly overrepresented with genes hypomethylated after status epilepticus. Surprisingly, only few genes displayed differential hypermethylation in epileptic tolerance. Nevertheless, gene ontology analysis emphasized the majority of differential methylation events between the groups occurred in genes associated with nuclear functions, such as DNA binding and transcriptional regulation. The present study reports select, genome-wide DNA methylation changes after status epilepticus and in epileptic tolerance, which may contribute to regulating the gene expression environment of the seizure-damaged hippocampus.