Serial Capture Affinity Purification and Integrated Structural Modeling of the H3K4me3 Binding and DNA Damage Related WDR76:SPIN1 Complex.

WDR76 is a multifunctional protein involved in many cellular functions. With a diverse and complicated protein interaction network, dissecting the structure and function of specific WDR76 complexes is needed. We previously demonstrated the ability of the Serial Capture Affinity Purification (SCAP) method to isolate specific complexes by introducing two proteins of interest as baits at the same time. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone marker reader that specifically recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to the SCAP analysis of the SPIN1:SPINDOC complex, H3K4me3 was copurified with the WDR76:SPIN1 complex. In combination with crosslinking mass spectrometry, we built an integrated structural model of the complex which revealed that SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Lastly, interaction network analysis of copurifying proteins revealed the potential role of the WDR76:SPIN1 complex in the DNA damage response. Teaser: In contrast to the SPINDOC/SPIN1 complex, analyses reveal that the WDR76/SPIN1 complex interacts with core histones and is involved in DNA damage.

SARS-CoV-2 disrupts host epigenetic regulation via histone mimicry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and caused the devastating global pandemic of coronavirus disease 2019 (COVID-19), in part because of its ability to effectively suppress host cell responses1-3. In rare cases, viral proteins dampen antiviral responses by mimicking critical regions of human histone proteins4-8, particularly those containing post-translational modifications required for transcriptional regulation9-11. Recent work has demonstrated that SARS-CoV-2 markedly disrupts host cell epigenetic regulation12-14. However, how SARS-CoV-2 controls the host cell epigenome and whether it uses histone mimicry to do so remain unclear. Here we show that the SARS-CoV-2 protein encoded by ORF8 (ORF8) functions as a histone mimic of the ARKS motifs in histone H3 to disrupt host cell epigenetic regulation. ORF8 is associated with chromatin, disrupts regulation of critical histone post-translational modifications and promotes chromatin compaction. Deletion of either the ORF8 gene or the histone mimic site attenuates the ability of SARS-CoV-2 to disrupt host cell chromatin, affects the transcriptional response to infection and attenuates viral genome copy number. These findings demonstrate a new function of ORF8 and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation. Further, this work provides a molecular basis for the finding that SARS-CoV-2 lacking ORF8 is associated with decreased severity of COVID-19.

Assessing the differences in operative and patient-reported outcomes between lateral approaches for lumbar fusion: a systematic review and indirect meta-analysis.

OBJECTIVE: Anterior-to-psoas lumbar interbody fusion (ATP-LIF), more commonly referred to as oblique lateral interbody fusion, and lateral transpsoas lumbar interbody fusion (LTP-LIF), also known as extreme lateral interbody fusion, are the two commonly used lateral approaches for performing a lumbar fusion procedure. These approaches help overcome some of the technical challenges associated with traditional approaches for lumbar fusion. In this systematic review and indirect meta-analysis, the authors compared operative and patient-reported outcomes between these two select approaches using available studies. METHODS: Using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) approach, the authors conducted an electronic search using the PubMed, EMBASE, and Scopus databases for studies published before May 1, 2019. Indirect meta-analysis was conducted on fusion rate, cage movement (subsidence plus migration), permanent deficits, and transient deficits; results were depicted as forest plots of proportions (effect size [ES]). RESULTS: A total of 63 studies were included in this review after applying the exclusion criteria, of which 26 studies investigated the outcomes of ATP-LIF, while 37 studied the outcomes of LTP-LIF. The average fusion rate was found to be similar between the two groups (ES 0.97, 95% CI 0.84-1.00 vs ES 0.94, 95% CI 0.91-0.97; p = 0.561). The mean incidence of cage movement was significantly higher in the ATP-LIF group compared with the LTP-LIF group (stand-alone: ES 0.15, 95% CI 0.06-0.27 vs ES 0.09, 95% CI 0.04-0.16 [p = 0.317]; combined: ES 0.18, 95% CI 0.07-0.32 vs ES 0.02, 95% CI 0.00-0.05 [p = 0.002]). The mean incidence of reoperations was significantly higher in patients undergoing ATP-LIF than in those undergoing LTP-LIF (ES 0.02, 95% CI 0.01-0.03 vs ES 0.04, 95% CI 0.02-0.07; p = 0.012). The mean incidence of permanent deficits was similar between the two groups (stand-alone: ES 0.03, 95% CI 0.01-0.06 vs ES 0.05, 95% CI 0.01-0.12 [p = 0.204]; combined: ES 0.03, 95% CI 0.01-0.06 vs ES 0.03, 95% CI 0.00-0.08 [p = 0.595]). The postoperative changes in visual analog scale (VAS) and Oswestry Disability Index (ODI) scores were both found to be higher for ATP-LIF relative to LTP-LIF (VAS: weighted average 4.11 [SD 2.03] vs weighted average 3.75 [SD 1.94] [p = 0.004]; ODI: weighted average 28.3 [SD 5.33] vs weighted average 24.3 [SD 4.94] [p < 0.001]). CONCLUSIONS: These analyses indicate that while both approaches are associated with similar fusion rates, ATP-LIF may be related to higher odds of cage movement and reoperations as compared with LTP-LIF. Furthermore, there is no difference in rates of permanent deficits between the two procedures.

Truncated titin proteins in dilated cardiomyopathy.

Truncating variants in TTN (TTNtvs) are the most common known cause of nonischemic dilated cardiomyopathy (DCM), but how TTNtvs cause disease has remained controversial. Efforts to detect truncated titin proteins in affected human DCM hearts have failed, suggesting that disease is caused by haploinsufficiency, but reduced amounts of titin protein have not yet been demonstrated. Here, we leveraged a collection of 184 explanted posttransplant DCM hearts to show, using specialized electrophoretic gels, Western blotting, allelic phasing, and unbiased proteomics, that truncated titin proteins can quantitatively be detected in human DCM hearts. The sizes of truncated proteins corresponded to that predicted by their respective TTNtvs; the truncated proteins were encoded by the TTNtv-bearing allele; and no degradation fragments from protein encoded by either allele were detectable. In parallel, full-length titin was less abundant in TTNtv+ than in TTNtv− DCM hearts. Disease severity or need for transplantation did not correlate with TTNtv location. Transcriptomic profiling revealed few differences in splicing or allelic imbalance of the TTN transcript between TTNtv+ and TTNtv− DCM hearts. Studies with isolated human adult cardiomyocytes revealed no defects in contractility in cells from TTNtv+ compared to TTNtv− DCM hearts. Together, these data demonstrate the presence of truncated titin protein in human TTNtv+ DCM, show reduced amounts of full-length titin protein in TTNtv+ DCM hearts, and support combined dominant-negative and haploinsufficiency contributions to disease.

Longitudinal Large-Scale Semiquantitative Proteomic Data Stability Across Multiple Instrument Platforms.

With the rapid developments in mass spectrometry (MS)-based proteomics methods, label-free semiquantitative proteomics has become an increasingly popular tool for profiling global protein abundances in an unbiased manner. However, the reproducibility of these data across time and LC-MS platforms is not well characterized. Here, we evaluate the performance of three LC-MS platforms (Orbitrap Elite, Q Exactive HF, and Orbitrap Fusion) in label-free semiquantitative analysis of cell surface proteins over a six-year period. Sucrose gradient ultracentrifugation was used for surfaceome enrichment, following gel separation for in-depth protein identification. With our established workflow, we consistently detected and reproducibly quantified >2300 putative cell surface proteins in a human acute myeloid leukemia (AML) cell line on all three platforms. To our knowledge this is the first study reporting highly reproducible semiquantitative proteomic data collection of biological replicates across multiple years and LC-MS platforms. These data provide experimental justification for semiquantitative proteomic study designs that are executed over multiyear time intervals and on different platforms. Multiyear and multiplatform experimental designs will likely enable larger scale proteomic studies and facilitate longitudinal proteomic studies by investigators lacking access to high throughput MS facilities. Data are available via ProteomeXchange with identifier PXD022721.

Biochemical and functional characterization of mutant KRAS epitopes validates this oncoprotein for immunological targeting.

Activating RAS missense mutations are among the most prevalent genomic alterations observed in human cancers and drive oncogenesis in the three most lethal tumor types. Emerging evidence suggests mutant KRAS (mKRAS) may be targeted immunologically, but mKRAS epitopes remain poorly defined. Here we employ a multi-omics approach to characterize HLA class I-restricted mKRAS epitopes. We provide proteomic evidence of mKRAS epitope processing and presentation by high prevalence HLA class I alleles. Select epitopes are immunogenic enabling mKRAS-specific TCRαß isolation. TCR transfer to primary CD8+ T cells confers cytotoxicity against mKRAS tumor cell lines independent of histologic origin, and the kinetics of lytic activity correlates with mKRAS peptide-HLA class I complex abundance. Adoptive transfer of mKRAS-TCR engineered CD8+ T cells leads to tumor eradication in a xenograft model of metastatic lung cancer. This study validates mKRAS peptides as bona fide epitopes facilitating the development of immune therapies targeting this oncoprotein.

Outcomes following surgical versus endovascular treatment of spinal dural arteriovenous fistula: a systematic review and meta-analysis.

Although surgical resection is associated with a complete cure in most cases of spinal dural arteriovenous fistulas (SDAVF), there has been an increasing trend towards embolisation. We performed a systematic review and meta-analysis comparing surgical resection with endovascular treatment in terms of success of treatment, rate of recurrence and complications. A literature search was conducted using Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Strength of evidence was assessed using the Grading of Recommendations Assessment, Development and Evaluation Working Group system. Surgical outcomes such as initial treatment failure, late recurrence, neurological improvement and complications were compared between the two approaches. We included 57 studies with 2029 patients, of which 32 studies with 1341 patients directly compared surgery (n=590) and embolisation (n=751). Surgery was found to be associated with significantly lower odds of initial treatment failure (OR: 0.15, 95% CI 0.09 to 0.24, I2 0%, p<0.001) and late recurrence (OR 0.18, 95% CI 0.09 to 0.39, I2 0%, p<0.001). The odds of neurological improvement following surgery were also significantly higher compared with embolisation alone (OR: 2.73, CI:1.67 to 4.48, I2 :49.5%, p<0.001). No difference in complication rates was observed between the two approaches (OR 1.78, 95% CI 0.97 to 3.26, I2 0%, p=0.063). Onyx was associated with significantly higher odds of initial failure/late recurrence as compared with n-butyl 2-cyanoacrylate (OR: 3.87, CI: 1.73 to 8.68, I2 :0%, p<0.001). Surgery may be associated with superior outcomes for SDAVFs in comparison to endovascular occlusion. Newer embolisation agents like Onyx have not conferred a significant improvement in occlusion rate.

Comparing outcomes of fusion versus repeat discectomy for recurrent lumbar disc herniation: A systematic review and meta-analysis.

OBJECTIVES: Current surgical treatment options for one-time recurrent lumbar disc herniation (RLDH) include repeat discectomy or discectomy supplemented with fusion. Significant contention exists within the surgical spine community with regard to the most effective treatment modality. The objective of this study is to compare reoperation rates and patient reported outcomes following fusion versus repeat discectomy for RLDH. PATIENTS AND METHODS: The electronic literature search was performed in Ovid Medline/Pubmed, EMBASE and Cochrane, Scopus and China National Knowledge Infrastructure for human studies directly comparing repeat discectomy with fusion for ipsilateral or contralateral RLDH. Random effects meta-analysis was conducted to pool the estimates of effect, using mean differences (MD) and odds ratios (OR) for continuous and categorical outcomes, respectively. RESULTS: A total of 1405 patients with RLDH (746 fusions and 659 repeat discectomies) from 15 studies (13 observational and 2 randomized controlled trials) were analyzed. Mean time to reherniation was 54.4 ±â€¯30.4 months, while average follow-up time was 40 ±â€¯11.7 months (range: 12-92.6). No difference was found between fusions and repeat discectomies with regards to related reoperations (OR: 0.68; 95% C.I: 0.14-3.2). Changes in PRO scores from baseline to last follow-up were also similar between the two groups, including VAS-back pain (MD, -0.3; 95% CI, -1.4 to 0.7), VAS-leg pain (MD, -0.3; 95% CI, -1.4 to 0.7), ODI (MD, 0.6; 95% CI, -0.2 to 1.4), JOA (MD: 1.0; 95% CI: 0.02 to 2.0) and MacNab satisfaction (OR: 1.5; 95% CI, 0.9 to 2.3). CONCLUSION: Available evidence shows that in treating one-time recurrent disc herniations, repeat discectomy and fusion are associated with comparable reoperation rates, incidence of dural tears, functional outcomes as well as satisfaction with surgical treatment at last follow-up. Future longitudinal, randomized controlled trials should be completed to validate any associations found in this study.

Effects of sex and DTNBP1 (dysbindin) null gene mutation on the developmental GluN2B-GluN2A switch in the mouse cortex and hippocampus.

BACKGROUND: Neurodevelopmental disorders such as autism spectrum disorders and schizophrenia differentially impact males and females and are highly heritable. The ways in which sex and genetic vulnerability influence the pathogenesis of these disorders are not clearly understood. The n-methyl-d-aspartate (NMDA) receptor pathway has been implicated in schizophrenia and autism spectrum disorders and changes dramatically across postnatal development at the level of the GluN2B-GluN2A subunit "switch" (a shift from reliance on GluN2B-containing receptors to reliance on GluN2A-containing receptors). We investigated whether sex and genetic vulnerability (specifically, null mutation of DTNBP1 [dysbindin; a possible susceptibility gene for schizophrenia]) influence the developmental GluN2B-GluN2A switch. METHODS: Subcellular fractionation to enrich for postsynaptic density (PSD), together with Western blotting and kinase assay, were used to investigate the GluN2B-GluN2A switch in the cortex and hippocampus of male and female DTNBP1 null mutant mice and their wild-type littermates. Main effects of sex and DTNBP1 genotype, and interactions with age, were assessed using factorial ANOVA. RESULTS: Sex differences in the GluN2B-GluN2A switch emerged across development at the frontal cortical synapse, in parameters related to GluN2B. Males across genotypes displayed higher GluN2B:GluN2A and GluN2B:GluN1 ratios (p < 0.05 and p < 0.01, respectively), higher GluN2B phosphorylation at Y1472 (p < 0.01), and greater abundance of PLCγ (p < 0.01) and Fyn (p = 0.055) relative to females. In contrast, effects of DTNBP1 were evident exclusively in the hippocampus. The developmental trajectory of GluN2B was disrupted in DTNBP1 null mice (genotype × age interaction p < 0.05), which also displayed an increased synaptic GluN2A:GluN1 ratio (p < 0.05) and decreased PLCγ (p < 0.05) and Fyn (only in females; p < 0.0005) compared to wild-types. CONCLUSIONS: Sex and DTNBP1 mutation influence the GluN2B-GluN2A switch at the synapse in a brain-region-specific fashion involving pY1472-GluN2B, Fyn, and PLCγ. This highlights the possible mechanisms through which risk factors may mediate their effects on vulnerability to disorders of NMDA receptor dysfunction.