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The rapid emergence and spread of escaping mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly challenged our efforts in fighting against the COVID-19 pandemic. A broadly neutralizing reagent against these concerning variants is thus highly desirable for the prophylactic and therapeutic treatments of SARS-CoV-2 infection. We herein report a covalent engineering strategy on protein minibinders for potent neutralization of the escaping variants such as B.1.617.2 (Delta), B.1.617.1 (Kappa), and B.1.1.529 (Omicron) through in situ cross-linking with the spike receptor binding domain (RBD). The resulting covalent minibinder (GlueBinder) exhibited enhanced blockage of RBD-human angiotensin-converting enzyme 2 (huACE2) interaction and more potent neutralization effect against the Delta variant than its noncovalent counterpart as demonstrated on authentic virus. By leveraging the covalent chemistry against escaping mutations, our strategy may be generally applicable for restoring and enhancing the potency of neutralizing antibodies to SARS-CoV-2 and other rapidly evolving viral targets.

Introduction: Brain arteriovenous malformations (bAVMs) are a major risk factor for cerebral hemorrhages in young patients. Recent clinical studies have identified somatic activating KRAS mutations in human bAVMs (~76%), and we confirmed the causal role of endothelial KRAS G12V mutation in bAVM development using a mouse model. However, the pathomechanisms underlying bAVM pathology remain to be determined. CD36 is highly expressed in microglia/macrophages (MMs) and microvascular endothelial cells (ECs) and plays various roles in inflammation and angiogenesis, closely related to vascular development and functions. In this study, we determined if CD36 is involved in bAVM pathophysiology. Methods: Mice were injected with either AAV-BR1 carrying GFP (control) or KRAS G12V . We determined CD36 expression in the control or bAVM tissues dissected from the mouse brains. We also determined CD36 levels in the in vitro cultured ECs overexpressing KRAS G12V . Finally, we determined bAVM numbers and size in CD36 knock-out (ko) mice injected with AAV-BR1-KRAS G12V . Results: We found that gene expression of CD36 and the ligands, thrombospondin-1 and -2 were significantly increased in the KRAS G12V -induced bAVMs. However, the CD36 levels were slightly but significantly reduced in KRAS G12V transfected ECs. In the CD36 ko mice, we found that the number of large bAVMs was significantly reduced compared to the control (C57BL/6). Conclusion: Our results indicate that CD36 expression is dysregulated in KRAS G12V -induced bAVMs and contributes to bAVM development. As CD36 has distinctive roles depending on cell types, the role of CD36 in each cell type (e.g., MMs or ECs) on bAVM pathology should be further determined.

Introduction: Plavix resistance is an independent risk factor for thromboembolic complications following carotid artery stent (CAS) placements. The VerifyNow P2Y12 assay is the best studied in gauging for Plavix responsiveness. Its performance is affected when hematocrit (HCT) value is Methods: We included symptomatic carotid artery patients who suffered ischemic strokes and received CAS during the same hospitalization with PRU measured via VerifyNow assay. Therapeutic PRU is defined as ≤180. Primary outcomes were the comparisons of PRU and the proportion of non-responders between low ( Results: 78 of 127 symptomatic carotid artery patients met inclusion criteria. Mean age was 70.5 with 38.5% females. 23 of 78 had HCT < 33. Baseline characteristics between low and high HCT groups were similar except for hemoglobin (9.4 vs. 12.7, p Conclusion: In symptomatic carotid patients receiving CAS, low HCT is associated with increased PRU, resulting in some to be falsely categorized as non-responders receiving more aggressive therapy unnecessarily.

ZBP1 is an interferon-induced cytosolic nucleic acid sensor that facilitates antiviral responses via RIPK3. Although ZBP1-mediated programmed cell death is widely described, whether and how it promotes inflammatory signaling is unclear. Here, we report a ZBP1-induced inflammatory signaling pathway mediated by K63- and M1-linked ubiquitin chains, which depends on RIPK1 and RIPK3 as scaffolds independently of cell death. In human HT29 cells, ZBP1 associated with RIPK1 and RIPK3 as well as ubiquitin ligases cIAP1 and LUBAC. ZBP1-induced K63- and M1-linked ubiquitination of RIPK1 and ZBP1 to promote TAK1- and IKK-mediated inflammatory signaling and cytokine production. Inhibition of caspase activity suppressed ZBP1-induced cell death but enhanced cytokine production in a RIPK1- and RIPK3 kinase activity-dependent manner. Lastly, we provide evidence that ZBP1 signaling contributes to SARS-CoV-2-induced cytokine production. Taken together, we describe a ZBP1-RIPK3-RIPK1-mediated inflammatory signaling pathway relayed by the scaffolding role of RIPKs and regulated by caspases, which may induce inflammation when ZBP1 is activated below the threshold needed to trigger a cell death response.

Identification of the tyrosine phosphorylation (pY)-dependent interactome of immune co-receptors is crucial for understanding signal pathways involved in immunotherapy. However, identifying the motif-specific interactome for each pY commonly found on these multi-phosphorylated membrane proteins remains challenging. Here, we describe a photoaffinity-based chemical proteomic approach to dissect the motif-specific cytoplasmic interactomes of the critical immune co-receptor CD28. Various full-length CD28 cytoplasmic tails (CD28cyto) with defined pY and selectively replaced photo-methionine were synthesized and applied to explore three pY-motif-dependent CD28cyto interactomes. We identified a stand-alone interaction of phospholipase PLCG1 with the Y191 motif with enhanced affinity for the sequence neighboring the transmembrane domain. Importantly, taking advantage of native top-down mass spectrometry with a 193-nm laser, we discovered the direct association of a previously undefined pY218 motif with the kinase PKCθ through its C2 domain. This synthetic CD28cyto-based photoaffinity proteomic approach is generically applicable to the study of other immune co-receptors with multiple pY sites on their linear cytoplasmic tail.

Objective A high rate of cerebral aneurysm recurrence following endovascular coiling has prompted the use of digital subtraction angiography (DSA) for interval follow-up. However, the utility of skull x-rays as an alternative screening method for aneurysm recurrence is unproperly characterized. Methods Retrospective review of a prospective registry of ruptured and unruptured cerebral aneurysms. Anteroposterior and lateral skull x-rays were obtained immediately at the end of the procedure and at 6-month follow-up. Aneurysm recurrence was defined by comparing post-procedure and 6-month DSA imaging. A true positive was defined as a change in coil mass morphology on at least one projection with aneurysm recurrence on DSA, and a true negative defined as a stable coil mass on both projections and no recurrence on DSA. Receiver operating characteristic area under the curve (AUC) statistics was used to assess the performance of skull x-rays in identifying aneurysm recurrence. Results A total of 118 cerebral aneurysms were evaluated with DSA imaging and skull x-rays. A change in coil mass morphology on one projection of skull x-rays correctly detected all true recurrences with a sensitivity of 100% (95% confidence interval [CI], 91-100%). Skull x-rays failed to identify a stable aneurysm coil mass in 15 cases, with a specificity of 79% (68-88%). Skull x-rays performed with AUC 0.8958 (95% CI, 0.8490-0.9431) in identifying aneurysm recurrence. Conclusions The findings of our study suggest that skull x-rays may represent a low-cost, non-invasive screening tool to rule out aneurysm recurrence, which can potentially aid in decreasing the utilization of DSA in the follow-up of patients with coiled cerebral aneurysms.

Objective Brain arteriovenous malformations (bAVMs) are a leading cause of hemorrhagic stroke and neurological deficits in children and young adults, however, no pharmacological intervention is available to treat these patients. Although more than 95% of bAVMs are sporadic without family history, the pathogenesis of sporadic bAVMs is largely unknown, which may account for the lack of therapeutic options. KRAS mutations are frequently observed in cancer, and a recent unprecedented finding of these mutations in human sporadic bAVMs offers a new direction in the bAVM research. Using a novel adeno-associated virus targeting brain endothelium (AAV-BR1), the current study tested if endothelial KRASG12V mutation induces sporadic bAVMs in mice. Methods Five-week-old mice were systemically injected with either AAV-BR1-GFP or -KRASG12V . At 8 weeks after the AAV injection, bAVM formation and characteristics were addressed by histological and molecular analyses. The effect of MEK/ERK inhibition on KRASG12V -induced bAVMs was determined by treatment of trametinib, a US Food and Drug Administration (FDA)-approved MEK/ERK inhibitor. Results The viral-mediated KRASG12V overexpression induced bAVMs, which were composed of a tangled nidus mirroring the distinctive morphology of human bAVMs. The bAVMs were accompanied by focal angiogenesis, intracerebral hemorrhages, altered vascular constituents, neuroinflammation, and impaired sensory/cognitive/motor functions. Finally, we confirmed that bAVM growth was inhibited by trametinib treatment. Interpretation Our innovative approach using AAV-BR1 confirms that KRAS mutations promote bAVM development via the MEK/ERK pathway, and provides a novel preclinical mouse model of bAVMs which will be useful to develop a therapeutic strategy for patients with bAVM. ANN NEUROL 2021;89:926-941.

Background and Purpose: The optimal endovascular stroke therapy (EVT) care delivery structure is unknown. Here, we present our experience in creating an integrated stroke system (ISS) to expand EVT availability throughout our region while maintaining hospital and physician quality standards. Methods: We identified all consecutive patients with large vessel occlusion acute ischemic stroke treated with EVT from January 2014 to February 2019 in our health care system. In October 2017, we implemented the ISS, in which 3 additional hospitals (4 total) became EVT-performing hospitals (EPHs) and physicians were rotated between all centers. The cohort was divided by time into pre-ISS and post-ISS, and the primary outcome was time from stroke onset to EPH arrival. Secondary outcomes included hospital and procedural quality metrics. We performed an external validation using data from the Southeast Texas Regional Advisory Council. Results: Among 513 patients with large vessel occlusion acute ischemic stroke treated with EVT, 58% were treated pre-ISS and 43% post-ISS. Over the study period, EVT procedural volume increased overall but remained relatively low at the 3 new EPHs (P P P Conclusions: In our system, increasing EVT availability decreased time from stroke onset to EPH arrival. The ISS provides a framework to maintain quality in lower volume hospitals.

CONTEXT: It is well known that thyroid hormones are important, being involved in affects the metabolism of carbohydrate, protein, lipids. The relationship between thyroid hormones and lipid metabolism is the focus of recent research. OBJECTIVE: To investigate the relationship between subclinical hypothyroidism and lipid metabolism in women. DESIGN: We conducted an epidemiological survey of thyroid diseases among women in Northeast China from September 2014 to December 2014. SUBJECTS AND METHODS: A total of 1397 women underwent physical examinations and laboratory tests for thyroid function and lipid metabolism. RESULTS: We found that the detection rate of subclinical hypothyroidism was 13.03%. Patients with subclinical hypothyroidism showed significantly higher levels of triglyceride (1.69±1.9 vs. 1.45±1.4) and the risk of hyper triglyceridemia in women with thyroid stimulating hormone (TSH) levels ≥10mIU/L was 4.96-fold higher compared with that in the normal population (P

OBJECTIVETraditionally, stent-assisted coiling and balloon remodeling have been the primary endovascular treatments for wide-necked intracranial aneurysms with complex morphologies. PulseRider is an aneurysm neck reconstruction device that provides parent vessel protection for aneurysm coiling. The objective of this study was to report early postmarket results with the PulseRider device.METHODSThis study was a prospective registry of patients treated with PulseRider at 13 American neurointerventional centers following FDA approval of this device. Data collected included clinical presentation, aneurysm characteristics, treatment details, and perioperative events. Follow-up data included degree of aneurysm occlusion and delayed (> 30 days after the procedure) complications.RESULTSA total of 54 aneurysms were treated, with the same number of PulseRider devices, across 13 centers. Fourteen cases were in off-label locations (7 anterior communicating artery, 6 middle cerebral artery, and 1 A1 segment anterior cerebral artery aneurysms). The average dome/neck ratio was 1.2. Technical success was achieved in 52 cases (96.2%). Major complications included the following: 3 procedure-related posterior cerebral artery strokes, a device-related intraoperative aneurysm rupture, and a delayed device thrombosis. Immediately postoperative Raymond-Roy occlusion classification (RROC) class 1 was achieved in 21 cases (40.3%), class 2 in 15 (28.8%), and class 3 in 16 cases (30.7%). Additional devices were used in 3 aneurysms. For those patients with 3- or 6-month angiographic follow-up (28 patients), 18 aneurysms (64.2%) were RROC class 1 and 8 (28.5%) were RROC class 2.CONCLUSIONSPulseRider is being used in both on- and off-label cases following FDA approval. The clinical and radiographic outcomes are comparable in real-world experience to the outcomes observed in earlier studies. Further experience is needed with the device to determine its role in the neurointerventionalist’s armamentarium, especially with regard to its off-label use.

The dynamic interactions between RNAs and proteins play crucial roles in regulating diverse cellular processes. Proteome-wide characterization of these interactions in their native cellular context remains desirable but challenging. Herein, we developed a photocatalytic crosslinking (PhotoCAX) strategy coupled with mass spectrometry (PhotoCAX-MS) and RNA sequencing (PhotoCAX-seq) for the study of the composition and dynamics of protein-RNA interactions. By integrating the blue light-triggered photocatalyst with a dual-functional RNA-protein crosslinker (RP-linker) and the phase separation-based enrichment strategy, PhotoCAX-MS revealed a total of 2044 RBPs in human HEK293 cells. We further employed PhotoCAX to investigate the dynamic change of RBPome in macrophage cells upon LPS-stimulation, as well as the identification of RBPs interacting directly with the 5' untranslated regions of SARS-CoV-2 RNA.

Quantitative detection and characterization of antigen-specific T cells are crucial to our understanding of immune responses as well as the development of new immunotherapies. Herein, we report a spatiotemporally resolved method for the detection and quantification of cell-cell interactions via

Xue-Song Bai,1,* Chi Zhang,2,* Rui Peng,2,* Guo-Qing Jiang,2 Sheng-Jie Jin,2 Qian Wang,2 Ai-Wu Ke,3 Dou-Sheng Bai2 1The First Clinical Medical College, Dalian Medical University, Dalian, Liaoning 116044, People’s Republic of China; 2Department of Hepatobiliary Surgery, Clinical Medical College, Yangzhou University, Yangzhou 225009, Jiangsu, People’s Republic of China; 3Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai 200032, People’s Republic of China*These authors contributed equally to this workCorrespondence: Dou-Sheng BaiClinical Medical College, Yangzhou University, 98 West Nantong Road, Yangzhou, Jiangsu 225000, People’s Republic of ChinaTel +86-514-87373385Fax +86-514-87990188Email drbaidousheng@163.comBackground: The ubiquitin-proteasome system participates in the pathogenesis and progression of hepatocellular carcinoma (HCC). As an E3 ubiquitin ligase, RNF128 has been proved vital in carcinogenesis, whereas, little is known about the oncogenic mechanisms of RNF128 in HCC.Materials and Methods: Through tissue microarray from HCC patients, we analyzed RNF128 expression and its relationship with clinical outcomes in HCC. Western blot and quantitativerealtime polymerase chain reaction (qRT-PCR) were performed to examine expression levels of RNF128 in HCC tissues and cell lines. Effects of RNF128 on HCC cellular biological functions and the potential mechanism were evaluated through knockdown and overexpression assays in vitro and in vivo methods.Results: RNF128 expression was found to be remarkably elevated in HCC tissues compared with adjacent normal tissues. Furthermore, the overexpression of RNF128 enhanced hepatoma cells proliferation, colony formation, migration, invasion, and apoptotic resistance both in vitro and in vivo. Mechanistically, RNF128 activated EGFR/MEK/ERK signaling pathway and the EGFR inhibitor, gefitinib partially reversed RNF128-enhanced proliferation, invasion, and migration in hepatoma cells.Conclusion: RNF128 promotes HCC progression by activating EGFR/MEK/ERK signaling pathway, which might function as a novel prognostic molecular signature with the potential to be a candidate therapeutic target for HCC patients.Keywords: hepatocellular carcinoma, RNF128, ubiquitination, EGFR/MEK/ERK signaling pathway, prognosis

Despite the increasing evidence of the association between breast cancer and meningioma in women, the relationship between these tumors remains improperly examined. In this study, we aim to identify the sociodemographic and clinicopathologic features of women with breast cancer associated with a higher risk of developing a meningioma.The SEER (Surveillance Epidemiology and End Results) database (18 registries) was used to identify women with breast cancer as their first neoplasm. The risk of subsequent meningioma was reported as the standardized incidence ratio (SIR) and was analyzed by sociodemographic and clinicopathologic subgroups. Results are given as SIR (95% confidence interval [CI]).A total of 564,516 women diagnosed with breast cancer between 2004 and 2016 were included for analysis. A 26% increased risk of meningioma development (SIR, 1.26; 95% CI, 1.19-1.33; P0.05) was found in the cohort compared with the general population. Patients aged between 18 and 49 years (SIR, 2.16; 95% CI, 1.78-2.61; P0.05) and those with a more advanced tumor stage (stage IV; SIR, 2.39; 95% CI, 1.71-3.25; P0.05) were at a higher risk. Hormone receptor expression and treatment modality subgroups were at a similar risk compared with the general population.Our study corroborated the known association between these tumors and found a 26% risk of meningioma development in women with breast cancer, with younger patients and those with a more aggressive disease having a higher than expected risk.

Patients with cerebral vasospasm caused by aneurysmal subarachnoid hemorrhage (aSAH) are often treated with intra-arterial (IA) vasodilator infusion. However, the optimal drug regimen is yet to be elucidated. A retrospective review of patients with aSAH and cerebral vasospasm treated with IA vasodilator infusion was performed. Patients in group 1 (2008–2011) were treated with a single agent, either nicardipine or verapamil, and patients in group 2 (2010–2016) were treated with a regimen of nitroglycerin, verapamil, and nicardipine. The post-infusion improvement ratio (PIIR) was compared between groups. Adjusted multivariate logistic regression models were utilized to determine whether patients treated with multiple vasodilators had an improved functional outcome, defined by the modified Rankin Scale, at discharge and 90-day follow-up. Among 116 patients from group 1 (N = 47) and group 2 (N = 69), the median age was 54.5 years [IQR, 46–53 years] and 78% were female. Use of multiple-agent therapy resulted in a 24.36% improvement in vessel diameter over single-agent therapy (median PIIR: group 1, 10.5% [IQR, 5.3–21.1%] vs group 2, 34.9% [IQR, 21.4–66.0%]; p < 0.0001). In the adjusted multivariate logistic regression, the use of multiple-agent therapy was associated with a better functional outcome at discharge (OR 0.15, 95% CI [0.04–0.55]; p < 0.01) and at 90-day follow-up (OR 0.20, 95% CI [0.05–0.77]; p < 0.05) when compared to single-agent therapy. In this study, we found that patients treated for cerebral vasospasm with IA infusion of multiple vasodilators had an increased vessel response and better functional outcomes compared to those treated with a single agent.

Introduction: Brain arteriovenous malformations (bAVMs) is a high risk factor for intracerebral hemorrhage (ICH). We recently confirmed that KRAS G12V mutation is sufficient to induce sporadic bAVMs that recapitulate the characteristics of human bAVMs including a tangled vasculature and ICH. However, the bAVMs form in random location in the mouse brain. Therefore, it will be important to establish non-invasive methods to detect bAVMs and bAVM-associated ICH for further studies identifying pathophysiology of bAVMs and testing drug efficacy on bAVM pathology. Methods: We induced bAVMs in mice by adeno-associated virus (AAV)-BR1 carrying KRAS G12V . Magnetic resonance angiography (MRA) and T2*-weighted MR imaging (MRI) were performed to assess bAVM and ICH. The bAVM and ICH were graded by the volume (e.g., for bAVMs, grade 13 , 1mm 3 3 ...grade 9>9mm 3 ; for ICH, grade 13 , 0.5mm 3 3 ...grade 9>4.5mm 3 ). The ICHs smaller than 0.5 mm 3 and round shape were considered as microbleeding (ITK-SNAP and ImageJ). The MRI/MRA-visualized bAVM and ICHs were confirmed by histological analyses (latex casting and H&E and Ter-119 immunostaining). Results: By MRA, bAVMs were first detected at 7 weeks after AAV-BR1-KRAS G12V injection and showed robust growth between 7 to 8 weeks. Various size of bAVMs were detected at 9 weeks after the AAV injection. The grade 1 of microbleeding preceded bAVM formation and was detected 2-3 weeks after AAV-BR1-KRAS G12V injection. We confirmed that the locations of bAVM/ICH detected by MRI/MRA imaging overlapped with the ones detected by vascular casting and histological analysis. Conclusions: We successfully established the non-invasive protocol for detecting bAVM growth and ICH in our sporadic bAVM mice model using MRI/MRA technique. With further improvement of the imaging resolution to detect smaller and earlier bAVMs, our non-invasive bAVM/ICH imaging methods will be a useful tool for bAVM study.

Introduction: Brain arteriovenous malformations (bAVMs) are a major risk factor of cerebral hemorrhages in young patients. Recently, somatic KRAS mutation has identified ~60% of human bAVMs, and we confirmed the causal role of endothelial KRAS G12V mutation in bAVM development using a mouse model. Excessive uncontrolled angiogenesis is associated with the abnormal vascular outgrowth in bAVMs. While abnormal VEGF signaling is involved in pathologic angiogenesis, the role in mutant KRAS-induced bAVMs has not been studied. Here, we hypothesized that KRAS mutation induces bAVMs via activation of VEGF signaling, and tested if VEGF inhibition prevents KRAS G12V -induced bAVM development. Methods: We induced bAVMs in mice by systemic injection of AAV-BR1 carrying KRAS G12V mutation (KRAS G12V mice). To determine the effect of KRAS mutation on VEGF signaling, we tested the expression of VEGF-A and VEGFR2 in KRAS G12V -induced bAVMs in vivo and cultured mouse endothelial cells (ECs) overexpressing KRAS G12V in vitro . Furthermore, we performed the tube formation assay to test angiogenesis by KRAS G12V mutation in the cultured EC. Finally, we treated VEGFR2 inhibitor (SU5416) in the KRAS G12V mice and measured the bAVM size and numbers in the latex-casted brains. Results: We found that the expression of VEGF-A, VEGFR2, and phospho(p)-VEGFR2 were significantly increased in KRAS G12V -induced bAVMs in mice. We also found that KRAS G12V transfection up-regulates expression of VEGF-A, VEGFR2, and p-VEGFR2, and increased tube formation in cultured ECs. Finally, we confirmed that SU5416 treatment significantly reduced the numbers and size of bAVMs in KRAS G12V mice. Conclusions: Our data show that endogenous VEGF signaling is activated by KRAS mutation. The VEGF inhibition study suggests that the endogenously activated VEGF signaling would be essential to mediate the mutant KRAS-induced bAVM development.