Monocytes release cystatin F dimer to associate with Aß and aggravate amyloid pathology and cognitive deficits in Alzheimer's disease.

BACKGROUND: Understanding the molecular mechanisms of Alzheimer's disease (AD) has important clinical implications for guiding therapy. Impaired amyloid beta (Aß) clearance is critical in the pathogenesis of sporadic AD, and blood monocytes play an important role in Aß clearance in the periphery. However, the mechanism underlying the defective phagocytosis of Aß by monocytes in AD remains unclear. METHODS: Initially, we collected whole blood samples from sporadic AD patients and isolated the monocytes for RNA sequencing analysis. By establishing APP/PS1 transgenic model mice with monocyte-specific cystatin F overexpression, we assessed the influence of monocyte-derived cystatin F on AD development. We further used a nondenaturing gel to identify the structure of the secreted cystatin F in plasma. Flow cytometry, enzyme-linked immunosorbent assays and laser scanning confocal microscopy were used to analyse the internalization of Aß by monocytes. Pull down assays, bimolecular fluorescence complementation assays and total internal reflection fluorescence microscopy were used to determine the interactions and potential interactional amino acids between the cystatin F protein and Aß. Finally, the cystatin F protein was purified and injected via the tail vein into 5XFAD mice to assess AD pathology. RESULTS: Our results demonstrated that the expression of the cystatin F protein was specifically increased in the monocytes of AD patients. Monocyte-derived cystatin F increased Aß deposition and exacerbated cognitive deficits in APP/PS1 mice. Furthermore, secreted cystatin F in the plasma of AD patients has a dimeric structure that is closely related to clinical signs of AD. Moreover, we noted that the cystatin F dimer blocks the phagocytosis of Aß by monocytes. Mechanistically, the cystatin F dimer physically interacts with Aß to inhibit its recognition and internalization by monocytes through certain amino acid interactions between the cystatin F dimer and Aß. We found that high levels of the cystatin F dimer protein in blood contributed to amyloid pathology and cognitive deficits as a risk factor in 5XFAD mice. CONCLUSIONS: Our findings highlight that the cystatin F dimer plays a crucial role in regulating Aß metabolism via its peripheral clearance pathway, providing us with a potential biomarker for diagnosis and potential target for therapeutic intervention.

Endothelial lincRNA-p21 alleviates cerebral ischemia/reperfusion injury by maintaining blood-brain barrier integrity.

Blood-brain barrier (BBB) disruption is increasingly recognized as an early contributor to the pathophysiology of cerebral ischemia/reperfusion (I/R) injury, and is also a key event in triggering secondary damage to the central nervous system. Recently, long non-coding RNA (lncRNA) have been found to be associated with ischemic stroke. However, the roles of lncRNA in BBB homeostasis remain largely unknown. Here, we report that long intergenic non-coding RNA-p21 (lincRNA-p21) was the most significantly down-regulated lncRNA in human brain microvascular endothelial cells (HBMECs) after oxygen and glucose deprivation/reoxygenation (OGD/R) treatment among candidate lncRNA, which were both sensitive to hypoxia and involved in atherosclerosis. Exogenous brain-endothelium-specific overexpression of lincRNA-p21 could alleviate BBB disruption, diminish infarction volume and attenuate motor function deficits in middle cerebral artery occlusion/reperfusion (MCAO/R) mice. Further results showed that lincRNA-p21 was critical to maintain BBB integrity by inhibiting the degradation of junction proteins under MCAO/R and OGD/R conditions. Specifically, lincRNA-p21 could inhibit autophagy-dependent degradation of occludin by activating PI3K/AKT/mTOR signaling pathway. Besides, lincRNA-p21 could inhibit VE-cadherin degradation by binding with miR-101-3p. Together, we identify that lincRNA-p21 is critical for BBB integrity maintenance, and endothelial lincRNA-p21 overexpression could alleviate cerebral I/R injury in mice, pointing to a potential strategy to treat cerebral I/R injury.

Monocytes Release Pro-Cathepsin D to Drive Blood-to-Brain Transcytosis in Diabetes.

BACKGROUND: Microvascular complications are the major outcome of type 2 diabetes progression, and the underlying mechanism remains to be determined. METHODS: High-throughput RNA sequencing was performed using human monocyte samples from controls and diabetes. The transgenic mice expressing human CTSD (cathepsin D) in the monocytes was constructed using CD68 promoter. In vivo 2-photon imaging, behavioral tests, immunofluorescence, transmission electron microscopy, Western blot analysis, vascular leakage assay, and single-cell RNA sequencing were performed to clarify the phenotype and elucidate the molecular mechanism. RESULTS: Monocytes expressed high-level CTSD in patients with type 2 diabetes. The transgenic mice expressing human CTSD in the monocytes showed increased brain microvascular permeability resembling the diabetic microvascular phenotype, accompanied by cognitive deficit. Mechanistically, the monocytes release nonenzymatic pro-CTSD to upregulate caveolin expression in brain endothelium triggering caveolae-mediated transcytosis, without affecting the paracellular route of brain microvasculature. The circulating pro-CTSD activated the caveolae-mediated transcytosis in brain endothelial cells via its binding with low-density LRP1 (lipoprotein receptor-related protein 1). Importantly, genetic ablation of CTSD in the monocytes exhibited a protective effect against the diabetes-enhanced brain microvascular transcytosis and the diabetes-induced cognitive impairment. CONCLUSIONS: These findings uncover the novel role of circulatory pro-CTSD from monocytes in the pathogenesis of cerebral microvascular lesions in diabetes. The circulatory pro-CTSD is a potential target for the intervention of microvascular complications in diabetes.

Endothelial depletion of Atg7 triggers astrocyte-microvascular disassociation at blood-brain barrier.

Microvascular basement membrane (BM) plays a pivotal role in the interactions of astrocyte with endothelium to maintain the blood-brain barrier (BBB) homeostasis; however, the significance and precise regulation of the endothelial cell-derived BM component in the BBB remain incompletely understood. Here, we report that conditional knockout of Atg7 in endothelial cells (Atg7-ECKO) leads to astrocyte-microvascular disassociation in the brain. Our results reveal astrocytic endfeet detachment from microvessels and BBB leakage in Atg7-ECKO mice. Furthermore, we find that the absence of endothelial Atg7 downregulates the expression of fibronectin, a major BM component of the BBB, causing significantly reduced coverage of astrocytes along cerebral microvessels. We reveal Atg7 triggers the expression of endothelial fibronectin via regulating PKA activity to affect the phosphorylation of cAMP-responsive element-binding protein. These results suggest that Atg7-regulated endothelial fibronectin production is required for astrocytes adhesion to microvascular wall for maintaining the BBB homeostasis. Thus, endothelial Atg7 plays an essential role in astrocyte-endothelium interactions to maintain the BBB integrity.

Lpp of Escherichia coli K1 inhibits host ROS production to counteract neutrophil-mediated elimination.

Escherichia coli (E. coli) is the most common Gram-negative bacterial organism causing neonatal meningitis. The pathogenesis of E. coli meningitis, especially how E. coli escape the host immune defenses, remains to be clarified. Here we show that deletion of bacterial Lpp encoding lipoprotein significantly reduces the pathogenicity of E. coli K1 to induce high-degree of bacteremia necessary for meningitis. The Lpp-deleted E. coli K1 is found to be susceptible to the intracellular bactericidal activity of neutrophils, without affecting the release of neutrophil extracellular traps. The production of reactive oxygen species (ROS), representing the primary antimicrobial mechanism in neutrophils, is significantly increased in response to Lpp-deleted E. coli. We find this enhanced ROS response is associated with the membrane translocation of NADPH oxidase p47phox and p67phox in neutrophils. Then we constructed p47phox knockout mice and we found the incidence of bacteremia and meningitis in neonatal mice induced by Lpp-deleted E. coli is significantly recovered by p47phox knockout. Proteomic profile analysis show that Lpp deficiency induces upregulation of flagellar protein FliC in E. coli. We further demonstrate that FliC is required for the ROS induction in neutrophils by Lpp-deleted E. coli. Taken together, these data uncover the novel role of Lpp in facilitating intracellular survival of E. coli K1 within neutrophils. It can be inferred that Lpp of E. coli K1 is able to suppress FliC expression to restrain the activation of NADPH oxidase in neutrophils resulting in diminished bactericidal activity, thus protecting E. coli K1 from the elimination by neutrophils.

Delayed Facial Nerve Paralysis After Vestibular Schwannoma Resection.

OBJECTIVE: To report the long-term outcome of delayed facial nerve paralysis (DFNP) after surgical resection of vestibular schwannoma and evaluate the influence of various factors on the prognosis of facial nerve function. METHODS: Of 265 patients who underwent surgical excision of VS through a retrosigmoid approach between April 2019 and October 2021, 15 (5.7%) developed DFNP and were retrospectively studied. Preoperative and postoperative data were collected and analyzed. RESULTS: The mean age of patients with DFNP was 42.6 years (range, 27-63 years), and 11 (73.3%) were male. Tumor size ranged from 12 to 37 mm (mean 24 mm) in largest dimension. All patients had normal (House-Brackmann [HB] I) facial nerve function preoperatively. Immediate postoperative facial nerve function was HB I in 12 patients (80%) and HB II in 3 patients (20%). The mean severity of DFNP onset was HB 4.7 (range, HB III-V). The average day of onset was postoperative day 12.6 (range, day 5-28). At 1-year follow-up, 12 patients (80%) were HB I, 1 patient (6.7%) was HB III, and 2 patients (13.3%) were HB IV. All patients who were HB III and IV at the last follow-up had immediate postoperative facial nerve function of HB II. CONCLUSIONS: Most patients who develop DFNP have a favorable prognosis. However, a small proportion of patients with deteriorated facial nerve function immediately after surgery have poor long-term outcomes, despite confirmation of their facial nerve integrity anatomically and by electrical stimulation.

Causal effects of systemic lupus erythematosus on endometrial cancer: A univariable and multivariable Mendelian randomization study.

Objective: Systemic lupus erythematosus (SLE) has been observationally associated with endometrial cancer, but the causality remains unclear. Here, we investigated for the first time the causal links between SLE and endometrial cancer risk. Methods: Univariable and multivariable Mendelian randomization (MR) analyses were conducted to disentangle the causality of SLE with endometrial cancer. Apart from the inverse-variance weighted (IVW) method as the primary MR estimate, three complementary MR techniques including weighted median, weighted mode, and MR-Egger regression in univariable MR were conducted to clarify the robustness of the causal estimate and mediation effects of the body mass index (BMI) and were investigated within multivariable MR-IVW and MR-Egger analyses. Results: All univariable MR analyses consistently suggested that SLE has a protective effect on the risk of overall endometrial cancer (IVW: OR = 0.956, 95% CI = 0.932-0.981, P = 0.001) and endometrioid endometrial cancer (IVW: OR = 0.965, 95% CI = 0.933-0.999, P = 0.043). More compelling, after adjustment for BMI within the multivariable MR setting, the association between SLE and decreased risk of overall endometrial cancer was significantly stronger (IVW: OR = 0.952, 95% CI = 0.931-0.973, P = 9.58E-06). Conclusions: Our findings provide evidence of a significant causal relationship between SLE and decreased endometrial cancer risk. Further understanding of the underlying mechanisms linking SLE with endometrial cancer is therefore needed.

Characteristics of human movement and injury in a side collision between the front of a small car and a bicycle.

OBJECTIVE: Our research groups have studied the movement and injury characteristics of the human body in a side collision between the front of a small car and a pedestrian. This study discusses the movement and injury characteristics of the human body in a side collision between the front of a small car and bicycle. METHODS: A total of 31 cases of traffic accidents caused by small car collisions when riding a bicycle across a road were collected. Through on-site inspection and trace inspection of the accident vehicles and bicycles, the speed of the car during the collision was calculated, the collision relationship between the small car and bicycle was determined, and the injury site and degree were determined through autopsy. The car speed was divided into two groups: <60 km/h and >60 km/h. Injuries of the skull, cervical spine, ribs, pelvis, femur and tibiofibular were analysed, and the correlations with the height of the bicycle controller, the height of the bicycle seat, the height of the car hood and the length of hood were discussed. PC-Crash was used for simulation analysis to further clarify the injury process. RESULTS: The ratio of the height of the bicycle seat to the height of the hood plus the length of the hood in the windshield-damaged group was larger than that in the undamaged windshield group (P < 0.05). No cervical fracture was found when V < 60 km/h, and 52.94% of cases had cervical fracture when V > 60 km/h. The ratio of the height of the bicycle seat to the height of the hood in the pelvic fracture group was smaller than that in the nonpelvic fracture group (P < 0.05). The incidence of tibiofibular fracture was less than 65%. CONCLUSIONS: When a side impact between a car front and a bicycle occurs, the resulting human injury is related not only to the speed but also to the height of the bicycle seat and the height and length of the hood of the car. The incidence of tibiofibular fractures was significantly lower than that of small car front-pedestrian side impacts.

Effect of Body Composition on Outcomes in Patients with Hepatocellular Carcinoma Undergoing Radiotherapy: A Retrospective Study.

Computed tomography (CT)-assessed body composition is considered a novel prognostic factor for cancer patients. Owing to the need for new prognostic markers for hepatocellular carcinoma (HCC) patients undergoing radiotherapy, we investigated the impact of body composition on outcomes in this patient population. We retrospectively evaluated 109 HCC patients receiving radiotherapy. The skeletal muscle index, subcutaneous adipose tissue index (SATI), and visceral adipose tissue index within 1 mo, before radiotherapy were assessed based on a single CT image slice at the level of the third lumbar (L3) vertebra. The impact of body composition parameters on progression-free survival (PFS) and overall survival (OS) was assessed. Overall, 62 (56.9%) patients died, and 47 (43.1%) patients experienced recurrence during a median follow-up period of 20.5 mo. Multivariate analysis revealed that SATI was an independent prognostic factor for both PFS (hazard ratio [HR] 0.542, P = 0.025) and OS (HR 0.385, P = 0.005). Patients with high SATI (n = 43) had significantly better PFS (P = 0.0093) and OS (P = 0.032) than those with low SATI (n = 66). CT-assessed SATI is an independent prognostic factor in HCC patients receiving radiotherapy. Further validation is warranted to determine whether this finding can be translated into other study populations.

Chemoradiotherapy Versus Chemotherapy Alone for Advanced Esophageal Squamous Cell Carcinoma: The Role of Definitive Radiotherapy for Primary Tumor in the Metastatic Setting.

Introduction: The role of definitive radiotherapy in advanced esophageal squamous cell carcinoma (ESCC), especially in the metastatic setting, remains unclear. Therefore, we aimed to investigate the efficacy of chemoradiotherapy (CRT) versus chemotherapy (CT) alone in these selected patients. Methods: We retrospectively evaluated 194 newly diagnosed advanced ESCC who underwent definitive CRT or CT alone, including 97 patients with locally advanced and 97 patients with distant metastatic disease. Cumulative overall survival (OS) and progression-free survival (PFS) were evaluated with a log-rank test. Propensity score matching was used to simulate random allocation. In addition, we performed subgroup analysis in the locally advanced and metastatic disease. Results: After matching, 63 well-paired patients were selected. The adjusted median OS (12.5 vs. 7.6 months, p = 0.002) and PFS (9.0 vs. 4.8 months, p = 0.0025) in the CRT group were superior to that in the CT-alone group. Further subgroup analysis revealed that CRT conferred survival benefits to both locally advanced and metastatic cohorts. For patients with distant metastasis, median OS (12.9 vs. 9.3 months, p = 0.029) and PFS (9.9 vs. 4.0 months, p =0.0032) in the CRT group were superior to that in the CT-alone group. In a multivariate Cox regression analysis of the entire cohort, additional definitive radiotherapy was independently associated with better OS (p = 0.041) and PFS (p = 0.007). Conclusions: In both locally advanced and metastatic ESCC, additional definitive-dose radiotherapy was associated with improved clinical outcomes. Therefore, more consideration should be given to its application in the metastatic setting.

An oxygen-adaptive interaction between SNHG12 and occludin maintains blood-brain barrier integrity.

Tight junctions (TJs) of brain microvascular endothelial cells (BMECs) play a pivotal role in maintaining the blood-brain barrier (BBB) integrity; however, precise regulation of TJs stability in response to physiological and pathological stimuli remains elusive. Here, using RNA immunoprecipitation with next-generation sequencing (RIP-seq) and functional characterization, we identify SNHG12, a long non-coding RNA (lncRNA), as being critical for maintaining the BBB integrity by directly interacting with TJ protein occludin. The interaction between SNHG12 and occludin is oxygen adaptive and could block Itch (an E3 ubiquitin ligase)-mediated ubiquitination and degradation of occludin in human BMECs. Genetic ablation of endothelial Snhg12 in mice results in occludin reduction and BBB leakage and significantly aggravates hypoxia-induced BBB disruption. The detrimental effects of hypoxia on BBB could be alleviated by exogenous SNHG12 overexpression in brain endothelium. Together, we identify a direct TJ modulator lncRNA SNHG12 that is critical for the BBB integrity maintenance and oxygen adaption.

Prognostic Value of Combined Lactate Dehydrogenase, C-Reactive Protein, Cancer Antigen 153 and Cancer Antigen 125 in Metastatic Breast Cancer.

BACKGROUND: Breast cancer (BC), especially metastatic BC, is one of the most lethal diseases in women. CA 125 and CA 15-3 are commonly used indicators for diagnosis and prognosis of BC. Some serological indicators, such as lactate dehydrogenase (LDH) and C-reactive protein (CRP), can also be used to assess the prognosis and progression in BC. METHODS: Univariate Cox regression analysis and LASSO regression analysis were performed to identify prognostic factors and build prognostic models. We distributed the patients into 2 groups based on the median risk score, analyzed prognosis by Kaplan-Meier curve, and screened independent prognostic factors by multivariate Cox regression analysis. RESULT: We identified 4 indicators-LDH, CRP, CA 15-3, and CA 125-related to the prognosis in BC and established a prognostic model. The high LDH group showed worse overall survival (OS) than low LDH group (P = .017; hazard ratio (HR), 1.528; 95% confidence interval (CI), 1.055-2.215). The high CRP group showed worse OS than low CRP group (P = .004; HR, 1.666; 95% CI, 1.143-2.429). The high CA153 group showed worse OS than low CA 15-3 group (P=.011; HR, 1.563; 95% CI, 1.075-2.274). The high CA 125 group showed worse OS than low CA 125 group (P = .021; HR, 1.499; 95% CI, 1.031-2.181). The area under the curve for risk score was .824, Ki-67 was .628, age was .511, and grade was .545. Risk score was found to be an independent prognostic factor using multivariate Cox regression analysis. CONCLUSION: We successfully established an optimization model by combining 4 prognosis-related indicators to assess the prognosis in patients with metastatic BC.

Analysis of Pedestrian Fractures in Collisions Between Small Cars and Pedestrians Based on Surveillance Videos.

OBJECTIVE: To discuss the collision relationship and the cause of the fracture caused by traffic accidents in which the front of a small car collides with the side of a pedestrian while braking. METHODS: The surveillance videos of 42 traffic accidents involving the front of a small car colliding with the side of a pedestrian while braking were collected. By analyzing the surveillance videos and the paths, the speed of the collision, the relationship between the vehicle and the pedestrian upon collision, and the movement trajectory of the human body were clearly identified. The type and severity of the injuries were also determined through autopsy. The characteristics of the human injuries and vehicle paths were analyzed according to the collision speed (<40 km/h, 40-60 km/h, 60-90 km/h), and the correlations between the fracture and the height of the pedestrian, the height of the hood and the length of the hood were discussed. RESULTS: When a small car hits the side of a pedestrian, the front bumper first hits the lower limbs of the pedestrian, and then, the human body falls to the side of the vehicle, causing a secondary collision with the hood and front windshield; thus, the pedestrian is thrown at a speed similar to the speed of the vehicle, finally falling to the ground and sliding forward a certain distance. (1) When V is less than 40 km/h (n = 10), the pedestrian's head did not collide with the windshield, and the fatal injuries were caused by the individual striking the ground. (2) When V is greater than 40 km/h (n = 32), the majority (97%) of cases showed collision with the windshield. (3) When 40 to 60 km/h (n = 16), the pedestrian's head collided with the windshield, which can cause fatal injuries, and pelvic fractures and rib fractures occurred in 56.25% of patients. (4) When V is less than 60 km/h (n = 26), the ratio of the height of the pedestrian to the height of the hood was significantly smaller in the pelvic fracture group than in the nonpelvic fracture group (P < 0.01). (5) When 60 to 90 km/h (n = 16), there were holes in the windshield, and the pedestrians experienced severe head injuries, with cervical spine fracture occurring in 37.5% of patients, pelvic fractures occurring in 43.75% of patients, and rib fractures occurring in 31.25% of patients. CONCLUSIONS: When V is less than 40 km/h, the vehicle does not cause severe injuries in pedestrians; when V is greater than 40 km/h, the collisions of the pedestrian's head with the windshield lead to severe head injuries and the accident can cause severe pelvic and rib fractures; when V is greater than 60 km/h, the collisions of the pedestrian's head with the windshield can cause cervical spine fracture in addition to head injuries. The occurrence of human injuries is related to not only the vehicle speed but also factors such as the height of the pedestrian, the height of the hood and the length of the hood.

Roles of RAGE/ROCK1 Pathway in HMGB1-Induced Early Changes in Barrier Permeability of Human Pulmonary Microvascular Endothelial Cell.

Background: High mobility group box 1 (HMGB1) causes microvascular endothelial cell barrier dysfunction during acute lung injury (ALI) in sepsis, but the mechanisms have not been well understood. We studied the roles of RAGE and Rho kinase 1 (ROCK1) in HMGB1-induced human pulmonary endothelial barrier disruption. Methods: In the present study, the recombinant human high mobility group box 1 (rhHMGB1) was used to stimulate human pulmonary microvascular endothelial cells (HPMECs). The endothelial cell (EC) barrier permeability was examined by detecting FITC-dextran flux. CCK-8 assay was used to detect cell viability under rhHMGB1 treatments. The expression of related molecules involved in RhoA/ROCK1 pathway, phosphorylation of myosin light chain (MLC), F-actin, VE-cadherin and ZO-1 of different treated groups were measured by pull-down assay, western blot and immunofluorescence. Furthermore, we studied the effects of Rho kinase inhibitor (Y-27632), ROCK1/2 siRNA, RAGE-specific blocker (FPS-ZM1) and RAGE siRNA on endothelial barrier properties to elucidate the related mechanisms. Results: In the present study, we demonstrated that rhHMGB1 induced EC barrier hyperpermeability in a dose-dependent and time-dependent manner by measuring FITC-dextran flux, a reflection of the loss of EC barrier integrity. Moreover, rhHMGB1 induced a dose-dependent and time-dependent increases in paracellular gap formation accompanied by the development of stress fiber rearrangement and disruption of VE-cadherin and ZO-1, a phenotypic change related to increased endothelial contractility and endothelial barrier permeability. Using inhibitors and siRNAs directed against RAGE and ROCK1/2, we systematically determined that RAGE mediated the rhHMGB1-induced stress fiber reorganization via RhoA/ROCK1 signaling activation and the subsequent MLC phosphorylation in ECs. Conclusion: HMGB1 is capable of disrupting the endothelial barrier integrity. This study demonstrates that HMGB1 activates RhoA/ROCK1 pathway via RAGE, which phosphorylates MLC inducing stress fiber formation at short time, and HMGB1/RAGE reduces AJ/TJ expression at long term independently of RhoA/ROCK1 signaling pathway.

Dynamin-2 mediates clathrin-dependent endocytosis for amyloid-ß internalization in brain microvascular endothelial cells.

Dynamin is recognized as a crucial regulator for membrane fission and has three isoforms in mammals. But the expression patterns of dynamin isoforms and their roles in non-neuronal cells are incompletely understood. In this study, the expression profiles of dynamin isoforms and their roles in endocytosis was investigated in brain endothelial cells. We found that Dyn2 was expressed at highest levels, whereas the expression of Dyn1 and Dyn3 were far less than Dyn2. Live-cell imaging was used to investigate the effects of siRNA-mediated knockdown of individual dynamin isoforms on transferrin uptake, and we found that Dyn2, but not Dyn1 or Dyn3, is required for the endocytosis in brain endothelial cells. Results of dextran uptake assay showed that dynamin isoforms are not involved in the clathrin-independent fluid-phase internalization of brain endothelial cells, suggesting the specificity of the role of Dyn2 in clathrin-dependent endocytosis. Immunofluorescence and electron microscopy analysis showed that Dyn2 co-localizes with clathrin and acts at the late stage of vesicle fission in the process of endocytosis. Further results showed that Dyn2 is necessary for the basolateral-to-apical internalization of amyloid-ß into brain endothelial cells. We concluded that Dyn2, but not Dyn1 or Dyn3, mediates the clathrin-dependent endocytosis for amyloid-ß internalization particularly from basolateral to apical side into brain endothelial cells.

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli.

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Surgical management of intraoperatively diagnosed facial nerve schwannoma located at internal auditory canal and cerebellopontine angle - our experiences of 14 cases.

BACKGROUND: Facial nerve schwannomas located at internal auditory canal and cerebellopontine angle (IAC/CPA FNS) were diagnosed intraoperatively, it poses a therapeutic dilemma to the surgeon. OBJECTIVE: To report our experience in managing IAC/CPA FNS and to propose a treatment strategy. METHODS: A total of 14 patients with IAC/CPA FNS who were diagnosed intraoperatively and treated by operation between 2015 and 2019 were retrospectively studied. RESULTS: Unilateral hearing loss was the most common symptom and all these patients had normal facial nerve function preoperatively. Surgical approaches used in these patients including translabyrinthine (2 cases), retrosigmoid (RS) (11 cases), and middle cranial fossa (MCF) approach (1 case). Eight patients underwent partial resection, three patients underwent subtotal resection and three patients had complete tumor removal with facial nerve reconstruction. All partial resection patients and two patients underwent subtotal resection achieved a long-term HB grade I facial nerve function. The long-term facial nerve function of patients underwent complete resection and nerve grafting was no better than HB grade III.1 of the eight patients underwent partial resection experienced tumor regrowth during the follow-up. CONCLUSIONS: Partial or subtotal resection for IAC/CPA FNS may provide an opportunity of retaining excellent facial nerve function. Regular postoperative imaging is helpful to monitor the recurrence.

Development and Validation of a Prognostic Signature Based on Immune Genes in Cervical Cancer.

BACKGROUND: Cervical cancer is one of the most common types of gynecological malignancies worldwide. This study aims to develop an immune signature to predict survival in cervical cancer. METHOD: The gene expression data of 296 patients with cervical cancer from The Cancer Genome Atlas database (TCGA) and immune-related genes from the Immunology Database and Analysis Portal (ImmPort) database were included in this study. The immune signature was developed based on prognostic genes. The validation dataset was downloaded from the Gene Expression Omnibus (GEO) database. RESULT: The immune signature namely immune-based prognostic score (IPRS) was developed with 229 genes. Multivariate analysis revealed that the IPRS was an independent prognostic factor for overall survival (OS) and progression-free survival (PFS) in patients with cervical cancer. Patients were stratified into high IPRS and low IPRS groups, and those in the high IPRS group were associated with better survival, which was validated in the validation set. A nomogram with IPRS and stage was constructed to predict mortality in cervical cancer. CONCLUSIONS: We developed a robust prognostic signature IPRS that could be used to predict patients' survival outcome.

PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates.

Tyrosine phosphorylation of secretion machinery proteins is a crucial regulatory mechanism for exocytosis. However, the participation of protein tyrosine phosphatases (PTPs) in different exocytosis stages has not been defined. Here we demonstrate that PTP-MEG2 controls multiple steps of catecholamine secretion. Biochemical and crystallographic analyses reveal key residues that govern the interaction between PTP-MEG2 and its substrate, a peptide containing the phosphorylated NSF-pY83 site, specify PTP-MEG2 substrate selectivity, and modulate the fusion of catecholamine-containing vesicles. Unexpectedly, delineation of PTP-MEG2 mutants along with the NSF binding interface reveals that PTP-MEG2 controls the fusion pore opening through NSF independent mechanisms. Utilizing bioinformatics search and biochemical and electrochemical screening approaches, we uncover that PTP-MEG2 regulates the opening and extension of the fusion pore by dephosphorylating the DYNAMIN2-pY125 and MUNC18-1-pY145 sites. Further structural and biochemical analyses confirmed the interaction of PTP-MEG2 with MUNC18-1-pY145 or DYNAMIN2-pY125 through a distinct structural basis compared with that of the NSF-pY83 site. Our studies thus provide mechanistic insights in complex exocytosis processes.

The transcriptome characteristics of vestibular organs from delayed endolymphatic hydrops patients (Meniere's disease).

OBJECTIVES: To identify genes that are related to delayed endolymphatic hydrops (DEH) in patients by RNA-Seq analysis. DESIGN: Observational study. SETTING: Eye & ENT Hospital, Fudan University (Shanghai, China). PARTICIPANTS: We collected the entire vestibular system from four patients with DEH who underwent labyrinthectomy. Three control samples were collected from patients with acoustic neuroma or facial neuroma treated via the translabyrinthine approach. High-throughput RNA-Seq analysis was performed to investigate gene expression in the pathological vestibular system. MAIN OUTCOME MEASURES: Our bioinformatic analysis identified 17 genes that were upregulated and eight genes that were downregulated in patients with DEH compared with the controls. RESULTS: The altered gene expression profile suggested that DEH is closely related to neuropathy and autoimmune disease. In addition, many of the differentially regulated genes were involved in cell adhesion, suggesting a role of cell adhesion in DEH. Immunofluorescence analysis confirmed the expression of PMP2 and CLDN19 in the cytoplasm of hair cells and scattered expression of MPZ at cell junctions. The protein expression levels were higher in specimens from patients with Ménière's disease and DEH compared with controls. CONCLUSIONS: The protein expression profile of vestibular organs in patients with endolymphatic hydrops exhibited a degree of similarity to that of Ménière's disease. Endolymphatic hydrops is characterised by autoimmune abnormalities. DEH and Ménière's disease are likely to be different manifestations of the same disease, with disparate clinical symptoms. RNA-Seq is a useful analytical tool to characterise the vestibular pathology based on its transcriptome.