Hypervirulent Klebsiella pneumoniae Mediated Hepatic Infarction Septic Shock After Rectal Cancer Surgery: A Case Report.

The liver receives blood from both the hepatic artery and portal vein. Hepatic infarction is rare in clinical practice as both the hepatic artery and portal vein can supply blood to the liver. Here, we reported a case of a 75-year-old man who underwent radical laparoscopic surgery for rectal cancer and subsequently developed hepatic infarction. The patient experienced severe infection, as well as circulatory and respiratory failure on the third day after surgery. The patient presented with high fever, chest tightness, shortness of breath, decreased blood oxygen saturation and blood pressure. The leukocyte count decreased from 8.10 × 10^9/L to 1.75 × 10^9/L. Procalcitonin (PCT) levels increased from 1.02 ng/mL to 67.14 ng/mL, and eventually reaching levels over 200 ng/mL. Enhanced abdominal computed tomography (CT) confirmed the presence of hepatic infarction, but no thrombosis was observed in the hepatic artery or portal vein. Metagenomic next-generation sequencing (mNGS) identified hypervirulent Klebsiella pneumoniae (hvKp) in the patient's blood and ascites, one day earlier than the detection results using traditional culture methods. The patient was diagnosed with hepatic infarction combined with septic shock caused by hvKp. This case emphasizes that in the high-risk group of thrombosis, infection can trigger exacerbated hepatic infarction events, particularly in cases after surgical procedures. For severely ill patients with infectious diseases who are admitted to the ICU with worsening symptoms, it is important to collect appropriate samples and send them for pathogen detection using mNGS in a timely manner. This may aid in early intervention and improve clinical outcomes.

Clinical performance of metagenomic next-generation sequencing for diagnosis of pulmonary Aspergillus infection and colonization.

Background: Investigations assessing the value of metagenomic next-generation sequencing (mNGS) for distinguish Aspergillus infection from colonization are currently insufficient. Methods: The performance of mNGS in distinguishing Aspergillus infection from colonization, along with the differences in patients' characteristics, antibiotic adjustment, and lung microbiota, were analyzed. Results: The abundance of Aspergillus significantly differed between patients with Aspergillus infection (n=36) and colonization (n=32) (P < 0.0001). Receiver operating characteristic (ROC) curve result for bronchoalveolar lavage fluid (BALF) mNGS indicated an area under the curve of 0.894 (95%CI: 0.811-0.976), with an optimal threshold value of 23 for discriminating between Aspergillus infection and colonization. The infection group exhibited a higher proportion of antibiotic adjustments in comparison to the colonization group (50% vs. 12.5%, P = 0.001), with antibiotic escalation being more dominant. Age, length of hospital stay, hemoglobin, cough and chest distress were significantly positively correlated with Aspergillus infection. The abundance of A. fumigatus and Epstein-Barr virus (EBV) significantly increased in the infection group, whereas the colonization group exhibited higher abundance of A. niger. Conclusion: BALF mNGS is a valuable tool for differentiating between colonization and infection of Aspergillus. Variations in patients' age, length of hospital stay, hemoglobin, cough and chest distress are observable between patients with Aspergillus infection and colonization.

Disseminated Histoplasmosis Infection Diagnosed by Metagenomic Next-Generation Sequencing: A Case Report.

Histoplasmosis is an endemic disease caused by Histoplasma capsulatum. This systemic disease can affect various organs beyond the lungs, such as the liver, spleen, adrenal gland, and lymph nodes. The clinical symptoms can range from asymptomatic to severe, life-threatening conditions, depending on the state of the patient's immune system. This report describes a 40-year-old male who presented with reports of weight loss, low back pain, and progressively worsening movement disorder of the bilateral lower extremities for months. Computed tomography (CT) examination showed multiple lytic lesions of vertebral bodies, bilateral ribs, and pelvic bone, histopathological examination and tumor-related serum markers exclude tumors. mNGS was employed to identify H. capsulatum var. capsulatum as the etiological agent of the lesions in the bone biopsy. Through phylogenetic tree analysis, Histoplasma capsulatum var. Capsulatum (Hcc) was the main responsible pathogen, rarely reported in bone lesions. The patient underwent spinal surgery and was successfully treated with liposomal amphotericin B and itraconazole. Based on the diagnosis and treatment of this case, we discuss the epidemiologic status, clinical presentations, diagnostic criteria, and treatment guidelines of histoplasmosis to provide additional information about this disease. mNGS is utilized in this case, and it appears to be a reliable method for early and accurate diagnosis of this disease.

The noncoding RNAs regulating pyroptosis in colon adenocarcinoma were derived from the construction of a ceRNA network and used to develop a prognostic model.

BACKGROUND: Noncoding RNAs (ncRNAs), pyroptosis and tumours are all hot topics in current research, but there are very limited studies on pyroptosis and its regulated ncRNAs in colon adenocarcinoma (COAD). METHODS: The COAD transcription profile dataset from TCGA was used for differential expression analysis. Pyroptosis-related genes (PRGs), the top 200 long noncoding RNAs (lncRNAs) and circular RNA (circRNAs) were selected from the results to construct an endogenous competitive RNA (ceRNA) network. Moreover, the expression of the ceRNAs was used for consensus cluster analysis of COAD and developing a risk model after combining clinical follow-up data by the least absolute shrinkage and selection operator method. The stability and independent prognostic ability of the risk model were evaluated. Finally, gene set enrichment analysis (GSEA) and immune score comparisons between the high-risk and low-risk groups were performed. RESULTS: There were 87 PRGs with significant differences, among which casp3/8, NLRP1/3, and IL-1α/1ß were at the core of the interactions. The ceRNA network consisted of 58 lncRNAs, 6 circRNAs, 25 PRGs, and 55 microRNAs. We speculated that KCNQ1OT1-miRNAs-SQSTM1 and HSA_CIRC_0001495-miRNAs-PTEN have great potential and value in the pyroptosis mechanism of COAD. Nine RNAs were involved in the risk score, which had excellent independent prognostic ability. Survival analyses were significant between the high-risk (HR) and low-risk (LR) groups (training cohort: P < 0.001; test cohort: P = 0.037). GSEA was mainly enriched in tumour proliferation and metastasis related pathways, while differences in immune activity showed a bipolar distribution between the HR and LR groups. CONCLUSIONS: The overall mechanism of pyroptosis in COAD was revealed. CeRNAs most closely related to the pyroptosis mechanism of COAD were selected and used to develop a prognostic model. The results may present new regulatory sites and potential targets for COAD pyroptosis mechanisms.

Parasite Circulating Cell-free DNA in the Blood of Alveolar Echinococcosis Patients as a Diagnostic and Treatment-Status Indicator.

BACKGROUND: Alveolar echinococcosis (AE) is a serious parasitic disease caused by the larvae of Echinococcus multilocularis. It is the less common but substantially more deadly of the 2 major echinococcosis diseases that can occur globally but are concentrated in central Asia. METHODS: We analyzed parasite circulating cell-free DNA (cfDNA) in 149 plasma samples using a DNA sequencing-based method (105 AE, 16 cystic echinococcosis, 4 liver cancer, 4 gallstones, and 20 healthy volunteers). After identifying the Echinococcus-specific cfDNA (Em-cfDNA) sequences in the samples, we determined whether Em-cfDNA could be used for AE diagnosis and as a potential indicator of the effectiveness of surgical treatment. We also examined potential associations between Em-cfDNA levels and clinical features of AE patients. RESULTS: Our work demonstrates that varying reads of Em-cfDNA were detectable in the plasma of 100% of preoperative AE patients and that all of the non-AE patients and healthy volunteers were negative. Em-cfDNA has good sensitivity and specificity for the diagnosis of AE. We also found that Em-cfDNA levels apparently have reference value for evaluating the therapeutic efficacy of surgery interventions for AE lesions. Finally, our analysis revealed that Em-cfDNA levels can reflect meaningful information about lesion size in preoperative AE patients. CONCLUSIONS: We demonstrate that sequencing-based monitoring of Em-cfDNA can be used in the clinic as a powerful diagnostic indicator for AE. We also note that there is a strong potential for use of this liquid-biopsy method to monitor ongoing disease status in postintervention AE patients.

Oxide modified aluminum for removal of methyl orange and methyl blue in aqueous solution.

In this work, pristine aluminum (Al) powder was soaked in deionized water for a time period and then it was dried and heat-treated at 400 °C such that a layer of fine Al2O3 grains covered the Al particle surfaces, forming oxide modified Al powder (OM-Al). It was found that OM-Al greatly enhanced the efficiency in removing methyl orange (M-orange) and methyl blue (M-blue) in aqueous solution. The time to completely degrade M-orange and M-blue by OM-Al is about one third of that by pristine Al powder, and decreases with increasing dosage of OM-Al. The enhancement in dye removal rate by oxide modification is much better than that with ultrasonic assistance, especially for M-blue. LC/MS spectrum analyses revealed that large dye molecules are broken into small biodegradable organic molecules after reaction with OM-Al. It is deduced that the promotion of fine Al2O3 on the hydration process of Al surface passive oxide film is the main mechanism responsible for the enhancement of dye removal by OM-Al. Furthermore, OM-Al has a good recyclability and 80% of M-orange and M-blue can be removed even when it was reused for up to three cycles. These results indicate that oxide modification is an effective way to activate Al for the removal of organic dyes.

A community-acquired lung abscess attributable to odontogenic flora.

A lung abscess is an infectious pulmonary disease characterized by pus-filled cavity formation and often an air-fluid level. In this article, we described an indolent community-acquired lung abscess suspected as a tumor previously. A 56-year-old male presented with cough and expectoration for 2 months and hemoptysis for 2 weeks. His physical examinations, whole blood count and C-reactive protein level were normal. The chest computed tomography (CT) scan showed a 40×38×39 mm high-density mass in the right upper pulmonary lobe, with irregular borders. The pathology of a CT-guided percutaneous needle aspiration biopsy showed numerous inflammatory cells and bacteria infiltration without tumor lesions. Bacteriological detection of lung tissue revealed the cause was odontogenic flora. A next-generation sequencing demonstrated the etiologic correlation between lung abscess and periodontitis. After a 2-month pathogen-directed oral antibiotics therapy combined with chlorhexidine gargle oral care, this patient showed a remarkable improvement. Periodontitis can be a cause of a lung abscess, which would be taken into account in the treatment regimes preventing infectious recurrence.

Multiband mucosectomy versus endoscopic submucosal dissection and endoscopic submucosal excavation for GI submucosal tumors: short and long term follow-up.

AIMS: To evaluate the short- and long-term outcomes of 3 different endoscopic dissection techniques for upper gastrointestinal (GI) submucosal tumours (SMTs). METHODS: Data for 135 patients withGI SMTs who underwent multiband mucosectomy (MBM), endoscopic submucosal dissection (ESD), or endoscopic submucosal excavation (ESE) were retrospectively assessed. The en bloc resection rate, endoscopic complete resection rate, operation time, potential complications and local recurrence rate were compared. RESULTS: No significant differences were observed in the rate of endoscopic complete resections and pathologic complete resections among the three groups. For SMTs > 15 mm in width, the lowest en bloc resection rate was found for MBM (P = 0.000). MBM was also associated with the shortest procedure time, lowest perforation rate and lowest rate of major bleeding. ESE was the most effective procedure for muscularis propria (MP) lesions but was associated with the longest operation time (P < 0.01). The ESD and ESE groups had similar perforation rates (P > 0.05). No differences were observed in 4-year local recurrence rates among the groups (P = 0.945). CONCLUSIONS: MBM is a simple and effective method for the treatment of small SMTs and achieves clinical success rates similar to those of ESD and ESE. However, ESD and ESE are preferable for larger and deep lesions and are associated with a longer operation time. Nonetheless, all 3 techniques resulted in a low 4-year local recurrence rate. Large-scale randomized clinical trials are needed to further investigate these results.

Next-generation-sequencing technology used for the detection of Mycoplasma hominis in renal cyst fluid: a case report.

Background: Mycoplasma is an opportunistic pathogen causing both urogenital and extragenital infections. The lack of cell wall renders Mycoplasma difficult to culture and identify with ordinary methods. Next-generation sequencing (NGS) is a new technology helping a lot in the diagnosis of infective diseases. In this case, NGS played a key role in the diagnosis of Mycoplasma infection. Case presentation: A mid-aged man suffering from renal cyst underwent cyst incision followed by invasive treatments to eliminate hematoma caused by renal artery hemorrhage. After the cyst incision operation, the patient had a persistent high temperature. The persistent increase of blood neutrophile granulocyte count and C-reaction protein suggested an unresolved infection. The empirically chosen anti-infective agents were meropenem and linezolid since the ordinary bacterial cultures of surgical site drainage and blood yielded a negative result. At postoperation day (POD) 17, NGS result of his drainage clearly indicated the pathogen was Mycoplasma hominis. At POD 24, the drug sensitivity test showed resistance to quinolones, clarithromycin and erythromycin, but intermediate to azithromycin. Since then, the antimicrobial agents were changed into azithromycin and kept unchanged until the patient was fully recovered and discharged at POD 39. Conclusion: When the ordinary laboratory diagnostic methods failed, NGS diagnosis could reduce the hospitalization expenses and shorten the lengths of hospital stay.

Efficacy of narrow-band imaging in detecting early esophageal cancer and risk factors for its occurrence.

OBJECTIVE: The aim of this study is to evaluate the efficacy of narrow-band imaging (NBI) in the detecting early esophageal cancer and precancerous lesions and to investigate the risk factors for its occurrence. METHODS: The esophagus was examined with ordinary endoscopy, NBI, and iodine staining. All the lesions were confirmed by histopathologically as the gold standard; NBI and intrapapillary capillary scale (IPCL) scale were compared with pathologic diagnosis. The accuracy, sensitivity, specificity, positive and negative predictive values (PPV, NPV) were calculated. Subgroup analysis was performed between the elderly vs. younger group, and head and neck squamous cell cancer (HNSCC) vs. non-HNSCC patients. RESULTS: Ninety lesions were detected with ordinary endoscopy, 108 with NBI, and 120 with iodine staining. All esophageal cancers were detected both by NBI and by iodine staining. Accuracy, sensitivity, and specificity for esophageal cancer and precancerous lesion were 67.8%, 58.1%, and 76.6%; 92%, 89.7%, and 96%; 93.4%, 93.4%, and 93.2%, respectively. NBI endoscopy and iodine staining were superior to ordinary endoscopy for detecting esophageal cancer and precancerous lesions (p < 0.05). NBI showed better detection of esophageal neoplasms in the elderly patients (p < 0.001). The incidence of multiple squamous cell cancers (SCCs) was significantly higher in non-elderly group (p = 0.009). NBI can also detect more esophageal neoplastic lesions in patients with head and neck cancers (p = 0.003). CONCLUSIONS: NBI endoscopy appears as effective as Lugol staining to detect and screen the early esophageal cancer. NBI shows better detection of esophageal neoplasms in the elderly patients. The incidence of multiple SCCs was much higher in non-elderly patients.

Comparison of Endoscopic Resection and Minimally Invasive Esophagectomy in Patients With Early Esophageal Cancer.

OBJECTIVE: To determine whether endoscopic resection (ER) and minimally invasive esophagectomy (MIE) are safe and effective for treating squamous intraepithelial neoplasia of the esophagus. MATERIALS AND METHODS: This study retrospectively analyzed a total of 99 consecutive patients with pathologically confirmed early esophageal cancer between December 2007 and 2011. ER was performed in 59 patients, whereas MIE was performed in 40 patients. We compared the 2 groups according to R0 resection rates, treatment-related complications, mean hospital stay, local recurrence rates, and 3- and 4-year overall survival. RESULTS: No significant differences were found in the R0 resection rates between ER and MIE (94.9% vs. 97.5%, P>0.05). The occurrence rate of minor complications in the ER group was significantly lower than that in the thoracoscopic esophagectomy group (11.8% vs. 32.5%, P>0.05). The mean operative time in the ER group was 74±23 minutes, which was significantly shorter than that in the MIE group (298±46 min). The average length of hospital stay in the ER group was significantly shorter than that in the MIE group (P<0.001). No significant differences were observed in the local recurrence rates between the 2 groups (P>0.05). Similarly, no differences were found in the 3-year survival rate (ER: 96.6%, vs. MIE: 97.5%, P>0.05) and 4-year survival rate (ER: 91.5% vs. MIE: 90%, P>0.05) between the 2 groups. CONCLUSIONS: ER achieves the same positive results as MIE in the treatment of early esophageal cancer and is associated with a lower complication rate, a shorter recovery time, and a similar survival rate. However, multiple ER procedures were required for several patients in this study.

Caspase-1 causes truncation and aggregation of the Parkinson's disease-associated protein α-synuclein.

The aggregation of α-synuclein (aSyn) leading to the formation of Lewy bodies is the defining pathological hallmark of Parkinson's disease (PD). Rare familial PD-associated mutations in aSyn render it aggregation-prone; however, PD patients carrying wild type (WT) aSyn also have aggregated aSyn in Lewy bodies. The mechanisms by which WT aSyn aggregates are unclear. Here, we report that inflammation can play a role in causing the aggregation of WT aSyn. We show that activation of the inflammasome with known stimuli results in the aggregation of aSyn in a neuronal cell model of PD. The insoluble aggregates are enriched with truncated aSyn as found in Lewy bodies of the PD brain. Inhibition of the inflammasome enzyme caspase-1 by chemical inhibition or genetic knockdown with shRNA abated aSyn truncation. In vitro characterization confirmed that caspase-1 directly cleaves aSyn, generating a highly aggregation-prone species. The truncation-induced aggregation of aSyn is toxic to neuronal culture, and inhibition of caspase-1 by shRNA or a specific chemical inhibitor improved the survival of a neuronal PD cell model. This study provides a molecular link for the role of inflammation in aSyn aggregation, and perhaps in the pathogenesis of sporadic PD as well.

Crystal structures of SIRT3 reveal that the α2-α3 loop and α3-helix affect the interaction with long-chain acyl lysine.

SIRT1-7 play important roles in many biological processes and age-related diseases. In addition to a NAD(+) -dependent deacetylase activity, they can catalyze several other reactions, including the hydrolysis of long-chain fatty acyl lysine. To study the binding modes of sirtuins to long-chain acyl lysines, we solved the crystal structures of SIRT3 bound to either a H3K9-myristoylated- or a H3K9-palmitoylated peptide. Interaction of SIRT3 with the palmitoyl group led to unfolding of the α3-helix. The myristoyl and palmitoyl groups bind to the C-pocket and an allosteric site near the α3-helix, respectively. We found that the residues preceding the α3-helix determine the size of the C-pocket. The flexibility of the α2-α3 loop and the plasticity of the α3-helix affect the interaction with long-chain acyl lysine.

Multiband Mucosectomy Versus Endoscopic Submucosal Dissection for Treatment of Squamous Intraepithelial Neoplasia of the Esophagus.

BACKGROUND & AIMS: We compared the efficacy and safety of multiband mucosectomy (MBM) vs endoscopic submucosal dissection (ESD) for the treatment of squamous intraepithelial neoplasia of the esophagus. METHODS: We performed a retrospective study of 78 patients with squamous intraepithelial neoplasia of the esophagus who received either ESD or MBM between January 2009 and January 2011 at the Tengzhou Central People's Hospital in China. We compared rates of bloc resection and curative resection, as well as complications and local recurrence, between groups. RESULTS: Overall, there was no statistical difference in the rate of complete resection between patients who received ESD (95.8%) vs MBM (93%) (P > .05). For tumors less than 15 mm in width, ESD produced a significantly higher rate of en bloc resection (100%) and curative resection (92.3%) than MBM (44.8% and 41%; P < .05). No significant differences were found between lesions less than 15 mm. MBM had a significantly shorter procedure time (38 ± 11 min) than ESD (84 ± 35 min) (P < .05). Major bleeding occurred in 1.85% of MBM procedures and in 16.7% of ESD procedures (P > .05). ESD led to perforations in 8.3% of cases, whereas MBM did not lead to any perforations (P < .05). No significant differences were found between groups in proportions of cases with postoperative esophageal strictures (16.7% vs 14.8%; P > .05) or the 3-year rate of local recurrence (P > .05). CONCLUSIONS: Based on a retrospective comparison of patients who underwent ESD vs MBM for squamous intraepithelial neoplasia of the esophagus, ESD should be reserved for patients with larger neoplastic lesions (>15 mm), with respect to the success of attempted en bloc resection and the number of curative resections achieved. However, ESD has longer procedure times and higher rates of complication. MBM allows for safe and easy piecemeal resections, and is associated with similar levels of clinical success as ESD for lesions less than 15 mm. Large, randomized, controlled studies are needed to determine which endoscopic resection modality is superior for patients with high-grade intraepithelia neoplasms.

Quantitative protein profiling of hippocampus during human aging.

The hippocampus appears commonly affected by aging and various neurologic disorders in humans, whereas little is known about age-related change in overall protein expression in this brain structure. Using the 4-plex tandem mass tag labeling, we carried out a quantitative proteomic study of the hippocampus during normal aging using postmortem brains from Chinese subjects. Hippocampal samples from 16 subjects died of non-neurological/psychiatric diseases were divided into 4 age groups: 22-49, 50-69, 70-89, and >90. Among 4582 proteins analyzed, 35 proteins were significantly elevated, whereas 25 proteins were downregulated, along with increasing age. Several upregulated proteins, including transgelin, vimentin, myosin regulatory light polypeptide 9, and calcyphosin, were further verified by quantitative Western blot analysis of hippocampal tissues from additional normal subjects. Bioinformatic analysis showed that the upregulated and downregulated proteins were largely involved in several important protein-protein interaction networks. Proteins in the electron transport chain and synaptic vesicle fusion pathway were consistently downregulated with aging, whereas proteins associated with Alzheimer's disease showed little change. Our study demonstrates substantial protein profile changes in the human hippocampus during aging, which could be of relevance to age-related loss of hippocampal functions.

Generation and characterization of novel conformation-specific monoclonal antibodies for α-synuclein pathology.

α-Synuclein (α-syn), a small protein that has the intrinsic propensity to aggregate, is implicated in several neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), which are collectively known as synucleinopathies. Genetic, pathological, biochemical, and animal modeling studies provided compelling evidence that α-syn aggregation plays a key role in the pathogenesis of PD and related synucleinopathies. It is therefore of utmost importance to develop reliable tools that can detect the aggregated forms of α-syn. We describe here the generation and characterization of six novel conformation-specific monoclonal antibodies that recognize specifically α-syn aggregates but not the soluble, monomeric form of the protein. The antibodies described herein did not recognize monomers or fibrils generated from other amyloidogenic proteins including ß-syn, γ-syn, ß-amyloid, tau protein, islet amyloid polypeptide and ABri. Interestingly, the antibodies did not react to overlapping linear peptides spanning the entire sequence of α-syn, confirming further that they only detect α-syn aggregates. In immunohistochemical studies, the new conformation-specific monoclonal antibodies showed underappreciated small micro-aggregates and very thin neurites in PD and DLB cases that were not observed with generic pan antibodies that recognize linear epitope. Furthermore, employing one of our conformation-specific antibodies in a sandwich based ELISA, we observed an increase in levels of α-syn oligomers in brain lysates from DLB compared to Alzheimer's disease and control samples. Therefore, the conformation-specific antibodies portrayed herein represent useful tools for research, biomarkers development, diagnosis and even immunotherapy for PD and related pathologies.

Non-neuronal and neuronal BACE1 elevation in association with angiopathic and leptomeningeal ß-amyloid deposition in the human brain.

BACKGROUND: Cerebral amyloid angiopathy (CAA) refers to the deposition of ß-amyloid (Aß) peptides in the wall of brain vasculature, commonly involving capillaries and arterioles. Also being considered a part of CAA is the Aß deposition in leptomeninge. The cellular origin of angiopathic Aß and the pathogenic course of CAA remain incompletely understood. METHODS: The present study was aimed to explore the pathogenic course of CAA in the human cerebrum via examination of changes in ß-secretase-1 (BACE1), the obligatory Aß producing enzyme, relative to Aß and other cellular markers, by neuroanatomical and biochemical characterizations with postmortem brain samples and primary cell cultures. RESULTS: Immunoreactivity (IR) for BACE1 was essentially not visible at vasculature in cases without cerebral amyloidosis (control group, n = 15, age = 86.1 ± 10.3 year). In cases with brain amyloid pathology (n = 15, age = 78.7 ± 12.7 year), increased BACE1 IR was identified locally at capillaries, arterioles and along the pia, localizing to endothelia, perivascular dystrophic neurites and meningeal cells, and often coexisting with vascular iron deposition. Double immunofluorescence with densitometric analysis confirmed a site-specific BACE1 elevation at cerebral arterioles in the development of vascular Aß deposition. Levels of BACE1 protein, activity and its immediate product (C99) were elevated in leptomeningeal lysates from cases with CAA relative to controls. The expression of BACE1 and other amyloidogenic proteins in the endothelial and meningeal cells was confirmed in primary cultures prepared from human leptomeningeal and arteriolar biopsies. CONCLUSION: These results suggest that BACE1 elevation in the endothelia and perivascular neurites may be involved in angiopathic Aß deposition, while BACE1 elevation in meningeal cells might contribute Aß to leptomeningeal amyloidosis.

Production and characterization of a fusion peptide derived from the rabies virus glycoprotein (RVG29).

Gene therapy targeting the brain holds great promise in curing nervous system degenerative diseases in clinical applications. With this in mind, in a previous study a 29 amino-acid peptide derived from the rabies virus glycoprotein (RVG29) with a nonamer stretch of arginine residues (RVG29-9R) at its carboxy-terminus was exploited as a ligand for brain-targeting gene delivery. Importantly, the report demonstrated that the RVG29-9R vector was able to cross the blood-brain barrier. RVG29-9R is currently synthesized by commercial companies with high associated costs. In this study, in order to reduce the costs of producing RVG29-9R, we have expressed and purified 6mg of a recombinant peptide (RVG29-9R-6His) from 0.4g of cultured Escherichia coli. We assessed the physiochemical properties of RVG29-9R-6His, its cytotoxicity, and the in vitro transfection efficiency in Neuro 2a cells (which express the acetylcholine receptor). Our results reveal that the RVG29-9R-6His peptide recognized Neuro 2a cells in a dose-dependent manner and it was also able to bind plasmid DNA and deliver it into the Neuro 2a cells effectively. Therefore, our study has demonstrated that the recombinant RVG29-9R-6His peptide retains the functions of RVG29-9R and so may provide an economically viable and alternative production method for the manufacture of RVG29-9R.

An inactivated recombinant rabies CVS-11 virus expressing two copies of the glycoprotein elicits a higher level of neutralizing antibodies and provides better protection in mice.

The rabies virus (RABV) G protein is the primary contributor to the pathogenicity and protective immunity of RABV. In this study, we generated a recombinant rCVS-11-G strain containing two copies of the G protein derived from the pathogenic wild-type (wt) CVS-11 strain and based on its infectious clone. Compared with the wtCVS-11 strain, the rCVS-11-G strain possessed a larger virion and 1.4-fold more G protein, but it exhibited a similar growth property to the rCVS-11 strain, including passaging stability in vitro. qPCR results showed that the two G genes were over-expressed in BHK-21 cells infected with the rCVS-11-G strain. However, the rCVS-11-G strain presented an 80 % lower LD50 than the wtCVS-11 strain when intracranially (i.c.) inoculated in adult mice. Adult mice that were either intracranially (i.c.) or intramuscularly (i.m.) inoculated with rCVS-11-G strain developed more acute neurological symptoms and greater mortality than those inoculated with the wtCVS-11 strain. Furthermore, the rCVS-11-G strain was more easily and rapidly taken up by neuroblastoma cells. These data indicated that the rCVS-11-G strain might have increased neurotropism because of the over-expression of the pathogenic G protein. The inactivated rCVS-11-G strain induced significantly higher levels of virus neutralization antibodies and provided better protection from street rabies virus challenge in mice. Therefore, the rCVS-11-G strain may be a promising inactivated vaccine strain due to its better immunogenicity.

Evidence that the LRRK2 ROC domain Parkinson's disease-associated mutants A1442P and R1441C exhibit increased intracellular degradation.

Mutations in the leucine-rich repeat kinase 2 (lrrk2) gene are the leading genetic cause of Parkinson's disease (PD). In characterizing the novel ROC domain mutant A1442P, we compared its steady-state protein levels, propensity to aggregate, and toxicity with the pathogenic R1441C mutant and wild-type (WT) LRRK2. Mutant (R1441C and A1442P) and WT LRRK2 fused to green fluorescent protein (GFP) and FLAG were transiently expressed in HEK293 cells using plasmid constructs. Western analysis and fluorescence microscopy consistently demonstrated lower mutant LRRK2 protein levels compared with WT. A time-course expression study using flow cytometry showed that WT LRRK2 expression increased initially but then plateaued by 72 hr. Conversely, R1441C and A1442P mutant expression attained 85% and 74% of WT levels at 24 hr but fell to 68% and 55% of WT levels by 72 hr, respectively. We found that proteasome inhibition markedly increased mutant LRRK2 to levels approaching those of WT. Taken together, our findings reveal increased intracellular degradation for both mutants. Furthermore, the impact of mutant and WT LRRK2 expression on HEK293 cell viability was assessed under normative and oxidative (hydrogen peroxide) conditions and found not to differ. Expression of WT and mutant LRRK2 protein gave rise to intracellular aggregates of similar appearance and cellular localization. In summary, we provide evidence that the novel A1442P mutant and the previously investigated R1441C pathogenic mutant exhibit increased intracellular degradation, a property reportedly demonstrated for the pathogenic LRRK2 kinase domain mutant I2020T.