A skin-specific α-Synuclein seeding amplification assay for diagnosing Parkinson's disease.

The seeding amplification assay (SAA) has recently emerged as a valuable tool for detecting α-synuclein (αSyn) aggregates in various clinically accessible biospecimens. Despite its efficiency and specificity, optimal tissue-specific conditions for distinguishing Parkinson's disease (PD) from non-PD outside the brain remain underexplored. This study systematically evaluated 150 reaction conditions to identify the one with the highest discriminatory potential between PD and non-synucleinopathy controls using skin samples, resulting in a modified SAA. The streamlined SAA achieved an overall sensitivity of 92.46% and specificity of 93.33% on biopsy skin samples from 332 PD patients and 285 controls within 24 h. Inter-laboratory reproducibility demonstrated a Cohen's kappa value of 0.87 (95% CI 0.69-1.00), indicating nearly perfect agreement. Additionally, αSyn seeds in the skin were stable at -80 °C but were vulnerable to short-term exposure to non-ultra-low temperatures and grinding. This study thoroughly investigated procedures for sample preprocessing, seed amplification, and storage, introducing a well-structured experimental framework for PD diagnosis using skin samples.

Ultrasensitive detection of aggregated α-synuclein using quiescent seed amplification assay for the diagnosis of Parkinson's disease.

BACKGROUND: Seed amplification assays (SAA) enable the amplification of pathological misfolded proteins, including α-synuclein (αSyn), in both tissue homogenates and body fluids of Parkinson's disease (PD) patients. SAA involves repeated cycles of shaking or sonication coupled with incubation periods. However, this amplification scheme has limitations in tracking protein propagation due to repeated fragmentation. METHODS: We introduced a modified form of SAA, known as Quiescent SAA (QSAA), and evaluated biopsy and autopsy samples from individuals clinically diagnosed with PD and those without synucleinopathies (control group). Brain biopsy samples were obtained from 14 PD patients and 6 controls without synucleinopathies. Additionally, skin samples were collected from 214 PD patients and 208 control subjects. Data were analyzed from April 2019 to May 2023. RESULTS: QSAA successfully amplified αSyn aggregates in brain tissue sections from mice inoculated with pre-formed fibrils. In the skin samples from 214 PD cases and 208 non-PD cases, QSAA demonstrated high sensitivity (90.2%) and specificity (91.4%) in differentiating between PD and non-PD cases. Notably, more αSyn aggregates were detected by QSAA compared to immunofluorescence with the pS129-αSyn antibody in consecutive slices of both brain and skin samples. CONCLUSION: We introduced the new QSAA method tailored for in situ amplification of αSyn aggregates in brain and skin samples while maintaining tissue integrity, providing a streamlined approach to diagnosing PD with individual variability. The integration of seeding activities with the location of deposition of αSyn seeds advances our understanding of the mechanism underlying αSyn misfolding in PD.

Multifaceted roles of lymphatic and blood endothelial cells in the tumor microenvironment of hepatocellular carcinoma: A comprehensive review.

The tumor microenvironment is a complex network of cells, extracellular matrix, and signaling molecules that plays a critical role in tumor progression and metastasis. Lymphatic and blood vessels are major routes for solid tumor metastasis and essential parts of tumor drainage conduits. However, recent studies have shown that lymphatic endothelial cells (LECs) and blood endothelial cells (BECs) also play multifaceted roles in the tumor microenvironment beyond their structural functions, particularly in hepatocellular carcinoma (HCC). This comprehensive review summarizes the diverse roles played by LECs and BECs in HCC, including their involvement in angiogenesis, immune modulation, lymphangiogenesis, and metastasis. By providing a detailed account of the complex interplay between LECs, BECs, and tumor cells, this review aims to shed light on future research directions regarding the immune regulatory function of LECs and potential therapeutic targets for HCC.

Dichotomous roles of ADAR1 in liver hepatocellular carcinoma and kidney renal cell carcinoma: Unraveling the complex tumor microenvironment and prognostic significance.

BACKGROUND: Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA-editing enzyme that significantly impacts cancer progression and various biological processes. The expression of ADAR1 mRNA has been examined in multiple cancer types using The Cancer Genome Atlas (TCGA) dataset, revealing distinct patterns in kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), and liver hepatocellular carcinoma (LIHC) compared to normal controls. However, the reasons for these differential expressions remain unclear. METHODS: In this study, we performed RT-PCR and western blotting (WB) to validate ADAR1 expression patterns in clinical tissue samples. Survival analysis and immune microenvironment analysis (including immune score and stromal score) were conducted using TCGA data to determine the specific cell types associated with ADAR1, as well as the key genes in those cell types. The relationship between ADAR1 and specific cell types' key genes was verified by immunohistochemistry (IHC), using clinical liver and kidney cancer samples. RESULTS: Our validation analysis revealed that ADAR1 expression was downregulated in KICH, KIRC, and KIRP, while upregulated in LIHC compared to normal tissues. Notably, a significant correlation was found between ADAR1 mRNA expression and patient prognosis, particularly in KIRC, KIRP, and LIHC. Interestingly, we observed a positive correlation between ADAR1 expression and stromal scores in KIRC, whereas a negative correlation was observed in LIHC. Cell type analysis highlighted distinct relationships between ADAR1 expression and the two stromal cell types, blood endothelial cells (BECs) and lymphatic endothelial cells (LECs), and further determined the signature gene claudin-5 (CLDN5), in KIRC and LIHC. Moreover, ADAR1 was inversely related with CLDN5 in KIRC (n = 26) and LIHC (n = 30) samples, verified via IHC. CONCLUSIONS: ADAR1 plays contrasting roles in LIHC and KIRC, associated with the enrichment of BECs and LECs within tumors. This study sheds light on the significant roles of stromal cells within the complex tumor microenvironment (TME) and provides new insights for future research in tumor immunotherapy and precision medicine.

Influence of Three-Dimensional Visual Reconstruction Technology Combined with Virtual Surgical Planning of CTA Images on Precise Resection of Liver Cancer in Hepatobiliary Surgery.

Hepatobiliary malignancies, such as hepatocellular carcinoma (HCC) and biliary tract cancers, namely, gallbladder carcinoma and cholangiocarcinoma, are linked to a high rate of morbidity and mortality, depending on the phase of the disease. The intricate hepatobiliary anatomy and the need for accurate peroperative management, especially in patients with advanced liver disease, make these tumors difficult to treat. Surgical resection is a notable therapy for hepatobiliary cancers. Unnecessary or excessive liver excision influences patient rehabilitation, normal liver function, and postoperative complications. Hepatobiliary operations must therefore include accurate liver removal. The present advancements in imaging technology are aimed at improving the diagnostic efficacy of liver injury even more. Three-dimensional visual reconstruction is becoming more important in the diagnosis as well as treatment of a variety of disorders. In this paper, we proposed a novel three-dimensional visual reconstruction technology using enhanced nonuniform rational basis spline (ENURBS) combined with virtual surgical planning of Computed Tomography Angiography (CTA) images for precise liver cancer resection. The purpose of this project is to rebuild 2D CTA scan images of liver cancer into a 3D reconstructed model for efficient visualization and diagnosis of liver cancer and to prepare an effective preoperative surgical plan for precise liver excision based on a 3D recreated liver model. This method's performance is compared to that of 2D planning in terms of accuracy and time taken to complete the plan. It is concluded that our proposed technique outperforms the planning technique based on 2D images.

CHCHD2 maintains mitochondrial contact site and cristae organizing system stability and protects against mitochondrial dysfunction in an experimental model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's dementia. Mitochondrial dysfunction is involved in the pathology of PD. Coiled-coil-helix-coiled-coil-helix domain-containing 2 (CHCHD2) was identified as associated with autosomal dominant PD. However, the mechanism of CHCHD2 in PD remains unclear. METHODS: Short hairpin RNA (ShRNA)-mediated CHCHD2 knockdown or lentivirus-mediated CHCHD2 overexpression was performed to investigate the impact of CHCHD2 on mitochondrial morphology and function in neuronal tumor cell lines represented with human neuroblastoma (SHSY5Y) and HeLa cells. Blue-native polyacrylamide gel electrophoresis (PAGE) and two-dimensional sodium dodecyl sulfate-PAGE analysis were used to illustrate the role of CHCHD2 in mitochondrial contact site and cristae organizing system (MICOS). Co-immunoprecipitation and immunoblotting were used to address the interaction between CHCHD2 and Mic10. Serotype injection of adeno-associated vector-mediated CHCHD2 and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration were used to examine the influence of CHCHD2 in vivo. RESULTS: We found that the overexpression of CHCHD2 can protect against methyl-4-phenylpyridinium (MPP+)-induced mitochondrial dysfunction and inhibit the loss of dopaminergic neurons in the MPTP-induced mouse model. Furthermore, we identified that CHCHD2 interacted with Mic10, and overexpression of CHCHD2 can protect against MPP+-induced MICOS impairment, while knockdown of CHCHD2 impaired the stability of MICOS. CONCLUSION: This study indicated that CHCHD2 could interact with Mic10 and maintain the stability of the MICOS complex, which contributes to protecting mitochondrial function in PD.

Chaperone-mediated Autophagy Regulates Cell Growth by Targeting SMAD3 in Glioma.

Previous studies suggest that the reduction of SMAD3 (mothers against decapentaplegic homolog 3) has a great impact on tumor development, but its exact pathological function remains unclear. In this study, we found that the protein level of SMAD3 was greatly reduced in human-grade IV glioblastoma tissues, in which LAMP2A (lysosome-associated membrane protein type 2A) was significantly up-regulated. LAMP2A is a key rate-limiting protein of chaperone-mediated autophagy (CMA), a lysosome pathway of protein degradation that is activated in glioma. We carefully analyzed the amino-acid sequence of SMAD3 and found that it contained a pentapeptide motif biochemically related to KFERQ, which has been proposed to be a targeting sequence for CMA. In vitro, we confirmed that SMAD3 was degraded in either serum-free or KFERQ motif deleted condition, which was regulated by LAMP2A and interacted with HSC70 (heat shock cognate 71 kDa protein). Using isolated lysosomes, amino-acid residues 75 and 128 of SMAD3 were found to be of importance for this process, which affected the CMA pathway in which SMAD3 was involved. Similarly, down-regulating SMAD3 or up-regulating LAMP2A in cultured glioma cells enhanced their proliferation and invasion. Taken together, these results suggest that excessive activation of CMA regulates glioma cell growth by promoting the degradation of SMAD3. Therefore, targeting the SMAD3-LAMP2A-mediated CMA-lysosome pathway may be a promising approach in anti-cancer therapy.

Effect of four modification methods on adsorption capacities and in vitro hypoglycemic properties of millet bran dietary fibre.

To improve the adsorption capacities and hypoglycemic properties of millet bran dietary fibre (MBDF), four methods including acrylate-grafting, carboxymethylation, heat assisted with cellulase hydrolysis, and enzymatic hydrolysis combined with acrylate-grafting were used. The results demonstrated that all carboxymethylation, acrylate-grafting, and enzymatic hydrolysis combined with acrylate-grafting improved soluble dietary fibre content, water swelling ability and α-amylase-inhibition activity of MBDF. They also increased oil, cholesterol, sodium cholate, copper ion and nitrite ion adsorption capacities of MBDF. But carboxymethylation, acrylate-grafting and enzymatic hydrolysis combined with acrylate-grafting decreased polyphenol content, glucose-binding ability and glucose dialysis retardation index of MBDF (p < 0.05). The heat assisted with cellulase hydrolysis increased soluble dietary fibre content, polyphenol content, sodium cholate-adsorption capacity, and hypoglycemic properties of MBDF including glucose-binding ability, glucose dialysis retardation index and α-amylase-inhibition activity; but reduced adsorption capacity of MBDF on cholesterol and copper ion (p < 0.05). Changes in structure of MBDF caused by these modification methods were proved by the results of scanning electron microscopy and Fourier-transformed infrared spectroscopy analysis. These results highlight potential applications of these modified MBDFs as ingredients of hypolipidemic and hypoglycemic foods, or scavenger of nitrite and copper ion.

Merged hepatopulmonary features in hepatoid adenocarcinoma of the lung: a systematic review.

This study aimed to provide diagnostic clues for patients with elevated serum alpha-fetoprotein (AFP) in the absence of liver tumors and rectify some previously confused concepts about hepatoid carcinoma of the lung through a systematic review on hepatoid adenocarcinoma of the lung (HAL). A thorough search for original articles on HAL published prior to November 2020 was performed using the PubMed, EBSCOhost, Embase, WanFang Data, and China National Knowledge Infrastructure (CNKI) databases. Ninety-four patients from 88 studies met the eligibility criteria. HAL was rare and mainly occurred among male Asian smokers in their 60 s, presenting with cough, hemoptysis, chest pain, dyspnea and/or weight loss, as well as elevated serum AFP with a mass usually in the right upper lung lobe but no liver masses. Hepatoid differentiation regions, acinar or papillary structures in tumor tissues, and positive immunohistochemical expression of AFP, HepPar-1, and CK8/18 were crucial indicators for the diagnosis of HAL. Surgery-based strategies were recommended for stage I-III patients, while stage IV patients were mainly treated with chemotherapy-based strategy. The 1-, 3-, and 5-year overall survival rates were 40%, 35%, and 19%, respectively. The 1-year relapse-free survival rate was 58%. The postoperative monitoring of AFP contributed to the early detection of tumor recurrence, with a positive rate of 71.43%. In conclusion, patients with elevated serum AFP levels without any detectable hepatic lesions should be evaluated for the possibility of HAL.

Machine learning approach to predict acute kidney injury after liver surgery.

BACKGROUND: Acute kidney injury (AKI) after surgery appears to increase the risk of death in patients with liver cancer. In recent years, machine learning algorithms have been shown to offer higher discriminative efficiency than classical statistical analysis. AIM: To develop prediction models for AKI after liver cancer resection using machine learning techniques. METHODS: We screened a total of 2450 patients who had undergone primary hepatocellular carcinoma resection at Changzheng Hospital, Shanghai City, China, from January 1, 2015 to August 31, 2020. The AKI definition used was consistent with the Kidney Disease: Improving Global Outcomes. We included in our analysis preoperative data such as demographic characteristics, laboratory findings, comorbidities, and medication, as well as perioperative data such as duration of surgery. Computerized algorithms used for model development included logistic regression (LR), support vector machine (SVM), random forest (RF), extreme gradient boosting (XGboost), and decision tree (DT). Feature importance was also ranked according to its contribution to model development. RESULTS: AKI events occurred in 296 patients (12.1%) within 7 d after surgery. Among the original models based on machine learning techniques, the RF algorithm had optimal discrimination with an area under the curve value of 0.92, compared to 0.87 for XGBoost, 0.90 for DT, 0.90 for SVM, and 0.85 for LR. The RF algorithm also had the highest concordance-index (0.86) and the lowest Brier score (0.076). The variable that contributed the most in the RF algorithm was age, followed by cholesterol, and surgery time. CONCLUSION: Machine learning algorithms are highly effective in discriminating patients at high risk of developing AKI. The successful application of machine learning models may help guide clinical decisions and help improve the long-term prognosis of patients.

Functional validation of a human GLUD2 variant in a murine model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by Lewy body formation and progressive dopaminergic neuron death in the substantia nigra (SN). Genetic susceptibility is a strong risk factor for PD. Previously, a rare gain-of-function variant of GLUD2 glutamate dehydrogenase (T1492G) was reported to be associated with early onset in male PD patients; however, the function and underlying mechanism of this variant remains elusive. In the present study, we generated adeno-associated virus expressing GLUD2 and its mutant under the control of the glial fibrillary acidic protein promotor and injected the virus into the SN pars compacta of either untreated mice or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model mice. Our results demonstrate that GLUD2 mutation in MPTP-induced PD mice exacerbates movement deficits and nigral dopaminergic neuron death and reduces glutamate transporters expression and function. Using GC-Q-TOF/MS-based metabolomics, we determined that GLUD2 mutation damages mitochondrial function by decreasing succinate dehydrogenase activity to impede the tricarboxylic acid cycle in the SN of MPTP-induced PD mice. Accordingly, GLUD2 mutant mice had reduced energy metabolism and increased apoptosis, possibly due to downregulation of brain-derived neurotrophic factor/nuclear factor E2-related factor 2 signaling in in vitro and in vivo PD models. Collectively, our findings verify the function of GLUD2 in PD and unravel a mechanism by which a genetic variant in human GLUD2 may contribute to disease onset.

Risk of adverse events in advanced hepatocellular carcinoma with immune checkpoint therapy: A systematic review and meta-analysis.

AIMS: To evaluate risk of adverse events (AEs) in advanced hepatocellular carcinoma (AHCC) with immune checkpoint therapy in this setting. METHODS: A systematic search of original articles published until November 2019 was performed using PubMed, Embase, Web of Science, and the Cochrane Central Register of Controlled Trials. And a meta-analysis was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. RESULTS: A total of eight studies, including 597 patients, met the eligibility criteria. The median cohort size of patients was 75 (range: 18-267). The pooled incidence rates of grade≥3 AEs and fatal adverse events (FAEs) at last follow-up were 20.87 per 100 person-years (95% CI: 11.00-39.59, I2=91.0%) and 4.98 per 100 person-years (95% CI: 1.83-13.56, I2=82.8%). Subgroup analyses showed that pembrolizumab had a lower risk of grade≥3 AEs, but a higher risk of FAEs, when compared with nivolumab and tremelimumab. Meta-regression showed significant correlation between grade≥3 AEs rate and proportion of Child-Pugh A stage. Fatigue (16.9%), adrenal insufficiency (8.5%) and rash (6.8%) were involved in common non-laboratory AEs. CONCLUSIONS: Immune checkpoint therapy significantly increases the risk of AEs in AHCC patients. And the risk of grade≥3 AEs is associated with Child-Pugh classification. Future retrospective analyses and prospective cohort studies are warranted to evaluate the safety of immune checkpoint therapy in AHCC.

Acute rejection after liver transplantation is less common, but predicts better prognosis in HBV-related hepatocellular carcinoma patients.

BACKGROUND: With a novel finding of significantly lower incidence of acute rejection (AR) in patients with hepatocellular carcinoma (HCC) after liver transplantation, compared with those with benign end-stage liver disease (BESLD), in a large national cohort, we analyzed the correlations among the perioperative immuno-inflammation status, postoperative AR, and prognosis in HCC and BESLD patients with same etiology of hepatitis B virus (HBV), who underwent liver transplantation. METHODS: Patients who underwent liver transplantation due to HBV-related HCC or BESLD and experienced AR between September 2008 and April 2017 were analyzed retrospectively and followed up until April 2018. HCC patients with AR were matched with those without AR according to tumor stage and immunosuppressant concentration, at a 1:3 ratio. Preoperative immuno-inflammation status and prognosis of patients in both groups were compared. RESULTS: The overall incidences of AR in patients with HCC and BESLD were 8.60% and 10.61%, respectively. The postoperative 28-day incidence of AR was significantly lower in HCC compared with BESLD patients (3.23% vs 7.08%, p = 0.031). Compared with BESLD patients, the rejection activity index and perioperative CD4/CD8 ratio were significantly lower (p = 0.047 and p < 0.001, respectively), while platelet/lymphocyte ratio was significantly higher in HCC patients (p = 0.041). Later tumor stage in HCC patients was associated with higher systemic immuno-inflammation index, neutrophil/lymphocyte ratio, monocyte/lymphocyte ratio, platelet/lymphocyte ratio, aspartate aminotransferase/lymphocyte ratio, C-reactive protein/albumin ratio and fibrinogen level, and lower CD4/CD8 ratio before transplantation. In HCC patients with AR, the percentage of regulatory T cells (CD4+/CD25+) and the level of IL-10 significantly decreased (p = 0.0023, < 0.0001, respectively), while Th1/Th2 ratio, levels of IFN-γ and IL-2 markedly increased before transplantation (p = 0.0018, 0.0059, 0.0416, respectively). Preoperative monocyte/lymphocyte ratio was an independent risk factor for overall and recurrence-free survival after liver transplantation in HCC patients (p = 0.025, < 0.001, respectively). The 1-, 3-, and 5-year survival rates were 76%, 71% and 53% in the AR group, and 67%, 37% and 25% in the non-AR group (p = 0.042). CONCLUSION: Preoperative tumor-related immunosuppression may persist after liver transplantation in HCC patients, and reduce the incidence of AR. AR after liver transplantation may indicate a better prognosis in HCC patients.

microRNA-29a regulates liver tumor-initiating cells expansion via Bcl-2 pathway.

MicroRNAs (miRNAs) participate in tumorigenesis, progression, recurrence and drug resistance of hepatocellular carcinoma (HCC). However, few miRNAs have been identified and entered clinical practice. Herein, we report that miR-29a is downregulated in tumor-initiating cells (T-ICs) and has an important function in liver T-ICs. Functional studies revealed that miR-29a knockdown promotes liver T-ICs self-renewal and tumorigenesis. Conversely, a forced miR-29a expression inhibits liver T-ICs self-renewal and tumorigenesis. Mechanistically, we find that miR-29a downregulates Bcl-2 via binding its mRNA 3'UTR in liver T-ICs. The correlation between miR-29a and Bcl-2 is validated in human HCC tissues. Furthermore, the miR-29a expression determines the responses of hepatoma cells to sorafenib treatment. Analysis of patient-derived xenografts (PDXs) further demonstrated that the miR-29a high patients are more sensitive to sorafenib treatment. In conclusion, our findings revealed the crucial role of the miR-29a in liver T-ICs expansion and sorafenib response, rendering miR-29a as an optimal target for the prevention and intervention of HCC.

Inhibition of allogeneic islet graft rejection by VISTA-conjugated liposome.

The Ig superfamily member V-domain Ig-containing suppressor of T-cell activation (VISTA) is a negative regulator with broad-spectrum activities and has reported that blockade of VISTA or combination with other negative checkpoint receptors sufficiently break tumor tolerance. However, it remains unclear whether VISTA could induce allogeneic T-cell hyporesponsiveness and inhibit allograft rejection. Here we found VISTA treatment significantly inhibited lymphocyte proliferation and activation in allogeneic MLR assay through impairing SYK-VAV pathway. Interestingly, though neither VISTA protein nor VISTA-Fc fusion protein administration exerted satisfactory immunosuppressive effect on allograft survival due to their short half-life in circulation, this problem was solved by conjugating VISTA protein on liposome by biotin-streptavidin system, which markedly prolonged its circulating half-life to 60 h. With islet transplant model, administration of VISTA-conjugated liposome could markedly prolong allograft survival by inhibition of SYK-VAV pathway, thus maintained the normal blood glucose level of recipients during treatment period. The results indicate VISTA is a promising therapeutic target to treat allograft rejection of islet transplantation.