Semiautomated design and soluble expression of a chimeric antigen TbpAB01 from Glaesserella parasuis.

Pathogenic bacterial membrane proteins (MPs) are a class of vaccine and antibiotic development targets with widespread clinical application. However, the inherent hydrophobicity of MPs poses a challenge to fold correctly in living cells. Herein, we present a comprehensive method to improve the soluble form of MP antigen by rationally designing multi-epitope chimeric antigen (ChA) and screening two classes of protein-assisting folding element. The study uses a homologous protein antigen as a functional scaffold to generate a ChA possessing four epitopes from transferrin-binding protein A of Glaesserella parasuis. Our engineered strain, which co-expresses P17 tagged-ChA and endogenous chaperones groEL-ES, yields a 0.346 g/L highly soluble ChA with the property of HPS-positive serum reaction. Moreover, the protein titer of ChA reaches 4.27 g/L with >90% soluble proportion in 5-L bioreactor, which is the highest titer reported so far. The results highlight a timely approach to design and improve the soluble expression of MP antigen in industrially viable applications.

A novel non-chemically amplified resist based on polystyrene-iodonium derivatives for electron beam lithography.

To break the resolution limitation of traditional resists, more work is needed on non-chemically amplified resists (non-CARs). Non-CARs based on iodonium salt modified polystyrene (PS-I) were prepared with controllable molecular weight and structure. The properties of the resist can be adjusted by the uploading of iodonium salts on the polymer chain, the materials with a higher proportion of iodonium salts show better lithography performance. By comparing contrast curves and quality of the lithographic patterns, the optimum developing condition of 4-methyl-2-pentanone and ethyl alcohol (v:v = 1:7) was selected. The high-resolution stripes of 15 nm half-pitch (HP) can be achieved by PS-I0.58in e-beam lithography (EBL). PS-I0.58shows the advanced lithography performance in the patterns of 16 nm HP and 18 nm HP stripes with low line edge roughness (3.0 nm and 2.4 nm). The resist shows excellent potential for further pattern transfer, the etch selectivity of resist PS-I0.58to the silicon was close to 12:1. The lithographic mechanism of PS-I was investigated by experimental and theoretical calculation, which indicates the polarity of materials changes results in the solubility switch. This work provides a new option and useful guidelines for the development of high-resolution resist.

Clinical application of combination [11C]C-methionine and [13N]N-ammonia PET/CT in recurrent functional pituitary adenomas with negative MRI or [18F]F-FDG PET/CT.

BACKGROUND: We assessed the value of positron emission tomography/computed tomography (PET/CT) with [13N]N-ammonia ([13N]N-NH3) and [11C]C-methionine ([11C]C-MET) for the evaluation and management of recurrent secreting pituitary adenoma, which could not be detected by magnetic resonance imaging (MRI) or fluorine-18 fluorodeoxyglucose ([18F]F-FDG) PET. METHODS: Nine consecutive patients with biochemical and clinical evidence of active recurrent tumor not detected by MRI and [18F]F-FDG PET were enrolled in this study. All of the patients underwent [13N]N-NH3 and [11C]C-MET PET/CT, after which the pattern of tracer uptake was studied, the tumor position was located, and a clinical decision was made. RESULTS: In general, [11C]C-MET had a higher uptake in pituitary adenomas (PAs) than that in pituitary tissues, while [13N]N-NH3 had a higher uptake in pituitary tissue than in pituitary adenomas. Increased [11C]C-MET uptake was observed in all nine PAs and three pituitary tissues, while all pituitary tissues and only one pituitary adenoma showed increased [13N]N-NH3 uptake. Four patients had concordant imaging and surgical findings indicative of biochemical remission without hypopituitarism after treatment. Radiotherapy was adopted in two patients, medication in another two, and follow-up observation in one case. CONCLUSION: Combined [11C]C-MET and [13N]N-NH3 PET/CT is effective in the differentiation of PAs from pituitary tissue in recurrent functional PAs with negative MRI or [18F]F-FDG PET. These results provide a valuable reference for further disease management.

New insights of bacterial and eukaryotic phenotypes on the plastics collected from the typical natural habitat of the endangered crocodile lizard.

Although accumulating evidence indicates that endangered animals suffer from plastic pollution, this has been largely overlooked. Here, we explored the bacteria and eukaryotes living in the plastics gathered from the natural habitat of the highly endangered crocodile lizard. The results demonstrated that the bacterial and eukaryotic communities on plastics formed a unique ecosystem that exhibited lower diversity than those in the surrounding water and soil. However, microbes displayed a more complex and stable network on plastic than that in water or soil, implying unique mechanisms of stabilization. These mechanisms enhanced their resilience and contributed to the provision of stable ecological services. Eukaryotes formed a simpler and smaller network than bacteria, indicating different survival strategies. The bacteria residing on the plastics played a significant role in carbon transformation and sequestration, which likely impacted carbon cycling in the habitat. Furthermore, microbial exchange between plastics and the crocodile lizard was observed, suggesting that plastisphere serves as a mobile gene bank for the exchange of information, including potentially harmful substances. Overall, microbes on plastic appear to significantly impact the crocodile lizard and its natural habitat via various pathways. These results provided novel insights into risks evaluation of plastic pollution and valuable guidance for government efforts in plastic pollutant control in nature reserves.

Development of a time-resolved immunochromatographic test strip for rapid and quantitative determination of retinol-binding protein 4 in urine.

Urine retinol-binding protein 4 (RBP4) has recently been reported as a novel earlier biomarker of chronic kidney disease (CKD) which is a global public health problem with high morbidity and mortality. Accurate and rapid detection of urine RBP4 is essential for early monitor of impaired kidney function and prevention of CKD progression. In the present study, we developed a time-resolved fluorescence immunochromatographic test strip (TRFIS) for the quantitative and rapid detection of urine RBP4. This TRFIS possessed excellent linearity ranging from 0.024 to 12.50 ng/mL for the detection of urine RBP4, and displayed a good linearity (Y = 239,581 × X + 617,238, R2 = 0.9902), with the lowest visual detection limit of 0.049 ng/mL. This TRFIS allows for quantitative detection of urine RBP4 within 15 min and shows high specificity. The intra-batch coefficient of variation (CV) and the inter-batch CV were both < 8%, respectively. Additionally, this TRFIS was applied to detect RBP4 in the urine samples from healthy donors and patients with CKD, and the results of TRFIS could efficiently discern the patients with CKD from the healthy donors. The developed TRFIS has the characteristics of high sensitivity, high accuracy, and a wide linear range, and is suitable for rapid and quantitative determination of urine RBP4.

Correction: Are pangolins the intermediate host of the 2019 novel coronavirus (SARS-CoV-2)?

[This corrects the article DOI: 10.1371/journal.ppat.1008421.].

Improving the soluble expression of difficult-to-express proteins in prokaryotic expression system via protein engineering and synthetic biology strategies.

Solubility and folding stability are key concerns for difficult-to-express proteins (DEPs) restricted by amino acid sequences and superarchitecture, resolved by the precise distribution of amino acids and molecular interactions as well as the assistance of the expression system. Therefore, an increasing number of tools are available to achieve efficient expression of DEPs, including directed evolution, solubilization partners, chaperones, and affluent expression hosts, among others. Furthermore, genome editing tools, such as transposons and CRISPR Cas9/dCas9, have been developed and expanded to construct engineered expression hosts capable of efficient expression ability of soluble proteins. Accounting for the accumulated knowledge of the pivotal factors in the solubility and folding stability of proteins, this review focuses on advanced technologies and tools of protein engineering, protein quality control systems, and the redesign of expression platforms in prokaryotic expression systems, as well as advances of the cell-free expression technologies for membrane proteins production.

A unified global tractography framework for automatic visual pathway reconstruction.

The human visual pathway starts from the retina, passes through the retinogeniculate visual pathway, the optic radiation, and finally connects to the primary visual cortex. Diffusion MRI tractography is the only technology that can noninvasively reconstruct the visual pathway. However, complete and accurate visual pathway reconstruction is challenging because of the skull base environment and complex fiber geometries. Specifically, the optic nerve within the complex skull base environment can cause abnormal diffusion signals. The crossing and fanning fibers at the optic chiasm, and a sharp turn of Meyer's loop at the optic radiation, contribute to complex fiber geometries of the visual pathway. A fiber trajectory distribution (FTD) function-based tractography method of our previous work and several high sensitivity tractography methods can reveal these complex fiber geometries, but are accompanied by false-positive fibers. Thus, the related studies of the visual pathway mostly applied the expert region of interest selection strategy. However, interobserver variability is an issue in reconstructing an accurate visual pathway. In this paper, we propose a unified global tractography framework to automatically reconstruct the visual pathway. We first extend the FTD function to a high-order streamline differential equation for global trajectory estimation. At the global level, the tractography process is simplified as the estimation of global trajectory distribution coefficients by minimizing the cost between trajectory distribution and the selected directions under the prior guidance by introducing the tractography template as anatomic priors. Furthermore, we use a deep learning-based method and tractography template prior information to automatically generate the mask for tractography. The experimental results demonstrate that our proposed method can successfully reconstruct the visual pathway with high accuracy.

Propensity score-matched analysis comparing hippocampus-avoidance whole-brain radiotherapy plus simultaneous integrated boost with hippocampus­avoidance whole-brain radiotherapy alone for multiple brain metastases-a retrospective study in multiple institutions.

BACKGROUND: The optimal treatment for multiple brain metastases has been recently controversially discussed.This study was aimed to explore the feasibility of Hippocampus-Avoidance Whole-Brain Radiotherapy plus a simultaneous integrated boost (HA-WBRT + SIB) in patients with multiple brain metastases and assess tumor control in comparison with Hippocampus-Avoidance Whole-Brain Radiotherapy (HA-WBRT) alone for brain metastases. METHODS: In this study, 63 patients with multiple brain metastases (≥ 4 metastases) had undergone HA-WBRT + SIB between January 2016 and December 2020 in the observation group:HA-WBRT (30 Gy in 12 fractions, the maximum dose of the hippocampus ≤ 14 Gy) plus a simultaneous integrated boost (48 Gy in 12 fractions) for brain metastases.Overall Survival (OS), Median survival,intracranial control (IC = control within the entire brain), intracranial progression-free survival (iPFS) and adverse events were compared with the control group (a HA-WBRT retrospective cohort) by propensity score matching analysis. RESULTS: After 1:1 propensity score matching,there were 56 patients in each group (the observation group, the control group). OS, median survival and iPFS were significantly longer in the observation group (18.4 vs. 10.9 months, P<0.001), (13.0 vs. 8.0 months, P<0.001), (13.9 vs.7.8 months, P<0.001). In comparison of 1-year-IC rates, the observation group also demonstrated higher than the control group (51.8% vs. 21.4%, P = 0.002), respectively. Seven hippocampal metastases were found in the control group (4/56,7.1%) and the observation group (3/56,5.4%) after HA-WBRT. The death rate of intracranial progression were 23.2% in the observation group and 37.5% in the control group.All adverse events were not significant difference between the two groups (P>0.05). CONCLUSIONS: HA-WBRT + SIB resulted in better OS,median survival, IC, iPFS, an acceptable risk of radiation response, and a potential way of declining neurocognitive adverse events, which may be a better treatment for patients with multiple brain metastases.

Hyperglycemia induces PFKFB3 overexpression and promotes malignant phenotype of breast cancer through RAS/MAPK activation.

BACKGROUND: Breast cancer is the most common tumor in women worldwide. Diabetes mellitus is a global chronic metabolic disease with increasing incidence. Diabetes mellitus has been reported to positively regulate the development of many tumors. However, the specific mechanism of hyperglycemic environment regulating breast cancer remains unclear. PFKFB3 (6-phosphofructose-2-kinase/fructose-2, 6-bisphosphatase 3) is a key regulatory factor of the glycolysis process in diabetes mellitus, as well as a promoter of breast cancer. So, we want to explore the potential link between PFKFB3 and the poor prognosis of breast cancer patients with hyperglycemia in this study. METHODS: Cell culture was utilized to construct different-glucose breast cancer cell lines. Immunohistochemistry was adopted to analyze the protein level of PFKFB3 in benign breast tissues, invasive ductal carcinoma with diabetes and invasive ductal carcinoma without diabetes. The Kaplan-Meier plotter database and GEO database (GSE61304) was adopted to analyze the survival of breast cancer patients with different PFKFB3 expression. Western blot was adopted to analyze the protein level of PFKFB3, epithelial-mesenchymal transition (EMT)-related protein and extracellular regulated protein kinases (ERK) in breast cancer cells. Gene Set Cancer Analysis (GSCA) was utilized to investigate the potential downstream signaling pathways of PFKFB3. TargetScan and OncomiR were utilized to explore the potential mechanism of PFKFB3 overexpression by hyperglycemia. Transfections (including siRNAs and miRNA transfection premiers) was utilized to restrain or mimic the expression of the corresponding RNA. Cell functional assays (including cell counting, MTT, colony formation, wound-healing, and cell migration assays) were utilized to explore the proliferation and migration of breast cancer cells. RESULTS: In this study, we demonstrated that the expression of PFKFB3 in breast cancer complicated with hyperglycemia was higher than that in breast cancer with euglycemia through cell experiment in vitro and histological experiment. PFKFB3 overexpression decreased the survival period of breast cancer patients and was correlated with a number of clinicopathological parameters of breast cancer complicated with diabetes. PFKFB3 promoted the proliferation and migration of breast cancer in a hyperglycemic environment and might be regulated by miR-26. In addition, PFKFB3 stimulated epithelial-mesenchymal transition of breast cancer in a hyperglycemic environment. In terms of downstream mechanism exploration, we predicted and verified the cancer-promoting effect of PFKFB3 in breast cancer complicated with hyperglycemia through RAS/MAPK pathway. CONCLUSIONS: In conclusion, PFKFB3 could be overexpressed by hyperglycemia and might be a potential therapeutic target for breast cancer complicated with diabetes.

Biogeography and diversity patterns of antibiotic resistome in the sediments of global lakes.

Lakes act as one of the reservoirs and dispersal routes of antibiotic resistance genes (ARGs) and pathogenic resistant bacteria in aquatic environments. Previous studies reported the occurrence and distribution of ARGs in lakes worldwide; however, few investigated the biogeography and diversity patterns of antibiotic resistome in the environment. To fill this gap, a large-scale data set of sediment metagenomes was collected from globally distributed lakes and characterized comprehensively using metagenomic assembly-based analysis, aiming to shed light on the biogeography and diversity patterns of ARGs in lake ecosystems from a global perspective. Our analyses showed that abundant and diverse ARGs were found in the global lake sediments, including a set of emerging ARGs such as mcr-type and carbapenem-resistant Enterobacteriaceae related genes. Most of the identified ARGs were generally associated with the commonly used antibiotics, suggesting the role of increasing antibiotic consumptions on the resistome prevalence. Spatially, the composition and diversity of ARGs varied across geographical distances and exhibited a scale-dependent distance-decay relationship. Notably, the composition of ARGs was largely shaped by bacterial community structure, and their diversities were co-governed by stochastic process (∼48%) and deterministic process (∼52%). Findings provide a valuable insight to better understand ecological mechanisms of ARGs in lake ecosystems and have important implication for the prevention and control of resistome risk.

Are pangolins the intermediate host of the 2019 novel coronavirus (SARS-CoV-2)?

The outbreak of a novel corona Virus Disease 2019 (COVID-19) in the city of Wuhan, China has resulted in more than 1.7 million laboratory confirmed cases all over the world. Recent studies showed that SARS-CoV-2 was likely originated from bats, but its intermediate hosts are still largely unknown. In this study, we assembled the complete genome of a coronavirus identified in 3 sick Malayan pangolins. The molecular and phylogenetic analyses showed that this pangolin coronavirus (pangolin-CoV-2020) is genetically related to the SARS-CoV-2 as well as a group of bat coronaviruses but do not support the SARS-CoV-2 emerged directly from the pangolin-CoV-2020. Our study suggests that pangolins are natural hosts of Betacoronaviruses. Large surveillance of coronaviruses in pangolins could improve our understanding of the spectrum of coronaviruses in pangolins. In addition to conservation of wildlife, minimizing the exposures of humans to wildlife will be important to reduce the spillover risks of coronaviruses from wild animals to humans.

MiR-34b/c play a role in early sex differentiation of Amur sturgeon, Acipenser schrenckii.

BACKGROUND: Sex differentiation can be viewed as a controlled regulatory balance between sex differentiation-related mRNAs and post-transcriptional mechanisms mediated by non-coding RNAs. In mammals, increasing evidence has been reported regarding the importance of gonad-specific microRNAs (miRNAs) in sex differentiation. Although many fishes express a large number of gonadal miRNAs, the effects of these sex-biased miRNAs on sex differentiation in teleost fish remain unknown. Previous studies have shown the exclusive and sexually dimorphic expression of miR-34b/c in the gonads of the Amur sturgeon (Acipenser schrenckii), suggesting its potential role in the sex differentiation process. RESULTS: Using quantitative real-time PCR (qPCR), we observed that miR-34b/c showed consistent spatiotemporal expression patterns; the expression levels significantly increased during early sex differentiation. Using in situ hybridization, miR-34c was found to be located in the germ cells. In primary germ cells in vitro, the group subjected to overexpression and inhibition of miR-34c showed significantly higher proliferation ability and lower apoptosis, respectively, compared to the corresponding control group. Luciferase reporter assays using the ar-3'UTR-psiCHECK-2 luciferase vector suggested a targeted regulatory interaction between miR-34b/c and the 3'UTR of the androgen receptor (ar) mRNA. Furthermore, miR-34b/c and ar showed negative expression patterns during early sex differentiation. Additionally, a negative feedback regulation pattern was observed between foxl2 expression in the ovaries and amh and sox9 expression in the testes during early sex differentiation. CONCLUSIONS: This study sheds new light on the roles of miR-34b/c in gonad development of Amur sturgeon, and provides the first comprehensive evidence that the gonad-predominant microRNAs may have a major role in sex differentiation in teleost fish.

An enzyme cascade fluorescence-based assay for the quantification of phenylalanine in serum.

The quantification of phenylalanine in clinical samples is essential for the diagnosis and treatment of neonatal phenylketonuria. In this report, an enzyme cascade strategy was proposed and a high-efficiency fluorescence assay was established for rapid and convenient phenylalanine quantification. The assay involves phenylalanine dehydrogenase for the quantitative metabolization of phenylalanine and the formation of NADH, as well as nitroreductase combining a nitroaromatic substrate for the fluorescent quantification of NADH and subsequently phenylalanine. The phenylalanine levels in clinical serum determined by this fluorescence assay are consistent with those from HPLC. This straightforward approach provides a versatile strategy for the development of cost-effective and convenient assays for mass screening and metabolite monitoring.

Retracted: Polyacrylamide Hydrogel Injection for Breast Augmentation: Another Injectable Failure.

This publication has been retracted by the Editor as it erroneously describes the effects of an injectable polyacrylamide gel.Reference:Zhenxiang Wang, Shirong Li, Lingli Wang, Shu Zhang, Yan Jiang, Jinping Chen, Donglin Luo. Polyacrylamide Hydrogel Injection for Breast Augmentation: Another Injectable Failure. Med Sci Monit, 2012; 18(6): CR399-408. DOI: 10.12659/MSM.882910.

Simultaneous determination of seven anthraquinones in Cassiae semen by natural deep eutectic solvent extraction.

INTRODUCTION: Anthraquinones are considered to be an important class of bioactive substances in Cassiae semen, and the content of anthraquinones is an essential indicator of the quality of Cassiae semen raw herbal materials. OBJECTIVES: The present study aimed to propose a novel, efficient and effective ultra-high-performance liquid chromatography (UHPLC) method for the simultaneous determination of aurantio-obtusin, aloe-emodin, rhein, obtusin, emodin, chrysophanol and physcion, with the help of natural deep eutectic solvents (NADESs) as extraction solvents. METHODOLOGY: NADESs were introduced to the simultaneous extraction of anthraquinones from Cassiae semen samples. Several NADESs were designed by menthol, choline chloride, d-glucose as hydrogen bond acceptors, with nine different acids and appropriate water as hydrogen bond donors. The parameters affecting the extraction efficiency of seven anthraquinones were demonstrated in detail. RESULTS: Among the obtained NADESs, the highest extraction efficiency was demonstrated by a solution consisting of d-glucose, lactic acid and water with a molar ratio of 1:5:4. The seven anthraquinones were separated on an ACQUITY UPLC® BEH C18 column (2.1 mm × 100 mm, 1.8 µm) and detected within 12 min by a photodiode array (PDA) detector at 254 and 284 nm. The limits of detection and quantitation were from 1.00 to 7.26 µg/l and 3.29 to 24.22 µg/l, respectively. And Cassiae semen sample-based recoveries ranged from 81.13% to 113.78% with the relative standard deviation (RSD) (n = 6) of 1.4% to 10.1%. CONCLUSION: The developed method demonstrated that NADESs were applied successfully to analyse the anthraquinones in Cassiae semen samples collected from different regions in China.

Full-length gonad transcriptome analysis of Amur sturgeon Dmrt family genes: identification, characterization, and expression patterns during gonadal differentiation.

The regulatory mechanisms that govern sex differentiation in sturgeon are still poorly understood. The doublesex and Mab-3-related transcription factor (Dmrt) gene family is known for its extensive roles in sex determination and differentiation across vertebrates. This study aimed to identify new members of sturgeon Dmrt family genes and core actors in the gonadal differentiation of Amur sturgeon. A full-length gonad transcriptome database was exploited to identify Dmrt gene orthologs. Analyses of phylogenetic relationships and selection pressure were performed, and tissue expression profiles and spatiotemporal expression patterns in gonads were then analyzed using real-time PCR. In total, five Dmrt family genes were identified from the full-length gonad transcriptome, including Dmrt2, DmrtA1, DmrtA2, DmrtB1a, and DmrtB1b. Phylogenetic analysis showed that these genes were clustered into clades corresponding to the doublesex/Mav-3 (DM) genes of vertebrates. Furthermore, the analysis of evolutionary selective pressure indicated that DmrtB1a and DmrtB1b were subject to positive selection, suggesting the existence of adaptive evolution in sturgeon. The extensive tissue expression profiling of each Dmrt family gene revealed typical characteristics. Remarkably, according to a spatiotemporal expression pattern analysis, in later stages, DmrtB1b expression increased in testes and was significantly higher in testes than in ovaries at 24 months after hatching (M) and 36 M. This study provides a genetic resource of full-length Dmrt family genes and increases the understanding of Dmrt functions in sex differentiation in sturgeon.

308-nm excimer lamp with 0.03% tacrolimus ointment is safe and effective to treat children with non-segmental vitiligo.

Introduction: A 308-nm excimer lamp combined with topical medicines has been used to treat vitiligo. However, few studies have evaluated its efficacy and influencing factors in children. Aim: We investigated the clinical effects and factors influencing the effectiveness of a 308-nm excimer lamp combined with 0.03% tacrolimus ointment in treating children with non-segmental vitiligo. Material and methods: A retrospective interventional case-series study was performed on 73 patients with non-segmental vitiligo treated with combination therapy. The duration of treatment ranged from 1 to 24 months, and the total number of treatments ranged from 4 to 78 sessions. We evaluated different treatment factors, including the number of treatments with a 308-nm excimer lamp, location, dose, disease course, and adverse reactions. Results: Overall, 105 leukodermas were treated: 36.2% had disappeared completely. The efficiency rate was 81.9% after a median treatment duration of 10.5 months. The treatment was most effective for the face and neck. Patients with a short disease duration showed a better response (disease duration shorter than 12 months). Reported adverse reactions were mild. Conclusions: Treatment using a 308-nm excimer lamp and 0.03% tacrolimus ointment is a safe and valuable treatment for children with non-segmental vitiligo.

Comparative analysis of the mitochondrial proteins reveals complex structural and functional relationships in Fasciola species.

Mitochondria is a cellular source of energy, appears to play an essential role in dealing with cellular stress induced by environmental stimuli. The genetic diversity of mitochondrial genes involved in oxidative phosphorylation affecting the production of cellular energy and regional adaptation to various ecological (climatic) pressures affecting amino acid sequences (variants of protein). However, little is known about the combined effect of protein changes on cell-level metabolic alterations in simultaneous exposure to various environmental conditions, including mitochondrial dysfunction and oxidative stress induction. The present study was designed to address this issue by analyzing the mitochondrial proteins in Fasciola species including Cytochrome oxidase (COX1, COX2, COX3, and CYTB) and NADH dehydrogenase (ND1, ND2, ND3, ND4, ND5, and ND6). Mitochondrial proteins were used for detailed computational investigation, using available standard bioinformatics tools to exploit structural and functional relationships. These proteins in Fasciola hepatica, Fasciola gigentica, and Fasciola jacksoni were functionally annotated using public databases. The results showed that the protein of COX1 of F. hepatica, F. gigantica, and F. jacksoni consist of 510, 513, and 517 amino acids, respectively. The alignment of proteins showed that these proteins are conserved in the same regions at ten positions in COX and CYTB proteins while at twelve locations in NADH. Three-dimensional structure of COX, CYTB, and NADH proteins were compared and showed differences in additional conserved and binding sites in COX and CYTB proteins as compared to NADH in three species of Fasciola. These results based on the amino acid diversity pattern were used to identify sites in the enzyme and the variations in mitochondrial proteins among Fasciola species. Our study provides valuable information for future experimental studies, including identification of therapeutic, diagnostic, and immunoprophylactic interests with novel mitochondrial proteins.

Which Staging System Is More Suitable for Gastric Neuroendocrine Cancer and Mixed Adenoneuroendocrine Carcinomas? A Multicenter Cohort Study.

PURPOSE: To evaluate whether the European Neuroendocrine Tumor Society (ENETS) system or the 8th American Joint Committee on Cancer (AJCC) staging manual are suitable for gastric neuroendocrine carcinomas and/or mixed adenoneuroendocrine carcinomas (G-NECs/MANECs). METHODS: Patients in a multicentric series with G-NEC/MANEC who underwent curative-intent surgical resection for a primary tumor were included. An optimal staging system was proposed base on analysis of the T and N status and validated by the SEER database. RESULTS: Compared with the ENETS system, the survival curves of the T category and N category in the 8th AJCC system were better separated and distributed in a more balanced way, but the survival curves of T2 vs. T3, N0 vs. N1, and N3a vs. N3b overlapped. For the T category, the 8th AJCC T category was modified by combining T2 and T3, which was consistent with the T category in the 6th AJCC manual for GC. For the N category, the optimal cut-off values of metastatic lymph nodes using X-tile were also similar to those of the N category in the 6th AJCC system. The Kaplan-Meier plots of the 6th AJCC system showed statistically significant differences between individual substages. Compared with the other 2 classifications, the 6th AJCC system also showed superior prognostic stratification. Similar results were obtained in both multicentric and SEER validation sets. CONCLUSIONS: Compared to the 8th AJCC and ENETS systems, the 6th AJCC staging system for GC is more suitable for G-NEC/MANEC and can be adopted in clinical practice.