De Novo Discovery of Cysteine Frameworks for Developing Multicyclic Peptide Libraries for Ligand Discovery.

Conserved cysteine frameworks are essential components of disulfide-rich peptides (DRPs), which dominantly define the structural diversity of both naturally occurring and de novo-designed DRPs. However, there are only very limited numbers of conserved cysteine frameworks, and general methods enabling de novo discovery of cysteine frameworks with robust foldability are still not available. Here, we devised a "touchstone"-based strategy that relies on chasing oxidative foldability between two individual disulfide-rich folds on the phage surface to discover new cysteine frameworks from random sequences. Unique cysteine frameworks with a high degree of compatibility with phage display systems and broad sequence tolerance were successfully identified, which were subsequently exploited for the development of multicyclic DRP libraries, enabling the rapid discovery of new peptide ligands with low-nanomolar and picomolar binding affinity. This study provides an unprecedented method for exploring and exploiting the sequence and structure space of DRPs that is not readily accessible by existing strategies, holding the potential to revolutionize the study of DRPs and significantly advance the design and discovery of multicyclic peptide ligands and drugs.

Design and Synthesis of Cross-Link-Dense Peptides by Manipulating Regioselective Bisthioether Cross-Linking and Orthogonal Disulfide Pairing.

Existing disulfide-rich peptides, both naturally occurring and de novo designed, only represent a tiny amount of the possible sequence space because natural evolution and de novo design only keep sequences that are structurally approachable by correct disulfide pairings. To bypass this limitation for designing new peptide scaffolds beyond the natural sequence space, we dedicate to developing novel disulfide-rich peptides with predefined disulfide pairing patterns irrelevant to primary sequences. However, most of these designed peptides still suffer from disulfide rearrangements to at least one to three possible isomers. Here, we report a general and reliable strategy for the design and synthesis of a range of structurally diverse cross-link-dense peptide (CDP) scaffolds with two orthogonal disulfide bonds and a bisthioether bridge that are not subject to disulfide isomerizations. Altering the pattern of cysteine and penicillamine generates hundreds of different CDP scaffolds tolerant to extensive sequence manipulations. This work thus provides many useful scaffolds for the design of functional molecules such as protein binders with improved proteolytic stability (e.g., designed by epitope grafting).

Immune Responses and Protection Profiles in Mice Induced by Subunit Vaccine Candidates Based on the Extracellular Domain Antigen of Respiratory Syncytial Virus G Protein Combined with Different Adjuvants.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease of infants and older people. There is an urgent need for safe and effective vaccines against RSV infection. In this study, we analyzed the effects of the immune response and protection with the RSV recombinant G protein extracellular domain (Gecto) combined with various adjuvants as novel subunit vaccines in mice. All groups receiving RSV Gecto combined with adjuvants exhibited robust humoral and cellular immunity compared to those receiving an adjuvant alone or inactivated RSV vaccine. The greatest effect was observed in mice receiving Gecto combined with a CpG ODN + Alum salt adjuvant, resulting in the highest production of neutralizing antibodies against both RSV A and B subtypes, G-specific IgG and IFN-γ production in splenocytes, and interleukin-2 and interferon-γ expression in CD4+ T cells. Significant humoral and cellular immune responses were observed in mice immunized with Gecto combined with AddaS03™ or cyclosporin A adjuvants. The vaccine containing the AddaS03™ adjuvant showed significantly high expression of interleukin-4 in CD4+ T cells. Cross-protection against a challenge with either RSV A or B subtypes was observed in the Gecto plus adjuvant groups, resulting in a significant decrease in viral load and reduced pathological damage in the mouse lungs. These findings offer valuable insights into the development and application of recombinant RSV G-subunit vaccines with adjuvants.

Comparison of different genetic testing modalities applied in paediatric patients with steroid-resistant nephrotic syndrome.

BACKGROUND: Steroid-resistant nephrotic syndrome (SRNS) are monogenic in some cases, however, there are still no clear guidelines on genetic testing in the clinical practice of SRNS in children. METHODS: Three hundred thirty-two children were diagnosed with SRNS, and all children underwent genetic testing, including gene panels and/or whole-exome/genome sequencing (WES/WGS), during treatment. We analysed the relationship between clinical manifestation and genotype, and compared different genetic testing methods' detection rates and prices. RESULTS: In this study, 30.12% (100/332) of children diagnosed with SRNS had monogenic causes of the disease. With 33.7% (122/332) of children achieving complete remission, 88.5% (108/122) received steroids combined with tacrolimus (TAC). In detectability, WES increased by 8.69% (4/46) on gene panel testing, while WGS increased by 4.27% (5/117) on WES, and WES was approximately 1/7 of the price of WGS for every further 1% increase in pathogenicity. CONCLUSIONS: We verified that steroids combined with TAC were the most effective option in paediatric SRNS. In detection efficiency, we found that WGS was the highest, followed by WES. The panel was the lowest, but the most cost-effective method when considering the economic-benefit ratio, and thus it should be recommended first in SRNS.

LncRNA LOC105369504 inhibits tumor proliferation and metastasis in colorectal cancer by regulating PSPC1.

There is growing evidence that long non-coding RNAs (lncRNAs) are significant contributors to the epigenetic mechanisms implicated in the emergence, progression and metastasis of the colorectal cancer (CRC), but many remain underexplored. A novel lncRNA LOC105369504, was identified to be a potential functional lncRNA by microarray analysis. In CRC, the expression of LOC105369504 was markedly decreased and resulted in distinct variations in proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) in vivo and in vitro. This study showed that LOC105369504 bound to the protein of paraspeckles compound 1 (PSPC1) directly and regulated its stability using the ubiquitin-proteasome pathway in CRC cells. The suppression of CRC by LOC105369504 could be reversed through PSPC1 overexpression.This study showed that in CRC, LOC105369504 was under-regulated and as a novel lncRNA, LOC105369504 exerted tumor suppressive activity to suppress the proliferation together with metastasis in CRC cells through the regulation of PSPC1. These results offer new perspectives on the lncRNA effect on the progression of CRC.

Frequency-Specific Analysis of the Dynamic Reconfiguration of the Brain in Patients with Schizophrenia.

The analysis of resting-state fMRI signals usually focuses on the low-frequency range/band (0.01−0.1 Hz), which does not cover all aspects of brain activity. Studies have shown that distinct frequency bands can capture unique fluctuations in brain activity, with high-frequency signals (>0.1 Hz) providing valuable information for the diagnosis of schizophrenia. We hypothesized that it is meaningful to study the dynamic reconfiguration of schizophrenia through different frequencies. Therefore, this study used resting-state functional magnetic resonance (RS-fMRI) data from 42 schizophrenia and 40 normal controls to investigate dynamic network reconfiguration in multiple frequency bands (0.01−0.25 Hz, 0.01−0.027 Hz, 0.027−0.073 Hz, 0.073−0.198 Hz, 0.198−0.25 Hz). Based on the time-varying dynamic network constructed for each frequency band, we compared the dynamic reconfiguration of schizophrenia and normal controls by calculating the recruitment and integration. The experimental results showed that the differences between schizophrenia and normal controls are observed in the full frequency, which is more significant in slow3. In addition, as visual network, attention network, and default mode network differ a lot from each other, they can show a high degree of connectivity, which indicates that the functional network of schizophrenia is affected by the abnormal brain state in these areas. These shreds of evidence provide a new perspective and promote the current understanding of the characteristics of dynamic brain networks in schizophrenia.

PBK/TOPK Inhibitor Suppresses the Progression of Prolactinomas.

Background: Prolactinoma is the most common type of pituitary tumors, and its resultant tumor occupying and hormone disturbance greatly damage the health of patients. In this study, we investigated a protein kinase-PDZ Binding Kinase (PBK)/T-LAK Cell-Originated Protein Kinase (TOPK) as a candidate protein regulating prolactin (PRL) secretion and tumor growth of prolactinomas. Methods: Downloaded prolactinoma transcriptome dataset from Gene Expression Omnibus (GEO) database, and screened differentially expressed genes (DEGs) between normal pituitary tissues and prolactinoma tissues. Then, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs were performed, a protein-protein interaction (PPI) network was constructed and the hub genes were identified. After a literature search, TOPK was presumed as an candidate target regulating the prolactinoma. We found a specific inhibitor of TOPK to investigate its effects on the proliferation, migration, apoptosis and PRL secretion of pituitary tumor cells. Finally, the regulation of TOPK inhibitor on its downstream target-p38 Mitogen Activated Protein Kinase (p38 MAPK) was detected to explore the potential mechanism. Results: A total of 361 DEGs were identified, and 20 hub genes were screened out. TOPK inhibitor HI-TOPK-032 could suppress the proliferation & migration and induce apoptosis of pituitary tumor cells in vitro, and reduce PRL secretion and tumor growth in vivo. HI-TOPK-032 also inhibited the phosphorylation level of the downstream target p38 MAPK, suggesting that TOPK inhibitors regulate the development of prolactinoma by mediating p38 MAPK. Conclusion: Our study of identification and functional validation of TOPK suggests that this candidate can be a promising molecular target for prolactinoma treatment.

Clinical Characteristics of 195 Cases of COVID-19 with Gastrointestinal Symptoms COVID-19 with Gastrointestinal Symptoms.

BACKGROUND: Patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), have fever, dry cough, dyspnea, and fatigue. The disease has now become a global pandemic. The purpose of this study was to explore the relationship between COVID-19 and gastrointestinal (GI) symptoms. METHODS: We collected and analyzed data on patients with laboratory-confirmed COVID-19 by high-throughput sequencing or reverse transcription-polymerase chain reaction. We reviewed electronic medical records of 405 hospitalized COVID-19 patients in the Third Hospital of Wuhan. RESULTS: Among the 405 confirmed patients, 210 had no GI symptoms, 195 had GI symptoms, and the first symptom of 155 patients was GI. The prevalence of vascular and digestive diseases in the group with GI symptoms was significantly higher than in the group without GI symptoms. In patients with GI symptoms, the proportion with fever, cough, dysphoria, chest tightness, poor appetite, chest pain, and pharyngeal pain was significantly higher than in those without GI symptoms. There was no significant difference in imaging between the 2 groups. In patients with GI symptoms, the proportion with increased procalcitonin (PCT) level and decreased lymphocyte count was significantly higher than in those without GI symptoms. CONCLUSION: COVID-19 patients with GI symptoms had significantly more vascular and digestive system diseases and were more likely to have clinical manifestations of fever, cough, poor appetite, chest tightness, chest pain, insomnia, and pharyngeal pain. There were more patients with diarrhea, nausea, and vomiting. Patients with GI symptoms were more likely to have increased PCT and decreased lymphocyte count.

Retrospective analysis of clinical characteristics of 405 patients with COVID-19.

OBJECTIVE: This study was performed to investigate the clinical characteristics of patients with coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: We analyzed the electronic medical records of 405 hospitalized patients with laboratory-confirmed COVID-19 in the Third Hospital of Wuhan. RESULTS: The patients' median age was 56 years, 54.1% were female, 11.4% had a history of smoking, and 10.6% had a history of drinking. All cases of COVID-19 were community-acquired. Fever (76.8%) and cough (53.3%) were the most common clinical manifestations, and circulatory system diseases were the most common comorbidities. Gastrointestinal symptoms were present in 61.2% of the patients, and 2.9% of the patients were asymptomatic. Computed tomography showed ground-glass opacities in most patients (72.6%) and consolidation in 30.9%. Lymphopenia (72.3%) and hypoproteinemia (71.6%) were observed in most patients. About 20% of patients had abnormal liver function. Patients with severe disease had significantly more prominent laboratory abnormalities, including an abnormal lymphocyte count and abnormal C-reactive protein, procalcitonin, alanine aminotransferase, aspartate aminotransferase, D-dimer, and albumin levels. CONCLUSION: SARS-CoV-2 causes a variety of severe respiratory illnesses similar to those caused by SARS-CoV-1. Older age, chronic comorbidities, and laboratory abnormalities are associated with disease severity.

miR-324-5p Contributes to Cell Proliferation and Apoptosis in Pancreatic Cancer by Targeting KLF3.

Pancreatic cancer cells are characterized by high cell proliferation and low cell apoptosis, but the factors involved in these processes remain to be further studied. In this study, we report that miR-324-5p regulates the proliferation and apoptosis of pancreatic cancer cells through regulating the expression of Krüppel-like factor 3 (KLF3). In both pancreatic cancer tissues and cell lines, the levels of miR-324-5p are significantly increased. Inhibition of miR-324-5p represses cell proliferation but promotes cell apoptosis, whereas overexpression of miR-324-5p exerts the opposite effect. Furthermore, we identified KLF3, a factor regulating pancreatic cancer cell proliferation and apoptosis, as a new direct downstream target of miR-324-5p. Our results suggest that miR-324-5p plays an important role in pancreatic cancer cell proliferation and apoptosis via downregulating the expression of KLF3.

MiR-331-3p Links to Drug Resistance of Pancreatic Cancer Cells by Activating WNT/ß-Catenin Signal via ST7L.

BACKGROUND: Pancreatic cancer is an aggressive type of cancer with poor prognosis, short survival rate, and high mortality. Drug resistance is a major cause of treatment failure in the disease. MiR-331-3p has been reported to play an important role in several cancers. We previously showed that miR-331-3p is upregulated in pancreatic cancer and promotes pancreatic cancer cell proliferation and epithelial-to-mesenchymal transition-mediated metastasis by targeting ST7L. However, it is uncertain whether miR-331-3p is involved in drug resistance. METHODS: We investigated the relationship between miR-331-3p and pancreatic cancer drug resistance. As part of this, microRNA mimics or inhibitors were transfected into pancreatic cancer cells. Quantitative polymerase chain reaction was used to detect miR-331-3p expression, and flow cytometry was used to detect cell apoptosis. The Cell Counting Kit-8 assay was used to measure the IC50 values of gemcitabine in pancreatic cancer cells. The expression of multidrug resistance protein 1, multidrug resistance-related protein 1, breast cancer resistance protein, ß-Catenin, c-Myc, Cyclin D1, Bcl-2, and Caspase-3 was evaluated by Western blotting. RESULTS: We confirmed that miR-331-3p is upregulated in gemcitabine-treated pancreatic cancer cells and plasma from chemotherapy patients. We also confirmed that miR-331-3p inhibition decreased drug resistance by regulating cell apoptosis and multidrug resistance protein 1, multidrug resistance-related protein 1, and breast cancer resistance protein expression in pancreatic cancer cells, whereas miR-331-3p overexpression had the opposite effect. We further demonstrated that miR-331-3p effects in drug resistance were partially reversed by ST7L overexpression. In addition, overexpression of miR-331-3p activated Wnt/ß-catenin signaling in pancreatic cancer cells, and ST7L overexpression restored activation of Wnt/ß-catenin signaling. CONCLUSIONS: Taken together, our data demonstrate that miR-331-3p contributes to drug resistance by activating Wnt/ß-catenin signaling via ST7L in pancreatic cancer cells. These data provide a theoretical basis for new targeted therapies in the future.

PIWIL2 may serve as a prognostic predictor in cancers: A systematic review and meta-analysis.

PURPOSE: PIWIL2, one of the PIWI gene subfamily, is now thought to be closely related to poor clinical outcomes in various cancers. The aim of this research was to comprehensively estimate its predictive value in the prognosis of cancer patients. METHODS: We thoroughly searched PubMed, Web of Science and Embase databases for eligible articles published until April 4th 2019, in which the association between cancer prognosis and PIWIL2 expression level was studied. Study qualities were assessed using NOS criteria. We performed analyses by Stata SE 12.0 and RevMan 5.3. The primary endpoints contained overall survival (OS), cancer-specific survival (CSS), metastasis-free survival (MFS), recurrence-free survival (RFS) and disease-free survival (DFS). RESULTS: Ten studies containing 2116 patients with 8 various solid cancers were finally included. The outcomes indicated that cancer patients with higher PIWIL2 expression level had significant shorter OS (HR:2.20, 95%CI:1.25-3.88, p=0.006), DFS/RFS/MFS (HR:2.96, 95%CI:1.68-5.23, p<0.001), CSS (HR: 2.12, 95%CI: 1.40-3.23, p<0.001) than cancer patients with lower PIWIL2 expression level. What's more, PIWIL2 over-expression was significantly correlated to more lymph node metastasis (LNM) (OR:1.61, 95%CI:1.28-2.02, p<0.001). And PIWIL2 expression was not significantly correlated with age, gender, differentiation, tumor invasion, tumor size, TNM stage and distant metastasis (DM). CONCLUSIONS: A higher expression level of PIWIL2 may predict a poorer prognosis of cancer patients. And its prognostic values are not significantly influenced by clinicopathological characters. Therefore, PIWIL2 could serve as a personalized prognostic predictor in cancers in the future.

Efficient elimination of sulfonamides by an anaerobic/anoxic/oxic-membrane bioreactor process: Performance and influence of redox condition.

Membrane bioreactors (MBRs) were shown contradictory results for the removal of antibiotics, such as sulfonamides (SAs), from wastewater in different studies, which highlighted the necessity for comprehensive investigation on removal mechanisms of sulfonamides in well-controlled lab-scale MBRs. In the present study, the removal performance of nine SAs by a lab-scale anaerobic/anoxic/oxic-membrane bioreactor (A1/A2/O-MBR) was studied at environmental relevant concentrations. The results showed that all the SAs were efficiently eliminated (93.9%-97.5%) in the A1/A2/O-MBR, much more efficiently than the previously reported MBR-based processes. The largest contribution to the total removal was made by the aerobic reactor (71.1%-85.3%) A small portion of SAs (7.1%-22.5%) were removed by anoxic reactor. Activated sludge in the A1/A2/O-MBR was harvested to conduct batch experiments to further study the removal and degradation kinetics of SAs under anaerobic, anoxic and aerobic conditions. The results indicated that only sulfisoxazole could be removed under anaerobic condition. Modest biodegradation of individual SAs (15-33%) was observed under anoxic condition. Under aerobic condition, most investigated SAs underwent an efficient and fast removal (68-77%) in 6h without a lag phase; while sulfisomidine and sulfamethazine were removed less efficiently (approximately 47% after 6h reaction). The aerobic and anoxic degradation of SAs fitted the first-order kinetics model well, and the obtained biodegradation rate constants (k1) were reliable to predict removal efficiencies of SAs in the anoxic and aerobic reactor of A1/A2/O-MBR based on their HRTs.