Incongruence between confirmed and suspected clinical cases of Japanese encephalitis virus infection.

Background: Japanese encephalitis (JE) is a notifiable infectious disease in China. Information on every case of JE is reported to the superior health administration department. However, reported cases include both laboratory-confirmed and clinically diagnosed cases. This study aimed to differentiate between clinical and laboratory-confirmed cases of Japanese encephalitis virus (JEV) infection, and improve the accuracy of reported JE cases by analyzing the acute-phase serum and cerebrospinal fluid of all reported JE cases in the Sichuan province from 2012 to 2022. Methods: All acute-phase serum and/or cerebrospinal fluid samples of the reported JE cases were screened for IgM(ImmunoglobulinM)to JEV using the enzyme-linked immunosorbent assay (ELISA), and the detection of the viral genes of JEV and 9 other pathogens including enterovirus (EV), using reverse transcription PCR was attempted. Epidemiological analyses of JE and non-JE cases based on sex, age, onset time, and geographical distribution were also performed. Results: From 2012 to 2022, 1558 JE cases were reported in the Sichuan province. The results of serological (JEV-specific IgM) and genetic testing for JEV showed that 81% (1262/1558) of the reported cases were confirmed as JEV infection cases (laboratory-confirmed cases). Among the 296 cases of non-JEV infection, 6 viruses were detected in the cerebrospinal fluid in 62 cases, including EV and the Epstein-Barr virus (EBV), constituting 21% (62/296) of all non-JE cases. Among the 62 non-JEV infection cases with confirmed pathogens, infections with EV and EBV included 17 cases each, herpes simplex virus (HSV-1/2) included 14 cases, varicella- zoster virus included 6 cases, mumps virus included 2 cases, and human herpes viruses-6 included 1 case. Additionally, there were five cases involving mixed infections (two cases of EV/EBV, one case of HSV-1/HSV-2, one case of EBV/HSV-1, and one case of EV/herpes viruses-6). The remaining 234 cases were classified as unknown viral encephalitis cases. Our analysis indicated that those aged 0-15 y were the majority of the patients among the 1558 reported JE cases. However, the incidence of laboratory-confirmed JE cases in the >40 y age group has increased in recent years. The temporal distribution of laboratory-confirmed cases of JE revealed that the majority of cases occurred from May to September each year, with the highest incidence in August. Conclusion: The results of this study indicate that there is a certain discrepancy between clinically diagnosed and laboratory-confirmed cases of JE. Each reported case should be based on laboratory detection results, which is of great importance in improving the accuracy of case diagnosis and reducing misreporting. Our results are not only important for addressing JE endemic to the Sichuan province, but also provide a valuable reference for the laboratory detection of various notifiable infectious diseases in China and other regions outside China.

Carbon-reinforced Ni3S2/Ti3C2Tx MXene composite as an anode for superior-performance lithium-ion capacitors.

Lithium ion capacitors (LICs) are a new generation of energy storage devices that combine the super energy storage capability of lithium ion batteries with the satisfactory power density of supercapacitors. The development of high-performance LICs still faces great challenges due to the unbalanced reaction kinetics at the anode and cathode. Therefore, it is an inevitable need to enhance the electron/ion transfer capability of the anode materials. In this paper, to obtain a superior-rate and high-capacity Ni3S2-based anode, highly conductive Ti3C2Tx MXene sheets were introduced to sever as the carrier of Ni3S2 nanoparticles and simultaneously an amorphous carbon layer which coats onto the surface of Ni3S2 nanoparticles was in-situ generated by the carbonization of dopamine reactant. The as-synthesized Ni3S2/Ti3C2Tx/C composite exhibits a high specific surface area (112.6 m2/g) because of the addition of Ti3C2Tx that can reduce the aggregation of Ni3S2 nanoparticles and the in-situ generated amorphous carbon layer that can suppress the growth of Ni3S2 nanoparticles. The Ni3S2/Ti3C2Tx/C anode possesses a remarkable reversible discharge specific capacity (626.0 mAh/g under 0.2 A/g current density), which increases to 1150.8 mAh/g after 400-cycle charge/discharge measurement at the same measurement condition indicating eminent cyclability, along with superior rate capability. To construct a superior-performance LIC device, a sterculiae lychnophorae derived porous carbon (SLPC) cathode with an average discharge specific capacity of 73.4 mAh/g@0.1A/g was prepared. The Ni3S2/Ti3C2Tx/C//SLPC LIC device with optimal cathode/anode mass ratio has a satisfactory energy density ranging from 32.8 to 119.1 Wh kg-1 at the corresponding power density of 8799.4 to 157.5 W kg-1, together with a prominent capacity retention (95.5 %@1 A/g after 10,000 cycles).

LncTarD 2.0: an updated comprehensive database for experimentally-supported functional lncRNA-target regulations in human diseases.

An updated LncTarD 2.0 database provides a comprehensive resource on key lncRNA-target regulations, their influenced functions and lncRNA-mediated regulatory mechanisms in human diseases. LncTarD 2.0 is freely available at (http://bio-bigdata.hrbmu.edu.cn/LncTarD or https://lnctard.bio-database.com/). LncTarD 2.0 was updated with several new features, including (i) an increased number of disease-associated lncRNA entries, where the current release provides 8360 key lncRNA-target regulations, with 419 disease subtypes and 1355 lncRNAs; (ii) predicted 3312 out of 8360 lncRNA-target regulations as potential diagnostic or therapeutic biomarkers in circulating tumor cells (CTCs); (iii) addition of 536 new, experimentally supported lncRNA-target regulations that modulate properties of cancer stem cells; (iv) addition of an experimentally supported clinical application section of 2894 lncRNA-target regulations for potential clinical application. Importantly, LncTarD 2.0 provides RNA-seq/microarray and single-cell web tools for customizable analysis and visualization of lncRNA-target regulations in diseases. RNA-seq/microarray web tool was used to mining lncRNA-target regulations in both disease tissue samples and CTCs blood samples. The single-cell web tools provide single-cell lncRNA-target annotation from the perspectives of pan-cancer analysis and cancer-specific analysis at the single-cell level. LncTarD 2.0 will be a useful resource and mining tool for the investigation of the functions and mechanisms of lncRNA deregulation in human disease.

The dynamic dysregulated network identifies stage-specific markers during lung adenocarcinoma malignant progression and metastasis.

Brain metastasis occurs in approximately 30% of patients with lung adenocarcinoma (LUAD) and is closely associated with poor prognosis, recurrence, and death. However, dynamic gene regulation and molecular mechanism driving LUAD progression remain poorly understood. In this study, we performed a comprehensive single-cell transcriptome analysis using data from normal, early stage, advanced stage, and brain metastasis LUAD. Our single-cell-level analysis reveals the cellular composition heterogeneity at different stages during LUAD progression. We identified stage-specific risk genes that could contribute to LUAD progression and metastasis by reprogramming immune-related and metabolic-related functions. We constructed an early advanced metastatic dysregulated network and revealed the dynamic changes in gene regulations during LUAD progression. We identified 6 early advanced (HLA-DRB1, HLA-DQB1, SFTPB, SFTPC, PLA2G1B, and FOLR1), 8 advanced metastasis (RPS15, RPS11, RPL13A, RPS24, HLA-DRB5, LYPLA1, KCNJ15, and PSMA3), and 2 common risk genes in different stages (SFTPD and HLA-DRA) as prognostic markers in LUAD. Particularly, decreased expression of HLA-DRA, HLA-DRB1, HLA-DQB1, and HLA-DRB5 refer poor prognosis in LUAD by controlling antigen processing and presentation and T cell activation. Increased expression of PSMA3 and LYPLA1 refer poor prognosis by reprogramming fatty acid metabolism and RNA catabolic process. Our findings will help further understanding the pathobiology of brain metastases in LUAD.

Identifying enhancer-driven subtype-specific prognostic markers in breast cancer based on multi-omics data.

Breast cancer is a cancer of high complexity and heterogeneity, with differences in prognosis and survival among patients of different subtypes. Copy number variations (CNVs) within enhancers are crucial drivers of tumorigenesis by influencing expression of their targets. In this study, we performed an integrative approach to identify CNA-driven enhancers and their effect on expression of target genes in four breast cancer subtypes by integrating expression data, copy number data and H3K27ac data. We identified 672, 555, 531, 361 CNA-driven enhancer-gene pairs and 280, 189, 113 and 98 CNA-driven enhancer-lncRNA pairs in the Basal-like, Her2, LumA and LumB subtypes, respectively. We then reconstructed a CNV-driven enhancer-lncRNA-mRNA regulatory network in each subtype. Functional analysis showed CNA-driven enhancers play an important role in the progression of breast cancer subtypes by influencing P53 signaling pathway, PPAR signaling pathway, systemic lupus erythematosus and MAPK signaling pathway in the Basal-like, Her2, LumA and LumB subtypes, respectively. We characterized the potentially prognostic value of target genes of CNV-driven enhancer and lncRNA-mRNA pairs in the subtype-specific network. We identified MUM1 and AC016876.1 as prognostic biomarkers in LumA and Basal-like subtypes, respectively. Higher expression of MUM1 with an amplified enhancer exhibited poorer prognosis in LumA patients. Lower expression of AC016876.1 with a deleted enhancer exhibited poorer survival outcomes of Basal-like patients. We also identified enhancer-related lncRNA-mRNA pairs as prognostic biomarkers, including AC012313.2-MUM1 in the LumA, AC026471.4-PLK5 in the LumB, AC027307.2-OAZ1 in the Basal-like and AC022431.1-HCN2 in the Her2 subtypes. Finally, our results highlighted target genes of CNA-driven enhancers and enhancer-related lncRNA-mRNA pairs could act as prognostic markers and potential therapeutic targets in breast cancer subtypes.

Association of admission hyperglycemia and all-cause mortality in acute myocardial infarction with percutaneous coronary intervention: A dose-response meta-analysis.

Objective: The aim of this study is to evaluate the associations between admission hyperglycemia and the risk of all-cause mortality in patients with acute myocardial infarction (AMI) with or without diabetes, to find optimal admission glucose intervention cut-offs, and to clarify the shape of the dose-response relations. Methods: Medline/PubMed and EMBASE were searched from inception to 1 April 2022. Cohort studies reporting estimates of all-cause mortality risk in patients with admission hyperglycemia with AMI were included. The outcomes of interest include mortality and major adverse cardiac events (MACEs). A random effect dose-response meta-analysis was conducted to access linear trend estimations. A one-stage linear mixed effect meta-analysis was used for estimating dose-response curves. Relative risks and 95% confidence intervals were pooled using a random-effects model. Results: Of 1,222 studies screened, 47 full texts were fully reviewed for eligibility. The final analyses consisted of 23 cohort studies with 47,177 participants. In short-term follow-up, admission hyperglycemia was associated with an increased risk of all-cause mortality (relative risk: 3.12, 95% confidence interval 2.42-4.02) and MACEs (2.34, 1.77-3.09). In long-term follow-up, admission hyperglycemia was associated with an increased risk of all-cause mortality (1.97, 1.61-2.41) and MACEs (1.95, 1.21-3.14). A linear dose-response association was found between admission hyperglycemia and the risk of all-cause mortality in patients with or without diabetes. Conclusion: Admission hyperglycemia was significantly associated with higher all-cause mortality risk and rates of MACEs. However, the association between admission hyperglycemia and long-term mortality risk needs to be determined with caution. Compared with current guidelines recommendations, a lower intervention cut-off and more stringent targets for admission hyperglycemia may be appropriate. Systematic review registration: [https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022317280], identifier [CRD42022317280].

Comprehensive characterization of clonality of driver genes revealing their clinical relevance in colorectal cancer.

BACKGROUND: Genomic studies of colorectal cancer have revealed the complex genomic heterogeneity of the tumor. The acquisition and selection of genomic alterations may be critical to understanding the initiation and progression of this disease. METHODS: In this study, we have systematically characterized the clonal architecture of 97 driver genes in 536 colorectal cancer patients from TCGA. RESULTS: A high proportion of clonal mutations in 93 driver genes were observed. 40 genes showed significant associations between their clonality and multiple clinicopathologic factors. Kaplan-Meier analysis suggested that the mutation clonality of ANK1, CASP8, SMAD2, and ARID1A had a significant impact on the CRC patients' outcomes. Multivariable analysis revealed that subclonal ANK1 mutations, clonal CASP8 mutations, and clonal SMAD2 mutations independently predicted for shorter overall survival after adjusting for clinicopathological factors. The poor outcome of the subclonal ANK1 mutation may be caused by upregulation of IL4I1, IDO1, IFNG and MAPK12 which showed potential roles in tumor immune evasion through accumulation of immunosuppressive cells such as regulatory T cells and myeloid derived suppressor cells. CONCLUSION: These results suggested that the clonality of driver genes could act as prognostic markers and potential therapeutic targets in human colorectal cancer.

Laparoscopic-assisted endoscopic full-thickness resection of a large gastric schwannoma: A case report.

BACKGROUND: Schwannomas, also known as neurinomas, are benign tumors derived from Schwann cells. Gastrointestinal schwannomas are rare and are most frequently reported in the stomach. They are usually asymptomatic and are difficult to diagnose preoperatively; however, endoscopy and imaging modalities can provide beneficial preliminary diagnostic data. There are various surgical options for management. Here, we present a case of a large gastric schwannoma (GS) managed by combined laparoscopic and endoscopic surgery. CASE SUMMARY: A 28-year-old woman presented with a 2-mo history of epigastric discomfort and a feeling of abdominal fullness. On upper gastrointestinal endoscopy and endoscopic ultrasonography, a hypoechogenic submucosal mass was detected in the gastric antrum: It emerged from the muscularis propria and projected intraluminally. Computed tomography showed a nodular lesion (4 cm × 3.5 cm), which exhibited uniform enhancement, on the gastric antrum wall. Based on these findings, a preliminary diagnosis of gastrointestinal stromal tumor was established, with schwannoma as a differential. Considering the large tumor size, we planned to perform endoscopic resection and to convert to laparoscopic treatment, if necessary. Eventually, the patient underwent combined laparoscopic and gastroscopic surgery. Immunohistochemically, the resected specimen showed positivity for S-100 and negativity for desmin, DOG-1, α-smooth muscle actin, CD34, CD117, and p53. The Ki-67 index was 3%, and a final diagnosis of GS was established. CONCLUSION: Combined laparoscopic and endoscopic surgery is a minimally invasive and effective treatment option for large GSs.

Functional regulations between genetic alteration-driven genes and drug target genes acting as prognostic biomarkers in breast cancer.

Differences in genetic molecular features including mutation, copy number alterations and DNA methylation, can explain interindividual variability in response to anti-cancer drugs in cancer patients. However, identifying genetic alteration-driven genes and characterizing their functional mechanisms in different cancer types are still major challenges for cancer studies. Here, we systematically identified functional regulations between genetic alteration-driven genes and drug target genes and their potential prognostic roles in breast cancer. We identified two mutation and copy number-driven gene pairs (PARP1-ACSL1 and PARP1-SRD5A3), three DNA methylation-driven gene pairs (PRLR-CDKN1C, PRLR-PODXL2 and PRLR-SRD5A3), six gene pairs between mutation-driven genes and drug target genes (SLC19A1-SLC47A2, SLC19A1-SRD5A3, AKR1C3-SLC19A1, ABCB1-SRD5A3, NR3C2-SRD5A3 and AKR1C3-SRD5A3), and four copy number-driven gene pairs (ADIPOR2-SRD5A3, CASP12-SRD5A3, SLC39A11-SRD5A3 and GALNT2-SRD5A3) that all served as prognostic biomarkers of breast cancer. In particular, RARP1 was found to be upregulated by simultaneous copy number amplification and gene mutation. Copy number deletion and downregulated expression of ACSL1 and upregulation of SRD5A3 both were observed in breast cancers. Moreover, copy number deletion of ACSL1 was associated with increased resistance to PARP inhibitors. PARP1-ACSL1 pair significantly correlated with poor overall survival in breast cancer owing to the suppression of the MAPK, mTOR and NF-kB signaling pathways, which induces apoptosis, autophagy and prevents inflammatory processes. Loss of SRD5A3 expression was also associated with increased sensitivity to PARP inhibitors. The PARP1-SRD5A3 pair significantly correlated with poor overall survival in breast cancer through regulating androgen receptors to induce cell proliferation. These results demonstrate that genetic alteration-driven gene pairs might serve as potential biomarkers for the prognosis of breast cancer and facilitate the identification of combination therapeutic targets for breast cancers.

Improved process intermediate stability through the identification and elimination of reactive glycation residues - a monoclonal antibody case study.

The manufacturing of therapeutic biologics can result in a heterogeneous population of charge variants, encompassing many quality attributes which could impact activity and pharmacokinetics. Monitoring the relative abundance of these charge variants to demonstrate process consistency is an expectation of regulatory agencies. Control of the relative abundance of charge variants is also necessary to ensure product comparability across the product lifecycle. We have observed a significant shift in the relative abundance of charged species, as measured by capillary isoelectric focusing, during clarified cell culture fluid holds for several monoclonal antibodies. This lack of stability requires that the hold time for this process intermediate be significantly curtailed, eliminating manufacturing flexibility. We have identified the cause of this shift in relative abundance of charged species as changes in glycation levels, focused predominantly on three conserved, solvent accessible, lysine residues. Mutants of a model protein were generated that show increased charge state stability can be gained by eliminating these reactive lysines. Further, characterization studies were conducted on these mutants to determine the impact to biological activity and stability of the molecule, with no detrimental effects observed. Incorporating this knowledge into the assessments of candidate drugs could allow for the selection of molecules less susceptible to this product degradation pathway, allowing for greater manufacturing flexibility. This process of identifying and removing reactive lysine residues could be useful in the design of drug candidates with improved charge state stability, across a range of modalities.

Characterization of N-Terminal Glutamate Cyclization in Monoclonal Antibody and Bispecific Antibody Using Charge Heterogeneity Assays and Hydrophobic Interaction Chromatography.

N-terminal glutamate (E) cyclization to form pyroglutamate (pE) generates charge heterogeneities for mAbs and proteins. Thus far, pE formation rate in lyophilized formulation as compared to in liquid formulation has not been reported. Impact of pE on antibody biological activity has only been predicted or assessed using stressed samples that may contain other confounding degradations besides pE. Additionally, application of hydrophobic interaction chromatography (HIC) to separate pE has not been reported. In our study, N-terminal E cyclization was identified as the major degradation pathway in lyophilized formulation at elevated temperature for both monoclonal antibody (mAb-A) and IgG-like bispecific antibody (bsAb-A). pE was enriched in salt-gradient ion exchange chromatography (IEC) as pre-peak and in HIC as post-peak for both mAb-A and bsAb-A. Structure-function studies with pE-enriched IEC and HIC fractions confirmed that pE did not affect binding activities for mAb-A and bsAb-A. In vitro incubation of bsAb-A in serum and PBS revealed that the serum matrix may play a role in pE conversion in human serum, in contrast to the chemical reaction mechanism reported. These techniques can help in characterization of N-terminal E-to-pE cyclization and quality attribute severity assessment during therapeutic protein product development.

Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells.

Bispecific antibodies (BsAbs) or fusion proteins (BsAbFPs) present a promising strategy for cancer immunotherapy. Numerous BsAbs targeting coinhibitory and costimulatory pathways have been developed for retargeting T cells and antigen presenting cells (APCs). It is challenging to assess the potency of BsAb that engages two different signaling pathways simultaneously in a single assay format, especially when the two antigen targets are expressed on different cells. To explore the potency of anti-PD-L1/CD40L BsAbFP, a fusion protein that binds to human CD40 and PD-L1, we engineered CHO cells as surrogate APCs that express T cell receptor activator and PD-L1, Jurkat cells with PD-1 and NFAT-luciferase reporter as effector T cells, and Raji cell with NFkB-luciferase that endogenously expresses CD40 as accessory B cells. A novel reporter gene bioassay was developed using these cell lines that allows anti-PD-L1/CD40L BsAbFP to engages both PD-1/PD-L1 and CD40/CD40L signaling pathways in one assay. As both reporters use firefly luciferase, the effects of activating both signaling pathways is observed as an increase in luminescence, either as a higher upper asymptote, a lower EC50, or both. This dual target reporter gene bioassay system reflects potential mechanism of action and demonstrated the ability of anti-PD-L1/CD40L BsAbFP to synergistically induce biological response compared to the combination of anti-PD-L1 monovalent monoclonal antibody and agonist CD40L fusion protein, or either treatment alone. The results also showed a strong correlation between the drug dose and biological response within the tested potency range with good linearity, accuracy, precision, specificity and stability indicating properties, suggesting that this "three-cell-in-one" dual target reporter gene bioassay is suitable for assessing potency, structure-function and critical quality attributes of anti-PD-L1/CD40L BsAbFP. This approach could be used for developing dual target bioassays for other BsAbs and antibodies used for combination therapy.

A single homogeneous assay for simultaneous measurement of bispecific antibody target binding.

Bispecific antibodies (BsAbs) are engineered to simultaneously bind two different antigens, and offer promising clinical outcomes for various diseases. The dual binding properties of BsAbs may enable superior efficacies and/or potencies compared to standard monoclonal antibodies (mAbs) or combination mAb therapies. Characterizing BsAb binding properties is critical during biotherapeutic development, where data is leveraged to predict efficacy and potency, assess critical quality attributes and improve antibody design. Traditional single-target, single-readout approaches (e.g., ELISA) have limited usefulness for interpreting complex bispecific binding, and double the benchwork. To address these deficiencies, we developed and implemented a new dual-target/readout binding assay that accurately dissects the affinities of both BsAb binding domains directly and simultaneously. This new assay uses AlphaPlex® technology, which eliminates traditional ELISA wash steps and can be miniaturized for automated workflows. The optimized BsAb AlphaPlex assay demonstrates 99-107% accuracy within a 50-150% linear range, and detected >50% binding degradation from photo- and thermal stress conditions. To the best of our knowledge, this is the first instance of a dual-target/readout BsAb AlphaPlex assay with GMP-suitable linear range, accuracy, specificity, and stability-indicating properties. As a highly customizable and efficient assay, BsAb AlphaPlex may be applicable to numerous bispecific formats and/or co-formulations against a variety of antigens beyond the clinical therapeutic space.

Development of a mechanism of action-reflective, dual target cell-based reporter bioassay for a bispecific monoclonal antibody targeting human CTLA-4 and PD-1.

T-cell-mediated immunotherapy has generated much excitement after the success of therapeutic biologics targeting immune checkpoint molecules. Bispecific antibodies (BsAbs) that recognize two antigen targets are a fast-growing class of biologics offering promising clinical benefits for cancer immunotherapy. Due to the complexity of the molecule structure and the potential mechanism of action (MOA) that involves more than one signaling pathway, it is critical to develop appropriate bioassays for measuring potency and characterizing the biological properties of BsAbs. Here, we present a dual target, cell-based reporter bioassay for a BsAb that binds human CTLA-4 and PD-1 and targets two subsequent signaling pathways that negatively regulate T-cell activation. This reporter bioassay is capable of measuring the potency of both antigen target arms in one assay, which would not be achievable using two single target bioassays. This dual target reporter bioassay demonstrates good performance characteristics suitable for lot release, stability testing, critical quality attribute assessment, and biological properties characterization of the CTLA-4/PD-1 BsAb. Furthermore, this assay can capture the synergistic effect of anti-CTLA-4 and anti-PD-1 activity of the BsAb. Compared to single target assays, this dual target bioassay could better reflect the potential MOA of BsAbs and could be used for evaluation of other bispecific biologics, as well as antibody combination therapies.

Comprehensive landscape of epigenetic-dysregulated lncRNAs reveals a profound role of enhancers in carcinogenesis in BC subtypes.

Aberrant expression of long non-coding RNAs (lncRNA) is associated with altered DNA methylation and histone modifications during carcinogenesis. However, identifying epigenetically dysregulated lncRNAs and characterizing their functional mechanisms in different cancer subtypes are still major challenges for cancer studies. In this study, we systematically analyzed the epigenetic alterations of lncRNAs at important regulatory elements in three breast cancer subtypes. We identified 87, 691, and 1,197 epigenetically dysregulated lncRNAs in luminal, basal, and claudin-low subtypes of breast cancer, respectively. The landscape of epigenetically dysregulated lncRNAs at enhancer elements revealed that epigenetic changes of the majority of lncRNAs occurred in a subtype-specific manner and contributed to subtype-specific biological functions. We identified six acetylation of lysine 27 on histone H3 (H3K27ac)-dysregulated lncRNAs and three DNA methylation-dysregulated lncRNAs (CTC-303L1.2, RP11-738B7.1, and SLC26A4-AS1) as prognostic biomarkers of basal subtype. These lncRNAs were involved in immune response-related biological functions. Treatment of the basal breast cancer cell line MDA-MB-468 with CREBBP/EP300 bromodomain inhibitors downregulated H3K27 acetylation levels and caused a decrease in the expression of five H3K27ac-dysregulated lncRNAs (LINC00393, KB-1836B5.1, RP1-140K8.5, AC005162.1, and AC020916.2) and inhibition of the growth of breast cancer cells. One epigenetically dysregulated lncRNA (LINC01983) and four lncRNA regulators (UCA1, RP11-221J22.2, RP11-221J22.1, and RP1-212P9.3) were identified as prognostic biomarkers of the luminal molecular subtype of breast cancer by controlling the tumor necrosis factor (TNF) signaling pathway, T helper (Th)17 cell differentiation, and T cell migration. Finally, our results highlighted a profound role of enhancer-related H3K27ac-dysregulated lncRNAs, DNA methylation-dysregulated lncRNAs, and lncRNA regulators in breast cancer subtype carcinogenesis and their potential prognostic value.

Comparative physiological and transcriptomic analysis of pear leaves under distinct training systems.

Canopy architecture is critical in determining the light interception and distribution, and subsequently the photosynthetic efficiency and productivity. However, the physiological responses and molecular mechanisms by which pear canopy architectural traits impact on photosynthesis remain poorly understood. Here, physiological investigations coupled with comparative transcriptomic analyses were performed in pear leaves under distinct training systems. Compared with traditional freestanding system, flat-type trellis system (DP) showed higher net photosynthetic rate (PN) levels at the most time points throughout the entire monitored period, especially for the interior of the canopy in sunny side. Gene ontology analysis revealed that photosynthesis, carbohydrate derivative catabolic process and fatty acid metabolic process were over-represented in leaves of DP system with open-canopy characteristics. Weighted gene co-expression network analysis uncovered a significant network module positive correlated with PN value. The hub genes (PpFKF1 and PpPRR5) of the module were enriched in circadian rhythm pathway, suggesting a functional role for circadian clock genes in mediating photosynthetic performance under distinct training systems. These results draw a link between pear photosynthetic response and specific canopy architectural traits, and highlight light harvesting and circadian clock network as potential targets for the input signals from the fluctuating light availability under distinct training systems.

Prevention of Fab-arm exchange and antibody reduction via stabilization of the IgG4 hinge region.

IgG4s are dynamic molecules that undergo a process called Fab-arm exchange. Disulfide bonds between heavy chains are transiently reduced, resulting in half antibodies that reform intact antibodies with other IgG4 half antibodies. In vivo, therapeutic IgG4s can recombine with endogenous IgG4s, resulting in a heterogeneous mixture of bispecific antibodies. A related issue that can occur for any therapeutic protein during manufacturing is interchain disulfide bond reduction. For IgG4s, this primarily results in high levels of half-mAb that persist through purification processes. The S228P mutation has been used to prevent half-mAb formation. However, we demonstrated that IgG4s with the S228P mutation are subject to half-mAb formation and Fab-arm exchange in reducing environments. We identified two novel mutations that stabilize the heavy-heavy chain interaction via incorporation of additional disulfide bonds in the hinge region. Individually, these mutations increase stability toward reduction and lessen Fab-arm exchange. Combination of all three mutations, Y219C, G220C, and S228P, has an additive benefit resulting in an IgG4 with ˃7-fold increase in stability toward reduction while preventing Fab-arm exchange. Importantly, the mutations do not affect antigen binding or Fc effector function. These mutations hold great promise for solving mAb reduction during manufacturing and preventing Fab-arm exchange in vivo.

Deamidation in Moxetumomab Pasudotox Leading to Conformational Change and Immunotoxin Activity Loss.

Asparagine (Asn) deamidation is a common posttranslational modification in which Asn is converted to aspartic acid or isoaspartic acid. By introducing a negative charge, deamidation could potentially impact the binding interface and biological activities of protein therapeutics. We identified a deamidation variant in moxetumomab pasudotox, an immunotoxin Fv fusion protein drug derived from a 38-kDa truncated Pseudomonas exotoxin A (PE38) for the treatment of hairy-cell leukemia. Although the deamidation site, Asn-358, was outside of the binding interface, the modification had a significant impact on the biological activity of moxetumomab pasudotox. Surprisingly, the variant eluted earlier than its unmodified form on anion exchange chromatography, which often leads to the conclusion that it has a higher positive charge. Here we describe the characterization of the deamidation variant with differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry, which revealed that the Asn-358 deamidation caused the conformational changes in the catalytic domain of the PE38 region. These results provide an explanation for why the deamidation affected the biological activity of moxetumomab pasudotox and suggest the approach that can be used for process control to ensure product quality and process consistency.

Identifying functions and prognostic biomarkers of network motifs marked by diverse chromatin states in human cell lines.

Epigenetic modifications play critical roles in modulating gene expression, yet their roles in regulatory networks in human cell lines remain poorly characterized. We integrated multiomics data to construct directed regulatory networks with nodes and edges labeled with chromatin states in human cell lines. We observed extensive association of diverse chromatin states and network motifs. The gene expression analysis showed that diverse chromatin states of coherent type-1 feedforward loop (C1-FFL) and incoherent type-1 feedforward loops (I1-FFL) contributed to the dynamic expression patterns of targets. Notably, diverse chromatin state compositions could help C1- or I1-FFL to control a large number of distinct biological functions in human cell lines, such as four different types of chromatin state compositions cooperating with K562-associated C1-FFLs controlling "regulation of cytokinesis," "G1/S transition of mitotic cell cycle," "DNA recombination," and "telomere maintenance," respectively. Remarkably, we identified six chromatin state-marked C1-FFL instances (HCFC1-NFYA-ABL1, THAP1-USF1-BRCA2, ZNF263-USF1-UBA52, MYC-ATF1-UBA52, ELK1-EGR1-CCT4, and YY1-EGR1-INO80C) could act as prognostic biomarkers of acute myelogenous leukemia though influencing cancer-related biological functions, such as cell proliferation, telomere maintenance, and DNA recombination. Our results will provide novel insight for better understanding of chromatin state-mediated gene regulation and facilitate the identification of novel diagnostic and therapeutic biomarkers of human cancers.