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Mitogen-activated protein kinase kinase kinase 3 (MEKK3) is an evolutionarily conserved Ser/Thr protein kinase of the MEKK family that is essential for the host immune response to pathogen challenges in mammals. However, the immune function of MEKK3s in lower vertebrate species, especially in bony fish, remains largely unknown. In this study, a fish MEKK3 (designated CiMEKK3) gene was cloned and identified from grass carp (Ctenopharyngodon idella). The present CiMEKK3 cDNA encoded a 620 amino acid polypeptide containing a conserved S-TKc domain and a typical PB1 domain. Several potential immune-related transcription factor-binding sites, including activating protein 1 (AP-1), nuclear factor kappa B (NF-κB) and signal transducer and activator of downstream transcription 3 (STAT3), were observed in the 5’ upstream DNA sequence of CiMEKK3. A phylogenetic tree showed that CiMEKK3 exhibits a close evolutionary relationship with MEKK3s from Cyprinus carpio and Carassius auratus. Quantitative real-time PCR analysis revealed that CiMEKK3 transcripts were widely distributed in all selected tissues of healthy grass carp, with a relatively high levels observed in the gill, head kidney and intestine. Upon in vitro challenge with bacterial pathogens (Aeromonas hydrophila and Aeromonas veronii) and pathogen-associated molecular patterns (PAMPs) (lipopolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-mDAP (Tri-DAP) and muramyl dipeptide (MDP)), the expression levels of CiMEKK3 in the intestinal cells of grass carp were shown to be significantly upregulated in a time-dependent manner. In vivo injection experiments revealed that CiMEKK3 transcripts were significantly induced by MDP challenge in the intestine; however, these effects could be inhibited by the nutritional dipeptides carnosine and Ala-Gln. Moreover, subcellular localization analysis and luciferase reporter assays indicated that CiMEKK3 could act as a cytoplasmic signal-transducing activator involved in the regulation of NF-κB and MAPK/AP-1 signaling cascades in HEK293T cells. Taken together, these findings strongly suggest that CiMEKK3 plays vital roles in the intestinal immune response to bacterial challenges, which will aid in understanding the pathogenesis of inflammatory bowel disease in bony fish.

Myeloid differentiation factor 88 (MyD88) is a key adapter molecule in Toll-like receptor signal transduction that triggers downstream immune cascades involved in the host defense response to exogenous pathogens. However, the function of MyD88s in mollusks, especially in freshwater shellfish, remains poorly understood. In this study, a novel freshwater shellfish MyD88 (denoted AwMyD88) was characterized from Anodonta woodiana. The present AwMyD88 protein consists of 474 amino acids and contains a conserved a typical death domain (DD) and a conservative Toll/IL-1R (TIR) domain with three typical boxes. Quantitative real-time PCR (qRT-PCR) analysis showed that AwMyD88 was broadly expressed in all the examined tissues, and the highest expression level was observed in hemocytes of A. woodiana. When challenged with Aeromonas hydrophila and lipopolysaccharide (LPS), the mRNA expression levels of AwMyD88 were significantly induced in hemocytes of A. woodiana in vivo and in vitro. In addition, in vivo injection experiments revealed that MyD88 signaling pathway genes showed strong responsiveness to A. hydrophila challenge, and their expression levels were significantly upregulated in hemocytes. Knockdown of AwMyD88 reduced the transcript levels of immune related transcription factors (AwNF-κB and AwAP-1) and effectors (AwTNF, AwLYZ, AwDefense and AwAIF) during A. hydrophila infection. Moreover, subcellular localization analysis indicated that AwMyD88 was mainly localized to the cytoplasm in HEK293T cells. Finally, luciferase reporter assays revealed that AwMyD88 associates with AwTLR to activate the NF-κB and AP-1 signaling pathways in HEK293T cells. These results suggested that AwMyD88 might be involved in the host defense response to bacterial challenge, providing new insight into the immune function of the MyD88 signaling pathway in freshwater shellfish.

Coronavirus disease 2019 (COVID-19) is an acute respiratory infection caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). However, there are still no specific treatments. Recently, traditional Chinese medicine (TCM) was proposed as a potential therapeutic strategy for the prevention and treatment of COVID-19. The present study aims to explore the potential of Hypericum japonicum Thunb. ex Murray against COVID-19 based on network pharmacology and molecular docking methods. Potential targets of H. japonicum were collected from available databases for protein–protein interaction analysis. Further GO and KEGG pathway analysis were performed to reveal the corresponding mechanism. Molecular docking was employed to evaluate the binding energy between the components in H. japonicum and three target proteins (ACE2, Mpro and PLP). Finally, a total of 45 core targets were screened. GO and KEGG pathway analysis revealed that several key pathways were involved in the potential beneficial effects of H. japonicum against COVID-19. Molecular docking results showed that most components in H. japonicum have a binding affinity to ACE2, Mpro and PLP molecules less than −5 kcal/mol, indicating good binding capacity to these target proteins. Vincetoxicoside B, bijaponicaxanthone and quercetin possess the top binding activity to ACE2, Mpro and PLP, respectively. The molecular docking results and the traditionally known possible functions of H. japonicum, including stabilizing blood oxygen environment, preventing inflammatory storms, regulating immune response and inhibiting viral infections, hereby indicate the potential of H. japonicum against COVID-19.

Background Accurate detection of anaplastic lymphoma kinase (ALK) rearrangement is the prerequisite for anti‐ALK therapy for the patient with non‐small cell lung cancer (NSCLC). Fusion‐induced asymmetric transcription assay (FIATA)‐based reverse transcription droplet digital PCR (RT‐ddPCR) was developed and performed for ALK status survey in NSCLC samples. Methods A total of 269 cases of formalin‐fixed paraffin‐embedded (FFPE) specimens from NSCLC, in which ALK status was confirmed by both fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), were analyzed by FIATA‐based RT‐ddPCR. Results In the ALK‐positive group, the 3′ ALK transcript copies range was 336.6–107 955.4, and the R3 [(the ratio of the 3′ ALK transcript copy numbers to the internal reference gene transcript copy numbers) × 100] was 17.23–672.77. In the ALK‐negative group, the 3′ ALK transcript copies range was 3.7–1370.6, and the R3 range was 0.10–15.57. The lowest R3 level in the ALK‐positive group was significantly higher than the highest R3 level in the ALK‐negative group. A positive correlation between the proportion of cancer cells in the tissue section and ALK RNA expression level (R3) was found (P

Abstract Background Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating rice disease in Southeast Asia and West Africa. OsSWEET14, encoding a sugar transporter, is known to be a major susceptible gene of bacterial blight targeted by four different transcription activator-like (TAL) effectors from either Asian or African Xoo strains. However, the OsSWEET14 single knockout or promoter mutants in the Kitaake background are moderately resistant or even susceptible to African Xoo strains. Therefore, in this study, we knocked out OsSWEET14 in rice cv. Zhonghua 11 background for disease assessment. Results In this study, CRISPR/Cas9 was utilized to disrupt the function of OsSWEET14 by modifying its corresponding coding region in the genome of rice cv. Zhonghua 11 (CR-S14). In total, we obtained nine different OsSWEET14-mutant alleles. Besides conferring broad-spectrum resistance to Asian Xoo strains, tested mutant alleles also showed strong resistance to African Xoo strain AXO1947. Moreover, the expression of OsSWEET14 was detected in vascular tissues, including the stem, leaf sheath, leaf blade and root. The disruption of OsSWEET14 led to increased plant height without a reduction in yield. Conclusions Disruption of OsSWEET14 in the Zhonghua 11 background is able to confer strong resistance to African Xoo strain AXO1947 and Asian Xoo strain PXO86. CR-S14 has normal reproductive growth and enhanced plant height under normal growth conditions. These results imply that CR-S14 may serve as a better tester line than sweet14 single-knockout mutant in the Kitaake background for the diagnostic kit for rice blight resistance. The genetic background and increased plant height need to be taken into consideration when utilizing OsSWEET14 for resistant rice breeding.

Immune checkpoint blockade (ICB) immunotherapy efficacy has still limitations. Here, the authors generate a vaccine that integrates CD274 siRNA into the L1 protein of human papillomavirus, which cooperates with ICB by activating innate immunity in breast cancer models.

Abstract Objectives Non-Hodgkin’s lymphoma (NHL) primarily derived from the base of the tongue, is rare. Human papillomavirus (HPV) and Epstein-Barr virus (EBV) are important aetiological risk factors for tumours of the head and neck. This study describes the clinicopathological features of NHL in the tongue base and the status of HPV and EBV in these cases. Methods Seven cases were identified from the Pathological Registry Database at Peking Union Medical College Hospital (PUMCH). The study utilized immunochemistry, in situ hybridization (ISH), and gene rearrangement to confirm the disease and and performed a clinical follow up for each case. Results All 7 lymphomas were localized at the base of the tongue. Six of the cases exhibited tongue base masses with smooth surface membranes. One case presented as multiple deep ulcers. The most common histologic subtype was diffuse large B-cell lymphoma (DLBCL), which occurred in five cases. The other two cases were mantle cell lymphoma (MCL) and peripheral T cell lymphoma, not otherwise specified (PTCL, NOS). One of the DLBCL cases was positive for HPV DNA and diffusely expressed P16 protein. During the follow up period, the MCL patient and an elderly DLBCL patient died. The remaining five patients were alive through the end of follow up. Conclusions Most lymphomas of the tongue base manifest as an endogenous mass without membranous change. The most common subtype of NHLs of the tongue base is DLBCL, and the occurrence at this site may have a good prognosis. With proper therapy, even late stage tongue base lymphomas can be suppressed and remain in remission.

Naoxintong Capsule (NXT), a Chinese medicine, has been widely used for the treatment of cardiovascular diseases in clinic. The present study was designed to clarify the effects of NXT on haemorheology, blood coagulation, hepatic function, antioxidant capacity and inflammatory responses in a rat model of blood stasis syndrome. Rats were placed in ice-cold water for 5 min during the time interval of two adrenaline injections to induce blood circulation disorders after intragastric administration of 10 consecutive days. Blood was collected from abdominal aorta to determine whole blood viscosity (WBV), plasma viscosity (PV), activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen (FIB), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), malonaldehyde (MDA), alanine transaminase (ALT), aspartate transaminase (AST), interleukin-1β (IL-1β), interleukin-6 (IL-6), platelet activating factor (PAF). The results showed that NXT could significantly decrease the WBV, PV, FIB and prolong the APTT and PT. Meanwhile, NXT significantly decreased the serum contents of MDA, IL-1β, IL-6, ALT, AST and PAF, while elevating T-AOC and SOD levels. These results indicate that NXT could improve coagulation function, enhance antioxidant capacity and suppress inflammation, which might partly account for its mechanisms of action in preventing and treating cardiovascular diseases.

Early life stress is thought to be a risk factor for emotional disorders, particularly depression and anxiety. Although the excitation/inhibition (E/I) imbalance has been implicated in neuropsychiatric disorders, whether early life stress affects the E/I balance in the medial prefrontal cortex at various developmental stages is unclear. In this study, rats exposed to maternal separation (MS) that exhibited a well-established early life stress paradigm were used to evaluate the E/I balance in adolescence (postnatal day P43–60) and adulthood (P82–100) by behavior tests, whole-cell recordings, and microdialysis coupled with high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. First, the behavioral tests revealed that MS induced both anxiety- and depressive-like behaviors in adolescent rats but only depressive-like behavior in adult rats. Second, MS increased the action potential frequency and E/I balance of synaptic transmission onto L5 pyramidal neurons in the prelimbic (PrL) brain region of adolescent rats while decreasing the action potential frequency and E/I balance in adult rats. Finally, MS increases extracellular glutamate levels and decreased the paired-pulse ratio of evoked excitatory postsynaptic currents (EPSCs) of pyramidal neurons in the PrL of adolescent rats. In contrast, MS decreased extracellular glutamate levels and increased the paired-pulse ratio of evoked EPSCs of pyramidal neurons in the PrL of adult rats. The present results reveal a key role of E/I balance in different MS-induced disorders may related to the altered probability of presynaptic glutamate release at different developmental stages.

Purpose. Pancreatic cancer (PC) is a common, highly lethal cancer with a low survival rate. Autophagy is involved in the occurrence and progression of PC. This study aims to explore the feasibility of using an autophagy-related long noncoding RNA (lncRNA) signature for assessing PC patient survival. Methods. We obtained RNA sequencing and clinical data of patients from the TCGA website. Autophagy genes were obtained from the Human Autophagy Database. The prognostic model, generated through univariate and multivariate Cox regression analyses, included 10 autophagy-related lncRNAs. Receiver operating characteristic (ROC) curves and forest plots were generated for univariate and multivariate Cox regression analyses, to examine the predictive feasibility of the risk model. Gene set enrichment analysis (GSEA) was used to screen enriched gene sets. Results. Twenty-eight autophagy-related lncRNAs were filtered out through univariate Cox regression analysis (P

Naringin is a dietary flavonoid glycoside with broad bioactivities, and it has been found to undergo extensive microbial metabolism in human gut. Microbial metabolites are believed to play an important role in the overall bioactivity of naringin. However, knowledge is scarce about its microbial metabolism in laboratory rats, which are the most commonly used animal model for naringin-related biomedical studies. Herein, we profiled the microbial metabolism of naringin in rat by an in vitro anaerobic fermentation combined with LC-MS/MS methods. A total of 35 microbial metabolites were identified, and corresponding metabolic pathways were proposed. Naringin and its metabolites were further quantified in fermentation samples. Rhoifolin, neoeriocitrin, neohesperidin, naringenin, methylated naringin, and hydroxylated naringin were detected as the primary microbial metabolites. Moreover, antioxidant capacity assays suggested that fermentation-associated microbial metabolites exhibited higher antioxidant activity than original naringin. Obtained results contribute to a more comprehensive understanding of the microbial metabolism and antioxidant capacity of naringin.

Background Following drug resistance in patients with lung cancer treated by EGFR TKIs, a biopsy is required to obtain sufficient cancer tissue for T790M detection in order to select potential beneficiaries suitable for third‐generation EGFR TKIs, such as osimertinib. The purpose of this study was to explore the feasibility of using a new in situ analysis technique based on RNA target sequences to detect EGFR T790M in lung cancer. Methods A total of 28 formalin‐fixed paraffin‐embedded (FFPE) samples from 24 lung adenocarcinoma patients archived in Peking Union Medical College Hospital from 2015 to 2016 were collected. The BaseScope T790M detection technique by in situ hybridization on FFPE slides was used to analyze the mutation of EGFR T790M, and then the results were compared with the data acquired by Scorpions ARMS assay, which is the so‐called gold standard for EGFR gene mutation testing. The sensitivity and specificity of the BaseScope T790M detection technique were preliminarily evaluated. Results Of the 28 FFPE specimens, the average proportion of T790M‐positive cells was 35.78% ± 17.68% in 18 samples with EGFR T790M, confirmed by Scorpions ARMS assay, Compared with real‐time PCR assay, the sensitivity and specificity of BaseScope T790M were all 100% in our cohort. Conclusion BaseScope T790M assay could be completed on only one FFPE slide and the visualized molecular result overplayed with histomorphological information perfectly, so it may be the alternative method for EGFR T790M evaluation. BaseScope assay has potential clinical utility, and it will be necessary to carry out validation with a large number of cases.

Abstract Background Hearing loss is one of the most common modifiable factors associated with cognitive and functional decline in geriatric populations. An accurate, easy-to-apply, and inexpensive hearing screening method is needed to detect hearing loss in community-dwelling elderly people, intervene early and reduce the negative consequences and burden of untreated hearing loss on individuals, families and society. However, available hearing screening tools do not adequately meet the need for large-scale geriatric hearing detection due to several barriers, including time, personnel training and equipment costs. This study aimed to propose an efficient method that could potentially satisfy this need. Methods In total, 1793 participants (≥60 years) were recruited to undertake a standard audiometric air conduction pure tone test at 4 frequencies (0.5–4 kHz). Audiometric data from one community were used to train the decision tree model and generate a pure tone screening rule to classify people with or without moderate or more serious hearing impairment. Audiometric data from another community were used to validate the tree model. Results In the decision tree analysis, 2 kHz and 0.5 kHz were found to be the most important frequencies for hearing severity classification. The tree model suggested a simple two-step screening procedure in which a 42 dB HL tone at 2 kHz is presented first, followed by a 47 dB HL tone at 0.5 kHz, depending on the individual’s response to the first tone. This approach achieved an accuracy of 91.20% (91.92%), a sensitivity of 95.35% (93.50%) and a specificity of 86.85% (90.56%) in the training dataset (testing dataset). Conclusions A simple two-step screening procedure using the two tones (2 kHz and 0.5 kHz) selected by the decision tree analysis can be applied to screen moderate-to-profound hearing loss in a community-based geriatric population in Shanghai. The decision tree analysis is useful in determining the optimal hearing screening criteria for local elderly populations. Implanting the pair of tones into a well-calibrated sound generator may create a simple, practical and time-efficient screening tool with high accuracy that is readily available at healthcare centers of all levels, thereby facilitating the initiation of extensive nationwide hearing screening in older adults.

Objective: Dry Weight (DW) is a typical hemodialysis (HD) prescription for End-Stage Renal Disease (ESRD) patients. However, an accurate DW assessment is difficult due to the complication of body components and individual variations. Our objective is to model a clinically practicable DW estimator. Method: We proposed a time series-based regression method to evaluate the weight fluctuation of HD patients according to Electronic Health Record (EHR). A total of 34 patients with 5100 HD sessions data were selected and partitioned into three groups; in HD-stabilized, HD-intolerant, and near-death. Each group's most recent 150 HD sessions data were adopted to evaluate the proposed model. Results: Within a 0.5 kg absolute error margin, our model achieved 95.44%, 91.95%, and 83.12% post-dialysis weight prediction accuracies for the HD-stabilized, HD-intolerant, and near-death groups, respectively. Within a 1%relative error margin, the proposed method achieved 97.99%, 95.36%, and 66.38% accuracies. For HD-stabilized patients, the Mean Absolute Error (MAE) of the proposed method was 0.17 kg ± 0.04 kg. In the model comparison experiment, the performance test showed that the quality of the proposed model was superior to those of the state-of-the-art models. Conclusion: The outcome of this research indicates that the proposed model could potentially automate the clinical weight management for HD patients. Clinical Impact: This work can aid physicians to monitor and estimate DW. It can also be a health risk indicator for HD patients.