Capsaicin functions as a selective degrader of STAT3 to enhance host resistance to viral infection.

Although STAT3 has been reported as a negative regulator of type I interferon (IFN) signaling, the effects of pharmacologically inhibiting STAT3 on innate antiviral immunity are not well known. Capsaicin, approved for the treatment of postherpetic neuralgia and diabetic peripheral nerve pain, is an agonist of transient receptor potential vanilloid subtype 1 (TRPV1), with additional recognized potencies in anticancer, anti-inflammatory, and metabolic diseases. We investigated the effects of capsaicin on viral replication and innate antiviral immune response and discovered that capsaicin dose-dependently inhibited the replication of VSV, EMCV, and H1N1. In VSV-infected mice, pretreatment with capsaicin improved the survival rate and suppressed inflammatory responses accompanied by attenuated VSV replication in the liver, lung, and spleen. The inhibition of viral replication by capsaicin was independent of TRPV1 and occurred mainly at postviral entry steps. We further revealed that capsaicin directly bound to STAT3 protein and selectively promoted its lysosomal degradation. As a result, the negative regulation of STAT3 on the type I IFN response was attenuated, and host resistance to viral infection was enhanced. Our results suggest that capsaicin is a promising small-molecule drug candidate, and offer a feasible pharmacological strategy for strengthening host resistance to viral infection.

Simplified plasmid cloning with a universal MCS design and bacterial in vivo assembly.

BACKGROUND: The ability to clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. The recent development of seamless cloning technologies has made significant improvements in plasmid construction, but simple and reliable tools are always desirable for time- and labor-saving purposes. RESULTS: We developed and standardized a plasmid cloning protocol based on a universal MCS (Multiple Cloning Site) design and bacterial in vivo assembly. With this method, the vector is linearized first by PCR (Polymerase Chain Reaction) or restriction digestion. Then a small amount (10 ~ 20 ng) of this linear vector can be mixed with a PCR-amplified insert (5× molar ratio against vector) and transformed directly into competent E. coli cells to obtain the desired clones through in vivo assembly. Since we used a 36-bp universal MCS as the homologous linker, any PCR-amplified insert with ~ 15 bp compatible termini can be cloned into the vector with high fidelity and efficiency. Thus, the need for redesigning insert-amplifying primers according to various vector sequences and the following PCR procedures was eliminated. CONCLUSIONS: Our protocol significantly reduced hands-on time for preparing transformation reactions, had excellent reliability, and was confirmed to be a rapid and versatile plasmid cloning technique. The protocol contains mostly mixing steps, making it an extremely automation-friendly and promising tool in modern biology studies.

Gastrointestinal Symptoms Associated With Unfavorable Prognosis of COVID-19 Patients: A Retrospective Study.

Background and Aim: The global pandemic of COVID-19 has posed an enormous threat to the economy and people's lives across various countries. Patients with COVID-19 most commonly present with respiratory symptoms. However, gastrointestinal (GI) symptoms can also occur. We aimed to study the relationship between GI symptoms and disease prognosis in patients with COVID-19. Methods: In a single-center and retrospective cohort study, the outcomes in COVID-19 patients with or without GI symptoms were compared. The propensity score is a conditional probability of having a particular exposure (COVID-19 patients with GI symptoms vs. without GI symptoms) given a set of baseline measured covariates. Survival was estimated using the Kaplan-Meier method, and any differences in survival were evaluated with a stratified log-rank-test. To explore the GI symptoms associated with ARDS, non-invasive ventilator treatment, tracheal intubation, tracheotomy, and CRRT, univariable and multivariable COX regression models were used. Results: Among 1,113 eligible patients, 359 patients with GI symptoms and 718 without GI symptoms had similar propensity scores and were included in the analyses. Patients with GI symptoms, as compared with those without GI symptoms, were associated with a similar risk of death, but with higher risks of ARDS, non-invasive mechanical ventilation in COVID-19 patients, respectively. Conclusions: The presence of GI symptoms was associated with a high risk of ARDS, non-invasive mechanical ventilation and tracheal intubation in patients with COVID-19 but not mortality.

Red nucleus IL-6 mediates the maintenance of neuropathic pain by inducing the productions of TNF-α and IL-1ß through the JAK2/STAT3 and ERK signaling pathways.

We previously reported that interleukin (IL)-6 in the red nucleus (RN) is involved in the maintenance of neuropathic pain induced by spared nerve injury (SNI), and exerts a facilitatory effect via Janus-activated kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) and extracellular signal-regulated kinase (ERK) signal transduction pathways. The present study aimed at investigating the roles of tumor necrosis factor-α (TNF-α) and IL-1ß in RN IL-6-mediated maintenance of neuropathic pain and related signal transduction pathways. Being similar to the elevation of RN IL-6 three weeks after SNI, increased protein levels of both TNF-α and IL-1ß were also observed in the contralateral RN three weeks after the nerve injury. The upregulations of TNF-α and IL-1ß were closely correlative with IL-6 and suppressed by intrarubral injection of a neutralizing antibody against IL-6. Administration of either the JAK2 antagonist AG490 or the ERK antagonist PD98059 to the RN of rats with SNI remarkably increased the paw withdrawal threshold (PWT) and inhibited the up-regulations of local TNF-α and IL-1ß. Further experiments indicated that intrarubral injection of exogenous IL-6 in naive rats apparently lowered the PWT of the contralateral hindpaw and boosted the local expressions of TNF-α and IL-1ß. Pretreatment with AG490 could block IL-6-induced tactile hypersensitivity and suppress the up-regulations of both TNF-α and IL-1ß. However, injection of PD98059 in advance only inhibited the upregulation of IL-1ß, but not TNF-α. These findings indicate that RN IL-6 mediates the maintenance of neuropathic pain by inducing the productions of TNF-α and IL-1ß. IL-6 induces the expression of TNF-α through the JAK2/STAT3 pathway, and the production of IL-1ß through the JAK2/STAT3 and ERK pathways.

Prolyl 4-hydroxylase subunit alpha 3 presents a cancer promotive function in head and neck squamous cell carcinoma via regulating epithelial-mesenchymal transition.

OBJECTIVE: This study aimed to explore the prognostic value and functional role of Prolyl 4-Hydroxylase Subunit Alpha 3 (P4HA3) in head and neck squamous cell carcinoma. METHODS: We downloaded the RNA-Seq dataset of head and neck squamous cell carcinoma for analyzing the expression of P4HA3 and determining its prognostic value. Head and neck squamous cell carcinoma cell lines CAL27 and FaDu were chose for gain- and loss-of-function of P4HA3 tests. mRNA and protein levels of P4HA3 in head and neck squamous cell carcinoma cells were tested by quantitative real-time PCR and western blot respectively. Cell counting Kit-8, clone formation assay, Transwell assay were used to determine the effect of P4HA3 on the proliferative, invasive and migratory capacities of head and neck squamous cell carcinoma cells. RESULTS: Bioinformatics analysis showed that P4HA3 was up-regulated in head and neck squamous cell carcinoma tissues, and had an independent prognostic value for head and neck squamous cell carcinoma patients. The outcomes of gain- and loss-of-function tests illustrated that P4HA3 significantly boosted head and neck squamous cell carcinoma cell proliferative, invasive and migratory abilities. Besides, western blot assay demonstrated that P4HA3 could remarkably activate the epithelial-mesenchymal transition process, with reduced the level of E-cadherin and up-regulated the levels of N-cadherin, Vimentin and Snail. CONCLUSION: We deduce that P4HA3 acts as an oncogene that raises tumor cell viability and metastasis partly by inducing the epithelial-mesenchymal transition process, suggesting that P4HA3 may be considered as a valuable biomarker for head and neck squamous cell carcinoma treatment.

[A reduction technique for the treatment of refractory femoral trochanteric fracture].

OBJECTIVE: To explore the effect of periosteal stripper through spiral blade incision combined with lateral sight bracket prying method in the treatment of irreducible femoral trochanteric fractures with proximal upwarping of fracture. METHODS: From March 2015 to October 2017, 26 patients with refractory femoral trochanteric fractures with proximal upwarp of fracture were treated by periosteal dissection through a spiral blade incision and lateral sight bracket prying, included 9 males and 17 females with an average age of (76.3±8.4) years old ranged from 63 to 85 years old. According to OA/OTA classification, 5 cases were type A1.3 fracture, 6 cases were A2.1, 7 cases were A2.2, 8 cases were A2.3. All cases were closed fractures. The amount of bleeding during operation and the time of fracture healing were observed and recorded. The Harris score of the hip joint function was performed. RESULTS: All cases were followed up from 7 to 26 months with an average of (17.4±4.7) months. Bone union time was from 9 to 15 months with an average of(12.4±3.5)weeks according to X-rays. At the final follow-up, Harris scores were 86 to 93 points with an average of (85.8±5.6) points, 23 cases were classified as excellent, 2 as good, and 1 as fair. CONCLUSIONS: Using periosteum stripper through spiral blade incision combined lateral sight bracket prying for the treatment of irreducible femoral trochanteric fracture, the reduction effect is accurate, the operation time is shortened obviously, no auxiliary small incision is needed, the clinical effect is good, the reduction technique is simple and practical.

AZD1480 can inhibit the biological behavior of ovarian cancer SKOV3 cells in vitro.

OBJECTIVE: To study the mechanism of effects of AZD1480 on the SKOV3 ovarian cancer cell line. METHODS: The MTT method was used to assess cellular proliferation, flow cytometry for cellular apoptosis, the scratch test to determine migration, transwell chamber assays to detect cellular invasion, plate clone experiments to detect the clone forming ability and Western blotting to determine p-STAT3 protein levels. RESULTS: The proliferation rate, migration ability, invasiveness and the clone forming ability of SKOV3 cells were reduced after treatment with AZD1480, while apoptosis rate and chemotherapeutic susceptibility were increased. After treatment with AZD1480 plus cisplatin, the apoptosis rate increased significantly while the expression level of p-STAT3 protein was decreased. CONCLUSION: AZD1480 can inhibit the proliferation, invasion, metastasis and clone formation of SKOV3 cells, induce cellulsar apoptosis, increase the chemotherapeutic sensitivity and reduce the expression level of p-STAT3 protein.

Tissue-specific expression of the PNZIP promoter is mediated by combinatorial interaction of different cis-elements and a novel transcriptional factor.

Recent studies demonstrated that PNZIP and its homologs encode a special cyclase and play an important role in chlorophyll biosynthesis in higher plants. To investigate the molecular mechanism governing the PNZIP gene, the PNZIP promoter was isolated and analyzed. Deletion analysis indicated that G-box is an important element in the regulation of the reporter gene expression. Further mutation assay demonstrated that G-box and GATACT elements are necessary and sufficient for the high and tissue-specific expression of the GUS gene. Using yeast one-hybrid screening, we have isolated a novel tobacco bZIP protein, NtbZIP, which can specifically recognize the G-box of the PNZIP promoter. The NtbZIP protein shares a limited amino acid homology to Arabidopsis ABI5 and AtAREB1 and very low homology to other bZIP proteins. Northern blot analysis showed that the NtbZIP gene is not induced by exogenous ABA and is expressed in different tobacco organs. Cotransformation assays showed that the NtbZIP protein could activate the transcription of the GUS gene driven by the PNZIP promoter. Transgenic tobaccos analysis demonstrated that constitutively expressing antisense NtbZIP gene resulted in a lower NTZIP synthesis and reduced chlorophyll levels. We suggest that NTZIP is a target gene of NtbZIP, which is involved in the regulation of chlorophyll biosynthesis.