RPTOR blockade suppresses brain metastases of NSCLC by interfering the ceramide metabolism via hijacking YY1 binding.

BACKGROUND: Ceramide metabolism is crucial in the progress of brain metastasis (BM). However, it remains unexplored whether targeting ceramide metabolism may arrest BM. METHODS: RNA sequencing was applied to screen different genes in primary and metastatic foci and whole-exome sequencing (WES) to seek crucial abnormal pathway in BM + and BM-patients. Cellular arrays were applied to analyze the permeability of blood-brain barrier (BBB) and the activation or inhibition of pathway. Database and Co-Immunoprecipitation (Co-IP) assay were adopted to verify the protein-protein interaction. Xenograft and zebrafish model were further employed to verify the cellular results. RESULTS: RNA sequencing and WES reported the involvement of RPTOR and ceramide metabolism in BM progress. RPTOR was significantly upregulated in BM foci and increased the permeability of BBB, while RPTOR deficiency attenuated the cell invasiveness and protected extracellular matrix. Exogenous RPTOR boosted the SPHK2/S1P/STAT3 cascades by binding YY1, in which YY1 bound to the regions of SPHK2 promoter (at -353 ~ -365 nt), further promoting the expression of SPHK2. The latter was rescued by YY1 RNAi. Xenograft and zebrafish model showed that RPTOR blockade suppressed BM of non-small cell lung cancer (NSCLC) and impaired the SPHK2/S1P/STAT3 pathway. CONCLUSION: RPTOR is a key driver gene in the brain metastasis of lung cancer, which signifies that RPTOR blockade may serve as a promising therapeutic candidate for clinical application.

The effects of acupuncture therapy in migraine: An activation likelihood estimation meta-analysis.

Background: Previous functional magnetic resonance imaging studies indicated that acupuncture could activate the brain regions in patients with migraine. However, these studies showed inconsistent results. This activation likelihood estimation (ALE) meta-analysis aimed to investigate the consistent activated change of brain regions between pre- and post-acupuncture treatment in migraineurs. Methods: We conducted a literature search in PubMed, Embase, Web of Science, the Cochrane Library, the China National Knowledge Infrastructure, the Chinese Science and Technology Periodical Database, the Wanfang Database, and the Chinese Biomedical Literature Database from their inception to 18 August, 2022, to obtain articles assessing the functional magnetic resonance imaging changes of acupuncture for migraine. Two investigators independently performed literature selection, data extraction, and quality assessment. The methodological quality was assessed with a modified version of the checklist. The reporting quality of interventions among included studies was evaluated by the Revised Standards for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA). Our meta-analysis was conducted according to the GingerALE software. The Jackknife sensitivity analysis was used to assess the robustness of the results. Results: 14 articles were finally included according to the eligible criteria. Regarding the immediate effect of acupuncture on migraine, the ALE meta-analysis demonstrated that the deactivation regions were mainly located in the superior frontal gyrus, and middle frontal gyrus (uncorrected P < 0.001). The ALE meta-analysis of the cumulative effect showed that the activation regions were the thalamus, superior frontal gyrus, posterior lobe of the cerebellum, insula, middle frontal gyrus, precentral gyrus, anterior cingulate, and the deactivation brain regions were located in the transverse temporal gyrus, postcentral gyrus, superior temporal gyrus, anterior cingulate, parahippocampal gyrus, inferior parietal lobule, and inferior occipital gyrus (uncorrected P < 0.001). Conclusion: Acupuncture could activate multiple brain areas related with the regulation of pain conduction, processing, emotion, cognition, and other brain regions in patients with migraine. In the future, the combination of multiple imaging technologies could be a new approach to deeply investigate the central mechanism of acupuncture for migraine.

Microbiota-Derived Short-Chain Fatty Acids Promote LAMTOR2-Mediated Immune Responses in Macrophages.

ABSRTACTKlebsiella pneumoniae is a common cause of human-pneumonia-derived sepsis with high morbidity and mortality. The microbiota promotes and maintains host immune homeostasis. The mechanisms by which the gut microbiota affects the host defenses in the respiratory system systematically, however, remain poorly understood. Here, we show that gut microbiota depletion increases susceptibility to extracellular K. pneumoniae infections in terms of increased bacterial burdens in lung and decreased survival rates. Oral supplementation with gut microbiota-derived short-chain fatty acids (SCFAs), subsequently activating G protein-coupled receptor 43 (GPCR43), enhances a macrophage's capacity to phagocytose invading K. pneumoniae Furthermore, SCFAs and GPR43 increase macrophage bacterial clearance by upregulating LAMTOR2, which is further identified as an antibacterial effector and elucidated to facilitate phagosome-lysosome fusion and extracellular signal-regulated kinase (ERK) phosphorylation. Lastly, conditional ablation of Lamtor2 in macrophages decreases their antimicrobial activity, even though mice were pretreated with exogenous SCFA supplementation.IMPORTANCE These observations highlight that SCFAs promote macrophage elimination of K. pneumoniae via a LAMTOR2-dependent signal pathway and suggest that it is possible to intervene in K. pneumoniae pneumonia by targeting the gut microbiota.

In Vitro and In Vivo Effects of the Polymyxin-Vorinostat Combination Therapy Against Multidrug-Resistant Gram-Negative Pathogens.

With the stagnancy of antibiotics development, polymyxins have become the last defense for treatment of multidrug-resistant (MDR) Gram-negative bacteria, whereas the effect of polymyxin monotherapy is limited by resistance. The objective of this study was to evaluate the effects of polymyxin B (PMNB)-vorinostat (SAHA) combination therapy against Gram-negative pathogens in vitro and in vivo. The antibacterial activities of PMNB and SAHA were evaluated by susceptibility testing. The synergistic effect was assessed by checkerboard tests and time-killing kinetics experiments. Cellular morphology studies and reactive oxygen species (ROS) assay were conducted to explore potential mechanisms. Also, Galleria mellonella models were made to evaluate the antibacterial effects in vivo. PMNB-SAHA had the synergistic effect against all tested isolates, reducing >2 log10 colony-forming units (CFU)/mL at 40 minutes, and showed more powerful antibacterial effects than PMNB alone in the 24-hour window. Cellular morphology study showed the change of membrane and disruption of integrity. ROS assay showed more oxidative stress in combination than PMNB or SAHA monotherapy. In animal models, PMNB-SAHA showed a higher survival rate than that of monotherapy. This study is the first to report the synergistic antibacterial effect of PMNB-SAHA therapy against MDR Gram-negative bacteria. Further clinical research is needed to confirm the results.

[Effect on follicular development and pregnancy outcome treated with acupuncture and moxibustion therapy of Tiaochongren Gushenyuan in patients with luteal phase defect].

OBJECTIVE: To observe the effect of acupuncture and moxibustion therapy of Tiaochongren Gushenyuan on ovulation and embryo implantation in luteal phase defect patients with spleen-kidney yang deficiency. METHODS: A total of 80 patients were randomly divided into an observation group and a control group, 40 cases in each one.In the observation group,acupuncture was applied at Shenting (GV 24), Shenque (CV 8), Guanyuan (CV 4), Qixue (KI 13), Lieque (LU 7), Gongsun (SP 4), Taixi (KI 3), Zusanli (ST 36) and Taichong (LR 3). And moxibustion was given at Taixi (KI 3) using moxibustion box during follicular phase, the stimulation of Taichong (LR 3) was strengthened during ovulatory phase, moxibustion was adopted at Shenque (CV 8) to Guanyuan (CV 4), Zusanli (ST 36) and Taixi (KI 3) during luteal phase. In the control group, acupuncture was applied at Guanyuan (CV 4), Dahe (KI 12), Sanyinjiao (SP 6), Ciliao (BL 32), Zhibian (BL 54) and Shenque (CV 8). Moxibustion was given at Sanyinjiao (SP 6) using moxibustion box during follicular phase, and moxibustion was adopted at Shenque (CV 8) to Guanyuan (CV 4) during luteal phase. The treatment were given every Monday, Wednesday and Friday, and the treatment were stoped during menstrual period in the two groups. Totally 3 menstrual cycle treatment were required, and 3 menstrual cycles were followed up. The pregnancy rate was observed after treatment, the ovulation rate, maximum folliclular diameter and difference of maximum folliclular diameters in ovulatory phase, serum progesterone (P) and basal body temperature (BBT) were compared before and after treatment in the two groups. RESULTS: In the observation group, 6 cases of successful pregnancy during treatment,10 cases in follow-up, the clinical pregnancy rate was 40.0% (16/40). In the control group, 1 case of successful pregnancy during treatment, 5 cases in follow-up, the clinical pregnancy rate was 15.0% (6/40). The clinical pregnancy rate in the observation group was higher than the control group (P<0.05). The ovulation rate after treatment in the observation group was 90.0% (36/40), and the control group was 70.0% (28/40), compared before treatment, the ovulation rates were increased after treatment in the two groups (P<0.05). The observation group was higher than the control group, but there was no significant difference between the two groups (P>0.05). Compared before treatment, the maximum folliclular diameter and difference of maximum folliclular diameters in ovulatory phase, serum P after treatment were improved in the two groups (P<0.05), and the improvements of the observation group were significant compared with the control group (P<0.05). The BBT after treatment were superior to before treatment in the two groups (P<0.05). After treatment, the normal BBT in the observation group was 33 cases, while the control group was 22 cases (P<0.05). CONCLUSION: Acupuncture and moxibustion therapy of Tiaochongren Gushenyuan can promote folliclar development, improve dominant follicle morphology, increase the level of serum P. The therapeutic effect is superior to routine acupuncture in increasing ovulation rate and improving pregnancy outcome.

[Discussion on opposing needling combined with dragon-tiger fighting needling for chronic pelvic inflammation].

Opposing needling is an acupoint selection method of acupuncture recorded in Huangdi Neijing (Yellow Emperor 's Internal Classic). And the first record of dragon-tiger fighting needling is found in the Zhenjiu Daquan (Great Compendium on Acupuncture and Moxibustion), it is a compound reinforcing and reducing manipulation of tonification-purgation method. Both of them are widely used in the treatment of pain syndrome, which are mainly for nerve system and musculoskeletal diseases and seldom for gynecological diseases. By analyzing the pathogenic characteristics of chronic pelvic inflammation, the clinical application of opposing needling combined with dragon-tiger fighting needling for chronic pelvic inflammation is expounded based on the theory of treating different diseases with the same treatment in TCM.

A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry.

Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors that block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes.

[Study on chromatographic fingerprint of extractive from Tripterygium wilfordii with supercritical carbon dioxide].

OBJECTIVE: To establish the chromatographic fingerprint of supercritical carbon dioxide extract of Tripterygium wilfordii. METHOD: HPLC method was applied for quality assessment of T. Wilfordii, HPLC analysis was performed on Kromasil C18 (4. 6 mm x 250 mm, 5 microm) with the mixture of acetonitrile-1% per thousand H3PO4, as mobile phase in gradient mode. The samples were detected at UV of 267 nm with column temperature of 35 degrees C, analytic time was 80 min; Flow-rate was 1.0 mL x min(-1). The chromatographic fingerprint of ten batches of samples was determined, for establishing the chromatographic fingerprint of T. Wilfordii. RESULT: Indicating 27 peaks in common, identified 21 peaks with chemical reference and HPLC-MS, and the HPLC fingerprint was established. CONCLUSION: The method is steady and accurate with a good repeatability and can be used as a quality control method for T. Wilfordii.

[Preparation of liposomes containing extracts of Tripterygium wilfordii and evaluation of its stability].

OBJECTIVE: The liposomes containing extracts of Tripterygium wilfordii were prepared and the possibility of entrapment of complex chemicals by liposomes were studied. METHOD: The liposomes containing extracts of T. wilfordii were prepared by thin-film dispersion method, the effect of process parameters and composition of materials on the entrapment efficiency of the main components were studied. The stability of the liposomes dispersion was also evaluated. RESULT: The liposomes made by thin-film dispersion method were mostly small unilamellar vesicles and their particle size was 30 nm to approximately 50 nm. The optimum entrapment efficiency of tripterine and the total alkaloids were respectively 98.10% and 88.63% but the liposomes dispersion was unstable when kept at 4 degrees C. CONCLUSION: The complex chemicals can be entrapped by the liposomes, but its stability need to be improved furtherly.