SMR peptide antagonizes Staphylococcus aureus biofilm formation.

The emergence and international dissemination of multi-drug resistant Staphylococcus aureus (S. aureus) strains challenge current antibiotic-based therapies, representing an urgent threat to public health worldwide. In the U.S. alone, S. aureus infections are responsible for 11,000 deaths and 500,000 hospitalizations annually. Biofilm formation is a major contributor to antibiotic tolerance and resistance-induced delays in empirical therapy with increased infection severity, frequency, treatment failure, and mortality. Developing novel treatment strategies to prevent and disrupt biofilm formation is imperative. In this article, we test the Secretion Modification Region (SMR) peptides for inhibitory effects on resistant S. aureus biofilm-forming capacity by targeting the molecular chaperone DnaK. The dose effect of SMR peptides on biofilm formation was assessed using microtiter plate methods and confocal microscopy. Interaction between the antagonist and DnaK was determined by immune precipitation with anti-Flag M2 Affinity and Western blot analysis. Increasing SMR peptide concentrations exhibited increasing blockade of S. aureus biofilm formation with significant inhibition found at 18 µM, 36 µM, and 72 µM. This work supports the potential therapeutic benefit of SMR peptides in reducing biofilm viability and could improve the susceptibility to antimicrobial agents.IMPORTANCEThe development of anti-biofilm agents is critical to restoring bacterial sensitivity, directly combating the evolution of resistance, and overall reducing the clinical burden related to pervasive biofilm-mediated infections. Thus, in this study, the SMR peptide, a novel small molecule derived from the HIV Nef protein, was preliminarily explored for anti-biofilm properties. The SMR peptide was shown to effectively target the molecular chaperone DnaK and inhibit biofilm formation in a dose-dependent manner. These results support further investigation into the mechanism of SMR peptide-mediated biofilm formation and inhibition to benefit rational drug design and the identification of therapeutic targets.

Novel secretion modification region (SMR) peptide exhibits anti-metastatic properties in human breast cancer cells.

Breast cancer is the second leading cause of cancer-related mortality in women worldwide, with nearly 90% attributed to metastatic progression. Exosomes containing epithelial-mesenchymal transition (EMT) 'programs' transmit pro-metastatic phenotypes. Our group discovered and developed a novel anti-cancer SMR peptide that antagonizes breast cancer cell exosome release resulting in cell cycle arrest and tumor growth suppression. This study aims to evaluate the anti-metastatic capabilities of the SMR peptide, focusing on exosomes and EMT. Breast cancer cell lines MDA-MB-231 and MCF-7 were treated with the SMRwt peptide, and the following assays were performed: cell wound-healing, migration, invasion. The SMRwt peptide consists of the following amino acid sequence VGFPVAAVGFPVDYKDDDDK and contains the SMR domain (66VGFPV70) of the HIV-1 Nef protein. Western blot analysis detected epithelial and mesenchymal markers to evaluate EMT progression. Extracellular vesicle type and quantity were assessed through NanoSight analysis. Mortalin and Vimentin knockdown was achieved through antibody targeting and miRNAs. Data gathered demonstrated that the SMR peptide interacts with Mortalin and Vimentin to inhibit pro-EMT exosome release and induce EMT tumor suppressor protein expression. Specifically, SMRwt treatment reduced mesenchymal markers Mortalin and Vimentin expression, while the epithelial marker E-cadherin expression was increased in breast cancer cells and breast cancer-derived exosomes. The SMR peptide specificity was identified as no effect was observed for MCF-10A exosome release or function. Direct Mortalin knockdown paralleled the results of SMR peptide treatment with an effective blockade of breast cancer cell migration. Conversely, the invasion assay differed between breast cancer cell lines with invasion blocked for in MCF-7 but not in MDA-MB-231. These results reinforce the therapeutic value of targeting breast cancer exosome release and reinforce Mortalin and Vimentin as critical regulators and therapeutic targets in breast cancer cell progression, EMT, and metastatic potential. A greater understanding of the SMR peptide mechanism of action will benefit the therapeutic design of anti-metastatic agents.

Improved Aitongxiao prescription (I-ATXP) induces apoptosis, cell cycle arrest and blocks exosomes release in hepatocellular carcinoma (HCC) cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second most common malignancy globally, after lung cancer, accounting for 85-90% of primary liver cancer. Hepatitis B virus (HBV) infection is considered the leading risk factor for HCC development in China. HCC is a highly malignant cancer whose metastasis is primarily influenced by the tumor microenvironment. The role of exosomes in cancer development has become the focus of much research due to the many newly described contents of exosomes, which may contribute to tumorigenesis. However, the possible role exosomes play in the interactions between HCC cells and their surrounding hepatic milieu is mainly unknown. We discovered an Improved Aitongxiao Prescription (I-ATXP): an 80% alcohol extract from a mix of 15 specific plant and animal compounds, which had been shown to have an anticancer effect through inducing apoptosis and cell cycle arrest and blocking exosomes release in HCC cells. However, the anticancer mechanism of I-ATXP on human liver carcinoma is still unclear. OBJECTIVE: Due to its inhibitory effects on chemical carcinogenesis and inflammation, I-ATXP has been proposed as an effective agent for preventing or treating human liver carcinoma. In this study, we aimed to explore the effect of I-ATXP on proliferation, apoptosis, and cell cycles of different HCC cell lines. We investigated the impact of I-ATXP on exosomes' secretion derived from these HCC cells. METHODS: The inhibitory effect of I-ATXP on proliferation and cytotoxicity of HepG2, SMMC7721, HKCL-C3 HCC cell lines, and MIHA immortalized hepatocyte cell line was assessed by CCK-8 assay. The cell cycle distribution and cell apoptosis were determined by flow cytometry using Annexin V-FITC/PI staining. The expression of Alix and CD63 of exosome marker proteins was detected by western blotting. The exosome protein concentration was measured by a fluorescent plate reader. The exosome-specific enzyme activity was measured by acetylcholinesterase (AchE) assay, and exosome morphological characteristics were identified by transmission electron microscopy (TEM). RESULTS: I-ATXP inhibited the growth of HCC cells in a dose and time-dependent manner. Flow cytometry analysis showed that I-ATXP induced G0/G1 phase arrest and cell apoptosis. The I-ATX reduced HepG2, SMMC7721, and HKCI-C HCC cell lines exosomes release and low-dose I-ATXP significantly enhanced the growth inhibition induced by 5-Fu. Western blot analysis shows that after HCC cell lines were treated with various concentrations of I-ATXP (0.125-1 mg/ml) for 24 h, exosomes derived from three different HCC cells expressed exosome-specific proteins Alix and CD63. Compared with the untreated group, with the increment of the concentration of I-ATXP, the expression of exosome-specific proteins Alix and CD63 were reduced. These results suggest that I-ATXP can inhibit the release of exosomes with Alix and CD63 protein from HCC cells. CONCLUSIONS: I-ATXP is a traditional Chinese medicine that acts as an effective agent for preventing or treating human liver carcinoma. (i) I-ATXP can effectively inhibit cell proliferation of different HCC cells in a time and dose-dependent manner. Compared with 5-Fu, I-ATXP exhibited more selective proliferation inhibition in HCC cells, displaying traditional Chinese medicine advantages on tumor therapy and providing the experimental basis for I-ATXP clinical application. (ii) I-ATXP can induce apoptosis and cell cycle arrest in HCC cells. The CCK-8 assay results indicated that I-ATXP could inhibit HCC cell proliferation mediated by apoptosis and cell cycle arrest. (iii) I-ATXP can inhibit both the exosome releases and expression of CD63, and Alix derived from HCC cells, but the exosomes derived from liver cancer cells affect liver cancer cells' biological properties such as proliferation, invasion, and migration. These suggest that I-ATXP may affect HCC cells via regulation of exosomes of HCC cells, further indicating the potential clinical values of I-ATXP for the prevention or treatment of human liver carcinoma.

SMR peptide antagonizes mortalin promoted release of extracellular vesicles and affects mortalin protection from complement-dependent cytotoxicity in breast cancer cells and leukemia cells.

Background: Mortalin/GRP-75/mt-hsp70 is a mitochondrial chaperone protein, found in the cytoplasm, endoplasmic reticulum and cytoplasmic vesicles. It functions in many cellular processes such as mitochondrial biogenesis, intracellular trafficking, cell proliferation, signaling, immortalization and tumorigenesis. Thus, inhibition of mortalin is a promising avenue for cancer therapy. Previous studies in our lab have suggested that mortalin contributes to breast cancer development and progression. We showed that tumor extracellular vesicle secretion was decreased by knockdown of mortalin expression using HIV-1 Nef SMR peptides. Specifically, these peptides can block extracellular vesicle secretion and mediate cell cycle arrest in MDA-MB-231 and MCF-7 breast cancer cells. Aims: This study aims to investigate further the function and mechanism of interaction of PEG-SMR-CLU and SMR-CPP peptides with the chaperone protein mortalin and to explore the effect of SMR-derived peptides and mortalin expression on extracellular vesicle release and complement dependent cell toxicity in human breast cancer and leukemia cell lines. Results: Our results demonstrated additional effects reversing the tumorigenicity of these cells. First, the modified SMRwt peptides reduced the expression of the mesenchymal marker vimentin (VIM). Second, exposure to the SMRwt peptide inhibited mortalin and complement C9 expression in MDA-MB-231, MCF-7 breast cancer cells and K562 leukemia cells as measured by the Western blot analysis. Third, the SMRwt peptides blocked the cancer cells' ability to release extracellular vesicles, which we observed blocked extracellular vesicle-mediated release of complement, re-establishing complements mediated cell death in those peptide-treated cells. Methods: We developed a series of peptides derived from the Secretion Modification Region (SMR) of HIV-1 Nef protein, modified by the addition of either a cell-penetrating peptide (CPP), a positively charged arginine-rich peptide derived from HIV-1 regulatory protein Tat, or a Clusterin-binding peptide (CLU), a molecular chaperone involved in protein secretion. Both CPP and CLU peptide sequences were added at the C-terminus of the Nef SMR peptide. The CLU-containing peptides were also modified with polyethylene glycol (PEG) to enhance solubility. After treatment of cells with the peptides, we used the MTT cell viability and complement-mediated cytotoxicity assays to confirm the inhibitory role of modified SMRwt peptides on the proliferation of MDA-MB-231 and MCF-7 breast cancer cells and K562 leukemia cells. Flow cytometry was used to determine complement mediated cell apoptosis and death. Western blot analysis was used to track SMR peptides impact on expression of mortalin, vimentin and complement C9 and to measure the expression of extracellular vesicle proteins. NanoSight analysis and acetylcholinesterase (AChE) assay were used for measuring extracellular vesicles particle size and concentration and acetylcholinesterase. Conclusions: Mortalin promotes cell proliferation, metastasis, angiogenesis, downregulate apoptotic signaling. Thus, mortalin is a potential therapeutic target for cancer immunotherapy. The novel SMRwt peptides antagonize the functions of mortalin, blocking tumor extracellular vesicle release and extracellular vesicle-mediated release of complement. This leads to decreases in breast cancer cell metastasis and allows standard treatment of these late stage tumor cells, thus having important clinical implications for late stage breast cancer chemotherapy. These findings support further investigation into the therapeutic value of the SMR peptide in cancer metastasis.

Characterization of Exosomes in Plasma of Patients with Breast, Ovarian, Prostate, Hepatic, Gastric, Colon, and Pancreatic Cancers.

Detection of circulating tumor-specific DNA, RNA or proteins can be difficult due to relative scarcity. Exosomes are extracellular vesicles, 30 - 150 nm in diameter derived from fusion of multivesicular bodies with the plasma membrane. They are composed of a lipid bilayer membrane and contain proteins, mRNA and miRNA. Exosomes are secreted by multiple cell types, including cancer cells. However, there is a relative lack of information concerning the contents of exosomes secreted by various tumor cell types. To examine exosomes in cancer, we collected blood plasma samples from patients with breast, ovarian, prostate, hepatic, gastric, colon, and pancreatic cancers. Exosomes were isolated from plasma and confirmed by AchE assay, transmission electron microscopy and expression of the CD63 exosomal marker. Expression of AFP, CA724, CA153, CEA, CA125, CA199 and PSA antigens were determined using an automated electro-chemiluminescence assay. Expression of the tumor-related chaperone protein, mortalin, was determined by Western blot analysis. Levels of exosome secretion were variable among the different tumor types. Both exosome levels and mortalin expression within tumor cell exosomes were higher than in healthy donors, except in pancreatic carcinoma, where exosomes were elevated but mortalin expression was not significantly different from healthy donors. Exosomes provide unique opportunities for the enrichment of tumor-specific materials and may be useful as biomarkers and possibly as tools of cancer therapies. Mortalin, which has been linked to cell proliferation and induction of epithelial-mesenchymal transition of cancer cells, may be useful as a prognostic bio-marker and as a possible therapeutic target.

Postoperative Conversion Disorder Presenting as Inspiratory Stridor and Hemiparesis in a Pediatric Patient.

BACKGROUND Postoperative conversion disorder is rare and has been reported. The diagnosis is usually made after all major organic causes have been ruled out. CASE REPORT We describe a case of a 13-year-old female who presented in the post-anesthesia care unit with acute-onset inspiratory stridor and unresponsiveness to verbal or painful stimuli after receiving a general anesthetic for upper endoscopy. Later in the post-anesthesia care unit, she presented with acute-onset right hemiplegia and sensory loss. She was first evaluated for causes of her stridor and unresponsiveness. The evaluation revealed paradoxical vocal cord movement, and all laboratory test values were normal. For her hemiplegia and sensory loss, she was evaluated for stroke with head MRI and CT scans, which were normal. CONCLUSIONS After extensive workup and consideration of multiple etiologies for her presenting signs and symptoms, the most likely diagnosis was conversion disorder.

Effectiveness of telephone counselling by a pharmacist in reducing mortality in patients receiving polypharmacy: randomised controlled trial.

OBJECTIVE: To investigate the effects of compliance and periodic telephone counselling by a pharmacist on mortality in patients receiving polypharmacy. DESIGN: Two year randomised controlled trial. SETTING: Hospital medical clinic. PARTICIPANTS: 502 of 1011 patients receiving five or more drugs for chronic disease found to be non-compliant at the screening visit were invited for randomisation to either the telephone counselling group (n = 219) or control group (n = 223) at enrollment 12-16 weeks later. MAIN OUTCOME MEASURES: Primary outcome was all cause mortality in randomised patients. Associations between compliance and mortality in the entire cohort of 1011 patients were also examined. Patients were defined as compliant with a drug if they took 80-120% of the prescribed daily dose. To calculate a compliance score for the whole treatment regimen, the number of drugs that the patient was fully compliant with was divided by the total number of prescribed drugs and expressed as a percentage. Only patients who complied with all recommended drugs were considered compliant (100% score). RESULTS: 60 of the 502 eligible patients defaulted and only 442 patients were randomised. After two years, 31 (52%) of the defaulters had died, 38 (17%) of the control group had died, and 25 (11%) of the intervention group had died. After adjustment for confounders, telephone counselling was associated with a 41% reduction in the risk of death (relative risk 0.59, 95% confidence interval 0.35 to 0.97; P = 0.039). The number needed to treat to prevent one death at two years was 16. Other predictors included old age, living alone, rate of admission to hospital, compliance score, number of drugs for chronic disease, and non-treatment with lipid lowering drugs at screening visit. In the cohort of 1011 patients, the adjusted relative risk for death was 1.61 (1.05 to 2.48; P = 0.029) and 2.87 (1.80 to 2.57; P < 0.001) in patients with compliance scores of 34-66% and 0-33%, respectively, compared with those who had a compliance score of 67% or more. CONCLUSION: In patients receiving polypharmacy, poor compliance was associated with increased mortality. Periodic telephone counselling by a pharmacist improved compliance and reduced mortality. TRIAL REGISTRATION: International Standard Randomised Controlled Trial Number Register: SRCTN48076318.