The Effect of Decreased Ca++/Mg++ ATPase Activity on Lactobacillus delbrueckii subsp. bulgaricus sp1.1 Survival during Spray Drying.

Compared with the commonly used technique of freeze-drying, spray drying has lower energy costs. However, spray drying also has a fatal disadvantage: a lower survival rate. In this study, the survival of bacteria in a spray-drying tower decreased as the water content was reduced. The water content of 21.10% was the critical point for spray drying Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) sp1.1 based on sampling in the tower. Based on the relationship between the moisture content of spray drying and the survival rate, the water content of 21.10% was also the critical point for the change in the survival rate during spray drying. Proteomic analysis was used to investigate the reasons for L. bulgaricus sp1.1 inactivation during and after spray drying. Gene Ontology (GO) enrichment revealed that differentially expressed proteins were mainly associated with the cell membrane and transport. In particular, proteins related to metal ion transport included those involved in the transport of potassium, calcium and magnesium ions. The protein-protein interaction (PPI) network revealed that Ca++/Mg++ adenosine triphosphatase (ATPase) may be a key protein. Ca++/Mg++ ATPase activity decreased substantially during spray drying (p < 0.05). Supplementation with Ca++ and Mg++ significantly increased the expression of ATPase-related genes and enzyme activity (p < 0.05). The Ca++/Mg++ ATPase activity of L. bulgaricus sp1.1 was enhanced by increasing the intracellular Ca++ or Mg++ concentration, thus increasing the survival of spray-dried LAB. Bacterial survival rates were increased to 43.06% with the addition of Ca++ and to 42.64% with the addition of Mg++, respectively. Ca++/Mg++ ATPase may be the key to the damage observed in spray-dried bacteria. Furthermore, the addition of Ca++ or Mg++ also reduced bacterial injury during spray drying by enhancing the activity of Ca++/Mg++ ATPase.

Sorafenib-Loaded Cu2-xSe Nanoparticles Boost Photothermal-Synergistic Targeted Therapy against Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) accounts for the predominant form of liver malignancy and presents a leading cause of cancer-related death globally. Sorafenib (SOR), a first-line targeted drug for advanced HCC treatment, has a battery of untoward side effects. Photothermal therapy (PTT) has been utilized as an effective adjuvant in synergy with other approaches. However, little is known about the tumoricidal efficacy of combining SOR with PTT for HCC. Herein, a novel versatile nanoparticle, Cu2-xSe@SOR@PEG (CSP), that is based on a photothermal Cu2-xSe core and SOR for simultaneously reinforcing PTT and reducing the adverse effects of SOR was constructed. The synthesized CSP exhibited a remarkably enhanced therapeutic effect upon 808 nm laser irradiation via dampening HCC cell propagation and metastasis and propelling cell apoptosis. The intravenous administration of CSP substantially suppressed tumor growth in a xenograft tumor mouse model. It was noted that the CSP manifested low toxicity and excellent biocompatibility. Together, this work indicates a promising and versatile tool that is based on synergistic PTT and molecular-targeted therapy for HCC management.

Iridium Complex-Loaded Sorafenib Nanocomposites for Synergistic Chemo-photodynamic Therapy of Hepatocellular Carcinoma.

Although sorafenib, a multi-kinase inhibitor, has provided noteworthy benefits in patients with hepatocellular carcinoma (HCC), the inevitable side effects, narrow therapeutic window, and low bioavailability seriously affect its clinical application. To be clinically distinctive, innovative drugs must meet the needs of reaching tumor tissues and cause limited side effects to normal organs and tissues. Recently, photodynamic therapy, utilizing a combination of a photosensitizer and light irradiation, was selectively accumulated at the tumor site and taken up effectively via inducing apoptosis or necrosis of cancer cells. In this study, a nano-chemo-phototherapy drug was fabricated to compose an iridium-based photosensitizer combined with sorafenib (IPS) via a self-assembly process. Compared to the free iridium photosensitizer or sorafenib, the IPS exhibited significantly improved therapeutic efficacy against tumor cells because of the increased cellular uptake and the subsequent simultaneous release of sorafenib and generation of reactive oxygen species production upon 532 nm laser irradiation. To evaluate the effect of synergistic treatment, cytotoxicity detection, live/dead staining, cell proliferative and apoptotic assay, and Western blot were performed. The IPS exhibited sufficient biocompatibility by hemolysis and serum biochemical tests. Also, the results suggested that IPS significantly inhibited HCC cell proliferation and promoted cell apoptosis. More importantly, marked anti-tumor growth effects via inhibiting cell proliferation and promoting tumor cell death were observed in an orthotopic xenograft HCC model. Therefore, our newly proposed nanotheranostic agent for combined chemotherapeutic and photodynamic therapy notably improves the therapeutic effect of sorafenib and has the potential to be a new alternative option for HCC treatment.

Self-administered acupressure for chronic severe functional constipation: A study protocol for a randomized controlled trial.

BACKGROUND: Functional constipation is a disease with a high incidence, which has a bad effect on general health, mental health, and social functioning. However, current treatment is sometimes unsatisfactory. Acupuncture has been proven effective in some randomized controlled trials. Acupressure is a subtype of acupuncture and can be manipulated by the patients at home. But the evidence is limited now. This study aims to provide some strict evidence for the use of self-administered acupressure in the treatment of functional constipation. METHODS: This 2-armed, parallel, nonspecific controlled, randomized trial will be conducted at The Third Affiliated Hospital of Zhejiang Chinese Medical University in Hangzhou. A total of 154 FC patients will be enrolled into the acupoint group and the sham acupoint group with a ratio of 1:1 into this trial and it will consist of a 2-week run-in period, an 8-week intervention period, and an 8-week follow-up period. The treatment will be done by the patients themselves at home twice a day and they should sign in on the WeChat APP every day to make sure they have done the acupressure. The outcome will also be collected in WeChat APP through the diary and questionnaires. For the one who is unable to use the WeChat, the print edition of the diary and questionnaires are provided and the supervision will be done by the short message. The primary outcome will be the proportion of participants whose CSBM≥3 during week 3 to 10. The secondary outcome will be the proportion of participants whose CSBM ≥3 between 2 groups in week 11 to 18, Spontaneous bowel movements, Bristol Stool Form Scale, Straining severity scores, Patient assessment of constipation quality of life, and Medicine use. DISCUSSION: Acupressure is not an invasive method and can be done by the patient itself at home. We hope this trial will provide credible evidence to the application of self-acupressure for the management of severe chronic functional constipation. TRIAL REGISTRATION: This trial has been registered at the Chinese Clinical Trial Registry (ChiCTR2000038594).

Development of a Simple In Vitro Assay To Identify and Evaluate Nucleotide Analogs against SARS-CoV-2 RNA-Dependent RNA Polymerase.

Nucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust in vitro nonradioactive primer extension assay to quantitatively evaluate the efficiency of incorporation of nucleotide analogs by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analogs over those of natural nucleotides were measured to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RdRp. In agreement with the data published in the literature, we found that the incorporation efficiency of remdesivir-TP is higher than that of ATP and incorporation of remdesivir-TP caused delayed chain termination, which can be overcome by higher concentrations of the next nucleotide to be incorporated. Our data also showed that the delayed chain termination pattern caused by remdesivir-TP incorporation is different for different template sequences. Multiple incorporations of remdesivir-TP caused chain termination under our assay conditions. Incorporation of sofosbuvir-TP is very low, suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2'-C-methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2'-C-methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful for evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp and for studying the mechanism of action of selected nucleotide analogs.

Actinidia Chinensis Planch Root extract attenuates proliferation and metastasis of hepatocellular carcinoma by inhibiting the DLX2/TARBP2/JNK/AKT pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Many studies have confirmed that traditional Chinese herbs exert potential anti-tumor effects. Actinidia Chinensis Planch root has been used as a traditional Chinese medicine (TCM) for thousands of years. However, the mechanism of anti-tumor effects of Actinidia Chinensis Planch root has not been clearly clarified. AIM OF THE STUDY: To explore the molecular biological mechanisms underlying the inhibitory effect of Actinidia Chinensis Planch root extract (acRoots) on hepatocellular carcinoma (HCC). MATERIALS AND METHODS: In our previous study, we used mRNA chip analyses to identify genes regulated by acRoots. Further analyses of altered genes led to the identification of a key regulator of genes that responds to acRoots. We explored the effects of acRoots on the proliferation and invasion of HCC cells via cell counting as well as transwell assays, and further explored the molecular mechanisms underlying the effects of acRoots on HCC cells using qRT-PCR, western blot, and Chip-PCR. RESULTS: Increasing the concentration of acRoots as well as prolonging its action time enhanced the inhibitory activity of acRoots as well as its cytotoxicity against HCC cells. High TARBP2 expression in HCC cells, which is associated with advanced-stage HCC and poor prognoses in HCC patients, was downregulated by treatment with acRoots. Furthermore, acRoots inhibited proliferation, invasion, and epithelial-to-mesenchymal transition by downregulating TARBP2 expression. HCC cells with higher TARBP2 expression were more sensitive to acRoots. The expression of TARBP2 and DLX2 in HCC patients and HCC cell lines was significantly positively correlated, and DLX2 as a transcription factor may promote TARBP2 expression, thereby further activating the JNK/AKT signaling pathway leading to the inhibition of HCC. CONCLUSIONS: acRoots inhibited the malignant behavior of HCC cells by inhibiting TARBP2 expression, which is affected by the transcription factor DLX2, leading to a reduction in JNK/AKT signaling pathway activation.

Prunin suppresses viral IRES activity and is a potential candidate for treating enterovirus A71 infection.

Human enterovirus A71 (HEVA71) causes hand, foot, and mouth disease (HFMD) in young children and is considered a major neurotropic pathogen but lacks effective antivirals. To identify potential therapeutic agents against HFMD, we screened a 502-compound flavonoid library for compounds targeting the HEVA71 internal ribosome entry site (IRES) that facilitates translation of the HEVA71 genome and is vital for the production of HEVA71 viral particles. We validated hits using cell viability and viral plaque assays and found that prunin was the most potent inhibitor of HEVA71. Downstream assays affirmed that prunin disrupted viral protein and RNA synthesis and acted as a narrow-spectrum antiviral against enteroviruses A and B, but not enterovirus C, rhinovirus A, herpes simplex 1, or chikungunya virus. Continuous HEVA71 passaging with prunin yielded HEVA71-resistant mutants with five mutations that mapped to the viral IRES. Knockdown studies showed that the mutations allowed HEVA71 to overcome treatment-induced suppression by differentially regulating recruitment of the IRES trans-acting factors Sam68 and hnRNPK without affecting the hnRNPA1-IRES interaction required for IRES translation. Furthermore, prunin effectively reduced HEVA71-associated clinical symptoms and mortality in HEVA71-infected BALB/c mice and suppressed hepatitis C virus at higher concentrations, suggesting a similar mechanism of prunin-mediated IRES inhibition for both viruses. These studies establish prunin as a candidate for further development as a HEVA71 therapeutic agent.