LFchimera protects HeLa cells from invasion by <Emphasis Type="Italic">Yersinia</Emphasis> spp. in vitro

Yersinia pestis is the causative agent of plague. As adequate antibiotic treatment falls short and currently no effective vaccine is available, alternative therapeutic strategies are needed. In order to contribute to solving this problem we investigated the therapeutic potential of the peptide construct LFchimera against the safer-to-handle Y. pestis simulants Yersinia enterocolitica and Yersinia pseudotuberculosis in vitro. LFchimera is a heterodimeric peptide construct mimicking two antimicrobial domains of bovine lactoferrin, i.e. lactoferrampin and lactoferricin. LFchimera has been shown to be a potent antimicrobial peptide against a variety of bacteria in vitro and in vivo. Also Y. enterocolitica and Y. pseudotuberculosis have been shown to be susceptible for LFchimera in vitro. As Yersiniae spp. adhere to and invade host cells upon infection, we here investigated the effects of LFchimera on these processes. It was found that LFchimera has the capacity to inhibit host-cell invasion by Yersiniae spp. in vitro. This effect appeared to be host-cell mediated, not bacteria-mediated. Furthermore it was found that exposure of human HeLa epithelial cells to both LFchimera and the bacterial strains evoked a pro-inflammatory cytokine release from the cells in vitro.     

Proteomic characterization of the outer membrane vesicle of the halophilic marine bacterium <Emphasis Type="Italic">Novosphingobium pentaromativorans</Emphasis> US6-1

Novosphingobium pentaromativorans US6-1 is a Gram-negative halophilic marine bacterium able to utilize several polycyclic aromatic hydrocarbons such as phenanthrene, pyrene, and benzo[a]pyrene. In this study, using transmission electron microscopy, we confirmed that N. pentaromativorans US6-1 produces outer membrane vesicles (OMVs). N. pentaromativorans OMVs (hereafter OMV_Novo) are spherical in shape, and the average diameter of OMV_Novo is 25–70 nm. Proteomic analysis revealed that outer membrane proteins and periplasmic proteins of N. pentaromativorans are the major protein components of OMV_Novo. Comparative proteomic analysis with the membrane-associated protein fraction and correlation analysis demonstrated that the outer membrane proteins of OMV_Novo originated from the membrane- associated protein fraction. To the best of our knowledge, this study is the first to characterize OMV purified from halophilic marine bacteria.     

A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.     

Antibacterial and antitumor activity of Bogorol B-JX isolated from <Emphasis Type="Italic">Brevibacillus laterosporus</Emphasis> JX-5

Antimicrobial peptides are promising anti-infective agent candidates because they have a broad antimicrobial spectrum and bioactivity and are unlikely to elicit antibiotic resistance. The bogorols represent a new cationic antibiotic peptide and possess great therapeutic potential because of their bioactivity and precise mode of action. Here, we report that Bogorol B-JX (BBJX), a peptide previously isolated from Brevibacillus laterosporus JX-5 by us, has significant antibacterial and antitumor activities in vitro. BBJX was found to inhibit methicillin-resistant Staphylococcus aureus (MRSA) at 2.5 µg/mL with distinct mechanisms of action from those against Bacillus bombyseptieus and Escherichia coli. It penetrates MRSA membrane with little visible destruction and binds to genomic DNA. BBJX could inhibit the proliferation of human histiocytic lymphoma cell line U-937 and ConA-activated spleen cells at 5 µg/mL, but was not cytotoxic to the Jurkat cells, resting spleen cells or differentiated macrophage-like U-937 immunocytes. Moreover, BBJX caused apoptosis of U-937 cells by opening the mitochondrial permeability transition pore and stimulating the production of reactive oxygen species. Taken together, these studies provided basis for future medical application of the bogorols.     

Antitumor Activity of Probiotic Bacteria and the Bacterial Association in the Tumor Growth Model and in Cell Culture

Strains of lactic acid and propionic acid bacteria and the A-5 bacterial association composed of the Lactobacillus brevis B-3/A-26, L. plantarum 2v/А-6, L. plantarum 14d/I, and Propionibacterium shermanii 2/10 cultures in equal proportion exhibiting high antitumor activity have been selected using the fungus Fusarium bulbigenum var. blasticola tumorlike growth model. L. acidophilus 27w, L. plantarum 2v/I, 2v/A-6, and 22A-310, L. fermentum 127A-4, L. cellobiosus 7n, L. salivarius 39n, P. shermanii 2/10, and the А-5 association were shown to inhibit completely the tumorlike growth of the fungus when present as 2% native cultures in the nutrient medium. The high antitumor activity of the A-5 association comparable with that of the therapeutic agents doxorubicin and cyclophosphamide was confirmed using human tumor cell lines originating from different organs (HepG2, RD, and H9). It has been demonstrated that the bacterial strains comprising the A-5 association were resistant to doxorubicin and methotrexate, the antitumor drugs tested.     

<Emphasis Type="Italic">In vivo</Emphasis> fluorescence imaging to assess early therapeutic response to tumor progression in a xenograft cancer model

Bioimaging technology plays an important role in assessment of therapeutic response in cancer therapy. Here, we report a non-invasive monitoring system for measuring tumor growth by in vivo fluorescence imaging. Target cells for xenograft tumor induction were manipulated by cell-surface fluorescence labelling. Fluorescence was clearly detected in vitro and in vivo without affecting cytotoxicity. Anti-tumor efficacy was evaluated by directly measuring the fluorescence signal of a progressive tumor in a xenograft model. This non-invasive in vivo monitoring system can be used to assess the early response to antitumor therapeutics and may be a valuable tool to replace or complement traditional caliper-based methods for preclinical studies.     

Quasi-adaptive response to alkylating agents and Ada-protein functions in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In the exponentially growing E. coli cells we have described in 2005 a new fundamental genetic phenomenon, namely quasi-adaptive response to alkylating compounds, “quasi-Ada”. Phenotypic expression of “quasi-Ada” is similar to the true Ada response, however in contrast it develops in the course of pretreatment of the cells by sublethal dose of non-alkylating agent, an NO-containing dinitrosyl iron complex with glutathione (DNIC_glu). To reveal the mechanisms of quasi-adaptation and its association with the function of the regulatory protein Ada here we used a unique property of dual gene expression regulation of aidB1 gene, a part of Ada-regulon, namely its relative independence from Ada protein in anaerobic conditions. Based on the results of aidB1 gene expression analysis an EPR spectra of E.coli MV2176 cells (aidB1::lacZ) in aerobic and anaerobic conditions after the corresponding treatments we concluded that the function and the spatial structure of ^meAda and [(Cys^?)₂Fe⁺(NO⁺)₂]Ada are identical and thus the nitrosylated protein represents an Ada regulon genes expression regulator during quasi-adaptation development.     

Intralesional uridine-5′-triphosphate (UTP) treatment induced resistance to <Emphasis Type="Italic">Leishmania amazonensis</Emphasis> infection by boosting Th<Subscript>1</Subscript> immune responses and reactive oxygen species production

Leishmania amazonensis is the etiologic agent of cutaneous leishmaniasis, an immune-driven disease causing a range of clinical symptoms. Infections caused by L. amazonensis suppress the activation and function of immune cells, including macrophages, dendritic cells, and CD4⁺ T cells. In this study, we analyzed the course of infection as well as the leishmanicidal effect of intralesional UTP treatment in L. amazonensis-infected BALB/c mice. We found that UTP treatment reduced the parasitic load in both footpad and lymph node sites of infection. UTP also boosted Th1 immune responses, increasing CD4⁺ T cell recruitment and production of IFN-γ, IL-1β, IL-12, and TNF-α. In addition, the role of UTP during innate immune response against L. amazonensis was evaluated using the air pouch model. We observed that UTP augmented neutrophil chemoattraction and activated microbicidal mechanisms, including ROS production. In conclusion, our data suggested an important role for this physiological nucleotide in controlling L. amazonensis infection, and its possible use as a therapeutic agent for shifting immune responses to Th1 and increasing host resistance against L. amazonensis infection.     

Immunostimulatory characteristics of a novel adjuvant on the basis of cucumarioside A<Subscript>2</Subscript>-2 and monogalactosyldiacylgycerol

A novel antigen carrier has been formulated on the basis of a cucumarioside-A₂-2 triterpene glycoside (CD) complex with cholesterol and monogalactosyldiacylglycerol from Ahnfeltia tobuchiensis (MGDGAt) and Ulva fenestrate (MGDGUf). Morphological and immunostimulative characteristics of the carrier were studied. Electron microscopy experiments demonstrated the formation of homogeneous tubular structures in a mixture of CD, cholesterol, and MGDG in molar ratio of 1: 2: 3. In animals immunized by the carrier bearing pore forming protein monomer of pseudotuberculosis agent CD and MGDG synergically affected synthesis of specific antibodies, interleukin-2, and γ-interferon and delayed hypersensitivity reaction when compared to Freund’s complete adjuvant or to immunostimulatory complexes between Quillaja saponaria saponins and phosphatidylcholine from egg yolk. The immunostimulatory effect depends upon the composition of polyunsaturated fatty acids of MGDG. The new tubular adjuvant carrier is a competitive adjuvant, as it includes CD obtained from far-eastern sea cucumber commercial species Cucumaria japonica, and MGDG from seaweed.     

Ceramide-Fabricated Co-Loaded Liposomes for the Synergistic Treatment of Hepatocellular Carcinoma

Combination therapy is one of the important methods to improve therapeutic effect on the treatment of hepatocellular carcinoma (HCC). Sorafenib (SF) is a canonical US Food and Drug Administration-approved multikinase molecule inhibitor against HCC. However, therapeutic benefit with Sorafenib alone was usually unsatisfactory. Ceramide (CE) is an endogenous bioactive sphingolipid, which has a strong potential to suppress various tumors. The combination of SF and CE was hoping to exert maximum synergistic antitumor effect through different tumor-suppressible mechanisms. In this respect, SF and CE co-loaded liposomes (SF/CE-liposomes) were developed to verify synergistic antitumor efficacy. The optimal molar ratio of SF and CE was determined through combination index. SF/CE-liposomes were prepared by thin-film hydration method, which exhibited spherical or ellipsoidal shape. Particle size of SF/CE-liposomes was 174?±?4 nm with homogeneous distribution. Release profile of SF demonstrated that addition of CE imposed no significant impact on the release of SF. SF/CE-liposomes exhibited acceptable stability in different media and desirable storage stability over 30 days at 4°C. In vitro cellular uptake confirmed that SF/CE-liposomes could be efficiently internalized into HepG2 cells. In vitro cytotoxicity evaluation indicated that SF/CE-liposomes exhibited higher cytotoxicity on HepG2 cells. IC₅₀ value of SF/CE-liposomes was 11.5?±?0.44 μM, which was significantly lower than that of SF-liposomes (^**p < 0.01). Evaluation of in vivo synergistic effect on H22-bearing mice verified that SF/CE-liposomes achieved robust antitumor activity in preventing tumor growth. All results suggested that SF/CE-liposomes might be served as an efficient co-delivery system for improving therapeutic efficacy of HCC.     

Interferon-mediated antiviral activities of Angelica tenuissima Nakai and its active components

Angelica tenuissima Nakai is a widely used commodity in traditional medicine. Nevertheless, no study has been conducted on the antiviral and immune-modulatory properties of an aqueous extract of Angelica tenuissima Nakai. In the present study, we evaluated the antiviral activities and the mechanism of action of an aqueous extract of Angelica tenuissima Nakai both in vitro and in vivo. In vitro, an effective dose of Angelica tenuissima Nakai markedly inhibited the replication of Influenza A virus (PR8), Vesicular stomatitis virus (VSV), Herpes simplex virus (HSV), Coxsackie virus, and Enterovirus (EV-71) on epithelial (HEK293T/HeLa) and immune (RAW264.7) cells. Such inhibition can be described by the induction of the antiviral state in cells by antiviral, IFNrelated gene induction and secretion of IFNs and pro-inflammatory cytokines. In vivo, Angelica tenuissima Nakai treated BALB/c mice displayed higher survivability and lower lung viral titers when challenged with lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2, H7N3, and H9N2). We also found that Angelica tenuissima Nakai can induce the secretion of IL-6, IFN-λ, and local IgA in bronchoalveolar lavage fluid (BALF) of Angelica tenuissima Nakai treated mice, which correlating with the observed prophylactic effects. In HPLC analysis, we found the presence of several compounds in the aqueous fraction and among them; we evaluated antiviral properties of ferulic acid. Therefore, an extract of Angelica tenuissima Nakai and its components, including ferulic acid, play roles as immunomodulators and may be potential candidates for novel anti-viral/anti-influenza agents.     

Novel phenotypes of <Emphasis Type="Italic">Drosophila spinster</Emphasis> locus on the head formation during embryogenesis

spinster (spin) is a late endosome/lysosome membrane protein with the amino acid sequence of a lysosomal sugar carrier and expressed in the glial cells. Spin is required for autophagy and lysosome reformation by releasing lysosomal degradation products of autolysosome into the cytosol in Drosophila larvae and adults. However, such kind of function has not been investigated in embryos yet. In this study, for the first time, we examined the effects of spin mutation on the endocytic pathway and autophagy during embryogenesis. Loss-of-function spin mutation led to the abnormal process of early endosome/recycling endosome and the accumulation of enlarged autophagosome/autolysosome. These abnormal endocytic pathway and autophagy subsequently caused the malformation of head at embryonic stages. These results show that Spin is involved in the endocytic pathway and autophagy during embryogenesis as well as larval and adult stages.     

In silico structural characterization of protein targets for drug development against <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Trypanosoma cruzi is the protozoan pathogen responsible for Chagas disease, which is a major public health problem in tropical and subtropical regions of developing countries and particularly in Brazil. Despite many studies, there is no efficient treatment against Chagas disease, and the search for new therapeutic targets specific to T. cruzi is critical for drug development. Here, we have revisited 41 protein sequences proposed by the analogous enzyme pipeline, and found that it is possible to provide structures for T. cruzi sequences with clear homologs or analogs in H. sapiens and likely associated with trypanothione reductase, cysteine synthase, and ATPase functions, and structures for sequences specific to T. cruzi and absent in H. sapiens associated with 2,4-dienoyl-CoA reductase, and leishmanolysin activities. The implications of our structures refined by atomistic molecular dynamics (monomer or dimer states) in their in vitro environments (aqueous solution or membrane bilayers) are discussed for drug development and suggest that all protein targets, except cysteine synthase, merit further investigation.     

Antibacterial and immunomodulatory activities of bovine lactoferrin against <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 infections in cattle

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen that causes food-borne disease in humans ranging from watery diarrhea to bloody diarrhea and severe hemorrhagic colitis, renal failure and hemolytic uremic syndrome. Cattle, the most important source of E. coli O157:H7 transmission to humans, harbor the bacteria in their gastrointestinal tract without showing clinical symptoms. Prevention of E. coli O157:H7 infections in ruminants could diminish the public health risk. However, there is no specific treatment available nor a vaccine or a therapeutic agent which completely prevents E. coli O157:H7 infections in cattle. This paper provides an overview of latest research data on eradicating enterohemorrhagic E. coli O157:H7 in ruminants by use of bovine lactoferrin administration. The article provides insights into the anti-microbial and immunomodulatory activities of bovine lactoferrin against E. coli O157:H7 infections in cattle.