A synthetic globotriaosylceramide analogue inhibits HIV-1 infection <Emphasis Type="Italic">in vitro</Emphasis> by two mechanisms

Previously, it was shown that the cell-membrane-expressed glycosphingolipid, globotriaosylceramide (Gb₃/P^k/CD77), protects against HIV-1 infection and may be a newly described natural resistance factor against HIV infection. We have now investigated the potential of a novel, water soluble, non-toxic and completely synthetic analogue of Gb₃/P^k (FSL-Gb₃) to inhibit HIV-1 infection in vitro. A uniquely designed analogue, FSL-Gb₃, of the natural Gb₃/P^k molecule was synthesized. HIV-1_IIIB (X4 virus) and HIV-1_Ba-L (R5 virus) infection of PHA/interleukin-2-activated, peripheral blood mononuclear cells (PBMCs) and Jurkat T cells in vitro was assessed, as well as infection of U87.CD4.CCR5 by various clinical R5 tropic viruses after treatment with FSL-Gb₃. We monitored Gb₃, CD4 and CXCR4 expression by fluorescent antibody cell sorting and viral replication by p24 ^gag ELISA. Total cellular Gb₃ was examined by glycosphingolipid extraction and thin layer chromatography. In vivo toxicity was monitored in mice by histological assessment of vital organs and lymphoid tissue. FSL-Gb₃ blocked X4 and R5 of both lab and clinical viral strains in activated PBMCs or the U87.CD4.CCR5 cell line with a 50% inhibitory concentration (IC₅₀) of approximately 200–250 μM. FACS and TLC overlay showed that FSL-Gb₃ can insert itself into cellular plasma membranes and that cellular membrane-absorbed FSL-Gb₃ is able to inhibit subsequent HIV-1 infection. There was no effect of FSL-Gb₃ on cell surface levels of CD4 or CXCR4. Thus, FSL-Gb₃ can inhibit HIV-1 by two mechanisms: direct inhibition of virus and inhibition of viral entry. Infusion of FSL-Gb₃ into laboratory mice at doses well in excess of theoretical therapeutic doses was tolerated with no untoward reactions. Our results demonstrate the potential utility of using a completely synthetic, water soluble globotriaosylceramide analogue, FSL-Gb₃, having low toxicity, for possible future use as a novel therapeutic approach for the systemic treatment of HIV/AIDS.     

Ultrasensitive detection of L-cysteine using gold-5-amino-2-mercapto-1,3,4-thiadiazole core-shell nanoparticles film modified electrode

We are reporting the selective, sensitive and stable determination of L-cysteine (CY) at physiological pH (pH=7.2) using a gold-aminomercaptothiadiazole core-shell nanoparticles (p-GAMCS NPs) film modified GC electrode. The p-GAMCS NPs film was fabricated on GC electrode by potentiodynamic method using 5-amino-2-mercapto-1,3,4-thiadiazole stabilized gold nanoparticles (AMT-AuNPs). The fabricated p-GAMCS NPs film was characterized by cyclic voltammetry and atomic force microscopy (AFM) techniques. The AFM image of the p-GAMCS NPs film showed that it contains a homogeneously distributed AuNPs with a spherical shape of ~10 nm. The p-GAMCS NPs film modified GC electrode was exploited for the determination of CY. The bare GC electrode failed to show any response for CY (pH=7.2) whereas p-GAMCS NPs film on GC electrode showed a well-defined oxidation peak for CY at 0.51 V. Further, p-GAMCS NPs film modified electrode successfully resolved the voltammetric signals of ascorbic acid (AA) and CY with a peak separation of 500 mV. This is the first report for the large voltammetric peak separation between CY and AA to the best of our knowledge. The amperometric current was increased linearly from 10 nM to 140 nM CY with a detection limit of 3 pM (S/N=3). The present modified electrode showed better recoveries for spiked CY into the human blood serum and urine samples.     

Differential susceptibility of interneurons expressing neuropeptide Y or parvalbumin in the aged hippocampus to acute seizure activity

Acute seizure (AS) activity in old age has an increased predisposition for evolving into temporal lobe epilepsy (TLE). Furthermore, spontaneous seizures and cognitive dysfunction after AS activity are often intense in the aged population than in young adults. This could be due to an increased vulnerability of inhibitory interneurons in the aged hippocampus to AS activity. We investigated this issue by comparing the survival of hippocampal GABA-ergic interneurons that contain the neuropeptide Y (NPY) or the calcium binding protein parvalbumin (PV) between young adult (5-months old) and aged (22-months old) F344 rats at 12 days after three-hours of AS activity. Graded intraperitoneal injections of the kainic acid (KA) induced AS activity and a diazepam injection at 3 hours after the onset terminated AS-activity. Measurement of interneuron numbers in different hippocampal subfields revealed that NPY+ interneurons were relatively resistant to AS activity in the aged hippocampus in comparison to the young adult hippocampus. Whereas, PV+ interneurons were highly susceptible to AS activity in both age groups. However, as aging alone substantially depleted these populations, the aged hippocampus after three-hours of AS activity exhibited 48% reductions in NPY+ interneurons and 70% reductions in PV+ interneurons, in comparison to the young hippocampus after similar AS activity. Thus, AS activity-induced TLE in old age is associated with far fewer hippocampal NPY+ and PV+ interneuron numbers than AS-induced TLE in the young adult age. This discrepancy likely underlies the severe spontaneous seizures and cognitive dysfunction observed in the aged people after AS activity.     

Development of a Mouse Model of Abdominal Cutaneous Flaps for Breast Reconstruction

Autologous tissue transfer, in addition to replacing tissue that was lost during injury or surgery, offers women an excellent option to improve cosmetic appearance and self-confidence following mastectomy due to breast cancer. However, flap necrosis is a complication in obese patients undergoing this procedure. We created a mouse model to study the flap-related complications that leads to decreased flap survival in autologous breast reconstruction.

Methods

Left superficial inferior epigastric (SIE) pedicle abdominal-cutaneous flaps were elevated in 8 week-old, obese ob/ob male mice and their lean littermates. Flaps were followed by serial photography. Area of flap necrosis was measured at 7 days. Statistical analysis was performed.

Results

Necrosis was observed at the distal margin of the flaps, in both lean and obese groups. Lean left SIE flaps (n = 8) had a total area flap necrosis of 9.1% at 7 days whereas obese left SIE flaps (n = 8) had a total area flap necrosis of 45.5% at 7 days. Obese flaps had a statistically significant increase in necrosis compared to the lean flaps, p = 0.001.

Conclusions

There was a significant difference between flap survival in lean and obese SIE pedicle flaps in our mouse model. We have developed the first flap model of obesity utilizing the superficial epigastric pedicle in the mouse. This model is optimal for future studies to dissect out mechanisms that lead to the complications related to flap survival for breast reconstruction, especially in obese subjects.     

Picomonas judraskeda Gen. Et Sp. Nov.: The First Identified Member of the Picozoa Phylum Nov., a Widespread Group of Picoeukaryotes,Formerly Known as ‘Picobiliphytes’

In 2007, a novel, putatively photosynthetic picoeukaryotic lineage, the ‘picobiliphytes’, with no known close eukaryotic relatives, was reported from 18S environmental clone library sequences and fluorescence in situ hybridization. Although single cell genomics later showed these organisms to be heterotrophic rather than photosynthetic, until now this apparently widespread group of pico-(or nano-)eukaryotes has remained uncultured and the organisms could not be formally recognized. Here, we describe Picomonas judraskeda gen. et sp. nov., from marine coastal surface waters, which has a ‘picobiliphyte’ 18S rDNA signature. Using vital mitochondrial staining and cell sorting by flow cytometry, a single cell-derived culture was established. The cells are biflagellate, 2.5–3.8×2–2.5 µm in size, lack plastids and display a novel stereotypic cycle of cell motility (described as the “jump, drag, and skedaddle”-cycle). They consist of two hemispherical parts separated by a deep cleft, an anterior part that contains all major cell organelles including the flagellar apparatus, and a posterior part housing vacuoles/vesicles and the feeding apparatus, both parts separated by a large vacuolar cisterna. From serial section analyses of cells, fixed at putative stages of the feeding cycle, it is concluded that cells are not bacterivorous, but feed on small marine colloids of less than 150 nm diameter by fluid-phase, bulk flow endocytosis. Based on the novel features of cell motility, ultrastructure and feeding, and their isolated phylogenetic position, we establish a new phylum, Picozoa, for Picomonas judraskeda, representing an apparently widespread and ecologically important group of heterotrophic picoeukaryotes, formerly known as ‘picobiliphytes’.     

Monoclonal antibody therapeutics with up to five specificities: Functional enhancement through fusion of target-specific peptides

The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvβ3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model.     

A murrel interferon regulatory factor-1: molecular characterization,gene expression and cell protection activity

In this study, we have reported a first murrel interferon regulatory factor-1 (designated as Murrel IRF-1) which is identified from a constructed cDNA library of striped murrel Channa striatus. The identified sequence was obtained by internal sequencing method from the library. The Murrel IRF-1 varies in size of the polypeptide from the earlier reported fish IRF-1. It contains a DNA binding domain along with a tryptophan pentad repeats, a nuclear localization signal and a transactivation domain. The homologous analysis showed that the Murrel IRF-1 had a significant sequence similarity with other known fish IRF-1 groups. The phylogenetic analysis exhibited that the Murrel IRF-1 clustered together with IRF-1 members, but the other members including IRF-2, 3, 4, 5, 6, 7, 8, 9 and 10 were clustered individually. The secondary structure of Murrel IRF-1 contains 27 % α-helices (85 aa residues), 5.7 % β-sheets (19 aa residues) and 67.19 % random coils (210 aa residues). Furthermore, we predicted a tertiary structure of Murrel IRF-1 using I-Tasser program and analyzed the structure on PyMol surface view. The RNA structure of the Murrel IRF-1 along with its minimum free energy (?284.43 kcal/mol) was also predicted. The highest gene expression was observed in spleen and its expression was inducted with pathogenic microbes which cause epizootic ulcerative syndrome in murrels such as fungus, Aphanomyces invadans and bacteria, Aeromonas hydrophila, and poly I:C, a viral RNA analog. The results of cell protection assay suggested that the Murrel IRF-1 regulates the early defense response in C. striatus. Moreover, it showed Murrel IRF-1 as a potential candidate which can be developed as a therapeutic agent to control microbial infections in striped murrel. Overall, these results indicate the immune importance of IRF-1, however, the interferon signaling mechanism in murrels upon infection is yet to be studied at proteomic level.     

Intereukin-10 and Kupffer cells protect steatotic mice livers from ischemia-reperfusion injury

Steatotic livers are more sensitive to ischemia/reperfusion (I/R) and are thus routinely rejected for transplantation because of their increased rate of primary nonfunction (PNF). Lean livers have less I/R-induced damage and inflammation due toKupffer cells (KC), which are protective after total, warm, hepatic I/R with associated bowel congestion. This protection has been linked to KC-dependent expression of the potent anti-inflammatory cytokine interleukin-10 (IL-10).We hypothesized that pretreatment with exogenous IL-10would protect the steatotic livers of genetically obese (ob/ob) mice from inflammation and injury induced by I/R. Lean and ob/ob mice were pretreated with either IL-10 or liposomally-encapsulated bisphosphonate clodronate (shown to deplete KC) prior to total, warm, hepatic I/R. IL-10 pretreatment increased survival of ob/ob animals at 24 hrs post-I/R from 30% to 100%, and significantly decreased serum ALT levels. At six hrs post-I/R, IL-10 pretreatment increased IL-10 mRNA expression, but suppressed up-regulation of the pro-inflammatory cytokine IL-1β mRNA. However, ALT levels were elevated at six hrs post-I/R in KC-depleted animals. These data reveal that pretreatment with IL-10 protects steatotic livers undergoing I/R, and that phagocytically active KC retain a hepatoprotective role in the steatotic environment.     

Influenza A Virus Encoding Secreted Gaussia Luciferase as Useful Tool to Analyze Viral Replication and Its Inhibition by Antiviral Compounds and Cellular Proteins

Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1–3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.