The use of computer-aided methods have continued to propel accelerated drug discovery across various disease models, interestingly allowing the specific inhibition of pathogenic targets. Chloride Intracellular Channel Protein 4 (CLIC4) is a novel class of intracellular ion channel highly implicated in tumor and vascular biology. It regulates cell proliferation, apoptosis and angiogenesis; and is involved in multiple pathologic signaling pathways. Absence of specific inhibitors however impedes its advancement to translational research. Here, we integrate structural bioinformatics and experimental research approaches for the discovery and validation of small-molecule inhibitors of CLIC4. High-affinity allosteric binders were identified from a library of 1615 Food and Drug Administration (FDA)-approved drugs via a high-performance computing-powered blind-docking approach, resulting in the selection of amphotericin B and rapamycin. NMR assays confirmed the binding and conformational disruptive effects of both drugs while they also reversed stress-induced membrane translocation of CLIC4 and inhibited endothelial cell migration. Structural and dynamics simulation studies further revealed that the inhibitory mechanisms of these compounds were hinged on the allosteric modulation of the catalytic glutathione (GSH)-like site loop and the extended catalytic β loop which may elicit interference with the catalytic activities of CLIC4. Structure-based insights from this study provide the basis for the selective targeting of CLIC4 to treat the associated pathologies.

Progranulin (PGRN) is a growth factor that is involved in the progression of multiple tumors. However, the effects and molecular mechanisms by which PGRN induces lung cancer remain unclear. The expression level of PGRN was analyzed by conducting immunohistochemistry of the histological sections of lung tissues from non-small-cell lung carcinoma (NSCLC) patients. The proliferation, apoptosis, migration, and invasion of NSCLC cells were assessed by the MTT assay, Western blot, degree of wound healing, and Transwell assays. A nude mouse xenograft model was used to validate the role of PGRN in vivo. The expression level of PGRN was higher in male patients with lung adenocarcinoma than in those with lung squamous cell carcinoma; by contrast, no difference was observed in female patients. The overexpression of PGRN promoted the proliferation and anti-apoptosis of H520 (derived from lung squamous cell carcinoma) cells, whereas knockdown of PGRN inhibited the proliferation and anti-apoptosis of A549 (derived from lung adenocarcinoma) cells. Copanlisib (targeting PI3K) inhibited the increase in the expression of cell anti-apoptosis marker Bcl-2 induced by rhPGRN protein; the PI3K agonist 740 Y-P partially reversed the decrease in Bcl-2 expression induced by PGRN deficiency in both A549 and H520 cells. PGRN increased the expression of Ki-67, PCNA, and Bcl-2 in vivo. PGRN inhibited cell apoptosis depending on the PI3K/Akt/Bcl-2 signaling axis; PGRN positivity correlated with lung adenocarcinoma. PGRN is a potential biomarker for the treatment and diagnosis of NSCLC, especially in lung adenocarcinoma.

Development of intranasal vaccines for HIV-1 and other mucosal pathogens has been hampered by the lack of adjuvants that can be given safely to humans. We have found that an intranasal Shigella vaccine (Invaplex) which is well tolerated in humans can also function as an adjuvant for intranasal protein and DNA vaccines in mice. To determine whether Invaplex could potentially adjuvant similar vaccines in humans, we simultaneously administered a simian immunodeficiency virus (SIV) envelope (Env) protein and DNA encoding simian-human immunodeficiency virus (SHIV) with or without Invaplex in the nasal cavity of female rhesus macaques. Animals were intranasally boosted with adenoviral vectors expressing SIV env or gag,pol to evaluate memory responses. Anti-SIV antibodies in sera and nasal, genital tract and rectal secretions were quantitated by ELISA. Intracellular cytokine staining was used to measure Th1-type T cells in blood. Macaques given DNA/protein immunizations with 0.5 mg Invaplex developed greater serum IgG, nasal IgA and cervicovaginal IgA responses to SIV Env and SHIV Gag,Pol proteins when compared to non-adjuvanted controls. Rectal IgA responses to Env were only briefly elevated and not observed to Gag,Pol. Invaplex increased frequencies of IFNγ-producing CD4 and CD8 T cells to the Env protein, but not T cell responses induced by the DNA. Ad-SIV boosting increased Env-specific polyfunctional T cells and Env- and Gag,Pol-specific antibodies in serum and all secretions. The data suggest that Invaplex could be highly effective as an adjuvant for intranasal protein vaccines in humans, especially those intended to prevent infections in the genital or respiratory tract.

Gene therapy is becoming an increasingly valuable tool to treat many genetic diseases with no or limited treatment options. This is the case for hundreds of monogenic metabolic disorders of hepatic origin, for which liver transplantation remains the only cure. Furthermore, the liver contains 10-15% of the body\'s total blood volume, making it ideal for use as a factory to secrete proteins into the circulation. In recent decades, an expanding toolbox has become available for liver-directed gene delivery. Although viral vectors have long been the preferred approach to target hepatocytes, an increasing number of non-viral vectors are emerging as highly efficient vehicles for the delivery of genetic material. Herein, we review advances in gene delivery vectors targeting the liver and more specifically hepatocytes, covering strategies based on gene addition and gene editing, as well as the exciting results obtained with the use of RNA as a therapeutic molecule. Moreover, we will briefly summarise some of the limitations of current liver-directed gene therapy approaches and potential ways of overcoming them.

UNASSIGNED: Development of new and more effective therapies against hepatitis B virus (HBV) is limited by the lack of suitable small animal models. The HBV transgenic mouse model containing an integrated overlength 1.3-mer construct has yielded crucial insights, but this model unfortunately lacks covalently closed circular DNA (cccDNA), the episomal HBV transcriptional template, and cannot be cured given that HBV is integrated in every cell.
UNASSIGNED: To solve these 2 problems, we generated a novel transgenic mouse (HBV1.1X), which generates an excisable circular HBV genome using Cre/LoxP technology. This model possesses a HBV1.1-mer cassette knocked into the ROSA26 locus and is designed for stable expression of viral proteins from birth, like the current HBV transgenic mouse model, before genomic excision with the introduction of Cre recombinase.
UNASSIGNED: We demonstrated induction of recombinant cccDNA (rcccDNA) formation via viral or transgenic Cre expression in HBV1.1X mice, and the ability to regulate HBsAg and HBc expression with Cre in mice. Tamoxifen-inducible Cre could markedly downregulate baseline HBsAg levels from the integrated HBV genome. To demonstrate clearance of HBV from HBV1.1X mice, we administered adenovirus expressing Cre, which permanently and significantly reduced HBsAg and core antigen levels in the murine liver via rcccDNA excision and a subsequent immune response.
UNASSIGNED: The HBV1.1X model is the first Cre-regulatable HBV transgenic mouse model and should be of value to mimic chronic HBV infection, with neonatal expression and tolerance of HBV antigens, and on-demand modulation of HBV expression.
UNASSIGNED: Hepatitis B virus (HBV) can only naturally infect humans and chimpanzees. Mouse models have been developed with the HBV genome integrated into mouse chromosomes, but this prevents mice from being cured. We developed a new transgenic mouse model that allows for HBV to be excised from mouse chromosomes to form a recombinant circular DNA molecule resembling the natural circular HBV genome. HBV expression could be reduced in these mice, enabling curative therapies to be tested in this new mouse model.

UNASSIGNED: Acute lower respiratory tract infection in children causes significant morbidity in the developing countries. Documentation of virus infection using PCR and clinical characteristics of patients affected with viral pneumonia are reviewed in this study.
UNASSIGNED: 51 children less than three years admitted to the Pediatric Hospital, Cairo University with viral pneumonia were included. All patients had undergone nasopharyngeal aspirate for PCR viral detection.
UNASSIGNED: A total of 51 cases were enrolled in the study, of which 7 cases were negative while 44 children were positive for viruses. The most common respiratory virus was Rhinovirus in 32 patients (72.2%), then parainfluenza virus (PIV) in 12 (27.3%), of which subtypes PIV1 were 2 (4.5%), PIV3 were 5 (11.4%) and PIV4 were 5 (11.4%) cases. The third common viruses were respiratory syncytial virus (RSV) in 9 (20.5%) cases of which 3 (6.8%) were RSVA and 6 (13.6%) were RSVB and adenovirus in 9 cases (20.5%). Boca virus was found in 8 (18.2%) patients, corona virus 2 (4.5%) patients, H1N1 2 (4.5%) patients, enterovirus 2 patients (4.5%) and human metapneumovirus in one case (2.3%). Influenza B and PIV2 were not detected. Coinfection was found in 28 (63.7%). Mortality occurred in 12 (23.5%). There was no significant relation between virus type or coinfection with disease severity.
UNASSIGNED: RV was the most commonly detected virus in children under 3 years admitted with acute lower respiratory tract infections. Coinfection was present in the majority of our patients; however it was not related significantly to parameters of disease severity.

Developing endothelial-protective, nonthrombogenic antirestenotic treatments has been a challenge. A major hurdle to this has been the identification of a common molecular target in both smooth muscle cells and endothelial cells, inhibition of which blocks dysfunction of both cell types. The authors\' findings suggest that the PERK kinase could be such a target. Importantly, PERK inhibition mitigated both restenosis and thrombosis in preclinical models, implicating a low-thrombogenic antirestenotic paradigm.

Interferon Regulatory Factor (IRF)-1, originally identified as a transcription factor of the human interferon (IFN)-β gene, mediates tumor suppression and may inhibit oncogenesis. We have shown that IRF-1 in human breast cancer cells results in the down-regulation of survivin, tumor cell death, and the inhibition of tumor growth in vivo in xenogeneic mouse models. In this current report, we initiate studies comparing the effect of IRF-1 in human nonmalignant breast cell and breast cancer cell lines. While IRF-1 in breast cancer cells results in growth inhibition and cell death, profound growth inhibition and cell death are not observed in nonmalignant human breast cells. We show that TNF-α or IFN-γ induces IRF-1 in breast cancer cells and results in enhanced cell death. Abrogation of IRF-1 diminishes TNF-α and IFN-γ-induced apoptosis. We test the hypothesis that IRF-1 augments TNF-α-induced apoptosis in breast cancer cells. Potential signaling networks elicited by IRF-1 are investigated by evaluating the NF-κB pathway. TNF-α and/or IFN-γ results in decreased presence of NF-κB p65 in the nucleus of breast cancer cells. While TNF-α and/or IFN-γ can induce IRF-1 in nonmalignant breast cells, a marked change in NF-κB p65 is not observed. Moreover, the ectopic expression of IRF-1 in breast cancer cells results in caspase-3, -7, -8 cleavage, inhibits NF-κB activity, and suppresses the expression of molecules involved in the NF-κB pathway. These data show that IRF-1 in human breast cancer cells elicits multiple signaling networks including intrinsic and extrinsic cell death and down-regulates molecules involved in the NF-κB pathway.

OBJECTIVE: Pancreatic beta-cells express three Munc18 isoforms. Much is known about the roles of Munc18a (pre-docked secretory granules-SGs) and Munc18b (newcomer SGs and SG-SG fusion) in insulin exocytosis. Although shown to influence glucose-stimulated insulin secretion (GSIS) in rodents the precise role of Munc18c in insulin SG exocytosis has not been elucidated. We here examined the role of Munc18c in human pancreatic beta-cells.
METHODS: Munc18c-shRNA/RFP lenti-virus (versus control virus) was used to knock down the expression level of Munc18c in human islets or single beta-cells. Insulin secretion and granule exocytosis were measured by performing islets perifusion, single-cell patch clamp capacitance measurements and total internal reflection fluorescence microscopy (TIRFM).
RESULTS: Munc18c is most abundant in the cytosol of human beta-cells. Endogenous function of Munc18c was assessed by knocking down (KD) its islet expression by 70% employing lenti-shRNA virus. Munc18c-KD caused reduction in cognate syntaxin-4 islet expression but not in other exocytotic proteins, resulting in the reduction in GSIS in first- (by 42%) and second phases (by 35%). Single cell analyses of RFP-tagged Munc18c-KD beta-cells by patch clamp capacitance measurements showed inhibition in both readily-releasable pool (by 52%) and mobilization from the reserve pool (by 57%). TIRFM to assess single SG behavior showed that Munc18c-KD inhibition of first phase GSIS was attributed to reduction in exocytosis of pre-docked and newcomer SGs, and second phase inhibition attributed entirely to reduction in newcomer SG fusion (SGs that undergo minimal residence or docking time at the plasma membrane before fusion).
CONCLUSIONS: Munc18c is involved in the distinct molecular machineries that affect exocytosis of both predocked and newcomer SG pools that underlie Munc18c\'s role in first and second phases of GSIS, respectively.

The present studies sought to determine whether the anti-folate pemetrexed (Alimta) and the sphingosine-1-phosphate receptor modulator FTY720 (Fingolimod, Gilenya) interacted to kill tumor cells. FTY720 and pemetrexed interacted in a greater than additive fashion to kill breast, brain and colorectal cancer cells. Loss of p53 function weakly enhanced the toxicity of FTY720 whereas deletion of activated RAS strongly or expression of catalytically inactive AKT facilitated killing. Combined drug exposure reduced the activity of AKT, p70 S6K and mTOR and activated JNK and p38 MAPK. Expression of activated forms of AKT, p70 S6K and mTOR or inhibition of JNK and p38 MAPK suppressed the interaction between FTY720 and pemetrexed. Treatment of cells with FTY720 and pemetrexed increased the numbers of early autophagosomes but not autolysosomes, which correlated with increased LC3II processing and increased p62 levels, suggestive of stalled autophagic flux. Knock down of ATG5 or Beclin1 suppressed autophagosome formation and cell killing. Knock down of ceramide synthase 6 suppressed autophagosome production and cell killing whereas knock down of ceramide synthase 2 enhanced vesicle formation and facilitated death. Collectively our findings argue that pemetrexed and FTY720 could be a novel adjunct modality for breast cancer treatment.