Intravital Fluorescence Excitation in Whole-Animal Optical Imaging

Whole-animal fluorescence imaging with recombinant or fluorescently-tagged pathogens or cells enables real-time analysis of disease progression and treatment response in live animals. Tissue absorption limits penetration of fluorescence excitation light, particularly in the visible wavelength range, resulting in reduced sensitivity to deep targets. Here, we demonstrate the use of an optical fiber bundle to deliver light into the mouse lung to excite fluorescent bacteria, circumventing tissue absorption of excitation light in whole-animal imaging. We present the use of this technology to improve detection of recombinant reporter strains of tdTomato-expressing Mycobacterium bovis BCG (Bacillus Calmette Guerin) bacteria in the mouse lung. A microendoscope was integrated into a whole-animal fluorescence imager to enable intravital excitation in the mouse lung with whole-animal detection. Using this technique, the threshold of detection was measured as 10³ colony forming units (CFU) during pulmonary infection. In comparison, the threshold of detection for whole-animal fluorescence imaging using standard epi-illumination was greater than 10⁶ CFU.     

Extracellular micro/nanovesicles rescue kidney from ischemia-reperfusion injury

Acute renal failure (ARF) is a clinical challenge that is highly resistant to treatment, and its high rate of mortality is alarming. Ischemia–reperfusion injury (IRI) is the most common cause of ARF. Especially IRI is implicated in kidney transplantation and can determine graft survival. Although the exact pathophysiology of renal IRI is unknown, the role of inflammatory responses has been elucidated. Because mesenchymal stromal cells (MSCs) have strong immunomodulatory properties, they are under extensive investigation as a therapeutic modality for renal IRI. Extracellular vesicles (EVs) play an integral role in cell-to-cell communication. Because the regenerative potential of the MSCs can be recapitulated by their EVs, the therapeutic appeal of MSC-derived EVs has dramatically increased in the past decade. Higher safety profile and ease of preservation without losing function are other advantages of EVs compared with their producing cells. In the current review, the preliminary results and potential of MSC-derived EVs to alleviate kidney IRI are summarized. We might be heading toward a cell-free approach to treat renal IRI.     

Harnessing Bioinformatic Approaches to Design Novel Multi-epitope Subunit Vaccine Against Leishmania infantum

International Journal of Peptide Research and Therapeutics - Kala-azar or visceral leishmaniasis (VL) is the most important vector-borne protozoan disease and a life-threatening problem in the...     

In silico prediction of exposure amino acid sequences of outer inflammatory protein A of Helicobacter pylori for surface display on Eschierchia coli

BACKGROUND:

Outer inflammatory protein A (OipA) is an outer membrane protein of Helicobacter pylori that is involved in inducing IL-8 and intracellular signaling. In this study, we have predicted exposure amino acid sequences of OipA for insertion in permissive sites of CstH subunit of Eschierchia coli CS3 pilli for bacterial surface display.

MATERIALS AND METHODS:

Databases: National Center for Biotechnology Institute and Protein Data Bank. Servers: PHD, SABLE, GOR 4, SignalP3.0, TBBpred, PRODIV-TMHMM, TMRPres2D, CPH Models, PHYRE, GETAREA, VADAR, Pep state and pep window. Software: Swiss PDB viewer and Discovery studio.

RESULTS:

In silico prediction of exposure amino acid sequences of OipA led to detection of six sequences of amino acid, 76-87, 106-112, 170-182, 222-230, 242-258, and 278-290. These sequences inserted between amino acid sequences 66-67, 100-101, and 109-110 of CstH that were predicted by Eskandari et al. as permissive sites of CstH.

CONCLUSION:

OipA has the ability to induce IL-8 from gastric epithelial cells and some papers are mentioned that this outer membrane protein involve to attachment and intracellular signaling. Receptor of OipA and adhesion motifs on this protein is unknown. Detection of exposure motifs aids to recognition of adhesion motifs and receptor of OipA on gastric epithelial cells. In this study, we have predicted exposure amino acid sequences for insert to subunit CstH of CS3 pilli E. coli for surface display.     

Two Novel Mutations in LAMC2 Gene in Iranian Families Affected by Junctional Epidermolysis Bullosa

Background:Junctional epidermolysis bullosa (JEB) is an autosomal recessive skin disorder with defective adhesion of dermal- epidermal within the lamina lucida region of the basement membrane zone. The main characterization of JEB is blistering and fragile skin and mucous membrane. Laminins are noncollagenous part of basement membrane and classified as a family of extracellular matrix glycoprotein. Laminins contain three chains: Laminin α, Laminin β and Laminin γ. LAMC2 (laminin subunit gamma 2) gene encodes γ subunit of laminin and its mutation contributes to JEB. Here, we report a disease-causing nonsense mutation and a large deletion mutation in LAMC2 gene in two families affected by JEB.Methods:Whole exome sequencing (WES) was carried out on the mother of patient in family I and the patient himself in family II to detect the underlying mutations. Then, sanger sequencing was performed to confirm the identified mutations.Results:Next generation sequencing (NGS) data analysis of the first family showed a novel, nonsense mutation in LAMC2 gene (LAMC2: {"type":"entrez-nucleotide","attrs":{"text":"NM_005562","term_id":"1653960840"}}NM_005562: exon14:c.C2143T: p.R715X). The heterozygous state of the mutation was confirmed by sanger sequencing in the parents and unaffected brother. In Family II, NGS data had no coverage in the large area of LAMC2 gene. Thus, to confirm the possible deletion sanger sequencing was done and blasting of sequence showed the deleted region of 9.4 kb (exon10-17) in LAMC2 gene.Conclusion:In summary, current study reported a novel disease-causing premature termination codon (PTC) mutation in LAMC2 gene and a large deletion mutation in patients affected by JEB.Key Words: Junctional Epidermolysis Bullosa, LAMC2 gene, Novel mutation, Skin disorder     

Novel Predicted B-Cell Epitopes of PSMA for Development of Prostate Cancer Vaccine

International Journal of Peptide Research and Therapeutics - Prostate cancer is one of the most common cancers around the world. Vaccines are a new hope for prevention of this cancer. In the case...     

Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli

The human epidermal growth factor receptor (HER) family plays an important role in cell growth and signaling and alteration of its function has been demonstrated in many different kinds of cancer. Receptor dimerization is necessary for the HER signal transduction pathway and tyrosine kinase activity. Recently, several monoclonal antibodies have been developed to directly interfere with ligand–HER receptor binding and receptor dimerization. A single chain variable fragment (ScFv) is a valuable alternative to an intact antibody. This report describes the production and purification of an ScFv specific for domain II of the HER2 receptor in Escherichia coli BL21 (DE3) cytoplasm. The majority of expressed of anti-her2his-ScFv protein was produced as inclusion bodies. A Ni-NTA affinity column was used to purify the anti-her2his-ScFv protein. The molecular weight of anti-her2his-ScFv protein was estimated to be approximately 27 kDa, as confirmed by SDS-PAGE and Western blotting assay. The anti-her2his-ScFv showed near 95 % purity and reached a yield of approximately 29 mg/l in flask fermentation. The purified anti-her2his-ScFv showed its biological activity by binding to HER2 receptor on the surface of BT-474 cells. This ScFv may be a potential pharmaceutical candidate for targeting tumour cells overexpressing HER2 receptor.     

Effect of Molecular Chaperone on the Soluble Expression of Recombinant Fab Fragment in E. coli

International Journal of Peptide Research and Therapeutics - Molecular chaperones are folding modulators that play a pivotal role in conformational quality control of proteome and assist proper...     

Preparation of an injectable doxorubicin surface modified cellulose nanofiber gel and evaluation of its anti‐tumor and anti‐metastasis activity in melanoma

Cellulose nanofibers (Cel‐NFs) gel can be considered as a useful drug carrier because of its biocompatibility, high specific surface area, and high loading capacity of drugs. Injectable Cel‐NFs gel could deliver doxorubicin (DOX) for localized chemotherapy of melanoma and suppress melanoma cells migration because of the physical barrier property of Cel‐NFs. We prepared DOX surface modified Cel‐NFs (DOX‐Cel‐NFs) gel by the electrostatic attachment of DOX molecules on the surface of Cel‐NFs. The increase in the zeta potential of nanofibers and the changes in the FTIR spectra of DOX‐Cel‐NFs compared to Cel‐NFs proved this attachment. DOX‐Cel‐NFs showed nano‐fibrous structure with an average diameter of 22.32 ± 10.66 nm after analyzing using field emission scanning electron microscopy. The suitable injectability of DOX‐Cel‐NFs gel verified its promising application for the localized chemotherapy. DOX‐Cel‐NFs gel exhibited a sustained drug release manner. The cytotoxicity results showed that DOX‐Cel‐NFs were more cytotoxic against melanoma cancer cells than the free DOX during 48 h incubation period. Moreover, DOX‐Cel‐NFs gel can suppress the melanoma cancer cells migration efficiently. Thus our results emphasize the potential of DOX‐Cel‐NFs gel as a chemotherapeutic agent for local delivery of DOX in order to treat melanoma and prevent its metastasis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:537–545, 2018