IgG subclass specificity to C1q determined by surface plasmon resonance using Protein L capture technique

Recombinant monoclonal antibodies (mAbs) have become an important category of biological therapeutics. mAbs share the same structures and biological functions as endogenous IgG molecules. One function is complement-dependent cytotoxicity (CDC) initiation by binding of C1q. Traditionally, ELISA methods have been utilized to measure C1q binding. A new robust capture method was established in this study to measure the binding affinity of C1q to antibodies by surface plasmon resonance (SPR). The utility of this method was demonstrated by determination of the difference in IgG subclass specificity of C1q binding.     

CyberKnife beam output factor measurements: A multi-site and multi-detector study

PurposeNew promising detectors are available for measuring small field size output factors (OFs). This study focused on a multicenter evaluation of two new generation detectors for OF measurements on CyberKnife systems.MethodsPTW-60019 microDiamond and W1 plastic scintillation detector (PSD) were used to measure OFs on eight CyberKnife units of various generations for 5–60 mm fixed cones. MicroDiamond and PSD OF were compared to routinely used silicon diodes data corrected applying published Monte Carlo (MC) factors. PSD data were corrected for Čerenkov Light Ratio (CLR). The uncertainties related to CLR determination were estimated.ResultsConsidering OF values averaged over all centers, the differences between MC corrected diode and the other two detectors were within 1.5%. MicroDiamond exhibited an over-response of 1.3% at 7.5 mm and a trend inversion at 5 mm with a difference of 0.2%. This behavior was consistent among the different units. OFs measured by PSD slightly under-responded compared to MC corrected diode for the smaller cones and the differences were within 1%. The observed CLR variability was 2.5% and the related variation in OF values was 1.9%.ConclusionThis study indicates that CyberKnife microDiamond OF require corrections below 2%. The results are enhanced by the consistency observed among different units. Scintillator shows a good agreement to MC corrected diode but CLR determination remains critical requiring further investigations. The results emphasized the value of a multi-center validation over a single center approach.     

A critical role of interleukin-1 in preterm labor

Preterm birth (PTB) is a leading cause of neonatal mortality and morbidity worldwide, and represents a heavy economic and social burden. Despite its broad etiology, PTB has been firmly linked to inflammatory processes. Pro-inflammatory cytokines are produced in gestational tissues in response to stressors and can prematurely induce uterine activation, which precedes the onset of preterm labor. Of all cytokines implicated, interleukin (IL)-1 has been largely studied, revealing a central role in preterm labor. However, currently approved IL-1-targeting therapies have failed to show expected efficacy in pre-clinical studies of preterm labor. Herein, we (a) summarize animal and human studies in which IL-1 or IL-1-targeting therapeutics are implicated with preterm labor, (b) focus on novel IL-1-targeting therapies and diagnostic tests, and (c) develop the case for commercialization and translation means to hasten their development.     

Inhibition of Cell Proliferation and Migration by miR-509-3p That Targets CDK2, Rac1, and PIK3C2A

CDK2 is a key regulator of cell cycle progression. In this study, we screened for miRNAs targeting CDK2 using a luciferase-3′-untranslated region reporter assay. Among 11 hit miRNAs, miR-509-3p reduced CDK2 protein levels and significantly inhibited cancer cell growth. Microarray, Western blotting, and luciferase reporter analyses revealed additional targets of miR-509-3p, including Rac1 and PIK3C2A. Overexpression of miR-509-3p induced G1 cell-cycle arrest and inhibited colony formation and migration. RNAi experiments indicated that the growth-inhibitory effects of miR-509-3p may occur through down-regulation of CDK2, Rac1, and PIK3C2A. Targeting of multiple growth regulatory genes by miR-509-3p may contribute to effective anti-cancer therapy.     

Synthesis and biological evaluation of 4-oxoquinoline-3-carboxamides derivatives as potent anti-fibrosis agents

Thirty-one 4-oxoquinoline-3-carboxamides derivatives were synthesized and evaluated for their anti-fibrotic activities by the inhibition of TGF-β1-induced total collagen accumulation and anti-inflammatory activities by the inhibition of LPS-stimulated TNF-α production. Among them, three compounds (10a, 10l and 11g) exhibited potent inhibitory effects on both TGF-β1-induced total collagen accumulation and LPS-stimulated TNF-α production. Furthermore, oral administrations of 10l at a dose of 20 mg/kg/day for 4 weeks effectively alleviated lung inflammation and injury, and decreased lung collagen accumulation in bleomycin-induced pulmonary fibrosis model. Histopathological evaluation of lung tissue confirmed 10l as a potential, orally active agent for the treatment of pulmonary fibrosis.     

Identifying novel targets in renal cell carcinoma: Design and synthesis of affinity chromatography reagents

Two novel scaffolds, 4-pyridylanilinothiazoles (PAT) and 3-pyridylphenylsulfonyl benzamides (PPB), previously identified as selective cytotoxins for von Hippel–Lindau-deficient Renal Carcinoma cells, were used as templates to prepare affinity chromatography reagents to aid the identification of the molecular targets of these two classes. Structure–activity data and computational models were used to predict possible points of attachment for linker chains. In the PAT class, Click coupling of long chain azides with 2- and 3-pyridylanilinothiazoleacetylenes gave triazole-linked pyridylanilinothiazoles which did not retain the VHL-dependent selectivity of parent analogues. For the PPB class, Sonagashira coupling of 4-iodo-(3-pyridylphenylsulfonyl)benzamide with a propargyl hexaethylene glycol carbamate gave an acetylene which was reduced to the corresponding alkyl 3-pyridylphenylsulfonylbenzamide. This reagent retained the VHL-dependent selectivity of the parent analogues and was successfully utilized as an affinity reagent.     

A role for Rev in the association of HIV-1 gag mRNA with cytoskeletal β-actin and viral protein expression

Human immunodeficiency virus type-1 (HIV-1) Rev acts by inducing the specific nucleocytoplasmic transport of a class of incompletely spliced RNAs that encodes the viral structural proteins. The transfection of HeLA cells with a rev-defective HIV-1 expression plasmid, however, resulted in the export of overexpressed, intron-containing species of viral RNAs, possibly through a default process of nuclear retention. Thus, this system enabled us to directly compare Rev⁺ and Rev⁻ cells as to the usage of RRE-containing mRNAs by the cellular translational machinery. Biochemical examination of the transfected cells revealed that although significant levels of gag and env mRNAs were detected in both the presence and absence of Rev, efficient production of viral proteins was strictly dependent on the presence of Rev. A fluoroscence in situ hybridisation assay confirmed these findings and provided further evidence that even in the presence of Rev, not all of the viral mRNA was equally translated. At the early phase of RNA export in Rev⁺ cells, gag mRNA was observed throughout both the cytoplasm and nucleoplasm as uniform fine stippling. In addition, the mRNA formed clusters mainly in the perinuclear region, which were not observed in Rev⁻ cells. In the presence of Rev, expression of the gag protein was limited to these perinuclear sites where the mRNA accumulated. Subsequent staining of the cytoskeletal proteins demonstrated that in Rev⁺ cells gag mRNA is colocalized with β-actin in the sites where the RNA formed clusters. In the absence of Rev, in contrast, the gag mRNA failed to associate with the cytoskeletal proteins. These results suggest that in addition to promoting the emergence of intron-containing RNA from the nucleus, Rev plays an important role in the compartmentation of translation by directing RRE-containing mRNAs to the β-actin to form the perinuclear clusters at which the synthesis of viral structural proteins begins.     

Isolation of an Aspergillus terreus mutant impaired in arginine biosynthesis and its complementation with the argB gene from Aspergillus nidulans

Using filtration enrichment techniques, an Aspergillus terreus arginine auxotrophic strain which contains a mutation that abolishes ornithine transcarbamylase (OTCase) activity has been isolated. This mutant has been genetically transformed with the cloned Aspergillus nidulans OTCase gene. Prototrophic transformants arose at a frequency of about 50 transformants per microgram of plasmid DNA. Southern blot analysis of DNA from the transformants showed that the transforming DNA was ectopically integrated at different locations in the A. terreus genome, often in multiple tandem copies. The transformants were phenotypically stable for several mitotic divisions and retained their capacity to produce extracellular enzymes.     

Dissecting the signaling pathways involved in the function of sperm flagellum

The mammalian flagellum is a specific type of motile cilium required for sperm motility and male fertility. Effective flagellar movement is dependent on axonemal function, which in turn relies on proper ion homeostasis within the flagellar compartment. This ion homeostasis is maintained by the concerted function of ion channels and transporters that initiate signal transduction pathways resulting in motility changes. Advances in electrophysiology and super-resolution microscopy have helped to identify and characterize new regulatory modalities of the mammalian flagellum. Here, we discuss what is currently known about the regulation of flagellar ion channels and transporters that maintain sodium, potassium, calcium, and proton homeostasis. Identification of new regulatory elements and their specific roles in sperm motility is imperative for improving diagnostics of male infertility.