Identifying nonselective hits from a homogeneous calcium assay screen

The authors used a homogeneous calcium dye kit with a cell line transfected using a recombinant protein construct to screen a 50,000 compound library for G-protein coupled receptor (GPCR) agonists. Only 1 of the 365 primary hits activated Gq-coupled GPCRs, as shown using IP-ONE HTRF. Furthermore, an agonist screen against the entire compound library and same heterologous cell line using AequoScreen technology generated no false positives and identified the same positive hit. Next, a multiplex assay composed of both Fluo-3 and Fura-2-loaded cells identified 1 false positive and the same true-positive hit out of the 365 primary hits. Finally, rescreening the 365 primary hits against the parental cell line loaded using the homogeneous calcium dye kit confirmed the specificity of the same true-positive hit only. In summary, the results suggest that AequoScreen technology, IP-ONE HTRF, and multiplex assays are unique, orthogonal technologies to identify nonspecific hits.     

The vitamin D receptor gene variants,ApaI, TaqI,BsmI, and FokI in diabetic foot ulcer and their association with oxidative stress

Molecular Biology Reports - To date, numerous disorders have been linked to vitamin D deficiency. Several lines of evidence indicate a relationship between vitamin D deficiency and the risk of...     

Improving the accumulation of recombinant human serum albumin (HSA) in transgenic tobacco plants by fusion with the N-terminal proline-rich domain of γ-zein (Zera)

In Vitro Cellular & Developmental Biology - Plant - Plants have long played a major role in human health as a source of herbal remedies. Recently, transgenic plants have become convenient...     

Hemispheric asymmetry in white matter connectivity of the temporoparietal junction with the insula and prefrontal cortex

The temporoparietal junction (TPJ) is a key node in the brain's ventral attention network (VAN) that is involved in spatial awareness and detection of salient sensory stimuli, including pain. The anatomical basis of this network's right-lateralized organization is poorly understood. Here we used diffusion-weighted MRI and probabilistic tractography to compare the strength of white matter connections emanating from the right versus left TPJ to target regions in both hemispheres. Symmetry of structural connectivity was evaluated for connections between TPJ and target regions that are key cortical nodes in the right VAN (insula and inferior frontal gyrus) as well as target regions that are involved in salience and/or pain (putamen, cingulate cortex, thalamus). We found a rightward asymmetry in connectivity strength between the TPJ and insula in healthy human subjects who were scanned with two different sets of diffusion-weighted MRI acquisition parameters. This rightward asymmetry in TPJ-insula connectivity was stronger in females than in males. There was also a leftward asymmetry in connectivity strength between the TPJ and inferior frontal gyrus, consistent with previously described lateralization of language pathways. The rightward lateralization of the pathway between the TPJ and insula supports previous findings on the roles of these regions in stimulus-driven attention, sensory awareness, interoception and pain. The findings also have implications for our understanding of acute and chronic pains and stroke-induced spatial hemineglect.     

The eicosanoid response to high dose UVR exposure of individuals prone and resistant to sunburn

High personal UVR doses can be gained during leisure activities, causing intense self-resolving inflammation (sunburn) of unprotected skin. UVR activates release of membrane fatty acids and upregulates their metabolism by cyclooxygenases (COX) and lipoxygenases (LOX) to different eicosanoids. While COX-derived prostaglandin (PG)E(2) is a potent mediator of sunburn vasodilatation, LOX-derived 15-hydroxyeicosatetraenoic acid (HETE) and its lipoxin metabolites may contribute to sunburn limitation. We explored the relationships between expression of these lipid mediators and the clinical and histological outcomes, comparing responses of individuals prone and more resistant to sunburn. An acute UVR exposure of 12 SED (standard erythema dose) was applied to buttock skin of 32 white Caucasians (n = 16 phototype I/II, n = 16 phototype III/IV), and over the subsequent 72 h assessments were made of skin erythema, immunohistochemical expression of leukocyte markers, COX-2, 12-LOX, 15-LOX and nitric oxide synthase (NOS), and eicosanoid levels by LC/ESI-MS/MS. Evidence of a significant inflammatory response was seen earlier in phototype I/II with regard to expression of erythema (4 h, p < 0.001), neutrophil infiltration (24 h, p = 0.01), epidermal COX-2 (24 h, p < 0.05) and 12-LOX (24 h, p < 0.01), and dermal eNOS (24 h, p < 0.05) proteins, although CD3+ lymphocyte infiltration showed an earlier increase in phototype III/IV (24 h, p < 0.05). Although erythema was equivalent at 72 h in both groups, phototype I/II showed higher PGE(2) accompanied by elevated 15-HETE, and a strong positive correlation was seen between these mediators (n = 18, r = 0.805, p = 0.0001). Hence anti-inflammatory eicosanoid 15-HETE may temper the pro-inflammatory milieu in sunburn, having greater influence in those prone to sunburn than those more resistant, given the same high UVR exposure conditions.     

In vivo efficacy of a phosphodiester TLR-9 aptamer and its beneficial effect in a pulmonary anthrax infection model

Immunostimulatory oligonucleotide (ISS-ODN) used as adjuvants are commonly modified with phosphorothioate (PS). The PS backbone prevents nuclease degradation, but confers undesired side effects, including systemic cytokine release. Previously, R10–60, a phosphodiester (PO) ISS-ODN, was structurally optimized as an intracellular Toll-like receptor-9 agonist. Here intravenous, intradermal and intranasal administration of PO R10–60 elicit local or adaptive immune responses with minimal systemic effects compared to a prototypic PS ISS-ODN in mice. Furthermore, prophylactic intranasal administration of PO R10–60 significantly delayed death in mice exposed to respiratory anthrax comparable to the PS ISS-ODN. The pattern of cytokine release suggested that early IL-1β production might contribute to this protective effect, which was replicated with recombinant IL-1β injections during infection. Hence, the transient effects from a PO TLR-9 agonist may be beneficial for protection in a bacterial bioterrorism attack, by delaying the onset of systemic infection without the induction of a cytokine syndrome.     

DRhoGEF2 and diaphanous regulate contractile force during segmental groove morphogenesis in the Drosophila embryo

Morphogenesis of the Drosophila embryo is associated with dynamic rearrangement of the actin cytoskeleton mediated by small GTPases of the Rho family. These GTPases act as molecular switches that are activated by guanine nucleotide exchange factors. One of these factors, DRhoGEF2, plays an important role in the constriction of actin filaments during pole cell formation, blastoderm cellularization, and invagination of the germ layers. Here, we show that DRhoGEF2 is equally important during morphogenesis of segmental grooves, which become apparent as tissue infoldings during mid-embryogenesis. Examination of DRhoGEF2-mutant embryos indicates a role for DRhoGEF2 in the control of cell shape changes during segmental groove morphogenesis. Overexpression of DRhoGEF2 in the ectoderm recruits myosin II to the cell cortex and induces cell contraction. At groove regression, DRhoGEF2 is enriched in cells posterior to the groove that undergo apical constriction, indicating that groove regression is an active process. We further show that the Formin Diaphanous is required for groove formation and strengthens cell junctions in the epidermis. Morphological analysis suggests that Dia regulates cell shape in a way distinct from DRhoGEF2. We propose that DRhoGEF2 acts through Rho1 to regulate acto-myosin constriction but not Diaphanous-mediated F-actin nucleation during segmental groove morphogenesis.     

Identification of antigens from nosocomial <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> clinical isolates in sera from ICU staff and infected patients using the antigenome technique

Nosocomial infections with a bacterial origin are considered one of the most dangerous threats to global health. Among the causes of these infections, Acinetobacter baumannii is playing a significant role, and the present study aimed? to determine the immunogenic proteins of this bacteria. Clinical isolates of A. baumannii were obtained from positive sputum cultures of intensive care unit (ICU) patients confirmed by Polymerase chain reaction (PCR) of the OXA-51 gene, and sera was obtained from 20 colonized patients. In addition, 20 and 30 serum samples were collected from ICU nurses and healthy controls, respectively. All the samples were screened in the presence of antibodies against A. baumannii by enzyme-linked immunosorbent assay (ELISA). IgG purified from the serum samples by affinity chromatography was used to isolate the bacteria by the Magnetic-activated cell sorting (MACS) procedure. After the bacteria were cultured, the identified antigen proteins were studied by western blotting and Mass spectrometry (MS). The MS results were analyzed with MASCOT software and revealed a 35 KD protein, which corresponds to outer membrane protein A (OmpA) of A. baumannii, a 25 KD band, which is a carbapenem-associated resistance protein precursor, and a 60 KD protein band, identified as a stress-induced bacterial acidophilic repeat motif protein. According to the properties of immunogen antigens and bio informatics tools, the outer membrane proteins (OMPs) can be used as a vaccine candidate in animal models.     

A Significant Reduction in the Plasma Levels and Gene Expression of CCL2 in Patients with Osteoarthritis following Intervention with Krocina™

Background:inflammatory chemokines such as CCL2 and CCL5 are involved in the progress of osteoarthritis. Crocin with antioxidant and anti-inflammatory properties can reduce the symptoms of osteoarthritis (OA). This study was performed investigate the effect of Krocina™, on the gene expressions and plasma levels of CCL2 and CCL5 in OA patients.Methods:The study included 35 patients that were randomized in the Krocina™ and placebo groups. The intervention was Krocina™ 15mg daily for four months. Clinical and paraclinical parameters were measured. CCL2 and CCL5 genes expression and plasma levels were determined using the SYBR Green Real-Time RT-PCR and Enzyme-linked Immunosorbent Assay (ELISA) techniques.Results:The C-reactive protein (CRP) value in the Krocina™ group and the visual analogue scale (VAS) value in the Krocina™ and placebo groups decreased significantly after the intervention. The gene expression of CCL2 in the Krocina™ and placebo groups decreased significantly. On the contrary, the gene expression of CCL5 in the Krocina™ and placebo groups increased significantly. Moreover, the plasma levels of CCL2 in the Krocina™ and placebo groups decreased meaningfully. There was no difference regarding the plasma levels of CCL5 within the Krocina™ and placebo groups before and after the intervention in either of the groups.Conclusion:Administration of Krocina™ reduced the clinical signs of inflammation and CRP and VAS value. Also, Krocina™ significantly decreased the plasma levels and gene expression of CCL2 in osteoarthritis patients.Key Words: CCL2, CCL5, Krocina™, Osteoarthritis