A simple, no-wash cell adhesion-based high-throughput assay for the discovery of small-molecule regulators of the integrin CD11b/CD18

The leukocyte-specific integrin CD11b/CD18 plays a key role in the biological function of these cells and represents a validated therapeutic target for inflammatory diseases. Currently, the low affinity interaction between CD11b/CD18 integrin and its respective ligand poses a challenge in the development of cell-based adhesion assays for the high-throughput screening (HTS) environment. Here the authors describe a simple cell-based adhesion assay that can be readily used for HTS for the discovery of functional regulators of CD11b/CD18. The assay consistently produces acceptable Z' values (> 0.5) for HTS. After testing the assay using 2 established blocking antibodies as reference biologicals, the authors performed a proof-of-concept primary screen using a library of 6612 compounds and identified both agonist and antagonist hits.     

ABCA1, ABCG1 and SR-BI: hormonal regulation in primary rat hepatocytes and human cell lines

ATM and PARP-1 are two of the most important players in the cell's response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after γ-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced γ-H2AX foci formation in response to γ-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced γ H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase.     

Fibrillarin, a nucleolar protein, is required for normal nuclear morphology and cellular growth in HeLa cells

Fibrillarin is a key small nucleolar protein in eukaryotes, which has an important role in pre-rRNA processing during ribosomal biogenesis. Though several functions of fibrillarin are known, its function during the cell cycle is still unknown. In this study, we confirmed the dynamic localization of fibrillarin during the cell cycle of HeLa cells and also performed functional studies by using a combination of immunofluorescence microscopy and RNAi technique. We observed that depletion of fibrillarin has almost no effect on the nucleolar structure. However, fibrillarin-depleted cells showed abnormal nuclear morphology. Moreover, fibrillarin depletion resulted in the reduction of the cellular growth and modest accumulation of cells with 4n DNA content. Our data suggest that fibrillarin would play a critical role in the maintenance of nuclear shape and cellular growth.     

Dual role of mesenchymal stem/stromal cells and their cell-free extracellular vesicles in colorectal cancer

Colorectal cancer (CRC) is one of the main causes of cancer-related deaths. However, the surgical control of the CRC progression is difficult, and in most cases, the metastasis leads to cancer-related mortality. Mesenchymal stem/stromal cells (MSCs) with potential translational applications in regenerative medicine have been widely researched for several years. MSCs could affect tumor development through secreting exosomes. The beneficial properties of stem cells are attributed to their cell–cell interactions as well as the secretion of paracrine factors in the tissue microenvironment. For several years, exosomes have been used as a cell-free therapy to regulate the fate of tumor cells in a tumor microenvironment. This review discusses the recent advances and current understanding of assessing MSC-derived exosomes for possible cell-free therapy in CRC.     

Kaolin and Jasmonic acid improved cotton productivity under water stress conditions

Drought is one of the most emerging threat that causes a severe reduction in cotton plant growth and development. Being cotton is a major cash crop has great threat to prevailing drought events in Pakistan. A field experiment was conducted in Kharif season 2018 at Research Area of MNS-University of Agriculture, Multan, Pakistan to assess the role of foliar applied kaolin and jasmonic acid on vegetative growth, gas exchange and reproductive traits of cotton under normal irrigated and artificial water deficit conditions. The experiment was laid -out in a factorial randomized complete block design with split – split plot arrangement. Main plots were allocated for irrigation levels, sub-plots for two -cotton genotypes viz. NIAB − 878 and SLH − 19 while sub – sub plots for treatments of kaolin and Jasmonic acid. Water deficit stress was created by skipping irrigation at flowering for 21 days. Foliar sprays of Kaolin (5%, w/v) and Jasmonic acid (100 μM) were applied alone or in combination at 60 days after planntinon both to normal irrigated and water-stresse skip irrigation while irrigation water alone was sprayed in control plots. Both cotton genotypes responded variably to normal irrigated and skip conditions. Skipping irrigation for up to 21 days at flowering caused a significant decrease in leaf relative water content, SPAD values, net photosynthetic rate and seed cotton yield in both the genotypes. Seed cotton yield showed an overall decline of 24.7% in skip over Normal irrigated crop. The genotype NIAB − 878 produced maximum seed cotton yield of 3.304 Mg ha⁻¹ in normal that dropped to 2.579 Mg ha⁻¹ in skip, thus showing an average decline of 21.9 %. Similarly, SLH − 19 produced 2.537 Mg ha⁻¹ seed cotton under normal that dropped to 1.822 Mg ha⁻¹ in skip, showing an average decline of 28.2%. The Application of Kaolin and JA Jasmonic acid, either applied individually or in combination, improved vegetative and reproductive development of both cotton varieties in normal and skip regimes. However, combined kaolin and Jasmonic Acid application proved to be more beneficial in terms of seed cotton production and other parameters studied.     

LncRNA‐miRNA network analysis across the Th17 cell line reveals biomarker potency of lncRNA NEAT1 and KCNQ1OT1 in multiple sclerosis

Differentiation of CD⁴⁺ T cells into Th17 cells is an important factor in the onset and progression of multiple sclerosis (MS) and Th17/Treg imbalance. Little is known about the role of lncRNAs in the differentiation of CD⁴⁺ cells from Th17 cells. This study aimed to analyse the lncRNA‐miRNAs network involved in MS disease and its role in the differentiation of Th17 cells. The lncRNAs in Th17 differentiation were obtained from {"type":"entrez-geo","attrs":{"text":"GSE66261","term_id":"66261"}}GSE66261 using the GEO datasets. Differential expression of lncRNAs in Th17 primary cells compared to Th17 effector cells was investigated by RNA‐seq analysis. Next, the most highlighted lncRNAs in autoimmune diseases were downloaded from the lncRNAs disease database, and the most critical miRNA was extracted by literature search. Then, the lncRNA‐miRNA interaction was achieved by the Starbase database, and the ceRNA network was designed by Cytoscape. Finally, using the CytoHubba application, two hub lncRNAs with the most interactions with miRNAs were identified by the MCODE plug‐in. The expression level of genes was measured by qPCR, and the plasma level of cytokines was analysed by ELISA kits. The results showed an increase in the expression of NEAT1, KCNQ1OT1 and RORC and a decrease in the expression of FOXP3. In plasma, an upregulation of IL17 and a downregulation of TGFB inflammatory cytokines were detected. The dysregulated expression of these genes could be attributed to relapsing‐remitting MS (RR‐MS) patients and help us understand MS pathogenesis better.     

Solution structure of the (+)-cis-anti-benzo[a]pyrene-dA ([BP]dA) adduct opposite dT in a DNA duplex.

Minor adducts, derived from the covalent binding of anti-benzo[a]pyrene-7,8-dihydroxy-9,10-epoxide to cellular DNA, may play an important role in generating mutations and initiating cancer. We have applied a combined NMR-computational approach including intensity based refinement to determine the solution structure of the minor (+)-cis-anti-[BP]dA adduct positioned opposite dT in the d(C1-T2-C3-T4-C5-[BP]A6-C7-T8-T9-C10-C11). (d(G12-G13-A14-A15-G16-T17-G18-A19-G20+ ++-A21-G22) 11-mer duplex. The BP ring system is intercalated toward the 5'-side of the [BP]dA6 lesion site without disrupting the flanking Watson-Crick dC5.dG18 and [BP]dA6.dT17 base pairs. This structure of the (+)-cis-anti-[BP]dA.dT 11-mer duplex, containing a bay region benzo[a]pyrenyl [BP]dA adduct, is compared with the corresponding structure of the (+)-trans-anti-[BPh]dA.dT 11-mer duplex (Cosman et al., Biochemistry 32, 12488-12497, 1993), which contains a fjord region benzo[c]phenanthrenyl [BPh]dA adduct with the same R stereochemistry at the linkage site. The carcinogen intercalates toward the 5'-direction of the modified strand in both duplexes (the adduct is embedded within the same sequence context) with the buckling of the Watson-Crick [BP]dA6.dT17 base pair more pronounced in the (+)-cis-anti-[BP]dA.dT 11-mer duplex compared to its Watson-Crick [BPh]dA.dT17 base pair in the (+)-trans-anti-[BPh]dA.dT 11-mer duplex. The available structural studies of covalent polycyclic aromatic hydrocarbon (PAH) carcinogen-DNA adducts point toward the emergence of a general theme where distinct alignments are adopted by PAH adducts covalently linked to the N(6) of adenine when compared to the N(2) of guanine in DNA duplexes. The [BPh]dA and [BP]dA N(6)-adenine adducts intercalate their polycyclic aromatic rings into the helix without disruption of their modified base pairs. This may reflect the potential flexibility associated with the positioning of the covalent tether and the benzylic ring of the carcinogen in the sterically spacious major groove. By contrast, such an intercalation without modified base pair disruption option appears not to be available to [BP]dG N(2)-guanine adducts where the covalent tether and the benzylic ring are positioned in the more sterically crowded minor groove. In the case of [BP]dG adducts, the benzopyrenyl ring is either positioned in the minor groove without base pair disruption, or if intercalated into the helix, requires disruption of the modified base pair and displacement of the bases out of the helix.