Extensive chromatin fragmentation improves enrichment of protein binding sites in chromatin immunoprecipitation experiments

Extensive sonication of formaldehyde-crosslinked chromatin can generate DNA fragments averaging 200 bp in length (range 75–300 bp). Fragmentation is largely random with respect to genomic region and nucleosome position. ChIP experiments employing such extensively fragmented samples show 2- to 4-fold increased enrichment of protein binding sites over control genomic regions, when compared to samples sonicated to a more conventional size range (300–500 bp). The basis of improved fold enrichments is that immunoprecipitation of protein-bound regions is unaffected by fragment size, whereas immunoprecipitation of control genomic regions decreases progressively along with reduced fragment size due to fewer nonspecific binding sites. The use of extensively sonicated samples improves mapping of protein binding sites, and it extends the dynamic range for quantitative measurements of histone density. We show that many yeast promoter regions are virtually devoid of histones

Cross-validation of ELISA and a portable surface plasmon resonance instrument for IgG antibody serology with SARS-CoV-2 positive individuals.

We report on the development of surface plasmon resonance (SPR) sensors and matching ELISAs for the detection of nucleocapsid and spike antibodies specific to the novel coronavirus 2019 (SARS-CoV-2) in human serum, plasma and dried blood spots (DBS)

Significance testing testate amoeba water table reconstructions

Transfer functions are valuable tools in palaeoecology, but their output may not always be meaningful. A recently-developed statistical test ('randomTF') offers the potential to distinguish among reconstructions which are more likely to be useful, and those less so. We applied this test to a large number of reconstructions of peatland water table depth based on testate amoebae. Contrary to our expectations, a substantial majority (25 of 30) of these reconstructions gave non-significant results (P > 0.05). The underlying reasons for this outcome are unclear. We found no significant correlation between randomTF P-value and transfer function performance, the properties of the training set and reconstruction, or measures of transfer function fit. These results give cause for concern but we believe it would be extremely premature to discount the results of non-significant reconstructions. We stress the need for more critical assessment of transfer function output, replication of results and ecologically-informed interpretation of palaeoecological data

A new class of glycomimetic drugs to prevent free fatty acid-induced endothelial dysfunction

Background: Carbohydrates play a major role in cell signaling in many biological processes. We have developed a set of glycomimetic drugs that mimic the structure of carbohydrates and represent a novel source of therapeutics for endothelial dysfunction, a key initiating factor in cardiovascular complications. Purpose: Our objective was to determine the protective effects of small molecule glycomimetics against free fatty acid­induced endothelial dysfunction, focusing on nitric oxide (NO) and oxidative stress pathways. Methods: Four glycomimetics were synthesized by the stepwise transformation of 2,5­dihydroxybenzoic acid to a range of 2,5­substituted benzoic acid derivatives, incorporating the key sulfate groups to mimic the interactions of heparan sulfate. Endothelial function was assessed using acetylcholine­induced, endotheliumdependent relaxation in mouse thoracic aortic rings using wire myography. Human umbilical vein endothelial cell (HUVEC) behavior was evaluated in the presence or absence of the free fatty acid, palmitate, with or without glycomimetics (1µM). DAF­2 and H2DCF­DA assays were used to determine nitric oxide (NO) and reactive oxygen species (ROS) production, respectively. Lipid peroxidation colorimetric and antioxidant enzyme activity assays were also carried out. RT­PCR and western blotting were utilized to measure Akt, eNOS, Nrf­2, NQO­1 and HO­1 expression. Results: Ex vivo endothelium­dependent relaxation was significantly improved by the glycomimetics under palmitate­induced oxidative stress. In vitro studies showed that the glycomimetics protected HUVECs against the palmitate­induced oxidative stress and enhanced NO production. We demonstrate that the protective effects of pre­incubation with glycomimetics occurred via upregulation of Akt/eNOS signaling, activation of the Nrf2/ARE pathway, and suppression of ROS­induced lipid peroxidation. Conclusion: We have developed a novel set of small molecule glycomimetics that protect against free fatty acidinduced endothelial dysfunction and thus, represent a new category of therapeutic drugs to target endothelial damage, the first line of defense against cardiovascular disease

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Mapping accessible chromatin regions using Sono-Seq

Disruptions in local chromatin structure often indicate features of biological interest such as regulatory regions. We find that sonication of cross-linked chromatin, when combined with a size-selection step and massively parallel short-read sequencing, can be used as a method (Sono-Seq) to map locations of high chromatin accessibility in promoter regions. Sono-Seq sites frequently correspond to actively transcribed promoter regions, as evidenced by their co-association with RNA Polymerase II ChIP regions, transcription start sites, histone H3 lysine 4 trimethylation (H3K4me3) marks, and CpG islands; signals over other sites, such as those bound by the CTCF insulator, are also observed. The pattern of breakage by Sono-Seq overlaps with, but is distinct from, that observed for FAIRE and DNase I hypersensitive sites. Our results demonstrate that Sono-Seq can be a useful and simple method by which to map many local alterations in chromatin structure. Furthermore, our results provide insights into the mapping of binding sites by using ChIP–Seq experiments and the value of reference samples that should be used in such experiments

Cross-Validation of ELISA and a Portable Surface Plasmon Resonance Instrument for IgG Antibodies Serology with SARS-CoV-2 Positive Individuals

We report on the development of surface plasmon resonance (SPR) sensors and matching ELISAs for the detection of nucleocapsid and spike antibodies specific against the novel coronavirus 2019 (SARS-CoV-2) in human serum, plasma and dried blood spots (DBS). When exposed to SARS-CoV-2 or a vaccine against SARS-CoV-2, the immune system responds by expressing antibodies at levels that can be detected and monitored to identify the fraction of the population potentially immunized against SARS-CoV-2 and support efforts to deploy a vaccine strategically. A SPR sensor coated with a peptide monolayer and functionalized with various sources of SARS-CoV-2 recombinant proteins expressed in different cell lines detected human anti-SARS-CoV-2 IgG in the nanomolar range. Nucleocapsid expressed in different cell lines did not significantly change the sensitivity of the assays, whereas the use of a CHO cell line to express spike ectodomain led to excellent performance. This bioassay was performed on a portable SPR instrument capable of measuring 4 biological samples within 30 minutes of sample/sensor contact and the chip could be regenerated at least 9 times. Multi-site validation was then performed with in-house and commercial ELISA, which revealed excellent cross-correlations with Pearson’s coefficients exceeding 0.85 in all cases, for measurements in DBS and plasma. This strategy paves the way to point-of-care and rapid testing for antibodies in the context of viral infection and vaccine efficacy monitoring