Averyebbesen8522
Current models of gene expression, which are based on single-molecule localization microscopy, acknowledge protein clustering and the formation of transcriptional condensates as a driving force of gene expression. However, these models largely omit the role of nuclear lipids and amongst them nuclear phosphatidylinositol phosphates (PIPs) in particular. Moreover, the precise distribution of nuclear PIPs in the functional sub-nuclear domains remains elusive. The direct stochastic optical reconstruction microscopy (dSTORM) provides an unprecedented resolution in biological imaging. Therefore, its use for imaging in the densely crowded cell nucleus is desired but also challenging. Here we present a dual-color dSTORM imaging and image analysis of nuclear PI(4,5)P2, PI(3,4)P2 and PI(4)P distribution while preserving the context of nuclear architecture. In the nucleoplasm, PI(4,5)P2 and PI(3,4)P2 co-pattern in close proximity with the subset of RNA polymerase II foci. PI(4,5)P2 is surrounded by fibrillarin in the nucleoli and all three PIPs are dispersed within the matrix formed by the nuclear speckle protein SON. PI(4,5)P2 is the most abundant nuclear PIP, while PI(4)P is a precursor for the biosynthesis of PI(4,5)P2 and PI(3,4)P2. Therefore, our data are relevant for the understanding the roles of nuclear PIPs and provide further evidence for the model in which nuclear PIPs represent a localization signal for the formation of lipo-ribonucleoprotein hubs in the nucleus. The discussed experimental pipeline is applicable for further functional studies on the role of other nuclear PIPs in the regulation of gene expression and beyond.Human membrane bound O-acyltransferase domain-containing 7 (MBOAT7), also known as lysophosphatidylinositol acyltransferase 1 (LPIAT1), is an enzyme involved in the acyl-chain remodeling of phospholipids via the Lands' cycle. The MBOAT7 rs641738 variant has been associated with the entire spectrum of fatty liver disease (FLD) and neurodevelopmental disorders, but the exact enzymatic activity and the catalytic site of the protein are still unestablished. Human wild type MBOAT7 and three MBOAT7 mutants missing in the putative catalytic residues (N321A, H356A, N321A + H356A) were produced into Pichia pastoris, and purified using Ni-affinity chromatography. The enzymatic activity of MBOAT7 wild type and mutants was assessed measuring the incorporation of radiolabeled fatty acids into lipid acceptors. MBOAT7 preferentially transferred 204 and 205 polyunsaturated fatty acids (PUFAs) to lysophosphatidylinositol (LPI). On the contrary, MBOAT7 showed weak enzymatic activity for transferring saturated and unsaturated fatty acids, regardless the lipid substrate. Missense mutations in the putative catalytic residues (N321A, H356A, N321A + H356A) result in a loss of O-acyltransferase activity. Thus, MBOAT7 catalyzes the transfer of PUFAs to lipid acceptors. MBOAT7 shows the highest affinity for LPI, and missense mutations at the MBOAT7 putative catalytic dyad inhibit the O-acyltransferase activity of the protein. Our findings support the hypothesis that the association between the MBOAT7 rs641738 variant and the increased risk of NAFLD is mediated by changes in the hepatic phosphatidylinositol acyl-chain remodeling. Taken together, the increased knowledge of the enzymatic activity of MBOAT7 gives insights into the understanding on the basis of FLD.
Multimodal monitoring of intracranial pressure and brain tissue oxygen tension (PbtO
) have been increasingly used to detect delayed cerebral ischemia (DCI) after subarachnoid hemorrhage. At our center, patients who cannot be easily assessed clinically will undergo intracranial pressure and PbtO
monitoring via a NEUROVENT-PTO bolt. We aimed to determine whether the Lindegaard ratios (LRs) computed from transcranial Doppler ultrasonography (TCDU) would correlate with, or can predict, the simultaneously recorded PbtO
value.
Patients with aneurysmal subarachnoid hemorrhage, PbtO
recordings from the middle cerebral artery territory, and simultaneous TCDU scans available from the ipsilateral middle cerebral artery and internal carotid artery from August 2018 to 2019 were included in the present study. The index test result was vasospasm (LR of ≥3) found on TCDU. The reference standard was the presence of regional hypoxia (PbtO
<20 mm Hg). bpV chemical structure The PbtO
results were compared with those from computed toymal probe). Therefore, it could be a useful and noninvasive tool in the context of preventative DCI monitoring. However, given the low sensitivity, the lack of vasospasm on TCDU should not preclude the possibility of the presence of evolving DCI.Mitochondrial dysfunction has emerged to be associated with a broad spectrum of diseases, and there is an increasing demand for accurate detection of mitochondrial DNA (mtDNA) variants. Whole genome sequencing (WGS) has been the dominant sequencing approach to identify genetic variants in recent decades, but most studies focus on variants on the nuclear genome. Whole genome sequencing is also costly and time consuming. Sequencing specifically targeted for mtDNA is commonly used in the diagnostic settings and has lower costs. However, there is a lack of pairwise comparisons between these two sequencing approaches for calling mtDNA variants on a population basis. In this study, we compared WGS and mtDNA-targeted sequencing (targeted-seq) in analyzing mitochondrial DNA from 1499 participants recruited into the Severe Asthma Research Program (SARP). Our study reveals that targeted-sequencing and WGS have comparable capacity to determine genotypes and to call haplogroups and homoplasmies on mtDNA. However, there exists a large variability in calling heteroplasmies, especially for low-frequency heteroplasmies, which indicates that investigators should be cautious about heteroplasmies acquired from different sequencing methods. Further research is highly desired to improve variant detection methods for mitochondrial DNA.
Candida albicans is a yeast that causes fungal infections with high mortality and is typically resistant to azole drugs. To overcome this resistance, we explored the combined use of oridonin (ORI) and three azole drugs, namely fluconazole (FLC), itraconazole (ITR) and voriconazole (VOR). Azole-resistant C. albicans strains were obtained from cancer patients and the reversal of drug resistance in these strains was investigated.
The synergistic antifungal activity of ORI and azole drugs was measured by checkerboard microdilution and time-kill assays. The resistance reversal mechanisms, namely inhibition of drug efflux and induction of apoptosis, were investigated by flow cytometry. Expression levels of the efflux pump-related genesCDR1 and CDR2 were assessed by RT-qPCR.
The efflux pump inhibition assay with ORI showed that the minimum inhibitory concentrations (MICs) of FLC (128-fold), ITR (64-fold) and VOR (250-fold) decreased significantly. Upregulation of genes encodingCDR1 and CDR2 was confirmed in the resistant strain.
