The interplay of membrane fluidity, acyl chain order and area per lipid on the partitioning of two antidepressants paroxetine and sertraline.

Selective serotonin reuptake inhibitors (SSRIs) are among the popular drugs for treating depression and mental disorders. Membrane fluidity has previously been considered as the main factor in modulating the membrane partitioning of SSRIs, while other biophysical properties, such as the acyl chain order and area per lipid, were often neglected. Varying the lipid membrane composition and temperature can significantly modify the physical phase and, in turn, affect its fluidity, acyl chain order and area per lipid. Here, we investigate the role of membrane fluidity, acyl chain order and area per lipid in the partitioning of two SSRIs, paroxetine (PAX) and sertraline (SER). The model membranes were either POPC : SM (1 : 1 mol ratio) or POPC : SM : Chol (1 : 1 : 1 mol ratio) and studied in the temperature range of 25-45 °C. The order parameters and area per lipid in the two lipid mixtures were calculated using molecular dynamics simulations. The membrane partitioning of PAX and SER was determined via second derivative spectrophotometry. In a lower temperature range (25-32 °C), membrane fluidity favors the SSRI partitioning into Lo/Ld POPC:SM:Chol. In a higher temperature range (37-45 °C), the interplay between membrane fluidity, acyl chain order and area per lipid favors drug partitioning into Ld POPC:SM. The findings offer indication for the inconsistent distribution of SSRIs in tissues as well as the possible interaction of SSRIs with lipid domains and membrane-bound proteins.

Formation of lipid raft nanodomains in homogeneous ternary lipid mixture of POPC/DPSM/cholesterol: Theoretical insights.

Lipid rafts, in biological membranes, are cholesterol-rich nanodomains that regulate many protein activities and cellular processes. Understanding the formation of the lipid-raft nanodomains helps us elucidate many complex interactions in the cell. In this study, the formation of lipid-raft nanodomains in a ternary palmitoyl-oleoyl-phosphatidylcholine/stearoyl-sphingomyelin/cholesterol (POPC/DPSM/Chol) lipid mixture, the most realistic surrogate model for biological membranes, has been successfully observed for the first time in-silico using microsecond timescale molecular dynamics simulations. The model reveals the formation of cholesterol-induced nanodomains with raft-like characteristics and their underlying mechanism: the cholesterol molecules segregate themselves into cholesterol nanodomains and then enrich the cholesterol-rich domain with sphingomyelin molecules to form a lipid raft thanks to the weak bonding of cholesterol with sphingomyelin. Besides, it is found that the increase in cholesterol concentration enhances the biophysical properties (e.g., bilayer thickness, area per lipid headgroup, and order parameter) of the lipid raft nanodomains. Such findings suggest that the POPC/DPSM/Chol bilayer is a suitable model to fundamentally extend the nanodomain evolution to investigate their lifetime and kinetics as well as to study protein-lipid interaction, protein-protein interaction, and selection of therapeutic molecules in the presence of lipid rafts.

MAPK inhibitors induce serine peptidase inhibitor Kazal type 1 (SPINK1) secretion in BRAF V600E-mutant colorectal adenocarcinoma.

The mitogen-activated protein kinase (MAPK) pathway plays a central role in colorectal cancers (CRC). In particular, BRAF V600E-mutant tumors, which represent around 10% of CRCs, are refractory to current therapies. Overexpression and secretion of serine peptidase inhibitor Kazal type 1 (SPINK1) are observed in around 50% of CRCs, and its serum level can be used as a biomarker for poor prognosis. Utilizing a recently developed extendable blocking probe assay, we analyzed the BRAF mutation status in a CRC patient cohort (N = 571) using tissue-derived RNA as the starting material. From the same RNA samples, we measured the relative SPINK1 expression levels using a quantitative real-time PCR method. Expression of mutant BRAF V600E correlated with poor prognosis, as did low expression of SPINK1 mRNA. Further, BRAF V600E correlated negatively with SPINK1 levels. In order to investigate the effect of MAPK pathway-targeted therapies on SPINK1 secretion, we conducted in vitro studies using both wild-type and V600E CRC cell lines. BRAF inhibitor vemurafenib, and subsequent MAPK pathway inhibitors trametinib and SCH772984, significantly increased SPINK1 secretion in V600E CRC cell lines Colo205 and HT-29 with a concomitant decrease in trypsin-1 and -2 secretion. Notably, no SPINK1 increase or trypsin-1 decrease was observed in BRAF wild-type CRC cell line Caco-2 in response to MAPK pathway inhibitors. In further mechanistic studies, we observed that only trametinib was able to diminish completely both MEK and ERK phosphorylation in the V600E CRC cells. Furthermore, the key regulator of integrated stress response, activating transcription factor 4 (ATF-4), was downregulated both at mRNA and at protein level in response to trametinib treatment. In conclusion, these data suggest that sustained inhibition of not only MAPK pathway activation, but also ATF-4 and trypsin, might be beneficial in the therapy of BRAF V600E-mutant CRC and that SPINK1 levels may serve as an indicator of therapy response.

Extendable blocking probe in reverse transcription for analysis of RNA variants with superior selectivity.

Here we provide the first strategy to use a competitive Extendable Blocking Probe (ExBP) for allele-specific priming with superior selectivity at the stage of reverse transcription. In order to analyze highly similar RNA variants, a reverse-transcriptase primer whose sequence matches a specific variant selectively primes only that variant, whereas mismatch priming to the alternative variant is suppressed by virtue of hybridization and subsequent extension of the perfectly matched ExBP on that alternative variant template to form a cDNA-RNA hybrid. This hybrid will render the alternative RNA template unavailable for mismatch priming initiated by the specific primer in a hot-start protocol of reverse transcription when the temperature decreases to a level where such mismatch priming could occur. The ExBP-based reverse transcription assay detected BRAF and KRAS mutations in at least 1000-fold excess of wild-type RNA and detection was linear over a 4-log dynamic range. This novel strategy not only reveals the presence or absence of rare mutations with an exceptionally high selectivity, but also provides a convenient tool for accurate determination of RNA variants in different settings, such as quantification of allele-specific expression.

Novel heat pulse extension-PCR-based method for detection of large CTG-repeat expansions in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is an autosomal-dominant disease caused by an expansion of CTG repeats in the 3' untranslated region of the Dystrophia Myotonica Protein Kinase (DMPK) gene. Detection and accurate sizing of the CTG-repeat expansions is clinically important, because the number of CTG repeats correlates with the disease severity. Because difficulties in PCR amplification over large expansions, molecular diagnosis of DM1 is still primarily based on Southern blotting, which is technically demanding and time consuming and requires large amounts of genomic DNA samples. We have recently discovered that the use of multiple heat pulses during Heat Pulse Extension PCR (HPE-PCR) enables efficient amplification over repetitive and GC-rich sequences. Based on this principle, we have developed an assay for efficient amplification of large CTG-repeat expansions seen in DM1 patients. The HPE-PCR method was able to amplify different DMPK1 repeat expansions of up to 1750 CTG repeats in 78 clinical samples with a varying degree of tissue heterogeneity, even in the presence of the short wild-type allele. The CTG-repeat lengths and fragmentation patterns obtained with HPE-PCR were fully concordant with the original diagnostic Southern blotting results. This novel technique provides a PCR-based platform for molecular diagnosis of DM1, and it has been adopted for routine diagnostic use.

Multiple heat pulses during PCR extension enabling amplification of GC-rich sequences and reducing amplification bias.

PCR amplification over GC-rich and/or long repetitive sequences is challenging because of thermo-stable structures resulting from incomplete denaturation, reannealing, and self-annealing of target sequences. These structures block the DNA polymerase during the extension step, leading to formation of incomplete extension products and favoring amplification of nonspecific products rather than specific ones. We have introduced multiple heat pulses in the extension step of a PCR cycling protocol to temporarily destabilize such blocking structures, in order to enhance DNA polymerase extension over GC-rich sequences. With this novel type of protocol, we were able to amplify all expansions of CGG repeats in five Fragile X cell lines, as well as extremely GC-rich nonrepetitive segments of the GNAQ and GP1BB genes. The longest Fragile X expansion contained 940 CGG repeats, corresponding to about 2.8 kilo bases of 100% GC content. For the GNAQ and GP1BB genes, different length PCR products in the range of 700 bases to 2 kilobases could be amplified without addition of cosolvents. As this technique improves the balance of amplification efficiencies between GC-rich target sequences of different length, we were able to amplify all of the allelic expansions even in the presence of the unexpanded allele.