We Have Always Lived in the Freak Show: Alienating the Perverse and Seeking Out the Alien in Shirley Jackson’s We Have Always Lived in the Castle

We Have Always Lived in the Castle tells the story of two sisters, Merricat and Constance Blackwood, who have been brought up by a wealthy family, most of whose members are now deceased. The family always isolated itself from the village, as evidenced by their gated mansion. In Shirley Jackson\u27s gothic novel, the villagers\u27 greed, jealousy, and patriarchal social structures cause Merricat and Constance to be perceived as a threat to the villagers\u27 egos, as the sisters challenge their ideologies with their matriarchal structure, one that keeps their riches in the hands of women who live comfortably secluded from the villagers. After the mysterious deaths of Merricat and Constance\u27s family, the villagers use their `fear\u27 of the Blackwoods to deem them monstrous and to justify their collective hatred; they also attempt to gain control over their independent family structure. Ultimately, though, the villagers are unable to assume power over the Blackwood girls as their matriarchy is not based on their material wealth but on their defiance of social norms. Their power lies in their self-isolation, rejection of conformity and male control, and self-sufficiency

Racial disparities in access to pain management services during COVID pandemic at an urban safety-net hospital

Background: Race has been identified as one of the great divisions of mankind, associated with differences in healthcare access in the US. The COVID pandemic has shed light on a variety of disparities, including access to pain management services. Method: All the adult patients who were referred to the pain clinic at Boston Medical Center (BMC) from December 2019- December 2020 were included in this study. A total of 2023 cases (1243 White Race and 790 People of Color) were recruited. Result: Patients of WR received care at a younger age. COVID-19 increased the average age of WR by 2.5 years, while it didn’t affect POC. The average gap between the date of the consult and the procedure was not significantly different between the 2 groups. It also was not significantly different pre-COVID and post-COVID. There were no differences in the racial makeup of the referred patients before and after the pandemic, although the percentage of patients being POC (39.3%) was significantly lower than the racial makeup of the representing population (47.18%). Patients of WR were significantly more likely to have completed visits (66.3%) than POC (60.5%). Conclusion: Our study demonstrated disparities between the WR and POC that were evident even before starting the COVID-19 pandemic. It also showed that these disparities continued to be the same as before COVID-19. Although many disparities are rooted in the society itself, we noticed that in numerous instances, pain management services were offered equally at our institution. We suggest that multiracial administrative staff living in the same community and hospital-sponsored support systems were the main contributors

Detection of Crosslinks within and between Proteins by LC-MALDI-TOFTOF and the Software FINDX to Reduce the MSMS-Data to Acquire for Validation

Lysine-specific chemical crosslinking in combination with mass spectrometry is emerging as a tool for the structural characterization of protein complexes and protein-protein interactions. After tryptic digestion of crosslinked proteins there are thousands of peptides amenable to MSMS, of which only very few are crosslinked peptides of interest. Here we describe how the advantage offered by off-line LC-MALDI-TOF/TOF mass spectrometry is exploited in a two-step workflow to focus the MSMS-acquisition on crosslinks mainly. In a first step, MS-data are acquired and all the peak list files from the LC-separated fractions are merged by the FINDX software and screened for presence of crosslinks which are recognized as isotope-labeled doublet peaks. Information on the isotope doublet peak mass and intensity can be used as search constraints to reduce the number of false positives that match randomly to the observed peak masses. Based on the MS-data a precursor ion inclusion list is generated and used in a second step, where a restricted number of MSMS-spectra are acquired for crosslink validation. The decoupling of MS and MSMS and the peptide sorting with FINDX based on MS-data has the advantage that MSMS can be restricted to and focused on crosslinks of Type 2, which are of highest biological interest but often lowest in abundance. The LC-MALDI TOF/TOF workflow here described is applicable to protein multisubunit complexes and using 14N/15N mixed isotope strategy for the detection of inter-protein crosslinks within protein oligomers

The extracellular matrix and insulin resistance

The extracellular matrix (ECM) is a highly dynamic compartment that undergoes remodeling as a result of injury and repair. Over the past decade, mounting evidence in humans and rodents suggest that ECM remodeling is associated with diet-induced insulin resistance in several metabolic tissues. Additionally, integrin receptors for the ECM have also been implicated in the regulation of insulin action. This review will address what is currently known about the ECM, integrins and insulin action in the muscle, liver and adipose tissue. Understanding how ECM remodeling and integrin signaling regulates insulin action may aid in the development of new therapeutic targets for the treatment of insulin resistance and type 2 diabetes

Integrin β3 Crosstalk with VEGFR Accommodating Tyrosine Phosphorylation as a Regulatory Switch

Integrins mediate cell adhesion, migration, and survival by connecting intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the importance of the interaction between β3 integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. Here we present in vitro evidence of the direct association between the cytoplasmic tails (CTs) of β3 and VEGFR2. Specifically, the membrane-proximal motif around 801YLSI in VEGFR2 mediates its binding to non-phosphorylated β3CT, accommodating an α-helical turn in integrin bound conformation. We also show that Y747 phosphorylation of β3 enhances the above interaction. To demonstrate the importance of β3 phosphorylation in endothelial cell functions, we synthesized β3CT-mimicking Y747 phosphorylated and unphosphorylated membrane permeable peptides. We show that a peptide containing phospho-Y747 but not F747 significantly inhibits VEGF-induced signaling and angiogenesis. Moreover, phospho-Y747 peptide exhibits inhibitory effect only in WT but not in β3 integrin knock-out or β3 integrin knock-in cells expressing β3 with two tyrosines substituted for phenylalanines, demonstrating its specificity. Importantly, these peptides have no effect on fibroblast growth factor receptor signaling. Collectively these data provide novel mechanistic insights into phosphorylation dependent cross-talk between integrin and VEGFR2

High-resolution structure determination of the CylR2 homodimer using paramagnetic relaxation enhancement and structure-based prediction of molecular alignment

Structure determination of homooligomeric proteins by NMR spectroscopy is difficult due to the lack of chemical shift perturbation data, which is very effective in restricting the binding interface in heterooligomeric systems, and the difficulty of obtaining a sufficient number of intermonomer distance restraints. Here we solved the high-resolution solution structure of the 15.4 kDa homodimer CylR2, the regulator of cytolysin production from Enterococcus faecalis, which deviates by 1.1 Å from the previously determined X-ray structure. We studied the influence of different experimental information such as long-range distances derived from paramagnetic relaxation enhancement, residual dipolar couplings, symmetry restraints and intermonomer Nuclear Overhauser Effect restraints on the accuracy of the derived structure. In addition, we show that it is useful to combine experimental information with methods of ab initio docking when the available experimental data are not sufficient to obtain convergence to the correct homodimeric structure. In particular, intermonomer distances may not be required when residual dipolar couplings are compared to values predicted on the basis of the charge distribution and the shape of ab initio docking solutions

Structure and Novel Functional Mechanism of Drosophila SNF in Sex-Lethal Splicing

Sans-fille (SNF) is the Drosophila homologue of mammalian general splicing factors U1A and U2B″, and it is essential in Drosophila sex determination. We found that, besides its ability to bind U1 snRNA, SNF can also bind polyuridine RNA tracts flanking the male-specific exon of the master switch gene Sex-lethal (Sxl) pre-mRNA specifically, similar to Sex-lethal protein (SXL). The polyuridine RNA binding enables SNF directly inhibit Sxl exon 3 splicing, as the dominant negative mutant SNF1621 binds U1 snRNA but not polyuridine RNA. Unlike U1A, both RNA recognition motifs (RRMs) of SNF can recognize polyuridine RNA tracts independently, even though SNF and U1A share very high sequence identity and overall structure similarity. As SNF RRM1 tends to self-associate on the opposite side of the RNA binding surface, it is possible for SNF to bridge the formation of super-complexes between two introns flanking Sxl exon 3 or between a intron and U1 snRNP, which serves the molecular basis for SNF to directly regulate Sxl splicing. Taken together, a new functional model for SNF in Drosophila sex determination is proposed. The key of the new model is that SXL and SNF function similarly in promoting Sxl male-specific exon skipping with SNF being an auxiliary or backup to SXL, and it is the combined dose of SXL and SNF governs Drosophila sex determination

Regulation of RhoGEF activity by intramolecular and intermolecular SH3 domain interactions

RhoGEFs are central controllers of small G-proteins in cells and are regulated by several mechanisms. There are at least 22 human RhoGEFs that contain SH3 domains, raising the possibility that, like several other enzymes, SH3 domains control the enzymatic activity of guanine nucleotide exchange factor (GEF) domains through intra- and/or intermolecular interactions. The structure of the N-terminal SH3 domain of Kalirin was solved using NMR spectroscopy, and it folds much like other SH3 domains. However, NMR chemical shift mapping experiments showed that this Kalirin SH3 domain is unique, containing novel cooperative binding site(s) for intramolecular PXXP ligands. Intramolecular Kalirin SH3 domain/ligand interactions, as well as binding of the Kalirin SH3 domain to the adaptor protein Crk, inhibit the GEF activity of Kalirin. This study establishes a novel molecular mechanism whereby intramolecular and intermolecular Kalirin SH3 domain/ligand interactions modulate GEF activity, a regulatory mechanism that is likely used by other RhoGEF family members