Sexual Deviations from Islamic Perspective: Sexual Harassment as An Example

This research addresses the pressing global issue of sexual harassment by examining its prevalence and underlying factors. Drawing on international data, our study reveals alarming rates of sexual harassment in various contexts, emphasizing its urgency. Employing a descriptive analysis method, the research investigates the motives and consequences of this behavioral deviation. Findings underscore the roles of education, economics, and the absence of a proper Islamic perspective on sexuality in perpetuating harassment. Educational institutions emerge as pivotal in both occurrence and prevention, while media portrayal of women and insufficient sexual education contribute to societal deviations. The study highlights the multifaceted impact of harassment on mental well-being and advocates for effective punitive measures to curb sexual deviancy and restore social harmony

HPLC-FosfoChip una nueva tecnología para el análisis selectivo de fosfopéptidos mediante LC/MS

Comunicaciones a congreso

Proteomics as a tool for live attenuated influenza vaccine characterisation

Many viral vaccines, including the majority of influenza vaccines, are grown in embryonated chicken eggs and purified by sucrose gradient ultracentrifugation. For influenza vaccines this process is well established, but the viral strains recommended for use in vaccines are updated frequently. As viral strains can have different growth properties and responses to purification, these updates risk changes in the composition of the vaccine product. Changes of this sort are hard to assess, as influenza virions are complex structures containing variable ratios of both viral and host proteins. To address this, we used liquid chromatography and tandem mass spectrometry (LC-MS/MS), a flexible and sensitive method ideally suited to identifying and quantifying the proteins present in complex mixtures. By applying LC-MS/MS to the pilot scale manufacturing process of the live attenuated influenza vaccine (LAIV) FluMist® Quadrivalent vaccine (AstraZeneca), we were able to obtain a detailed description of how viral and host proteins are removed or retained at each stage of LAIV purification. LC-MS/MS allowed us to quantify the removal of individual host proteins at each stage of the purification process, confirming that LAIV purification efficiently depletes the majority of host proteins and identifying the small subset of host proteins which are associated with intact virions. LC-MS/MS also identified substantial differences in the retention of the immunosuppressive viral protein NS1 in purified virions. Finally, LC-MS/MS allowed us to detect subtle variations in the LAIV production process, both upstream of purification and during downstream purification stages. This demonstrates the potential utility of LC-MS/MS for optimising the purification of complex biological mixtures and shows that it is a promising approach for process optimisation in a wide variety of vaccine manufacturing platforms

Uncovering viral RNA-host cell interactions on a proteome-wide scale

RNA viruses interact with a wide range of cellular RNA-binding proteins (RBPs) during their life cycle. The prevalence of these host–virus interactions has been highlighted by new methods that elucidate the composition of viral ribonucleoproteins (vRNPs). Applied to 11 viruses so far, these approaches have revealed hundreds of cellular RBPs that interact with viral (v)RNA in infected cells. However, consistency across methods is limited, raising questions about methodological considerations when designing and interpreting these studies. Here, we discuss these caveats and, through comparing available vRNA interactomes, describe RBPs that are consistently identified as vRNP components and outline their potential roles in infection. In summary, these novel approaches have uncovered a new universe of host–virus interactions holding great therapeutic potential

Peptide Orientation Affects Selectivity in Ion-Exchange Chromatography

Here we demonstrate that separation of proteolytic peptides, having the same net charge and one basic residue, is affected by their specific orientation toward the stationary phase in ion-exchange chromatography. In electrostatic repulsion−hydrophilic interaction chromatography (ERLIC) with an anion-exchange material, the C-terminus of the peptides is, on average, oriented toward the stationary phase. In cation exchange, the average peptide orientation is the opposite. Data with synthetic peptides, serving as orientation probes, indicate that in tryptic/Lys-C peptides the C-terminal carboxyl group appears to be in a zwitterionic bond with the side chain of the C-terminal Lys/Arg residue. In effect, the side chain is then less basic than the N-terminus, accounting for the specific orientation of tryptic and Lys-C peptides. Analyses of larger sets of peptides, generated from lysates by either Lys-N, Lys-C, or trypsin, reveal that specific peptide orientation affects the ability of charged side chains, such as phosphate residues, to influence retention. Phosphorylated residues that are remote in the sequence from the binding site affect retention less than those that are closer. When a peptide contains multiple charged sites, then orientation is observed to be less rigid and retention tends to be governed by the peptide’s net charge rather than its sequence. These general observations could be of value in confirming a peptide’s identification and, in particular, phosphosite assignments in proteomics analyses. More generally, orientation accounts for the ability of chromatography to separate peptides of the same composition but different sequence

The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration

Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542 of Shp2 mainly stained ALK-positive cells. In ALCL cell lines, Shp2-constitutive phosphorylation was dependent on NPM-ALK, as it significantly decreased after short hairpin RNA (shRNA)-mediated NPM-ALK knock down. In addition, only the constitutively active NPM-ALK, but not the kinase dead NPM-ALK(K210R), formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416 in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration

Doxorubicin-induced DNA Damage Causes Extensive Ubiquitination of Ribosomal Proteins Associated with a Decrease in Protein Translation

Protein posttranslational modifications (PTMs) play a central role in the DNA damage response. In particular, protein phosphorylation and ubiquitination have been shown to be essential in the signaling cascade that coordinates break repair with cell cycle progression. Here, we performed whole-cell quantitative proteomics to identify global changes in protein ubiquitination that are induced by DNA double-strand breaks. In total, we quantified more than 9,400 ubiquitin sites and found that the relative abundance of similar to 10% of these sites was altered in response to DNA double-strand breaks. Interestingly, a large proportion of ribosomal proteins, including those from the 40S as well as the 60S subunit, were ubiquitinated in response to DNA damage. In parallel, we discovered that DNA damage leads to the inhibition of ribosome function. Taken together, these data uncover the ribosome as a major target of the DNA damage response.This work is funded by a TOP-GO grant from the Netherlands Organization for Scientific Research (NWO ZonMW 912100651 to R.H.M., S.M., and V.A.H.). I.G.S. was supported with a postdoctoral fellowship from the Basque Country Government (Spain). We thank Christian Frese and Teck Yew Low for fruitful discussions. We also thank Teck Yew Low for submitting the raw files and annotated spectra to PRIDE. We thank Fabricio Loayza-Puch for his technical help with the sucrose gradients

Discovering the RNA-binding proteome of plant leaves with an improved RNA interactome capture method

RNA-binding proteins (RBPs) play a crucial role in regulating RNA function and fate. However, the full complement of RBPs has only recently begun to be uncovered through proteome-wide approaches such as RNA interactome capture (RIC). RIC has been applied to various cell lines and organisms, including plants, greatly expanding the repertoire of RBPs. However, several technical challenges have limited the efficacy of RIC when applied to plant tissues. Here, we report an improved version of RIC that overcomes the difficulties imposed by leaf tissue. Using this improved RIC method in Arabidopsis leaves, we identified 717 RBPs, generating a deep RNA-binding proteome for leaf tissues. While 75% of these RBPs can be linked to RNA biology, the remaining 25% were previously not known to interact with RNA. Interestingly, we observed that a large number of proteins related to photosynthesis associate with RNA in vivo, including proteins from the four major photosynthetic supercomplexes. As has previously been reported for mammals, a large proportion of leaf RBPs lack known RNA-binding domains, suggesting unconventional modes of RNA binding. We anticipate that this improved RIC method will provide critical insights into RNA metabolism in plants, including how cellular RBPs respond to environmental, physiological and pathological cues

Rapid Analyses of Proteomes and Interactomes Using an Integrated Solid-Phase Extraction–Liquid Chromatography–MS/MS System

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Phosphorylation of Not4p Functions Parallel to BUR2 to Regulate Resistance to Cellular Stresses in Saccharomyces cerevisiae

Background The evolutionarily conserved Ccr4-Not and Bur1/2 kinase complexes are functionally related in Saccharomyces cerevisiae. In this study, we further explore the relationship between the subunits Not4p and Bur2p. Methodology/Principal Findings First, we investigated the presence of post-translational modifications on the Ccr4-Not complex. Using mass spectrometry analyses we identified several SP/TP phosphorylation sites on its Not4p, Not1p and Caf1p subunits. Secondly, the influence of Not4p phosphorylation on global H3K4 tri-methylation status was examined by immunoblotting. This histone mark is severely diminished in the absence of Not4p or of Bur2p, but did not require the five identified Not4p phosphorylation sites. Thirdly, we found that Not4p phosphorylation is not affected by the kinase-defective bur1-23 mutant. Finally, phenotypic analyses of the Not4p phosphomutant (not4S/T5A) and bur2Δ strains showed overlapping sensitivities to drugs that abolish cellular stress responses. The double-mutant not4S/T5A and bur2Δ strain even revealed enhanced phenotypes, indicating that phosphorylation of Not4p and BUR2 are active in parallel pathways for drug tolerance. Conclusions Not4p is a phospho-protein with five identified phosphorylation sites that are likely targets of a cyclin-dependent kinase(s) other than the Bur1/2p complex. Not4p phosphorylation on the five Not4 S/T sites is not required for global H3K4 tri-methylation. In contrast, Not4p phosphorylation is involved in tolerance to cellular stresses and acts in pathways parallel to BUR2 to affect stress responses in Saccharomyces cerevisiae