19F NMR spectroscopy monitors ligand binding to recombinantly fluorine-labelled b'x from human protein disulphide isomerase (hPDI)

We report a protein-observe (19)F NMR-based ligand titration binding study of human PDI b'x with ?-somatostatin that also emphasises the need to optimise recombinant protein fluorination when using 5- or 6-fluoroindole. This study highlights a recombinant preference for 5-fluoroindole over 6-fluoroindole; most likely due to the influence of fluorine atomic packing within the folded protein structure. Fluorination affords a single (19)F resonance probe to follow displacement of the protein x-linker as ligand is titrated and provides a dissociation constant of 23 ± 4 ?M

Inactivation of mammalian Ero 1α is catalysed by specific protein disulfide isomerases

Disulfide formation within the endoplasmic reticulum is a complex process requiring a disulfide exchange protein such as protein disulfide isomerase and a mechanism to form disulfides de novo. In mammalian cells, the major pathway for de novo disulfide formation involves the enzyme Ero1α which couples oxidation of thiols to the reduction of molecular oxygen to form hydrogen peroxide. Ero1α activity is tightly regulated by a mechanism that requires the formation of regulatory disulfides. These regulatory disulfides are reduced to activate and reform to inactive the enzyme. To investigate the mechanism of inactivation we analysed regulatory disulfide formation in the presence of various oxidants under controlled oxygen concentration. Neither molecular oxygen, nor hydrogen peroxide was able to oxidise Ero1α efficiently to form the correct regulatory disulfides. However, specific members of the PDI family such as PDI or ERp46 were able to catalyse this process. Further studies showed that both active sites of PDI contribute to the formation of regulatory disulfides in Ero1α and that the PDI substrate binding domain is crucial to allow electron transfer between the two enzymes. These results demonstrate a simple feedback mechanism of regulation of mammalian Ero1α involving its primary substrate

Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway

In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a Kd of ca. 10−5 M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI's interaction with a partly-folded protein, and the first to analyze this folding catalyst's changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding – differential affinity, rapid ligand exchange and conformational flexibility

Family Problems Experienced by Students of the University of Jordan

The study has aimed at identifying the family problems among the students of the University of Jordan. The descriptive survey method has been used, and the questionnaire has been used as a tool for the study. (604) students have been selected in a stratified random manner from the University of Jordan as a sample for the study. The study results have showed that the most important and prominent family problems experienced by the University of Jordan students are: Problems in communication between family members, problems with emotional expression, lack of respect among family members, and lack of trust in the relationship with the parents. The results also showed that there are no statistically significant differences in family problems experienced by the University of Jordan students from the viewpoint of the students themselves due to the variables of gender, college, and the interaction between them both

On the Analysis of the Disproportionate Structural Collapse in RC Buildings

Increasing structural robustness is the goal which is of interest for structural engineering community. The partial collapse of RC buildings is subject of this dissertation. Understanding the robustness of RC buildings will guide the development of safer structures against abnormal loading scenarios such as; explosions, earthquakes, fine, and/or long-term accumulation effects leading to deterioration or fatigue. Any of these may result in local immediate structural damage, that can propagate to the rest of the structure causing what is known by the disproportionate collapse. This work handels collapse propagation through various analytical approaches which simplifies the mechanical description of damaged reinfoced concrete structures due to extreme acidental event

Dual Role for DsbA in Attacking and Targeted Bacterial Cells during Type VI Secretion System-Mediated Competition

Incorporation of disulfide bonds into proteins can be critical for function or stability. In bacterial cells, the periplasmic enzyme DsbA is responsible for disulfide incorporation into many extra-cytoplasmic proteins. The type VI secretion system (T6SS) is a widely occurring nanomachine that delivers toxic effector proteins directly into rival bacterial cells, playing a key role in inter-bacterial competition. We report that two redundant DsbA proteins are required for virulence and for proper deployment of the T6SS in the opportunistic pathogen Serratia marcescens, with several T6SS components being subject to the action of DsbA in secreting cells. Importantly, we demonstrate that DsbA also plays a critical role in recipient target cells, being required for the toxicity of certain incoming effector proteins. Thus we reveal that target cell functions can be hijacked by T6SS effectors for effector activation, adding a further level of complexity to the T6SS-mediated inter-bacterial interactions which define varied microbial communities

Retarded PDI diffusion and a reductive shift in poise of the calcium depleted endoplasmic reticulum

Background: Endoplasmic reticulum (ER) lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases. Lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a rapid and reversible shift towards a more reducing poise. Calcium depletion induced ER redox alterations are relevant to physiological conditions associated with calcium signaling, such as the response of pancreatic cells to secretagogues and neuronal activity. The core components of the ER redox machinery are well characterized; however, the molecular basis for the calcium-depletion induced shift in redox balance is presently obscure. Results: In vitro, the core machinery for generating disulfides, consisting of ERO1 and the oxidizing protein disulfide isomerase, PDI1A, was indifferent to variation in calcium concentration within the physiological range. However, ER calcium depletion in vivo led to a selective 2.5-fold decline in PDI1A mobility, whereas the mobility of the reducing PDI family member, ERdj5 was unaffected. In vivo, fluorescence resonance energy transfer measurements revealed that declining PDI1A mobility correlated with formation of a complex with the abundant ER chaperone calreticulin, whose mobility was also inhibited by calcium depletion and the calcium depletion-mediated reductive shift was attenuated in cells lacking calreticulin. Measurements with purified proteins confirmed that the PDI1A-calreticulin complex dissociated as Ca2+ concentrations approached those normally found in the ER lumen ([Ca2+] K-0.5max = 190 mu M). Conclusions: Our findings suggest that selective sequestration of PDI1A in a calcium depletion-mediated complex with the abundant chaperone calreticulin attenuates the effective concentration of this major lumenal thiol oxidant, providing a plausible and simple mechanism for the observed shift in ER lumenal redox poise upon physiological calcium depletion.Wellcome Trust [Wellcome 084812/Z/08/Z]; European Commission (EU FP7 Beta-Bat) [277713]; Fundacao para a Ciencia e Tecnologia, Portugal [PTDC/QUI-BIQ/119677/2010]info:eu-repo/semantics/publishedVersio

هياكل الحوكمة المحلية والاستجابات الإنسانية للزلزال في شمال غرب سوريا

This paper scrutinises the complexities of aid delivery in rebel-held areas in northern Aleppo and Idlib governorate in Syria. Looking at the response to the February 2023 earthquake that struck northern Syria, it compares the approaches of the Syrian Interim Government (SIG) and the Salvation Government (SSG) in a context of limited resources, weak governance and lack of international recognition. It highlights the significant challenges impacting the effectiveness of the early response, such as the politicisation of aid, fragmented governance and the influence of armed factions. The pivotal role of non-governmental organisations in the crisis response despite significant logistical challenges is acknowledged. The paper critiques the delayed international response and emphasises the need for improved preparedness and cooperation between local and international actors to enhance the efficacy of future disaster responses.تبحث هذه الورقة في تعقيدات إيصال المساعدات في المناطق الخاضعة لسيطرة الثوار في كلّ من شمال حلب ومحافظة إدلب في سوريا. فتنظر في الاستجابة لزلزال شباط 2023 الذي ضرب شمال سوريا، وتقارن ما بين مقاربتَي الحكومة السورية المؤقّتة وحكومة الإنقاذ السورية في سياقٍ من الموارد المحدودة، والحوكمة الضعيفة، وفي ظلّ غيابٍ للاعتراف الدولي بهما. كذلك تسلّط الورقة الضوء على التحدّيات البارزة التي تؤثّر في مدى فعالية الاستجابة المبكرة، مثل تسييس المساعدات، والحوكمة المجزَّأة، ونفوذ الفصائل المسلحة. وإذ تعترف الورقة بالدور المحوري الذي اضطّلعت به المنظمات غير الحكومية في الاستجابة للأزمة على الرغم من التحدّيات اللوجستية الكبيرة، تنتقد الاستجابة الدولية المتأخّرة، مشدّدةً على ضرورة تحسين التأهّب والتعاون بين الجهات الفاعلة المحلية والدولية بغية تعزيز فعالية الاستجابة للكوارث في المستقبل

Human brain harbors single nucleotide somatic variations in functionally relevant genes possibly mediated by oxidative stress

Somatic variation in DNA can cause cells to deviate from the preordained genomic path in both disease and healthy conditions. Here, using exome sequencing of paired tissue samples, we show that the normal human brain harbors somatic single base variations measuring up to 0.48% of the total variations. Interestingly, about 64% of these somatic variations in the brain are expected to lead to non-synonymous changes, and as much as 87% of these represent G:C>T:A transversion events. Further, the transversion events in the brain were mostly found in the frontal cortex, whereas the corpus callosum from the same individuals harbors the reference genotype. We found a significantly higher amount of 8-OHdG (oxidative stress marker) in the frontal cortex compared to the corpus callosum of the same subjects (p<0.01), correlating with the higher G:C>T:A transversions in the cortex. We found significant enrichment for axon guidance and related pathways for genes harbouring somatic variations. This could represent either a directed selection of genetic variations in these pathways or increased susceptibility of some loci towards oxidative stress. This study highlights that oxidative stress possibly influence single nucleotide somatic variations in normal human brain