Molecular insights into the transport lectin function of ERGIC-53

Secretion of proteins is an essential function of eukaryotic cells. The secretory proteins’ journey along the organelles of the exocytic pathway is initiated by the exit from the endoplasmic reticulum (ER), which defines a major rate-limiting step for protein secretion. ER-exit is subject to tight quality control. Selective, receptor-mediated cargo capture is one of the mechanisms thought to contribute to the elaborate proof-reading system of the ER. The recycling mannose lectin ERGIC-53 operates as an ER-export receptor of a subset of secretory glycoproteins. The required signals for this transport step, however, remain poorly described. Experiments in this thesis show that ERGIC-53 assisted ER-exit of procathepsin Z depends on a novel transport motif that is composed of a high-mannose type oligosaccharide and a peptide β-hairpin loop. Deletion of either determinant compromises ERGIC-53 association and slows procathepsin Z transport. An equivalent carbohydrate/hairpin structure is identified in cathepsin C, another cargo of ERGIC-53, reflecting the general nature of this ER-export signal. Further experiments reveal that the Nglycans of loop-deficient procathepsin Z become efficiently mannose 6-phosphorylated, but undergo increased carbohydrate processing in the Golgi including complex glycosylation. Strikingly, cathepsin Z lacking the peptide loop is not targeted to its normal destination, the lysosome, suggesting that it lacks the correct carbohydrate signal for lysosomal delivery. The presented data describe the first ER-exit signal on a secretory protein and establish an unexpected link between lectin-mediated export from the ER and post-Golgi sorting. This thesis also provides the molecular basis for ERGIC-53/cargo dissociation in the ERGIC. In vitro mannose binding experiments reveal that the lectin only displays its full activity at pH 7.4 – the pH of the ER – but not at slightly lower pH. The acid-sensitivity is modulated by the calcium concentration indicating a molecular link between pH-sensing and calcium complexation. This link is spotted by the identification of His178 that is conserved throughout the family of animal L-type lectins and – in its deprotonated form – binds a calcium ion in the carbohydrate recognition domain (CRD) of ERGIC-53. pH-induced inactivation of ERGIC-53 is also shown in cell culture. Glycoprotein binding is inhibited, if the ER is acidified, and the kinetics of glycoprotein dissociation are slowed, if the ERGIC is neutralized. The results establish the ERGIC as the earliest acid compartment of the secretory pathway and suggest that pH-induced glycoprotein dissociation may be backed by a mechanism that maintains lower levels of free calcium in the ERGIC. The organelles of the secretory pathway operate as intracellular calcium stores. High concentrations of calcium have been measured in the lumen of the ER and the Golgi, but the calcium concentration in the ERGIC is not known. Therefore, a strategy was developed to quantitatively assess the free calcium concentration of the ERGIC in vivo using the green fluorescent protein-based calcium-indicator yellow cameleon. Targeting of the indicator to the ERGIC is achieved by fusing it to an inert variant of the ER-Golgi SNARE Sec22b. The fusion protein dynamically localizes to the ERGIC without disturbing the function of the endogenous SNARE machinery. It will in the future provide a valuable tool for calcium measurements in the ERGIC

Clinical parameters and biomarkers predicting spontaneous operational tolerance after liver transplantation: a scoping review protocol

Objective:; This scoping review aims at systematically mapping reported prognostic factors for spontaneous immunosuppression (IS) free allograft tolerance (operational tolerance, OT) in non-viral hepatitis and non-autoimmune disease liver transplant (LT) recipients who are undergoing immunosuppression withdrawal (ISW). The results may inform the subsequent conduct of a systematic review with a more specific review question.; Background:; LT is currently the most effective treatment for end-stage liver diseases. Whereas the short-term outcomes after LT have dramatically improved over the last decades, the long-term outcomes remain unsatisfactory, mainly because of side effects of lifelong IS, such as infections, cardiovascular diseases, malignancies, and nephrotoxicity. ISW studies have shown that OT can be achieved by a subset of LT recipients and recent research has identified biomarkers of OT in these patients. However, an evidence-based selection algorithm for patients that can predictably benefit from ISW is not available to date. The planned review will, therefore, map existing knowledge on prognostic clinical parameters and biomarkers for OT.; Inclusion criteria:; We will consider studies that record any clinical parameter or biomarker before the initiation of ISW in paediatric or adult non-viral hepatitis and non-autoimmune disease LT recipients and analyse their possible association with ISW outcomes (OT or non-tolerance). Studies addressing the effectiveness of OT-inducing treatments will be excluded.; Methods:; Embase, MEDLINE, and Cochrane Library will be searched for relevant articles or conference abstracts. Full-texts of selected abstracts will be independently screened for inclusion by two reviewers. References and citing articles of included records will be screened for additional relevant records. Clinical trial registries will be searched for ongoing studies, and their investigators contacted for the sharing of unpublished data. Data from included records will be independently extracted by two reviewers using a prespecified data extraction table and presented in both tabular and narrative form

Disulphide production by Ero1alpha-PDI relay is rapid and effectively regulated

The molecular networks that control endoplasmic reticulum (ER) redox conditions in mammalian cells are incompletely understood. Here, we show that after reductive challenge the ER steady-state disulphide content is restored on a time scale of seconds. Both the oxidase Ero1alpha and the oxidoreductase protein disulphide isomerase (PDI) strongly contribute to the rapid recovery kinetics, but experiments in ERO1-deficient cells indicate the existence of parallel pathways for disulphide generation. We find PDI to be the main substrate of Ero1alpha, and mixed-disulphide complexes of Ero1 primarily form with PDI, to a lesser extent with the PDI-family members ERp57 and ERp72, but are not detectable with another homologue TMX3. We also show for the first time that the oxidation level of PDIs and glutathione is precisely regulated. Apparently, this is achieved neither through ER import of thiols nor by transport of disulphides to the Golgi apparatus. Instead, our data suggest that a dynamic equilibrium between Ero1- and glutathione disulphide-mediated oxidation of PDIs constitutes an important element of ER redox homeostasis

Using citation tracking for systematic literature searching - study protocol for a scoping review of methodological studies and a Delphi study

Background: Up-to-date guidance on comprehensive study identification for systematic reviews is crucial. According to current recommendations, systematic searching should combine electronic database searching with supplementary search methods. One such supplementary search method is citation tracking. It aims at collecting directly and/or indirectly cited and citing references from "seed references". Tailored and evidence-guided recommendations concerning the use of citation tracking are strongly needed. Objective: We intend to develop recommendations for the use of citation tracking in systematic literature searching for health-related topics. Our study will be guided by the following research questions: What is the benefit of citation tracking for systematic literature searching for health-related topics? Which methods, citation indexes, and other tools are used for citation tracking? What terminology is used for citation tracking methods? Methods: Our study will have two parts: a scoping review and a Delphi study. The scoping review aims at identifying methodological studies on the benefit and use of citation tracking in systematic literature searching for health-related topics with no restrictions on study design, language, and publication date. We will perform database searching in MEDLINE (Ovid), CINAHL (EBSCOhost), Web of Science Core Collection, two information science databases, web searching, and contact experts in the field. Two reviewers will independently perform study selection. We will conduct direct backward and forward citation tracking on included articles. Data from included studies will be extracted using a prespecified extraction sheet and presented in both tabular and narrative form. The results of the scoping review will inform the subsequent Delphi study through which we aim to derive consensus recommendations for the future practice and research of citation tracking

Citation tracking for systematic literature searching: A scoping review

Citation tracking (CT) collects references with citation relationships to pertinent references that are already known. This scoping review maps the benefit of and the tools and terminology used for CT in health-related systematic literature searching. We included methodological studies on evidence retrieval by CT in health-related literature searching without restrictions on study design, language, or publication date. We searched MEDLINE/Ovid, Web of Science Core Collection, CINAHL/EBSCOhost, LLISFT/EBSCOhost, LISTA/EBSCOhost, conducted web searching via Google Scholar, backward/forward CT of included studies and pertinent reviews, and contacting of experts. Two reviewers independently assessed eligibility. Data extraction and analysis were performed by one reviewer and checked by another. We screened 11,861 references and included 47 studies published between 1985 and 2021. Most studies (96%) assessed the benefit of CT either as supplementary or primary/stand-alone search method. Added value of CT for evidence retrieval was found by 96% of them. Science Citation Index and Social Sciences Citation Index were the most common citation indexes used. Application of multiple citation indexes in parallel, co-citing or co-cited references, CT iterations, or software tools was rare. CT terminology was heterogeneous and frequently ambiguous. The use of CT showed an added value in most of the identified studies; however, the benefit of CT in health-related systematic literature searching likely depends on multiple factors that could not be assessed with certainty. Application, terminology, and reporting are heterogeneous. Based on our results, we plan a Delphi study to develop recommendations for the use and reporting of CT

Abductor muscle strength deficit in patients after total hip arthroplasty for hip osteoarthritis: a protocol for a systematic review and meta-analysis

Conservation of abductor muscle strength is directly associated with physical function after total hip replacement (THA). Although many studies have tried to explore and quantify a potential abductor muscle strength deficit after THA as well as identify possible causes and treatment options, this topic has not been addressed systematically.; Human-based studies reporting measurements of hip abductor strength will be included in this review. Studies reporting on hip abductor strength measured manually or isometric measurements at an abduction angle other than 0° will not be considered. No restriction will be placed on study design, publication date operative approach, prosthesis design, age and sex of the patients or severity of OA. Data sources will be Embase via embase.com, Medline ALL via Ovid and the Cochrane Central Register of Controlled Trials. The preliminary search was conducted on 5 May 2019. Data regarding absolute values or torque ratio of hip abductor torque between sides as well as patient demographic data, surgical approaches and rehabilitation protocols will be extracted. The assessment of quality and risk of bias will be performed with the modified Newcastle-Ottawa Scale. The screening, data extraction and quality assessment will be performed by two reviewers independently. Where necessary, a third review author will make a final judgement. Narrative synthesis as well as tabular presentation of the extracted data will be included. Whenever possible, metaregression and subgroup specific meta-analyses will be used to investigate the influence of time since THA and type of measurement (isokinetic or isometric) on the different outcomes. In case of sufficient information, these analyses will be extended to include characteristics such as age, sex, surgical approach or rehabilitation programme.; No ethics approval is required. The results will be disseminated through peer-reviewed publications and conference presentations.; CRD42020153185

Hip abductor muscle strength in patients after total or unicompartmental knee arthroplasty for knee osteoarthritis or avascular necrosis: a systematic review and meta-analysis protocol

Reduced hip abductor strength may indirectly lead to changes in knee kinematics and functional impairment and has been reported in patients with patellofemoral pain and knee osteoarthritis (OA). Limited information is available regarding hip abductor strength following total or unicompartmental knee arthroplasty (TKA/UKA). The aims of this systematic review are to synthesise the evidence of hip abductor muscle strength deficits in patients following TKA/UKA and to determine influencing factors for these deficits.; Embase, Medline, SportDiscus, the Web of Science Core Collection and Scopus will be searched for human-based clinical studies investigating hip abductor muscle strength after TKA/UKA for knee OA or avascular necrosis (AVN). Articles studying hip abductor strength after knee arthroplasty for post-traumatic OA will not be considered. No restriction on study design, prosthesis design, surgical approach, patient characteristics or severity of OA/AVN will be applied. We will search articles published between 1 January 1990 and the date of our last search. Only articles in English or German language will be considered for inclusion. Studies reporting manually measured muscle strength or measurements performed at hip abduction angles other than 0° will be excluded. References will be screened by two reviewers independently. Where necessary, a third author will make the final decision. The assessment of quality and risk of bias will be performed with the modified Newcastle-Ottawa scale. Data will be extracted and presented in a tabular form. Depending on availability, comparable subgroup and meta-analyses will be conducted. Patient characteristics such as age, sex and surgical approach or rehabilitation programme will be analysed, if sufficient data are available.; No ethics approval is required. The results will be published in a peer-reviewed journal and as conference presentation

Identification of the PDI-Family Member ERp90 as an Interaction Partner of ERFAD

In the endoplasmic reticulum (ER), members of the protein disulfide isomerase (PDI) family perform critical functions during protein maturation. Herein, we identify the previously uncharacterized PDI-family member ERp90. In cultured human cells, we find ERp90 to be a soluble ER-luminal glycoprotein that comprises five potential thioredoxin (Trx)-like domains. Mature ERp90 contains 10 cysteine residues, of which at least some form intramolecular disulfides. While none of the Trx domains contain a canonical Cys-Xaa-Xaa-Cys active-site motif, other conserved cysteines could endow the protein with redox activity. Importantly, we show that ERp90 co-immunoprecipitates with ERFAD, a flavoprotein involved in ER-associated degradation (ERAD), through what is most likely a direct interaction. We propose that the function of ERp90 is related to substrate recruitment or delivery to the ERAD retrotranslocation machinery by ERFAD

Methodological approaches for conducting follow-up research with clinical trial participants: a scoping review and expert interviews

Evidence-based establishment and implementation of best principles, laws and ordinances that regulate clinical research depend on the consultation and involvement of trial participants. Yet, guidance on methodological approaches to obtain trial participants' perspectives is currently missing. This scoping review therefore aimed at identifying, describing and evaluating research approaches to obtain trial participants' feedback on their views and experiences.; We searched the electronic databases Medline and PsycInfo via Ovid and the Web of Science Core Collection. Clinical trials were included that involved adult participants that were conducted in selected high-income countries and that were published in peer-reviewed journals between 1985 and 2018. In addition, 29 expert interviews were conducted between March and May 2019.; Out of 5994 identified records, 23 articles were included in this review. Twelve studies used a qualitative approach, 10 were quantitative and one study used a mixed-method design. More than 75% of all work was conducted in the USA and the UK. The scoping review and the expert interviews highlighted that recruitment of participants was generally done through direct contact by principal investigators and/or study nurses or through searches in de-identified patient databases. Authors used surveys, interviews or focus group discussions. The tools used were either based on existing validated ones or developed and verified de novo with the support of experts and/or patient representatives.; To our knowledge, this is the first methodological literature review of approaches to researching experiences of clinical trial participants where findings were triangulated with expert interviews. Covering a range of indications, trial phases and study settings, it demonstrates that clinical trial participant perspectives and experience is heavily under-researched. This casts doubt on the overall robustness of available insight into trial participants' views and experiences. Our results demonstrate that the methodology for studying participant opinion, perception and experience should be adapted to the measure of interest and conform to the study population. Using valid patient experience data is the basis to evaluate existing legal and regulatory human subject research frameworks for their appropriateness from a patient perspective. Such an evaluation will be critical to empower research participants

ER-Mitochondria contact sites : a new regulator of cellular calcium flux comes into play

Endoplasmic reticulum (ER)-mitochondria membrane contacts are hotspots for calcium signaling. In this issue, Raturi et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201512077) show that the thioredoxin TMX1 inhibits the calcium pump SERCA2b at ER-mitochondria contact sites, thereby affecting ER-mitochondrial calcium transfer and mitochondrial bioenergetics