Structural basis of unidirectional export of lipopolysaccharide to the cell surface.

Gram-negative bacteria are surrounded by an inner cytoplasmic membrane and by an outer membrane, which serves as a protective barrier to limit entry of many antibiotics. The distinctive properties of the outer membrane are due to the presence of lipopolysaccharide1. This large glycolipid, which contains numerous sugars, is made in the cytoplasm; a complex of proteins forms a membrane-to-membrane bridge that mediates transport of lipopolysaccharide from the inner membrane to the cell surface1. The inner-membrane components of the protein bridge comprise an ATP-binding cassette transporter that powers transport, but how this transporter ensures unidirectional lipopolysaccharide movement across the bridge to the outer membrane is unknown2. Here we describe two crystal structures of a five-component inner-membrane complex that contains all the proteins required to extract lipopolysaccharide from the membrane and pass it to the protein bridge. Analysis of these structures, combined with biochemical and genetic experiments, identifies the path of lipopolysaccharide entry into the cavity of the transporter and up to the bridge. We also identify a protein gate that must open to allow movement of substrate from the cavity onto the bridge. Lipopolysaccharide entry into the cavity is ATP-independent, but ATP is required for lipopolysaccharide movement past the gate and onto the bridge. Our findings explain how the inner-membrane transport complex controls efficient unidirectional transport of lipopolysaccharide against its concentration gradient.

Determination of Initial Rates of Lipopolysaccharide Transport.

Nonvesicular lipid trafficking pathways are an important process in every domain of life. The mechanisms of these processes are poorly understood in part due to the difficulty in kinetic characterization. One important class of glycolipids, lipopolysaccharides (LPS), are the primary lipidic component of the outer membrane of Gram-negative bacteria. LPS are synthesized in the inner membrane and then trafficked to the cell surface by the lipopolysaccharide transport proteins, LptB2FGCADE. By characterizing the interaction of a fluorescent probe and LPS, we establish a quantitative assay to monitor the flux of LPS between proteoliposomes on the time scale of seconds. We then incorporate photocaged ATP into this system, which allows for light-based control of the initiation of LPS transport. This control allows us to measure the initial rate of LPS transport (3.0 min-1 per LptDE). We also find that the rate of LPS transport by the Lpt complex is independent of the structure of LPS. In contrast, we find the rate of LPS transport is dependent on the proper function of the LptDE complex. Mutants of the outer membrane Lpt components, LptDE, that cause defective LPS assembly in live cells display attenuated transport rates and slower ATP hydrolysis compared to wild type proteins. Analysis of these mutants reveals that the rates of ATP hydrolysis and LPS transport are correlated such that 1.2 ± 0.2 ATP are hydrolyzed for each LPS transported. This correlation suggests a model where the outer membrane components ensure the coupling of ATP hydrolysis and LPS transport by stabilizing a transport-active state of the Lpt bridge.

Association Between Fibrosis Stage and Outcomes of Patients With Nonalcoholic Fatty Liver Disease: A Systematic Review and Meta-Analysis.

BACKGROUND & AIMS: Biopsy-confirmed liver fibrosis is a prognostic factor for patients with nonalcoholic fatty liver disease (NAFLD). We performed a systematic review to quantify the prognostic value of fibrosis stage in patients with NAFLD and the subgroup of patients with nonalcoholic steatohepatitis (NASH) and to assess the evidence that change in fibrosis stage is a surrogate endpoint. METHODS: We searched the MEDLINE, Embase, Cochrane Library, and trial registry databases through August 2018 for prospective or retrospective cohort studies of liver-related clinical events and outcomes in adults with NAFLD or NASH. We collected data on mortality (all cause and liver related) and morbidity (cirrhosis, liver cancer, and all liver-related events) by stage of fibrosis, determined by biopsy, for patients with NAFLD or NASH. Using fibrosis stage 0 as a reference population, we calculated fibrosis stage-specific relative risk (RR) and 95% confidence interval (CI) values for mortality and morbidities. We performed fixed-effect and random-effect model meta-analyses. Metaregression was used to examine associations among study design (prospective vs retrospective cohort), overall risk of bias (medium or high), and mean duration of follow-up (in years). RESULTS: Our meta-analysis included 13 studies, comprising 4428 patients with NAFLD; 2875 of these were reported to have NASH. Compared with no fibrosis (stage 0), unadjusted risk increased with increasing stage of fibrosis (stage 0 vs 4): all-cause mortality RR, 3.42 (95% CI, 2.63-4.46); liver-related mortality RR, 11.13 (95% CI, 4.15-29.84); liver transplant RR, 5.42 (95% CI, 1.05-27.89); and liver-related events RR, 12.78 (95% CI, 6.85-23.85). The magnitude of RR did not differ significantly after adjustment for confounders, including age or sex in the subgroup of NAFLD patients with NASH. Three studies examined the effects of increasing fibrosis on quality of life had inconsistent findings. CONCLUSIONS: In a systematic review and meta-analysis, we found biopsy-confirmed fibrosis to be associated with risk of mortality and liver-related morbidity in patients with NAFLD, with and without adjustment for confounding factors and in patients with reported NASH. Further studies are needed to assess the association between fibrosis stage and patient quality of life and establish that change in liver fibrosis stage is a valid endpoint for use in clinical trials.

Navigating Identity: The Intersection of Social and Biological Identity from the World War II Battle of Tarawa.

The 1943 Battle of Tarawa resulted in the loss of approximately 1,000 US service members on or around Betio Island, Tarawa Atoll, Republic of Kiribati. Nearly half these casualties were accounted for after the battle. The Defense POW/MIA Accounting Agency (DPAA) has worked to identify the remaining ∼510 unaccounted-for service members and has successfully identified ∼160 service members to date. Demographic data pulled from historical documentation of the US losses indicate a relatively homogeneous population (99% White, 81% 17-23 years of age, and only two individuals with a documented religious preference other than Protestant or Catholic). Using this demographic data as a framework, three case studies are presented to demonstrate how a holistic biosocial approach to building identity could facilitate forensic identifications. The temporal and sociocultural contextualization of analyses enables anthropologists to navigate inconsistencies between 21st-century and historical (1940s) social identity concepts to overcome challenges to identification. The case studies demonstrate how biological evidence, genetic evidence, and material evidence (material culture) differently contribute to the social identity of an individual and can impact identification efforts when analytical conclusions are incongruent with historical documentation. The first case of US Battle of Tarawa casualties examines how morphometric biological affinity assessments are biased by the fluidity of social identity concepts when complex morphological and metric indicators of biological affinity are not represented in historical race categories. The second case demonstrates how biogeographic genetic affinity predictions, through a discussion of the G2a4 haplogroup, need to be examined holistically in the context of other lines of evidence. The third case highlights how material evidence can further define social identity beyond physicality, genetic structure, and race. The challenges of interpreting identity from human remains, as highlighted through these examples, are commonly encountered by anthropologists working in disaster victim identification and other humanitarian contexts. Thus, it is imperative for anthropologists to be self-aware of implicit biases toward the current prevailing definitions of biological and social identity and to consider historical perceptions of identity when working in these contexts.

XBP1s activation can globally remodel N-glycan structure distribution patterns.

Classically, the unfolded protein response (UPR) safeguards secretory pathway proteostasis. The most ancient arm of the UPR, the IRE1-activated spliced X-box binding protein 1 (XBP1s)-mediated response, has roles in secretory pathway maturation beyond resolving proteostatic stress. Understanding the consequences of XBP1s activation for cellular processes is critical for elucidating mechanistic connections between XBP1s and development, immunity, and disease. Here, we show that a key functional output of XBP1s activation is a cell type-dependent shift in the distribution of N-glycan structures on endogenous membrane and secreted proteomes. For example, XBP1s activity decreased levels of sialylation and bisecting GlcNAc in the HEK293 membrane proteome and secretome, while substantially increasing the population of oligomannose N-glycans only in the secretome. In HeLa cell membranes, stress-independent XBP1s activation increased the population of high-mannose and tetraantennary N-glycans, and also enhanced core fucosylation. mRNA profiling experiments suggest that XBP1s-mediated remodeling of the N-glycome is, at least in part, a consequence of coordinated transcriptional resculpting of N-glycan maturation pathways by XBP1s. The discovery of XBP1s-induced N-glycan structural remodeling on a glycome-wide scale suggests that XBP1s can act as a master regulator of N-glycan maturation. Moreover, because the sugars on cell-surface proteins or on proteins secreted from an XBP1s-activated cell can be molecularly distinct from those of an unactivated cell, these findings reveal a potential new mechanism for translating intracellular stress signaling into altered interactions with the extracellular environment.

A cluster of residues in the lipopolysaccharide exporter that selects substrate variants for transport to the outer membrane.

Most Gram-negative bacteria assemble lipopolysaccharides (LPS) on their surface to form a permeability barrier against many antimicrobials. LPS is synthesized at the inner membrane and then transported to the outer leaflet of the outer membrane. Although the overall LPS structure is conserved, LPS molecules can differ in composition at the species and strain level. Some bacteria also regulate when to modify phosphates on LPS at the inner membrane in order to become resistant to cationic antimicrobial peptides. The multi-protein Lpt trans-envelope machine, which transports LPS from the inner to the outer membrane, must therefore handle a variety of substrates. The most poorly understood step in LPS transport is how the ATP-binding cassette LptB2 FG transporter extracts LPS from the inner membrane. Here, we define residue K34 in LptG as a site within the structural cavity of the Escherichia coli LptB2 FG transporter that interacts electrostatically with phosphates on unmodified LPS. Alterations to this residue cause transport defects that are suppressed by the activation of the BasSR two-component signaling system, which results in modifications to the LPS phosphates. We also show this residue is part of a larger site in LptG that differentially contributes to the transport of unmodified and modified LPS.

Outer Membrane Translocon Communicates with Inner Membrane ATPase To Stop Lipopolysaccharide Transport.

The survival of Gram-negative bacteria depends on assembly of the asymmetric outer membrane, which creates a barrier that prevents entry of toxic molecules including antibiotics. The outer leaflet of the outer membrane is composed of lipopolysaccharide, which is made at the inner membrane and pushed across a protein bridge that spans the inner and outer membranes. We have developed a fluorescent assay to follow lipopolysaccharide (LPS) transport across a bridge linking proteoliposomes that mimic the inner and outer membranes. We show that LPS is delivered to the leaflet of the outer membrane proteoliposome that corresponds to the outer leaflet of the membrane in a cell. Transport stops long before substrates at the inner membrane are exhausted. Using mutants of the transport machinery, we find that the final amount of LPS delivered into the membrane depends on the affinity of the outer membrane translocon for LPS. Furthermore, ATP hydrolysis depends on delivery of LPS into the outer membrane. Therefore, the transport process is regulated by the outer membrane translocon causing ATP hydrolysis in the inner membrane proteoliposome to stop. Negative feedback from the outer membrane to the inner membrane provides a mechanism for long distance control over LPS transport.

Predictors of pain relief following spinal cord stimulation in chronic back and leg pain and failed back surgery syndrome: a systematic review and meta-regression analysis.

We sought to assess the extent to which pain relief in chronic back and leg pain (CBLP) following spinal cord stimulation (SCS) is influenced by patient-related factors, including pain location, and technology factors. A number of electronic databases were searched with citation searching of included papers and recent systematic reviews. All study designs were included. The primary outcome was pain relief following SCS, we also sought pain score (pre- and post-SCS). Multiple predictive factors were examined: location of pain, history of back surgery, initial level of pain, litigation/worker's compensation, age, gender, duration of pain, duration of follow-up, publication year, continent of data collection, study design, quality score, method of SCS lead implant, and type of SCS lead. Between-study association in predictive factors and pain relief were assessed by meta-regression. Seventy-four studies (N = 3,025 patients with CBLP) met the inclusion criteria; 63 reported data to allow inclusion in a quantitative analysis. Evidence of substantial statistical heterogeneity (P < 0.0001) in level of pain relief following SCS was noted. The mean level of pain relief across studies was 58% (95% CI: 53% to 64%, random effects) at an average follow-up of 24 months. Multivariable meta-regression analysis showed no predictive patient or technology factors. SCS was effective in reducing pain irrespective of the location of CBLP. This review supports SCS as an effective pain relieving treatment for CBLP with predominant leg pain with or without a prior history of back surgery. Randomized controlled trials need to confirm the effectiveness and cost-effectiveness of SCS in the CLBP population with predominant low back pain.

The impact of early postoperative pain on health-related quality of life.

OBJECTIVES: To examine how the severity of postoperative pain affects patient's health-related quality of life (HRQoL) at 1 week following surgery and to compare two generic validated HRQoL instruments. METHODS: Patients undergoing general or orthopaedic surgery at the Royal London Hospital were randomly sampled. The following patient outcome data were collected EQ-5D (EuroQoL) pre-operatively and the Revised American Pain Society Patient Outcome Questionnaire (APS-POQ-R) at 24 hours postoperation; and EQ-5D, Short-Form-12 (SF-12) and APS-POQ-R at 7 days postoperation. The degree of association between pain and HRQoL was assessed using Pearson's correlation coefficient and multivariate generalized linear regression models. RESULTS: Of the 228 patients included, 166 patients provided data at 7 days. Sixteen percent reported severe pain ≥ 50% of the day at 7 days. The severity of pain on both the APS-POQ-R pain severity and interference and affective impairment domains at 7 days was highly correlated with a decrease in HRQoL as assessed by the SF-12 Physical Component Score (PCS), SF-12 Mental Component Score (MCS), and EQ-5D scores (r = -0.34 to -0.61, P < 0.0001). Multivariate regression analyses showed that irrespective of confounding factors (eg, age, gender, and pre-operative HRQoL) patients with severe postoperative pain experience important reductions in both physical and mental well-being domains of their HRQoL. CONCLUSIONS: A proportion of patients continue to experience severe pain at 7 days postoperatively, even after minor surgery. HRQoL is strongly associated with the level of pain and provides additional data on the impact of postsurgery pain on patient's function and well-being. Additional studies are needed to elucidate the interaction between pain severity and HRQoL during the peri-operative period.

The structure of COPI vesicles and regulation of vesicle turnover.

COPI-coated vesicles mediate transport between Golgi stacks and retrograde transport from the Golgi to the endoplasmic reticulum. The COPI coat exists as a stable heptameric complex in the cytosol termed coatomer and is recruited en bloc to the membrane for vesicle formation. Recruitment of COPI onto membranes is mediated by the Arf family of small GTPases, which, in their GTP-bound state, bind both membrane and coatomer. Arf GTPases also influence cargo selection, vesicle scission and vesicle uncoating. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) regulate nucleotide binding by Arf GTPases. To understand the mechanism of COPI-coated vesicle trafficking, it is necessary to characterize the interplay between coatomer and Arf GTPases and their effectors. It is also necessary to understand interactions between coatomer and cargo, cargo adaptors/receptors and tethers facilitating binding to the target membrane. Here, we summarize current knowledge of COPI coat protein structure; we describe how structural and biochemical studies contributed to this knowledge; we review mechanistic insights into COPI vesicle biogenesis and disassembly; and we discuss the potential to answer open questions in the field.

Trotter and Gleser's (1958) equations outperform Trotter and Gleser's (1952) equations in stature estimation of the US White males.

Trotter and Gleser presented two sets of stature estimation equations for the US White males in their 1952 and 1958 studies. Following Trotter's suggestion favouring the 1952 equations simply due to the smaller standard errors, the 1958 equations have been seldom used and have gone without additional systematic validation tests. This study aims to assess the performance of the Trotter and Gleser 1952, Trotter and Gleser 1958, and FORDISC equations for the White males in a quantitative and systematic way, particularly when applied to the WWII and Korean War casualties. In sum, 27 equations (7 from the 1952 study, 10 from the 1958 study, and 10 from FORDISC) were applied to the osteometric data of 240 accounted-for White male casualties of the WWII and Korean War. Then, the bias, accuracy, and Bayes factor for each set of stature estimates were calculated. The results show that, overall, Trotter and Gleser's 1958 equations outperform the 1952 and FORDISC equations in terms of all three measures. Particularly, the equations with higher Bayes factors produced stature estimates where distributions were closer to that of the reported statures than those with lower Bayes factors. When considering Bayes factors, the best performing equation was the "Radius" equation from the 1958 study (BF = 15.34) followed by the "Humerus+Radius" equation from FORDISC (BF = 14.42) and the "Fibula" equation from the 1958 study (BF = 13.82). The results of this study will provide researchers and practitioners applying the Trotter and Gleser stature estimation method with a practical guide for equation selection. Key Points: The performance of three stature estimation methods was compared quantitatively.Trotter and Gleser's (1952, 1958) and FORDISC White male equations were included.Overall, Trotter and Gleser's 1958 method outperformed the other methods.This study provides a practical guide for stature estimation equation selection.

Suppressor Mutations in LptF Bypass Essentiality of LptC by Forming a Six-Protein Transenvelope Bridge That Efficiently Transports Lipopolysaccharide.

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) of many Gram-negative bacteria, providing a barrier against the entry of toxic molecules. In Escherichia coli, LPS is exported to the cell surface by seven essential proteins (LptA-G) that form a transenvelope complex. At the inner membrane, the ATP-binding cassette (ABC) transporter LptB2FG associates with LptC to power LPS extraction from the membrane and transfer to the periplasmic LptA protein, which is in complex with the OM translocon LptDE. LptC interacts both with LptB2FG and LptADE to mediate the formation of the transenvelope bridge and regulates the ATPase activity of LptB2FG. A genetic screen has previously identified suppressor mutants at a residue (R212) of LptF that are viable in the absence of LptC. Here, we present in vivo evidence that the LptF R212G mutant assembles a six-protein transenvelope complex in which LptA mediates interactions with LptF and LptD in the absence of LptC. Furthermore, we present in vitro evidence that the mutant LptB2FG complexes restore the regulation of ATP hydrolysis as it occurs in the LptB2FGC complex to achieve wild-type efficient coupling of ATP hydrolysis and LPS movement. We also show the suppressor mutations restore the wild-type levels of LPS transport both in vivo and in vitro, but remarkably, without restoring the affinity of the inner membrane complex for LptA. Based on the sensitivity of lptF suppressor mutants to selected stress conditions relative to wild-type cells, we show that there are additional regulatory functions of LptF and LptC that had not been identified. IMPORTANCE The presence of an external LPS layer in the outer membrane makes Gram-negative bacteria intrinsically resistant to many antibiotics. Millions of LPS molecules are transported to the cell surface per generation by the Lpt molecular machine made, in E. coli, by seven essential proteins. LptC is the unconventional regulatory subunit of the LptB2FGC ABC transporter, involved in coordinating energy production and LPS transport. Surprisingly, despite being essential for bacterial growth, LptC can be deleted, provided that a specific residue in the periplasmic domain of LptF is mutated and LptA is overexpressed. Here, we apply biochemical techniques to investigate the suppression mechanism. The data produced in this work disclose an unknown regulatory function of LptF in the transporter that not only expands the knowledge about the Lpt complex but can also be targeted by novel LPS biogenesis inhibitors.

Predictors of health-related quality of life and costs in adults with epilepsy: a systematic review.

PURPOSE: Given the high burden of epilepsy on both health-related quality of life (HRQoL) and costs, identification of factors that are predictive of either reduced HRQoL or increased expenditure is central to the better future targeting and optimization of existing and emerging interventions and management strategies for epilepsy. METHODS: Searches of Medline, Embase, and Cochrane Library (up to July 2010) to identify studies examining the association between demographic, psychosocial, and condition-related factors and HRQoL, resource utilization or costs in adults with epilepsy. For each study, predictor factor associations were summarized on the basis of statistical significance and direction; the results were then combined across studies. KEY FINDINGS: Ninety-three HRQoL and 16 resource utilization/cost studies were included. Increases in seizure frequency, seizure severity, level of depression, and level of anxiety and presence of comorbidity were strongly associated with reduced HRQoL. The majority of studies were cross-sectional in design and had an overall methodologic quality that was judged to be "moderate" for HRQoL studies and "poor" for health care resource or costs studies. In the 53 multivariate studies, age, gender, marital status, type of seizure, age at diagnosis, and duration of epilepsy did not appear to be associated with HRQoL, whereas the predictive influence of educational and employment status, number of antiepileptic drugs (AEDs) and AED side effects was unclear. The association between predictive factors and HRQoL appeared to be consistent across individuals whether refractory or seizures controlled or managed by AEDs. There were insufficient multivariate studies (five) to reliably comment on the predictors of resource utilization or cost in epilepsy. SIGNIFICANCE: In addition to seizure control, effective epilepsy management requires the early detection of those most at risk of psychological dysfunction and comorbidity, and the targeting of appropriate interventions. There is need for more rigorous studies with appropriate multivariate statistical methods that prospectively investigate the predictors of HRQoL, resource utilization, and costs in epilepsy.

Lipopolysaccharide is transported to the cell surface by a membrane-to-membrane protein bridge.

Gram-negative bacteria have an outer membrane that serves as a barrier to noxious agents in the environment. This protective function is dependent on lipopolysaccharide, a large glycolipid located in the outer leaflet of the outer membrane. Lipopolysaccharide is synthesized at the cytoplasmic membrane and must be transported to the cell surface. To understand this transport process, we reconstituted membrane-to-membrane movement of lipopolysaccharide by incorporating purified inner and outer membrane transport complexes into separate proteoliposomes. Transport involved stable association between the inner and outer membrane proteoliposomes. Our results support a model in which lipopolysaccharide molecules are pushed one after the other in a PEZ dispenser-like manner across a protein bridge that connects the inner and outer membranes.

A cysteine-based molecular code informs collagen C-propeptide assembly.

Fundamental questions regarding collagen biosynthesis, especially with respect to the molecular origins of homotrimeric versus heterotrimeric assembly, remain unanswered. Here, we demonstrate that the presence or absence of a single cysteine in type-I collagen's C-propeptide domain is a key factor governing the ability of a given collagen polypeptide to stably homotrimerize. We also identify a critical role for Ca2+ in non-covalent collagen C-propeptide trimerization, thereby priming the protein for disulfide-mediated covalent immortalization. The resulting cysteine-based code for stable assembly provides a molecular model that can be used to predict, a priori, the identity of not just collagen homotrimers, but also naturally occurring 2:1 and 1:1:1 heterotrimers. Moreover, the code applies across all of the sequence-diverse fibrillar collagens. These results provide new insight into how evolution leverages disulfide networks to fine-tune protein assembly, and will inform the ongoing development of designer proteins that assemble into specific oligomeric forms.

Resident physician mentoring program in ophthalmology: the Tennessee experience.

OBJECTIVE: To establish a mentoring program to provide resident physicians in ophthalmology with career guidance in practice management and to identify new and creative ways to involve future eye physicians in the legislative and political process. METHODS: A multicenter prospective study was conducted of the mentorship experiences of 24 (88.9%) of 27 resident physicians in Tennessee during the 2000-2001 academic year. Participants were assigned into 1 of 3 groups: an active mentorship group, a passive mentorship group, and a no mentorship group. The active mentorship group participated in preceptorship activities with "mentor" community-based eye physicians and scheduled meetings with state legislators and regulators. The active mentorship and passive mentorship groups attended a 1-day practice management seminar, but the no mentorship group received no formal mentorship during the 4-month study period. A survey instrument was given to all participants before and after the 4-month study period. RESULTS: Following completion of the mentorship program, the active mentorship group had favorable changes in perceptions and attitudes toward medical organizations (P<.03) when compared with baseline prementorship responses. Compared with the no mentorship group, the active mentorship group also reported an increased willingness to make political campaign donations (P<.05) and expressed an increased desire for the Tennessee Academy of Ophthalmology to offer practice management programs (P<.02). CONCLUSION: A short 4-month mentorship program can elicit favorable changes in residents' perceptions and attitudes toward medical professional organizations. Additional opportunities may lie with a lengthier and more intensive mentoring program.

Storm-Related Postmortem Damage to Skeletal Remains.

In April 2011, human skeletons were exposed to heavy storms at the outdoor Anthropology Research Facility (ARF) in Knoxville, Tennessee. Of the approximate 125 skeletons at the ARF in April 2011, 30 donations exhibited postmortem damage that could be attributed to the storms. At least 20 of the affected donations exhibit postmortem damage clearly associated with hailstones due to the oval shape and similar small size of the defects observed. The irregular shape and larger size of other defects may be a product of other falling objects (e.g., tree branches) associated with the storms. Storm-related damage was observed throughout the skeleton, with the most commonly damaged skeletal elements being the scapula and ilium, but more robust elements (i.e., femora and tibiae) also displayed characteristic features of hailstone damage. Thus, hailstone damage should be considered when forensic practitioners observe unusual postmortem damage in skeletal remains recovered from the outdoor context.

Mapping and Exploring the Collagen-I Proteostasis Network.

Collagen-I is the most abundant protein in the human body, yet our understanding of how the endoplasmic reticulum regulates collagen-I proteostasis (folding, quality control, and secretion) remains immature. Of particular importance, interactomic studies to map the collagen-I proteostasis network have never been performed. Such studies would provide insight into mechanisms of collagen-I folding and misfolding in cells, an area that is particularly important owing to the prominence of the collagen misfolding-related diseases. Here, we overcome key roadblocks to progress in this area by generating stable fibrosarcoma cells that inducibly express properly folded and modified collagen-I strands tagged with distinctive antibody epitopes. Selective immunoprecipitation of collagen-I from these cells integrated with quantitative mass spectrometry-based proteomics permits the first mapping of the collagen-I proteostasis network. Biochemical validation of the resulting map leads to the assignment of numerous new players in collagen-I proteostasis, and the unanticipated discovery of apparent aspartyl-hydroxylation as a new post-translational modification in the N-propeptide of collagen-I. Furthermore, quantitative analyses reveal that Erp29, an abundant endoplasmic reticulum proteostasis machinery component with few known functions, plays a key role in collagen-I retention under ascorbate-deficient conditions. In summary, the work here provides fresh insights into the molecular mechanisms of collagen-I proteostasis, yielding a detailed roadmap for future investigations. Straightforward adaptations of the cellular platform developed will also enable hypothesis-driven, comparative research on the likely distinctive proteostasis mechanisms engaged by normal and disease-causing, misfolding collagen-I variants, potentially motivating new therapeutic strategies for currently incurable collagenopathies.

XBP1s Links the Unfolded Protein Response to the Molecular Architecture of Mature N-Glycans.

The molecular architecture of the mature N-glycome is dynamic, with consequences for both normal and pathologic processes. Elucidating cellular mechanisms that modulate the N-linked glycome is, therefore, crucial. The unfolded protein response (UPR) is classically responsible for maintaining proteostasis in the secretory pathway by defining levels of chaperones and quality control proteins. Here, we employ chemical biology methods for UPR regulation to show that stress-independent activation of the UPR's XBP1s transcription factor also induces a panel of N-glycan maturation-related enzymes. The downstream consequence is a distinctive shift toward specific hybrid and complex N-glycans on N-glycoproteins produced from XBP1s-activated cells, which we characterize by mass spectrometry. Pulse-chase studies attribute this shift specifically to altered N-glycan processing, rather than to changes in degradation or secretion rates. Our findings implicate XBP1s in a new role for N-glycoprotein biosynthesis, unveiling an important link between intracellular stress responses and the molecular architecture of extracellular N-glycoproteins.

The cost effectiveness of spinal cord stimulation in the treatment of pain: a systematic review of the literature.

In this systematic review, we identified and evaluated studies of the cost effectiveness of spinal cord stimulation (SCS) for the treatment of chronic pain. Published reports were identified from a systematic search of a number of general medical electronic databases (Medline, CINAHL, and EMBASE), and specialist economic databases (NHS Centre for Reviews and Dissemination Economic Evaluation Database, and Health Economics Evaluation Database). Reference lists of retrieved reports were also searched, and contact was made with experts in the field. Of the 99 abstracts identified, 14 studies were considered to meet the inclusion criteria of the review. We found that across a range of medical indications, the initial healthcare acquisition costs of SCS implantation are consistently offset by a reduction in post-implant healthcare resource demand and costs. Further research is required to formally examine the cost effectiveness of SCS.