Glial sulfatides and neuronal complex gangliosides are functionally interdependent in maintaining myelinating axon integrity

Sulfatides and gangliosides are raft-associated glycolipids essential for maintaining myelinated nerve integrity. Mice deficient in sulfatide (cerebroside sulfotransferase knockout, CST-/- ) or complex gangliosides (β-1,4-N-acetylegalactosaminyltransferase1 knockout, GalNAc-T-/- ) display prominent disorganization of proteins at the node of Ranvier (NoR) in early life, and age-dependent neurodegeneration. Loss of neuronal rather than glial complex gangliosides underpins the GalNAc-T-/- phenotype, as shown by neuron or glial-specific rescue, whereas sulfatide is principally expressed and functional in glial membranes. The similarities in NoR phenotype of CST-/- , GalNAc-T-/- and axo-glial protein deficient mice suggests these glycolipids stabilise membrane proteins including neurofascin155 (NF155) and myelin-associated glycoprotein (MAG) at axo-glial junctions. To assess the functional interactions between sulfatide and gangliosides, CST-/- and GalNAc-T-/- genotypes were interbred. CST-/- x GalNAc-T-/- mice develop normally to P10, but all die between P20-P25, coinciding with peak myelination. Ultrastructural, immunohistological and biochemical analysis of either sex reveals widespread axonal degeneration and disruption to the axo-glial junction at the NoR. In addition to sulfatide-dependent loss of NF155, CST-/-x GalNAc-T-/- mice exhibited a major reduction in MAG protein levels in CNS myelin, compared to wild type and single lipid deficient mice. The CST-/- x GalNAc-T-/- phenotype was fully restored to that of CST-/- mice by neuron-specific expression of complex gangliosides, but not by their glial-specific expression nor by the global expression of a-series gangliosides. These data indicate that sulfatide and complex b-series gangliosides on the glial and neuronal membranes respectively act in concert to promote NF155 and MAG in maintaining the stable axo-glial interactions essential for normal nerve function.SIGNIFICANCE STATEMENTSulfatides and complex gangliosides are membrane glycolipids with important roles in maintaining nervous system integrity. Node of Ranvier maintenance in particular requires stable compartmentalisation of multiple membrane proteins. The axo-glial adhesion molecules neurofascin 155 and myelin-associated glycoprotein require membrane microdomains containing either sulfatides or complex gangliosides to localise and function effectively. The co-operative roles of these microdomains and associated proteins are unknown. Here we show vital interdependent roles for sulfatides and complex gangliosides as double (but not single) deficiency causes a rapidly lethal phenotype in early age. These findings suggests that sulfatides and complex gangliosides on opposing axo-glial membranes are responsible for essential tethering of the axo-glial junction proteins, neurofascin155 and myelin-associated glycoprotein that interact to maintain the nodal complex

The potential for research-based information in public health: Identifying unrecognised information needs

OBJECTIVE: To explore whether there is a potential for greater use of research-based information in public health practice in a local setting. Secondly, if research-based information is relevant, to explore the extent to which this generates questioning behaviour. DESIGN: Qualitative study using focus group discussions, observation and interviews. SETTING: Public health practices in Norway. PARTICIPANTS: 52 public health practitioners. RESULTS: In general, the public health practitioners had a positive attitude towards research-based information, but believed that they had few cases requiring this type of information. They did say, however, that there might be a potential for greater use. During five focus groups and six observation days we identified 28 questions/cases where it would have been appropriate to seek out research evidence according to our definition. Three of the public health practitioners identified three of these 28 cases as questions for which research-based information could have been relevant. This gap is interpreted as representing unrecognised information needs. CONCLUSIONS: There is an unrealised potential in public health practice for more frequent and extensive use of research-based information. The practitioners did not appear to reflect on the need for scientific information when faced with new cases and few questions of this type were generated

Iron therapy for preoperative anaemia

Preoperative anaemia is common and occurs in 5% to 76% of patients preoperatively. It is associated with an increased risk of perioperative allogeneic blood transfusion, longer hospital stay, and increased morbidity and mortality. Iron deficiency is one of the most common causes of anaemia. Oral and intravenous iron therapy can be used to treat anaemia. Parenteral iron preparations have been shown to be more effective in conditions such as inflammatory bowel disease, chronic heart failure and postpartum haemorrhage due to rapid correction of iron stores. A limited number of studies has investigated iron therapy for the treatment of preoperative anaemia. The aim of this Cochrane Review is to summarise the evidence for iron supplementation, both enteral and parenteral, for the management of preoperative anaemia. Objectives To evaluate the effects of preoperative iron therapy (enteral or parenteral) in reducing the need for allogeneic blood transfusions in anaemic patients undergoing surgery. Search methods We ran the search on 30 July 2018. We searched the Cochrane Injuries Group's Specialised Register, Cochrane Central Register of Controlled Trials (CENTRAL, the Cochrane Library), Ovid MEDLINE(R), Ovid MEDLINE(R) In‐Process & Other Non‐Indexed Citations, Ovid MEDLINE(R) Daily and Ovid OLDMEDLINE(R), Embase Classic and Embase (Ovid), CINAHL Plus (EBSCO), PubMed, and clinical trials registries, and we screened reference lists. We ran a top‐up search on 28 November 2019; one study is now awaiting classification. Selection criteria We included all randomised controlled trials (RCTs) that compared preoperative iron monotherapy to placebo, no treatment, standard care or another form of iron therapy for anaemic adults undergoing surgery. We defined anaemia as haemoglobin values less than 13 g/dL for males and 12 g/dL for non‐pregnant females. Data collection and analysis Two review authors collected data and a third review author checked all collected data. Data were collected on the proportion of patients who receive a blood transfusion, the amount of blood transfused per patient (units), quality of life, ferritin levels and haemoglobin levels, measured as continuous variables at the following predetermined time points: pretreatment (baseline), preoperatively but postintervention, and postoperatively. We performed statistical analysis using the Cochrane software, Review Manager 5. We summarised outcome data in tables and forest plots. We used the GRADE approach to describe the quality of the body of evidence. Main results Six RCTs, with a total of 372 participants, evaluated preoperative iron therapy to correct anaemia before planned surgery. Four studies compared iron therapy (either oral (one study) or intravenous (three studies)) with no treatment, placebo or usual care, and two studies compared intravenous iron therapy with oral iron therapy. Iron therapy was delivered over a range of periods that varied from 48 hours to three weeks prior to surgery. The 372 participants in our analysis fall far short of the 819 required ‐ as calculated by our information size calculation ‐ to detect a 30% reduction in blood transfusions. Five trials, involving 310 people, reported the proportion of participants who received allogeneic blood transfusions. Meta‐analysis of iron therapy versus placebo or standard care showed no difference in the proportion of participants who received a blood transfusion (risk ratio (RR) 1.21, 95% confidence interval (CI) 0.87 to 1.70; 4 studies, 200 participants; moderate‐quality evidence). Only one study that compared oral versus intravenous iron therapy measured this outcome, and reported no difference in risk of transfusion between groups. There was no difference between the iron therapy and placebo/standard care groups for haemoglobin level preoperatively at the end of the intervention (mean difference (MD) 0.63 g/dL, 95% CI ‐0.07 to 1.34; 2 studies, 83 participants; low‐quality evidence). However, intravenous iron therapy produced an increase in preoperative postintervention haemoglobin levels compared with oral iron (MD 1.23 g/dL, 95% CI 0.80 to 1.65; 2 studies, 172 participants; low‐quality evidence). Ferritin levels were increased by intravenous iron, both when compared to standard care ((MD 149.00, 95% CI 25.84 to 272.16; 1 study, 63 participants; low‐quality evidence) or to oral iron (MD 395.03 ng/mL, 95% CI 227.72 to 562.35; 2 studies, 151 participants; low‐quality evidence). Not all studies measured quality of life, short‐term mortality or postoperative morbidity. Some measured the outcomes, but did not report the data, and the studies which did report the data were underpowered. Therefore, uncertainty remains regarding these outcomes. The inclusion of new research in the future is very likely to change these results. Authors' conclusions The use of iron therapy for preoperative anaemia does not show a clinically significant reduction in the proportion of trial participants who received an allogeneic blood transfusion compared to no iron therapy. Results for intravenous iron are consistent with a greater increase in haemoglobin and ferritin when compared to oral iron, but do not provide reliable evidence. These conclusions are drawn from six studies, three of which included very small numbers of participants. Further, well‐designed, adequately powered, RCTs are required to determine the true effectiveness of iron therapy for preoperative anaemia. Two studies are currently in progress, and will include 1500 randomised participants.Published onlin

Mechanism-Based Screen for G1/S Checkpoint Activators Identifies a Selective Activator of EIF2AK3/PERK Signalling

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty

Gag Mutations Strongly Contribute to HIV-1 Resistance to Protease Inhibitors in Highly Drug-Experienced Patients besides Compensating for Fitness Loss

Human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors (PI) results from mutations in the viral protease (PR) that reduce PI binding but also decrease viral replicative capacity (RC). Additional mutations compensating for the RC loss subsequently accumulate within PR and in Gag substrate cleavage sites. We examined the respective contribution of mutations in PR and Gag to PI resistance and RC and their interdependence using a panel of HIV-1 molecular clones carrying different sequences from six patients who had failed multiple lines of treatment. Mutations in Gag strongly and directly contributed to PI resistance besides compensating for fitness loss. This effect was essentially carried by the C-terminal region of Gag (containing NC-SP2-p6) with little or no contribution from MA, CA, and SP1. The effect of Gag on resistance depended on the presence of cleavage site mutations A431V or I437V in NC-SP2-p6 and correlated with processing of the NC/SP2 cleavage site. By contrast, reverting the A431V or I437V mutation in these highly evolved sequences had little effect on RC. Mutations in the NC-SP2-p6 region of Gag can be dually selected as compensatory and as direct PI resistance mutations, with cleavage at the NC-SP2 site behaving as a rate-limiting step in PI resistance. Further compensatory mutations render viral RC independent of the A431V or I437V mutations while their effect on resistance persists

Behavioral states may be associated with distinct spatial patterns in electrocorticogram

To determine if behavioral states are associated with unique spatial electrocorticographic (ECoG) patterns, we obtained recordings with a microgrid electrode array applied to the cortical surface of a human subject. The array was constructed with the intent of extracting maximal spatial information by optimizing interelectrode distances. A 34-year-old patient with intractable epilepsy underwent intracranial ECoG monitoring after standard methods failed to reveal localization of seizures. During the 8-day period of invasive recording, in addition to standard clinical electrodes a square 1 × 1 cm microgrid array with 64 electrodes (1.25 mm separation) was placed on the right inferior temporal gyrus. Careful review of video recordings identified four extended naturalistic behaviors: reading, conversing on the telephone, looking at photographs, and face-to-face interactions. ECoG activity recorded with the microgrid that corresponded to these behaviors was collected and ECoG spatial patterns were analyzed. During periods of ECoG selected for analysis, no electrographic seizures or epileptiform patterns were present. Moments of maximal spatial variance are shown to cluster by behavior. Comparisons between conditions using a permutation test reveal significantly different spatial patterns for each behavior. We conclude that ECoG recordings obtained on the cortical surface with optimal high spatial frequency resolution reveal distinct local spatial patterns that reflect different behavioral states, and we predict that similar patterns will be found in many if not most cortical areas on which a microgrid is placed

Herpes Simplex Virus-Induced Epithelial Damage and Susceptibility to Human Immunodeficiency Virus Type 1 Infection in Human Cervical Organ Culture

Normal human premenopausal cervical tissue has been used to derive primary cell populations and to establish ex vivo organ culture systems to study infections with herpes simplex virus (HSV-1 or HSV-2) and human immunodeficiency virus type 1 (HIV-1). Infection with either HSV-1 or HSV-2 rapidly induced multinuclear giant cell formation and widespread damage in mucosal epithelial cells. Subsequent exposure of the damaged mucosal surfaces to HIV-1 revealed frequent co-localization of HSV and HIV-1 antigens. The short-term organ culture system provides direct experimental support for the epidemiological findings that pre-existing sexually transmitted infections, including primary and recurrent herpes virus infections at mucosal surfaces, represent major risk factors for acquisition of primary HIV-1 infection. Epithelial damage in combination with pre-existing inflammation, as described here for overtly normal human premenopausal cervix, creates a highly susceptible environment for the initiation and establishment of primary HIV-1 infection in the sub-mucosa of the cervical transformation zone