Membrane Fusion Mediated by Non-covalent Binding of Re-engineered Cholera Toxin Assemblies to Glycolipids

Membrane fusion is essential for the transport of macromolecules and viruses across membranes. While glycan-binding proteins (lectins) often initiate cellular adhesion, subsequent fusion events require additional protein machinery. No mechanism for membrane fusion arising from simply a protein binding to membrane glycolipids has been described thus far. Herein, we report that a biotinylated protein derived from cholera toxin becomes a fusogenic lectin upon cross-linking with streptavidin. This novel reengineered protein brings about hemifusion and fusion of vesicles as demonstrated by mixing of fluorescently labeled lipids between vesicles as well as content mixing of liposomes filled with fluorescently labeled dextran. Exclusion of the complex at vesicle–vesicle interfaces could also be observed, indicating the formation of hemifusion diaphragms. Discovery of this fusogenic lectin complex demonstrates that new emergent properties can arise from simple changes in protein architecture and provides insights into new mechanisms of lipid-driven fusion

Novel tau fragments in cerebrospinal fluid: relation to tangle pathology and cognitive decline in Alzheimer's disease

Tau is an axonal microtubule-binding protein. Tau pathology in brain and increased tau concentration in the cerebrospinal fluid (CSF) are hallmarks of Alzheimer's disease (AD). Most of tau in CSF is present as fragments. We immunoprecipitated tau from CSF and identified several endogenous peptides ending at amino acid (aa) 123 or 224 using high-resolution mass spectrometry. We raised neo-epitope-specific antibodies against tau fragments specifically ending at aa 123 and 224, respectively. With these antibodies, we performed immunohistochemistry on brain tissue and designed immunoassays measuring N-123, N-224, and x-224 tau. Immunoassays were applied to soluble brain fractions from pathologically confirmed subjects (81 AD patients, 33 controls), CSF from three cross-sectional and two longitudinal cohorts (a total of 133 AD, 38 MCI, 20 MCI-AD, 31 PSP, 15 CBS patients, and 91 controls), and neuronally- and peripherally-derived extracellular vesicles (NDEVs and PDEVs, respectively) in serum from four AD patients and four controls. Anti-tau 224 antibody stained neurofibrillary tangles and neuropil threads, while anti-tau 123 only showed weak cytoplasmic staining in AD. N-224 tau was lower in the AD soluble brain fraction compared to controls, while N-123 tau showed similar levels. N-224 tau was higher in AD compared to controls in all CSF cohorts (p < 0.001), but not N-123 tau. Decrease in cognitive performance and conversion from MCI to AD were associated with increased baseline CSF levels of N-224 tau (p < 0.0001). N-224 tau concentrations in PSP and CBS were significantly lower than in AD (p < 0.0001) and did not correlate to t-tau and p-tau. In a longitudinal cohort, CSF N-224 tau levels were stable over 6 months, with no significant effect of treatment with AChE inhibitors. N-224 tau was present in NDEVs, while N-123 tau showed comparable concentrations in both vesicle types. We suggest that N-123 tau is produced both in CNS and PNS and represents a general marker of tau metabolism, while N-224 tau is neuron-specific, present in the tangles, secreted in CSF, and upregulated in AD, suggesting a link between tau cleavage and propagation, tangle pathology, and cognitive decline

In Vivo Human Apolipoprotein E Isoform Fractional Turnover Rates in the CNS

Apolipoprotein E (ApoE) is the strongest genetic risk factor for Alzheimer’s disease and has been implicated in the risk for other neurological disorders. The three common ApoE isoforms (ApoE2, E3, and E4) each differ by a single amino acid, with ApoE4 increasing and ApoE2 decreasing the risk of Alzheimer’s disease (AD). Both the isoform and amount of ApoE in the brain modulate AD pathology by altering the extent of amyloid beta (Aβ) peptide deposition. Therefore, quantifying ApoE isoform production and clearance rates may advance our understanding of the role of ApoE in health and disease. To measure the kinetics of ApoE in the central nervous system (CNS), we applied in vivo stable isotope labeling to quantify the fractional turnover rates of ApoE isoforms in 18 cognitively-normal adults and in ApoE3 and ApoE4 targeted-replacement mice. No isoform-specific differences in CNS ApoE3 and ApoE4 turnover rates were observed when measured in human CSF or mouse brain. However, CNS and peripheral ApoE isoform turnover rates differed substantially, which is consistent with previous reports and suggests that the pathways responsible for ApoE metabolism are different in the CNS and the periphery. We also demonstrate a slower turnover rate for CSF ApoE than that for amyloid beta, another molecule critically important in AD pathogenesis

Lesson from the Stoichiometry Determination of the Cohesin Complex: A Short Protease Mediated Elution Increases the Recovery from Cross-Linked Antibody-Conjugated Beads

Affinity purification of proteins using antibodies coupled to beads and subsequent mass spectrometric analysis has become a standard technique for the identification of protein complexes. With the recent transfer of the isotope dilution mass spectrometry principle (IDMS) to the field of proteomics, quantitative analysesssuch as the stoichiometry determination of protein complexesshave become achievable. Traditionally proteins were eluted from antibody-conjugated beads using glycine at low pH or using diluted acids such as HCl, TFA, or FA, but elution was often found to be incomplete. Using the cohesin complex and the anaphase promoting complex/cyclosome (APC/C) as examples, we show that a short 15-60 min predigestion with a protease such as LysC (modified on-bead digest termed protease elution) increases the elution efficiency 2- to 3-fold compared to standard acid elution protocols. While longer incubation periodssas performed in standard on-bead digestionsled to partial proteolysis of the cross-linked antibodies, no or only insignificant cleavage was observed after 15-60 min protease mediated elution. Using the protease elution method, we successfully determined the stoichiometry of the cohesin complex by absolute quantification of the four core subunits using LC-SRM analysis and 19 reference peptides generated with the EtEP strategy. Protease elution was 3-fold more efficient compared to HCl elution, but measurements using both elution techniques are in agreement with

Taxonomic Distinctness of Demersal Fishes of the California Current: Moving Beyond Simple Measures of Diversity for Marine Ecosystem-Based Management

BACKGROUND: Large-scale patterns or trends in species diversity have long interested ecologists. The classic pattern is for diversity (e.g., species richness) to decrease with increasing latitude. Taxonomic distinctness is a diversity measure based on the relatedness of the species within a sample. Here we examined patterns of taxonomic distinctness in relation to latitude (ca. 32-48 degrees N) and depth (ca. 50-1220 m) for demersal fishes on the continental shelf and slope of the US Pacific coast. METHODOLOGY/PRINCIPAL FINDINGS: Both average taxonomic distinctness (AvTD) and variation in taxonomic distinctness (VarTD) changed with latitude and depth. AvTD was highest at approximately 500 m and lowest at around 200 m bottom depth. Latitudinal trends in AvTD were somewhat weaker and were depth-specific. AvTD increased with latitude on the shelf (50-150 m) but tended to decrease with latitude at deeper depths. Variation in taxonomic distinctness (VarTD) was highest around 300 m. As with AvTD, latitudinal trends in VarTD were depth-specific. On the shelf (50-150 m), VarTD increased with latitude, while in deeper areas the patterns were more complex. Closer inspection of the data showed that the number and distribution of species within the class Chondrichthyes were the primary drivers of the overall patterns seen in AvTD and VarTD, while the relatedness and distribution of species in the order Scorpaeniformes appeared to cause the relatively low observed values of AvTD at around 200 m. CONCLUSIONS/SIGNIFICANCE: These trends contrast to some extent the patterns seen in earlier studies for species richness and evenness in demersal fishes along this coast and add to our understanding of diversity of the demersal fishes of the California Current

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

Background: Many patients with COVID-19 have been treated with plasma containing anti-SARS-CoV-2 antibodies. We aimed to evaluate the safety and efficacy of convalescent plasma therapy in patients admitted to hospital with COVID-19. Methods: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]) is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. The trial is underway at 177 NHS hospitals from across the UK. Eligible and consenting patients were randomly assigned (1:1) to receive either usual care alone (usual care group) or usual care plus high-titre convalescent plasma (convalescent plasma group). The primary outcome was 28-day mortality, analysed on an intention-to-treat basis. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936. Findings: Between May 28, 2020, and Jan 15, 2021, 11558 (71%) of 16287 patients enrolled in RECOVERY were eligible to receive convalescent plasma and were assigned to either the convalescent plasma group or the usual care group. There was no significant difference in 28-day mortality between the two groups: 1399 (24%) of 5795 patients in the convalescent plasma group and 1408 (24%) of 5763 patients in the usual care group died within 28 days (rate ratio 1·00, 95% CI 0·93–1·07; p=0·95). The 28-day mortality rate ratio was similar in all prespecified subgroups of patients, including in those patients without detectable SARS-CoV-2 antibodies at randomisation. Allocation to convalescent plasma had no significant effect on the proportion of patients discharged from hospital within 28 days (3832 [66%] patients in the convalescent plasma group vs 3822 [66%] patients in the usual care group; rate ratio 0·99, 95% CI 0·94–1·03; p=0·57). Among those not on invasive mechanical ventilation at randomisation, there was no significant difference in the proportion of patients meeting the composite endpoint of progression to invasive mechanical ventilation or death (1568 [29%] of 5493 patients in the convalescent plasma group vs 1568 [29%] of 5448 patients in the usual care group; rate ratio 0·99, 95% CI 0·93–1·05; p=0·79). Interpretation: In patients hospitalised with COVID-19, high-titre convalescent plasma did not improve survival or other prespecified clinical outcomes. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health Research

Tocilizumab in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Background: In this study, we aimed to evaluate the effects of tocilizumab in adult patients admitted to hospital with COVID-19 with both hypoxia and systemic inflammation. Methods: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. Those trial participants with hypoxia (oxygen saturation &lt;92% on air or requiring oxygen therapy) and evidence of systemic inflammation (C-reactive protein ≥75 mg/L) were eligible for random assignment in a 1:1 ratio to usual standard of care alone versus usual standard of care plus tocilizumab at a dose of 400 mg–800 mg (depending on weight) given intravenously. A second dose could be given 12–24 h later if the patient's condition had not improved. The primary outcome was 28-day mortality, assessed in the intention-to-treat population. The trial is registered with ISRCTN (50189673) and ClinicalTrials.gov (NCT04381936). Findings: Between April 23, 2020, and Jan 24, 2021, 4116 adults of 21 550 patients enrolled into the RECOVERY trial were included in the assessment of tocilizumab, including 3385 (82%) patients receiving systemic corticosteroids. Overall, 621 (31%) of the 2022 patients allocated tocilizumab and 729 (35%) of the 2094 patients allocated to usual care died within 28 days (rate ratio 0·85; 95% CI 0·76–0·94; p=0·0028). Consistent results were seen in all prespecified subgroups of patients, including those receiving systemic corticosteroids. Patients allocated to tocilizumab were more likely to be discharged from hospital within 28 days (57% vs 50%; rate ratio 1·22; 1·12–1·33; p&lt;0·0001). Among those not receiving invasive mechanical ventilation at baseline, patients allocated tocilizumab were less likely to reach the composite endpoint of invasive mechanical ventilation or death (35% vs 42%; risk ratio 0·84; 95% CI 0·77–0·92; p&lt;0·0001). Interpretation: In hospitalised COVID-19 patients with hypoxia and systemic inflammation, tocilizumab improved survival and other clinical outcomes. These benefits were seen regardless of the amount of respiratory support and were additional to the benefits of systemic corticosteroids. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health Research