Multiscale Modeling of Macromolecular Interactions between Tau-Amylin Oligomers and Asymmetric Lipid Nanodomains That Link Alzheimer's and Diabetic Diseases.

The molecular events of protein misfolding and self-aggregation of tau and amylin are associated with the progression of Alzheimer's and diabetes, respectively. Recent studies suggest that tau and amylin can form hetero-tau-amylin oligomers. Those hetero-oligomers are more neurotoxic than homo-tau oligomers. So far, the detailed interactions between the hetero-oligomers and the neuronal membrane are unknown. Using multiscale MD simulations, the lipid binding and protein folding behaviors of hetero-oligomers on asymmetric lipid nanodomains or raft membranes were examined. Our raft membranes contain phase-separated phosphatidylcholine (PC), cholesterol, and anionic phosphatidylserine (PS) or ganglioside (GM1) in one leaflet of the lipid bilayer. The hetero-oligomers bound more strongly to the PS and GM1 than other lipids via the hydrophobic and hydrophilic interactions, respectively, in the raft membranes. The hetero-tetramer disrupted the acyl chain orders of both PC and PS in the PS-containing raft membrane, but only the GM1 in the GM1-containing raft membrane as effectively as the homo-tau-tetramer. We discovered that the alpha-helical content in the heterodimer was greater than the sum of alpha-helical contents from isolated tau and amylin monomers on both raft membranes, indicative of a synergetic effect of tau-amylin interactions in surface-induced protein folding. Our results provide new molecular insights into understanding the cross-talk between Alzheimer's and diabetes.

Comparing Catastrophic Costs: Active vs. Passive Tuberculosis Case Finding in Urban Vietnam.

Active case finding (ACF) is a strategy that aims to identify people with tuberculosis (TB) earlier in their disease. This outreach approach may lead to a reduction in catastrophic cost incurrence (costs exceeding 20% of annual household income), a main target of WHO's End TB Strategy. Our study assessed the socio-economic impact of ACF by comparing patient costs in actively and passively detected people with TB. Longitudinal patient cost surveys were prospectively fielded for people with drug-sensitive pulmonary TB, with 105 detected through ACF and 107 passively detected. Data were collected in four Vietnamese cities between October 2020 and March 2022. ACF reduced pre-treatment (USD 10 vs. 101, p < 0.001) and treatment costs (USD 888 vs. 1213, p < 0.001) in TB-affected individuals. Furthermore, it reduced the occurrence of job loss (15.2% vs. 35.5%, p = 0.001) and use of coping strategies (28.6% vs. 45.7%, p = 0.004). However, catastrophic cost incurrence was high at 52.8% and did not differ between cohorts. ACF did not significantly decrease indirect costs, the largest contributor to catastrophic costs. ACF reduces costs but cannot sufficiently reduce the risk of catastrophic costs. As income loss is the largest driver of costs during TB treatment, social protection schemes need to be expanded.

Exploring the Role of Anionic Lipid Nanodomains in the Membrane Disruption and Protein Folding of Human Islet Amyloid Polypeptide Oligomers on Lipid Membrane Surfaces Using Multiscale Molecular Dynamics Simulations.

The aggregation of human Islet Amyloid Polypeptide (hIAPP) on cell membranes is linked to amyloid diseases. However, the physio-chemical mechanisms of how these hIAPP aggregates trigger membrane damage are unclear. Using coarse-grained and all-atom molecular dynamics simulations, we investigated the role of lipid nanodomains in the presence or absence of anionic lipids, phosphatidylserine (PS), and a ganglioside (GM1), in the membrane disruption and protein folding behaviors of hIAPP aggregates on phase-separated raft membranes. Our raft membranes contain liquid-ordered (Lo), liquid-disordered (Ld), mixed Lo/Ld (Lod), PS-cluster, and GM1-cluster nanosized domains. We observed that hIAPP aggregates bound to the Lod domain in the absence of anionic lipids, but also to the GM1-cluster- and PS-cluster-containing domains, with stronger affinity in the presence of anionic lipids. We discovered that L16 and I26 are the lipid anchoring residues of hIAPP binding to the Lod and PS-cluster domains. Finally, significant lipid acyl chain order disruption in the annular lipid shells surrounding the membrane-bound hIAPP aggregates and protein folding, particularly beta-sheet formation, in larger protein aggregates were evident. We propose that the interactions of hIAPP and both non-anionic and anionic lipid nanodomains represent key molecular events of membrane damage associated with the pathogenesis of amyloid diseases.

Lipid domain boundary triggers membrane damage and protein folding of human islet amyloid polypeptide in the early pathogenesis of amyloid diseases.

The misfolding and self-aggregation of human Islet Amyloid Polypeptide (hIAPP) are linked to the onset of type 2 diabetes (T2D). However, the mechanism of how the disordered hIAPP aggregates trigger membrane damage leading to the loss of Islet cells in T2D is unknown. Using coarse-grained (CG) and all-atom (AA) molecular dynamics simulations, we have investigated the membrane-disruption behaviors of hIAPP oligomers on the phase-separated lipid nanodomains that mimic the highly heterogeneous lipid raft structures of cell membranes. Our results revealed that hIAPP oligomers preferentially bind to the liquid-ordered and liquid-disordered domain boundary around two hydrophobic residues at L16 and I26, and lipid acyl chain order disruption and beta-sheet formation occur upon hIAPP binding to the membrane surface. We propose that the lipid order disruption and surface-induced beta-sheet formation on the lipid domain boundary represent the early molecular events of membrane damage associated with the early pathogenesis of T2D.

Exploring Membrane Binding Targets of Disordered Human Tau Aggregates on Lipid Rafts Using Multiscale Molecular Dynamics Simulations.

The self-aggregation of tau, a microtubule-binding protein, has been linked to the onset of Alzheimer's Disease. Recent studies indicate that the disordered tau aggregates, or oligomers, are more toxic than the ordered fibrils found in the intracellular neurofibrillary tangles of tau. At present, details of tau oligomer interactions with lipid rafts, a model of neuronal membranes, are not known. Using molecular dynamics simulations, the lipid-binding events, membrane-damage, and protein folding of tau oligomers on various lipid raft surfaces were investigated. Tau oligomers preferred to bind to the boundary domains (Lod) created by the coexisting liquid-ordered (Lo) and liquid-disordered (Ld) domains in the lipid rafts. Additionally, stronger binding of tau oligomers to the ganglioside (GM1) and phosphatidylserine (PS) domains, and subsequent protein-induced lipid chain order disruption and beta-sheet formation were detected. Our results suggest that GM1 and PS domains, located exclusively in the outer and inner leaflets, respectively, of the neuronal membranes, are specific membrane domain targets, whereas the Lod domains are non-specific targets, of tau oligomers binding to neurons. The molecular details of these specific and non-specific tau bindings to lipid rafts may provide new insights into understanding membrane-associated tauopathies leading to Alzheimer's Disease.

Exploring the binding kinetics and behaviors of self-aggregated beta-amyloid oligomers to phase-separated lipid rafts with or without ganglioside-clusters.

Lipid binding kinetics and energetics of self-aggregated and disordered beta-amyloid oligomers of various sizes, from solution to lipid raft surfaces, were investigated using MD simulations. Our systems include small (monomers to tetramers) and larger (octamers and dodecamers) oligomers. Our lipid rafts contain saturated and unsaturated phosphatidylcholine (PC), cholesterol, and with or without asymmetrically distributed monosialotetrahexosylganglioside (GM1). All rafts exhibited dynamic and structurally diversified domains including liquid-ordered (Lo), liquid-disordered (Ld), and interfacial Lod domains. For rafts without GM1, all oligomers bound to the Lod domain. For GM1-containing rafts, all small oligomers and most larger oligomers bound specifically to the GM1-clusters embedded in the Lo domain. Lipid-protein binding energies followed an order of GM1 >> unsaturated PC > saturated PC > cholesterol for all rafts. In addition, protein-induced membrane structural disruption increased progressively with the size of the oligomer for the annular lipids surrounding the membrane-bound protein in non-GM1-containing rafts. We propose that the tight binding of beta-amyloid oligomers to the GM1-clusters and the structural perturbation of lipids surrounding the membrane-bound proteins at the Lod domain are early molecular events of the beta-amyloid aggregation process on neuronal membrane surfaces that trigger the onset of Alzheimer's.

Survival outcomes of the combination of extrafascial extrahepatic and extrafascial intrahepatic pedicle approaches in hepatectomy for hepatocellular carcinoma.

Introduction: This study was conducted to evaluate the results of hepatectomy for hepatocellular carcinoma (HCC) by the combination of extrafascial extrahepatic (Takasaki method) and extrafascial intrahepatic pedicle approaches (Ton That Tung method). Material and methods: A longitudinal follow-up study was conducted on 83 patients undergoing hepatectomy for HCC using the combination of extrafascial extrahepatic (Takasaki method) and extrafascial intrahepatic pedicle approaches at Nghe An Provincial Hospital from April 2017 to July 2021. Survival analysis was applied. Results: The cumulative overall survival (OS) rates after 1, 2, 3 and 4 years were 88.4%, 76.3%, 69.5% and 56.9%, respectively. The mean OS time was 40.68 ±2.17 months. The 1-, 2-, 3- and 4-year disease-free survival (DFS) rates were 67.1%, 56%, 53.1% and 50%, respectively. The mean DFS time was 32.58 ±2.56 months. Surgical margin > 1 cm was an independent predictor of both overall and disease-free survival (HR = 5.194, 95% CI = 1.467-18,385, p = 0.011 for OS; HR = 2.822, 95% CI = 1.231-6.468, p = 0.014 for DFS). Conclusions: Hepatectomy for HCC by a combination of extrafascial extrahepatic (Takasaki method) and extrafascial intrahepatic pedicle approaches (Ton That Tung method) is effective and safe, and side effects and complications can be controlled. Patient selection is a key issue and plays a very important role in the outcome of treatment.

Four-Month Rifapentine Regimens with or without Moxifloxacin for Tuberculosis.

BACKGROUND: Rifapentine-based regimens have potent antimycobacterial activity that may allow for a shorter course in patients with drug-susceptible pulmonary tuberculosis. METHODS: In an open-label, phase 3, randomized, controlled trial involving persons with newly diagnosed pulmonary tuberculosis from 13 countries, we compared two 4-month rifapentine-based regimens with a standard 6-month regimen consisting of rifampin, isoniazid, pyrazinamide, and ethambutol (control) using a noninferiority margin of 6.6 percentage points. In one 4-month regimen, rifampin was replaced with rifapentine; in the other, rifampin was replaced with rifapentine and ethambutol with moxifloxacin. The primary efficacy outcome was survival free of tuberculosis at 12 months. RESULTS: Among 2516 participants who had undergone randomization, 2343 had a culture positive for Mycobacterium tuberculosis that was not resistant to isoniazid, rifampin, or fluoroquinolones (microbiologically eligible population; 768 in the control group, 791 in the rifapentine-moxifloxacin group, and 784 in the rifapentine group), of whom 194 were coinfected with human immunodeficiency virus and 1703 had cavitation on chest radiography. A total of 2234 participants could be assessed for the primary outcome (assessable population; 726 in the control group, 756 in the rifapentine-moxifloxacin group, and 752 in the rifapentine group). Rifapentine with moxifloxacin was noninferior to the control in the microbiologically eligible population (15.5% vs. 14.6% had an unfavorable outcome; difference, 1.0 percentage point; 95% confidence interval [CI], -2.6 to 4.5) and in the assessable population (11.6% vs. 9.6%; difference, 2.0 percentage points; 95% CI, -1.1 to 5.1). Noninferiority was shown in the secondary and sensitivity analyses. Rifapentine without moxifloxacin was not shown to be noninferior to the control in either population (17.7% vs. 14.6% with an unfavorable outcome in the microbiologically eligible population; difference, 3.0 percentage points [95% CI, -0.6 to 6.6]; and 14.2% vs. 9.6% in the assessable population; difference, 4.4 percentage points [95% CI, 1.2 to 7.7]). Adverse events of grade 3 or higher occurred during the on-treatment period in 19.3% of participants in the control group, 18.8% in the rifapentine-moxifloxacin group, and 14.3% in the rifapentine group. CONCLUSIONS: The efficacy of a 4-month rifapentine-based regimen containing moxifloxacin was noninferior to the standard 6-month regimen in the treatment of tuberculosis. (Funded by the Centers for Disease Control and Prevention and others; Study 31/A5349 ClinicalTrials.gov number, NCT02410772.).

Functional Comparison between VP64-dCas9-VP64 and dCas9-VP192 CRISPR Activators in Human Embryonic Kidney Cells.

Reversal in the transcriptional status of desired genes has been exploited for multiple research, therapeutic, and biotechnological purposes. CRISPR/dCas9-based activators can activate transcriptionally silenced genes after being guided by gene-specific gRNA(s). Here, we performed a functional comparison between two such activators, VP64-dCas9-VP64 and dCas9-VP192, in human embryonic kidney cells by the concomitant targeting of POU5F1 and SOX2. We found 22- and 6-fold upregulations in the mRNA level of POU5F1 by dCas9-VP192 and VP64-dCas9-VP64, respectively. Likewise, SOX2 was up-regulated 4- and 2-fold using dCas9-VP192 and VP64dCas9VP64, respectively. For the POU5F1 protein level, we observed 3.7- and 2.2-fold increases with dCas9-VP192 and VP64-dCas9-VP64, respectively. Similarly, the SOX2 expression was 2.4- and 2-fold higher with dCas9-VP192 and VP64-dCas9-VP64, respectively. We also confirmed that activation only happened upon co-transfecting an activator plasmid with multiplex gRNA plasmid with a high specificity to the reference genes. Our data revealed that dCas9-VP192 is more efficient than VP64-dCas9-VP64 for activating reference genes.

A peptide derived from the core ß-sheet region of TIRAP decoys TLR4 and reduces inflammatory and autoimmune symptoms in murine models.

BACKGROUND: TLRs are some of the actively pursued drug-targets in immune disorders. Owing to a recent surge in the cognizance of TLR structural biology and signalling pathways, numerous therapeutic modulators, ranging from low-molecular-weight organic compounds to polypeptides and nucleic acid agents have been developed. METHODS: A penetratin-conjugated small peptide (TIP3), derived from the core ß-sheet of TIRAP, was evaluated in vitro by monitoring the TLR-mediated cytokine induction and quantifying the protein expression using western blot. The therapeutic potential of TIP3 was further evaluated in TLR-dependent in vivo disease models. FINDINGS: TIP3 blocks the TLR4-mediated cytokine production through both the MyD88- and TRIF-dependent pathways. A similar inhibitory-effect was exhibited for TLR3 but not on other TLRs. A profound therapeutic effect was observed in vivo, where TIP3 successfully alleviated the inflammatory response in mice model of collagen-induced arthritis and ameliorated the disease symptoms in psoriasis and SLE models. INTERPRETATION: Our data suggest that TIP3 may be a potential lead candidate for the development of effective therapeutics against TLR-mediated autoimmune disorders. FUNDING: This work was supported by the National Research Foundation of Korea (NRF-2019M3A9A8065098, 2019M3D1A1078940 and 2019R1A6A1A11051471). The funders did not have any role in the design of the present study, data collection, data analysis, interpretation, or the writing of the manuscript.

Feasibility of Direct Sputum Molecular Testing for Drug Resistance as Part of Tuberculosis Clinical Trials Eligibility Screening.

A rapid diagnosis of drug-resistant tuberculosis (TB) is critical for early initiation of effective therapy. Molecular testing with line probe assays (MTBDRplus and MTBDRsl) on culture isolates has been available for some time and significantly reduces the time to diagnosis of drug resistance. However, routine use of this test directly on sputum is less common. As part of enrollment screening procedures for tuberculosis clinical trials conducted in Hanoi, Vietnam, we evaluated the feasibility and performance of line probe assay (LPA) testing directly on sputum samples from 315 participants with no prior history of TB treatment. Test performance characteristics for the detection of rifampin (RIF) and isoniazid (INH) drug resistance as compared to culture-based drug susceptibility testing (DST) reference standard were calculated. LPA demonstrated high sensitivity and specificity for the diagnosis of drug resistance. Scaling up molecular testing on sputum as part of time-sensitive clinical trial screening procedures in high TB burden settings is feasible and will reduce both time to initiation of appropriate therapy and the risk of late exclusions due to microbiologic ineligibility.