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Autophagy degrades impaired organelles and toxic proteins to maintain cellular homeostasis. Dysregulated autophagy is a pathogenic participant in Alzheimer's disease (AD) progression. In early-stage AD, autophagy is beneficially initiated by mild endoplasmic reticulum (ER) stress to alleviate cellular damage and inflammation. However, chronic overproduction of toxic Aβ oligomers eventually causes Ca

The novel coronavirus SARS-CoV-2 has become a global health concern. The morbidity and mortality of the potentially lethal infection caused by this virus arise from the initial viral infection and the subsequent host inflammatory response. The latter may lead to excessive release of pro-inflammatory cytokines, IL-6 and IL-8, as well as TNF-α ultimately culminating in hypercytokinemia (“cytokine storm”). To address this immuno-inflammatory pathogenesis, multiple clinical trials have been proposed to evaluate anti-inflammatory biologic therapies targeting specific cytokines. However, despite the obvious clinical utility of such biologics, their specific applicability to COVID-19 has multiple drawbacks, including they target only one of the multiple cytokines involved in COVID-19’s immunopathy. Therefore, we set out to identify a small molecule with broad-spectrum anti-inflammatory mechanism of action targeting multiple cytokines of innate immunity. In this study, a library of small molecules endogenous to the human body was assembled, subjected to in silico molecular docking simulations and a focused in vitro screen to identify anti-pro-inflammatory activity via interleukin inhibition. This has enabled us to identify the loop diuretic furosemide as a candidate molecule. To pre-clinically evaluate furosemide as a putative COVID-19 therapeutic, we studied its anti-inflammatory activity on RAW264.7, THP-1 and SIM-A9 cell lines stimulated by lipopolysaccharide (LPS). Upon treatment with furosemide, LPS-induced production of pro-inflammatory cytokines was reduced, indicating that furosemide suppresses the M1 polarization, including IL-6 and TNF-α release. In addition, we found that furosemide promotes the production of anti-inflammatory cytokine products (IL-1RA, arginase), indicating M2 polarization. Accordingly, we conclude that furosemide is a reasonably potent inhibitor of IL-6 and TNF-α that is also safe, inexpensive and well-studied. Our pre-clinical data suggest that it may be a candidate for repurposing as an inhaled therapy against COVID-19.

The semiconductor industry is one of the key industries of Korea, which has continued growing at a steady annual growth rate. Important technology for the semiconductor industry is high integration of devices. This is to increase the memory capacity for unit area, of which key is photolithography. The photolithography refers to a technique for printing the shadow of light lit on the mask surface on to wafer, which is the most important process in a semiconductor manufacturing process. In this study, the width and step-height of wafers patterned through this process were measured to ensure uniformity. The widths and inter-plate heights of the specimens patterned using photolithography were measured using transmissive digital holography. A transmissive digital holographic interferometer was configured, and nine arbitrary points were set on the specimens as measured points. The measurement of each point was compared with the measurements performed using a commercial device called scanning electron microscope (SEM) and Alpha Step. Transmission digital holography requires a short measurement time, which is an advantage compared to other techniques. Furthermore, it uses magnification lenses, allowing the flexibility of changing between high and low magnifications. The test results confirmed that transmissive digital holography is a useful technique for measuring patterns printed using photolithography.

Purpose: The deformation characteristics induced by non-destructive stresses using piezoelectric transducer(PZT) were analyzed for 3-unit fixed partial dentures manufactured PFM, Everest(CAD/CAM) and Zirkonzahn(copy milling, MAD/MAM) by electron speckle pattern interferometery(ESPI). Methods: The ESPI analysis after loading the restoration with PZT by applying electric voltage of 900mV at the points of 10 mm above the base of the prostheses. Results: PFM and All-Ceramic Everest prostheses showed about 0.1 while that of All- Ceramic Zirkonzahn prostheses showed 0.085 , demonstrating that Zirkonzahn displaced less. For PFM and All-Ceramic Zirkonzahn prostheses, the displacements were large at just below the loading point, while generalize displacement was shown over the loading point and weak connector areas for All-Ceramic Everest prostheses. Conclusion: We could find that the deformation characteristics induced by non-destructive stresses using PZT analyzed by ESPI were similar to the fracture strengths evaluated using universal testing machine.

It is unclear whether mutations in fused in sarcoma (FUS) cause familial amyotrophic lateral sclerosis via a loss-of-function effect due to titrating FUS from the nucleus or a gain-of-function effect from cytoplasmic overabundance. To investigate this question, we generated a series of independent Caenorhabditis elegans lines expressing mutant or wild-type (WT) human FUS. We show that mutant FUS, but not WT-FUS, causes cytoplasmic mislocalization associated with progressive motor dysfunction and reduced lifespan. The severity of the mutant phenotype in C. elegans was directly correlated with the severity of the illness caused by the same mutation in humans, arguing that this model closely replicates key features of the human illness. Importantly, the mutant phenotype could not be rescued by overexpression of WT-FUS, even though WT-FUS had physiological intracellular localization, and was not recruited to the cytoplasmic mutant FUS aggregates. Our data suggest that FUS mutants cause neuronal dysfunction by a dominant gain-of-function effect related either to neurotoxic aggregates of mutant FUS in the cytoplasm or to dysfunction in its RNA-binding functions.

Vascular endothelial growth factor (VEGF), which has neurotrophic and neuroprotective effects in addition to its major role in angiogenesis, interacts with Aβ and accumulates in the senile plaques of Alzheimer's disease (AD) patients' brains. It is known that Aβ binds to the heparin-binding domain (HBD) of the 165-amino acid VEGF variant, VEGF(165). In this study, we showed that triamterene (Trm) inhibits VEGF--Aβ interaction without affecting other biological activities of VEGF or Aβ. We investigated the importance of structural and dynamic features of HBD for its molecular-recognition processes. The binding model of HBD and Trm was constructed based on measurements of chemical shift changes and docking study. The results showed that the loop region (S11-L17) and F18 at the beginning of the first β-sheet in the HBD constitute the inhibitor binding site. The N1 atom of pteridine ring of Trm forms hydrogen bonding with backbone amide proton of R13, and the phenyl ring took part in a hydrophobic interaction with the aromatic ring of F18. To investigate the functional importance of the inherent structural flexibility of the HBD in VEGF, the dynamic properties of free HBD and HBD--Trm complex were assessed by measuring spin relaxation rates, and the backbone dynamics were investigated by model-free analysis. The residues in the disordered loop region of the N-terminus exhibited conformational exchanges in free HBD, and flexibility of this loop region decreased dramatically upon binding to Trm, suggesting that Aβ as well as inhibitor may recognize these unique dynamic features of the HBD. Furthermore, C-terminal residues continued to exhibit slow conformational motions, even in the HBD--Trm complex, implying that these motions at the C-terminus of the HBD might be important for interactions with heparin molecules. The flexibility of HBD demonstrated here should be essential for VEGF function and interaction with other protein partners.

International audience; Complexes involved in the gamma/epsilon-secretase-regulated intramembranous proteolysis of substrates such as the amyloid-beta precursor protein are composed primarily of presenilin (PS1 or PS2), nicastrin, anterior pharynx defective-1 (APH1), and PEN2. The presenilin aspartyl residues form the catalytic site, and similar potentially functional polar transmembrane residues in APH1 have been identified. Substitution of charged (E84A, R87A) or polar (Q83A) residues in TM3 had no effect on complex assembly or activity. In contrast, changes to either of two highly conserved histidines (H171A, H197A) located in TM5 and TM6 negatively affected PS1 cleavage and altered binding to other secretase components, resulting in decreased amyloid generating activity. Charge replacement with His-to-Lys substitutions rescued nicastrin maturation and PS1 endoproteolysis leading to assembly of the formation of structurally normal but proteolytically inactive gamma-secretase complexes. Substitution with a negatively charged side chain (His-to-Asp) or altering the structural location of the histidines also disrupted gamma-secretase binding and abolished functionality of APH1. These results suggest that the conserved transmembrane histidine residues contribute to APH1 function and can affect presenilin catalytic activity.

beta-amyloid (Abeta) is a major component of senile plaques that is commonly found in the brain of Alzheimer's disease (AD) patient. In the previous report, we showed that an important angiogenic factor, vascular endothelial growth factor (VEGF) interacts with Abeta and is accumulated in the senile plaques of AD patients' brains. Here we show that Abeta interacts with VEGF(165) isoform, but not with VEGF(121). Abeta binds to the heparin-binding domain (HBD) of VEGF(165) with similar affinity as that of intact VEGF(165). Abeta binds mostly to the C-terminal subdomain of HBD, but with greatly reduced affinity than HBD. Therefore, the full length of HBD appears to be required for maximal binding of Abeta. Although Abeta binds to heparin-binding sequence of VEGF, it does not bind to other heparin-binding growth factors except midkine. Thus it seems that Abeta recognizes unique structural features of VEGF HBD. VEGF(165) prevents aggregation of Abeta through its HBD. We localized the core VEGF binding site of Abeta at around 26-35 region of the peptide. VEGF(165) and HBD protect PC12 cells from the Abeta-induced cytotoxicity. The mechanism of protection appears to be inhibition of both Abeta-induced formation of reactive oxygen species and Abeta aggregation.

Background and Hypothesis: Both beta amyloid and tau produce neurotoxic protein misfolding co-conspirators most often implicated in the molecular causation of AD. Moreover, since the clinical presentation of AD is heterogeneous (e.g. extrapyramidal symptoms in some people with AD and dementia symptoms in some people with PD), the clinical overlap amongst neurodegenerative disorders implies that AD is more of a syndrome than a disease and that the suppression of other aberrantly misfolded protein (e.g. alpha synuclein) may afford additional therapeutic benefits. Accordingly, we are seeking to design and develop brain-penetrable small molecule new chemical entities as anti-protein misfolding agents targeting three proteins: beta amyloid 40 and 42; tau and alpha synuclein. Based upon extensive in silico modeling, a family of novel compounds has been identified. A representative compound is TRV101. Methods: Efficacy of TRV101 was measured in a variety of in vitro assays including Thioflavin T beta amyloid aggregation, Thioflavin S tau aggregation, and biotin-beta amyloid 42 oligomerization. TRV101 was incubated with full length tau and the resultant solution was examined by electron microscopy. Additionally, data was collected on blood brain penetrance, protein binding, and longevity in mouse liver microsomes. Results: TRV101 showed anti-aggregation activity against both beta amyloid and tau. TRV has drug like properties, has high brain penetrance, and is stable in various ADME tests. Conclusion: We have developed a new class of compounds capable of inhibiting aggregation of both beta amyloid and tau proteins. Our small molecules are pharmacokinetically stable and are able to reach the target tissue. Our compounds are currently being tested in animal models of both beta amyloid and tau pathologies. In Vivo PK (Mouse, p.o.)  AUC (brain) = 8692 hr*ng/ mL  T1/2 = 5.4h  Cmax = 1217 ng/mL (brain)  Bioavailability = 78.3 %  B/P = 1 MLM T1/2 > 60 min Clint = 0.008 mL/min/mg HLM T1/2 = 57 min Clint = 0.024 mL/min/mg Physical Chemistry Properties  LogD = 3.15  MW: 446 g/mol  tPSA = 60.6 Protein binding  98.9 % (mouse)  97.6 % (human) 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 A b s o rb a n c e ( 4 5 0 n m ) In vitro assays Thioflavin S aggregation assay – Thioflavin S fluoresces when in the presence of aggregated tau. The reaction is initiated by addition of heparin and the reaction is followed by fluorescence until the signal change plateaus. Test compounds are measured by change in fluorescence compared to a vehicle control. TRV101 inhibits tau-441 aggregation with an IC50 of 2.5μM Oligomer formation assay – Biotinylated amyloid-β is allowed to self-associate in the presence of compound, vehicle, and 0.1% Tween20. The reaction is stopped by addition of 0.1% Tween-20 and the sample is applied to a neutravidin ELISA plate. Oligomers are detected by addition of streptavidin-HRP, which will only detect unbound biotin label, i.e., oligomers. TRV101 inhibits biotinylated amyloidβ (1-40) oligomerization with an IC50 of 5μM Animal models APP/PS1 (amyloid-β model), n=13 The mice were dosed at 30mg/kg (p.o., qd) commencing at 6 weeks of age with behavioral testing (2d-RAWM and fear conditioning) at ~3.5 months of age followed by ex vivo LTP analysis performed at the CA3/CA1 synapse in the hippocampus.

Objective To delineate the role of neuropilin-1 (NP-1), a vascular endothelial growth factor receptor (VEGFR), in rheumatoid inflammation and to determine whether blockade of NP-1 could suppress synoviocyte survival and angiogenesis. Methods VEGF(111-165) peptide, which encompasses the NP-1 binding domain of VEGF(165), was generated by cleaving VEGF(165) with plasmin. The effect of this peptide on the interaction between VEGF(165) and its receptor was determined by (125)I-VEGFR binding assay. Assays to determine synoviocyte apoptosis, adhesion, and migration were performed in the presence of VEGF(165) and/or the peptide. VEGF(165)-induced angiogenesis was assessed by measuring the proliferation, tube formation, and wounding migration of endothelial cells (ECs). Mice were immunized with type II collagen to induce experimental arthritis. Results VEGF(111-165) peptide specifically inhibited the binding of (125)I-VEGF(165) to NP-1 on rheumatoid synoviocytes and ECs. The peptide eliminated the VEGF(165)-mediated increase in synoviocyte survival and activation of p-ERK and Bcl-2. The peptide also completely inhibited a VEGF(165)-induced increase in synoviocyte adhesion and migration. In addition, the anti-NP-1 peptide blocked VEGF(165)-stimulated proliferation, capillary tube formation, and wounding migration of ECs in vitro. VEGF(165)-induced neovascularization in a Matrigel plug in mice was also blocked by treatment with the peptide. Finally, subcutaneous injection of anti-NP-1 peptide suppressed arthritis severity and autoantibody formation in mice with experimental arthritis and inhibited synoviocyte hyperplasia and angiogenesis in arthritic joints. Conclusion Anti-NP-1 peptide suppressed VEGF(165)-induced increases in synoviocyte survival and angiogenesis, and thereby blocked experimental arthritis. Our findings suggest that anti-NP-1 peptide could be useful in alleviating chronic arthritis.