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Despite much effort, antibody therapies for Alzheimers disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.

Objective To investigate the impact of metabolic labeling on Porphyromonas gingivalis (Pg) and compare the imaging effects of two fluorescent probes. Methods This study was reviewed by the unit Ethics Committee and was approved by the Experimental Animal Welfare Ethics Branch of the Unit Experimental Biomedical Ethics Committee. Pg integrated N-azidoacetylgalactosamine (Ac4GalNAz) via a bioorthogonal reaction and was labeled with Cy5-DBCO or Cy7-DBCO via a click chemistry reaction. The bacteria were divided into Pg group (control, not fluorescently labeled), Cy5-Pg group (tagged by Cy5-DBCO), and Cy7-Pg group (tagged by Cy7-DBCO). A live/dead staining kit was applied to test the viability of Pg, Cy5-Pg, and Cy7-Pg. The mRNA levels of interleukin-6 (IL-6) and IL-8 and cell proliferation were examined in human gingival fibroblasts (HGFs) after the challenge of Cy5-Pg, Cy7-Pg, or Pg. To investigate the stability of metabolic labeling, Cy5-Pg or Cy7-Pg was cocultured with Escherichia coli (E. coli). Cy5-Pg and Cy7-Pg signal intensity with serial dilutions were examined using an in vivo imaging system (IVIS). Finally, C57BL/6J mice were orally administered Cy5-Pg or Cy7-Pg for IVIS detection, and the signal-to-background ratios were calculated. Results Metabolic labeling could be applied to label live Pg in vitro. The optimal labeling concentrations for Cy5 and Cy7 were 20 μmol/L and 30 μmol/L, respectively. The area ratios of live to dead bacteria were approximately 2.0 in the three groups (F = 0.318, P>0.05). After a 6-h challenge with Cy5-Pg, Cy7-Pg, or Pg, the mRNA levels of HGFs increased by 7.86-, 7.46-, and 6.56-fold for IL-6, respectively (F = 40.886, PF = 18.781, PP>0.05). HGFs were further challenged by Cy5-Pg, Cy7-Pg, or Pg at different MOIs, and cell proliferation was significantly inhibited (MOI = 104∶1, F = 153.52, P5∶1, F = 331.21, P6∶1, F = 533.65, PP>0.05). Within 24 h of co-culturing Cy5-Pg or Cy7-Pg with E. coli, minimal E. coli was detected. The intensities of Cy5 and Cy7 remained stable for 3 h. Additionally, the fluorescence signal intensities of Cy5 and Cy7 were linearly correlated with the concentration (R2 = 0.97). After oral gavage of Cy5-Pg or Cy7-Pg in mice for the abdominal region at 1 h and 3 h, the signal-to-background ratios of Cy7-Pg were approximately 4.24-fold (t = 6.893, Pt = 4.407, PPg. For the isolated gastrointestinal tracts at 3 h, the signal-to-background ratio of Cy7-Pg was 5.19-fold higher than that of Cy5-Pg (t = 4.418, P

La-related protein 7 (LARP7) are a family of RNA chaperones that protect the 3'-end of RNA and are components of specific ribonucleoprotein complexes (RNP). In Tetrahymena thermophila telomerase, LARP7 protein p65 together with telomerase reverse transcriptase (TERT) and telomerase RNA (TER) form the core RNP. p65 has four known domains-N-terminal domain (NTD), La motif (LaM), RNA recognition motif 1 (RRM1), and C-terminal xRRM2. To date, only the xRRM2 and LaM and their interactions with TER have been structurally characterized. Conformational dynamics leading to low resolution in cryo-EM density maps have limited our understanding of how full-length p65 specifically recognizes and remodels TER for telomerase assembly. Here, we combined focused classification of Tetrahymena telomerase cryo-EM maps with NMR spectroscopy to determine the structure of p65-TER. Three previously unknown helices are identified, one in the otherwise intrinsically disordered NTD that binds the La module, one that extends RRM1, and another preceding xRRM2, that stabilize p65-TER interactions. The extended La module (αN, LaM and RRM1) interacts with the four 3' terminal U nucleotides, while LaM and αN additionally interact with TER pseudoknot, and LaM with stem 1 and 5' end. Our results reveal the extensive p65-TER interactions that promote TER 3'-end protection, TER folding, and core RNP assembly and stabilization. The structure of full-length p65 with TER also sheds light on the biological roles of genuine La and LARP7 proteins as RNA chaperones and core RNP components.

The self-assembly of the Nucleocapsid protein (NCAP) of SARS-CoV-2 is crucial for its function. Computational analysis of the amino acid sequence of NCAP reveals low-complexity domains (LCDs) akin to LCDs in other proteins known to self-assemble as phase separation droplets and amyloid fibrils. Previous reports have described NCAP's propensity to phase-separate. Here we show that the central LCD of NCAP is capable of both, phase separation and amyloid formation. Within this central LCD we identified three adhesive segments and determined the atomic structure of the fibrils formed by each. Those structures guided the design of G12, a peptide that interferes with the self-assembly of NCAP and demonstrates antiviral activity in SARS-CoV-2 infected cells. Our work, therefore, demonstrates the amyloid form of the central LCD of NCAP and suggests that amyloidogenic segments of NCAP could be targeted for drug development.

Neurodegenerative diseases are characterized by the pathologic accumulation of aggregated proteins. Known as amyloid, these fibrillar aggregates include proteins such as tau and amyloid-β (Aβ) in Alzheimer's disease (AD) and alpha-synuclein (αSyn) in Parkinson's disease (PD). The development and spread of amyloid fibrils within the brain correlates with disease onset and progression, and inhibiting amyloid formation is a possible route toward therapeutic development. Recent advances have enabled the determination of amyloid fibril structures to atomic-level resolution, improving the possibility of structure-based inhibitor design. In this work, we use these amyloid structures to design inhibitors that bind to the ends of fibrils, "capping" them so as to prevent further growth. Using de novo protein design, we develop a library of miniprotein inhibitors of 35 to 48 residues that target the amyloid structures of tau, Aβ, and αSyn. Biophysical characterization of top in silico designed inhibitors shows they form stable folds, have no sequence similarity to naturally occurring proteins, and specifically prevent the aggregation of their targeted amyloid-prone proteins in vitro. The inhibitors also prevent the seeded aggregation and toxicity of fibrils in cells. In vivo evaluation reveals their ability to reduce aggregation and rescue motor deficits in Caenorhabditis elegans models of PD and AD.

Alzheimer's disease (AD) is the consequence of neuronal death and brain atrophy associated with the aggregation of protein tau into fibrils. Thus disaggregation of tau fibrils could be a therapeutic approach to AD. The small molecule EGCG, abundant in green tea, has long been known to disaggregate tau and other amyloid fibrils, but EGCG has poor drug-like properties, failing to fully penetrate the brain. Here we have cryogenically trapped an intermediate of brain-extracted tau fibrils on the kinetic pathway to EGCG-induced disaggregation and have determined its cryoEM structure. The structure reveals that EGCG molecules stack in polar clefts between the paired helical protofilaments that pathologically define AD. Treating the EGCG binding position as a pharmacophore, we computationally screened thousands of drug-like compounds for compatibility for the pharmacophore, discovering several that experimentally disaggregate brain-derived tau fibrils in vitro. This work suggests the potential of structure-based, small-molecule drug discovery for amyloid diseases.

Many voltammetry methods have been developed to monitor brain extracellular dopamine levels. Fewer approaches have been successful in detecting serotonin in vivo. No voltammetric techniques are currently available to monitor both neurotransmitters simultaneously across timescales, even though they play integrated roles in modulating behavior. We provide proof-of-concept for rapid pulse voltammetry coupled with partial least squares regression (RPV-PLSR), an approach adapted from multi-electrode systems (i.e., electronic tongues) used to identify multiple components in complex environments. We exploited small differences in analyte redox profiles to select pulse steps for RPV waveforms. Using an intentionally designed pulse strategy combined with custom instrumentation and analysis software, we monitored basal and stimulated levels of dopamine and serotonin. In addition to faradaic currents, capacitive currents were important factors in analyte identification arguing against background subtraction. Compared to fast-scan cyclic voltammetry-principal components regression (FSCV-PCR), RPV-PLSR better differentiated and quantified basal and stimulated dopamine and serotonin associated with striatal recording electrode position, optical stimulation frequency, and serotonin reuptake inhibition. The RPV-PLSR approach can be generalized to other electrochemically active neurotransmitters and provides a feedback pipeline for future optimization of multi-analyte, fit-for-purpose waveforms and machine learning approaches to data analysis.

Objective. Develop a prototype on-line positron emission tomography (PET) scanner and evaluate its capability of on-line imaging and intra-fractionated proton-induced radioactivity range measurement. Approach. Each detector consists of 32 × 32 array of 2 × 2 × 30 mm3 Lutetium–Yttrium Oxyorthosilicate scintillators with single-scintillator-end readout through a 20 × 20 array of 3 × 3 mm2 Silicon Photomultipliers. The PET can be configurated with a full-ring of 20 detectors for conventional PET imaging or a partial-ring of 18 detectors for on-line imaging and range measurement. All detector-level readout and processing electronics are attached to the backside of the system gantry and their output signals are transferred to a field-programable-gate-array based system electronics and data acquisition that can be placed 2 m away from the gantry. The PET imaging performance and radioactivity range measurement capability were evaluated by both the offline study that placed a radioactive source with known intensity and distribution within a phantom and the online study that irradiated a phantom with proton beams under different radiation and imaging conditions. Main results. The PET has 32 cm diameter and 6.5 cm axial length field-of-view (FOV), ∼2.3–5.0 mm spatial resolution within FOV, 3% sensitivity at the FOV center, 18%–30% energy resolution, and ∼9 ns coincidence time resolution. The offline study shows the PET can determine the shift of distal falloff edge position of a known radioactivity distribution with the accuracy of 0.3 ± 0.3 mm even without attenuation and scatter corrections, and online study shows the PET can measure the shift of proton-induced positron radioactive range with the accuracy of 0.6 ± 0.3 mm from the data acquired with a short-acquisition (60 s) and low-dose (5 MU) proton radiation to a human head phantom. Significance. This study demonstrated the capability of intra-fractionated PET imaging and radioactivity range measurement and will enable the investigation on the feasibility of intra-fractionated, range-shift compensated adaptive proton therapy. [ABSTRACT FROM AUTHOR]

Purtscher-like retinopathy is a rare and vision-threatening eye disease, which is usually associated with acute pancreatitis, autoimmune diseases, and renal failure. We here reported a case of Purtscher-like retinopathy after dupilumab treatment in atopic dermatitis. A 48-year-old woman with a history of severe atopic dermatitis developed ocular manifestations after second exposure to dupilumab. Best-corrected visual acuity (BCVA) was count fingers at 2 feet in the right eye and 20/133 in the left eye when she referred to our hospital. Fundus examination demonstrated bilateral serous macular off, thickening of papillomacular bundle, intraretinal hemorrhage, cotton-wool spots and Purtscher flecken surrounding the optic disc. Optical coherence tomography (OCT) angiography showed flow void in the superficial and deep capillary plexuses. And early hypofluorescence corresponding to grayish-white retinal thickening was observed in fundus fluorescein angiography. Laboratory tests revealed hypereosinophilia after symptoms appeared. A diagnosis of Purtscher-like retinopathy was made, which may associate with dupilumab. The patient accepted the treatment with glucocorticoids. At six months follow up, BCVA was 20/40 in the right eye and 20/25 in the left eye. The Purtscher-like retinopathy we report may be the adverse event of dupilumab. Rare adverse reactions to biologics deserve the attention of ophthalmologists. [ABSTRACT FROM AUTHOR]

Systems neuroscience research is increasingly possible through the use of integrated molecular and circuit-level analyses. These studies depend on the use of animal models and, in many cases, molecular and circuit-level analyses. Associated with genetic, pharmacologic, epigenetic, and other types of environmental manipulations. We illustrate typical pitfalls resulting from poor validation of behavior tests. We describe experimental designs and enumerate controls needed to improve reproducibility in investigating and reporting of behavioral phenotypes.

Cellulose is used widely in antimicrobial packaging due to its abundance in nature, biodegradability, renewability, non-toxicity, and low cost. However, how efficiently and rapidly it imparts high antimicrobial activity to cellulose-based packaging materials remains a challenge. In this work, Ag NPs were deposited on the surface of carboxymethyl cellulose/starch/N'N Methylenebisacrylamide film using ultrasonic radiation. Morphology and structure analysis of as-prepared films were conducted, and the antibacterial effects under different ultrasonic times and reductant contents were investigated. These results showed that Ag NPs were distributed uniformly on the film surface under an ultrasonic time of 45 min. The size of Ag NPs changes as the reducing agent content decreases. The composite film demonstrated a slightly better antibacterial effect against E. coli than against S. aureus. Therefore, this work can provide valuable insights for the research on antimicrobial packaging. [ABSTRACT FROM AUTHOR]

In recent years, the rapid development of the domestic projection industry, the projector industry will usher in more development opportunities, 4K LCD projection technology has formed the LCD projection full link self‐research design and manufacturing capabilities, It includes the industry's first 4K LCD optical machine, LCD projection display panel, self‐designed display motherboard and reliable ODM machine manufacturing capabilities, giving 4K LCD projectors a richer built‐in ultra HD keystone correction technology, the solution uses external hard decoding dedicated processor, ultra‐low latency and does not occupy the GPU soft decoding resources of SoC in the traditional solution. The GPU processing power of the SoC can be fully released. And support edge‐blending image quality adjustment. The unique image fusion technology can achieve a seamless visual experience and perfect picture quality improvement when multiple projectors are used together, creating a high‐quality UHD projection audio‐visual experience for users.. [ABSTRACT FROM AUTHOR]