Brain regions differences in amyloid-ß and gene expression in early APP/PS1 mice and identification of Npas4 as a key molecule in Alzheimer's disease.

Distinct brain regions are differentially affected during the various stages of Alzheimer's disease (AD). While the hippocampus and cortex are known to play significant roles, the involvement of the cerebellum has received less attention. Understanding the changes in diverse brain regions is essential to unravel the neuropathological mechanism in early-stage AD. Our research aimed to explore and compare amyloid-ß (Aß) pathology and gene expression profiles across the hippocampus, cortex, and cerebellum in the early stages of the Amyloid Precursor Protein/Presenilin-1 (APP/PS1) mouse model. By 7 months of age, significant Aß plaque accumulation was observed in the hippocampus and cortex of APP/PS1 mice, while no such deposits were found in the cerebellum. Gene expression analysis revealed predominant effects on immune response pathways in the hippocampus and cortex. Even in the absence of Aß deposition, notable gene expression changes were observed in the cerebellum of APP/PS1 mice. Intriguingly, Neuronal PAS Domain protein 4 (Npas4) expression was consistently down-regulated across all brain regions, independent of Aß plaque presence. Our findings reveal distinct transcriptomic alterations and Aß pathology in select cerebral regions during the initial phase of AD. Notably, the diminished expression of the Npas4 across three brain regions implies that Npas4 could play a pivotal role in the early pathogenesis of AD.

PepExplainer: An explainable deep learning model for selection-based macrocyclic peptide bioactivity prediction and optimization.

Macrocyclic peptides possess unique features, making them highly promising as a drug modality. However, evaluating their bioactivity through wet lab experiments is generally resource-intensive and time-consuming. Despite advancements in artificial intelligence (AI) for bioactivity prediction, challenges remain due to limited data availability and the interpretability issues in deep learning models, often leading to less-than-ideal predictions. To address these challenges, we developed PepExplainer, an explainable graph neural network based on substructure mask explanation (SME). This model excels at deciphering amino acid substructures, translating macrocyclic peptides into detailed molecular graphs at the atomic level, and efficiently handling non-canonical amino acids and complex macrocyclic peptide structures. PepExplainer's effectiveness is enhanced by utilizing the correlation between peptide enrichment data from selection-based focused library and bioactivity data, and employing transfer learning to improve bioactivity predictions of macrocyclic peptides against IL-17C/IL-17 RE interaction. Additionally, PepExplainer underwent further validation for bioactivity prediction using an additional set of thirteen newly synthesized macrocyclic peptides. Moreover, it enabled the optimization of the IC50 of a macrocyclic peptide, reducing it from 15 nM to 5.6 nM based on the contribution score provided by PepExplainer. This achievement underscores PepExplainer's skill in deciphering complex molecular patterns, highlighting its potential to accelerate the discovery and optimization of macrocyclic peptides.

PepScaf: Harnessing Machine Learning with In Vitro Selection toward De Novo Macrocyclic Peptides against IL-17C/IL-17RE Interaction.

The combination of library-based screening and artificial intelligence (AI) has been accelerating the discovery and optimization of hit ligands. However, the potential of AI to assist in de novo macrocyclic peptide ligand discovery has yet to be fully explored. In this study, an integrated AI framework called PepScaf was developed to extract the critical scaffold relative to bioactivity based on a vast dataset from an initial in vitro selection campaign against a model protein target, interleukin-17C (IL-17C). Taking the generated scaffold, a focused macrocyclic peptide library was rationally constructed to target IL-17C, yielding over 20 potent peptides that effectively inhibited IL-17C/IL-17RE interaction. Notably, the top two peptides displayed exceptional potency with IC50 values of 1.4 nM. This approach presents a viable methodology for more efficient macrocyclic peptide discovery, offering potential time and cost savings. Additionally, this is also the first report regarding the discovery of macrocyclic peptides against IL-17C/IL-17RE interaction.

Implications of N7-hydrogen and C8-keto on the base pairing, mutagenic potential and repair of 8-oxo-2'-deoxy-adenosine: Investigation by nucleotide analogues.

Oxidative lesions, such as 8-oxo-dG and 8-oxo-dA, are continuously generated from exposure to reactive oxygen species. While 8-oxo-dG has been extensively studied, 8-oxo-dA has not received as much attention until recently. Herein, we report the synthesis of duplex DNAs incorporating dA, 8-oxo-dA, 7-deaza-dA, 8-Br-dA, and 8-Br-7-deaza-dA, which have different substitutions at 7- and 8-position, for the investigation into the implications of N7-hydrogen and C8-keto on the base pairing preference, mutagenic potential and repair of 8-oxo-dA. Base pairing study suggested that the polar N7-hydrogen and C8-keto of 8-oxo-dA, rather than the syn-preference, might be essential for 8-oxo-dA to form a stable base pair with dG. Insertion and extension studies using KF-exo- and human DNA polymerase ß indicated that the efficient dGTP insertion opposite 8-oxo-dA and extension past 8-oxo-dA:dG are contingent upon not only the stable base pair with dG, but also the flexibility of the active site in polymerase. The N7-hydrogen in 8-oxo-dA or C7-hydrogen in 7-deaza-dA and 8-Br-7-deaza-dA was suggested to be important for the recognition by hOGG1, although the excision efficiencies of 7-deaza-dA and 8-Br-7-deaza-dA were much lower than 8-oxo-dA. This study provides an insight into the structure-function relationship of 8-oxo-dA by nucleotide analogues.

The impairment of the hippocampal neuro-vascular unit precedes changes in spatial cognition in naturally aged rats.

Ageing is the major risk factor for the most neurodegenerative diseases, such as Alzheimer's disease (AD). Damage to neurovascular components (microvessels, glia, and neurons) occurs with ageing and is suspected to exacerbate or cause mild cognitive impairment (MCI), vascular dementia, and AD. However, whether vascular cells, glia, and neurons change synchronously or asynchronously during ageing is unclear, and the relationship between complex dynamic pathophysiological changes in the brain and cognitive ability needs to be further studied. We used male Sprague-Dawley (SD) rats of three different ages (2 months, 12 months, and 24 months) and explored changes in the neurovascular unit (cerebral vessels, microglia, astrocytes, and neurons) and spatial memory upon normal ageing by the Morris water maze (MWM) test and immunofluorescence staining. We found that the impairments of microvessels, glia, neurons, and spatial memory were age-dependent in the rat hippocampus. In middle-aged (12-month-old) rats, some neurovascular unit components have become abnormal: the density and length of microvessels, pyramidal neuron, and SST (Somatostatin) neuron number was decreased, the number of astrocytes was increased in the hippocampus. The diameter of microvessels and PV (Parvalbumin) neuron numbers were decreased, the microglial number was increased and spatial learning was deficit at 24 months of age. In conclusion, we found that the impairment of the hippocampal neuro-vascular unit precedes changes in spatial cognition in naturally aged rats.

Cytophaga hutchinsonii chu_2177, encoding the O-antigen ligase, is essential for cellulose degradation.

Cytophaga hutchinsonii can efficiently degrade crystalline cellulose, in which the cell surface cellulases secreted by the type IX secretion system (T9SS) play important roles, but the degradation mechanism remains unclear, and the anchor mechanism of cellulases on the outer membrane in C. hutchinsonii has not been studied. Here, chu_2177 was identified by transposon mutagenesis and was proved to be indispensable for cellulose utilization in C. hutchinsonii. Disruption of chu_2177 resulted in O-antigen deficiency and chu_177 could confer O-antigen ligase activity upon an Escherichia coli waal mutant, indicating that chu_2177 encoded the O-ntigen ligase. Moreover, deletion of chu_2177 caused defects in cellulose utilization, cell motility, biofilm formation, and stress resistance. Further study showed that the endoglucanase activity was markedly decreased in the outer membrane but was increased in the culture fluid without chu_2177. Western blot proved that endoglucanase CHU_1336 was not located on the outer membrane but was released in the culture fluid of the Δ2177 mutant. Further proteomics analysis showed that many cargo proteins of T9SS were missing in the outer membrane of the Δ2177 mutant. Our study revealed that the deletion of chu_2177 affected the localization of many T9SS cargo proteins including cellulases on the outer membrane of C. hutchinsonii.

N-Glycosylation of a Cargo Protein C-Terminal Domain Recognized by the Type IX Secretion System in Cytophaga hutchinsonii Affects Protein Secretion and Localization.

Cytophaga hutchinsonii is a Gram-negative bacterium belonging to the phylum Bacteroidetes. It digests crystalline cellulose with an unknown mechanism and possesses a type IX secretion system (T9SS) that can recognize the C-terminal domain (CTD) of the cargo protein as a signal. In this study, the functions of the CTD in the secretion and localization of T9SS substrates in C. hutchinsonii were studied by fusing the green fluorescent protein (GFP) with the CTD from CHU_2708. The CTD is necessary for the secretion of GFP by C. hutchinsonii T9SS. The GFP-CTDCHU_2708 fusion protein was found to be glycosylated in the periplasm, with a molecular mass about 5 kDa higher than that predicted from its sequence. The glycosylated protein was sensitive to peptide-N-glycosidase F, which can hydrolyze N-linked oligosaccharides. Analyses of mutants obtained by site-directed mutagenesis of asparagine residues in the N-X-S/T motif of CTDCHU_2708 suggested that N-glycosylation occurred on the CTD. CTD N-glycosylation is important for the secretion and localization of GFP-CTD recombinant proteins in C. hutchinsonii. Glycosyltransferase-encoding gene chu_3842, a homologous gene of Campylobacter jejuni pglA, was found to participate in the N-glycosylation of C. hutchinsonii. Deletion of chu_3842 affected cell motility, cellulose degradation, and cell resistance to some chemicals. Our study provided evidence that the CTD as the signal of T9SS was N-glycosylated in the periplasm of C. hutchinsonii. IMPORTANCE The bacterial N-glycosylation system has previously been found only in several species of Proteobacteria and Campylobacterota, and the role of N-linked glycans in bacteria is still not fully understood. C. hutchinsonii has a unique cell contact cellulose degradation mechanism, and many cell surface proteins, including cellulases, are secreted by the T9SS. In this study, we found that C. hutchinsonii, a member of the phylum Bacteroidetes, has an N-glycosylation system. Glycosyltransferase CHU_3842 was found to participate in the N-glycosylation of C. hutchinsonii proteins and had effects on cell resistance to some chemicals, cell motility, and cellulose degradation. Moreover, N-glycosylation occurs on the CTD translocation signal of T9SS. The glycosylation of the CTD appears to play an important role in affecting T9SS substrate transportation and localization. This study enriched our understanding of the widespread existence and multiple biological roles of N-glycosylation in bacteria.

Merging the Versatile Functionalities of Boronic Acid with Peptides.

Peptides inherently feature the favorable properties of being easily synthesized, water-soluble, biocompatible, and typically non-toxic. Thus, boronic acid has been widely integrated with peptides with the goal of discovering peptide ligands with novel biological activities, and this effort has led to broad applications. Taking the integration between boronic acid and peptide as a starting point, we provide an overview of the latest research advances and highlight the versatile and robust functionalities of boronic acid. In this review, we summarize the diverse applications of peptide boronic acids in medicinal chemistry and chemical biology, including the identification of covalent reversible enzyme inhibitors, recognition, and detection of glycans on proteins or cancer cell surface, delivery of siRNAs, development of pH responsive devices, and recognition of RNA or bacterial surfaces. Additionally, we discuss boronic acid-mediated peptide cyclization and peptide modifications, as well as the facile chemical synthesis of peptide boronic acids, which paved the way for developing a growing number of peptide boronic acids.

Ferulic Acid Ameliorates Alzheimer's Disease-like Pathology and Repairs Cognitive Decline by Preventing Capillary Hypofunction in APP/PS1 Mice.

Brain capillaries are crucial for cognitive functions by supplying oxygen and other nutrients to and removing metabolic wastes from the brain. Recent studies have demonstrated that constriction of brain capillaries is triggered by beta-amyloid (Aß) oligomers via endothelin-1 (ET1)-mediated action on the ET1 receptor A (ETRA), potentially exacerbating Aß plaque deposition, the primary pathophysiology of Alzheimer's disease (AD). However, direct evidence is still lacking whether changes in brain capillaries are causally involved in the pathophysiology of AD. Using APP/PS1 mouse model of AD (AD mice) relative to age-matched negative littermates, we identified that reductions of density and diameter of hippocampal capillaries occurred from 4 to 7 months old while Aß plaque deposition and spatial memory deficit developed at 7 months old. Notably, the injection of ET1 into the hippocampus induced early Aß plaque deposition at 5 months old in AD mice. Conversely, treatment of ferulic acid against the ETRA to counteract the ET1-mediated vasoconstriction for 30 days prevented reductions of density and diameter of hippocampal capillaries as well as ameliorated Aß plaque deposition and spatial memory deficit at 7 months old in AD mice. Thus, these data suggest that reductions of density and diameter of hippocampal capillaries are crucial for initiating Aß plaque deposition and spatial memory deficit at the early stages, implicating the development of new therapies for halting or curing memory decline in AD.

Cytophaga hutchinsonii SprA and SprT Are Essential Components of the Type IX Secretion System Required for Ca2+ Acquisition, Cellulose Degradation, and Cell Motility.

The type IX secretion system (T9SS) is a novel protein secretion system, which is found in and confined to the phylum Bacteroidetes. T9SS is involved in the secretion of virulence factors, cell surface adhesins, and complex biopolymer degrading enzymes to the cell surface or extracellular medium. Cytophaga hutchinsonii is a widely distributed bacterium, which is able to efficiently digest cellulose and rapidly glide along the solid surfaces. C. hutchinsonii has a full set of orthologs of T9SS components. However, the functions of most homologous proteins have not been verified. In C. hutchinsonii, CHU_0029 and CHU_2709 are similar in sequence to Flavobacterium johnsoniae T9SS components SprA and SprT, respectively. In this study, the single deletion mutants of chu_0029 (sprA) and chu_2709 (sprT) were obtained using a complex medium with the addition of Ca2+ and Mg2+. Single deletion of sprA or sprT resulted in defects in cellulose utilization and gliding motility. Moreover, the ΔsprA and ΔsprT mutants showed growth defects in Ca2+- and Mg2+-deficient media. The results of ICP-MS test showed that both the whole cell and intracellular concentrations of Ca2+ were dramatically reduced in the ΔsprA and ΔsprT mutants, indicating that SprA and SprT are both important for the assimilation of trace amount of Ca2+. While the assimilation of Mg2+ was not obviously influenced in the ΔsprA and ΔsprT mutants. Through proteomics analysis of the cell surface proteins of the wild type and mutants, we found that the ΔsprA and ΔsprT mutants were defective in secretion of the majority of T9SS substrates. Together, these results indicate that SprA and SprT are both essential components of C. hutchinsonii T9SS, which is required for protein secretion, Ca2+ acquisition, cellulose degradation, and gliding motility in C. hutchinsonii. Our study shed more light on the functions of SprA and SprT in T9SS, and further proved the link between the T9SS and Ca2+ uptake system.