In vivo characterization of cerebrovascular impairment induced by amyloid ß peptide overload in glymphatic clearance system using swept-source optical coherence tomography.

Significance: Antiamyloid ß ( A ß ) immunotherapy is a promising therapeutic strategy for Alzheimer's disease (AD) but generates large amounts of soluble A ß peptides that could overwhelm the clearance pathway, leading to serious side effects. Direct implications of A ß in glymphatic drainage transport for cerebral vasculature and tissue are not well known. Studies are needed to resolve this issue and pave the way to better monitoring abnormal vascular events that may occur in A ß -modifying therapies for AD. Aim: The objective is to characterize the modification of cerebral vasculature and tissue induced by soluble A ß abundantly present in the glymphatic clearance system. Approach: A ß 1 - 42 peptide was injected intracerebroventricularly and swept-source optical coherence tomography (SS-OCT) was used to monitor the progression of changes in the brain microvascular network and tissue in vivo over 14 days. Parameters reflecting vascular morphology and structure as well as tissue status were quantified and compared before treatment. Results: Vascular perfusion density, vessel length, and branch density decreased sharply and persistently following peptide administration. In comparison, vascular average diameter and vascular tortuosity were moderately increased at the late stage of monitoring. Endpoint density gradually increased, and the global optical attenuation coefficient value decreased significantly over time. Conclusions: A ß burden in the glymphatic system directly contributes to cerebrovascular structural and morphological abnormalities and global brain tissue damage, suggesting severe deleterious properties of soluble cerebrospinal fluid- A ß . We also show that OCT can be used as an effective tool to monitor cerebrovascular dynamics and tissue property changes in response to therapeutic treatments in drug discovery research.

Identification of Microproteins in Saccharomyces cerevisiae under Different Stress Conditions.

Small open reading frame-encoded peptides (SEPs) are microproteins with a length of 100 amino acids or less, which may play a critical role in maintaining cell homeostasis under stress. Therefore, we used mass spectrometry-based proteomics to explore microproteins potentially involved in cellular stress responses in Saccharomyces cerevisiae. A total of 225 microproteins with 1920 unique peptides were identified under six culture conditions: normal, oxidation, starvation, ultraviolet radiation, heat shock, and heat shock with starvation. Among these microproteins, we found 70 SEPs with 75 unique peptides. The annotated microproteins are involved in stress-related processes, such as cell redox reactions, cell wall modification, protein folding and degradation, and DNA damage repair. It suggests that SEPs may also play similar functions under stress conditions. For example, SEP IP_008057, translated from a short coding sequence of YJL159W, may play a role in heat shock. This study identified stress-responsive SEPs in S. cerevisiae and provided valuable information to determine the functions of these proteins, which enrich the genome and proteome of S. cerevisiae and show clues to improving the stress tolerance of S. cerevisiae.

Comparative Network Biology Discovers Protein Complexes That Underline Cellular Differentiation in Anabaena sp.

The filamentous cyanobacterium Anabaena sp. PCC 7120 can differentiate into heterocysts to fix atmospheric nitrogen. During cell differentiation, cellular morphology and gene expression undergo a series of significant changes. To uncover the mechanisms responsible for these alterations, we built protein-protein interaction (PPI) networks for these two cell types by cofractionation coupled with mass spectrometry. We predicted 280 and 215 protein complexes, with 6322 and 2791 high-confidence PPIs in vegetative cells and heterocysts, respectively. Most of the proteins in both types of cells presented similar elution profiles, whereas the elution peaks of 438 proteins showed significant changes. We observed that some well-known complexes recruited new members in heterocysts, such as ribosomes, diflavin flavoprotein, and cytochrome c oxidase. Photosynthetic complexes, including photosystem I, photosystem II, and phycobilisome, remained in both vegetative cells and heterocysts for electron transfer and energy generation. Besides that, PPI data also reveal new functions of proteins. For example, the hypothetical protein Alr4359 was found to interact with FraH and Alr4119 in heterocysts and was located on heterocyst poles, thereby influencing the diazotrophic growth of filaments. The overexpression of Alr4359 suspended heterocyst formation and altered the pigment composition and filament length. This work demonstrates the differences in protein assemblies and provides insight into physiological regulation during cell differentiation.

Improved Identification of Small Open Reading Frames Encoded Peptides by Top-Down Proteomic Approaches and De Novo Sequencing.

Small open reading frames (sORFs) have translational potential to produce peptides that play essential roles in various biological processes. Nevertheless, many sORF-encoded peptides (SEPs) are still on the prediction level. Here, we construct a strategy to analyze SEPs by combining top-down and de novo sequencing to improve SEP identification and sequence coverage. With de novo sequencing, we identified 1682 peptides mapping to 2544 human sORFs, which were all first characterized in this work. Two-thirds of these new sORFs have reading frame shifts and use a non-ATG start codon. The top-down approach identified 241 human SEPs, with high sequence coverage. The average length of the peptides from the bottom-up database search was 19 amino acids (AA); from de novo sequencing, it was 9 AA; and from the top-down approach, it was 25 AA. The longer peptide positively boosts the sequence coverage, more efficiently distinguishing SEPs from the known gene coding sequence. Top-down has the advantage of identifying peptides with sequential K/R or high K/R content, which is unfavorable in the bottom-up approach. Our method can explore new coding sORFs and obtain highly accurate sequences of their SEPs, which can also benefit future function research.

In vivo monitoring of thrombosis in mice by optical coherence tomography.

The objective of this study is to establish a novel method for continuously monitoring thrombus progression with various outcome measures and to assess the efficacy of antithrombotic drugs in murine thrombosis model in mice. In the study, thrombus was induced in the femoral vein of mice by FeCl3 and monitored over time by spectral-domain optical coherence tomography (OCT). Three-dimensional images of thrombi with or without heparin as an antithrombotic agent were obtained from OCT angiography. In addition, several parameters of thrombi were analyzed and compared between control and anticoagulant groups. By using OCT, we were able to trace thrombus generation in the same mouse in real time. We found that in our model heparin reduced thrombus size by ~60% and thrombus cross-sectional area by 50%. OCT results also show that both time to thrombus size (>0.02mm3 ) and time to occlusion (>30%) were significantly reduced after heparin addition. This study demonstrates that OCT reliably monitors thrombus generation and progression from various aspects including thrombus size. This enables us to measure the kinetic of thrombosis more accurately, and effectively evaluate the efficacy and activities of antithrombotic drugs. This model may represent a useful tool in antithrombotic drug discoveries in preclinical studies.

[Effects of beta1-integrin, fibronectin and laminin on invasive behavior of human gliomas].

OBJECTIVE: To investigate the effects of beta1-integrin, fibronectin (FN) and laminin (LN) on the invasive behavior of human gliomas. METHODS: Functional impacts of beta1-integrin, fibronectin and laminin on cell adhesion, migration and metastasis of U251 malignant glioblastoma cells were investigated by in vitro adhesion, migration and invasion assays. The amount and distributions of cellular microfilaments and pseudopodia were studied by fluorescent cytochemistry, confocal laser scanning microscope and scanning electron microscope. Lastly, beta1-integrin, fibronectin and laminin were investigated for their roles in cellular microfilament skeleton. RESULTS: (1) Fibronectin did not affect cell adhesion of U251MG cells, but anti-beta1 integrin antibodies inhibited cell adhesion (P < 0.01); Laminin stimulated cell adhesion of U251MG cells (P < 0.01) but anti-beta1 integrin antibodies had little effect on the laminin-mediated cell adhesion. (2) The migration of U251MG cells on dishes coated with FN was inhibited by anti-beta1 integrin antibodies (P < 0.05). (3) F-actins formed strong and dense stress fibers in U251MG cells on dishes coated with FN and LN. Anti-beta1 integrin antibodies disrupted the microfilament network and F-actin aggregation. (4) FN and LN increased the number of pseudopodia on cell surface, whereas anti-beta1 integrin antibodies reversed this function. (5) FN and anti-beta1 integrin antibodies had little effects on the invasive ability of U251MG cells in vitro. The invasion was increased by LN, but inhibited by anti-beta1 integrin antibodies. CONCLUSIONS: (1) The interaction between beta1-integrin, FN may stimulate U251MG cell migration via changing the structures of microfilament skeleton and the number of pseudopodia. (2) beta1-integrin may play a role in the LN-mediated in vitro invasion of U251MG cells.