Benchmarking and Automating the Biotinylation Proteomics Workflow.

Protein biotinylation has been widely used in biotechnology with various labeling and enrichment strategies. However, different enrichment strategies have not been systematically evaluated due to the lack of a benchmarking model for fair comparison. Most biotinylation proteomics workflows suffer from lengthy experimental steps, non-specific bindings, limited throughput, and experimental variability. To address these challenges, we designed a two-proteome model, where biotinylated yeast proteins were spiked in unlabeled human proteins, allowing us to distinguish true enrichment from non-specific bindings. Using this benchmarking model, we compared common biotinylation proteomics methods and provided practical selection guidelines. We significantly optimized and shortened sample preparation from 3 days to 9 hours, enabling fully-automated 96-well plate sample processing. Next, we applied this optimized and automated workflow for proximity labeling to investigate the intricate interplay between mitochondria and lysosomes in living cells under both healthy state and mitochondrial damage. Our results suggested a time-dependent proteome remodeling and dynamic translocation within mitochondria and between mitochondria and lysosomes upon mitochondrial damage. This newly established benchmarking model and the fully-automated 9-hour workflow can be readily applied to the broad fields of protein biotinylation to study protein interaction and organelle dynamics.

Changes in physiology, antioxidant system, and gene expression in Microcystis aeruginosa under fenoxaprop-p-ethyl stress.

Fenoxaprop-p-ethyl (FE) is one of the typical aryloxyphenoxypropionate herbicides. FE has been widely applied in agriculture in recent years. Human health and aquatic ecosystems are threatened by the cyanobacteria blooms caused by Microcystis aeruginosa, which is one of the most common cyanobacteria responsible for freshwater blooming. Few studies have been reported on the physiological effects of FE on M. aeruginosa. This study analyzed the growth curves, the contents of chlorophyll a and protein, the oxidative stress, and the microcystin-LR (MC-LR) levels of M. aeruginosa exposed to various FE concentrations (i.e., 0, 0.5, 1, 2, and 5 mg/L). FE was observed to stimulate the cell density, chlorophyll a content, and protein content of M. aeruginosa at 0.5- and 1-mg/L FE concentrations but inhibit them at 2 and 5 mg/L FE concentrations. The superoxide dismutase and catalase activities were enhanced and the malondialdehyde concentration was increased by FE. The intracellular (intra-) and extracellular (extra-) MC-LR contents were also affected by FE. The expression levels of photosynthesis-related genes psbD1, psaB, and rbcL varied in response to FE exposure. Moreover, the expressions of microcystin synthase-related genes mcyA and mcyD and microcystin transportation-related gene mcyH were significantly inhibited by the treatment with 2 and 5 mg/L FE concentrations. These results might be helpful in evaluating the ecotoxicity of FE and guiding the rational application of herbicides in modern agriculture.

Toxicity effects of disinfection byproduct chloroacetic acid to Microcystis aeruginosa: Cytotoxicity and mechanisms.

Chlorine-based disinfectants are widely used for disinfection in wastewater treatment. The mechanism of the effects of chlorinated disinfection by-products on cyanobacteria was unclear. Herein, the physiological effects of chloroacetic acid (CAA) on Microcystis aeruginosa (M. aeruginosa), including acute toxicity, oxidative stress, apoptosis, production of microcystin-LR (MC-LR), and the microcystin transportation-related gene mcyH transcript abundance have been investigated. CAA exposure resulted in a significant change in the cell ultrastructure, including thylakoid damage, disappearance of nucleoid, production of gas vacuoles, increase in starch granule, accumulation of lipid droplets, and disruption of cytoplasm membranes. Meanwhile, the apoptosis rate of M. aeruginosa increased with CAA concentration. The production of MC-LR was affected by CAA, and the transcript abundance of mcyH decreased. Our results suggested that CAA poses acute toxicity to M. aeruginosa, and it could cause oxidative damage, stimulate MC-LR production, and damage cell ultrastructure. This study may provide information about the minimum concentration of CAA in the water environment, which is safe for aquatic organisms, especially during the global coronavirus disease 2019 pandemic period.

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics.

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to human brain function, as neurons are postmitotic and rely heavily on the autophagy-lysosome pathway to maintain cellular homeostasis. Despite advancements in the understanding of various lysosomal functions, capturing the highly dynamic communications between lysosomes and other cellular components is technically challenging, particularly in a high-throughput fashion. Here, a detailed protocol is provided for the recently published endogenous (knock-in) lysosome proximity labeling proteomic method in human induced pluripotent stem cell (hiPSC)-derived neurons. Both lysosomal membrane proteins and proteins surrounding lysosomes within a 10-20 nm radius can be confidently identified and accurately quantified in live human neurons. Each step of the protocol is described in detail, i.e., hiPSC-neuron culture, proximity labeling, neuron harvest, fluorescence microscopy, biotinylated protein enrichment, protein digestion, LC-MS analysis, and data analysis. In summary, this unique endogenous lysosomal proximity labeling proteomics method provides a high-throughput and robust analytical tool to study the highly dynamic lysosomal activities in live human neurons.

Protein Contaminants Matter: Building Universal Protein Contaminant Libraries for DDA and DIA Proteomics.

Mass spectrometry-based proteomics is constantly challenged by the presence of contaminant background signals. In particular, protein contaminants from reagents and sample handling are almost impossible to avoid. For data-dependent acquisition (DDA) proteomics, an exclusion list can be used to reduce the influence of protein contaminants. However, protein contamination has not been evaluated and is rarely addressed in data-independent acquisition (DIA). How protein contaminants influence proteomic data is also unclear. In this study, we established new protein contaminant FASTA and spectral libraries that are applicable to all proteomic workflows and evaluated the impact of protein contaminants on both DDA and DIA proteomics. We demonstrated that including our contaminant libraries can reduce false discoveries and increase protein identifications, without influencing the quantification accuracy in various proteomic software platforms. With the pressing need to standardize proteomic workflow in the research community, we highly recommend including our contaminant FASTA and spectral libraries in all bottom-up proteomic data analysis. Our contaminant libraries and a step-by-step tutorial to incorporate these libraries in various DDA and DIA data analysis platforms can be valuable resources for proteomic researchers, freely accessible at https://github.com/HaoGroup-ProtContLib.

Clinical study on swallowing function of brainstem stroke by tDCS.

OBJECTIVE: To investigate the effect of transcranial direct current stimulation (tDCS) combined with conventional comprehensive rehabilitation on dysphagia after brainstem stroke. MATERIALS AND METHODS: Forty brainstem stroke patients were randomly divided into tDCS group and conventional comprehensive treatment group, including 20 patients in each group. Both groups were given routine swallowing function training, and tDCS group added transcranial direct current stimulation (tDCS). The Dysphagia Outcome and Severity Scale (DOSS) and Functional Dysphagia Scale (FDS) were evaluated respectively before and after 8 weeks of continuous treatment with VFSS. The white blood cell (WBC), c-reactive protein, prealbumin (PAB), albumin (Alb), and hemoglobin (Hb) were also compared between the two groups before and after 8 weeks of continuous treatment. RESULTS: After 8 consecutive weeks of treatment, the score of DOSS scale and FDS scale in both groups was improved (P < 0.05), WBC and CRP were decreased (P < 0.05), and Alb and Hb were improved (P < 0.05), and PAB had no differences (P=0.474). The tDCS group was superior to conventional comprehensive group in improving the swallowing function and nutritional indexes (P < 0.05). CONCLUSIONS: tDCS therapy combined with routine training can improve the swallowing function and nutritional status of patients, and reduce infection.

Effects of mirror neuron system-based training on rehabilitation of stroke patients.

OBJECTIVE: To investigate the clinical effects of the mirror neuron system (MNS)-based training on upper extremity motor function and cognitive function in stroke patients. METHODS: Sixty stroke patients (time from stroke onset 3-9 months) with upper extremity paresis (Brunnstrom stage II-IV) and cognitive impairment (MoCA score ≥ 15) were enrolled in this study. Patients were randomly allocated into MNS treatment group (N = 30) and control group (N = 30). Both groups underwent regular training for upper extremity motor function and cognitive function, and the MNS group was trained with a therapeutic apparatus named mirror neuron system training (MNST) including different levels of action observation training (AOT). Training lasted 20 min/day, 5 days/week for 8 weeks. MoCA, reaction time, and Wisconsin Card Sorting Test (WCST) were assessed at baseline and 8 weeks after training. Furthermore, Fugl-Meyer assessment (FMA) and Modified Barthel index (MBI) were adopted to evaluated upper extremity motor function and daily life ability. RESULTS: After 8 consecutive weeks' training, both groups showed significant improvements on the upper extremity motor function, cognitive function, and daily life ability score after training (p < .05). The MNS group showed significantly improved upper extremity motor function and cognitive function (p < .05) compared with control group. CONCLUSIONS: Combining MNS-based and conventional training can improve upper extremity motor function and cognitive function in stroke patients.

Antiviral Activity of Benzoic Acid Derivative NC-5 Against Influenza A Virus and Its Neuraminidase Inhibition.

The currently available drugs against influenza A virus primarily target neuraminidase (NA) or the matrix protein 2 (M2) ion channel. The emergence of drug-resistant viruses requires the development of new antiviral chemicals. Our study applied a cell-based approach to evaluate the antiviral activity of a series of newly synthesized benzoic acid derivatives, and 4-(2,2-Bis(hydroxymethyl)-5-oxopyrrolidin-l-yl)-3-(5-cyclohexyl-4H-1,2,4-triazol-3-yl)amino). benzoic acid, termed NC-5, was found to possess antiviral activity. NC-5 inhibited influenza A viruses A/FM/1/47 (H1N1), A/Beijing/32/92 (H3N2) and oseltamivir-resistant mutant A/FM/1/47-H275Y (H1N1-H275Y) in a dose-dependent manner. The 50% effective concentrations (EC50) for H1N1 and H1N1-H275Y were 33.6 µM and 32.8 µM, respectively, which showed that NC-5 had a great advantage over oseltamivir in drug-resistant virus infections. The 50% cytotoxic concentration (CC50) of NC-5 was greater than 640 µM. Orally administered NC-5 protected mice infected with H1N1 and H1N1-H275Y, conferring 80% and 60% survival at 100 mg/kg/d, reducing body weight loss, and alleviating virus-induced lung injury. NC-5 could suppress NP and M1 protein expression levels during the late stages of viral biosynthesis and inhibit NA activity, which may influence virus release. Our study proved that NC-5 has potent anti-influenza activity in vivo and in vitro, meaning that it could be regarded as a promising drug candidate to treat infection with influenza viruses, including oseltamivir-resistant viruses.