Data_Sheet_1_A novel detection method for the pathogenic Aeromonas hydrophila expressing aerA gene and/or hlyA gene based on dualplex RAA and CRISPR/Cas12a.PDF

Aeromonas hydrophila is an emerging waterborne and foodborne pathogen with pathogenicity to humans and warm water fishes, which severely threatens human health, food safety and aquaculture. A novel method for the rapid, accurate, and sensitive detection of pathogenic A. hydrophila is still needed to reduce the impact on human health and aquaculture. In this work, we developed a rapid, accurate, sensitive, and visual detection method (dRAA-CRISPR/Cas12a), without elaborate instruments, integrating the dualplex recombinase-assisted amplification (dRAA) assay and CRISPR/Cas12a system to detect pathogenic A. hydrophila expressing aerA and/or hlyA virulence genes. The dRAA-CRISPR/Cas12a method has high sensitivity, which can rapidly detect (about 45 min) A. hydrophila with the limit of detection in 2 copies of genomic DNA per reaction, and has high specificity for three pathogenic A. hydrophila strains (aerA+hlyA−, aerA−hlyA+, and aerA+hlyA+). Moreover, dRAA-CRISPR/Cas12a method shows satisfactory practicability in the analysis of the spiked human blood and stool and fish samples. These results demonstrate that our developed pathogenic A. hydrophila detection method, dRAA-CRISPR/Cas12a, is a promising potential method for the early diagnosis of human A. hydrophila infection and on-site detection of A. hydrophila in food and aquaculture.</p

Table_1_Rapid and Sensitive Detection of Vibrio vulnificus Using CRISPR/Cas12a Combined With a Recombinase-Aided Amplification Assay.DOC

Vibrio vulnificus is an important zoonotic and aquatic pathogen and can cause vibriosis in humans and aquatic animals (especially farmed fish and shrimp species). Rapid and sensitive detection methods for V. vulnificus are still required to diagnose human vibriosis early and reduce aquaculture losses. Herein, we developed a rapid and sensitive diagnostic method comprising a recombinase-aided amplification (RAA) assay and the CRISPR/Cas12a system (named RAA-CRISPR/Cas12a) to detect V. vulnificus. The RAA-CRISPR/Cas12a method allows rapid and sensitive detection of V. vulnificus in 40 min without a sophisticated instrument, and the limit of detection is two copies of V. vulnificus genomic DNA per reaction. Meanwhile, the method shows satisfactory specificity toward non-target bacteria and high accuracy in the spiked blood, stool, and shrimp samples. Therefore, our proposed rapid and sensitive V. vulnificus detection method, RAA-CRISPR/Cas12a, has great potential for early diagnosis of human vibriosis and on-site V. vulnificus detection in aquaculture and food safety control.</p

Quantitative proteomic profiling of USP6-regulated proteins and signaling pathways.

(A) Schematic diagram depicting the quantitative proteomics pipeline used to identify differentially ubiquitinated proteins in CAM-USP6 Tg mouse brain. (B) Volcano plot indicating ubiquitinated protein species identified in CAM-USP6 compared with WT mouse brain cortex; colored plots represent significantly down-regulated (blue) and up-regulated (red) proteins. Log10 P value (t test, y-axis) and FC (log2FC, CAM-USP6 versus WT, x-axis) are shown. Significance cutoffs were set to P 1.2. (C) KEGG analysis of 175 differentially regulated proteins identified in CAM-USP6 versus WT mouse cortex. (D) Differentially ubiquitinated synaptic protein components identified in CAM-USP6 (compared with WT) mouse cortex. The underlying data for this figure can be found in S1 Data. Calm1, Calmodulin 1; CAM, CamK2a; CamK2a, calcium/calmodulin dependent protein kinase II alpha; FC, fold change; FDR, false discovery rate; Glu, glutamate ionotropic receptor; IP, immunoprecipitation; KEGG, Kyoto encyclopedia of genes and genomes; KGG, anti-di-glycine remnant; PSD, postsynaptic density; Rac1, Rac family small GTPase 1; Stx1b, syntaxin 1B; Syt1, synaptotagmin 1; Tg, transgenic; USP, ubiquitin-specific protease; WT, wild-type.</p

Tissue sample information from fetal and adult donors.

Tissue sample information from fetal and adult donors.</p

A schematic model depicting USP6-dependent regulation of synaptic function.

The hominoid-specific gene USP6 is required for maintaining synaptic function by stabilizing NMDARs. USP6 deubiquitinates NMDAR at the PSD, thereby facilitating NMDAR stabilization, resulting in increased NMDAR function and synaptic activity and enhanced cognition. AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; LTP, long-term potentiation; NMDAR, N-methyl-D-aspartate-type glutamate receptor; PSD, postsynaptic density; USP, ubiquitin-specific protease.</p

USP6 depletion reduces GluN1 expression in ESC-derived human excitatory neurons.

(A) Schematic diagram depicting the differentiation timescale and experimental timeline in ESC-derived human excitatory neurons. (B) Immunostaining for vGluT1 and MAP2 at day 44 of differentiation. (C) Action potential at day 60 of differentiation in ESC-derived human excitatory neurons. (D) Knockdown efficiency of USP6 shRNAs in induced human excitatory neurons as quantified by qRT-PCR analysis. Data represent means ± SEM. n = 3. **P P n = 43 neurites from 36 neurons), USP6 shRNA1 (n = 36 neurites from 19 neurons), and USP6 shRNA2 (n = 21 neurites from 15 neurons); quantification of PSD95 puncta: control shRNA (n = 61 neurites from 44 neurons), USP6 shRNA1 (n = 29 neurites from 23 neurons), and USP6 shRNA2 (n = 21 neurites from 15 neurons). The data represent means ± SEM. *P P P n = 3. *P S1 Data. B27, B-27 serum-free supplement; BDNF, brain-derived neurotrophic factor; EGF, epidermal growth factor receptor; ESC, embryonic stem cell; FGF2, fibroblast growth factor 2; GDNF, glial cell–derived neurotrophic factor; Glu, glutamate ionotropic receptor; MAP2, microtubule-associated protein 2; mEPSC, miniature excitatory postsynaptic current; N2aa, DMEM-F12 medium with N2-supplement and ascorbic acid; NMDA, N-methyl-D-aspartate; NPC, neural progenitor cell; PSD, postsynaptic density; qRT-PCR, quantitative reverse transcription PCR; shRNA, short hairpin RNA; USP, ubiquitin-specific protease; vGluT1, vesicular glutamate transporter.</p

Transgenic USP6 expression enhances learning and memory in mice.

(A) USP6 mRNA levels in the cortex of fetal and adult human brains were quantified by qRT-PCR. The data represent means ± SEM, n = 5. ***P 0.001 determined by Student t test. (B) USP6 mRNA levels in H9 hESCs, human astrocytes, induced human excitatory neurons, and interneurons were quantified by qRT-PCR. Data represent means ± SEM, n = 15. **P 0.01, ****P 0.0001 determined by nonparametric test (Kruskal-Wallis test) with Dunn’s post hoc analysis. (C) Schematic diagram of the USP6 transgene under the regulation of a CamK2a promoter (CAM-USP6). (D) Immunoblot analysis of USP6-HA expression in the cortex, hippocampus, and cerebellum of CAM-USP6 mice. (E) Schematic diagram and results from NOR test analysis. Data represent means ± SEM. WT: n = 16 mice, CAM-USP6: n = 16 mice. **P 0.01 determined by Student t test. (F) MWM assessment of spatial memory and reversal learning. (G) MWM test results depicting escape latency as defined by the time taken to find a hidden platform. Data represent means ± SEM. WT: n = 16 mice, CAM-USP6: n = 16 mice. *P n = 16 mice, CAM-USP6: n = 16 mice. *P P t test. The underlying data for this figure can be found in S1 Data. CAM, CamK2a; CamK2a, calcium/calmodulin dependent protein kinase II alpha; Cb, cerebellum; Ctx, cortex; HA, hemagglutinin; hESC, human embryonic stem cell; Hip, hippocampus; MWM, Morris water maze; NE, northeast; NOR, novel object recognition; NS, not significant; NW, northwest; qRT-PCR, quantitative reverse transcription PCR; SE, southeast; SW, southwest; Tg, transgenic; USP, ubiquitin-specific protease; WT, wild-type.</p

siRNA sequences.

siRNA sequences.</p

Primer sequences.

Primer sequences.</p

USP6 interacts with NMDA receptors.

(A) Characterizing interactions between USP6-HA and glutamate receptor subunits by co-IP. WT and CAM-USP6 mouse brain lysates were precipitated with anti-HA magnetic beads, and precipitates were subsequently immunoblotted with antibodies to detect GluN1, GluN2B, GluA1, GluA2, APP, and β-actin. (B) co-IP interactions between exogenously expressed GluN1-Myc and Flag-tagged USP6, TBC1D3, or USP32. GluN1-Myc and USP6-Flag/TBC1D3-Flag/USP32-Flag were detected in lysates from cotransfected HEK293T cells; anti-Flag complexes were immunoprecipitated from lysates with an anti-Flag antibody and immunoblotted with a Myc antibody. (C) co-IP interactions detected between exogenously expressed GluN2B-Myc and USP6-Flag/TBC1D3-Flag/USP32-Flag. GluN1-Myc and USP6-Flag/TBC1D3-Flag/USP32-Flag were cotransfected into HEK293T cells, and complexes were immunoprecipitated from lysates with an anti-Flag antibody and immunoblotted with a Myc antibody. APP, amyloid precursor protein; CAM, CamK2a; co-IP, coimmunoprecipitation; Glu, glutamate ionotropic receptor; HA, hemagglutinin; IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitation; NMDA, N-methyl-D-aspartate; TBC1D3, TBC1 domain family member 3; USP, ubiquitin-specific protease; WT, wild-type.</p