Proteomic Analysis of Differences in the Freezability of Porcine Sperm Identifies α-Amylase As a Key Protein.

To investigate the mechanisms underlying the differences in the freezability of boar semen, Yorkshire boars with freezing-tolerant semen (YT, n = 3), Yorkshire boars with freezing-sensitive semen (YS, n = 3), Landrace boars with freezing-tolerant semen (LT, n = 3), and Landrace boars with freezing-sensitive semen (LS, n = 3) were selected for this study. Their sperm was subjected to protein extraction, followed by data-independent acquisition proteomics and functional bioinformatics analysis. A total of 3042 proteins were identified, of which 2810 were quantified. Some key KEGG pathways were enriched, such as starch and sucrose metabolism, carbohydrate digestion and absorption, mineral absorption, the HIF-1 signaling pathway, and the necroptosis pathways. Through PRM verification, we found that several proteins, such as α-amylase and epididymal sperm-binding protein 1, can be used as molecular markers of the freezing resistance of boar semen. Furthermore, we found that the addition of α-amylase to cryoprotective extender could significantly improve the post-thaw motility and quality of boar semen. In summary, this study revealed some molecular markers and potential molecular pathways contributing to the high or low freezability of boar sperm, identifying α-amylase as a key protein. This study is valuable for optimizing boar semen cryopreservation technology.

Mesophilic Argonaute-Mediated Polydisperse Droplet Biosensor for Amplification-Free, One-Pot, and Multiplexed Nucleic Acid Detection Using Deep Learning.

Detection of nucleic acids from a single multiplexed and amplification-free test is critical for ensuring food safety, clinical diagnostics, and environmental monitoring. In this study, we introduced a mesophilic Argonaute protein from Clostridium butyricum (CbAgo), which exhibits nucleic acid endonuclease activity, to achieve a programmable, amplification-free system (PASS) for rapid nucleic acid quantification at ambient temperatures in one pot. By using CbAgo-mediated binding with specific guide DNA (gDNA) and subsequent targeted cleavage of wild-type target DNAs complementary to gDNA, PASS can detect multiple foodborne pathogen DNA (<102 CFU/mL) simultaneously. The fluorescence signals were then transferred to polydisperse emulsions and analyzed by using deep learning. This simplifies the process and increases the suitability of polydisperse emulsions compared to traditional digital PCR, which requires homogeneous droplets for accurate detection. We believe that PASS has the potential to become a next-generation point-of-care digital nucleic acid detection method.

Artificial Intelligence-Based Imaging Transcoding System for Multiplex Screening of Viable Foodborne Pathogens.

Multiplex detection of viable foodborne pathogens is critical for food safety and public health, yet current assays suffer trade-offs between cost, assay complexity, sensitivities, and the specificity between live and dead bacteria. We herein developed a sensing method using artificial intelligence transcoding (SMART) for rapid, sensitive, and multiplex profiling of foodborne pathogens. The assay utilizes the programmable polystyrene (PS) microspheres to encode different pathogens, inducing subsequent visible signals under conventional microscopy that can be analyzed using a customized, artificial intelligence-computer vision, which was trained to decode the intrinsic properties of PS microspheres to reveal the numbers and types of pathogens. Our approach enabled the rapid and simultaneous detection of multiple bacteria from egg samples of <102 CFU/mL without DNA amplification and showed strong consistency with the standard microbiologic and genotypic methods. We adopted our assay through phage-guided targeting to enable the discrimination between live and dead bacteria.

Early-stage diagnosis of ovarian cancer via digital immunoassay on a SlipChip.

Ovarian cancer (OC) is one of the three major gynecologic malignancies and has the highest mortality rate because of the late diagnosis. Liquid biopsy based on serum protein biomarkers has demonstrated great potential for early diagnosis but remains limited by the analysis performance of conventional immunoassay technologies, such as chemiluminescence, and biomarkers, such as CA125. To address this challenge and achieve accurate early-stage diagnosis of OC, we developed a digital immunoassay on a SlipChip (DiSC) for quantitative analysis of a potential serum protein biomarker, Spondin-1 (SPON1). The DiSC system achieved a limit of detection (LoD) of 23 fg/µL for digital quantification of SPON1. The DiSC system was utilized to quantify the serum level of SPON1 in 357 clinical serum samples, including 63 from patients with benign ovarian tumors and 294 from patients with malignant ovarian cancer, ranging from stages I to IV. SPON1 concentrations were significantly different in samples from patients with malignant ovarian cancer. Notably, significantly different SPON1 levels were observed in early stages (I and II), in lymph node-negative cases (N0), and before metastasis (M0), suggesting that SPON1 could serve as a sensitive diagnostic biomarker for early-stage OC. The differential diagnostic model based on SPON1 levels quantified using DiSC demonstrated an area under the curve (AUC) of 0.8187 for early-stage OC, a significant improvement over CA-125 (AUC = 0.6967). For OC of all stages, the AUC was 0.8225, which could be further increased to 0.8750 when combined with CA-125. This showcases the potential of SPON1 as a novel biomarker for sensitive early-stage diagnosis of ovarian cancer and the capability of the DiSC system in discovering low-abundance biomarkers for disease diagnosis.

Multiplexed and DNA amplification-free detection of foodborne pathogens in egg samples: Combining electrical resistance-based microsphere counting and DNA hybridization reaction.

A multiplex and DNA amplification-free sensor based on electrical resistance microsphere counter (ERMC) and DNA hybridization reaction has been developed for simultaneous detection of Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus in egg samples. In this strategy, magnetic nanoparticles (MNPs) and polystyrene microspheres (PMs) were labeled with DNA probes, the PMs with different sizes (3 µm, 4 µm and 6 µm) were used as multiplex signal reporters due to the high resolution of ERMC. After magnetic separation, the number of unbound PM-probe was related to the concentration of pathogens, and their sizes were attributed to types of pathogens. The limit of detection of this sensor was 20 CFU/mL for Listeria monocytogenes, 89 CFU/mL for Salmonella, and 94 CFU/mL for Staphylococcus aureus, respectively. The DNA amplification-free, simplicity and low-cost of this assay make it a promising tool for the rapid screening of multiple pathogens in food samples.

Polydopamine-mediated quantity-based magnetic relaxation sensing for the rapid and sensitive detection of chloramphenicol in fish samples.

Chloramphenicol is an antibiotic that cause adverse effects in humans. In this work, a novel quantity-based magnetic relaxation switching (sMRS) sensor using polydopamine (PDA) for signal amplification was successfully developed for the rapid and sensitive detection of chloramphenicol in fish samples. We first prepared the conjugation of large magnetic nanoparticles (MNPs) and CAP antigen (MNP-antigen), which performed a competitive immune reaction with chloramphenicol and chloramphenicol antibody. Horseradish peroxidase, that can bind to antibody at the surface of large MNPs, was employed to catalyze the rapid polymerization of dopamine to PDA, which was easily deposited onto the surface of large MNPs. The concentration of chloramphenicol is inversely proportional to the content of HRP after reaction, in other words, it is inversely proportional to the content of PDA. PDA then reacted with 30 nm-diameter amine-functionalized MNPs (NH2-MNP30), the consumption of which resulted in a decrease in the concentration of free NH2-MNP30 particles in solution. After magnetic separation, the remaining free NH2-MNP30 served as the magnetic probe for signal readout. The limit of detection of the sMRS assay for detecting chloramphenicol was 16.6 pg/mL, which was 49-fold lower than MRS sensor without signal amplification. This quantity-based MRS sensor can provide a powerful platform for enabling the rapid and sensitive detection of chloramphenicol in food samples.

Advances in the role of auxin for transcriptional regulation of lignin biosynthesis.

Lignin is a natural polymer interlaced with cellulose and hemicellulose in secondary cell walls (SCWs). Auxin acts via its signalling transduction to regulate most of plant physiological processes. Lignification responds to auxin signals likewise and affects the development of anther and secondary xylem in plants. In this review, the research advances of AUXIN RESPONSE FACTOR (ARF)-dependent signalling pathways regulating lignin formation are discussed in detail. In an effort to facilitate the understanding of several key regulators in this process, we present a regulatory framework that comprises protein-protein interactions at the top and protein-gene regulation divided into five tiers. This characterises the regulatory roles of auxin in lignin biosynthesis and links auxin signalling transduction to transcriptional cascade of lignin biosynthesis. Our works further point to several of significant problems that need to be resolved in the future to gain a better understanding of the underlying mechanisms through which auxin regulates lignin biosynthesis.

Protective effect of effective composite of Chinese medicine prescription naodesheng against focal cerebral ischemia in rats.

OBJECTIVE: To study the effects and possible mechanisms of effective composite of Naodesheng (NDS) on permanent cerebral ischemia-induced injury in rats. METHODS: Male Sprague-Dawley rats: with middle cerebral artery occlusion (MCAO) were established with the modified suture method, and they were randomly divided into the following groups: the sham-operated group, the model group, the Nimodipine group (0.012 g/kg), the NDS group (1.075 g/kg), the total extracts group (0.23 g/kg), the high-dose NEC group (0.07 g/kg), the middle-dose NEC group (0.02 g/kg), and the low-dose NEC group (0.007 g/kg). The aforesaid medicines were administered at the 2nd, 4th, and 24th h after focal cerebral ischemia, and the infarction size and water content in the brain were evaluated at the 26th h after MCAO. Then, after oral administration once daily for 7 successive days, the changes in the degree of neurological deficit, oxidative stress, and apoptosis were measured on the 7th day. RESULTS: NEC could significantly reduce the infarction size after focal cerebral ischemia, and slightly relieve water content in the brain, significantly alleviate neurological function impairment, increase the levels of superoxide dismutase (SOD) and adenosine triphosphate enzyme (ATPase) activity, and decrease the content of malondialdehyde (MDA). NEC could also extenuate Bax and caspase-3 expression in the hippocampus tissue of the ischemic region. As compared with the three NEC treated groups, the high-dose NEC showed better efficacy. CONCLUSIONS: NEC could significantly reduce brain injury induced by ischemia;: its mechanism was closely associated with hindering oxidative stress and apoptosis. The effective composite-guided methodology is a feasible tool to improve the neuro-protective properties of the Chinese medicine guided prescription NDS against focal cerebral ischemia in rats.

Neuroprotective effects of tetramethylpyrazine on hydrogen peroxide-induced apoptosis in PC12 cells.

In the present study, we investigated the effects of tetramethylpyrazine (TMP) on hydrogen peroxide (H2O2)-induced apoptosis in PC12 cells. The apoptosis in H2O2-induced PC12 cells was accompanied by a decrease in Bcl-2/Bax protein ratio, release of cytochrome c to cytosol and the activation of caspase-3. TMP not only suppressed the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol, but also attenuated caspase-3 activation and eventually protected against H2O2-induced apoptosis. These results indicated that TMP blocked H2O2-induced apoptosis by the regulation of Bcl-2 family members, suppression of cytochrome c release, and caspase cascade activation in PC12 cells.