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DNA test is broadly used in diagnosis of crop disease and identification of genetic modified organism (GMO) in agriculture. However, rapid, low-cost, user-friendly, and field-deployable DNA test method is still limit. Recently, the RNA programmable nuclease of CRISPR/Cas is engineered as a new nucleic acid detection platform, but their application in plant remains to explore. In this study, we evaluated the Cas12a-based DNA detection for crop disease diagnosis and GMO test. A total of 14 crRNAs were designed to target two Magnaporthe oryzae genes and a synthetic Cry1C gene which encodes Bacillus thuringiensis δ -endotoxin and has been used to develop transgenic rice cultivar (Bt-rice) in China. Using a fluorescent reporter, targeted genes were easily detected by LbCas12a after recombinase polymerase amplification (RPA) for all crRNAs, despite that the signal strength varied 2–3-folds between different crRNAs. We further combined the filter paper-based DNA extraction and lateral flow assay (LFA) with RPA-Cas12a for DNA detection. This optimized Cas12a diagnostics method is carried at body temperature and does not require extra instrument except filter paper and LFA strip. Our data show that rice blast pathogen and Bt-rice were efficiently identified from leaf disc samples using this optimized DNA test method with highly active crRNAs. Moreover, LbCas12a exhibited variable nuclease activities on different targets; therefore, highly active crRNA is critical for successful DNA test using Cas12a and LFA. Owing to its simplicity, efficiency, and low-cost, DNA test using CRISPR/Cas12a would be easily applied in field for crop disease diagnosis and GMO administration.

Recent interest in the control of bone metabolism has focused on a specialized subset of CD31 hi endomucin hi vessels, which are reported to couple angiogenesis with osteogenesis. However, the underlying mechanisms that link these processes together remain largely undefined. Here we show that the zinc-finger transcription factor ZEB1 is predominantly expressed in CD31 hi endomucin hi endothelium in human and mouse bone. Endothelial cell-specific deletion of ZEB1 in mice impairs CD31 hi endomucin hi vessel formation in the bone, resulting in reduced osteogenesis. Mechanistically, ZEB1 deletion reduces histone acetylation on Dll 4 and Notch1 promoters, thereby epigenetically suppressing Notch signaling, a critical pathway that controls bone angiogenesis and osteogenesis. ZEB1 expression in skeletal endothelium declines in osteoporotic mice and humans. Administration of Zeb1 -packaged liposomes in osteoporotic mice restores impaired Notch activity in skeletal endothelium, thereby promoting angiogenesis-dependent osteogenesis and ameliorating bone loss. Pharmacological reversal of the low ZEB1/Notch signaling may exert therapeutic benefit in osteoporotic patients by promoting angiogenesis-dependent bone formation. An endothelial cell subtype, expressing endomucin and CD31, has been reported to couple angiogenesis with osteogenesis. Here, the authors show that loss of ZEB1 in these cells epigenetically suppresses Notch signaling, leading to impaired angiogenesis and osteogenesis, and that Zeb1 delivery via liposomes ameliorates bone loss in osteoporotic mice

The ability to remember conspecifics is critical for adaptive cognitive functioning and social communication, and impairments of this ability are hallmarks of autism spectrum disorders (ASDs). Although hippocampal ventral CA1 (vCA1) neurons are known to store social memories, how their activities are coordinated remains unclear. Here we show that vCA1 social memory neurons, characterized by enhanced activity in response to memorized individuals, were preferentially reactivated during sharp-wave ripples (SPW-Rs). Spike sequences of these social replays reflected the temporal orders of neuronal activities within theta cycles during social experiences. In ASD model Shank3 knockout mice, the proportion of social memory neurons was reduced, and neuronal ensemble spike sequences during SPW-Rs were disrupted, which correlated with impaired discriminatory social behavior. These results suggest that SPW-R-mediated sequential reactivation of neuronal ensembles is a canonical mechanism for coordinating hippocampus-dependent social memories and its disruption underlie the pathophysiology of social memory defects associated with ASD.

Individual recognition (IR) is essential for maintaining various social interactions in a group, and face recognition is one of the most specialised cognitive abilities in IR. We used both a mating preference system and an electric shock conditioning experiment to test IR ability in medaka, and found that signals near the face are important. Medaka required more time to discriminate vertically inverted faces, but not horizontally shifted faces or inverted non-face objects. The ability may be comparable to the classic ‘face inversion effect’ in humans and some other mammals. Extra patterns added to the face also did not influence the IR. These findings suggest the possibility that the process of face recognition may differ from that used for other objects. The complex form of recognition may promote specific processing adaptations, although the mechanisms and neurological bases might differ in mammals and medaka. The ability to recognise other individuals is important for shaping animal societies. Being able to recognize each other is crucial for social interactions in humans, as well as many other animals. To humans, faces are the most important body part to differentiate between one another. Humans read the face as a whole, rather than look at parts of the face, which is why it is harder to recognise a face when we see it upside-down, but not when we see an upside-down object. Some other mammals also identify each other by the face and take longer to recognise an upside-down face, but this ability has never been observed in animals other than mammals. Previous research has shown that some fish species can distinguish between individuals. For example, female medaka fish prefer males they have seen before to ‘strangers’. However, until now, it was not known if they can recognize individual faces, nor how they distinguish a specific male from many others. To see if medaka fish use vision, smell or both cues to recognise mates, Wang and Takeuchi familiarised the fish before the mating test in different settings. In the first group, the male and the female could see each other but were kept in different tanks; in the second group to test odour cues, the male and the female were in the same tank but could not see each other; in the third group, the fish were in the same tank and could see each other; the fish in the fourth group were kept in different tanks and could not see each other. To make sure the fish can recognise and distinguish between fish or objects, Wang and Takeuchi also performed negative conditioning experiments, in which the females had to learn to form an association between a negative stimulus and a specific situation. Wang and Takeuchi found that medaka fish use both vision and smell to distinguish between other fish, but could recognise each other based on vision alone. More specifically, the fish looked at the faces to tell others apart, and even when spots were added to their faces, the fish could still recognise the other. The mekada fish were also able to discriminate between two fish and two objects, but failed the task when the fish images were presented upside-down. However, when two objects were inverted, they were still able to tell the difference. This suggests that just like humans, faces may be special for fish too. This is the first study that shows the face inversion effect in animals other than mammals. A next step will be to compare the different mechanisms between species, and identify the underlying genes and nerve cells responsible for face recognition. This will enable us to better understand social interactions in fish, and enhance our knowledge of how our own ability to recognize faces has changed from an evolutionary point of view.

Objective. Patients with severe asthma respond poorly to corticosteroids, and their care accounts for more than 60% of the total costs attributed to asthma. Neutrophils form neutrophil extracellular traps (NETs), which play a crucial role in severe asthma. Statins have shown anti-inflammatory effects by reducing NETosis. In this study, we investigate if simvastatin can attenuate severe asthma by reducing NETosis and the underlying mechanism. Methods. Mice were concomitantly sensitized with ovalbumin (OVA), house dust mite (HDM), and lipopolysaccharide (LPS) during sensitization to establish a mouse model of severe asthma with neutrophil predominant inflammation (OVA+LPS mice) and treated with or without simvastatin. In inflammatory response, proportions of Th2, Th17, and Treg cells in lung tissue were detected by flow cytometry, and the levels of cytokines, dsDNA, and MPO-DNA in bronchoalveolar lavage fluid (BALF) were analyzed by ELISA. Citrullinated histone H3 (CitH3) and peptidyl arginine deiminase 4 (PAD4) in lung tissue were determined by Western blot and immunofluorescence imaging. PAD4 mRNA was determined by quantitative PCR (qPCR). HL-60 cells were differentiated into neutrophil-like cells by 1.25% DMSO. The neutrophil-like cells were treated with or without LPS, and simvastatin was then stimulated with PMA. CitH3 and PAD4 expressions were determined. Results. Sensitization with OVA, HDM, and LPS resulted in neutrophilic inflammation and the formation of NETs in the lungs. Simvastatin treatment reduced the inflammation score, cytokine levels, total cells, and neutrophil counts in the BALF and reduced proportions of Th2 and Th17 but increased Treg cells in lungs of OVA+LPS mice. Simvastatin-treated OVA+LPS mice show reduced NET formation in BALF and lung tissue compared to control mice. Adoptive transfer of neutrophils was sufficient to restore NETosis and neutrophilic inflammation in simvastatin-treated OVA+LPS mice. Simvastatin reduced PAD4 mRNA and protein expression in lung tissues and neutrophils isolated from lungs of OVA+LPS mice and consequent NET formation. In vitro, simvastatin reduced LPS-induced PAD4 upregulation and NETosis in HL-60-differentiated neutrophil-like cells. Furthermore, PAD4-overexpressed lentiviral transduction was sufficient to restore PAD4 protein expression and NETosis in simvastatin-treated HL-60-differentiated neutrophil-like cells. Conclusions. Simvastatin reduces Th17-mediated neutrophilic inflammation and airway hyperreactivity by reducing PAD4 expression and inhibiting NETosis in a mouse model of severe asthma. Severe asthmatic patients with high levels of circulating NETs or sputum NETs may show improved responses to statin treatment.

Image pre-processing is crucial for large fleet management. Many traffic videos are collected by closed-circuit television (CCTV), which has a fixed area monitoring for image analysis. This paper adopts the front camera installed in large vehicles to obtain moving traffic images, whereas CCTV is more limited. In practice, fleets often install cameras with different resolutions due to cost considerations. The cameras evaluate the front images with traffic lights. This paper proposes fuzzy enhancement with RGB and CIELAB conversions to handle multiple resolutions. This study provided image pre-processing adjustment comparisons, enabling further model training and analysis. This paper proposed fuzzy enhancement to deal with multiple resolutions. The fuzzy enhancement and fuzzy with brightness adjustment produced images with lower MSE and higher PSNR for the images of the front view. Fuzzy enhancement can also be used to enhance traffic light image adjustments. Moreover, this study employed You Only Look Once Version 9 (YOLOv9) for model training. YOLOv9 with fuzzy enhancement obtained better detection performance. This fuzzy enhancement made more flexible adjustments for pre-processing tasks and provided guidance for fleet managers to perform consistent image-enhancement adjustments for handling multiple resolutions.

Perception of fear induced by others in danger elicits complex vicarious fear responses and behavioral outputs. In rodents, observing a conspecific receive aversive stimuli leads to escape and freezing behavior. It remains unclear how these behavioral self-states in response to others in fear are neurophysiologically represented. Here, we assess such representations in the ventromedial prefrontal cortex (vmPFC), an essential site for empathy, in an observational fear (OF) paradigm in male mice. We classify the observer mouse’s stereotypic behaviors during OF using a machine-learning approach. Optogenetic inhibition of the vmPFC specifically disrupts OF-induced escape behavior. In vivo Ca 2+ imaging reveals that vmPFC neural populations represent intermingled information of other- and self-states. Distinct subpopulations are activated and suppressed by others’ fear responses, simultaneously representing self-freezing states. This mixed selectivity requires inputs from the anterior cingulate cortex and the basolateral amygdala to regulate OF-induced escape behavior. Observational fear is accompanied by both freezing and escape behavior in rodents. Here, the authors show that ventromedial prefrontal cortex (vmPFC) inhibition disrupts escape behavior specifically, and that vmPFC neural activity represents intermingled information of other- and self-states.

Epstein-Barr Virus (EBV) is a globally prevalent herpesvirus associated with infectious mononucleosis and many malignancies. The survey on EBV prevalence appears to be important to study EBV-related diseases and determine when to administer prophylactic vaccine. The purpose of this retrospective study was to collect baseline information about the prevalence of EBV infection in Chinese children. We collected 1778 serum samples from healthy children aged 0 to 10, who were enrolled in conventional health and nutrition examinations without any EBV-related symptom in 2012 and 2013 in North China (n = 973) and South China (n = 805). We detected four EBV-specific antibodies, i.e., anti-VCA-IgG and IgM, anti-EBNA-IgG and anti-EA-IgG, by ELISA, representing all of the phases of EBV infection. The overall EBV seroprevalence in samples from North and South China were 80.78% and 79.38% respectively. The EBV seropositivity rates dropped slightly at age 2, and then increased gradually with age. The seroprevalence became stabilized at over 90% after age 8. In this study, the seroprevalence trends between North and South China showed no difference (P>0.05), and the trends of average antibody concentrations were similar as well (P>0.05). EBV seroprevalence became more than 50% before age 3 in Chinese children, and exceed 90% after age 8. This study can be helpful to study the relationship between EBV and EBV-associated diseases, and supportive to EBV vaccine development and implementation.

ObjectiveCurrently, there is no cure for chronic pancreatitis (CP). Germline loss-of-function variants in SPINK1 (encoding trypsin inhibitor) are common in patients with CP and are associated with acute attacks and progression of the disease. This preclinical study was conducted to explore the potential of adeno-associated virus type 8 (AAV8)-mediated overexpression of human SPINK1 (hSPINK1) for pancreatitis therapy in mice.DesignA capsid-optimised AAV8-mediated hSPINK1 expression vector (AAV8-hSPINK1) to target the pancreas was constructed. Mice were treated with AAV8-hSPINK1 by intraperitoneal injection. Pancreatic transduction efficiency and safety of AAV8-hSPINK1 were dynamically evaluated in infected mice. The effectiveness of AAV8-hSPINK1 on pancreatitis prevention and treatment was studied in three mouse models (caerulein-induced pancreatitis, pancreatic duct ligation and Spink1 c.194+2T>C mouse models).ResultsThe constructed AAV8-hSPINK1 vector specifically and safely targeted the pancreas, had low organ tropism for the heart, lungs, spleen, liver and kidneys and had a high transduction efficiency (the optimal expression dose was 2×1011 vg/animal). The expression and efficacy of hSPINK1 peaked at 4 weeks after injection and remained at significant level for up to at least 8 weeks. In all three mouse models, a single dose of AAV8-hSPINK1 before disease onset significantly alleviated the severity of pancreatitis, reduced the progression of fibrosis, decreased the levels of apoptosis and autophagy in the pancreas and accelerated the pancreatitis recovery process.ConclusionOne-time injection of AAV8-hSPINK1 safely targets the pancreas with high transduction efficiency and effectively ameliorates pancreatitis phenotypes in mice. This approach is promising for the prevention and treatment of CP.