Protocol for automated N-glycan sequencing using mass spectrometry and computer-assisted intelligent fragmentation.

Biological functions of glycans are intimately linked to fine details in branches and linkages, which make structural identification extremely challenging. Here, we present a protocol for automated N-glycan sequencing using multi-stage mass spectrometry (MSn). We describe steps for release/purification and derivation of glycans and procedures for MSn scanning. We then detail "glycan intelligent precursor selection" to computationally guide MSn experiments. The protocol can be used for both discrete individual glycans and isomeric glycan mixtures. For complete details on the use and execution of this protocol, please refer to Sun et al.,1 Huang et al.,2 and Huang et al.3.

The proper timing of Atoh1 expression is pivotal for hair cell subtype differentiation and the establishment of inner ear function.

Atoh1 overexpression is essential for hair cell (HC) regeneration in the sensory epithelium of mammalian auditory and vestibular organs. However, Atoh1 overexpression alone cannot induce fully mature and functional HCs in the mammalian inner ear. In the current study, we investigated the effect of Atoh1 constitutive overexpression in native HCs by manipulating Atoh1 expression at different developmental stages. We demonstrated that constitutive overexpression of Atoh1 in native vestibular HCs did not affect cell survival but did impair vestibular function by interfering with the subtype differentiation of HCs and hair bundle development. In contrast, Atoh1 overexpression in cochlear HCs impeded their maturation, eventually leading to gradual HC loss in the cochlea and hearing dysfunction. Our study suggests that time-restricted Atoh1 expression is essential for the differentiation and survival of HCs in the inner ear, and this is pivotal for both hearing and vestibular function re-establishment through Atoh1 overexpression-induced HC regeneration strategies.

Generation of p27icreER transgenic mice: A tool for inducible gene expression in supporting cells in the cochlea.

The loss of cochlear hair cells (HCs) is an important cause of sensorineural hearing loss, and finding ways to regenerate HCs would be the ideal way forward for restoring hearing. In this research field, tamoxifen-inducible Cre recombinase (iCreER) transgenic mice and the Cre-loxp system are widely used to manipulate gene expression in supporting cells (SCs), which lie beneath the sensory HCs and are a natural source for HC regeneration. However, many iCreER transgenic lines are of limited utility because they cannot target all subtypes of SCs or they cannot be used in the adult stage. In this study, a new line of iCreER transgenic mice, the p27-P2A-iCreERT2 knock-in mouse strain, was generated by inserting the P2A-iCreERT2 cassette immediately in front of the stop codon of p27, which kept the endogenous expression and function of p27 intact. Using a reporter mouse line with tdTomato fluorescence, we showed that the p27iCreER transgenic line can target all subtypes of cochlear SCs, including Claudius cells. p27-CreER activity in SCs was observed in both the postnatal and the adult stage, suggesting that this mouse strain can be useful for research work in adult cochlear HC regeneration. We then overexpressed Gfi1, Pou4f3, and Atoh1 in p27+ SCs of P6/7 mice using this strain and successfully induced many new Myo7a/tdTomato double-positive cells, further confirming that the p27-P2A-iCreERT2 mouse strain is a new and reliable tool for cochlear HC regeneration and hearing restoration.

The expression of p27 in the adult vestibular sensory organs and its possible roles.

Vestibular hair cells (HCs) located in the inner ear are the receptors of vestibular sensory, which facilitates the human sense of balance. The detailed differentiation pattern and maturation process of the vestibular HCs are unclear now. p27, a cyclin/CDK inhibitor, plays a critical role in regulating the exit of cell cycle. We found that p27 was continuously expressed in the terminally differentiated and mature vestibular HCs using p27-P2A-iCreER/+; Rosa26-LSL-tdTomato/+ mice, suggesting p27 might have novel roles independent of its CDK inhibitory action. p27 is also reported to be associated with cell differentiation, cell migration and cell survival. We further explored the difference of p27 expression between two subtypes of vestibular HCs, and found that the proportion of p27-tdTomato positive type I vestibular HCs increased gradually along the subtype determination and maturation of vestibular HCs, suggesting that p27 might play a role in the HC subtype differentiation, maturation and function acquirement.

A High-Precision Method for 100-Day-Old Classification of Chickens in Edge Computing Scenarios Based on Federated Computing.

Due to the booming development of computer vision technology and artificial intelligence algorithms, it has become more feasible to implement artificial rearing of animals in real production scenarios. Improving the accuracy of day-age detection of chickens is one of the examples and is of great importance for chicken rearing. This paper focuses on the problem of classifying the age of chickens within 100 days. Due to the huge amount of data and the different computing power of different devices in practical application scenarios, it is important to maximize the computing power of edge computing devices without sacrificing accuracy. This paper proposes a high-precision federated learning-based model that can be applied to edge computing scenarios. In order to accommodate different computing power in different scenarios, this paper proposes a dual-ended adaptive federated learning framework; in order to adapt to low computing power scenarios, this paper performs lightweighting operations on the mainstream model; and in order to verify the effectiveness of the model, this paper conducts a number of targeted experiments. Compared with AlexNet, VGG, ResNet and GoogLeNet, this model improves the classification accuracy to 96.1%, which is 14.4% better than the baseline model and improves the Recall and Precision by 14.8% and 14.2%, respectively. In addition, by lightening the network, our methods reduce the inference latency and transmission latency by 24.4 ms and 10.5 ms, respectively. Finally, this model is deployed in a real-world application and an application is developed based on the wechat SDK.

A High-Performance Day-Age Classification and Detection Model for Chick Based on Attention Encoder and Convolutional Neural Network.

Thanks to the boom of computer vision techniques and artificial intelligence algorithms, it is more available to achieve artificial rearing for animals in real production scenarios. Improving the accuracy of chicken day-age detection is one of the instances, which is of great importance for chicken rearing. To solve this problem, we proposed an attention encoder structure to extract chicken image features, trying to improve the detection accuracy. To cope with the imbalance of the dataset, various data enhancement schemes such as Cutout, CutMix, and MixUp were proposed to verify the effectiveness of the proposed attention encoder. This paper put the structure into various mainstream CNN networks for comparison and multiple ablation experiments. The final experimental results show that by applying the attention encoder structure, ResNet-50 can improve the accuracy of chicken age detection to 95.2%. Finally, this paper also designed a complete image acquisition system for chicken houses and a detection application configured for mobile devices.

Regeneration of Hair Cells in the Human Vestibular System.

The vestibular system is a critical part of the human balance system, malfunction of this system will lead to balance disorders, such as vertigo. Mammalian vestibular hair cells, the mechanical receptors for vestibular function, are sensitive to ototoxic drugs and virus infection, and have a limited restorative capacity after damage. Considering that no artificial device can be used to replace vestibular hair cells, promoting vestibular hair cell regeneration is an ideal way for vestibular function recovery. In this manuscript, the development of human vestibular hair cells during the whole embryonic stage and the latest research on human vestibular hair cell regeneration is summarized. The limitations of current studies are emphasized and future directions are discussed.

Levels, sources and influence mechanisms of heavy metal contamination in topsoils in Mirror Peninsula, East Antarctica.

Heavy metal contaminants in Mirror Peninsula, East Antarctica, have rarely been studied and the source and influencing factors are poorly understood. We sampled a grid of 189 topsoil samples from Mirror Peninsula and analyzed the concentrations of Zn, Cu, U, Cr, Ga, Pb, Hg, Se and As; we also calculated the chemical index of alteration (CIA), a proxy of weathering. The results show that the distributions of Cr, Ga, Cu, and Zn are associated with weathering; the distributions of As and Pb are related to vehicle use and unloading activities at the wharfs, respectively; and the distribution of Hg is likely associated with both anthropogenic impacts and biological activity. The contamination level of these heavy metals in Mirror Peninsula is relatively low and within the controllable range. Both weathering processes and anthropogenic impacts can cause the enrichment of heavy metals; thus reliable source apportionment is crucial in studying heavy metal enrichment and contamination.