Computer-assisted semi-rational design enhanced the enzymatic activity and protein stability of Proteinase K in calcium-free conditions.

Wild-type Proteinase K binds to two Ca2+ ions, which play an important role in regulating enzymaticactivity and maintaining protein stability. Therefore, a predetermined concentration of Ca2+ must be added during the use of Proteinase K, which increases its commercial cost. Herein, we addressed this challenge using a computational strategy to engineer a Proteinase K mutant that does not require Ca2+ and exhibits high enzymatic activity and protein stability. In the absence of Ca2+, the best mutant, MT24 (S17W-S176N-D260F), displayed an activity approximately 9.2-fold higher than that of wild-type Proteinase K. It also exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 °C for 5 days. The residual enzymatic activity was 65-fold higher than that of the wild-type Proteinase K under the same storage conditions. Structural analysis and molecular dynamics simulations suggest that the introduction of new hydrogen bond and π-π stacking at the Ca2+ binding sites due to the mutation may be the reasons for the increased enzymatic activity and stability of MT24.

Score-based Counterfactual Generation for Interpretable Medical Image Classification and Lesion Localization.

Deep neural networks (DNNs) have immense potential for precise clinical decision-making in the field of biomedical imaging. However, accessing high-quality data is crucial for ensuring the high-performance of DNNs. Obtaining medical imaging data is often challenging in terms of both quantity and quality. To address these issues, we propose a score-based counterfactual generation (SCG) framework to create counterfactual images from latent space, to compensate for scarcity and imbalance of data. In addition, some uncertainties in external physical factors may introduce unnatural features and further affect the estimation of the true data distribution. Therefore, we integrated a learnable FuzzyBlock into the classifier of the proposed framework to manage these uncertainties. The proposed SCG framework can be applied to both classification and lesion localization tasks. The experimental results revealed a remarkable performance boost in classification tasks, achieving an average performance enhancement of 3-5% compared to previous state-of-the-art (SOTA) methods in interpretable lesion localization.

Maturation of induced pluripotent stem cell-derived cardiomyocytes and its therapeutic effect on myocardial infarction in mouse.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have an irreplaceable role in the treatment of myocardial infarction (MI), which can be injected into the transplanted area with new cardiomyocytes (Cardiomyocytes, CMs), and improve myocardial function. However, the immaturity of the structure and function of iPSC-CMs is the main bottleneck at present. Since collagen participates in the formation of extracellular matrix (ECM), we synthesized nano colloidal gelatin (Gel) with collagen as the main component, and confirmed that the biomaterial has good biocompatibility and is suitable for cellular in vitro growth. Subsequently, we combined the PI3K/AKT/mTOR pathway inhibitor BEZ-235 with Gel and found that the two combined increased the sarcomere length and action potential amplitude (APA) of iPSC-CMs, and improved the Ca2+ processing ability, the maturation of mitochondrial morphological structure and metabolic function. Not only that, Gel can also prolong the retention rate of iPSC-CMs in the myocardium and increase the expression of Cx43 and angiogenesis in the transplanted area of mature iPSC-CMs, which also provides a reliable basis for the subsequent treatment of mature iPSC-CMs.

Essential role of MESP1-RING1A complex in cardiac differentiation.

Heart development is controlled by a complex transcriptional network composed of transcription factors and epigenetic regulators. Mutations in key developmental transcription factor MESP1 and chromatin factors, such as PRC1 and cohesin components, have been found in human congenital heart diseases (CHDs), although their functional mechanism during heart development remains elusive. Here, we find that MESP1 interacts with RING1A/RING1, the core component of PRC1. RING1A depletion impairs human cardiomyocyte differentiation, and cardiac abnormalities similar to those in patients with MESP1 mutations were observed in Ring1A knockout mice. Mechanistically, MESP1 associates with RING1A to activate cardiogenic genes through promoter-enhancer interactions regulated by cohesin and CTCF and histone acetylation mediated by p300. Importantly, CHD mutations of MESP1 significantly affect such mechanisms and impair target gene activation. Together, our results demonstrate the importance of MESP1-RING1A complex in heart development and provide insights into the pathogenic mechanisms of CHDs caused by mutations in MESP1, PRC1, and cohesin components.

Downregulated ALKBH5 contributes to myocardial ischemia/reperfusion injury by increasing m6A modification of Trio mRNA.

Background: The modification of N6-methyladenosine (m6A) is a dynamic and reversible course that might play a role in cardiovascular disease. However, the mechanisms of m6A modification in myocardial ischemia/reperfusion injury (MIRI) remain unclear. Methods: A mouse model of MIRI and a cell model of oxygen-glucose deprivation/reperfusion (OGD/R) HL-1 cells were employed. In an in vivo study, the total RNA m6A modification levels were determined by dot blot, and the key genes related to m6A modification were screened by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. In an in vitro study, the effects of AlkB homolog 5 (ALKBH5), an RNA demethylase, on cell proliferation, cell injury, and apoptosis were detected by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, lactate dehydrogenase (LDH) and cardiac troponin-I (cTnI) levels, and flow cytometry. Besides, the m6A modification-changed and differentially expressed messenger RNA (mRNA) were determined by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) in ALKBH5-overexpressed HL-1 cells. Finally, the mRNA levels of the promising targeted gene were examined by RT-qPCR and its m6A modification levels were examined by MeRIP-qPCR. Results: Our results showed that RNA m6A modification was involved in MIRI, in which ALKBH5 was downregulated. Functionally, by overexpressing or silencing ALKBH5 in experimental cells, we verified its protective properties on cell proliferation, cell injury, and apoptosis in the process of MIRI. Besides, we provided a mass of latent different mRNAs with m6A modification variation in ALKBH5-overexpressed HL-1 cells. Mechanistically, we further screened the most potential targeted mRNAs and suggested that triple functional domain (Trio) mRNA could be upregulated by ALKBH5 by reducing m6A level of Trio. Conclusions: This study demonstrated that the downregulated ALKBH5 might contribute to MIRI process by increasing the m6A modification of Trio mRNA and downregulating Trio.

Immunomodulatory effects of icariin in a myocardial infarction mouse model.

Myocardial infarction (MI) is a prevalent cardiovascular disease defined by myocardial ischemia and hypoxic damage caused by plaque rupture, thrombosis, lumen stenosis, or blockage in the coronary artery. However, the development of emergency percutaneous coronary interventional therapy has enabled the rapid restoration of blood perfusion to ischemic myocardium and the rescue of dying myocardium cells. Some dying myocardium cells have caused irreversible damage and impaired cardiac function recovery in recent years. Icariin has been utilized to treat various ailments as a natural chemical extract. In this study, we employed a variety of approaches to observe MI, including western blotting, quantitative RT-PCR, immunohistochemistry, and flow cytometric analysis using icariin. As demonstrated by the research findings, icariin may prevent MI-induced cell apoptosis. This is accomplished by inhibiting proinflammatory factors via the Nrf2/HO-1 signaling pathways. These data imply that icariin may be an effective treatment for MI.

Engineered Schwann Cell-Based Therapies for Injury Peripheral Nerve Reconstruction.

Compared with the central nervous system, the adult peripheral nervous system possesses a remarkable regenerative capacity, which is due to the strong plasticity of Schwann cells (SCs) in peripheral nerves. After peripheral nervous injury, SCs de-differentiate and transform into repair phenotypes, and play a critical role in axonal regeneration, myelin formation, and clearance of axonal and myelin debris. In view of the limited self-repair capability of SCs for long segment defects of peripheral nerve defects, it is of great clinical value to supplement SCs in necrotic areas through gene modification or stem cell transplantation or to construct tissue-engineered nerve combined with bioactive scaffolds to repair such tissue defects. Based on the developmental lineage of SCs and the gene regulation network after peripheral nerve injury (PNI), this review summarizes the possibility of using SCs constructed by the latest gene modification technology to repair PNI. The therapeutic effects of tissue-engineered nerve constructed by materials combined with Schwann cells resembles autologous transplantation, which is the gold standard for PNI repair. Therefore, this review generalizes the research progress of biomaterials combined with Schwann cells for PNI repair. Based on the difficulty of donor sources, this review also discusses the potential of "unlimited" provision of pluripotent stem cells capable of directing differentiation or transforming existing somatic cells into induced SCs. The summary of these concepts and therapeutic strategies makes it possible for SCs to be used more effectively in the repair of PNI.

Learning Feature Channel Weighting for Real-Time Visual Tracking.

Recently, the siamese convolutional neural network plays an important role in the field of visual tracking, which can obtain high tracking accuracy and good real-time performance. However, the requirement of offline training a specific neural network results in the hardware source and time consumption. In order to improve the tracking efficiency and save computation resources, we adopt pre-trained densely connected neural network to extract robust target features. Since the pre-trained model is mainly used for classification task, it is not appropriate to directly adopt these deep features for visual tracking. We design a regression network to measure the importance of each channel to the target, and then propose a weighting fusion strategy to select the suitable features for visual tracking. Besides, we provide deep analysis about the proposed channel weighting method to demonstrate the superiority of this method through visualization of feature heatmaps. Extensive experiments on four classical benckmarks show that compared with state-of-the-art methods, our algorithm achieves the best results on several standard indicators and comparable results on other indicators.

Feature Map Distillation of Thin Nets for Low-Resolution Object Recognition.

Intelligent video surveillance is an important computer vision application in natural environments. Since detected objects under surveillance are usually low-resolution and noisy, their accurate recognition represents a huge challenge. Knowledge distillation is an effective method to deal with it, but existing related work usually focuses on reducing the channel count of a student network, not feature map size. As a result, they cannot transfer "privilege information" hidden in feature maps of a wide and deep teacher network into a thin and shallow student one, leading to the latter's poor performance. To address this issue, we propose a Feature Map Distillation (FMD) framework under which the feature map size of teacher and student networks is different. FMD consists of two main components: Feature Decoder Distillation (FDD) and Feature Map Consistency-enforcement (FMC). FDD reconstructs the shallow texture features of a thin student network to approximate the corresponding samples in a teacher network, which allows the high-resolution ones to directly guide the learning of the shallow features of the student network. FMC makes the size and direction of each deep feature map consistent between student and teacher networks, which constrains each pair of feature maps to produce the same feature distribution. FDD and FMC allow a thin student network to learn rich "privilege information" in feature maps of a wide teacher network. The overall performance of FMD is verified in multiple recognition tasks by comparing it with state-of-the-art knowledge distillation methods on low-resolution and noisy objects.

[Analysis of P gene variations among fourteen patients with oculocutaneous albinism type II].

OBJECTIVE: To analyze variations of TYR and P genes among 14 patients with clinically diagnosed oculocutaneous albinism. METHODS: Potential variations of the TYR and P genes were detected by Sanger sequencing. Novel variations were predicted with bioinformatics software including SIFT and PolyPhen-2. RESULTS: No variation was found in the TYR gene, while 9 types of variations were found in the P gene among the 14 patients, which included c.803-3C>G (7/26), c.1327G>A (p.Val443Ile) (5/26), c.632C>T (p.Pro211Leu) (4/26), c.1832T>C (p.Leu611Pro) (3/26), c.1349C>A (p.Thr450Lys) (2/26), c.2363C>T (p.Ser788Leu) (2/26), c.2228C>T (p.Pro743Leu) (1/26), c.1525A>G (p.Thr509Ala) (1/26), and c.1349C>T (p.Thr450Met) (1/26). Only 1 heterozygous variation was detected in 2 families. c.2363C>T (p.Ser788Leu), c.1832T>C (p.Leu611Pro) and c.1525A>G (p.Thr509Ala) were not reported previously and predicted as "harmful" to the protein function. CONCLUSION: The main type of ocular albinism is oculocutaneous albinism type II in Liuzhou region, where the most common variations of the P gene were c.803-3C>G and c.1327G>A (p.Val443Ile). Above finding has enriched the variation spectrum of the P gene.

Online Multi-expert Learning for Visual Tracking.

The correlation filters based trackers have achieved an excellent performance for object tracking in recent years. However, most existing methods use only one filter but ignore the information of the previous filters. In this paper, we propose a novel online multi-expert learning algorithm for visual tracking. In our proposed scheme, there are former trackers which retain the previous filters, and those trackers will give their predictions in each frame. The current tracker represents the filter of current frame, and both the current tracker and the former trackers constitute our expert ensemble. We use an adaptive Second-order Quantile strategy to learn the weights of each expert, which can take full advantage of all the experts. To simplify our model and remove some bad experts, we prune our models via a minimum entropy criterion. Finally, we propose a new update strategy to avoid the model corruption problem. Extensive experimental results on both OTB2013 and OTB2015 benchmarks demonstrate that our proposed tracker performs favorably against state-of-the-art methods.

[Analysis of TRPM6 gene variant in a pedigree affected with hypocalcemia secondary to hypomagnesemia].

OBJECTIVE: To explore the molecular pathogenesis for a pedigree affected with hypocalcemia secondary to hypomagnesemia. METHODS: Sanger sequencing was used to detect potential variant of the TRPM6 gene in the patient and their parents. RESULTS: The results showed that the patient has carried novel homozygous c.3311C>T (p.Pro1104Leu) variant of the TRMP6 gene, for which both of his parents were heterozygous carriers. Analysis of protein functions using software predicted high risk of pathogenicity. CONCLUSION: The homozygous c.3311C>T (p.Pro1104Leu) variant of the TRPM6 gene probably underlies the disease in this patient.

[SLC22A5 gene mutation analysis and prenatal diagnosis for a family with primary carnitine deficiency].

OBJECTIVE: To carry out mutation analysis and prenatal diagnosis for a family affected with primary carnitine deficiency. METHODS: Genomic DNA of the proband was extracted from peripheral blood sample 10 days after birth. The 10 exons and intron/exon boundaries of the SLC22A5 gene were subjected to PCR amplification and Sanger sequencing. The proband's mother was pregnant again two years after his birth. Fetal DNA was extracted from amniocytes and subjected to PCR and Sanger sequencing. RESULTS: Tandem mass spectrometric analysis of the proband revealed low level of plasma-free carnitine whilst organic acids in urine was normal. Compound heterozygous SLC22A5 mutations c.1195C>T (inherited from his father) and c.517delC (inherited from his mother) were detected in the proband. Prenatal diagnosis has detected no mutation in the fetus. The plasma-free carnitine was normal after birth. CONCLUSION: Appropriate genetic testing and prenatal diagnosis can prevent further child with carnitine deficiency. The identification of c.517delC, a novel mutation, enriched the spectrum of SLC22A5 mutations.

[Clinical and genetic analysis of a child with Noonan syndrome].

OBJECTIVE: To identify potential mutation in a child clinically diagnosed as Noonan syndrome and to provide genetic counseling and prenatal diagnosis for his family. METHODS: Genomic DNA was extracted from peripheral blood samples of the patient and his parents, and amniotic fluid was taken from the mother during the second trimester. Next generation sequencing (NGS) was used to screen potential mutations from genomic DNA. Suspected mutation was verified by Sanger sequencing. RESULTS: A heterozygous c.4A>G (p.Ser2Gly) mutation of the SHOC2 gene was identified in the patient but not among other family members including the fetus. CONCLUSION: The Noonan syndrome is probably caused by the c.4A>G mutation of the SHOC2 gene. NGS is helpful for the diagnosis of complicated genetic diseases. SHOC2 gene mutation screening is recommended for patient suspected for Noonan syndrome.

Copy number variations in the GATA4, NKX2-5, TBX5, BMP4 CRELD1, and 22q11.2 gene regions in Chinese children with sporadic congenital heart disease.

BACKGROUND: Congenital heart disease (CHD) is a common birth defect originating from both environmental and genetic factors. An overabundance of copy number variations (CNVs) affecting cardiac-related genes has previously been detected in individuals with CHD. OBJECTIVE: To evaluate if the presence of CNVs in the 22q11.2 region, and to determine whether GATA4, NKX2-5, TBX5, BMP, and CRELD1 genes contributed toward the pathogenesis of isolated incidences of CHDs in southwest China. METHODS: In total 167 patients from southwest China with sporadic CHD were studied, including 121 patients with ventricular septal defect (VSD), 24 with atrial septal defect (ASD), 12 with tetralogy of fallot (TOF), six VSD cases with TOF, two cases with patent ductus arteriosus (PDA), and two VSD cases with ASD. 22q11.2, GATA4, NKX2-5, TBX5, BMP4, and CRELD1 regions were screened using MLPA and copy number variation sequencing (CNV-Seq). RESULTS: A 2.5-2.8 Mb deletion in the 22q11.2 region was identified in 5 patients with CHD. Two of these patients were diagnosed with VSD, while two had VSD and ASD, and the other had TOF. 5 patients correspond to the same classical DiGeorge syndrome. A 0.86 Mb duplication in the 22q11.2 region was identified in a PDA patient, whom was without extracardiac symptoms. CONCLUSION: These data suggest that copy number variation in the 22q11.2 region is common in CHD patients in southwest China. Regardless of the presence or absence of extracardiac symptoms, results also indicate that it is necessary to perform prenatal screening for CHD.

Visual Tracking With Weighted Adaptive Local Sparse Appearance Model via Spatio-Temporal Context Learning.

Sparse representation has been widely exploited to develop an effective appearance model for object tracking due to its well discriminative capability in distinguishing the target from its surrounding background. However, most of these methods only consider either the holistic representation or the local one for each patch with equal importance, and hence may fail when the target suffers from severe occlusion or large-scale pose variation. In this paper, we propose a simple yet effective approach that exploits rich feature information from reliable patches based on weighted local sparse representation that takes into account the importance of each patch. Specifically, we design a reconstruction-error based weight function with the reconstruction error of each patch via sparse coding to measure the patch reliability. Moreover, we explore spatio-temporal context information to enhance the robustness of the appearance model, in which the global temporal context is learned via incremental subspace and sparse representation learning with a novel dynamic template update strategy to update the dictionary, while the local spatial context considers the correlation between the target and its surrounding background via measuring the similarity among their sparse coefficients. Extensive experimental evaluations on two large tracking benchmarks demonstrate favorable performance of the proposed method over some state-of-the-art trackers.

POI Summarization by Aesthetics Evaluation From Crowd Source Social Media.

Place-of-Interest (POI) summarization by aesthetics evaluation can recommend a set of POI images to the user and it is significant in image retrieval. In this paper, we propose a system that summarizes a collection of POI images regarding both aesthetics and diversity of the distribution of cameras. First, we generate visual albums by a coarse-to-fine POI clustering approach and then generate 3D models for each album by the collected images from social media. Second, based on the 3D to 2D projection relationship, we select candidate photos in terms of the proposed crowd source saliency model. Third, in order to improve the performance of aesthetic measurement model, we propose a crowd-sourced saliency detection approach by exploring the distribution of salient regions in the 3D model. Then, we measure the composition aesthetics of each image and we explore crowd source salient feature to yield saliency map, based on which, we propose an adaptive image adoption approach. Finally, we combine the diversity and the aesthetics to recommend aesthetic pictures. Experimental results show that the proposed POI summarization approach can return images with diverse camera distributions and aesthetics.

[Genetic diagnosis of 10 neonates with primary carnitine deficiency].

OBJECTIVE: To study the gene mutation profile of primary carnitine deficiency (PCD) in neonates, and to provide a theoretical basis for early diagnosis and treatment, genetic counseling, and prenatal diagnosis of PCD. METHODS: Acylcarnitine profile analysis was performed by tandem mass spectrometry using 34 167 dry blood spots on filter paper. The SLC22A5 gene was sequenced and analyzed in neonates with free carnitine (C0) levels lower than 10 µmol/L as well as their parents. RESULTS: In the acylcarnitine profile analysis, a C0 level lower than 10 µmol/L was found in 10 neonates, but C0 level was not reduced in their mothers. The 10 neonates had 10 types of mutations at 20 different sites in the SLC22A5 gene, which included 4 previously unreported mutations: c.976C>T, c.919delG, c.517delC, and c.338G>A. Bioinformatics analysis showed that the four new mutations were associated with a risk of high pathogenicity. CONCLUSIONS: Tandem mass spectrometry combined with SLC22A5 gene sequencing may be useful for the early diagnosis of PCD. Identification of new mutations enriches the SLC22A5 gene mutation profile.

[CPT2 gene mutation analysis and prenatal diagnosis in a family with carnitine palmitoyltransferase II deficiency].

This study aimed to identify the type of carnitine palmitoyltransferase 2 (CPT2) gene mutation in the child with carnitine palmitoyltransferase II (CPT II) deficiency and her parents and to provide the genetic counseling and prenatal diagnosis for the family members. As the proband, a 3-month-old female baby was admitted to the hospital due to fever which had lasted for 8 hours. Tandem mass spectrometric analysis for blood showed an elevated plasma level of acylcarnitine, which suggested CPT II deficiency. The genomic DNA was extracted from peripheral blood of the patient and her parents. Five exon coding regions and some intron regions at the exon/intron boundaries of the CPT2 gene were analyzed by PCR and Sanger sequencing. Amniotic fluid was taken from the mother during the second trimester, and DNA was extracted to analyze the type of CPT2 gene mutation. Sanger sequencing results showed that two mutations were identified in the CPT2 gene of the proband: c.886C>T (p.R296X) and c.1148T>A (p.F383Y), which were inherited from the parents; the second child of the mother inherited the mutation of c.886C>T (p.R296X) and showed normal acylcarnitine spectrum and normal development after birth. It is concluded that the analysis of CPT2 gene mutations in the family suggested that the proband died of CPT II deficiency and that the identification of the mutations was helpful in prenatal diagnosis in the second pregnancy.

[Pancytopenia and metabolic decompensation in a neonate].

A 9-day-old male patient was admitted to the hospital because of cough, anhelation, feeding difficulty and lethargy. The diagnostic examinations indicated pulmonary infection, severe metabolic acidosis, hyperglycemia, hyperammonemia and pancytopenia in the patient. Blood and urine screening and isovaleryl-CoA dehydrogenase (IVD) gene detection for inherited metabolic diseases were performed to clarify the etiology. Tandem mass spectrometric screening for blood showed an elevated isovalerylcarnitine (C5) level. The organic acid analysis of urine by gas chromatography-mass spectrometry showed significantly increased levels in isovaleryl glycine and 3-hydroxyisovaleric acid. Homozygous mutations (c.1208A>G, p.Tyr403Cys) in the IVD gene were identified in the patient. His parents were heterozygous carriers. After the treatment with low-leucine diets and L-carnitine for 3 days, the patient showed a significant improvement in symptoms, but he died one week later. It is concluded that the neonates with pneumonia and metabolic decompensation of unknown etiology should be screened for genetic metabolic disease.