A first-in-class inhibitor of Hsp110 molecular chaperones of pathogenic fungi.

Proteins of the Hsp110 family are molecular chaperones that play important roles in protein homeostasis in eukaryotes. The pathogenic fungus Candida albicans, which causes infections in humans, has a single Hsp110, termed Msi3. Here, we provide proof-of-principle evidence supporting fungal Hsp110s as targets for the development of new antifungal drugs. We identify a pyrazolo[3,4-b] pyridine derivative, termed HLQ2H (or 2H), that inhibits the biochemical and chaperone activities of Msi3, as well as the growth and viability of C. albicans. Moreover, the fungicidal activity of 2H correlates with its inhibition of in vivo protein folding. We propose 2H and related compounds as promising leads for development of new antifungals and as pharmacological tools for the study of the molecular mechanisms and functions of Hsp110s.

Language-Aware Spatial-Temporal Collaboration for Referring Video Segmentation.

Given a natural language referring expression, the goal of referring video segmentation task is to predict the segmentation mask of the referred object in the video. Previous methods only adopt 3D CNNs upon the video clip as a single encoder to extract a mixed spatio-temporal feature for the target frame. Though 3D convolutions are able to recognize which object is performing the described actions, they still introduce misaligned spatial information from adjacent frames, which inevitably confuses features of the target frame and leads to inaccurate segmentation. To tackle this issue, we propose a language-aware spatial-temporal collaboration framework that contains a 3D temporal encoder upon the video clip to recognize the described actions, and a 2D spatial encoder upon the target frame to provide undisturbed spatial features of the referred object. For multimodal features extraction, we propose a Cross-Modal Adaptive Modulation (CMAM) module and its improved version CMAM+ to conduct adaptive cross-modal interaction in the encoders with spatial- or temporal-relevant language features which are also updated progressively to enrich linguistic global context. In addition, we also propose a Language-Aware Semantic Propagation (LASP) module in the decoder to propagate semantic information from deep stages to the shallow stages with language-aware sampling and assignment, which is able to highlight language-compatible foreground visual features and suppress language-incompatible background visual features for better facilitating the spatial-temporal collaboration. Extensive experiments on four popular referring video segmentation benchmarks demonstrate the superiority of our method over the previous state-of-the-art methods.

Transcription factor YY1 mediates self-renewal of glioblastoma stem cells through regulation of the SENP1/METTL3/MYC axis.

Glioma is a primary brain tumor with limited treatment approaches and glioblastoma stem cells (GSCs) are manifested with the self-renewal capability and high tumorigenic capacity. This study was performed to investigate the regulatory effect of the SUMO-specific protease 1 (SENP1)/methyltransferase-like 3 (METTL3)/MYC axis on the self-renewal of GSCs mediated by transcription factor Yin Yang 1 (YY1). Following bioinformatics analysis and clinical and cellular experiments, we found that YY1 was highly expressed in GBM tissues and cells, while silencing its expression reduced the self-renewal ability of GSCs. Functionally, YY1 promoted the transcriptional expression of SENP1 by binding to the promoter region of SENP1, while the deSUMOase SENP1 facilitated the methylase activity of m6A through deSUMOylation of the methylase METTL3, thereby promoting the m6A modification of MYC mRNA via METL3 and promoting the expression of MYC. A nude mouse xenograft model of GBM was also constructed to examine the tumorigenicity of GSCs. The obtained findings demonstrated that YY1 promoted tumorigenicity of GSCs by promoting the expression of MYC in vivo. Conclusively, YY1 can transcriptionally upregulate the SUMOylase SENP1 and enhance the methylase activity of METTL3, resulting in the increased m6A modification level of MYC mRNA, thereby promoting the self-renewal of GSCs.

Reduced PTCH2 expression is associated with glioma development through its regulation of the PTEN/AKT signaling pathway.

Mutations in the human protein patched homolog (PTCH) gene have been demonstrated to be associated with cancer development in several types of malignancy. However, the underlying mechanism of PTCH-associated cancer development remains poorly understood, to the best of our knowledge. In the present study, the expression of PTCH2 in glioma tumor tissues from The Cancer Genome Atlas (TCGA) database and clinical patients with glioma were measured. Reduced expression levels of PTCH2 were observed in patients with glioma with poor prognose. In vitro, overexpression of PTCH2 significantly suppressed the proliferation and invasion of the glioma cell lines, LN229 and U87-MG. Mechanistically, PTCH2 upregulated the expression of tumor suppressor PTEN, thereby leading to the suppression of pro-survival AKT signals in glioma. Reduced expression of PTEN and enhanced expression of AKT promoted glioma development in vitro and in vivo. Blockade of PTCH2/AKT signals efficiently strengthened the anticancer effects of chemotherapy and prolonged the survival time in tumor-bearing mice, which provided a novel insight into potential treatment strategies for glioma in the clinic.

Acidic preconditioning reduces lipopolysaccharide-induced acute lung injury by upregulating the expression of angiotensin-converting enzyme 2.

Acid preconditioning (APC) through carbon dioxide inhalation can exert protective effects during acute lung injury (ALI) triggered by ischemia-reperfusion. Angiotensin-converting enzyme 2 (ACE2) has been identified as a receptor for severe acute respiratory syndrome coronavirus and the novel coronavirus disease-19. Downregulation of ACE2 plays an important role in the pathogenesis of severe lung failure after viral or bacterial infections. The aim of the present study was to examine the anti-inflammatory mechanism through which APC alleviates lipopolysaccharide (LPS)-induced ALI in vivo and in vitro. The present results demonstrated that LPS significantly downregulated the expression of ACE2, while APC significantly reduced LPS-induced ALI and provided beneficial effects. In addition, bioinformatics analysis indicated that microRNA (miR)-200c-3p directly targeted the 3'untranslated region of ACE2 and regulated the expression of ACE2 protein. LPS exposure inhibited the expression of ACE2 protein in A549 cells by upregulating the levels of miR-200c-3p. However, APC inhibited the upregulation of miR-200c-3p induced by LPS, as well as the downregulation of ACE2 protein, through the NF-κB pathway. In conclusion, although LPS can inhibit the expression of ACE2 by upregulating the levels of miR-200c-3p through the NF-κB pathway, APC inhibited this effect, thus reducing inflammation during LPS-induced ALI.

Scene Graph Generation With Hierarchical Context.

Scene graph generation has received increasing attention in recent years. Enhancing the predicate representations is an important entry point to this task. There are various methods to fully investigate the context of representation enhancement. In this brief, we analyze the decisive factors that can significantly affect the relation detection results. Our analysis shows that spatial correlations between objects, focused regions of objects, and global hints related to the relations have strong influences in relation prediction and contradiction elimination. Based on our analysis, we propose a hierarchical context network (HCNet) to generate a scene graph. HCNet consists of three contexts, including interaction context, depression context, and global context, which integrates information from pair, object, and graph levels. The experiments show that our method outperforms the state-of-the-art methods on the Visual Genome (VG) data set.

Galangin (GLN) Suppresses Proliferation, Migration, and Invasion of Human Glioblastoma Cells by Targeting Skp2-Induced Epithelial-Mesenchymal Transition (EMT).

BACKGROUND: Galangin (GLN), a pure natural flavonoid compound found in plants, has been shown to exert anti-cancer effects against multiple cancer types, including glioma. However, its underlying molecular mechanism remains unclear. Epithelial-to-mesenchymal transition (EMT) performs an important function in the genesis and development of cancer. Skp2, a pivotal component of SCFSkp2 E3 ubiquitin ligase, has been shown to function as an oncogene in GBM invasion that contributes to the EMT process. Thus, we explored whether GLN inhibited Skp2-mediated EMT and the mechanism underlying the Skp2 degradation pathway. METHODS: CCK-8 assay, wound healing assay and transwell assay were used to examine cell proliferation, migration, and invasion after treatment with or without GLN. RT-PCR and Western blotting analysis were performed to evaluate mRNA and protein expression, respectively. Co-immunoprecipitation was conducted to detect ubiquitinated Skp2 levels in vitro and in vivo after GLN treatment. Bioluminescence imaging was performed to examine the intracranial tumor size of U87 xenograft mice. Microscale thermophoresis (MST) experiment was used to detect interactions between Skp2 and GLN. RESULTS: GLN suppressed GBM cell growth, migration, and invasion, and also downregulated the expression of Skp2 and mesenchymal markers (Zeb1, N-cadherin, snail, vimentin) in vitro. Moreover, the overexpression of Skp2 in GBM cells decreased the effect of GLN on EMT. Furthermore, we demonstrated that GLN degraded skp2 protein through the ubiquitination proteasome pathway and directly interacted with skp2 protein, as shown through the MST assay. CONCLUSION: This study is the first to identify Skp2 as a novel target of GLN for the treatment of GBM and report of Skp2 protein degradation in a ubiquitination proteasome pathway. Results from our study indicated the potential of GLN for the treatment of GBM through ubiquitin-mediated degradation of Skp2.

ORDNet: Capturing Omni-Range Dependencies for Scene Parsing.

Learning to capture dependencies between spatial positions is essential to many visual tasks, especially the dense labeling problems like scene parsing. Existing methods can effectively capture long-range dependencies with self-attention mechanism while short ones by local convolution. However, there is still much gap between long-range and short-range dependencies, which largely reduces the models' flexibility in application to diverse spatial scales and relationships in complicated natural scene images. To fill such a gap, we develop a Middle-Range (MR) branch to capture middle-range dependencies by restricting self-attention into local patches. Also, we observe that the spatial regions which have large correlations with others can be emphasized to exploit long-range dependencies more accurately, and thus propose a Reweighed Long-Range (RLR) branch. Based on the proposed MR and RLR branches, we build an Omni-Range Dependencies Network (ORDNet) which can effectively capture short-, middle- and long-range dependencies. Our ORDNet is able to extract more comprehensive context information and well adapt to complex spatial variance in scene images. Extensive experiments show that our proposed ORDNet outperforms previous state-of-the-art methods on three scene parsing benchmarks including PASCAL Context, COCO Stuff and ADE20K, demonstrating the superiority of capturing omni-range dependencies in deep models for scene parsing task.

Photodynamic Modification of Native HCN Channels Expressed in Thalamocortical Neurons.

The photodynamic process requires three elements: light, oxygen, and photosensitizer, and involves the formation of singlet oxygen, the molecular oxygen in excited electronic states. Previously, we reported that heterologously expressed hyperpolarization-activated cAMP-gated (HCN) channels in excised membrane patches are sensitive to photodynamic modification (PDM). Here we extend this study to native HCN channels expressed in thalamocortical (TC) neurons in the ventrobasal (VB) complex of the thalamus and dopaminergic neurons (DA) of the ventral tegmental area (VTA). To do this, we introduced the photosensitizer FITC-cAMP into TCs or DAs of rodent brain slices via a whole-cell patch-clamp recording pipette. After illumination with blue light pulses, we observed an increase in the voltage-insensitive, instantaneous Iinst component, accompanied by a long-lasting decrease in the hyperpolarization-dependent Ih component. Both Ih and the increased Iinst after PDM could be blocked by the HCN blockers Cs+ and ZD7288. When FITC and cAMP were dissociated and loaded into neurons as two separate chemicals, light application did not result in any long-lasting changes of the HCN currents. In contrast, light pulses applied to HCN2-/- neurons loaded with FITC-cAMP generated a much greater reduction in the Iinst component compared to that of WT neurons. Next, we investigated the impact of the long-lasting increases in Iinst after PDM on the cellular physiology of VB neurons. Consistent with an upregulation of HCN channel function, PDM elicited a depolarization of the resting membrane potential (RMP). Importantly, Trolox-C, an effective quencher for singlet oxygen, could block the PDM-dependent increase in Iinst and depolarization of the RMP. We propose that PDM of native HCN channels under physiological conditions may provide a photodynamic approach to alleviate HCN channelopathy in certain pathological conditions.

Heterologous expression of AoD9D enhances salt tolerance with increased accumulation of unsaturated fatty acid in transgenic Saccharomyces cerevisiae.

Salt stress can trigger several physiological responses in microorganisms such as the increasing accumulation of unsaturated fatty acid, which was biosynthesized by delta-9 fatty acid desaturases (D9D) at the first step. In the present study, two D9D genes, designated AoD9D1 and AoD9D2, were isolated from Aspergillus oryzae. The expression analysis showed that AoD9D1 and AoD9D2 were upregulated under salt stress. To investigate the function of AoD9D, transgenic Saccharomyces cerevisiae strains that heterologously expressed AoD9D were exposed to salinity condition. These transgenic strains exhibited greater tolerance to salt stress than wild-type strains, and the heterologous expression of AoD9D increased the content in unsaturated fatty acids as compared to control cells. Moreover, AoD9D1 and AoD9D2 both contained fatty acid desaturase (FAD) and cytochrome b5-like Heme/Steroid-binding domains (Cyt-b5). S. cerevisiae separately transformed with the gene fragments coding for the FAD and Cyt-b5 domains in the AoD9D1 protein grew better and accumulated a higher concentration of unsaturated FAs than the control. Altogether, the heterologous expression of AoD9D enhanced the tolerance of transgenic S. cerevisiae to high salinity stress with increased accumulation of unsaturated fatty acid. The results provide some practical basis for the successful development of salt-tolerant fermentation microorganisms.

Gene transcription profiling of Aspergillus oryzae 3.042 treated with ergosterol biosynthesis inhibitors.

Ergosterol, a unique component of fungal cells, is not only important for fungal growth and stress responses but also holds great economic value. Limited studies have been performed on ergosterol biosynthesis in Aspergillus oryzae, a safe filamentous fungus that has been used for the manufacture of oriental fermented foods. This study revealed that the ergosterol biosynthesis pathway is conserved between Saccharomyces cerevisiae and A. oryzae 3.042 by treatment with ergosterol biosynthesis inhibitors and bioinformatics analysis. However, the ergosterol biosynthesis pathway in A. oryzae 3.042 is more complicated than that in S. cerevisiae as there are multiple paralogs encoding the same biosynthetic enzymes. Using RNA-seq, this study identified 138 and 104 differentially expressed genes (DEG) in response to the ergosterol biosynthesis inhibitors tebuconazole and terbinafine, respectively. The results showed that the most common DEGs were transport- and metabolism-related genes. There were only 17 DEGs regulated by both tebuconazole and terbinafine treatments and there were 256 DEGs between tebuconazole and terbinafine treatments. These results provide new information on A. oryzae ergosterol biosynthesis and regulation mechanisms, which may lay the foundation for genetic modification of the ergosterol biosynthesis pathway in A. oryzae.

A Cross-Domain Collaborative Filtering Algorithm Based on Feature Construction and Locally Weighted Linear Regression.

Cross-domain collaborative filtering (CDCF) solves the sparsity problem by transferring rating knowledge from auxiliary domains. Obviously, different auxiliary domains have different importance to the target domain. However, previous works cannot evaluate effectively the significance of different auxiliary domains. To overcome this drawback, we propose a cross-domain collaborative filtering algorithm based on Feature Construction and Locally Weighted Linear Regression (FCLWLR). We first construct features in different domains and use these features to represent different auxiliary domains. Thus the weight computation across different domains can be converted as the weight computation across different features. Then we combine the features in the target domain and in the auxiliary domains together and convert the cross-domain recommendation problem into a regression problem. Finally, we employ a Locally Weighted Linear Regression (LWLR) model to solve the regression problem. As LWLR is a nonparametric regression method, it can effectively avoid underfitting or overfitting problem occurring in parametric regression methods. We conduct extensive experiments to show that the proposed FCLWLR algorithm is effective in addressing the data sparsity problem by transferring the useful knowledge from the auxiliary domains, as compared to many state-of-the-art single-domain or cross-domain CF methods.

Human Embryonic Kidney 293 Cells: A Vehicle for Biopharmaceutical Manufacturing, Structural Biology, and Electrophysiology.

Mammalian cells, e.g., CHO, BHK, HEK293, HT-1080, and NS0 cells, represent important manufacturing platforms in bioengineering. They are widely used for the production of recombinant therapeutic proteins, vaccines, anticancer agents, and other clinically relevant drugs. HEK293 (human embryonic kidney 293) cells and their derived cell lines provide an attractive heterologous system for the development of recombinant proteins or adenovirus productions, not least due to their human-like posttranslational modification of protein molecules to provide the desired biological activity. Secondly, they also exhibit high transfection efficiency yielding high-quality recombinant proteins. They are easy to maintain and express with high fidelity membrane proteins, such as ion channels and transporters, and thus are attractive for structural biology and electrophysiology studies. In this article, we review the literature on HEK293 cells regarding their origins but also stress their advancements into the different cell lines engineered and discuss some significant aspects which make them versatile systems for biopharmaceutical manufacturing, drug screening, structural biology research, and electrophysiology applications.

Transcriptome analysis of different growth stages of Aspergillus oryzae reveals dynamic changes of distinct classes of genes during growth.

BACKGROUND: The gene expression profile and metabolic pathways of Aspergillus oryzae underlying the anatomical and morphological differentiation across different growth stages have not been fully characterized. The rapid development of next-generation sequencing technologies provides advanced knowledge of the genomic organization of A. oryzae. RESULTS: In this study, we characterized the growth and development of A. oryzae at different growth stages, including the adaptive phase, logarithmic phase, and stationary phase. Our results revealed that A. oryzae undergoes physiological and morphological differentiation across the different stages. RNA-seq was employed to analyze the three stages of A. oryzae, which generated more than 27 million high-quality reads per sample. The analysis of differential gene expression showed more genes expressed differentially upon transition from the adaptive phase to the logarithmic and stationary phases, while relatively steady trend was observed during the transition from the logarithmic phase to the stationary phase. GO classification of the differentially expressed genes among different growth stages revealed that most of these genes were enriched for single-organism process, metabolic process, and catalytic activity. These genes were then subjected to a clustering analysis. The results showed that the cluster with the majority of genes with increased expression upon transition from the adaptive phase to the logarithmic phase, and steady expression from the logarithmic phase to the stationary phase was mainly involved in the carbohydrate and amino acid metabolism. CONCLUSION: Our results provide a foundation for identifying developmentally important genes and understanding the biological processes across various growth stages.