To understand the role of Bcl-2 overexpression in oral tongue squamous cell carcinoma (OTSCC) patients and investigate the efficacy of targeting Bcl-2 in OTSCC.

Virus production in the deep-sea environment has been found to be high, and viruses have been suggested to play significant roles in the overall functioning of this ecosystem. Nevertheless, little is known about these viruses, including the mechanisms that control their production, which makes them one of the least understood biological entities on Earth. Previously, we isolated the filamentous phage SW1, whose virus production and gene transcription were found to be active at low temperatures, from a deep-sea bacterium, Shewanella piezotolerans WP3. In this study, the operon structure of phage SW1 is presented, which shows two operons with exceptionally long 5' and 3' untranslated regions (UTRs). In addition, the 5'UTR was confirmed to significantly influence the RNA stability of the SW1 transcripts. Our study revealed novel regulation of the operon and led us to propose a unique regulatory mechanism for Inoviruses. This type of RNA-based regulation may represent a mechanism for significant viral production in the cold deep biosphere.

Parathyroid hormone (PTH) plays critical, but distinct, roles in bone remodeling, including bone formation (anabolic response) and resorption (catabolic response). Although its signaling and function have been extensively investigated, it just began to be understood how distinct functions are induced by PTH activating a common receptor, the PTH type 1 receptor (PTH1R), and how PTH1R signaling is terminated. Here, we provide evidence for vacuolar protein sorting 35 (VPS35), a major component of retromer, in regulating PTH1R trafficking, turning off PTH signaling, and promoting its catabolic function. VPS35 is expressed in osteoblast (OB)-lineage cells. VPS35-deficiency in OBs impaired PTH(1-34)-promoted PTH1R translocation to the trans-Golgi network, enhanced PTH(1-34)-driven signaling, and reduced PTH(1-34)'s catabolic response in culture and in mice. Further mechanical studies revealed that VPS35 interacts with not only PTH1R, but also protein phosphatase 1 regulatory subunit 14C (PPP1R14C), an inhibitory subunit of PP1 phosphatase. PPP1R14C also interacts with PTH1R, which is necessary for the increased endosomal PTH1R signaling and decreased PTH(1-34)'s catabolic response in VPS35-deficient OB-lineage cells. Taken together, these results suggest that VPS35 deregulates PTH1R-signaling likely by its interaction with PTH1R and PPP1R14C. This event is critical for the control of PTH(1-34)-signaling dynamics, which may underlie PTH-induced catabolic response and adequate bone remodeling.

Neurotransmission requires precise control of neurotransmitter release from axon terminals. This process is regulated by glial cells; however, the underlying mechanisms are not fully understood. We found that glutamate release in the brain was impaired in mice lacking low-density lipoprotein receptor-related protein 4 (Lrp4), a protein that is critical for neuromuscular junction formation. Electrophysiological studies revealed compromised release probability in astrocyte-specific Lrp4 knockout mice. Lrp4 mutant astrocytes suppressed glutamatergic transmission by enhancing the release of ATP, whose level was elevated in the hippocampus of Lrp4 mutant mice. Consequently, the mutant mice were impaired in locomotor activity and spatial memory and were resistant to seizure induction. These impairments could be ameliorated by blocking the adenosine A1 receptor. The results reveal a critical role for Lrp4, in response to agrin, in modulating astrocytic ATP release and synaptic transmission. Our findings provide insight into the interaction between neurons and astrocytes for synaptic homeostasis and/or plasticity.

Insulin-like growth factor 1 receptor (IGF-1R) is important in cancer cell growth and survival and has been implicated in cancer pathophysiology and treatment. Here we report a novel function for IGF-1R in p53-dependent apoptotic response. We show that inhibition or loss of IGF-1R activity reduces translational synthesis of p53 and Mdm2 protein. Notably, IGF-1R inhibition increases p53 protein stability by reducing p53 ubiquitination and maintains p53 at low levels by decreasing p53 synthesis, thus rendering p53 insensitive to stabilization after DNA damage. The accumulation and apoptosis of DNA-damage-induced p53 is therefore reduced in Igf-1r(-/-) mouse embryonic fibroblasts or tumor cells treated with the IGF-1R inhibitor. Furthermore, we find that inhibition of IGF-1R reduces p53 and Mdm2 translation through a gene-specific mechanism mediated by the respective 5' untranslated region of p53 and mdm2 messenger RNA. The eukaryotic translation initiation factor 4F complex is also involved in this translational inhibition. These results demonstrate an unexpected role for translational control by IGF-1R in p53-mediated apoptosis.

Tissue sampling of biliary tract carcinomas (BTCs) for molecular characterization is challenging. The aim of this study was to investigate the possibility of identifying individual actionable mutations derived from bile cell‑free DNA (cfDNA) using targeted deep sequencing. Ten BTC patients, four with gallbladder carcinomas and six with cholangiocarcinomas, were enrolled in the present study. Using targeted deep sequencing with a panel of 150 tumor‑related genes, paired bile cfDNA and tumor DNA were analyzed for mutational variants individually and then compared. The present study, to the best of our knowledge, is the first to reveal that bile cfDNA is predominantly comprised of long DNA fragments, which is not the case for plasma cfDNA. Herein, paired bile cfDNA and tumors from ten BTC patients were examined using targeted deep sequencing. When comparing bile cfDNA and tumor DNA for single nucleotide variation (SNV)/insertion and deletion (Indel), the results using targeted deep sequencing revealed high sensitivity (94.7%) and specificity (99.9%). Additionally, the sensitivity of detecting a copy number variation (CNV) was 75.0%, with a specificity of 98.9%. When comparing two bile extraction methods, including percutaneous transhepatic cholangial drainage and operation, no significant difference in SNV/Indel or CNV detection sensitivity was noted. Moreover, when examining the tumor stage and incidence site, AJCC stage II and the distal bile duct both had significantly decreased CNV detection sensitivities. The present study revealed that targeted deep sequencing can reliably detect mutational variants within bile cfDNA obtained from BTC patients. These preliminary results may shed light on bile cfDNA as a promising liquid biopsy for BTC patients.

Mauremys reevesii is a classical organism with temperature-dependent sex determination (TSD). Gonad development in early life has recently received considerable attention but gonadal maintenance after sex differentiation in turtles with TSD remains a mystery. In this study, we sequenced the transcriptomes for the adult testis and ovary using RNA-seq, and 36,221 transcripts were identified. In total, 1,594 differentially expressed genes (DEGs) were identified where 756 DEGs were upregulated in the testis and 838 DEGs were upregulated in the ovary. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that the TGF-beta signaling pathway and Hedgehog signaling pathway have important roles in testis maintenance and spermatogenesis, whereas the Hippo signaling pathway and Wnt signaling pathway are likely to participate in ovary maintenance. We determined the existence of antagonistic networks containing significant specific-expressed genes and pathways related to gonadal maintenance and gametogenesis in the adult gonads of M. reevesii. The candidate gene Fibronectin type 3 and ankyrin repeat domains 1 (FANK1) might be involved with the regulation of testis spermatogenesis.

YAP (yes-associated protein) is a transcriptional factor that is negatively regulated by Hippo pathway, a conserved pathway for the development and size control of multiple organs. The exact function of YAP in bone homeostasis remains controversial. Here we provide evidence for YAP's function in promoting osteogenesis, suppressing adipogenesis, and thus maintaining bone homeostasis. YAP is selectively expressed in osteoblast (OB)-lineage cells. Conditionally knocking out Yap in the OB lineage in mice reduces cell proliferation and OB differentiation and increases adipocyte formation, resulting in a trabecular bone loss. Mechanistically, YAP interacts with β-catenin and is necessary for maintenance of nuclear β-catenin level and Wnt/β-catenin signaling. Expression of β-catenin in YAP-deficient BMSCs (bone marrow stromal cells) diminishes the osteogenesis deficit. These results thus identify YAP-β-catenin as an important pathway for osteogenesis during adult bone remodeling and uncover a mechanism underlying YAP regulation of bone homeostasis.

Amyloid precursor protein (APP) is ubiquitously expressed in various types of cells including bone cells. Mutations in App gene result in early-onset Alzheimer's disease (AD). However, little is known about its physiological function in bone homeostasis. Here, we provide evidence for APP's role in promoting bone formation. Mice that knocked out App gene (APP-/-) exhibit osteoporotic-like deficit, including reduced trabecular and cortical bone mass. Such a deficit is likely due in large to a decrease in osteoblast (OB)-mediated bone formation, as little change in bone resorption was detected in the mutant mice. Further mechanical studies of APP-/- OBs showed an impairment in mitochondrial function, accompanied with increased reactive oxygen species (ROS) and apoptosis. Intriguingly, these deficits, resemble to those in Tg2576 animal model of AD that expresses Swedish mutant APP (APPswe), were diminished by treatment with an anti-oxidant NAC (n-acetyl-l-cysteine), uncovering ROS as a critical underlying mechanism. Taken together, these results identify an unrecognized physiological function of APP in promoting OB survival and bone formation, implicate APPswe acting as a dominant negative factor, and reveal a potential clinical value of NAC in treatment of AD-associated osteoporotic deficits.

The lotus (Nelumbo Adans.) is an important aquatic plant with ornamental, medicinal and edible values and cultural connotations. It has single-, semi-double-, double- and thousand-petalled types of flower shape and is an ideal material for developmental research of flower doubling. The lotus is a basal eudicot species without a morphological difference between the sepals and petals and occupies a critical phylogenetic position in flowering plants. In order to investigate the genetic relationship between the sepals and petals in the lotus, the class E genes which affect sepal formation were focused on and analyzed. Here, SEPALLATA 1(NnSEP1) and its homologous genes AGAMOUS-LIKE MADS-BOXAGL9 (NnAGL9) and MADS-BOX TRANSCRIPTION FACTOR 6-like (NnMADS6-like) of the class E gene family were isolated from the flower buds of the Asian lotus (Nelumbo nucifera Gaertn.). The protein structure, subcellular localization and expression patterns of these three genes were investigated. All three genes were verified to locate in the nucleus and had typical MADS-box characteristics. NnSEP1 and NnMADS6-like were specifically expressed in the sepals, while NnAGL9 was highly expressed in the petals, suggesting that different developmental mechanisms exist in the formation of the sepals and petals in the lotus. The significant functional differences between NnSEP1, NnMADS6-like and NnAGL9 were also confirmed by a yeast two-hybrid assay. These results expand our knowledge on the class E gene family in sepal formation and will benefit fundamental research on the development of floral organs in Nelumbo.

Gallbladder carcinoma (GBC) is a lethal biliary tract malignant neoplasm. Patient-derived primary cancer cell lines (PDPCs) are appropriate models to explore biological characteristics and potential therapeutics; however, there is a lack of PDPCs in GBC. In this study, we aimed to establish and characterize the GBC PDPCs, and further investigated the intra-tumor heterogeneity (ITH). Multi-region sampling (3-9 regions) of the operable tumor tissue samples was used to establish PDPCs. Short tandem repeat genotyping for cell authentication and karyotyping was performed, followed by whole-exome sequencing and RNA sequencing to assess the ITH at the genetic and transcriptional levels, respectively. Thirty-eight PDPCs were successfully established from seven GBC patients and characterized. ITH was observed with a median of 38.3% mutations being heterogeneous (range, 26.6-59.4%) across all patients. Similar with other tumor types, TP53 mutations were always truncal. In addition, there were three genes, KMT2C, CDKN2A, and ARID1A, with truncal mutations in at least two patients. A median of 370 differentially expressed genes (DEGs) was identified per patient. Distinct expression patterns were observed between major histocompatibility complex (MHC) class I and II genes. We found the expression of MHC class II genes in the PDPC samples was closely regulated by CIITA, while that of MHC class I genes were not correlated with CIITA expression. The PDPCs established from GBC patients can serve as novel in vitro models to identify the ITH, which may pave a crucial molecular foundation for enhanced understanding of tumorigenesis and progression.

Phosphorothioate (PT) modification by the dnd gene cluster is the first identified DNA backbone modification and constitute an epigenetic system with multiple functions, including antioxidant ability, restriction modification, and virus resistance. Despite these advantages for hosting dnd systems, they are surprisingly distributed sporadically among contemporary prokaryotic genomes. To address this ecological paradox, we systematically investigate the occurrence and phylogeny of dnd systems, and they are suggested to have originated in ancient Cyanobacteria after the Great Oxygenation Event. Interestingly, the occurrence of dnd systems and prophages is significantly negatively correlated. Further, we experimentally confirm that PT modification activates the filamentous phage SW1 by altering the binding affinity of repressor and the transcription level of its encoding gene. Competition assays, concurrent epigenomic and transcriptomic sequencing subsequently show that PT modification affects the expression of a variety of metabolic genes, which reduces the competitive fitness of the marine bacterium Shewanella piezotolerans WP3. Our findings strongly suggest that a series of negative effects on microorganisms caused by dnd systems limit horizontal gene transfer, thus leading to their sporadic distribution. Overall, our study reveals putative evolutionary scenario of the dnd system and provides novel insights into the physiological and ecological influences of PT modification.

The differential diagnosis of pancreatic ductal adenocarcinoma (PDAC) from chronic pancreatitis (CP) is clinically challenging due to a lack of minimally invasive diagnosis methods. MicroRNAs (miRNAs) derived from small extracellular vesicles (EVs) in the blood have been reported as a promising diagnosis biomarker for various types of cancer. However, blood small EV miRNA signatures and their diagnostic value to differentiate between PDAC and CP remain to be determined.

The rapid development of insecticide resistance has hampered the use of Bacillus thuringiensis (Bt), a widely used bio-pesticide. Plutella xylostella (L.) is a globally distributed lepidopteran pest of cruciferous vegetables and has developed severe field resistance to the Bt toxin. Vacuolar H+-ATPases (VHA) are multi-subunit complexes and participate in multiple physiological processes. However, the characterization and functional studies of VHA genes are lacking in insects. This study performed a genome-wide analysis and identified 35 VHA gene family members divided into 15 subfamilies in P. xylostella. We cloned a V-ATPase subunit G gene, PxVHA-G1, in our previous midgut transcriptome profiles. Quantitative reverse transcriptase-polymerase chain reaction results showed that PxVHA-G1 was upregulated in the Cry1S1000-resistant strain than in the G88-susceptible strain, and its expression profile revealed that the midgut, Malpighian tubules, and larva stages generally showed high expression levels. RNAi-mediated knockdown of the PxVHA-G1 gene increased the susceptibility of P. xylostella (G88 and Cry1S1000) to Cry1Ac toxin. Our study is the first to explore the role of PxVHA-G1 on regulating Cry1Ac toxicity in P. xylostella, thus, providing new insights into the role of VHAs in the development of Cry1Ac resistance and sustainable development of pest management.

Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with an extremely poor prognosis. Effective targets for anticancer therapy confirmed in PDAC are limited. However, the characteristics of genomics have not been fully elucidated in large-scale patients with PDAC from China.

Brown adipose tissue (BAT) is functionally linked to skeletal muscle because both tissues originate from a common progenitor cell, but the precise mechanism controlling muscle-to-brown-fat communication is insufficiently understood. This report demonstrates that the immunoglobulin superfamily containing leucine-rich repeat (Islr), a marker of mesenchymal stromal/stem cells, is critical for the control of BAT mitochondrial function and whole-body energy homeostasis. The mice loss of Islr in BAT after cardiotoxin injury resulted in improved mitochondrial function, increased energy expenditure, and enhanced thermogenesis. Importantly, it was found that interleukin-6 (IL-6), as a myokine, participates in this process. Mechanistically, Islr interacts with NADH: Ubiquinone Oxidoreductase Core Subunit S2 (Ndufs2) to regulate IL-6 signaling; consequently, Islr functions as a brake that prevents IL-6 from promoting BAT activity. Together, these findings reveal a previously unrecognized mechanism for muscle-BAT cross talk driven by Islr, Ndufs2, and IL-6 to regulate energy homeostasis, which may be used as a potential therapeutic target in obesity.

Methionine aminopeptidases (MetAPs) catalyze the cleavage of the N-terminal initiator methionine (iMet) in new peptide chains and arylamides, which is essential for protein and peptide synthesis. MetAP is differentially expressed in two diamondback moth (DBM; Plutella xylostella) strains: the G88 susceptible strain and the Cry1S1000 strain, which are resistant to the Bt toxin Cry1Ac, implicating that MetAP expression might be associated with Bt resistance. In this study, we identified and cloned a MetAP gene from DBMs, named PxMetAP1, which has a CDS of 1140 bp and encodes a 379 amino acid protein. The relative expression of PxMetAP1 was found to be ~2.2-fold lower in the Cry1S1000 strain compared to that in the G88 strain. PxMetAP1 presents a stage- and tissue-specific expression pattern, with higher levels in the eggs, adults, integument, and fatbody of DBMs. The linkage between PxMetAP1 and Cry1Ac resistance is verified by genetic linkage analysis. The knockout of PxMetAP1 in G88 by CRISPR/Cas9 leads to a ~5.6-fold decrease in sensitivity to the Cry1Ac toxin, further supporting the association between the PxMetAP1 gene and Bt tolerance. Our research sheds light on the role of MetAP genes in the development of Bt tolerance in P. xylostella and enriches the knowledge for the management of such a cosmopolitan pest.

Many insects, including the Plutella xylostella (L.), have developed varying degrees of resistance to many insecticides, including Bacillus thuringiensis (Bt) toxins, the bioinsecticides derived from Bt. The polycalin protein is one of the potential receptors for Bt toxins, and previous studies have confirmed that the Cry1Ac toxin can bind to the polycalin protein of P. xylostella, but whether polycalin is associated with the resistance of Bt toxins remains controversial. In this study, we compared the midgut of larvae from Cry1Ac-susceptible and -resistant strains, and found that the expression of the Pxpolycalin gene was largely reduced in the midgut of the resistant strains. Moreover, the spatial and temporal expression patterns of Pxpolycalin showed that it was mainly expressed in the larval stage and midgut tissue. However, genetic linkage experiments showed that the Pxpolycalin gene and its transcript level were not linked to Cry1Ac resistance, whereas both the PxABCC2 gene and its transcript levels were linked to Cry1Ac resistance. The larvae fed on a diet containing the Cry1Ac toxin showed no significant change in the expression of the Pxpolycalin gene in a short term. Furthermore, the knockout of polycalin and ATP-binding cassette transporter subfamily C2 (ABCC2) genes separately by CRISPR/Cas9 technology resulted in resistance to decreased susceptibility to Cry1Ac toxin. Our results provide new insights into the potential role of polycalin and ABCC2 proteins in Cry1Ac resistance and the mechanism underlying the resistance of insects to Bt toxins.

Patients of Alzheimer's disease (AD) frequently have lower bone mineral density and higher rate of hip fracture. Tg2576, a well characterized AD animal model that ubiquitously express Swedish mutant amyloid precursor protein (APPswe), displays not only AD-relevant neuropathology, but also age-dependent bone deficits. However, the underlying mechanisms remain poorly understood. As APP is implicated as a regulator of iron export, and the metal chelation is considered as a potential therapeutic strategy for AD, we examined iron chelation's effect on the osteoporotic deficit in Tg2576 mice. Remarkably, in vivo treatment with iron chelator, clinoquinol (CQ), increased both trabecular and cortical bone-mass, selectively in Tg2576, but not wild type (WT) mice. Further in vitro studies showed that low concentrations of CQ as well as deferoxamine (DFO), another iron chelator, selectively inhibited osteoclast (OC) differentiation, without an obvious effect on osteoblast (OB) differentiation. Intriguingly, both CQ and DFO's inhibitory effect on OC was more potent in bone marrow macrophages (BMMs) from Tg2576 mice than that of wild type controls. The reduction of intracellular iron levels in BMMs by CQ was also more dramatic in APPswe-expressing BMMs. Taken together, these results demonstrate a potent inhibition on OC formation and activation in APPswe-expressing BMMs by iron chelation, and reveal a potential therapeutic value of CQ in treating AD-associated osteoporotic deficits.

Bacteria with a dual flagellar system, which consists of a polar flagellum (PF) and several lateral flagella (LF), have been identified in diverse environments. Nevertheless, whether and how these two flagellar systems interact with each other is largely unknown. In the present study, the relationship between the structural genes for the PF and LF of the deep-sea bacterium Shewanella piezotolerans WP3 was investigated by genetic, phenotypic and phylogenetic analyses. The mutation of PF genes induced the expression of LF genes and the production of LF in liquid medium, while the defective LF genes led to a decrease in PF gene transcription. However, the level of PF flagellin remained unchanged in LF gene mutants. Further investigation showed that the flgH2 gene (encoding LF L-ring protein) can compensate for mutations of the flgH1 gene (encoding PF L-ring protein), but this compensation does not occur between the flagellar hook-filament junction proteins (FlgL1, FlgL2). Swarming motility was shown to specifically require LF genes, and PF genes cannot substitute for the LF genes in the lateral flagella synthesis. Considering the importance of flagella-dependent motility for bacterial survival in the abyssal sediment, our study thus provided a better understanding of the adaptation strategy of benthic bacteria.