Cunninghamia lanceolata (Lamb.) Hook.) is one of the most important coniferous evergreen tree species in South China due to its desirable attributes of fast growth and production of strong and hardy wood. However, the yield of Chinese fir is often inhibited by aluminum (Al) toxicity in acidic soils of South China. Understanding the molecular mechanisms of Chinese fir root responses to Al toxicity might help to further increase its productivity. Here we used the Illumina Hiseq4000 platform to carry out transcriptome analysis of Chinese fir roots subjected to Al toxicity conditions. A total of 88.88 Gb of clean data was generated from 12 samples and assembled into 105,732 distinct unigenes. The average length and N50 length of these unigenes were 839 bp and 1411 bp, respectively. Among them, 58362 unigenes were annotated through searches of five public databases , which led to association of unigenes with 44 GO terms. Plus, 1615 transcription factors (TFs) were functionally classified. Then, differentially expressed genes | ≥ 1 and FDR ≤ 0.05) were identified in comparisons labelled TC1 (CK-72 h/CK-1 h) and TC2 (Al-72 h/Al-1 h). A large number of TC2 DEGs group were identified, with most being down-regulated under Al stress, while TC1 DEGs were primarily up-regulated. Combining GO, KEGG, and MapMan pathway analysis indicated that many DEGs are involved in primary metabolism, including cell wall metabolism and lipid metabolism, while other DEGs are associated with signaling pathways and secondary metabolism, including flavonoids and phenylpropanoids metabolism. Furthermore, TFs identified in TC1 and TC2 DEGs represented 21 and 40 transcription factor families, respectively. Among them, expression of bHLH, C2H2, ERF, bZIP, GRAS, and MYB TFs changed considerably under Al stress, which suggests that these TFs might play crucial roles in Chinese fir root responses to Al toxicity. These differentially expressed TFs might act in concert with flavonoid and phenylpropanoid pathway genes in fulfilling of key roles in Chinese fir roots responding to Al toxicity.Chinese fir ( Cunninghamia lanceolata (Lamb.) Hook.) is an important coniferous evergreen tree species that is widely cultivated in South China due to its desirable attributes of fast growth and production of strong and hardy wood. Based on planting area data in China’s ninth national forest inventory, Chinese fir is the second most dominant tree species in China, with cultivation occurring on approximately 11 million ha of plantations that account for about 6% of all forested land and 14.2% of total plantation area in China. This tree species, which has been planted in southeastern Asia for more than 1000 years [Chinese fir . Moreover, transcriptome sequencing represents an attractive alternative to whole genome sequencing because it only analyzes transcribed portions of the genome, while avoiding non-coding and repeat sequences that can make up much of the whole genome. When observed at specific developmental stages or under controlled physiological conditions, the transcriptome can provide a complete and quantitative picture of cell transcription at a given moment.3+ concentrations.In this study, to further understand the mechanisms of Al stress tolerance in Chinese fir at the molecular level, RNA transcriptome sequencing (RNA-seq) was conducted to identify Al-associated genes in the roots of Chinese fir using the Illumina Hiseq4000 platform. Based on bioinformatics analysis of this transcriptome, pathways involved in phenolic, phenylpropanoid and flavonoid metabolism were identified as important players in Chinese fir roots subjected to Al toxicity. Furthermore, numerous transcription factors (TFs), including bHLH, C2H2, ERF, bZIP, GRAS and MYB, were identified as potential regulators of the metabolic responses through changes in transcription observed in Chinese fir roots growing under Al stress. This transcriptome sequencing of Chinese fir roots in response to Al stress complements past physiology work and may help to elucidate previously unidentified Al resistance and tolerance mechanisms, all of which might be applied in future marker-based breeding efforts geared to genetically improving tree growth in the presence of high Al2 solution (pH = 4.0) and grown 7 days; and then the uniform seedlings were transplanted into 0.5 mmol/L CaCl2 with or without 1 mmol/L AlCl3 (pH = 4.0) solution, respectively. The hydroponics experiment was carried out in a temperature controlled room at 25 ± 3 °C, with the photoperiod set to 12 h light and 12 h dark cycles, constant relative humidity of 60%, 2000 lux of light intensity during the day, and continuously aerated growth media. Each treatment was observed in three biological replicates. Four cm root tips were sampled at 1 h and 72 h after initiation of Al treatments, which consisted of exposing roots to growth media (pH = 4.0) with (Al) or without  1 mmol/L AlCl3 added. Root samples weighing 0.1 g were individually inserted into 2.0 mL RNase/DNase-free tubes containing two sterile 5 mm steel balls, and immediately frozen in liquid nitrogen. All samples were stored at −80 °C prior to extracting RNA.One-year-old seedlings of Chinese fir were provided by the State Forestry Administration Engineering Research Center of Chinese Fir. Firstly, the seedlings were transplanted into 0.5 mmol/L CaClTotal RNA was isolated from root samples using the RNApre Pure Plant Kit  according to the manufacturer’s instructions. The quality and concentration of extracted RNA were determined on the Agilent 2100 Bio-analyzer  and NanoDrop 2000 spectrophotometer , respectively.TM Magnetic Kit . Enriched mRNA was then fragmented into short fragments using fragmentation buffer prior to being reverse transcribed into cDNA using random primers. Second-strand cDNA was synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then, cDNA fragments were purified with 1.8x Agencourt AMPure XP Beads, end repaired, lengthened with poly(A) tails, and ligated to Illumina sequencing adapters. Ligation products were size selected in agarose gel electrophoresis, PCR amplified, and sequenced using the Illumina HiSeqTM 4000 platform as operated by Gene Denovo Biotechnology Co. .After total RNA was extracted, mRNA was enriched using Oligo (dT) beads, while rRNA was removed using the Ribo-Zerohttp://www.ncbi.nlm.nih.gov/BLAST/) searching of four databases, including the NCBI non-redundant protein (Nr) database (http://www.ncbi.nlm.nih.gov), the Swiss-Prot protein database (http://www.expasy.ch/sprot), the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg), and the COG/KOG database (http://www.ncbi.nlm.nih.gov/COG). All BLASTx searches were conducted with an E-value threshold of 10−5.After removing adapters and low-quality reads, and filtering contaminant rRNA reads, clean reads were de novo assembled in the short read assembling program Trinity . Expresshttp://www.r-project.org/) was used, and raw counts were inputted in edgeR. Transcripts responding to Al stress with a fold change ≥ 2 and a false discovery rate (FDR) ≤ 0.05 were considered differentially expressed. All of the DEGs identified in groups TC1 (control roots at 72 h relative to control roots at 1 h) and TC2 (Al treated roots at 72 relative to Al treated roots at 1 h) were further annotated and analyzed using GO functional annotation, KEGG pathway analysis and pathway visualization in MapMan (https://mapman.gabipd.org/mapman-download).To identify differentially expressed genes across samples or groups, the edgeR package (http://planttfdb.cbi.pku.edu.cn/) sequences with potential TFs filtered to those matching database sequences with an E-value threshold of 10−5.The predicted plant protein coding sequences of assembled unigenes were aligned by BLASTp to Plant TFdb   following the manufacturer’s protocol. The primers used in qRT-PCR for validation of DEGs are listed in  under the following conditions: 94 °C for 30 s, and 40 cycles of 95 °C for 5 s, 58 °C for 15 s, and 72 °C for 10 s, followed by melting curve generation (60 °C to 95 °C), and a final step of 94 °C for 30 s. All treatments included three biological replicates, and the analysis of gene expression was calculated using 2-△△Cq values.Twelve RNA samples were reverse transcribed into cDNA using the reverse transcript reagent TransScriptTo understand potential Al tolerance mechanisms active in Chinese fir roots, 12 root samples from control or Al stress treatments and collected 1 h or 72 h after transferring to treatment media were sequenced on the Illumina HiSeqTM 4000 platform. After removing adapters, low-quality reads and contaminant rRNA reads, the remaining high-quality RNA-seq datasets contained 5.3–10.4 Gb of clean data from each root sample . The Q20Since no reference genome is available for Chinese fir, transcriptome de novo assembly was carried out in the short read assembly program Trinity . This yi-5 led to annotation of 58,362 unigenes, including 56,577 with matches in the NCBI non-redundant (Nr) protein database, 46,225 matching Swiss-Prot protein database entries, 39,868 with matches in the eukaryotic Orthologous Groups (KOG) database, and 26,914 unigenes aligning with sequences in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database  to plant proteins, while 47.40% showed strong homology  and the remaining 24.19% displayed very strong homology  to available plant sequences  was used to identify differentially expressed genes (DEGs) between control and Al treated roots. Transcripts with at least a 2-fold change in RPKM expression values and a false discovery rate (FDR ≤ 0.05) corrected t-test p-value corr in pairwise comparisons were considered significant DEGs. These DEGs were then subjected to enrichment analysis of GO categories and KEGG pathways. Only 3 up-regulated DEGs were identified in the T1 group (Al-1 h/ CK-1 h), while 140 up-regulated DEGs and 6244 down-regulated DEGs were identified in the T2 group (Al-72 h/ CK-72 h) (The edgeR package (CK-72 h) . In timeCK-72 h) . The numInterestingly, 785 DEGs were identified in both TC1 and TC2 comparisons , the majTo determine the main biological functions filled by TC1 and TC2 DEGs, these transcripts were mapped to GO terms in BLAST2GO analysis.In the TC1 group, a total of 4314 DEGs, including 3918 up-regulated and 396 down-regulated DEGs, were categorized to 35 GO terms . Among tIn the TC2 group, a total of 23,268 DEGs, including 3130 up-regulated and 20,138 down-regulated DEGs, were categorized to 41 GO terms. Among them, the top 3 biological process GO terms were metabolic process , cellular process , and single-organism process . The top 4 cellular component GO terms were cell , cell part , organelle , and macromolecular complex . The top 2 molecular function GO terms were catalytic activity , and binding  .To better understand the metabolic functions of DEGs identified in Chinese fir roots under Al stress, TC1 and TC2 group DEGs were queried against the KEGG database.TC1 DEGs mapped into 127 KEGG pathways . The topTC2 DEGs, most of which were down-regulated, mapped into 134 KEGG pathways . The KEGEnrichment analysis of KEGG terms associated with DEGs was conducted to further understand potential metabolic roles played by these gene products in Chinese fir roots subjected to Al stress treatments for 1 h and 72 h. TC1 DEGs were significantly enriched in Citrate cycle, Lysine biosynthesis, Oxidative phosphorylation, and specific amino acid biosynthesis and metabolism pathway, while TC2 DEGs were significantly enriched in different pathways, including Flavone and flavonol biosynthesis, Terpenoid backbone biosynthesis, Steroid biosynthesis, Phenylpropanoid biosynthesis, and Linoleic acid metabolism . These dThe software MapMan was used to display the global overview of DEGs in Chinese fir roots responding to Al stress. This visualization helps to understand the contrasting roles between primarily up-regulated TC1 DEGs A and preThis contrast of DEG response patterns was also observed between TC1 and TC2 comparisons in analysis of DEGS associated with secondary metabolism, including DEGs annotated to Phenylpropanoid and Phenolic biosynthesis, Flavonoid biosynthesis, Amino acid synthesis and degradation, Ammonia metabolism and Sulfur metabolism, and Nucleotide metabolism . Among TBased on the KEGG pathway enrichment results for TC1 and TC2 DEGs, TC2 DEGs were found to be significantly enriched in different pathways than TC1 DEGs, with TC2 DEGs being enriched in flavone and flavonol biosynthesis, terpenoid backbone biosynthesis, steroid biosynthesis, phenylpropanoid biosynthesis, and linoleic acid metabolism . TherefoPAL), 4-coumarate-CoA ligase (4CL), cinnamic acid 4-hydroxylase (C4H), cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), 4-coumarate 3-hydroxylase (C3H), and caffeic acid 3-O-methyltransferase (COMT). Similarly, several DEGs possibly involved in flavonoid biosynthesis were also observed, including DEGs annotated as chalcone synthase  (CHS), flavonol synthase (FLS), dihydroflavonol-4-reductase (DFR), anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR), and caffeoyl-CoA O-methyltransferase (CCOAOMT). Among them, 4CL, CHS, FLS, ANS, and CAD were all up-regulated in the TC1 comparison, while CCR was down-regulated  . In thisTo validate the accuracy of RNA-Seq, qRT-PCR was conducted to measure the expression of the 21 selected DEGs, including one Al-activated malate transporter 10-like protein (Unigene0062724), two auxin/Al responsive-like proteins (Unigene0067300 and Unigene0067301), two peroxidase-like proteins (Unigene0007466 and Unigene0075814), five phosphate transporters , and 10 DEGs involved in flavonoid and phenylpropanoid pathways  . The resIn this study, transcriptome analysis was employed to investigate mechanisms of Al tolerance in Chinese fir. As such, this study constitutes the first investigation of transcriptomic analysis of Chinese fir root responses to Al toxicity. After subjecting Chinese fir roots to 1 mM Al and control treatments for 1 h or 72 h, RNA-seq analysis returned a total of 105,732 unigenes, with an average length of 839 bp and an N50 length of 1411 bp. Compared with previous studies of transcriptomes obtained from Chinese fir vascular cambium  or immatQueries of four sequence databases with the 105732 assembled unigenes returned annotations for 58,362 of them, of which 56,577 were homologous with sequences in the Nr database. To maximize the possible functional predictions of transcripts isolated from Chinese fir roots responding to Al stress, further analysis was limited to these 56,577 unigenes aligning to Nr annotated sequences. Furthermore, DEG analysis was also limited to this set of 56,577 unigenes. Upon exposure to 1 mM Al for 1 h or 72 h, Chinese fir roots yielded only 3 DEGs in the T1 comparison group, but 6384, 4314 and 23,268 DEGs in T2, TC1 and TC2 comparison groups, respectively A. The faIn this study, we also identified hundreds of Al responsive transcription factors belonging to diverse families, including bHLH, C2H2, ERF, bZIP, and MYB members identified among TC2 DEGs, along with other, largely distinct sets of TFs identified among TC1 DEGs. These results indicate that the TFs found among TC2 DEGs might be involved in Chinese fir root responses to Al toxicity . AtALMT1 [All of the TF families illuminated in this study are known to participate in plant abiotic and biotic stress responses . In a reAtALMT1 . In addiSeveral groups have reported that ERF Tfs regulate many key functions, including developmental processes and responses to abiotic and biotic stresses ,44. In tLittle is known about the molecular mechanisms underlying Chinese fir responses to Al stress. This study is the first attempt to investigate changes of Chinese fir roots in response to Al toxicity at the transcriptional level, which also provided indications of which biochemical mechanisms underlie Al tolerance in Chinese fir. Several DEGs associated with phenylpropanoid and flavonoid metabolism were identified in this research as strongly down-regulated in Chinese fir roots responding to Al toxicity, suggesting that phenylpropanoid and flavonoid metabolism could participate in regulating responses to Al toxicity in Chinese fir. In addition, bHLH, C2H2, ERF, bZIP, MYB and the other TFs were differentially expressed among Al treatments over time, and, therefore, may play important roles in Chinese fir root responses to Al stress.Overall, this research represents a first step toward understanding the molecular mechanisms behind Chinese fir response to Al stress. The transcriptome data in this study will help to further develop novel Al-resistance strategies in Chinese fir and other tree species.


Signal transducer and activator of transcription 3 (STAT3), a previously accepted tumor-promoting protein in various malignancies, plays a key role in the process of cancer glycolysis. However, the role and potential mechanism of STAT3 in aerobic glycolysis and progression of oral squamous cell carcinoma (OSCC) has not been explored. In the present study, we demonstrated that STAT3 knockdown remarkably inhibited migration, invasion, expressions of epithelial-mesenchymal transition (EMT) markers, and aerobic glycolysis of OSCC cells by up-regulation of FoxO1. Consistently, the expression of nuclear Tyr705-phosphorylated STAT3, an active form of STAT3, was significantly elevated in OSCC tissues compared with adjacent normal tissues, and increased nuclear staining of Tyr705-phosphorylated STAT3 was associated with metastasis and shorter overall survival. Moreover, FoxO1, which was also mainly expressed in OSCC specimens, decreased in poorly-differentiated tissues compared with the relatively well-differentiated ones, and inversely correlated with the expression of nuclear Tyr705-phosphorylated STAT3 from patients with OSCC. Hence, our findings collectively characterized the contributing role of STAT3/FoxO1 in invasion and aerobic glycolysis of OSCC cells, which may lead to the worse clinical outcome. Oral squamous cell carcinoma (OSCC), which includes epithelial malignancies of the oral cavity and oropharynx, is one of the most prevalent problems in many parts of the world , 2. DespMetastasis, a dynamic process which is multifactorial and involves the detachment of cancer cells from the primary site, cell mobility, entry of lymphatic and vascular vessels, and recolonization conceptually, is a major cause of poor prognosis , 6. DuriSignal transducer and activator of transcription 3 (STAT3) is a member of the STAT protein family, and is known for its roles in promoting tumor cells proliferation, survival, tumor invasion, angiogenesis, and immunosuppression , 12. ActIn the present study, we characterized the function of STAT3 in OSCC cells, and carried out immunohistochemical analysis of pY-STAT3 protein (at the 705 residue) in OSCC tissues to determine its relationships with clinicopathological features and prognosis. The data revealed that STAT3 promoted migration, invasion, EMT and aerobic glycolysis of OSCC cells by inhibiting FoxO1 transcription. Then, we found that Tyr705-phosphorylated STAT3 was correlated with metastasis and poor prognosis of OSCC patients, and inversely correlated with FoxO1 expression, demonstrating the clinical significance of STAT3/FoxO1 during OSCC progression.The present study was approved by the Institutional Ethics Committee of the West China Medical Center, Sichuan University, China. Sixty eight patients diagnosed with OSCC in West China School of Stomatology, Sichuan University between 2000 and 2003 were recruited after giving informed consent. Primary resection and no prior treatment had been giving to patients in the present study. Besides, 10 cases of normal oral mucosae were included in this study. All specimens were fixed in 10% formaldehyde, then embedded in paraffin, and confirmed by hematoxylin-eosin staining.Monoclonal mouse anti-human antibodies to STAT3 (sc-293151), FoxO1 (H-128), β-catenin (sc-1496) were purchased from Santa Cruz Biotechnology Inc. . Monoclonal mouse anti-human antibodies to tyrosine phosphorylated STAT3  (9145S), E-cadherin (3195S) were obtained from Cell Signaling Technology Inc. . Monoclonal mouse anti-human antibodies to N-cadherin (ab98952), Vimentin (ab8978) were purchased from Abcam . Monoclonal mouse anti-human antibody to β-actin (ab008-100) was purchased from MultiSciences .All sections were deparaffinized in xylene and rehydrated through a gradient alcohol series. After endogenous peroxidase activity blocking by incubation with 3% hydrogen peroxide for 20 min, antigen retrieval by 0.01 M citrate buffer (pH = 6.0) in a microwave oven for 3 min, and non-specific binding blocking by incubation with 5% normal goat serum for 25 min, sections were incubated with primary pY-STAT3 antibody (1:100) at 4°C overnight in a moist chamber. The sections were then incubated with biotinylated anti-mouse IgG and streptavidin-biotin peroxidase. The primary antibody was detected using diaminobenzidine tetrahydrochloride (DAB), and sections were counterstained with hematoxylin. Six microscopic fields at 200× magnification per section were observed and at least 1,000 cells were counted independently by three of us. The percentage of cells showing positive staining was classified into lower and higher part due to the median for all cases. For representing negative expression, weakly positive expression, moderately positive expression and strongly positive expression, the percentage was categorized into (–), <5%; (+), 5–25%; (++), 25–50%; (+ + +), >50%. In negative controls, the primary antibody was replaced with isotype-specific immunoglobulin (IgG) to ensure specificity.In present study, human OSCC cell lines Cal-27, Tca8113, and SCC25 were obtained from State Key Laboratory of Oral Diseases, Sichuan University. Cells were cultured in Dulbecco's modified Eagle's medium  containing 10% FBS in a humidified atmosphere of 95% air and 5% CO2 at 37°C. For transfections, targeted sequences of shRNAs were as follows: STAT3: GCACAATCTACGAAGAATCAA (sh-1-S), GGTTGCTGGTCAAATTCCCTGAGTT (sh-2-S), and AAGGAGGAGGCATTCGGAAAGTATT (sh-3-S); FoxO1: GCATGTTCATTGAGCGCTTAG (sh-1-F), CAATTCGTCATAATCTGTCCCTACA (sh-2-F), and CAACCTTCTCTCATCACCAACATCA (sh-3-F); scramble: TTCTCCGAACGTGTCACGTAA. The shRNA lentiviral vector pHBLV-U6-mCherry-Puro and the human STAT3 gene stably overexpressing vector using vector pDC316-mCMV-mCherry-Puro was from Hibio , and all constructs were verified by sequencing. Cells were transfected with shRNAs in the presence of 5 μg/ml of polybrenen and incubated for 12 h. Then, 2 μg/ml puromycin was used to select transfected cells for 7 days. Transfected cells were collected and verified by qRT-PCR and Western blot, and thus for further experiments.−ΔΔCT method.Total RNA was extracted using Trizol reagent . The reverse transcription was performed with HiScript II Q RT SuperMix for qPCR . The real-time PCR reaction was carried out with ChamQ™ SYBR Color qPCR Master Mix (Vazyme) on CFX Connect Real-Time System  according to the protocol. Specific primers were listed in The whole cells extracts were lysed using 200 μl RIPA lysis buffer (Santa Cruz) for 30 min. Equal amounts of protein were then separated on SDS-PAGE, transferred to PVDF membranes , and immunoblotted with primary antibody. Antibodies against STAT3, pY-STAT3, FoxO1, E-cadherin, N-cadherin, β-catenin, Vimentin, and β-actin were used at a 1:1,000 dilution. Immunoblots were visualized after using goat anti-mouse antibody (MultiSciences) with the chemiluminescence (ECL) reagent  and ChemiDoc XRS+ System (Bio-Rad).Cells were seeded in a 6-well plate until forming confluent monolayers. Twelve hours prior to the experiment, cells were starved in serum-free DMEM medium. All cells were then scratched with a pipette tip to form a gap space. Finally, photomicrographs were taken immediately after the scratch, and after 24 h. The closed scratch areas were measured using ImageJ software. Each assay was carried out in triplicate.4 cells were seeded in the upper chamber coated with Matrigel . Complete medium was added to the lower chamber. After 24 h incubation and 0.1% crystal violet staining, cells that had invaded through the Matrigel were counted under a microscope in five pre-determined fields (×200). Experiments were carried out in triplicate.Cell invasion assay was performed using 24-well plates . Cells were starved in serum-free DMEM medium for 12 h. Then 5.0 × 10Cell apoptosis was measured using the Annexin V-FITC Apoptosis Detection Kit (Invitrogen). Cells were grown in 6-well plates and resuspended in 1× Binding Buffer after 48 h. Five microliter of Annexin FITC Conjugate and 10 μl of Propidium Iodide Solution were added into each cell suspension, separately. Then, we analyzed the stained cells using a flow cytometry .5 cells.The glucose consumption and lactate production levels were detected using Glucose  and Lactate (Abcam) assay kits according to the protocol. Results were normalized to 10t-test or one-way ANOVA. Experimental values were expressed as means ± SD. P < 0.05 was considered to be statistically significant.Chi-square was used to analyze associations between related factors. Kaplan-Meier method was used to estimate survival curves, and differences between two groups were evaluated using log-rank test. The correlation between pY-STAT3 and FoxO1 expression in OSCC specimens was assessed by 2-tailed Pearson's statistics. Means comparisons were analyzed using Student To investigate the role of STAT3 in migration, invasion and EMT of OSCC cells, we firstly assessed the relative STAT3 mRNA expression in five OSCC cell lines, including SCC25, Cal-27, HSC3, Tca8113, and UM1 . Cal-27 As EMT is a key process for cancer cell migration and invasion, defined by loss of epithelial cell polarity and reorganization of the cytoskeleton, and STAT3 is activated in tumors as the form of pY-STAT3 , we nextAerobic glycolysis, widely recognized as a central hallmark of human cancer, facilitates survival of aggressive cancer cells as well as their metastasis through metabolic pattern shifting . Using GAs a downstream protein of STAT3 signaling pathway, FoxO1 has been demonstrated to play important biological roles as tumor suppressor in regulating cancer cell growth, apoptosis, DNA damage repair, and so on , 20. EmeTo further evaluate the role of FoxO1, we applied shRNAs to knockdown FoxO1 expression in Cal-27 and Tca8113 cells, and using Western blot and qRT-PCR, we selected sh-1-FoxO1 which had the highest knockdown efficiency for further experiments . As showThen we investigated the effect of FoxO1 knockdown on aerobic glycolysis of OSCC cells. As shown in p = 0.041) (p = 0.048), poorer differentiation (p < 0.000), higher clinical stage (p = 0.002), and the presence of lymph node metastasis (p = 0.010), while no obvious relations were found between Tyr705-phosphorylated STAT3 and age, gender, drink, smoke, and recurrence in this study  . Furtheris study .n = 34) in tumor tissues had a significantly increased overall survival (p = 0.0003) , 48.5% (33/68), 8.8% (6/68), 2.9% (2/68), respectively ; Table 1 0.0003) . Furtherin vitro and in vivo (STAT3 has a contributing role in most human malignancies, where it not only promotes proliferation, survival, angiogenesis, and metastasis, but also interferes with apoptosis and immunosuppression , 23. It  in vivo .STAT3 exerts effects mainly by translocating into the nucleus and interacting with specific DNA binding elements, thus impacting transcription. The process of nuclear accumulation is strongly dependent on Tyr705 phosphorylation, while other reports also showed that STAT3 could enter the nucleus independent of its phosphorylation , 29. HowDespite a high genetic diversity, cancer cells still exhibit a common set of characteristics, continuous high glucose uptake and a higher rate of aerobic glycolysis than that in normal cells, to meet the rapid energy requirements for activities like EMT and progression , 32. In FoxO1, one of the members of the Forkhead box O-class proteins, was considered as a downstream factor of STAT3 . In a stAs a tumor-suppressor protein which has pleiotropic functions including inhibiting cell proliferation, inducing apoptosis, protecting cells from oxidative stress and DNA damage, and regulating immune response, FoxO1 is also a critical glucose metabolism-related protein , 37, 38.In conclusion, we clarified that STAT3 promoted migratory and invasive phenotypes, EMT, and aerobic glycolysis of OSCC cells by inhibiting FoxO1 transcription. Consistently, clinical studies have revealed that Tyr705-phosphorylated STAT3 was up-regulated in cancer tissues and correlated with metastasis and poor prognosis. These findings provided the evidence of STAT3/FoxO1 for the first time in modulating glycolysis to promote the malignant transformation of OSCC. Considering that tumors are generally typified by phenotypic diversity, it would be interesting in the future to unravel the metabolic heterogeneity and flexibility that will contribute to the utility of STAT3/FoxO1-targeted therapy for better OSCC management.The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.The studies involving human participants were reviewed and approved by the Institutional Ethics Committee of the West China Medical Center, Sichuan University, China. The patients/participants provided their written informed consent to participate in this study.MZ, MC, XY, YT, and XL designed the research study. MZ, MC, XY, and LL conducted the cell experiments. MZ, MC, XY, SW, HW, and Y-JT conducted the experiments on the human samples. MZ, MC, YT, and XL were involved in data analysis. MZ and MC were responsible for writing of manuscript. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bone remodeling represents a physiological phenomenon of continuous bone tissue renewal that requires fine orchestration of multiple cell types, which is critical for the understanding of bone disease but not yet clarified in precise detail. Exosomes, which are cell-secreted nanovesicles drawing increasing attention for their broad biosignaling functions, can shed new light on how multiple heterogeneous cells communicate for the purpose of bone remodeling. In the healthy bone, exosomes transmit signals favoring both bone synthesis and resorption, regulating the differentiation, recruitment, and activity of most cell types involved in bone remodeling and even assuming an active role in extracellular matrix mineralization. Additionally, in the ailing bone, they actively participate in pathogenic processes constituting also potential therapeutic agents and drug vectors. The present review summarizes the current knowledge on bone exosomes and bone remodeling in health and disease. Although grossly rigid and motionless, the bone is active, subject to an incessant, lifelong process of remodeling, i.e., renewal of aging, microdamaged tissue. This serves multiple purposes: bone maintenance or repair and adaptation to changing mechanical loads, as well as homeostasis of blood calcium and phosphorus levels. The bone remodeling seems to be triggered by osteocyte apoptosis, takes place simultaneously in multiple microscopic foci throughout the skeleton termed “bone remodeling compartments,” and requires the formation cell groups termed “bone multicellular units,” which are composed of chiefly three cell types: osteoclasts, osteoblasts, and osteocytes . The ostExosomes are cell-secreted, membrane-bound particles measuring 40-120 nm in diameter, which belong to “extracellular vesicles” along with microvesicles and apoptotic bodies . AlthougUnlike the most extensive part of the literature on exosomes, which deals predominantly with cancer, we intend to feature their function in physiology and nonneoplastic pathophysiology of the bone. Recent reviews have elucidated their role in primary bone cancers  or in bobone AND (exosome OR “extracellular vesicle”) AND (remodeling OR osteogenesis OR osteogenic OR “bone formation” OR osteoclast OR osteoblast OR fracture), which produced 148 results. When no reference to the skeletal system was made in the abstract, the article was excluded. The most common reason was the item bone appearing only as part of the term bone marrow. Articles referring to the teeth or—as explained above—cancer were also excluded. If the generic term “extracellular vesicles” instead of “exosomes” was used, the study was included only if isolation methods specific to exosomes had been applied. The paragraph dedicated to exosomes in the introduction was based on results from the relevant literature searches we conducted recently [We searched the computerized MEDLINE® database of the U.S. National Library of Medicine with the complex term recently , 21. Ultβ1, interleukin- (IL-) 8, hepatocyte growth factor (HGF), human T-cell factor 4 (TCF4), and multiple miRNAs) and promyogenic molecules  has been found in exosomes, mostly shed by MSCs [The orchestration of bone remodeling is an archetypal complex physiological process highly dependent on finely tuned intercellular communication, which is only incompletely explained on the basis of the cytokine and the hormone model. Since their emergence as intercellular messengers, exosomes have come to disclose further details of bone physiology. Skeletal health is, certainly, also contingent upon successful angiogenesis and myogenesis, which are physiological processes also involving exosomal signaling. In the context of skeletal physiology, an abundance of proangiogenic   by MSCs . The folACIN1, DDX6, DGKA, DKK2, Lsm2, RPS2, Xsox17, and the NF-κB-related ADAM17 and NF-κB1) are differentially expressed over time. These represent for the most part modulation of mRNA surveillance, Wnt signaling, and RNA transport pathways and also less prominent changes in numerous other intracellular pathways  [As bone marrow MSCs differentiate into osteoblasts, their exosomal cargo is modified accordingly , 24: somgnaling) .FGF2 [BMP2 [GDF10 [PHEX [ALPL [TGF-β1 [RUNX2 [(OSX) [(OCN) [OPN [VEGFA [COL1 [BMP9 [BMP6 [GAPDH [B2M [BSP [BMP2, OSX, SPP1, OSC, IBSP , and ALP ) can steer other MSCs towards the same phenotype [Exosomes secreted by MSCs , either FGF2 , BMP2 [2F2 [BMP2 , 27, GDF2 [GDF10 , PHEX [210 [PHEX , ALPL [2EX [ALPL –27, 29,  [TGF-β1 , RUNX2 [1 [RUNX2 –29, oste2 [(OSX) , 27, ost) [(OCN) –27, OPN CN) [OPN , 26, 28,N [VEGFA , COL1 [2FA [COL1 , 27–29, L1 [BMP9 , BMP6 [2P9 [BMP6 , GAPDH [6 [GAPDH , B2M [25B2M [BSP . The altB2M [BSP . Exosomehenotype , 31. MSChenotype .Specific exosomal contents suggested to be responsible for their proosteogenic properties include miRNAs   and, in Conditioning with MSC-derived exosomes induces tissue changes, such as increased matrix mineralization , 27 and in vitro [Although all current evidence overwhelmingly supports that MSC-derived exosomes favor osteogenesis, an animal model study focused on alveolar bone deterioration showed that exosomes from bone marrow MSCs could enhance the differentiation of osteoclast precursor cells in vitro .SOX9 upregulation and metalloproteinase MMP13 downregulation [Lastly, exosomes seem to be also implicated in cartilage development. Bone marrow MSCs with an induced chondrogenic phenotype produce exosomes with an altered miRNA cargo , which further favor chondrogenesis . The mosgulation .Ample data suggest that exosomes shed by osteoblasts occupy a role in bone physiology. They contain a wide range of proteins, the majority of which, unremarkably, participate in vesicle-related molecular processes, while others are more closely related to the functions of their parent cell  , 37. ForExosome-mediated mechanisms favoring bone synthesis involve miRNAs  and the On the contrary, TRAP , RANKL , 43, andAlthough traditionally perceived as relatively inert and trapped in bone lacunae, osteocytes remain active and, what is more, employ exosomes in order to perform not only paracrine but also systemic functions. In fact, they possess cytoplasmic projections reaching the vascular-facing surface of the osteoblast layer , which cAn exosome-related mechanism has been proposed to elucidate how mechanical stimuli activate bone synthesis, based on the observation that mechanically induced calcium oscillations bring about not only actomyosin contractions in the osteocyte's cytoskeleton but also increased exosome release . Other dRunx2 in osteoblasts and increase in calcium salt deposition [in vitro [Osteoclast-derived exosomes seem to have an overall proosteoblastic effect, witnessed as overexpression of position . A subseposition , 50, speposition , a phenoposition  , seposition  apparentposition , and canposition ; semaphoin vitro ; ephrin-in vitro , binds tExosomes shed by mature osteoclasts differ significantly from those shed by osteoclast precursors, in that only the former contain RANK  and inhiRunx2 [BMP2 [MMP3 and MMP1 [Monocyte-derived exosomes exert proosteogenic effects on MSCs. They induce the expression of Runx2 , BMP2 [5x2 [BMP2 , and matand MMP1  but not and MMP1  and boosand MMP1 . These cand MMP1 .Runx2 overexpression and an increase in ALK activity following MSC treatment with exosomes harvested from dendritic cells [Dendritic-cell-derived exosomes carry a variety of chemotactic agents and can recruit MSCs , a cruciic cells .PPARγ, CEBPα, and CEBPδ) and miRNAs  that target the transcripts of osteogenic genes  and cause downregulation of osteocalcin (OSC) and osteopontin, all indications of an antiosteogenic impact [in vitro and in vivo [β, mimicking the pathology of osteoarthritis, and include, in the case of chondrocytes, upregulation of MMP13 and ADAMTS5 and downregulation of COL2A1 and ACAN and in the case of endothelial cells increased migration and tube formation activity [APC gene and consequently activates the β-catenin pathway, a critical intracellular event for their differentiation into osteoblasts [Osteoblasts and mature fat lie in close proximity and share a common progenitor, the MSC, whose differentiation can be shifted either way under the influence of various factors , in partc impact . Preadipc impact . Exosome in vivo . Normal activity . Conversactivity . Nonseneactivity . Exosomeactivity . Myoblaseoblasts . Indireceoblasts .Matrix vesicles are small, osteoblast-secreted vesicular particles enveloped by a lipid bilayer membrane, critical for the formation of calcifying nodules in primary mineralization , 70. TheExosome-based intercellular communication is pivotal to the efficient orchestration of bone tissue repair. Mice with impaired exosome formation (CD9 knockout) also exhibit defective fracture repair in terms of chondrocyte and woven bone formation, vascularization, and healing time, all largely attributable to the lack of MSC-produced exosomes .β-TCP) [in vivo acceleration of bone regeneration [Exosomes, engineered or not, can be used to enhance bioactive materials for therapeutic purposes , 74. Theβ-TCP) , 75 or pβ-TCP)  and theiβ-TCP)  with mulβ-TCP) , 75, altβ-TCP) , and in neration .Runx2 and Dlx5 genes, has been shown to promote the production of RANK-containing exosomes from bone marrow MSCs. These exosomes can foster osteoblastic differentiation [In addition, exosomes illuminate parts of the mechanism of action of some osteoprotective drugs. Clodronate, a bisphosphonate, besides mediating proosteogenic molecular alterations involving ALP activity and ntiation . Of notentiation . Liragluntiation .In the following paragraphs, disease-specific data are presented regarding the role of exosomes in the development of new therapies for bone regeneration and the elucidation of pathophysiological aspects of bone disease.2O2 administration) [Bone health deterioration is a universal feature of old age with a multifactorial etiology, largely involving osteoporosis and osteoarthritis, which are discussed in more detail in Sections tration) . Interestration) . The agetration) .Osteoporosis is a very common condition characterized by the loss of bone mass and alteration of bone architecture with consequent increased bone fragility and fracture risk . Xie et ALP, OCN, and RUNX2 [A multitude of miRNAs are deregulated in osteoporosis in a cell-free, vesicle-free circulating form . Howevernd RUNX2 . Shen etnd RUNX2 , suggestnd RUNX2 .β-catenin signaling pathway [Regarding potential therapeutic applications, studies on animal models demonstrated recently that endothelial progenitor cell-derived exosomes block osteoclast induction and inhibit osteoporosis via miR-155  and prom pathway .ACAN (aggrecan) and COL2A1  [MMP13 and ADAMTS-5 [β simulating the inflammatory milieu of osteoarthritis [The exosome concept casts new light on the pathophysiology of osteoarthritis. Exosomes isolated from human synovial fluid carry miRNA profiles specific to osteoarthritis , 90 and,alpha 1) , 90, uprADAMTS-5 , and prorthritis .in vitro or animal models. In a way, MSC-derived exosomes seem to exert largely opposite effects compared to synovial fibroblast-derived ones. These effects include maintaining the synthesis-degradation equilibrium of chondrocyte ECM [β [TGF-β1, Survivin, Bcl-2, FGF-2, PCNA [SOX9 [β [α [β/SMAD pathway and/or COL2A1 and SOX9 upregulation, rather than produce foci of endochondral ossification [From a therapeutic point of view, exosomes produced by the bone marrow or embryonic MSCs can effect chondroprotective and anti-inflammatory changes in osteoarthritis according to data drawn from cyte ECM  or even cyte ECM  by increcyte ECM –94, cartcyte ECM , aggrecacyte ECM , 93, andcyte ECM , 94) ande ECM [β , 93, 94.e ECM [β , which i-2, PCNA , and SOXNA [SOX9  and the NA [SOX9 , as well[SOX9 [β , TNF-α [X9 [β [α , and iNOX9 [β [α . Protectfication . Howeverfication . Many offication . The ausfication .CALCR, CTSK, and RANK gene expression. The latter stimulatory effect is not reproduced on monocytes from other individuals [Extracellular vesicles are known to participate in the pathogenesis of rheumatoid arthritis as carriers of autoantigens, proinflammatory proteins and miRNAs, and matrix degradation enzymes . Exosomeividuals .Exosomes derived from bone marrow dendritic cells possess anti-inflammatory and immunosuppressive properties, potentially exploitable for the treatment of inflammatory arthritis, based on a murine model of collagen-induced arthritis . PeriartlprFas mutation, exosomes were used to supply Fas-impaired cells with Fas protein so as to reduce intracellular miR-29b levels; this prevented Dnmt1-mediated hypermethylation of Notch1 promoter and maintained the ability of MSCs to differentiate into osteoblasts. Thus, multiple parameters were improved, including trabecular bone volume, bone mineral density, and the bone volume/total volume ratio [Systemic lupus erythematosus (SLE), besides being a cause of inflammatory arthritis, is also characterized by impaired bone density and a higher fracture risk, a phenomenon with multifactorial, disease-specific, therapy-related, and comorbidity-related causes . In a mome ratio .Avascular bone necrosis, or osteonecrosis, is the result of diminished blood flow to the bone occurring in various diseases and is often a complication of medication, particularly of corticosteroids .Bcl-2 upregulation, Bax downregulation, and reduction of cytochrome c, cleaved caspase-9, and cleaved caspase-3 protein levels, as well as antiosteoclastogenic effects mediated by the modulation of the RANKL/OPG ratio [In an experimental model of hypoxia-induced bone damage, adipose tissue MSC-derived exosomes showed antiapoptotic effects attributable to the attenuation of reactive oxygen species (ROS) production, PG ratio .α (HIF-1α), which maintains function in normoxic conditions, from transfected bone marrow MSC. The exosome-bound mutant HIF-1α can increase trabecular reconstruction and microvascular density, indications of bone regeneration and angiogenesis [β/BMP signaling pathway, among which SOX9 upregulation might be the most vital [In the case of corticosteroid-induced osteonecrosis, three exosome-based strategies have yielded positive results in animal models. The first strategy involves the engineering of exosomes that bear a mutated variant of hypoxia-inducible factor 1ogenesis . The secogenesis . The thist vital .β, activin A, annexin II, adiponectin, Bruton's tyrosine kinase (BTK), stromal cell-derived factor-1α (SDF-1α), chemokines, and interleukins [RUNX2, OSX, and COL1A1 in osteoblasts [RUNX2 translation [in vivo [Osteolysis, a clinical hallmark of multiple myeloma, results from changes in several intra- and intercellular pathways such as RANK/RANKL/osteoprotegerin, Notch, Wnt, RUNX2, EphrinB2/EphB4, and the TNF pathway, involving signaling molecules such as Dickkopf-1 (DKK1), sclerostin, periostin, osteopontin, growth factor independence-1 (GFI1), bone morphogenetic proteins, TGF-rleukins . Amid thrleukins , 111 andrleukins . On the nslation . It is w[in vivo  and even[in vivo  and enhaThe cell-to-cell communication for the coordination of bone remodeling occurs in part through exosomal exchange. Steering the MSCs towards or away from osteoblastic differentiation is pivotal in this regard , 65, 68.In impaired bone repair, e.g., in fractures complicated by delayed healing or nonunion, the use of autologous bone graft is the “gold standard” practice , althougin vivo [Given the above disadvantages in current practice, exosomes constitute a potential therapeutic alternative. From a pharmacological point of view, the advantages of exosomes include the ability to carry at once multiple, both hydrophilic and lipophilic molecules, clinical safety , as wellin vivo  and in sin vivo . These bin vivo . In bonein vivo , 107, enin vivo , 75, vecin vivo , 106 or in vivo , or evenin vivo , 96. Whain vivo , 74, tenin vivo  or peripCurrent knowledge on exosomes in bone remodeling is based on data from almost exclusively preclinical experiments; clinical trials involving exosomes as diagnostic or therapeutic agents are practically limited to cancer and none has focused on bone repair . Despite


The endoplasmic reticulum (ER) has diverse functions, and especially misfolded protein modification is in the focus of this review paper. With a highly regulatory mechanism, called unfolded protein response (UPR), it protects cells from the accumulation of misfolded proteins. Nevertheless, not only does UPR modify improper proteins, but it also degrades proteins that are unable to recover. Three pathways of UPR, namely PERK, IRE-1, and ATF6, have a significant role in regulating stress-induced physiological responses in cells. The dysregulated UPR may be involved in diseases, such as atherosclerosis, heart diseases, amyotrophic lateral sclerosis (ALS), and cancer. Here, we discuss the relation between UPR and cancer, considering several aspects including survival, dormancy, immunosuppression, angiogenesis, and metastasis of cancer cells. Although several moderate adversities can subject cancer cells to a hostile environment, UPR can ensure their survival. Excessive unfavorable conditions, such as overloading with misfolded proteins and nutrient deprivation, tend to trigger cancer cell death signaling. Regarding dormancy and immunosuppression, cancer cells can survive chemotherapies and acquire drug resistance through dormancy and immunosuppression. Cancer cells can also regulate the downstream of UPR to modulate angiogenesis and promote metastasis. In the end, regulating UPR through different molecular mechanisms may provide promising anticancer treatment options by suppressing cancer proliferation and progression. It has diverse functions, such as in the synthesis of fats, metabolism of glucose, detoxification, Cafication ,2. ER isfication . ER-trigfication . Besidesfication , which cThe function of UPR is to maintain homeostasis by strengthening the ability of protein folding in the ER via chaperones. Furthermore, it can also promote ERAD (ER-associated degradation) pathways or chaperones to cope with abnormal proteins and even suppress other mRNA translation to attenuate more protein synthesis . ERAD alUPR protects cells in two respects. In general, it plays a crucial role in maintaining cell survival by ensuring the quality of secretory proteins. However, overloaded stressors, such as persistent hypoxia and nutrient deprivation of cells or even increased the accumulation of reactive oxygen species, may activate cell death signals, not cell adaptation . NormallIn mammalian cells, there are three integral ER membrane proteins: (1) IRE-1-XBP1; (2) PERK-ATF4-CHOP; and (3) ATF6  Figure . These pHowever, IRE-1β is merely expressed in the epithelial cells of the gastrointestinal tract. IRE-1α has two enzymatic domains: one is the Ser/Thr kinase domain, and the other is the site-specific endoribonuclease (RNase), which autoregulates its mRNA for UPR ,16. WithThe IRE-1 pathway also suppresses the expression of other mRNAs to reduce excessive unfolded proteins. It can also lead to microRNA degradation by RIDD (regulating an IRE1-dependent decay), including miR-17, miR-34a, miR96, and miR-125b . PERK seFurthermore, it also activates CHOP to trigger cell death signals. CHOP, also known as DNA damage-inducible transcript 3, is a pro-apoptotic transcription factor . During ATF6 as the other transmembrane protein is a basic leucine zipper transcription factor. Once Grp78/Bip dissociates, it gets transported to the Golgi body and is activated by RIP (Regulated intramembrane proteolysis), including S1P (Site-1 protease) and S2P (Site-2 protease), to form an active transcription factor that translocates to the nucleus. The active ATF6 induces the expression of CHOP, chaperones, and ERAD components . FurtherIt is significantly revealed that ER stress and UPR activation play vital roles in the development of cancer . UPR sigWhen using UPR inhibitors, such as 4-PBA (4-Phenylbutyric acid) or TUD-CA (Tauroursodeoxycholic acid), tumor progression and metastasis are delayed . Therefo−/− under hypoxia, have lower viability and lost their ability of angiogenesis [Besides, hypoxia and nutrient deprivation can induce breast cancer cell line growth viability through XBP1 splicing . Anotherogenesis .PERK mRNA than wild-type cells, which leads to a decreased level of phosphorylation of eIF2α [CHOP knockout leads to lung lesion in an immunocompetent K-RasG12V mutation-driven murine model of lung cancer [When the PERK gene in mouse embryonic stem cells is mutated, the cells with homozygous mutation express 10-fold less of eIF2α . Furtherof eIF2α . It is rg cancer . Furtherg cancer . On the g cancer   and subsequently induces Bim and inhibits Bcl-2, Bcl-XL and Mcl-1 (anti-apoptotic proteins) ,40. FurtCancer dormancy can roughly refer to two different types: One is tumor mass dormancy, and the other is cellular dormancy . Tumor mHowever, the dormant cancer cells are difficult to be detected and to be treated. Several studies supported that DCTs (dissemination tumor cells) enter the dormant state by establishing unstable interactions with the ECM (extracellular matrix) . There aHowever, in former studies, it is manifested that the wrestling between MAPK p38 and ERK (Extracellular regulated protein kinases) plays a crucial role in cell dormancy . When tuRegarding tumor cell dormancy, UPR might play a crucial role. As mentioned above, MAPK p38 not only inhibits ERK but also induces the downstream of UPR, including PERK and IRE-1α. PERK can trigger Nrf2 and phosphorylated-elF2α, which contribute to cell cycle G0–G1 arrest. Besides, IRE-1α can induce XBP1s and subsequently stimulate Grp78/Bip for cell survival. The combination of cell cycle G0–G1 arrest and cell survival leads to tumor cell dormancy and drug resistance .It is widely considered that dormant cancer cells are resistant to chemotherapies owing to their proliferative suppression. Furthermore, it was shown that HEp3, head, and neck squamous carcinoma cells up-regulate all three transmembrane proteins of UPR: ATF-6α, IRE-1α, and PERK . AdditioNevertheless, ATF6α can play another role that induces HEp3 cell survival via the activation of mTOR  signaling, which is regulated by the upregulation of the small GTPase Rheb (Ras homolog enriched in brain). However, the overexpression of Rheb is observed in multiple carcinomas. Thus, inhibiting Rheb and suppressing mTOR signaling provide promising treatments for cancer cells ,53. FurtAs described above, IRE-1α-XBP1 signaling plays a significant role in cancer cell survival. However, the mechanism by which IRE-1α-XBP1 signaling inhibits immune response is not completely understood . In the However, immunosuppression may provide an opportunity for the formation of cancer cells. Furthermore, cancer cells sometimes protect themselves from elimination through UPR-driven immunosuppression. Aggressive cancers can not only recruit immune cells, such as dendritic cells and Tc lymphocytes but also eliminate their original functions, such as cytotoxic effects and antigen presentation . TherefoMoreover, active XBP1 not only up-regulates the XBP1-associated genes involved in ER stress but also promotes triglyceride biosynthesis, which leads to the abnormal accumulation of lipids, such as phosphatidylcholine, the primary phospholipid of the ER membrane ,59. AnotUnder stressful conditions, especially hypoxic microenvironments, cancer cells are inclined to take several means to maintain energy balance, including HIF (hypoxia-inducible factor), UPR and macroautophagy. However, angiogenesis is a significant mechanism for tumor cells to maintain their metabolic balance. There are several known factors regulating angiogenesis, including FGF (fibroblast growth factor), PDGF (platelet-derived growth factor), IL-8, and VEGF  .When tumor cells are under hypoxic conditions, it is necessary for them to vascularize to survive and proliferate. Under hypoxic conditions, tumor cells induce HIFs (the activated form of HIF) to promote synthesis and expression of pro-angiogenic factors, such as VEGF and EPO (erythropoietin) . Recent Insufficient vascular supply causes hypoxia, nutrient deprivation, and consequently a lowered ATP production. These factors are also associated with misfolded proteins, and ER stress and UPR will be triggered. Interestingly, UPR can induce both pro-angiogenesis and anti-angiogenesis.As for pro-angiogenesis, the transcription factors from the arms of UPR (XBP1s and ATF4) can bind with the VEGF promoter and activate it . Accordi−/− mice, which shows ischemia-induced neovascularization.Furthermore, activated Nrf2 (NF-E2-related nuclear factors) can cooperate with UPR to trigger a pro-angiogenic response under oxidative stress. Then, HIF-α will be stabilized through UPR signaling, which promotes VEGF expression to make tumor cells deal with hypoxic conditions. By contrast, as for anti-angiogenesis, there are several known mechanisms mentioned below: The activation of TRAF2-JNK by IRE-1α induces cytochrome c-mediated apoptotic pathways through phosphorylating specific Bcl-2 family proteins, such as Bim . BesidesEvidence also shows that a reduction in apoptosis and increased expression of eNOS were observed in the CHOP10 (C/EBP homologous protein-10) knockout mice model compared with wild-type mice . Upon ERMetastasis is cancer cells proliferating from the original colony to a distant site . It resuWe mentioned that there are three integral ER membrane proteins in the UPR, including IRE-1, PERK, and ATF6. However, PERK is most significant for stimulating metastasis because it can maintain endothelial cell survival and promote angiogenesis by VEGF. Epithelial-to-mesenchymal transition (EMT) is a cell transdifferentiation program usually used by cancer cells to migrate and invade. Cancer cells that have undergone an EMT tend to employ the PERK-ATF4 branch of UPR for metastasis . FurtherWe summarize the anti-cancer drugs targeting on UPR proteins and their mechanisms . The catChaperone Grp78/Bip plays a significant role in cancer cell survival, metastasis, and drug resistance. An example is provided by cisplatin, an anti-cancer agent that interferes with DNA replication to suppress tumor cell proliferation . HoweverAnother example is Epigallocatechin-3-gallate (EGCG), which binds with the ATP-binding domain of Grp78 in glioma cells, strengthening the sensitivity of cancer cells to temozolomide or etoposide . AccumulFocusing on Grp78-associated angiogenesis or tumor resistance, Grp78 is highly expressed on the surface of proliferative endothelial cells, with the assistance of T-cadherin, and thus endothelial cells can maintain survival . BesidesAnother promising therapy targeting Grp78/Bip is PAT-SM6, a monoclonal IgM antibody . It recoATF6α, which upregulates the expression of foldases and chaperones, is activated by proteolysis and translocates from the ER to the Golgi body. Besides, the activation of ATF6α is crucial for cancer cell survival . ATF6α c®). However, 16F16, also a PDI inhibitor, triggers a molecular mechanism similar to that of PACMA31, as well as reducing the chemo-resistance and promoting the sensitivity of cancer cells to Imatinib [Moreover, indirect inhibitors that mainly target ATF6α-associated enzymes, such as PDI, S1P, and S2P. 4-(2-aminoethyl) benzene-sulfonyl fluoride (AEBSP), a serine protease inhibitor, inhibits the proteases S1P and S2P in the Golgi body, which leads to the blockade of ATF6α downstream signaling pathway . AnotherImatinib .As mentioned in the Introduction, IRE-1α downstream signaling can either promote adaptive signals via XBP1 mRNA splicing or trigger cell death signals via RIDD. Therefore, promising therapies targeting IRE-1 can be mainly classified into two categories. One stimulates RIDD for apoptosis, and the other focuses on blocking XBP1 mRNA splicing. Moreover, the therapies related to XBP1 mRNA splicing can fall into two classes, including ATP binding kinase domain inhibitors and IRE-1 RNase inhibitors . ATP binIn contrast, IRE-1 endoribonuclease inhibitors, such as MKC-3946 and 8-formyl-7-hydroxy-4-methylcoumarin (4μ8C), protect the formation of XBP1s (active XBP1) through interacting with the catalytic core of the RNase domain of IRE-1α ,97. QuerAs mentioned above, PERK can not only activate its downstream transcription factor, ATF4, to increase cancer cell survival but can also trigger Nrf2, also a transcription factor, to block expression of CHOP, which induces cell death signaling. Thus, GSK2656157, a small molecule PERK inhibitor, possesses ATP-competitive activity to interact with elF2α and inactivates it (phosphorylated elF2α) . FurtherTwo synthetic compounds, Salubrinal and Guanabenz, target the GADD34/PP1c complex and protect eIF2α from dephosphorylation, which stimulates eIF2α phosphorylation, induces expression of CHOP and triggers apoptosis ,100. EviERAD, a member of the proteasome, is accessible to eliminate the accumulation of misfolded proteins. Bortezomib, an ERAD inhibitor, is widely used in multiple myeloma and mantle cell lymphoma for retarding the proliferation of cancer cells . It inhiThe chaperone inhibitor MAL3-101 interferes with the ATPase activity of the HSP70 protein and brings about the accumulation of misfolded protein and apoptosis . For exaER stress plays a significant role in both normal and tumor cells. As for cancer development, UPR is a double-edged sword, as it can not only suppress tumor cell proliferation but also induce tumor cell survival, progression, and metastasis under unfavorable conditions, such as hypemic hypoxia, accumulation of reactive oxygen species, and ATP as well as nutrient deprivation. Tumor cells take advantages of the UPR pathway for their survival. For example, they decrease mRNA stabilities of CHOP via PERK and its downstream signals, including ATF4 and Nrf2 for survival. Besides, they can upregulate the mTOR signaling pathway through ATF6, which increases survival. They also trigger cell cycle G0–G1 arrest by IRE-1α and PERK signaling to escape from chemotherapies and adversities.Moreover, they make use of IRE-1α-XBP1 downstream signaling to suppress the function of dendritic cells and the maturation of the immune system. As for metastasis, tumor cells transfer themselves to distant sites via PERK-ATF4. After metastasis, tumor cells overcome hypoxia and nutrient deprivation through IRE-1α-induced and PERK-driven angiogenesis. With increasing knowledge of the relation between UPR and tumor cells, several anti-cancer therapies will become feasible. This holds particularly for drugs targeting the UPR signaling pathway that combined with other already available anti-cancer therapies can enhance anticancer therapeutic effects while decreasing side effects.


Heterocysts arise from photosynthetic active vegetative cells by multiple morphological and physiological rearrangements including the absence of O2 evolution and CO2 fixation. The key function of this cell type is carried out by the metalloprotein complex known as nitrogenase. Additionally, many other important processes in heterocysts also depend on metalloproteins. This leads to a high metal demand exceeding the one of other bacteria in content and concentration during heterocyst development and in mature heterocysts. This review provides an overview on the current knowledge of the transition metals and metalloproteins required by heterocysts in heterocyst-forming cyanobacteria. It discusses the molecular, physiological, and physicochemical properties of metalloproteins involved in N2 fixation, H2 metabolism, electron transport chains, oxidative stress management, storage, energy metabolism, and metabolic networks in the diazotrophic filament. This provides a detailed and comprehensive picture on the heterocyst demands for Fe, Cu, Mo, Ni, Mn, V, and Zn as cofactors for metalloproteins and highlights the importance of such metalloproteins for the biology of cyanobacterial heterocysts.Cyanobacteria are photoautotrophic microorganisms present in almost all ecologically niches on Earth. They exist as single-cell or filamentous forms and the latter often contain specialized cells for N Proteins are involved in a broad spectrum of biological functions and catalyze a wide range of chemical reactions in cells . Howevermetalloprotein is used to designate transient or permanent metal‒protein complexes that contain one or more metal atoms in their structure, either directly attached to the polypeptide chain or inserted into a non-protein organic molecule, which is bound subsequently to the polypeptide chain . Th. Thd-bloCyanobacteria are one of the largest, most ecologically diverse, and important groups of bacteria on Earth  and the 2 via the reductive pentose phosphate pathway, yet sugars can support chemoheterotrophic and photoheterotrophic growth in some species  hydrogenase module that is formed by HoxYH, and a HoxEFU diaphorase moiety which serves as an enzymatic redox subcomplex . HoxEFU  cluster ,208. In chondria ,208,209.chondria . In the catalytic moiety, HoxH is the α subunit involved in the reaction catalysis and harbors the [Ni–Fe] active site. HoxY is the β subunit that coordinates a proximal [4Fe–4S] cluster to the [Ni–Fe] active site in the catalytic moiety and facilitates electron transfer to and from the hydrogenase active site ,211,212.+ and FMN (flavin mononucleotide) binding motifs  cluster and one [4Fe–4S] cluster and contains NAD(P)H/NAD(P)g motifs ,209. Eacg motifs ,211,212.g motifs ,211. Eacg motifs ,211,212.g motifs ,213,214.g motifs ,209. Thi2 gas from NAD(P)H or NAD(P)+ as the electron donor or acceptor as shown in the following reaction:The bidirectional hydrogenase is a soluble enzyme located in the cytoplasm that lacks membrane spanning domains ,208, but+/NAD(P)H-dependent enzyme, recent results show that it is a Fd-dependent enzyme in Synechocystis sp. PCC 6803  cluster  clusters FX, FA, and FB [B to Fd and ultimately transferred to the enzyme FNR. Whereas the [4Fe–4S] cluster FX is localized between subunits PsaA and PsaB and coordinated to two Cys residues from each subunit [A and FB and exhibits a pseudo-two-fold symmetry similar to that of bacterial 2[4Fe–4S] ferredoxins (B) . H. H0 (Chlxins (B) . Thus, t FB → Fd   protein (PetC)  ; Table 3 ligands ,246,247, ligands ,248. Hemproteins ,244,249. complex . Althoug→ x → PQ ,250.b6f, the photosynthetic electron transport chain in heterocysts also requires metal-dependent small, mobile electron carriers to operate, such as PC, cytochrome c6, and Fd  alternative oxidases, and (iv) several components that are shared with the photosynthetic electron transport chain, such as the PQ/PQH2 pools, the cytochrome b6f, and the soluble electron carriers PC and cytochrome c6  a type-I NAD(P)H dehydrogenase (NDH-1) in the thylakoid membranes, (ii) terminal oxidases, such as a cytochrome hrome c6  85]. Ho. Hoc oxib6f and the luminal electron carriers PC or cytochrome c6 + or Fd . C. Cflv geeduce O2 ,338.FDP structures display a head-to-tail arrangement where the Fe–Fe site of one monomer and the FMN of the other monomer closely contact each other, ensuring a fast electron transfer between the two redox centers ,340,341.2 into H2O without the formation of reactive oxygen species  cluster, which in turn is reduced by NAD(P)H, but a concerted electron transfer process to the catalytic site favors the two-electron reduction reaction of H2O2 over a one-electron, Fenton-type redox reaction . T. T8-barrn sphere M, right molecule M, left a , which can provide the Krebs cycle with oxaloacetate through the enzyme PEP carboxylase (PEPC) present in heterocysts ,421,435.4+  and an enzyme similar to PEPSs  , w, wAnabaeate 2-OG  [442,445ve cells  [442,4452 fixation and, thus, they require a massive reprogramming of the cellular ultrastructure and proteome to efficiently carry out this function. This also includes a further increase of transition metal requirements involved as cofactors in a wide range of metalloproteins, since the chemical composition of amino acid residues does not provide the capability to perform all chemical reactions required to maintain key cellular functions in heterocysts (2 [2-sensitive process of N2 fixation as a consequence of the cyanobacterial success in O2 photosynthesis [2 and radical oxygen species (2-fixation apparatus (d-block metals, namely Fe, Cu, Mo, Ni, Mn, V, and Zn, are found as central components of metalloproteins required for heterocyst function. Thus, the extreme metal requirements of heterocysts far exceed those of vegetative cells and most groups of microorganisms, making heterocysts highly metal-dependent and prolific cells in the management and use of transition metals. While the physiological requirements on metals for the heterocyst function has been explored under limiting and toxic metal levels, future research on the protein composition and the biochemical pathways of this special bacterial cell might uncover not only additional metal-containing proteins but also novel metalloproteins dependent on other d-block metals.Photoautotrophic bacteria exhibit higher transition metal demands than other microorganisms. These raised demands are partially attributed to the photosynthetic apparatus. However, filamentous heterocyst-forming cyanobacteria are able to transform vegetative cells into heterocysts . These cerocysts . It has cysts (2 . Moreoveynthesis ,467. Thi species  that, otpparatus . The wea


Raoultella planticola is a gram-negative, aerobic, nonmotile mostly found in environments with high prevalence in soil and water. This organism is a very rare human pathogen as only 29 cases of Raoultella planticola–related infections have been reported until 2017, with only 7 cases in the United States. Only 3 cases of urinary tract infection secondary to R planticola have been reported, 1 in a pediatric patient and 2 in adults. In this article, we present a case of R planticola urinary tract infection in a 65-year-old male with immunoglobulin A nephropathy. On investigation, the patient was found to be septic and empirical antibiotic was started for gram-negative coverage. The patient showed remarkable improvement and discharged on oral antibiotic for 7 days. R planticola rarely cause infection in humans, with overall good prognosis. Raoultella planticola is a gram-negative, aerobic, nonmotile mostly found in environments with high prevalence in soil and water. It was first described in the 1980s as Klebsiella planticola and Klebsiella trevisanii.1 It was reclassified into a new genus in 2001 as Raoultella planticola.2 This organism is a very rare human pathogen as only 29 cases of R planticola–related infections have been reported until 2017, with only 7 cases in the United States. Only 3 cases of urinary tract infection secondary to R planticola have been reported, 1 in a pediatric patient and 2 in adults.4 In this article, we present a case of R planticola urinary tract infection in 65-year-old male with immunoglobulin A nephropathy.R planticola sensitive to all main-line antibiotics, including ceftriaxone, ciprofloxacin, nitrofurantoin, cefazolin, gentamicin, and trimethoprim-sulfamethoxazole. The patient was subsequently discharged on the fourth day on ciprofloxacin renal dose 250 mg orally every 12 hours for 4 more days to complete 7 days of treatment. The patient was seen in clinic after 2 weeks with complete resolution of symptoms.A 65-year-old male with past medical history of hypertension, diabetes mellitus type 2, hyperlipidemia, and end-stage renal diseases secondary to biopsy-proven immunoglobulin A nephropathy came to our hospital with dysuria, dark urine, and fever going on for the last 3 days. His vitals showed a temperature of 39.3°C, blood pressure of 164/89 mm Hg, pulse of 99 beats per minute, and respiratory rate of 20 breaths per minute. The examination was unremarkable except for mild lower abdominal tenderness. Initial laboratory workup was remarkable for white blood cells (WBCs) of 10 900/µL (3100-8500/µL), neutrophil percentage of 87% (25% to 62%), absolute neutrophils of 9500/µL (1700-6300/µL), platelets of 174 000/µL (140 00-440 000/µL), sodium of 135 mmol/L (136-145 mmol/L), lactic acid of 1.1 mmol/L (0.5-2.2 mmol/L), blood urea nitrogen of 47 mg/dL (9-21 mg/dL), and creatinine of 4.5 mg/dL (0.6-1.1 mg/dL). Urine analysis showed 1+ bacteria, large leukocytes, WBC >50/high-power field (HPF), and squamous epithelial cells 0 to 5/HPF. Keeping in mind urosepsis, blood cultures were drawn from 2 peripheral sites along with urine culture, and he was started on intravenous ceftriaxone and intravenous fluid as per sepsis protocol. Nephrology was consulted and the patient got dialysis the next day secondary to end-stage renal diseases. The patient started showing improvement. On day 2, the patient was afebrile, and WBC started trending down along with resolution of dysuria. Blood cultures did not grow anything, but 3 days later urine culture grew Raoultella planticola is an encapsulated, nonmotile, aerobic, gram-negative rod predominantly found in water and soil. Although R planticola is mainly an aquatic and soil bacterium, it has been clinically isolated from human sputum, stool, wound, and urine. To date, 29 cases of human infection with R planticola has been reported with only 3 urinary tract infections.4R planticola is difficult to isolate and to identify, as it can easily be confused with other genera, especially klebsiella.5R planticola rarely cause infection in healthy individuals. Malignancy, transplant recipients, dialysis-dependent patients, diabetes mellitus, and immunocompromised state also put them at high risk.69Raoultella planticola has been associated with cases of pneumonia, urinary tract infection, cholangitis, conjunctivitis, peritonitis, necrotizing fasciitis, bacteremia, cellulitis, and soft tissue infection. On literature review of 29 reported cases, 3 patients died, 22 patients had full recovery, and 4 patients had an unknown outcome. Mortality is high in immunocompromised patients.3 The First reported human infection due to Klebsiella trevisanii (later reclassified as R planticola) was in 1986, which included bacteremia in a 69-year-old patient.10 In 2014, a case of R Planticola bacteremia in a 56-year-old female was reported following consumption of seafood salad containing squid and octopus.5R planticola has the ability to change histidine to histamine leading to scombroid poisoning when poorly cooked sea food eaten in a large quality.5 Variety of human organ systems had been affected, with no predilection for a particular organ system.As there is limited data regarding this pathogen, especially in humans, the mechanism of its pathogenesis remains unclear. Immunocompromised state, proton pump inhibitor use, and chemotherapy increase the chances of infection. R planticola but is also highly sensitive in differentiating between Raoultella and Klebsiella.Culture along with VITEK-2 (bioMerieux) automated bacterial identification system not only help in identification of R planticola urinary tract is mainly empiric antibiotic for gram-negative coverage and should be narrowed accordingly when further microbiologic information is available. Usually R planticola is sensitive to all main-line gram-negative covering antibiotics; however, multidrug-resistant strains of R planticola have been isolated from both patients and the environment.1113 Our patient did not show resistance to any antibiotic.Treatment of R planticola has been on the rise recently. The organism has been involved in infecting multiple organ systems. Individuals who are immunocompromised, have multiple comorbid diseases, and dialysis patients are at risk. Avoidance of contaminated water is the key to prevention. R planticola infection has good prognosis overall. It is prudent to be aware of this human pathogen and early initiation of antibiotics is the main treatment. As with any other human pathogen, we recommend closely monitoring its pattern of resistance.In conclusion, infection due to


CCAR2 (cell cycle and apoptosis regulator 2) controls a variety of cellular functions; however, its main function is to regulate cell survival and cell death in response to genotoxic and metabolic stresses. Recently, we reported that CCAR2 protects cells from apoptosis following mitochondrial stress, possibly by co-operating with Hsp60. However, it is not clear how CCAR2 and Hsp60 control cell survival and death. Here, we found that depleting CCAR2 and Hsp60 downregulated expression of survivin, a member of the inhibitor of apoptosis (IAP) family. Survivin expression in neuroblastoma tissues and human cancer cell lines correlated positively with expression of CCAR2 and Hsp60. Furthermore, high expression of CCAR2, Hsp60, and survivin was associated with poor survival of neuroblastoma patients. In summary, both CCAR2 and Hsp60 are required for expression of survivin, and both promote cancer cell survival, at least in part, by maintaining survivin expression. Therefore, CCAR2, Hsp60, and survivin are candidate tumor biomarkers and prognostic markers in neuroblastomas. Cell cycle and apoptosis regulator 2 (CCAR2), formerly known as deleted in breast cancer 1 (DBC1), is an emerging key regulator of multiple cellular functions. CCAR2 mediates positive and negative regulation of several transcription factors, including ERα/β, GR, TR, AR, Rev-Erbα, and BRCA1, and affects transcription, metabolism, circadian cycles, and aging . CCAR2 aRecently, we showed that CCAR2 interacts with Hsp60 in mitochondria . The resHsp60 mediates pro-survival and pro-apoptotic effects via numerous Hsp60-interacting proteins, including survivin , pro-casSurvivin, one of Hsp60-interacting proteins, is a member of the inhibitor of apoptosis (IAP) family . Hsp60 hNeuroblastoma is a common pediatric tumor that is usually diagnosed after 18 months of age, at which time it has usually metastasized. Neuroblastoma is genetically heterogeneous . In partA growing body of evidence suggests that CCAR2 and Hsp60 function as pro-survival factors; therefore, it is important to investigate how CCAR2 and Hsp60 react to cellular stress. Hsp60 interacts with several apoptosis regulators in mitochondria to inhibit apoptosis; these regulators include survivin, an anti-apoptotic protein . RecentlA previous report shows that Hsp60 is required for expression of mitochondrial survivin and inhibition of apoptosis . The resThe next question we asked was how do CCAR2 and Hsp60 control survivin expression? First, to investigate whether depleting CCAR2 and Hsp60 induces proteasome-mediated degradation of survivin, we examined survivin levels in siRNA-transfected SH-SY5Y cells treated with MG132 prior to cell lysis A. Ghosh The above data demonstrate that CCAR2 and Hsp60 are required for expression of survivin; therefore, it is of interest to examine the correlation between expression of CCAR2, Hsp60, and survivin in vivo. First, we examined expression of mRNAs in 675 commonly used human cancer cell lines (data obtained from the ArrayExpress dataset E-MTAB-2706). Expression of CCAR2 and Hsp60 showed a weak positive relationship (0.2 < r < 0.4) with that of survivin A. Next, The above data suggest that both CCAR2 and Hsp60 promote tumor cell survival by upregulating expression of survivin. This implies that tumors expressing high levels of CCAR2 and Hsp60 would be more aggressive because survivin is anti-apoptotic. Here, we used the R2 platform to examine the association between expression of mRNA encoding CCAR2, Hsp60, and survivin and survival of neuroblastoma patients. In the Kocak dataset , only 47Here, we show that both CCAR2 and Hsp60 are required for maintenance of survivin expression. Furthermore, expression of mRNA encoding CCAR2 and Hsp60 correlates positively with that of survivin in neuroblastoma tissues. High expression of each mRNA was associated with poor survival of neuroblastoma patients, suggesting that tumor cells with high expression proliferate more rapidly. Taken together, the results demonstrate that CCAR2 and Hsp60 act as pro-survival factors in neuroblastoma.Survivin exerts its anti-apoptotic function in the cytosol and mitochondria. First, following apoptotic stress, survivin is released from the mitochondria into the cytosol, where it interacts with HBXIP (Hepatitis B X-interacting protein)  and XIAPGhosh et al. showed that Hsp60 stabilizes mitochondrial survivin . Cohen-SThe data presented herein demonstrate that Hsp60 is not only a key regulator of survivin protein stability, but also a regulator of survivin mRNA expression, although the latter probably occurs via an indirect pathway. Expression of survivin mRNA is regulated by several factors ,53. The Overall, survivin is regulated by CCAR2 and Hsp60 in two different modes. Once survivin is translated and translocated to mitochondria, it is stabilized by Hsp60, which blocks the release of survivin from mitochondria to cytosol and its proteasomal degradation. Considering that CCAR2 binds survivin in the absence of mitochondrial stress  and thatCCAR2 is overexpressed by several cancers, and expression is related to prognosis . UpregulIn summary, CCAR2 and Hsp60 act as pro-survival factors by upregulating survivin expression. The results also suggest that CCAR2 and Hsp60, as well as survivin, are useful therapeutic targets for cancer.2 incubator.SH-SY5Y and BE(2)-M17 human neuroblastoma cells established from metastatic bone marrow were maintained in DMEM and DMEM/F-12, respectively, supplemented with 10% FBS, 100 U/mL penicillin G sodium, 100 μg/mL streptomycin sulfate, and 0.25 μg/mL amphotericin B. Cells were incubated at 37 °C in 5% COUniversal, CCAR2, and Hsp60 siRNAs were synthesized by ST Pharm. Co., LTD. . The siRNA duplexes were as follows: universal (control) siRNA, AUGAACGUGAAUUGCUCAAdTdT; CCAR2 (NM_021174) siRNA #1, CAGCUUGCAUGACUACUUUdTdT; CCAR2 siRNA #2, CAGCGGGUCUUCACUGGUAdTdT; Hsp60 (NM_002156) siRNA #1, UGAAGAAAUUGCACAGGUUdTdT; Hsp60 siRNA #2, UGAAUGAACGGCUUGCAAAdTdT. Transfection was performed with 20 nM siRNA using Lipofectamine RNAiMax . Forty-eight hours after transfection, all experiments were performed.3VO4) containing a protease inhibitor cocktail  on ice for 10 min. After centrifugation at 12,000× g for 5 min, the supernatant was saved as whole cell lysates. For the immunoprecipitation, the whole cell lysates were incubated with rabbit IgG , anti-survivin  or anti-CCAR2 antibody  and centrifuged again at 10,000× g for 10 min at 4 °C. To avoid disruption of the mitochondria, the mitochondrial suspension was transferred using a wide-bore tip. The crude mitochondrial pellet from the last centrifugation was resuspended in ice-cold buffer . GAPDH and Hsp90 are cytosolic markers, and cytochrome c is a mitochondrial marker.The crude mitochondrial fraction was prepared as described ,60. BrieTM reverse transcriptase . The sequences of each forward (F) and reverse (R) primer used for PCR were as follows: β-actin-F, GCTCGTCGTCGACAACGGCT; β-actin-R, CAAACATGATCTGGGTCATCTTCTC; CCAR2-F, CAAACATCCCACACACTTCAC; CCAR2-R, GACCTGGATCCGGCTTGGATG; Hsp60-F, CCCACAGTCTTTCGCCAGAT; Hsp60-R, CTTGGCTATAGAGCGTGCCA; survivin-F, GCATGGGTGCCCCGACGTTG; survivin-R, GCTCCGGCCAGAGGCCTCAA.Total RNA was isolated using Trizol reagent  and was used to synthesize cDNA using PrimeScriptwww.ebi.ac.uk/arrayexpress) (access on 14 June 2017) (Accession No. E-MTAB-2706) and the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (Accession No. GSE45547 [r) was calculated to determine the correlation between the two transcripts.Microarray data are available in the ArrayExpress database (GSE45547  and GSE6GSE45547 ). Publichttp://r2.amc.nl]) (access on 13 June 2017). The median value for each transcript was used as the cut-off point, and expression was defined as high  or low . The association between transcript levels and overall survival was visualized using Kaplan–Meier curves, and the significance of differences was assessed using the log-rank test [Expressions of CCAR2 (KIAA1967), Hsp60 (HSPD1), and survivin (BIRC5) mRNA were examined in neuroblastoma tissues of 476 (Kocak dataset)  and 498 ank test .


Yet, residual aortic regurgitation (AR) secondary toparavalvular leaks (PVL) remains a procedural limitation.3,5-7Therefore, other methods for proper AR assessment have been evaluated in recent years,such as 3D echocardiography, hemodynamic AR index, aortography and even cardiovascularmagnetic resonance, each one with its specific advantages and disadvantages.1,5,7While Doppler echocardiography has been the most common method to assess AR followingTAVR, its accurate quantification is challenging since AR jets are often multiple andeccentric.8 investigate a quantitative angiographic assessment of AR byvideodensitometry (VD-AR) before and after BPD was performed. VD-AR was shown todecrease significantly from 24.0 [18.0-30.5] % to 12.0 [5.5-19.0] % (p < 0.001) afterBPD, with some degree of AR grade improvement for up to 70% of patients treated. Ofnote, significant AR (VD-AR > 17%) was observed in 47 patients (77%) before and in 19patients (31%) after BPD; moreover, in up to a quarter of these, pre-BPD VD-AR was below17%, indicating that this additional maneuver could have been avoided. The study has itsinherent limitations, e.g., the cohort was relatively small, with retrospective patientselection and imaging acquisition, and the decision whether or not to perform BPD wasleft to the discretion of the operators. Accordingly, the study only comprises caseswhere BPD was deemed necessary, and only aortograms with good quality imaging wereselected.In the current issue of the journal, Miyazaki et al.9,10 This technique provides an accurate assessment of theseverity of PVL, and a VD-AR index greater than 17% correlated with increased mortalityand with impaired cardiac reverse remodeling after TAVR.11,12 And while VD-AR measurements are performed offline only,real-time online assessment is underway so as to enable this method to help guiding TAVRin the near future. After all, BPD is currently performed in about 10% to 20% ofpatients following TAVR, and it reduces the severity of PVL by at least one grade inmore than two thirds of patients.13,14Nevertheless, BPD may be associated with an increased risk of cerebrovascular events andannular trauma, therefore judicious utilization of this procedure isrecommended.13,14Notably, the technique used to quantify VD-AR is a novel method that can accuratelydetermine the regurgitation fraction in aortograms performed during TAVR; it usesdedicated software and showed excellent reproducibility and accuracy.In conclusion, since PVL has a negative impact on clinical outcomes after TAVR, itsproper assessment through a multimodality, multiparametric, integrative approach isfundamental. Priority should be given to PVL prevention through accurate sizing ofaortic annulus by 3D imaging techniques, THV devices with improved sealing features, andoptimal THV sizing and positioning. Still, if PVL does occur after TAVR, theinterventional cardiologist can consider corrective procedures such as BPD,valve-in-valve, or leak closure. The novel VD-AR after TAVR also allows quantitativelyassessing post-TAVR regurgitation and may assist decision making on whether or not toperform BPD, as well as determining its efficacy. Future prospective studies arewarranted to further confirm the present results.


To diagnose ≥90% HIV-infected residents (diagnostic coverage), the Bukoba Combination Prevention Evaluation (BCPE) implemented provider-initiated (PITC), home- (HBHTC), and venue-based (VBHTC) HIV testing and counseling (HTC) intervention in Bukoba Municipal Council, a mixed urban and rural lake zone community of 150,000 residents in Tanzania. This paper describes the methods, outcomes, and incremental costs of these HTC interventions. PITC was implemented in outpatient department clinics in all eight public and three faith-based health facilities. In clinics, lay counselors routinely screened and referred eligible patients for HIV testing conducted by HTC-dedicated healthcare workers. In all 14 wards, community teams offered HTC to eligible persons encountered at 31,293 home visits and at 79 male- and youth-frequented venues. HTC was recommended for persons who were not in HIV care or had not tested in the prior 90 days. BCPE conducted 133,695 HIV tests during the 2.5 year intervention . Compared with other strategies, PITC conducted proportionally more tests among females (65%), and VBHTC conducted proportionally more tests among males (69%) and young-adults aged 15–24 years (42%). Of 5,550  HIV-positive tests, 4,143 (75%) clients were newly HIV diagnosed, including 1,583 males and 881 young adults aged 15–24 years. Of HIV tests conducted 3.7%, 1.8%, and 2.1% of PITC, HBHTC, and VBHTC clients, respectively, were newly HIV diagnosed; PITC accounted for 79% of all new diagnoses. Cost per test (per new diagnosis) was $4.55 ($123.66), $6.45 ($354.44), and $7.98 ($372.67) for PITC, HBHTC, and VBHTC, respectively. In a task-shifting analysis in which lay counselors replaced healthcare workers, estimated costs per test (per new diagnosis) would have been $3.06 ($83.15), $ 4.81 ($264.04), and $5.45 ($254.52), for PITC, HBHTC, and VBHTC, respectively. BCPE models reached different target groups, including men and young adults, two groups with consistently low coverage. Implementation of multiple models is likely necessary to achieve ≥90% diagnostic coverage. The Joint United Nations Programme on HIV/AIDS (UNAIDS) 90-90-90 targets proposes that epidemic control will be achieved by 2030 if 90% of all people living with HIV (PLHIV) are diagnosed (diagnostic coverage), 90% of diagnosed PLHIV receive sustained antiretroviral therapy (ART), and 90% of PLHIV on ART are virally suppressed by 2020 . DiagnosAchieving 90-90-90 is particularly important in Tanzania, a country of 51.3 million persons and with an estimated 1.4 million adult PLHIV, aged 15–64 years   4 2014 through Q1 2017), an average of 13,365 tests were conducted per quarter . After tOf 133,695 tests conducted, 5,550 (4.2%) were HIV-positive. Of HIV-positive tests, 4,143 (75%) clients were newly diagnosed, and 588 (11%) and 891 (14%) had been previously diagnosed, and were out-of-care and in-care, respectively. Over the 2.5 year intervention period, 1,583 males (2.8%), 2,560 females (3.3%), and 3,705 persons aged 15–49 years (3.5%) were newly diagnosed . The proPITC identified 79% of all new diagnoses and >70% of new diagnoses across all demographic groups, with the exception of young adult males aged 15–24 years. PITC, HBHTC, and VBHTC identified 55%, 15%, and 30% of new diagnoses among young adult males, respectively . HBHTC , respectOf HIV tests conducted, the proportion of new diagnoses was highest in the first two quarters of implementation and then steadily declined overall and for all strategies on the mainland . Of HTC Of HIV-positive tests, the proportion of clients who were newly and previously-diagnosed, out-of-care decreased slightly over time from 78% (294/376) and 13% (50/376) in Q4 2014, respectively, to 72% (260/363) and 8% (28/363) in Q1 2017, respectively. The proportion of previously diagnosed, in-care clients increased from 9% (32/376) of positive tests in Q4 2014 to 21% (75/363) in Q1 2017 .The estimated incremental costs of implementing the three HTC strategies for 2.5 years was $720,607.67. Personnel costs accounted for 57%, 46%, and 51% of PITC, HBHTC, and VBHTC costs, respectively . The secOver a 2.5 year intervention period, BCPE comprehensively implemented facility- and community-based HTC throughout Bukoba Municipal Council in accordance with Tanzania national testing guidelines using a standard screening method, eligibility criteria, and testing algorithm. In 11 health facilities, during 31,000 home visits, and at 79 venues, BCPE conducted over 133,000 tests and newly diagnosed 4,143 PLHIV, of whom 1,583 and 881 were males and young adults aged 15–24 years, two groups with consistently low diagnostic coverage . An addiSince recommended by World Health Organization in 2007, routine PITC has been successfully implemented in many antenatal and tuberculosis clinics, but in few OPD clinics in sub-Saharan Africa, underscoring the need to evaluate barriers and costs for providing PITC as recommended by national guidelines , 36–38. Underscoring the effectiveness of PITC reported elsewhere, in our evaluation, PITC accounted for >75% of all new HIV diagnoses among young women aged 15–24 years and >82% of all new diagnoses among women of all other ages , 41, 42.BCPE PITC costs per test ($4.55) were lower than reported costs from other studies ($6.83-$13.73 per test in 2017 USD) , 45–47. Although PITC dominated CBHTC strategies in identifying new HIV diagnoses, CBHTC is critical for testing persons who have limited access to or who do not regularly access health care. In rural settings with limited or remote facility-based services, door-to-door HBHTC or HTC campaigns can substantially increase testing and diagnostic coverage. Prior systematic reviews have also shown that community-based strategies identified HIV-positive individuals at higher CD4 counts and thus earlier in the course of their HIV disease than HIV-positive clients identified through facility-based HTC , 51. In In addition to rural residents, men and young adults are less likely than women and older adults, respectively, to access healthcare services and test for HIV , 52, 53.Our costs per HBHTC ($6.45) and VBHTC $7.98) test were lower than those reported in most studies from sub-Saharan Africa  .98 test , 58, 59.As reported elsewhere, our findings suggest that a combination of facility- and community-based HTC strategies will probably be necessary for countries to achieve 90% diagnostic coverage in all geographic settings and subgroups , 51. In Because PITC costs per new diagnosis were approximately one-third lower than CBHTC, our findings suggest that PITC should reasonably be optimized first at high-volume OPDs and potentially other clinics in healthcare facilities, before resources are committed to CBHTC strategies for the general population. Targeted community-based HTC strategies are also needed in areas with limited or remote health facilities, to test partners and biological children of PLHIV, and to serve key and vulnerable populations who are unlikely to go to a health facility, especially when they are asymptomatic . ScalingBCPE used a very inclusive eligibility criteria in accordance with national testing guidelines with the goal of diagnosing as many PLHIV as possible during the 2.5 year intervention. Yield of new HIV diagnoses achieved through facility- and community-based strategies could have been higher had we employed a more restrictive HTC eligibility criteria. However, at the time of our study, a highly sensitive and specific screening form was not available and not authorized for use per national testing guidelines. To improve the cost-effectiveness of HTC strategies, it is important for programs to assess and implement suitable HTC eligibility screening forms. Screening for symptoms of sexually transmitted infections and TB, for example, might improve yield considerably and reduce cost per new HIV diagnosis , 60. IntSupporting prior reports that many PLHIV retest for HIV after diagnosis, BCPE staff used a standard form to identify 1,407 testers who had been previously diagnosed, of whom 819 were currently in HIV care , 73. ThuOur methods and findings have several important limitations. Some HTC test and diagnostic-status data may not have been accurately recorded on national registers and study forms, or compiled and transcribed accurately on monthly reporting forms. However, comprehensive quality-assurance procedures were employed throughout the intervention, and senior-investigator audits found few discrepancies between source forms , registers, and monthly reporting forms. We are also unable to report the number of residents who tested and who were newly diagnosed during the 2.5 year intervention because tests conducted do not represent unique individuals and we did not collect information on BMC resident status. Another limitation is that prior diagnostic and HIV-care status were self-reported and thus subject to social-desirability bias. Although all clients were screened with standard instruments twice by HIV-positive, peer lay counselors (once before and after testing), some clients may have withheld their prior diagnosis. To minimize this bias, lay counselors tried to establish rapport with clients during posttest counseling and assure clients that their information would be kept confidential. Although testing of partners and biological children of PLHIV were conducted through BCPE and at HIV care and treatment clinics, the national register did not measure index-client testing. Index-client testing outcomes, however, were tracked throughout the intervention and reported as part of BCPE linkage case management . AlthougDespite these limitations, BCPE PITC, VBHTC, and HBHTC models are promising strategies that may help communities achieve 90% HIV diagnostic coverage among all PLHIV. The BCPE models reached different target groups, including men and young adults, two populations with consistently low diagnostic coverage. In 2017, the Tanzania MoHCDGEC identified the BCPE PITC as a new service delivery model . ThroughS1 Tables(XLSX)Click here for additional data file.S2 Tables(XLSX)Click here for additional data file.