AKR1C3-dependent lipid droplet formation confers hepatocellular carcinoma cell adaptability to targeted therapy.

Rationale: Increased lipid droplet (LD) formation has been linked to tumor metastasis, stemness, and chemoresistance in various types of cancer. Here, we revealed that LD formation is critical for the adaptation to sorafenib in hepatocellular carcinoma (HCC) cells. We aim to investigate the LD function and its regulatory mechanisms in HCC. Methods: The key proteins responsible for LD formation were screened by both metabolomics and proteomics in sorafenib-resistant HCC cells and further validated by immunoblotting and immunofluorescence staining. Biological function of AKR1C3 was evaluated by CRISPR/Cas9-based gene editing. Isotopic tracing analysis with deuterium3-labeled palmitate or carbon13-labeled glucose was conducted to investigate fatty acid (FA) and glucose carbon flux. Seahorse analysis was performed to assess the glycolytic flux and mitochondrial function. Selective AKR1C3 inhibitors were used to evaluate the effect of AKR1C3 inhibition on HCC tumor growth and induction of autophagy. Results: We found that long-term sorafenib treatment impairs fatty acid oxidation (FAO), leading to LD accumulation in HCC cells. Using multi-omics analysis in cultured HCC cells, we identified that aldo-keto reductase AKR1C3 is responsible for LD accumulation in HCC. Genetic loss of AKR1C3 fully depletes LD contents, navigating FA flux to phospholipids, sphingolipids, and mitochondria. Furthermore, we found that AKR1C3-dependent LD accumulation is required for mitigating sorafenib-induced mitochondrial lipotoxicity and dysfunction. Pharmacologic inhibition of AKR1C3 activity instantly induces autophagy-dependent LD catabolism, resulting in mitochondrial fission and apoptosis in sorafenib-resistant HCC clones. Notably, manipulation of AKR1C3 expression is sufficient to drive the metabolic switch between FAO and glycolysis. Conclusions: Our findings revealed that AKR1C3-dependent LD formation is critical for the adaptation to sorafenib in HCC through regulating lipid and energy homeostasis. AKR1C3-dependent LD accumulation protects HCC cells from sorafenib-induced mitochondrial lipotoxicity by regulating lipophagy. Targeting AKR1C3 might be a promising therapeutic strategy for HCC tumors.

Inhibition of PP2A by LB100 sensitizes bladder cancer cells to chemotherapy by inducing p21 degradation.

PURPOSE: Bladder carcinoma (BLCA) is the most common urinary tract malignancy and exhibits a poor response to chemotherapy. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase involved in a wide variety of regulatory cellular processes, including apoptosis and the DNA-damage response (DDR). LB100, a small molecule inhibitor of PP2A, has been shown to act as a chemo-sensitizer in multiple types of cancer. However, the anti-tumor effect and mode of action of LB100 in BLCA have yet to be identified. METHODS: In vitro and in vivo experiments were performed to assess the anti-tumor effect of LB100 alone or in combination with gemcitabine. Mass spectrometry (MS)-based phosphoproteomics analysis was used to identify the downstream substrates of PP2A and to explore the mechanism underlying LB100-induced DNA damage and apoptosis. In addition, we established a chemo-resistant BLCA cell line (RT-112-R) by prolonged drug exposure and determined the effect of LB100 in enhancing genotoxicity in BLCA cell lines and xenograft mouse models. RESULTS: We found that LB100 is sufficient to induce an anti-tumor response in BLCA cells by inducing DNA damage and apoptosis both in vitro and in vivo. Furthermore, we found that PP2A potentially dephosphorylates p-p21-ser130 to stabilize p21. Inhibition of PP2A by LB100 increased the level of p-p21-ser130, subsequently leading to a reduction in p21 level in a dose-dependent manner. In addition, we found that treatment of LB100 abrogated the G1/S cell cycle checkpoint, resulting in increased phosphorylation of γH2AX in BLCA cells. Moreover, LB100 enhanced genotoxicity in chemo-resistant BLCA cells by inducing DNA damage and apoptosis in vitro and in vivo. CONCLUSION: Our findings indicate that PP2A may serve as a potential therapeutic target in BLCA through regulating p21 stability.

Generation of an induced pluripotent stem cell (iPSC) line from a diabetic patient with glucagon receptor (GCGR) p.W83X mutation.

Induced pluripotent stem cell (iPSC) line, SJHi001-A, was generated from a diabetic patient carrying a heterozygous c.G248A mutation in GCGR gene that generated the p.W83X. The PBMCs from her father (no diabetes and no mutation site) were also prepared into iPSC (SJHi002-A) as a control. Both two cell lines had normal karyotype, expressed pluripotency markers and could differentiate into the three germ layers in vivo.

The anatomy of the palate in Early Triassic Chaohusaurus brevifemoralis (Reptilia: Ichthyosauriformes) based on digital reconstruction.

The palatal anatomy of ichthyosauriforms remains largely unknown. Here, the complete palate of the early-branching ichthyosauriform Chaohusaurus brevifemoralis is reconstructed and described for the first time with the assistance of high-resolution X-ray computed tomography (CT) scanning on the basis of the three-dimensionally preserved skull of its paratype (GMPKU-P-3086) from the Lower Triassic of South China. The reconstruction reveals new palatal features of C. brevifemoralis. The palatine contacts the jugal directly, which is observed in ichthyosauriforms for the first time. A single row of denticles is present on each side of the palate. The vomer exceeds the anterior and posterior margins of the internal naris. The pterygoid is posterior to the internal naris. The epipterygoid is present and the ectopterygoid is absent.

EGFR signaling confers resistance to BET inhibition in hepatocellular carcinoma through stabilizing oncogenic MYC.

BACKGROUND: The bromodomain and extra-terminal domain (BET) inhibitor is a type of anti-tumor agent, currently being evaluated in phase I and II clinical trials for cancer therapy. It can decrease MYC expression levels and cause effective anti-tumor effects in diverse human cancers. However, its cytotoxic effect and related mechanisms of drug resistance are poorly understood in hepatocellular carcinomas (HCC). Here, we investigated the anti-tumor effects of BET inhibitor on HCC and the molecular mechanisms involved in its associated drug resistance. METHODS: We assessed the cytotoxicity of BET inhibitor on HCC cells compared with sorafenib by cell viability assay, metastasis assay and reproduced the anti-tumor effect in xenograft mouse model. In addition, the molecular mechanisms involved in drug resistance on JQ1-resistant HCC cells were revealed by western blotting, qRT-PCR, whole exome-sequencing and gene-editing technology. Finally, with specific inhibition of EGFR or ERK activity by interference RNAs or inhibitors, the efficacy of the synergistic treatment was investigated using cell viability assay, colony formation, apoptosis and xenograft mouse model. RESULTS: We found that JQ1, a commonly used BET bromo-domain inhibitor, offered a better anti-tumor response than sorafenib in MYC-positive HCC cells by inducing apoptosis in vitro and in vivo. Unlike sorafenib, JQ1 treatment significantly impaired mitochondrial respiration and glycolysis in HCC cells. Importantly, we revealed that MAPK activation by a previously undescribed activating mutation of EGFR-I645L, was critical for JQ1 sensitivity through stabilizing oncogenic MYC protein in JQ1-resistant HCC cells. Inhibition of either EGFR or ERK activity overcame the JQ1 resistance and significantly decreased MYC protein level in vitro and in vivo. CONCLUSION: Since MYC amplification is frequently identified in HCC, co-occurring with EGFR amplification, our findings suggest that targeting EGFR signaling might be essential for JQ1 therapy in advanced HCC.

Cranial morphology of Sinovenator changii (Theropoda: Troodontidae) on the new material from the Yixian Formation of western Liaoning, China.

A new three-dimensionally preserved troodontid specimen consisting of most of the skull, partial mandibles and six articulated cervical vertebrae (PMOL-AD00102) from the Early Cretaceous Yixian Formation of Beipiao, western Liaoning, China is identified as Sinovenator changii on the basis of a surangular with a "T"-shaped cross-section. High-resolution computed tomographic data for the skull of this new specimen facilitated a detailed description of the cranial anatomy of S. changii. New diagnostic features of S. changii include a well-developed medial shelf on the jugal, a slender bar in the parasphenoid recess, a lateral groove on the pterygoid flange of the ectopterygoid, and the lateral surface of the anterior cervical vertebrae bearing two pneumatic foramina. Our new observation confirms that the braincase of Sinovenator is not as primitive as previously suggested, although it still shows an intermediate state between derived troodontids and non-troodontid paravians in having an initial stage of the subotic recess and the otosphenoidal crest. Additionally, this new specimen reveals some novel and valuable anatomical information of troodontids regarding the quadrate-quadratojugal articulation, the stapes, the epipterygoid and the atlantal ribs.

The synchronous TAG production with the growth by the expression of chloroplast transit peptide-fused ScPDAT in Chlamydomonas reinhardtii.

BACKGROUND: The synchronous triacylglycerol (TAG) production with the growth is a key step to lower the cost of the microalgae-based biofuel production. Phospholipid: diacylglycerol acyltransferase (PDAT) has been identified recently and catalyzes the phospholipid contributing acyl group to diacylglycerol to synthesize TAG, and is considered as the important source of TAG in Chlamydomonas reinhardtii. RESULTS: Using a chimeric Hsp70A-RbcS2 promoter, exogenous PDAT form Saccharomyces cerevisiae fused with a chloroplast transit peptide was expressed in C. reinhardtii CC-137. Proved by western blot, the expression of ScPDAT showed a synchronous trend to the growth in the exponential phase. Compared to the wild type, the strain of Scpdat achieved 22% increase in the content of total fatty acids and 32% increase in TAG content. In addition, the fluctuation of C16 series fatty acid in monogalactosyldiacylglycerol, diacylglyceryltrimethylhomoserine and TAG indicated an enhancement in the TAG accumulation pathway. CONCLUSION: The TAG production was enhanced in the regular cultivation without the nutrient stress by strengthening the conversion of polar lipid to TAG in C. reinhardtii and the findings provide a candidate strategy for rational engineered strain to overcome the decline in the growth during the TAG accumulation triggered by nitrogen starvation.

A novel palatal implant surgery combined with uvulopalatopharyngoplasty and inferior turbinate radiofrequency for the treatment of moderate to severe obstructive sleep apnea: a pilot study.

The efficacy of Pillar implant system for the treatment of obstructive sleep apnea-hypopnea syndrome (OSAHS) is limited. The aim of this study is to explore a new type of soft palatal surgery for the treatment of adult OSAHS, and describe the subjective and objective results of this new surgery combined with uvulopharyngoplasty and inferior turbinate radiofrequency, and assess its safety and feasibility. Pre-operative preparation and risk assessment were conducted following the guidelines for uvulopalatopharyngoplasty surgery. At the follow-up visits 6 and 12 months after surgery, polysomnography and video laryngoscope examination were performed, and the snoring visual analog scale (VAS) and Epworth Sleepiness Scale (ESS) were used to assess the success of the treatment. The median apnea-hypopnea index (AHI) was 16.0 at 6 months and 20.9 at 12 months after the operation. The pre-operative median LSaO2, 74.0, was significantly increased to 82 at 6 months and 80 at 12 months after the operation. VAS was significantly decreased from the pre-operative median of 8.0 to 3.0 and 3.0 at 6 and 12 months after the operation, respectively. ESS was significantly decreased from the pre-operative median of 10.0 to 5.0 and 6.0 at 6 and 12 months after the operation, respectively. The soft palate support surgery is less invasive and has a mild postoperative reaction, few complications and adverse reactions, and good graft safety. Its short-term efficacy is good in patients with moderate to severe OSAHS and velopharyngeal obstruction.

Removal of transgene-expressing cells by a specific immune response induced by sustained transgene expression.

BACKGROUND: Induction of the immune response to transgene products is a serious concern in gene therapy, and is generally known to be influenced by the transgene expression profile, as well as the types of cells that express the transgene. However, the exact nature of the association between the transgene expression profile and immune induction following gene transfer is unclear. METHODS: In the present study, plasmids, pCpG-fLuc or pCMV-fLuc, used for driving long- or short-term expression of firefly luciferase, respectively, were injected into mice by hydrodynamic injections along with a reporter plasmid expressing Gaussia luciferase (pROSA-gLuc) to evaluate the transgene expression profile in the liver. Single pROSA-gLuc administration resulted in stable gLuc activity in serum for more than 1 year; thus, gLuc activity was used for monitoring immune responses to the liver cells expressing both gLuc and fLuc after co-injection. RESULTS: A significant reduction in gLuc activity was observed 2 weeks after co-injection of pROSA-gLuc with pCpG-fLuc, whereas stable gLuc activity was observed when pROSA-gLuc was co-injected with pCMV-fLuc. A high level of fLuc-specific immunoglobulin G was detectable in pCpG-fLuc-injected mice; furthermore, histological analysis of the liver sections of these mice indicated CD8(+) cell infiltration, implying that the transgene-expressing hepatocytes were removed by the infiltrating cells. CONCLUSIONS: Our results demonstrate that sustained transgene expression in hepatocytes triggers antigen-specific immune responses, although short-term expression of the same transgene product elicits little, if any, immune response.

Sequence and structure prediction of RNA-dependent RNA polymerase of lily symptomless virus isolated from L. × 'Casablanca'.

The DNA sequence of the RNA-dependent RNA polymerase (RdRp) gene of lily symptomless virus (LSV), a lily-infecting member of the genus Carlavirus, was determined from nine overlapping cDNA fragments of different sizes. The complete sequence of this RdRp gene (HM070294) consisted of 5,847 nucleotides coding for a protein of 220 kDa. It had 97-98% sequence identity with RdRps of other known isolates at both the DNA and the amino acid level. Phylogenetic analysis indicated that this RdRp (designated as RdRp-DL) was closely related to the RdRp of the Korean isolate (AM516059), as well as to the RdRps from Passiflora latent virus (PLV) and Kalanchoe latent virus (KLV) of the genus Carlavirus. Hydrophobic analysis of RdRp-DL revealed a hydrophobic N-terminus and a hydrophilic C-terminus. Helices and Loops were the major secondary structures of RdRp-DL. In addition, RdRp-DL also had three coil structures. Four conserved domains were identified: typoviral methyltransferase, RNA-dependent RNA polymerase, P-loop-containing nucleoside triphosphate hydrolases and carlavirus endopeptidase. A model of the tertiary structure predicted by I-TASSER was obtained for each of these conserved domains. This is the first report of a detailed phylogenetic analysis of LSV RdRp with those of other members of the genus Carlavirus, and the first to predict the domain structures of LSV RdRp.