Loss of ceramide kinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts.

Arabidopsis thaliana plants that lack ceramide kinase, encoded by ACCELERATED CELL DEATH5 (ACD5), display spontaneous programmed cell death late in development and accumulate substrates of ACD5. Here, we compared ceramide accumulation kinetics, defense responses, ultrastructural features, and sites of reactive oxygen species (ROS) production in wild-type and acd5 plants during development and/or Botrytis cinerea infection. Quantitative sphingolipid profiling indicated that ceramide accumulation in acd5 paralleled the appearance of spontaneous cell death, and it was accompanied by autophagy and mitochondrial ROS accumulation. Plants lacking ACD5 differed significantly from the wild type in their responses to B. cinerea, showing earlier and higher increases in ceramides, greater disease, smaller cell wall appositions (papillae), reduced callose deposition and apoplastic ROS, and increased mitochondrial ROS. Together, these data show that ceramide kinase greatly affects sphingolipid metabolism and the site of ROS accumulation during development and infection, which likely explains the developmental and infection-related cell death phenotypes. The acd5 plants also showed an early defect in restricting B. cinerea germination and growth, which occurred prior to the onset of cell death. This early defect in B. cinerea restriction in acd5 points to a role for ceramide phosphate and/or the balance of ceramides in mediating early antifungal responses that are independent of cell death.

High-level expression of periostin is significantly correlated with tumour angiogenesis and poor prognosis in osteosarcoma.

Periostin (PN), originally named as osteoblast-specific factor-2 (OSF-2), has been involved in regulating adhesion and differentiation of osteoblasts. Recently many studies have shown that high-level expression of PN is correlated significantly with tumour angiogenesis and prognosis in many kinds of human cancer. However, whether and how periostin expression influences prognosis in osteosarcoma remains unknown. This study aimed to examine the expression of PN in patients with osteosarcoma and explore the relationship of PN expression with clinicopathologic factors, tumour angiogenesis and prognosis. Immunohistochemistry was performed to determine the expression of PN in osteosarcoma and osteochondroma respectively. Vascular endothelial growth factor (VEGF) and CD34 were also examined in tissues from the osteosarcoma patients mentioned above. The results showed that PN expression was significantly (P < 0.05) higher in osteosarcoma (80.9%) than in osteochondroma (14.7%). Increased PN protein expression was associated with histological subtype (P = 0.000), Enneking stage (P = 0.027) and tumour size (P = 0.009). The result also showed that high expression of PN correlated with VEGF expression (r = 0.285; P = 0.019) and that tumours with PN-positive expression significantly had higher microvessal density (44.6 ± 13.7 vs. 20.6 ± 6.5; P = 0.000) compared to those in normal bone tissues. Additionally, the expression of PN was found to be an independent prognostic factor in osteosarcoma patients. In conclusion, our findings suggest that PN may have an important role in tumour progression and may be used as a prognostic biomarker for patients with osteosarcoma.

Visualizing the replicating HSV-1 virus using STED super-resolution microscopy.

BACKGROUND: Replication of viral genome is the central event during the lytic infectious cycle of herpes simplex virus 1 (HSV-1). However, the details of HSV-1 replication process are still elusive due to the limitations of current molecular and conventional fluorescent microscopy methods. Stimulated emission depletion (STED) microscopy is one of the recently available super-resolution techniques allowing observation at sub-diffraction resolution. METHODS: To gain new insight into HSV-1 replication, we used a combination of stimulated emission depletion microscopy, fluorescence in situ hybridization (FISH) and immunofluorescence (IF) to observe the HSV-1 replication process. RESULTS: Using two colored probes labeling the same region of HSV-1 genome, the two probes highly correlated in both pre-replication and replicating genomes. In comparison, when probes from different regions were used, the average distance between the two probes increased after the virus enters replication, suggesting that the HSV-1 genome undergoes dynamic structure changes from a compact to a relaxed formation and occupies larger space as it enters replication. Using FISH and IF, viral single strand binding protein ICP8 was seen closely positioned with HSV-1 genome. In contrast, ICP8 and host RNA polymerase II were less related. This result suggests that ICP8 marked regions of DNA replication are spatially separated from regions of active transcription, represented by the elongating form of RNA polymerase II within the viral replication compartments. Comparing HSV-1 genomes at early stage of replication with that in later stage, we also noted overall increases among different values. These results suggest stimulated emission depletion microscopy is capable of investigating events during HSV-1 replication. CONCLUSION: 1) Replicating HSV-1 genome could be observed by super-resolution microscopy; 2) Viral genome expands spatially during replication; 3) Viral replication and transcription are partitioned into different sub-structures within the replication compartments.

[Outcome analysis of total hip arthroplasty for traumatic avascular necrosis of femoral head].

OBJECTIVE: To analyze the operative modalities and outcomes of total hip arthroplasty for traumatic avascular necrosis of femoral head (ANFH). METHODS: A retrospective study was conducted for 40 ANFH patients undergoing total hip arthroplasty from January 2007 to October 2011. There were 31 males and 9 females with a mean age of 49 (39-65) years. Their scores of Harris hip score and visual analogue scale (VAS) at pre- and post-operation were recorded to evaluate the clinical outcomes and assess the location of prosthesis by radiology. RESULTS: All of them were followed for an average of 31 (19-51) months. All incisions healed with first intention. There was no prosthetic infection, hip dislocation, deep vein thrombosis or nerve injury. The scores of Harris hip and VAS at Month 1 post-operation and the last follow-up were significantly higher than those preoperative ones (P < 0.05). And the Harris hip score of the last follow-up was also significantly higher than that at Month 1 post-operation (P < 0.05). However, no significant difference existed in VAS score (P > 0.05). The outcomes were excellent (n = 28), good (n = 9) and fair (n = 3) with an excellent and good rate of 92.5%. Possible loosing occurred in 1/11 cemented cases while bone stability was achieved in all 29 cementless cases. And 2 case had Brooker type I heterotopic bone without treatment. CONCLUSION: Total hip arthroplasty for traumatic ANFH can achieve satisfactory clinical efficacies with careful preoperative planning and intraoperative precision.

[Nondestructive analysis of chemical composition, structure and mineral constitution of jadeite jade].

The techniques of portable energy-dispersive X-ray fluorescence analysis (PXRF), proton induced X-ray emission spectroscopy (LRS) were employed to analyze the chemical composition, structure and mineral constitution of 12 samples. The results indicated that the chemical compositions determined by PXRF and PIXE are well comparable and most samples are constituted by almost pure jadeite with low concentration of secondary elements. One sample contains a little omphacite and one sample is composed only by omphacite. Raman characteristic peaks of jadeite occurred at 201, 372, 698, 985 and 1 037 cm(-1), while those of omphacite located at 680 and 1 017 cm(-1). By using laser Raman spectroscopy for testing the fissures of the samples, wax in 3 samples and epoxy resin in one sample were found. Raman characteristic peaks of wax located at 2 846 and 2 880 cm(-1) and those of epoxy resin occurred at 2 924 and 3 065 c(-1). The application of nondestructive techniques in jadeite jade broadens the range of samples for future study and provides technical support for jadeite jade's further study, identification and classification.

AC0010, an Irreversible EGFR Inhibitor Selectively Targeting Mutated EGFR and Overcoming T790M-Induced Resistance in Animal Models and Lung Cancer Patients.

AC0010 is a pyrrolopyrimidine-based irreversible EGFR inhibitor, structurally distinct from previously reported pyrimidine-based irreversible EGFR inhibitors, such as osimertinib and rociletinib. AC0010 selectively inhibits EGFR-active and T790M mutations with up to 298-fold increase in potency compared with wild-type EGFR. In a xenograft model, oral administration of AC0010 at a daily dose of 500 mg/kg resulted in complete remission of tumors with EGFR-active and T790M mutations for over 143 days with no weight loss. Three major metabolites of AC0010 were tested and showed no wild-type EGFR inhibition or off-target effects, such as inhibition of IGF-1R. AC0010 is safe in non-small cell lung cancer (NSCLC) patients at a dose range between 50 and 550 mg once per day, and no hyperglycemia or other severe adverse effects were detected, such as grade 3 QT prolongation. The objective responses were observed in NSCLC patients with EGFR T790M mutation. Mol Cancer Ther; 15(11); 2586-97. ©2016 AACR.

A detrimental mitochondrial-nuclear interaction causes cytoplasmic male sterility in rice.

Plant cytoplasmic male sterility (CMS) results from incompatibilities between the organellar and nuclear genomes and prevents self pollination, enabling hybrid crop breeding to increase yields. The Wild Abortive CMS (CMS-WA) has been exploited in the majority of 'three-line' hybrid rice production since the 1970s, but the molecular basis of this trait remains unknown. Here we report that a new mitochondrial gene, WA352, which originated recently in wild rice, confers CMS-WA because the protein it encodes interacts with the nuclear-encoded mitochondrial protein COX11. In CMS-WA lines, WA352 accumulates preferentially in the anther tapetum, thereby inhibiting COX11 function in peroxide metabolism and triggering premature tapetal programmed cell death and consequent pollen abortion. WA352-induced sterility can be suppressed by two restorer-of-fertility (Rf) genes, suggesting the existence of different mechanisms to counteract deleterious cytoplasmic factors. Thus, CMS-related cytoplasmic-nuclear incompatibility is driven by a detrimental interaction between a newly evolved mitochondrial gene and a conserved, essential nuclear gene.

A conserved cysteine motif is critical for rice ceramide kinase activity and function.

BACKGROUND: Ceramide kinase (CERK) is a key regulator of cell survival in dicotyledonous plants and animals. Much less is known about the roles of CERK and ceramides in mediating cellular processes in monocot plants. Here, we report the characterization of a ceramide kinase, OsCERK, from rice (Oryza sativa spp. Japonica cv. Nipponbare) and investigate the effects of ceramides on rice cell viability. PRINCIPAL FINDINGS: OsCERK can complement the Arabidopsis CERK mutant acd5. Recombinant OsCERK has ceramide kinase activity with Michaelis-Menten kinetics and optimal activity at 7.0 pH and 40°C. Mg2+ activates OsCERK in a concentration-dependent manner. Importantly, a CXXXCXXC motif, conserved in all ceramide kinases and important for the activity of the human enzyme, is critical for OsCERK enzyme activity and in planta function. In a rice protoplast system, inhibition of CERK leads to cell death and the ratio of added ceramide and ceramide-1-phosphate, CERK's substrate and product, respectively, influences cell survival. Ceramide-induced rice cell death has apoptotic features and is an active process that requires both de novo protein synthesis and phosphorylation, respectively. Finally, mitochondria membrane potential loss previously associated with ceramide-induced cell death in Arabidopsis was also found in rice, but it occurred with different timing. CONCLUSIONS: OsCERK is a bona fide ceramide kinase with a functionally and evolutionarily conserved Cys-rich motif that plays an important role in modulating cell fate in plants. The vital function of the conserved motif in both human and rice CERKs suggests that the biochemical mechanism of CERKs is similar in animals and plants. Furthermore, ceramides induce cell death with similar features in monocot and dicot plants.