ABSTRACT
Argonaute (AGO) proteins recruit small RNAs to form the core of RNAi effector complexes. Arabidopsis encodes ten AGO proteins and a large network of small RNAs. How these small RNAs are sorted into specific AGO complexes remains largely unknown. We have cataloged small RNAs resident in four AGO complexes. We found that AGO2 and AGO4 preferentially recruit small RNAs with a 5' terminal adenosine, whereas AGO1 harbors microRNAs (miRNAs) that favor a 5' terminal uridine. AGO5 predominantly binds small RNAs that initiate with cytosine. Changing the 5' terminal nucleotide of an miRNA predictably redirected it into a different AGO complex and alters its biological activity. These results reveal a role for small RNA sequences in assorting among AGO complexes. This suggests that specialization of AGO complexes might involve remodeling the 5' end-binding pocket to accept certain small RNA sequences, perhaps explaining the evolutionary drive for miRNAs to initiate with uridine.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , MicroRNAs/metabolism , RNA, Plant/metabolism , RNA, Untranslated/metabolism , RNA-Induced Silencing Complex/metabolism , Arabidopsis/chemistry , Arabidopsis/genetics , Argonaute Proteins , MicroRNAs/chemistry , Nucleotides/analysis , Nucleotides/metabolism , RNA, Plant/chemistry , RNA, Plant/isolation & purification , RNA, Small Interfering/metabolism , RNA, Untranslated/isolation & purification , RNA-Induced Silencing Complex/chemistryABSTRACT
OBJECTIVE: To evaluate application of a computed tomography (CT)-ultrasound fusion imaging technique to unilateral percutaneous vertebroplasty (PVP) for treating patients with osteoporotic thoracolumbar compression fracture. METHODS: Fourteen patients with osteoporotic thoracolumbar compression fractures were included, randomly divided into CT-ultrasound fusion imaging (nâ=â7) and traditional X-ray fluoroscopy groups (nâ=â7). Patients in the first group underwent unilateral PVP using real-time CT-ultrasound fusion imaging. A body surface locator was placed on the side contralateral to the scheduled puncture site (2-3âcm from the spinous process). Patient CT image information was recorded in the ultrasound system for registration during real-time ultrasound and CT fusion imaging, and one-click automatic registration was then performed. The puncture point and target point at which the puncture needle arrived were determined on CT images, with the puncture being performed under ultrasound guidance. Patients in the second group underwent X-ray fluoroscopy-guided PVP. Bone cement injection was injected under monitoring using a C-arm X-ray system. Patients' X-ray exposure and puncture times were recorded and compared between the two groups. RESULTS: The average puncture times in the CT-ultrasound fusion imaging and traditional X-ray fluoroscopy groups were 2.50±0.31âmin (without exposing patients and operators to radiation) and 5.00±0.65âmin (with the same duration of radiation exposure), respectively. The average times for bone cement injection were 3.29±0.81âmin and 3.50±0.86âmin, respectively. The mean visual analog scale (VAS) scores were 2.10±0.11 and 2.20±0.21, respectively. The bone cement was evenly distributed without cement leakage in patients in the CT-ultrasound fusion imaging group, but a poor distribution of bone cement and bone cement leakage were found in one patient in the traditional X-ray fluoroscopy group. CONCLUSIONS: Real-time CT-ultrasound fusion imaging is easy to perform, and provides precise localization of the puncture point, path, and target point. The selected puncture path was reasonable, and the needle had reached the target point accurately, which increased the success rate of puncture without radiation exposure.
Subject(s)
Fractures, Compression/diagnostic imaging , Fractures, Compression/therapy , Osteoporotic Fractures/diagnostic imaging , Osteoporotic Fractures/therapy , Tomography, X-Ray Computed/methods , Ultrasonography/methods , Vertebroplasty/methods , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Multimodal Imaging/methods , Treatment OutcomeABSTRACT
The evasion of anti-growth signaling is an important characteristic of cancer cells. In order to continue to proliferate, cancer cells must somehow uncouple themselves from the many signals that exist to slow down cell growth. Here, we define the anti-growth signaling process, and review several important pathways involved in growth signaling: p53, phosphatase and tensin homolog (PTEN), retinoblastoma protein (Rb), Hippo, growth differentiation factor 15 (GDF15), AT-rich interactive domain 1A (ARID1A), Notch, insulin-like growth factor (IGF), and Krüppel-like factor 5 (KLF5) pathways. Aberrations in these processes in cancer cells involve mutations and thus the suppression of genes that prevent growth, as well as mutation and activation of genes involved in driving cell growth. Using these pathways as examples, we prioritize molecular targets that might be leveraged to promote anti-growth signaling in cancer cells. Interestingly, naturally occurring phytochemicals found in human diets (either singly or as mixtures) may promote anti-growth signaling, and do so without the potentially adverse effects associated with synthetic chemicals. We review examples of naturally occurring phytochemicals that may be applied to prevent cancer by antagonizing growth signaling, and propose one phytochemical for each pathway. These are: epigallocatechin-3-gallate (EGCG) for the Rb pathway, luteolin for p53, curcumin for PTEN, porphyrins for Hippo, genistein for GDF15, resveratrol for ARID1A, withaferin A for Notch and diguelin for the IGF1-receptor pathway. The coordination of anti-growth signaling and natural compound studies will provide insight into the future application of these compounds in the clinical setting.
Subject(s)
Carcinogenesis/genetics , Cell Proliferation/genetics , Neoplasms/genetics , Neoplasms/therapy , Signal Transduction , DNA-Binding Proteins , Growth Differentiation Factor 15/genetics , Hippo Signaling Pathway , Humans , Kruppel-Like Transcription Factors/genetics , Molecular Targeted Therapy , Nuclear Proteins/genetics , PTEN Phosphohydrolase/genetics , Protein Serine-Threonine Kinases/genetics , Retinoblastoma Protein/genetics , Somatomedins/genetics , Transcription Factors/genetics , Tumor Suppressor Protein p53/geneticsABSTRACT
Tumour mutational burden (TMB) is used to predict response to immunotherapies. Although several groups have proposed calculation methods for TMB, a clear consensus has not yet emerged. In this study, we explored TMB calculation approaches with a 586-gene cancer panel (1.75 Mb) benchmarked to TMB measured by whole-exome sequencing (WES), using 30 samples across a range of tumour types. We explored variant allelic fraction (VAF) cut-offs of 5% and 10%, population database filtering at 0.001, 0.0001 and 0.000025, as well as different combinations of synonymous, insertion/deletion and intronic (splice site) variants, as well as exclusion of hotspot mutations, and examined the effect on TMB correlation. Good correlation (Spearman, range 0.66-0.78) between WES and panel TMB was seen across all methods evaluated. Each method of TMB calculation evaluated showed good positive per cent agreement and negative per cent agreement using 10 mutations/Mb as a cut-off, suggesting that multiple TMB calculation approaches may yield comparable results.
Subject(s)
Benchmarking , Neoplasms , Humans , Exome Sequencing , Neoplasms/genetics , Neoplasms/pathology , Biomarkers, Tumor/genetics , Mutation , Computational Biology , High-Throughput Nucleotide Sequencing/methodsABSTRACT
Background: The aim of this study was to investigate the application value of laparoscopic ultrasound (LU) in retroperitoneal radical nephrectomy for renal cell carcinoma with Type II inferior vena cava tumor thrombectomy (RRN-RCC-TII-IVCTT). Methods: The clinical data (operative time, length of tumor thrombus, tumor length, intraoperative bleeding, clinical stage, histological type, residual tumor tissue, and postoperative follow-up) of 6 patients who underwent LU-guided RRN-RCC-TII-IVCTT were retrospectively analyzed, and the intraoperative experience of LU was also summarized. Results: All 6 patients recovered well with liver and kidney functions returning to normal, and no tumor recurrence, metastasis, or vena cava tumor thrombus. Conclusions: LU-guided RRN-RCC-TII-IVCTT is a feasible treatment option, which locates the tumor accurately by retroperitoneal approach and provides the additional benefit of reduced intraoperative bleeding and shortened operative time, also achieving the much sought-after goal of precision.
ABSTRACT
An a-galactosidase-producing fungus was screened out of 26 filamentous fungi isolated from soil by us. Phylogenetic analysis based on the alignment of 18S rDNA sequences, combined with the morphological identification, indicated that the strain F63 was a member of the genus Penicillium. The a-galactosidase from Penicillium sp. F63 was purified to apparent homogeneity by ammonium sulfate precipitation, ion-exchange and gel filtration chromatography. The molecular size of the purified enzyme is approximately 82kDa estimated by SDS-PAGE. The a-galactosidase has an optimum pH of 5.0 and an optimum temperature of 45 degrees C. The enzyme is stable between pH5.0 and 6.0 below 40 degrees C. The a-galactosidase activity is slightly inhibited by Ag+ , which is dissimilar to other a-galactosidases. Kinetic studies of the a-galactosidase showed that the Km and the Vmax for pNPG are 1.4mmol/L and 1.556mmol/L. min(-1) x mg- 1, respectively. The enzyme is able to degrade natural substrates such as melibiose, raffinose and stachyose but not galactose-containing polysaccharides. The alpha-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The results show that the a-galactosidase is a novel one.
Subject(s)
Penicillium/enzymology , alpha-Galactosidase/isolation & purification , Amino Acid Sequence , Hydrogen-Ion Concentration , Molecular Sequence Data , Temperature , alpha-Galactosidase/chemistry , alpha-Galactosidase/metabolismABSTRACT
PURPOSE: Uterine carcinosarcoma is a rare aggressive malignancy frequently presenting at advanced stage of disease with extrauterine metastases. Median survival is less than 2 years due to high relapse rates after surgery and poor response to chemotherapy or radiotherapy. The goal of this study was to identify novel therapeutic targets. EXPERIMENTAL DESIGN: We applied RNA-seq analysis to prospectively collected uterine carcinosarcoma tumor samples from patients undergoing primary surgical resection and for comparison, normal endometrial tissues from postmenopausal women undergoing hysterectomy for benign indications. Functional assays were done in primary carcinosarcoma cell lines developed from patients and in established cell lines, as well as a cell line-derived xenograft model. Validation was done by analysis of an independent cohort of patients with uterine carcinosarcoma from The Cancer Genome Atlas (TCGA). RESULTS: Rac GTPase-activating protein 1 (RACGAP1) was identified to be highly upregulated in uterine carcinosarcoma. Functional assays showed that RACGAP1 mediates motility and invasion via regulation of STAT3 phosphorylation and survivin expression. RACGAP1 depletion or survivin inhibition abrogated motility and invasiveness of carcinosarcoma cells, while RACGAP1 overexpression conferred invasiveness to endometrial adenocarcinoma cells. In the TCGA cohort, RACGAP1 expression correlated with survivin expression and extrauterine spread of disease. CONCLUSIONS: The RACGAP1-STAT3-survivin signaling pathway is required for the invasive phenotype of uterine carcinosarcoma and is a newly identified therapeutic target in this lethal disease. Clin Cancer Res; 22(18); 4676-86. ©2016 AACR.
Subject(s)
Carcinosarcoma/genetics , Carcinosarcoma/pathology , GTPase-Activating Proteins/genetics , Uterine Neoplasms/genetics , Uterine Neoplasms/pathology , Aged , Aged, 80 and over , Carcinosarcoma/therapy , Case-Control Studies , Cell Movement , Female , Gene Expression , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Humans , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Inhibitor of Apoptosis Proteins/genetics , Inhibitor of Apoptosis Proteins/metabolism , Middle Aged , Neoplasm Metastasis , Neoplasm Staging , STAT3 Transcription Factor/metabolism , Survivin , Transcriptome , Uterine Neoplasms/therapyABSTRACT
OBJECTIVE: To evaluate the effectiveness of percutaneous vertebroplasty (PVP) in the treatment of osteoporotic vertebral compression fractures with or without intravertebral clefts by unilateral approach and the impact of intravertebral clefts on the effectiveness. METHODS: The clinical data of 65 patients who met the inclusion criteria of osteoporotic vertebral compression fracture were retrospectively analyzed. According to having intravertebral clefts or not, the patients were divided into 2 groups: cleft group (group A, n=25) and non-cleft group (group B, n=40). There was no significant difference in gender, age, cause of injury, the level of fracture vertebrae, degree of damage, and interval of injury and operation between 2 groups (P > 0.05). All patients were given PVP procedure by unilateral approach. The operation time, the injected volume of bone cement, time to ambulate, complications, and adjacent vertebral re-fracture were recorded. The height of anterior and middle column and the posterior convex Cobb angle of injured spine were measured on the lateral X-ray film in standing position at preoperation and 1, 48 weeks after operation. The visual analogue scale (VAS) score and Oswestry disability index (ODI) system were used to evaluate the pain relief and improvement of daily activity function respectively at preoperation and 1, 4, and 48 weeks after operation. RESULTS: There was no significant difference in the operation time and time to ambulate between 2 groups (P > 0.05). The injected volume of bone cement in group B was significantly less than that in group A (t=1.833, P=0.034). Asymptomatic cement leakage occurred in 6 patients (4 in group A and 2 in group B), in group A including 1 case of venous leakage, 2 cases of paravertebral leakage, and 1 case of intradiscal leakage; in group B including 2 cases of venous leakage. No symptomatic pulmonary embolism was observed. The vital sign was stable during operation and postoperatively. All patients were followed up 12-30 months (mean, 18.5 months). No re-fracture of the vertebrae occurred during the follow-up. The postoperative VAS score, ODI, the height of anterior and middle column, and the posterior convex Cobb angle of injured spine were improved significantly when compared with the preoperative ones in 2 groups (P < 0.05), but no significant difference was found between 2 groups at pre- and post-operation (P > 0.05). CONCLUSION: PVP by unilateral approach is safty and efficacy in the treatment of osteoporosis vertebral compression fracture combined with intravertebral clefts. Intravertebral clefts have no significant impact on the effectiveness in the pain relief and function improvement.
Subject(s)
Fractures, Compression/surgery , Osteoporotic Fractures/surgery , Spinal Fractures/surgery , Vertebroplasty/methods , Aged , Aged, 80 and over , Bone Cements/therapeutic use , Extravasation of Diagnostic and Therapeutic Materials/epidemiology , Female , Fractures, Compression/pathology , Humans , Male , Middle Aged , Osteoporotic Fractures/pathology , Pain/etiology , Pain Measurement , Retrospective Studies , Spinal Fractures/pathology , Spine/pathology , Spine/surgery , Treatment OutcomeABSTRACT
Argonaute (AGO) proteins recruit small RNAs to form effector complexes of RNA interference (RNAi), collectively termed RNA-induced silencing complexes (RISCs). Here, we describe detailed protocols for the purification of AGO complexes and their associated small RNAs, using Arabidopsis AGO1 as an example.
Subject(s)
RNA, Small Interfering/chemistry , RNA, Small Interfering/metabolism , RNA-Induced Silencing Complex/isolation & purification , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Argonaute Proteins , Blotting, Western , DNA Methylation , RNA-Induced Silencing Complex/chemistryABSTRACT
OBJECTIVE: To explore the effectiveness and methods of intervention assistant operation in the treatment of phalanx closed fracture combined with artery crisis. METHODS: Between August 2002 and December 2008, 24 cases (31 toes) of phalanx closed fracture combined with artery crisis were treated. There were 17 males (22 toes) and 7 females (9 toes), aged from 16 to 62 years (mean, 38 years). The causes of injury included crush and bruise (20 cases), traffic accident (3 cases), and machine twist (1 case). The locations were the first toe (19 toes), the second toe (10 toes), and the third toe (2 toes). The period between injury and hospitalization was 1-10 hours (mean, 6.8 hours). Phalanx angiography was performed by using venous indwelling needle for dorsalis pedis artery and posterior tibial artery puncture; according to angiography results, proper treatment could be done, then the contrast medium was injected to the artery to observe the blood supply. According to different types and locations of fracture, Kirschner wire and plate were chosen to fix fracture after the blood supply were recovered. RESULTS: Two cases (2 toes) received amputation due to necrosis at 4 days and 6 days after interventional therapy, respectively. Twenty-two cases (29 toes) survived. Incision healed primarily in 21 cases. Exudation occurred at wound of 1 case and was cured at 3 weeks after dressing change. Twenty-two cases (29 toes) were followed up 1-6 years (mean, 3.5 years) postoperatively. Two cases (3 toes) felt cool or anaesthesia and could not tolerate even in cold environment. The other toes had no senses of cold pain and paresthesia. Two cases (2 toes) had nonunion and achieved fracture healing after grafting bone. The mean union time was 4.5 months (range, 3-6 months) in other cases. CONCLUSION: Intervention assistant operation is an effective measure in the treatment of phalanx closed fracture combined with artery crisis.
Subject(s)
Arteries/injuries , Fractures, Closed/therapy , Toe Phalanges/injuries , Adolescent , Adult , Catheterization, Peripheral , Female , Fracture Fixation, Internal/methods , Fractures, Closed/surgery , Humans , Male , Middle Aged , Young AdultABSTRACT
OBJECTIVE: To explore the feasibility of percutaneous vertebroplasty for the treatment of acute burst thoracolumbar fracture. METHODS: Fifty-eight patients (male 38 and female 20, ranging in age from 38 to 70 years, with an average of 56.8 years) with acute burst thoracolumbar fracture were treated by percutaneous vertebroplasty. The injuried vertebrae were T11 in 3 cases, T12 18 cases, L1 29 cases, L2 5 cases and L3 3 cases. All suited cases were classified into 3 types according to injuried vertebral shapes,type I (safe type 26 cases), type II (risk type 21 cases), and type III (marginal type 11 cases). RESULTS: All the patients were followed up ranging from 1 to 2.5 years (mean 1.6 years). Fifty-three patients could walk in 1 to 3 days after operation. Among 55 patients who obtained complete recovery (CR), 39 patients could do daily works and 16 patients could do houseworks. The CR rate was 95%. Three patients who obtained partial recovery (PR), could live by themselves and felt slight lumbago after movements. The PR rate was 5%. CONCLUSION: Percutaneous vertebroplasty for the treatment of acute burst thoracolumbar fracture is a feasible and effective method even for particular risks.
Subject(s)
Lumbar Vertebrae/injuries , Spinal Fractures/surgery , Thoracic Vertebrae/injuries , Vertebroplasty/methods , Acute Disease , Adult , Aged , Female , Humans , Lumbar Vertebrae/surgery , Male , Middle Aged , Radiography , Spinal Fractures/diagnostic imaging , Thoracic Vertebrae/surgeryABSTRACT
INTRODUCTION: Non-compression osteoporotic vertebral pain (NCOVP) can also cause pain and severe immobilization, such as typical vertebral compression fracture (VCF). The aim of this study was to evaluate whether patients with NCOVP refractory to medical treatment and severely affecting normal daily activities could be offered therapeutic benefit with percutaneous vertebroplasty. METHODS: We conducted a retrospective review of the records of consecutive percutaneous vertebroplasty procedures performed at our institutions during a 28-month period to define a population of patients who suffered from severe NCOVP. Nine such patients were identified based on physical examination, computed tomography, magnetic resonance (MR) imaging, and bone scans. Initial clinical outcomes were assessed by comparing quantitative measurements of pain (10-point scale) and mobility (5-point scale) 1 day before the operation with those 1 day post-operation. A second follow-up took place between 2 weeks and 1 month after the operation, with a third follow-up between 6 and 10 months post-operative. Biopsy was taken in each case. RESULTS: Each patient demonstrated point tenderness over radiographically normal-shaped vertebra. Every patient showed a low signal on T1W images, and seven cases showed a high signal on T2W images inside the vertebra, indicating bone marrow edema. All patients experienced a reduction in pain and an increase in mobility after percutaneous vertebroplasty, with a mean pain reduction of 7.0 points and an average improved mobility of 2.8 points. Biopsy results indicated necrotic and/or degenerative changes in eight cases. CONCLUSION: The clinical outcomes of our patients suggest that NCOVP, mainly verified by abnormal MR signals and biopsy results, can be successfully treated by percutaneous vertebroplasty.
Subject(s)
Osteoporosis/complications , Pain/etiology , Pain/prevention & control , Spine , Vertebroplasty , Aged , Aged, 80 and over , Female , Humans , Male , Retrospective StudiesABSTRACT
Endogenous small RNAs function in RNA interference (RNAi) pathways to control gene expression through mRNA cleavage, translational repression, or chromatin modification. Plants and animals contain many microRNAs (miRNAs) that play vital roles in development, including helping to specify cell type and tissue identity. To date, no miRNAs have been reported in unicellular organisms. Here we show that Chlamydomonas reinhardtii, a unicellular green alga, encodes many miRNAs. We also show that a Chlamydomonas miRNA can direct the cleavage of its target mRNA in vivo and in vitro. We further show that the expression of some miRNAs/Candidates increases or decreases during Chlamydomonas gametogenesis. In addition to miRNAs, Chlamydomonas harbors other types of small RNAs including phased small interfering RNAs (siRNAs) that are reminiscent of plant trans-acting siRNAs, as well as siRNAs originating from protein-coding genes and transposons. Our findings suggest that the miRNA pathway and some siRNA pathways are ancient mechanisms of gene regulation that evolved prior to the emergence of multicellularity.