SLX4 interacts with several endonucleases to resolve structural barriers in DNA metabolism. SLX4 also interacts with telomeric protein TRF2 in human cells. The molecular mechanism of these interactions at telomeres remains unknown. Here, we report the crystal structure of the TRF2-binding motif of SLX4 (SLX4TBM) in complex with the TRFH domain of TRF2 (TRF2TRFH) and map the interactions of SLX4 with endonucleases SLX1, XPF, and MUS81. TRF2 recognizes a unique HxLxP motif on SLX4 via the peptide-binding site in its TRFH domain. Telomeric localization of SLX4 and associated nucleases depend on the SLX4-endonuclease and SLX4-TRF2 interactions and the protein levels of SLX4 and TRF2. SLX4 assembles an endonuclease toolkit that negatively regulates telomere length via SLX1-catalyzed nucleolytic resolution of telomere DNA structures. We propose that the SLX4-TRF2 complex serves as a double-layer scaffold bridging multiple endonucleases with telomeres for recombination-based telomere maintenance.
To determine the effect of 1% cyclopentolate on the refractive status of children aged 4 to 18 years.
Using a random cluster sampling in a cross-sectional school-based study design, children with an age of 4–18 years were selected from kindergardens, primary schools, junior and senior high schools in a rural county and a city. Auto-refractometry was performed before and after inducing cycloplegia which was achieved by 1% cyclopentolate eye drops.
Out of 6364 eligible children, data of 5999 (94.3%) children were included in the statistical analysis. Mean age was 10.0±3.3 years (range: 4–18 years). Mean difference between cycloplegic and non-cycloplegic refractive error (DIFF) was 0.78±0.79D (median: 0.50D; range: -1.00D to +10.75D). In univariate analysis, DIFF decreased significantly with older age (P<0.001;correlation coefficient r:-0.24), more hyperopic non-cycloplegic refractive error (P<0.001;r = 0.13) and more hyperopic cycloplegic refractive error (P<0.001;r = 0.49). In multivariate analysis, higher DIFF was associated with higher cycloplegic refractive error (P<0.001; standardized regression coefficient beta:0.50; regression coefficient B: 0.19; 95% confidence interval (CI): 0.18, 0.20), followed by lower intraocular pressure (P<0.001; beta: -0.06; B: -0.02; 95%CI: -0.03, -0.01), rural region of habitation (P = 0.001; beta: -0.04; B: -0.07; 95%CI: -0.11, -0.03), and, to a minor degree, with age (P = 0.006; beta: 0.04; B: 0.009; 95%CI: 0.003, 0.016). 66.4% of all eyes with non-cycloplegic myopia (≤-0.50D) remained myopic after cycloplegia while the remaining 33.6% of eyes became emmetropic (18.0%) or hyperopic (15.7%) under cycloplegia. Prevalence of emmetropia decreased from 37.5% before cycloplegia to 19.8% after cycloplegia while the remaining eyes became hyperopic under cycloplegia.
The error committed by using non-cycloplegic versus cycloplegic refractometry in children with mid to dark-brown iris color decreased with older age, and in parallel manner, with more myopic cycloplegic refractive error. Non-cycloplegic refractometric measures lead to a misclassification of refractive error in a significant proportion of children.
To investigate the distribution of the (CCR) and its associated factors in children.
Using a random cluster sampling method, the school-based, cross-sectional Shandong Children Eye Study included children aged 4 to 18 years from the rural county of Guanxian and the city of Weihai in the province of Shandong in East China. CCR was measured by ocular biometry.
CCR measurements were available for 5913 (92.9%) out of 6364 eligible children. Mean age was 10.0±3.3 years, and mean CCR was 7.84±0.27 mm (range: 6.98 to 9.35 mm). In multivariate linear regression analysis, longer CCR (i.e. flatter cornea) was significantly associated with the systemic parameters of male sex (P<0001;standardized regression coefficient beta: -0.08;regression coefficient B:-0.04; 95% Confidence Interval (CI):-0.05,-0.03), younger age (P<0.001;beta:-0.37;B:-0.03;95%CI:-0.04,-0.03), taller body height (P = 0.002;beta:0.06;B:0.001;95%CI:0.000,0.001), lower level of education of the father (P = 0.001;beta:-0.04;B:-0.01;95%CI:-0.02,-0.01) and maternal myopia (P<0.001;beta:-0.07;B:-0.04;95%CI:-0.06,-0.03), and with the ocular parameters of longer ocular axial length (P<0.001;beta:0.59;B:0.13;95%CI:0.12,0.14), larger horizontal corneal diameter (P<0.001;beta:0.19;B:0.13;95%CI:0.11,0.14), and smaller amount of cylindrical refractive error (P = 0.001;beta:-0.09;B:-0.05;95%CI:-0.06,-0.04).
Longer CCR (i.e., flatter corneas) (mean:7.84±0.27mm) was correlated with male sex, younger age, taller body height, lower paternal educational level, maternal myopia, longer axial length, larger corneas (i.e., longer horizontal corneal diameter), and smaller amount of cylindrical refractive error. These findings may be of interest for elucidation of the process of emmetropization and myopization and for corneal refractive surgery.
AIM: To investigate the clinicopathological signiﬁcance and prognostic value of caveolin-1 (CAV-1) in both tumor and stromal cells in colorectal cancer (CRC).
METHODS: A total of 178 patients with CRC were included in this study. The correlation between CAV-1 expression and clinicopathologic features and survival was studied.
RESULTS: CAV-1 expression was detected in tumor and stromal cells. The expression of stromal CAV-1 was closely associated with histological type (P = 0.022), pathologic tumor-node-metastasis stage (P = 0.047), pathologic N stage (P = 0.035) and recurrence (P = 0.000). However, tumor cell CAV-1 did not show any correlation with clinical parameters. Additionally, the loss of stromal CAV-1 expression was associated with shorter disease-free survival (P = 0.000) and overall survival (P = 0.000). Multivariate analysis revealed that the loss of stromal CAV-1 expression was an independent prognostic factor for both overall survival (P = 0.014) and disease-free survival (P = 0.006).
CONCLUSION: The loss of stromal CAV-1 expression in CRC was associated with poor prognosis and could be a prognostic factor for CRC patients.
CAV-1; Stroma; Colorectal cancer; Prognosis
AIM: To evaluate the efficacy of centralized culture and possible influencing factors.
METHODS: From January 2010 to July 2012, 66452 patients with suspected Helicobacter pylori (H. pylori) infection from 26 hospitals in Zhejiang and Jiangsu Provinces in China underwent gastrointestinal endoscopy. Gastric mucosal biopsies were taken from the antrum for culture. These biopsies were transported under natural environmental temperature to the central laboratory in Hangzhou city and divided into three groups based on their transport time: 5, 24 and 48 h. The culture results were reported after 72 h and the positive culture rates were analyzed by a χ2 test. An additional 5736 biopsies from H. pylori-positive patients (5646 rapid urease test-positive and 90 14C-urease breath test-positive) were also cultured for quality control in the central laboratory setting.
RESULTS: The positive culture rate was 31.66% (21036/66452) for the patient samples and 71.72% (4114/5736) for the H. pylori-positive quality control specimens. In the 5 h transport group, the positive culture rate was 30.99% (3865/12471), and 32.84% (14960/45553) in the 24 h transport group. In contrast, the positive culture rate declined significantly in the 48 h transport group (26.25%; P < 0.001). During transportation, the average natural temperature increased from 4.67 to 29.14 °C, while the positive culture rate declined from 36.67% (1462/3987) to 24.12% (1799/7459). When the temperature exceeded 24 °C, the positive culture rate decreased significantly, especially in the 48 h transport group (23.17%).
CONCLUSION: Transportation of specimens within 24 h and below 24 °C is reasonable and acceptable for centralized culture of multicenter H. pylori samples.
Centralized isolation; Helicobacter pylori; Influencing factor; Multiple centers; Personalized treatment
High-mobility group protein 2 (HMGA2) and epithelial–mesenchymal transition (EMT)-associated proteins play key roles in cancer progression and metastasis. However, the clinical significance of HMGA2 and its relationship with EMT markers in nasopharyngeal carcinoma (NPC) is unclear. This study aimed to assess the clinicopathological significance and prognostic value of HMGA2, E-cadherin, and vimentin in NPC.
Using immunohistochemistry, HMGA2, E-cadherin, and vimentin expression levels were evaluated in NPC (n=124) and non-tumoral inflammatory nasopharynx (n=20) tissues. The association of HMGA2 and EMT markers with clinicopathological characteristics and relationships between the protein levels and overall survival were analyzed.
Compared with non-tumorous tissues, HMGA2 and vimentin levels were markedly increased in NPC tissues, whereas decreased E-cadherin levels were observed (P<0.001). Moreover, HMGA2 expression was positively correlated with vimentin levels (r=0.431, P<0.001) and negatively correlated with E-cadherin amounts (r=−0.413, P<0.001) in NPC tissues. The expression of all three proteins correlated significantly with tumor N stage, TNM stage, and 2-year metastasis. Furthermore, significant correlations were found for T stage, N stage, TNM stage, HMGA2, E-cadherin, and vimentin (all P<0.013) with poor prognosis (univariate analysis). However, multivariate analyses showed that only HMGA2 (hazard ratio [HR]: 2.683, 95% confidence interval [CI]: 1.185–6.077, P=0.018) and N stage (HR: 7.892, 95% CI: 2.731–22.807, P<0.001) were independent predictors of poor prognosis.
These results demonstrated that HMGA2, an independent prognostic factor, may promote NPC progression and metastasis, and is significantly associated with EMT proteins. Therefore, HMGA2 may be considered a potential therapeutic target in NPC.
EMT; NPC; high-mobility group protein 2
As an alternative to fluorescence-based DNA sequencing by synthesis (SBS), we report here an approach using an azido moiety (N3) that has an intense, narrow and unique Raman shift at 2125 cm−1, where virtually all biological molecules are transparent, as a label for SBS. We first demonstrated that the four 3′-O-azidomethyl nucleotide reversible terminators (3′-O-azidomethyl-dNTPs) displayed surface enhanced Raman scattering (SERS) at 2125 cm−1. Using these 4 nucleotide analogues as substrates, we then performed a complete 4-step SBS reaction. We used SERS to monitor the appearance of the azide-specific Raman peak at 2125 cm−1 as a result of polymerase extension by a single 3′-O-azidomethyl-dNTP into the growing DNA strand and disappearance of this Raman peak with cleavage of the azido label to permit the next nucleotide incorporation, thereby continuously determining the DNA sequence. Due to the small size of the azido label, the 3′-O-azidomethyl-dNTPs are efficient substrates for the DNA polymerase. In the SBS cycles, the natural nucleotides are restored after each incorporation and cleavage, producing a growing DNA strand that bears no modifications and will not impede further polymerase reactions. Thus, with further improvements in SERS for the azido moiety, this approach has the potential to provide an attractive alternative to fluorescence-based SBS.
In many traditional Chinese medicine (TCM) hospitals, most patients are elderly with chronic diseases. Nosocomial bloodstream infections (nBSIs) are an important cause of morbidity and mortality. A retrospective surveillance study was performed to examine the epidemiology and microbiology of nBSIs in a TCM hospital from 2009 to 2011. A total of 482 patients with nBSIs were included in the study period. The incidence rate was 5.7/1000 admissions. Escherichia coli (25.5%) was the most common Gram-negative and coagulase-negative staphylococcus (CoNS) (14.1%) was the most common Gram-positive organism isolated. One-third of the E. coli and Klebsiella pneumoniae isolated from the nBSIs were the third-generation cephalosporin-resistant. Half of the Acinetobacter species isolates were resistant to imipenem. Of all the CoNS isolates, 90.7% were resistant to methicillin. Carbapenems and glycopeptide were the most frequently used for nBSI therapy. Only about one-third of patients (157/482) received appropriate empirical therapy. Septic shock, hemodialysis, Pitt bacteremia score >4, urinary tract infection, and appropriate empirical therapy were most strongly associated with 28-d mortality. The incidence of nBSIs was low in the TCM hospital but the proportion of nBSIs due to antibiotic-resistant organisms was high. A high Pitt bacteremia score was one of the most important risk factors for mortality in nBSIs. Therefore, the implementation of appropriate empirical therapy is crucial to improve the clinical outcome of nBSIs.
Nosocomial bloodstream infection; Traditional Chinese medicine hospital; Epidemiology; Microbiology
Methylthioalkylmalate synthases (MAMs) encoded by MAM genes are central to the diversification of the glucosinolates, which are important secondary metabolites in Brassicaceae species. However, the evolutionary pathway of MAM genes is poorly understood. We analyzed the phylogenetic and synteny relationships of MAM genes from 13 sequenced Brassicaceae species. Based on these analyses, we propose that the syntenic loci of MAM genes, which underwent frequent tandem duplications, divided into two independent lineage-specific evolution routes and were driven by positive selection after the divergence from Aethionema arabicum. In the lineage I species Capsella rubella, Camelina sativa, Arabidopsis lyrata, and A. thaliana, the MAM loci evolved three tandem genes encoding enzymes responsible for the biosynthesis of aliphatic glucosinolates with different carbon chain-lengths. In lineage II species, the MAM loci encode enzymes responsible for the biosynthesis of short-chain aliphatic glucosinolates. Our proposed model of the evolutionary pathway of MAM genes will be useful for understanding the specific function of these genes in Brassicaceae species.
glucosinolates; MAM genes; syntenic; evolution; Brassicaceae
The prostaglandin E2 receptor, EP2 (E-prostanoid 2), plays an important role in mice glomerular MCs (mesangial cells) damage induced by TGFβ1 (transforming growth factor-β1); however, the molecular mechanisms for this remain unknown. The present study examined the role of the EP2 signalling pathway in TGFβ1-induced MCs proliferation, ECM (extracellular matrix) accumulation and expression of PGES (prostaglandin E2 synthase). We generated primary mice MCs. Results showed MCs proliferation promoted by TGFβ1 were increased; however, the production of cAMP and PGE2 (prostaglandin E2) was decreased. EP2 deficiency in these MCs augmented FN (fibronectin), Col I (collagen type I), COX2 (cyclooxygenase-2), mPGES-1 (membrane-associated prostaglandin E1), CTGF (connective tissue growth factor) and CyclinD1 expression stimulated by TGFβ1. Silencing of EP2 also strengthened TGFβ1-induced p38MAPK (mitogen-activated protein kinase), ERK1/2 (extracellular-signal-regulated kinase 1/2) and CREB1 (cAMP responsive element-binding protein 1) phosphorylation. In contrast, Adenovirus-mediated EP2 overexpression reversed the effects of EP2-siRNA (small interfering RNA). Collectively, the investigation indicates that EP2 may block p38MAPK, ERK1/2 and CREB1 phosphorylation via activation of cAMP production and stimulation of PGE2 through EP2 receptors which prevent TGFβ1-induced MCs damage. Our findings also suggest that pharmacological targeting of EP2 receptors may provide new inroads to antagonize the damage induced by TGFβ1.
adenovirus; EP2; ERK1/2; PGE2; siRNA; TGFβ1; BP, blood pressure; CCK, cholecystokinin; CKD, chronic kidney disease; Col I, collagen type I; COX2, cyclooxygenase-2; CRE, CREB, cAMP responsive element binding protein; CTGF, connective tissue growth factor; DMEM, Dulbecco’s modified Eagle’s medium; ECM, extracellular matrix; EP2, E-prostanoid 2; ERK, extracellular-signal-regulated kinase; FBS, fetal bovine serum; FN, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MC, mesangial cell; MOI, multiplicity of infection; mPGES-1, membrane associated prostaglandin E1; PGE2, prostaglandin E2; PGES, prostaglandin E2 synthase; PKA, protein kinase A; RT–PCR, reverse transcription–PCR; siRNA, small interfering RNA; TGFβ1, transforming growth factor-β1
A 47-year-old male presented with a six-month history of fatigue and a four-month history of alanine and aspartate aminopherase elevation. Laboratory examination revealed that the serum α-fetoprotein (AFP) level was 371.51 μg/l (normal range, 0–20 μg/l), and a computed tomography scan revealed a hypodense lesion in the left hepatic lobe. During laparotomy, a dark red-colored soft tumor (1.5×1.7 cm in diameter) was found in segment eight of the liver. Intra-operative pathology and post-operative histopathology examinations revealed that the tumor was a hepatic cavernous hemangioma. The serum AFP level was decreased to 24.45 μg/l by the second post-operative week. The literature was searched and only three similar cases were found. A brief review of this rare disease entity was produced, which attempted to explain this appearance reasonably.
hepatic cavernous hemangioma; α-fetoprotein; cancer stem cell
Coculture of mesenchymal stem cells with chondrocytes increases production of cartilaginous matrix. Chondrocytes isolated from late stage osteoarthritic patients usually lost their phenotype of producing cartilaginous matrix. Fibroblast growth factor 18 is believed to redifferentiate OA chondrocyte into functionally active chondrocytes. The aim of this study is to investigate the supportive effects of MSCs on OA chondrocytes and test if FGF18 could enhance the responsiveness of OA chondrocytes to the support of MSCs in a coculture system. Both pellet and transwell co-cultures were used. GAG quantification, hydroxyproline assay, and qPCR were performed. An ectopic models of cartilage formation was also applied. Our data indicated that, in pellets coculture of MSCs and OA chondrocytes, matrix production was increased in the presence of FGF18, comparing to the monoculture of chondrocytes. Results from transwell coculture study showed that expression of matrix producing genes in OA chondrocytes increased when cocultured with MSCs with FGF18 in culture medium, while hypertrophic genes were not changed by coculture. Finally, coimplantation of MSCs with OA chondrocytes produces more matrix than chondrocytes only. In conclusion, FGF18 can restore the responsiveness of OA chondrocytes to the trophic effects of MSCs. Coimplantation of MSCs and OA chondrocytes treated with FGF18 may be a good alternative cell source for regenerating cartilage tissue that is degraded during OA pathological changes.
Guan Chang Fu Fang (GCFF) is a natural compound, which is extracted from three medicinal plants, Agrimonia pilosa Ledeb., Patrinia scabiosaefolia and Solanum nigrum L. GCFF has demonstrated clinical efficacy in the treatment of colon cancer. At present, 5-fluorouracil (5-FU) is the primary active chemotherapeutic agent used for treating colon cancer. Using median-effect and apoptosis analyses, fluorescence microscopy and western blotting, the present study analyzed the association between GCFF and 5-FU in the human colon adenocarcinoma LoVo cell line. The effect of GCFF on the expression of chemotherapeutic agent-associated genes was also investigated. The results of the synergistic analysis revealed that GCFF exhibited a significant effect upon 5-FU-associated cytotoxicity within the LoVo cell line. This effect was observed over a broad dose-inhibition range (5–95%), but was particularly significant in the lower concentrations. The flow cytometry results revealed that low doses of GCFF or 5-FU induced S-phase arrest, as did a low-dose combination of the two drugs. After 48 h, GCFF significantly suppressed the expression levels of the chemotherapeutic agent resistance-associated genes within the colon cancer cells. The western blot analysis revealed that the combined effects of 5-FU and GCFF were due to a regulation of the B-cell lymphoma-2 family of proteins. The findings of the present study suggested that GCFF, when combined with 5-FU, has the potential to be a novel, chemotherapeutic compound for the treatment of colon cancer.
Guan Chang Fu Fang; colon cancer cell; apoptosis; synergy
Although the stomach is the most common location for gastrointestinal stromal tumor (GIST) with co-primary tumors, the synchronous appearance of a poorly differentiated neuroendocrine carcinoma (NEC) and GIST in the stomach is extremely rare. To the best of our knowledge, this is the first case of gastric GIST coexisting with gastric NEC to be reported in the literature. The current study reports the case of a 71-year-old male with gastric poorly differentiated NEC and GIST discovered incidentally during surgical treatment of the NEC. Immunohistochemistry analysis showed that the NEC tumor cells were positive for CK (cytokeratin), CD57, synaptophysin, chromogranin, CD117 (KIT protein), Dog-1 (discovered on GIST-1 protein) and CD34. The synchronous GIST immunophenotype showed positivity for CD117, Dog-1 and CD34 (100%), whereas staining for CK, SMA, desmin and S100 was negative. Ki-67 labeling of proliferating cells was 90% in NEC and 1% in GIST. An accurate diagnosis was confirmed by immunohistochemical findings. Furthermore, genetic analysis using PCR direct sequencing identified no mutations in the KIT (exons 9, 11, 13 and 17) and PDGFRA (exons 12 and 18) genes. The patient developed lymph node metastases and underwent cisplatin-based chemotherapy after the operation. This is the first documented case of synchronous gastric GIST and NEC with the examination of protein expression and gene mutations in KIT and PDGFRA, which will help to further understand the etiology and pathogenesis of NEC coexisting with GIST in a gastric location.
Snchronous tumor; neuroendocrine; carcinoma; GIST; gastric
A previous model suggested doubling of Sfi1 as the first step of SPB assembly. Here it is shown that Sfi1 is gradually recruited to SPBs throughout the cell cycle. Conserved tryptophans in Sfi1 are required for its equal partitioning during mitosis, and unequal partitioning of Sfi1 underlies SPB assembly and mitotic defects in the next cell cycle.
Centrosomes play critical roles in the cell division cycle and ciliogenesis. Sfi1 is a centrin-binding protein conserved from yeast to humans. Budding yeast Sfi1 is essential for the initiation of spindle pole body (SPB; yeast centrosome) duplication. However, the recruitment and partitioning of Sfi1 to centrosomal structures have never been fully investigated in any organism, and the presumed importance of the conserved tryptophans in the internal repeats of Sfi1 remains untested. Here we report that in fission yeast, instead of doubling abruptly at the initiation of SPB duplication and remaining at a constant level thereafter, Sfi1 is gradually recruited to SPBs throughout the cell cycle. Like an sfi1Δ mutant, a Trp-to-Arg mutant (sfi1-M46) forms monopolar spindles and exhibits mitosis and cytokinesis defects. Sfi1-M46 protein associates preferentially with one of the two daughter SPBs during mitosis, resulting in a failure of new SPB assembly in the SPB receiving insufficient Sfi1. Although all five conserved tryptophans tested are involved in Sfi1 partitioning, the importance of the individual repeats in Sfi1 differs. In summary, our results reveal a link between the conserved tryptophans and Sfi1 partitioning and suggest a revision of the model for SPB assembly.
PVT1, which maps to chromosome 8q24, is a copy number amplification-associated long non-coding RNA. Overexpression of PVT1 is a powerful predictor of tumor progression and patient survival in a diverse range of cancer types. However, the association between PVT1 and hepatocellular carcinoma (HCC) remains unclear. The aim of the present study was to examine the expression pattern of PVT1, and its clinical significance in HCC. Between 2003 and 2012, reverse transcription-quantitative polymerase chain reaction was used to determine the expression levels of PVT1 in two independent cohorts: Cohort one, 58 HCC resection samples; and cohort 2, 214 HCC transplant samples. Additionally, the correlation between PVT1 expression levels and clinical parameters and outcomes was analyzed. The relative expression levels of PVT1 were significantly higher in cancerous tissues compared with the corresponding non-cancerous tissues (cohort one, P=0.0016; cohort two, P=0.0274). Furthermore, overexpression of PVT1 was associated with a higher serum α-fetoprotein expression level (P=0.011) and a higher recurrence rate (P=0.004). Kaplan-Meier analysis indicated that the patients with high PVT1 expression exhibited poor recurrence-free survival (P=0.021), and multivariate analysis demonstrated that high levels of PVT1 expression are an independent predictor for HCC recurrence (P=0.042; hazard ratio, 1.653). Thus, the high expression levels of PVT1 in HCC may serve as a novel biomarker for predicting tumor recurrence in HCC patients, and as a potential therapeutic target.
hepatocellular carcinoma; long non-coding RNA; PVT1; recurrence; progression
New sequencing technologies and high-resolution microarray analysis have revealed genome-wide pervasive transcription in many eukaryotes, generating a large number of RNAs of no coding capacity. The focus of current debate is whether many of these non-coding RNAs are functional, and if so, what their function is. In this review, we describe recent discoveries in the field of non-coding RNAs in the yeast Saccharomyces cerevisiae. Newly identified non-coding RNAs in this budding yeast, their functions in gene regulation and possible mechanisms of action are discussed.
Non-coding RNAs; pervasive transcription; non-coding RNA regulation; Saccharomyces cerevisiae
The methyltransferase Skb1 and the previously uncharacterized protein Slf1 physically interact and colocalize at a 1:1 molar ratio in cortical nodes. Skb1 and Slf1 are mutually dependent for node localization, and the Slf1 C-terminus binds to lipids for anchoring nodes at the plasma membrane.
The plasma membrane contains both dynamic and static microdomains. Given the growing appreciation of cortical microdomains in cell biology, it is important to determine the organizational principles that underlie assembly of compartmentalized structures at the plasma membrane. The fission yeast plasma membrane is highly compartmentalized by distinct sets of cortical nodes, which control signaling for cell cycle progression and cytokinesis. The mitotic inhibitor Skb1 localizes to a set of cortical nodes that provide spatial control over signaling for entry into mitosis. However, it has been unclear whether these nodes contain other proteins and how they might be organized and tethered to the plasma membrane. Here we show that Skb1 forms nodes by interacting with the novel protein Slf1, which is a limiting factor for node formation in cells. Using quantitative fluorescence microscopy and in vitro assays, we demonstrate that Skb1-Slf1 nodes are megadalton structures that are anchored to the membrane by a lipid-binding region in the Slf1 C-terminus. We propose a mechanism for higher-order node formation by Skb1 and Slf1, with implications for macromolecular assemblies in diverse cell types.
Cyclin-dependent kinase 4 (CDK4) is known to be a 33 kD protein that drives G1 phase progression of the cell cycle by binding to a CCND protein to phosphorylate RB proteins. Using different CDK4 antibodies in western blot, we detected 2 groups of proteins around 40 and 33 kD, respectively, in human and mouse cells; each group often appeared as a duplet or triplet of bands. Some CDK4 shRNAs could decrease the 33 kD wild-type (wt) CDK4 but increase some 40 kD proteins, whereas some other shRNAs had the opposite effects. Liquid chromatography–mass spectrometry/mass spectrometry analysis confirmed the existence of CDK4 isoforms smaller than 33 kD but failed to identify CDK4 at 40 kD. We cloned one CDK4 mRNA variant that lacks exon 2 and encodes a 26 kD protein without the first 74 amino acids of the wt CDK4, thus lacking the ATP binding sequence and the PISTVRE domain required for binding to CCND. Co-IP assay confirmed that this ΔE2 protein lost CCND1- and RB1-binding ability. Moreover, we found, surprisingly, that the wt CDK4 and the ΔE2 could inhibit G1–S progression, accelerate S–G2/M progression, and enhance or delay apoptosis in a cell line-specific manner in a situation where the cells were treated with a CDK4 inhibitor or the cells were serum-starved and then replenished. Hence, CDK4 seems to be expressed as multiple proteins that react differently to different CDK4 antibodies, respond differently to different shRNAs, and, in some situations, have previously unrecognized functions at the S–G2/M phases of the cell cycle via mechanisms independent of binding to CCND and RB.
CDK4; alternative splicing; CCND1; RB1; cell cycle
Aims: Nasopharyngeal carcinoma (NPC) is the most common primary malignancy of the nasopharynx. Due to its local recurrence and distant metastasis, conventional therapy is usually ineffective. MDA-7/IL-24 (melanoma differentiation associated gene 7), a member of the IL10 family of cytokines, inhibits growth of various human cancer cells, but the underlying mechanism is largely unknown. There is no report of mda-7 in nasopharyngeal carcinoma. We aimed to investigate the role of MDA-7/IL-24 in NPC. Methods: Immune defective adenoviral vector carrying the gene was produced, infected NPC CNE cells and observed its growth, cell proliferation, apoptosis and the effect of combination with chemotherapy. Results: The results showed that (1) MDA-7/IL-24 inhibited NPC CNE cell growth and survival; (2) mda-7 induced cell apoptosis and death; (3) MDA-7/IL-24 in collaboration with chemotherapy induced cell apoptosis significantly; (4) MDA-7/IL-24 induced cell apoptosis by down-regulation of anti-apoptosis molecules such as Bcl-2 and Bcl-xl and up-regulation of caspase 3. Conclusion: The results suggested that MDA-7/IL-24 had obvious therapeutic effect in NPC cells. It is verified that adenovirus mediated MDA-7/IL-24 represents a potentially important new approach to NPC therapy.
MDA-7/IL-24; nasopharyngeal carcinoma; cell survival
In fission yeast, Rng8 and Rng9 form oligomers and cluster multiple Myo51 dimers to regulate Myo51 localization and function during cytokinesis.
The myosin-V family of molecular motors is known to be under sophisticated regulation, but our knowledge of the roles and regulation of myosin-Vs in cytokinesis is limited. Here, we report that the myosin-V Myo51 affects contractile ring assembly and stability during fission yeast cytokinesis, and is regulated by two novel coiled-coil proteins, Rng8 and Rng9. Both rng8Δ and rng9Δ cells display similar defects as myo51Δ in cytokinesis. Rng8 and Rng9 are required for Myo51’s localizations to cytoplasmic puncta, actin cables, and the contractile ring. Myo51 puncta contain multiple Myo51 molecules and walk continuously on actin filaments in rng8+ cells, whereas Myo51 forms speckles containing only one dimer and does not move efficiently on actin tracks in rng8Δ. Consistently, Myo51 transports artificial cargos efficiently in vivo, and this activity is regulated by Rng8. Purified Rng8 and Rng9 form stable higher-order complexes. Collectively, we propose that Rng8 and Rng9 form oligomers and cluster multiple Myo51 dimers to regulate Myo51 localization and functions.
Mueller matrices can be used as a powerful tool to probe qualitatively the microstructures of biological tissues. Certain transformation processes can provide new sets of parameters which are functions of the Mueller matrix elements but represent more explicitly the characteristic features of the sample. In this paper, we take the backscattering Mueller matrices of a group of tissues with distinctive structural properties. Using both experiments and Monte Carlo simulations, we demonstrate qualitatively the characteristic features of Mueller matrices corresponding to different structural and optical properties. We also calculate two sets of transformed polarization parameters using the Mueller matrix transformation (MMT) and Mueller matrix polar decomposition (MMPD) techniques. We demonstrate that the new parameters can separate the effects due to sample orientation and present quantitatively certain characteristic features of these tissues. Finally, we apply the transformed polarization parameters to the unstained human cervix cancerous tissues. Preliminary results show that the transformed polarization parameters can provide characteristic information to distinguish the cancerous and healthy tissues.
(110.5405) Polarimetric imaging; (170.3880) Medical and biological imaging; (290.5855) Scattering, polarization
We demonstrate the application of polystyrene-functionalized gold nanorods (AuNRs) as a platform for surface enhanced Raman scattering (SERS) quantification of the exogenous cancer biomarker Acetyl Amantadine (AcAm). We utilize the hydrophobicity of the polystyrene attached to the AuNR surface to capture the hydrophobic AcAm from solution, followed by drying and detection using SERS. We achieve a detection limit of 16 ng/mL using this platform. This result shows clinical potential for low-cost early cancer detection.
(240.6695) Surface-enhanced Raman scattering; (240.6680) Surface plasmons; (280.4788) Optical sensing and sensors
Intraductal papillary mucinous neoplasms (IPMNs) are the most common cystic precursor lesions of invasive pancreatic cancer. The recent identification of activating GNAS mutations at codon 201 in IPMNs is a promising target for early detection and therapy. The purpose of this study was to explore clinicopathological correlates of GNAS mutational status in resected IPMNs.
Clinical and pathologic characteristics were retrieved on 54 patients in whom GNAS codon 201 mutational status was previously reported (“historical group”, Wu et al. Sci Transl Med 3:92ra66, 2011). In addition, a separate cohort of 32 patients (validation group) was included. After microdissection and DNA extraction, GNAS status was determined in the validation group by pyrosequencing.
GNAS activating mutations were found in 64 % of the 32 IPMNs included in the validation group, compared with a previously reported prevalence of 57 % in the historical group. Overall, 52 of 86 (61 %) of IPMNs demonstrated GNAS mutations in the two studies combined. Analysis of both groups confirmed that demographic characteristics, tumor location, ductal system involvement, focality, size, grade of dysplasia, presence of an associated cancer, and overall survival were not correlated with GNAS mutational status. Stratified by histological subtype, 100 % of intestinal type IPMNs demonstrated GNAS mutations compared to 51 % of gastric IPMN, 71 % of pancreatobiliary IPMNs, and 0 % of oncocytic IPMNs.
GNAS activating mutations can be reliably detected in IPMNs by pyrosequencing. In terms of clinicopathological parameters, only histological subtype was correlated with mutational frequency, with the intestinal phenotype always associated with GNAS mutations.
Colorectal cancer has become one of the leading cause of cancer morbidity and mortality throughout world. Hederagenin, a derivative of oleanolic acid isolated from the leaves of ivy (Hedera helix L.), has been shown to have potential anti-tumor activity. The study was conducted to evaluate whether hederagenin could induce apoptosis of human colon cancer LoVo cells and explore the possible mechanism.
MTT assay was used for evaluating cell viability while Annexin V-FITC/PI assay and Hoechst 33342 nuclear stainining were used for the determination of apoptosis and mitochondrial membrane potential. DCFH-DA fluorescence staining and flow cytometry were used to measure ROS generation. Real-time PCR and western blot analysis were performed for apoptosis-related protein expressions.
MTT assay showed that hederagenin could significantly inhibit the viability of LoVo cells in a concentration-dependent and time-dependent manner by IC50 of 1.39 μM at 24 h and 1.17 μM at 48 h. The apoptosis ratio was significantly increased to 32.46% and 81.78% by the induction of hederagenin (1 and 2 μM) in Annexin V-FITC/PI assay. Hederagenin could also induce the nuclear changes characteristic of apoptosis by Hoechst 33342 nuclear stainining under fluorescence microscopy. DCFH-DA fluorescence staining and flow cytometry showed that hederagenin could increase significantly ROS generation in LoVo cells. Real-time PCR showed that hederagenin induced the up-regulation of Bax and down-regulation of Bcl-2, Bcl-xL and Survivin. Western blotting analysis showed that hederagenin decreased the expressions of apoptosis-associated proteins Bcl-2, procaspase-9, procaspase-3, and polyADP- ribosepolymerase (PARP) were increased, while the expressions of Bax, caspase-3, caspase-9 were increased. However, there was no significant change on caspase-8.
These results indicated that the disruption of mitochondrial membrane potential might contribute to the apoptosis of hederagenin in LoVo cells. Our findings suggested that hederagenin might be a promising therapeutic candidate for human colon cancer.
Hederagenin; Apoptosi; Mitochondrial pathway; Colon cancer