The gelling effect of platelet-rich fibrin matrix when exposed to human tenocytes from the rotator cuff in small-diameter culture wells and the design of a co-culture device to overcome this phenomenon.

OBJECTIVES: Platelet-rich fibrin matrix (PRFM) has been proved to enhance tenocyte proliferation but has mixed results when used during rotator cuff repair. The optimal PRFM preparation protocol should be determined before clinical application. To screen the best PRFM to each individual's tenocytes effectively, small-diameter culture wells should be used to increase variables. The gelling effect of PRFM will occur when small-diameter culture wells are used. A co-culture device should be designed to avoid this effect. METHODS: Tenocytes harvested during rotator cuff repair and blood from a healthy volunteer were used. Tenocytes were seeded in 96-, 24-, 12-, and six-well plates and co-culture devices. Appropriate volumes of PRFM, according to the surface area of each culture well, were treated with tenocytes for seven days. The co-culture device was designed to avoid the gelling effect that occurred in the small-diameter culture well. Cell proliferation was analyzed by water soluble tetrazolium-1 (WST-1) bioassay. RESULTS: The relative quantification (condition/control) of WST-1 assay on day seven revealed a significant decrease in tenocyte proliferation in small-diameter culture wells (96 and 24 wells) due to the gelling effect. PRFM in large-diameter culture wells (12 and six wells) and co-culture systems induced a significant increase in tenocyte proliferation compared with the control group. The gelling effect of PRFM was avoided by the co-culture device. CONCLUSION: When PRFM and tenocytes are cultured in small-diameter culture wells, the gelling effect will occur and make screening of personalized best-fit PRFM difficult. This effect can be avoided with the co-culture device.Cite this article: C-H. Chiu, P. Chen, W-L. Yeh, A. C-Y. Chen, Y-S. Chan, K-Y. Hsu, K-F. Lei. The gelling effect of platelet-rich fibrin matrix when exposed to human tenocytes from the rotator cuff in small-diameter culture wells and the design of a co-culture device to overcome this phenomenon. Bone Joint Res 2019;8:216-223. DOI: 10.1302/2046-3758.85.BJR-2018-0258.R1.

Economic impact of preventing brain metastases with alectinib in ALK-positive non-small cell lung cancer.

OBJECTIVES: Despite improved progression-free survival, most patients treated with the first generation ALK inhibitor crizotinib ultimately experience central nervous system (CNS) progression. Brain metastases (BM) are associated with high clinical burden in patients with advanced anaplastic lymphoma kinase positive (ALK+) non-small cell lung cancer (NSCLC). In this study we estimate the real-world economic burden of BM in newly diagnosed ALK+ NSCLC patients and investigate whether alectinib, a second generation ALK inhibitor that delays CNS progression, may help reduce healthcare costs in patients with ALK+ NSCLC. MATERIALS AND METHODS: Cost of BM was measured in ALK+ NSCLC patients identified from a stacked PharMetrics Plus and MarketScan claims database from January 2008 to March 2016 and December 2015, respectively. Per patient per month (PPPM) cost of BM was calculated as the difference in baseline-adjusted total costs in patients with and without BM over a variable follow-up period of up to 24 months. Cumulative incidence of new BM was derived from 88 alectinib-treated and 93 crizotinib-treated patients without baseline BM in a randomized phase III clinical trial, ALEX (NCT02075840). Costs of BM per patient were then calculated by applying the PPPM BM cost to the number of incident BM patients in each treatment cohort. RESULTS: 207 patients with no BM and 198 with BM were selected from the claims database. Total cost of BM was estimated at $6,029 PPPM. 24-month cumulative incidence rates of BM from the clinical trial were 7.2% and 45.3% for alectinib and crizotinib, respectively. Over follow-up, alectinib was estimated to reduce BM-related costs by $41,434 per patient compared to crizotinib. CONCLUSION: BM is associated with substantial economic burden. Alectinib was estimated to reduce BM-related costs by preventing or delaying the occurrence of BM compared to crizotinib.

No Pharmacokinetic Interaction Between the Hepatitis C Virus Inhibitors Elbasvir/Grazoprevir and Famotidine or Pantoprazole.

Use of agents to suppress gastric acid secretion is common among patients with hepatitis C virus (HCV) infection. The aims of this open-label, three-period, fixed-sequence study were to evaluate the effect of famotidine and pantoprazole on the pharmacokinetics and safety of elbasvir/grazoprevir fixed-dose combination (FDC) in 16 healthy subjects. Elbasvir and grazoprevir each exhibited similar pharmacokinetics following single-dose administration of elbasvir/grazoprevir with or without famotidine or pantoprazole. Geometric mean ratios (GMRs) of grazoprevir AUC(0,∞), Cmax , and C24 (elbasvir/grazoprevir + famotidine or elbasvir/grazoprevir + pantoprazole vs. elbasvir/grazoprevir) ranged from 0.89-1.17. Similarly, GMRs of elbasvir AUC(0,∞), Cmax , and C24 (elbasvir/grazoprevir + famotidine or elbasvir/grazoprevir + pantoprazole vs. elbasvir/grazoprevir) ranged from 1.02-1.11. These results indicate that gastric acid-reducing agents do not modify the pharmacokinetics of elbasvir or grazoprevir in a clinically relevant manner and may be coadministered with elbasvir/grazoprevir in HCV-infected patients without restriction.

Agronomically important thrips: development of species-specific primers in multiplex PCR and microarray assay using internal transcribed spacer 1 (ITS1) sequences for identification.

Thrips, the sole vector of plant Tospovirus, are major pests of many agricultural crops throughout the world. Molecular approaches have been applied in recent decades to identify these minute and morphologically difficult to distinguish insects. In this study, sequences of internal transcribed spacer 1 (ITS1) region of 15 agronomically important thrips, including several virus transmission species, have been analyzed in order to design species-specific primers for multiplex PCR and probes for microarray assay. That the ITS1 sequence distances within species were smaller than those among species suggests that the ITS1 fragment can be used for thrips species identification. The specificity and stability of these primers, combined with universal paired primers, were tested and verified in multiplex PCR. Using these specific primers as probes, microarray assay showed that PCR products of all thrips species hybridized consistently to their corresponding probes, though some signals were weak. We have demonstrated that multiplex PCR using specific primers based on ITS1 sequences is a simple, reliable, and cost-effective diagnostic tool for thrips species identification. Moreover, the DNA microarray assay is expected to extend into a reliable high-throughput screening tool for the vast numbers of thrips.

Pain following double-bundle anterior cruciate ligament reconstruction: correlation with morphological graft findings and dynamic contrast-enhanced MRI.

AIM: To determine the relationship between knee pain following anterior cruciate ligament (ACL) graft placement with morphological graft findings and dynamic contrast enhancement as assessed at MRI. MATERIAL AND METHODS: Following institutional review board approval, 37 consecutive patients with double-bundle ACL reconstruction were enrolled. Thirteen patients had pain and 24 were asymptomatic. Imaging was performed using a 1.5 T MRI machine an average of 7.6 months after surgery. Graft-related (increase signal intensity, abnormal orientation, discontinuity, cystic degeneration, anterior translation of lateral tibia, arthrofibrosis), and non-graft related causes of knee pain (meniscal tear, cartilage injury, loose bodies, and synovitis) were evaluated. During dynamic contrast enhancement analysis, peak enhancement (ePeak) was calculated by placing a region of interest at the osteoligamentous interface of each bundle. Student's t-test was used for continuous variables analysis and chi-square or Fisher's exact test was used for categorical variables analysis. RESULTS: There was no difference between symptomatic and asymptomatic patients regarding morphological graft-related or non-graft-related causes of knee pain. For dynamic contrast enhancement analysis, symptomatic patients had significantly lower ePeak values than asymptomatic patients in the anteromedial (p = 0.008) and posterolateral (p = 0.001) bundles or when using the higher ePeak value in either bundle (p = 0.003). CONCLUSION: Morphological ACL graft findings as assessed at MRI could not be used to distinguish between symptomatic and asymptomatic patients. However, lower ePeak values had a significant association with knee pain. This may indicate poor neovascularization of the graft, potentially leading to graft failure.

Development of Species-Specific Primers for Agronomical Thrips and Multiplex Assay for Quarantine Identification of Western Flower Thrips.

While morphological identification of thrips species has been difficult because of their minute size and a lack of easily recognizable characteristics, molecular identification based on the development of specific molecular markers can be easily and reliably carried out. Among the known molecular markers, the nuclear internal transcribed spacer (ITS) exhibits distinguishable variations among thrips species. In this study, sequences of ITS2 region of 10 agriculturally important thrips were established to design species-specific primers for polymerase chain reaction (PCR). ITS2 sequence variations within these species were far less than those among species, indicating the suitability of this marker for species-specific primers design. These primers, though with one or two sporadically variable positions, showed a good efficacy within species. The specificity of these primers, examined on thrips species belonging to 15 genera, proved satisfactory. Furthermore, a multiplex PCR was used successfully for identifying Frankliniella occidentalis (Pergande), an insect pest monitored for quarantine purpose, and three additional thrips also commonly found in imported agricultural products and field samples, i.e., Thrips tabaci Lindeman, Thrips hawaiiensis (Morgan), and Frankliniella intonsa (Trybom). This study has demonstrated that specific primers and multiplex PCR based on ITS2 are reliable, convenient, and diagnostic tool to discriminate thrips species of quarantine and agricultural importance.

A self produced mother wavelet feature extraction method for motor imagery brain-computer interface.

Motor imagery base brain-computer interface (BCI) is an appropriate solution for stroke patient to rehabilitate and communicate with external world. For such applications speculating whether the subjects are doing motor imagery is our primary mission. So the problem turns into how to precisely classify the two tasks, motor imagery and idle state, by using the subjects' electroencephalographic (EEG) signals. Feature extraction is a factor that significantly affects the classification result. Based on the concept of Continuous Wavelet Transform, we proposed a wavelet-liked feature extraction method for motor imagery discrimination. And to compensate the problem that the feature varies between subjects, we use the subjects' own EEG signals as the mother wavelet. After determining the feature vector, we choose Bayes linear discriminant analysis (LDA) as our classifier. The BCI competition III dataset IVa is used to evaluate the classification performance. Comparing with variance and fast Fourier transform (FFT) methods in feature extraction, 2.02% and 16.96% improvement in classification accuracy are obtained in this work respectively.

Murine TMJ loading causes increased proliferation and chondrocyte maturation.

The purpose of this study was to examine the effects of forced mouth opening on murine mandibular condylar head remodeling. We hypothesized that forced mouth opening would cause an anabolic response in the mandibular condylar cartilage. Six-week-old female C57BL/6 mice were divided into 3 groups: (1) control, (2) 0.25 N, and (3) 0.50 N of forced mouth opening. Gene expression, micro-CT, and proliferation were analyzed. 0.5 N of forced mouth opening caused a significant increase in mRNA expression of Pthrp, Sox9, and Collagen2a1, a significant increase in proliferation, and a significant increase in trabecular spacing in the subchondral bone, whereas 0.25 N of forced mouth opening did not cause any significant changes in any of the parameters examined. Forced mouth opening causes an increase in the expression of chondrocyte maturation markers and an increase in subchondral trabecular spacing.

Phylogeny of Glaucosomatidae inferred from molecular evidence.

Most species of glaucosomatids (Teleostei: Glaucosomatidae) are endemic to Australia, except Glaucosoma buergeri that is widely distributed from Australia to Japan. This study elucidated phylogenetic relationships among glaucosomatids based on the morphological characters of the saccular-otolith sagitta, in addition to molecular evidence of mitochondrial 16S rDNA, cytochrome oxidase I (COI) and cytochrome b (cyt b) sequences, and nuclear rhodopsin sequences. The topologies of individuals' phylogenetic trees, based on 16S rDNA, COI and cyt b sequences, were statistically indistinguishable from one another, and were only slightly different from a tree based on rhodopsin sequences. These molecular tree topologies, however, differed from species relationships in morphology-based phylogenetic hypothesis proposed in previous studies. Specimens of G. buergeri from Australia and Taiwan showed differences in the sagitta and molecular differentiation at the four genes, suggesting a possible speciation event. Both molecular and morphological evidences indicate that Glaucosoma magnificum is the plesiomorphic sister species of other glaucosomatid species. Glaucosoma hebraicum is the sister species of a clade composed of G. buergeri and Glaucosoma scapulare. Molecular and morphological evidences also support the species status of G. hebraicum.

M(1)-like muscarinic acetylcholine receptors regulate fast-spiking interneuron excitability in rat dentate gyrus.

Cholinergic transmission through muscarinic acetylcholine receptors (mAChRs) plays a key role in cortical oscillations. Although fast-spiking (FS), parvalbumin-expressing basket cells (BCs) are proposed to be the cellular substrates of gamma oscillations, previous studies reported that FS nonpyramidal cells in neocortical areas are unresponsive to cholinergic modulation. Dentate gyrus (DG) is an independent gamma oscillator in the hippocampal formation. However, in contrast to other cortical regions, the direct impact of mAChR activation on FS BC excitability in this area has not been investigated. Here, we show that bath-applied muscarine or carbachol, two mAChR agonists, depolarize DG BCs in the acute brain slices, leading to action potential firing in the theta-gamma bands in the presence of blockers of ionotropic glutamate and gamma-aminobutyric acid type A receptors at physiological temperatures. The depolarizing action persists in the presence of tetrodotoxin, a voltage-gated Na(+) channel blocker. In voltage-clamp recordings, muscarine markedly reduces background K(+) currents. These effects are mimicked by oxotremorine methiodide, an mAChR-specific agonist, and largely reversed by atropine, a non-selective mAChR antagonist, or pirenzepine, an M(1) receptor antagonist, but not by gallamine, an M(2/4) receptor antagonist. Interestingly, in contrast to M(1)-receptor-mediated depolarization, M(2) receptor activation by the specific agonist arecaidine but-2-ynyl ester tosylate down-regulates GABA release at BC axons-the effect is occluded by gallamine, an M(2) receptor antagonist. Overall, muscarinic activation results in a net increase in phasic inhibitory output to the target cells. Thus, cholinergic activation through M(1)-like receptor enhances BC activity and promotes the generation of nested theta and gamma rhythms, thereby enhancing hippocampal function and associated performance.

Analysis of microarchitectural changes in a mouse temporomandibular joint osteoarthritis model.

OBJECTIVE: Little is known about the natural progression of the disease process of temporomandibular joint (TMJ) osteoarthritis (OA), which affects approximately 1% of the US population. The goal of this study was to examine the early microarchitectural and molecular changes in the condylar cartilage and subchondral bone in biglycan/fibromodulin (Bgn/Fmod) double-deficient mice, which develop TMJ-OA at 6 months. METHODS: TMJs from 3-month-old (n=44) and 9-month-old (n=52) wild-type (WT n=46) and Bgn/Fmod (n=50) double-deficient mice were evaluated. Micro-CT analysis of the subchondral bone (n=24), transmission electron microscopy for condylar cartilage fibril diameters (n=26), and real-time PCR analysis for gene expression for bone and cartilage maturation markers (n=45) was performed. RESULTS: A statistically significant increase in collagen fibril diameter of the condylar cartilage and a decrease in expression of Parathyroid related protein in the mandibular condylar head were observed in the 3-month Bgn/Fmod double-deficient mice compared to WT controls. The 9-month Bgn/Fmod double-deficient mouse demonstrated an increase in bone volume and total volume in subchondral bone, and an increase in the expression of Collagen Type X and Aggrecan in the mandibular condylar head compared to the WT controls. CONCLUSION: We found that changes in the microarchitecture of the condylar cartilage preceded changes in the subchondral bone during OA in the TMJ in Bgn/Fmod double-deficient mice.

Impaired TNFalpha-induced A20 expression in E1A/Ras-transformed cells.

BACKGROUND: Tumour necrosis factor (TNF) is capable of activating the cell death pathway, and has been implicated in killing transformed cells. However, TNF also activates survival signals, including NF-kappaB activation and the subsequent expression of anti-apoptotic genes, leading to protection against TNF toxicity. METHODS: In this study, we show that, although untransformed mouse embryonic fibroblasts (MEFs) were resistant to TNF killing, E1A/Ras-transformed MEFs were susceptible to extensive apoptosis induced by TNF. The key factors for determining TNF sensitivity were explored by comparing wild-type and E1A/Ras-transformed MEFs. RESULTS: TNF signalling to NF-kappaB and to its target genes such as IkappaBalpha seemed to be mostly intact in E1A/Ras-transformed cells. Instead, the induction of A20 was completely abolished in E1A/Ras-transformed MEFs, although A20 is known to be NF-kappaB dependent. Reintroduction of A20 into E1A/Ras-transformed MEFs rescued these cells from TNF-induced death and reduced the formation of the FADD/caspase-8 complex. This impaired A20 induction in E1A/Ras MEFs was not because of the stabilisation of p53 or a defective TNF-induced p38 and Jun N-terminal kinase (JNK) signalling. Consistently, we found a reduced A20 promoter activity but normal NF-kappaB activity in TNF-treated E1A/Ras MEFs. However, Bcl-3 seemed to have a role in the transactivation of the A20 promoter in E1A/Ras cells. CONCLUSIONS: Our results suggest that specific inhibition of certain survival factors, such as A20, may determine the sensitivity to TNF-induced apoptosis in transformed cells such as E1A/Ras MEFs.

Molecular differentiation and diversity of Forcipomyia taiwana (Diptera: Ceratopogonidae) based on the mitochondrial cytochrome oxidase II sequence.

Forcipomyia taiwana (Shiraki), a biting midge, is one of the most annoying blood-sucking pests in Taiwan. In this study, partial DNA sequences of cytochrome c oxidase II from 113 individuals collected from 11 locations around the island were analyzed to delineate the differentiation pattern and possible dispersal processes of F. taiwana in Taiwan. The uncorrected nucleotide divergences, composed of mostly transition substitutions, were high (up to 2.7%) among the samples. Average comparable variations (approximately equal to 0.7%) were found within and between populations. Phylogenetic analysis suggested that several distinct lineages exist and some can be found simultaneously in some populations. A relationship between sequence divergences among populations and their relative geographical distances was observed. Moreover, haplotype diversity was high in all populations, and low to middle levels (Fst = 0.004-0.288) of genetic differentiation were found among populations. Linearized calibration from sequence divergences and phylogenetic analysis showed that different ancestral lineages of F. taiwana possibly emerged as early as 0.6 million years ago. Taken together, genetic exchanges among these divergently ancestral lineages, likely caused by recent artificial events, have possibly led to the similarly diversified compositions of F. taiwana populations all around Taiwan nowadays.

Functional consequences of cyclin D1/BRCA1 interaction in breast cancer cells.

The inheritance of one defective BRCA1 or BRCA2 allele predisposes an individual to developing breast and ovarian cancers. BRCA1 is a multifunctional tumor suppressor protein, which through interaction with a vast array of proteins has implications in processes such as cell cycle, transcription, DNA damage response and chromatin remodeling. Conversely, the oncogene, cyclin D1 is overexpressed in about 35% of all breast cancer cases. In this study, we provide detailed analyses on the phosphorylation state of BRCA1 by cyclin D1/cdk4 complexes. In particular, we have identified Ser 632 of BRCA1 as a cyclin D1/cdk4 phosphorylation site in vitro. Using chromatin immunoprecipitation assays, we observed that the inhibition of cyclin D1/cdk4 activity resulted in increased BRCA1 DNA binding at particular promoters in vivo. In addition, we identified multiple novel genes that are bound by BRCA1 in vivo. Collectively, these results indicate that cyclin D1/cdk4-mediated phosphorylation of BRCA1 inhibits the ability of BRCA1 to be recruited to particular promoters in vivo. Therefore, cyclin D1/Cdk4 phosphorylation of BRCA1 could provide a mechanism to interfere with the DNA-dependent activities of BRCA1.

Mechanism of ASC-mediated apoptosis: bid-dependent apoptosis in type II cells.

Apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor molecule that mediates apoptotic and inflammatory signals, and implicated in tumor suppression. However, the mechanism of ASC-mediated apoptosis has not been well elucidated. Here, we investigated the molecular mechanisms of ASC-mediated apoptosis in several cell lines using a caspase recruitment domain 12-Nod2 chimeric protein that transduces the signal from muramyl dipeptide into ASC-mediated apoptosis. Experiments using dominant-negative mutants, small-interfering RNAs and peptide inhibitors for caspases indicated that caspase-8 was generally required for ASC-mediated apoptosis, whereas a requirement for caspase-9 depended on the cell type. In addition, caspase-like apoptosis-regulatory protein (CLARP)/Fas-like inhibitor protein, a natural caspase-8 inhibitor, suppressed ASC-mediated apoptosis, and Clarp-/- mouse embryonic fibroblasts were highly sensitive to ASC-mediated apoptosis. Bax-deficient HCT116 cells were resistant to ASC-mediated apoptosis as reported previously, although we failed to observe colocalization of ASC and Bax in cells. Like Fas-ligand-induced apoptosis, the ASC-mediated apoptosis was inhibited by Bcl-2 and/or Bcl-XL in type-II but not type-I cell lines. Bid was cleaved upon ASC activation, and suppression of endogenous Bid expression using small-interfering RNAs in type-II cells reduced the ASC-mediated apoptosis. These results indicate that ASC, like death receptors, mediates two types of apoptosis depending on the cell type, in a manner involving caspase-8.

Do centrally obese Chinese with normal BMI have increased risk of metabolic disorders?

BACKGROUND: Body mass index (BMI) and waist circumference are highly correlated. One or the other predicts the metabolic syndromes better, depending on characteristic of the population studied, such as age, gender, and ethnicity. We examined the impact of isolated central obesity, isolated BMI elevation, and the combined type of obesity on metabolic disorders, in order to shed lights on the strategy of obesity screening. METHODS: The study subjects were Chinese aged 20 or above residing in Taiwan. Their data were derived from two large-scale studies: the Nutrition and Health Survey in Taiwan (NAHSIT 1993-1996) and the Cardiovascular Disease Risk Factor Two-township Study (CVDFACTS, 1994-1997). In evaluating the relations between obesity and health risks, the cut-points of BMI (> or =24 kg/m(2) for overweight) and waist circumference (> or =80 cm for women and > or =90 cm for men) recommended by Department of Health in Taiwan for Taiwanese people were used to define various types of obesity. RESULTS: We found that there was a small but nontrivial proportion (1.7% for men and 4.0% for women) of Taiwanese people for whom BMI was in the normal range but their waist circumferences were above normal. These people were at a higher risk of developing metabolic syndromes than those with isolated BMI elevation. Their risks were close to that of the combined type. CONCLUSIONS: In order to screen out high-risk obese individuals, isolated centrally obese subjects should not be overlooked. Therefore, we recommend to assess waist circumference in parallel to, not just sequential to the measurement of BMI in Chinese.