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Loop-mediated isothermal amplification (LAMP) has received considerable attention for decentralized (point-of-care and on-site) nucleic acid testing in view of its simple temperature control (60-65 °C) and short assay time (15-60 min). There remains a challenge in its wide adoption and acceptance due to the limitations of the existing amplification result reporter probes, e.g., photobleaching of organic fluorophore and reduced sensitivity of the pH-sensitive colorimetric dye. Herein, we demonstrate CdSeS/ZnS quantum dots (semiconductor fluorescent nanocrystals with superior photostability than organic fluorophore) with surface modification of cysteamine (amine-QDs) as a new reporter probe for LAMP that enabled single-copy sensitivity (limit of detection of 83 zM; 20 μL reaction volume). For a negative LAMP sample (absence of target sequence), positively charged amine-QDs remained dispersed due to interparticle electrostatic repulsion. While for a positive LAMP sample (presence of target sequence), amine-QDs became precipitated. The characterization data showed that amine-QDs were embedded in magnesium pyrophosphate crystals (generated during positive LAMP), thus leading to their coprecipitation. This amine-QD-based one-step LAMP assay advances the field of QD-based nucleic acid amplification assays in two aspects: (1) compatibility─one-step amplification and detection (versus separation of amplification and detection steps); and (2) universality─the same amine-QDs for different target sequences (versus different oligonucleotide-modified QDs for different target sequences).

Contains fulltext : 292919.pdf (Publisher’s version ) (Open Access) BACKGROUND: High myopia (HM), defined as a spherical equivalent refractive error (SER) ≤ -6.00 diopters (D), is a leading cause of sight impairment, through myopic macular degeneration (MMD). We aimed to derive an improved polygenic score (PGS) for predicting children at risk of HM and to test if a PGS is predictive of MMD after accounting for SER. METHODS: The PGS was derived from genome-wide association studies in participants of UK Biobank, CREAM Consortium, and Genetic Epidemiology Research on Adult Health and Aging. MMD severity was quantified by a deep learning algorithm. Prediction of HM was quantified as the area under the receiver operating curve (AUROC). Prediction of severe MMD was assessed by logistic regression. FINDINGS: In independent samples of European, African, South Asian and East Asian ancestry, the PGS explained 19% (95% confidence interval 17-21%), 2% (1-3%), 8% (7-10%) and 6% (3-9%) of the variation in SER, respectively. The AUROC for HM in these samples was 0.78 (0.75-0.81), 0.58 (0.53-0.64), 0.71 (0.69-0.74) and 0.67 (0.62-0.72), respectively. The PGS was not associated with the risk of MMD after accounting for SER: OR = 1.07 (0.92-1.24). INTERPRETATION: Performance of the PGS approached the level required for clinical utility in Europeans but not in other ancestries. A PGS for refractive error was not predictive of MMD risk once SER was accounted for. FUNDING: Supported by the Welsh Government and Fight for Sight (24WG201).

Gene expression of the chick retina was examined during the early development of lens-induced myopia (LIM) using whole transcriptome sequencing.

BACKGROUND: This study investigated the feasibility of using a computer-assisted method to evaluate and differentiate the carotid plaque characteristics in radiation-induced and non-radiation-induced carotid atherosclerosis. METHODS: This study included 107 post-radiotherapy (post-RT) nasopharyngeal carcinoma (NPC) patients and 110 subjects with cardiovascular risk factors (CVRFs). Each participant had a carotid ultrasound examination, and carotid plaques and carotid intima-media thickness (CIMT) were evaluated with grey scale ultrasound. The carotid plaque characteristics were evaluated for grey-scale median (GSM) and detailed plaque texture analysis (DPTA) using specific computer software. In DPTA, five different intra-plaque components were colour-coded according to different grey scale ranges. A multivariate linear regression model was used to evaluate the correlation of risk factors and carotid plaque characteristics. RESULTS: Post-RT NPC patients have significantly higher CIMT (748±15.1 µm, P=0.001), more patients had a plaque formation (80.4%, P

Myopia is the commonest eye disorder in the world. High myopes are predisposed to ocular pathologies. The vasoactive intestinal peptide receptor 2 (VIPR2) gene was identified as a myopia susceptibility locus by our group and another group. We continued to fine-map this locus. A case-control study was performed in 4 sequential stages with a total of 941 highly myopic subjects and 846 control subjects, all unrelated Chinese. Stage 1 experimentally genotyped 64.4% of the entire cohort for 152 single-nucleotide polymorphisms (SNPs) and Stage 2 the remaining subjects for 21 SNPs. Stage 3 combined the genotypes for 21 SNPs for the entire cohort, and identified one group of high-risk haplotypes and one group of protective haplotypes significantly associated with high myopia. Stage 4 imputed genotypes for variants in the VIPR2 region and identified two independent groups of variants: one group with high-risk minor alleles and another with protective minor alleles. Variants within each group were generally in strong linkage disequilibrium among themselves while high-risk variants were in linkage equilibrium with protective variants. Therefore, the VIPR2 locus seems to contain variants with opposite effects. This is the first study that has examined the genetic architecture of a myopia susceptibility locus in detail.

Recent research has focused on the mechanisms by which long non-coding RNAs (lncRNAs) modulate diverse cellular processes such as tumorigenesis. However, the functional characteristics of these non-coding elements in the genome are poorly understood at present. In this study, we have explored several mechanisms that involve the novel lncRNA and microRNA (miRNA) axis participating in modulation of drug response and the tumor microenvironment of myeloproliferative neoplasms (MPNs). We identified novel lncRNAs via mRNA sequencing that was applied to leukemic cell lines derived from BCR-ABL1-positive and JAK2-mutant MPNs under treatment with therapeutic tyrosine kinase inhibitors (TKI). The expression and sequence of novel LNC000093 were further validated in both leukemic cells and normal primary and pluripotent cells isolated from human blood, including samples from patients with chronic myelogenous leukemia (CML). Downregulation of LNC000093 was validated in TKI-resistant CML while a converse expression pattern was observed in blood cells isolated from TKI-sensitive CML cases. In addition to BCR-ABL1-positive CML cells, the driver mutation JAK2-V617F-regulated lncRNA BANCR axis was further identified in BCR-ABL1-negative MPNs. Further genome-wide validation using MPN patient specimens identified 23 unique copy number variants including the 7 differentially expressed lncRNAs from our database. The newly identified LNC000093 served as a competitive endogenous RNA for miR-675-5p and reversed the imatinib resistance in CML cells through regulating RUNX1 expression. The extrinsic function of LNC000093 in exosomal H19/miR-675-induced modulation for the microenvironment was also determined with significant effect on VEGF expression.

MicroRNAs (miRNAs) are critical regulators of gene expression that may be used to identify the pathological pathways influenced by disease and cellular interactions. Viral miRNAs (v-miRNAs) encoded by both DNA and RNA viruses induce immune dysregulation, virus production, and disease pathogenesis. Given the absence of effective treatment and the prevalence of highly infective SARS-CoV-2 strains, improved understanding of viral-associated miRNAs could provide novel mechanistic insights into the pathogenesis of COVID-19. In this study, SARS-CoV-2 v-miRNAs were identified by deep sequencing in infected Calu-3 and Vero E6 cell lines. Among the ~0.1% small RNA sequences mapped to the SARS-CoV-2 genome, the top ten SARS-CoV-2 v-miRNAs (including three encoded by the N gene; v-miRNA-N) were selected. After initial screening of conserved v-miRNA-N-28612, which was identified in both SARS-CoV and SARS-CoV-2, its expression was shown to be positively associated with viral load in COVID-19 patients. Further in silico analysis and synthetic-mimic transfection of validated SARS-CoV-2 v-miRNAs revealed novel functional targets and associations with mechanisms of cellular metabolism and biosynthesis. Our findings support the development of v-miRNA-based biomarkers and therapeutic strategies based on improved understanding of the pathophysiology of COVID-19.

Coronavirus disease 2019 (COVID-19) is the most devastating infectious disease in the 21st century with more than 2 million lives lost in less than a year. The activation of inflammasome in the host infected by SARS-CoV-2 is highly related to cytokine storm and hypercoagulopathy, which significantly contribute to the poor prognosis of COVID-19 patients. Even though many studies have shown the host defense mechanism induced by inflammasome against various viral infections, mechanistic interactions leading to downstream cellular responses and pathogenesis in COVID-19 remain unclear. The SARS-CoV-2 infection has been associated with numerous cardiovascular disorders including acute myocardial injury, myocarditis, arrhythmias, and venous thromboembolism. The inflammatory response triggered by the activation of NLRP3 inflammasome under certain cardiovascular conditions resulted in hyperinflammation or the modulation of angiotensin-converting enzyme 2 signaling pathways. Perturbations of several target cells and tissues have been described in inflammasome activation, including pneumocytes, macrophages, endothelial cells, and dendritic cells. The interplay between inflammasome activation and hypercoagulopathy in COVID-19 patients is an emerging area to be further addressed. Targeted therapeutics to suppress inflammasome activation may have a positive effect on the reduction of hyperinflammation-induced hypercoagulopathy and cardiovascular disorders occurring as COVID-19 complications.

Initial cases of coronavirus disease in Hong Kong were imported from mainland China. A dramatic increase in case numbers was seen in February 2020. Most case-patients had no recent travel history, suggesting the presence of transmission chains in the local community. We collected demographic, clinical, and epidemiologic data from 50 patients, who accounted for 53.8% of total reported case-patients as of February 28, 2020. We performed whole-genome sequencing to determine phylogenetic relationship and transmission dynamics of severe acute respiratory syndrome coronavirus 2 infections. By using phylogenetic analysis, we attributed the community outbreak to 2 lineages; 1 harbored a common mutation, Orf3a-G251V, and accounted for 88.0% of the cases in our study. The estimated time to the most recent common ancestor of local coronavirus disease outbreak was December 24, 2019, with an evolutionary rate of 3.04 × 10−3 substitutions/site/year. The reproduction number was 1.84, indicating ongoing community spread.

Hong Kong has already been hit by three waves of the coronavirus disease 2019 (COVID-19) as of October 2020. More than 120 people were infected in the first round, which was triggered mainly by tou...

The common occurrence of cardiovascular diseases and the lack of proper autologous tissues prompt and promote the pressing development of tissue-engineered vascular grafts (TEVGs). Current progress on scaffold production, genetically modified cells, and use of nanotechnology-based monitoring has considerably improved the long-term patency of engineered tissue grafts. However, challenges abound in the autologous materials and manipulation of genes and cells for tissue engineering. This review overviews current development in TEVGs and discusses recent improvements in scaffolding techniques and the efficiency of gene-editing tools and their ability to fill the existing gaps in stem cell and regenerative therapies. Current advances in three-dimensional printing approaches for fabrication of engineered tissues are also reviewed together with specific biomaterials for vascular tissues. In addition, the natural and synthetic polymers that hold increasing significance for vascular tissue engineering are highlighted. Both animal models and nanotechnology-based monitoring are proposed for preclinical evaluation of engineered grafts in view of their historical significance in tissue engineering. The ultimate success of tissue regeneration, which is yet to be fully realized, depends on the optimal performance of culture systems, biomaterial constructs, and stem cells in a suitable artificial physiological environment. Impact statement The main goal of this review is to provide a broadened insight on recent advances in stem cell-based vascular tissue engineering and the development of gene-editing and nanotechnology systems in this field. Moreover, the uses of autologous cells to prepare personalized hydrogels offer promising methods for scaffold fabrication. These new approaches not only improve the efficacy of tissue-engineered vascular grafts, but also offer potential therapeutic options with long-term beneficial effects on patients.

In this work, we developed a simple and sensitive colorimetric detection platform for specific DNA sequences by using peroxidase mimetics of platinum nanoparticles supported on reduced graphene oxide. This nanocomposite possessed the combined advantages of platinum nanoparticles (superior peroxidase-like activity) and reduced graphene oxide (π-stacking interaction with single-stranded but not double-stranded DNA). The catalytic activity was strongly dependent on the chloroplatinic acid-to-graphene oxide mass ratio during the synthesis step, with an optimum ratio of 7 : 1. Unlike natural peroxidase, the nanocomposite had excellent stability over wide ranges of temperature (4-90 °C) and pH (1-13). For DNA detection, the nanocomposite had higher affinity for the single-stranded probe (in the absence of target) than the probe-target duplex. The probe-bound nanocomposite was stabilized against salt-induced aggregation and thus upon the addition of 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide to the supernatant, an intense blue color was generated. The linear range and limit of detection of this assay platform were 0.5-10 nM and 0.4 nM, respectively. Moreover, this platform featured high specificity that 3-base-mismatched sequence could be distinguished with the naked eye and 1-base-mismatched sequence with absorbance measurement. Furthermore, the applicability for real sample detection was demonstrated by polymerase chain reaction product analysis. Taken together, this new platform is well suited for point-of-care and on-site nucleic acid testing.

Background Carotid atherosclerosis is common in post-radiotherapy (post-RT) patients and subjects with cardiovascular risk factors (CVRFs). The associated development of carotid plaques (CPs), particularly unstable plaques, is a major cause of cerebrovascular disease. Therefore, accurate detection and evaluation of CP characteristics is of essence. This study investigated the feasibility of using a computer-assisted method to evaluate and differentiate the CP characteristics in radiation-induced and non-radiation-induced CA. Methods 107 post-RT NPC and 110 CVRF subjects were recruited. Each participant had a carotid ultrasound examination of CPs and carotid intima-media thickness (CIMT). The carotid plaque characteristics were evaluated for grey-scale median (GSM) and detailed plaque texture analysis (DPTA) using specific computer software. In DPTA, five different intra-plaque components were color-coded according to different grey scale ranges. Multivariate regression model was used to evaluate the correlation of risk factors and carotid plaque characteristics. Results Post-RT NPC patients have significantly higher CIMT (748±15.1µm, p=0.001), more incidence of plaque formation (80.4%, p

Background: Initial cases of coronavirus disease 2019 (COVID-19) reported in Hong Kong were mostly imported cases from Mainland China. However, most cases reported in February 2020 were local infections with unknown source, indicating local community transmissions. This study aimed to report the clinical, epidemiological and phylogenomic characteristics of the local cases of COVID-19 in our community. Methods: We extracted the demographic, clinical and epidemiological data from 50 COVID-19 patients, who accounted for 53.8% of the cases in Hong Kong by the end of February 2020. We used both Nanopore and Illumina platforms to perform whole-genome sequencing (WGS) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from these patients. Phylogenetic relatedness among these local cases and their placement in the global phylogeny were examined. The evolutionary rate and divergence time of transmission were also determined. Findings: Of these 50 patients, only three (6.0%) had visited Wuhan while 43 (86.0%) did not have recent travel records. The average interval from symptom onset to hospital admission was 8.5 days. The most common signs and symptoms on admission were cough (74.0%) and fever (58.0%). Radiographic abnormality was found in 46 (92.0%) patients. Three (6.0%) patients required ICU admission. Phylogenetic analysis concurred with epidemiological investigation that 42 (84.0%) cases could be grouped into six transmission clusters. Forty-four (88.0%) cases harboured a common mutation Orf3a G251V. Global phylogeny of SARS-CoV-2 revealed that most (88.0%) cases in Hong Kong were clustered in two subclades with the strains from other countries. The estimated time to the most recent common ancestor (tMRCA) of COVID-2019 outbreak in Hong Kong was December 24, 2019 with an evolutionary rate of 3.04x10-3 substitutions per site per year. The reproduction number value was 1.84 as of February 28, 2020 in Hong Kong. Interpretation: We provided a territory-wide overview of COVID-19 in Hong Kong, which has borders connecting to Mainland China. Transmission in closed settings especially during family and religious gatherings is a hallmark of the recently reported cases. The reproduction number value indicated an ongoing outbreak in the community. Social distancing and vigilant epidemiological control are crucial to the containment of COVID-19 transmission

Background: Initial cases of coronavirus disease 2019 (COVID-19) reported in Hong Kong were mostly imported cases from Mainland China. However, most cases reported in February 2020 were local infections with unknown source, indicating local community transmissions. This study aimed to report the clinical, epidemiological and phylogenomic characteristics of the local cases of COVID-19 in our community. Methods: We extracted the demographic, clinical and epidemiological data from 50 COVID-19 patients, who accounted for 53·8% of the cases in Hong Kong by the end of February 2020. We used both Nanopore and Illumina platforms to perform whole-genome sequencing (WGS) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from these patients. Phylogenetic relatedness among these local cases and their placement in the global phylogeny were examined. The evolutionary rate and divergence time of transmission were also determined. Findings: Of these 50 patients, only three (6·0%) had visited Wuhan while 43 (86·0%) did not have recent travel records. The average interval from symptom onset to hospital admission was 8·5 days. The most common signs and symptoms on admission were cough (74·0%) and fever (58·0%). Radiographic abnormality was found in 46 (92·0%) patients. Three (6·0%) patients required ICU admission. Phylogenetic analysis concurred with epidemiological investigation that 42 (84·0%) cases could be grouped into six transmission clusters. Forty-four (88·0%) cases harboured a common mutation Orf3aG251V. Global phylogeny of SARS-CoV-2 revealed that most (88·0%) cases in Hong Kong were clustered in two subclades with the strains from other countries. The estimated time to the most recent common ancestor (tMRCA) of COVID-2019 outbreak in Hong Kong was December 24, 2019 with an evolutionary rate of 3.04×10-3 substitutions per site per year. The reproduction number value was 1·84 as of February 28, 2020 in Hong Kong. Interpretation: We provided a territory-wide overview of COVID-19 in Hong Kong, which has borders connecting to Mainland China. Transmission in closed settings especially during family and religious gatherings is a hallmark of the recently reported cases. The reproduction number value indicated an ongoing outbreak in the community. Rapid isolation of suspected or confirmed cases and their close contacts coupled with robust laboratory surveillance is crucial to the containment of COVID-19 transmission. Funding Statement: The project was supported by internal funds from the Faculty of Health and Social Science, and Department of Health Technology and Informatics of The Hong Kong Polytechnic University. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: This study was approved by the Institutional Review Boards of The Hong Kong Polytechnic University (RSA20021) and the public hospitals involved (HKECREC-20200014; KCC/KEC2020-0070).

Recent studies have revealed that non-coding regions comprise the vast majority of the human genome and long non-coding RNAs (lncRNAs) are a diverse class of non-coding RNAs that has been implicated in a variety of biological processes. Abnormal expression of lncRNAs has also been linked to different human diseases including cancers, yet the regulatory mechanisms and functional effects of lncRNAs are still ambiguous, and the molecular details also need to be confirmed. Unlike protein-coding gene, it is much more challenging to unravel the roles of lncRNAs owing to their unique and complex features such as functional diversity and low conservation among species, which greatly hamper their experimental characterization. In this review, we summarize and discuss both conventional and advanced approaches for the identification and functional characterization of lncRNAs related to hematological malignancies. In particular, the utility and advancement of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system as gene-editing tools are envisioned to facilitate the molecular dissection of lncRNAs via different knock-in/out strategies. Besides experimental considerations specific to lncRNAs, the roles of lncRNAs in the pathogenesis and progression of leukemia are also highlighted in the review. We expect that these insights may ultimately lead to clinical applications including development of biomarkers and novel therapeutic approaches targeting lncRNAs.

Pressure-induced injury (PI), such as a pressure ulcer, in patients with limited mobility is a healthcare issue worldwide. PI is an injury to skin and its underlying tissue such as skeletal muscle. Muscle compression, composed of mechanical deformation of muscle and external load, leads to localized ischemia and subsequent unloading reperfusion and, hence, a pressure ulcer in bed-bound patients. Although the gross factors involved in PI have been identified, little is known about the exact disease mechanism or its links to apoptosis, autophagy and inflammation. Here, we report that PI is mediated by intrinsic apoptosis and exacerbated by autophagy. Conditional ablation of Bax and Bak activates the Akt-mTOR pathway and Bnip3-mediated mitophagy and preserves mitochondrial contents in compressed muscle. Moreover, we find that the presence/absence of Bax and Bak alters the roles and functions of autophagy in PI. Our results suggest that manipulating apoptosis and autophagy are potential therapeutic targets for treatment and prevention of PI.

Color Doppler vascular index (VI) was assessed alone and in combination with grey-scale ultrasound (GSU) in regionally subdivided thyroid nodules in diagnosing thyroid cancer. Color Doppler sonograms of 111 thyroid nodules were evaluated by a home-developed algorithm that performed “offsetting” (algorithm for changing the area of a region of interest, ROI, without distorting the ROI’s contour) and assessed peripheral, central and overall VI of thyroid nodules. Results showed that the optimum offset for dividing peripheral and central regions of nodule was 22%. At the optimum offset, the mean VI of peripheral, central, and overall regions of malignant nodules were significantly higher than those of benign nodules (26.5 ± 16.2%, 21.7 ± 19.6%, 23.8 ± 4.6% v/s 18.2 ± 16.7%, 11.9 ± 15.1% and 16.6 ± 1.8% respectively, P P

Background Genome-wide association studies (GWAS) is a major method for studying the genetics of complex diseases. Finding all sequence variants to explain fully the aetiology of a disease is difficult because of their small effect sizes. To better explain disease mechanisms, pathway analysis is used to consolidate the effects of multiple variants, and hence increase the power of the study. While pathway analysis has previously been performed within GWAS only, it can now be extended to examining rare variants, other “-omics” and interaction data. Scope of review 1. Factors to consider in the choice of software for GWAS pathway analysis. 2. Examples of how pathway analysis is used to analyse rare variants, other “-omics” and interaction data. Major conclusions To choose appropriate software tools, factors for consideration include covariate compatibility, null hypothesis, one- or two-step analysis required, curation method of gene sets, size of pathways, and size of flanking regions to define gene boundaries. For rare variants, analysis performance depends on consistency between assumed and actual effect distribution of variants. Integration of other “-omics” data and interaction can better explain gene functions. General significance Pathway analysis methods will be more readily used for integration of multiple sources of data, and enable more accurate prediction of phenotypes.

Identification of genetic variations related to high myopia may advance our knowledge of the etiopathogenesis of refractive error. This study investigated the role of potassium channel gene (KCNQ5) polymorphisms in high myopia. We performed a case-control study of 1563 unrelated Han Chinese subjects (809 cases of high myopia and 754 emmetropic controls). Five tag single-nucleotide polymorphisms (SNPs) of KCNQ5 were genotyped, and association testing with high myopia was conducted using logistic regression analysis adjusted for sex and age to give Pasym values, and multiple comparisons were corrected by permutation test to give Pemp values. All five noncoding SNPs were associated with high myopia. The SNP rs7744813, previously shown to be associated with refractive error and myopia in two GWAS, showed an odds ratio of 0.75 (95% CI 0.63–0.90; Pemp = 0.0058) for the minor allele. The top SNP rs9342979 showed an odds ratio of 0.75 (95% CI 0.64–0.89; Pemp = 0.0045) for the minor allele. Both SNPs are located within enhancer histone marks and DNase-hypersensitive sites. Our data support the involvement of KCNQ5 gene polymorphisms in the genetic susceptibility to high myopia and further exploration of KCNQ5 as a risk factor for high myopia.

Purpose Gap junctions provide a conduit between the intracellular fluids of the pigmented (PE) and non-pigmented (NPE) ciliary epithelial cells, and are therefore critical in the secretion of the aqueous humor (AH). However, opinions differ concerning the connexin (Cx) composition of the gap junctions. Therefore, we aimed to characterize the expression of Cx in the porcine ciliary epithelium (CE), a favorable model for humans; and determine the contribution of the highest expressed Cx to AH secretion. Methods Freshly-harvested porcine CE cells were used. The mRNA and protein expressions of gap junctions were assessed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (WB), respectively. The relative gene expressions of various Cx were determined by quantitative PCR. The gap junction permeability of isolated PE-NPE cell couplets was evaluated by Lucifer Yellow dye transfer. Results Using RT-PCR and WB, Cx43, Cx45, Cx47, Cx50, and Cx60 were present in porcine CE, with Cx43 being the most abundant isoform, having over 200-fold higher expression than other Cx. Cx43 was primarily localized in the PE-NPE interface and the basolateral membranes of PE cells. Knockdown of Cx43 by siRNA significantly reduced gene and protein expressions, resulting in reduction of transcellular fluid flow by 90%. Conclusions Cx43 was found to be the major component of gap junctions in porcine CE. Consistent with results from a bovine model, our results support the important role of Cx43 in mediating AH secretion. This finding may shed light on the development of a novel ocular hypotensive agent.

Study of the exacerbating effects of chemotherapeutics, such as doxorubicin, on the impairment of insulin metabolic signaling in aged skeletal muscle is very limited. Here, we tested the hypothesis that activation of sirtuin 1 deacetylase activity by resveratrol would prevent the disruption of insulin signaling and augmentation of catabolic markers induced by doxorubicin in aged skeletal muscle. Two- and 10-month-old senescence-accelerated mice (prone 8) were randomized to receive saline, doxorubicin, doxorubicin and resveratrol, or a combination of doxorubicin, resveratrol, and sirtinol or EX527. Doxorubicin reduced the sirtuin 1 activity without affecting the phosphorylation levels of IRS1(Ser307), mTOR(Ser2481), Akt(Thr308/Ser473), membranous glucose transporter 4, protein abundance of PDK4, and enzymatic activity of pyruvate dehydrogenase in aged muscles. Intriguingly, resveratrol attenuated the doxorubicin-induced elevations of apoptotic and catabolic markers measured as Bax, caspase 3 activity, apoptotic DNA fragmentation, MuRF-1, ubiquitinated proteins, and proteasomal activity in aged muscles, whereas these beneficial effects were abolished on inhibition of sirtuin 1 by sirtinol or EX527. Markers of insulin signaling were not affected by doxorubicin or resveratrol in the senescent skeletal muscle. Nevertheless, the antiapoptotic and anticatabolic effects of resveratrol in aged skeletal muscle treated with doxorubicin were mediated in a sirtuin 1-dependent signaling manner.

Impairment of insulin signaling in skeletal muscle detrimentally affects insulin-stimulated disposal of glucose. Restoration of insulin signaling in skeletal muscle is important as muscle is one of the major sites for disposal of blood glucose. Recently, unacylated ghrelin (UnAG) has received attention in diabetic research due to its favorable actions on improving glucose tolerance, glycemic control, and insulin sensitivity. The investigation of UnAG has entered phase Ib clinical trial in type 2 diabetes and phase II clinical trial in hyperphagia in Prader-Willi syndrome. Nonetheless, the precise mechanisms responsible for the anti-diabetic actions of UnAG remain incompletely understood. In this study, we examined the effects of UnAG on restoring the impaired insulin signaling in skeletal muscle of db/db diabetic mice. Our results demonstrated that UnAG effectively restored the impaired insulin signaling in diabetic muscle. UnAG decreased insulin receptor substrate (IRS) phosphorylation, increased protein kinase B (Akt) phosphorylation, and, hence, suppressed mTOR signaling. Consequently, UnAG enhanced Glut4 localization and increased PDH activity in the diabetic skeletal muscle. Intriguingly, our data indicated that UnAG normalized the suppressed autophagic signaling in diabetic muscle. In conclusion, our findings illustrated that UnAG restored the impaired insulin and autophagic signaling in skeletal muscle of diabetic mice, which are valuable to understand the underlying mechanisms of the anti-diabetic action of UnAG at peripheral skeletal muscle level.

Activation of Foxo1 is known to promote apoptosis and disturbances to insulin signalling. However, their modulating roles in aged skeletal muscle are not clear. The present study tested the hypothesis that long-term (i.e. 8 month) resveratrol supplementation would improve physical traits including exercise capacity and basal voluntary activity of aged mice and modulate insulin/apoptotic signalling in aged skeletal muscle. This study also examined whether these resveratrol-associated alterations would involve orchestration of the SIRT1-Foxo1 signalling axis.Two-month-old SAMP8 mice were randomly assigned to young, aged and aged with resveratrol treatment (AR) groups. The AR mice were supplemented with 4.9 mg(-1) kg(-1) day(-1) resveratrol for 8 months. All animals were subject to endurance capacity test and voluntary motor behaviour assessment. The lateral gastrocnemius muscle tissues were harvested for further analyses.Long-term resveratrol treatment significantly alleviated the age-associated reductions in exercise capacity and voluntary motor behaviour. The protein content, but not the deacetylase activity of SIRT1 was increased with concomitant elevations of p300 acetylase and acetylation of Foxo1 in aged muscle. The aged muscle also manifested signs of impaired insulin signalling including attenuated phosphorylation of Akt, activity of pyruvate dehydrogenase and membrane trafficking of GLUT4 and elevated levels of phosphorylated IRS1 and iNOS and apoptotic activation measured as Bim, p53 and apoptotic DNA fragmentation. Intriguingly, all these age-related adverse changes were mitigated with the activation of SIRT1 deacetylase activity after long-term resveratrol treatment.These data suggest that modulation of the SIRT1-Foxo1 axis by long-term resveratrol treatment enhances physical traits and alleviates the unfavourable changes in insulin and apoptotic signalling in aged muscle.

Purpose To comprehensively evaluate and compare the degree of carotid atherosclerosis in patients treated with radiotherapy (RT) for nasopharyngeal carcinoma (NPC) and in patients with type 2 diabetes mellitus (DM), and using healthy subjects as controls. Materials and Methods The present study recruited 69 post-RT NPC patients without conventional cardiovascular risk factors, 70 type 2 diabetic patients without previous RT, and 76 healthy controls without conventional cardiovascular risk factors and previous RT. For each participant, 5 carotid atherosclerotic parameters, namely carotid intima-media thickness (CIMT), carotid arterial stiffness (CAS), presence of carotid plaque, carotid plaque score, and presence of ≥ 50 % carotid stenosis, were assessed using ultrasonography. The differences in these carotid atherosclerotic parameters between study groups were compared using ANCOVA or logistic regression after the adjustment for age and gender. Multiple comparisons were corrected using the Benjamini-Hochberg false discovery rate. Results Post-RT NPC patients and type 2 diabetics had a significantly higher CIMT, CAS and carotid plaque burden compared to the healthy subjects (corrected P-value, Pcor Conclusion Neck RT for NPC is an independent risk factor of carotid atherosclerosis, and radiation induces more severe carotid atherosclerosis in post-RT NPC patients. Thus, assessment of carotid atherosclerosis using ultrasonography may be necessary for these patients and should be indicated in the routine follow-up of NPC.

Key points Doxorubicin induced functional deteriorations and elevations of USP7-related apoptotic/catabolic signalling in the senescent heart Resveratrol protects against doxorubicin-induced alterations through the restoration of SIRT1 deacetylase activity Abstract A compromised cardiac function is often seen in elderly cancer patients receiving doxorubicin therapy. The present study tested the hypothesis that acute intervention with resveratrol, a natural anti-oxidant found in grapes and red wine, reduces the cardiotoxicity of doxorubicin through restoration of sirtuin 1 (SIRT1) deacetylase activity, and attenuation of the catabolic/apoptotic pathways orchestrated by USP7, a p53 deubiquitinating protein, using young (aged 2 months) and old (aged 10 months) senescence-accelerated mice prone 8 (SAMP8). Animals were randomised to receive saline, doxorubicin, and doxorubicin in combination with resveratrol, in the presence or absence of SIRT1 inhibitors, sirtinol or EX527. Resveratrol alone, but not in combination with either of the SIRT1 inhibitors, suppressed the doxorubicin-induced impairment of cardiac systolic function in aged animals. Doxorubicin reduced SIRT1 deacetylase activity, and elevated proteasomal activity and USP7; it also increased the protein level of p300 and ubiquitinated proteins in hearts from aged SAMP8. These doxorubicin-induced alterations were prevented by resveratrol, whereas the protective action of resveratrol was antagonised by sirtinol and EX527. In young SAMP8 hearts, resveratrol attenuated the doxorubicin-induced increases in acetylation of Foxo1 and transactivation of MuRF-1, whereas these mitigations were not found after treatment with SIRT1 inhibitors. However, the protein contents of acetylated Foxo1 and MuRF-1 were not affected by any of the drugs studied in aged SAMP8 hearts. Resveratrol also ameliorated the augmentation of pro-apoptotic markers including p53, Bax, caspase 3 activity and apoptotic DNA fragmentation induced by doxorubicin in hearts from aged animals, whereas these reductions were diminished by combined treatment with SIRT1 inhibitors. These data demonstrate that resveratrol ameliorates doxorubicin-induced cardiotoxicity in aged hearts through the restoration of SIRT1 activity to attenuate USP7-related catabolic/pro-apoptotic signalling.

[D-Lys3]-GHRP-6 is regarded as a highly selective growth-hormone secretagogue receptor (GHSR) antagonist and has been widely used to investigate the dependency of GHSR-1a signalling mediated by acylated ghrelin. However, [D-Lys3]-GHRP-6 has been reported to influence other cellular processes which are unrelated to GHSR-1a. This study aimed to examine the effects of [D-Lys3]-GHRP-6 on autophagic and apoptotic cellular signalling in skeletal muscle. [D-Lys3]-GHRP-6 enhanced the autophagic signalling demonstrated by the increases in protein abundances of beclin-1 and LC3 II-to-LC3 1 ratio in both normal muscle and doxorubicin-injured muscle. [D-Lys3]-GHRP-6 reduced the activation of muscle apoptosis induced by doxorubicin. No histological abnormalities were observed in the [D-Lys3]-GHRP-6-treated muscle. Intriguingly, the doxorubicin-induced increase in centronucleated muscle fibres was not observed in muscle treated with [D-Lys3]-GHRP-6, suggesting the myoprotective effects of [D-Lys3]-GHRP-6 against doxorubicin injury. The [D-Lys3]-GHRP-6-induced activation of autophagy was found to be abolished by the co-treatment of CXCR4 antagonist, suggesting that the pro-autophagic effects of [D-Lys3]-GHRP-6 might be mediated through CXCR4. In conclusion, [D-Lys3]-GHRP-6 exhibits pro-autophagic effects on skeletal muscle under both normal and doxorubicin-injured conditions.

EGFR signaling pathway and microRNAs (miRNAs) are two important factors for development and treatment in non-small cell lung cancer (NSCLC). Microarray analysis enables the genome-wide expression profiling. However, the information from microarray data may not be fully deciphered through the existing approaches. In this study we present an mRNA:miRNA stepwise regression model supported by miRNA target prediction databases. This model is applied to explore the roles of miRNAs in the EGFR signaling pathway. The results show that miR-145 is positively associated with epidermal growth factor (EGF) in the pre-surgery NSCLC group and miR-199a-5p is positively associated with EGF in the post-surgery NSCLC group. Surprisingly, miR-495 is positively associated with protein tyrosine kinase 2 (PTK2) in both groups. The coefficient of determination (R(2)) and leave-one-out cross-validation (LOOCV) demonstrate good performance of our regression model, indicating that it can identify the miRNA roles as oncomirs and tumor suppressor mirs in NSCLC.

Shear wave elastography provides information about the stiffness of thyroid nodules that could be a new indicator of malignancy. The current study aimed to investigate the feasibility of using shear wave elastography (SWE) alone and in conjunction with grey scale ultrasound (GSU) to predict malignancy in 111 solitary thyroid nodules. Malignant thyroid nodules tended to have microcalcification, hypoechogenicity, tall to width ratio >1, and irregular borders (p < 0.05). SWE indices (Emaximum and Emean) of malignant nodules (median ± standard error: 85.2 ± 8.1 kPa and 26.6 ± 2.5 kPa) were significantly higher than those of benign nodules (median ± standard error: 50.3 ± 3.1 kPa and 20.2 ± 1 kPa) (p < 0.05). The optimal cut-off of Emaximum and Emean for distinguishing benign and malignant nodules was 67.3 kPa and 23.1 kPa, respectively. Diagnostic performances for GSU + Emaximum, GSU + Emean, GSU, Emaximum and Emean were: 70.4%, 74.1%, 96.3%, 70.4% and 74.1% for sensitivity, 83.3%, 79.8%, 46.4%, 70.2%, and 66.7% for specificity, and 80.2%, 78.4%, 58.5%, 70.3%, and 68.5% for accuracy, respectively. Our results suggested that combining GSU with SWE (using Emaximum or Emean) increased the overall diagnostic accuracy in distinguishing benign and malignant thyroid nodules.

Myopia and astigmatism, two common refractive errors frequently co-exist, are affecting vision at all working distances in the affected populations worldwide. Eyeballs having these refractive errors are known to exhibit abnormal eye shape at the anterior and posterior eye segments, but whether the outer coats of these abnormal eyeballs, cornea anteriorly and sclera posteriorly, are regulated by region-specific molecular mechanism remains unclear. Here we presented the changes in mRNA expression levels of three genes (MMP2, TIMP2, and TGFB2), all known to participate in extracellular matrix organization, at five regions of the cornea and sclera in chickens developing high myopia and astigmatism induced by form deprivation. We found that, compared to normal chicks, the highly myopic-astigmatic chicks had significantly higher expression of all three genes in the superior sclera (Mann-Whitney tests, all p ≤ 0.05), as well as higher TIMP2 expression in the central cornea and nasal sclera (Mann-Whitney tests, both p ≤ 0.05). Strikingly, the superior scleral region stood out as showing the strongest and most widespread correlations between mRNA expression and biometry parameters including axial and astigmatic components (r = + 0.52~ + 0.85, all p