Method for the elucidation of LAMP products captured on lateral flow strips in a point of care test for HPV 16.

Loop-mediated amplification (LAMP) is an isothermal amplification technique favored in diagnostics and point-of-care work due to its high sensitivity and ability to run in isothermal conditions. In addition, a visual readout by lateral flow strips (LFS) can be used in conjunction with LAMP, making the assay accessible at the point-of-care. However, the amplicons resulting from a LAMP reaction varied in length and shape, making them undiscernible on a double-stranded DNA intercalating dye stained gel. Standard characterization techniques also do not identify which amplicons specifically bind to the LFS, which generate the visual readout. We aimed to standardize our characterization of LAMP products during assay development by using fluorescein amidite (FAM) and biotin-tagged loop forward and backward primers during assay development. A pvuII restriction enzyme digest is applied to the LAMP products. FAM-tagged bands are directly correlated with the LFS visual readout. We applied this assay development workflow for an HPV 16 assay using both plasmid DNA and clinical samples to demonstrate proof of concept for generalized assay development work.

A Sec14 domain protein is required for photoautotrophic growth and chloroplast vesicle formation in Arabidopsis thaliana.

In eukaryotic photosynthetic organisms, the conversion of solar into chemical energy occurs in thylakoid membranes in the chloroplast. How thylakoid membranes are formed and maintained is poorly understood. However, previous observations of vesicles adjacent to the stromal side of the inner envelope membrane of the chloroplast suggest a possible role of membrane transport via vesicle trafficking from the inner envelope to the thylakoids. Here we show that the model plant Arabidopsis thaliana has a chloroplast-localized Sec14-like protein (CPSFL1) that is necessary for photoautotrophic growth and vesicle formation at the inner envelope membrane of the chloroplast. The cpsfl1 mutants are seedling lethal, show a defect in thylakoid structure, and lack chloroplast vesicles. Sec14 domain proteins are found only in eukaryotes and have been well characterized in yeast, where they regulate vesicle budding at the trans-Golgi network. Like the yeast Sec14p, CPSFL1 binds phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA) and acts as a phosphatidylinositol transfer protein in vitro, and expression of Arabidopsis CPSFL1 can complement the yeast sec14 mutation. CPSFL1 can transfer PIP into PA-rich membrane bilayers in vitro, suggesting that CPSFL1 potentially facilitates vesicle formation by trafficking PA and/or PIP, known regulators of membrane trafficking between organellar subcompartments. These results underscore the role of vesicles in thylakoid biogenesis and/or maintenance. CPSFL1 appears to be an example of a eukaryotic cytosolic protein that has been coopted for a function in the chloroplast, an organelle derived from endosymbiosis of a cyanobacterium.

Experimental and numerical evaluation of a genetically engineered M13 bacteriophage with high sensitivity and selectivity for 2,4,6-trinitrotoluene.

Selective and sensitive detection of desired targets is very critical in sensor design. Here, we report a genetically engineered M13 bacteriophage-based sensor system evaluated by quantum mechanics (QM) calculations. Phage display is a facile way to develop the desired peptide sequences, but the resulting sequences can be imperfect peptides for binding of target molecules. A TNT binding peptide (WHW) carrying phage was self-assembled to fabricate thin films and tested for the sensitive and selective surface plasmon resonance-based detection of TNT molecules at the 500 femtomole level. SPR studies performed with the WHW peptide and control peptides (WAW, WHA, AHW) were well-matched with those of the QM calculations. Our combined method between phage engineering and QM calculation will significantly enhance our ability to design selective and sensitive sensors.

Prevalence of neutralising antibodies to interferon-beta and clinical response in Chinese patients with relapsing multiple sclerosis.

BACKGROUND: There are no data on neutralising antibodies to interferon-beta and its clinical implications in Chinese patients with multiple sclerosis (MS). OBJECTIVES: The objectives of this study were to investigate the prevalence of neutralising antibodies among Chinese patients with relapsing MS receiving interferon-beta (1a or 1b) and to study the association between neutralising antibodies and the clinical-radiological response. METHODS: We performed a cross-sectional study on MS patients who received interferon-beta for 9 months or more, and evaluated the clinical response by relapses and magnetic resonance imaging lesions. Blood samples were evaluated for myxovirus resistance protein A (MxA) gene expression by polymerase chain reaction, anti-interferon-beta binding antibodies by enzyme-linked immunosorbent assay, and neutralising antibodies by cell-based MxA protein induction and luciferase reporter gene assays. Assay performances were evaluated by receiver operating characteristic analysis. RESULTS: Among 78 subjects recruited, 61/77 (79%) had anti-interferon-beta binding antibodies, and 22/78 (28%) had neutralising antibodies by MxA protein induction assay. The presence of high-titre neutralising antibodies was associated with poor clinical outcome (odds ratio 6.1, 95% confidence interval 1.5-25.6, P = 0.013). The sensitivity and specificity for neutralising antibodies using MxA gene expression assay (cut-off 0.20) was 80% and 68%, respectively (area under the curve 0.71). CONCLUSIONS: Neutralising antibodies are associated with poor clinical outcome in Chinese patients with relapsing MS. MxA gene expression and protein induction assays are complimentary assays for neutralising antibody detection.

Outcomes of post-prostatectomy radiotherapy at a Regional Cancer Centre.

INTRODUCTION: To investigate the efficacy and toxicity of radiation therapy (RT) after radical prostatectomy (RP) for prostate cancer at Radiation Oncology Centres, Toowoomba. METHODS: The electronic medical records of 130 consecutive patients with histologically proven prostate adenocarcinoma who underwent post-prostatectomy RT between January 2008 and December 2014 were analysed. Primary endpoint was Biochemical Recurrence (BCR) after RT. BCR was defined by PSA > 0.2 ng/mL and BCR endpoints were analysed using Kaplan-Meier methods. The impact of RT technique and the rates of acute and late toxicities are also reported. Toxicities were graded according to Radiation Therapy Oncology Group (RTOG) criteria. RESULTS: Median follow-up time after RT (regardless of technique) was 28 months. BCR occurred in 32 of the 126 patients (25%) whose prostate specific antigen (PSA) levels have been monitored post-RT. At 24 and 36 months, 85% and 75% of patients were BCR-free, respectively. Patients with a pre-RT PSA above 0.2 ng/mL had a higher probability of recurrence than patients with values below 0.2 ng/mL (P = 0.03). RT technique, pelvic nodal irradiation, androgen deprivation therapy, T staging or surgical margin did not significantly impact BCR results. No patient experienced acute toxicities greater than grade 2. Grade 1 or 2 late gastrointestinal (GI) toxicity occurred in 11% and 1 patient experienced a grade 3 event. 12% of patients developed grade 1 or 2 late genitourinary (GU) toxicity, with evidence of grade 3 severity in only 1 patient. Evidence of a trend in reduction in late GI toxicity with the use of intensity modulated radiation therapy (IMRT) or volumetric modulated arc therapy (VMAT) was apparent but not with late GU toxicity. CONCLUSION: At our regional centre, early RT (PSA < 0.2 ng/mL) was associated with significant improvement in BCR-free survival. Rates of toxicity mirror those of landmark trials which suggest no detriment for our regional prostate cancer patients. The use of IMRT/VMAT techniques was associated with a trend towards reduced rates of GI toxicity.

Coordinated regulation of acid resistance in Escherichia coli.

BACKGROUND: Enteric Escherichia coli survives the highly acidic environment of the stomach through multiple acid resistance (AR) mechanisms. The most effective system, AR2, decarboxylates externally-derived glutamate to remove cytoplasmic protons and excrete GABA. The first described system, AR1, does not require an external amino acid. Its mechanism has not been determined. The regulation of the multiple AR systems and their coordination with broader cellular metabolism has not been fully explored. RESULTS: We utilized a combination of ChIP-Seq and gene expression analysis to experimentally map the regulatory interactions of four TFs: nac, ntrC, ompR, and csiR. Our data identified all previously in vivo confirmed direct interactions and revealed several others previously inferred from gene expression data. Our data demonstrate that nac and csiR directly modulate AR, and leads to a regulatory network model in which all four TFs participate in coordinating acid resistance, glutamate metabolism, and nitrogen metabolism. This model predicts a novel mechanism for AR1 by which the decarboxylation enzymes of AR2 are used with internally derived glutamate. This hypothesis makes several testable predictions that we confirmed experimentally. CONCLUSIONS: Our data suggest that the regulatory network underlying AR is complex and deeply interconnected with the regulation of GABA and glutamate metabolism, nitrogen metabolism. These connections underlie and experimentally validated model of AR1 in which the decarboxylation enzymes of AR2 are used with internally derived glutamate.

Epigallocatechin Gallate (EGCG) Inhibits Alpha-Synuclein Aggregation: A Potential Agent for Parkinson's Disease.

Protein aggregation is a prominent feature of many neurodegenerative disorders including Parkinson's disease (PD). Aggregation of alpha-synuclein (SNCA) may underlie the pathology of PD. They are the main components of Lewy bodies and dystrophic neurites that are the intraneuronal inclusions characteristic of the disease. We have demonstrated that the polyphenol (-)-epi-gallocatechine gallate (EGCG) inhibited SNCA aggregation, which made it a candidate for therapeutic intervention in PD. Three methods were used: SNCA fibril formation inhibition by EGCG in incubates; inhibition of the SNCA fluorophore A-Syn-HiLyte488 binding to plated SNCA in microwells; and inhibition of the A-Syn-HiLyte488 probe binding to aggregated SNCA in postmortem PD tissue. Recombinant human SNCA was incubated under conditions that result in fibril formation. The aggregation was blocked by 100 nM EGCG in a concentration-dependent manner, as shown by an absence of thioflavin T binding. In the microplate assay system, the ED50 of EGCG inhibition of A-Syn-HiLyte488 binding to coated SNCA was 250 nM. In the PD tissue based assay, SNCA aggregates were recognized by incubation with 7 nM of A-Syn-HiLyte488. This binding was blocked by EGCG in a concentration dependent manner. The SNCA amino acid sites, which potentially interacted with EGCG, were detected on peptide membranes. It was implicated that EGCG binds to SNCA by instable hydrophobic interactions. In this study, we suggested that EGCG could be a potent remodeling agent of SNCA aggregates and a potential disease modifying drug for the treatment of PD and other α-synucleinopathies.

Rapid Detection of Bacteria from Blood with Surface-Enhanced Raman Spectroscopy.

Traditional methods for identifying pathogens in bacteremic patients are slow (24-48+ h). This can lead to physicians making treatment decisions based on an incomplete diagnosis and potentially increasing the patient's mortality risk. To decrease time to diagnosis, we have developed a novel technology that can recover viable bacteria directly from whole blood and identify them in less than 7 h. Our technology combines a sample preparation process with surface-enhanced Raman spectroscopy (SERS). The sample preparation process enriches viable microorganisms from 10 mL of whole blood into a 200 µL aliquot. After a short incubation period, SERS is used to identify the microorganisms. We further demonstrated that SERS can be used as a broad detection method, as it identified a model set of 17 clinical blood culture isolates and microbial reference strains with 100% identification agreement. By applying the integrated technology of sample preparation and SERS to spiked whole blood samples, we were able to correctly identify both Staphylococcus aureus and Escherichia coli 97% of the time with 97% specificity and 88% sensitivity.

A fully integrated paperfluidic molecular diagnostic chip for the extraction, amplification, and detection of nucleic acids from clinical samples.

Paper diagnostics have successfully been employed to detect the presence of antigens or small molecules in clinical samples through immunoassays; however, the detection of many disease targets relies on the much higher sensitivity and specificity achieved via nucleic acid amplification tests (NAAT). The steps involved in NAAT have recently begun to be explored in paper matrices, and our group, among others, has reported on paper-based extraction, amplification, and detection of DNA and RNA targets. Here, we integrate these paper-based NAAT steps into a single paperfluidic chip in a modular, foldable system that allows for fully integrated fluidic handling from sample to result. We showcase the functionality of the chip by combining nucleic acid isolation, isothermal amplification, and lateral flow detection of human papillomavirus (HPV) 16 DNA directly from crude cervical specimens in less than 1 hour for rapid, early detection of cervical cancer. The chip is made entirely of paper and adhesive sheets, making it low-cost, portable, and disposable, and offering the potential for a point-of-care molecular diagnostic platform even in remote and resource-limited settings.

Overexpression of Tyro3 and its implications on hepatocellular carcinoma progression.

While various tyrosine kinases have been associated with the pathogenesis of hepatocellular carcinoma (HCC), the identification of a dominant therapeutic target among them remains a challenge. Here, we investigated the role of Tyro3, a relatively uncharacterized member of the TAM (Tyro3, Axl and Mer) receptor family. The present study aimed to profile and identify potential association between Tyro3 expression in HCC and cancer phenotypes. RNAs obtained from 55 HCC patients were quantified for Tyro3 expression in both cancerous tissue and the adjacent normal tissue. Expression profile was correlated with clinical data. These observations were further substantiated with in vitro HCC cell culture investigations.Tyro3 was strongly upregulated (>2-fold elevation) in the tumor tissue of ~42% of the patients. It was shown that higher expression level of Tyro3 was associated with the key tumor marker AFP, and the tumor diameter and liver injury marker ALT. Subsequent cell culture models indicated high expression in various HCC cell lines, in particular Hep3B. Gene silencing of Tyro3 in Hep3B effectively reduced cell proliferation, ERK phosphorylation and cyclin D1 expression, indicating a key in maintaining the proliferative state of these cells. Notably, silencing also suppressed the transcriptional and translational expression of HCC tumor marker AFP. Overall, these data suggest that Tyro3 contributes significantly to tumor growth, aggressiveness and liver dysfunction. Inhibition of Tyro3 and its aberrant signaling in tumors with high expression could present new opportunities for HCC treatment.

Selective and Sensitive Sensing of Flame Retardant Chemicals Through Phage Display Discovered Recognition Peptide.

We report a highly selective and sensitive biosensor for the detection of an environmentally toxic molecule, decabrominated diphenyl ether (DBDE), one of the most common congeners of the polybrominated frame retardants (polybrominated diphenyl ether (PBDE)), using newly discovered DBDE peptide receptors integrated with carbon nanotube field-effect transistors (CNT-FET). The specific DBDE peptide receptor was identified using a high-throughput screening process of phage library display. The resulting binding peptide carries an interesting consensus binding pocket with two Trp-His/Asn-Trp repeats, which binds to the DBDE in a multivalent manner. We integrated the novel DBDE binding peptide onto the CNT-FET using polydiacetylene coating materials linked through cysteine-maleimide click chemistry. The resulting biosensor could detect the desired DBDE selectively with a 1 fM detection limit. Our combined approaches of selective receptor discovery, material nanocoating through click chemistry, and integration onto a sensitive CNT-FET electronic sensor for desired target chemicals will pave the way toward the rapid development of portable and easy-to-use biosensors for desired chemicals to protect our health and environment.

Increased number of structured diabetes education attendance was not associated with the improvement in patient-reported health-related quality of life: results from Patient Empowerment Programme (PEP).

AIMS: To assess the effect of a structured education intervention, Patient Empowerment Programme (PEP) patient-reported health-related quality of life (HRQOL) among type 2 diabetes mellitus (T2DM) patients, and if positive effect is confirmed, to further explore any association between frequency of sessions attendance and HRQOL. METHODS: A total of 298 T2DM patients were recruited when they attended the first session of PEP, between March and September 2010, and were followed over a one-year period from baseline. HRQOL data were assessed using Short Form-12 Health Survey version 2 (SF-12) and Short Form-6 Dimension (SF-6D) at baseline and one-year follow-up. Individuals' anthropometric and biomedical data were extracted from an administrative database in Hong Kong. Unadjusted and adjusted analyses of linear regression models were performed to examine the impact of PEP session attendance on the change in the HRQOL scores, accounting for the socio-demographic and clinical characteristics at baseline. RESULTS: Of the 298 eligible patients, 257 (86.2%) participated in the baseline assessment and 179 (60.1%) patients completed the follow-up assessment, respectively. Overall, PEP resulted in a significant improvement in SF-12 bodily pain and role emotional subscales and SF-6D utility scores. These positive changes were not associated with the level of participation as shown in both unadjusted and adjusted analyses. CONCLUSIONS: The PEP made significant improvement in bodily pain, role emotional and overall aspects of HRQOL. Higher number of session attendance was not associated with improvement in HRQOL in primary care real-world setting. Key Messages ● Participants with type 2 diabetes mellitus who participated in structured diabetes education programme made significant improvement in bodily pain and role emotional subscales and SF-6D scores. ● There was no association between the number of sessions attended and any aspect of HRQOL.

Longitudinal Changes in Retinal Nerve Fibre Layer Thickness after an Isolated Unilateral Retrobulbar Optic Neuritis: 1-Year Results.

The objective of this study was to investigate the longitudinal changes in retinal nerve fibre layer (RNFL) thickness 1 year after an episode of unilateral acute optic neuritis. This prospective cohort study recruited consecutive patients with a first episode of isolated, unilateral acute optic neuritis from October 2010 to June 2013. RNFL thickness of the attack and normal fellow eyes was measured by optical coherence tomography on presentation and 3, 6, and 12 months post attack in both the treatment and non-treatment groups. The treatment group consisted of subjects that opted for systemic steroids to hasten recovery time. In 20 subjects, 11 received systemic steroids and 9 were treated conservatively. The baseline RNFL thickness was similar in the attack and fellow eyes (p ≥ 0.4). Progressive RNFL thinning was seen in the attack eye over the 12-month period, with significant differences for baseline versus 3 months; baseline versus 12 months; and 3 versus 12 months (all p < 0.0001). At 12 months, the attack eye had a thinner average RNFL than the fellow eye (100.9 ± 6.1 versus 107.3 ± 5.5 µm; p = 0.002). The 12-month RNFL was similar between the treatment and non-treatment groups (p ≥ 0.6). A single episode of optic neuritis triggered an accelerated, progressive RNFL thinning up to 6 months post attack. Initial treatment with systemic steroids did not seem to alter the degree of RNFL loss at 12 months.

Glycemia and cognitive function in metabolic syndrome and coronary heart disease.

OBJECTIVE: Higher hemoglobin A1c (HbA1c) is associated with lower cognitive function in type 2 diabetes. To determine whether associations persist at lower levels of dysglycemia in patients who have established cardiovascular disease, cognitive performance was assessed in the Targeting INflammation Using SALsalate in CardioVascular Disease (TINSAL-CVD) trial. METHODS: The age-adjusted relationships between HbA1c and cognitive performance measured by the Mini-Mental State Examination, Digit Symbol Substitution Test, Rey Auditory Verbal Learning Test, Trail Making Test, and Categorical Verbal Fluency were assessed in 226 men with metabolic syndrome and established stable coronary artery disease. RESULTS: Of the participants, 61.5% had normoglycemia, 20.8% had impaired fasting glucose, and 17.7% had type 2 diabetes. HbA1c was associated with cognitive function tests of Digit Symbol Substitution Test, Rey Auditory Verbal Learning Test, Trail Making Test, and Categorical Verbal Fluency (all P < .02), but not the Mini-Mental State Examination. In an age-adjusted model, a 1% (11 mmol/mol) higher HbA1c value was associated with a 5.9 lower Digit Symbol Substitution Test score (95% confidence interval [CI], -9.58 to -2.21; P < .0001); a 2.44 lower Rey Auditory Verbal Learning Test score (95% CI, -4.00 to -0.87; P < .0001); a 15.6 higher Trail Making Test score (95% CI, 5.73 to 25.6; P < .0001); and a 3.71 lower Categorical Verbal Fluency score (95% CI, -6.41 to -1.01; P < .02). In a multivariate model adjusting for age, education, and cardiovascular covariates, HbA1c remained associated with cognitive function tests of Rey Auditory Verbal Learning Test (R(2) = 0.27, P < .0001), Trail Making Test (R(2) = 0.18, P < .0001), and Categorical Verbal Fluency (R(2) = 0.20, P < .0001), although association with the Digit Symbol Substitution Test was reduced. CONCLUSIONS: Higher HbA1c is associated with lower cognitive function performance scores across multiple domain tests in men with metabolic syndrome and coronary artery disease. Future studies may demonstrate whether glucose lowering within the normative range improves cognitive health.

Optic neuritis in Hong Kong: a 1-year follow-up study.

To investigate the etiology and prevalence of optic neuritis in a Chinese population. This was a single centre prospective cohort study. Consecutive patients with either a first or recurrent attack of optic neuritis from November 2010 to December 2011 were recruited from a district hospital in Hong Kong Special Administrative Region, China. All patients underwent serology testing for NMO (neuromyelitis optica) IgG; oligoclonal bands from lumbar puncture; computer tomography and contrast magnetic resonance imaging (MRI) of the brain and orbit as well as visual field; and optical coherence tomography testing. Patients were followed up for 1 year after the initial attack. 30 optic neuritis subjects were recruited. 73.3 % (22/30) remain as clinical isolated syndrome (CIS) after 1-year follow-up. 10 % (3/30) patients developed multiple sclerosis. 10 % (3/30) were diagnosed with NMO and 6.7 % (2/30) with NMO-spectrum disorder. The majority of acute unilateral optic neuritis in Chinese was CIS in origin although a fraction does progress to develop MS or NMO-related disorders. Clinicians should be aware of the associations and offer appropriate systemic workups.

Energy weighted x-ray dark-field imaging.

The dark-field image obtained in grating-based x-ray phase-contrast imaging can provide information about the objects' microstructures on a scale smaller than the pixel size even with low geometric magnification. In this publication we demonstrate that the dark-field image quality can be enhanced with an energy-resolving pixel detector. Energy-resolved x-ray dark-field images were acquired with a 16-energy-channel photon-counting pixel detector with a 1 mm thick CdTe sensor in a Talbot-Lau x-ray interferometer. A method for contrast-noise-ratio (CNR) enhancement is proposed and validated experimentally. In measurements, a CNR improvement by a factor of 1.14 was obtained. This is equivalent to a possible radiation dose reduction of 23%.

Incremental changes verses a technological quantum leap: the additional value of intensity-modulated radiotherapy beyond image-guided radiotherapy for prostate irradiation.

INTRODUCTION: Both intensity-modulated radiotherapy (IMRT) and image-guided radiotherapy (IGRT) have been shown to independently reduce late rectal toxicity for men with prostate cancer (PC) treated with radiotherapy. We explore whether IMRT offers further reductions in late rectal toxicity for men already being managed with IGRT and compare this with more gradual changes over time. METHODS: Between 2007 and 2009, 103 patients with PC were treated with three-dimensional conformal radiotherapy (3D-CRT n = 52) or IMRT (n = 51) with doses of 74­78 Gy at 2 Gy per fraction. All men had daily IGRT using intra-prostatic gold fiducials. The primary endpoint was incidence of grade ≥2 late rectal toxicity as graded by the Radiation Therapy Oncology Group scale. RESULTS: The relative risk of late grade ≥2 rectal toxicity in patients treated with IMRT was 68% less than seen with image-guided 3D-CRT at 36 months post-treatment (7% versus 22%; hazard ratio = 0.32, P = 0.03). IMRT remained a significant protective factor in a multivariate analysis. A discriminant analysis showed that the relative volume of rectal wall exposed to doses over 50 Gy was most strongly associated with late rectal toxicity. Controlling for duration of follow-up, a later year of treatment was the strongest clinical predictor of late rectal toxicity in multivariate modelling (P = 0.03). CONCLUSION: For men with PC managed to doses of 74­78 Gy with IGRT, IMRT leads to reduced rectal toxicity compared with 3D-CRT. Incremental improvements in treatment delivery over time also appear to have an independently strong beneficial effect.

Mesenchymal stem cell-derived exosomes promote hepatic regeneration in drug-induced liver injury models.

INTRODUCTION: Mesenchymal stem cell-conditioned medium (MSC-CM) has been shown to have protective effects against various cellular-injury models. This mechanism of protection, however, has yet to be elucidated. Recently, exosomes were identified as the active component in MSC-CM. The aim of this study is to investigate the effect of MSC-derived exosomes in an established carbon tetrachloride (CCl4)-induced liver injury mouse model. This potential effect is then validated by using in vitro xenobiotic-induced liver-injury assays: (1) acetaminophen (APAP)- and (2) hydrogen peroxide (H2O2)-induced liver injury. METHODS: The exosomes were introduced concurrent with CCl4 into a mouse model through different routes of administration. Biochemical analysis was performed based on the blood and liver tissues. Subsequently the exosomes were treated in APAP and H2O2-toxicants with in vitro models. Cell viability was measured, and biomarkers indicative of regenerative and oxidative biochemical responses were determined to probe the mechanism of any hepatoprotective activity observed. RESULTS: In contrast to mice treated with phosphate-buffered saline, CCl4 injury in mice was attenuated by concurrent-treatment exosomes, and characterized by an increase in hepatocyte proliferation, as demonstrated with proliferating cell nuclear antigen (PCNA) elevation. Significantly higher cell viability was demonstrated in the exosomes-treated group compared with the non-exosome-treated group in both injury models. The higher survival rate was associated with upregulation of the priming-phase genes during liver regeneration, which subsequently led to higher expression of proliferation proteins (PCNA and cyclin D1) in the exosomes-treated group. Exosomes also inhibited the APAP- and H2O2-induced hepatocytes apoptosis through upregulation of Bcl-xL protein expression. However, exosomes do not mitigate hepatocyte injury via modulation of oxidative stress. CONCLUSIONS: In summary, these results suggest that MSC-derived exosomes can elicit hepatoprotective effects against toxicants-induced injury, mainly through activation of proliferative and regenerative responses.

Benzylidene-indolinones are effective as multi-targeted kinase inhibitor therapeutics against hepatocellular carcinoma.

Effective pharmacological intervention of advanced hepatocellular carcinoma (HCC) is currently lacking. Despite the use of tyrosine kinase inhibitors (TKIs) for the targeted therapy of several malignancies, no agent has been developed to specifically interfere with the oncogenic tyrosine kinase signaling aberrations found in HCC. Therefore, we adopted an orthogonal biological phenotypic screening approach to uncover candidate compounds: based on a potent cytotoxicity toward HCC-derived cell lines, and minimal toxicity toward normal liver cells. Given the success of indolinone as a chemical scaffold in deriving potent multi-kinase inhibitors (e.g. sunitinib), we screened a group of newly synthesized benzylidene-indolinones. Among the candidates, E/Z 6-Chloro-3-(3-trifluoromethyl-benzyliden)-1,3-dihydroindol-2-one (compound 47) exhibited potent anti-proliferative, anti-migratory, pro-apoptotic properties and good safety profile as compared to known multi-targeted tyrosine kinase inhibitors sunitinib and sorafenib. Additionally, an accompanying suppression of alpha-fetoprotein (AFP) transcription, an HCC tumor marker, implies a favorable selectivity and efficacy on HCC. The in vivo efficacy was demonstrated in an HCC xenograft where 47 was administered once weekly (60 mg/kg) and suppressed tumor burden to the same extent as sorafenib (30 mg/kg daily). A receptor tyrosine kinase (RTK) array study revealed promising inhibition of multiple tyrosine kinases such as IGF-1R, Tyro3 and EphA2 phosphorylation. Gene silencing of these targets ameliorated the cytotoxic potential of 47 on the HuH7 cell line, thereby implicating their contribution to the tumorigenicity of HCC. Hence, 47 exhibits potent anti-cancer effects on HCC cell lines, and is a suitable lead for developing multi-targeted kinase inhibitors of relevance to HCC.

Effects of Patient Empowerment Programme (PEP) on clinical outcomes and health service utilization in type 2 diabetes mellitus in primary care: an observational matched cohort study.

BACKGROUND: To evaluate the effects of a large population-based patient empowerment programme (PEP) on clinical outcomes and health service utilization rates in type 2 diabetes mellitus (T2DM) patients in the primary care setting. RESEARCH DESIGN AND SUBJECTS: A stratified random sample of 1,141 patients with T2DM enrolled to PEP between March and September 2010 were selected from general outpatient clinics (GOPC) across Hong Kong and compared with an equal number of T2DM patients who had not participated in the PEP (non-PEP group) matched by age, sex and HbA1C level group. MEASURES: Clinical outcomes of HbA1c, SBP, DBP and LDL-C levels, and health service utilization rates including numbers of visits to GOPC, specialist outpatient clinics (SOPC), emergency department (ED) and inpatient admissions, were measured at baseline and at 12-month post-recruitment. The effects of PEP on clinical outcomes and health service utilization rates were assessed by the difference-in-difference estimation, using the generalized estimating equation models. RESULTS: Compared with non-PEP group, PEP group achieved additional improvements in clinical outcomes over the 12-month period. A significantly greater percentage of patients in the PEP group attained HbA1C≤7% or LDL-C≤2.6 mmol/L at 12-month follow-up compared with the non-PEP group. PEP group had a mean 0.813 fewer GOPC visits in comparison with the non-PEP group. CONCLUSIONS: PEP was effective in improving the clinical outcomes and reduced the general outpatient clinic utilization rate over a 12-month period. Empowering T2DM patients on self-management of their disease can enhance the quality of diabetes care in primary care. TRIAL REGISTRATION: ClinicalTrials.gov NCT01935349.