Extracellular vesicle-carried Jagged-1 inhibits HUVEC sprouting in a 3D microenvironment.

NOTCH signalling is an evolutionarily conserved juxtacrine signalling pathway that is essential in development. Jagged1 (JAG1) and Delta-like ligand 4 (DLL4) are transmembrane NOTCH ligands that regulate angiogenesis by controlling endothelial cell (EC) differentiation, vascular development and maturation. In addition, DLL4 could bypass its canonical cell-cell contact-dependent signalling to influence NOTCH signalling and angiogenesis at a distance when it is packaged into extracellular vesicles (EVs). However, it is not clear whether JAG1 could also be packaged into EVs to influence NOTCH signalling and angiogenesis. In this work, we demonstrate that JAG1 is also packaged into EVs. We present evidence that JAG1-EVs inhibit NOTCH signalling and regulate EC behaviour and function. JAG1-EVs inhibited VEGF-induced HUVEC proliferation and migration in 2D culture condition and suppressed sprouting in a 3D microfluidic microenvironment. JAG1-EV treatment of HUVECs leads to a reduction of Notch1 intracellular domain (N1-ICD), and the proteasome and the intracellular domain of JAG1 (JAG1-ICD) are both required for this reduction to occur. These findings reveal a novel mechanism of JAG1 function in NOTCH signalling and ECs through EVs.

Long-chain glucosylceramides crosstalk with LYN mediates endometrial cell migration.

Endometriosis is a disease characterized by regurgitated lesions which are invasive and migratory, embedding at ectopic, extra-uterine locations. Extracellular glucosylceramides (GlcCers), bioactive sphingolipids potentiating signals for cell migration, are found in elevated levels in endometriosis; however underlying mechanisms that result in cellular migration are poorly defined. Here, we demonstrated that internalized GlcCer induced migratory activity in immortalized human endometrial stromal cells (HESCs), with highest potency observed in long-chain GlcCer. Long-chain ceramide (Cer) similarly induced cellular migration and mass spectrometry results revealed that the migratory behavior was contributed through glycosylation of ceramides. Cells treated with GlcCer synthase inhibitor, or RNAi-mediated knockdown of glucosylceramide synthase (GCS), the enzyme catalyzing GlcCer production attenuated cell motility. Mechanistic studies showed that GlcCer acts through stromal cell-derived factor-1 alpha and its receptor, CXC chemokine receptor 4 (SDF-1α-CXCR4) signaling axis and is dependent on phosphorylation of LYN kinase at Tyr396, and dephosphorylation of Tyr507. Migration was prominently attenuated in cells exposed to CXCR4 antagonist, AMD3100, yet can be rescued with diprotin A, which prevents the degradation of SDF-1α. Furthermore, blocking of LYN kinase activity in the presence of SDF-1α and GlcCer reduced HESC migration, suggesting that LYN acts downstream of GlcCer-SDF-1α-CXCR4 axis as part of its intracellular signal transduction. Our results reveal a novel role of long-chain GlcCer and the dialog between GlcCer, LYNpTyr396 and SDF-1α-CXCR4 in inducing HESC migration. This finding may improve our understanding how endometriotic lesions invade to their ectopic sites, and the possibility of using GlcCer to modulate the SDF-1α-CXCR4-LYNpTyr396 axis in endometriosis.

Entrapment of the deep peroneal nerve and anterior tibial vessels by a spiral tibial fracture causing partial non-union: a case report.

We report a case of entrapment of the deep peroneal nerve as well as the anterior tibial artery and vein by a spiral distal tibial shaft fracture, causing partial non-union. The authors describe the utility of MRI in making the diagnosis of this post-traumatic complication, which may potentially result in a permanent neurovascular deficit and adverse functional outcome if left undetected. The importance of recognizing the distinct possibility of entrapment and injury to the deep peroneal nerve as well as the anterior tibial vessels, when managing a fracture involving the distal third of the tibial shaft is emphasized. Absence of clinical symptoms or signs of neurovascular entrapment should not deter one from performing the relevant investigations to exclude this complication, in particular when surgical fixation is being contemplated, or in the presence of a non-healing fracture.

Neurobehavioural correlates in older children and adolescents with obesity and obstructive sleep apnoea.

AIM: The relationship between obstructive sleep apnoea (OSA) and poorer neurobehavioural outcomes in school-age children is well established, but the relationship in obese children and adolescents, in whom OSA is more common, is not so well established. We aimed to investigate this relationship in 10-18-year-olds. METHODS: Thirty-one participants with a mean body mass index (BMI) of 32.3 ± 4.9 enrolled. BMI-for-age cut-offs were used to define obesity. Participants underwent polysomnography and were classified into OSA (apnoea-hypopnoea index (AHI) > 2 per hour) and non-OSA (AHI ≤ 2) groups. Intelligence, memory and learning, academic achievement, behaviour and executive functioning were assessed using the Wechsler Abbreviated Scale of Intelligence, Wide Range Assessment of Memory and Learning 2, Wechsler Individual Achievement Test II (WIAT-II), Behavioural Assessment System for Children 2 and Behaviour Rating Inventory of Executive Function, respectively. RESULTS: Forty-eight per cent (15/31) were classified as having OSA, and 52% (16/31) as non-OSA. The obese cohort performed below the average of normative data on several neurobehavioural measures. WIAT-II maths scores were significantly lower (P = 0.034) in the OSA group than in the non-OSA group (means 84.5 vs. 94.6, respectively), losing significance after adjustment for IQ, age and gender. Self-reported school problems were significantly worse in the OSA group before and after multivariate adjustment (P = 0.010, Cohen's d = 1.02). No other significant differences were found. CONCLUSIONS: Results suggest that OSA may increase risk for some poorer educational and behavioural outcomes. The findings are reasonably consistent with and add to the evidence base of the few studies that have explored this relationship.

Sleep hygiene intervention for youth aged 10 to 18 years with problematic sleep: a before-after pilot study.

BACKGROUND: The current study aimed to examine the changes following a sleep hygiene intervention on sleep hygiene practices, sleep quality, and daytime symptoms in youth. METHODS: Participants aged 10-18 years with self-identified sleep problems completed our age-appropriate F.E.R.R.E.T (an acronym for the categories of Food, Emotions, Routine, Restrict, Environment and Timing) sleep hygiene programme; each category has three simple rules to encourage good sleep. Participants (and parents as appropriate) completed the Adolescent Sleep Hygiene Scale (ASHS), Pittsburgh Sleep Quality Index (PSQI), Sleep Disturbance Scale for Children (SDSC), Pediatric Daytime Sleepiness Scale (PDSS), and wore Actical® monitors twice before (1 and 2 weeks) and three times after (6, 12 and 20 weeks) the intervention. Anthropometric data were collected two weeks before and 20 weeks post-intervention. RESULTS: Thirty-three youths (mean age 12.9 years; M/F = 0.8) enrolled, and retention was 100%. ASHS scores significantly improved (p = 0.005) from a baseline mean (SD) of 4.70 (0.41) to 4.95 (0.31) post-intervention, as did PSQI scores [7.47 (2.43) to 4.47 (2.37); p < 0.001] and SDSC scores [53.4 (9.0) to 39.2 (9.2); p < 0.001]. PDSS scores improved from a baseline of 16.5 (6.0) to 11.3 (6.0) post- intervention (p < 0.001). BMI z-scores with a baseline of 0.79 (1.18) decreased significantly (p = 0.001) post-intervention to 0.66 (1.19). Despite these improvements, sleep duration as estimated by Actical accelerometry did not change. There was however a significant decrease in daytime sedentary/light energy expenditure. CONCLUSIONS: Our findings suggest the F.E.R.R.E.T sleep hygiene education programme might be effective in improving sleep in children and adolescents. However because this was a before and after study and a pilot study with several limitations, the findings need to be addressed with caution, and would need to be replicated within a randomised controlled trial to prove efficacy. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry: ACTRN12612000649819.

Lower socioeconomic status is associated with increased co-morbidity burden and independently associated with time to surgery, length of hospitalisation, and readmission rates of hip fracture patients.

This study examines the relationship between socioeconomic status, comorbidities, and clinical outcomes of hip fracture patients. Lower socioeconomic status is not only associated with poorer comorbidities but is also independently impacting surgical access and outcomes. This can be considered a "double setback" in the management of hip fractures. PURPOSE: The effect of socioeconomic status on hip fracture outcomes remains controversial. We examine the relationship between SES and patient comorbidity, care access, and clinical outcomes of surgically managed hip fracture patients. METHODS: Using healthcare payor status as a surrogate for SES, patients operated for fragility hip fractures between 2013 and 2016 were dichotomised based on payor status, namely private healthcare (PRIV) versus subsidised healthcare (SUB). PRIV patients were compared with SUB patients in terms of demographic data, ASA scores, co-morbidity burden (Charlson comorbidity index, CCI), time to surgery, length of acute hospitalisation, and 90-day readmission rates. RESULTS: A total of 145 patients in group PRIV and 1146 patients in group SUB were included. SUB patients had a higher mean Charlson Co-morbidity Index (CCI) (p = 0.01), a longer length of hospitalisation (p = 0.001), an increased delay in surgery (p = 0.005), and higher 90-day readmission rates (p = 0.013). Lower SES (p = 0.01), older age (p = 0.01), higher CCI (p < 0.01), and a higher American Society of Anaesthesiologists score (ASA) (p = 0.03) were predictive of time to surgery. Lower SES (p = 0.02) and higher CCI (p < 0.001) were predictive of the length of hospitalisation. Lower SES (p = 0.04) and higher CCI (p < 0.001) were predictive of 90-day readmission rates. CONCLUSIONS: Low SES is associated with higher CCI in surgically treated hip fracture patients. However, it is independently associated with slower access to surgery, a longer hospital stay, and higher readmission rates. Hence, lower SES, with its associated higher CCI and independent impact on surgical access and outcomes, can be considered a "double setback" in the management of fragility hip fractures.

HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors.

Animal cell-based expression platforms enable the production of complex biomolecules such as recombinant proteins and viral vectors. Although most biotherapeutics are produced in animal cell lines, production in human cell lines is expanding. One important advantage of using human cell lines is the increased potential that the resulting biotherapeutics would carry more "human-like" post-translational modifications. Among the human cell lines, HEK293 is widely utilized due to its high transfectivity, rapid growth rate, and ability to grow in a serum-free, suspension culture. In this review, we discuss the use of HEK293 cells and its subtypes in the production of biotherapeutics. We also compare their usage against other commonly used host cell lines in each category of biotherapeutics and summarise the factors influencing the choice of host cell lines used.

Higher Charlson Comorbidity Index Increases 90-Day Readmission Rate with Poorer Functional Outcomes in Surgically Treated Hip Fracture Patients.

INTRODUCTION: The associated mortality and morbidity in hip fracture patients pose a major healthcare burden for ageing populations worldwide. We aim to analyse how an individual's comorbidity profile based on age-adjusted Charlson Comorbidity Index (CCI) may impact on functional outcomes and 90-day readmission rates after hip fracture surgery. MATERIALS AND METHODS: Surgically treated hip fracture patients between 2013 and 2016 were followed up for 1-year and assessed using Parker Mobility Score (PMS), EuroQol-5D (EQ-5D) and Physical and Mental Component Scores (PCS and MCS, respectively) of Short Form-36 (SF-36). Statistical analysis was done by categorising 444 patients into three groups based on their CCI: (1) CCI 0-3, (2) CCI 4-5 and (3) CCI ≥ 6. RESULTS: PMS, EQ-5D and SF-36 PCS were significantly different amongst the CCI groups pre-operatively and post-operatively at 3, 6 and 12 months (all P < 0.05), with CCI ≥ 6 predicting for poorer outcomes. In terms of 90-day readmission rates, patients who have been readmitted have poorer outcome scores. Multivariate analysis showed that high CCI scores and 90-day readmission rate both remained independent predictors of worse outcomes for SF-36 PCS, PMS and EQ-5D. DISCUSSION: CCI scores ≥6 predict for higher 90-day readmission rates, poorer quality of life and show poor potential for functional recovery 1-year post-operation in hip fracture patients. 90-day readmission rates are also independently associated with poorer functional outcomes. Peri-operatively, surgical teams should liaise with medical specialists to optimise patients' comorbidities and ensure their comorbidities remain well managed beyond hospital discharge to reduce readmission rates. With earlier identification of patient groups at risk of poorer functional outcomes, more planning can be directed towards appropriate management and subsequent rehabilitation. CONCLUSION: Further research should focus on development of a stratified, peri-operative multidisciplinary, hip-fracture care pathway treatment regime based on CCI scores to determine its effectiveness in improving functional outcomes.

An Electromagnetic System for Inducing a Localized Force Gradient in an ECM and Its Influence on HMVEC Sprouting.

Mechanical properties of the extracellular matrix (ECM) have been observed to influence the behavior of cells. Investigations on such an influence commonly rely on using soluble cues to alter the global intrinsic ECM properties in order to study the subsequent response of cells. This article presents an electromagnetic system for inducing a localized force gradient in an ECM, and reports the experimentally observed effect of such a force gradient on in vitro angiogenic sprouting of human microvascular endothelial cells (HMVECs). This force gradient is realized through the induction of magnetic forces on the superparamagnetic microparticle-embedded ECM ( sECM). Both analytical and statistically meaningful experimental results demonstrate the effectiveness of this approach in influencing the behavior of a targeted HMVEC sprout without affecting that of other sprouts nearby. These results suggest the possibility of selectively controlling the in vitro behavior of cells by the induction of a localized force gradient in the ECM.

Serum periostin levels in adults of Chinese descent: an observational study.

BACKGROUND: Periostin has been shown to be a marker of Type 2 airway inflammation, associated with airway eosinophilia. It has a potential role in identifying asthmatics who may be responsive to treatment with monoclonal antibody therapy directed against Type 2 cytokines, such as interleukin (IL)-13, IL-4 receptor subunit-α and immunoglobulin E. The clinical utility of periostin measurements depends on better understanding of factors that may affect serum periostin levels, such as race. We aimed to identify the ranges of serum periostin in Chinese adults both with and without asthma, and compare them with those previously identified in Caucasian adults. METHODS: A two-centred cross-sectional study, recruiting 188 Chinese adults, aged 18 to 75 years. 120 participants had no history of asthma or chronic obstructive pulmonary disease. 68 participants had a doctor's diagnosis of asthma and were on current treatment. Univariate comparisons of periostin by dichotomous variables were made using t-tests with logarithmic transformation as the distribution of periostin was skewed. RESULTS: In the Chinese non-asthma group, periostin levels were sex-, but not age-dependent, with females having higher periostin levels. The individual predicted (90% CI) reference range for periostin in females was 61.1 ng/ml (41.6 to 89.8) ng/ml and in males was 53.2 ng/ml (36.1 to 78.3) ng/ml. There was no difference in median serum periostin levels between Chinese non-asthmatics and Chinese asthmatics, 57.0 versus 56.8 ng/ml, difference (95% CI) 0.1 (- 4.2 to 4.2) ng/ml, P = 0.94. The median serum periostin levels were higher in Chinese non-asthmatics than Caucasian non-asthmatics, 57.0 versus 49.7 ng/ml, difference (95% CI) 8.2 (5.8-10.6) ng/ml, P < 0.001. CONCLUSIONS: Serum periostin does not discriminate between asthmatics and non-asthmatics and is therefore not a good biomarker to diagnose asthma. Serum periostin levels were higher in the Chinese compared to the Caucasian non-asthma group, and also sex dependent in the Chinese participants. There was no difference in serum periostin levels between Chinese non-asthma and asthma groups. This suggests that ethnicity should be considered in the interpretation of periostin levels in asthma patients and sex is an additional consideration in Chinese patients.Trial registration This trial was prospectively registered with Australian New Zealand Clinical Trials Registry (ACTRN12614000122651).

Pulse transit time and blood pressure changes following auditory-evoked subcortical arousal and waking of infants.

STUDY OBJECTIVES: To establish a normal range of data in 3-month-old infants in relation to changes in cardiovascular measurements, with particular reference to pulse transit time (PTT), following subcortical arousals and awakenings from sleep. DESIGN: Prospective study. SETTING: Sleep laboratory, Dunedin Hospital PARTICIPANTS: Twenty healthy infants aged 9-12 weeks. METHODS: Nap studies were performed using a standard polysomnographic setup with the addition of a Portapres blood pressure (BP) cuff (wrist application) and a piezoelectric sensor on the foot. PTT was measured from the ECG-R waveform to the arrival of the pulse peripherally. Infants were exposed to white noise from 50 to 100 dB at 10 dB intervals within REM and NREM sleep. RESULTS: Awakening thresholds were higher (P = 0.01) in NREM (>90 dB) than REM sleep (mean +/- SD; 74.3 +/- 9.4dB). Subcortical thresholds were always 10 dB below waking thresholds. Following awakening, there was an immediate increase in HR, SBP, and DBP of 21%, 14%, and 17%, respectively, and a 13% decrease in PTT returning to baseline within 25-30 seconds. PTT at baseline measured 140 +/- 11 and 139 +/- 9 msec in NREM and REM sleep, respectively, and decreased approximately 20 msec with waking. PTT changes were negatively correlated with heart rate (HR) but not BP, although a trend was evident. CONCLUSIONS: At 3 months of age, infants provoked to arouse from sleep showed PTT changes that inversely mimicked BP trends, suggesting that PTT could be useful in infant studies as a marker for autonomic perturbations that occur during sleep in both clinical and research settings.

Dll4-containing exosomes induce capillary sprout retraction in a 3D microenvironment.

Delta-like 4 (Dll4), a membrane-bound Notch ligand, plays a fundamental role in vascular development and angiogenesis. Dll4 is highly expressed in capillary endothelial tip cells and is involved in suppressing neighboring stalk cells to become tip cells during angiogenesis. Dll4-Notch signaling is mediated either by direct cell-cell contact or by Dll4-containing exosomes from a distance. However, whether Dll4-containing exosomes influence tip cells of existing capillaries is unknown. Using a 3D microfluidic device and time-lapse confocal microscopy, we show here for the first time that Dll4-containing exosomes causes tip cells to lose their filopodia and trigger capillary sprout retraction in collagen matrix. We demonstrate that Dll4 exosomes can freely travel through 3D collagen matrix and transfer Dll4 protein to distant tip cells. Upon reaching endothelial sprout, it causes filopodia and tip cell retraction. Continuous application of Dll4 exosomes from a distance lead to significant reduction of sprout formation. This effect correlates with Notch signaling activation upon Dll4-containing exosome interaction with recipient endothelial cells. Furthermore, we show that Dll4-containing exosomes increase endothelial cell motility while suppressing their proliferation. These data revealed novel functions of Dll4 in angiogenesis through exosomes.

Exosomes in Cancer Microenvironment and Beyond: have we Overlooked these Extracellular Messengers?

Cancer is a complex organ whose behavior is not only influenced by genetic and epigenetic changes in cancer cells but also by stromal cells, local extracellular matrix and specific tissue architecture. Intercellular communications within the cancer microenvironment are critical to coordinate the assembly of multiple cell types for an amalgamated form and function of a cancer. Exosomes are small membrane vesicles with an endosome origin that are released by cells into the extracellular environment. They carry a cargo of proteins, lipids, and nucleic acids and transfer their cargo to recipient cells and altering the recipient cells' biochemical composition, signaling pathways, and gene regulation. Exosomes can thus serve as extracellular messengers mediating cell-cell communication. Both cancer cells and stromal cells release exosomes not only into the cancer microenvironment but also into the circulation. In this review, we summarize the research done so far on cancer-derived exosomes and assess their roles as extracellular messengers facilitating cancer progression and metastasis.

Pulse transit time and assessment of childhood sleep disordered breathing.

OBJECTIVES: To compare nocturnal polysomnography (PSG) with pulse transit time (PTT) recordings and structured clinical assessments and assess the reliability of these methods as a surrogate for the apnea-hypopnea index (AHI; calculated as the number of apneas/hypopneas per hour of total sleep time) and to test the associations between the clinical assessments and sleep disordered breathing (SDB). DESIGN: Prospective observational study. The parents of 51 children and adolescents filled out a questionnaire on SDB and the participants underwent examination. Scores from questionnaire and examination items were weighted according to their association with SDB. A total clinical score was assigned combining questionnaire and examination scores. SETTING: Hospital pediatrics department. PATIENTS: Children and adolescents aged 5 to 17 years undergoing standard PSG with the addition of PTT as part of a clinical investigation for SDB. MAIN OUTCOME MEASURES: The AHI and associations between the AHI and PTT arousal index (PTT-AI) and questionnaire, examination, and total clinical scores. RESULTS: We found a significant correlation between the AHI and PTT-AI (r = 0.55; P < .001). The relationship between the AHI and PTT-AI was stronger when the AHI was greater than 3. We also found significant correlations between the PTT-AI and the total clinical score (r = 0.38; P = .008) and the examination score (r = 0.44; P = .002) but not the questionnaire score (r = 0.23; P = .12). There was an association between the AHI and examination score in particular when the AHI was greater than 3. CONCLUSIONS: Pulse transit time shows promise as a screening test for SDB associated with an AHI greater than 3. For less severe SDB, the validity of using the PTT to separate these conditions from primary snoring has not been demonstrated in a clinical setting.