Amphiphysin is a component of clathrin coats formed during synaptic vesicle recycling at the lamprey giant synapse

Amphiphysin is a protein enriched at mammalian synapses thought to function as a clathrin accessory factor in synaptic vesicle endocytosis. Here we examine the involvement of amphiphysin in synaptic vesicle recycling at the giant synapse in the lamprey. We show that amphiphysin resides in the synaptic vesicle cluster at rest and relocates to sites of endocytosis during synaptic activity. It accumulates at coated pits where its SH3 domain, but not its central clathrin/AP-2-binding (CLAP) region, is accessible for antibody binding. Microinjection of antibodies specifically directed against the CLAP region inhibited recycling of synaptic vesicles and caused accumulation of clathrin-coated intermediates with distorted morphology, including flat patches of coated presynaptic membrane. Our data provide evidence for an activity-dependent redistribution of amphiphysin in intact nerve terminals and show that amphiphysin is a component of presynaptic clathrin-coated intermediates formed during synaptic vesicle recycling.

Effects of LL-37 on Human Gingival Fibroblast Function

Background and Objectives: Gingival fibroblasts play a significant role in the innate immune response of the periodontium to bacterial stimulation. A number of microorganisms and their by-products induce a host response that commonly leads to tissue destruction and periodontal disease progression. LL-37 is an antimicrobial peptide which has multiple roles in host defence including immunomodulation and wound-healing. We have investigated the role of LL-37 on the responsiveness of human gingival fibroblasts to microbial challenge from E. coli lipopolysaccharide (LPS) and P. gingivalis LPS, as well as exploring the direct effects of LL-37 on human gingival fibroblasts. Methods: The effect of LL-37 on bacterial LPS-induced expression of IL-6 and IL-8 by gingival fibroblasts was determined by ELISA. The influence of LL-37 on bacterial LPS-induced IκBα degradation in human gingival fibroblasts was investigated by western blot. The direct effects of LL-37 on modulating gingival fibroblasts gene expression were initially determined by DNA microarray analysis and subsequently confirmed by quantitative polymerase chain reaction (Q-PCR) and ELISA analysis of 9 selected genes. Results: Bacterial LPS-induced IL-8 and IL-6 production by human gingival fibroblasts were significantly reduced in the presence of LL-37 at concentrations in the range of 1-10 µg/ml (p<0.05). The presence of LL-37 at a concentration of 5 µg/ml led to a reduction in LPS-induced IκBα degradation by E. coli LPS (100 ng/ml) and P. gingivalis LPS (10 µg/ml). LL-37 (50 µg/ml) significantly altered the gene expression of 367 genes in human gingival fibroblasts by at least 2-fold. CXCL1, CXCL2, CXCL3, IL-24, IL-8, CCL2, and SOCS3 mRNA were significantly upregulated by LL-37 (p<0.05). LL-37 also significantly stimulated expression of IL-8, hepatocyte growth factor (HGF) and CXCL1 (p<0.05) at the protein level. Discussion: LL-37 plays an important role in the innate immune response due to its broad spectrum antimicrobial and immunomodulatory activity. The ability of LL-37 to directly regulate expression of a range of genes, central to the pathogenesis of periodontitis, identifies multiple roles for the peptide in host homeostasis.

Is the raised volume rapid thoracic compression technique ready for use in clinical trials in infants with cystic fibrosis?

The European Cystic Fibrosis Society Clinical Trial Network (ECFS-CTN) has established a Standardization Committee to undertake a rigorous evaluation of promising outcome measures with regard to use in multicentre clinical trials in cystic fibrosis (CF). The aim of this article is to present a review of literature on clinimetric properties of the infant raised-volume rapid thoracic compression (RVRTC) technique in the context of CF, to summarise the consensus amongst the group on feasibility and answer key questions regarding the promotion of this technique to surrogate endpoint status.

METHODS: A literature search (from 1985 onwards) identified 20 papers that met inclusion criteria of RVRTC use in infants with CF. Data were extracted and tabulated regarding repeatability, validity, correlation with other outcome measures, responsiveness and reference values. A working group discussed the tables and answered 4 key questions.

RESULTS: Overall, RVRTC in particular forced expiratory volume in 0.5s, showed good clinimetric properties despite presence of individual variability. Few studies showed a relationship between RVRTC and inflammation and infection, and to date, data remains limited regarding the responsiveness of RVRTC after an intervention. Concerns were raised regarding feasibility in multi-centre studies and availability of reference values.

CONCLUSION: The ECFS-CTN Working Group considers that RVRTC cannot be used as a primary outcome in clinical trials in infants with CF before universal standardization of this measurement is achieved and implementation of inter-institutional networking is in place. We advise its use currently in phase I/II trials and as a secondary endpoint in phase III studies. We emphasise the need for (1) more short-term variability and longitudinal 'natural history' studies, and (2) robust reference values for commercially available devices.

Is the Schwabe organ a retained larval eye? Anatomical and behavioural studies of a novel sense organ in adult leptochiton asellus (Mollusca, Polyplacophora) indicate links to larval photoreceptors

The discovery of a sensory organ, the Schwabe organ, was recently reported as a unifying feature of chitons in the order Lepidopleurida. It is a patch of pigmented tissue located on the roof of the pallial cavity, beneath the velum on either side of the mouth. The epithelium is densely innervated and contains two types of potential sensory cells. As the function of the Schwabe organ remains unknown, we have taken a cross-disciplinary approach, using anatomical, histological and behavioural techniques to understand it. In general, the pigmentation that characterises this sensory structure gradually fades after death; however, one particular concentrated pigment dot persists. This dot is positionally homologous to the larval eye in chiton trochophores, found in the same neuroanatomical location, and furthermore the metamorphic migration of the larval eye is ventral in species known to possess Schwabe organs. Here we report the presence of a discrete subsurface epithelial structure in the region of the Schwabe organ in Leptochiton asellus that histologically resembles the chiton larval eye. Behavioural experiments demonstrate that Leptochiton asellus with intact Schwabe organs actively avoid an upwelling light source, while Leptochiton asellus with surgically ablated Schwabe organs and a control species lacking the organ (members of the other extant order, Chitonida) do not (Kruskal-Wallis, H = 24.82, df = 3, p < 0.0001). We propose that the Schwabe organ represents the adult expression of the chiton larval eye, being retained and elaborated in adult lepidopleurans.

Effects of LL-37 Peptide Mimetics on Gingival Fibroblast Function

Host defence peptides, including the cathelicidin LL-37, play an important role in mucosal immunity, functioning as both antimicrobial agents and modulators of the inflammatory response. In the current climate of antibiotic resistance, the idea of using naturally occurring antimicrobial peptides, or their synthetic mimetics, to combat oral infection is particularly appealing. Objectives: The aim of this study was to investigate the effects of parent LL-37, and two peptide mimetics (KR-12 and KE-18), on cytokine expression and response to bacterial challenge by gingival fibroblasts. Methods: KR-12 and KE-18 are peptide mimetics of the biologically active, mid-region sequence of LL-37. The effects of commercially available LL-37, KR-12 and KE-18 on gingival fibroblast response to E coli and P gingivalis LPS challenge, analysed by IL-6 and IL-8 expression, were determined in cell culture by ELISA. The direct effects of each peptide on IL-6, IL-8, CXCL-1 and HGF expression were also determined by ELISA. The MTT assay was used to evaluate peptide effects on fibroblast viability. Results: LL-37 and KE-18, but not KR-12, inhibited LPS induction of inflammatory cytokine expression and directly stimulated CXCL-1 production by fibroblasts. All 3 peptides stimulated production of IL-8 and HGF. Neither LL-37 nor KE-12 affected cell viability, while KE-18, at higher concentrations, induced cell death. Conclusions: Shorter, peptide mimetics of LL-37, in particular KE-18, retain the immunomodulatory effects of the parent molecule and possess excellent potential as therapeutic agents in the treatment of oral infections including periodontal disease.

Correction of moderate myopia is associated with improvement in self-reported visual functioning among Mexican school-aged children.

PURPOSE: To quantify the impact on self-reported visual functioning of spectacle provision for school-aged children in Oaxaca, Mexico. METHODS: The Refractive Status Vision Profile (RSVP), a previously validated tool to measure the impact of refractive correction on visual functioning, was adapted for use in rural children and administered at baseline and 4 weeks (27.3 +/- 4.4 days) after the provision of free spectacles. Visual acuity with and without correction, age, sex, and spherical equivalent refraction were recorded at the time of follow-up. RESULTS: Among 88 children (mean age, 12 years; 55.7% girls), the median presenting acuity (uncorrected or with original spectacles), tested 4 weeks after the provision of free spectacles, was 6/9 (range, 6/6-6/120). Significant improvements in the following subscales of the RSVP were seen for the group as a whole after the provision of free spectacles: function, 11.2 points (P = 0.0001); symptoms, 14.3 points (P < 0.0001); total score, 10.3 points (P = 0.0001). After stratification by presenting vision in the better-seeing eye, children with 6/6 acuity (n = 22) did not have significant improvement in any subscale; those with acuity of 6/7.5 to 6/9 (n = 34) improved only on function (P = 0.02), symptoms (P = 0.005), and total score (P = 0.003); and those with acuity of 6/12 or worse improved on total score (P < 0.0001) and all subscales. Subjects (n = 31) with uncorrected myopia of -1.25 D or more had a mean improvement in total score of 15.9 points (P < 0.0001), whereas those with uncorrected myopia between -0.50 and -1.00 D inclusive (n = 53) had a mean improvement of 8 points (P = 0.01). CONCLUSIONS: Provision of spectacles to children in this setting had a significant impact on self-reported function, even at modest levels of baseline visual disability. The correlation between presenting vision/refraction and improvement and the failure of children 6/6 at baseline to improve offer evidence for a real effect.

Ocular comorbidities among cataract-operated patients in rural China: the caring is hip Study of Cataract Outcomes and Uptake of Services (SCOUTS), report No. 3.

PURPOSE: To determine the prevalence and impact on vision and visual function of ocular comorbidities in a rural Chinese cataract surgical program, and to devise strategies to reduce their associated burden. DESIGN: Cross-sectional cohort study. PARTICIPANTS: Persons undergoing cataract surgery by one of two recently trained local surgeons at a government-run village-level hospital in rural Guangdong between August 8 and December 31, 2005. INTERVENTIONS: Eligible subjects were invited to return for a comprehensive ocular examination and visual function interview 10 to 14 months after surgery. Prevalent ocular comorbid conditions were identified. MAIN OUTCOME MEASURES: Presenting and best-corrected vision, visual function, and treatability of the comorbidity. RESULTS: Of 313 persons operated within the study window, 242 (77%) could be contacted by telephone; study examinations and interviews were performed on 176 (74%). Examined subjects had a mean age of 69.4+/-10.5 years, 116 (66%) were female, and 149 (85%) had been blind (presenting vision < or = 6/60) in the operative eye before surgery. Among unoperated eyes, 89 of 109 (81.7%) had > or =1 ocular comorbidities, whereas for operated eyes the corresponding proportion was 72 of 211 (34.1%). The leading comorbidity among operated eyes was refractive error (43/72 [59.7%]), followed by glaucoma/glaucoma suspect (14/72 [19.4%]), whereas for unoperated eyes, it was cataract (80/92 [87.0%]), followed by refractive error (12/92 [13.0%]). Among operated eyes with comorbidities, 90.3% (65/72) had > or =1 comorbidities that were treatable. In separate models adjusting for age and gender, persons with > or =1 comorbidities in the operated eye had significantly worse presenting vision (P<0.001) than those without such findings, but visual function (P = 0.197) and satisfaction with surgery (P = 0.796) were unassociated with comorbidities. CONCLUSIONS: Ocular comorbidities are highly prevalent among persons undergoing cataract surgery in this rural Asian setting, and their presence is significantly associated with poorer visual outcomes. The fact that the great majority of comorbidities encountered in this program are treatable suggests that strategies to reduce their impact can be successful.

Cataract surgical outcomes, visual function and quality of life in four rural districts in Vietnam.

BACKGROUND: To evaluate cataract surgical outcomes in four rural districts of Ha Tinh Province, Vietnam. DESIGN: Cross-sectional study. PARTICIPANTS: Post-cataract surgery patients sampled randomly from facilities in four rural districts of Ha Tinh Province >3 months after surgery. MAIN OUTCOME MEASURES: Postoperative visual acuity (VA), visual function and quality of life. RESULTS: Among 412 patients, the mean age was 74.5 ± 9.4 years, 67% (276) were female, and 377 (91.5%) received intraocular lenses (IOL). Nearly two-thirds of patients had no postoperative visits after discharge. Postoperatively, more than 40% of eyes had presenting VA <6/18, while 20% remained <6/60. The mean self-reported visual function and quality of life for all patients were 68.7 ± 23.8 and 73.8 ± 21.6, respectively. Most patients (89.5%) were satisfied with surgery and the majority (94.4%) would recommend surgery to others. One-third of patients paid ≥$US50 for surgery. In multiple regression modelling, older age (P < 0.01), intraoperative complications (P < 0.01) and failure to receive an IOL (P < 0.01) were associated with postoperative VA <6/60. CONCLUSION: Satisfaction with surgery was high, and many patients were willing to pay for their operations. Poor visual outcomes were common; however, and better surgical training is needed to reduce complications and their impact on visual outcomes. More intensive postoperative follow-up may also be beneficial. © 2011 The Authors. Clinical and Experimental Ophthalmology © 2011 Royal Australian and New Zealand College of Ophthalmologists.

INFLUENCE OF NOX4 NADPH OXIDASE ON GENERATION AND ANGIOGENIC FUNCTION OF INDUCED PLURIPOTENT STEM CELL-DERIVED ENDOTHELIAL CELLS

Human induced pluripotent stem (iPS) cell-derived endothelial cells (ECs) hold clear potential for therapeutic angiogenesis as a novel strategy for ischaemic disease. Recently, we have developed a novel method for direct reprogramming of partial iPS (PiPS) cells, which unlike iPS cells, are generated before pluripotency so do not form tumours, and may be differentiated into ECs with characteristic morphology and pro-angiogenic actions. Our previous work showed that PiPS-derived ECs are capable of forming vascular-like tubes both in vitro and in vivo and promoting re-endothelialisation of ischemic tissue, with greater effectiveness versus mature ECs.

Interestingly, our preliminary data demonstrate that Nox NADPH oxidases, which are reported to influence stem cell function, are progressively induced during PiPs/PiPS-EC differentiation and in response to hypoxia, with Nox4 demonstrating highest expression. As this isoform is an established regulator of angiogenesis, we hypothesize that Nox4 plays a key role in modulating PiPS-EC generation and angiogenic function.

The aim of this project is therefore to investigate: (1) the specific role of Nox4 in direct reprogramming of PiPS cells and differentiation to PiPS-ECs; (2) whether genetic manipulation of Nox4 influences in vitro function of PiPs-ECs and their ability to promote in vivo angiogenesis. This will be achieved by employing established in vitro functional assays and an experimental model of hindlimb ischaemia with assessment of relevant end-points. Identification of a key role for Nox4 in regulating PiPS-EC generation/function may inform selective targeting of this isoform to enhance the efficiency of PiPS-EC differentiation and their capacity to treat ischemic disease.

A hybrid mixed cost-function TEQ initialization algorithm for ADSL modems

In this paper, we present a hybrid mixed cost-function adaptive initialization algorithm for the time domain equalizer in a discrete multitone (DMT)-based asymmetric digital subscriber loop. Using our approach, a higher convergence rate than that of the commonly used least-mean square algorithm is obtained, whilst attaining bit rates close to the optimum maximum shortening SNR and the upper bound SNR. Moreover, our proposed method outperforms the minimum mean-squared error design for a range of TEQ filter lengths.

Endothelial Function in Hypertension: Victim or Culprit?

Far from simply lining the inner surface of blood vessels, the cellular monolayer that comprises the endothelium is a highly active organ that regulates vascular tone. In health, the endothelium maintains the balance between opposing dilator and constrictor influences, while in disease, it is the common ground on which cardiovascular risk factors act to initiate the atherosclerotic process. As such, it is the site at which cardiovascular disease begins and consequently acts as a barometer of an individual's likely future cardiovascular health. The vascular endothelium is a very active organ responsible for the regulation of vascular tone through the effects of locally synthesized mediators, predominantly nitric oxide (NO), endothelial NO synthase (eNOS), and superoxide. NO is abundantly evident in normally functioning vasculature where it acts as a vasodilator, inhibits inflammation, and has an antiaggregant effect on platelets. Its depletion is both a sign and cause of endothelial dysfunction resulting from reduced activity of eNOS and amplified production of nicotinamide adenine dinucleotide oxidase, which, in turn, results in raised levels of reactive oxygen species. This cascade is the basis for reduced vascular compliance through an imbalanced regulation of tone with a predominance of vasoconstrictive elements. Further, structural changes in the microvasculature are a critical early step in the loss of normal function. This microvascular dysfunction is known to be highly predictive of future macrovascular events and is consequently a very attractive target for intervention in the hypertensive population in order to prevent cardiovascular events.

Inhibition of protease-ENaC signaling improves mucociliary function in cystic fibrosis airways

Rationale: In cystic fibrosis (CF) a reduction in airway surface liquid (ASL) height
compromises mucociliary clearance, favoring mucus plugging and chronic bacterial infection. Inhibitors of ENaC have therapeutic potential in CF airways to reduce the hyperstimulated sodium and fluid absorption to levels which can restore airways hydration.

Objectives: To determine whether a novel compound (QUB-TL1) designed to inhibit protease/ENaC signaling in CF airways restores ASL volume and mucociliary function.

Methods: Protease activity was measured using fluorogenic activity assays. Differentiated primary airway epithelial cell cultures (F508del homozygotes) were used to determined ENaC activity (Ussing chamber recordings), ASL height (confocal microscopy) and mucociliary function (by tracking the surface flow of apically applied microbeads). Cell toxicity was measured by LDH assay.

Measurements and Results: QUB-TL1 inhibits extracellularly-located CAPs, including prostasin, matriptase and furin, the activities of which are observed at excessive levels at the apical surface of CF airway epithelial cells (AECs). QUB-TL1-mediated CAPs inhibition results in diminished ENaC-mediated Na+ absorption in CF AECs due to internalization of a prominent pool of cleaved (active) ENaCγ from the cell surface. Importantly, diminished ENaC activity correlates with improved airway hydration status and mucociliary clearance. We further demonstrate QUB-TL1-mediated furin inhibition, which is in contrast to other serine protease inhibitors (camostat mesylate and aprotinin), affords protection against neutrophil elastase-mediated ENaC activation and Pseudomonas aeruginosa exotoxin A induced cell death.

Conclusions: QUB-TL1 corrects aberrant CAP activities providing a mechanism to delay or prevent the development of CF lung disease in a manner independent of CFTR mutation.

Selective Multi-Carrier Index Keying OFDM: Error Propagation Rate with Moment Generating Function

We propose a new selective multi-carrier index keying in orthogonal frequency division multiplexing (OFDM) systems that opportunistically modulate both a small subset of sub-carriers and their indices. Particularly, we investigate the performance enhancement in two cases of error propagation sensitive and compromised deviceto-device (D2D) communications. For the performance evaluation, we focus on analyzing the error propagation probability (EPP) introducing the exact and upper bound expressions on the detection error probability, in the presence of both imperfect and perfect detection of active multi-carrier indices. The average EPP results in closedform are generalized for various fading distribution using the moment generating function, and our numerical results clearly show that the proposed approach is desirable for reliable and energy-efficient D2D applications.

Addiction to Runx1 is partially attenuated by loss of p53 in the Eµ-Myc lymphoma model

The Runx genes function as dominant oncogenes that collaborate potently with Myc or loss of p53 to induce lymphoma when over-expressed. Here we examined the requirement for basal Runx1 activity for tumor maintenance in the Eµ-Myc model of Burkitt's lymphoma. While normal Runx1fl/fl lymphoid cells permit mono-allelic deletion, primary Eµ-Myc lymphomas showed selection for retention of both alleles and attempts to enforce deletion in vivo led to compensatory expansion of p53null blasts retaining Runx1. Surprisingly, Runx1 could be excised completely from established Eµ-Myc lymphoma cell lines in vitro without obvious effects on cell phenotype. Established lines lacked functional p53, and were sensitive to death induced by introduction of a temperature-sensitive p53 (Val135) allele. Transcriptome analysis of Runx1-deleted cells revealed a gene signature associated with lymphoid proliferation, survival and differentiation, and included strong de-repression of recombination-activating (Rag) genes, an observation that was mirrored in a panel of human acute leukemias where RUNX1 and RAG1,2 mRNA expression were negatively correlated. Notably, despite their continued growth and tumorigenic potential, Runx1null lymphoma cells displayed impaired proliferation and markedly increased sensitivity to DNA damage and dexamethasone-induced apoptosis, validating Runx1 function as a potential therapeutic target in Myc-driven lymphomas regardless of their p53 status.

Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in the regulation of metabolic activity in cancer and immune cells, and affects whole-body metabolism by regulating ghrelin-signalling in the hypothalamus. This has led to efforts to develop specific CaMKK2 inhibitors, and STO-609 is the standardly used CaMKK2 inhibitor to date. We have developed a novel fluorescence-based assay by exploiting the intrinsic fluorescence properties of STO-609. Here, we report an in vitro binding constant of KD ∼17 nM between STO-609 and purified CaMKK2 or CaMKK2:Calmodulin complex. Whereas high concentrations of ATP were able to displace STO-609 from the kinase, GTP was unable to achieve this confirming the specificity of this association. Recent structural studies on the kinase domain of CaMKK2 had implicated a number of amino acids involved in the binding of STO-609. Our fluorescent assay enabled us to confirm that Phe(267) is critically important for this association since mutation of this residue to a glycine abolished the binding of STO-609. An ATP replacement assay, as well as the mutation of the 'gatekeeper' amino acid Phe(267)Gly, confirmed the specificity of the assay and once more confirmed the strong binding of STO-609 to the kinase. In further characterising the purified kinase and kinase-calmodulin complex we identified a number of phosphorylation sites some of which corroborated previously reported CaMKK2 phosphorylation and some of which, particularly in the activation segment, were novel phosphorylation events. In conclusion, the intrinsic fluorescent properties of STO-609 provide a great opportunity to utilise this drug to label the ATP-binding pocket and probe the impact of mutations and other regulatory modifications and interactions on the pocket. It is however clear that the number of phosphorylation sites on CaMKK2 will pose a challenge in studying the impact of phosphorylation on the pocket unless the field can develop approaches to control the spectrum of modifications that occur during recombinant protein expression in E. coli.