Mutational Analysis of the Rhodopsin Gene in Sector Retinitis Pigmentosa

Background: To determine the role of rhodopsin (RHO) gene mutations in patients with sector retinitis pigmentosa (RP) from Northern Ireland.

Design: A case series of sector RP in a tertiary ocular genetics clinic.

Participants: Four patients with sector RP were recruited from the Royal Victoria Hospital (Belfast, Northern Ireland) and Altnagelvin Hospital (Londonderry, Northern Ireland) following informed consent.

Methods: The diagnosis of sector RP was based on clinical examination, International Society for Clinical Electrophysiology of Vision (ISCEV) standard electrophysiology, and visual field analysis. DNA was extracted from peripheral blood leucocytes and the coding regions and adjacent flanking intronic sequences of the RHO gene were polymerase chain reaction (PCR) amplified and cycle sequenced.

Main Outcome Measure: Rhodopsin mutational status.

Results: A heterozygous missense mutation in RHO (c.173C > T) resulting in a non-conservative substitution of threonine to methionine (p. Thr58Met) was identified in one patient and was absent from 360 control individuals. This non-conservative substitution (p.Thr58Met) replaces a highly evolutionary conserved polar hydrophilic threonine residue with a non-polar hydrophobic methionine residue at position 58 near the cytoplasmic border of helix A of RHO.

Conclusions: The study identified a RHO gene mutation (p.Thr58Met) not previously reported in RP in a patient with sector RP. These findings outline the phenotypic variability associated with RHO mutations. It has been proposed that the regional effects of RHO mutations are likely to result from interplay between mutant alleles and other genetic, epigenetic and environmental factors.

Validity of the Global Physical Activity Questionnaire (GPAQ) in assessing levels and change in moderate-vigorous physical activity and sedentary behaviour

Background

Feasible, cost-effective instruments are required for the surveillance of moderate-to-vigorous physical activity (MVPA) and sedentary behaviour (SB) and to assess the effects of interventions. However, the evidence base for the validity and reliability of the World Health Organisation-endorsed Global Physical Activity Questionnaire (GPAQ) is limited. We aimed to assess the validity of the GPAQ, compared to accelerometer data in measuring and assessing change in MVPA and SB.

Participants (n = 101) were selected randomly from an on-going research study, stratified by level of physical activity (low, moderate or highly active, based on the GPAQ) and sex. Participants wore an accelerometer (Actigraph GT3X) for seven days and completed a GPAQ on Day 7. This protocol was repeated for a random sub-sample at a second time point, 3–6 months later. Analysis involved Wilcoxon-signed rank tests for differences in measures, Bland-Altman analysis for the agreement between measures for median MVPA and SB mins/day, and Spearman’s rho coefficient for criterion validity and extent of change.

95 participants completed baseline measurements (44 females, 51 males; mean age 44 years, (SD 14); measurements of change were calculated for 41 (21 females, 20 males; mean age 46 years, (SD 14). There was moderate agreement between GPAQ and accelerometer for MVPA mins/day (r = 0.48) and poor agreement for SB (r = 0.19). The absolute mean difference (self-report minus accelerometer) for MVPA was −0.8 mins/day and 348.7 mins/day for SB; and negative bias was found to exist, with those people who were more physically active over-reporting their level of MVPA: those who were more sedentary were less likely to under-report their level of SB. Results for agreement in change over time showed moderate correlation (r = 0.52, p = 0.12) for MVPA and poor correlation for SB (r = −0.024, p = 0.916).

Levels of agreement with objective measurements indicate the GPAQ is a valid measure of MVPA and change in MVPA but is a less valid measure of current levels and change in SB. Thus, GPAQ appears to be an appropriate measure for assessing the effectiveness of interventions to promote MVPA.

Diagenetic Alterations and Reservoir Quality Evolution of Lower Cretaceous Fluvial Sandstones: Nubian Formation, Sirt Basin, North-Central Libya

Lower Cretaceous meandering and braided fluvial sandstones of the Nubian Formation form some of the most important subsurface reservoir rocks in the Sirt Basin, north-central Libya. Mineralogical, petrographical and geochemical analyses of sandstone samples from well BB6-59, Sarir oilfield, indicate that the meandering fluvial sandstones are fine- to very fine-grained subarkosic arenites (av. Q91F5L4), and that braided fluvial sandstones are medium- to very coarse-grained quartz arenites (av. Q96F3L1). The reservoir qualities of these sandstones were modified during both eodiagenesis (ca. <70oC; <2 km) and mesodiagenesis (ca. >70oC; >2km). Reservoir quality evolution was controlled primarily by the dissolution and kaolinitization of feldspars, micas and mud intraclasts during eodiagenesis, and by the amount and thicknessof grain-coating clays, chemical compaction and quartz overgrowths during mesodiagenesis. However, dissolution and kaolinitization of feldspars, micas and mud intraclasts resulted in the creation of intercrystalline micro- and mouldic macro-porosity and permeability during eodiagenesis, which were more widespread in braided fluvial than in meandering fluvial sandstones. This was because of the greater depositional porosity and permeability in the braided fluvial sandstones which enhanced percolation of meteoric waters. The development of only limited quartz overgrowths in the braided fluvial sandstones, in which quartz grains are coated by thick illite layers, retained high porosity and permeability (12-23 % and 30- 600 mD). By contrast, meandering fluvial sandstones underwent porosity loss as a result of quartz overgrowth development on quartz grains which lack or have thin and incomplete grain-coating illite (2-15 % and 0-0.1mD). Further loss of porosity in the meandering fluvial sandstones occurred as a result of chemical compaction (pressuredissolution) induced by the occurrence of micas along grains contacts. Otherdiagenetic alterations, such as the growth of pyrite, siderite, dolomite/ankerite and albitization, had little impact on reservoir quality. The albitization of feldspars may have had minor positive influence on reservoir quality throughthe creation of intercrystalline micro-porosity between albite crystals.The results of this study show that diagenetic modifications of the braided and meandering fluvial sandstones in the Nubian Formation, and resulting changes in reservoir quality, are closely linked to depositional porosity and permeability. They are also linked to the thickness of grain-coating infiltrated clays, and to variations in detrital composition, particularly the amounts of mud intraclasts, feldspars and mica grains as well as climatic conditions.

Electrical conduction and rheological behaviour of composites of poly(epsilon-caprolactone) and MWCNTs

Two mechanisms of conduction were identified from temperature dependent (120 K-340 K) DC electrical resistivity measurements of composites of poly(c-caprolactone) (PCL) and multi-walled carbon nanotubes (MWCNTs). Activation of variable range hopping (VRH) occurred at lower temperatures than that for temperature fluctuation induced tunneling (TFIT). Experimental data was in good agreement with the VRH model in contrast to the TFIT model, where broadening of tunnel junctions and increasing electrical resistivity at T > T-g is a consequence of a large difference in the coefficients of thermal expansion of PCL and MWCNTs. A numerical model was developed to explain this behavior accounting for a thermal expansion effect by supposing the large increase in electrical resistivity corresponds to the larger relative deformation due to thermal expansion associated with disintegration of the conductive MWCNT network. MWCNTs had a significant nucleating effect on PCL resulting in increased PCL crystallinity and an electrically insulating layer between MWCNTs. The onset of rheological percolation at similar to 0.18 vol% MWCNTs was clearly evident as storage modulus, G' and complex viscosity, vertical bar eta*vertical bar increased by several orders of magnitude. From Cole-Cole and Van Gurp-Palmen plots, and extraction of crossover points (G(c)) from overlaying plots of G' and G '' as a function of frequency, the onset of rheological percolation at 0.18 vol% MWCNTs was confirmed, a similar MWCNT loading to that determined for electrical percolation. 

Melt Processing and Properties of Polyamide 6/Graphene Nanoplatelet Composites

Graphene, due to its outstanding properties, has become the topic of much research activity in recent years. Much of that work has been on a laboratory scale however, if we are to introduce graphene into real product applications it is necessary to examine how the material behaves under industrial processing conditions. In this paper the melt processing of polyamide 6/graphene nanoplatelet composites via twin screw extrusion is investigated and structure–property relationships are examined for mechanical and electrical properties. Graphene nanoplatelets (GNPs) with two aspect ratios (700 and 1000) were used in order to examine the influence of particle dimensions on composite properties. It was found that the introduction of GNPs had a nucleating effect on polyamide 6 (PA6) crystallization and substantially increased crystallinity by up to 120% for a 20% loading in PA6. A small increase in crystallinity was observed when extruder screw speed increased from 50 rpm to 200 rpm which could be attributed to better dispersion and more nucleation sites for crystallization. A maximum enhancement of 412% in Young's modulus was achieved at 20 wt% loading of GNPs. This is the highest reported enhancement in modulus achieved to date for a melt mixed thermoplastic/GNPs composite. A further result of importance here is that the modulus continued to increase as the loading of GNPs increased even at 20 wt% loading and results are in excellent agreement with theoretical predictions for modulus enhancement. Electrical percolation was achieved between 10–15 wt% loading for both aspect ratios of GNPs with an increase in conductivity of approximately 6 orders of magnitude compared to the unfilled PA6.

Melt Processing and Properties of Polyamide 6/Graphene Nanoplatelet Composites

In this paper, the processing and characterization of Polyamide 6 (PA6) / graphite nanoplatelets
(GNPs) composites is reported. PA6/GNPs composites were prepared by melt-mixing using an
industrial, co-rotating, intermeshing, twin-screw extruder. A bespoke screw configuration was used
that was designed in-house to enhance nanoparticle dispersion into a polymer matrix. The effects of
GNPs type (xGnP® M-5 and xGnP® C-500), GNPs content, and extruder screw speed on the bulk
properties of the PA6/GNPs nanocomposites were investigated. Results show a considerable
improvement in the thermal and mechanical properties of PA6/GNPs composites, as compared with
the unfilled PA6 polymer. An increase in crystallinity (%Xc) with increasing GNPs content, and a
change in shape of the crystallization exotherms (broadening) and melting endotherms, both suggest a
change in the crystal type and perfection. An increase in tensile modulus of as much as 376% and
412% was observed for PA6/M-5 xGnP® and PA6/C-500 xGnP® composites, respectively, at filler
contents of 20wt%. The enhancement of Young’s modulus and yield stress can be attributed to the
reinforcing effect of GNPs and their uniform dispersion in the PA6 matrix. The rheological response
of the composite resembles that of a ‘pseudo-solid’, rather than a molten liquid, and analysis of the
rheological data indicates that a percolation threshold was reached at GNPs contents of between 10–
15wt%. The electrical conductivity of the composite also increased with increasing GNPs content,
with an addition of 15wt% GNPs resulting in a 6 order-of-magnitude increase in conductivity. The
electrical percolation thresholds of all composites were between 10–15wt%.

Characterisation of melt processed nanocomposites of Polyamide 6 subjected to uniaxial-drawing

In this paper, the processing and characterization of Polyamide 6 (PA6) nanocomposites containing graphite nanoplatelets (GNPs) is reported. PA6 nanocomposites were prepared by melt-mixing using an industrial, co-rotating, intermeshing, twin-screw extruder. A bespoke screw configuration was used that was designed in-house to enhance nanoparticle dispersion into a polymer matrix. The effects of nano-filler type (xGnPTM M-5 and xGnPTM C-500), nano-filler content, and extruder screw speed on the bulk properties of the PA6 nanocomposites were investigated. The crystalline structures of PA6 nanocomposites are related to thermal treatment, stress history and the presence of moisture and nanofillers. DSC, Raman and XRD studies show an increase in crystallinity with increasing GNP content and a phase transformation between α-form to γ-form crystals as a result of the heterophase nucleation effect. The effect of uniaxial stretching on PA6 nanocomposites was investigated by drawing specimens heated at temperatures below the melting temperature. DSC and Raman studies on the drawn samples show an increase in yield stress as the GNP content increases due to the strain induced crystallization and γ—β transition during stretching. The rheological response of the nanocomposites resemble that of a ‘pseudo-solid’, rather than a molten liquid, and analysis of the rheological data indicates that a percolation threshold was reached at GNP contents of between 10–15wt%. An increase in tensile modulus of as much as 412% was observed for PA6/C-500 xGnPTM composites, at a filler content of 20wt%. The enhancement of Young’s modulus and yield stress can be attributed to the reinforcing effect of GNPs and their uniform dispersion in the PA6 matrix. The electrical conductivity of the composite also increased with increasing GNP content, with an addition of 15wt% GNP resulting in a 6 order-of-magnitude increase in conductivity. The effects of uniaxial-drawing and the inclusion of multiple nano-filler varieties on the electrical and mechanical properties are currently under investigation.

Processability, structural evolution and properties of melt processed biaxially stretched HDPE/MWCNT nanocomposites

Biaxial stretching of melt mixed high density polyethylene (HDPE)/multiwalled carbon nanotube (MWCNT) nanocomposites was conducted in the melt state at different stretching ratios (SRs). The addition of MWCNTs leads to significant strain hardening in the HDPE, greatly improving the stability and thus processability of the stretching process. Scanning electron microscopy shows that the MWCNTs in the polymer matrix are gradually disentangled and randomly oriented in the stretching plane with increasing SRs. All the stretched samples exhibit an increase in crystallinity (about 10%) due to strain induced crystallization and a broadened distribution of crystallite size according to the XRD and DSC results. The mechanical properties of the composites improve with increasing SRs, while they drop off after a SR of 2.5 for the neat HDPE which is likely to be due to the relaxation of polymer chains prior to solidification. The presence of the MWCNTs appears to inhibit this relaxation thus helping to maintain the orientation and mechanical properties at high SRs. The modulus, yield strength and breaking strength of stretched composites with 8 wt% MWCNTs increase by approximately 54%, 85% and 193% respectively compared with the neat HDPE at a SR of 3. The electrical percolation threshold for the unstretched material occurs at 1.9 wt% MWCNTs. As SR increases, the values of critical concentration increase from 1.9 wt% to 4.9 wt% implying the destruction of conductive networks due to an increased inter-particle distance. A loading of 6 wt% MWCNTs is sufficient to ensure that the sheet conductivity is robust to changes in the SR. Decreased values of critical exponent from 1.9 to 1.1 and morphological investigation reveal a transformation of the system structure from three dimensional to two dimensional as SR increases.

Characterization and structure–property relationship of melt-mixed high density polyethylene/multi-walled carbon nanotube composites under extensional deformation

Melt-mixed high density polyethylene (HDPE)/multi-walled carbon nanotube (MWCNT) nanocomposites with 1–10 wt% MWCNTs were prepared by twin screw extrusion and compression moulded into sheet form. The compression moulded nanocomposites exhibit a 112% increase in modulus at a MWCNT loading of 4 wt%, and a low electrical percolation threshold of 1.9 wt%. Subsequently, uniaxial, sequential (seq-) biaxial and simultaneous (sim-) biaxial stretching of the virgin HDPE and nanocomposite sheets was conducted at different strain rates and stretching temperatures to investigate the processability of HDPE with the addition of nanotubes and the influence of deformation on the structure and final properties of nanocomposites. The results show that the processability of HDPE is improved under all the uniaxial and biaxial deformation conditions due to a strengthened strain hardening behaviour with the addition of MWCNTs. Extensional deformation is observed to disentangle nanotube agglomerates and the disentanglement degree is shown to depend on the stretching mode, strain rate and stretching temperatures applied. The disentanglement effectiveness is: uniaxial stretching < sim-biaxial stretching < seq-biaxial stretching, under the same deformation parameters. In sim-biaxial stretching, reducing the strain rate and stretching temperature can lead to more nanotube agglomerate breakup. Enhanced nanotube agglomerate disentanglement exhibits a positive effect on the mechanical properties and a negative effect on the electrical properties of the deformed nanocomposites. The ultimate stress of the composite containing 4 wt% MWCNTs increased by ∼492% after seq-biaxial stretching, while the resistivity increased by ∼1012 Ω cm.

Rethinking Rice Preparation for Highly Efficient Removal of Inorganic Arsenic Using Percolating Cooking Water

A novel way of cooking rice to maximize the removal of the carcinogen inorganic arsenic (Asi) is presented here. In conventional rice cooking water and grain are in continuous contact, and it is known that the larger the water:rice cooking ratio, the more Asi removed by cooking, suggesting that the Asi in the grain is mobile in water. Experiments were designed where rice is cooked in a continual stream of percolating near boiling water, either low in Asi, or Asi free. This has the advantage of not only exposing grain to large volumes of cooking water, but also physically removes any Asi leached from the grain into the water receiving vessel. The relationship between cooking water volume and Asi removal in conventional rice cooking was demonstrated for the rice types under study. At a water-to-rice cooking ratio of 12:1, 57±5% of Asi could be removed, average of 6 wholegrain and 6 polished rice samples. Two types of percolating technology were tested, one where the cooking water was recycled through condensing boiling water steam and passing the freshly distilled hot water through the grain in a laboratory setting, and one where tap water was used to cook the rice held in an off-the-shelf coffee percolator in a domestic setting. Both approaches proved highly effective in removing Asi from the cooking rice, with up to 85% of Asi removed from individual rice types. For the recycled water experiment 59±8% and 69±10% of Asi was removed, on average, compared to uncooked rice for polished (n=27) and wholegrain (n=13) rice, respectively. For coffee percolation there was no difference between wholegrain and polished rice, and the effectiveness of Asi removal was 49±7% across 6 wholegrain and 6 polished rice samples. The manuscript explores the potential applications and further optimization of this percolating cooking water, high Asi removal, discovery.

Effect of cooling rate on the properties of high density polyethylene/multi-walled carbon nanotube composites

High density polyethylene (HDPE)/multi-walled carbon nanotube (MWCNT) nanocomposites were prepared by melt mixing using twin-screw extrusion. The extruded pellets were compression moulded at 200°C for 5min followed by cooling at different cooling rates (20°C/min and 300°C/min respectively) to produce sheets for characterization. Scanning electron microscopy (SEM) shows that the MWCNTs are uniformly dispersed in the HDPE. At 4 wt% addition of MWCNTs composite modulus increased by over 110% compared with the unfilled HDPE (regardless of the cooling rate). The yield strength of both unfilled and filled HDPE decreased after rapid cooling by about 10% due to a lower crystallinity and imperfect crystallites. The electrical percolation threshold of composites, irrespective of the cooling rate, is between a MWCNT concentration of 1∼2 wt%. Interestingly, the electrical resistivity of the rapidly cooled composite with 2 wt% MWCNTs is lower than that of the slowly cooled composites with the same MWCNT loading. This may be due to the lower crystallinity and smaller crystallites facilitating the formation of conductive pathways. This result may have significant implications for both process control and the tailoring of electrical conductivity in the manufacture of conductive HDPE/MWCNT nanocomposites.

Autosomal dominant retinitis pigmentosa:a new multi-allelic marker (D3S621) genetically linked to the disease locus (RP4)

We report the characterization of a new eight-allele microsatellite (D3S621) isolated from a human chromosome 3 library. Two-point and multi-locus genetic linkage analysis have shown D3S621 to co-segregate with the previously mapped RP4 (theta m = 0.12, Zm = 4.34) and with other genetic markers on the long arm of the chromosome, including D3S14 (R208) (theta m = 0.00, Zm = 15.10), D3S47 (C17) (theta m = 0.11, Zm = 4.95), Rho (theta m = 0.07, Zm = 1.37), D3S21 (L182) (theta m = 0.07, Zm = 2.40) and D3S19 (U1) (theta m = 0.13, Zm = 2.78). This highly informative marker, with a polymorphic information content of 0.78, should be of considerable value in the extension of linkage data for autosomal dominant retinitis pigmentosa with respect to locii on the long arm of chromosome 3.

Quantification of Type VI secretion system activity in macrophages infected with Burkholderia cenocepacia

The Gram-negative bacterial type VI Secretion System (T6SS) delivers toxins to kill orinhibit the growth of susceptible bacteria, while others target eukaryotic cells. Deletionof atsR, a negative regulator of virulence factors in B. cenocepacia K56-2, increasesT6SS activity. Macrophages infected with a K56-2 ΔatsR mutant display dramaticalterations in their actin cytoskeleton architecture that rely on the T6SS, which isresponsible for the inactivation of multiple Rho-family GTPases by an unknownmechanism. We employed a strategy to standardize the bacterial infection ofmacrophages and densitometrically quantify the T6SS-associated cellular phenotype,which allowed us to characterize the phenotype of systematic deletions of each genewithin the T6SS cluster and ten vgrG encoding genes in K56-2 ΔatsR. None of thegenes from the T6SS core cluster and the individual vgrGs were directly responsiblefor the cytoskeletal changes in infected cells. However, a mutant strain with all vgrGgenes deleted was unable to cause macrophage alterations. Despite not being able toidentify a specific effector protein responsible for the cytoskeletal defects inmacrophages, our strategy resulted in the identification of the critical core componentsand accessory proteins of the T6SS assembly machinery and provides a screeningmethod to detect T6SS effectors targeting the actin cytoskeleton in macrophages byrandom mutagenesis.