Tailored Microcantilever Optimization for Multifrequency Force Microscopy.

Microcantilevers are at the heart of atomic force microscopy (AFM) and play a significant role in AFM-based techniques. Recent advancements in multifrequency AFM require the simultaneous excitation and detection of multiple eigenfrequencies of microcantilevers to assess more data channels to quantify the material properties. However, to achieve higher spatiotemporal resolution there is a need to optimize the structure of microcantilevers. In this study, the architecture of the cantilever with gold nanoparticles using a dip-coating method is modified, aiming to tune the higher eigenmodes of the microcantilever as integer multiples of its fundamental frequency. Through the theoretical methodology and simulative model, that integer harmonics improve the coupling in multifrequency AFM measurements is demonstrated, leading to enhanced image quality and resolution. Furthermore, via the combined theoretical-experimental approach, the interplay between induced mass and stiffness change of the modified cantilever depending on the attached particle location, size, mass, and geometry is found. To validate the results of this predictive model, tapping-mode AFM is utilized and bimodal Amplitude Modulation AFM techniques to examine and quantify the impact of tuning higher-order eigenmodes on the imaging quality of a polystyrene-polymethylmethacrylate (PS-PMMA) block co-polymer assembly deposited on a glass slide and Highly Ordered Pyrolytic Graphite (HOPG).

Nanotopography of Polystyrene/Poly(methyl methacrylate) for the Promotion of Patient Specific Von Willebrand Factor Entrapment and Platelet Adhesion in a Whole Blood Microfluidic Assay.

Platelet function testing is essential for the diagnosis of patients with bleeding disorders. Specifically, there is a need for a whole blood assay that is capable of analysing platelet behaviour in contact with a patient-specific autologous von Willebrand factor (vWF), under physiologically relevant conditions. The creation of surface topography capable of entrapping and uncoiling vWF for the support of subsequent platelet adhesion within the same blood sample offers a potential basis for such an assay. In this study, spin coating of polystyrene/poly (methyl methacrylate) (PS/PMMA) demixed solutions onto glass substrates in air has been used to attain surfaces with well-defined topographical features. The effect of augmenting the PS/PMMA solution with uniform 50 µm PS microspheres that can moderate the demixing process on the resultant surface features has also been investigated. The topographical features created here by spin coating under ambient air pressure conditions, rather than in nitrogen, which previous work reports, produces substrate surfaces with the ability to entrap vWF from flowing blood and facilitate platelet adhesion. The direct optical visualisation of fluorescently-labelled platelets indicates that topography resulting from inclusion of PS microspheres in the PS/PMMA spin coating solution increases the total number of platelets that adhere to the substrate surface over the period of the microfluidic assay. However, a detailed analysis of the adhesion rate, mean translocating velocity, mean translocation distance, and fraction of the stably adhered platelets measured during blood flow under arterial equivalent mechanical shear conditions indicates no significant difference for topographies created with or without inclusion of the PS microspheres.

3D Fabrication and Characterisation of Electrically Receptive PCL-Graphene Scaffolds for Bioengineered In Vitro Tissue Models.

Polycaprolactone (PCL) is a well-established biomaterial, offering extensive mechanical attributes along with low cost, biocompatibility, and biodegradability; however, it lacks hydrophilicity, bioactivity, and electrical conductivity. Advances in 3D fabrication technologies allow for these sought-after attributes to be incorporated into the scaffolds during fabrication. In this study, solvent-free Fused Deposition Modelling was employed to fabricate 3D scaffolds from PCL with increasing amounts of graphene (G), in the concentrations of 0.75, 1.5, 3, and 6% (w/w). The PCL+G scaffolds created were characterised physico-chemically, electrically, and biologically. Raman spectroscopy demonstrated that the scaffold outer surface contained both PCL and G, with the G component relatively uniformly distributed. Water contact angle measurement demonstrated that as the amount of G in the scaffold increases (0.75-6% w/w), hydrophobicity decreases; mean contact angle for pure PCL was recorded as 107.22 ± 9.39°, and that with 6% G (PCL+6G) as 77.56 ± 6.75°. Electrochemical Impedance Spectroscopy demonstrated a marked increase in electroactivity potential with increasing G concentration. Cell viability results indicated that even the smallest addition of G (0.75%) resulted in a significant improvement in electroactivity potential and bioactivity compared with that for pure PCL, with 1.5 and 3% exhibiting the highest statistically significant increases in cell proliferation.

Gene expression analysis of the Xenopus laevis early limb bud proximodistal axis.

BACKGROUND: Limb buds develop as bilateral outgrowths of the lateral plate mesoderm and are patterned along three axes. Current models of proximal to distal patterning of early amniote limb buds suggest that two signals, a distal organizing signal from the apical epithelial ridge (AER, Fgfs) and an opposing proximal (retinoic acid [RA]) act early on pattern this axis. RESULTS: Transcriptional analysis of stage 51 Xenopus laevis hindlimb buds sectioned along the proximal-distal axis showed that the distal region is distinct from the rest of the limb. Expression of capn8.3, a novel calpain, was located in cells immediately flanking the AER. The Wnt antagonist Dkk1 was AER-specific in Xenopus limbs. Two transcription factors, sall1 and zic5, were expressed in distal mesenchyme. Zic5 has no described association with limb development. We also describe expression of two proximal genes, gata5 and tnn, not previously associated with limb development. Differentially expressed genes were associated with Fgf, Wnt, and RA signaling as well as differential cell adhesion and proliferation. CONCLUSIONS: We identify new candidate genes for early proximodistal limb patterning. Our analysis of RA-regulated genes supports a role for transient RA gradients in early limb bud in proximal-to-distal patterning in this anamniote model organism.

Nanoindentation and nano-scratching of hydroxyapatite coatings for resorbable magnesium alloy bone implant applications.

The corrosion rate of Mg alloys is currently too high for viable resorbable implant applications. One possible solution is to coat the alloy with a hydroxyapatite (HA) layer to slow the corrosion and promote bone growth. As such coatings can be under severe stresses during implant insertion, we present a nano-mechanical and nano-tribological investigation of RF-sputtered HA films on AZ31 Mg alloy substrates. EDX and XRD analysis indicate that as-deposited coatings are amorphous and Ca-deficient whereas rapid thermal annealing results in c-axis orientation and near-stoichiometric composition. Analysis of the nanoindentation data using a thin film model shows that annealing increases the coating's intrinsic hardness (H) and strain at break (H/E) values, from 2.7 GPa to 9.4 GPa and from 0.043 to 0.079, respectively. In addition, despite being rougher, the annealed samples display better wear resistance; a sign that the rapid thermal annealing does not compromise their interfacial strength and that these systems have potential for resorbable bone implant applications.

Manipulating the microbiome alters regenerative outcomes in Xenopus laevis tadpoles via lipopolysaccharide signalling.

Xenopus laevis tadpoles can regenerate functional tails, containing the spinal cord, notochord, muscle, fin, blood vessels and nerves, except for a brief refractory period at around 1 week of age. At this stage, amputation of the tadpole's tail may either result in scarless wound healing or the activation of a regeneration programme, which replaces the lost tissues. We recently demonstrated a link between bacterial lipopolysaccharides and successful tail regeneration in refractory stage tadpoles and proposed that this could result from lipopolysaccharides binding to Toll-like receptor 4 (TLR4). Here, we have used 16S rRNA sequencing to show that the tadpole skin microbiome is highly variable between sibships and that the community can be altered by raising embryos in the antibiotic gentamicin. Six Gram-negative genera, including Delftia and Chryseobacterium, were over-represented in tadpoles that underwent tail regeneration. Lipopolysaccharides purified from a commensal Chryseobacterium spp. XDS4, an exogenous Delftia spp. or Escherichia coli, could significantly increase the number of antibiotic-raised tadpoles that attempted regeneration. Conversely, the quality of regeneration was impaired in native-raised tadpoles exposed to the antagonistic lipopolysaccharide of Rhodobacter sphaeroides. Editing TLR4 using CRISPR/Cas9 also reduced regeneration quality, but not quantity, at the level of the cohort. However, we found that the editing level of individual tadpoles was a poor predictor of regenerative outcome. In conclusion, our results suggest that variable regeneration in refractory stage tadpoles depends at least in part on the skin microbiome and lipopolysaccharide signalling, but that signalling via TLR4 cannot account for all of this effect.

The Surface Characterisation of Fused Filament Fabricated (FFF) 3D Printed PEEK/Hydroxyapatite Composites.

Polyetheretherketone (PEEK) is a high-performance thermoplastic polymer which has found increasing application in orthopaedics and has shown a lot of promise for 'made-to-measure' implants via additive manufacturing approaches. However, PEEK is bioinert and needs to undergo surface modification to make it at least osteoconductive to ensure a more rapid, improved, and stable fixation that will last longer in vivo. One approach to solving this issue is to modify PEEK with bioactive agents such as hydroxyapatite (HA). The work reported in this study demonstrates the direct 3D printing of PEEK/HA composites of up to 30 weight percent (wt%) HA using a Fused Filament Fabrication (FFF) approach. The surface characteristics and in vitro properties of the composite materials were investigated. X-ray diffraction revealed the samples to be semi-crystalline in nature, with X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry revealing HA materials were available in the uppermost surface of all the 3D printed samples. In vitro testing of the samples at 7 days demonstrated that the PEEK/HA composite surfaces supported the adherence and growth of viable U-2 OS osteoblast like cells. These results demonstrate that FFF can deliver bioactive HA on the surface of PEEK bio-composites in a one-step 3D printing process.

11-Ketotestosterone induces oocyte growth, but does not affect oocyte cytology in pre-vitellogenic captive beluga, Huso huso L.

An effect of 11-ketotestosterone (11-KT) on growth of previtellogenic (PV) ovaries of eel, salmon and Atlantic cod has been demonstrated. The purpose of this study was to investigate the effects of 11-KT treatment (in vivo) on ovarian growth, on hormonal and biochemical changes in blood, and on ovarian mRNA levels of lipidation-related genes in captive beluga with PV oocytes. In addition, the potential involvement of lipoprotein lipase (Lpl), an important enzyme for extracellular hydrolysis of lipoprotein-associated lipids, was evaluated. Twelve beluga (4-year olds) were treated with an intraperitoneal slow-release implant of either 11-KT (2.5 mg) or a compressed matrix (control). Ovarian biopsy was done to obtain pre- (day 0: T0) and post-treatment (day 21: T21) data on histology and target gene expression. Three weeks of exposure resulted in an increase in serum 11-KT levels from 2.2 ng/mL to 83 ng/mL but did not yield significant changes in serum levels of triacylglycerides and cholesterol. Furthermore, 11-KT implantation increased oocyte diameters from 259 µm (T0) to 309 µm by T21. Regardless of the increase in oocyte size, ovaries remained in the PV stage, mostly as late perinucleolar oocytes. Meanwhile, at the molecular level, the expression of lipidation-related transcripts [lpl, apolipoprotein E (apoe), very low density lipoprotein receptors (vldlr), low-density lipoprotein receptor-related protein 8-like (lrp8)] was significantly up-regulated after three weeks. Immunostaining for Lpl by Western blotting indicated three immunoreactive bands (70, 58 and 37 kDa) in ovarian homogenates from beluga, but signal intensity was not affected by treatment. Altogether, the administration of 11-KT increased 11-KT serum levels, oocyte size, and the expression of genes associated with lipid uptake. However, this treatment did not advance ovarian development beyond the PV stage.

Owner personality and the wellbeing of their cats share parallels with the parent-child relationship.

Human personality may substantially affect the nature of care provided to dependants. This link has been well researched in parents and children, however, relatively little is known about this dynamic with regards to humans' relationships with non-human animals. Owner interactions with companion animals may provide valuable insight into the wider phenomenon of familial interactions, as owners usually adopt the role of primary caregiver and potentially surrogate parent. This study, using cats as an exemplar, explored the relationship between owner personality and the lifestyles to which cats are exposed. In addition, it explored owner personality as it related to reported cat behaviour and wellbeing. Cat owners (n = 3331) responded to an online survey examining their personality and the health, behaviour and management of their cats. Owner personality was measured using the Big Five Inventory (BFI) to assess: Agreeableness, Conscientiousness, Extroversion, Neuroticism and Openness. Owners also provided information concerning the physical health, breed type, management and behavioural styles of their cats. Generalised linear mixed models were used to identify relationships between owner personality and a range of factors that may have welfare implications for the wider companion animal population, and specifically, cats. Higher owner Neuroticism was associated with an increased likelihood of non-pedigree rather than pedigree cat ownership, a decreased likelihood of ad libitum access to the outdoors, cats being reported as having a 'behavioural problem', displaying more aggressive and anxious/fearful behavioural styles and more stress-related sickness behaviours, as well as having an ongoing medical condition and being overweight. Other owner personality traits were generally found to correlate more positively with various lifestyle, behaviour and welfare parameters. For example, higher owner Extroversion was associated with an increased likelihood that the cat would be provided ad libitum access to the outdoors; higher owner Agreeableness was associated with a higher level of owner reported satisfaction with their cat, and with a greater likelihood of owners reporting their cats as being of a normal weight. Finally higher owner Conscientiousness was associated with the cat displaying less anxious/fearful, aggressive, aloof/avoidant, but more gregarious behavioural styles. These findings demonstrate that the relationship between carer personality and the care received by a dependent, may extend beyond the human family to animal-owner relationships, with significant implications for the choice of management, behaviour and potentially the broader wellbeing of companion animals.

Entrapment of Autologous von Willebrand Factor on Polystyrene/Poly(methyl methacrylate) Demixed Surfaces.

Human platelets play a vital role in haemostasis, pathological bleeding and thrombosis. The haemostatic mechanism is concerned with the control of bleeding from injured blood vessels, whereby platelets interact with the damaged inner vessel wall to form a clot (thrombus) at the site of injury. This adhesion of platelets and their subsequent aggregation is dependent on the presence of the blood protein von Willebrand Factor (vWF). It is proposed here that the entrapment of vWF on a substrate surface offers the opportunity to assess an individual's platelet function in a clinical diagnostic context. Spin coating from demixed solutions of polystyrene (PS) and poly(methyl methacrylate) (PMMA) onto glass slides has been shown previously to support platelet adhesion but the mechanism by which this interaction occurs, including the role of vWF, is not fully understood. In this work, we report a study of the interaction of platelets in whole blood with surfaces produced by spin coating from a solution of a weight/weight mixture of a 25% PS and 75% PMMA (25PS/75PMMA) in chloroform in the context of the properties required for their use as a Dynamic Platelet Function Assay (DPFA) substrate. Atomic Force Microscopy (AFM) indicates the presence of topographical features on the polymer demixed surfaces in the sub-micron to nanometer range. X-ray Photoelectron Spectroscopy (XPS) analysis confirms that the uppermost surface chemistry of the coatings is solely that of PMMA. The deliberate addition of various amounts of 50 µm diameter PS microspheres to the 25PS/75PMMA system has been shown to maintain the PMMA chemistry, but to significantly change the surface topography and to subsequently effect the scale of the resultant platelet interactions. By blocking specific platelet binding sites, it has been shown that their interaction with these surfaces is a consequence of the entrapment and build-up of vWF from the same whole blood sample.

Murine norovirus replication induces G0/G1 cell cycle arrest in asynchronously growing cells.

UNLABELLED: Many viruses replicate most efficiently in specific phases of the cell cycle, establishing or exploiting favorable conditions for viral replication, although little is known about the relationship between caliciviruses and the cell cycle. Microarray and Western blot analysis of murine norovirus 1 (MNV-1)-infected cells showed changes in cyclin transcript and protein levels indicative of a G1 phase arrest. Cell cycle analysis confirmed that MNV-1 infection caused a prolonging of the G1 phase and an accumulation of cells in the G0/G1 phase. The accumulation in G0/G1 phase was caused by a reduction in cell cycle progression through the G1/S restriction point, with MNV-1-infected cells released from a G1 arrest showing reduced cell cycle progression compared to mock-infected cells. MNV-1 replication was compared in populations of cells synchronized into specific cell cycle phases and in asynchronously growing cells. Cells actively progressing through the G1 phase had a 2-fold or higher increase in virus progeny and capsid protein expression over cells in other phases of the cell cycle or in unsynchronized populations. These findings suggest that MNV-1 infection leads to prolonging of the G1 phase and a reduction in S phase entry in host cells, establishing favorable conditions for viral protein production and viral replication. There is limited information on the interactions between noroviruses and the cell cycle, and this observation of increased replication in the G1 phase may be representative of other members of the Caliciviridae. IMPORTANCE: Noroviruses have proven recalcitrant to growth in cell culture, limiting our understanding of the interaction between these viruses and the infected cell. In this study, we used the cell-culturable MNV-1 to show that infection of murine macrophages affects the G1/S cell cycle phase transition, leading to an arrest in cell cycle progression and an accumulation of cells in the G0/G1 phase. Furthermore, we show that MNV replication is enhanced in the G1 phase compared to other stages of the cell cycle. Manipulating the cell cycle or adapting to cell cycle responses of the host cell is a mechanism to enhance virus replication. To the best of our knowledge, this is the first report of a norovirus interacting with the host cell cycle and exploiting the favorable conditions of the G0/G1 phase for RNA virus replication.

Do recycled spectacles meet the refractive needs of a developing country?

PURPOSE: The aim was to compare the power of spectacles donated to a recycled spectacle program to the custom-made spectacle refractive prescriptions dispensed in a developing country. METHODS: Two hundred consecutive prescriptions were audited in an optical dispensary in Timor-Leste, a developing nation. These refractions were compared against measurements of 2,075 wearable donated spectacles. We determined how many of the 200 prescriptions could be matched to a donated spectacle measurement, how many donated spectacles could be tried for each prescription and how long it would take to find the matched spectacles. RESULTS: There were 1,854 donated spectacles identified as being suitable for comparison with the 200 refractive prescriptions. Twenty-nine out of 200 prescriptions (14.5 per cent) were matched to at least one pair of donated spectacles. CONCLUSION: Recycling all spectacles is not cost-effective in a developing country that has the ability to make custom-made spectacles and dispense ready-made spectacles.

Electrospray ionization mass spectrometry identifies substrates and products of lipoprotein-associated phospholipase A2 in oxidized human low density lipoprotein.

There is increasing evidence that modified phospholipid products of low density lipoprotein (LDL) oxidation mediate inflammatory processes within vulnerable atherosclerotic lesions. Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is present in vulnerable plaque regions where it acts on phospholipid oxidation products to generate the pro-inflammatory lysophsopholipids and oxidized non-esterified fatty acids. This association together with identification of circulating Lp-PLA(2) levels as an independent predictor of cardiovascular disease provides a rationale for development of Lp-PLA(2) inhibitors as therapy for atherosclerosis. Here we report a systematic analysis of the effects of in vitro oxidation in the absence and presence of an Lp-PLA(2) inhibitor on the phosphatidylcholine (PC) composition of human LDL. Mass spectrometry identifies three classes of PC whose concentration is significantly enhanced during LDL oxidation. Of these, a series of molecules, represented by peaks in the m/z range 594-666 and identified as truncated PC oxidation products by accurate mass measurements using an LTQ Orbitrap mass spectrometer, are the predominant substrates for Lp-PLA(2). A second series of oxidation products, represented by peaks in the m/z range 746-830 and identified by LTQ Orbitrap analysis as non-truncated oxidized PCs, are quantitatively more abundant but are less efficient Lp-PLA(2) substrates. The major PC products of Lp-PLA(2), saturated and mono-unsaturated lyso-PC, constitute the third class. Mass spectrometric analysis confirms the presence of many of these PCs within human atherosclerotic lesions, suggesting that they could potentially be used as in vivo markers of atherosclerotic disease progression and response to Lp-PLA(2) inhibitor therapy.

Glucose transporters in human renal proximal tubular cells isolated from the urine of patients with non-insulin-dependent diabetes.

The bulk of glucose that is filtered by the renal glomerulus is reabsorbed by the glucose transporters of the proximal convoluted tubular epithelium. However, it has been difficult to investigate this in diseases such as type 2 diabetes because of the inability to isolate primary renal cells from patients without a renal biopsy. We report here a method for the immunomagnetic isolation and novel primary culture of human exfoliated proximal tubular epithelial cells (HEPTECs) from fresh urine. The primary isolates are highly enriched and differentiated and express characteristic proximal tubular phenotypic markers. They continue to express the proximal tubular markers CD13/aminopeptidase-N, sodium glucose cotransporter (SGLT) 2, and alkaline phosphatase through up to six subsequent subcultures in a similar way to human proximal cells isolated from renal biopsies. In a hyperglycemic environment, HEPTECs isolated from patients with type 2 diabetes expressed significantly more SGLT2 and the facilitative glucose transporter GLUT2 than cells from healthy individuals. We also demonstrated a markedly increased renal glucose uptake in HEPTECs isolated from patients with type 2 diabetes compared with healthy control subjects. Our findings indicate for the first time in a human cellular model that increased renal glucose transporter expression and activity is associated with type 2 diabetes.

BRCA1 and BRCA2 mutations in a population-based study of male breast cancer.

BACKGROUND: The contribution of BRCA1 and BRCA2 to the incidence of male breast cancer (MBC) in the United Kingdom is not known, and the importance of these genes in the increased risk of female breast cancer associated with a family history of breast cancer in a male first-degree relative is unclear. METHODS: We have carried out a population-based study of 94 MBC cases collected in the UK. We screened genomic DNA for mutations in BRCA1 and BRCA2 and used family history data from these cases to calculate the risk of breast cancer to female relatives of MBC cases. We also estimated the contribution of BRCA1 and BRCA2 to this risk. RESULTS: Nineteen cases (20%) reported a first-degree relative with breast cancer, of whom seven also had an affected second-degree relative. The breast cancer risk in female first-degree relatives was 2.4 times (95% confidence interval [CI] = 1.4-4.0) the risk in the general population. No BRCA1 mutation carriers were identified and five cases were found to carry a mutation in BRCA2. Allowing for a mutation detection sensitivity frequency of 70%, the carrier frequency for BRCA2 mutations was 8% (95% CI = 3-19). All the mutation carriers had a family history of breast, ovarian, prostate or pancreatic cancer. However, BRCA2 accounted for only 15% of the excess familial risk of breast cancer in female first-degree relatives. CONCLUSION: These data suggest that other genes that confer an increased risk for both female and male breast cancer have yet to be found.