A polymerase chain reaction-based method for isolating clones from a complimentary DNA library in sheep

The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes.

Defining a developmental subtype of bipolar disorder in a sample of nonreferred adults by age at onset

Objective To test the hypothesis that the age at onset of bipolar disorder would identify a developmental subtype of bipolar disorder in adults characterized by increased levels of irritability, chronic course, rapid cycling, and comorbidity with attention deficit hyperactivity disorder. Methods Forty-four adult subjects diagnosed with bipolar disorder were selected from large family studies of youth with and without attention deficit hyperactivity disorder. These subjects were stratified by the age at onset in childhood (younger than 13 years; n = 8, 18%), adolescence (13–18 years; n = 12, 27%, or adulthood (older than 19 years; n = 24, 55%). All subjects were administered structure diagnostic interviews and a brief cognitive battery. Results In contrast with adult-onset bipolar disorder, child-onset bipolar disorder was associated with a longer duration of illness, more irritability than euphoria, a mixed presentation, a more chronic or rapid-cycling course, and increased comorbidity with childhood disruptive behavior disorders and anxiety disorders. Conclusion Stratification by age at onset of bipolar disorder identified subgroups of adult subjects with differing clinical correlates. This pattern of correlates is consistent with findings documented in children with pediatric bipolar disorder and supports the hypothesis that child-onset bipolar disorder may represent a developmental subtype of the disorder.

Analysis of complex molecular systems: The impact of multivariate analysis for resolving the interactions of small molecules with biopolymers - a review

This review is focused on the impact of chemometrics for resolving data sets collected from investigations of the interactions of small molecules with biopolymers. These samples have been analyzed with various instrumental techniques, such as fluorescence, ultraviolet–visible spectroscopy, and voltammetry. The impact of two powerful and demonstrably useful multivariate methods for resolution of complex data—multivariate curve resolution–alternating least squares (MCR–ALS) and parallel factor analysis (PARAFAC)—is highlighted through analysis of applications involving the interactions of small molecules with the biopolymers, serum albumin, and deoxyribonucleic acid. The outcomes illustrated that significant information extracted by the chemometric methods was unattainable by simple, univariate data analysis. In addition, although the techniques used to collect data were confined to ultraviolet–visible spectroscopy, fluorescence spectroscopy, circular dichroism, and voltammetry, data profiles produced by other techniques may also be processed. Topics considered including binding sites and modes, cooperative and competitive small molecule binding, kinetics, and thermodynamics of ligand binding, and the folding and unfolding of biopolymers. Applications of the MCR–ALS and PARAFAC methods reviewed were primarily published between 2008 and 2013.

Nanofiber orientation and surface functionalization modulate human mesenchymal stem cell behavior in vitro

Electrospun nanofiber meshes have emerged as a new generation of scaffold membranes possessing a number of features suitable for tissue regeneration. One of these features is the flexibility to modify their structure and composition to orchestrate specific cellular responses. In this study, we investigated the effects of nanofiber orientation and surface functionalization on human mesenchymal stem cell (hMSC) migration and osteogenic differentiation. We used an in vitro model to examine hMSC migration into a cell-free zone on nanofiber meshes and mitomycin C treatment to assess the contribution of proliferation to the observed migration. Poly (ɛ-caprolactone) meshes with oriented topography were created by electrospinning aligned nanofibers on a rotating mandrel, while randomly oriented controls were collected on a stationary collector. Both aligned and random meshes were coated with a triple-helical, type I collagen-mimetic peptide, containing the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) motif. Our results indicate that nanofiber GFOGER peptide functionalization and orientation modulate cellular behavior, individually, and in combination. GFOGER significantly enhanced the migration, proliferation, and osteogenic differentiation of hMSCs on nanofiber meshes. Aligned nanofiber meshes displayed increased cell migration along the direction of fiber orientation compared to random meshes; however, fiber alignment did not influence osteogenic differentiation. Compared to each other, GFOGER coating resulted in a higher proliferation-driven cell migration, whereas fiber orientation appeared to generate a larger direct migratory effect. This study demonstrates that peptide surface modification and topographical cues associated with fiber alignment can be used to direct cellular behavior on nanofiber mesh scaffolds, which may be exploited for tissue regeneration.

Behavior of sprayed tricyclazole in rice paddy lysimeters

The behavior of sprayed tricyclazole in rice paddy lysimeters was studied. Tricyclazole residues were measured from rice leaves and paddy water after tricyclazole spraying in paddy lysimeters. The rate of photolysis and hydrolysis of tricyclazole on the surface of rice leaves was also determined in a laboratory experiment. Tricyclazole was extracted from leaf and water samples and determined by liquid chromatography with UV or mass spectrometry. The hydrolysis half-lives of tricyclazole on rice leaves were 11.9 and 5.1 d for the formulated product and standard, respectively. The photolysis half-lives were longer, 16.4 d for the formulated product and 20.9 d for the standard. In the paddy lysimeter, tricyclazole dissipation on leaves involved either biphasic first-order kinetics or single-phase first-order kinetics, depending on the rainfall pattern. Half-lives of tricyclazole on lysimeter rice leaves were from 3.0 to 5.7 d. The dissipation of tricyclazole in paddy water followed single-phase first-order kinetics with half-lives ranging from 2.1 to 5.0 d.

Can nurses do more for patients to reduce vascular access and cardiac complications following PCI?

Percutaneous coronary interventions have increased 50% in Australia, yet vascular and cardiac complications remain ongoing outcome issues for patients. Managing complications is confounded by reduced length of patient stay, yet is an integral component of a cardiac nurses’ scope of practice. The aim of this study was to highlight in and out of hospital vascular and cardiac complications, for twelve months post patient discharge after PCI. Prospective data was collected from the hospital angioplasty database from 1089 consecutive patients who had PCI procedures from 1 January 2005 to 31 December 2006. In hospital vascular complications were reported by 391 (35%) of the 1089 patients, following PCI. Of these, 22.4% had haemorrhage only, 7.1% haematoma only. Cardiac complications in hospital were, one death (0.09%) following PCI, three deaths (0.27%) during the same admission and no incidence of myocardial infarction or bypass surgery. Patients who had PCI in 2005 (525) were telephone followed up after discharge at one and twelve months. Surprisingly, ongoing vascular outcomes were noted, with a 2.5% incidence at one month and 4% at 12 months. Cardiac complications were also identified, 51 (9.7%) patients requiring readmission for repeat angiogram, 19 (3.6%) a repeat PCI and 7 (1.3%) patients undergoing bypass surgery. This review highlights that vascular and cardiac problems are ongoing issues for PCI patients both in and out of hospital. The results suggest that cardiac nurses focus more on improving the monitoring and discharge care of patients and families for recovery after PCI.

Impaired physical function associated with childhood obesity: How should we intervene?

- Background This study examined relationships between adiposity, physical functioning and physical activity. - Methods Obese (N=107) and healthy-weight (N=132) children aged 10-13 years underwent assessments of percent body fat (%BF, dual energy X-ray absorptiometry), knee extensor strength (KE, isokinetic dynamometry), cardiorespiratory fitness (CRF, peak oxygen uptake by cycle ergometry), physical health-related quality of life (HRQOL), worst pain intensity and walking capacity [six-minute walk (6MWT)]. Structural equation modelling was used to assess relationships between variables. - Results Moderate relationships were observed between %BF and 6MWT, KE strength corrected for mass and CRF relative to mass (r -.36 to -.69, P≤.007). Weak relationships were found between: %BF and physical HRQOL (r -.27, P=.008); CRF relative to mass and physical HRQOL (r -.24, P=.003); physical activity and 6MWT (r .17, P=.004). Squared multiple correlations showed that 29.6% variance in physical HRQOL was explained by %BF, pain and CRF relative to mass, while 28% variance in 6MWT was explained by %BF and physical activity. - Conclusions It appears that children with a higher body fat percentage have poorer KE strength, CRF and overall physical functioning. Reducing percent fat appears to be the best target to improve functioning. However, a combined approach to intervention, targeting reductions in body fat percentage, pain and improvements in physical activity and CRF may assist physical functioning.

Proinflammatory cytokines regulate cementogenic differentiation of periodontal ligament cells by Wnt/Ca2+ signaling pathway

Periodontal inflammation can inhibit cell differentiation of periodontal ligament cells (PDLCs), resulting in decreased bone/cementum regeneration ability. The Wnt signaling pathway, including canonical Wnt/β-catenin signaling and noncanonical Wnt/Ca2+ signaling, plays essential roles in cell proliferation and differentiation during tooth development. However, little is still known whether noncanonical Wnt/Ca2+ signaling cascade could regulate cementogenic/osteogenic differentiation capability of PDLCs within an inflammatory environment. Therefore, in this study, human PDLCs (hPDLCs) and their cementogenic differentiation potential were investigated in the presence of cytokines. The data demonstrated that both cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) inhibited cell proliferation, relative alkaline phosphatase activity, bone/cementum-related gene/protein expression, and canonical Wnt pathway-related gene/protein expression in hPDLCs. Interestingly, both cytokines upregulated the noncanonical Wnt/Ca2+ signaling-related gene and protein expression in hPDLCs. When the Wnt/Ca2+ pathway was blocked by Ca2+/calmodulin-dependent protein kinase II inhibitor KN93, even in the presence of IL-6 and TNF-α, cementogenesis could be stimulated in hPDLCs. Our data indicate that the Wnt/Ca2+ pathway plays an inhibitory role on PDLC cementogenic differentiation in inflammatory microenvironments. Therefore, targeting the Wnt/Ca2+ pathway may provide a novel therapeutic approach to improve periodontal regeneration for periodontal diseases.

Monitoring healing progression and characterizing the mechanical environment in preclinical models for bone tissue engineering

The treatment of large segmental bone defects remains a significant clinical challenge. Due to limitations surrounding the use of bone grafts, tissue-engineered constructs for the repair of large bone defects could offer an alternative. Before translation of any newly developed tissue engineering (TE) approach to the clinic, efficacy of the treatment must be shown in a validated preclinical large animal model. Currently, biomechanical testing, histology, and microcomputed tomography are performed to assess the quality and quantity of the regenerated bone. However, in vivo monitoring of the progression of healing is seldom performed, which could reveal important information regarding time to restoration of mechanical function and acceleration of regeneration. Furthermore, since the mechanical environment is known to influence bone regeneration, and limb loading of the animals can poorly be controlled, characterizing activity and load history could provide the ability to explain variability in the acquired data sets and potentially outliers based on abnormal loading. Many approaches have been devised to monitor the progression of healing and characterize the mechanical environment in fracture healing studies. In this article, we review previous methods and share results of recent work of our group toward developing and implementing a comprehensive biomechanical monitoring system to study bone regeneration in preclinical TE studies.

Drug discovery approaches utilizing three-dimensional cell culture

Historically, two-dimensional (2D) cell culture has been the preferred method of producing disease models in vitro. Recently, there has been a move away from 2D culture in favor of generating three-dimensional (3D) multicellular structures, which are thought to be more representative of the in vivo environment. This transition has brought with it an influx of technologies capable of producing these structures in various ways. However, it is becoming evident that many of these technologies do not perform well in automated in vitro drug discovery units. We believe that this is a result of their incompatibility with high-throughput screening (HTS). In this study, we review a number of technologies, which are currently available for producing in vitro 3D disease models. We assess their amenability with high-content screening and HTS and highlight our own work in attempting to address many of the practical problems that are hampering the successful deployment of 3D cell systems in mainstream research.

Diagnostic values for skin temperature assessment to detect diabetes-related foot complications

Background Skin temperature assessment is a promising modality for early detection of diabetic foot problems, but its diagnostic value has not been studied. Our aims were to investigate the diagnostic value of different cutoff skin temperature values for detecting diabetes-related foot complications such as ulceration, infection, and Charcot foot and to determine urgency of treatment in case of diagnosed infection or a red-hot swollen foot. Materials and Methods The plantar foot surfaces of 54 patients with diabetes visiting the outpatient foot clinic were imaged with an infrared camera. Nine patients had complications requiring immediate treatment, 25 patients had complications requiring non-immediate treatment, and 20 patients had no complications requiring treatment. Average pixel temperature was calculated for six predefined spots and for the whole foot. We calculated the area under the receiver operating characteristic curve for different cutoff skin temperature values using clinical assessment as reference and defined the sensitivity and specificity for the most optimal cutoff temperature value. Mean temperature difference between feet was analyzed using the Kruskal–Wallis tests. Results The most optimal cutoff skin temperature value for detection of diabetes-related foot complications was a 2.2°C difference between contralateral spots (sensitivity, 76%; specificity, 40%). The most optimal cutoff skin temperature value for determining urgency of treatment was a 1.35°C difference between the mean temperature of the left and right foot (sensitivity, 89%; specificity, 78%). Conclusions Detection of diabetes-related foot complications based on local skin temperature assessment is hindered by low diagnostic values. Mean temperature difference between two feet may be an adequate marker for determining urgency of treatment.

Assessment of signs of foot infection in diabetes patients using photographic foot imaging and infrared thermography

Background Patients with diabetic foot disease require frequent screening to prevent complications and may be helped through telemedical home monitoring. Within this context, the goal was to determine the validity and reliability of assessing diabetic foot infection using photographic foot imaging and infrared thermography. Subjects and Methods For 38 patients with diabetes who presented with a foot infection or were admitted to the hospital with a foot-related complication, photographs of the plantar foot surface using a photographic imaging device and temperature data from six plantar regions using an infrared thermometer were obtained. A temperature difference between feet of > 2.2 °C defined a ''hotspot.'' Two independent observers assessed each foot for presence of foot infection, both live (using the Perfusion-Extent-Depth- Infection-Sensation classification) and from photographs 2 and 4 weeks later (for presence of erythema and ulcers). Agreement in diagnosis between live assessment and (the combination of ) photographic assessment and temperature recordings was calculated. Results Diagnosis of infection from photographs was specific (> 85%) but not very sensitive (< 60%). Diagnosis based on hotspots present was sensitive (> 90%) but not very specific (<25%). Diagnosis based on the combination of photographic and temperature assessments was both sensitive (> 60%) and specific (> 79%). Intra-observer agreement between photographic assessments was good (Cohen's j = 0.77 and 0.52 for both observers). Conclusions Diagnosis of foot infection in patients with diabetes seems valid and reliable using photographic imaging in combination with infrared thermography. This supports the intended use of these modalities for the home monitoring of high-risk patients with diabetes to facilitate early diagnosis of signs of foot infection.

Growth factor-loaded microparticles for tissue engineering: The discrepancies of in vitro characterization assays

Efficient and effective growth factor (GF) delivery is an ongoing challenge for tissue regeneration therapies. The accurate quantification of complex molecules such as GFs, encapsulated in polymeric delivery devices, is equally critical and just as complex as achieving efficient delivery of active GFs. In this study, GFs relevant to bone tissue formation, vascular endothelial growth factor (VEGF) and bone morphogenetic protein 7 (BMP-7), were encapsulated, using the technique of electrospraying, into poly(lactic-co-glycolic acid) microparticles that contained poly(ethylene glycol) and trehalose to assist GF bioactivity. Typical quantification procedures, such as extraction and release assays using saline buffer, generated a significant degree of GF interactions, which impaired accurate assessment by enzyme-linked immunosorbent assay (ELISA). When both dry BMP-7 and VEGF were processed with chloroform, as is the case during the electrospraying process, reduced concentrations of the GFs were detected by ELISA; however, the biological effect on myoblast cells (C2C12) or endothelial cells (HUVECs) was unaffected. When electrosprayed particles containing BMP-7 were cultured with preosteoblasts (MC3T3-E1), significant cell differentiation into osteoblasts was observed up to 3 weeks in culture, as assessed by measuring alkaline phosphatase. In conclusion, this study showed how electrosprayed microparticles ensured efficient delivery of fully active GFs relevant to bone tissue engineering. Critically, it also highlights major discrepancies in quantifying GFs in polymeric microparticle systems when comparing ELISA with cell-based assays.

Sign and share: What influences our participation in online microvolunteering

Microvolunteering is bite-size volunteering with no commitment to repeat and minimum formality, involving short and specific actions. Online microvolunteering occurs through an internet-connected device. University students' online microvolunteering decisions were investigated using an extended theory of planned behavior (TPB) comprising attitudes and normative and control perceptions, with the additional variables of moral norm and group norm. Participants (N = 303) completed the main TPB questionnaire and 1-month follow-up survey (N = 171) assessing engagement in online microvolunteering. Results generally supported standard and additional TPB constructs predicting intention. Intention predicted behavior. The findings suggest an important role for attitudes and moral considerations in understanding what influences this increasingly popular form of online activity.