Method for 3D fibre reconstruction on a microrobotic platform.

Automated handling of a natural fibrous object requires a method for acquiring the three-dimensional geometry of the object, because its dimensions cannot be known beforehand. This paper presents a method for calculating the three-dimensional reconstruction of a paper fibre on a microrobotic platform that contains two microscope cameras. The method is based on detecting curvature changes in the fibre centreline, and using them as the corresponding points between the different views of the images. We test the developed method with four fibre samples and compare the results with the references measured with an X-ray microtomography device. We rotate the samples through 16 different orientations on the platform and calculate the three-dimensional reconstruction to test the repeatability of the algorithm and its sensitivity to the orientation of the sample. We also test the noise sensitivity of the algorithm, and record the mismatch rate of the correspondences provided. We use the iterative closest point algorithm to align the measured three-dimensional reconstructions with the references. The average point-to-point distances between the reconstructed fibre centrelines and the references are 20-30 µm, and the mismatch rate is low. Given the manipulation tolerance, this shows that the method is well suited to automated fibre grasping. This has also been demonstrated with actual grasping experiments.

A flexible microrobotic platform for handling microscale specimens of fibrous materials for microscopic studies.

One of the most challenging issues faced in handling specimens for microscopy, is avoiding artefacts and structural changes in the samples caused by human errors. In addition, specimen handling is a laborious and time-consuming task and requires skilful and experienced personnel. This paper introduces a flexible microrobotic platform for the handling of microscale specimens of fibrous materials for various microscopic studies such as scanning electron microscopy and nanotomography. The platform is capable of handling various fibres with diameters ranging from 10 to 1000 µm and lengths of 100 µm-15 mm, and mounting them on different types of specimen holders without damaging them. This tele-operated microrobotic platform minimizes human interaction with the samples, which is one of the main sources contributory to introducing artefacts into the specimens. The platform also grants a higher throughput and an improved success rate of specimen handling, when compared to the manual processes. The operator does not need extensive experience of microscale manipulation and only a short training period is sufficient to operate the platform. The requirement of easy configurability for various samples and sample holders is typical in the research and development of materials in this field. Therefore, one of the main criteria for the design of the microrobotic platform was the ability to adapt the platform to different specimen handling methods required for microscopic studies. To demonstrate this, three experiments are carried out using the microrobotic platform. In the first experiment, individual paper fibres are mounted successfully on scanning electron microscopy specimen holders for the in situ scanning electron microscopy diagonal compression test of paper fibres. The performance of the microrobotic platform is compared with a skilled laboratory worker performing the same experiment. In the second experiment, a strand of human hair and an individual paper fibre bond are mounted on a specimen holder for nanotomography studies. In the third experiment, individual paper fibre bonds with controlled crossing and vertical angles are made using the microrobotic platform. If an industrial application requires less flexibility but a higher speed when handling one type of sample to a specific holder, then the platform can be automated in the future.

Parent-reported food allergy requiring an avoidance diet in children starting elementary school.

AIM: To evaluate the prevalence of parent-reported food allergies requiring avoidance diet at early school age. METHODS: The school health nurses interviewed, by using a structured questionnaire on the required diet at school, the parents of all the 1542 children starting elementary school in a Finnish town with 210,000 inhabitants. RESULTS: An allergy to basic foods was found in 41 (2.7%) children: 1.5% to milk, 1.1% to eggs and 1.0% to grains. An allergy to nuts was present in 3.1% and to fruits and vegetables in 5.8%, both with known cross-sensitization to pollens. In all, 9.2% of the children reported some allergy. Milk, egg and grain allergies were associated with soy, nut and spice allergies. CONCLUSION: Over 2% of the 1542 Finnish first-graders reported allergies to basic foods (milk, eggs or grains) requiring special avoidance diets at school. The figure suggests that avoidance diets started in the first years of life still unnecessarily continued.

Dietary carbohydrate modification alters serum metabolic profiles in individuals with the metabolic syndrome.

BACKGROUND AND AIMS: Whole-grain cereals and diets with a low glycemic index may protect against the development of type 2 diabetes and heart disease, but the mechanisms are poorly understood. We studied the effect of carbohydrate modification on serum metabolic profiles, including lipids and branched chain amino acids, and dependencies between these and specific gene expression pathways in adipose tissue. METHODS AND RESULTS: Twenty subjects with metabolic syndrome were selected from the larger FUNGENUT study population, randomized either to a diet high in oat and wheat bread and potato (OWP) or rye bread and pasta (RP). Serum metabolomics analyses were performed using ultra-performance liquid chromatography coupled to electrospray ionization mass spectrometry (UPLC/MS), gas chromatography (GC) and UPLC. In the OWP group multiple proinflammatory lysophosphatidylcholines increased, while in the RP group docosahexaenoic acid (DHA 22:6n-3) increased and isoleucine decreased. mRNA expression of stress reactions- and adipose tissue differentiation-related genes were up-regulated in adipose tissue in the OWP group. In the RP group, however, pathways related to stress reactions and insulin signaling and energy metabolism were down-regulated. The lipid profiles had the strongest association with the changes in the adipose tissue differentiation pathway when using the elastic net regression model of the lipidomic profiles on selected pathways. CONCLUSION: Our results suggest that the dietary carbohydrate modification alters the serum metabolic profile, especially in lysoPC species, and may, thus, contribute to proinflammatory processes which in turn promote adverse changes in insulin and glucose metabolism.

Experimental and numerical dataset of Microbond test using optical fibres for strain.

This data article provides useful information often required for numerical modeling of the so-called microbond tests. It includes the experimental and simulation data of the microbond testing using Fibre Bragg Grating (FBG) fibres for optical strains. Microbond testing was performed on five different droplets of varying embedded length and diameter to collect the data. Finite element simulation was carried out and modelling was validated, by using two variables force and strain, to collect the data. The output data of the fitted models is given and is also visualized via graphs of force-strain derivative curves. The data of the simulations is provided for different finite element mesh densities. Here, to clarify the type and form of the data for the use by readers, the energy distribution curves describing various functionalities of the droplet, fibre and interface are presented. For further reading, the interpretation and analysis of this data can be found in a research article titled "3D interfacial debonding during microbond testing: Advantages of local strain recording" (R. Dsouza et al., 2020) [1].

Association of sequence variations in the gene encoding insulin-like growth factor binding protein 5 with adiponectin.

BACKGROUND: Insulin-like growth factor binding protein 5 (IGFBP5) binds to IGF and thus modulates IGF signaling pathway. We have shown earlier that the IGFBP5 gene was downregulated in the adipose tissue after 12-week carbohydrate diet with low insulinemic response. OBJECTIVE: The aim was to examine the putative contribution of genetic variation of the IGFBP5 gene to the characteristics of metabolic syndrome and incidence of type 2 diabetes (T2DM) in the Finnish Diabetes Prevention Study (DPS). METHODS: DPS is a longitudinal study where 522 subjects with impaired glucose tolerance were randomized to either lifestyle intervention group or control group. DNA was available from 507 subjects (mean body mass index (BMI) 31.2+/-4.5 kg/m(2), age 55+/-7 years). The eight single-nucleotide polymorphisms (SNPs) were selected from HapMap database and genotyped by Taqman allelic discrimination protocol. The main results were confirmed in a larger cross-sectional study population (METSIM). In addition, the gene expression of IGFBP5 was studied in two previously published study populations (FUNGENUT and GENOBIN) of 124 subjects with insulin resistance (BMI 32.2+/-3.5 kg/m(2), age 57.7+/-7.4 years). RESULTS: Three out of eight IGFBP5 markers (rs9341234, rs3276 and rs11575134) were significantly associated with circulating adiponectin concentrations in men. Furthermore, mRNA expression studies of subcutaneous adipose tissue showed that mRNA concentrations of IGFBP5 correlated with adiponectin concentrations in all subjects and in women. None of the IGFBP5 SNPs were associated with T2DM. CONCLUSIONS: Our findings show that IGFBP5 has a gender-specific association with adiponectin, which may modulate the development of metabolic syndrome.

Weight reduction modulates expression of genes involved in extracellular matrix and cell death: the GENOBIN study.

OBJECTIVE: Lifestyle and genetic factors interact in the development of obesity and the metabolic syndrome. The molecular mechanisms underlying the beneficial dietary modifications are, however, unclear. We aimed to examine the effect of the long-term moderate weight reduction on gene expression in adipose tissue (AT) and to identify genes and gene clusters responsive to treatment and thereby likely contributing to the development of the metabolic syndrome. DESIGN: Randomized controlled and individualized weight reduction intervention. SUBJECTS: Forty-six subjects with impaired fasting glycemia or impaired glucose tolerance and features of metabolic syndrome, aged 60+/-7 years were randomized either to a weight reduction (WR) (n=28) or a control (n=18) group lasting for 33 weeks. MEASUREMENTS: Oral and intravenous glucose tolerance tests and subcutaneous AT biopsies were performed before and after the intervention. Gene expression of AT was studied using microarray technology in subgroups of WR (with weight reduction > or =5%, n=9) and control group (n=10). The results were confirmed using quantitative PCR. RESULTS: In the WR group, glucose metabolism improved. Moreover, an inverse correlation between the change in S (I) and the change in body weight was found (r=-0.44, P=0.026). Downregulation of gene expression (P<0.01) involving gene ontology groups of extracellular matrix and cell death was seen. Such changes did not occur in the control group. The tenomodulin-gene was one of the most downregulated genes (-39+/-16%, P<0.0001). Moreover, its expression correlated with insulin sensitivity (r=-0.34, P=0.005) before the intervention and with body adiposity both before (r=0.42, P=0.007) and after (r=0.30, P=0.056) the intervention. CONCLUSION: Genes regulating the extracellular matrix and cell death showed a strong downregulation after long-term weight reduction. This likely reflects a new stable state at the molecular level in AT. Further studies are warranted to elucidate the mechanisms of these genetic factors.

Kick Scooter Injuries in Children and Adolescents: Minor Fractures and Bruise.

BACKGROUND:: Kick scooters are popular among children in both transportation and recreational activities. The aim of this retrospective study was to assess the incidence of and injury patterns associated with kick scooter accidents in school-aged children and adolescents. METHODS:: All 171 patients at the age of 7-15 years who were treated for kick scooter-related injuries in the metropolitan Helsinki area, Southern Finland from January 2008 to December 2013 were included. Electronic medical records were reviewed and Pediatric Trauma Scores and Injury Severity Scores were utilized to assess the injuries. RESULTS:: The annual number of patients increased from 7 in 2008 to 55 in 2013. Almost all patients (94%, n = 161) were injured after a fall from their own height. Most patients (n = 118; 69%) were diagnosed with a fracture but only 26 patients (15%) required surgical procedures under general anesthesia. Pediatric Trauma Scores were low and only one patient had an Injury Severity Score > 15 which can be considered major trauma. CONCLUSION:: Most injuries acquired from kick scooter injuries were easily treatable fractures and bruises. Considering the background population of 105,000 in the respective age group and the 6-year period of data collection from tertiary care, scooting seems a safe means of increasing the physical activity levels of school-aged children and adolescents.

Dominant negative regulation of trans-activation by the rat androgen receptor: roles of the N-terminal domain and heterodimer formation.

A series of deletion mutants was constructed for the rat androgen receptor (AR) to delineate sequences involved in transcriptional activation. Using transient expression conditions in CV-1 cells and in vitro DNA-binding studies, the amino-terminal domain of the receptor was shown to contain a region (residues 147-296) that is mandatory for trans-activation. Receptors with deletions (residues 147-408) in the N-terminal domain, but with intact DNA- and ligand-binding domains, interacted in vitro with androgen-responsive elements albeit with affinities lower than that of the wild type receptor. Coexpression of N-terminal deletion mutants (delta 46-408 and delta 38-296) with the wild type AR blunted trans-activation by the latter protein in a dominant fashion. By contrast, a hormone-binding-deficient receptor (delta 788-902) that had poor intrinsic activity potentiated the trans-activation by the native receptor. Mechanisms by which deletion mutants in the N-terminal region abolish the function of the wild type protein appear to involve heterodimer formation during interaction with DNA and direct competition for available binding sites on DNA, rather than squelching of accessory proteins. In contrast to impaired trans-activation, binding of the ligand to N-terminal deletion mutants brought about conformational changes that were comparable in wild type and mutant forms, as judged by electrophoretic mobility shift assays. Taken together, these data have specified a region in the N-terminal domain of the AR that plays a decisive role in transcriptional regulation.

Androgen receptor-mediated transcriptional regulation in the absence of direct interaction with a specific DNA element.

Androgen receptor (AR) brings about a ligand-dependent inhibition of low-affinity neurotrophin receptor (p75) promoter constructs in cultured cells, with the greatest inhibition being achieved with a reporter gene containing 1050 nucleotides (nt) of the promoter. The receptor domain critical for trans-repression localizes to the same region (amino acids 147-296) as that mandatory for transactivation. In contrast to trans-activation, AR does not interact directly with specific DNA elements to elicit trans-repression of p75 promoter constructs, although an intact DNA-binding domain of the receptor is required for both actions. In a search for interacting partners, both extensively purified full-length AR and AR-DNA binding domain were found to inhibit c-Jun/AP-1 site interaction without themselves binding to the AP-1 element. Prior binding of c-Jun to the AP-1 element protected the complex from the receptor's interference. Repression was not mutual, as c-Jun did not inhibit AR-androgen response element interaction or trans-activation through an androgen response element-containing promoter. The 1050-nt-long p75 promoter sequence does not contain an AP-1 element; an AP-1-like site in the vector backbone mediates the trans-repression by the AR in recipient cells. Intriguingly, an AR form with a large N-terminal deletion (the delta 46-408 mutant) behaved as a transcriptional activator of the p75 promoter through a mechanism that was also independent of specific DNA binding. Collectively, these data indicate that, in a proper context, AR is able to elicit both transrepression and trans-activation without interacting directly with specific DNA elements. Sequences responsible for the down-regulation of p75 mRNA by androgens in vivo are, however, not located in the proximal 1050 nt of the p75 promoter.

Specificity of the monoclonal anti-I antibody (Hy).

In previous work we found that a monoclonal cold hemagglutinin from patient Hy strongly bound antigens contained in stage IV breast cancer sera. To infer the chemical structure of the antigens expressed in the cancer sera, we studied the specificity of the antibody (Hy). The antibody (Hy) had I specificity, based on agglutination scores with adult and cord red blood cells. The binding of the antibody to synthetic and milk oligosaccharides was determined using a solid phase enzyme immunoassay (EIA). The anti-I antibody (Hy) strongly bound LacNAc0-Me, LacNAc1----6Gal, LacNAc1----6 (LacNAc1----3)Gal, LacNAc-1----6 alpha GalNAc, LacNAc1----3LacNAc, and LacNAc, 0.05, 0.06, 0.09, 0.22, 0.35 and 0.75 mM giving 50% inhibition, respectively. The anti-I antibody (Hy), similar to the anti-I antibody (Ma), strongly bound LacNAc1----6Gal, but it differed from the anti-I antibody (Ma) in its cross-reactivity with the i sequence. The anti-I antibody (Hy) showed similar reactivities as the hybridoma monoclonal antibodies M18 and M39 with LacNAc1----6Gal and with the i-active sequence. The EIA procedure is a useful alternative to either radioimmunoassay or immunoprecipitation method in the study of anti-I,i specificities.

Agonists, but not antagonists, alter the conformation of the hormone-binding domain of androgen receptor.

Androgen receptors synthesized by translation in vitro form dimeric aporeceptor complexes with an androgen response element (ARE). Physiological and synthetic androgens elicit a conformational change in the receptor, which increases the mobility of receptor-ARE complexes in gel retardation assays. Neither a steroidal (cyproterone acetate) nor a non-steroidal (casodex) antiandrogen brings about the same effect and, at high concentrations, reverse the action of androgen agonists. When receptor-agonist complexes are subjected to a limited trypsin or chymotrypsin digestion, a protease-resistant 30-kDa fragment corresponding to the entire ligand-binding domain is formed. A similar fragment is not protected in the presence of antiandrogens. The C-terminal origin of the protected region was verified using mutated receptor forms: a mutant with a large N-terminal deletion behaves like the wild-type protein, but the properties of a hormone binding-negative receptor, due to a single-base substitution at codon 807, are not influenced by androgen agonists or antagonists.

Stimulation of androgen-regulated transactivation by modulators of protein phosphorylation.

The effect of modulators of protein phosphorylation on the transcriptional activity of the androgen receptor (AR) was studied under transient expression conditions. Activators of protein kinase-A [8-bromo-cAMP (8-Br-cAMP)] and protein kinase-C (phorbol 12-myristate 13-acetate) or an inhibitor of protein phosphatase-1 and -2A (okadaic acid) influenced minimally pMMTV-chloramphenicol acetyl-transferase (CAT) activity in CV-1 cells cotransfected with an AR expression plasmid in the absence of androgen. In the presence of testosterone, however, all compounds enhanced AR-mediated transactivation by 2- to 4-fold. A nonsteroidal antiandrogen, Casodex, behaved as a pure antagonist; it blunted the action of testosterone and was not rendered agonistic by activators of protein kinase-A. A reporter plasmid containing two androgen response elements (AREs) in front of the thymidine kinase promoter (pARE2tk-CAT) was also used to examine promoter specificity. It was activated by 8-Br-cAMP, forskolin, or okadaic acid even without AR or androgen. However, when forskolin or okadaic acid was used together with androgen and AR, the resulting AR-dependent transactivation of pARE2tk-CAT was more than additive. Intact DNA- and ligand-binding domains, but not the N-terminal amino acid residues 40-147, of the receptor were mandatory for the synergism between protein kinase-A activators and androgen. Immunoreactive AR content in transfected COS-1 cells was not influenced by exposure to 8-Br-cAMP. Similar results were obtained by ligand binding assays. Quantitative or qualitative differences were not observed in DNA-binding characteristics between receptors extracted from cells treated with testosterone with or without protein kinase-A activator. Collectively, the synergistic stimulation of AR-dependent transactivation by androgen and protein kinase activators is not due to changes in cellular AR content or affinity of the receptor for the cognate DNA element; rather, this phenomenon seems to result from altered interaction of ligand-activated AR with other proteins in the transcription machinery.

Construction and characterization of a novel cross-regulation system for regulating cloned gene expression in Escherichia coli.

A novel cross-regulation system employing two pairs of interacting promoter-repressor systems was constructed using the tac-lacI and lambda pL-cI promoter-operator-repressor systems. In particular, transcription of the cat gene and the fused cI gene is regulated by the tac promoter, while transcription of the lacI gene is controlled by the lambda pL promoter. In order to compare CAT production from this new system with a currently employed transcription control configuration, a control expression vector utilizing the constitutive repressor synthesis configuration was also constructed. In this construct, cat is under the control of the tac promoter, and lac repressor is provided from a single copy of the lacIq allele included in the plasmid. Induction results using different copy number vectors indicate that induced cat expression levels are at least twofold higher using the cross-regulation system which has very low basal expression. These results match well with previous mathematical modeling predictions indicating excellent control of basal expression and also higher cloned-gene expression post-induction over a broad range of copy numbers for a cross-regulation control configuration. Induction of the cross-regulation system both up-regulated the activation pathway and down-regulated the inhibition pathway, shifting the system steady-state from lac repressor expression to cat and cI expression. The control strategy presented here should be equally applicable to regulate transcription in diverse hosts.

Targeted expression of a synthetic codon optimized gene, encoding the spruce budworm antifreeze protein, leads to accumulation of antifreeze activity in the apoplasts of transgenic tobacco.

A synthetic gene based on the primary sequence of the mature spruce budworm antifreeze protein (sbwAFP) was constructed by primer overlap extension. The amino acid codons were chosen to mimic those of a highly expressed tobacco nuclear gene. A DNA sequence encoding the amino-terminal leader sequence from the tobacco pathogen related protein 1b (PR), which targets the protein to the apoplastic space, was fused in frame to the synthetic sbwAFP gene. This fusion was placed downstream of the cauliflower mosaic virus 35S promoter and upstream of the nopaline synthase terminator in a T-DNA binary vector. Transgenic tobacco lines transcribing PR-sbwAFP were selected by RT-PCR. The apoplastic protein fractions of sbwAFP expressing tobacco lines exhibited enhanced antifreeze activity as demonstrated by the ability to inhibit ice re-crystallization and increased thermal hysteresis.

Control of the initiation of sporulation in Bacillus subtilis by a phosphorelay.

Sporulation in Bacillus subtilis is a developmental process induced as a response to nutritional stress. Activation of sporulation-specific gene transcription is under the control of the spoOA gene product. The SpoOA protein and the SpoOF protein are both homologous to response regulator proteins of two-component regulatory systems which control bacterial responses to a variety of environmental challenges. Response regulators are activated by specific kinases which phosphorylate them. In this study, it was shown that phosphorylation of SpoOA occurs via a phosphotransferase which is the product of the spoOB locus. The phosphodonor in this reaction is the phosphorylated form of SpoOF. It is postulated that SpoOF acts as a secondary messenger that can be phosphorylated by a variety of kinases depending on the particular environmental stress. The series of phosphate transfer reactions in this system is called a phosphorelay. The end product of this series of reactions is SpoOA approximately P which is shown to have greater affinity for the DNA target, the OA box, of SpoOA on the abrB promoter than the unphosphorylated form. SpoOA approximately P, but not SpoOA, was shown to be an activator of transcription of the spoIIA operon which codes for the sporulation-specific sigma factor sigma F. Thus, the initiation of sporulation is dependent on SpoOA approximately P which arises through the phosphorelay and which acts as a transcription factor to repress certain genes, e.g. abrB, and activate others, e.g. spoIIA.

Scientific basis of a matrix metalloproteinase-8 specific chair-side test for monitoring periodontal and peri-implant health and disease.

Matrix metalloproteinases (MMPs), especially collagenase-2 (MMP-8), are key mediators of irreversible tissue destruction associated with periodontitis and peri-implantitis. MMP-8 is known to exist in elevated amounts and in active form in the gingival crevicular fluid (GCF) and peri-implant sulcular fluid (PISF) from progressing periodontitis and peri-implantitis lesions and sites, respectively. (Sorsa et al. Ann. N.Y. Acad. Sci. 737: 112-131 [1994]; Teronen et al. J. Dent. Res. 76: 1529-1537 [1997]). We have developed monoclonal antibodies to MMP-8 (Hanemaaijer et al. J. Biol. Chem. 272: 31504-31509 [1997]) that can be used in a chair-side dipstick test to monitor the course and treatment of periodontitis and peri-implantitis. Monoclonal and polyclonal antibody tests for MMP-8 coincided with the classical functional collagenase activity test from GCF and PISF (Sorsa et al. J. Periodont. Res. 22: 386-393 [1988]) in periodontal and peri-implant health and disease. In future a chair-side functional and/or immunological MMP-test can be useful to diagnose and monitor periodontal and peri-implant disease and health.

A simple method for isolation of DNA from formalin-fixed paraffin-embedded samples for PCR.

A simple method of processing formalin-fixed, paraffin-embedded tissue sections for DNA amplification by polymerase chain reaction (PCR) is described. In this procedure, deparaffinized sections are readily subjected to DNA isolation simply by boiling and the released DNA can be directly employed for PCR. The method allows analysis of single-copy genes or viral sequences at least up to 300 base pairs long in one working day. This method is particularly useful in analysing retrospective materials when the simplicity and low cost of the assay are preferable. Furthermore, the simplicity of the procedure reduces the risk of contamination.

The in vivo expression of the collagenolytic matrix metalloproteinases (MMP-2, -8, -13, and -14) and matrilysin (MMP-7) in adult and localized juvenile periodontitis.

Periodontal inflammation is characterized by irreversible degradation of periodontal ligament collagen fibers leading to loss of tooth attachment. Cultured gingival keratinocytes and fibroblasts express, in vitro, various matrix metalloproteinases (MMPs) which can degrade fibrillar collagens. We hypothesized that several MMPs are also synthesized in vivo by sulcular epithelium, and analyzed the collagenolytic MMPs (MMP-2, -8, -13, and -14) and matrilysin (MMP-7) in gingival tissue specimens and gingival crevicular fluid from adult and localized juvenile periodontitis patients by in situ hybridization, immunohistochemistry, and Western immunoblotting. MMP-2, -7, -8, and -13 were expressed in gingival sulcular epithelium. MMP-7 and -13 were also located in fibroblasts and macrophages, and MMP-8 in neutrophils. MMP-8- and -13-positive cells/mm2 were higher in periodontitis gingiva when compared with healthy control tissue (p < 0.01). In periodontal diseases, gingival sulcular epithelium expresses several, rather than a single, collagenolytic MMPs, and this proteolytic cascade is evidently responsible for the tissue destruction characteristic of adult and juvenile periodontitis.