Adaptable behavior such as triggered disintegration affords a broad scope and utility for (bio)materials in diverse applications in materials science and engineering. The impact of such materials continues to grow due to the increased importance of environmental considerations as well as the increased use of implants in medical practices. However, examples of such materials are still few. In this work, we engineer triggered liquefaction of hydrogel biomaterials in response to internal, localized heating, mediated by near-infrared light as external stimulus. This adaptable behavior is engineered into the readily available physical hydrogels based on poly(vinyl alcohol), using gold nanoparticles or an organic photothermal dye as heat generators. Upon laser light irradiation, engineered biomaterials underwent liquefaction within seconds. Pulsed laser light irradiation afforded controlled, on-demand release of the incorporated cargo, successful for small molecules as well as proteins (enzymes) in their biofunctional form.

Leptin, secreted mostly by the white adipose tissue and notably by the hypodermis, is an adipokine that regulates appetite and energy expenditure thus playing a role in obesity. Leptin also participates in inflammatory responses. Serum levels of leptin are increased in obese individuals similarly to levels observed in psoriasis patients. Moreover, leptin expression is increased in psoriatic skin. Psoriasis is often associated with obesity, metabolic syndrome, diabetes and cardiovascular disease and a chronic low-grade inflammation of the adipose tissue seems to trigger these co-morbidities. Strong evidence suggests that obesity predisposes to psoriasis and affects the efficacy of psoriasis treatment. Thus, understanding the association between obesity and psoriasis may enable the discovery of new targets to improve psoriasis treatment. We therefore hypothesized that leptin could play a role in psoriasis and results from our laboratory showed that, in the dermal fibroblast, leptin induced a pro-inflammatory response that was different whether cells were from psoriatic or healthy skin. The purpose of this study was to investigate how leptin mediates its effects in psoriatic skin. In particular, we aimed to investigate whether leptin's effect in the dermal fibroblast modulates or is modulated by other known pro-inflammatory cytokines associated with psoriasis. Dermal fibroblasts were isolated from psoriatic or from healthy skin, grown in vitro and assessed for a pro-inflammatory response after stimulation. Leptin stimulus was provided alone or in combination with IL-1alpha, TNF-alpha or IL-17 for 24 hours. The cell culture medium was then analyzed by ELISA for the expression level of the inflammatory marker IL-6. Our results suggest that leptin potentiates the effect of TNF-alpha and IL-17A in normal dermal fibroblasts. These data may explain the pro-inflammatory mechanism through which obesity exacerbates skin diseases such as psoriasis.

We report the first successful implementation of transfection agents to facilitate the delivery of non-nucleic acid based anti-inflammatory and anti-viral drugs. In doing so, we illustrate a new paradigm in the intracellular delivery of polyanionic drugs and also extend the scope and utility of successful tools of gene transfer into a new area of biomedical research.

Dimensioning of air conditioning plants for animal houses is traditionally based on steady-state conditions with constant animal heat and moisture production over a 24-hour cycle, despite the fact that the heat and moisture production varies diurnally. The heat production is normally highest in daytime and lowest during the night and therefore, there will be a discrepancy between the actual and the calculated indoor temperature and humidity, when using steady state models to control ventilation rate. This article deals with the two main factors influencing on the diurnal heat production, namely the animal activity and the digestion of food. In experiments carried out over three years in respiration chambers at Research Centre Foulum, Denmark, the diurnal influence of feeding and other behavioural activities have been analyzed for weaned pigs, growing pigs, dry sows and broilers. These experiments show that most of the diurnal variations in animal heat production can be explained by animal activity (R 2 between 0.58 and 0.89), for examined species but heat production due to digestion processes does also contribute. Because of coincidence between high animal activity and the feeding schedules, the animal heat production due to digestion is confounded with that caused by physical activity. By applying a model that takes into account both animal activity and the time after feeding for pigs fed twice a day, the correlation is improved (R 2 between 0.80 and 0.90). This model shows for growing pigs at 35, 65 and 90 kg, fed twice daily, that animal activity and digestion each can explain half of the animal heat production above basal heat production. For dry sows with low feed intake the impact of digestion on the heat production is negligible compared to the impact due to activity of the animals. The investigations also show that even under experimental conditions in climatic chambers, there is a typical diurnal variation in animal heat production. Expressed by a sinusoidal function, the diurnal variation in heat production (amplitude), measured in climatic chambers was in the range from 9 to 21%, which is lower than that found in other investigations for conventional animal houses with normal group size and human activities in the animal house during the daytime. The analysis shows that the measured animal heat production in the laboratory investigations was in fairly good agreement with the calculated total heat production according to CIGR Equations, with deviations from -6.3 to 6.8 % for fattening pigs and dry sows, but greater deviations were measured for piglets fed by an artificial sow and for broilers.

Zinc salt‐based fixation (ZBF) is a simple, cost‐effective, and nonhazardous fixation method for cell suspensions that preserves all cellular structures and enables flow cytometric analysis of both surface and intracellular proteins, DNA content profiles, and pulse‐labeling using the thymidine analog EdU in the same cell sample. ZBF performs equally well to formaldehyde in the preservation of surface epitope labeling and forward and side light scatter parameters, as measured by flow cytometry. DNA is maintained at high molecular weight, improving the quantification and allowing subsequent quantitative PCR analysis. Finally, ZBF treatment allows for long‐term storage of labeled cells with little change in these parameters. Curr. Protoc. Cytom. 57:7.40.1‐7.40.9. © 2011 by John Wiley & Sons, Inc.

Zinc salt‐based fixation (ZBF) has proved advantageous in histochemical analyses conducted on intact tissues but has not been exploited in flow cytometry procedures that focus on quantitative analysis of individual cells. Here, we show that ZBF performs equally well to paraformaldehyde in the preservation of surface epitope labeling and forward and side scatter parameters as measured by flow cytometry. ZBF‐fixed mouse epithelial keratinocytes exhibit a staining pattern for the surface markers Sca‐1, CD34 and α6 integrin that is highly analogous to live cells. Furthermore, ZBF also preserves DNA allowing subsequent quantitative PCR analysis or labeling for incorporation of the thymidine analog EdU following surface and intracellular epitope staining. Finally, ZBF treatment allows for long‐term storage of labeled cells with little change in these parameters. Thus, we present a protocol for zinc salt fixation of cells that allows for the simultaneous analysis of DNA and intracellular and cell surface proteins by flow cytometry. © 2010 International Society for Advancement of Cytometry

Arabidopsis and rice are the only two model plants whose finished phase genome sequence has been completed. Here we report the construction of an oligomer microarray based on the presently known and predicted gene models in the rice genome. This microarray was used to analyze the transcriptional activity of the gene models in representative rice organ types. Expression of 86% of the 41,754 known and predicted gene models was detected. A significant fraction of these expressed gene models are organized into chromosomal regions, about 100 kb in length, that exhibit a coexpression pattern. Compared with similar genome-wide surveys of the Arabidopsis transcriptome, our results indicate that similar proportions of the two genomes are expressed in their corresponding organ types. A large percentage of the rice gene models that lack significant Arabidopsis homologs are expressed. Furthermore, the expression patterns of rice and Arabidopsis best-matched homologous genes in distinct functional groups indicate dramatic differences in their degree of conservation between the two species. Thus, this initial comparative analysis reveals some basic similarities and differences between the Arabidopsis and rice transcriptomes.

Chromosome painting refers to the complete decoration of specific metaphase chromosomes with complex probe mixtures. The painting signal is obtained by fluorescence in situ hybridization (FISH) of such mixtures established from a number of different sources (1–4). This technique is a valuable supplemental method in the detailed analysis of specific chromosome aberrations, complex chromosome rearrangements, and chromosomes of poor quality, which have been unresolvable with the traditional chromosome banding techniques. In traditional chromosome painting, complex probe mixtures representing specific chromosomes are hybridized to metaphase spreads containing the chromosome aberration in question. If the hybridized probe mixtures are fluorescence labeled, the composition of the aberrant chromosome is revealed by the distribution of the fluorescence signals obtained with the different probes. The combination of different labeled probes and multicolor techniques simplifies the evaluation.

The effects of different dust control methods and their combinations were studied in a pig finishing building with two identical rooms: a control room, and a treatment room. The methods involved in the study were addition of animal fat in feed liquid feeding, spraying with water and spraying with a mixture of rapeseed oil and water: The last mentioned method was used with and without an animal activity sensor to coordinate the time of spraying with the levels of animal activity The combined method, spraying an oil-water mixture controlled by the animal activity sensor and adding animal fat to dry feed reduced the airborne dust concentrations and dust exposures of herdsmen by about 80 and 85%, respectively. The optimum rate of oil spraying was found to be about 3.3 g oil day(-1) pig(-1).

We have determined the cDNA sequence and exon/intron structure of the human CLPX gene encoding a human ortholog of the E. coli ClpX chaperone and protease subunit. The CLPX gene comprises 14 exons and encodes a 633-amino acid-long precursor polypeptide. The polypeptide contains an N-terminal putative mitochondrial transit peptide, and expression of a full-length ClpX cDNA tagged at its C-terminus (Myc-His) shows that the polypeptide is transported into mitochondria. FISH analysis localized the CLPX gene to human Chromosome (Chr) 15q22.1-22.32. This localization was refined by radiation hybrid mapping placing the CLPX gene 4.6 cR distal to D15S159. Murine ClpX cDNA was sequenced, and the mouse Clpx locus was mapped to a position between 31 and 42 cM offset from the centromere on mouse Chr 9. Experimental observations indicate the presence of a pseudogene in the mouse genome and sequence variability between mouse ClpX cDNAs from different strains. Alignment of the human and mouse ClpX amino acid sequences with ClpX sequences from other organisms shows that they display the typical modular organization of domains with one AAA+ domain common to a large group of ATPases and several other domains conserved in ClpX orthologs linked by non-conserved sequences. Notably, a C-4 zinc finger type motif is recognized in human and mouse ClpX. This motif of so far unknown function is present only in a subset of the known ClpX sequences.

Nucleoporins are the main components of nuclear pore complexes (NPCs) involved in nucleo-cytoplasmic transport. Starting with an expressed DNA fragment retrieved by exon trapping from pooled human BAC clones mapped to the short arm of chromosome 5, we identified a human nucleoporin cDNA sequence by PCR from a human testis cDNA library. The coding sequence showed high homology to that of the rat nucleoporin 155 (Nup155) cDNA. FISH analysis with the human BAC clone as probe localized the human NUP155 gene to chromosome band 5p13. Northern analysis showed that the human NUP155 gene was expressed at different levels in all tissues tested. Two species of transcripts were observed with estimated lengths of 5.4 and 4.7 kb, respectively, in concordance with the finding of two alternative polyadenylation sites in the cDNA. The genomic location of the human NUP155 gene suggests a possible role in the mental and developmental retardation associated with hemizygous deletions of the 5p13 region.

Antigen-mediated T-cell proliferation is a transient phenomenon. Like other somatic cells, T lymphocytes generally show replicative senescence in vitro. However, we here show that cytokine-dependent continuous (immortal) T-cell lines can be established from skin biopsy specimens of inflammatory skin diseases. Continuous growth can be obtained by culturing T cells in medium supplemented with interleukin-2 and interleukin-4, but without antigen or antigen-presenting cells added. Loss of the T-cell antigen receptor complex is observed in some of the continuous T-cell lines. Most T-cell lines develop clonal chromosome aberrations during continuous growth. Aberrations for chromosomes 1, 2, 8, 16, and 18 are most commonly observed.

We currently use direct and reverse chromosome painting in prenatal diagnosis. In a family with a subtle 12;16 translocation, adjacent 1 segregation was diagnosed in the first child, a boy, in whom symptoms compatible with partial trisomy 16p and partial monosomy 12q were seen. In the next pregnancy, a chorionic villus biopsy was tested using chromosome painting. Only by supplementing conventional cytogenetic methods with molecular cytogenetic techniques could the true karyotype be unequivocally determined. Reverse painting, using DOP-PCR amplified, flow sorted paternal derivative chromosomes as a DNA library to paint the chorionic villus cells, was especially informative.

Primed in situ labeling (PRINS) and fluorescence in situ hybridization (FISH) are methods for visualizing specific DNA sequences directly on chromosome spreads, thereby demonstrating the presence of a specific sequence in a certain cell and at the same time localizing the sequence at a specific site on the chromosome. This chapter describes protocols for the fluorescence labeling of specific sequences in chromosomes fixed to a microscope slide using either PRINS or FISH. For the labeling it uses either the biotin-avidin or the digoxigenin-antidigoxigenin system, conjugated with various fluorochromes. PRINS and in situ hybridization are both methods originally developed for microscopy of standard metaphase chromosome spreads. The two techniques have been used in many situations, in gene mapping, in the deciphering of complex chromosome aberrations, and in the simple determination of aneuploidy. For a number of applications PRINS and traditional in situ hybridization are equally effective. This applies to situations where cloned probes are used for detection of highly repeated sequences.

We report on a newborn white male infant with marked dysmorphic features and various congenital malformations. The initial clinical evaluation showed Crouzon-like features as well as some features of trisomy 18 syndrome and trisomy 13 syndrome. The results from conventional cytogenetic analysis showed a structurally abnormal chromosome replacing one normal chromosome 18, but only by applying molecular cytogenetic methods could the architecture of this abnormal chromosome be characterised clearly. The primed in situ labelling (PRINS) technique, using a newly synthesised alpha 18 oligonucleotide, showed the dicentric pattern and direct chromosome painting established the origin to be from chromosome 18. The combination of conventional cytogenetics and molecular cytogenetics showed the karyotype in the proband to be 45,XY,-14,-18,-21,+t(14;21),+psu dic(18) (qter-->cen-->p11.3: :p11.3-->psu cen-->qter). This was supported by molecular analysis using chromosome 18 specific DNA markers, which showed the paternal origin of the abnormal chromosome.

A case of a supernumerary der(18) marker chromosome is presented. The chromosomal origin of the marker chromosome was not evident by traditional chromosome analysis, but was determined by PRimed IN Situ labelling (PRINS) with chromosome specific centromere probes as primers for chain elongation in situ . For this purpose a strategy was developed which, in a few simple reactions, makes it possible unequivocally to determine the origin of any small marker chromosome. The approach does not require any hints about the origin of the chromosome prior to the analysis, since the chromosomal origin of the marker is established through PRINS reactions with pooled and single chromosome‐specific centromere probes. Identification, mosaic screening and structural analysis require a total of 8–9 such reactions and may, due to the extreme speed of the PRINS reaction, be obtained within a single working day.

To illustrate the advantage of fluorescence in situ hybridization (FISH) in clinical genetics, we have chosen six clinical cases from our routine laboratory where we have used molecular cytogenetic techniques to optimise the cytogenetic analysis. Using centromere-specific DNA probes and whole chromosome DNA libraries either obtained from somatic cell hybrids or generated from flow-sorted chromosomes, we have been able to identify small marker chromosomes, chromosomal duplications and inversions, to determine the ploidy in interphase nuclei, and to characterize subtle chromosomal translocations. We conclude that molecular cytogenetics is a valuable technique that should be used as a supplement to conventional cytogenetics to reduce the number of unresolved karyotypes in clinical genetics.

A new version of our previously published PRINS (PRimed IN Situ labeling) method is presented. It represents a significant improvement in the detection of specific DNA sequences in situ. The idea is to perform the reaction repeatedly, rather than just once. This change of strategy results in a localized accumulation of sequence-specific labeled DNA, resulting in up to a 15-fold amplification of the signal as compared to the standard PRINS method. Interestingly, the retention of the labeled DNA is so good that it stays within the chromosomal band where it is synthesized, provided that the reaction is not performed an excessive number of times. The key trick is the performance of the procedure on small glass slides in PCR tubes, thereby avoiding the use of cover slips. In addition, use of the small glass slides seems to give less variance when the signals are quantified in a fluorescence microscope.

The Polymerase Chain Reaction (PCR) can be performed on isolated cells or chromosomes and the product can be analyzed by DNA technology or by FISH to test metaphases. The authors have good experiences analyzing aberrant chromosomes by FACS sorting, PCR with degenerated primers and painting of test metaphases with the PCR product. They also utilize polymerases for PRimed IN Situ labelling (PRINS) of specific nucleic acid sequences. In PRINS oligonucleotides are hybridized to their target sequences and labeled nucleotides are incorporated at the site of hybridization with the oligonucleotide as primer. PRINS may eventually allow the study of individual genes, gene expression and even somatic mutations (in mRNA) in single cells.