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Paper-based packaging is abundant, non-toxic, from renewable resource, biodegradable material and of low marketing cost compared to other conventional materials. However, paper offers low resistance to water vapor, oils and gases, which may restrict its use for perishable products. As an alternative, biopolymers are used for coating papers, such as cellulose derivatives, starches, chitosan or chitin, proteins (animal or vegetable) and lipids. These biopolymers offer advanced and enhanced properties when used for coating papers. The objective of this review is to address the main biopolymers studied and tested for coating paper in food packaging, describing their main characteristics, in addition to presenting works that show that the mentioned biopolymers have shown to be promising, having compatible properties for the use on a large scale in the coating of paper in packaging. With this work, we hope to contribute to the advancement of scientific research and projects that promote the use of biopolymers in paper coating for use in production of food packaging. Los envases a base de papel destacan por ser un material abundante, no tóxico, biodegradable y de bajo coste de comercialización frente a otros materiales convencionales. Sin embargo, el papel ofrece una baja resistencia al vapor de agua, aceites y gases, lo que puede hacer que los envases sean frágiles, imposibilitando su uso para productos perecederos. Como alternativa, para el recubrimiento de papeles se utilizan biopolímeros, como derivados de la celulosa, almidones, quitosano o quitina, proteínas (animales o vegetales) y lípidos. Estos biopolímeros ofrecen propiedades avanzadas y mejoradas cuando se usan para recubrir papeles. El objetivo de esta revisión es abordar los principales biopolímeros estudiados y probados para el recubrimiento de papel en envases de alimentos, describiendo sus principales características, además de presentar trabajos que demuestren que los biopolímeros mencionados han mostrado ser promisorios, teniendo propiedades compatibles para el uso en a gran escala en el recubrimiento de papel en los embalajes. Con este trabajo, esperamos contribuir al avance de investigaciones científicas y proyectos que promuevan el uso de biopolímeros en el recubrimiento de papeles utilizados en la producción de envases para alimentos. As embalagens à base de papel se destacam por ser um material abundante, atóxico, biodegradável e de baixo custo de comercialização em relação a outros materiais convencionais. Entretanto, o papel oferece baixa resistêcia ao vapor de água, óleos e gases, o que pode tornar frágil as embalagens, impossibilitando o seu uso para produtos perecíveis. Como alternativa, são utilizados biopolímeros para recobrimento de papéis, tais como derivados da celulose, amidos, quitosana ou quitina, proteínas (animal ou vegetal) e lipídeos. Esses biopolímeros oferecem propriedades avançadas e aprimoradas quando utilizados para recobrimento de papéis. O objetivo desta revi são é abordar sobre os principais biopolímeros estudados e testados para o recobrimento de papéis em embalagens alimentícias, sendo descritas suas principais características, além de apresentar trabalhos que mostram que os biopolímeros mencionados têm-se mostrado promissores, possuindo propriedades compatíveis para o uso em larga escala no recobrimento de papéis em embalagens. Com este trabalho, esperamos contribuir no avanço de pesquisas científicas e projetos que promovam a utilização de biopolímeros no recobrimento de papéis utilizados na produção de embalagens alimentícias.

Proteinuria is a condition in which an excessive amount of protein is excreted in urine. It is, among others, an indicator of kidney disease or risk of cardiovascular disease. Rapid and reliable diagnosis and monitoring of proteinuria is of great importance for both patients and their physicians. For that reason, a paper-based sensor for proteinuria diagnosis was designed, optimized, and validated utilizing smartphone-assisted signal acquisition. In the first step, a few commonly employed protein assays were optimized and compared in terms of analytical performance on paper matrix. The tetrabromophenol blue method was selected as the one providing a sufficiently low limit of detection (39 mg·L−1) on the one hand and appropriate long-term stability (up to 3 months) on the other hand. The optimized assay was employed for protein-to-creatinine ratio (PCR) determination on a single paper-based sensor. For both analytes the linear ranges were within the clinically relevant range. The analytical usefulness of the developed sensors was demonstrated by a PCR recovery study in artificial urine. The obtained PCR recoveries were from ca. 80 to 150%.

The present review article reports the most innovative methods to detect proteins in historical and archeological samples as well as to characterize proteins used as binders in artworks. Difficulties to ascribe proteins to a certain animal species are often due to post-translational modifications originated by chemical or microbial deterioration during aging. Combining different techniques such as peptide mass fingerprinting and tandem mass spectrometry can solve some of these problems and also allow discrimination between taxonomically related species like sheep and goat. The most studied proteins in bones and textile samples are osteocalcin, collagen and keratin, whereas egg yolk and white proteins, casein and collagen are the most relevant for binders used in old paintings. With the suitable approaches (immune-based methods, DOT-blot, etcaEuro broken vertical bar) it is also possible to obtain in situ characterization or analyze the samples directly in the museum laboratories, with the advantage of avoiding artwork damage and expensive external commitments. Recent cutting-edge strategies allowed detection of proteinaceous infection markers that, for instance, were used to establish the cause of death of old Inca mummies and also proved the presence of Yersinia pestis in old documents dating from the period in 17th century in which the plague ravaged Europe.

For the past 20 years, the Clusters of Orthologous Genes (COG) database had been a popular tool for microbial genome annotation and comparative genomics. Initially created for the purpose of evolutionary classification of protein families, the COG have been used, apart from straightforward functional annotation of sequenced genomes, for such tasks as (i) unification of genome annotation in groups of related organisms; (ii) identification of missing and/or undetected genes in complete microbial genomes; (iii) analysis of genomic neighborhoods, in many cases allowing prediction of novel functional systems; (iv) analysis of metabolic pathways and prediction of alternative forms of enzymes; (v) comparison of organisms by COG functional categories; and (vi) prioritization of targets for structural and functional characterization. Here we review the principles of the COG approach and discuss its key advantages and drawbacks in microbial genome analysis.

We describe the design and characteristics of a paper-based analytical device for analyte concentration enrichment. The device, called a hybrid paper-based analytical device (hyPAD), uses faradaic electrochemistry to create an ion depletion zone (IDZ), and hence a local electric field, within a nitrocellulose flow channel. Charged analytes are concentrated near the IDZ when their electrophoretic and electroosmotic velocities balance. This process is called faradaic ion concentration polarization. The hyPAD is simple to construct and uses only low-cost materials. The hyPAD can be tuned for optimal performance by adjusting the applied voltage or changing the electrode design. Moreover, the throughput of hyPAD is 2 orders of magnitude higher than that of conventional, micron-scale microfluidic devices. The hyPAD is able to concentrate a range of analytes, including small molecules, DNA, proteins, and nanoparticles, in the range of 200−500-fold within 5 min.

pTwo-photon microscopes have been successfully translated into clinical imaging tools to obtain high-resolution optical biopsies foritalicin vivo/italichistology. We report on clinical multiphoton coherent anti-Stokes Raman spectroscopy (CARS) tomography based on two tunable ultrashort near-infrared laser beams for label-freeitalicin vivo/italicmultimodal skin imaging. The multiphoton biopsies were obtained with the compact tomograph "MPTflex-CARS" using a photonic crystal fiber, an optomechanical articulated arm, and a four-detector-360 deg measurement head. The multiphoton tomograph has been employed to patients in a hospital with diseased skin. The clinical study involved 16 subjects, 8 patients with atopic dermatitis, 4 patients with psoriasis vulgaris, and 4 volunteers served as control. Two-photon cellular autofluorescence lifetime, second harmonic generation (SHG) of collagen, and CARS of intratissue lipids/proteins have been detected with single-photon sensitivity, submicron spatial resolution, and picosecond temporal resolution. The most important signal was the autofluorescence from nicotinamide adenine dinucleotide [NAD(P)H]. The SHG signal from collagen was mainly used to detect the epidermal-dermal junction and to calculate the ratio elastin/collagen. The CARS/Raman signal provided add-on information. Based on this view on the disease-affected skin on a subcellular level, skin areas affected by dermatitis and by psoriasis could be clearly identified. Multimodal multiphoton tomographs may become important label-free clinical high-resolution imaging tools foritalicin vivo/italicskin histology to realize rapid early diagnosis as well as treatment control./p.

Paper Spray Ionization (PSI) is commonly applied for the analysis of small molecules, including drugs, metabolites, and pesticides in biological fluids, due to its high versatility, simplicity, and low costs. In this study, a new setup called Solvent Assisted Paper Spray Ionization (SAPSI), able to increase data acquisition time, signal stability, and repeatability, is proposed to overcome common PSI drawbacks. The setup relies on an integrated solution to provide ionization potential and constant solvent flow to the paper tip. Specifically, the ion source was connected to the instrument fluidics along with the voltage supply systems, ensuring a close control over the ionization conditions. SAPSI was successfully applied for the analysis of different classes of biomolecules: amyloidogenic peptides, proteins, and N-glycans. The prolonged analysis time allowed real-time monitoring of processes taking places on the paper tip, such as amyloid peptides aggregation and disaggregation phenomena. The enhanced signal stability allowed to discriminate protein species characterized by different post translational modifications and adducts with electrophilic compounds, both in aqueous solutions and in biofluids, such as serum and cerebrospinal fluid, without any sample pretreatment. In the next future, application to clinical relevant modifications, could lead to the development of quick and cost-effective diagnostic tools.

Paper-based fluorescence resonance energy transfer assay (FRET) is gaining great interest in detecting macro-biological molecule. It is difficult to achieve conveniently and fast detection for macro-biological molecule. Herein, a graphene oxide (GO)-based paper chip (glass fiber) integrated with fluorescence labeled single-stranded DNA (ssDNA) for fast, inexpensive and direct detection of biological macromolecules (proteins and nucleic acids) has been developed. In this paper, we employed the Cy3/FAM-labeled ssDNA as the reporter and the GO as quencher and the original glass fiber paper as data acquisition substrates. The chip which was designed and fabricated by a cutting machine is a miniature biosensor that monitors fluorescence recovery from resonance energy transfer. The hybridization assays and fluorescence detection were all simplified, and the surface of the chip did not require immobilization or washing. A Nikon Eclipse was employed as excited resource and a commercial digital camera was employed for capturing digital images. This paper-based microfluidics chip has been applied in the detection of proteins and nucleic acids. The biosensing capability meets many potential requirements for disease diagnosis and biological analysis.

Ricin is a highly-toxic compound derived from castor plant beans. Several incidents involving contamination of residences and buildings due to ricin production or dissemination have occurred in recent years. The goal of this study was to determine whether ricin bioactivity could be attenuated in reasonable time via simple modifications of the indoor environment. Attenuation was assessed on six different materials as a function of temperature, relative humidity (RH), and contact time, using both a pure and crude preparation of the toxin. Ricin bioactivity was quantified via a cytotoxicity assay, and attenuation determined as the difference in ricin recovered from test and positive controls. The results showed that pure ricin could be attenuated successfully, while the crude ricin was generally more persistent and results more variable. We found no significant attenuation in crude ricin after two weeks at typical indoor environmental conditions, except on steel. Attenuation mostly improved with increasing temperature, but the effect of RH varied. For pure ricin, heat treatments at 40°C for 5 days or 50°C for 2–3 days achieved greater than 96% attenuation on steel. In contrast, appreciable recovery of the crude ricin preparation still occurred at 40°C after two weeks.

This work describes a device for electrochemical enzyme-linked immunosorbent assay (ELISA) designed for low-resource settings and diagnostics at the point of care. The device is fabricated entirely in hydrophobic paper, produced by silanization of paper with decyl trichlorosilane, and comprises two zones separated by a central crease: an embossed microwell, on the surface of which the antigen or antibody immobilization and recognition events occur, and a detection zone where the electrodes are printed. The two zones are brought in contact by folding the device along this central crease; the analytical signal is recorded from the folded configuration. Two proof-of-concept applications, an electrochemical direct ELISA for the detection of rabbit IgG as a model antigen in buffer and an electrochemical sandwich ELISA for the detection of malarial histidine-rich protein from Plasmodium falciparum (Pf HRP2) in spiked human serum, show the versatility of this device. The limit of detection of the electrochemical sandwich ELISA for the quantification of Pf HRP2 in spiked human serum was 4 ng mL(-1) (10(2) pmol L(-1)), a value within the range of clinically relevant concentrations.

A concept integrating sampling and protein digestion is introduced here combining fast and simple fabrication by wax printing on filter paper with trypsin immobilized polymer beads. The paper reactors showed promising results with a high degree of protein digestion within fifty minutes in model protein mixtures as well as in human blood. The model protein mixture was used for the evaluation of performance both with and without a reduction and alkylation step. The paper reactors without reduction and alkylation showed between 46% and 75% protein sequence coverage and between five and 20 high confidence peptides (one and five zero missed cleavage peptides, respectively). Compared to a conventional in-solution approach, the paper reactor showed 10% less protein sequence coverage, 29% fewer high confidence peptides and 19% fewer high confidence peptides with zero missed cleavages. Placement of the protein reduction and alkylation step (before or after protein digestion) was shown to be of low importance. The storage stability of the paper reactors with (six weeks) and without (twelve weeks) tryptic peptides was satisfactory. The ability of the paper reactors to digest complex biological samples was investigated by comparison with human whole blood samples prepared using a conventional dried blood spot (DBS) procedure with overnight digestion in non-targeted analysis. The reactors showed a comparable performance with 75 ± 25 for the protein groups compared to 76 ± 5 for the DBS samples. Additionally, 267 ± 72 and 335 ± 11 unique peptides (high confidence) were identified for on-paper digestion and DBS, respectively.

X-ray crystallography is the main tool for structural determination of proteins. Yet, the underlying crystallization process is costly, has a high attrition rate and involves a series of trial-and-error attempts to obtain diffraction-quality crystals. The Structural Genomics Consortium aims to systematically solve representative structures of major protein-fold classes using primarily high-throughput X-ray crystallography. The attrition rate of these efforts can be improved by selection of proteins that are potentially easier to be crystallized. In this context, bioinformatics approaches have been developed to predict crystallization propensities based on protein sequences. These approaches are used to facilitate prioritization of the most promising target proteins, search for alternative structural orthologues of the target proteins and suggest designs of constructs capable of potentially enhancing the likelihood of successful crystallization. We reviewed and compared nine predictors of protein crystallization propensity. Moreover, we demonstrated that integrating selected outputs from multiple predictors as candidate input features to build the predictive model results in a significantly higher predictive performance when compared to using these predictors individually. Furthermore, we also introduced a new and accurate predictor of protein crystallization propensity, Crysf, which uses functional features extracted from UniProt as inputs. This comprehensive review will assist structural biologists in selecting the most appropriate predictor, and is also beneficial for bioinformaticians to develop a new generation of predictive algorithms.

We have demonstrated a multistep 2-dimensional paper network immunoassay based on controlled rehydration of patterned, dried reagents. Previous work has shown that signal enhancement improves the limit of detection in 2-dimensional paper network assays, but until now, reagents have only been included as wet or dried in separate conjugate pads placed at the upstream end of the assay device. Wet reagents are not ideal for point-of-care because they must be refrigerated and typically limit automation and require more user steps. Conjugate pads allow drying but do not offer any control of the reagent distribution upon rehydration and can be a source of error when pads do not contact the assay membrane uniformly. Furthermore, each reagent is dried on a separate pad, increasing the fabrication complexity when implementing multistep assays that require several different reagents. Conversely, our novel method allows for consistent, controlled rehydration from patterned reagent storage depots directly within the paper membrane. In this assay demonstration, four separate reagents were patterned in different regions of the assay device: a gold-antibody conjugate used for antigen detection and three different signal enhancement components that must not be mixed until immediately before use. To show the viability of patterning and drying reagents directly onto a paper device for dry reagent storage and subsequent controlled release, we tested this device with the malaria antigen Plasmodium falciparum histidine-rich protein 2 (PfHRP2) as an example of target analyte. In this demonstration, the signal enhancement step increases the visible signal by roughly 3-fold and decreases the analytical limit of detection by 2.75-fold.

The first step in curing a disease is being able to detect the disease effectively. Paper-based microfluidic devices are biodegradable and can make diagnosing diseases cost-effective and easy in almost all environments. We created a three-dimesnional (3D) paper device using wax printing fabrication technique and basic principles of origami. This design allows for a versatile fabrication technique over previously reported patterning of SU-8 photoresist on chromatography paper by employing a readily available wax printer. The design also utilizes multiple colorimetric assays that can accommodate one or more analytes including urine, blood, and saliva. In this case to demonstrate the functionality of the 3D paper-based microfluidic system, a urinalysis of protein and glucose assays is conducted. The amounts of glucose and protein introduced to the device are found to be proportional to the color change of each assay. This color change was quantified by use of Adobe Photoshop. Urine samples from participants with no pre-existing health conditions and one person with diabetes were collected and compared against synthetic urine samples with predetermined glucose and protein levels. Utilizing this method, we were able to confirm that both protein and glucose levels were in fact within healthy ranges for healthy participants. For the participant with diabetes, glucose was found to be above the healthy range while the protein level was in the healthy range.

Made available in DSpace on 2018-12-11T16:45:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-03-08 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) This paper describes the fabrication of 2D and 3D microfluidic paper-based analytical devices (μPADs) for monitoring glucose, total protein, and nitrite in blood serum and artificial urine. A new method of cutting and sealing filter paper to construct μPADs was demonstrated. Using an inexpensive home cutter printer soft cellulose-based filter paper was easily and precisely cut to produce pattern hydrophilic microchannels. 2D and 3D μPADs were designed with three detection zones each for the colorimetric detection of the analytes. A small volume of samples was added to the μPADs, which was photographed after 15 min using a digital camera. Both μPADs presented an excellent analytical performance for all analytes. The 2D device was applied in artificial urine samples and reached limits of detection (LODs) of 0.54 mM, 5.19 μM, and 2.34 μM for glucose, protein, and nitrite, respectively. The corresponding LODs of the 3D device applied for detecting the same analytes in artificial blood serum were 0.44 mM, 1.26 μM, and 4.35 μM. Departamento de Química Universidade Federal de São Carlos Intituto de Química-UNESP, Av. Francisco Degni s/n Intituto de Química-UNESP, Av. Francisco Degni s/n CNPq: 140484/2013-2 FAPESP: 2015/19890-1 CNPq: 427727/2016-2

Increase in global population and growing disease burden due to the emergence of infectious diseases (Zika virus), multidrug-resistant pathogens, drug-resistant cancers (cisplatin-resistant ovarian cancer) and chronic diseases (arterial hypertension) necessitate effective therapies to improve health outcomes. However, the rapid increase in drug development cost demands innovative and sustainable drug discovery approaches. Drug repositioning, the discovery of new or improved therapies by reevaluation of approved or investigational compounds, solves a significant gap in the public health setting and improves the productivity of drug development. As the number of drug repurposing investigations increases, a new opportunity has emerged to understand factors driving drug repositioning through systematic analyses of drugs, drug targets and associated disease indications. However, such analyses have so far been hampered by the lack of a centralized knowledgebase, benchmarking data sets and reporting standards. To address these knowledge and clinical needs, here, we present RepurposeDB, a collection of repurposed drugs, drug targets and diseases, which was assembled, indexed and annotated from public data. RepurposeDB combines information on 253 drugs [small molecules (74.30%) and protein drugs (25.29%)] and 1125 diseases. Using RepurposeDB data, we identified pharmacological (chemical descriptors, physicochemical features and absorption, distribution, metabolism, excretion and toxicity properties), biological (protein domains, functional process, molecular mechanisms and pathway cross talks) and epidemiological (shared genetic architectures, disease comorbidities and clinical phenotype similarities) factors mediating drug repositioning. Collectively, RepurposeDB is developed as the reference database for drug repositioning investigations. The pharmacological, biological and epidemiological principles of drug repositioning identified from the meta-analyses could augment therapeutic development.

Samples of bleached kraft pine cellulosic pulp, either treated with an enzyme preparation (a Thermomyces lanuginosus xylanase, an Aspergillus sp. cellulase, and a multienzyme preparation NS-22086 containing both these activities) or untreated, were refined in a laboratory PFI mill. The treatment with cellulases contained in the last two preparations significantly improved the pulp’s susceptibility to refining (the target freeness value of 30°SR was achieved in a significantly shorter time), increased water retention value (WRV) and fines contents while the weighted average fiber length was significantly reduced. These changes of pulp parameters caused deterioration of paper strength properties. The treatment with the xylanase, which partially hydrolyzed xylan, small amounts of which are associated with cellulose fibers, only slightly loosened the structure of fibers. These subtle changes positively affected the susceptibility of the pulp to refining (refining energy was significantly reduced) and improved the static strength properties of paper. Thus, the treatment of kraft pulps with xylanases may lead to substantial savings of refining energy without negative effects on paper characteristics.

Sea urchin teeth grow continuously and develop a complex mineralized structure consisting of spatially separate but crystallographically aligned first stage calcitic elements of high Mg content (5–15 mol% mineral). These become cemented together by epitaxially oriented second stage very high Mg calcite (30–40 mol% mineral). In the tooth plumula, ingressing preodontoblasts create layered cellular syncytia. Mineral deposits develop within membrane-bound compartments between cellular syncytial layers. We seek to understand how this complex tooth architecture is developed, how individual crystalline calcitic elements become crystallographically aligned, and how their Mg composition is regulated. Synchrotron microbeam X-ray scattering was performed on live, freshly dissected teeth. We observed that the initial diffracting crystals lie within independent syncytial spaces in the plumula. These diffraction patterns match those of mature tooth calcite. Thus, the spatially separate crystallites grow with the same crystallographic orientation seen in the mature tooth. Mineral-related proteins from regions with differing Mg contents were isolated, sequenced, and characterized. A tooth cDNA library was constructed, and selected matrix-related proteins were cloned. Antibodies were prepared and used for immunolocaliztion. Matrix-related proteins are acidic, phosphorylated, and associated with the syncytial membranes. Time-of-flight secondary ion mass spectroscopy of various crystal elements shows unique amino acid, Mg, and Ca ion distributions. High and very high Mg calcites differ in Asp content. Matrix-related proteins are phosphorylated. Very high Mg calcite is associated with Asp-rich protein, and it is restricted to the second stage mineral. Thus, the composition at each part of the tooth is related to architecture and function.

Paper has been proposed as an inexpensive and versatile carrier for microfluidics devices with abilities well beyond simple capillary action for pregnancy tests and the like. Unlike standard microfluidics devices, extracting a fluid from the paper is a challenge and a drawback to its broader use. Here, we extract fluid from narrow paper strips using surface acoustic wave (SAW) irradiation that subsequently atomizes the extracted fluid into a monodisperse aerosol for use in mass spectroscopy, medical diagnostics, and drug delivery applications. Two protein molecules, ovalbumin and bovine serum albumin (BSA), have been preserved in paper and then extracted using atomized mist through SAW excitation; protein electrophoresis shows there is less than 1% degradation of either protein molecule in this process. Finally, a solution of live yeast cells was infused into paper, which was subsequently dried for preservation then remoistened to extract the cells via SAW atomization, yielding live cells at the completion of the process. The successful preservation and extraction of fluids, proteins and yeast cells significantly expands the usefulness of paper in microfluidics.

Aquatic habitat is affected by paper mill effluent discharge in many ways. The effect of paper mill effluent on the gill ATPases was studied in freshwater fish Channa punctata (Bloch) exposed to 1%(v/v) of effluent for 15, 30, and 60 days. There was a time-dependent significant (P0.05-0.001) decrease in all the ATPase activities measured, viz., total, Na(+), K(+)--and ouabain-insensitive ATPase in gill. ATPases play an important role in maintenance of functional integrity of plasma membrane and in several intracellular functions and are considered to be a sensitive indicator of toxicity. In addition to this, branchial ATPases are intimately involved in osmoregulation, acid-base regulation, and respiration of fish. The inhibition of ATPases in gills by, e.g., paper mill effluent could cause disruption of these processes. It is suggested that measurement of ATPases could also be used as a surrogate biomarker of exposure to chemical pollutants.

Objective: To determine the rate and correlates of weight change in a large, well characterised sample of patients with Huntington’s disease followed at 44 sites by the Huntington Study Group. Participants and methods: Weight change was assessed in 927 adults with a definite diagnosis of Huntington’s disease who were followed prospectively for (mean (SD)) 3.4 (1.4) years. The unified Huntington’s disease rating scale was used to assess weight, motor dysfunction (including chorea and dystonia), depressive symptoms, and functional decline. Results: Random effects modelling determined that patients gained an average of 0.11 (1.7) kg/year and their chorea scores increased by 0.36 (0.78) points/year. There were significant but weak relations between weight loss and increasingly severe chorea ( r = −0.13), worse baseline motor performance ( r = −0.12), less severe baseline depressed mood ( r = 0.14), and poorer baseline independence ratings ( r = 0.07). Patients who were within 0 to 2 years of symptom onset at the time of the baseline visit gained more weight than those with longer disease duration. Conclusions: Weight loss following symptom onset is not a consistent feature of Huntington’s disease. The mechanisms contributing to weight change in this condition are unclear and probably multifactorial. Future studies examining asymptomatic carriers of the mutation could be helpful in identifying incipience of low body weight and may be better suited for identifying clinical correlates of weight loss than studies in symptomatic patients.

Objectives: To use the technique of dermal microdialysis to examine sensitivity of skin vessels to noradrenaline (NA) in patients with familial dysautonomia (FD) and in healthy controls. Methods: In 14 patients with FD and 12 healthy controls, plasma extravasation, local laser Doppler blood flow, and skin blanching were observed before, during, and after application of 10-6 M NA through a microdialysis membrane, located intradermally in the skin of the lower leg. Results: Maximum local vasoconstriction measured by laser Doppler blood flow did not differ between patients with FD and controls. In contrast, patients with FD had an earlier onset of vasoconstriction (p = 0.02). Moreover, reaction to NA was more prominent and prolonged in FD, shown by a larger zone of skin blanching around the microdialysis membrane (p < 0.001) and delayed reduction of the protein content in the dialysate after termination of NA application (p = 0.03). Conclusion: These data support the hypothesis that peripheral blood vessels of patients with FD show a denervation hypersensitivity to catecholamines. This may be one mechanism contributing to the major hypertension that frequently occurs during "dysautonomic crises" in FD.

Owing to the emerging impact of bioinformatics and computational biology, in this article, we present an overview of the history and current state of the research on this field in Latin America (LA). It will be difficult to cover without inequality all the efforts, initiatives and works that have happened for the past two decades in this vast region (that includes >19 million km2 and >600 million people). Despite the difficulty, we have done an analytical search looking for publications in the field made by researchers from 19 LA countries in the past 25 years. In this way, we find that research in bioinformatics in this region should develop twice to approach the average world scientific production in the field. We also found some of the pioneering scientists who initiated and led bioinformatics in the region and were promoters of this new scientific field. Our analysis also reveals that spin-off began around some specific areas within the biomolecular sciences: studies on genomes (anchored in the new generation of deep sequencing technologies, followed by developments in proteomics) and studies on protein structures (supported by three-dimensional structural determination technologies and their computational advancement). Finally, we show that the contribution to this endeavour of the Iberoamerican Society for Bioinformatics, founded in Mexico in 2009, has been significant, as it is a leading forum to join efforts of many scientists from LA interested in promoting research, training and education in bioinformatics., The publication charges for this article were funded by the research grant PI15/00328, given by the Instituto de Salud Carlos III (ISCiii, MINECO, Spain) and co-funded by the Fondo Europeo de Desarrollo Regional (FEDER, Europe).

Aims - To analyse the similarities between the Twisted gastrulation (TSG) proteins known to date; in addition, to determine phylogenetic relations among the TSG proteins, and between the TSGs and other protein families - the CCN (for example, CCN2 (CTGF), CCN1 (CYR61), and CCN3 (NOV)) and IGFBP (insulin-like growth factor binding protein) families. Methods - TBLASTN and FASTA3 were used to identify new tsg genes and relatives of the TSG family. The sequences were aligned with Clusta1W. The predictions of sites for signal peptide cleavage, post-translational modifications, and putative protein domains were carried out with software available at various databases. Unrooted phylogenetic trees were calculated using the UPGMA method. Results - Several tsg genes from vertebrates and invertebrates were compared. Alignment of protein sequences revealed a highly conserved family of TSG proteins present in both vertebrates and invertebrates, whereas the slightly less well conserved IGFBP and CCN proteins are apparently present only in vertebrates. The TSG proteins display strong homology among themselves and they are composed of a putative signal peptide at the N-terminus followed by a cysteine rich (CR) region, a conserved domain devoid of cysteines, a variable midregion, and a C-terminal CR region. The most striking similarity between the TSGs and the IGFBP and CCN proteins occurs in the N-terminal conserved cysteine rich domain and the characteristic 5′ cysteine rich domain(s), spacer region, and 3′ cysteine rich domain structure. Conclusion - The family of highly conserved TSG proteins, together with the IGFBP and CCN families, constitute an emerging multigene superfamily of secreted cysteine rich factors. The TSG branch of the superfamily appears to predate the others because it is present in all species examined, whereas the CCN and IGFBP genes are found only in vertebrates.

BACKGROUND: The development in the deinking process has made recycled fiber a major part of the raw material for pulp and paper industry. Enzymes have revolutionized the deinking process obtaining brightness levels surpassing conventional deinking processes. This study explores the deinking efficiencies of bacterial alkalophilic laccase (L) and xylanase (X) enzymes along with physical deinking methods of microwaving (MW) and sonication (S) for recycling of old newsprint (ONP). METHODS AND RESULTS: The operational parameters viz. enzyme dose, pH and treatment time for X and L deinking were optimized statistically using Response Surface Methodology. Laccase did not require any mediator supplementation for deinking. Deinking of ONP pulp with a combination of xylanase and laccase enzymes was investigated, and fiber surface composition and morphological changes were studied using X-ray diffraction, fourier transform infrared spectroscopy and scanning electron microscopy. Compared to the pulp deinked with xylanase (47.9%) or laccase (62.2%) individually, the percentage reduction of effective residual ink concentration (ERIC) was higher for the combined xylanase/laccase-deinked pulp (65.8%). An increase in brightness (21.6%), breaking length (16.5%), burst factor (4.2%) tear factor (6.9%), viscosity (13%) and cellulose crystallinity (10.3%) along with decrease in kappa number (22%) and chemical consumption (50%) were also observed. Surface appeared more fibrillar along with changes in surface functional groups. A combination of physical and enzymatic processes (S-MW-XL) for deinking further improved brightness (28.8%) and decreased ERIC (73.9%) substantially. CONCLUSION: This is the first report on deinking of ONP with laccase without any mediator supplementation. XL pretreatment resulted in marked improvement in paper quality and a new sequence being reported for deinking (S-MW-XL) will contribute further in decreasing chemical consumption and making the process commercially feasible.

In this review we discuss how nanomaterials can be integrated in diagnostic paper-based biosensors for the detection of proteins, nucleic acids and cells. In particular first the different types and properties of paper-based nanobiosensors and nanomaterials are briefly explained. Then several examples of their application in diagnostics of several biomarkers are reported. Finally our opinions regarding future trends in this field are discussed. © The Royal Society of Chemistry 2013., We acknowledge MEC (Madrid) for the project MAT2011–25870 and E.U. for the FP7 ‘‘NADINE’’ project (contract number 246513). C.P. also thanks the Generalitat de Catalunya for the PhD scholarship.

Novel methods are demonstrated that enable controlled spatial and temporal rehydration of dried reagents in a porous matrix. These methods can be used in paper-based microfluidic assays to define reagent concentrations over time at zones downstream for improved performance, and can reduce costs by simplifying the manufacturing process with the use of a single porous substrate. First, the creation of uniform reagent pulses from patterned arrays of dried reagent is demonstrated. Second, reagents are stored dry in separate regions of the porous matrix so that they can be combined upon rehydration for immediate use in the device. Third, reagents are reconstituted sequentially from dry storage depots with tunable delivery times. Fourth, the total time for dissolution is varied to achieve a range of reagent delivery times to a downstream region. Finally, the utility of these control methods is demonstrated in the context of real-time reagent rehydration and mixing on a porous device.

Paper-based analytical devices are the subject of growing interest for the development of low-cost point-of-care diagnostics, environmental monitoring technologies, and research tools for limited-resource settings. However, there are limited chemistries available for the conjugation of biomolecules to cellulose for use in biomedical applications. Herein, divinyl sulfone (DVS) chemistry was demonstrated to immobilize small molecules, proteins, and DNA covalently onto the hydroxyl groups of cellulose membranes through nucleophilic addition. Assays on modified cellulose using protein-carbohydrate and protein-glycoprotein interactions as well as oligonucleotide hybridization showed that the membrane's bioactivity was specific, dose-dependent, and stable over a long period of time. The use of an inkjet printer to form patterns of biomolecules on DVS-activated cellulose illustrates the adaptability of the DVS functionalization technique to pattern sophisticated designs, with potential applications in cellulose-based lateral flow devices.

Surface treatment of biological tissue sections improves detection of peptides and proteins for mass spectrometry imaging. However, liquid surface treatments can result in diffusion of surface analytes and fragile tissue sections can be easily damaged by typical washing solvents. Here, we present a new surface washing procedure for mass spectrometry imaging. This procedure uses solvent wetted fiber-free paper to enable local washing of tissue sections for mass spectrometry imaging and tissue profiling experiments. In addition, the method allows fragile tissues that cannot be treated by conventional washing techniques to be analyzed by mass spectrometry imaging.

Calcium-containing spherical bodies (calcospherulites) exist along the mineralization front of bone and are thought to play a role in bone formation. Existing methods to isolate calcospherulites involve harsh treatments that remove much of their organic matter. This study sought to isolate them using a less destructive approach to better preserve their organic components. Juvenile rats were injected with a low dose of calcein to label the newly formed mineral at the mineralization front of bone in vivo. Periosteum was completely dissected from the tibial diaphysis and unmineralized osteoid matrix was removed by collagenase in order to expose calcospherulites. Calcein-labeled calcospherulites of approximately 0.5 μm average diameter were observed all along the mineralization front and they exhibited a Ca/P ratio of 1.3 in situ. Calcospherulites were released from the mineralization front by a short dispase digestion and isolated via fluorescence flow sorting. X-ray diffraction revealed they contained apatite crystals (c-axis length of 17.5 ± 0.2 nm) and their Ca/P ratio was preserved during isolation. Calcospherulites treated with ice-cold ethanol exhibited a Ca/P ratio of 1.6, suggesting the presence of some extractable phospholipids. Proteins extracted from isolated calcospherulites were resolved by SDS-PAGE into more than 20 distinct bands. Western blot analyses showed the presence of matrix proteins in these preparations. These results indicate that calcospherulites can be isolated from the mineralization front of bone in a form that can be used to study their proteome and lipid composition.

This paper describes the use of a modified x,y-plotter to generate hydrophilic channels by printing a solution of hydrophobic polymer (poly(dimethyl siloxane; PDMS) dissolved in hexanes onto filter paper. The PDMS penetrates the depth of the paper, and forms a hydrophobic wall that aqueous solutions cannot cross. The minimum size of printed features is approximately 1 mm; this resolution is adequate for the rapid prototyping of hand-held, visually read, diagnostic assays (and other microfluidic systems) based on paper. After curing the printed PDMS, the paper-based devices can be bent or folded to generate three-dimensional (3D) systems of channels. Capillary action pulls aqueous samples into the paper channels. Colorimetric assays for the presence of glucose and protein are demonstrated in the printed devices; spots of Bromothymol Blue distinguished samples with slightly basic pH (6.5) from samples with slightly acidic pH (8.0). The work also describes using printed devices that can be loaded using multipipettes, and printed flexible, foldable channels in paper over areas larger than 100 cm2.

Objective: Spinocerebellar ataxia type 2 (SCA2) has been reported as the commonest dominant hereditary ataxia in India. However, India is an ethnically and religiously diverse population. Previous studies have not clearly indicated exact ethnic and religious origins, and must therefore be interpreted with caution. The purpose of this study was to determine the prevalence of different SCA mutations in a relatively homogeneous population from eastern India. Methods: We identified 28 families with autosomal dominant cerebellar ataxia from eastern India. Each underwent full clinical evaluation and were analysed for the presence of SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA12, and SCA17 mutations. In addition, haplotype analysis was carried out in seven of the 16 families with SCA2. Results: Seven patients from four (14%) families were positive for an expansion in SCA1 and 26 patients from 16 (57%) families were positive for an expansion in SCA2. No mutations were detected in the remaining eight families (29%). Most of the SCA1 and SCA2 families were Hindu from the state of Bihar. Five out of 26 SCA2 patients in this study did not have slow saccades. In addition, four of seven SCA1 patients had slow saccades. We found an association between the SCA2 CAG repeat expansion and the 285 base pair (bp) allele of microsatellite marker D12S1672, and also data supportive of the association between the expansion and the 225 bp allele of D12S1333, which has been previously described. Conclusions: We conclude that (1) although slow ocular saccades are highly suggestive of SCA2, that they are not universal, nor are they exclusive to this disorder and (2) SCA2 is likely to be the commonest dominant ataxia in eastern India, with further evidence for a founder effect.

Aims—Chromosome 3p deletions and loss of heterozygosity (LOH) for 3p markers are features of clear cell renal cell carcinoma but are rare in non-clear cell renal cell carcinoma. The VHL tumour suppressor gene, which maps to 3p25, is a major gatekeeper gene for clear cell renal cell carcinoma and is inactivated in most sporadic cases of this disease. However, it has been suggested that inactivation of other 3p tumour suppressor genes might be crucial for clear cell renal cell carcinoma tumorigenesis, with inactivation (VHL negative) and without inactivation (VHL positive) of the VHL tumour suppressor gene. This study set out to investigate the role of non-VHL tumour suppressor genes in VHL negative and VHL positive clear cell renal cell carcinoma. Methods—Eighty two clear cell renal cell carcinomas of known VHL inactivation status were analysed for LOH at polymorphic loci within the candidate crucial regions for chromosome 3p tumour suppressor genes (3p25, LCTSGR1 at 3p21.3, LCTSGR2 at 3p12 and at 3p14.2). Results—Chromosome 3p12–p21 LOH was frequent both in VHL negative and VHL positive clear cell renal cell carcinoma. However, although the frequency of 3p25 LOH in VHL negative clear cell renal cell carcinoma was similar to that at 3p12–p21, VHL positive tumours demonstrated significantly less LOH at 3p25 than at 3p12–p21. Although there was evidence of LOH for clear cell renal cell carcinoma tumour suppressor genes at 3p21, 3p14.2, and 3p12, both in VHL negative and VHL positive tumours, the major clear cell renal cell carcinoma LOH region mapped to 3p21.3, close to the lung cancer tumour suppressor gene region 1 (LCTSGR1). There was no association between tumour VHL status and tumour grade and stage. Conclusions—These findings further indicate that VHL inactivation is not sufficient to initiate clear cell renal cell carcinoma and that loss of a gatekeeper 3p21 tumour suppressor gene is a crucial event for renal cell carcinoma development in both VHL negative and VHL positive clear cell renal cell carcinoma.

This communication describes a simple method for printing aqueous solutions with millimeter-scale patterns on a variety of substrates using an easily fabricated, paper-based microfluidic device (a paper-based "stamp") as a contact printing device. The device is made from inexpensive materials, and it is easily assembled by hand; this method is thus accessible to a wide range of laboratories and budgets. A single device was used to print over 2500 spots in less than three minutes at a density of 16 spots per square centimetre. This method provides a new tool to pattern biochemicals-reagents, antigens, proteins, and DNA-on planar substrates. The accuracy of the volume of fluid delivered in simple paper-to-paper printing is low, and although the pattern transfer is rapid, it is better suited for qualitative than accurate, quantitative work. By patterning the paper to which the transfer occurs using wax printing or an equivalent technique, accuracy increases substantially.

A method for patterning narrow lines of biomolecules onto nitrocellulose membranes using laboratory syringe pumps is described. One syringe pump is used to drive the biomolecule solution through a needle, while another modified syringe pump acts as a one-dimensional translation stage, moving the needle across the membrane much like a pen. This method consumes very small volumes of reagents, and is a viable option for laboratory-scale fabrication and prototyping of point-of-care rapid diagnostic test strips.

We describe a method by which complex protein mixtures are fractionated by standard one-dimensional Na-DodSO4/polyacrylamide gel electrophoresis or O'Farrell two-dimensional gel electrophoresis and then are efficiently and rapidly transferred electrophoretically to diazobenzyloxymethyl- or diazophenylthioether-paper and analyzed by immunoautoradiography. The method is illustrated with protein extracts of human KB cells infected with adenovirus type 2. Proteins were transferred from gels without decrease in resolution and with an increase in the sensitivity of detection by autoradiography when [35S]-methionine-labeled proteins were used. When unlabeled proteins were transferred, low levels of virus encoded proteins could be detected by sequential treatment of diazobenzyloxymethyl-paper with anti-adenovirus type 2 virion or anti-73,000 DNA binding protein and 125I-labeled Staphyloccus aureus protein A. Covalently bound viral proteins retained immunologic reactivity after dissociation of the protein A and antibody, By one-dimensional gel transfer/immunoautoradiography, seven virion proteins were detected as prominent bands and several others as weaker bands. By two-dimensional gel transfer/immunoautoradiography, several additional viral proteins were detected. By use of anti-DNA binding protein serum, the Mr 73,000 protein and Mr 41,000-48,000 subspecies were detected. A protein present at a concentration of approximately 1 part in 100,000 of the total protein can be identified in cell extracts. This method may be applicable to various biological problems requiring resolution and detection of small amounts of specific proteins that can be recognized immunologically or that can be detected by binding to specific radiolabeled DNA or RNA sequences or hormones.

Rainbow trout ( Salmo gairdneri ) were exposed for 10 and 30 days to microbiologically treated effluent of a bleached kraft pulp and paper mill (BKME). Throughout the experiments, the quality of dilution water, taken from an upstream location close to the mill, was allowed to vary both diurnally and seasonally (September–October) in a natural manner. The test concentrations averaged 0 (control), 0.6, 1.2, 2.0, and 5.1% (v/v) of BKME. Conjugates of all major resin acids and chlorinated phenolics of BKME, except chlorocatechols, were detected in trout bile. They comprised 93.1–99.9% of the total amounts in the bile. The level of conjugated resin acids increased linearly with the content of BKME in test water. On the other hand, no increase in concentration of conjugated phenolics was seen above 2.0% BKME. In the blood plasma no conjugates were analyzable. Concentrations of free resin acids in the blood plasma, but not in the bile, significantly increased at high test concentrations of BKME. No active excretion of free resin acids from plasma to bile can be inferred, but some of the phenolics—particularly 3,4,5,6-tetrachloroguaiacol—seem to be concentrated in the bile as a free compound. Changes in several biochemical parameters did not display any simple relationship with the external concentrations of BKME. In intermediate exposure concentrations, however, liver RNA and protein concentrations were decreased after 10 days but increased after 30 days. Condition of fish, relative size of the liver, and concentrations of liver glycogen and water were unresponsive to BKME at the dilutions investigated.

Link protein (Mr = 42,000) is an integral component of cartilage as well as of some noncartilagenous tissues. In cartilage, it forms a macromolecular complex with cartilage proteoglycan and hyaluronic acid, but its function in other tissues is unknown. We provide evidence here that the link protein of cartilage binds well to native collagen types I and III. The binding occurs only if both link protein and collagen are native. The binding of link protein to collagen type fibrils is higher than to monomeric collagen. Link protein binding to collagen fibrils is saturable and occurs at molar ratio of collagen to link protein of 7-13:1. These data suggest that the link protein binds to collagen and that the binding requires the collagen to be in its native triple helical structure. This interaction may play a role in collagen fibril formation.