Protein Identification and Quantification Using Porous Silicon Arrays, Optical Measurements, and Machine Learning.

We report a versatile platform based on an array of porous silicon (PSi) thin films that can identify analytes based on their physical and chemical properties without the use of specific capture agents. The ability of this system to reproducibly classify, quantify, and discriminate three proteins separately is demonstrated by probing the reflectance of PSi array elements with a unique combination of pore size and buffer pH, and by analyzing the optical signals using machine learning. Protein identification and discrimination are reported over a concentration range of two orders of magnitude. This work represents a significant first step towards a low-cost, simple, versatile, and robust sensor platform that is able to detect biomolecules without the added expense and limitations of using capture agents.

Porous Silicon-Based Aptasensors: Toward Cancer Protein Biomarker Detection.

The anterior gradient homologue-2 (AGR2) protein is an attractive biomarker for various types of cancer. In pancreatic cancer, it is secreted to the pancreatic juice by premalignant lesions, which would be an ideal stage for diagnosis. Thus, designing assays for the sensitive detection of AGR2 would be highly valuable for the potential early diagnosis of pancreatic and other types of cancer. Herein, we present a biosensor for label-free AGR2 detection and investigate approaches for enhancing the aptasensor sensitivity by accelerating the target mass transfer rate and reducing the system noise. The biosensor is based on a nanostructured porous silicon thin film that is decorated with anti-AGR2 aptamers, where real-time monitoring of the reflectance changes enables the detection and quantification of AGR2, as well as the study of the diffusion and target-aptamer binding kinetics. The aptasensor is highly selective for AGR2 and can detect the protein in simulated pancreatic juice, where its concentration is outnumbered by orders of magnitude by numerous proteins. The aptasensor's analytical performance is characterized with a linear detection range of 0.05-2 mg mL-1, an apparent dissociation constant of 21 ± 1 µM, and a limit of detection of 9.2 µg mL-1 (0.2 µM), which is attributed to mass transfer limitations. To improve the latter, we applied different strategies to increase the diffusion flux to and within the nanostructure, such as the application of isotachophoresis for the preconcentration of AGR2 on the aptasensor, mixing, or integration with microchannels. By combining these approaches with a new signal processing technique that employs Morlet wavelet filtering and phase analysis, we achieve a limit of detection of 15 nM without compromising the biosensor's selectivity and specificity.

Morlet Wavelet Filtering and Phase Analysis to Reduce the Limit of Detection for Thin Film Optical Biosensors.

The ultimate detection limit of optical biosensors is often limited by various noise sources, including those introduced by the optical measurement setup. While sophisticated modifications to instrumentation may reduce noise, a simpler approach that can benefit all sensor platforms is the application of signal processing to minimize the deleterious effects of noise. In this work, we show that applying complex Morlet wavelet convolution to Fabry-Pérot interference fringes characteristic of thin film reflectometric biosensors effectively filters out white noise and low-frequency reflectance variations. Subsequent calculation of the average difference in extracted phase between the filtered analyte and reference signals enables a significant reduction in the limit of detection (LOD). This method is applied on experimental data sets of thin film porous silicon sensors (PSi) in buffered solution and complex media obtained from two different laboratories. The demonstrated improvement in the LOD achieved using wavelet convolution and average phase difference paves the way for PSi optical biosensors to operate with clinically relevant detection limits for medical diagnostics, environmental monitoring, and food safety.

Habitat as a mediator of mesopredator-driven mammal extinction.

A prevailing view in dryland systems is that mammals are constrained by the scarcity of fertile soils and primary productivity. An alternative view is that predation is a primary driver of mammal assemblages, especially in Australia, where 2 introduced mesopredators-feral cat (Felis catus) and red fox (Vulpes vulpes)-are responsible for severe declines of dryland mammals. We evaluated productivity and predation as drivers of native mammal assemblage structure in dryland Australia. We used new data from 90 sites to examine the divers of extant mammal species richness and reconstructed historic mammal assemblages to determine proportional loss of mammal species across broad habitat types (landform and vegetation communities). Predation was supported as a major driver of extant mammal richness, but its effect was strongly mediated by habitat. Areas that were rugged or had dense grass cover supported more mammal species than the more productive and topographically simple areas. Twelve species in the critical weight range (CWR) (35-5500 g) that is most vulnerable to mesopredator predation were extirpated from the continent's central region, and the severity of loss of species correlated negatively with ruggedness and positively with productivity. Based on previous studies, we expect that habitat mediates predation from red foxes and feral cats because it affects these species' densities and foraging efficiency. Large areas of rugged terrain provided vital refuge for Australian dryland mammals, and we predict such areas will support the persistence of CWR species in the face of ongoing mammal declines elsewhere in Australia.

Females Choose Mates Based on Genetic Relatedness in a Small Dasyurid Marsupial, the Agile Antechinus (Antechinus agilis).

Females in a variety of taxa mate with more than one male during a single oestrus and exhibit mate preferences for genetically compatible males, but the influence of female mate choice on siring success is not clearly understood. Whether females choose to mate with more than one male or endure forced copulations is also often unknown. Here, we examined the effects of genetic relatedness on female mate choice and siring success in a small semelparous carnivorous marsupial, the agile antechinus (Antechinus agilis), during two consecutive breeding seasons. Experimental trials were conducted in captivity over periods of 72 hours using interconnected enclosures in which female antechinus could choose to access any of four separated males, but males were only able to access females that entered their quarters. Females had access to two genetically similar and two genetically dissimilar males simultaneously and all behavioural interactions were observed and scored from continuous video recordings. Genetic similarity between mates and paternity of young was determined by microsatellite analyses. Some females chose to enter and mate with more than one male during a single oestrus period. Although females investigated all males, they spent significantly more time visiting, and mated more times with, genetically dissimilar males. Males that were genetically dissimilar to the female sired 88% of subsequent offspring. Whilst males mated readily with most females, they rejected the advances of some receptive females, indicating a previously unexpected level of male mate choice. The results show that genetic relatedness between mates has a significant influence on mate choice, breeding and siring success in the agile antechinus.

Epidermal barrier dysfunction in atopic dermatitis.

Atopic dermatitis (AD) is a multifactorial, heterogenous disease that arises as a result of the interaction between both environmental and genetic factors. Changes in at least three groups of genes encoding structural proteins, epidermal proteases, and protease inhibitors predispose to a defective epidermal barrier and increase the risk of developing AD. Loss-of-function mutations found within the FLG gene encoding the structural protein, filaggrin, represent the most significant genetic factor predisposing to AD identified to date. Enhanced protease activity and decreased synthesis of the lipid lamellae lead to exacerbated breakdown of the epidermal barrier. Environmental factors, including the use of soap and detergents, exacerbate epidermal barrier breakdown, attributed to the elevation of stratum corneum pH. A sustained increase in pH enhances the activity of degradatory proteases and decreases the activity of the lipid synthesis enzymes. The strong association between both genetic barrier defects and environmental insults to the barrier with AD suggests that epidermal barrier dysfunction is a primary event in the development of this disease. Our understanding of gene-environment interactions should lead to a better use of some topical products, avoidance of others, and the increased use and development of products that can repair the skin barrier.

Patterns of expression of retinoic acid receptor beta 2 (RAR-beta2)-LacZ reporter gene in the embryonic foregut.

Vitamin A and its active form retinoic acid (RA) are essential for normal embryonic development. Maternal vitamin A deficiency in experimental animals is known to produce various developmental anomalies including foregut malformations and lung hypoplasia. However, there is a little known about the role of RA receptors in the developing foregut. Our aim was to study the pattern of expression of retinoic acid receptor beta 2 (RAR-beta2) in the region of the foregut during the early stages of embryonic development. Normal mouse embryos homozygous for the lacZ-fused RAR-beta2 promoter transgene were studied to detect the expression of RAR-beta2 in the embryonic foregut. Transverse and sagittal sections of the embryos were taken at the region of the foregut to observe for The normal pattern of expression of RARbeta2-LacZ between 9.5-12.5 days post conception. RAR-beta2-LacZ was expressed in the foregut tube on 9.5 and 10.5 dpc, mainly in the oesophageal part and maximally in the region of tracheo-oesophageal fold formation. This expression faded on day 11.5, and was not seen on 12.5 dpc. The change of RAR-beta2 expression between 9.5-11.5 dpc coincided with the time of tracheo-esophageal separation of the foregut. Our study has shown a possible RA-driven genetic activity during embryonic foregut development.

In vitro effects of adriamycin: a dose-response study.

The in vivo effects of adriamycin (ADR) on the mouse and rat embryos are well described in the literature. However, there is a lack of knowledge about the in vitro effects of ADR. The aim of this study was to investigate the effects of ADR on the developing mouse embryo and to identify a dose of ADR, which could be used for further studies of ADR effects in vitro. CD1 mouse embryos were collected at day 8.5 post conception. They were cultured in the presence of different doses of ADR (0, 125, 250, 375 and 500 microM). After 24 h, the culture was stopped and the embryos (n = 77) were scored morphologically using the Brown-Fabro scoring system and the mean score for each organ was calculated. Dose-response plots were generated and the effective dose 50 (ED50) for each organ was identified from the plots. The effects of ADR on the developing embryo were found to be dose related and there is a dose-response relationship in most of the plots. The dose-response plots were found to be parallel for some organs. A dose of 250 microM ADR was identified as the appropriate dose for further in vitro studies. The effects of ADR on the embryos were dose related and there is a dose-response relationship in most of the developing systems. The presence of parallel dose-response plots for some regions is suggestive of similar mechanism of action of ADR on these regions. A dose of 250 microM of ADR was identified for the first time in the literature and could be used for further studies of the effects of ADR on the mouse foregut in vitro.

New perspectives on epidermal barrier dysfunction in atopic dermatitis: gene-environment interactions.

Atopic dermatitis (AD) is a multifactorial, chronic inflammatory skin disorder in which genetic mutations and cutaneous hyperreactivity to environmental stimuli play a causative role. Genetic mutations alone might not be enough to cause clinical manifestations of AD, and this review will propose a new perspective on the importance of epidermal barrier dysfunction in genetically predisposed individuals, predisposing them to the harmful effects of environmental agents. The skin barrier is known to be damaged in patients with AD, both in acute eczematous lesions and also in clinically unaffected skin. Skin barrier function can be impaired first by a genetic predisposition to produce increased levels of stratum corneum chymotryptic enzyme. This protease enzyme causes premature breakdown of corneodesmosomes, leading to impairment of the epidermal barrier. The addition of environmental interactions, such as washing with soap and detergents, or long-term application of topical corticosteroids can further increase production of stratum corneum chymotryptic enzyme and impair epidermal barrier function. The epidermal barrier can also be damaged by exogenous proteases from house dust mites and Staphylococcus aureus. One or more of these factors in combination might lead to a defective barrier, thereby increasing the risk of allergen penetration and succeeding inflammatory reaction, thus contributing to exacerbations of this disease.

Predisposition to sensitive skin and atopic eczema.

Genes that control the thickness of our skin and its vulnerability to chemicals in the environment play a role in the development of contact dermatitis and atopic eczema. Sensitive skin manifests itself as a burning, stinging or itching sensation following the application of topical products such as soap, bubble baths and cosmetics. The skin may become red and dry after repeated application of these products. New insights into the skin barrier can help us improve treatment of the skin and prevent problems associated with atopic eczema and sensitive skin

Single-point haplotype scores telomeric to human leukocyte antigen-C give a high susceptibility major histocompatibility complex haplotype for psoriasis in a Caucasian population.

Psoriasis is a chronic inflammatory skin disease that affects 0.1%-5% depending on the population. PSORS1 is the major susceptibility locus, accounting for approximately 33%-50% of the genetic component of psoriasis among Caucasians. PSORS1 is located within the major histocompatibility complex (MHC) locus on 6p21.3. Its position has been refined to hundreds of kilobase and the region located at approximately 100-200 kb telomeric to human leukocyte antigen (HLA)-C is a very strong candidate. To determine the MHC psoriasis risk haplotype, we screened the whole 46 kb interval for single-nucleotide polymorphisms (SNP) and identified 138 SNP. We genotyped 29 SNP throughout this region in psoriatic nuclear families. We calculated the frequency of haplotypes generated by the 29 SNP using all genotyped founder individuals and found four common haplotype with frequency >0.10. We then used SNPtagger to derive the best six SNP and fed these into Transmit using 148 nuclear families. We found that CTGGAC haplotype is a single-point score haplotypes telomeric to HLA-C and gives a 1 df, chi2 of 50.27 (p<0.0001). Most importantly the six selected SNP accurately tagged the most common haplotype found in this region. Moreover, using the same program (Transmit) we show that the association with CTGGAC is higher than the one with HLA-Cw6 (chi2=10.53; p=0.0051). Our results give scores as high as the highest single-point scores suggesting that it is unlikely to be able to discriminate the origin of the association on this analysis on strength of association.

Eye birth defects in humans may be caused by a recessively-inherited genetic predisposition to the effects of maternal vitamin A deficiency during pregnancy.

Congenital malformations of the eye can cause blindness in children. They occur throughout the world and in most cases the aetiology is unknown. Linkage studies have largely been unsuccessful and the risk to siblings is generally low. Epidemiological and laboratory evidence support a hypothesis that there may be genetic (recessive) predisposition to the teratogenetic effects of mild to moderate maternal vitamin A deficiency (VAD) during pregnancy. This may explain the higher prevalence of congenital eye anomalies in a part of Asian countries, where maternal VAD is common and consanguineous marriages are popular. Other congenital malformations commonly found in association with ocular coloboma (e.g. oesophageal fistulae and heart defects in CHARGE association) may also be VAD related. Mutations in a gene involved in the cellular access to vitamin A that normally protects the tissue or embryo from natural variation in dietary vitamin A intake, could render that individual intolerant of conditions of VAD. An interaction of this kind could also explain a proportion of "sporadic" cases in locations where VAD is uncommon. If this interaction is shown to be true, there are public health implications for the prevention of blindness due to congenital eye malformations. The hypotheses proposed above are reminiscent of the research leading to the discovery that folic acid supplementation could prevent neural tube defects. However, this form of intervention would be much more difficult with vitamin A, which is itself a powerful teratogen if present in excess.

Identification of known and novel genes whose expression is regulated by endogenous retinoic acid during early embryonic development of the mouse.

Retinoic acid (RA) derived from vitamin A is necessary for, among other things, mammalian embryonic development. Although the impact of RA-dependent gene-regulation on embryonic development has been examined through genetic disruption of the retinoid receptors, the understanding of the underlying molecular mechanism remain unclear, in part, due to the difficulty in identifying RA-regulated genes in an intact embryo. We report here that RA-regulated genes can be identified from total RA-deficient embryos created by retinol-binding protein antisense (RBP-AS) oligodeoxynucleotide treatment in conjunction with differential display. Of the 28 genes isolated, 15 genes matched known genes in the GenBank database and the others either represented EST sequences or encoded novel genes. Semi-quantitative reverse transcriptase-polymerase chain reaction verified that the mRNA levels of mouse DN 38, COL VI 3 alpha, cul-1, alpha-tropomyosin, and PP2A-C alpha were substantially increased, whereas mouse Msh 2, Ndufa2, Ribosomal protein S19, sFRP-1, GDAP-10 and mSmcD were significantly decreased in vitamin A deficient (VAD) embryos compared to the control embryos. The utility of the method is exemplified by our finding that several genes in the Wnt signaling pathway are vitamin A regulated in day 9.0 post coitum (p.c.) embryos.

Environmental risk factors in congenital malformations of the eye.

Developmental eye defects such as coloboma are a significant cause of visual morbidity in children, and are more common in India than elsewhere. The possible role of environmental factors in the aetiology of these conditions was investigated by studying birth order, symptoms of vitamin A deficiency (night blindness), drug use and maternal illness in pregnancy, rubella antibodies and exposure to agricultural chemicals. Through hospital records and community-based rehabilitation programmes in Andhra Pradesh, children with colobomata were recruited from schools for the blind. Eighty-three mothers of affected children were interviewed. The results showed that 43% of parents were consanguineous, that 19% had a positive family history and that the frequency of coloboma was highest in second-born children. Eleven (16%) mothers had a history of night blindness while pregnant with the affected child; seven (8%) took medication during the 1st trimester, abortifacients in two cases; three reported fever in the 1st trimester; and 11 (13%) reported exposure to agricultural chemicals.