Array-based sequencing of filaggrin gene for comprehensive detection of disease-associated variants

The filaggrin gene (FLG) is essential for skin differentiation and epidermal barrier formation. FLG loss-of-function (LoF) variants are associated with ichthyosis vulgaris and the major genetic risk factor for developing atopic dermatitis (AD).1, 2, 3 Genetic stratification of patients with AD according to FLG LoF risk is a common practice for both research and clinical studies; however, few studies comprehensively sequence the entire FLG coding region. Most studies that include FLG genotyping have screened for common predominant LoF variants to report allele frequencies after full Sanger sequencing of a smaller batch of test patient samples or previously published data. This strategy potentially results in underreporting of the genetic contribution especially in ethnicities where FLG LoF variants are highly diverse.4 Distinct LoF variants have been reported for most ethnicities studied to date. For example, 2 predominant sequence variants (p.R501X and c.2282del4) make up approximately 80% of the mutation burden in northern Europeans,5 whereas in East Asian ethnicities, a larger FLG LoF mutation spectrum is found with fewer predominating variants.6, 7 However, routinely Sanger sequencing the entire FLG coding region for large cohorts is not always feasible, although desirable as it is essential to correctly stratify patients. To address this, we developed a robust and cost-effective high-throughput PCR-based method for analyzing the entire coding region of FLG using Fluidigm microfluidics technology and next-generation sequencing (NGS). We have applied this method to fully resequence cohorts of Chinese, Malay, and Indian patients with AD from the Singaporean population.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio

Field Longevity of a Fluorescent Protein Marker in an Engineered Strain of the Pink Bollworm, Pectinophora gossypiella (Saunders)

The cotton pest, pink bollworm (Pectinophora gossypiella (Saunders)), is a significant pest in most cotton-growing areas around the world. In southwestern USA and northern Mexico, pink bollworm is the target of the sterile insect technique (SIT), which relies on the mass-release of sterile pink bollworm adults to over-flood the wild population and thereby reduce it over time. Sterile moths reared for release are currently marked with a dye provided in their larval diet. There are concerns, however, that this marker fails from time to time, leading to sterile moths being misidentified in monitoring traps as wild moths. This can lead to expensive reactionary releases of sterile moths. We have developed a genetically marked strain that is engineered to express a fluorescent protein, DsRed2, which is easily screened under a specialised microscope. In order to test this marker under field conditions, we placed wild-type and genetically marked moths on traps and placed them in field cages. The moths were then screened, in a double-blind fashion, for DsRed2 fluorescence at regular intervals to determine marker reliability over time. The marker was shown to be robust in very high temperatures and generally proved reliable for a week or longer. More importantly, genotyping of moths on traps by PCR screening of the moths was 100% correct. Our findings indicate that this strain - and fluorescent protein markers in general - could make a valuable contribution to SIT

Tortricid Moths Reared from the Invasive Weed Mexican Palo Verde, Parkinsonia aculeata, with Comments on their Host Specificity, Biology, Geographic Distribution, and Systematics

As part of efforts to identify native herbivores of Mexican palo verde, Parkinsonia aculeata L. (Leguminosae: Caesalpinioideae), as potential biological control agents against this invasive weed in Australia, ten species of Tortricidae (Lepidoptera) were reared from Guatemala, Mexico, Nicaragua, and Venezuela: Amorbia concavana (Zeller), Platynota rostrana (Walker), Platynota helianthes (Meyrick), Platynota stultana Walsingham (all Tortricinae: Sparganothini), Rudenia leguminana (Busck), Cochylis sp. (both Tortricinae: Cochylini), Ofatulena duodecemstriata (Walsingham), O. luminosa Heinrich, Ofatulena sp. (all Olethreutinae: Grapholitini), and Crocidosema lantana Busck (Olethreutinae: Eucosmini). Significant geographic range extensions are provided for O. duodecemstriata and R. leguminana. These are the first documented records of P. aculeata as a host plant for all but O. luminosa. The four species of Sparganothini are polyphagous; in contrast, the two Cochylini and three Grapholitini likely are specialists on Leguminosae. Ofatulena luminosa is possibly host specific on P. aculeata. Host trials with Rudenia leguminana also provide some evidence of specificity, in contrast to historical rearing records. To examine the possibility that R. leguminana is a complex of species, two data sets of molecular markers were examined: (1) a combined data set of two mitochondrial markers (a 781-basepair region of cytochrome c oxidase I (COI) and a 685-basepair region of cytochrome c oxidase II) and one nuclear marker (a 531-basepair region of the 28S domain 2); and (2) the 650-basepair “barcode” region of COI. Analyses of both data sets strongly suggest that individuals examined in this study belong to more than one species

Prognostic model to predict postoperative acute kidney injury in patients undergoing major gastrointestinal surgery based on a national prospective observational cohort study.

Background: Acute illness, existing co-morbidities and surgical stress response can all contribute to postoperative acute kidney injury (AKI) in patients undergoing major gastrointestinal surgery. The aim of this study was prospectively to develop a pragmatic prognostic model to stratify patients according to risk of developing AKI after major gastrointestinal surgery. Methods: This prospective multicentre cohort study included consecutive adults undergoing elective or emergency gastrointestinal resection, liver resection or stoma reversal in 2-week blocks over a continuous 3-month period. The primary outcome was the rate of AKI within 7 days of surgery. Bootstrap stability was used to select clinically plausible risk factors into the model. Internal model validation was carried out by bootstrap validation. Results: A total of 4544 patients were included across 173 centres in the UK and Ireland. The overall rate of AKI was 14·2 per cent (646 of 4544) and the 30-day mortality rate was 1·8 per cent (84 of 4544). Stage 1 AKI was significantly associated with 30-day mortality (unadjusted odds ratio 7·61, 95 per cent c.i. 4·49 to 12·90; P < 0·001), with increasing odds of death with each AKI stage. Six variables were selected for inclusion in the prognostic model: age, sex, ASA grade, preoperative estimated glomerular filtration rate, planned open surgery and preoperative use of either an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker. Internal validation demonstrated good model discrimination (c-statistic 0·65). Discussion: Following major gastrointestinal surgery, AKI occurred in one in seven patients. This preoperative prognostic model identified patients at high risk of postoperative AKI. Validation in an independent data set is required to ensure generalizability

Obesity and the food system transformation in Latin America

The Latin America and the Caribbean (LAC) region faces a major diet-related health problem accompanied by enormous economic and social costs. The shifts in diet are profound: major shifts in intake of less-healthful low-nutrient-density foods and sugary beverages, changes in away-from-home eating and snacking and rapid shifts towards very high levels of overweight and obesity among all ages along with, in some countries, high burdens of stunting. Diet changes have occurred in parallel to, and in two-way causality with, changes in the broad food system – the set of supply chains from farms, through midstream segments of processing, wholesale and logistics, to downstream segments of retail and food service (restaurants and fast food chains). An essential contribution of this piece is to marry and integrate the nutrition transition literature with the literature on the economics of food system transformation. These two literatures and debates have been to date largely ‘two ships passing in the night’. This review documents in-depth the recent history of rapid growth and transformation of that broad food system in LAC, with the rapid rise of supermarkets, large processors, fast food chains and food logistics firms. The transformation is the story of a ‘double-edged sword’, showing its links to various negative diet side trends, e.g. the rise of consumption of fast food and highly processed food, as well as in parallel, to various positive trends, e.g. the reduction of the cost of food, de-seasonalization, increase of convenience of food preparation reducing women's time associated with that and increase of availability of some nutritious foods like meat and dairy. We view the transformation of the food system, as well as certain aspects of diet change linked to long-run changes in employment and demographics (e.g. the quest for convenience), as broad parameters that will endure for the next decades without truly major regulatory and fiscal changes. We then focus in on what are the steps that are being and can be taken to curb the negative effects on diet of these changes. We show that countries in LAC are already among the global leaders in initiating demand-related solutions via taxation and marketing controls. But we also show that this is only a small step forward. To shift LAC's food supply towards prices that incentivize consumption of healthier diets and demand away from the less healthy component is not simple and will not happen immediately. We must be cognizant that ultimately, food industry firms must be incentivized to market the components of healthy diets. This will primarily need to be via selective taxes and subsidies, marketing controls, as well as food quality regulations, consumer education and, in the medium term, consumers' desires to combine healthier foods with their ongoing quest for convenience in the face of busy lives. In the end, the food industry in LAC will orient itself towards profitable solutions, ie those demanded by the broad mass of consumers

A cell-based drug discovery assay identifies inhibition of cell stress responses as a new approach to treatment of epidermolysis bullosa simplex

In the skin fragility disorder epidermolysis bullosa simplex (EBS), mutations in keratin 14 (K14, also known as KRT14) or keratin 5 (K5, also known as KRT5) lead to keratinocyte rupture and skin blistering. Severe forms of EBS are associated with cytoplasmic protein aggregates, with elevated kinase activation of ERK1 and ERK2 (ERK1/2; also known as MAPK3 and MAPK1, respectively), suggesting intrinsic stress caused by misfolded keratin protein. Human keratinocyte EBS reporter cells stably expressing GFP-tagged EBS-mimetic mutant K14 were used to optimize a semi-automated system to quantify the effects of test compounds on keratin aggregates. Screening of a protein kinase inhibitor library identified several candidates that reduced aggregates and impacted on epidermal growth factor receptor (EGFR) signalling. EGF ligand exposure induced keratin aggregates in EBS reporter keratinocytes, which was reversible by EGFR inhibition. EBS keratinocytes treated with a known EGFR inhibitor, afatinib, were driven out of activation and towards quiescence with minimal cell death. Aggregate reduction was accompanied by denser keratin filament networks with enhanced intercellular cohesion and resilience, which when extrapolated to a whole tissue context would predict reduced epidermal fragility in EBS patients. This assay system provides a powerful tool for discovery and development of new pathway intervention therapeutic avenues for EBS.Published versionThis work was supported by DEBRA International grants LANE2/LANE3 to E.B.L., and by grants IAF311011 and SPF2013/004 to E.B.L. and J.E.A.C. from the Biomedical Research Council of Singapore. The funding sources were not involved in the conduct of the research, or writing of the manuscript. No payment was received from any pharmaceutical company or other for-profit agency to write the manuscript. Deposited in PMC for immediate release

Investigating PEGDA and GelMA Microgel Models for Sustained 3D Heterotypic Dermal Papilla and Keratinocyte Co-Cultures

10.3390/ijms22042143INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES22

Plau and Tgfbr3 are YAP-regulated genes that promote keratinocyte proliferation

Yes-associated protein (YAP) is a mechanosensor protein and a downstream effector of the Hippo kinase pathway, which controls organ growth, cell proliferation, survival, maintenance and regeneration. Unphosphorylated YAP translocates to the nucleus where it acts as a cofactor of primarily the TEAD transcription factors to activate target gene transcription and cell proliferation. Perturbed YAP activation results in tumorigenesis. The pathways downstream of activated YAP that drive cell proliferation remain relatively unexplored. In this study, we employed YAP2-5SA-∆C transgenic mice, which overexpress a mildly activated YAP mutant protein in basal keratinocytes leading to increased proliferation of the epidermal stem/progenitor cell populations. We performed massively-parallel sequencing of skin biopsy mRNA (RNA-Seq) and found dysregulation of 1491 genes in YAP2-5SA-∆C skin, including many with roles in cell activation and proliferation. Furthermore, we found that 150 of these dysregulated genes harbored YAP/TEAD binding motifs in the 3' UTR, suggesting that these may be direct YAP/TEAD target genes in the control of epidermal regeneration. Further validation and functional characterization assays identified Plau and Tgfbr3 as prime candidate genes that may be activated by epidermal YAP activity in the mouse skin in vivo to promote keratinocyte proliferation. This study provides novel insights into the mechanisms regulated by YAP that control tissue homeostasis, and in particular in conditions where YAP is aberrantly activated such as in neoplastic and regenerative skin disease