Genetic Mechanism of Non-Targeted-Site Resistance to Diquat in Spirodela polyrhiza.

Understanding non-target-site resistance (NTSR) to herbicides represents a pressing challenge as NTSR is widespread in many weeds. Using giant duckweed (Spirodela polyrhiza) as a model, we systematically investigated genetic and molecular mechanisms of diquat resistance, which can only be achieved via NTSR. Quantifying the diquat resistance of 138 genotypes, we revealed an 8.5-fold difference in resistance levels between the most resistant and most susceptible genotypes. Further experiments suggested that diquat uptake and antioxidant-related processes jointly contributed to diquat resistance in S. polyrhiza. Using a genome-wide association approach, we identified several candidate genes, including a homolog of dienelactone hydrolase, that are associated with diquat resistance in S. polyrhiza. Together, these results provide new insights into the mechanisms and evolution of NTSR in plants.

Development of a three-colour digital PCR for early and quantitative detection of benzimidazole resistance-associated single nucleotide polymorphisms in Haemonchus contortus.

Haemonchus contortus is the most pathogenic nematode in small ruminants and anthelmintic resistance (AR) hampers its efficient control. Early detection of AR status is required to reduce selection for AR and cannot be achieved using phenotypic tests. For benzimidazoles (BZs), the detection of AR-associated alleles characterised by single nucleotide polymorphisms (SNPs) in the isotype 1 ß-tubulin gene allows early AR detection in strongyles. The F200Y, F167Y, E198A and E198L polymorphisms have been described in BZ-resistant populations with a clear variation in frequencies between regions. A novel digital PCR (dPCR) enables the detection of all of the above-described polymorphisms in H. contortus. Assays were validated using synthetic DNA fragments containing these SNPs. Then, larvae obtained and pooled at farm level from 26 Austrian and 10 Italian sheep farms were analysed. For all assays a detection limit of 15 copies/µl of resistance alleles and a high level of accuracy were demonstrated, allowing to detect allele frequencies of 1% in most samples. In Austrian samples, elevated frequencies of F200Y resistance alleles were detected on all farms. Polymorphisms in codon 167 and codon 198 were identified in H. contortus from Austria for the first time. In Italian samples, the frequency of resistance alleles was still comparatively low, but F200Y resistance alleles were traceable. In conclusion we developed for the first time dPCR assays that target all SNPs of relevance associated with BZ-resistance in H. contortus. Future research on AR development could benefit from an early onset of SNP-based surveillance that would include the developed assays for all SNPs of relevance. Improved surveillance in the long term will include other important, though less pathogenic, nematode genera in the analyses.

Adult alcohol drinking and emotional tone are mediated by neutral sphingomyelinase during development in males.

Alcohol use, abuse, and addiction, and resulting health hazards are highly sex-dependent with unknown mechanisms. Previously, strong links between the SMPD3 gene and its coded protein neutral sphingomyelinase 2 (NSM) and alcohol abuse, emotional behavior, and bone defects were discovered and multiple mechanisms were identified for females. Here we report strong sex-dimorphisms for central, but not for peripheral mechanisms of NSM action in mouse models. Reduced NSM activity resulted in enhanced alcohol consumption in males, but delayed conditioned rewarding effects. It enhanced the acute dopamine response to alcohol, but decreased monoaminergic systems adaptations to chronic alcohol. Reduced NSM activity increased depression- and anxiety-like behavior, but was not involved in alcohol use for the self-management of the emotional state. Constitutively reduced NSM activity impaired structural development in the brain and enhanced lipidomic sensitivity to chronic alcohol. While the central effects were mostly opposite to NSM function in females, similar roles in bone-mediated osteocalcin release and its effects on alcohol drinking and emotional behavior were observed. These findings support the view that the NSM and multiple downstream mechanism may be a source of the sex-differences in alcohol use and emotional behavior.

Estimating global economic well-being with unlit settlements.

It is well established that nighttime radiance, measured from satellites, correlates with economic prosperity across the globe. In developing countries, areas with low levels of detected radiance generally indicate limited development - with unlit areas typically being disregarded. Here we combine satellite nighttime lights and the world settlement footprint for the year 2015 to show that 19% of the total settlement footprint of the planet had no detectable artificial radiance associated with it. The majority of unlit settlement footprints are found in Africa (39%), rising to 65% if we consider only rural settlement areas, along with numerous countries in the Middle East and Asia. Significant areas of unlit settlements are also located in some developed countries. For 49 countries spread across Africa, Asia and the Americas we are able to predict and map the wealth class obtained from ~2,400,000 geo-located households based upon the percent of unlit settlements, with an overall accuracy of 87%.

Neutral sphingomyelinase mediates the co-morbidity trias of alcohol abuse, major depression and bone defects.

Mental disorders are highly comorbid and occur together with physical diseases, which are often considered to arise from separate pathogenic pathways. We observed in alcohol-dependent patients increased serum activity of neutral sphingomyelinase. A genetic association analysis in 456,693 volunteers found associations of haplotypes of SMPD3 coding for NSM-2 (NSM) with alcohol consumption, but also with affective state, and bone mineralisation. Functional analysis in mice showed that NSM controls alcohol consumption, affective behaviour, and their interaction by regulating hippocampal volume, cortical connectivity, and monoaminergic responses. Furthermore, NSM controlled bone-brain communication by enhancing osteocalcin signalling, which can independently supress alcohol consumption and reduce depressive behaviour. Altogether, we identified a single gene source for multiple pathways originating in the brain and bone, which interlink disorders of a mental-physical co-morbidity trias of alcohol abuse-depression/anxiety-bone disorder. Targeting NSM and osteocalcin signalling may, thus, provide a new systems approach in the treatment of a mental-physical co-morbidity trias.

Equine Parvovirus-Hepatitis Screening in Horses and Donkeys with Histopathologic Liver Abnormalities.

There is strong evidence that equine parvovirus-hepatitis (EqPV-H) is associated with the onset of Theiler's disease, an acute hepatic necrosis, in horses. However, the impact of this virus on other hepatopathies remains unknown. The objective of this retrospective study was to evaluate the prevalence and quantify the viral loads of EqPV-H in formalin-fixed, paraffin-embedded equine and donkey livers with various histopathologic abnormalities. The pathologies included cirrhosis, circulatory disorders of the liver, toxic and metabolic hepatic diseases as well as neoplastic and inflammatory diseases (n = 84). Eight normal liver samples were included for comparison as controls. EqPV-H DNA was qualitatively and quantitatively measured by real-time PCR and digital PCR, respectively. The virus was detected in two livers originating from horses diagnosed with abdominal neoplasia and liver metastasis (loads of 5 × 103 and 9.5 × 103 genome equivalents per million cells). The amount of viral nucleic acids measured indicates chronic infection or persistence of EqPV-H, which might have been facilitated by the neoplastic disease. In summary, this study did not provide evidence for EqPV-H being involved in hepatopathies other than Theiler's disease.

Transgenerational non-genetic inheritance has fitness costs and benefits under recurring stress in the clonal duckweed Spirodela polyrhiza.

Although non-genetic inheritance is thought to play an important role in plant ecology and evolution, evidence for adaptive transgenerational plasticity is scarce. Here, we investigated the consequences of copper excess on offspring defences and fitness under recurring stress in the duckweed Spirodela polyrhiza across multiple asexual generations. Growing large monoclonal populations (greater than 10 000 individuals) for 30 generations under copper excess had negative fitness effects after short and no fitness effect after prolonged growth under recurring stress. These time-dependent growth rates were likely influenced by environment-induced transgenerational responses, as propagating plants as single descendants for 2 to 10 generations under copper excess had positive, negative or neutral effects on offspring fitness depending on the interval between initial and recurring stress (5 to 15 generations). Fitness benefits under recurring stress were independent of flavonoid accumulations, which in turn were associated with altered plant copper concentrations. Copper excess modified offspring fitness under recurring stress in a genotype-specific manner, and increasing the interval between initial and recurring stress reversed these genotype-specific fitness effects. Taken together, these data demonstrate time- and genotype-dependent adaptive and non-adaptive transgenerational responses under recurring stress, which suggests that non-genetic inheritance alters the evolutionary trajectory of clonal plant lineages in fluctuating environments.

S100A4 mRNA-protein relationship uncovered by measurement noise reduction.

Intrinsic biological fluctuation and/or measurement error can obscure the association of gene expression patterns between RNA and protein levels. Appropriate normalization of reverse-transcription quantitative PCR (RT-qPCR) data can reduce technical noise in transcript measurement, thus uncovering such relationships. The accuracy of gene expression measurement is often challenged in the context of cancer due to the genetic instability and "splicing weakness" involved. Here, we sequenced the poly(A) cancer transcriptome of canine osteosarcoma using mRNA-Seq. Expressed sequences were resolved at the level of two consecutive exons to enable the design of exon-border spanning RT-qPCR assays and ranked for stability based on the coefficient of variation (CV). Using the same template type for RT-qPCR validation, i.e. poly(A) RNA, avoided skewing of stability assessment by circular RNAs (circRNAs) and/or rRNA deregulation. The strength of the relationship between mRNA expression of the tumour marker S100A4 and its proportion score of quantitative immunohistochemistry (qIHC) was introduced as an experimental readout to fine-tune the normalization choice. Together with the essential logit transformation of qIHC scores, this approach reduced the noise of measurement as demonstrated by uncovering a highly significant, strong association between mRNA and protein expressions of S100A4 (Spearman's coefficient ρ = 0.72 (p = 0.006)). KEY MESSAGES: • RNA-seq identifies stable pairs of consecutive exons in a heterogeneous tumour. • Poly(A) RNA templates for RT-qPCR avoid bias from circRNA and rRNA deregulation. • HNRNPL is stably expressed across various cancer tissues and osteosarcoma. • Logit transformed qIHC score better associates with mRNA amount. • Quantification of minor S100A4 mRNA species requires poly(A) RNA templates and dPCR.

Retinoic acid prevents immunogenicity of milk lipocalin Bos d 5 through binding to its immunodominant T-cell epitope.

The major cow's milk allergen Bos d 5 belongs to the lipocalin protein family, with an intramolecular pocket for hydrophobic ligands. We investigated whether Bos d 5 when loaded with the active vitamin A metabolite retinoic acid (RA), would elicit differential immune responses compared to the unloaded state. By in silico docking an affinity energy of -7.8 kcal/mol was calculated for RA into Bos d 5. Loading of RA to Bos d 5 could be achieved in vitro, as demonstrated by ANS displacement assay, but had no effect on serum IgE binding in tolerant or challenge-positive milk allergic children. Bioinformatic analysis revealed that RA binds to the immunodominant T-cell epitope region of Bos d 5. In accordance, Bos d 5 significantly suppressed the CD3+ CD4+ cell numbers, proliferative response and IL-10, IL-13 and IFN-γ secretion from stimulated human PBMCs only when complexed with RA. This phenomenon was neither associated with apoptosis of T-cells nor with the activation of Foxp3+ T-cells, but correlated likely with enhanced stability to lysosomal digestion due to a predicted overlap of Cathepsin S cleavage sites with the RA binding site. Taken together, proper loading of Bos d 5 with RA may suppress its immunogenicity and prevent its allergenicity.

Stem cell growth factor receptor in canine vs. feline osteosarcomas.

Osteosarcoma is considered the most common bone cancer in cats and dogs, with cats having a much better prognosis than dogs, since the great majority of dogs with osteosarcoma develop distant metastases. In search of a factor possibly contributing to this disparity, the stem cell growth factor receptor KIT was targeted, and the messenger (m)RNA and protein expression levels of KIT were compared in canine vs. feline osteosarcomas, as well as in normal bone. The mRNA expression of KIT was quantified by reverse transcription-quantitative polymerase chain reaction, and was observed to be significantly higher in canine (n=14) than in feline (n=5) osteosarcoma samples (P<0.001). KIT protein expression was evaluated by immunohistochemistry, which revealed that 21% of canine osteosarcoma samples did not exhibit KIT staining in their neoplastic cells, while in 14% of samples, a score of 1 (<10% positive tumour cells) was observed, and in 50% and 14% of samples, a score of 2 (10-50% positivity) and 3 (>50% positivity), respectively, was observed. By contrast, the cancer cells of all the feline bone tumour samples analysed were entirely negative for KIT. Notably, canine and feline osteocytes of healthy bone tissue lacked any KIT expression. These results could be the first evidence that KIT may be involved in the higher aggressiveness of canine osteosarcoma compared with feline osteosarcoma.

Trend of antihyperglycaemic therapy and glycaemic control in 184,864 adults with type 1 or 2 diabetes between 2002 and 2014: Analysis of real-life data from the DPV registry from Germany and Austria.

AIMS: To analyse time trends of antihyperglycaemic therapy and glycaemic control in adult subjects with type 1, or type 2 diabetes between 2002 and 2014 in Germany/Austria. METHODS: 184,864 adults with diabetes (35,144 type 1 diabetes (T1D), 149,720 type 2 diabetes (T2D)) from the DPV-database documented between 2002 and 2014 were included. Regression models were applied for antihyperglycaemic therapy in T2D (non-pharmacological, OADs only, insulin±OADs), insulin therapy in T1D (CT, ICT, CSII) and T2D (BOT, SIT, CT, ICT, CSII), for the use of insulin analogues, and for glycaemic control (HbA1C, severe hypoglycaemia), adjusting for confounders sex, age, and diabetes duration. RESULTS: In T1D, CT (2002:19.7%; 2014:16.0%) and ICT (2002:66.8%; 2014:52.4%) decreased, while CSII increased from 13.5% to 31.5%. In T2D, non-pharmacological treatment became less frequent (2002:36.0%, 2014:21.8%), the use of OADs (2002:19.3%, 2014:28.9%) and insulin±OADs (2002:44.6%, 2014:49.4%) increased. BOT increased from 7.9% to 18.9%, SIT decreased from 12.0% to 8.3%. ICT slightly increased (2002:44.0%, 2014:45.3%), CT decreased (2002:35.8%, 2014:27.2%). Insulin analogues were used more frequently in T1D (rapid-acting:2002:46.8%, 2014:84.8%; long-acting:2002:26.0%, 2014:54.8%) and in T2D (rapid-acting:2002:26.0%, 2014:43.5%; long-acting:2002:13.7%, 2014:53.6%). Until 2011, HbA1C increased in T1D and T2D, but then decreased again. High variability in the rate of hypoglycaemia was observed. CONCLUSIONS: This observational study indicates an increased use of insulin pumps in T1D. In T2D, non-pharmacological therapy decreased, and insulin therapy, particular as BOT, rose. An increase in the use of rapid- and long-acting insulin analogues was present in both patient-groups. Time trend was less clear in glycaemic control.

Allelic variation of the COMT gene in a despotic primate society: A haplotype is related to cortisol excretion in Macaca fuscata.

Sequence variations in genes of the monoamine neurotransmitter system and their common function in human and non-human primate species are an ongoing issue of investigation. However, the COMT gene, coding for the catechol-O-methyltransferase, has not yet attracted much scientific attention regarding its functional role in non-human primates. Considering that a polymorphism of the human COMT gene affects the enzyme activity and cortisol level in response to a social stressor, this study investigated the impact of COMT on endocrine stress and behavioural parameters in Japanese macaques (Macaca fuscata). The species exemplifies a despotic hierarchy in which males' social rank positions require an adaptation of behaviour strategies. During the mating period steroid secretion and the frequency of aggressive encounters between males increase. We addressed i) whether this species exhibits potential functional COMT variants, ii) whether these variants are associated with faecal cortisol excretion of males, iii) how they are distributed among different social rank positions and iv) whether they are associated with behavioural strategies during times of mate competition. By genotyping 26 males we identified three COMT haplotypes (HT), including a putative splice mutant (HT3). This variant was associated with increased cortisol excretion. Given the observed inverse correlation between cortisol and physical aggression, we assume that different COMT haplotypes may predispose individuals to pursue more or less aggressive strategies. How these gene-stress effects might favour a specific social role is discussed. Our study of non-invasive genotyping in combination with behavioural and endocrine parameters represents an important step towards the understanding of gene-stress effects in a hierarchically organised primate society.

Different metabolic activity in placental and reflected regions of the human amniotic membrane.

Cells of the human amniotic membrane (hAM) have stem cell characteristics with low immunogenicity and anti-inflammatory properties. While hAM is an excellent source for tissue engineering, so far, its sub-regions have not been taken into account. We show that placental and reflected hAM differ distinctly in morphology and functional activity, as the placental region has significantly higher mitochondrial activity, however significantly less reactive oxygen species. Since mitochondria may participate in processes such as cell rescue, we speculate that amniotic sub-regions may have different potential for tissue regeneration, which may be crucial for clinical applications.

Glyphosate-based herbicides reduce the activity and reproduction of earthworms and lead to increased soil nutrient concentrations.

Herbicide use is increasing worldwide both in agriculture and private gardens. However, our knowledge of potential side-effects on non-target soil organisms, even on such eminent ones as earthworms, is still very scarce. In a greenhouse experiment, we assessed the impact of the most widely used glyphosate-based herbicide Roundup on two earthworm species with different feeding strategies. We demonstrate, that the surface casting activity of vertically burrowing earthworms (Lumbricus terrestris) almost ceased three weeks after herbicide application, while the activity of soil dwelling earthworms (Aporrectodea caliginosa) was not affected. Reproduction of the soil dwellers was reduced by 56% within three months after herbicide application. Herbicide application led to increased soil concentrations of nitrate by 1592% and phosphate by 127%, pointing to potential risks for nutrient leaching into streams, lakes, or groundwater aquifers. These sizeable herbicide-induced impacts on agroecosystems are particularly worrisome because these herbicides have been globally used for decades.

Putative regulation mechanism for the MSTN gene by a CpG island generated by the SINE marker Ins227bp.

BACKGROUND: A single nucleotide polymorphism (SNP) in the first intron of the myostatin gene (MSTN) is associated with aptness of elite Thoroughbreds to race over sprint, middle or long distances. This intronic marker (g.66493737 T ≻ C), a short interspersed nuclear element (SINE) of 227 bp (Ins227bp) insertion polymorphism in the MSTN promoter, and the adjacent SNP BIEC2-417495 have not been studied for their association with racing aptness of the average Thoroughbreds raced in countries with lower status of the racing industry. This study investigated these markers regarding their prevalence and association with performance in common race horses. Markers were genotyped by amplification refractory mutation system-quantitative PCR (ARMS-qPCR) or amplicon melting. Furthermore, we asked whether the Ins227bp marker might theoretically regulate the expression of myostatin by generating a novel target for DNA methylation or by changing binding sites for transcription factors. Putative sites for DNA methylation or binding of transcription factors were predicted by MethPrimer and by the softwares JASPAR, MatInspector and UniPROBE, respectively. RESULTS: Pairwise linkage disequilibrium between g.66493737 T ≻ C and Ins227bp was high (r (2) = 0.93). A lower linkage was determined for g.66493737 T ≻ C and BIEC2-417495 (r (2) = 0.69) as well as for BIEC2-417495 and Ins227bp (r (2) = 0.76). The estimated frequencies for the presence of Ins227bp (I) indel and the C alleles at g.66493737 T ≻ C and BIEC2-417495 were 0.46, 0.47 and 0.43, respectively. Heterozygotes represented the most abundant genotype at each locus. The best racing distance (BRD) was significantly different between the homozygotes of each SNP (p = 0.01 to 0.03). C allele homozygotes at BIEC2-417495 or g.66493737 T ≻ C, as well as Ins227bp homozygotes earned most money on a mean distance ranging from 1211 to 1230 m. Heterozygotes earned most money on races over 1690 to 1709 m. The BRD for the T/T carriers at both SNP loci and for the SINE-free genotype was 1812 to 1854 m. Other performance parameters were not significantly different between the genotypes, except of the relative success score (RSS). The RSS was significantly slightly better on a distance of ≤ 1300 m for all carriers of the C allele and the Ins227bp compared to homozygous T genotypes and SINE-negative horses (p = 0.037 to 0.046). For distances of more than 1300 m the RSS was not significantly different between genotypes. In silico assessment indicated that the Ins227bp promoter insertion might have generated a CpG island and a few novel putative binding sites for transcription factors. CONCLUSIONS: All three target polymorphisms (Ins227bp, g.66493737 T ≻ C, BIEC2-417495) are suitable markers to assess the ability of non-elite Thoroughbreds to race at short or longer distances. The CpG island generated by Ins227bp may cause training-induced silencing of MSTN expression.

Routes to fiber delivery of ultra-short laser pulses in the 25 fs regime.

Femtosecond laser pulses came of age and found applications in many fields of life-sciences that call for dispersion-managed guiding of very short optical pulses. We investigate the potential for delivering 25-fs, nanojoule pulses from a Ti:Sapphire laser through optical fibers with lengths of up to 2m.

Acquired noncompaction associated with myopathy.

In a 40-year-old woman, hospitalised in May 2001 because of heart failure NYHA IV due to dilated cardiomyopathy, echocardiography had shown a normally sized but poorly contracting left ventricle with normal wall thickness. In October 2005, echocardiography in the same patient revealed a dilated left ventricle with extensive trabeculations and deep intertrabecular recesses in the lateral and posterior wall. The ratio of the noncompacted/compacted myocardium was 2. At clinical neurologic examination myopathy was suspected. Left ventricular hypertrabeculation/noncompaction may be associated with neuromuscular disorders and is not a congenital abnormality in each case.

Asymmetric dimethylarginine is associated with macrovascular disease and total homocysteine in patients with type 2 diabetes.

BACKGROUND: The endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) and total homocysteine (tHcy) are elevated in patients at increased cardiovascular risk. Patients with type 2 diabetes (T2DM) have higher incidence of macrovascular disease than the general population. Recent reports suggest a relationship between tHcy and ADMA. To evaluate the connection between ADMA and tHcy and macrovascular disease, we determined both risk factors in T2DM patients with and without macrovascular disease. SUBJECTS AND METHODS: Plasma concentrations of ADMA and tHcy were cross-sectionally determined in 136 T2DM patients. Fifty-five patients had macrovascular disease defined by history of stroke, myocardial infarction, coronary heart disease or peripheral arterial occlusive disease. Logistic regression analysis was performed to examine the relationship between macrovascular disease and these risk factors. Potential confounders were identified by significant Spearman rank correlation coefficients. RESULTS: In unadjusted models ADMA (per 0.1 micromol/l) and tHcy (per 5 micromol/l) were both significantly related to macrovascular disease (OR=1.63, 95% CI: 1.21-2.19 and OR=1.49, 95% CI: 1.04-2.14). In multivariate models, ADMA was significantly associated with macrovascular disease independent of l-arginine, albumin excretion rate, tHcy and glomerular filtration rate (GFR; OR=1.53, 95% CI: 1.04-2.26). The connection between tHcy and macrovascular disease was not independent of diastolic blood pressure, age, ADMA or GFR. Linear regression analyses revealed that ADMA, GFR and low-density lipoprotein cholesterol were independent predictors for tHcy. CONCLUSION: ADMA is associated with macrovascular disease independent of tHcy and traditional cardiovascular risk factors in patients with T2DM.