Assessment of selected pharmaceuticals in Riyadh wastewater treatment plants, Saudi Arabia: Mass loadings, seasonal variations, removal efficiency and environmental risk.

Despite increasing interest in pharmaceutical emissions worldwide, studies of environmental contamination with pharmaceuticals arising from wastewater discharges in Saudi Arabia are scarce. Therefore, this study examined occurrence, mass loads and removal efficiency for 15 pharmaceuticals and one metabolite (oxypurinol) from different therapeutic classes in three wastewater treatment plants (WWTPs), in Riyadh city in Saudi Arabia. A total of 144 samples were collected from the influents and effluents between March 2018 and July 2019 and analyzed using Solid Phase Extraction followed by triple quadrupole LC-MS/MS. The average concentrations in the influents and effluents were generally higher than their corresponding concentrations found either in previous Saudi Arabian or global studies. The four most dominant compounds in the influent were acetaminophen, ciprofloxacin, caffeine, and diclofenac, with caffeine and acetaminophen having the highest concentrations ranging between 943 and 2282 µg/L. Metformin and ciprofloxacin were the most frequently detected compounds in the effluents at concentrations as high as 33.2 µg/L. Ciprofloxacin had the highest mass load in the effluents of all three WWTPs, ranging between 0.20 and 20.7 mg/day/1000 inhabitants for different WWTPs. The overall average removal efficiency was estimated high (≥80), with no significant different (p > 0.05) between the treatment technology applied. Acetaminophen and caffeine were almost completely eliminated in all three WWTPs. The samples collected in the cold season generally had higher levels of detected compounds than those from the warm seasons, particularly for NSAID and antibiotic compounds. The estimated environmental risk from pharmaceutical compounds in the studied effluents was mostly low, except for antibiotic compounds. Thus, antibiotics should be considered for future monitoring programmes of the aquatic environment in Saudi Arabia.

Ectopic expression of T in the paraxial mesoderm disrupts somite maturation in the mouse.

T is the founding member of the T-box family of transcription factors; family members are critical for cell fate decisions and tissue morphogenesis throughout the animal kingdom. T is expressed in the primitive streak and notochord with mouse mutant studies revealing its critical role in mesoderm formation in the primitive streak and notochord integrity. We previously demonstrated that misexpression of Tbx6 in the paraxial and lateral plate mesoderm results in embryos resembling Tbx15 and Tbx18 nulls. This, together with results from in vitro transcriptional assays, suggested that ectopically expressed Tbx6 can compete with endogenously expressed Tbx15 and Tbx18 at the binding sites of target genes. Since T-box proteins share a similar DNA binding domain, we hypothesized that misexpressing T in the paraxial and lateral plate mesoderm would also interfere with the endogenous Tbx15 and Tbx18, causing embryonic phenotypes resembling those seen upon Tbx6 expression in the somites and limbs. Interestingly, ectopic T expression led to distinct embryonic phenotypes, specifically, reduced-sized somites in embryos expressing the highest levels of T, which ultimately affects axis length and neural tube morphogenesis. We further demonstrate that ectopic T leads to ectopic expression of Tbx6 and Mesogenin 1, known targets of T. These results suggests that ectopic T expression contributes to the phenotype by activating its own targets rather than via a straight competition with endogenous T-box factors.

A Multiple-Trait Bayesian Variable Selection Regression Method for Integrating Phenotypic Causal Networks in Genome-Wide Association Studies.

Bayesian regression methods that incorporate different mixture priors for marker effects are used in multi-trait genomic prediction. These methods can also be extended to genome-wide association studies (GWAS). In multiple-trait GWAS, incorporating the underlying causal structures among traits is essential for comprehensively understanding the relationship between genotypes and traits of interest. Therefore, we develop a GWAS methodology, SEM-Bayesian alphabet, which, by applying the structural equation model (SEM), can be used to incorporate causal structures into multi-trait Bayesian regression methods. SEM-Bayesian alphabet provides a more comprehensive understanding of the genotype-phenotype mapping than multi-trait GWAS by performing GWAS based on indirect, direct and overall marker effects. The superior performance of SEM-Bayesian alphabet was demonstrated by comparing its GWAS results with other similar multi-trait GWAS methods on real and simulated data. The software tool JWAS offers open-source routines to perform these analyses.

Functionally distinct roles for T and Tbx6 during mouse development.

The mouse T-box transcription factors T and Tbx6 are co-expressed in the primitive streak and have unique domains of expression; T is expressed in the notochord, while Tbx6 is expressed in the presomitic mesoderm. T-box factors are related through a shared DNA binding domain, the T-domain, and can therefore bind to similar DNA sequences at least in vitro We investigated the functional similarities and differences of T and Tbx6 DNA binding and transcriptional activity in vitro and their interaction genetically in vivo We show that at one target, Dll1, the T-domains of T and Tbx6 have different affinities for the binding sites present in the mesoderm enhancer. We further show using in vitro assays that T and Tbx6 differentially affect transcription with Tbx6 activating expression tenfold higher than T, that T and Tbx6 can compete at target gene enhancers, and that this competition requires a functional DNA binding domain. Next, we addressed whether T and Tbx6 can compete in vivo First, we generated embryos that express Tbx6 at greater than wild-type levels embryos and show that these embryos have short tails, resembling the T heterozygous phenotype. Next, using the dominant-negative TWis allele, we show that Tbx6+/- TWis/+ embryos share similarities with embryos homozygous for the Tbx6 hypomorphic allele rib-vertebrae, specifically fusions of several ribs and malformation of some vertebrae. Finally, we tested whether Tbx6 can functionally replace T using a knockin approach, which resulted in severe T null-like phenotypes in chimeric embryos generated with ES cells heterozygous for a Tbx6 knockin at the T locus. Altogether, our results of differences in affinity for DNA binding sites and transcriptional activity for T and Tbx6 provide a potential mechanism for the failure of Tbx6 to functionally replace T and possible competition phenotypes in vivo.

Capacity challenges in water quality monitoring: understanding the role of human development.

Monitoring the qualitative status of freshwaters is an important goal of the international community, as stated in the Sustainable Development Goal (SDGs) indicator 6.3.2 on good ambient water quality. Monitoring data are, however, lacking in many countries, allegedly because of capacity challenges of less-developed countries. So far, however, the relationship between human development and capacity challenges for water quality monitoring have not been analysed systematically. This hinders the implementation of fine-tuned capacity development programmes for water quality monitoring. Against this background, this study takes a global perspective in analysing the link between human development and the capacity challenges countries face in their national water quality monitoring programmes. The analysis is based on the latest data on the human development index and an international online survey amongst experts from science and practice. Results provide evidence of a negative relationship between human development and the capacity challenges to meet SDG 6.3.2 monitoring requirements. This negative relationship increases along the course of the monitoring process, from defining the enabling environment, choosing parameters for the collection of field data, to the analytics and analysis of five commonly used parameters (DO, EC, pH, TP and TN). Our assessment can be used to help practitioners improve technical capacity development activities and to identify and target investment in capacity development for monitoring.

Applying citizen science to monitor for the Sustainable Development Goal Indicator 6.3.2: a review.

The United Nations has called for increased public participation in scientific research, to benefit professionals, the public and the planet. Citizen science has been suggested as a cost-effective means by which this call can be met, and by which monitoring for the Sustainable Development Goals (SDGs) may be carried out. Indeed, citizen science has gained significant attention in recent years as the scale of environmental issues surpasses the monitoring resources that currently exist. However, many challenges continue to act as a barrier to the acceptance of citizen science as a reliable scientific approach. Here, the current state of knowledge on the use of citizen science in water quality monitoring is reviewed, and the potential for utilizing this approach to enhance monitoring for SDG Indicator 6.3.2 on the "proportion of bodies of water of good ambient water quality" is evaluated. The objective of this review is to identify key knowledge gaps and hurdles hindering the adoption of citizen science contributions to water quality monitoring under the SDGs, so that these gaps may be addressed in a timely manner for future monitoring programmes.

Water Quality Assessment: A Quali-Quantitative Method for Evaluation of Environmental Pressures Potentially Impacting on Groundwater, Developed under the M.I.N.O.Re. Project.

Background: At global level, the vulnerability of aquifers is deteriorating at an alarming rate due to environmental pollution and intensive human activities. In this context, Local Health Authority ASL Lecce has launched the M.I.N.O.Re. (Not Compulsory Water Monitoring Activities at Regional level) project, in order to assess the vulnerability of the aquifer in Salento area (Puglia Region) by performing several non-compulsory analyses on groundwater samples. This first paper describes the quali-quantitative approach adopted under the M.I.N.O.Re. project for the assessment of environmental pressures suffered by groundwater and determines the number of wells to be monitored in specific sampling areas on the basis of the local potential contamination and vulnerability of the aquifer. Methods: We created a map of the entire Lecce province, interpolating it with a grid that led to the subdivision of the study area in 32 quadrangular blocks measuring 10 km × 10 km. Based on current hydrogeological knowledge and institutional data, we used GIS techniques to represent on these 32 blocks the 12 different layers corresponding to the main anthropic or environmental type of pressures potentially impacting on the aquifer. To each kind of pressure, a score from 0 to 1 was attributed on the basis of the potential impact on groundwater. A total score was assigned to each of the 32 blocks. A higher number of wells was selected to be monitored in those blocks presenting higher risk scores for possible groundwater contamination due to anthropic/environmental pressures. Results: The range of total scores varied from 2.4 to 42.5. On the basis of total scores, the 10 km × 10 km blocks were divided into four classes of environmental pressure (1st class: from 0,1 to 10,00; 2nd class: from 10,01 to 20,00; 3rd class: from 20,1 to 30,00; 4th class: from 30,01 to 42,50). There were 11 areas in the 1st class, 9 areas in the 2nd class, 8 areas in the 3rd class and 4 areas in the 4th class. We assigned 1 monitoring well in 1st class areas, 2 monitoring wells in 2nd class areas, 3 monitoring wells in 3rd class areas and 4 monitoring wells in 4th class areas. Conclusion: The methodology developed under the M.I.N.O.Re. project could represent a useful model to be used in other areas to assess the environmental pressures suffered by aquifers and the quality of the groundwater.

Validating citizen science monitoring of ambient water quality for the United Nations sustainable development goals.

Citizen science (CS) may be described as research carried out by members of the public with the aim of gathering scientific information for the purpose of aiding in scientific projects. It has many potential advantages, including data collection at a scale not possible by professional scientists alone. The United Nations (UN) has recently recognized citizen science as a potential source of data that may contribute to the UN Sustainable Development Goals (SDGs). The availability of relatively inexpensive water quality monitoring field equipment suitable for CS suggests great potential for increased spatial coverage far beyond that of traditional, laboratory-based monitoring networks for water quality. In support of work towards the achievement of Sustainable Development Goal 6: "Clean Water and Sanitation", this study tested the use of such field equipment by citizen scientists for SDG Indicator 6.3.2: "Proportion of bodies of water with good ambient water quality". Data generated by 26 citizen scientists were compared with the results produced by an accredited laboratory. The results compared well for most parameters, suggesting that citizen science may be able to contribute towards monitoring ambient water quality for the Sustainable Development Goals.

Impaired intermediate formation in mouse embryos expressing reduced levels of Tbx6.

Intermediate mesoderm (IM) is the strip of tissue lying between the paraxial mesoderm (PAM) and the lateral plate mesoderm that gives rise to the kidneys and gonads. Chick fate mapping studies suggest that IM is specified shortly after cells leave the primitive streak and that these cells do not require external signals to express IM-specific genes. Surgical manipulations of the chick embryo, however, revealed that PAM-specific signals are required for IM differentiation into pronephros-the first kidney. Here, we use a genetic approach in mice to examine the dependency of IM on proper PAM formation. In Tbx6 null mutant embryos, which form 7-9 improperly patterned anterior somites, IM formation is severely compromised, while in Tbx6 hypomorphic embryos, where somites form but are improperly patterned along the axis, the impact to IM formation is lessened. These results suggest that IM and its derivatives, the kidneys and the gonads, are directly or indirectly dependent on proper PAM formation. This has implications for humans harboring Tbx6 mutations which are known to have somite-derived defects including congenital scoliosis.

TET-mediated DNA demethylation controls gastrulation by regulating Lefty-Nodal signalling.

Mammalian genomes undergo epigenetic modifications, including cytosine methylation by DNA methyltransferases (DNMTs). Oxidation of 5-methylcytosine by the Ten-eleven translocation (TET) family of dioxygenases can lead to demethylation. Although cytosine methylation has key roles in several processes such as genomic imprinting and X-chromosome inactivation, the functional significance of cytosine methylation and demethylation in mouse embryogenesis remains to be fully determined. Here we show that inactivation of all three Tet genes in mice leads to gastrulation phenotypes, including primitive streak patterning defects in association with impaired maturation of axial mesoderm and failed specification of paraxial mesoderm, mimicking phenotypes in embryos with gain-of-function Nodal signalling. Introduction of a single mutant allele of Nodal in the Tet mutant background partially restored patterning, suggesting that hyperactive Nodal signalling contributes to the gastrulation failure of Tet mutants. Increased Nodal signalling is probably due to diminished expression of the Lefty1 and Lefty2 genes, which encode inhibitors of Nodal signalling. Moreover, reduction in Lefty gene expression is linked to elevated DNA methylation, as both Lefty-Nodal signalling and normal morphogenesis are largely restored in Tet-deficient embryos when the Dnmt3a and Dnmt3b genes are disrupted. Additionally, a point mutation in Tet that specifically abolishes the dioxygenase activity causes similar morphological and molecular abnormalities as the null mutation. Taken together, our results show that TET-mediated oxidation of 5-methylcytosine modulates Lefty-Nodal signalling by promoting demethylation in opposition to methylation by DNMT3A and DNMT3B. These findings reveal a fundamental epigenetic mechanism featuring dynamic DNA methylation and demethylation crucial to regulation of key signalling pathways in early body plan formation.

Autosomal dominant spondylocostal dysostosis is caused by mutation in TBX6.

In humans, congenital spinal defects occur with an incidence of 0.5-1 per 1000 live births. One of the most severe syndromes with such defects is spondylocostal dysostosis (SCD). Over the past decade, the genetic basis of several forms of autosomal recessive SCD cases has been solved with the identification of four causative genes (DLL3, MESP2, LFNG and HES7). Autosomal dominant forms of SCD have also been reported, but to date no genetic etiology has been described for these. Here, we have used exome capture and next-generation sequencing to identify a stoploss mutation in TBX6 that segregates with disease in two generations of one family. We show that this mutation has a deleterious effect on the transcriptional activation activity of the TBX6 protein, likely due to haploinsufficiency. In mouse, Tbx6 is essential for the patterning of the vertebral precursor tissues, somites; thus, mutation of TBX6 is likely to be causative of SCD in this family. This is the first identification of the genetic cause of an autosomal dominant form of SCD, and also demonstrates the potential of exome sequencing to identify genetic causes of dominant diseases even in small families with few affected individuals.

Microbiological assessment of private drinking water supplies in Co. Cork, Ireland.

The microbiological quality of 75 private drinking water supply boreholes in Co. Cork, Ireland was assessed in order to determine the incidence of contamination and the potential pathways of such contamination. Microbiological analysis was carried out using the membrane filtration technique for the recovery of thermotolerant (faecal) coliforms. The sanitary protection of the supplies was evaluated by means of systematic inspections and subsequent qualitative sanitary risk assessment. Almost a quarter of all supplies investigated (24%, n = 18) was found positive for thermotolerant coliforms. Weather conditions had a significant impact on microbiological water quality, increasing both contamination incidence and gross contamination frequency. Over half of the supplies had nine or more sanitary hazards and most had rudimentary sanitary protection measures at the head of the borehole. These low sanitary protection measures suggest that boreholes can pose a significant hazard to valuable groundwater resources by providing direct contamination routes.

The Wnt3a/ß-catenin target gene Mesogenin1 controls the segmentation clock by activating a Notch signalling program.

Segmentation is an organizing principle of body plans. The segmentation clock, a molecular oscillator best illustrated by the cyclic expression of Notch signalling genes, controls the periodic cleavage of somites from unsegmented presomitic mesoderm during vertebrate segmentation. Wnt3a controls the spatiotemporal expression of cyclic Notch genes; however, the underlying mechanisms remain obscure. Here we show by transcriptional profiling of Wnt3a (-/-) embryos that the bHLH transcription factor, Mesogenin1 (Msgn1), is a direct target gene of Wnt3a. To identify Msgn1 targets, we conducted genome-wide studies of Msgn1 activity in embryonic stem cells. We show that Msgn1 is a major transcriptional activator of a Notch signalling program and synergizes with Notch to trigger clock gene expression. Msgn1 also indirectly regulates cyclic genes in the Fgf and Wnt pathways. Thus, Msgn1 is a central component of a transcriptional cascade that translates a spatial Wnt3a gradient into a temporal pattern of clock gene expression.

Tbx18 and Tbx15 null-like phenotypes in mouse embryos expressing Tbx6 in somitic and lateral plate mesoderm.

Members of the T-box family of transcription factors play essential roles in cell type specification, differentiation, and proliferation during embryonic development. All T-box family members share a common DNA binding domain - the T-domain - and can therefore recognize similar sequences. Consequently, T-box proteins that are co-expressed during development have the potential to compete for binding at downstream targets. In the mouse, Tbx6 is expressed in the primitive streak and presomitic mesoderm, and is sharply down-regulated upon segmentation of the paraxial mesoderm. We sought to determine the phenotypic and molecular consequences of ectopically expressing Tbx6 within the segmented paraxial mesoderm and its derivatives using a 3-component transgenic system. The vertebral column, ribs, and appendicular skeleton were all affected in these embryos, which resembled Tbx18 and Tbx15 null embryos. We hypothesize that these phenotypes result from competition between the ectopically expressed Tbx6 and the endogenously expressed Tbx18 and Tbx15 at the binding sites of target genes. In vitro luciferase transcriptional assays provide further support for this hypothesis.

Kinked tail mutation results in notochord defects in heterozygotes and distal visceral endoderm defects in homozygotes.

Proper formation of the anterior-posterior (AP) axis in the developing embryo is critical for the correct patterning and often survival of the organism. In the mouse, an initial step in axis establishment is the specification and migration of the distal visceral endoderm (DVE). We have identified a semi-dominant spontaneous mutation in mouse, named kinked tail (knk), which when heterozygous results in a kinky tail phenotype due to fusions and dysmorphology of the tail vertebrae. Vertebral fusions appear to be a secondary effect of notochord thickening and branching in the tail region. Homozygosity for knk results in early embryonic lethality by embryonic day 8.5 due to improper timing of DVE specification and migration, and subsequent failure to establish the AP axis.

Generation of transgenic mice expressing Cre recombinase under the control of the Dll1 mesoderm enhancer element.

To study paraxial mesoderm formation in the mouse, transgenic lines that can be used to either selectively delete or express genes of interest in the paraxial mesoderm are required. We have generated a transgenic mouse line that expresses Cre recombinase in the paraxial mesoderm (PAM) beginning at e7.5. A lacZ Cre recombinase reporter line showed that in addition to PAM and its derivatives, lateral plate and intermediate mesoderm derivatives were also exposed to Cre activity, while the node, notochord, and cardiac mesoderm were not. We further demonstrate that 70-75% of the fibroblasts generated from Dll1-msd Cre, ROSA26-rtTA embryos possess Cre recombinase activity. These mice can therefore be used in combination with tet-responsive transgenic lines to generate mesoderm-derived embryonic fibroblasts that inducibly express a gene of interest.

Mesp2 and Tbx6 cooperatively create periodic patterns coupled with the clock machinery during mouse somitogenesis.

The metameric structures in vertebrates are based on the periodicity of the somites that are formed one by one from the anterior end of the presomitic mesoderm (PSM). The timing and spacing of somitogenesis are regulated by the segmentation clock, which is characterized by the oscillation of several signaling pathways in mice. The temporal information needs to be translated into a spatial pattern in the so-called determination front, at which cells become responsive to the clock signal. The transcription factor Mesp2 plays a crucial role in this process, regulating segmental border formation and rostro-caudal patterning. However, the mechanisms regulating the spatially restricted and periodic expression of Mesp2 have remained elusive. Using high-resolution fluorescent in situ hybridization in conjunction with immunohistochemical analyses, we have found a clear link between Mesp2 transcription and the periodic waves of Notch activity. We also find that Mesp2 transcription is spatially defined by Tbx6: Mesp2 transcription and Tbx6 protein initially share an identical anterior border in the PSM, but once translated, Mesp2 protein leads to the suppression of Tbx6 protein expression post-translationally via rapid degradation mediated by the ubiquitin-proteasome pathway. This reciprocal regulation is the spatial mechanism that successively defines the position of the next anterior border of Mesp2. We further show that FGF signaling provides a spatial cue to position the expression domain of Mesp2. Taken together, we conclude that Mesp2 is the final output signal by which the temporal information from the segmentation clock is translated into segmental patterning during mouse somitogenesis.

Clinical laboratory specimen rejection--association with the site of patient care and patients' characteristics: findings from a single health care organization.

CONTEXT: Programs to track laboratory quality have reported aggregated specimen rejection rates ranging from 0.30% to 0.83%. Because the performance of the laboratory, rather than errors, has been the focus, reasons for specimen rejection or demographic characteristics of individuals at risk for specimens of poor quality may not be fully understood. OBJECTIVE: To calculate the proportions of rejected specimens stratified by point of collections and demographic information of patients. DESIGN: Retrospective cross-sectional study. Data were retrieved from the intrainstitutional electronic databases. RESULTS: The proportions of specimens that were rejected in the emergency department and inpatient services were 2-fold and more than 5-fold higher, respectively, than for the outpatient services. Assessment of data by patients' ethnic heritages yielded no significant differences among African Americans (0.38%), Caucasians (0.38%), or "Others" (0.35%) in the outpatient services (P = .07). In the emergency department, the proportions of rejected specimens for African Americans (2.24%) were almost twice that of Caucasians (1.39%) and 30% higher than for Others (1.70%). A similar finding was observed for the inpatient services. CONCLUSIONS: The effect of ethnicity on the proportions of rejected specimens was significant for samples that were collected in the emergency department and inpatient services, even after adjusting for the total number of specimens. A constellation of factors, that is, disease severity and seriousness, practice of blood sample collection, and lesser proficiency of the nursing staff in phlebotomy may be reasons for this observation. However, the likelihood of differential care, although unlikely, cannot be refuted by the present data.

3-[Substituted]-5-(5-pyridin-2-yl-2H-tetrazol-2-yl)benzonitriles: identification of highly potent and selective metabotropic glutamate subtype 5 receptor antagonists.

Structure-activity relationship studies on the phenyl ring of 3-(5-pyridin-2-yl-2H-tetrazol-2-yl)benzonitrile 2 led to the discovery that small, non-hydrogen bond donor substituents at the 3-position led to a substantial increase in in vitro potency. In particular, 3-fluoro-5-(5-pyridin-2-yl-2H-tetrazol-2-yl)benzonitrile (7) is a highly potent and selective mGlu5 receptor antagonist with good rat pharmacokinetics, brain penetration, and in vivo receptor occupancy.