Mapping the substrate landscape of protein phosphatase 2A catalytic subunit PPP2CA

Protein phosphatase 2A (PP2A) is an essential Ser/Thr phosphatase. The PP2A holoenzyme complex comprises a scaffolding (A), regulatory (B), and catalytic (C) subunit, with PPP2CA being the principal catalytic subunit. The full scope of PP2A substrates in cells remains to be defined. To address this, we employed dTAG proteolysis-targeting chimeras to efficiently and selectively degrade dTAG-PPP2CA in homozygous knock-in HEK293 cells. Unbiased global phospho-proteomics identified 2,204 proteins with significantly increased phosphorylation upon dTAG-PPP2CA degradation, implicating them as potential PPP2CA substrates. A vast majority of these are novel. Bioinformatic analyses revealed involvement of the potential PPP2CA substrates in spliceosome function, cell cycle, RNA transport, and ubiquitin-mediated proteolysis. We identify a pSP/pTP motif as a predominant target for PPP2CA and confirm some of our phospho-proteomic data with immunoblotting. We provide an in-depth atlas of potential PPP2CA substrates and establish targeted degradation as a robust tool to unveil phosphatase substrates in cells.</p

Mapping the substrate landscape of protein phosphatase 2A catalytic subunit PPP2CA

Protein phosphatase 2A (PP2A) is an essential Ser/Thr phosphatase. The PP2A holoenzyme complex comprises a scaffolding (A), regulatory (B), and catalytic (C) subunit, with PPP2CA being the principal catalytic subunit. The full scope of PP2A substrates in cells remains to be defined. To address this, we employed dTAG proteolysis-targeting chimeras to efficiently and selectively degrade dTAG-PPP2CA in homozygous knock-in HEK293 cells. Unbiased global phospho-proteomics identified 2,204 proteins with significantly increased phosphorylation upon dTAG-PPP2CA degradation, implicating them as potential PPP2CA substrates. A vast majority of these are novel. Bioinformatic analyses revealed involvement of the potential PPP2CA substrates in spliceosome function, cell cycle, RNA transport, and ubiquitin-mediated proteolysis. We identify a pSP/pTP motif as a predominant target for PPP2CA and confirm some of our phospho-proteomic data with immunoblotting. We provide an in-depth atlas of potential PPP2CA substrates and establish targeted degradation as a robust tool to unveil phosphatase substrates in cells.</p

Identifying Farming Strategies Associated With Achieving Global Agricultural Sustainability

Sustainable agroecosystems provide adequate food while supporting environmental and human wellbeing and are a key part of the United Nations Sustainable Development Goals (SDGs). Some strategies to promote sustainability include reducing inputs, substituting conventional crops with genetically modified (GM) alternatives, and using organic production. Here, we leveraged global databases covering 121 countries to determine which farming strategies—the amount of inputs per area (fertilizers, pesticides, and irrigation), GM crops, and percent agriculture in organic production—are most correlated with 12 sustainability metrics recognized by the United Nations Food and Agriculture Organization. Using quantile regression, we found that countries with higher Human Development Indices (HDI) (including education, income, and lifespan), higher-income equality, lower food insecurity, and higher cereal yields had the most organic production and inputs. However, input-intensive strategies were associated with greater agricultural greenhouse gas emissions. In contrast, countries with more GM crops were last on track to meeting the SDG of reduced inequalities. Using a longitudinal analysis spanning 2004–2018, we found that countries were generally decreasing inputs and increasing their share of agriculture in organic production. Also, in disentangling correlation vs. causation, we hypothesize that a country's development is more likely to drive changes in agricultural strategies than vice versa. Altogether, our correlative analyses suggest that countries with greater progress toward the SDGs of no poverty, zero hunger, good health and wellbeing, quality education, decent work, economic growth, and reduced inequalities had the highest production of organic agriculture and, to a lesser extent, intensive use of inputs

Silver–N-heterocyclic carbenes in π–Activation: Synergistic effects between the ligand ring size and the anion

A series of 12 silver­(I)–N-heterocyclic carbene (NHC) complexes were prepared featuring five- (both saturated and unsaturated backbone), six-, and seven-membered ring ligand scaffolds. The N-substituents of the NHCs were diisopropylphenyl in all cases, while the anion was varied between bromide, acetate, and triflate. The complexes were evaluated as catalysts in the spirocyclization of 1-(1H-indol-3-yl)-4-phenylbut-3-yn-2-one to give a spirocyclic indolenine product. To our knowledge, it is the first time that a systematic study has been conducted to examine the effects of both NHC ring size and anion in this type of silver-catalyzed reaction. While the acetate and triflate complexes catalyzed the reaction to 100% conversion, the bromide complexes exhibited a significant ligand/anion effect. Reactions catalyzed by both complexes bearing the five-membered ring NHC ligands and the complex bearing the seven-membered ring NHC ligand stalled after approximately two turnovers. However, the bromide complex bearing the six-membered ring NHC ligand catalyzes the reaction to almost full conversion, similarly to the acetate and triflate complexes. This demonstrates that the NHC ligand ring size can have a dramatic effect in these types of reactions and does not necessarily display a linear correlation

Microbial lineages in sarcoidosis. A metagenomic analysis tailored for low–microbial content samples

RATIONALE: The etiology of sarcoidosis is unknown, but microbial agents are suspected as triggers. OBJECTIVES: We sought to identify bacterial, fungal, or viral lineages in specimens from patients with sarcoidosis enriched relative to control subjects using metagenomic DNA sequencing. Because DNA from environmental contamination contributes disproportionately to samples with low authentic microbial content, we developed improved methods for filtering environmental contamination. METHODS: We analyzed specimens from subjects with sarcoidosis (n = 93), control subjects without sarcoidosis (n = 72), and various environmental controls (n = 150). Sarcoidosis specimens consisted of two independent sets of formalin-fixed, paraffin-embedded lymph node biopsies, BAL, Kveim reagent, and fresh granulomatous spleen from a patient with sarcoidosis. All specimens were analyzed by bacterial 16S and fungal internal transcribed spacer ribosomal RNA gene sequencing. In addition, BAL was analyzed by shotgun sequencing of fractions enriched for viral particles, and Kveim and spleen were subjected to whole-genome shotgun sequencing. MEASUREMENTS AND MAIN RESULTS: In one tissue set, fungi in the Cladosporiaceae family were enriched in sarcoidosis compared with nonsarcoidosis tissues; in the other tissue set, we detected enrichment of several bacterial lineages in sarcoidosis but not Cladosporiaceae. BAL showed limited enrichment of Aspergillus fungi. Several microbial lineages were detected in Kveim and spleen, including Cladosporium. No microbial lineage was enriched in more than one sample type after correction for multiple comparisons. CONCLUSIONS: Metagenomic sequencing revealed enrichment of microbes in single types of sarcoidosis samples but limited concordance across sample types. Statistical analysis accounting for environmental contamination was essential to avoiding false positives

Skeletal carbonate mineralogy of Scottish bryozoans

This paper describes the skeletal carbonate mineralogy of 156 bryozoan species collected from Scotland (sourced both from museum collections and from waters around Scotland) and collated from literature. This collection represents 79% of the species which inhabit Scottish waters and is a greater number and proportion of extant species than any previous regional study. The study is also of significance globally where the data augment the growing database of mineralogical analyses and offers first analyses for 26 genera and four families. Specimens were collated through a combination of field sampling and existing collections and were analysed by X-ray diffraction (XRD) and micro-XRD to determine wt% MgCO3 in calcite and wt% aragonite. Species distribution data and phylogenetic organisation were applied to understand distributional, taxonomic and phylo-mineralogical patterns. Analysis of the skeletal composition of Scottish bryozoans shows that the group is statistically different from neighbouring Arctic fauna but features a range of mineralogy comparable to other temperate regions. As has been previously reported, cyclostomes feature low Mg in calcite and very little aragonite, whereas cheilostomes show much more variability, including bimineralic species. Scotland is a highly variable region, open to biological and environmental influx from all directions, and bryozoans exhibit this in the wide range of within-species mineralogical variability they present. This plasticity in skeletal composition may be driven by a combination of environmentally-induced phenotypic variation, or physiological factors. A flexible response to environment, as manifested in a wide range of skeletal mineralogy within a species, may be one characteristic of successful invasive bryozoans

Reduced Diversity and High Sponge Abundance on a Sedimented Indo-Pacific Reef System: Implications for Future Changes in Environmental Quality

Although coral reef health across the globe is declining as a result of anthropogenic impacts, relatively little is known of how environmental variability influences reef organisms other than corals and fish. Sponges are an important component of coral reef fauna that perform many important functional roles and changes in their abundance and diversity as a result of environmental change has the potential to affect overall reef ecosystem functioning. In this study, we examined patterns of sponge biodiversity and abundance across a range of environments to assess the potential key drivers of differences in benthic community structure. We found that sponge assemblages were significantly different across the study sites, but were dominated by one species Lamellodysidea herbacea (42% of all sponges patches recorded) and that the differential rate of sediment deposition was the most important variable driving differences in abundance patterns. Lamellodysidea herbacea abundance was positively associated with sedimentation rates, while total sponge abundance excluding Lamellodysidea herbacea was negatively associated with rates of sedimentation. Overall variation in sponge assemblage composition was correlated with a number of variables although each variable explained only a small amount of the overall variation. Although sponge abundance remained similar across environments, diversity was negatively affected by sedimentation, with the most sedimented sites being dominated by a single sponge species. Our study shows how some sponge species are able to tolerate high levels of sediment and that any transition of coral reefs to more sedimented states may result in a shift to a low diversity sponge dominated system, which is likely to have subsequent effects on ecosystem functioning. © 2014 Powell et al

Level of agreement between frequently used cardiovascular risk calculators in people living with HIV

Objectives The aim of the study was to describe agreement between the QRISK2, Framingham and Data Collection on Adverse Events of Anti‐HIV Drugs (D:A:D) cardiovascular disease (CVD) risk calculators in a large UK study of people living with HIV (PLWH). Methods PLWH enrolled in the Pharmacokinetic and Clinical Observations in People over Fifty (POPPY) study without a prior CVD event were included in this study. QRISK2, Framingham CVD and the full and reduced D:A:D CVD scores were calculated; participants were stratified into ‘low’ ( 20%) categories for each. Agreement between scores was assessed using weighted kappas and Bland–Altman plots. Results The 730 included participants were predominantly male (636; 87.1%) and of white ethnicity (645; 88.5%), with a median age of 53 [interquartile range (IQR) 49–59] years. The median calculated 10‐year CVD risk was 11.9% (IQR 6.8–18.4%), 8.9% (IQR 4.6–15.0%), 8.5% (IQR 4.8–14.6%) and 6.9% (IQR 4.1–11.1%) when using the Framingham, QRISK2, and full and reduced D:A:D scores, respectively. Agreement between the different scores was generally moderate, with the highest level of agreement being between the Framingham and QRISK2 scores (weighted kappa = 0.65) but with most other kappa coefficients in the 0.50–0.60 range. Conclusions Estimates of predicted 10‐year CVD risk obtained with commonly used CVD risk prediction tools demonstrate, in general, only moderate agreement among PLWH in the UK. While further validation with clinical endpoints is required, our findings suggest that care should be taken when interpreting any score alone

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

Prevalence and architecture of de novo mutations in developmental disorders.

The genomes of individuals with severe, undiagnosed developmental disorders are enriched in damaging de novo mutations (DNMs) in developmentally important genes. Here we have sequenced the exomes of 4,293 families containing individuals with developmental disorders, and meta-analysed these data with data from another 3,287 individuals with similar disorders. We show that the most important factors influencing the diagnostic yield of DNMs are the sex of the affected individual, the relatedness of their parents, whether close relatives are affected and the parental ages. We identified 94 genes enriched in damaging DNMs, including 14 that previously lacked compelling evidence of involvement in developmental disorders. We have also characterized the phenotypic diversity among these disorders. We estimate that 42% of our cohort carry pathogenic DNMs in coding sequences; approximately half of these DNMs disrupt gene function and the remainder result in altered protein function. We estimate that developmental disorders caused by DNMs have an average prevalence of 1 in 213 to 1 in 448 births, depending on parental age. Given current global demographics, this equates to almost 400,000 children born per year