Suppression of Phospholipase Dγs Confers Increased Aluminum Resistance in Arabidopsis thaliana

Aluminum (Al) toxicity is the major stress in acidic soil that comprises about 50% of the world's arable land. The complex molecular mechanisms of Al toxicity have yet to be fully determined. As a barrier to Al entrance, plant cell membranes play essential roles in plant interaction with Al, and lipid composition and membrane integrity change significantly under Al stress. Here, we show that phospholipase Dγs (PLDγs) are induced by Al stress and contribute to Al-induced membrane lipid alterations. RNAi suppression of PLDγ resulted in a decrease in both PLDγ1 and PLDγ2 expression and an increase in Al resistance. Genetic disruption of PLDγ1 also led to an increased tolerance to Al while knockout of PLDγ2 did not. Both RNAi-suppressed and pldγ1-1 mutants displayed better root growth than wild-type under Al stress conditions, and PLDγ1-deficient plants had less accumulation of callose, less oxidative damage, and less lipid peroxidation compared to wild-type plants. Most phospholipids and glycolipids were altered in response to Al treatment of wild-type plants, whereas fewer changes in lipids occurred in response to Al stress in PLDγ mutant lines. Our results suggest that PLDγs play a role in membrane lipid modulation under Al stress and that high activities of PLDγs negatively modulate plant tolerance to Al

Recruiting men from across the socioeconomic spectrum via GP registers and community outreach to a weight management feasibility randomised controlled trial

Background Men, particularly those living in disadvantaged areas, are less likely to participate in weight management programmes than women despite similar levels of excess weight. Little is known about how best to recruit men to weight management interventions. This paper describes patient and public involvement in pre-trial decisions relevant to recruitment and aims to report on recruitment to the subsequent men-only weight management feasibility trial, including the: i) acceptability and feasibility of recruitment; and ii) baseline sample characteristics by recruitment strategy. Methods Men with BMI ≥30 kg/m2 and/or waist circumference ≥ 40 in. were recruited to the feasibility trial via two strategies; community outreach (venue information stands and word of mouth) and GP letters, targeting disadvantaged areas. Recruitment activities (e.g. letters sent, researcher venue hours) were recorded systematically, and baseline characteristics questionnaire data collated. Qualitative interviews (n = 50) were conducted three months post-recruitment. Analyses and reporting followed a complementary mixed methods approach. Results 105 men were recruited within four months (community n = 60, GP letter n = 45). Community outreach took 2.3 recruiter hours per participant and GP letters had an opt-in rate of 10.2% (n = 90/879). More men were interested than could be accommodated. Most participants (60%) lived in more disadvantaged areas. Compared to community outreach, men recruited via GP letters were older (mean = 57 vs 48 years); more likely to report an obesity-related co-morbidity (87% vs 44%); and less educated (no formal qualifications, 32% vs 10%, degree educated 11% vs 41%). Recruitment strategies were acceptable, a sensitive approach and trusting relationships with recruiters valued, and the ‘catchy’ study name drew attention. Conclusions Targeted community outreach and GP letters were acceptable strategies that successfully recruited participants to a men-only weight management feasibility trial. Both strategies engaged men from disadvantaged areas, a typically underserved population. Using two recruitment strategies produced samples with different health risk profiles, which could add value to research where either primary or secondary prevention is of interest. Further work is required to examine how these strategies could be implemented and sustained in practice

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Cell autonomy of DSCAM function in retinal development

Cell adhesion molecules (CAMs) provide identifying cues by which neural architecture is sculpted. The Down Syndrome Cell Adhesion Molecule (DSCAM) is required for many neurodevelopmental processes in different species and also has several potential mechanisms of activity, including homophilic adhesion, homophilic repulsion and heterophilic interactions. In the mouse retina, Dscam is expressed in many, but not all neuronal subtypes. Mutations in Dscam cause the fasciculation of dendrites of neighboring homotypic neurons, indicating a role in self-avoidance among cells of a given type, a disruption of the non-random patterning of their cell bodies, and a decrease in developmental cell death in affected cell populations. In order to address how DSCAM facilitates retinal pattering, we developed a conditional allele of Dscam to use alongside existing Dscam mutant mouse strains. Conditional deletion of Dscam reproduces cell spacing, cell number and dendrite arborization defects. Inducible deletion of Dscam and retinal ganglion cell depletion in Brn3b mutant retinas both indicate that these DSCAM-mediated phenotypes can occur independently. In chimeric retinas, in which wild type and Dscam mutant cells are comingled, Dscam mutant cells entangle adjacent wild type cells of the same type, as if both cells were lacking Dscam, consistent with DSCAM-dependent cell spacing and neurite arborization being mediated through homophilic binding cell-to-cell. Deletion of Dscam in specific cell types causes cell-type-autonomous cell body spacing defects, indicating that DSCAM mediates arborization and spacing by acting within given cell types. We also examine the cell autonomy of DSCAM in laminar stratification and find that laminar disorganization can be caused in a non-cell autonomous fashion. Finally, we find Dscam dosage-dependent defects in developmental cell death and amacrine cell spacing, relevant to the increased cell death and other disorders observed in Down syndrome mouse models and human patients, in which Dscam is present in three copies. ► Conditional allele developed to test DSCAM cell autonomy. ► DSCAM‐mediated processes were genetically separated and are distinct. ► Dscam regulates developmental cell death and spacing in a dose dependent manner. ► DSCAM deficient cells induce mutant phenotype on adjacent homotypic wild type cells. ► DSCAM mediates arborization and spacing by acting within cell types

A novel mouse Dscam mutation inhibits localization and shedding of DSCAM

The differential adhesion hypothesis of development states that patterning of organisms, organs and tissues is mediated in large part by expression of cell adhesion molecules. The cues provided by cell adhesion molecules are also hypothesized to facilitate specific connectivity within the nervous system. In this study we characterize a novel mouse mutation in the gene Dscam (Down Syndrome Cell Adhesion Molecule). Vertebrate DSCAM is required for normal development of the central nervous system and has been best characterized in the visual system. In the visual system DSCAM is required for regulation of cell number, mosaic formation, laminar specificity, and refinement of retinal-tectal projections. We have identified a novel mutation in Dscam that results in a single amino acid substitution, R1018P, in the extracellular domain of the DSCAM protein. Mice homozygous for the R1018P mutation develop a subset of defects observed in Dscam null mice. In vitro analysis identified defects in DSCAM(R1018P) localization to filopodia. We also find that wild type DSCAM protein is constitutively cleaved and shed from transfected cells. This secretion is inhibited by the R1018P mutation. We also characterized a novel splice isoform of Dscam and identified defects in lamination of type 2 and type 6 cone bipolar cells in Dscam mutant mice. The identification and characterization of partial loss of function mutations in genes such as Dscam will be helpful in predicting signs and symptoms that may be observed in human patients with partial loss of DSCAM function

Neurite laminar specificity is preserved in <i>Dscam^3J</i> mutant retina. A

<p>–<b>C</b>, Wild type, <i>Dscam^2J</i> and <i>Dscam^3J</i> retinal sections were stained with antibodies to ChAT, a marker of starburst amacrine cells and bNOS, a marker of bNOS-amacrine cells (A–C). Wild type laminar specificity of bNOS-positive neurites in s1, s3 and s5 is roughly preserved in the <i>Dscam^3J</i>, but not <i>Dscam^2J</i> retina. <b>D</b>–<b>F</b>, Wild type, <i>Dscam^2J</i> and <i>Dscam^3J</i> retinal sections were stained with antibodies to TH, a marker of dopaminergic amacrine cells and melanopsin, a marker of melanopsin-positive retinal ganglion cells. Although extensive fasciculation of neurites is observed, the laminar specificity pattern of cell types is maintained in mutant genotypes. <b>G</b>–<b>H</b>, Wild type, <i>Dscam^2J</i> and <i>Dscam^3J</i> retinal sections were stained with antibodies to ChAT, a marker of starburst amacrine cells and PKCα, a marker of rod bipolar cells (G–H). The gross disorganization of cholinergic amacrine cell neurites observed in the <i>Dscam^2J</i> retina is not observed in the <i>Dscam^3J</i> retina, although limited examples of disorganization do occur (E; arrow). <b>I</b>–<b>K</b>, Wild type, <i>Dscam^2J</i> and <i>Dscam^3J</i> retinal sections were stained with antibodies to ChAT, a marker of starburst amacrine cells and Syt2, a marker of type 2 and type 6 cone bipolar cells (I–K). The wild type laminar specificity pattern of type 2 and type 6 cone bipolar cell axons is disrupted in the <i>Dscam^2J</i> retina (arrowheads). The scale bar in (K) is equivalent to 112.5 µm.</p

Amino Acid substitution R1018P is genetic basis of nm2122. A

<p>, A spontaneous mutation, nm2122, occurred at The Jackson Laboratory and exhibited overt phenotypes, including a large dome shaped head and muscle stiffness, similar to previously characterized <i>Dscam</i> mutants. <b>B</b> and <b>C</b>, nm2122 mutant mice develop enlarged central and lateral ventricles compared to controls (arrows). <b>D</b>, After complementation tests with another <i>Dscam</i> mutant, NM992 (<i>Dscam^2J</i>), failed, the <i>Dscam</i> open reading frame was sequenced and a single nucleotide substitution was found resulting in substitution of proline at 1018 in place of the wild type arginine. The nm2122 mutation will henceforth be referred to as <i>Dscam^3J</i>. <b>E</b>, The <i>Dscam^3J</i> mutation is located in the second fibronectin domain (arrow). <b>F</b>, The mutation destroys a recognition site for the enzyme BstUI, allowing wild type, heterozygous and homozygous mutants to be identified based on PCR of the mutation-containing region followed by subsequent restriction enzyme digest. The wild type allele is digested by BstUI resulting in two bands of close to equivalent size, while the mutant allele remains intact. <b>G</b>, Western blot analysis of wild type, <i>Dscam^2J</i> and <i>Dscam^3J</i>. A polyclonal antibody to the N-terminus of DSCAM recognizes a single band of approximately 220 kD and a slightly smaller band in retinal extracts prepared from wild type mice while a single band of 220 kD was detected in <i>Dscam^3J</i> extracts. No protein product is observed from retina (not shown) or brain extract prepared from <i>Dscam^2J</i> mice. The scale bar in (C) is equivalent to 1 cm.</p

DSCAM is constitutively shed <i>in vitro</i> and the R1018P mutation inhibits this secretion. A

<p>, Antibodies recognizing the extracellular N-terminus of DSCAM (N-DSCAM) or the intracellular C-terminus (C-DSCAM) were used to monitor post-translational DSCAM processing. <b>B</b>, Lane 1; transfected with C-terminus of <i>Dscam</i>, lane 2; untransfected cells, lane 3; transfected with <i>Dscam</i> and lane 4; transfected with myc/his tagged <i>Dscam</i>. The N-terminal DSCAM antibody recognizes a band of approximately 220 kD and a band slightly smaller than 220 kD in lysates of DSCAM-transfected cells. The C-terminal DSCAM antibody recognized a band of approximately 220 kD and a band the same size of the DSCAM C-terminus, at about 45 kD (arrows). <b>C</b>, A band the same size as the smaller of the two bands recognized by the N-terminal DSCAM antibody was detected, and increases in abundance post-transfection, in conditioned media (CM) collected from cells transfected with <i>Dscam</i>, but not in CM from untransfected cells. This band is not detected by the C-terminal DSCAM antibody. <b>D</b>, Production of shed product is dependent on membrane trafficking, as addition of brefeldin A (BFA) inhibits production of shed product. Incubation of cells in brefeldin A (BFA) also inhibits production of the 45 kD band detected by the C-terminal antibody. <b>E</b> and <b>F</b>, Cell lysates and conditioned media from N2A or HeLa cells expressing either DSCAM or DSCAM^R1018P were analyzed. Wild type DSCAM protein was less abundant in cell lysates compared to DSCAM^R1018P. A large decrease in the amount of shed DSCAM^R1018P was observed compared to shed wild type DSCAM. <b>G</b>, The amount of shed DSCAM or DSCAM^R1018P was normalized to the amount of protein observed in cell lysates. <b>H</b>, Models of DSCAM cleavage and secretion. DSCAM^R1018P is cleaved into two products but is not abundantly shed. This indicates that cleavage is not necessarily directly linked to secretion. This could be because cleavage occurs directly at the membrane and is linked to secretion and occurs separately after internalization. Alternately, cleavage could occur after internalization of DSCAM, the N-terminal portion of which is then secreted, and DSCAM^R1018P could be deficient in this subsequent secretion.</p

<i>Dscam^3J</i> mutation reproduces some aspect of <i>Dscam</i> null retina. A

<p>–<b>C</b>, Retinal sections from wild type, <i>Dscam^2J</i> and <i>Dscam^3J</i> mice were stained with hematoxylin and eosin. In the wild type retina (A) the three cellular layers are neatly stacked and the synapse containing plexiform layers do not intrude within the cellular layers. <b>B</b>, Cell number is increased in the <i>Dscam^2J</i> mutant retina, with ectopic cells located in the inner plexiform layer, which projects into the inner nuclear layer. <b>C</b>, The <i>Dscam^3J</i> retina is hypercellular; however, cellular lamination is more neatly organized compared to the <i>Dscam^2J</i> retina. <b>D</b>–<b>F</b>, Retinal ganglion cell spacing and arborization is disrupted in both the <i>Dscam^2J</i> and <i>Dscam^3J</i> retina compared to wild type. <b>G</b>–<b>I</b>, Amacrine cell spacing and arborization is disrupted in the <i>Dscam^2J</i> mutant retina but this degree of disruption is not observed in the <i>Dscam^3J</i> mutant retina (<b>I</b>). <b>J</b>, Occasional loose fasciculation of <i>Dscam^3J</i> dopaminergic cell neurites was observed (arrows). <b>K</b> and <b>L</b>, Wild type and <i>Dscam^3J</i> retinas were stained with antibodies to bNOS, to detect bNOS-positive amacrine cells. The number of bNOS positive amacrine cells is increased in the <i>Dscam^3J</i> retina. The scale bar in (A-C) is equivalent to 132 µm. The scale bar in (I) is equivalent to 320 µm in D-I and K and L, and 100 µm in J.</p