Gonadal Hormone Substitution in People with Prader-Labhart-Willi Syndrome: An International Prader-Willi Syndrome Organisation Survey.

BACKGROUND: Prader-Labhart-Willi syndrome (PWS) is a rare genetic disorder characterized by intellectual disability, behavioural problems, hypothalamic dysfunction, and specific dysmorphisms. Hypothalamic dysfunction causes growth hormone deficiency, dysregulation of energy balance, and hypogonadism. Although hypogonadism is prevalent in PWS, there are no clear guidelines for diagnosis and treatment. In particular, gonadal hormone substitution is a matter of debate due to concerns associated with the potentially induced aggressive behaviour, foremost in males, by sex steroids. METHODS: In 2019, a workshop dedicated to hypogonadism was held prior to the 10th International PWS Organization Conference. In this context, we designed a questionnaire to assess "the current standard of care" of hypogonadism in children and adults with PWS, which was sent out to physicians caring for people with PWS worldwide. RESULTS: Responses were received from a total of 24 centres located in 19 countries. Participating centres treat a total number of at least 1,000 children and adults with PWS. Responses showed limited consensus on who should be treated or at what age treatment should commence. Remarkably, very few behavioural problems were attributed to hormone substitution. CONCLUSION: Based on our findings, we make recommendations to progress the knowledge on hypogonadism in PWS and improve daily practice.

Agrobacterium tumefaciens Small Lipoprotein Atu8019 Is Involved in Selective Outer Membrane Vesicle (OMV) Docking to Bacterial Cells.

Outer membrane vesicles (OMVs), released from Gram-negative bacteria, have been attributed to intra- and interspecies communication and pathogenicity in diverse bacteria. OMVs carry various components including genetic material, toxins, signaling molecules, or proteins. Although the molecular mechanism(s) of cargo delivery is not fully understood, recent studies showed that transfer of the OMV content to surrounding cells is mediated by selective interactions. Here, we show that the phytopathogen Agrobacterium tumefaciens, the causative agent of crown gall disease, releases OMVs, which attach to the cell surface of various Gram-negative bacteria. The OMVs contain the conserved small lipoprotein Atu8019. An atu8019-deletion mutant produced wildtype-like amounts of OMVs with a subtle but reproducible reduction in cell-attachment. Otherwise, loss of atu8019 did not alter growth, susceptibility against cations or antibiotics, attachment to plant cells, virulence, motility, or biofilm formation. In contrast, overproduction of Atu8019 in A. tumefaciens triggered cell aggregation and biofilm formation. Localization studies revealed that Atu8019 is surface exposed in Agrobacterium cells and in OMVs supporting a role in cell adhesion. Purified Atu8019 protein reconstituted into liposomes interacted with model membranes and with the surface of several Gram-negative bacteria. Collectively, our data suggest that the small lipoprotein Atu8019 is involved in OMV docking to specific bacteria.

Virulence of Agrobacterium tumefaciens requires lipid homeostasis mediated by the lysyl-phosphatidylglycerol hydrolase AcvB.

Agrobacterium tumefaciens transfers oncogenic T-DNA via the type IV secretion system (T4SS) into plants causing tumor formation. The acvB gene encodes a virulence factor of unknown function required for plant transformation. Here we specify AcvB as a periplasmic lysyl-phosphatidylglycerol (L-PG) hydrolase, which modulates L-PG homeostasis. Through functional characterization of recombinant AcvB variants, we showed that the C-terminal domain of AcvB (residues 232-456) is sufficient for full enzymatic activity and defined key residues for catalysis. Absence of the hydrolase resulted in ~10-fold increase in L-PG in Agrobacterium membranes and abolished T-DNA transfer and tumor formation. Overproduction of the L-PG synthase gene (lpiA) in wild-type A. tumefaciens resulted in a similar increase in the L-PG content (~7-fold) and a virulence defect even in the presence of intact AcvB. These results suggest that elevated L-PG amounts (either by overproduction of the synthase or absence of the hydrolase) are responsible for the virulence phenotype. Gradually increasing the L-PG content by complementation with different acvB variants revealed that cellular L-PG levels above 3% of total phospholipids interfere with T-DNA transfer. Cumulatively, this study identified AcvB as a novel virulence factor required for membrane lipid homeostasis and T-DNA transfer.

Less physically active children are shorter.

BACKGROUND: Current concepts of linear growth include genetic, endocrine and nutritional factors. Limited and controversial results exist regarding the effect of physical activity (PA) on linear growth. In 2009, we showed that PA promotes foot length in hypoactive children with Prader-Willi-Syndrome. In the present study we tested the hypothesis that PA related epiphyseal loading has a positive modulating effect on linear growth in healthy school children. METHODS: In 99 healthy schoolchildren, we measured height, foot length and PA by tri-axial accelerometry. PA related epiphyseal loading was expressed as the product between mass (body weight) and acceleration (vector magnitude). Correlation between height, foot length and PA were calculated taking into account co-variables age, sex, parental height, lean and fat mass measured by DEXA. RESULTS: Height SDS (P<0.015, r=0.245) as well as foot length SDS (P<0.001, r=0.363) correlated with PA. Multiple linear regression models showed that muscle mass expressed by lean body mass has higher correlation with PA, height SDS and foot length SDS than fat mass. CONCLUSIONS: This study shows that physically less active children are shorter and have shorter feet. In analogy to the "muscle bone unit", we propose a "muscle epiphyseal unit" which regulates local bone growth as long as epiphyseal plates are still open.

Should viral load thresholds be lowered?: Revisiting the WHO definition for virologic failure in patients on antiretroviral therapy in resource-limited settings.

The World Health Organization (WHO) guidelines on antiretroviral therapy (ART) define treatment failure as 2 consecutive viral loads (VLs) ≥1000 copies/mL. There is, however, little evidence supporting 1000 copies as an optimal threshold to define treatment failure. Objective of this study was to assess the correlation of the WHO definition with the presence of drug-resistance mutations in patients who present with 2 consecutive unsuppressed VL in a resource-limited setting.In 10 nurse-led clinics in rural Lesotho children and adults on first-line ART for ≥6 months received a first routine VL. Those with plasma VL ≥80 copies/mL were enrolled in a prospective study, receiving enhanced adherence counseling (EAC) and a follow-up VL after 3 months. After a second unsuppressed VL genotypic resistance testing was performed. Viruses with major mutations against ≥2 drugs of the current regimen were classified as "resistant".A total of 1563 adults and 191 children received a first routine VL. Of the 138 adults and 53 children with unsuppressed VL (≥80 copies/mL), 165 (116 adults; 49 children) had a follow-up VL after EAC; 108 (74 adults; 34 children) remained unsuppressed and resistance testing was successful. Ninety of them fulfilled the WHO definition of treatment failure (both VL ≥1000 copies/mL); for another 18 both VL were unsuppressed but with <1000 copies/mL. The positive predictive value (PPV) for the WHO failure definition was 81.1% (73/90) for the presence of resistant virus. Among the 18 with VL levels between 80 and 1000 copies/mL, thereby classified as "non-failures", 17 (94.4%) harbored resistant viruses. Lowering the VL threshold from 1000 copies/mL to 80 copies/mL at both determinations had no negative influence on the PPV (83.3%; 90/108).The current WHO-definition misclassifies patients who harbor resistant virus at VL below 1000 c/mL as "nonfailing." Lowering the threshold to VL ≥80 copies/mL identifies a significantly higher number of patients with treatment-resistant virus and should be considered.

Two Distinct Cardiolipin Synthases Operate in Agrobacterium tumefaciens.

Cardiolipin (CL) is a universal component of energy generating membranes. In most bacteria, it is synthesized via the condensation of two molecules phosphatidylglycerol (PG) by phospholipase D-type cardiolipin synthases (PLD-type Cls). In the plant pathogen and natural genetic engineer Agrobacterium tumefaciens CL comprises up to 15% of all phospholipids in late stationary growth phase. A. tumefaciens harbors two genes, atu1630 (cls1) and atu2486 (cls2), coding for PLD-type Cls. Heterologous expression of either cls1 or cls2 in Escherichia coli resulted in accumulation of CL supporting involvement of their products in CL synthesis. Expression of cls1 and cls2 in A. tumefaciens is constitutive and irrespective of the growth phase. Membrane lipid profiling of A. tumefaciens mutants suggested that Cls2 is required for CL synthesis at early exponential growth whereas both Cls equally contribute to CL production at later growth stages. Contrary to many bacteria, which suffer from CL depletion, A. tumefaciens tolerates large changes in CL content since the CL-deficient cls1/cls2 double mutant showed no apparent defects in growth, stress tolerance, motility, biofilm formation, UV-stress and tumor formation on plants.

Same day ART initiation versus clinic-based pre-ART assessment and counselling for individuals newly tested HIV-positive during community-based HIV testing in rural Lesotho - a randomized controlled trial (CASCADE trial).

BACKGROUND: Achievement of the UNAIDS 90-90-90 targets in Sub-Sahara Africa is challenged by a weak care-cascade with poor linkage to care and retention in care. Community-based HIV testing and counselling (HTC) is widely used in African countries. However, rates of linkage to care and initiation of antiretroviral therapy (ART) in individuals who tested HIV-positive are often very low. A frequently cited reason for non-linkage to care is the time-consuming pre-ART assessment often requiring several clinic visits before ART-initiation. METHODS: This two-armed open-label randomized controlled trial compares in individuals tested HIV-positive during community-based HTC the proposition of same-day community-based ART-initiation to the standard of care pre-ART assessment at the clinic. Home-based HTC campaigns will be conducted in catchment areas of six clinics in rural Lesotho. Households where at least one individual tested HIV positive will be randomized. In the standard of care group individuals receive post-test counselling and referral to the nearest clinic for pre-ART assessment and counselling. Once they have started ART the follow-up schedule foresees monthly clinic visits. Individuals randomized to the intervention group receive on the spot point-of-care pre-ART assessment and adherence counselling with the proposition to start ART that same day. Once they have started ART, follow-up clinic visits will be less frequent. First primary outcome is linkage to care (individual presents at the clinic at least once within 3 months after the HIV test). The second primary outcome is viral suppression 12 months after enrolment in the study. We plan to enrol a minimum of 260 households with 1:1 allocation and parallel assignment into both arms. DISCUSSION: This trial will show if in individuals tested HIV-positive during community-based HTC campaigns the proposition of same-day ART initiation in the community, combined with less frequent follow-up visits at the clinic could be a pragmatic approach to improve the care cascade in similar settings. TRIAL REGISTRATION: NCT02692027 , registered February 21, 2016.

Enzymatic properties and substrate specificity of a bacterial phosphatidylcholine synthase.

Phosphatidylcholine (PC) is a rare membrane lipid in bacteria, but is crucial for virulence of the plant pathogen Agrobacterium tumefaciens and various other pathogens. Agrobacterium tumefaciens uses two independent PC biosynthesis pathways. One is dependent on the integral membrane protein PC synthase (Pcs), which catalyzes the conversion of cytidine diphosphate-diacylglycerol (CDP-DAG) and choline to PC, thereby releasing a cytidine monophosphate (CMP). Here, we show that Pcs consists of eight transmembrane segments with its N- and C-termini located in the cytoplasm. A cytoplasmic loop between the second and third membrane helix contains the majority of the conserved amino acids of a CDP-alcohol phosphotransferase motif (DGX2 ARX12 GX3 DX3 D). Using point mutagenesis, we provide evidence for a crucial role of this motif in choline binding and enzyme activity. To study the catalytic features of the enzyme, we established a purification protocol for recombinant Pcs. The enzyme forms stable oligomers and exhibits broad substrate specificity towards choline derivatives. The presence of CDP-DAG and manganese is a prerequisite for cooperative binding of choline. PC formation by Pcs is reversible and proceeds via two successive reactions. In a first choline- and manganese-independent reaction, CDP-DAG is hydrolyzed releasing a CMP molecule. The resulting phosphatidyl intermediate reacts with choline in a second manganese-dependent step to form PC. STRUCTURED DIGITAL ABSTRACT: Pcs and Pcs bind by molecular sieving (1, 2, 3).

Discovery of a bifunctional cardiolipin/phosphatidylethanolamine synthase in bacteria.

Phosphatidylethanolamine (PE) and cardiolipin (CL) are major components of bacterial and eukaryotic membranes. In bacteria, synthesis of PE usually occurs via decarboxylation of phosphatidylserine (PS) by PS decarboxylases (Psd). CL is produced by various CL synthases (Cls). Membranes of the plant pathogen Xanthomonas campestris predominantly contain PE, phosphatidylglycerol (PG) and CL. The X. campestris genome encodes one Psd and six putative CLs. Deletion of psd resulted in loss of PE and accumulation of PS. The mutant was severely affected in growth and cell size. PE synthesis, growth and cell division were partially restored when cells were supplied with ethanolamine (EA) suggesting a previously unknown PE synthase activity. Via mutagenesis, we identified a Cls enzyme (Xc_0186) responsible for EA-dependent PE biosynthesis. Xanthomonas lacking xc_0186 not only lost its ability to utilize EA for PE synthesis but also produced less CL suggesting a bifunctional enzyme. Recombinant Xc_0186 in E. coli and in cell-free extracts uses cytidine diphosphate diacylglycerol (CDP-DAG) and PG for CL synthesis. It is also able to use CDP-DAG and EA for PE synthesis. Owing to its dual function in CL and PE production, we consider Xc_0186 the founding member of a new class of enzymes called CL/PE synthase (CL/PEs).

Spatial inattention abolishes voice adaptation.

Adaptation to male voices causes a subsequent voice to be perceived as more female, and vice versa. Similar contrastive aftereffects have been reported for phonetic perception, and in vision for face perception. However, while aftereffects in the perception of phonetic features of speech have been reported to persist even when adaptors were processed inattentively, face aftereffects were previously reported to be abolished by inattention to adaptors. Here we demonstrate that auditory aftereffects of adaptation to voice gender are eliminated when the male and female adaptor voices are spatially unattended. Participants simultaneously heard gender-specific male or female adaptor voices in one ear and gender-neutral (androgynous) adaptor voices in the contralateral ear. They selectively attended to the adaptor voices in a designated ear, by either classifying voice gender (Exp. 1) or spoken syllable (Exp. 2). Voice aftereffects were found only if the gender-specific voices were spatially attended, suggesting capacity limits in the processing of voice gender for the unattended ear. Remarkably, gender-specific adaptors in the attended ear elicited comparable aftereffects in test voices, regardless of prior attention to voice gender or phonetic content. Thus, within the attended ear, voice gender was processed even when it was irrelevant for the task at hand, suggesting automatic processing of gender along with linguistic information. Overall, voice gender adaptation requires spatial, but not dimensional, selective attention.

Outcomes of antiretroviral treatment programs in rural Southern Africa.

BACKGROUND: Data on outcomes of antiretroviral treatment (ART) programs in rural sub-Saharan African are scarce. We describe early losses and long-term outcomes in 6 rural programs in Southern Africa with limited access to viral load monitoring and second-line ART. METHODS: Patients aged ≥16 years starting ART in 2 programs each in Zimbabwe, Mozambique, and Lesotho were included. We evaluated risk factors for no follow-up after starting ART and mortality and loss to follow-up (LTFU) over 3 years of ART, using logistic regression and competing risk models. Odds ratios and subdistribution hazard ratios, adjusted for gender, age category, CD4 category, and World Health Organization stage at start of ART are reported. RESULTS: Among 7725 patients, 449 (5.8%) did not return after initiation of ART. During 9575 person-years, 698 (9.6%) of those with at least 1 follow-up visit died, and 1319 (18.1%) were LTFU. At 3 years, the cumulative incidence of death and LTFU were 12.5% (11.5%-13.5%) and 25.4% (24.0%-26.9%), respectively, with important differences between countries as follows: in Zimbabwe 75.1% (72.8%-77.3%) were alive and on ART at 3 years compared with 55.4% (52.8%-58.0%) in Lesotho and 51.6% (48.0%-55.2%) in Mozambique. In all settings, young age and male gender predicted LTFU, whereas advanced clinical stage and low baseline CD4 counts predicted death. CONCLUSIONS: In African ART programs with limited access to second-line treatment, mortality, and LTFU are high in the first 3 years of ART. Low retention in care is a major threat to the sustainability of ART delivery in Southern Africa, particularly in rural sites.

Choline uptake in Agrobacterium tumefaciens by the high-affinity ChoXWV transporter.

Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 µM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 µM; betaine, KD of ≈470 µM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called "Venus flytrap mechanism" of substrate binding.

Virulence of Agrobacterium tumefaciens requires phosphatidylcholine in the bacterial membrane.

Phosphatidylcholine (PC, lecithin) has long been considered a solely eukaryotic membrane lipid. Only a minority of all bacteria is able to synthesize PC. The plant-transforming bacterium Agrobacterium tumefaciens encodes two potential PC forming enzymes, a phospholipid N-methyltransferase (PmtA) and a PC synthase (Pcs). We show that PC biosynthesis and tumour formation on Kalanchoë plants was impaired in the double mutant. The virulence defect was due to a complete lack of the type IV secretion machinery in the Agrobacterium PC mutant. Our results strongly suggest that PC in bacterial membranes is an important determinant for the establishment of host-microbe interactions.