TMEM41B is a novel regulator of autophagy and lipid mobilization

Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens, or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well‐characterized autophagy‐related proteins orchestrates the biogenesis of autophagosomes; however, the origin of the required membranes is incompletely understood. Here, we have applied sensitized pooled CRISPR screens and identify the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2‐ and DFCP1‐positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and β‐oxidation of fatty acids. Immunostaining and interaction proteomics data suggest that TMEM41B localizes to the endoplasmic reticulum (ER). Taken together, we propose that TMEM41B is a novel ER‐localized regulator of autophagosome biogenesis and lipid mobilization.     

Lack of a Negative Effect of BCG-Vaccination on Child Psychomotor Development: Results from the Danish Calmette Study - A Randomised Clinical Trial

ObjectivesTo assess the non-specific effect of Bacillus Calmette-Guérin (BCG) vaccination at birth on psychomotor development.DesignThis is a pre-specified secondary outcome from a randomised, clinical trial.SettingMaternity units and paediatric wards at three university hospitals in Denmark.ParticipantsChildren born at gestational age (GA) 32 weeks and above. All women planning to give birth at the three sites were invited during the recruitment period. Out of 4262 randomised children, 144 were premature (GA < 37 weeks). There were 2129 children (71 premature) randomised to BCG and 2133 randomised (73 premature) to the control group.InterventionsBCG vaccination 0.05 ml was given intradermally in the upper left arm at the hospital within seven days of birth. Children in the control group did not receive any intervention. Parents were not blinded to allocation.ResultsThe mean difference in ASQ score at 12 months adjusted for age and prematurity was -0.7 points (BCG vs. control, 95% confidence interval; -3.7 to 2.4), p = 0.67, corresponding to an effect size of Cohen’s d = -0.015 (-0.082 to 0.052). The mean difference in ASQ score for premature children at 22 months was -7.8 points (-20.6 to 5.0, p = 0.23), d = -0.23 (-0.62 to 0.15).ConclusionsA negative non-specific effect of BCG vaccination at birth on psychomotor development was excluded in term children.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01694108","term_id":"NCT01694108"}}NCT01694108     

1-[4-[3-[4-[bis(4-fluorophenyl)hydroxymethyl]-1- piperidinyl]propoxy]-3-methoxyphenyl]ethanone(AHR-5333): a selective human blood neutrophil 5-lipoxygenase inhibitor

In this study we report the in vitro inhibition of leukotriene synthesis in calcium ionophore (A23187)-stimulated, intact human blood neutrophils by AHR-5333. The results showed that AHR-5333 inhibits 5-HETE, LTB4 and LTC4 synthesis with IC50 values of 13.9, 13.7 and 6.9 microM, respectively. Further examination of the effect of AHR-5333 on individual reactions of the 5-lipoxygenase pathway (i.e. conversion of LTA4 to LTB4, LTA4 to LTC4, and arachidonic acid to 5-HETE) showed that this agent was not inhibitory to LTA4 epoxyhydrolase and glutathione-S-transferase activity in neutrophil homogenates. However, conversion of arachidonic acid (30 microM) to 5-HETE was half maximally inhibited by 20 microM AHR-5333 in the cell-free system. The inhibition of LTB4 and LTC4 formation in intact neutrophils by AHR-5333 appears to be entirely due to a selective inhibition of 5-lipoxygenase activity and an impaired formation of LTA4, which serves as substrate for LTA4 epoxyhydrolase and glutathione-S-transferase. AHR-5333 did not affect the transformation of exogenous arachidonic acid to thromboxane B2, HHT and 12-HETE in preparations of washed human platelets, indicating that this agent has no effect on platelet prostaglandin H synthase, thromboxane synthase and 12-lipoxygenase activity. The lack of inhibitory activity of AHR-5333 on prostaglandin H synthase activity was confirmed with microsomal preparations of sheep vesicular glands.     

γ-Irradiation of malic acid in aqueous solutions

The -irradiation of malic acid in aqueous solutions was studied under initially oxygenated and oxygen-free conditions in an attempt to determine the possible interconversion of malic acid into other carboxylic acids, specifically those associated with Krebs cycle. The effect of dose on product formation of the system was investigated. Gas-liquid chromatography combined with mass spectrometry was used as the principal means of identification of the non-volatile products. Thin layer chromotography and direct probe mass spectroscopy were also employed.The findings show that a variety of carboxylic acids are formed, with malonic and succinic acids in greatest abundance. These products have all been identified as being formed in the -irradiation of acetic acid, suggesting a common intermediary. Since these molecules fit into a metabolic cycle, it is strongly suggestive that prebiotic pathways provided the basis for biological systems.     

A solid-phase method for the quantitation of protein in the presence of sodium dodecyl sulfate and other interfering substances

We describe a simple assay for small amounts of protein that is insensitive to sodium dodecyl sulfate (SDS) or many common interfering substances including Tris and reducing sugars. For this reason, it is particularly useful in the analysis of protein content of samples prior to SDS electrophoresis. The assay consists of the following steps: (i) absorption of protein to nitrocellulose; (ii) fixation of the bound protein with methanol; (iii) staining of the bound protein with amido black; and (iv) elution and spectrophotometric measurement of the bound dye. The assay is sensitive to as little as 0.5 microgram of protein in 1 microliter of solution. Although SDS does not interfere appreciably with measurement, Nonidet-P40 does.     

Effect of increasing duration of denervation on the rate of entry of inorganic phosphate into rat gastrocnemius muscle.

The increased inorganic phosphate flow, characteristic of denervated gastrocnemius muscle, is shown to have no direct relation with either the loss of muscle mass or with the concentrations of the acid-soluble phosphate fractions. It is shown to increase hyperbolically with the time elapsed since the nerve section. The asymptotic value reached after thirty days suggests the presence of a saturable mechanism.     

Genetic Determinants of Pelvic Organ Prolapse among African American and Hispanic Women in the Women’s Health Initiative

Current evidence suggests a multifactorial etiology to pelvic organ prolapse (POP), including genetic predisposition. We conducted a genome-wide association study of POP in African American (AA) and Hispanic (HP) women from the Women’s Health Initiative Hormone Therapy study. Cases were defined as any POP (grades 1–3) or moderate/severe POP (grades 2–3), while controls had grade 0 POP. We performed race-specific multiple logistic regression analyses between SNPs imputed to 1000 genomes in relation to POP (grade 0 vs 1–3; grade 0 vs 2–3) adjusting for age at diagnosis, body mass index, parity, and genetic ancestry. There were 1274 controls and 1427 cases of any POP and 317 cases of moderate/severe POP. Although none of the analyses reached genome-wide significance (p<5x10^-8), we noted variants in several loci that met p<10⁻⁶. In race-specific analysis of grade 0 vs 2–3, intronic SNPs in the CPE gene (rs28573326, OR:2.14; 95% CI 1.62–2.83; p = 1.0x10^-7) were associated with POP in AAs, and SNPs in the gene AL132709.5 (rs1950626, OR:2.96; 95% CI 1.96–4.48, p = 2.6x10^-7) were associated with POP in HPs. Inverse variance fixed-effect meta-analysis of the race-specific results showed suggestive signals for SNPs in the DPP6 gene (rs11243354, OR:1.36; p = 4.2x10^-7) in the grade 0 vs 1–3 analyses and for SNPs around PGBD5 (rs740494, OR:2.17; p = 8.6x10^-7) and SHC3 (rs2209875, OR:0.60; p = 9.3x10^-7) in the grade 0 vs 2–3 analyses. While we did not identify genome-wide significant findings, we document several SNPs reaching suggestive statistical significance. Further interrogation of POP in larger minority samples is warranted.     

Mapping the epitopes of neutralizing anti-human IL-3 monoclonal antibodies. Implications for structure-activity relationship.

The epitopes of neutralizing mAb were mapped in order to identify a receptor binding site on human IL-3 (huIL-3). To initiate this structure and activity analysis, four neutralizing mAb were selected on the basis of preventing rhuIL-3 stimulated proliferation of peripheral blood cells from a patient with chronic myelogenous leukemia (CML). In order to identify continuous epitopes, the neutralizing mAb were assayed in a solid-phase ELISA for their reactivity with either denatured rhuIL-3 or with the peptides generated by digestion of rhuIL-3 by using two different proteinases. Two of the neutralizing mAb recognized single fragments from both digestions. Amino acid (aa) sequence determination showed that these peptides overlap, defining a region of 22 aa (aa 29 to 50 of the mature rhuIL-3 protein). In a competition ELISA, the two continuous epitopes were shown to be linked to one another and to the two discontinuous epitopes, suggesting that all four neutralizing mAb bind to a discrete region of the IL-3 molecule, which might be involved in binding to the IL-3R.     

Early and late systemic effects of colchicine on muscle permeability to inorganic phosphate

A single systemic injection of 75 micrograms colchicine/100 g body weight in the lumbar muscles increases within 6 h the permeability of the extensor digitorum longus muscle to inorganic phosphate. Twenty four hours after the injection the specific activities and isotope uptakes of both inorganic and organic-bound acid-soluble phosphates are markedly increased. By the third day a maximal four-fold increase in rate of inorganic phosphate exchange is reached. The observed effects are slowly reversible, a near normal situation obtaining after 30 days. They are qualitatively different from those observed after administration of vincristine sulphate and bupivaca?ne (Marcaine).