Mutations in the leucine zipper motif and sterol-sensing domain inactivate the Niemann-Pick C1 glycoprotein.

Niemann-Pick type C (NPC) disease, characterized by accumulation of low density lipoprotein-derived free cholesterol in lysosomes, is caused by mutations in the NPC1 gene. We examined the ability of wild-type NPC1 and NPC1 mutants to correct the NPC sterol trafficking defect and their subcellular localization in CT60 cells. Cells transfected with wild-type NPC1 expressed 170- and 190-kDa proteins. Tunicamycin treatment resulted in a 140-kDa protein, the deduced size of NPC1, suggesting that NPC1 is N-glycosylated. Mutation of all four asparagines in potential N-terminal N-glycosylation sites to glutamines resulted in a 20-kDa reduction of the expressed protein. Proteins with a single N-glycosylation site mutation localized to late endosome/lysosomal compartments, as did wild-type NPC1, and each corrected the cholesterol trafficking defect. However, mutation of all four potential N-glycosylation sites reduced ability to correct the NPC phenotype commensurate with reduced expression of the protein. Mutations in the putative sterol-sensing domain resulted in inactive proteins targeted to lysosomal membranes encircling cholesterol-laden cores. N-terminal leucine zipper motif mutants could not correct the NPC defect, although they accumulated in lysosomal membranes. We conclude that NPC1 is a glycoprotein that must have an intact sterol-sensing domain and leucine zipper motif for cholesterol-mobilizing activity.     

Leveraging vibration of effects analysis for robust discovery in observational biomedical data science

Hypothesis generation in observational, biomedical data science often starts with computing an association or identifying the statistical relationship between a dependent and an independent variable. However, the outcome of this process depends fundamentally on modeling strategy, with differing strategies generating what can be called “vibration of effects” (VoE). VoE is defined by variation in associations that often lead to contradictory results. Here, we present a computational tool capable of modeling VoE in biomedical data by fitting millions of different models and comparing their output. We execute a VoE analysis on a series of widely reported associations (e.g., carrot intake associated with eyesight) with an extended additional focus on lifestyle exposures (e.g., physical activity) and components of the Framingham Risk Score for cardiovascular health (e.g., blood pressure). We leveraged our tool for potential confounder identification, investigating what adjusting variables are responsible for conflicting models. We propose modeling VoE as a critical step in navigating discovery in observational data, discerning robust associations, and cataloging adjusting variables that impact model output.

COVID positivity and vitamin D intake, red meat and heart disease; how can we discern when biomedical associations are reliable and when they are susceptible to our own arbitrary choices and assumptions? This study presents “quantvoe,” a software package for exploring the entirety of possible findings due to the multiverse of associations possible.     

FBXL20 promotes breast cancer malignancy by inhibiting apoptosis through degradation of PUMA and BAX

Apoptosis is a programmed cell death that efficiently removes damaged cells to maintain tissue homeostasis. Defect in apoptotic machinery can lead to tumor development, progression, and resistance to chemotherapy. PUMA (p53 upregulated modulator of apoptosis) and BAX (BCL2-associated X protein) are among the most well-known inducers of apoptosis. It has been reported that expression levels of BAX and PUMA are controlled at the posttranslational level by phosphorylation. However, the posttranslational regulation of these proapoptotic proteins remains largely unexplored. In this study, using biochemical, molecular biology, flow cytometric, and immunohistochemistry techniques, we show that PUMA and BAX are the direct target of the F-box protein FBXL20, which restricts their cellular levels. FBXL20 directs the proteasomal degradation of PUMA and BAX in a protein kinase AKT1-dependent manner to promote cancer cell proliferation and tumor growth. Interestingly, inactivation of AKT1 results in activation of another protein kinase GSK3α/β, which facilitates the proteasomal degradation of FBXL20 by another F-box protein, FBXO31. Thus, a switch between two signaling kinases AKT1 and GSK3α/β modulates the functional activity of these proapoptotic regulators, thereby determining cell survival or death. RNAi-mediated ablation of FBXL20 results in increased levels of PUMA as well as BAX, which further enhances the sensitivity of cancer cells to chemotherapeutic drugs. We showed that high level expression of FBXL20 in cancer cells reduces therapeutic drug-induced apoptosis and promotes chemoresistance. Overall, this study highlights the importance of targeting FBXL20 in cancers in conjunction with chemotherapy and may represent a promising anticancer strategy to overcome chemoresistance.     

The role of long range, local and direct signalling molecules during chick feather bud development involving the BMPs, follistatin and the Eph receptor tyrosine kinase Eph-A4.

The development of the feather buds during avian embryogenesis is a classic example of a spacing pattern. The regular arrangement of feather buds is achieved by a process of lateral inhibition whereby one developing feather bud prevents the formation of similar buds in the immediate vicinity. Lateral inhibition during feather formation implicates a role of long range signalling during this process. Recent work has shown that BMPs are able to enforce lateral inhibition during feather bud formation. However these results do not explain how the feather bud escapes the inhibition itself. We show that this could be achieved by the expression of the BMP antagonist, Follistatin. Furthermore we show that local application of Follistatin leads to the development of ectopic feather buds. We suggest that Follistatin locally antagonises the action of the BMPs and so permits the cellular changes associated with feather placode formation. We also provide evidence for the role of short range signalling during feather formation. We have correlated changes in cellular morphology in feather placodes with the expression of the gene Eph-A4 which encodes a receptor tyrosine kinase that requires direct cell-cell contact for activation. We show that the expression of this gene precedes cellular reorganisation required for feather bud formation.     

Solution conformation of the (+)-trans-anti-benzo[g]chrysene-dA adduct opposite dT in a DNA duplex.

The solution structure of the adduct derived from the covalent bonding of the fjord region (+)-(11S, 12R, 13R, 14S) stereoisomer of anti -11,12-dihydroxy-13,14-epoxy-11,12,13, 14-tetrahydrobenzo[g]chrysene, (+)- anti -B[g]CDE, to the exocyclic N(6)amino group of the adenine residue dA6, (designated (+)- trans-anti -(B[g]C)dA6), positioned opposite a thymine residue dT17 in the DNA sequence context d(C1-T2-C3-T4-C5-(B[g]C)A6-C7-T8-T9-C10-C11). d(G12-G13-A14-A15-G16-T17-G18-A19-G20++ +-A21-G22) (designated (B[g]C)dA. dT 11-mer duplex), has been studied using structural information derived from NMR data in combination with molecular dynamics (MD) calculations. The solution structure of the (+)- trans-anti -(B[g]C)dA.dT 11-mer duplex has been determined using an MD protocol where both interproton distance and dihedral angle restraints deduced from NOESY and COSY spectra are used during the refinement process, followed by additional relaxation matrix refinement to the observed NOESY intensities to account for spin diffusion effects. The results established that the covalently attached benzo[g]chrysene ring intercalates into the DNA helix directed towards the 5'-side of the modified strand and stacks predominantly with dT17 when intercalated between dC5.dG18 and (B[g]C)dA6.dT17 base-pairs. All base-pairs, including the modified (B[g]C)dA6.dT17 base-pair, are aligned through Watson-Crick pairing as in normal B -DNA. In addition, the potential strain associated with the highly sterically hindered fjord region of the aromatic portion of the benzo[g]chrysenyl ring is relieved through the adoption of a non-planar, propeller-like geometry within the chrysenyl ring system. This conformation shares common structural features with the related (+)- trans-anti -(B[c]Ph)dA adduct in the identical base sequence context, derived from the fjord region (+)-(1S,2R,3R,4S)-3, 4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene stereoisomer, in which intercalation is also observed towards the 5'-side of the modified dA6.dT17 base-pair.     

Class II MHC/peptide complexes are released from APC and are acquired by T cell responders during specific antigen recognition.

T cell expression of class II MHC/peptide complexes may be important for maintenance of peripheral self-tolerance, but mechanisms underlying the genesis of class II MHC glycoproteins on T cells are not well resolved. T cell APC (T-APC) used herein were transformed IL-2-dependent clones that constitutively synthesized class II MHC glycoproteins. When pulsed with myelin basic protein (MBP) and injected into Lewis rats, these T-APC reduced the severity of experimental autoimmune encephalomyelitis, whereas unpulsed T-APC were without activity. Normal MBP-reactive clones cultured without APC did not express class II MHC even when activated with mitogens and exposed to IFN-gamma. However, during a 4-h culture with T-APC or macrophage APC, recognition of MBP or mitogenic activation of responder T cells elicited high levels of I-A and I-E expression on responders. Acquisition of class II MHC glycoproteins by responders was resistant to the protein synthesis inhibitor cycloheximide, coincided with transfer of a PKH26 lipophilic dye from APC to responders, and resulted in the expression of syngeneic and allogeneic MHC glycoproteins on responders. Unlike rested I-A- T cell clones, rat thymic and splenic T cells expressed readily detectable levels of class II MHC glycoproteins. When preactivated with mitogens, naive T cells acquired APC-derived MHC class II molecules and other membrane-associated proteins when cultured with xenogeneic APC in the absence of Ag. In conclusion, this study provides evidence that APC donate membrane-bound peptide/MHC complexes to Ag-specific T cell responders by a mechanism associated with the induction of tolerance.     

Chronic wounds in Sierra Leone: Searching for Buruli ulcer,a NTD caused by Mycobacterium ulcerans,at Masanga Hospital

BackgroundChronic wounds pose a significant healthcare burden in low- and middle-income countries. Buruli ulcer (BU), caused by Mycobacterium ulcerans infection, causes wounds with high morbidity and financial burden. Although highly endemic in West and Central Africa, the presence of BU in Sierra Leone is not well described. This study aimed to confirm or exclude BU in suspected cases of chronic wounds presenting to Masanga Hospital, Sierra Leone.MethodologyDemographics, baseline clinical data, and quality of life scores were collected from patients with wounds suspected to be BU. Wound tissue samples were acquired and transported to the Swiss Tropical and Public Health Institute, Switzerland, for analysis to detect Mycobacterium ulcerans using qPCR, microscopic smear examination, and histopathology, as per World Health Organization (WHO) recommendations.FindingsTwenty-one participants with wounds suspected to be BU were enrolled over 4-weeks (Feb-March 2019). Participants were predominantly young working males (62% male, 38% female, mean 35yrs, 90% employed in an occupation or as a student) with large, single, ulcerating wounds (mean diameter 9.4cm, 86% single wound) exclusively of the lower limbs (60% foot, 40% lower leg) present for a mean 15 months. The majority reported frequent exposure to water outdoors (76%). Self-reports of over-the-counter antibiotic use prior to presentation was high (81%), as was history of trauma (38%) and surgical interventions prior to enrolment (48%). Regarding laboratory investigation, all samples were negative for BU by microscopy, histopathology, and qPCR. Histopathology analysis revealed heavy bacterial load in many of the samples. The study had excellent participant recruitment, however follow-up proved difficult.ConclusionsBU was not confirmed as a cause of chronic ulceration in our cohort of suspected cases, as judged by laboratory analysis according to WHO standards. This does not exclude the presence of BU in the region, and the definitive cause of these treatment-resistance chronic wounds is uncertain.     

Co-encapsulation of amyloglucosidase with starch and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> as basis for a long-lasting CO<Subscript>2</Subscript> release

CO₂ is known as a major attractant for many arthropod pests which can be exploited for pest control within novel attract-and-kill strategies. This study reports on the development of a slow-release system for CO₂ based on calcium alginate beads containing granular corn starch, amyloglucosidase and Saccharomyces cerevisiae. Our aim was to evaluate the conditions which influence the CO₂ release and to clarify the biochemical reactions taking place within the beads. The amyloglucosidase was immobilized with a high encapsulation efficiency of 87% in Ca-alginate beads supplemented with corn starch and S. cerevisiae biomass. The CO₂ release from the beads was shown to be significantly affected by the concentration of amyloglucosidase and corn starch within the beads as well as by the incubation temperature. Beads prepared with 0.1 amyloglucosidase units/g matrix solution led to a long-lasting CO₂ emission at temperatures between 6 and 25?°C. Starch degradation data correlated well with the CO₂ release from beads during incubation and scanning electron microscopy micrographs visualized the degradation of corn starch granules by the co-encapsulated amyloglucosidase. By implementing MALDI-ToF mass spectrometry imaging for the analysis of Ca-alginate beads, we verified that the encapsulated amyloglucosidase converts starch into glucose which is immediately consumed by S. cerevisiae cells. When applied into the soil, the beads increased the CO₂ concentration in soil significantly. Finally, we demonstrated that dried beads showed a CO₂ production in soil comparable to the moist beads. The long-lasting CO₂-releasing beads will pave the way towards novel attract-and-kill strategies in pest control.