An atypical GABARAP binding module drives the pro-autophagic potential of the AML-associated NPM1c variant.

The nucleolar scaffold protein NPM1 is a multifunctional regulator of cellular homeostasis, genome integrity, and stress response. NPM1 mutations, known as NPM1c variants promoting its aberrant cytoplasmic localization, are the most frequent genetic alterations in acute myeloid leukemia (AML). A hallmark of AML cells is their dependency on elevated autophagic flux. Here, we show that NPM1 and NPM1c induce the autophagy-lysosome pathway by activating the master transcription factor TFEB, thereby coordinating the expression of lysosomal proteins and autophagy regulators. Importantly, both NPM1 and NPM1c bind to autophagy modifiers of the GABARAP subfamily through an atypical binding module preserved within its N terminus. The propensity of NPM1c to induce autophagy depends on this module, likely indicating that NPM1c exerts its pro-autophagic activity by direct engagement with GABARAPL1. Our data report a non-canonical binding mode of GABARAP family members that drives the pro-autophagic potential of NPM1c, potentially enabling therapeutic options.

Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum.

The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.

Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy.

Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.

Identification of potential selective autophagy receptors from protein-content profiling of autophagosomes.

Selective autophagy receptors (SARs) are central to cellular homeostatic and organellar recycling pathways. Over the last two decades, more than 30 SARs have been discovered and validated using a variety of experimental approaches ranging from cell biology to biochemistry, including high-throughput imaging and screening methods. Yet, the extent of selective autophagy pathways operating under various cellular contexts, for example, under basal and starvation conditions, remains unresolved. Currently, our knowledge of all known SARs and their associated cargo components is fragmentary and limited by experimental data with varying degrees of resolution. Here, we use classical predictive and modeling approaches to integrate high-quality autophagosome content profiling data with disparate datasets. We identify a global set of potential SARs and their associated cargo components active under basal autophagy, starvation-induced, and proteasome-inhibition conditions. We provide a detailed account of cellular components, biochemical pathways, and molecular processes that are degraded via autophagy. Our analysis yields a catalog of new potential SARs that satisfy the characteristics of bonafide, well-characterized SARs. We categorize them by the subcellular compartments they emerge from and classify them based on their likely mode of action. Our structural modeling validates a large subset of predicted interactions with the human ATG8 family of proteins and shows characteristic, conserved LC3-interacting region (LIR)-LIR docking site (LDS) and ubiquitin-interacting motif (UIM)-UIM docking site (UDS) binding modes. Our analysis also revealed the most abundant cargo molecules targeted by these new SARs. Our findings expand the repertoire of SARs and provide unprecedented details into the global autophagic state of HeLa cells. Taken together, our findings provide motivation for the design of new experiments, testing the role of these novel factors in selective autophagy.

ESTIMATION OF INDOOR AND OUTDOOR EFFECTIVE DOSES AND LIFETIME CANCER RISK FROM GAMMA DOSE RATES IN AND AROUND MANDYA DISTRICT, KARNATAKA.

Indoor and outdoor gamma-ray dose rates have been measured in and around Mandya district (12° 20″ N and 77° 20″ E). The measurements were carried out from 45 locations of Mandya district at 1 m above the ground surface for radiometric, geophysical and environmental surveys using a lightweight portable radiation dosemeter ER­709. The indoor and outdoor absorbed gamma dose rate in air varied from 66.12±0.8 to 131.89±5.5 nGy per h with a geometrical mean value of 97.79±2.6 nGy per h and 45.94±0.7 to 80.39±2.6 nGy per h with a geometrical mean value of 58.75±1.5. The indoor and outdoor effective doses vary from 0.32 to 0.65 mSv per y with a geometric mean value of 0.48 mSv per y and 0.06 to 0.10 mSv per y with a geometric mean value of 0.07 mSv per y which is slightly higher when compared with the worldwide average of the effective doses. The indoor and outdoor excess lifetime cancer risk (ELCR) of residents along the different locations varies from 1.14 × 10-3 to 2.26 × 10-3 with a geometric mean value of 1.68 × 10-3 and 0.20 × 10-3 to 0.35 × 10-3 with a geometric mean value of 0.25 × 10-3 which is similar to the worldwide average of the ELCR.

The Ephrin B2 Receptor Tyrosine Kinase Is a Regulator of Proto-oncogene MYC and Molecular Programs Central to Barrett's Neoplasia.

BACKGROUND & AIMS: Mechanisms contributing to the onset and progression of Barrett's (BE)-associated esophageal adenocarcinoma (EAC) remain elusive. Here, we interrogated the major signaling pathways deregulated early in the development of Barrett's neoplasia. METHODS: Whole-transcriptome RNA sequencing analysis was performed in primary BE, EAC, normal esophageal squamous, and gastric biopsy tissues (n = 89). Select pathway components were confirmed by quantitative polymerase chain reaction in an independent cohort of premalignant and malignant biopsy tissues (n = 885). Functional impact of selected pathway was interrogated using transcriptomic, proteomic, and pharmacogenetic analyses in mammalian esophageal organotypic and patient-derived BE/EAC cell line models, in vitro and/or in vivo. RESULTS: The vast majority of primary BE/EAC tissues and cell line models showed hyperactivation of EphB2 signaling. Transcriptomic/proteomic analyses identified EphB2 as an endogenous binding partner of MYC binding protein 2, and an upstream regulator of c-MYC. Knockdown of EphB2 significantly impeded the viability/proliferation of EAC and BE cells in vitro/in vivo. Activation of EphB2 in normal esophageal squamous 3-dimensional organotypes disrupted epithelial maturation and promoted columnar differentiation programs, notably including MYC. EphB2 and MYC showed selective induction in esophageal submucosal glands with acinar ductal metaplasia, and in a porcine model of BE-like esophageal submucosal gland spheroids. Clinically approved inhibitors of MEK, a protein kinase that regulates MYC, effectively suppressed EAC tumor growth in vivo. CONCLUSIONS: The EphB2 signaling is frequently hyperactivated across the BE-EAC continuum. EphB2 is an upstream regulator of MYC, and activation of EphB2-MYC axis likely precedes BE development. Targeting EphB2/MYC could be a promising therapeutic strategy for this often refractory and aggressive cancer.

Role of FAM134 paralogues in endoplasmic reticulum remodeling, ER-phagy, and Collagen quality control.

Degradation of the endoplasmic reticulum (ER) via selective autophagy (ER-phagy) is vital for cellular homeostasis. We identify FAM134A/RETREG2 and FAM134C/RETREG3 as ER-phagy receptors, which predominantly exist in an inactive state under basal conditions. Upon autophagy induction and ER stress signal, they can induce significant ER fragmentation and subsequent lysosomal degradation. FAM134A, FAM134B/RETREG1, and FAM134C are essential for maintaining ER morphology in a LC3-interacting region (LIR)-dependent manner. Overexpression of any FAM134 paralogue has the capacity to significantly augment the general ER-phagy flux upon starvation or ER-stress. Global proteomic analysis of FAM134 overexpressing and knockout cell lines reveals several protein clusters that are distinctly regulated by each of the FAM134 paralogues as well as a cluster of commonly regulated ER-resident proteins. Utilizing pro-Collagen I, as a shared ER-phagy substrate, we observe that FAM134A acts in a LIR-independent manner and compensates for the loss of FAM134B and FAM134C, respectively. FAM134C instead is unable to compensate for the loss of its paralogues. Taken together, our data show that FAM134 paralogues contribute to common and unique ER-phagy pathways.

Membrane fusion and drug delivery with carbon nanotube porins.

Drug delivery mitigates toxic side effects and poor pharmacokinetics of life-saving therapeutics and enhances treatment efficacy. However, direct cytoplasmic delivery of drugs and vaccines into cells has remained out of reach. We find that liposomes studded with 0.8-nm-wide carbon nanotube porins (CNTPs) function as efficient vehicles for direct cytoplasmic drug delivery by facilitating fusion of lipid membranes and complete mixing of the membrane material and vesicle interior content. Fusion kinetics data and coarse-grained molecular dynamics simulations reveal an unusual mechanism where CNTP dimers tether the vesicles, pull the membranes into proximity, and then fuse their outer and inner leaflets. Liposomes containing CNTPs in their membranes and loaded with an anticancer drug, doxorubicin, were effective in delivering the drug to cancer cells, killing up to 90% of them. Our results open an avenue for designing efficient drug delivery carriers compatible with a wide range of therapeutics.

FAM134B-RHD Protein Clustering Drives Spontaneous Budding of Asymmetric Membranes.

Living cells constantly remodel the shape of their lipid membranes. In the endoplasmic reticulum (ER), the reticulon homology domain (RHD) of the reticulophagy regulator 1 (RETR1/FAM134B) forms dense autophagic puncta that are associated with membrane removal by ER-phagy. In molecular dynamics (MD) simulations, we find that FAM134B-RHD spontaneously forms clusters, driven in part by curvature-mediated attractions. At a critical size, as in a nucleation process, the FAM134B-RHD clusters induce the formation of membrane buds. The kinetics of budding depends sensitively on protein concentration and bilayer asymmetry. Our MD simulations shed light on the role of FAM134B-RHD in ER-phagy and show that membrane asymmetry can be used to modulate the kinetic barrier for membrane remodeling.

Data on identification of desertified regions in Anantapur district, Southern India by NDVI approach using remote sensing and GIS.

Present dataset aims at the inventory data on preparation of Desertification Status Maps (DSM) for the first time in semi-arid region of Anantapur district in the state of Andhra Pradesh, South India by applying Normalized Difference Vegetation Index (NDVI) with acquired Remote Sensing (RS) satellite imageries and processed in ERDAS Imagine and ArcGIS software's. The NDVI has been classified into five such as water body, vegetation, fallow land, degradation land, and desertified land. Further, degradation land has been decreased to 4.87% which lead to desertification in the study region. The current research data will be resourceful to the environmental scientists and planning agencies who can utilize optimum for sustainable development and good governance in land degradation, desertification, and conserve land resources.

TBK1-mediated phosphorylation of LC3C and GABARAP-L2 controls autophagosome shedding by ATG4 protease.

Autophagy is a highly conserved catabolic process through which defective or otherwise harmful cellular components are targeted for degradation via the lysosomal route. Regulatory pathways, involving post-translational modifications such as phosphorylation, play a critical role in controlling this tightly orchestrated process. Here, we demonstrate that TBK1 regulates autophagy by phosphorylating autophagy modifiers LC3C and GABARAP-L2 on surface-exposed serine residues (LC3C S93 and S96; GABARAP-L2 S87 and S88). This phosphorylation event impedes their binding to the processing enzyme ATG4 by destabilizing the complex. Phosphorylated LC3C/GABARAP-L2 cannot be removed from liposomes by ATG4 and are thus protected from ATG4-mediated premature removal from nascent autophagosomes. This ensures a steady coat of lipidated LC3C/GABARAP-L2 throughout the early steps in autophagosome formation and aids in maintaining a unidirectional flow of the autophagosome to the lysosome. Taken together, we present a new regulatory mechanism of autophagy, which influences the conjugation and de-conjugation of LC3C and GABARAP-L2 to autophagosomes by TBK1-mediated phosphorylation.

Phospholipid Scramblases Remodel the Shape of Asymmetric Membranes.

The cell membrane and many organellar membranes are asymmetric and highly curved. In experiments, it is challenging to reconstitute and characterize membranes that differ in the lipid composition of their leaflets. Here we use molecular dynamics simulations to study the large-scale membrane shape changes associated with lipid shuttling between asymmetric leaflets. We exploit leaflet asymmetry to create a stable, near-spherical vesicle bud connected to a flat bilayer under periodic boundary conditions. Then we demonstrate how the lipid scramblase nhTMEM16 relaxes the lipid-number asymmetry. By mediating the flipping of lipids, this transmembrane protein dissipates the mechanochemical gradient between the leaflets and drives a large-scale membrane reorganization, converting the vesicle bud into a flat membrane. Our procedure to exploit bilayer asymmetry for simulations of highly curved membranes can be used to study the function of other lipid transporters and membrane-shaping proteins.

Membrane perforation by the pore-forming toxin pneumolysin.

Pneumolysin (PLY), a major virulence factor of Streptococcus pneumoniae, perforates cholesterol-rich lipid membranes. PLY protomers oligomerize as rings on the membrane and then undergo a structural transition that triggers the formation of membrane pores. Structures of PLY rings in prepore and pore conformations define the beginning and end of this transition, but the detailed mechanism of pore formation remains unclear. With atomistic and coarse-grained molecular dynamics simulations, we resolve key steps during PLY pore formation. Our simulations confirm critical PLY membrane-binding sites identified previously by mutagenesis. The transmembrane ß-hairpins of the PLY pore conformation are stable only for oligomers, forming a curtain-like membrane-spanning ß-sheet. Its hydrophilic inner face draws water into the protein-lipid interface, forcing lipids to recede. For PLY rings, this zone of lipid clearance expands into a cylindrical membrane pore. The lipid plug caught inside the PLY ring can escape by lipid efflux via the lower leaflet. If this path is too slow or blocked, the pore opens by membrane buckling, driven by the line tension acting on the detached rim of the lipid plug. Interestingly, PLY rings are just wide enough for the plug to buckle spontaneously in mammalian membranes. In a survey of electron cryo-microscopy (cryo-EM) and atomic force microscopy images, we identify key intermediates along both the efflux and buckling pathways to pore formation, as seen in the simulations.

Curvature induction and membrane remodeling by FAM134B reticulon homology domain assist selective ER-phagy.

FAM134B/RETREG1 is a selective ER-phagy receptor that regulates the size and shape of the endoplasmic reticulum. The structure of its reticulon-homology domain (RHD), an element shared with other ER-shaping proteins, and the mechanism of membrane shaping remain poorly understood. Using molecular modeling and molecular dynamics (MD) simulations, we assemble a structural model for the RHD of FAM134B. Through MD simulations of FAM134B in flat and curved membranes, we relate the dynamic RHD structure with its two wedge-shaped transmembrane helical hairpins and two amphipathic helices to FAM134B functions in membrane-curvature induction and curvature-mediated protein sorting. FAM134B clustering, as expected to occur in autophagic puncta, amplifies the membrane-shaping effects. Electron microscopy of in vitro liposome remodeling experiments support the membrane remodeling functions of the different RHD structural elements. Disruption of the RHD structure affects selective autophagy flux and leads to disease states.

MEASUREMENT OF RADON SOIL GAS IN AND AROUND BHARATHINAGARA, MANDYA DISTRICT.

Radon activity concentration in soil gas has been studied in and around Bharathinagara, Mandya district (12° 13|| N and 77° 20|| E) using Solid State Nuclear Track Detectors with Twin cup dosimeter. The activity concentration of 222Rn in soil gas was studied at two depths. Radon in soil gas was found to increase with depth and decrease with increase in moisture content of the soil. Radon in soil gas was found to be higher in winter season which varies from 0.22 ± 0.01 to 1.31 ± 0.01 kBq/m3 with a Geometric mean value of 0.56 ± 0.01 kBq/m3 in 1 m depth and lower radon soil gas was found to be 0.16 ± 0.01 to 0.60 ± 0.01 kBq/m3 with a Geometric mean value of 0.30 ± 0.01 kBq/m3 in 0.5 m depth during summer season. The activity concentrations of radon soil gas from in and around Bharathinagara are lower compared to those in other parts of the world.

Data on comparative studies of lineaments extraction from ASTER DEM, SRTM, and Cartosat for Jilledubanderu River basin, Anantapur district, A.P, India by using remote sensing and GIS.

The data deals with the functions that automatically extracted lineaments from the Cartosat, ASTER and SRTM of Digital Elevation Model (DEM) of different spatial resolutions, in the software ArcGIS 10.4. The extracted lineaments result shows the ASTER (Advanced Spaceborne Thermal Emission and Reflection Radiometer) DEM gives the lowest number of lineaments reflects Cartosat and SRTM (Shuttle Radar Topography Mission) DEM shows a medium number of lineaments. Cartosat DEM is most appropriate for extraction of contours precisely rather than ASTER and SRTM. This study reveals the Cartosat DEM data is best to use extraction of lineaments in the Indian provinces, offers at most comprehensive geological structural info amongst all the data sets. The extracted lineaments lengths and densities are determined by the statistical method. Based on the data generated lineament density and rose diagram. Cartosat DEM data are the best suited for studying very small areas as through geological and structural information can be mined by using this data.

Cryo-EM structure of the bifunctional secretin complex of Thermus thermophilus.

Secretins form multimeric channels across the outer membrane of Gram-negative bacteria that mediate the import or export of substrates and/or extrusion of type IV pili. The secretin complex of Thermus thermophilus is an oligomer of the 757-residue PilQ protein, essential for DNA uptake and pilus extrusion. Here, we present the cryo-EM structure of this bifunctional complex at a resolution of ~7 Å using a new reconstruction protocol. Thirteen protomers form a large periplasmic domain of six stacked rings and a secretin domain in the outer membrane. A homology model of the PilQ protein was fitted into the cryo-EM map. A crown-like structure outside the outer membrane capping the secretin was found not to be part of PilQ. Mutations in the secretin domain disrupted the crown and abolished DNA uptake, suggesting a central role of the crown in natural transformation.

Simulation study of Hemodynamic in Bifurcations for Cerebral Arteriovenous Malformation using Electrical Analogy.

BACKGROUND AND OBJECTIVE: Cerebral Arteriovenous Malformation (CAVM) hemodynamic is disease condition, results changes in the flow and pressure level in cerebral blood vessels. Measuring flow and pressure without catheter intervention along the vessel is big challenge due to vessel bifurcations/complex bifurcations in Arteriovenous Malformation patients. The vessel geometry in CAVM patients are complex, composed of varying diameters, lengths, and bifurcations of various angles. The variations in the vessel diameter and bifurcation angle complicate the measurement and analysis of blood flow features invasively or non-invasively. METHODS: In this paper, we proposed a lumped model for the bifurcation for symmetrical and asymmetrical networks in CAVM patients. The models are created using MATLAB Simulation software for various bifurcation angles. Each bifurcation angle created using electrical network- RLC. The segmentation and pre-processing of bifurcation vessels are implemented using adaptive segmentation. The proposed network address clinicians problem by measuring hemodynamic non-invasively. The method is applicable for any types of bifurcation networks with different bifurcation angles in CAVM patients. RESULTS: In this work, we constructed a mathematical model, measured hemodynamic for 23 patients (actual and simulated cases) with 60 vessel bifurcation angles variations. The results indicate that comparisons evidenced highly significant correlations between values computed by the lumped model and simulated mechanical model for both networks with p < 0.0001. A P value of less than 0.05 considered statistically significant. CONCLUSION: In this paper, we have modelled different bifurcation types and automatically display pressure and flow non-invasively at different node and at different angles of bifurcation in the complex vessel with help of bifurcation parameters, using lumped parameter model. We have simulated for different bifurcation angles and diameters of vessel for various imaging modality and model extend for different organs. This will help clinicians to measure haemodynamic parameters noninvasively at various bifurcations, where even catheter cannot be reached.