Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis

The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N‐terminal ubiquitin‐like (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Å crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitin‐binding interface, provide a model for the phosphoubiquitin–parkin complex and show how phosphorylation of the Ubl domain primes parkin for optimal phosphoubiquitin binding. Furthermore, we demonstrate that the addition of phosphoubiquitin leads to displacement of the Ubl domain through loss of structure, unveiling a ubiquitin‐binding site used by the E2~Ub conjugate, thus leading to active parkin. We find the role of the Ubl domain is to prevent parkin activity in the absence of the phosphorylation signals, and propose a model for parkin inhibition, optimization for phosphoubiquitin recruitment, release of inhibition by the Ubl domain and engagement with an E2~Ub conjugate. Taken together, this model provides a mechanistic framework for activating parkin.     

Comparison of peripheral dose measurements using Ionization chamber and MOSFET detector

BackgroundIn radiation therapy, the peripheral dose (PD) – the dose outside the geometric boundaries of the radiation field – is of clinical importance. A metal oxide semiconductor field effect transistor (MOSFET) detector is used to estimate the peripheral dose.AimThe aim of this study is to investigate the ability of a MOSFET dosimetry system to accurately measure doses in peripheral regions of high energy X-ray beams.Materials & MethodsThe accuracy of the MOSFET system is evaluated by comparing peripheral region dose measurement with the results of standard ionization chamber measurements. Furthermore, the measurement of PD using a MOSFET detector helps us to keep the tolerance dose of any critical organ closer to the treatment field within the acceptable limits. The measurements were carried out using a 0.6 cc Farmer type ionization chamber and MOSFET 20 dosimetry system for field sizes ranging from 5 × 5 cm² to 20 × 20 cm² at three depths of 1.5 cm, 5 cm and 10 cm in a blue water phantom. PD were measured at distances varying from 1 cm to 30 cm from the field edges along the x axis for the open fields, with collimator rotation and with beam modifiers like 15 degree, 30 degree and 45 degree wedges.ResultsThe results show a good agreement of measured dose by both methods for various field sizes, collimator rotation and wedges.ConclusionThe MOSFET detector has a compact construction, provides instant readout, is of minimal weight and can be used on any surface.     

Development of a high-throughput Candida albicans biofilm chip

We have developed a high-density microarray platform consisting of nano-biofilms of Candida albicans. A robotic microarrayer was used to print yeast cells of C. albicans encapsulated in a collagen matrix at a volume as low as 50 nL onto surface-modified microscope slides. Upon incubation, the cells grow into fully formed "nano-biofilms". The morphological and architectural complexity of these biofilms were evaluated by scanning electron and confocal scanning laser microscopy. The extent of biofilm formation was determined using a microarray scanner from changes in fluorescence intensities due to FUN 1 metabolic processing. This staining technique was also adapted for antifungal susceptibility testing, which demonstrated that, similar to regular biofilms, cells within the on-chip biofilms displayed elevated levels of resistance against antifungal agents (fluconazole and amphotericin B). Thus, results from structural analyses and antifungal susceptibility testing indicated that despite miniaturization, these biofilms display the typical phenotypic properties associated with the biofilm mode of growth. In its final format, the C. albicans biofilm chip (CaBChip) is composed of 768 equivalent and spatially distinct nano-biofilms on a single slide; multiple chips can be printed and processed simultaneously. Compared to current methods for the formation of microbial biofilms, namely the 96-well microtiter plate model, this fungal biofilm chip has advantages in terms of miniaturization and automation, which combine to cut reagent use and analysis time, minimize labor intensive steps, and dramatically reduce assay costs. Such a chip should accelerate the antifungal drug discovery process by enabling rapid, convenient and inexpensive screening of hundreds-to-thousands of compounds simultaneously.     

Mangroves of Godavari – Analysis Through Remote Sensing Approach

The expansion of agriculture and aquaculture farms in the coastal areas has led to conversion of mangroves in the recent past. The extent of mangroves has also changed due to the erosion of mangroves along the coast and accretion near river mouths, leading to the formation of new mangrove areas. This study has been undertaken in the mangroves of the Godavari estuary, Andhra Pradesh, India to understand the changes in the extent of mangroves, namely accreted mangroves, erosion due to wave action and river water flow during floods, and changes due to forest restoration between 1986 and 2001, through remote sensing. The geomorphological changes due to river water flow in and around the mangroves have also been analysed. The changes in the vegetation due to forest restoration and natural regeneration are appreciable, while the changes in the area due to erosion and accretion are more or less equal. An analysis of the remote sensing images of 1986 and 2001 reveal that the mangroves outside the forest boundary have been converted to aquaculture. The sand spit of Hope Island has changed with time and has grown nearly 2.6 km between 1937 and 2001.     

Exosomal microRNAs from mesenchymal stem/stromal cells: Biology and applications in neuroprotection

Mesenchymal stem/stromal cells (MSCs) are extensively studied as cell-therapy agents for neurological diseases. Recent studies consider exosomes secreted by MSCs as important mediators for MSCs’ neuroprotective functions. Exosomes transfer functional molecules including proteins, lipids, metabolites, DNAs, and coding and non-coding RNAs from MSCs to their target cells. Emerging evidence shows that exosomal microRNAs (miRNAs) play a key role in the neuroprotective properties of these exosomes by targeting several genes and regulating various biological processes. Multiple exosomal miRNAs have been identified to have neuroprotective effects by promoting neurogenesis, neurite remodeling and survival, and neuroplasticity. Thus, exosomal miRNAs have significant therapeutic potential for neurological disorders such as stroke, traumatic brain injury, and neuroinflammatory or neurodegenerative diseases and disorders. This review discusses the neuroprotective effects of selected miRNAs (miR-21, miR-17-92, miR-133, miR-138, miR-124, miR-30, miR146a, and miR-29b) and explores their mechanisms of action and applications for the treatment of various neurological disease and disorders. It also provides an overview of state-of-the-art bioengineering approaches for isolating exosomes, optimizing their yield and manipulating the miRNA content of their cargo to improve their therapeutic potential.     

Hormones andCuscuta development: In vitro induction of haustoria by cytokinin and its inhibition by other hormones

Cytokinins induced haustoria formation in excised 10-mm segments ofCuscuta vine, the subapical 25-to-50-mm region being most responsive, producing a mean of 4–6 haustoria per segment. The order of effectiveness of cytokinins continuously applied (72 h) was 6-benzylaminopurine (BA) isopentenyladenine (iP) zeatin (Z). Ribosides of BA and Z were as effective as the bases, whereas riboside of iP ([9R]iP) was half as effective as iP. Haustoria induction was influenced by weather and seasonal conditions at the time of vine collection; materials obtained on warm, sunny days responded better than those obtained on rainy, cloudy, or cool days. Haustoria were induced equally well all around the segment, and no thigmostimulus was needed for induction. p ]A 10-min pulse of 100 M BA induced half as many haustoria as a 60-min pulse or continuous application of BA. White light inhibited haustoria induction elicited by a short (30-min) pulse of BA, whereas a longer (120-min) BA application overcame this light inhibition. Auxins (IAA or NAA, 1–10 M), gibberellin (GA₃, 1–10 M), ethylene (as ethrel, 10–100 M), and abscisic acid (ABA, 100 M) were individually inhibitory (60–80%) with respect to haustoria induction when given continuously with 50 M BA. A 60-min pulse of auxins (10 M), GA₃ (100 M), or ethrel (10 M), given at various time intervals during or after a 60-min pulse of 100 M BA, showed that inhibition was maximal (70–95%) between 4 and 16 h of BA application and negligible (GA₃) or much reduced (auxin, ethrel) at 20 h, indicating a commitment to haustoria formation by this time.     

Structure and mechanism of the type I-G CRISPR effector

Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3′ end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.     

The crystal structure of a plant 3-ketoacyl-CoA thiolase reveals the potential for redox control of peroxisomal fatty acid beta-oxidation

Crystal structures of peroxisomal Arabidopsis thaliana 3-ketoacyl-CoA thiolase (AtKAT), an enzyme of fatty acid beta-oxidation, are reported. The subunit, a typical thiolase, is a combination of two similar alpha/beta domains capped with a loop domain. The comparison of AtKAT with the Saccharomyces cerevisiae homologue (ScKAT) structure reveals a different placement of subunits within the functional dimers and that a polypeptide segment forming an extended loop around the open catalytic pocket of ScKAT converts to alpha-helix in AtKAT, and occludes the active site. A disulfide is formed between Cys192, on this helix, and Cys138, a catalytic residue. Access to Cys138 is determined by the structure of this polypeptide segment. AtKAT represents an oxidized, previously unknown inactive form, whilst ScKAT is the reduced and active enzyme. A high level of sequence conservation is observed, including Cys192, in eukaryotic peroxisomal, but not mitochondrial or prokaryotic KAT sequences, for this labile loop/helix segment. This indicates that KAT activity in peroxisomes is influenced by a disulfide/dithiol change linking fatty acid beta-oxidation with redox regulation.     

Meibum lipid hydrocarbon chain branching and rheology after hematopoietic stem cell transplantation

PurposeMeibum from donors who have had hematological stem cell transplantations (M_HSCT) are susceptible to severe dry eye symptoms and exhibit very high lipid order (stiffness) compared with meibum from donors without dry eye (M_n). Since lipid order could have functional consequences, we compared the rheology and composition of M_n and M_HSCT to measure meibum compositional, structural and functional relationships.MethodsThe rheology and composition was measured using Langmuir trough and ¹H NMR spectroscopy, respectively.ResultsM_HSCT and M_n was studied from 16 to 43 donors, respectively, using NMR spectroscopy. M_HSCT contained significantly 16% more straight chain and 24% less iso-chain hydrocarbons compared with M_n. The cholesteryl ester to wax ester molar ratio, and hydrocarbon chain unsaturation were not significantly different, for M_HSCT compared with M_n.Surface pressure-area isotherms of meibum from 30 donors without dry-eye were grouped into 4 pools (PC) and meibum from 32 donors with dry eye who had hematopoietic stem cell transplantation (PT) were grouped into 3 pools. Above 15 years of age the П_max and (C_s^?1)_max increased with age for both the PC and the PT cohorts. (C_s^?1)_max values were higher for PT samples compared with age matched PC samples, indicating they had higher elasticity and stiffness. A more ordered lipid could contribute to the formation of a discontinuous patchy tear film lipid layer, which in turn results in deteriorated spreading, and decreased surface elasticity.ConclusionsThe composition and rheology of meibum from donors with dry eye and who have had HSCT support the idea from other studies that more ordered meibum may contribute to or be a marker of dry eye.