Late Steps Research Articles

The translational activator Sov1 coordinates mitochondrial gene expression with mitoribosome biogenesis.

Mitoribosome biogenesis is an expensive metabolic process that is essential to maintain cellular respiratory capacity and requires the stoichiometric accumulation of rRNAs and proteins encoded in two distinct genomes. In yeast, the ribosomal protein Var1, alias uS3m, is mitochondrion-encoded. uS3m is a protein universally present in all ribosomes, where it forms part of the small subunit (SSU) mRNA entry channel and plays a pivotal role in ribosome loading onto the mRNA. However, despite its critical functional role, very little is known concerning VAR1 gene expression. Here, we demonstrate that the protein Sov1 is an in bona fide VAR1 mRNA translational activator and additionally interacts with newly synthesized Var1 polypeptide. Moreover, we show that Sov1 assists the late steps of mtSSU biogenesis involving the incorporation of Var1, an event necessary for uS14 and mS46 assembly. Notably, we have uncovered a translational regulatory mechanism by which Sov1 fine-tunes Var1 synthesis with its assembly into the mitoribosome.

Alix-Mediated Rescue of Feline Immunodeficiency Virus Budding Differs from That Observed with Human Immunodeficiency Virus.

The structural protein Gag is the only viral component required for retroviral budding from infected cells. Each of the three conserved domains-the matrix (MA), capsid (CA), and nucleocapsid (NC) domains-drives different phases of viral particle assembly and egress. Once virus assembly is complete, retroviruses, like most enveloped viruses, utilize host proteins to catalyze membrane fission and to free progeny virions. These proteins are members of the endosomal sorting complex required for transport (ESCRT), a cellular machinery that coats the inside of budding necks to perform membrane-modeling events necessary for particle abscission. The ESCRT is recruited through interactions with PTAP and LYPXnL, two highly conserved sequences named late (L) domains, which bind TSG101 and Alix, respectively. A TSG101-binding L-domain was identified in the p2 region of the feline immunodeficiency virus (FIV) Gag protein. Here, we show that the human protein Alix stimulates the release of virus from FIV-expressing human cells. Furthermore, we demonstrate that the Alix Bro1 domain rescues FIV mutants lacking a functional TSG101-interacting motif, independently of the entire p2 region and of the canonical Alix-binding L-domain(s) in FIV Gag. However, in contrast to the effect on human immunodeficiency virus type 1 (HIV-1), the C377,409S double mutation, which disrupts both CCHC zinc fingers in the NC domain, does not abrogate Alix-mediated virus rescue. These studies provide insight into conserved and divergent mechanisms of lentivirus-host interactions involved in virus budding.IMPORTANCE FIV is a nonprimate lentivirus that infects domestic cats and causes a syndrome that is reminiscent of AIDS in humans. Based on its similarity to HIV with regard to different molecular and biochemical properties, FIV represents an attractive model for the development of strategies to prevent and/or treat HIV infection. Here, we show that the Bro1 domain of the human cellular protein Alix is sufficient to rescue the budding of FIV mutants devoid of canonical L-domains. Furthermore, we demonstrate that the integrity of the CCHC motifs in the Gag NC domain is dispensable for Alix-mediated rescue of virus budding, suggesting the involvement of other regions of the Gag viral protein. Our research is pertinent to the identification of a conserved yet mechanistically divergent ESCRT-mediated lentivirus budding process in general, and to the role of Alix in particular, which underlies the complex viral-cellular network of interactions that promote late steps of the retroviral life cycle.

RNA-Seq Analyses Reveal That Endothelial Activation and Fibrosis Are Induced Early and Progressively by Besnoitia besnoiti Host Cell Invasion and Proliferation.

The pathogenesis of bovine besnoitiosis and the molecular bases that govern disease progression remain to be elucidated. Thus, we have employed an in vitro model of infection based on primary bovine aortic endothelial cells (BAEC), target cells during the acute infection. Host-parasite interactions were investigated by RNA-Seq at two post-infection (pi) time points: 12 hpi, when tachyzoites have already invaded host cells, and 32 hpi, when tachyzoites have replicated for at least two generations. Additionally, the gene expression profile of B. besnoiti tachyzoites was studied at both pi time points. Up to 446 differentially expressed B. taurus genes (DEGs) were found in BAEC between both pi time points: 249 DEGs were up-regulated and 197 DEGs were down-regulated at 32 hpi. Upregulation of different genes encoding cytokines, chemokines, leukocyte adhesion molecules predominantly at 12 hpi implies an activation of endothelial cells, whilst upregulation of genes involved in angiogenesis and extracellular matrix organization was detected at both time points. NF-κB and TNF-α signaling pathways appeared to be mainly modulated upon infection, coordinating the expression of several effector proteins with proinflammatory and pro-fibrotic phenotypes. These mediators are thought to be responsible for macrophage recruitment setting the basis for chronic inflammation and fibrosis characteristic of chronic besnoitiosis. Angiogenesis regulation also predominated, and this multistep process was evidenced by the upregulation of markers involved in both early (e.g., growth factors and matrix metalloproteinases) and late steps (e.g., integrins and vasohibin). Besnoitia besnoiti ortholog genes present in other Toxoplasmatinae members and involved in the lytic cycle have shown to be differentially expressed among the two time points studied, with a higher expression at 32 hpi (e.g., ROP40, ROP5B, MIC1, MIC10). This study gives molecular clues on B. besnoiti- BAECs interaction and shows the progression of type II endothelial cell activation upon parasite invasion and proliferation.

VAMP8 mediates TLR4-dependent TNF secretion in RBL-2H3 pro-inflammatory cells

Abstract Mast cells (MCs) participate in allergic diseases by producing pro-inflammatory mediators and regulatory cytokines. After high affinity IgE (FceRI) receptor activation, they release tryptase, b-hexosaminidase and other preformed pro-inflammatory substances by anaphylactic degranulation. Also, upon stimulation through Toll-like receptor 4 (TLR4), they release pro-inflammatory mediators (such as Tumor Necrosis Factor, TNF), by piecemeal degranulation, orchestrating protective reactions against pathogens. Reactions involved in anaphylactic degranulation require specific vesicular-associated membrane proteins (VAMPs) involved in the latest step of exocytosis. However, the particular roles of VAMP proteins on piecemeal secretion observed after TLR4 receptor activation are not known. Utilizing RBL-2H3 cells differentiated to a pro-inflammatory phenotype, we investigated the role of VAMP8 and VAMP3 on lipopolysaccharide (LPS)-induced mediator secretion in MCs. Confocal microscopy images showed that, in non-stimulated cells, TNF localizes in VAMP3 and VAMP8-positive vesicles. After TLR4 stimulation, we observed an increase in TNF and VAMP8 co-localization, without any change on TNF and VAMP3 co-localization. Remarkably, VAMP8 blockade with specific siRNA impaired TNF secretion after LPS-challenge, while VAMP3 siRNA-mediated down regulation did not. Our data indicate that, although TNF is maintained in VAMP3-containing vesicles, TLR4 triggering leads to the secretion of TNF in VAMP8-containing carriers. Our results contribute to the description of the molecular events involved in late steps of TNF secretion induced by innate immune stimuli in that important immune cell type.

Division of Labor between PCNA Loaders in DNA Replication and Sister Chromatid Cohesion Establishment

SummaryConcomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Several replication-fork-associated “cohesion establishment factors,” including the multifunctional Ctf18-RFC complex, aid this process in as yet unknown ways. Here, we show that Ctf18-RFC’s role in sister chromatid cohesion correlates with PCNA loading but is separable from its role in the replication checkpoint. Ctf18-RFC loads PCNA with a slight preference for the leading strand, which is dispensable for DNA replication. Conversely, the canonical Rfc1-RFC complex preferentially loads PCNA onto the lagging strand, which is crucial for DNA replication but dispensable for sister chromatid cohesion. The downstream effector of Ctf18-RFC is cohesin acetylation, which we place toward a late step during replication maturation. Our results suggest that Ctf18-RFC enriches and balances PCNA levels at the replication fork, beyond the needs of DNA replication, to promote establishment of sister chromatid cohesion and possibly other post-replicative processes.

Chlorophyllide a oxidoreductase Preferentially Catalyzes 8-Vinyl Reduction over B-Ring Reduction of 8-Vinyl Chlorophyllide a in the Late Steps of Bacteriochlorophyll Biosynthesis.

Bacteriochlorophyll a (BChl) is an essential pigment for anoxygenic photosynthesis. In late steps of the BChl biosynthesis of Rhodobacter capsulatus, the C8 vinyl group and C7=C8 double bond of 8-vinyl chlorophyllide a (8 V-Chlide) are reduced by a C8 vinyl reductase (8VR), BciA, and a nitrogenase-like enzyme, chlorophyllide a oxidoreductase (COR), respectively, to produce 3-vinyl-bacteriochlorphyllide a. Recently, we discovered 8VR activity in COR. However, the kinetic parameters of the COR 8VR activity remain unknown, while those of the COR C7=C8 reductase activity and BciA have been reported. Here, we determined the kinetic parameters of COR 8VR activity by using 8 V-Chlide. The Km value for 8 V-Chlide was 1.4 μM, which is much lower than the 6.2 μM determined for the C7=C8 reduction of Chlide. The kinetic parameters of the dual activities of COR suggest that COR catalyzes the reduction of the C8 vinyl group of 8 V-Chlide preferentially over C7=C8 reduction when both substrates are supplied during BChl biosynthesis.

Overexpression, Purification, and Preliminary Crystallographic Analysis of the Archaeosine Synthase GAT‐QueC

Archaeosine (G+) is a highly modified nucleosides found exclusively at position‐15 in the dihydrouridine loops (D‐loop) of archaeal tRNAs where it contributes to tRNA structural stability. In a late step in G+ biosynthesis, the precursor nucleoside 7‐cyano‐7‐deazaguanine (preQ0) is inserted into tRNA by the enzyme tRNA‐guanine transglycosylase (arcTGT) before conversion to G+ by archaeosine synthases that vary among archaeal phyla: ArcS in Euryarchaeota, and QueF‐like or GAT‐QueC in Chrenoarchaeota. GAT‐QueC is a two‐domain enzyme harboring an N‐terminal glutamine amidotransferase (GAT) domain, and a C‐terminal domain homologous to QueC, the enzyme that produces preQ0. Consistent with its 2‐domain structure, GAT‐QueC functions in vivo as both a preQ0 synthase and an amidinotransferase, generating G+‐modified tRNA. However, it remains unknown whether the GAT domain acts on preQ0 before or after its insertion into tRNA by arcTGT. Here we describe overexpression, purification and preliminary biochemical and crystallographic characterization of GAT‐QueC from Sulfolobus tokodaii (StGAT‐QueC). Analytical gel filtration chromatography and electrophoretic mobility shift analysis reveal that StGAT‐QueC is a monomeric protein with molecular weight of 55 kDa and no affinity for tRNA, suggesting that the enzyme acts on free preQ0, not preQ0‐modified tRNA. High throughput crystallization of the apoenzyme (and in the presence of preQ0) using the vapor diffusion method yielded crystals that grew in 21 days from solutions containing methyl pentanediol and sodium acetate, and diffracted to 5.8 Å without cryo protection. The crystals belong to cubic space group F23, exhibit unit cell length=292 Å and contain 4 protein molecules per asymmetric unit.Support or Funding InformationNIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).

Cancer Cell-Derived Matrisome Proteins Promote Metastasis in Pancreatic Ductal Adenocarcinoma.

The prognosis for pancreatic ductal adenocarcinoma (PDAC) remains poor despite decades of effort. The abundant extracellular matrix (ECM) in PDAC comprises a major fraction of the tumor mass and plays various roles in promoting resistance to therapies. However, nonselective depletion of ECM has led to poor patient outcomes. Consistent with that observation, we previously showed that individual matrisome proteins derived from stromal cells correlate with either long or short patient survival. In marked contrast, those derived from cancer cells correlate strongly with poor survival. Here, we studied three cancer cell-derived matrisome proteins that are significantly overrepresented during PDAC progression, AGRN (agrin), SERPINB5 (serine protease inhibitor B5), and CSTB (cystatin B). Using both overexpression and knockdown experiments, we demonstrate that all three are promoters of PDAC metastasis. Furthermore, these proteins operate at different metastatic steps. AGRN promoted epithelial-to-mesenchymal transition in primary tumors, whereas SERPINB5 and CSTB enhanced late steps in the metastatic cascade by elevating invadopodia formation and in vivo extravasation. All three genes were associated with a poor prognosis in human patients and high levels of SERPINB5, secreted by cancer cells and deposited in the ECM, correlated with poor patient prognosis. This study provides strong evidence that cancer cell-derived matrisome proteins can be causal in promoting tumorigenesis and metastasis and lead to poor patient survival. Therefore, compared with the bulk matrix, mostly made by stromal cells, precise interventions targeting cancer cell-derived matrisome proteins, such as AGRN, SERPINB5, and CSTB, may represent preferred potential therapeutic targets. SIGNIFICANCE: This study provides insights into the biological roles of cancer cell-derived matrisome proteins in PDAC and supports the notion that these proteins are protumorigenic and better therapeutic targets.

Phage capsid nanoparticles with defined ligand arrangement block influenza virus entry.

Multivalent interactions at biological interfaces occur frequently in nature and mediate recognition and interactions in essential physiological processes such as cell-to-cell adhesion. Multivalency is also a key principle that allows tight binding between pathogens and host cells during the initial stages of infection. One promising approach to prevent infection is the design of synthetic or semisynthetic multivalent binders that interfere with pathogen adhesion1-4. Here, we present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus. Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo and in vivo. Such highly functionalized capsids present an alternative to strategies that target virus entry by spike-inhibiting antibodies5 and peptides6 or that address late steps of the viral replication cycle7.

Role of Hepatitis B virus capsid phosphorylation in nucleocapsid disassembly and covalently closed circular DNA formation.

Hepatitis B virus (HBV) delivers a partially double-stranded, relaxed circular (RC) DNA genome in complete virions to the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, which establishes and sustains viral infection. An overlength pregenomic RNA (pgRNA) is then transcribed from CCC DNA and packaged into immature nucleocapsids (NCs) by the viral core (HBc) protein. pgRNA is reverse transcribed to produce RC DNA in mature NCs, which are then enveloped and secreted as complete virions, or delivered to the nucleus to replenish the nuclear CCC DNA pool. RC DNA, whether originating from extracellular virions or intracellular mature NCs, must be released upon NC disassembly (uncoating) for CCC DNA formation. HBc is known to undergo dynamic phosphorylation and dephosphorylation at its C-terminal domain (CTD) to facilitate pgRNA packaging and reverse transcription. Here, two putative phosphorylation sites in the HBc N-terminal domain (NTD), S44 and S49, were targeted for genetic and biochemical analysis to assess their potential roles in viral replication. The NTD mutant that mimics the non-phosphorylated state (N2A) was competent in all steps of viral replication tested from capsid assembly, pgRNA packaging, reverse transcription, to virion secretion, except for a decrease in CCC DNA formation. On the other hand, the phosphor-mimetic mutant N2E showed a defect in the early step of pgRNA packaging but enhanced the late step of mature NC uncoating and consequently, increased CCC DNA formation. N2E also enhanced phosphorylation in CTD and possibly elsewhere in HBc. Furthermore, inhibition of the cyclin-dependent kinase 2 (CDK2), which is packaged into viral capsids, could block CCC DNA formation. These results prompted us to propose a model whereby rephosphorylation of HBc at both NTD and CTD by the packaged CDK2, following CTD dephosphorylation during NC maturation, facilitates uncoating and CCC DNA formation by destabilizing mature NCs.

CesA6 and PGIP2 Endocytosis Involves Different Subpopulations of TGN-Related Endosomes.

Endocytosis is an essential process for the internalization of plasma membrane proteins, lipids and extracellular molecules into the cells. The mechanisms underlying endocytosis in plant cells involve several endosomal organelles whose origins and specific role needs still to be clarified. In this study we compare the internalization events of a GFP-tagged polygalacturonase-inhibiting protein of Phaseolus vulgaris (PGIP2-GFP) to that of a GFP-tagged subunit of cellulose synthase complex of Arabidopsis thaliana (secGFP-CesA6). Through the use of endocytic traffic chemical inhibitors (tyrphostin A23, salicylic acid, wortmannin, concanamycin A, Sortin 2, Endosidin 5 and BFA) it was evidenced that the two protein fusions were endocytosed through distinct endosomes with different mechanisms. PGIP2-GFP endocytosis is specifically sensitive to tyrphostin A23, salicylic acid and Sortin 2; furthermore, SYP51, a tSNARE with interfering effect on late steps of vacuolar traffic, affects its arrival in the central vacuole. SecGFP-CesA6, specifically sensitive to Endosidin 5, likely reaches the plasma membrane passing through the trans Golgi network (TGN), since the BFA treatment leads to the formation of BFA bodies, compatible with the aggregation of TGNs. BFA treatments determine the accumulation and tethering of the intracellular compartments labeled by both proteins, but PGIP2-GFP aggregated compartments overlap with those labeled by the endocytic dye FM4-64 while secGFP-CesA6 fills different compartments. Furthermore, secGFP-CesA6 co-localization with RFP-NIP1.1, marker of the direct ER-to-Vacuole traffic, in small compartments separated from ER suggests that secGFP-CesA6 is sorted through TGNs in which the direct contribution from the ER plays an important role. All together the data indicate the existence of a heterogeneous population of Golgi-independent TGNs.

Role of uL3 in the Crosstalk between Nucleolar Stress and Autophagy in Colon Cancer Cells.

The nucleolus is the site of ribosome biogenesis and has been recently described as important sensor for a variety of cellular stressors. In the last two decades, it has been largely demonstrated that many chemotherapeutics act by inhibiting early or late rRNA processing steps with consequent alteration of ribosome biogenesis and activation of nucleolar stress response. The overall result is cell cycle arrest and/or apoptotic cell death of cancer cells. Our previously data demonstrated that ribosomal protein uL3 is a key sensor of nucleolar stress activated by common chemotherapeutic agents in cancer cells lacking p53. We have also demonstrated that uL3 status is associated to chemoresistance; down-regulation of uL3 makes some chemotherapeutic drugs ineffective. Here, we demonstrate that in colon cancer cells, the uL3 status affects rRNA synthesis and processing with consequent activation of uL3-mediated nucleolar stress pathway. Transcriptome analysis of HCT 116p53−/− cells expressing uL3 and of a cell sub line stably depleted of uL3 treated with Actinomycin D suggests a new extra-ribosomal role of uL3 in the regulation of autophagic process. By using confocal microscopy and Western blotting experiments, we demonstrated that uL3 acts as inhibitory factor of autophagic process; the absence of uL3 is associated to increase of autophagic flux and to chemoresistance. Furthermore, experiments conducted in presence of chloroquine, a known inhibitor of autophagy, indicate a role of uL3 in chloroquine-mediated inhibition of autophagy. On the basis of these results and our previous findings, we hypothesize that the absence of uL3 in cancer cells might inhibit cancer cell response to drug treatment through the activation of cytoprotective autophagy. The restoration of uL3 could enhance the activity of many drugs thanks to its pro-apoptotic and anti-autophagic activity.

Identifying and Supporting Students to Prevent USMLE Step 1 Failures When Testing Follows Clerkships: Insights From 9 Schools

Several schools have moved the United States Medical Licensing Examination Step 1 exam after core clerkships, and others are considering this change. Delaying Step 1 may improve Step 1 performance and lower Step 1 failure rates. Schools considering moving Step 1 are particularly concerned about late identification of struggling students and late Step failures, which can be particularly problematic due to reduced time to remediate and accumulated debt if remediation is ultimately unsuccessful. In the literature published to date, little attention has been given to these students. In this article, authors from 9 medical schools with a postclerkship Step 1 exam share their experiences. The authors describe curricular policies, early warning and identification strategies, and interventions to enhance success for all students and struggling students in particular. Such learners can be identified by understanding challenges that place them "at risk" and by tracking performance outcomes, particularly on other standardized assessments. All learners can benefit from early coaching and advising, mechanisms to ensure early feedback on performance, commercial study tools, learning specialists or resources to enhance learning skills, and wellness programs. Some students may need intensive tutoring, neuropsychological testing and exam accommodations, board preparation courses, deceleration pathways, and options to postpone Step 1. In rare instances, a student may need a compassionate off-ramp from medical school. With the National Board of Medical Examiner's announcement that Step 1 scoring will change to pass/fail as early as January 2022, residency program directors might use failing Step 1 scores to screen out candidates. Institutions altering the timing of Step 1 can benefit from practical guidance by those who have made the change, to both prevent Step 1 failures and minimize adverse effects on those who fail.

Computational Modelling Framework to Study Ca2+ Activation of Synaptic Vesicle Fusion by Different Synaptotagmin Isoforms

Controlled release of neurotransmitters stored in synaptic vesicles (SVs) is central to information processing in the brain. This process relies on fast (sub-millisecond) coupling of SV fusion to the triggering signal - action potential (AP)-evoked presynaptic Ca2+ influx. During the last three decades the key proteins that mediate SV exocytosis have been identified. Yet,the molecular mechanisms that allow synchronisation of SV fusion to APs remain in the centre of debate. SV fusion is catalysed by synaptic SNARE proteins, VAMP2 on the vesicles (v-SNAREs) and syntaxin/SNAP25 on the pre-synaptic membrane (t-SNAREs). The current view is that the SNARE assembly process is arrested at a ‘half-zippered’ state to prevent continuous release of neurotransmitters and to allow for rapid and synchronous SV fusion following the Ca2+ influx. The key players that ‘clamp’ vesicle release and synchronise the late SV fusion steps are complexin proteins and isoforms of the presynaptic Ca2+ release sensor synaptotagmin (Syt). Here we describe a computational modelling framework that allows one to simulate AP-evoked synchronous and asynchronous release controlled by Syt1 and Syt7 isoforms under different conditions. The model is based on the recent structural data describing interaction of Syt1 with the SNARE complex and complexin. Ca2+ activation of SV fusion is simulated using the well-established Ca2+ and membrane-binding properties of Syt1 and Syt7 C2 domains. Conformational protein changes are represented by transitions between discrete states in a Markovian system, with parameters constrained by the known results for Ca2+ and membrane binding affinities. Our stochastic simulations demonstrate that this relatively simple model can successfully capture the kinetics and Ca2+ dependency of synchronous and asynchronous neurotransmitter release in central synapses.

Beyond Optical Depth: Future Determination of Ionization History from the Cosmic Microwave Background.

We explore the fundamental limits to which reionization histories can be constrained using only large-scale cosmic microwave background (CMB) anisotropy measurements. The redshift distribution of the fractional ionization x e (z) affects the angular distribution of CMB polarization. We project constraints on the reionization history of the universe using low-noise full-sky temperature and E-mode measurements of the CMB. We show that the measured TE power spectrum, , has roughly one quarter of the constraining power of on the reionization optical depth τ, and its addition improves the precision on τ by 20% over using only. We also use a two-step reionization model with an additional high-redshift step, parameterized by an early ionization fraction , and a late reionization step at z re. We find that future high signal-to-noise measurements of the multipoles 10 ⩽ ℓ < 20 are especially important for breaking the degeneracy between and z re. In addition, we show that the uncertainties on these parameters determined from a map with sensitivity 10 μK arcmin are less than 5% larger than the uncertainties in the noiseless case, making this noise level a natural target for future large sky area E-mode measurements.

Real-Time BODIPY-Binding Assay To Screen Inhibitors of the Early Oligomerization Process of Aβ1-42 Peptide.

Misfolding and aggregation of amyloid β1–42 peptide (Aβ1–42) play a central role in the pathogenesis of Alzheimer's disease (AD). Targeting the highly cytotoxic oligomeric species formed during the early stages of the aggregation process represents a promising therapeutic strategy to reduce the toxicity associated with Aβ1–42. Currently, the thioflavin T (ThT) assay is the only established spectrofluorometric method to screen aggregation inhibitors. The success of the ThT assay is that it can detect Aβ1–42 aggregates with high β‐sheet content, such as protofibrils or fibrils, which appear in the late aggregation steps. Unfortunately, by using the ThT assay, the detection of inhibitors of early soluble oligomers that present a low β‐sheet character is challenging. Herein, a new, facile, and robust boron‐dipyrromethene (BODIPY) real‐time assay suitable for 96‐well plate format, which allows screening of compounds as selective inhibitors of the formation of Aβ1–42 oligomers, is reported. These inhibitors decrease the cellular toxicity of Aβ1–42, although they fail in the ThT assay. The findings have been confirmed and validated by structural analysis and cell viability assays under comparable experimental conditions. It is demonstrated that the BODIPY assay is a convenient method to screen and discover new candidate compounds that slow down or stop the pathological early oligomerization process and are active in the cellular assay. Therefore, it is a suitable complementary screening method of the current ThT assay.

Aggregation of disease-related peptides.

Protein misfolding and aggregation of amyloid proteins is the fundamental cause of more than 20 diseases. Molecular mechanisms of the self-assembly and the formation of the toxic aggregates are still elusive. Computer simulations have been intensively used to study the aggregation of amyloid peptides of various amino acid lengths related to neurodegenerative diseases. We review atomistic and coarse-grained simulations of short amyloid peptides aimed at determining their transient oligomeric structures and the early and late aggregation steps.

Tubulin-binding cofactor E-like (TBCEL), the protein product of the mulet gene, is required in the germline for the regulation of inter-flagellar microtubule dynamics during spermatid individualization.

ABSTRACTIndividual sperm cells are resolved from a syncytium during late step of spermiogenesis known as individualization, which is accomplished by an Individualization Complex (IC) composed of 64 investment cones. mulet encodes Tubulin-binding cofactor E-like (TBCEL), suggesting a role for microtubule dynamics in individualization. Indeed, a population of ∼100 cytoplasmic microtubules fails to disappear in mulet mutant testes during spermatogenesis. This persistence, detected using epi-fluorescence and electron microscopy, suggests that removal of these microtubules by TBCEL is a prerequisite for individualization. Immunofluorescence reveals TBCEL expression in elongated spermatid cysts. In addition, testes from mulet mutant males were rescued to wild type using tubulin-Gal4 to drive TBCEL expression, indicating that the mutant phenotype is caused by the lack of TBCEL. Finally, RNAi driven by bam-GAL4 successfully phenocopied mulet, confirming that mulet is required in the germline for individualization. We propose a model in which the cytoplasmic microtubules serve as alternate tracks for investment cones in mulet mutant testes.This article has an associated First Person interview with the first author of the paper.

Characterization of Glaciecola sp. enzymes involved in the late steps of degradation of sulfated polysaccharide ulvan extracted from Ulva ohnoi

Ulvan is a complex water-soluble sulfated polysaccharide in the cell wall of green algae belonging to genus Ulva. It is composed of l-rhamnose-3-sulfate (Rha3S), glucuronic acid (GluA), iduronic acid (IduA), and d-xylose (Xyl) distributed in three repetition moieties. The first step of a bacterial ulvan degradation is the cleavage of the β-glycosidic bond between Rha3S and GluA/IduA through a β-elimination mechanism by a ulvan lyase to produce oligo-ulvans with unsaturated 4-deoxy-L-threo-hex-4-enopyranosiduronate (Δ) at the non-reducing end. We have identified an ulvan associated polysaccharide utilization locus (PUL) residing between two ulvan lyase genes belonging to families of polysaccharide lyase 24 (PL24) and PL25 in the genome of a ulvan-utilizing bacterium Glaciecola KUL10 strain. The PUL contains many genes responsible for oligo-ulvan degradation. Among them, we demonstrated that both KUL10_26540 and KUL10_26770 had an unsaturated β-glucuronyl hydrolase activity to produce Rha3S and oligosaccharides, such as Rha3S-GluA-Rha3S, Rha3S-IduA-Rha3S and, Rha3S-Xyl-Rha3S, by releasing 5-dehydro-4-deoxy-d-glucuronate. KUL10_26540 showed much higher activity than KUL10_26770 and was more active on disaccharide than tetrasaccharide. We also found a rhamnosidase activity on four KUL10 gene products, although they could not react on the sulfated rhamnose.

Art in Endoscopy: “The Whale”

This is a sessile serrated lesion after submucosal dye injection, just before endoscopic mucosal resection. The prominent adenomatous nodule reveals progression to cytologic dysplasia, which is a late step in the serrated neoplasia pathway to colorectal cancer. Often, an adenomatous nodule in a polyp is mistaken for a small tubular adenoma by the endoscopist, while the surrounding nondysplastic area of sessile serrated lesion is easily overlooked because of its subtle appearance. This might lead to resection of only the adenomatous nodule, leaving in situ the rest of the lesion.