Arv1 Regulates PM and ER Membrane Structure and Homeostasis But is Dispensable for Intracellular Sterol Transport

The pan‐eukaryotic endoplasmic reticulum (ER) membrane protein Arv1 has been suggested to play a role in intracellular sterol transport. We tested this proposal by comparing sterol traffic in wild‐type and Arv1‐deficient Saccharomyces cerevisiae. We used fluorescence microscopy to track the retrograde movement of exogenously supplied dehydroergosterol (DHE) from the plasma membrane (PM) to the ER and lipid droplets and high performance liquid chromatography to quantify, in parallel, the transport‐coupled formation of DHE esters. Metabolic labeling and subcellular fractionation were used to assay anterograde transport of ergosterol from the ER to the PM. We report that sterol transport between the ER and PM is unaffected by Arv1 deficiency. Instead, our results indicate differences in ER morphology and the organization of the PM lipid bilayer between wild‐type and arv1Δ cells suggesting a distinct role for Arv1 in membrane homeostasis. In arv1Δ cells, specific defects affecting single C‐terminal transmembrane domain proteins suggest that Arv1 might regulate membrane insertion of tail‐anchored proteins involved in membrane homoeostasis .     

Improved Detection of Remote Homologues Using Cascade PSI-BLAST: Influence of Neighbouring Protein Families on Sequence Coverage

Background

Development of sensitive sequence search procedures for the detection of distant relationships between proteins at superfamily/fold level is still a big challenge. The intermediate sequence search approach is the most frequently employed manner of identifying remote homologues effectively. In this study, examination of serine proteases of prolyl oligopeptidase, rhomboid and subtilisin protein families were carried out using plant serine proteases as queries from two genomes including A. thaliana and O. sativa and 13 other families of unrelated folds to identify the distant homologues which could not be obtained using PSI-BLAST.

Methodology/Principal Findings

We have proposed to start with multiple queries of classical serine protease members to identify remote homologues in families, using a rigorous approach like Cascade PSI-BLAST. We found that classical sequence based approaches, like PSI-BLAST, showed very low sequence coverage in identifying plant serine proteases. The algorithm was applied on enriched sequence database of homologous domains and we obtained overall average coverage of 88% at family, 77% at superfamily or fold level along with specificity of ∼100% and Mathew’s correlation coefficient of 0.91. Similar approach was also implemented on 13 other protein families representing every structural class in SCOP database. Further investigation with statistical tests, like jackknifing, helped us to better understand the influence of neighbouring protein families.

Conclusions/Significance

Our study suggests that employment of multiple queries of a family for the Cascade PSI-BLAST searches is useful for predicting distant relationships effectively even at superfamily level. We have proposed a generalized strategy to cover all the distant members of a particular family using multiple query sequences. Our findings reveal that prior selection of sequences as query and the presence of neighbouring families can be important for covering the search space effectively in minimal computational time. This study also provides an understanding of the ‘bridging’ role of related families.     

Factors,mechanisms and implications of chromatin condensation and chromosomal structural maintenance through the cell cycle

A series of well-orchestrated events help in the chromatin condensation and the formation of chromosomes. Apart from the formation of chromosomes, maintenance of their structure is important, especially for the cell division. The structural maintenance of chromosome (SMC) proteins, the non-SMC proteins and the SMC complexes are critical for the maintenance of chromosome structure. While condensins have roles for the DNA compaction, organization, and segregation, the cohesin functions in a cyclic manner through the cell cycle, as a “cohesin cycle.” Specific mechanisms maintain the architecture of the centromere, the kinetochore and the telomeres which are in tandem with the cell cycle checkpoints. The presence of chromosomal territories and compactness differences through the length of the chromosomes might have implications on selective susceptibility of specific chromosomes for induced genotoxicity.     

Environmental Factors Associated with Disease Progression after the First Demyelinating Event: Results from the Multi-Center SET Study

Objectives

To investigate the associations of environmental MS risk factors with clinical and MRI measures of progression in high-risk clinically isolated syndromes (CIS) after the first demyelinating event.

Methods

We analyzed 211 CIS patients (age: 28.9±7.8 years) enrolled in the SET study, a multi-center study of high-risk CIS patients. Pre-treatment samples were analyzed for IgG antibodies against cytomegalovirus (anti-CMV), Epstein Barr virus (EBV) early nuclear antigen-1 (EBNA-1), viral capsid antigen (VCA), early antigen-diffuse (EA-D), 25 hydroxy-vitamin D3 and cotinine levels and HLA DRB1*1501 status. The inclusion criteria required evaluation within 4 months of the initial demyelinating event, 2 or more brain MRI lesions and the presence of two or more oligoclonal bands in cerebrospinal fluid. All patients were treated with interferon-beta. Clinical and MRI assessments were obtained at baseline, 6, 12, and 24 months.

Results

The time to first relapse decreased and the number of relapses increased with anti-CMV IgG positivity. Smoking was associated with increased number and volume of contrast-enhancing lesions (CEL) during the 2-year period. The cumulative number of CEL and T2 lesions during the 2-year period was greater for individuals in the highest quartile of anti-EBV VCA IgG antibodies. The percent loss of brain volume was increased for those in the highest quartile of with anti-EBV VCA IgG antibodies.

Conclusions

Relapses in CIS patients were associated with CMV positivity whereas anti-EBV VCA positivity was associated with progression on MRI measures, including accumulation of CEL and T2 lesions and development of brain atrophy.     

Low‐Overpotential Electrocatalytic Water Splitting with Noble‐Metal‐Free Nanoparticles Supported in a sp3 N‐Rich Flexible COF

Covalent organic frameworks (COFs) are crystalline organic polymers with tunable structures. Here, a COF is prepared using building units with highly flexible tetrahedral sp³ nitrogens. This flexibility gives rise to structural changes which generate mesopores capable of confining very small (<2 nm sized) non‐noble‐metal‐based nanoparticles (NPs). This nanocomposite shows exceptional activity toward the oxygen‐evolution reaction from alkaline water with an overpotential of 258 mV at a current density of 10 mA cm^?2. The overpotential observed in the COF‐nanoparticle system is the best in class, and is close to the current record of ≈200 mV for any noble‐metal‐free electrocatalytic water splitting system—the Fe–Co–Ni metal‐oxide‐film system. Also, it possesses outstanding kinetics (Tafel slope of 38.9 mV dec^?1) for the reaction. The COF is able to stabilize such small‐sized NP in the absence of any capping agent because of the COF–Ni(OH)₂ interactions arising from the N‐rich backbone of the COF. Density‐functional‐theory modeling of the interaction between the hexagonal Ni(OH)₂ nanosheets and the COF shows that in the most favorable configuration the Ni(OH)₂ nanosheets are sandwiched between the sp³ nitrogens of the adjacent COF layers and this can be crucial to maximizing their synergistic interactions.     

B Cell,Th17, and Neutrophil Related Cerebrospinal Fluid Cytokine/Chemokines Are Elevated in MOG Antibody Associated Demyelination

Background

Myelin oligodendrocyte glycoprotein antibody (MOG Ab) associated demyelination represents a subgroup of autoimmune demyelination that is separate from multiple sclerosis and aquaporin 4 IgG-positive NMO, and can have a relapsing course. Unlike NMO and MS, there is a paucity of literature on immunopathology and CSF cytokine/chemokines in MOG Ab associated demyelination.

Aim

To study the differences in immunopathogenesis based on cytokine/chemokine profile in MOG Ab-positive (POS) and -negative (NEG) groups.

Methods

We measured 34 cytokines/chemokines using multiplex immunoassay in CSF collected from paediatric patients with serum MOG Ab POS [acute disseminated encephalomyelitis (ADEM = 8), transverse myelitis (TM = 2) n = 10] and serum MOG Ab NEG (ADEM = 5, TM = 4, n = 9) demyelination. We generated normative data using CSF from 20 non-inflammatory neurological controls.

Results

The CSF cytokine and chemokine levels were higher in both MOG Ab POS and MOG Ab NEG demyelination groups compared to controls. The CSF in MOG Ab POS patients showed predominant elevation of B cell related cytokines/chemokines (CXCL13, APRIL, BAFF and CCL19) as well as some of Th17 related cytokines (IL-6 AND G-CSF) compared to MOG Ab NEG group (all p<0.01). In addition, patients with elevated CSF MOG antibodies had higher CSF CXCL13, CXCL12, CCL19, IL-17A and G-CSF than patients without CSF MOG antibodies.

Conclusion

Our findings suggest that MOG Ab POS patients have a more pronounced CNS inflammatory response with elevation of predominant humoral associated cytokines/chemokines, as well as some Th 17 and neutrophil related cytokines/chemokines suggesting a differential inflammatory pathogenesis associated with MOG antibody seropositivity. This cytokine/chemokine profiling provides new insight into disease pathogenesis, and improves our ability to monitor inflammation and response to treatment. In addition, some of these molecules may represent potential immunomodulatory targets.     

Alirocumab,a Therapeutic Human Antibody to PCSK9, Does Not Affect CD81 Levels or Hepatitis C Virus Entry and Replication into Hepatocytes

BackgroundProprotein convertase subtilisin/kexin type 9 (PSCK9) is secreted mainly from the liver and binds to the low-density lipoprotein receptor (LDLR), reducing LDLR availability and thus resulting in an increase in LDL-cholesterol. While the LDLR has been implicated in the cell entry process of the hepatitis C virus (HCV), overexpression of an artificial non-secreted, cell membrane-bound form of PCSK9 has also been shown to reduce surface expression of CD81, a major component of the HCV entry complex, leading to concerns that pharmacological inhibition of PCSK9 may increase susceptibility to HCV infection by increasing either CD81 or LDLR availability. Here, we evaluated effects of PCSK9 and PCSK9 blockade on CD81 levels and HCV entry with a physiologically relevant model using native secreted PCSK9 and a monoclonal antibody to PCSK9, alirocumab.ConclusionThese results suggest that inhibition of PCSK9 with alirocumab has no effect on CD81 and does not result in increased susceptibility to HCV entry.     

A 12-Crown-4 Ether Containing Dipeptide Boc-12-Crown-4-<Emphasis Type="SmallCaps">l</Emphasis>-DOPA-Gly-OMe Induces Cell Cycle Arrest and Apoptosis in Rat Eggs Cultured In Vitro

The study was designed to investigate whether crown ether containing dipeptide Boc-12-crown-4-l-DOPA-Gly-OMe has potential to induce meiotic cell cycle arrest and apoptosis in rat eggs cultured in vitro. The immature female rats were subjected to superovulation induction protocol and ovulated eggs were collected from ampulla of the fallopian tube. Ovulated eggs arrested at metaphase-II (M-II) stage of meiotic cell cycle were cultured in media-199 with or without various concentrations (0.0, 0.025, 0.050, 0.10, and 0.20 mM) of dipeptide for 3 h in vitro. Morphological apoptotic changes, hydrogen peroxide (H₂O₂) concentration, cytochrome c level, caspase-3 level as well as activity and DNA fragmentation were analysed in eggs cultured in vitro. Culture of M-II arrested eggs in plain medium for 3 h in vitro induced meiotic exit from M-II arrest in majority of eggs as evidenced by initiation of extrusion of second polar body (II PB). The dipeptide induced maintenance of M-II arrest and morphological apoptotic features in a concentration-dependent manner prior to degeneration. The dipeptide-induced morphological features were associated with increased H₂O₂ and cytochrome c levels in treated eggs. The increased cytochrome c induced caspase-3 level and activity and thereby DNA fragmentation as evidenced by DAB positive staining in treated eggs. Our results suggest that dipeptide Boc-12-C-4-l-DOPA-Gly-OMe induces cell cycle arrest at M-II stage and apoptosis in rat eggs cultured in vitro.     

The characteristics of antibiotic resistance and phenotypes in 29 outer-membrane protein mutant strains in Aeromonas hydrophila

Although many typical outer-membrane proteins (OMPs) have been well characterized, the biological functions of many OMPs remain largely elusive. In this study, we successfully constructed 29 OMP knockout strains in the pathogen Aeromonas hydrophila, which account for about 50% of all predicted OMPs in this bacterial species. We then further validated the antibiotics' susceptibility characteristics against 20 antimicrobial reagents in these mutants considering several phenotypes. Our results showed that a total of 22 OMP mutants affected the susceptibility to at least one antibiotic. The deletion of some OMPs, such as ΔlamB and ΔbamA, revealed very important roles in the resistance to certain antibiotics. However, not a single OMP mutant presented a constant behaviour to all of the tested antibiotics, suggesting the existence of a complex intercellular regulation mechanism and a protein–protein interaction network underlying the OMP homeostasis in the presence of antibiotics. Meanwhile, some OMP mutants also affected biofilm formation, ECPase and haemolytic activity, and carbon resources utilization. This report demonstrates the biological functions of OMPs on a large scale and most of results have not been reported in A. hydrophila.