Analysis of the molecular determinants for furin cleavage of the spike protein S1/S2 site in defined strains of the prototype coronavirus murine hepatitis virus (MHV).

We have analyzed the spike protein S1/S2 cleavage site of selected strains of MHV by the cellular protease furin, in order to understand the structural requirements underlying the sequence selectivity of the scissile segment. The probability of cleavage of the various MHV strains was first evaluated from furin cleavage scores predicted by the ProP computer software, and then cleavage was measured experimentally with a fluorogenic peptide cleavage assay consisting of S1/S2 peptide mimics and purified furin. We found that in vitro cleavability varied across MHV strains in line with predicted results-but with the notable exception of MHV-A59, which was not cleaved despite a high score predicted for its sequence. Using the known X-Ray structure of furin in complex with a substrate-like inhibitor as an initial structural reference, we carried out molecular dynamics (MD) simulations to learn the modes of binding of the peptides in the furin active site, and the suitability of the complex for initiation of the enzymatic cleavage. We thus identified the 3D structural requirements of the furin active site configuration that enable bound peptides to undergo cleavage, and the way in which the various strains tested experimentally are fulfilling these requirements. We find that despite some flexibility in the organization of the peptide bound to the active site of the enzyme, the presence of a histidine at P2 of MHV-A59 fails to properly orient the sidechain of His194 of the furin catalytic triad and therefore produces a distortion that renders the peptide/complex structural configuration in the active site incompatible with requirements for cleavage initiation. The Ser/Thr in P1 of MHV-2 and MHV-S has a similar effect of distorting the conformation of the furin active site residues produced by the elimination of the canonical salt-bridge formed by arginine in P1 position. This work informs a study of coronavirus infection and pathogenesis with respect to the function of the viral spike protein, and suggests an important process of viral adaptation and evolution within the spike S1/S2 structural loop.

Lingacom muography.

Lingacom Ltd develops detectors for muography-imaging using cosmic-ray muons-together with imaging algorithms and tools. We present selected simulation results from muon imaging of cargo conta- iners, from a joint muon and X-ray imaging algorithm, and for ground surveys using borehole detectors.This article is part of the Theo Murphy meeting issue 'Cosmic-ray muography'.

Topical pimecrolimus for paediatric cutaneous mastocytosis.

BACKGROUND: Most cases of paediatric cutaneous mastocytosis (CM) occur before the age of 2 years, and regression occurs in only 67% of children. Given the absence of any specific therapy, CM is usually treated symptomatically. A few publications have reported the beneficial effect of calcineurin inhibitors for CM. AIM: We sought to evaluate the clinical effectiveness and safety profile of topical pimecrolimus cream for the treatment of CM. METHODS: We performed a retrospective study of all diagnosed cases of CM treated with topical pimecrolimus 1% cream between 2013 and 2015. All patients were evaluated in a paediatric dermatology unit of a tertiary medical centre. Epidemiological, clinical and treatment data, including effectiveness and safety, were reviewed. RESULTS: In total, 18 children (11 male, 7 female; age range 3-42 months) with CM were evaluated. Of the 146 treated lesions, 39 (26.7%) disappeared and 98 (67%) faded or developed postinflammatory hyperpigmentation. Of the 119 papular lesions, 24 (16.4%) showed partial flattening and 56 (47%) became macular. Darier sign became negative in 14 (82%) of 17 patients. No topical or systemic complications were observed. CONCLUSIONS: Topical therapy with pimecrolimus 1% cream should be considered in the treatment of CM.

Isotretinoin treatment of autosomal recessive congenital ichthyosis complicated by coexisting dysferlinopathy.

Consanguinity is known to be associated with an increase in the prevalence of autosomal recessive disorders such as autosomal recessive congenital ichthyosis (ARCI). ARCI often responds well to retinoid treatment. We describe a patient with ARCI who improved under isotretinoin treatment. The patient subsequently developed elevated levels of serum creatinine phosphokinase (CPK), which led to the diagnosis of a second autosomal recessive disorder, dysferlinopathy, a rare myopathy characterized by muscle weakness, decreased tendon reflexes and marked elevation of CPK levels. This report demonstrates the need for physicians to remain alert to the possible coexistence of rare and mutually relevant disorders in populations with a high rate of consanguinity.

Non-syndromic autosomal recessive congenital ichthyosis in the Israeli population.

BACKGROUND: Autosomal recessive congenital ichthyosis (ARCI) is the term given to a complex and heterogeneous group of cornification disorders associated with mutations in at least eight distinct genes. Mutation distribution and prevalence rates are instrumental for the design of diagnostic strategies in ARCI but have not yet been systematically explored in the Israeli population. Previous data suggest that the demographic features specific to Middle Eastern populations, such as a high frequency of consanguineous marriages, may have an effect on the molecular epidemiology of genodermatoses. METHODS: We systematically assessed all families with ARCI presenting at our clinics over a period of 9 years, using a combination of homozygosity mapping, direct sequencing and PCR-restriction fragment length polymorphism assays. RESULTS: In total, 20 families with ARCI were assessed, and causative mutations were identified in 7 genes: TGM1 (30% of patients), ALOX12B (20%), ABCA12 (5%), CYP4F22 (10%), ALOXE3 (10%), LIPN (5%) and NIPAL4 (5%) Two families (10%) had mutations mapped to an ARCI-associated locus on 12p11.2-q13, while no mutation was found for one additional kindred. In the subgroup of families of Arab Muslim origin, mutations were identified most frequently in ALOX12B and TGM1 (31%), whereas the other subgroups displayed a subtype distribution very similar to that previously reported in western populations. CONCLUSIONS: The present data point to the need for population-tailored mutation screening strategies in genetically heterogeneous genodermatoses, based on the relative prevalence of the disease subsets.

Type 2A phosphoprotein phosphatase is required for asexual development and pathogenesis of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a necrotrophic, omnivorous plant pathogen with worldwide distribution. Sclerotia of S. sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Plant infection depends on the formation of melanin-rich infection cushions, and secretion of hydrolytic enzymes and oxalic acid. Type 2A Ser/Thr phosphatases (PP2As) are involved in the regulation of a variety of cellular process. In the presence of cantharidin, a PP2A-specific inhibitor, hyphal elongation and sclerotia numbers were impaired whereas sclerotial size increased. We partially inactivated PP2A by antisense expression of the gene (pph1) encoding the PP2A catalytic subunit. When antisense expression was induced, almost complete cessation of fungal growth was observed, indicative of a crucial role for PP2A in fungal growth. RNAi-based gene silencing was employed to alter the expression of the 55-kDa R2 (B regulatory subunit). Isolates in which rgb1 RNA levels were decreased were slow growing, but viable. Melanin biosynthesis, infection-cushion production, and pathogenesis were significantly impaired in the rgb1 mutants, yet theses mutants were pathogenic on wounded leaves. Reduced ERK (extracellular signal-regulated kinases)-like mitogen-activated protein kinase (MAPK) function conferred a reduction in NADPH oxidase and PP2A activity levels, suggesting a functional link between MAPK, reactive oxygen species, and PP2A activity in S. sclerotiorum.

Calcineurin is required for sclerotial development and pathogenicity of Sclerotinia sclerotiorum in an oxalic acid-independent manner.

Sclerotinia sclerotiorum is a necrotrophic, omnivorous plant pathogen with worldwide distribution. Sclerotia of S. sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Calcineurin, a Ser/Thr phosphatase linked to several signal-transduction pathways, plays a key role in the regulation of cation homeostasis, morphogenesis, cell-wall integrity, and pathogenesis in fungi. We demonstrate that calcineurin expression in S. sclerotiorum is altered in a phase-specific manner during sclerotial development. Inhibition of calcineurin by FK506, cysclosporin A, or inducible antisense calcineurin expression impaired sclerotial development at the prematuration phase and increased germination of preformed sclerotia. Induction of antisense calcineurin expression in S. sclerotiorum resulted in reduced pathogenesis on tomato and Arabidopsis. However, secretion of oxalic acid, a key virulence factor of S. sclerotiorum, was not altered. Inhibition of calcineurin conferred a reduction in cell wall beta-1,3-glucan content and increased sensitivity to cell-wall-degrading enzymes and to the glucan synthase inhibitor caspofungin. Thus, calcineurin plays a major role in both sclerotial development and pathogenesis of S. sclerotiorum and, most likely, other phytopathogens.

Minocycline-induced cutaneous hyperpigmentation: confocal laser scanning microscope analysis.

BACKGROUND: Minocycline has a characteristic yellow-green fluorescent emission. This fluorescence has been previously demonstrated only in type 1 minocycline-induced skin hyperpigmentation. OBJECTIVE: To investigate whether the fluorescence can be detected in other types of minocycline-induced cutaneous hyperpigmentation, and to study the possible mechanisms. METHODS: Biopsies of pigmented and nonpigmented skin from 3 patients with different types of skin hyperpigmentation induced by minocycline were analysed by light microscopy and Confocal Laser Scanning Microscope (CLSM). RESULTS: A yellow-green fluorescence was observed in the hyperpigmented skin of two patients with type 2, and one patient with type 4 minocycline-induced cutaneous hyperpigmentation. No fluorescence was detected in the non-pigmented skin. CONCLUSION: Minocycline can possibly serve as a fluorescent probe in the diagnosis of all types of minocycline-induced cutaneous hyperpigmentation.

Changes in Protein Kinase A Activity Accompany Sclerotial Development in Sclerotinia sclerotiorum.

ABSTRACT Sclerotia of Sclerotinia sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Sclerotial formation has been shown to be affected by increased intracellular cAMP levels. Cyclic AMP (cAMP) is a key modulator of cAMP-dependent protein kinase A (PKA) and the latter may prove to play a significant role in sclerotial development. Therefore, we monitored changes in relative PKA activity levels during sclerotial development. To do so, we first developed conditions for near-synchronous sclerotial development in culture, based on hyphal maceration and filtering. Relative PKA activity levels increased during the white-sclerotium stage in the wild-type strain, while low levels were maintained in nonsclerotium-producing mutants. Furthermore, applying caffeine, an inducer of PKA activity, resulted in increased relative PKA activity levels and was correlated with the formation of sclerotial initial-like aggregates in cultures of the non-sclerotium-producing mutants. In addition, low PKA activities were found in an antisense smk1 strain, which exhibits low extracellular-signal-regulated kinase (ERK)-type mitogen-activated protein kinase (MAPK) activity, and does not produce sclerotia. The changes in PKA activity, as well as the abundance of phosphorylated MAPKs (ERK-like as well as p38-like) that accompany sclerotial development in a distinct developmental phase manner represent a potential target for antifungal intervention.

Differentiation of cultured mice bone marrow into osteoblast-like cells results in acquisition of sex-specific responsiveness to gonadal steroids.

We have previously demonstrated that mouse skeletal tissue, rat bone as well as rat or human derived bone cells in culture, show a sex-specific response to gonadal steroids in stimulation of the specific activity of the BB isozyme of creatine kinase (CK). This response could be modified by manipulation of the endocrine environment during early postnatal development. Moreover, pretreatment with vitamin D up-regulated the sex-specific responsiveness and sensitivity to gonadal steroids. In the present study we examine the differentiation pattern into osteoblast-like cells using dexamethasone (DEX) and 1,25 dihydroxy vitamin D3 (1,25D) and their effect on the acquisition of responsiveness to gonadal steroids by the differentiated cells. Cultured femoral bone marrow in the presence of DEX or 1,25D or both, were examined for their response to gonadal steroids by measuring the specific activities of alkaline phosphatase (AP) and CK BB. The constitutive level of CK in both male- and female-derived bone cells was decreased by DEX, by 1,25D or by both, whereas the constitutive level of AP was increased by DEX while decreased by 1,25D or by both. Following incubation of the bone marrow cultures with DEX, treatment with estradiol 17beta (E2, 30 nM, 24 h) stimulated CK activity in female derived bone cells, with no effect of treatment with dihydrotestosterone (DHT, 300 nM). In contrast, in male derived bone cells, DHT but not E2 increased CK activity. This sex-specific response was also achieved upon culturing with 1,25D and was significantly augmented by culturing with both. No response to gonadal steroids was seen with undifferentiated bone marrow cells. All cultures responded to IGF-I when cultured with or without DEX and/or 1,25D but with no augmentation by 1,25D. Gonadal steroids increased AP to a much lesser extent; but enzyme activity decreased in the presence of 1,25D. IGF-I stimulated AP slightly with no effect of 1,25D. These findings suggest that manipulation of the hormonal milieu in early stages of differentiation sequence of osteoblast-like cells, determines the subsequent selective responsiveness of the developing bone tissue to gonadal steroids.

Surveillance of methicillin-resistant Staphylococcus aureus (MRSA) and Enterobacteriaceae producing extended-spectrum beta-lactamase (ESBLE) in Northern France: a five-year multicentre incidence study.

In order to measure the incidence of methicillin-resistant Staphylococcus aureus (MRSA) and of Enterobacteriaceae producing extended-spectrum beta-lactamase (ESBLE), and to evaluate the impact of the national guidelines for multidrug-resistant bacteria (MDRB) prevention in hospitals of Northern France, a multicentre study was conducted for three months every year starting in 1996, in volunteer hospital laboratories. All clinical specimens positive for MRSA and ESBLE were prospectively surveyed. During the five-year surveillance period, the overall proportion of MRSA was 38.4% in the 28,534 strains of S. aureus, and that of ESBLE was 11.4% in the 6121 strains of Klebsiella pneumoniae and 47.7% in the 2353 strains of Enterobacter aerogenes. The overall incidence rates of clinical specimens positive for MRSA, ESBL-K. pneumoniae and E. aerogenes were 0.84. 0.05 and 0.12/1000 hospital-days (HD), respectively. In the 23 hospitals that participated in the survey every year, the proportion and incidence of ESBLE decreased. Hence, despite recommendations as for isolation precautions, MRSA remains poorly controlled and requires more effective measures.

The hormonal milieu in early stages of bone cell differentiation modifies the subsequent sex-specific responsiveness of the developing bone to gonadal steroids.

We have established previously that rat bone tissue, as well as rat and human-derived bone cells in culture, show a sex-specific response to gonadal steroids in stimulation of the specific activity of the BB isozyme of creatine kinase (CK) and DNA synthesis. This response could be modified by manipulation of the endocrine environment during early stages in rat development. To further examine the influence of changing hormonal steroid milieu and vitamin D status on the action of gonadal steroids in developing bone tissue, we used two models of ectopic bone formation: demineralized tooth matrix (DTM) implanted under the skin, and femoral bone marrow (BM) transplanted under the kidney capsule of a syngeneic recipient mouse. The response to gonadal steroids in ossicles developed from implanted DTM depended on the recipient's gender; injection of estradiol 17beta (E2; 5 microg) into young female mice 21 days after DTM implantation increased, 24 h later, CK activity in the newly formed ossicles by approximately 60%, whereas injection of dihydrotestosterone (DHT; 50 microg) had no effect on CK activity. In contrast, in male mice, DHT but not E2 increased CK activity in the ossicles by approximately 50%. This sex-specific response was abolished in gonadectomized mice resulting in a similar response of the ossicles to both E2 and DHT. When DTM was implanted into vitamin D- deficient female mice, there was a lower basal CK activity and a significantly diminished response to E2 in the newly formed bone tissues. When BM, which contains mesenchymal and stromal cells and committed osteoprogenitor cells, was transplanted into 6-week-old intact or gonadectomized female or male mice, the response of the newly formed bone ossicles, 21 days after transplantation, to E2 or to DHT was according to the gender of the donor. Bone formed from BM obtained from female mice responded to E2 only and those formed from male BM responded to DHT only. Ossicles developed from BM obtained from gonadectomized mice showed lack of response to either gonadal steroid. Furthermore, only approximately 25% of the BM transplants obtained from castrated (CAST) male donors developed into ossicles. Ossicles formed from BM obtained from vitamin D-deficient female donors showed lack of response to gonadal steroids. These findings suggest that the manipulation of the hormonal milieu in early stages of the differentiation sequence of bone cells modifies the subsequent selective responsiveness of the developing bone tissue to gonadal steroids.

Clinical significance of markedly elevated serum creatine kinase levels in patients with acne on isotretinoin.

Muscle-related complaints and high creatine kinase (CK) blood levels have been reported in 16-51% of patients with acne treated with isotretinoin. It has been suggested that this retinoid and exercise have a synergistic effect on muscle. The presence of marked hyperCKemia during the treatment raises concern about rhabdomyolysis. The objective of this report was to evaluate the incidence, course and clinical significance of severe hyperCKemia in isotretinoin therapy for acne. Out of 442 patients on isotretinoin, we reviewed 7 patients (1.58%) with CK values above 5,000 IU/l. Only two of them had myalgia. Physical activity or intramuscular injection prior to blood testing was reported in 6 patients. CK values returned to normal within 2 weeks and all subjects except 2, completed treatment. In conclusion, marked hyperCKemia with or without muscle-related complaints in isotretinoin-treated patients with acne is a benign phenomenon.

Review: nuclear lamins--structural proteins with fundamental functions.

The nuclear lamina is located between the inner nuclear membrane and the peripheral chromatin. It is composed of both peripheral and integral membrane proteins, including lamins and lamina-associated proteins. Lamins can interact with one another, with lamina-associated proteins, with nuclear scaffold proteins, and with chromatin. Likewise, most of the lamina-associated proteins are likely to interact directly with chromatin. The nuclear lamina is required for proper cell cycle regulation, chromatin organization, DNA replication, cell differentiation, and apoptosis. Mutations in proteins of the nuclear lamina can disrupt these activities and cause genetic diseases. The structure and assembly of the nuclear lamina proteins and their roles in chromatin organization and cell cycle regulation were recently reviewed. In this review, we discuss the roles of the nuclear lamina in DNA replication and apoptosis and analyze how mutations in nuclear lamina proteins might cause genetic diseases.

The tail domain of lamin Dm0 binds histones H2A and H2B.

In multicellular organisms, the higher order organization of chromatin during interphase and the reassembly of the nuclear envelope during mitosis are thought to involve an interaction between the nuclear lamina and chromatin. The nuclear distribution of lamins and of peripheral chromatin is highly correlated in vivo, and lamins bind specifically to chromatin in vitro. Deletion mutants of Drosophila lamin Dm0 were expressed to map regions of the protein that are required for its binding to chromosomes. The binding activity requires two regions in the lamin Dm0 tail domain. The apparent Kd of binding of the lamin Dm0 tail domain was found to be approximately 1 microM. Chromatin subfractions were examined to search for possible target molecules for the binding of lamin Dm0. Isolated polynucleosomes, nucleosomes, histone octamer, histone H2A/H2B dimer, and histones H2A or H2B displaced the binding of lamin Dm0 tail to chromosomes. This displacement was specific, because polyamines or proteins such as histones H1, H3, or H4 did not displace the binding of the lamin Dm0 tail to chromosomes. In addition, DNA sequences, including M/SARs, did not interfere with the binding of lamin Dm0 tail domain to chromosomes. Taken together, these results suggest that the interaction between the tail domain of lamin Dm0 and histones H2A and H2B may mediate the attachment of the nuclear lamina to chromosomes in vivo.

The nuclear lamina: molecular organization and interaction with chromatin.

The nuclear lamina is located between the inner nuclear membrane and the peripheral chromatin. It is composed mainly of nuclear lamins and lamina-associated proteins. The nuclear lamina is involved in nuclear organization, cell cycle regulation, and differentiation. As such, impairment in its architecture and/or function leads to genetic diseases and apoptosis. This article describes the molecular organization of the nuclear lamins, their assembly into filaments, their distribution within the nucleus, and the complex network of interactions between them and other proteins of the inner nuclear membrane. Recent findings unraveled evidence for specific interactions between proteins of the nuclear lamina and the chromatin. These include interactions between nuclear lamins and core histones, Lamina Associated Polypeptide 2 (LAP2), and the Barrier to Autointegration Factor (BAF) and interactions between lamin B receptor (LBR) and the chromodomain protein HP1. Taken together, these studies attribute a role for both the nuclear lamins and the lamina-associated proteins, LAP2 and LBR, in nuclear organization and nuclear assembly.

Modulation of circulatory residence of recombinant acetylcholinesterase through biochemical or genetic manipulation of sialylation levels.

Sialylation of N-glycans associated with recombinant human acetylcholinesterase (rHuAChE) has a central role in determining its circulatory clearance rate. Human embryonal kidney 293 (HEK-293) cells, which are widely used for the expression of recombinant proteins, seem to be limited in their ability to sialylate overexpressed rHuAChE. High-resolution N-glycan structural analysis, by gel permeation, HPLC anion-exchange chromatography and high-pH anion-exchange chromatography (HPAEC), revealed that the N-glycans associated with rHuAChE produced in HEK-293 cells belong mainly to the complex-biantennary class and are only partly sialylated, with approx. 60% of the glycans being monosialylated. This partial sialylation characterizes rHuAChE produced by cells selected for high-level expression of the recombinant protein. In low-level producer lines, the enzyme exhibits a higher sialic acid content, suggesting that undersialylation of rHuAChE in high-level producer lines stems from a limited endogenous glycosyltransferase activity. To improve sialylation in HEK-293 cells, rat liver beta-galactoside alpha-2,6-sialyltransferase cDNA was stably transfected into cells expressing high levels of rHuAChE. rHuAChE produced by the modified cells displayed a significantly higher proportion of fully sialylated glycans as shown by sialic acid incorporation assays, direct measurement of sialic acid, and HPAEC glycan profiling. Genetically modified sialylated rHuAChE exhibited increased circulatory retention (the slow-phase half-life, t12beta, was 130 min, compared with 80 min for the undersialylated enzyme). Interestingly, the same increase in circulatory residence was observed when rHuAChE was subjected to extensive sialylation in vitro. The engineered HEK-293 cells in which the glycosylation machinery was modified might represent a valuable tool for the high level of expression of recombinant glycoproteins whose sialic acid content is important for their function or for pharmacokinetic behaviour.

Nuclear membrane vesicle targeting to chromatin in a Drosophila embryo cell-free system.

A Drosophila cell-free system was used to characterize proteins that are required for targeting vesicles to chromatin and for fusion of vesicles to form nuclear envelopes. Treatment of vesicles with 1 M NaCl abolished their ability to bind to chromatin. Binding of salt-treated vesicles to chromatin could be restored by adding the dialyzed salt extract. Lamin Dm is one of the peripheral proteins whose activity was required, since supplying interphase lamin isoforms Dm1, and Dm2 to the assembly extract restored binding. As opposed to the findings in Xenopus, okadaic acid had no effect on vesicle binding. Trypsin digestion of the salt-stripped vesicles eliminated their association with chromatin even in the presence of the dialyzed salt extract. One vesicles attached to chromatin surface, fusion events took place were found to be sensitive to guanosine 5'-[gamma-thio]triphosphate (GTP gamma S). These chromatin-attached vesicles contained lamin Dm and otefin but not gp210. Thus, these results show that in Drosophila there are two populations of nuclear vesicles. The population that interacts first with chromatin contains lamin and otefin and requires both peripheral and integral membrane proteins, whereas fusion of vesicles requires GTPase activity.