Resting cells rely on the DNA helicase component MCM2 to build cilia

Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formation in human cells and zebrafish that is uncoupled from replication. Zebrafish depleted of MCM2 develop ciliopathy-phenotypes including microcephaly and aberrant heart looping due to malformed cilia. In non-cycling human fibroblasts, loss of MCM2 promotes transcription of a subset of genes, which cause cilia shortening and centriole overduplication. Chromatin immunoprecipitation experiments show that MCM2 binds to transcription start sites of cilia inhibiting genes. We propose that such binding may block RNA polymerase II-mediated transcription. Depletion of a second MCM (MCM7), which functions in complex with MCM2 during its canonical functions, reveals an overlapping cilia-deficiency phenotype likely unconnected to replication, although MCM7 appears to regulate a distinct subset of genes and pathways. Our data suggests that MCM2 and 7 exert a role in ciliogenesis in post-mitotic tissues

Msb2 Shedding Protects Candida albicans against Antimicrobial Peptides

Msb2 is a sensor protein in the plasma membrane of fungi. In the human fungal pathogen C. albicans Msb2 signals via the Cek1 MAP kinase pathway to maintain cell wall integrity and allow filamentous growth. Msb2 doubly epitope-tagged in its large extracellular and small cytoplasmic domain was efficiently cleaved during liquid and surface growth and the extracellular domain was almost quantitatively released into the growth medium. Msb2 cleavage was independent of proteases Sap9, Sap10 and Kex2. Secreted Msb2 was highly O-glycosylated by protein mannosyltransferases including Pmt1 resulting in an apparent molecular mass of >400 kDa. Deletion analyses revealed that the transmembrane region is required for Msb2 function, while the large N-terminal and the small cytoplasmic region function to downregulate Msb2 signaling or, respectively, allow its induction by tunicamycin. Purified extracellular Msb2 domain protected fungal and bacterial cells effectively from antimicrobial peptides (AMPs) histatin-5 and LL-37. AMP inactivation was not due to degradation but depended on the quantity and length of the Msb2 glycofragment. C. albicans msb2 mutants were supersensitive to LL-37 but not histatin-5, suggesting that secreted rather than cell-associated Msb2 determines AMP protection. Thus, in addition to its sensor function Msb2 has a second activity because shedding of its glycofragment generates AMP quorum resistance

Mucins and Pathogenic Mucin-Like Molecules Are Immunomodulators During Infection and Targets for Diagnostics and Vaccines

Mucins and mucin-like molecules are highly O-glycosylated proteins present on the cell surface of mammals and other organisms. These glycoproteins are highly diverse in the apoprotein and glycan cores and play a central role in many biological processes and diseases. Mucins are the most abundant macromolecules in mucus and are responsible for its biochemical and biophysical properties. Mucin-like molecules cover various protozoan parasites, fungi and viruses. In humans, modifications in mucin glycosylation are associated with tumors in epithelial tissue. These modifications allow the distinction between normal and abnormal cell conditions and represent important targets for vaccine development against some cancers. Mucins and mucin-like molecules derived from pathogens are potential diagnostic markers and targets for therapeutic agents. In this review, we summarize the distribution, structure, role as immunomodulators, and the correlation of human mucins with diseases and perform a comparative analysis of mucins with mucin-like molecules present in human pathogens. Furthermore, we review the methods to produce pathogenic and human mucins using chemical synthesis and expression systems. Finally, we present applications of mucin-like molecules in diagnosis and prevention of relevant human diseases

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

<i>C. albicans</i> strains.

<p><i>C. albicans</i> strains.</p

Msb2*-mediates protection of <i>C. albicans</i> and <i>E. coli</i> against histatin-5.

<p><i>C. albicans</i> strains CAF2-1 (wt), FCCa27 (<i>msb2</i>Δ<i>1</i>), REP18 (<i>msb2</i>Δ<i>0</i>) and <i>E. coli</i> DH5αF′ were allowed to react with the indicated amounts of histatin-5 for 1.5 h at 37°C, in the absence or presence of the affinity-purified secreted Msb2* protein. Colony-forming units were determined on YPD (<i>C. albicans</i> strains) or on LB medium (<i>E. coli</i>).</p

Secretion and processing of Msb2.

<p><i>C. albicans</i> strains grown in YPD medium to OD₆₀₀ = 6, centrifuged and cell extracts (50 µg protein derived from cells in 90 µl of medium) or medium (20 µl) were analyzed for epitope-tagged Msb2 protein. <b>A.</b> Immunoblot to detect HA-tagged Msb2. Proteins were separated by a 8% SDS-PAGE gel and immunoblots were reacted with rat anti-HA antibody. Strains tested included ESCa8 (<i>ACT1p</i>-<i>MSB2</i>^HA; lanes 3 and 8), ESCa3 (<i>ACT1p</i>-<i>MSB2</i>^HA-V5; lanes 4 and 9), ESCa9 (<i>MSB2p</i>-<i>MSB2</i>^HA; lanes 5 and 10) and ESCa10 (<i>MSB2p</i>-<i>MSB2</i>^HA-V5; lanes 6 and 11). Strains CAF2-1 (wt) and CIS23 (<i>PMT1</i>^HA) were used as negative and positive control strains, respectively. The migration of HA-tagged Msb2 and Pmt1 are indicated by the arrow and triangle, respectively. <b>B.</b> Immunoblot to detect V5-tagged Msb2. Proteins were separated by a 4–20% gradient SDS-PAGE gel and immunoblots were reacted with mouse monoclonal anti-V5 antibody. Identical strains and fractions as in (A) were tested. The migration of V5-tagged Msb2 and Pmt2 (strain CIS29) are indicated by the arrow and the triangle, respectively; a protein cross-reacting with the anti-V5 antibody is marked by the asterisk. <b>C.</b> Secretion of HA-tagged Msb2 protein during growth on agar. Cell suspensions were dropped on a membrane filter (pore diameter 0.45 µm) situated on a PVDF membrane, which had been placed on YPD agar (a). Colonies were allowed to grow for 15 h at 30°C (b). The membrane filter was removed and the PVDF membrane was probed by immunoblotting using rat anti-HA antibody (c). Strains tested were (1) CAF2-1 (wild-type), (2) CIS23 (<i>PMT1</i>^HA), (3) ESCa3 (<i>ACT1p</i>-<i>MSB2</i>^HA-V5) and ESCa10 (<i>MSB2p</i>-<i>MSB2</i>^HA-V5). <b>D.</b> Gel filtration chromatography of secreted Msb2. A Superdex 200 10/300 GL column was (a) calibrated using standard proteins of the indicated sizes (dotted lines) and (b) used to fractionate 500 µl of the medium of strain ESCa3 (Msb2^HA-V5), which had been grown at 30°C in SD medium to OD₆₀₀ = 10. The protein elution profiles were recorded by absorption at 280 nm. 200 µl fractions were collected and (c) tested by immunoblotting for the presence of HA-tagged Msb2. Fractions tested are placed at a position corresponding to the elution profile in b). <b>E.</b> Glycosylation of secreted Msb2. (a) Growth medium of strain ESCa3 (Msb2^HA-V5) was not treated (1) or treated with β-elimination reagent mixture over night (2,3); the sample in lane 3 was heated to 80°C before reagent addition in an attempt to increase deglycosylation. (b) The medium was not treated (1) or treated with TFMS (2). Samples were tested by immunoblotting as in A. The migration of glyosylated and deglycosylated Msb2* are indicated by the filled and open arrows, respectively. (c) Msb2 secreted by <i>pmt</i> mutants defective in protein-<i>O</i>-mannosyltransferases carrying carried pES11a (Msb2^HA-V5). Strains included ESca18 (<i>pmt1</i>), ESCa19 (<i>PMT2/pmt2</i>), ESCa20 (<i>pmt4</i>), ESCa21 (<i>pmt5</i>) and ESCa22 (<i>pmt6</i>) and were tested by immunblotting as in A.</p

Activity of Msb2 variants.

<p><b>A.</b> Structure of Msb2 protein variants. The positions of signal sequence (SS), transmembrane region (TM) and HA- and V5-epitope tags are indicated. Plasmids encoding variants were chromosomally integrated into strain FCCa28, which produces the inactive Msb2-Δ1 variant by the <i>msb2</i>Δ<i>1</i> allele. Resulting transformants (encoded variants) were strains ESCa3 (Msb2^HA-V5), ESCa25 (Msb2-ΔN), ESCa38 (Msb2-ΔC) and ESCa39 (Msb2-ΔTM-C). Corresponding phenotypes are summarized in the table and are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002501#ppat.1002501.s001" target="_blank">Figure S1</a>. +, wild-type phenotype; −, <i>msb2</i> mutant phenotype with regard to Msb2* release, hypha formation, caspofungin resistance (Cas^R) and Cek1 phosphorylation (Cek1-P). <b>B.</b> Cek1 activation by strains producing variant Msb2 proteins. Cells were grown to stationary phase (st), diluted in fresh YPD medium, grown to OD₆₀₀ = 0.8 at 37°C and incubated further for 1 h in the presence (+) or absence (−) of tunicamycin (2 µg/ml). Cells in stationary phase (st) and after 1 h incubation were harvested and assayed for the activation of MAPK Cek1 by immunoblottings; the Hog1 MAPK protein signal was used as the loading control. Strains as in A., in addition strains ESCa37 encoding the Msb2-tail variant and strain ESCa7 carrying an empty vector (control) were tested.</p