The innate immune receptor MDA5 limits rotavirus infection but promotes cell death and pancreatic inflammation

Melanoma differentiation‐associated protein 5 (MDA5) mediates the innate immune response to viral infection. Polymorphisms in IFIH1, the gene coding for MDA5, correlate with the risk of developing type 1 diabetes (T1D). Here, we demonstrate that MDA5 is crucial for the immune response to enteric rotavirus infection, a proposed etiological agent for T1D. MDA5 variants encoded by minor IFIH1 alleles associated with lower T1D risk exhibit reduced activity against rotavirus infection. We find that MDA5 activity limits rotavirus infection not only through the induction of antiviral interferons and pro‐inflammatory cytokines, but also by promoting cell death. Importantly, this MDA5‐dependent antiviral response is specific to the pancreas of rotavirus‐infected mice, similar to the autoimmunity associated with T1D. These findings imply that MDA5‐induced cell death and inflammation in the pancreas facilitate progression to autoimmune destruction of pancreatic β‐cells.     

Taphonomy and compositional fidelity of Quaternary fossil assemblages of terrestrial gastropods from carbonate-rich environments of the Canary Islands

Quaternary aeolian deposits of the Canary Islands contain well‐preserved terrestrial gastropods, providing a suitable setting for assessing the taphonomy and compositional fidelity of their fossil record over ~13 kyr. Nine beds (12, 513 shells) have been analysed in terms of multivariate taphonomic and palaeoecological variables, taxonomic composition, and the stratigraphic and palaeontological context. Shells are affected by carbonate coatings, colour loss and fragmentation. Shell preservation is size‐specific: juveniles are less fragmented and show colour preservation more commonly than adults. In palaeosols, the adult shell density correlates negatively with the proportion of fragmented adults, negatively with the proportion of juveniles, and positively with the proportion of adults with coatings. High bioturbation intensity in palaeosols is associated with low shell fragmentation and high proportion of shells with coatings. These relationships imply that high adult density in palaeosols was driven by an increase in shell production rate (related to a decrease in predation rates on adults and a decrease in juvenile mortality) and a decrease in shell destruction rate (related to an increase in durability enhanced by carbonate precipitation). In dunes, the relationships between taphonomic alteration, shell density and bioturbation are insignificant. However, dune assemblages are characterized by a lower frequency of shells with coatings and higher rates of colour loss, indicating lower shell durability in dunes than in palaeosols. Additionally, non‐random differences in the coating proportion among palaeosols imply substantial temporal variation in the rate of carbonate crust formation, reflecting long‐term changes in bioturbation intensity that covaries positively with shell preservation. Dunes and palaeosols do not differ in species abundances despite differences in the degree of shell alteration, suggesting that both weakly and strongly altered assemblages offer data with a high compositional fidelity. Carbonate‐rich terrestrial deposits originating in arid conditions can enhance the preservation of gastropods and result in fossil assemblages that are suitable for palaeoecological and palaeoenvironmental studies of terrestrial ecosystems.     

Efficacy of neem and diatomaceous earth against cowpea aphids and their deleterious effect on predating Coccinelidae

Philippine vegetable farmers commonly use synthetic insecticides to control insect pests on yardlong beans ( Vigna unguiculata ssp. sesquipedalis ). An important pest on yardlong beans is the cowpea aphid, Aphis craccivora Koch. Overuse of chemical insecticides and the adverse consequences for farmer health and for the environment have been reported. The natural enemies of A. craccivora , such as the coccinelidae beetle, Menochilus sexmaculatus (Fabr.), do not provide economic control on their own. In the present study the efficacy of the biological insecticide neem (both commercial and homemade) alone, and in combination with diatomaceous earth against A. craccivora was evaluated. The same insecticides were also examined to investigate their deleterious effect on M. sexmaculatus . The efficacies of different treatments with biological insecticides were compared with the use of the synthetic insecticide Hostathion (triazophos). Experiments were conducted under Philippine lowland conditions during the dry season when the occurrence of pest problems in yardlong beans is very great. Commercial neem, NeemAzal-T/S (Trifolio-M GmbH, Lahnau, Germany), significantly reduced the number of A. craccivora . NeemAzal-T/S and diatomaceous earth in combination produced the best control of A. craccivora and were less toxic to M. sexmaculatus than treatment with Hostathion (triazophos). Aqueous homemade neem solution did not control the A. craccivora populations.     

Glycosylphosphatidylinositol biosynthesis defects in Gpi11p- and Gpi13p-deficient yeast suggest a branched pathway and implicate gpi13p in phosphoethanolamine transfer to the third mannose

Glycosylphosphatidylinositols (GPIs) are critical for membrane anchoring and intracellular transport of certain secretory proteins. GPIs have a conserved trimannosyl core bearing a phosphoethanolamine (EthN-P) moiety on the third mannose (Man-3) through which the glycolipid is linked to protein, but diverse GPI precursors with EthN-Ps on Man-1 and Man-2 have also been described. We report on two essential yeast genes whose products are required late in GPI assembly. GPI11 (YDR302w) encodes a homologue of human Pig-Fp, a protein implicated in the addition of EthN-P to Man-3. PIG-F complements the gpi11 deletion, but the rescued haploids are temperature sensitive. Abolition of Gpi11p or Pig-Fp function in GPI11 disruptants blocks GPI anchoring and formation of complete GPI precursors and leads to accumulation of two GPIs whose glycan head groups contain four mannoses but differ in the positioning and number of side chains, probably EthN-Ps. The less polar GPI bears EthN-P on Man-2, whereas the more polar lipid has EthN-P on Man-3. The latter finding indicates that Gpi11p is not required for adding EthN-P to Man-3. Gpi13p (YLL031cp), a member of a family of phosphoryltransferases, is a candidate for the enzyme responsible for adding EthN-P to Man-3. Depletion of Gpi13p in a Gpi11p-defective strain prevents formation of the GPI bearing EthN-P on Man-3, and Gpi13p-deficient strains accumulate a Man(4)-GPI isoform that bears EthN-P on Man-1. We further show that the lipid accumulation phenotype of Gpi11p-deficient cells resembles that of cells lacking Gpi7p, a sequence homologue of Gpi13p known to add EthN-P to Man-2 of a late-stage GPI precursor. This result suggests that in yeast a Gpi11p-deficiency can affect EthN-P addition to Man-2 by Gpi7p, in contrast to the Pig-Fp defect in mammalian cells, which prevents EthN-P addition to Man-3. Because Gpi11p and Pig-Fp affect EthN-P transfer to Man-2 and Man-3, respectively, these proteins may act in partnership with the GPI-EthN-P transferases, although their involvement in a given EthN-P transfer reaction varies between species. Possible roles for Gpi11p in the supply of the EthN-P donor are discussed. Because Gpi11p- and Gpi13p-deficient cells accumulate isoforms of Man(4)-GPIs with EthN-P on Man-2 and on Man-1, respectively, and because the GPIs that accumulate in Gpi11p-defective strains are likely to have been generated independently of one another, we propose that the yeast GPI assembly pathway is branched.     

Acetamide selection of Kluyveromyces lactis cells transformed with an integrative vector leads to high-frequency formation of multicopy strains

The yeast Kluyveromyces lactis has been extensively used as a host for heterologous protein expression. A necessary step in the construction of a stable expression strain is the introduction of an integrative expression vector into K. lactis cells, followed by selection of transformed strains using either medium containing antibiotic (e.g., G418) or nitrogen-free medium containing acetamide. In this study, we show that selection using acetamide yields K. lactis transformant populations nearly completely comprised of strains bearing multiple tandem insertions of the expression vector pKLAC1 at the LAC4 chromosomal locus, whereas an average of 16% of G418-selected transformants are multiply integrated. Additionally, the average copy number within transformant populations doubled when acetamide was used for selection compared to G418. Finally, we demonstrate that the high frequency of multicopy integration associated with using acetamide selection can be exploited to rapidly construct expression strains that simultaneously produce multiple heterologous proteins or multisubunit proteins, such as Fab antibodies.     

曲酸生产菌的复合诱变选育*

以黄曲霉(Aspergillus flavus.)为出发菌株,经3次紫外线、1次^60Co、3次亚硝基胍多重复合诱变处理,选育获得曲酸生产菌UCN7—17,配以最佳培养条件,发酵7d,曲酸产量由原来的0．926％,提高到6．3％。实验证明采用多因子复合诱变,能有效改变菌株对诱变因素敏感性,提高变异率,逐步提高突变株的产酸水平。     

Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis

Endogenous proteins secreted from Kluyveromyces lactis were screened for their ability to bind to or to hydrolyze chitin. This analysis resulted in identification of a nucleus-encoded extracellular chitinase (KlCts1p) with a chitinolytic activity distinct from that of the plasmid-encoded killer toxin α-subunit. Sequence analysis of cloned KlCTS1 indicated that it encodes a 551-amino-acid chitinase having a secretion signal peptide, an amino-terminal family 18 chitinase catalytic domain, a serine-threonine-rich domain, and a carboxy-terminal type 2 chitin-binding domain. The association of purified KlCts1p with chitin is stable in the presence of high salt concentrations and pH 3 to 10 buffers; however, complete dissociation and release of fully active KlCts1p occur in 20 mM NaOH. Similarly, secreted human serum albumin harboring a carboxy-terminal fusion with the chitin-binding domain derived from KlCts1p also dissociates from chitin in 20 mM NaOH, demonstrating the domain's potential utility as an affinity tag for reversible chitin immobilization or purification of alkaliphilic or alkali-tolerant recombinant fusion proteins. Finally, haploid K. lactis cells harboring a cts1 null mutation are viable but exhibit a cell separation defect, suggesting that KlCts1p is required for normal cytokinesis, probably by facilitating the degradation of septum-localized chitin.     

Le Cortex de la Vacuole Contractile et son Ultrastructure chez les Ciliés

SYNOPSIS. In various ciliates the contractile vacuole is a permanent organelle, delimited by a differentiated cortex.
The cortex is made up of a dense reticulum of anastomosing tubules limited by a smooth membrane, and vesicles. This "spongiome" can be considered as a localized and specialized condensation of the endoplasmic reticulum.     

Mass Spectrometric and Glycan Microarray–Based Characterization of the Filarial Nematode Brugia malayi Glycome Reveals Anionic and Zwitterionic Glycan Antigens

Millions of people worldwide are infected with filarial nematodes, responsible for lymphatic filariasis (LF) and other diseases causing chronic disablement. Elimination programs have resulted in a substantial reduction of the rate of infection in certain areas creating a need for improved diagnostic tools to establish robust population surveillance and avoid LF resurgence. Glycans from parasitic helminths are emerging as potential antigens for use in diagnostic assays. However, despite its crucial role in host–parasite interactions, filarial glycosylation is still largely, structurally, and functionally uncharacterized. Therefore, we investigated the glycan repertoire of the filarial nematode Brugia malayi. Glycosphingolipid and N-linked glycans were extracted from several life-stages using enzymatic release and characterized using a combination of MALDI-TOF-MS and glycan sequencing techniques. Next, glycans were purified by HPLC and printed onto microarrays to assess the host anti-glycan antibody response. Comprehensive glycomic analysis of B. malayi revealed the presence of several putative antigenic motifs such as phosphorylcholine and terminal glucuronic acid. Glycan microarray screening showed a recognition of most B. malayi glycans by immunoglobulins from rhesus macaques at different time points after infection, which permitted the characterization of the dynamics of anti-glycan immunoglobulin G and M during the establishment of brugian filariasis. A significant level of IgG binding to the parasite glycans was also detected in infected human plasma, while IgG binding to glycans decreased after anthelmintic treatment. Altogether, our work identifies B. malayi glycan antigens and reveals antibody responses from the host that could be exploited as potential markers for LF.