Heteroresistance to the model antimicrobial peptide polymyxin B in the emerging Neisseria meningitidis lineage 11.2 urethritis clade: mutations in the pilMNOPQ operon

Clusters of Neisseria meningitidis (Nm) urethritis among primarily heterosexual males in multiple US cities have been attributed to a unique non‐encapsulated meningococcal clade (the US Nm urethritis clade, US_NmUC) within the hypervirulent clonal complex 11. Resistance to antimicrobial peptides (AMPs) is a key feature of urogenital pathogenesis of the closely related species, Neisseria gonorrhoeae. The US_NmUC isolates were found to be highly resistant to the model AMP, polymyxin B (PmB, MICs 64–256 µg ml^–1). The isolates also demonstrated stable subpopulations of heteroresistant colonies that showed near total resistant to PmB (MICs 384–1024 µg ml^–1) and colistin (MIC 256 µg ml^–1) as well as enhanced LL‐37 resistance. This is the first observation of heteroresistance in N. meningitidis. Consistent with previous findings, overall PmB resistance in US_NmUC isolates was due to active Mtr efflux and LptA‐mediated lipid A modification. However, whole genome sequencing, variant analyses and directed mutagenesis revealed that the heteroresistance phenotypes and very high‐level AMP resistance were the result of point mutations and IS1655 element movement in the pilMNOPQ operon, encoding the type IV pilin biogenesis apparatus. Cross‐resistance to other classes of antibiotics was also observed in the heteroresistant colonies. High‐level resistance to AMPs may contribute to the pathogenesis of US_NmUC.     

Protein-free transfection of CHO host cells with an IgG-fusion protein: selection and characterization of stable high producers and comparison to conventionally transfected clones

In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development.     

The tallysomycin biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 unveiling new insights into the biosynthesis of the bleomycin family of antitumor antibiotics

The tallysomycins (TLMs) belong to the bleomycin (BLM) family of antitumor antibiotics. The BLM biosynthetic gene cluster has been cloned and characterized previously from Streptomyces verticillus ATCC 15003, but engineering BLM biosynthesis for novel analogs has been hampered by the lack of a genetic system for S. verticillus. We now report the cloning and sequencing of the TLM biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 and the development of a genetic system for S. hindustanus, demonstrating the feasibility to manipulate TLM biosynthesis in S. hindustanus by gene inactivation and mutant complementation. Sequence analysis of the cloned 80.2 kb region revealed 40 open reading frames (ORFs), 30 of which were assigned to the TLM biosynthetic gene cluster. The TLM gene cluster consists of nonribosomal peptide synthetase (NRPS) genes encoding nine NRPS modules, a polyketide synthase (PKS) gene encoding one PKS module, genes encoding seven enzymes for deoxysugar biosynthesis and attachment, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The involvement of the cloned gene cluster in TLM biosynthesis was confirmed by inactivating the tlmE glycosyltransferase gene to generate a TLM non-producing mutant and by restoring TLM production to the DeltatlmE::ermE mutant strain upon expressing a functional copy of tlmE. The TLM gene cluster is highly homologous to the BLM cluster, with 25 of the 30 ORFs identified within the two clusters exhibiting striking similarities. The structural similarities and differences between TLM and BLM were reflected remarkably well by the genes and their organization in their respective biosynthetic gene clusters.     

Monocyte cholesterol homeostasis correlates with the presence of detergent resistant membrane microdomains.

BACKGROUND: Lipid membrane microdomains are involved in the regulation of biological functions of monocyte membrane proteins. These microdomains show a relative resistance to non-ionic detergents providing an easy analytical tool to study them. METHODS: Here, we applied a rapid detergent-based flow cytometric assay to investigate microdomain association of proteins on monocytes from whole blood samples. The association of known surface antigens with detergent resistant fraction of membranes (DRMs) was compared using monocytes from healthy blood donors, patients with genetic disorders affecting cellular cholesterol traffic and patients with systemic inflammatory response. RESULTS: All investigated surface antigens of Niemann-Pick type C (NPC)-mutant monocytes with impaired cholesterol influx and defective late endosome cholesterol trafficking, presented a strongly increased DRM-association. Though, membrane antigens of ATP binding cassette transporter A1 (ABCA1)-mutant monocytes with impaired cholesterol efflux did not show alterations in DRM-association. Differential CD14-dependent receptor clustering within microdomains was also investigated in response to in vivo lipopolysaccharide (LPS) and/or atherogenic lipoprotein activation. Increased DRM-association of the GPI-anchored proteins CD14, CD55, the Fcgamma receptor CD64, the scavenger receptors CD36, CD91 and CD163, the integrin CD11a, and complement receptor 3 complex CD11b/CD18 were observed from patients with systemic inflammatory response syndrome (SIRS)/sepsis or coronary artery disease (CAD)/myocardial infarction. Interestingly, the tetraspanin CD81 presented increased DRM-association in SIRS/sepsis patients, but not in CAD patients. Moreover, the pentaspanin CD47 and the Fcgamma RIII CD16 showed an increased DRM partition in CAD patients but disassembled from DRMs in SIRS/sepsis patients. CONCLUSIONS: Our results demonstrate that flow cytometric analysis of short time in situ detergent extraction provides a powerful tool for rapid screening of blood monocyte DRMs to preselect patients with potential raft/microdomain abnormalities for more detailed analysis.     

Comparing methods for estimating the abundance of Western Capercaillie Tetrao urogallus males in Pyrenean leks: singing counts versus genetic analysis of non-invasive samples

ABSTRACT

Population estimates of male Western Capercaillies Tetrao urogallus were carried out during the mating season using two methods: counts of singing males and non-invasive genetic analysis. Estimates of male numbers were 50% lower using the singing counts compared to the estimates obtained through genetic analysis, and underestimates were greatest when the number of Capercaillies was lowest.     

Pregnancy reduces critical thermal maximum,but not voluntary thermal maximum,in a viviparous skink

Journal of Comparative Physiology B - Upper thermal limits are commonly measured in ectotherms; however, the effects of life-history stages, and in particular pregnancy in viviparous species, are...     

Development of a SNP genotyping panel for genetic monitoring of the laboratory mouse

We have developed a genotyping system for detecting genetic contamination in the laboratory mouse based on assaying single-nucleotide polymorphism (SNP) markers positioned on all autosomes and the X chromosome. This system provides a fast, reliable, and cost-effective way for genetic monitoring, while maintaining a very high degree of confidence. We describe the allelic distribution of 235 SNPs in 48 mouse strains, thereby creating a database of polymorphisms useful for genotyping purposes. The SNP markers used in this study were chosen from publicly available SNP databases. Four genotyping methods were evaluated, and dynamic two-tube allele-specific PCR assays were developed for each marker and tested on a set of 48 inbred mouse strains. The minimal number of assays sufficient to distinguish groups consisting of different numbers of mouse strains was estimated, and a panel of 28 SNPs sufficient to distinguish virtually all of the inbred strains tested was selected. Amplifluor SNP detection assays were developed for these markers and tested on an extended list of 96 strains. This panel was used as a genetic quality control approach to monitor the genotypes of nearly 300 inbred, wild-derived, congenic, consomic, and recombinant inbred strains maintained at The Jackson Laboratory. We have concluded that this marker panel is sufficient for genetic contamination monitoring in colonies containing a large number of genetically diverse mouse strains and that reduced versions of the panel could be implemented in facilities housing a lower number of strains.     

Survival and growth of goat primordial follicles after in vitro culture of ovarian cortical slices in media containing coconut water

The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. We investigated the effectiveness of five culture media, two supplements and three culture periods on the survival and growth of goat primordial follicles after culturing ovarian cortex. The media were based on minimal essential minimum (MEM) and coconut water solution (CWS) added in the proportion of 0, 25, 50, 75 or 100%. The two supplements were none versus supplemented with insulin-transferrin-selenium, pyruvate, glutamine, hypoxanthine, and BSA. Pieces of goat ovarian cortex were cultured in the media for 1, 3 or 5 days and representative samples were evaluated at day 0 as non-cultured controls. The replicates were the two ovaries of five mixed breed goats. The number of primordial, intermediate, primary and secondary follicles at each period of culture and the number of degenerated follicles were evaluated. Mitotic activity of granulosa cells was studied by immunolocalization of proliferating cell nuclear antigen (PCNA). The number of follicles in each stage and degenerated follicles were statistically analyzed by ANOVA using a factorial design and the significance of differences assessed using Tukey test. Chi-square test was used to compare the percentage of follicles with PCNA positive granulosa cells. As the culture period progressed, the number of primordial follicles fell and there was a significant increase in the number of primary follicles. The fall in the number of primordial follicles was particularly marked after 1 day culture. No effect of media on the number of primordial and primary follicles was observed after culture, but MEM as well as supplements increased the number of intermediate follicles. Follicular degeneration was kept at the same level after culture in the media tested, except for pure CWS that increased the number of degenerated follicles. In contrast, addition of supplements to culture media reduced follicular degeneration. In non-cultured tissue, PCNA was expressed in granulosa cells of 31.6% of the growing follicles. This percentage had not significantly changed after 5 days culture in the various media, indicating the maintenance of proliferation activity of granulosa cells during culture. In conclusion, it is shown that goat primordial follicles may be successfully activated after in vitro culture in all media tested. However, when pure CWS is used the follicular degeneration is enhanced, but the addition of supplements to culture media decrease follicular degeneration.