Generalized substitution of isoencoding codons shortens the duration of papillomavirus L1 protein expression in transiently gene-transfected keratinocytes due to cell differentiation

Recently we reported that gene codon composition determines differentiation-dependent expression of the PV L1 genes in mouse primary keratinocytes (KCs) in vitro and in vivo (Zhao et al. 2005, Mol. Cell Biol. 25:8643–8655). Here, we investigated whether generalized substitution of isoencoding codons affects the duration of expression of PV L1 genes in mouse and human KCs in day 1 culture transiently transfected with native (Nat) and codon modified (Mod) L1 genes. Following transient transfection, KC continuously transcribed both Nat and Mod PV L1 genes for at least 12 days, with the levels of L1 mRNAs from the Mod L1 genes significantly higher than those from the Nat L1 genes. However, continuous L1 protein expression at day 9 post-transfection was observed for both mouse and human KCs transfected with the Nat L1 genes only. Further, aa-tRNAs prepared from D8 KC cultures enhanced translation of two PV Nat L1 DNAs in RRL lysate and PV Nat L1 mRNAs in D0 cell-free lysate, whereas aa-tRNAs from D0 KCs enhanced translation of PV Mod L1 mRNAs in D8 cell-free lysate. It appears that aa-tRNAs in less-differentiated and differentiated KCs differentially match the PV Nat and Mod L1 mRNAs to regulate their translations in vitro.     

Synthesis and structure-activity relationships of spirohydantoin-derived small-molecule antagonists of the melanin-concentrating hormone receptor-1 (MCH-R1)

The design, synthesis, and SAR of a series of substituted spirohydantoins are described. Optimization of an in-house screening hit gave compounds that exhibited potent binding affinity and functional activity at MCH-R1.     

A molecular assessment of northeast Pacific Alaria species (Laminariales, Phaeophyceae) with reference to the utility of DNA barcoding

Despite their relatively complex morphologies, species in the genus Alaria Greville are notoriously difficult to identify with certainty. Morphological characters, often influenced by environmental factors, make individuals in similar habitats artificially appear related. Species identification would, therefore, benefit greatly from the application of molecular tools. We applied DNA barcoding, using the 5' end of the cytochrome c oxidase I (coxI-5') gene from the mitochondrial genome, to define species limits and relationships in northeast Pacific populations of Alaria. This emerging technique is being employed to catalogue species diversity worldwide, particularly among animals, and it has been shown to be sensitive enough to discriminate between closely related species. However, the utility of this marker for identifying or categorizing the majority of life remains unclear. We compared the resolution obtained with this marker to two other molecular systems commonly used in algal research: the nuclear internal transcribed spacer (ITS) of the ribosomal cistron, and the plastid Rubisco operon spacer (rbcSp). In agreement with previous results, Alaria fistulosa Postels & Ruprecht, with its distinct morphological, ecological and molecular features, stands apart from the other species in the genus and we establish Druehlia gen. nov. to accommodate it. For the remaining isolates, distinct mitochondrial haplotypes resolved with the barcode data indicate a period of genetic isolation for at least three incipient species in the northeast Pacific, whereas unexpected levels and patterns of ITS variation, as well as the extreme morphological plasticity found among these isolates, have most probably resulted from a recent collapse in species barriers. The cloning of ITS amplicons revealed multiple ITS copies in several individuals, further supporting this hypothesis.     

Genetic variation in C57BL/6 ES cell lines and genetic instability in the Bruce4 C57BL/6 ES cell line

Genetically modified mouse strains derived from embryonic stem (ES) cells are powerful tools for gene function analysis. ES cells from the C57BL/6 mouse strain are not widely used to generate mouse models despite the advantage of a defined genetic background. We assessed genetic variation in six such ES cell lines with 275 SSLP markers. Compared to C57BL/6, Bruce4 differed at 34 SSLP markers and had significant heterozygosity on three chromosomes. BL/6#3 and Dale1 ES cell lines differed at only 3 SSLP makers. The C2 and WB6d ES cell lines differed at 6 SSLP markers. It is important to compare the efficiency of producing mouse models with available C57BL/6 ES cells relative to standard 129 mouse strain ES cells. We assessed genetic stability (the tendency of cells to become aneuploid) in 110 gene-targeted ES cell clones from the most widely used C57BL/6 ES cell line, Bruce4, and 710 targeted 129 ES cell clones. Bruce4 clones were more likely to be aneuploid and unsuitable for ES cell-mouse chimera production. Despite their tendency to aneuploidy and consequent inefficiency, use of Bruce4 ES cells can be valuable for models requiring behavioral studies and other mouse models that benefit from a defined C57BL/6 background. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

CLPB Variants Associated with Autosomal-Recessive Mitochondrial Disorder with Cataract,Neutropenia, Epilepsy,and Methylglutaconic Aciduria

3-methylglutaconic aciduria (3-MGA-uria) is a nonspecific finding associated with mitochondrial dysfunction, including defects of oxidative phosphorylation. 3-MGA-uria is classified into five groups, of which one, type IV, is genetically heterogeneous. Here we report five children with a form of type IV 3-MGA-uria characterized by cataracts, severe psychomotor regression during febrile episodes, epilepsy, neutropenia with frequent infections, and death in early childhood. Four of the individuals were of Greenlandic descent, and one was North American, of Northern European and Asian descent. Through a combination of homozygosity mapping in the Greenlandic individuals and exome sequencing in the North American, we identified biallelic variants in the caseinolytic peptidase B homolog (CLPB). The causative variants included one missense variant, c.803C>T (p.Thr268Met), and two nonsense variants, c.961A>T (p.Lys321^∗) and c.1249C>T (p.Arg417^∗). The level of CLPB protein was markedly decreased in fibroblasts and liver of affected individuals. CLPB is proposed to function as a mitochondrial chaperone involved in disaggregation of misfolded proteins, resulting from stress such as heat denaturation.     

Keystone resources available to wild pollinators in a winter‐flowering tree crop plantation

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Tropical herbivores provide resilience to a climate‐mediated phase shift on temperate reefs

Climate‐mediated changes to biotic interactions have the potential to fundamentally alter global ecosystems. However, the capacity for novel interactions to drive or maintain transitions in ecosystem states remains unresolved. We examined temperate reefs that recently underwent complete seaweed canopy loss and tested whether a concurrent increase in tropical herbivores could be maintaining the current canopy‐free state. Turf‐grazing herbivorous fishes increased in biomass and diversity, and displayed feeding rates comparable to global coral reefs. Canopy‐browsing herbivores displayed high (~ 10 000 g 100 m^?2) and stable biomass between 2006 and 2013. Tropical browsers had the highest abundance in 2013 and displayed feeding rates approximately three times higher than previously observed on coral reefs. These observations suggest that tropical herbivores are maintaining previously kelp‐dominated temperate reefs in an alternate canopy‐free state by grazing turfs and preventing kelp reestablishment. This remarkable ecosystem highlights the sensitivity of biotic interactions and ecosystem stability to warming and extreme disturbance events.     

Differential effects of cyclophosphamide and mycophenolate mofetil on cellular and serological parameters in patients with systemic lupus erythematosus

IntroductionDisease activity and therapy show an impact on cellular and serological parameters in patients with systemic lupus erythematosus (SLE). This study was performed to compare the influence of mycophenolate mofetil (MMF) and cyclophosphamide (CYC) therapy on these parameters in patients with flaring, organ-threatening disease.MethodsSLE patients currently receiving CYC (n = 20), MMF (n = 25) or no immunosuppressive drugs (n = 22) were compared using a cross-sectional design. Median disease activity and daily corticosteroid dose were similar in these treatment groups. Concurrent medication, organ manifestations, and disease activity were recorded, and cellular and serological parameters were determined by routine diagnostic tests or flow cytometric analysis. In addition follow-up data were obtained from different sets of patients (CYC n = 24; MMF n = 23).ResultsAlthough both drugs showed a significant effect on disease activity and circulating B cell subsets, only MMF reduced circulating plasmablasts and plasma cells as well as circulating free light chains within three months of induction therapy. Neither MMF nor CYC were able to reduce circulating memory B cells. MMF lowered IgA levels more markedly than CYC. We did not observe a significant difference in the reduction of IgG levels or anti-dsDNA antibodies comparing patients receiving MMF or CYC. In contrast to MMF, induction therapy with CYC was associated with a significant increase of circulating CD8+ effector T cells and plasmacytoid dendritic cells (PDCs) after three months.ConclusionsThe results indicate differences between MMF and CYC with regard to the mechanism of action. MMF, but not CYC, treatment leads to a fast and enduring reduction of surrogate markers of B cell activation, such as circulating plasmablasts, plasma cells and free light chains but a comparable rate of hypogammaglobulinemia.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0603-8) contains supplementary material, which is available to authorized users.     

Flanking domain stability modulates the aggregation kinetics of a polyglutamine disease protein

Spinocerebellar Ataxia Type 3 (SCA3) is one of nine polyglutamine (polyQ) diseases that are all characterized by progressive neuronal dysfunction and the presence of neuronal inclusions containing aggregated polyQ protein, suggesting that protein misfolding is a key part of this disease. Ataxin-3, the causative protein of SCA3, contains a globular, structured N-terminal domain (the Josephin domain) and a flexible polyQ-containing C-terminal tail, the repeat-length of which modulates pathogenicity. It has been suggested that the fibrillogenesis pathway of ataxin-3 begins with a non-polyQ-dependent step mediated by Josephin domain interactions, followed by a polyQ-dependent step. To test the involvement of the Josephin domain in ataxin-3 fibrillogenesis, we have created both pathogenic and nonpathogenic length ataxin-3 variants with a stabilized Josephin domain, and have both stabilized and destabilized the isolated Josephin domain. We show that changing the thermodynamic stability of the Josephin domain modulates ataxin-3 fibrillogenesis. These data support the hypothesis that the first stage of ataxin-3 fibrillogenesis is caused by interactions involving the non-polyQ containing Josephin domain and that the thermodynamic stability of this domain is linked to the aggregation propensity of ataxin-3.