MCM2-7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts

The MCM2-7 complex is believed to function as the eukaryotic replicative DNA helicase. It is recruited to chromatin by the origin recognition complex (ORC), Cdc6, and Cdt1, and it is activated at the G(1)/S transition by Cdc45 and the protein kinases Cdc7 and Cdk2. Paradoxically, the number of chromatin-bound MCM complexes greatly exceeds the number of bound ORC complexes. To understand how the high MCM2-7:ORC ratio comes about, we examined the binding of these proteins to immobilized linear DNA fragments in Xenopus egg extracts. The minimum length of DNA required to recruit ORC and MCM2-7 was approximately 80 bp, and the MCM2-7:ORC ratio on this fragment was approximately 1:1. With longer DNA fragments, the MCM2-7:ORC ratio increased dramatically, indicating that MCM complexes normally become distributed over a large region of DNA surrounding ORC. Only a small subset of the chromatin-bound MCM2-7 complexes recruited Cdc45 at the onset of DNA replication, and unlike Cdc45, MCM2-7 was not limiting for DNA replication. However, all the chromatin-bound MCM complexes may be functional, because they were phosphorylated in a Cdc7-dependent fashion, and because they could be induced to support Cdk2-dependent Cdc45 loading. The data suggest that in Xenopus egg extracts, origins of replication contain multiple, distributed, initiation-competent MCM2-7 complexes.     

The Sphagnum microbiome supports bog ecosystem functioning under extreme conditions

Sphagnum‐dominated bogs represent a unique yet widely distributed type of terrestrial ecosystem and strongly contribute to global biosphere functioning. Sphagnum is colonized by highly diverse microbial communities, but less is known about their function. We identified a high functional diversity within the Sphagnum microbiome applying an Illumina‐based metagenomic approach followed by de novo assembly and MG‐RAST annotation. An interenvironmental comparison revealed that the Sphagnum microbiome harbours specific genetic features that distinguish it significantly from microbiomes of higher plants and peat soils. The differential traits especially support ecosystem functioning by a symbiotic lifestyle under poikilohydric and ombrotrophic conditions. To realise a plasticity–stability balance, we found abundant subsystems responsible to cope with oxidative and drought stresses, to exchange (mobile) genetic elements, and genes that encode for resistance to detrimental environmental factors, repair and self‐controlling mechanisms. Multiple microbe–microbe and plant–microbe interactions were also found to play a crucial role as indicated by diverse genes necessary for biofilm formation, interaction via quorum sensing and nutrient exchange. A high proportion of genes involved in nitrogen cycle and recycling of organic material supported the role of bacteria for nutrient supply. 16S rDNA analysis indicated a higher structural diversity than that which had been previously detected using PCR‐dependent techniques. Altogether, the diverse Sphagnum microbiome has the ability to support the life of the host plant and the entire ecosystem under changing environmental conditions. Beyond this, the moss microbiome presents a promising bio‐resource for environmental biotechnology – with respect to novel enzymes or stress‐protecting bacteria.     

The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing

MHC class I-restricted epitopes, which carry a tumor-specific mutation resulting in improved MHC binding affinity, are preferred T cell receptor targets in innovative adoptive T cell therapies. However, T cell therapy requires efficient generation of the selected epitope. How such mutations may affect proteasome-mediated antigen processing has so far not been studied. Therefore, we analyzed by in vitro experiments the effect on antigen processing and recognition of a T210M exchange, which previously had been introduced into the melanoma gp100_209–217tumor epitope to improve the HLA-A*02:01 binding and its immunogenicity. A quantitative analysis of the main steps of antigen processing shows that the T210M exchange affects proteasomal cleavage site usage within the _mutgp100_201–230 polypeptide, leading to the generation of an unique set of cleavage products. The T210M substitution qualitatively affects the proteasome-catalyzed generation of spliced and non-spliced peptides predicted to bind HLA-A or -B complexes. The T210M substitution also induces an enhanced production of the _mutgp100_209–217 epitope and its N-terminally extended peptides. The T210M exchange revealed no effect on ERAP1-mediated N-terminal trimming of the precursor peptides. However, mutant N-terminally extended peptides exhibited significantly increased HLA-A*02:01 binding affinity and elicited CD8⁺ T cell stimulation in vitro similar to the _wtgp100_209–217 epitope. Thus, our experiments demonstrate that amino acid exchanges within an epitope can result in the generation of an altered peptide pool with new antigenic peptides and in a wider CD8⁺ T cell response also towards N-terminally extended versions of the minimal epitope.     

Cloning and functional characterization of inward-rectifying potassium (Kir) channels from Malpighian tubules of the mosquito Aedes aegypti

Inward-rectifying K⁺ (Kir) channels play critical physiological roles in a variety of vertebrate cells/tissues, including the regulation of membrane potential in nerve and muscle, and the transepithelial transport of ions in osmoregulatory epithelia, such as kidneys and gills. It remains to be determined whether Kir channels play similar physiological roles in insects. In the present study, we sought to 1) clone the cDNAs of Kir channel subunits expressed in the renal (Malpighian) tubules of the mosquito Aedes aegypti, and 2) characterize the electrophysiological properties of the cloned Kir subunits when expressed heterologously in oocytes of Xenopus laevis. Here, we reveal that three Kir subunits are expressed abundantly in Aedes Malpighian tubules (AeKir1, AeKir2B, and AeKir3); each of their full-length cDNAs was cloned. Heterologous expression of the AeKir1 or the AeKir2B subunits in Xenopus oocytes elicits inward-rectifying K⁺ currents that are blocked by barium. Relative to the AeKir2B-expressing oocytes, the AeKir1-expressing oocytes 1) produce larger macroscopic currents, and 2) exhibit a modulation of their conductive properties by extracellular Na⁺. Attempts to functionally characterize the AeKir3 subunit in Xenopus oocytes were unsuccessful. Lastly, we show that in isolated Aedes Malpighian tubules, the cation permeability sequence of the basolateral membrane of principal cells (Tl⁺ > K⁺ > Rb⁺ > NH₄⁺) is consistent with the presence of functional Kir channels. We conclude that in Aedes Malpighian tubules, Kir channels contribute to the majority of the barium-sensitive transepithelial transport of K⁺.     

Improved method for the determination of cyclic guanosine monophosphate (cGMP) in human plasma by LC–MS/MS

Cyclic guanosine monophosphate (cGMP) is an important second messenger molecule involved in gating ion channels and activating protein kinases. Here, we describe a validated LC–MS/MS method for the quantification of cGMP in human plasma, utilizing a stable isotope labeled analogue of cGMP as I.S. Plasma samples were extracted and concentrated by weak anion exchange solid phase extraction and the extracts were chromatographically separated on a porous graphitic carbon column. The analytes were detected by positive electrospray ionization and tandem mass spectrometry. The calibration function was linear in the range 1–20 nM and the intra- and inter-day precision showed relative standard deviations of better than 2 and 6%, respectively. The accuracy was always better than 4%. Plasma concentrations in healthy human subjects determined with this method were 3.92 ± 1.17 nM (n = 20). The method was, due to its isotope labeled I.S., matrix independent.     

The Syk Kinase SmTK4 of Schistosoma mansoni Is Involved in the Regulation of Spermatogenesis and Oogenesis

The signal transduction protein SmTK4 from Schistosoma mansoni belongs to the family of Syk kinases. In vertebrates, Syk kinases are known to play specialized roles in signaling pathways in cells of the hematopoietic system. Although Syk kinases were identified in some invertebrates, their role in this group of animals has not yet been elucidated. Since SmTK4 is the first Syk kinase from a parasitic helminth, shown to be predominantly expressed in the testes and ovary of adult worms, we investigated its function. To unravel signaling cascades in which SmTK4 is involved, yeast two-/three-hybrid library screenings were performed with either the tandem SH2-domain, or with the linker region including the tyrosine kinase domain of SmTK4. Besides the Src kinase SmTK3 we identified a new Src kinase (SmTK6) acting upstream of SmTK4 and a MAPK-activating protein, as well as mapmodulin acting downstream. Their identities and colocalization studies pointed to a role of SmTK4 in a signaling cascade regulating the proliferation and/or differentiation of cells in the gonads of schistosomes. To confirm this decisive role we performed biochemical and molecular approaches to knock down SmTK4 combined with a novel protocol for confocal laser scanning microscopy for morphological analyses. Using the Syk kinase-specific inhibitor Piceatannol or by RNAi treatment of adult schistosomes in vitro, corresponding phenotypes were detected in the testes and ovary. In the Xenopus oocyte system it was finally confirmed that Piceatannol suppressed the activity of the catalytic kinase domain of SmTK4. Our findings demonstrate a pivotal role of SmTK4 in gametogenesis, a new function for Syk kinases in eukaryotes.     

Technical viability of the YF MAC-HD ELISA kit for use in yellow fever-endemic regions

Yellow fever (YF), an arboviral disease, affects an estimated 200,000 people and causes 30,000 deaths per year and recently has caused major epidemics in Africa and South America. Timely and accurate diagnosis of YF is critical for managing outbreaks and implementing vaccination campaigns. A YF immunoglobulin M (IgM) antibody-capture (MAC) enzyme-linked immunosorbent assay (ELISA) kit, the YF MAC-HD, was successfully introduced starting in 2018 to laboratories in Africa and South America. The YF MAC-HD kit can be performed in 3.5 hours, test up to 24 samples, and includes all reagents necessary to perform the test, except for water used to dilute wash buffer. In 2018 and 2019, a total of 56 laboratory personnel from 39 countries in Africa and South America were trained to use the kit during workshops, followed by take-home YF IgM proficiency testing (PT) exercises. Participants received either a 10- or 20-sample YF PT panel and performed testing using the YF MAC-HD kit. All countries obtained 90% or higher correct results. These results verified the technical viability and transferability of YF MAC-HD kit use for laboratories in YF-endemic countries.