Inflammatory pre-conditioning of mesenchymal multipotent stromal cells improves their immunomodulatory potency in acute pyelonephritis in rats

Background aimsAcute pyelonephritis is one of the most frequent infectious diseases of the urinary tract and a leading cause of kidney failure worldwide. One strategy for modulating excessive inflammatory responses in pyelonephritis is administration of mesenchymal multipotent stromal cells (MMSCs).MethodsThe putative protective effect of injection of MMSCs against experimental acute pyelonephritis was examined. We used in vivo experimental model of APN where bacteria are introduced in the bladder of rat. Three days after, intravenous injection of MMSCs was done. On the 7th day blood samples and kidneys were taken for further analysis.ResultsWe found obvious signs of oxidative stress and inflammation in the kidney in acute pyelonephritis in rats. Particularly, pro-inflammatory cytokine tumor necrosis factor-α levels, malondialdehyde, nitrite and myeloperoxidase activity were significantly increased. Histologic evaluation revealed numerous attributes of inflammation and tissue damage in the kidney. Treatment with MMSCs caused a remarkable decrease of all of these pathologic signs in renal tissue. Also, activated leukocytes induced pre-conditioning-like signaling in MMSCs. We showed alterations of expression or activity of inducible nitric oxide synthase, transforming growth factor-β, matrix metalloproteinase-2 and glycogen synthase kinase-3β, which could mediate immunomodulation and protective effects of MMSCs. This signaling could be characterized as inflammatory pre-conditioning.ConclusionsThe beneficial capacity of MMSCs to alleviate renal inflammation was more pronounced when pre-conditioned MMSCs were used. This approach could be used to prime MMSCs with different inflammatory modulators to enhance their engraftment and function in an immunoprotected fashion.     

Longitudinal Study of Reproductive Performance of Female Cattle Produced by Somatic Cell Nuclear Transfer

The objective of this study was to determine whether or not reproductive performance in cattle produced by somatic cell nuclear transfer (SCNT) is significantly different from that of their genetic donors. To address this question, we directed two longitudinal studies using different embryo production procedures: (1) superovulation followed by artificial insemination (AI) and embryo collection and (2) ultrasound-guided ovum pick-up followed by in vitro fertilization (OPU-IVF). Collectively, these two studies represent the largest data set available for any species on the reproductive performance of female clones and their genetic donors as measured by their embryo production outcomes in commercial embryo production program. The large-scale study described herein was conducted over a six-year period of time and provides a unique comparison of 96 clones to the 40 corresponding genetic donors. To our knowledge, this is the first longitudinal study on the reproductive performance of cattle clones using OPU-IVF. With nearly 2,000 reproductive procedures performed and more than 9,200 transferable embryos produced, our observations show that the reproductive performance of cattle produced by SCNT is not different compared to their genetic donors for the production of transferable embryos after either AI followed by embryo collection (P = 0.77) or OPU-IVF (P = 0.97). These data are in agreement with previous reports showing that the reproductive capabilities of cloned cattle are equal to that of conventionally produced cattle. In conclusion, results of this longitudinal study once again demonstrate that cloning technology, in combination with superovulation, AI and embryo collection or OPU-IVF, provides a valuable tool for faster dissemination of superior maternal genetics.     

Assessment of SLX4 Mutations in Hereditary Breast Cancers

Background

SLX4 encodes a DNA repair protein that regulates three structure-specific endonucleases and is necessary for resistance to DNA crosslinking agents, topoisomerase I and poly (ADP-ribose) polymerase (PARP) inhibitors. Recent studies have reported mutations in SLX4 in a new subtype of Fanconi anemia (FA), FA-P. Monoallelic defects in several FA genes are known to confer susceptibility to breast and ovarian cancers.

Methods and Results

To determine if SLX4 is involved in breast cancer susceptibility, we sequenced the entire SLX4 coding region in 738 (270 Jewish and 468 non-Jewish) breast cancer patients with 2 or more family members affected by breast cancer and no known BRCA1 or BRCA2 mutations. We found a novel nonsense (c.2469G>A, p.W823*) mutation in one patient. In addition, we also found 51 missense variants [13 novel, 23 rare (MAF<0.1%), and 15 common (MAF>1%)], of which 22 (5 novel and 17 rare) were predicted to be damaging by Polyphen2 (score = 0.65–1). We performed functional complementation studies using p.W823* and 5 SLX4 variants (4 novel and 1 rare) cDNAs in a human SLX4-null fibroblast cell line, RA3331. While wild type SLX4 and all the other variants fully rescued the sensitivity to mitomycin C (MMC), campthothecin (CPT), and PARP inhibitor (Olaparib) the p.W823* SLX4 mutant failed to do so.

Conclusion

Loss-of-function mutations in SLX4 may contribute to the development of breast cancer in very rare cases.     

Low Genetic Diversity in Wide-Spread Eurasian Liver Fluke Opisthorchis felineus Suggests Special Demographic History of This Trematode Species

Opisthorchis felineus or Siberian liver fluke is a trematode parasite (Opisthorchiidae) that infects the hepato-biliary system of humans and other mammals. Despite its public health significance, this wide-spread Eurasian species is one of the most poorly studied human liver flukes and nothing is known about its population genetic structure and demographic history. In this paper, we attempt to fill this gap for the first time and to explore the genetic diversity in O. felineus populations from Eastern Europe (Ukraine, European part of Russia), Northern Asia (Siberia) and Central Asia (Northern Kazakhstan). Analysis of marker DNA fragments from O. felineus mitochondrial cytochrome c oxidase subunit 1 and 3 (cox1, cox3) and nuclear rDNA internal transcribed spacer 1 (ITS1) sequences revealed that genetic diversity is very low across the large geographic range of this species. Microevolutionary processes in populations of trematodes may well be influenced by their peculiar biology. Nevertheless, we suggest that lack of population genetics structure observed in O. felineus can be primarily explained by the Pleistocene glacial events and subsequent sudden population growth from a very limited group of founders. Rapid range expansion of O. felineus through Asian and European territories after severe bottleneck points to a high dispersal potential of this trematode species.     

The stoat Mustela erminea population decline in northern Belarus and its consequences for weasels Mustela nivalis

Abstract

In northern Belarus, we have documented a decline in the local stoat Mustela erminea population following the naturalisation of the American mink Mustela vison. The most likely cause is the reduction in the density and distribution of the main prey of stoats, the riparian voles (the water vole Arvicola terrestris and the root vole Microtus oeconomus), due to excessive predation by mink. Since the stoat population has declined, the number of weasels Mustela nivalis in marshlands has increased and their mean body mass has increased, correlated with the higher number and mean weights of rodents available for weasels in marshland compared with forest habitats.     

Ethidium Probing of the Parallel Double- and Four-stranded Structures Formed by the Telomeric DNA Sequences dG(GT)4G and d(GT)5

Abstract

Oligonucleotides 3′-d(GT)₅-(CH₂CH₂O)₃-d(GT)₅-3′ (parGT), containing GT repeats present in the telomeric DNA from Saccharomyces cerevisiae, had been demonstrated to form bimolecular structure, GT-quadruplex (qGT) [O. F. Borisova et al. FEBS Letters 306, 140–142 (1992)]. Four d(GT)₅ strands of the GT-quadruplex are parallel and form five G-quartets while thymines are bulged out. The four GT repeats when flanked by guanines, 3′-dG(TG)₄G-(CH₂CH₂O)₃- dG(GT)₄G-3′ (hp-GT), had been shown to form a novel parallel-stranded (ps) double helix with G·G and T·T base pairs (hp-GT ps-DNA) [A. K. Shchyolkina et al. J. Biomol. Struct. Dynam. 18, 493–503 (2001)]. In the present study the intercalator ethidium bromide (Et) was used for probing the two structures. The mode of Et binding and its effect on thermostability of qGT and hp-GT were compared. The quantum yield (q) and the fluorescence lifetime (τ) of Et:qGT (q = 0.15 ±0.01 and τ = 24 ±1 ns) and Et:hp-GT (q = 0.10 ± 0.01 and τ = 16.5 ± 1 ns) indicative of intercalation mode of Et binding were determined. Et binding to qGT was found to be cooperative with corresponding coefficient ω = 3.9 ± 0.1 and the binding constant K= (6.4 ± 0.1)·10M^?1. The maximum number of Et molecules intercalating into GT-quadruplex is as high as twice the number of inerspaces between G-quartets (eight in our case). The data conform to the model of Et association with GT-quadruplex suggested earlier [O. F. Borisova et al. Mol. Biol. (Russ) 35, 732–739 (2001)]. The anticooperative type of Et binding was observed in case of hp- GT ps-DNA, with the maximum number of bound Et molecules, N = 4 ÷ 5, and the association constant K = (1.5 ± 0.1)·10⁵ M^?1. Thermodynamic parameters of formation of Et:qGT and EtBr:hp-GT complexes were calculated from UV thermal denaturation profiles.     

Cdc45 Protein-Single-stranded DNA Interaction Is Important for Stalling the Helicase during Replication Stress

Replicative polymerase stalling is coordinated with replicative helicase stalling in eukaryotes, but the mechanism underlying this coordination is not known. Cdc45 activates the Mcm2-7 helicase. We report here that Cdc45 from budding yeast binds tightly to long (≥ 40 nucleotides) genomic single-stranded DNA (ssDNA) and that 60mer ssDNA specifically disrupts the interaction between Cdc45 and Mcm2-7. We identified a mutant of Cdc45 that does not bind to ssDNA. When this mutant of cdc45 is expressed in budding yeast cells exposed to hydroxyurea, cell growth is severely inhibited, and excess RPA accumulates at or near an origin. Chromatin immunoprecipitation suggests that helicase movement is uncoupled from polymerase movement for mutant cells exposed to hydroxyurea. These data suggest that Cdc45-ssDNA interaction is important for stalling the helicase during replication stress.     

Simultaneous Surface Display and Secretion of Proteins from Mammalian Cells Facilitate Efficient in Vitro Selection and Maturation of Antibodies

A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.