Borrelia burgdorferi outer surface protein C (OspC) binds complement component C4b and confers bloodstream survival

Borrelia burgdorferi (Bb) is the causative agent of Lyme disease in the United States, a disease that can result in carditis, and chronic and debilitating arthritis and/or neurologic symptoms if left untreated. Bb survives in the midgut of the Ixodes scapularis tick, or within tissues of immunocompetent hosts. In the early stages of infection, the bacteria are present in the bloodstream where they must resist clearance by the innate immune system of the host. We have found a novel role for outer surface protein C (OspC) from B. burgdorferi and B. garinii in interactions with the complement component C4b and bloodstream survival in vivo. Our data show that OspC inhibits the classical and lectin complement pathways and competes with complement protein C2 for C4b binding. Resistance to complement is important for maintenance of the lifecycle of Bb, enabling survival of the pathogen within the host as well as in the midgut of a feeding tick when ospC expression is induced.     

Cryopreservation of rat MSCs by use of a programmed freezer with magnetic field

Mesenchymal stem cells (MSCs) can be used for the regeneration of various tissues and cryopreservation of MSCs is so important for regenerative medicine. The purpose of this study was to evaluate the influences of cryopreservation on MSCs by use of a programmed freezer with a magnetic field (CAS freezer). MSCs were isolated from bone marrow of rat femora. The cells were frozen by a CAS freezer with 10% dimethyl sulfoxide (Me2SO) and cryopreserved for 7 days at a temperature of −150 °C. Immediately after thawing, the number of survived cells was counted. The cell proliferation also examined after 48 h culture. Next, MSCs were frozen by two different freezers; CAS freezer and a conventional programmed freezer without magnetic field. Then, osteogenic and adipogenic differentiations of cryopreserved cells were examined. As a result, survival and proliferation rates of MSCs were significantly higher in CAS freezer than in the non-magnetic freezer. Alizarin positive reaction, large amount of calcium quantification, and greater alkaline phosphatase activity were shown in both the non-cryopreserved and CAS groups after osteogenic differentiation. Moreover, Oil Red O staining positive reaction and high amount of PPARγ and FABP4 mRNAs were shown in both the non-cryopreserved and CAS groups after adipogenic differentiation. From these findings, it is shown that a CAS freezer can maintain high survival and proliferation rates of MSCs and maintain both adipogenic and osteogenic differentiation abilities. It is thus concluded that CAS freezer is available for cryopreservation of MSCs, which can be applied to various tissue regeneration.     

On the morphology of antennular sensory and attachment organs in cypris larvae of the deep‐sea vent/seep barnacles,Ashinkailepas and Neoverruca

Barnacle cypris larvae show high morphological variation in the organs used in search of and attaching to a substratum. This variation may represent adaptation to the habitat of the species. Here, we studied SEM level morphologies of cypris antennular sensory and attachment organs in a deep‐sea vent endemic species (Neoverruca sp.) and a vent/seep inhabiting species (Ashinkailepas seepiophila). We compare them with three species from other environments. The antennular morphologies of Neoverruca sp. and A. seepiophila were similar, which is consistent with recent molecular studies showing a close relationship of the two species. The setation pattern of the antennules was very conservative among species from various environments. In contrast, striking differences were observed in the structure of the attachment organ (the third antennular segment). Neoverruca sp. and A. seepiophila had no velum or a skirt surrounding the attachment disc on the third segment, while other cirripede cyprids almost always have either of these structures. In addition, both cyprids of A. seepiophila and Neoverruca sp. had the attachment disc angled toward the substratum, whereas it faces distally in cyprids from hard bottom inhabiting barnacles. We suggest that both velum/skirt and the angle of the attachment disc play an important role, when the antennules are contacting the substratum during surface exploration. Differences in attachment organ structures may be highly adaptive, enabling cirripede species to enter new habitats during evolution. J. Morphol. 277:594–602, 2016. © 2016 Wiley Periodicals, Inc.     

Ups1p, a conserved intermembrane space protein, regulates mitochondrial shape and alternative topogenesis of Mgm1p

Mgm1p is a conserved dynamin-related GTPase required for fusion, morphology, inheritance, and the genome maintenance of mitochondria in Saccharomyces cerevisiae. Mgm1p undergoes unconventional processing to produce two functional isoforms by alternative topogenesis. Alternative topogenesis involves bifurcate sorting in the inner membrane and intramembrane proteolysis by the rhomboid protease Pcp1p. Here, we identify Ups1p, a novel mitochondrial protein required for the unique processing of Mgm1p and for normal mitochondrial shape. Our results demonstrate that Ups1p regulates the sorting of Mgm1p in the inner membrane. Consistent with its function, Ups1p is peripherally associated with the inner membrane in the intermembrane space. Moreover, the human homologue of Ups1p, PRELI, can fully replace Ups1p in yeast cells. Together, our findings provide a conserved mechanism for the alternative topogenesis of Mgm1p and control of mitochondrial morphology.     

Phylogeographic insights into the invasion history and secondary spread of the signal crayfish in Japan

Successful invasion by nonindigenous species is often attributed to high propagule pressure, yet some foreign species become widespread despite showing reduced genetic variation due to founder effects. The signal crayfish (Pacifastacus leniusculus) is one such example, where rapid spread across Japan in recent decades is believed to be the result of only three founding populations. To infer the history and explore the success of this remarkable crayfish invasion, we combined detailed phylogeographical and morphological analyses conducted in both the introduced and native ranges. We sequenced 16S mitochondrial DNA of signal crayfish from across the introduced range in Japan (537 samples, 20 sites) and the native range in western North America (700 samples, 50 sites). Because chela size is often related to aggressive behavior in crayfish, and hence, their invasion success, we also measured chela size of a subset of specimens in both introduced and native ranges. Genetic diversity of introduced signal crayfish populations was as high as that of the dominant phylogeographic group in the native range, suggesting high propagule pressure during invasion. More recently established crayfish populations in Japan that originated through secondary spread from one of the founding populations exhibit reduced genetic diversity relative to older populations, probably as a result of founder effects. However, these newer populations also show larger chela size, consistent with expectations of rapid adaptations or phenotypic responses during the invasion process. Introduced signal crayfish populations in Japan originate from multiple source populations from a wide geographic range in the native range of western North America. A combination of high genetic diversity, especially for older populations in the invasive range, and rapid adaptation to colonization, manifested as larger chela in recent invasions, likely contribute to invasion success of signal crayfish in Japan.     

Water entry for the black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.) seeds observed by dedicated micro-magnetic resonance imaging

Water entry at germination for black locust (Robinia pseudoacacia L.) seeds which are known as hard seeds with impermeable seed coat to water, was examined using micro-magnetic resonance imaging (MRI). The MRI apparatus equipped with a low-field (1 T; Tesla) permanent magnet was used, which is open access, easy maintenance, operable and transportable. The excellent point of the apparatus is that T ₁-enhancement of water signals absorbed in dry seeds against steeping free water is stronger than the apparatuses with high-field superconducting magnets, which enabled clear detection of water entry. Water hardly penetrated into the seeds for more than 8 h but approximately 60 % of seeds germinated by incubating on wet filter papers for several days. Hot water treatments above 75 °C for 3 min effectively induced water gap; scarification was 70 % at 100 °C and 75 °C, declined to 15 % at 50 °C and decreased further at room temperature. Water entered into the scarified seeds exclusively through the lens, spread along the dorsal side of the seeds and reached the hypocotyl, whereas water migrated slowly through hilum side to radicle within 3 h.     

One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)

Background

The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently.

Results

Here, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice.

Conclusions

The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1432-5) contains supplementary material, which is available to authorized users.     

Two forms of secreted and thermostable luciferases from the marine copepod crustacean, Metridia pacifica

We cloned two forms of the secreted and thermostable luciferase genes, MpLuc1 and MpLuc2, from the marine copepod, Metridia pacifica. The 840-bp MpLuc1 cDNA comprised a 630-bp open reading frame encoding a 210-amino acid polypeptide (22.7 kDa). MpLuc1 had the closest homology with Metridia longa luciferase. The 753-bp MpLuc2 cDNA consisted of a 567-bp open reading frame (20.3 kDa), and it had the closest homology with Gaussia princeps luciferase. Single-specimen genomic PCR confirmed the presence of two luciferase genes in M. pacifica, and single-specimen RT-PCR revealed that both luciferase mRNAs were expressed. Both MpLuc1 and MpLuc2 (MpLucs) specifically reacted with the substrate coelenterazine producing identical bioluminescent spectra (lambdamax, 485 nm), but with different kinetics. Adding salt such as MgCl2 and CaCl2 to the reaction mixture significantly enhanced MpLuc1 and MpLuc2 activities. Wild-type MpLucs were remarkably thermostable; MpLuc1 retained about 60% of the original activity even after incubation at 90 degrees C for 30 min. MpLucs expressed in NIH-3T3 and HeLa cells were largely secreted into the culture medium. Continuous monitoring of secreted MpLuc1 driven by the c-fos promoter demonstrated the potential usefulness of MpLuc1 in nondisruptive reporter assays.     

In addition to the ARS core, the ARS box is necessary for autonomously replicating sequences

Summary Autonomously replicating sequences (ARSs) were cloned from nuclear and mitochondrial DNA of D. melanogaster using YIp5, which is composed of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^- yeast strain as the recipient. The nucleotide sequences of six ARSs, two from nuclear bulk, two from the nuclear 1.688 satellite, and two from mitochondorial DNA, were determined. The relationship between the transformation frequency and the inclusion of the ARS core, 5 _T ^A TT-TAT _A ^G TTT _T ^A 3, of these fragments was analysed. All the ARSs contained an ARS core or a single base change of it. However, not all the fragments that contained a single base change of the ARS core were able to transform the recipient cells, suggesting that certain bases in the ARS core were not exchangeable. It is suggested by transformation experiments with subfragments that in addition to an ARS core, an ARS box which is located within 25 bp upstream of the ARS core and whose sequence is composed of 5TNT _G ^A AA 3, is necessary for autonomous replication.     

l-Carnitine is essential to β-oxidation of quarried fatty acid from mitochondrial membrane by PLA2

Mitochondrial β-oxidation is an important system involved in the energy production of various cells. In this system, the function of l-carnitine is essential for the uptake of fatty acids to mitochondria. However, it is unclear whether or not endogenous respiration, ADP-induced O₂ consumption without substrates, is caused by l-carnitine treatment. In this study, we investigated whether l-carnitine is essential to the β-oxidation of quarried fatty acids from the mitochondrial membrane by phospholipase A₂ (PLA₂) using isolated mitochondria from the liver of rats. Intact mitochondria were incubated in a medium containing Pi, CoA and l-carnitine. The effect of l-carnitine treatment on ADP-induced mitochondrial respiration was observed without exogenous respiratory substrate. Increase in mitochondrial respiration was induced by treatment with l-carnitine in a concentration-dependent manner. Treatment with rotenone, a complex I blocker, completely inhibited ADP-induced oxygen consumption even in the presence of l-carnitine. Moreover, the l-carnitine dependent ADP-induced mitochondrial oxygen consumption did not increase when PLA₂ inhibitors were treated before ADP treatment. The l-carnitine-dependent ADP-induced oxygen consumption did contribute to ATP productions but not heat generation via an uncoupling system. These results suggest that l-carnitine might be essential to the β-oxidation of quarried fatty acids from the mitochondrial membrane by PLA₂.