Species diversity and phylogeography of Cornus kousa (Asian dogwood) captured by genomic and genic microsatellites

Cornus kousa (Asian dogwood), an East Asia native tree, is the most economically important species of the dogwood genus, owing to its desirable horticultural traits and ability to hybridize with North America‐native dogwoods. To assess the species genetic diversity and to better inform the ongoing and future breeding efforts, we assembled an herbarium and arboretum collection of 131 noncultivated C. kousa specimens. Genotyping and capillary electrophoresis analyses of our C. kousa collection with the newly developed genic and published nuclear genomic microsatellites permitted assessment of genetic diversity and evolutionary history of the species. Regardless of the microsatellite type used, the study yielded generally similar insights into the C. kousa diversity with subtle differences deriving from and underlining the marker used. The accrued evidence pointed to the species distinct genetic pools related to the plant country of origin. This can be helpful in the development of the commercial cultivars for this important ornamental crop with increased pyramided utility traits. Analyses of the C. kousa evolutionary history using the accrued genotyping datasets pointed to an unsampled ancestor population, possibly now extinct, as per the phylogeography of the region. To our knowledge, there are few studies utilizing the same gDNA collection to compare performance of genomic and genic microsatellites. This is the first detailed report on C. kousa species diversity and evolutionary history inference.     

Establishment of a mouse model for post‐inflammatory hyperpigmentation

Post‐inflammatory hyperpigmentation (PIH) is a common cutaneous condition that can cause a disfigured appearance. However, the pathophysiology of PIH remains poorly understood, at least in part, because an appropriate animal model for research has not been established. In order to analyze the pathomechanism of PIH, we successfully induced PIH in a hairless version of transgenic mice (hk14‐SCF Tg/HRM) that have a human‐type epidermis containing melanin by repeated hapten application of 2,4‐dinitrofluorobenzene. Histopathologic observation showed epidermal hyperplasia, predominant infiltrations of inflammatory cells, and melanin‐containing cells in the dermis just after elicitation of the atopic dermatitis‐like condition. At week 2, the findings were similar to the characteristics of PIH, that is, an increase of melanin without spongiosis or liquid degeneration in the epidermis and an increase in dermal melanophages. Dynamic analysis of melanin showed that the melanin in the dermis remained for a longer duration than in the epidermis. Furthermore, immunohistochemical staining revealed that the majority of cells containing melanin were positive for the anti‐CD68 antibody, but negative for the anti‐F4/80 antibody. These data suggest that novel treatments of PIH should be targeted against macrophages and should eventually lead to the development of new treatment modalities.     

Detection of Cryptomeria japonica roots with ground penetrating radar

Abstract

Coarse tree roots, which are responsible for most root carbon storage, are usually measured by destructive methods such as excavation and coring. Ground penetrating radar (GPR) is a non-destructive tool that could be used to detect coarse roots in forest soils. In this study, we examined whether the roots of Cryptomeria japonica, a major plantation species in Japan, can be detected with GPR. We also looked for factors that impact the analysis and detection of roots. Roots and wooden dowels of C. japonica were buried 30 cm deep in sandy granite soil. From GPR measurements with a 900 MHz antenna, the distribution and diameter of samples in several transects were recorded. The buried roots were detected clearly and could be distinguished at diameters of 1.1–5.2 cm. There were significant positive relationships between root diameter and parameters extracted from the resultant GPR waveform. The difference in water content between roots and soil is a crucial factor impacting the ability to detect roots with GPR. We conclude that GPR can be used as a non-destructive tool, but further investigation is needed to determine optimal conditions (e.g. water content) and analytical methods for using GPR to examine roots in forest sites.     

The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.     

Meningitis and bacteremia by nonhemolytic Group B Streptococcus strain: A whole genome analysis

Group B streptococcus (GBS) is a leading cause of neonatal infections. Most isolates are β-hemolytic, and their activity is considered to be pivotal for GBS pathogenicity. We report a case of a neonate with meningitis caused by nonhemolytic GBS. The patient developed meningitis 3 days after birth. Genotyping was performed and the characteristics of the strain (GCMC97051) identified by whole genome sequence using next generation sequencing. GCMC97051 possesses genetic alterations such as disruption of cylA by IS1381A insertion and a frameshift mutation in cylE, resulting in a lack of hemolysis. Thus, nonhemolytic GBS can retain the potential to cause invasive infections.     

Cold shock proteins improve E. coli cell-free synthesis in terms of soluble yields of aggregation-prone proteins

Protein folding is usually slowed-down at low temperatures, and thus low-temperature expression is an effective strategy to improve the soluble yield of aggregation-prone proteins. In this study, we investigated the effects of a variety of cold shock proteins and domains (Csps) on an Escherichia coli cell extract-based cell-free protein synthesis system (CF). Most of the 12 Csps that were successfully prepared dramatically improved the protein yields, by factors of more than 5 at 16°C and 2 at 23°C, to levels comparable to those obtained at 30°C. Their stimulatory effects were complementary to each other, while CspD and CspH were inhibitory. The Csps’ effects correlated well with their Pfam CSD family scores (PF00313.22). All of the investigated Csps, except CspH, similarly possessed RNA binding and chaperon activities and increased the messenger RNA amount irrespective of their effect, suggesting that the proper balance between these activities was required for the enhancement. Unexpectedly, the 5′-untranslated region of cspA was less effective as the leader sequence. Our results demonstrated that the use of the Csps presented in this study will provide a simple and highly effective strategy for the CF, to improve the soluble yields of aggregation-prone proteins.     

Treatment with β2‐Adrenoceptor Agonist in Vivo Induces Human Clock Gene,Per1, mRNA Expression in Peripheral Blood

This study examined whether in vivo exposure to a β₂‐adrenoceptor agonist, tulobuterol, induces human Period1 (hPer1) mRNA expression in cells from peripheral whole blood. In one experiment, oral tulobuterol was administered to five healthy volunteers at 22:00 h, while in another, a transdermally tulobuterol patch was applied to the same five subjects at 20:00 h. In each experiment, serum tulobuterol concentrations were measured at four time points, and total RNA was isolated from peripheral blood cells for determinations of hPer1 mRNA expression by real‐time polymerase chain reaction. Both the tulobuterol tablet and the transdermal patch increased hPer1 mRNA expression, suggesting that analyses of human peripheral blood cells could reliably represent peripheral clock gene mRNA expression in vivo.     

Root orientation can affect detection accuracy of ground-penetrating radar

Aim

Ground-penetrating radar (GPR) has been applied to detect coarse tree roots. The horizontal angle of a root crossing a scanning line is a factor that affects both root detection and waveform parameter values. The purpose of this study was to quantitatively evaluate the influence of root orientation (x, degree) on two major waveform parameters, amplitude area (A, dB × ns) and time interval between zero crossings (T, ns).

Methods

We scanned four diameter classes of dowels in a sandy bed as simulated roots using a 900 MHz antenna from multiple angles to clarify the relationships between the parameters and x.

Results

Angle x strongly affected reflection images and A values. The variation in A(x) fitted a sinusoidal waveform, whereas T was independent of x. The value of A scanning at 90° was estimated by A values of arbitrary x in two orthogonal transects. The sum of T in all reflected waveforms showed a significant linear correlation with dowel diameter.

Conclusions

We clarified that root orientation dramatically affected root detection and A values. The sum of T of all reflected waveforms was a suitable parameter for estimating root diameter. Applying grid transects can overcome the effects of root orientation.     

Focus Issue on Calcium Signaling: Analyses of a Gravistimulation-Specific Ca2+ Signature in Arabidopsis using Parabolic Flights

Gravity is a critical environmental factor affecting the morphology and functions of organisms on the Earth. Plants sense changes in the gravity vector (gravistimulation) and regulate their growth direction accordingly. In Arabidopsis (Arabidopsis thaliana) seedlings, gravistimulation, achieved by rotating the specimens under the ambient 1g of the Earth, is known to induce a biphasic (transient and sustained) increase in cytoplasmic calcium concentration ([Ca2+]_c). However, the [Ca2+]_c increase genuinely caused by gravistimulation has not been identified because gravistimulation is generally accompanied by rotation of specimens on the ground (1g), adding an additional mechanical signal to the treatment. Here, we demonstrate a gravistimulation-specific Ca²⁺ response in Arabidopsis seedlings by separating rotation from gravistimulation by using the microgravity (less than 10⁻⁴g) conditions provided by parabolic flights. Gravistimulation without rotating the specimen caused a sustained [Ca2+]_c increase, which corresponds closely to the second sustained [Ca2+]_c increase observed in ground experiments. The [Ca2+]_c increases were analyzed under a variety of gravity intensities (e.g. 0.5g, 1.5g, or 2g) combined with rapid switching between hypergravity and microgravity, demonstrating that Arabidopsis seedlings possess a very rapid gravity-sensing mechanism linearly transducing a wide range of gravitational changes (0.5g–2g) into Ca²⁺ signals on a subsecond time scale.Calcium ion (Ca²⁺) functions as an intracellular second messenger in many signaling pathways in plants (White and Broadley, 2003; Hetherington and Brownlee, 2004; McAinsh and Pittman, 2009; Spalding and Harper, 2011). Endogenous and exogenous signals are spatiotemporally encoded by changing the free cytoplasmic concentration of Ca²⁺ ([Ca2+]_c), which in turn triggers [Ca2+]_c-dependent downstream signaling (Sanders et al., 2002; Dodd et al., 2010). A variety of [Ca2+]_c increases induced by diverse environmental and developmental stimuli are reported, such as phytohormones (Allen et al., 2000), temperature (Plieth et al., 1999; Dodd et al., 2006), and touch (Knight et al., 1991; Monshausen et al., 2009). The [Ca2+]_c increase couples each stimulus and appropriate physiological responses. In the Ca²⁺ signaling pathways, the stimulus-specific [Ca2+]_c pattern (e.g. amplitude and oscillation) provide the critical information for cellular signaling (Scrase-Field and Knight, 2003; Dodd et al., 2010). Therefore, identification of the stimulus-specific [Ca2+]_c signature is crucial for an understanding of the intracellular signaling pathways and physiological responses triggered by each stimulus, as shown in the case of cold acclimation (Knight et al., 1996; Knight and Knight, 2000).Plants often exhibit biphasic [Ca2+]_c increases in response to environmental stimuli. Thus, slow cooling causes a fast [Ca2+]_c transient followed by a second, extended [Ca2+]_c increase in Arabidopsis (Arabidopsis thaliana; Plieth et al., 1999; Knight and Knight, 2000). The Ca²⁺ channel blocker lanthanum (La³⁺) attenuated the fast transient but not the following increase (Knight and Knight, 2000), suggesting that these two [Ca2+]_c peaks have different origins. Similarly, hypoosmotic shock caused a biphasic [Ca2+]_c increase in tobacco (Nicotiana tabacum) suspension-culture cells (Takahashi et al., 1997; Cessna et al., 1998). The first [Ca2+]_c peak was inhibited by gadolinium (Gd³⁺), La³⁺, and the Ca²⁺ chelator EGTA (Takahashi et al., 1997; Cessna et al., 1998), whereas the second [Ca2+]_c increase was inhibited by the intracellular Ca²⁺ store-depleting agent caffeine but not by EGTA (Cessna et al., 1998). The amplitude of the first [Ca2+]_c peak affected the amplitude of the second increase and vice versa (Cessna et al., 1998). These results suggest that even though the two [Ca2+]_c peaks originate from different Ca²⁺ fluxes (e.g. Ca²⁺ influx through the plasma membrane and Ca²⁺ release from subcellular stores, respectively), they are closely interrelated, showing the importance of the kinetic and pharmacological analyses of these [Ca2+]_c increases.Changes in the gravity vector (gravistimulation) could work as crucial environmental stimuli in plants and are generally achieved by rotating the specimens (e.g. +180°) in ground experiments. Use of Arabidopsis seedlings expressing apoaequorin, a Ca²⁺-reporting photoprotein (Plieth and Trewavas, 2002; Toyota et al., 2008a), has revealed that gravistimulation induces a biphasic [Ca2+]_c increase that may be involved in the sensory pathway for gravity perception/response (Pickard, 2007; Toyota and Gilroy, 2013) and the intracellular distribution of auxin transporters (Benjamins et al., 2003; Zhang et al., 2011). These two Ca²⁺ changes have different characteristics. The first transient [Ca2+]_c increase depends on the rotational velocity but not angle, whereas the second sustained [Ca2+]_c increase depends on the rotational angle but not velocity. The first [Ca2+]_c transient was inhibited by Gd³⁺, La³⁺, and the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid but not by ruthenium red (RR), whereas the second sustained [Ca2+]_c increase was inhibited by all these chemicals. These results suggest that the first transient and second sustained [Ca2+]_c increases are related to the rotational stimulation and the gravistimulation, respectively, and are mediated by distinct molecular mechanisms (Toyota et al., 2008a). However, it has not been demonstrated directly that the second sustained [Ca2+]_c increase is induced solely by gravistimulation; it could be influenced by other factors, such as an interaction with the first transient [Ca2+]_c increase (Cessna et al., 1998), vibration, and/or deformation of plants during the rotation.To elucidate the genuine Ca²⁺ signature in response to gravistimulation in plants, we separated rotation and gravistimulation under microgravity (μg; less than 10⁻⁴g) conditions provided by parabolic flight (PF). Using this approach, we were able to apply rotation and gravistimulation to plants separately (Fig. 1). When Arabidopsis seedlings were rotated +180° under μg conditions, the [Ca2+]_c response to the rotation was transient and almost totally attenuated in a few seconds. Gravistimulation (transition from μg to 1.5g) was then applied to these prerotated specimens at the terminating phase of the PF. This gravistimulation without simultaneous rotation induced a sustained [Ca2+]_c increase. The kinetic properties of this sustained [Ca2+]_c increase were examined under different gravity intensities (0.5g–2g) and sequences of gravity intensity changes (Fig. 2A). This analysis revealed that gravistimulation-specific Ca²⁺ response has an almost linear dependency on gravitational acceleration (0.5g–2g) and an extremely rapid responsiveness of less than 1 s.Open in a separate windowFigure 1.Diagram of the experimental procedures for applying separately rotation and gravistimulation to Arabidopsis seedlings. Rotatory stimulation (green arrow) was applied by rotating the seedlings 180° under μg conditions, and 1.5g 180° rotation gravistimulation (blue arrow) was applied to the prerotated seedlings after μg.Open in a separate windowFigure 2.Acceleration, temperature, humidity, and pressure in an aircraft during flight experiments. A, Accelerations along x, y, and z axes in the aircraft during PF. The direction of flight (FWD) and coordinates (x, y, and z) are indicated in the bottom graph. The inset shows an enlargement of the acceleration along the z axis (gravitational acceleration) during μg conditions lasting for approximately 20 s. B, Temperature, humidity, and pressure in the aircraft during PF. Shaded areas in graphs denote the μg condition.     

Modulation of chondrocyte migration and aggregation by insulin-like growth factor-1 in cultured cartilage

The effect of insulin-like growth factor-1 (IGF-1) on the behavior of rabbit chondrocytes in cultured collagen (CL) gels initially seeded with 2 × 10⁵ cells/ml was examined. On day 5, the frequency of migrating cells cultured in presence of 100 ng IGF-1/ml was 0.04, which was 54 % of the frequency in IGF-1-free culture. The presence of IGF-1 caused an increase in the frequency of dividing cells from 0.09 to 0.13. These results suggest that IGF-1 suppressed the migration of chondrocytes in the CL gels while stimulating cell division in the initial culture phase. The proteolytic migration of cells was thought to be suppressed by the down-regulation of membrane type 1 matrix metalloproteinase by IGF-1. This contributed to the formation of aggregates with spherical-shaped cells that produced collagen type II.