A receptor. This effect of myricetin was not blocked by the GABAA receptor benzodiazepine- (BZ-) binding site antagonist flumazenil, but by KN-62, a specific inhibitor of the Ca2+/calmodulin-stimulated protein kinase II (CaMK-II). Patch clamp and live Ca2+ imaging studies found that myricetin could increase Ca2+ current and intracellular Ca2+ concentration, respectively, via T- and L-type Ca2+ channels in rat PVN neurons and hypothalamic primary culture neurons. Immunofluorescence staining showed increased phosphorylation of CaMK-II after myricetin incubation in primary culture of rat hypothalamic neurons, and the myricetin-induced CaMK-II phosphorylation was further confirmed by Western blotting in PC-12 cells. The present results suggest that myricetin enhances GABAA receptor activity via calcium channel/CaMK-II dependent mechanism, which is distinctively different from that of most existing BZ-binding site agonists of GABAA receptor.The flavonoid myricetin is found in several sedative herbs, for example, the St. John's Wort, but its influence on sedation and its possible mechanism of action are unknown. Using patch-clamp technique on a brain slice preparation, the present study found that myricetin promoted GABAergic activity in the neurons of hypothalamic paraventricular nucleus (PVN) by increasing the decay time and frequency of the inhibitory currents mediated by GABA After 3 washes by PBS, samples were blocked by 5% normal goat serum for 1 hour, and then incubated with antiphospho-CaMKII  and antibeta tubulin  at 4°C overnight. Alexa Fluor 488F , and Alexa Fluor 568  were used as secondary antibodies. Nuclei were stained by Hoechst 33342.Hypothalamus neurons cultured on cover-slips were incubated with 0.5% DMSO or 50 μM), and zolpidem (5 μM) were added in the culture medium of PC-12 cells for 30 min. The cells were harvested in a lysis buffer . Total lysates (40 mg per lane) were subjected to SDS-PAGE and were transferred onto nitrocellulose membranes . The transferred membrane was blocked by incubating in TBST plus 5% fat-free milk. The membrane was then washed 3 times with TBST and incubated with rabbit antirat CaMK-II (1 : 500) polyclonal antibody, and rabbit antirat phosphor-CaMK-II (1 : 500) polyclonal antibody  in TBST plus 1.5% fat-free milk at 4°C for overnight. The membrane was subsequently washed with TBST and incubated for 1 hour with peroxidase-conjugated secondary antibody. The membrane was washed 3 times with TBST and then detected by enhanced chemiluminescence .0.1% DMSO, myricetin  and CNQX (20 μM) were used to block glutamate induced excitory post-synaptic currents. TTX (0.5 μM) was used to block the action potential dependent IPSCs. The recorded currents could be totally blocked by GABAA receptor antagonist bicuculine (10 μM), confirming that the currents were the GABAA-mediated mIPSCs (μg/mL) significantly increased the decay time of mIPSCs to 115 ± 7.29% compared with basal , among which 5 neurons also showed an decreased of the interevent interval, indicating increased frequency of presynaptic GABA release (P < 0.05). These data suggest that myricetin not only modulates the kinetics of post-synaptic GABAA receptors by increasing the decay time, but also influences the spontaneous pre-synaptic GABA release in some cases.We first examined whether myricetin had effect on GABAergic activity of PVN neurons using a brain slice preparation. The spontaneous synaptic currents were recorded at the holding potential of −70 mV. AP-5 , this effect could be totally blocked by preperfusion of 10 μM flumazenil (P = 0.32). On the contrary, flumazenil failed to block myricetin-induced increase of decay time of mIPSCs (P = 0.042), suggesting that the effect of myricetin on GABAA receptor was not through binding to the BZ site, but rather, through a different mechanism from that of the classic BZ hypnotics such as zolpidem.The BZ-binding site is the most important modulating site on the GABAreceptor . To invereceptor , 10 μM fA receptor can be phosphorylated by multiple protein kinases and thus regulate the channel functions [A receptor subunits in neurons, which in turn not only enhance the inhibitory GABA effects but also influence synaptic plasticity [μM KN-62, a CaMK-II inhibitor [P = 0.49), suggesting the involvement of CaMK-II in mediating the effect of myricetin on GABAA receptor.Apart from the direct regulatory binding sites on the extracellular domain, the intracellular domains of GABAunctions . CaMK-IIasticity –34. To inhibitor , into thnhibitor , 37. Aft2+ increase is the well-known primary trigger of CaMK-II. Previous studies have also demonstrated that myricetin could induce vasoconstriction by activating Ca2+ channels in rat vascular smooth muscles [2+ current of PVN neurons with or without the presence of myricetin. 2+ currents recorded in PVN neurons by depolarizing the membrane potential from −80 mV to −20 mV in order to active both T-type and L-type Ca2+ currents. Myricetin significantly increased the peak Ca2+ current by about 50% . The current-voltage relationship was also examined by applying a 500 ms voltage ramp test from −80 mV to +60 mV. Myricetin increased the inward current and shifted the peak current to the hyperpolarizing direction, while had no effect on the reversal potential . The second part of the current was inactivated slowly and blocked by 30 μM nifedipine, confirming high voltage dependent L-type Ca2+ current. In 12 cells examined, 4 neurons contained only the high voltage dependent Ca2+ currents, other neurons all expressed both L- and T-type Ca2+ currents. Intracellular Ca muscles , 39. A r muscles . We thusotential . The aveotential . To sepa current . The fir2+ levels in the hypothalamus primary culture neurons by live Ca2+ fluorescent imaging. We found that there were about 50% cells responded to 50 μM myricetin with a fast rise in Fura-2 AM fluorescent intensity, indicating an increase in intracellular Ca2+ level , and a L-type Ca2+ channel blocker, nifedipine (1 μM), indicating involvement of both T-type and L-type Ca2+ channels . Autophosphorylation of Thr286 in CaMK-II α subunit triggered by Ca2+ dramatically increases its enzyme activity and affinity to calmodulin, which are essential to many neural process such as synaptic transmissions [in vitro model in conjunction with immunofluorescence staining to further investigate if myricetin can indeed increase CaMK-II phosphorylation. We found that after 10 min of incubation with 50 μM myricetin, the phospho-CaMK-II (Thr286) immunoreactivity was significantly increased in the cell body of neurons . Both the immunostaining and western blot results confirmed enhanced CaMK-II phosphorylation by myricetin.The biological activities of CaMK-II are regulated by several phosphorylation sites on its four subunits  myricetin inhibits the calcium current [The present results suggest that the action of myricetin on PVN neurons is through an indirect pathway by first activating T- and/or L-type Caunctions . In the  current , suggest2+ channels in rat vascular smooth muscles [2+ channels in hypothalamic neurons, which may have important therapeutic implication for insomnia treatment. Activation of Ca2+ channels by myricetin in presynaptic neurons may also enhance GABA release as evidenced by the decrease in the inter-event interval, or increase in the frequency of mIPSC (While previous studies have demonstrated that myricetin could induce vasoconstriction by activating L-type Ca muscles , 39, theof mIPSC . Thus, m2+ channels, particularly T-type Ca2+ channels, and to induce changes in intracellular Ca2+ levels in hypothalamic neurons may indicate its potential as a novel hypnotic for improving sleep maintenance insomnia. In the thalamus, T-type Ca2+ channel activation could block transmission of arousal signals and thus stabilize sleep; deletion of which has been reported to result in fragmented sleep in mice [2+ channel in slow wave sleep generation and maintenance has also been documented [2+ channels are activated by small depolarization near the resting membrane potential and therefore regulate the excitability and threshold of neurons. Targeting T-type Ca2+ channels as a potential way to improve slow wave sleep in insomniacs has been proposed several years ago, but lacking of T-type Ca2+ channel modulators is the main obstacle [2+ channel, and modulate GABAA receptor activity makes it a promising new target for investigation. One of the major problems with existing hypnotics such as zolpidem is that despite their efficacy in promoting sleep initiation, they do not appear to improve sleep quality, with less deep sleep and REM sleep , 42. Fra in mice . The pivcumented . T-type obstacle . As a nain vivo, but the GABAergic action of myricetin appears to be distinctively different from that of currently available hypnotics, and its ability to activate Ca2+ channels, particularly T-type Ca2+ channels, makes it an attractive new candidate for further investigation as a possible sedative agent.The PVN has extend connections with GABAergic, hypocretinergic, cytokines and NPY systems , 28, whi


Dryopteris (Dryopteridaceae) is among the most common and species rich fern genera in temperate forests in the northern hemisphere containing 225–300 species worldwide. The circumscription of Dryopteris has been controversial and various related genera have, over the time, been included in and excluded from Dryopteris. The infrageneric phylogeny has largely remained unclear, and the placement of the majority of the supraspecific taxa of Dryopteris has never been tested using molecular data.The fern genus rbcL gene, rps4-trnS spacer, trnL intron, trnL-F spacer) were used to reconstruct the phylogeny of Dryopteris. A total of 122 accessions are sampled in our analysis and they represent 100 species of the expanded Dryopteris including Acrophorus, Acrorumohra, Diacalpe, Dryopsis, Nothoperanema, and Peranema. All four subgenera and 19 sections currently recognized in Dryopteris s.s. are included. One species each of Arachniodes, Leptorumohra, and Lithostegia of Dryopteridaceae are used as outgroups. Our study confirms the paraphyly of Dryopteris and provides the first strong molecular evidence on the monophyly of Acrophorus, Diacalpe, Dryopsis, Nothoperanema, and Peranema. However, all these monophyletic groups together with the paraphyletic Acrorumohra are suggested to be merged into Dryopteris based on both molecular and morphological evidence. Our analysis identified 13 well-supported monophyletic groups. Each of the 13 clades is additionally supported by morphological synapomophies and is inferred to represent a major evolutionary lineage in Dryopteris. In contrast, monophyly of the four subgenera and 15 out of 19 sections currently recognized in Dryopteris s.s is not supported by plastid data.In this study, DNA sequences of four plastid loci  in Dryopteris strongly suggests that the current taxonomy of this genus is in need of revision. The disagreement between the previous taxonomy and molecular results in Dryopteris may be due partly to interspecific hybridization and polyplodization. More morphological studies and molecular data, especially from the nuclear genome, are needed to thoroughly elucidate the evolutionary history of Dryopteris. The 13 well-supported clades identified based on our data represent 13 major evolutionary lineages in Dryopteris that are also supported by morphological synapomophies.The genera,  Dryopteris Adans. (Dryopteridaceae) is estimated to contain 225 to 300 species worldwide  resolving phylogenetic relationships between Dryopteris and Acrophorus, Acrorumohra, Diacalpe, Dryopsis, Dryopteris, Nothoperanema, and Peranema; [3] assessing the monophylies of supraspecific taxa at sectional and subgeneric ranks recognized in current classifications using relatively large sampling and DNA sequences of multiple loci; and [4] identifying major evolutionary lineages in Dryopteris.The goals of this study include: [1] vigorously testing the monophyly of trnL intron and trnL-F spacer yielded similar tree topologies in both MP and ML analyses (trees not shown). The most parsimonious, parsimony JK, and likelihood JK and BS trees for all analyses are available upon request from the first author. There were no well-supported  clades Dryopteris.The combined data matrix of four plastid regions consisted of 3,638 bases. A simultaneous analysis ,30 of nuUnweighted MP simultaneous analysis generated 1,785,800 most parsimonious trees with a length of 1,831 steps, a consistency index  of 0.83Dryopteris in its current circumscription is paraphyletic in relation to AcrophorusAcrorumohraDiacalpeDryopsisNothoperanema, and Peranema , and AcrorumohraDryopsis, and Nothoperanema, should all be merged into Dryopteris, though each is monophyletic except Acrorumohra. The expanded Dryopteris is supported as monophyletic with strong support , and Peranamea. Such a close relationship among all of them had not previously been suggested in the literature, although the close affinities among Peranemataceae, Dryopsis (previously in Tectariaceae), and Dryopteridaceae have partially long been noticed , and Dryopsis as independent genera, in addition to Dryopteris and another 24 or 25 genera (with one being Incertae Sedis), in his large subfamily Dryopteridoideae, one of the two subfamilies in Dryopteridaceae sensu lato (the other one is Athyrioideae). Recognition of these genera in Dryopteridaceae was largely followed by Smith et al.’s [The family Peranemataceae was established by Ching ; as “Per Peranema-35,37,38 Peranema recognizet al.’s  classifiet al.’s  are highet al.’s ,40).Nothoperanema, normally not viewed as a member of Peranemataceae, is embedded within Peranemataceae. In our analyses, Nothoperanema and Peranemataceae together formed a strongly supported monophyletic group in our analyses , sister to the Aemulae clade within Dryopteris (see below). Our molecular data unambiguously resolved the species of Peranemataceae as members of Dryopteris . Based on our study, Acrophorus belongs to Dryopteris, and represents a specialized group within Dryopteris with round indusia and cordate scales at costa base.The genus s  and Diacalpe and Peranema are paraphyletic in relation to Acrophorus and Nothoperanema, contrasting Nayar & Kaur’s [Diacalpe as a synonym of Peranema while recognizing Acrophorus. Our resolution of Diacalpe is consistent with Liu et al. [Diacalpe were sampled. Morphologically, Diacalpe is characterized by unstalked sori, a few single-celled long and clavate paraphyses on the lower portion of sporangiate stalk, and entire scales at the stipe bases [Sermolli , Wu [33,Sermolli , Wu & ChSermolli , and ChrSermolli , but is r & Kaur  who alsou et al.  and our u et al. , Kramer u et al. , and Smiu et al.  also tre& Kaur’s , Kramer’& Kaur’s , and Smi& Kaur’s  treatmenu et al.  where onpe bases .Diacalpe, D. annamensis Tagawa is resolved as sister to the rest of species, with D. chinensis Ching & S. H. Wu then sister to D. aspidioides + D. christensenae Ching  Fraser-Jenk. Acrorumohra is well defined morphologically. Its pinnules are all anadromous and the terminal pinnules have asymmetrical bases, different from Dryopteris. Ten accessions of three species of Acrorumohra, including the type, A. diffracta (Baker) H. Itô, are sampled in our study. Our analyses demonstrate, for the first time, that Acrorumohra is paraphyletic in relation to Dryopteris polita Rosenst. D. polita is sister to A. hasseltii (Blume) Ching plus A. subreflexipinna (Ogata) H. Itô, and together these three are sister to A. diffracta. Our results clearly show that Acrorumorha is a member of Dryopteris.Originally described as a subgenus, a H. Itô , and lata H. Itô , Acrorumd  Ching. For the first time, Nothoperanema is supported as monophyletic in our analyses  Ching and N. shikokianum (Makino) Ching needs further clarifications.Within Dryopsis was established by Holttum & Edwards [Ctenitis subgen. Dryopsis Ching”. It is now widely recognized [CtenitisDryopsis, and Dryopteris have been controversial. Morphologically, Dryopsis appears to be more distant from Ctenitis than from Dryopteris. Dryopsis has distinct venation on the abaxial surfaces, sori terminal on veinlets, and marginal, entire scales that are clathrate or not, but with long and dull areolae. Ctenitis has venation indistinct on both the adaxial and abaxial surfaces, sori middle on the veinlets, and scales ciliate on their margins, clathrate, and with nearly hexagonal and lustrous areolae [Dryopsis and Dryopteris is that the former has either shallow or deep rachis and costa grooves that are closed near their bases, as well as multi-celled hairs with a thickened base on the margins but not in the grooves of the rachis and costae. Dryopteris, in contrast, always has deep rachis and costa grooves that are connected near their bases, and normally has no hairs on the rachis or costae [The genus  Edwards  based oncognized ,39,53,54 areolae ,13,54. Tr costae ,13,54.Dryopsis contains about 22 species [Dryopsis should be a member of Dryopteridaceae but Liu et al. [DryopsisDryopteris, and Peranemataceae sensu Ching [Dryopsis is not closely related to Ctenitis. species  occurrin species ,55. With species  discoveru et al.  failed tsu Ching ,37, and su Ching . Liu et su Ching  also conDryopsis, including the type, D. apiciflora  Holttum & P. J. Edwards, are sampled in our analysis. Our results demonstrate that Dryopsis is monophyletic , in contrast to the resolution of Li & Lu [Dryopsis formed an unresolved trichotomy with two species of Dryopteris and one species of Acrorumohra. Our results also indicate that Dryopsis is nested within a paraphyletic Dryopteris .Six accessions of five species of  Li & Lu , where ts Figure , stronglDryopsis, the species sampled were resolved into two clades. Morphologically, species of the upper clade  Holttum & P.J. Edwards, and D. sp.) have bullate scales, while those of the lower clade (D. heterolaena (C. Chr.) Holttum & P.J. Edwards, D. mariformis (Rosenst.) Holttum & P.J. Edwards) have flat scales  were represented by two or more species in our study (Appendix I).Our 100-species sampling is still not dense enough to rigorously test the monophylies of all supraspecific taxa (sections or subgenera) recognized in recent classifications by Fraser-Jenkins ,24,25, aween 225 , and in Dryopteris strongly suggests that the current taxonomy of this genus is in need of revision. However, our data do not necessarily falsify the monophyly of these 19 sections. The disagreement between previous taxonomy and molecular results in Dryopteris may be due partly to interspecific hybridization and polyplodization [The non-monophyly of the 19 out of the 21 supraspecific taxa in dization ,58.Dryopteris that have evolved via inter-clade hybridization, based on plastid trnL-F sequences, nuclear PgiC sequences, and/or biochemical evidence. D. guanchica, limited to Spain, Portugal, and the Canary Islands, has been postulated to be of hybrid origin between D. aemula  and possibly D. intermedia (D. sect. Lophodium) [D. shibipedis Sa. Kurata, an obvious member of D. sect. Variae judging from the morphology [D. kinkiensis  and D. pacifica (Nakai) Tagawa  [D. corleyi, an endemic of northern Spain, is of hybrid origin between D. aemula (D. sect. Aemulae) and D. oreades Fomin . Our analyses based on plastid data and the resolution of D. corleyi as sister to D. aemula suggest that D. aemula is the maternal progenitor of D. corleyi. In addition, Sessa et al. [Dryopteris that has involved inter-continental long-distance dispersal as well as inter-clade hybridization. These examples of hybridization not only highlight the importance of reticulate evolution and thus the importance of nuclear data in understanding the evolutionary history of Dryopteris, but also strongly support the inclusion of these 13 lineages, including the small segregates, within Dryopteris, as opposed to breaking Dryopteris into many small genera.There are four well-documented allopolyploids in phodium) . The Japrphology ,42, has e clade) . Using ae clade)  concludea et al.  found evDryopteris : This clade contains species of Peranemataceae sensu Ching , Wu 33,,33,34, aDryopteris aemula and D. corleyi, based on the current sampling. These two species are different enough morphologically to have been placed in different sections by Fraser-Jenkins [D. aemula is consistent with those of Geiger & Ranker [D. corleyi, an endemic of northern Spain, is thought to be of hybrid origin between D. aemula (D. sect. Aemulae) and D. oreades Fomin based on allozyme data  Kuntze, and D. hawaiiensis (Hillebrand) W. Robinson, but D. guanchica is an allotetraploid (see blow) and D. hawaiiensis possibly an allotriploid [2. The Aemulae clade : The Aemulae clade, or the Hawaiian glabra group , contain& Ranker , Juslén & Ranker , and Ses& Ranker . This isme data , and D. polita (D. sect. Politae). The morphological synapomorphy is the flat scales in comparison with the Dryopsis clade, the Erythrovariae clade, and the Variae clade. The gain of bullate scales is considered here as the morphological synapomorphy of the expanded D. subgen. Erythrovariae including the Acrorumohra clade, the Dryopsis clade, the Erythrovariae clade, and the Variae clade.3. The Acrorumohra clade : This clade contains species of Dryopteris sect. Variae Fraser-Jenk. It is characterized by having slightly bullate-based scales and stiffly coriaceous lamina and pinnules with caudate apices and pointed lobes [4. The Variae clade : The Variae clade contains species of ed lobes ,26.Dryopteris sect. Erythrovariae plus D. podophylla. It is characterized by having more bullate scales and herbaceous lamina and pinnules with acute apices and rounded lobes [5. The Erythrovariae clade : This clade contains species of ed lobes ,26.Dryopsis. The relatively moderate branch support may be the result of some missing sequence data for members of this clade. The potential major morphological synapomorphies are the rachis and costa grooves that are closed near their bases and the multi-cellular hairs (see above).6. The Dryopsis clade : This clade contains species of the former genus Dryopteris sect. Cinnamomeae and one species of D. sect. Purpurascentes in our current sampling. This clade is defined by having pinnules angled acroscopically and usually with narrower bases and having linear scales on stipe base [7. The Cinnamomeae clade : The Cinnamomeae clade contains two species of Dryopteris crinalis (Hook. & Arn.) C. Chr., D. mauiensis C. Chr., D. sandwiciensis (Hook. & Arn.) C. Chr., D. tetrapinnata W. H. Wagner & Hobdy, and D. unidentata (Hook. & Arn.) C. Chr. The potential morphological synapomorphy is the absence of indusia [8. The Crinales clade : This clade was named the Hawaiian exindusiate group by Palmer  and it c indusia ,62.Dryopteris sect. Pallidae sensu Fraser-Jenkins [D. aitoniana Pic. Serm., D. odontoloma (Bedd.) C. Chr., D. pallida (Bory) C. Chr. ex Maire & Petitm., and D. mindshelkensis N. Pavl. (synonym: D. submontana (Fraser-Jenk. & Jermy) Fraser-Jenk.), and additional species from other sections, e.g., D. goldiana (Hook.) A. Gray, D. monticola (Makino) (D. sect. Dryopteris), D. oligodonta (Desv.) Pic. Serm. (D. sect. Marginatae), and D. tokyoensis (Matsum. & Makino) C. Chr. (D. sect. Pandae), based our current sampling and Juslén et al. [D. odontoloma in this clade needs further study. This clade is weakly supported as sister to the Crinales clade : This clade contains some species of -Jenkins , e.g., Dn et al. . The incDryopteris sect. Nephrocystis sensu Fraser-Jenkins [10. The Nephrocystis clade : This clade contains those species of -Jenkins .Dryopteris subgen. Dryopteris sensu Fraser-Jenkins [D. reflexosquamataD. rubrobrunnea, etc.) and are mainly distributed in the Sino-Japanese and Sino-Himalayan regions.11. The Dryopteris clade : This clade contains large portion of species of -Jenkins  and is tDryopteris sect. Lophodium Fraser-Jenk. and D. remota. The species of this clade share short-stalked pinnae, long-aristate ultimate segments, and minutely spinulose perispore sculpturing except D. remota[12. The Lophodium clade : This clade contains species of Dryopteris sect. Fragrantes (H. Itô) Seriz., D. fragrans (L.) Schott. Our work shows that D. fragrans is outside of D. subgen. Dryopteris where it was placed by Fraser-Jenkins [D. fragrans as sister to the rest of Dryopteris, though our statistical support values were low .Our data clearly show that the Dryopsis clade is sister to the Erythrovariae clade; these two together are sister to a clade containing the Acrorumohra clade and the Variae clade; these four clades together are sister to a clade containing the Aemulae clade and the Nothoperanema clade; and these six clades are strongly supported as monophyletic . The relationships among the remaining seven clades are resolved in the ML tree : This clade contains one of the two species of -Jenkins , a resol-Jenkins . Most no-Jenkins ,58 in reAcrophorus, Acrorumohra, Diacalpe, Dryopsis, Nothoperanema, and Peranema, should all be merged into Dryopteris. Most species of these genera share a short rhizome and catadromic arrangement of frond segments, unlike the sister genus of Dryopteris s.l., Arachniodes.The genera, Dryopteris strongly suggests that the current taxonomy of this genus is in need of revision. However, our data do not necessarily falsify the monophyly of these 19 sections. The disagreement between previous taxonomy and molecular results in Dryopteris may be due partly to interspecific hybridization and polyplodization.The non-monophyly of the 19 out of the 21 supraspecific taxa in Dryopteris that are supported by morphological synapomophies and may deserve circumscription as supraspecific entities within Dryopteris.The 13 well-supported clades identified with our data represent 13 major evolutionary lineages in Dryopteris recognized by Fraser-Jenkins [D. sect. Caespitosae S. G. Lu, D. sect. Chrysocomae S. G. Lu, D. sect. Indusiatae S. G. Lu) recognized by Lu [D. sect. Purpurascentes Fraser-Jenk. and the monotypic D. sect. Politae Fraser-Jenk. In total, 78 accessions representing 77 species of Dryopteris s.s. were sampled, including all four subgenera and 19 sections in the current classifications of Dryopteris s.s. by Fraser-Jenkins [All four subgenera and 14 out of 16 sections of ed by Lu ,24,25 aned by Lu , were re-Jenkins .Dryopteris and Acrophorus, Acrorumohra, Diacalpe, Dryopsis, Nothoperanema, and Peranema, further included are eight accessions representing five (63%) out of eight species of Acrophorus, 10 accessions representing three (43%) out of seven species of Acrorumohra, seven accessions representing four (40%) out of 10 species of Diacalpe, six accessions representing five (31%) out of 16 species of Dryopsis, 11 accessions representing six (75%) out of eight species of Nothoperanema, and two accessions representing both species of the bitypic Peranema. Type species of all these six genera, Acrophorus, Acrorumohra, Diacalpe, Dryopsis, Nothoperanema, and Peranema, are included. In total, 122 accessions representing 100 species of the expanded Dryopteris  are included in this study.To assess the phylogenetic relationships between Arachniodes Blume, Leptorumohra H. Itô, and Lithostegia Ching of Dryopteridaceae are used as outgroups based on Liu et al. [ArachniodesLeptorumohraLithostegia, and Phanerophlebiopsis Ching together were resolved as sister to a clade consisting of AcrophorusAcrorumohraDiacalpeDryopsisDryopterisNothoperanema, and Peranema. All sequences used in this study together with their GenBank accession numbers and/or voucher information are listed in Appendix II.One species each of u et al.  where ArrbcL gene, rps4-trnS spacer, trnL intron, and trnL-F spacer. The rbcL gene was amplified with primers F1  and BigDye® Terminator v3.1 Cycle Sequencing Kit .Total genomic DNA was extracted from silica-gel dried material or sometimes from herbarium specimens using Plant Genomic DNA Kits  and DNeasy Plant Mini Kits . The PCR protocols followed Zhang et al.  and Ebihy et al. ) and 137y et al. ). The prTCGAATC; ) and Li TCGAATC; ). The trGGRAACC; ) and theGGRAACC; . AmplifirbcLrps4-trnStrnL, and trnL-F, respectively, were included in our analyses. Some 151 DNA sequences are newly generated for this study (GenBank JX535813-JX535961).Additional sequences were obtained from Genbank and had originally been generated by Geiger & Ranker , Li & LurbcL data was manually obtained using Microsoft Wordpad. Preliminary alignments of rps4-trnS and trnL-F (trnL intron + trnL-F spacer) data were obtained using the default alignment parameters in Clustal X [The alignment of the lustal X  followedtrnL intron and trnL-F spacer were also conducted since these two linked regions are sometimes viewed as one locus.Equally weighted maximum parsimony (MP) tree searches were conducted for each data matrix using 1000 tree-bisection-reconnection (TBR) searches in PAUP* ver. 4.0b10  with MAXtrnL-F spacer), GTR + I (trnL intron), GTR + I + G , HKY + G (rps4-trnS spacer), and SYM + I + G (rbcL gene). The selected models and parameters estimated  with 1000 rapid bootstrap analyses followed by a search for the best-scoring tree in a single run.The simultaneous ML analyses of nucleotide characters and ML bootstrapping (BS) were performed using RAxML-HPC2 on TG ver. 7.2.8  Ching, are monophyletic. Sessa et al. [D. subgen Pycnopteris, for which they had insufficient sampling to test monophyly. In the current study, most of the members of D. subgen. Dryopteris are resolved in the Dryopteris clade, while others are placed in other major clades except the Acrorumohra, Dryopsis, Erythrovariae, Nothoperanema, and Variae clades. Members of D. subgen. Erythrovariae sensu Fraser-Jenkins [, Erythrovariae, and Variae clades, but these clades are paraphyletic in relation to the Dryopsis clade as well as D. chinensis (Baker) Koidz. and D. gymnophylla (Baker) C. Chr. (members of D. sect. Aemulae Fraser-Jenk.), and D. podophylla (Hook.) Kuntze (a member of D. subgen. Pycnopteris). Members of D. subgen. Erythrovariae sensu Wu & Lu [D. subgen. Pycnopteris is consistent with Li & Lu’s [rps4-trnS data. Of our three representatives of this subgenus, two fell in the Dryopteris clade (D. bodinieri (Christ) C. Chr. and D. sieboldii (Van Houtte ex Melt.) Kuntze), and one in the Erythrovariae clade (D. podophylla). Our sole sequence of D. podophylla was derived from Li & Lu [D. subgen. Nephrocystis is not monophyletic because Acrorumohra diffracta Baker (= D. diffracta (Baker) C. Chr.), A. hasseltii Blume (=D. hasseltii (Blume) C. Chr.), A. subreflexipinna (Ogata) Ching (= D. subreflexipinna Ogata), and D. futura A. R. Sm., a member of D. sect. Purpurascentes, are resolved in the Acrorumohra clade and the Cinnamomeae clade. Wu & Lu [D. subgen. Nephrocystis.Our results show that none of the four subgenera, a et al.  also rej-Jenkins  are reso Wu & Lu  are resoi & Lu’s ,20 findi Li & Lu . D. subg Wu & Lu  did not D. sect. Cinnamomeae Fraser-Jenk. and D. sect. Variae Fraser-Jenk., are resolved as monophyletic with our current sampling. This is at odds with Sessa et al. [D. sect. Cinnamomeae and D. sect. Variae, and rejected the monophly of all. Although these two sections are found to be monophyletic in the current study, the sampling for both was larger in Sessa et al. [Our results also demonstrated that 15 out of the 17 sections currently recognized ,24-26, fa et al. , which ta et al. , and theDryopteris sect. Aemulae Fraser-Jenk.: Represented by D. aemula (Aiton) Kuntze (Aemulae clade), D. chinensis, and D. gymnophylla (Acrorumohra clade).Dryopteris sect. Caespitosae S. G. Lu: Represented by D. alpestris Tagawa (Dryopteris clade) and D. fragrans (Fragrantes clade).Dryopteris sect. Chrysocomae S. G. Lu: Represented by D. chrysocoma (Christ) C. Chr. and D. himachalensis Fraser-Jenk. (Dryopteris clade).Dryopteris sect. Dryopteris: This section sensu Fraser-Jenkins [D. alpestrisD. filix-mas (L.) Schott, and D. sichotensis V. Komarov (Dryopteris clade).-Jenkins  is repreDryopteris sect. Fibrillosae Ching: Represented by D. affinis (Lowe) Fraser-Jenk. subsp. borreri Fraser-Jenk., D. polylepis  C. Chr., and D. rosthornii (Diels) C. Chr. (Dryopteris clade).Dryopteris sect. Erythrovariae: This section sensu Fraser-Jenkins [D. caudipinna Nakai, D. championii (Benth.) C. Chr., D. cordipinna Ching & Shing, D. decipiens (Hook.) Kuntze, D. erythrosora (D. Eaton) Kuntze, D. gymnosora (Makino) C. Chr., D. indusiata Makino & Yamam.  Makino following Fraser-Jenkins [D. ryo-itoana Kurata, and D. simasakii (H. Itô) Kurata and all are in the Erythrovariae clade. If D. podophylla (Hook.) Kuntze, a member of D. subgen. Pycnopteris, is included, this section sensu Fraser-Jenkins [-Jenkins  is repre-Jenkins ), D. kin-Jenkins ), D. ryo-Jenkins  becomes Dryopteris sect. Hirtipedes Fraser-Jenk.: Represented by D. atrata  Ching, D. commixta Tagawa, D. conjugata Ching, D. cycadina (Franchet & P. A. L. Savat.) C. Chr., D. dickinsii (Franchet & P. A. L. Savat.) C. Chr., D. handeliana C. Chr., D. hangchowensis Ching, D. lunanensis (Christ) C. Chr., D. scottii (Bedd.) Ching, D. stenolepis (Baker) C. Chr. and D. tsutsuiana Kurata, all of which are in the Dryopteris clade. This section becomes monophyletic if D. rosthornii (D. sect. Fibrillosae) and D. uniformis  are included.Dryopteris sect. Indusiatae S. G. Lu: Represented by D. gymnosora (Makino) C. Chr. and D. indusiata Makino & Yamam. (Erythrovariae clade).Dryopteris sect. Lophodium (Newman) C. Chr. ex H. Itô: Represented by D. amurensis Christ, D. expansa (C. Presl) Fraser-Jenk. & Jermy, D. intermedia (Muhlenb. ex Willd.) A. Gray, and D. dilatata (Hoffm.) A. Gray (Lophodium clade). These four species are paraphyletic in relation to D. remota (A. Braun ex Doell) Druce, the type of D. sect. Remotae. The close relationship between D. remota and species of D. sect. Lophodium based on our plastid data shows that D. remota, a triploid, is possibly originated through hybridization with one of the species in D. sect. Lophodium being the maternal donor.Dryopteris sect. Marginatae Fraser-Jenk.: Represented by D. aquilinoides (Desv.) C. Chr. and D. shiroumensis Kurata & Nakaike (Dryopteris clade). They are resolved as paraphyletic in relation to the rest of the Dryopteris clade.Dryopteris sect. Nephrocystis: Represented by Acrorumohra diffractaA. hasseltiiA. subreflexipinnaDryopteris hayatae Tagawa  C. Chr. by Fraser-Jenkins [D. melanocarpa Hayata (subsumed in D. platypus (Kunze) Kuntze by Fraser-Jenkins [D. maximowicziana (Miq.) C. Chr. (not recognized by Fraser-Jenkins [D. sabae (Franchet & P. A. L. Savat.) C. Chr., D. sparsa (Buch.-Ham. ex D. Don) Kuntze, and D. yakusilvicola Sa. Kurata (subsumed in D. cacaiana Tagawa by Fraser-Jenkins [-Jenkins ), D. mel-Jenkins ), D. max-Jenkins ), D. sab-Jenkins ). The fiDryopteris sect. Pallidae Fraser-Jenk.: Represented by D. juxtaposita Christ, D. lacera (Thunb.) Kuntze, D. sublacera Christ, D. uniformis (Makino) Makino (Dryopteris clade), D. odontoloma (Bedd.) C. Chr., and D. pallida (Bory) C. Chr. ex Maire & Petitm. .Dryopteris sect. Pandae Fraser-Jenk.: Represented by D. himachalensis Fraser-Jenk. (Acrorumohra clade) and D. tokyoensis (Matsum. & Makino) C. Chr. .Dryopteris sect. Remotae Fraser-Jenk.: Represented by D. corleyi Fraser-Jenk. (Aemulae clade) and D. remota (Lophodium clade).Dryopteris sect. Splendentes Fraser-Jenk.: Represented by D. reflexosquamata Hayata and D. rubrobrunnea W. M. Chu (Dryopteris clade). These two are paraphyletic in relation to three members of D. sect. Pallidae and two member of D. subgen. Pycnopteris.Acrophorus emeiensis Ching: rbcL zl1474, trnL JX535916, trnL-F JX535867, rps4-trnS JX535815. Acrophorus exstipellatus Ching & S. H. Wu: rbcL JX535857, trnL JX535914, trnL-F JX535865, rps4-trnS JX535813. Acrophorus macrocarpus Ching & S. H. Wu: rbcL DQ054522 (“Acrophorus emeiensis Ching”). Acrophorus nodosus C. Presl: rbcL AB575065, trnL JX535915, trnL-F JX535866, rps4-trnS JX535814. Acrophorus paleolatus Pic. Serm. (“Acrophorus stipellatus T. Moore”): rbcL DQ054510 DQ508756 EF463106, trnL-F DQ514500 EF540696 DQ480130.Acrorumohra diffracta (Baker) H. Itô: rbcL DQ508758 EF463108, trnL-F EU797681 EU797682 EU797683, rps4-trnS EU797685 EU797686 EU797687. Acrorumohra hasseltii (Blume) Ching: rbcL AB575136 DQ054519 DQ508757 EF463107, trnL-F DQ514479 EU797677 EU797679 EU797680, rps4-trnS DQ191888 EU797691 EU797692 EU797693. Acrorumohra subreflexipinna (Ogata) H. Itô: trnL -F EU797675 EU797676 EU797678, rps4-trnS EU797688 EU797689 EU797690.Arachniodes aristata (G. Forst.) Tindale: rbcL AY268851, trnL-F AY268782. Arachniodes assamica (Kuhn) Ohwi: rps4-trnS DQ191891.Diacalpe annamensis Tagawa: rbcL EF463125, trnL-F DQ480132 EF540698. Diacalpe aspidioides Blume: rbcL DQ054523 EF463126, trnL-F DQ514490. Diacalpe chinensis Ching & S. H. Wu: rbcL JX535864, trnL JX535956, trnL-F JX535908, rps4-trnS JX535854. Diacalpe christensenae Ching: rbcL DQ054518 EF540699, trnL-F DQ480131 EF540699, rps4-trnS DQ480131 EF540699.Dryopsis apiciflora  Holttum & P.J. Edwards: rbcL DQ054521, trnL JX535957, trnL-F JX535909. Dryopsis clarkei (Baker) Holttum & P.J. Edwards: trnL JX535958, trnL-F JX535910, rps4-trnS JX535855. Dryopsis heterolaena (C. Chr.) Holttum & P.J. Edwards: rbcL DQ508770, trnL-F DQ514492. Dryopsis mariformis (Rosenst.) Holttum & P.J. Edwards: rbcL DQ054520 EF460683, trnL JX535959, trnL-F JX535911. Dryopsis sp.: rbcL DQ054525.Dryopteris aemula (Aiton) Kuntze: rbcL AY268881, trnL-F AY268816, rps4-trnS JN189189. Dryopteris affinis (Lowe) Fraser-Jenk. subsp. borreri Fraser-Jenk.: rbcL AY268849, trnL-F AY268776, rps4-trnS JN189190. Dryopteris alpestris Tagawa: rbcL JX535858, trnL JX535917, trnL-F JX535868, rps4-trnS JXH11103. Dryopteris amurensis Christ: rbcL AB575112, trnL JX535918, trnL-F JX535869, rps4-trnS JX535816. Dryopteris aquilinoides (Desv.) C. Chr.: rbcL AY268868, trnL-F AY268803, rps4-trnS JN189211. Dryopteris atrata  Ching: rbcL AB575115, trnL JX535919, trnL-F JX535870, rps4-trnS JX535817. Dryopteris bissetiana (Baker) C. Chr.: rbcL AY268862, trnL-F AY268796, rps4-trnS DQ191829. Dryopteris bodinieri (Christ) C. Chr.: rbcL DQ508772, trnL-F DQ514494, rps4-trnS DQ191830. Dryopteris caudipinna Nakai: rbcL AB575117, trnL JX535920, trnL-F JX535871, rps4-trnS JX535818. Dryopteris championii (Benth.) C. Chr.: rbcL AY268863, trnL-F AY268797, rps4-trnS DQ151856. Dryopteris chinensis (Baker) Koidz.: rbcL JX535859, trnL JX535921, trnL-F JX535872, rps4-trnS JX535819. Dryopteris chrysocoma (Christ) C. Chr.: rbcL DQ508773, trnL-F DQ514495, rps4-trnS DQ191832. Dryopteris cinnamomea (Cav.) C. Chr.: rbcL JN189528, trnL-F FR731991, rps4-trnS JN189202. Dryopteris commixta Tagawa: rbcL AB575120, trnL JX535922, trnL-F JX535873, rps4-trnS JX535820. Dryopteris corleyi Fraser-Jenk.: rbcL AY268873, trnL-F AY268808. Dryopteris crinalis (Hook. &Arn.) C. Chr.: AY268835, trnL-F AY268774. Dryopteris cycadina (Franchet & P. A. L. Savat.) C. Chr.: rbcL EF463127, trnL-F AY278400, rps4-trnS DQ191835. Dryopteris decipiens (Hook.) Kuntze var. decipiens: rbcL AB575123, trnL JX535923, trnL-F JX535874, rps4-trnS JX535821. Dryopteris dickinsii (Franchet & P. A. L. Savat.) C. Chr.: rbcL AB575125, trnL JX535924, trnL-F JX535875, rps4-trnS JX535822. Dryopteris dilatata (Hoffm.) A. Gray: rbcL AY268848, trnL-F AY268779, rps4-trnS JN189248. Dryopteris erythrosora (D. Eaton) Kuntze: rbcL DQ508774, trnL-F DQ514496, rps4-trnS JN189255. Dryopteris expansa (C. Presl) Fraser-Jenk. & Jermy: rbcL AY268844, trnL-F AY268775, rps4-trnS JN189180. Dryopteris filix-mas (L.) Schott: rbcL AY268845, trnL-F AY268776, rps4-trnS JN189181. Dryopteris fragrans (L.) Schott: rbcL AB575129, AY268865, trnL-F FR731981 AY268800, rps4-trnS JN189185. Dryopteris futura A. R. Sm.: rbcL JN189534, trnL-F JN189103, rps4-trnS JN189208. Dryopteris gymnophylla (Baker) C. Chr.: rbcL JX535860, trnL JX535925, trnL-F JX535876, rps4-trnS JX535823. Dryopteris gymnosora (Makino) C. Chr.: rbcL AB575132, trnL JX535926, trnL-F JX535877, rps4-trnS JX535824. Dryopteris hadanoi Kurata: rbcL AB575133, trnL JX535927, trnL-F JX535878, rps4-trnS JX535825. Dryopteris handeliana C. Chr.: rbcL AB575134, trnL JX535928, trnL-F JX535879, rps4-trnS JX535826. Dryopteris hangchowensis Ching: rbcL AB575135, trnL JX535929, trnL-F JX535880, rps4-trnS JX535827. Dryopteris hayatae Tagawa: rbcL AB575137, trnL JX535930, trnL-F JX535881, rps4-trnS JX535828. Dryopteris himachalensis Fraser-Jenk.: rps4-trnS DQ191845. Dryopteris indusiata Makino & Yamam.: rbcL AB575140, trnL JX535931, trnL-F JX535882, rps4-trnS JX535829. Dryopteris intermedia (Muhlenb. ex Willd.) A. Gray subsp. maderensis (J. Milde ex Alston) Fraser-Jenkins: rbcL AB575143, trnL-F FR731985. Dryopteris juxtaposita Christ: rbcL AY268875, trnL-F AY268810, rps4-trnS DQ191848. Dryopteris kinkiensis Koidz. ex Tagawa: rbcL AB575144, trnL JX535932, trnL-F JX535883, rps4-trnS JX535830. Dryopteris lacera (Thunb.) Kuntze: rbcL AB575148, trnL JX535933, trnL-F JX535884, rps4-trnS JX535831. Dryopteris laeta (Kom.) C. Chr.: rbcL AB575149, trnL JX535934, trnL-F JX535885, rps4-trnS JX535832. Dryopteris lunanensis (Christ) C. Chr.: rbcL AB575150, trnL JX535935, trnL-F JX535886, rps4-trnS JX535833. Dryopteris mauiensis C. Chr.: rbcL AY268833, trnL-F AY268770. Dryopteris maximowicziana: rbcL AB575151, trnL JX535936, trnL-F JX535887, rps4-trnS JX535834. Dryopteris melanocarpa Hayata: rbcL AB575153, trnL JX535937, trnL-F JX535888, rps4-trnS JX535835. Dryopteris monticola (Makino) C. Chr.: rbcL AB575154, trnL JX535938, trnL-F JX535889, rps4-trnS JX535836. Dryopteris nipponensis Koidz.:AB575156, trnL JX535939, trnL-F JX535890, rps4-trnS JX535837. Dryopteris odontoloma (Bedd.) C. Chr.: rbcL AY268872, trnL-F AY268807, rps4-trnS DQ191859. Dryopteris pacifica (Nakai) Tagawa: rbcL AB575157, trnL JX535940, trnL-F JX535891, rps4-trnS JX535838. Dryopteris pallida (Bory) C. Chr. ex Maire & Petitm.: rbcL AY268874, trnL-F AY268809, rps4-trnS JN189266. Dryopteris patula (Sw.) L. Underw.: rbcL JN189500, trnL-F AY268823, rps4-trnS JN189176. Dryopteris podophylla (Hook.) Kuntze: rps4-trnS DQ191864. Dryopteris polita Rosenst.: rbcL AB575158, trnL-F EU797684, rps4-trnS EU797694. Dryopteris polylepis  C. Chr.: rbcL AY268864, trnL-F AY268798, rps4-trnS JN189263. Dryopteris reflexosquamata Hayata: rbcL JN189604, trnL-F JN189171, rps4-trnS DQ191870. Dryopteris remota (A. Braun ex Doell) Druce: rbcL AY268858, trnL-F AY268792, rps4-trnS JN189204. Dryopteris rosthornii (Diels) C. Chr.: rbcL JX535861, trnL JX535941, trnL-F JX535892, rps4-trnS JX535839. Dryopteris rubrobrunnea W. M. Chu: rbcL JX535862, trnL JX535942, trnL-F JX535893, rps4-trnS JX535840. Dryopteris ryo-itoana Kurata: rbcL AB575161, trnL JX535943, trnL-F JX535894, rps4-trnS JX535841. Dryopteris sabae (Franchet & P. A. L. Savat.) C. Chr.: rbcL AB575162, trnL JX535944, trnL-F JX535895, rps4-trnS JX535842. Dryopteris sacrosancta Koidz.: rbcL AB575163, trnL JX535945, trnL-F JX535896, rps4-trnS JX535843. Dryopteris sandwiciensis (Hook. & Arn.) C. Chr.: rbcL AY268827, trnL-F AY268762. Dryopteris saxifraga H. Itô: rbcL AB575164, trnL JX535946, trnL-F JX535897, rps4-trnS JX535844. Dryopteris scottii (Bedd.) Ching: rbcL JX535863, trnL-F JX535898, rps4-trnS DQ191872. Dryopteris shiroumensis Kurata & Nakaike: rbcL AB575168, trnL JX535947, trnL-F JX535899, rps4-trnS JX535845. Dryopteris sichotensis V. Komarov: rbcL AY268869, trnL-F AY268804. Dryopteris sieboldii (Van Houtte ex Melt.) Kuntze: rbcL AB575169, trnL JX535948, trnL-F JX535900, rps4-trnS JX535846. Dryopteris simasakii (H. Itô) Kurata var. simasakii: rbcL AB575170, trnL JX535949, trnL-F JX535901, rps4-trnS JX535847. Dryopteris sordidipes Tagawa: rbcL AB575172, trnL JX535950, trnL-F JX535902, rps4-trnS JX535848. Dryopteris sparsa (Buch.-Ham. ex D. Don) Kuntze: rbcL AB575173, trnL JX535951, trnL-F JX535903, rps4-trnS JX535849. Dryopteris stenolepis (Baker) C. Chr.: rbcL AY268889, trnL-F AY268824, rps4-trnS DQ191877. Dryopteris sublacera Christ: rbcL DQ508778, trnL-F DQ514501, rps4-trnS DQ191878. Dryopteris tetrapinnata W. H. Wagner & Hobdy: rbcL AY268838, trnL-F AY268772. Dryopteris tokyoensis (Matsum. & Makino) C. Chr.: rbcL AY268861, trnL-F AY268795, rps4-trnS JN189251. Dryopteris tsutsuiana Kurata: rbcL AB575176, trnL JX535952, trnL-F JX535904, rps4-trnS JX535850. Dryopteris unidentata (Hook. & Arn.) C. Chr. var. unidentata: rbcL AY268825, trnL-F AY268766. Dryopteris uniformis (Makino) Makino: rbcL AB575177, trnL JX535953, trnL-F JX535905, rps4-trnS JX535851. Dryopteris varia (L.) Kuntze: rbcL AB575178, trnL JX535954, trnL-F JX535906, rps4-trnS JX535852. Dryopteris xanthomelas (Christ) C. Chr.: rbcL AY587118, trnL-F DQ150394, rps4-trnS DQ151857. Dryopteris yakusilvicola Sa. Kurata: rbcL AB575180, trnL JX535955, trnL-F JX535907, rps4-trnS JX535853.Leptorumohra quadripinnata (Hayata) H. Itô: rbcL DQ508781, trnL-F DQ514505, rps4-trnS EF540707.Lithostegia foeniculacea (Hook.) Ching: rbcL DQ508782, trnL-F DQ514506, rps4-trnS EF540717.Nothoperanema diacalpioides Ching: rbcL DQ054511. Nothoperanema hendersonii (Bedd.) Ching: rbcL AB575138 DQ508783 EF463135 JN189547, trnL-F DQ514507 JN189116, rps4-trnS DQ191885 JN189221. Nothoperanema rubiginosum (Brack.) A. R. Sm. & D. R. Palmer: rbcL AY268836 DQ054511 (“Nothoperanema hendersonii (Bedd.) Ching”) EF463182 (“Nothoperanema squamisetum (Hook.) Ching”), trnL-F AY268771. Nothoperanema shikokianum (Makino) Ching: rbcL AB575167 DQ054509 EF463136, trnL JX535960, trnL-F JX535912, rps4-trnS DQ191886 JX535856. Nothoperanema squamisetum (Hook.) Ching: rbcL DQ054512, trnL JX535961, trnL-F JX535913, rps4-trnS DQ191887.Peranema cyatheoides D. Don: rbcL DQ054513. Peranema luzonicum Copel.: rbcL DQ508784 (“Peranema cyatheoides D. Don”), trnL-F DQ514509 (“Peranema cyatheoides D. Don”).The author(s) declare that they have no competing interests.LBZ desgined the study, conducted data analyses, and wrote the manuscript, LZ, EBS, and AE carried out the lab work, LZ, SYD, EBS, XFG, and AE collected and identified portion of the material. All authors contributed to the manuscript revision. All authors read and approved the final manuscript.


Chronic urticaria, defined as widespread daily or nearly daily wheals for at least 6 weeks, with or without angioedema, impacts on patients’ quality of life. Natural course is self-limited, with spontaneous remissions and occasional relapses. Antihistamines, leukotriene inhibitor, immunosuppressive agents are used. 60% are idiopathic and 40% are autoimmune due to presence of anti-IgE antibody or IgG autoantibodies against Fc∑R1. An association exists between chronic urticaria and autoimmune diseases. We report a case of a patient with autoimune urticaria, thyroid disease and vitiligo, who showed resolution of histamine releasing antibody (reflab) on remission.In February 2005, a 44 yr old healthy woman was referred to the Allergy outpatient clinic with two month history of daily hives, moderately controlled with antihistamines, with good response to oral steroid. Lesions were pruritic, raised, erythematous, lasting for < 24 hrs and resolved with purplish discoloration. Screening negative for malignancy, connective tissue disease. TSH normal. Histamine releasing antibody positive, maximum histamine release 27% (<16%). Skin biopsy confirmed chronic urticaria with neutrophils without vasculitis. Sulfasalazine was not tolerated, but control attained with antihistamines and leukotriene inhibitor. By one year, she failed trial of weaning medications.6 months after presentation she developed vitiligo. 5 years later, she was hypothyroid. By 3 years, the urticaria was in complete remission without medications. Repeat histamine releasing antibody was negative.There is a known association of severe chronic urticaria with auto antibody etiology and other autoimmune disease. Does resolution of antibody correlate with achieving remission? Further prospective studies are required to establish this relationship.


Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca2+ and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca2+ binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca2+ had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca2+ at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG) values in iTC data confirmed that the interaction between DNA and Ca2+ is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca2+ alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca2+ to eDNA thereby mediating bacterial aggregation and biofilm formation.Calcium (Ca The presence of various molecules such as proteins and DNA determines surface properties including cell surface hydrophobicity (measured using contact angles). Hydrophobicity determines surface energies and subsequently influences the physico-chemical interactions between interacting bacterial cell surfaces. The physico-chemical interactions include non-specific attractive Lifshitz van der Waals (LW) interactions, attractive or repulsive electrostatic double layer interactions, and Lewis acid-base interactions and hydrophobic forces/interactions (based on electron-donating and accepting properties of surfaces) 2+ and DNA we hypothesized that the combination of Ca2+ and eDNA promotes bacterial aggregation and subsequent biofilm formation. For this, we tested our hypothesis in aggregation and biofilm formation assays using a variety of Gram–negative  and Gram-positive  bacterial species by adding exogenous Ca2+ and before and after removal of eDNA by DNase I treatment. The thermodynamics of Ca2+ binding with DNA was determined by using isothermal titration calorimetry (iTC) at constant temperature and pressure. iTC determines the physico-chemical interactions that drive many biological processes including binding of biopolymers with ions, folding of proteins and bacterial aggregation by measuring the changes in Gibbs energy (ΔG) based on changes in enthalpy (ΔH) and entropy (ΔS) when the interactions occur Considering the importance of eDNA in bacterial biofilm formation and the interactions between CaP. aeruginosa PA14 has been chosen as a model organism to investigate the surface characteristics i.e. contact angle  and subsequent surface thermodynamics analysis in presence and absence of naturally occurring eDNA and added Ca2+ ions. The rationale/principle behind using polar liquids (e.g. water and formamide) was to determine electron accepting and electron donating parameters of acid-base interactions. This is because polar liquids/molecules have high surface tension/energy and tend to accept and donate electrons with interacting surfaces. In contrast the non-polar liquids (e.g. diiodomethane) have low surface energy and are not involved in electron donating or accepting parameters hence used for determining only Lifshitz-Van der Waals interactions.To this end 2+ ions in eDNA mediated bacterial aggregation and biofilm formation has not yet been investigated. Therefore, the present work investigates for the first time the concurrent role of Ca2+ ions and eDNA in mediating aggregation and biofilm formation in divergent bacterial strains.To the best of our knowledge, the role of Ca2 for 90 min at room temperature under static conditions and subsequently washed twice with phosphate buffered saline (PBS)  and finally resuspended in PBS.Bacterial strains listed in P. aeruginosa, A. hydrophilla, S. aureus and S. epidermidis cultures (over 24 h at 37°C in a static incubator) in LB medium were recorded at various growth times  using a Bio-Rad Smartspec 3000  at 600 nm using plain LB medium without bacterial cells as blank.The optical density of P. aeruginosa and S. aureus (used as model strains) cultures grown in LB medium for 24 h at 37°C in a static incubator were used as is without harvesting/centrifugation to compare differences in dead cells. A 1 ml aliquot of the bacterial culture (averaging OD 2.3) was allowed to adhere on 6 well polystyrene plates  for 30 min at room temperature in static condition. After 30 min the well plate surfaces were gently washed with PBS once in order to remove any loosely bound bacterial cells (since loosely bound cells possibly interfere with microscopy imaging). The remaining adhered bacteria to the well plate's surface were imaged after staining with the Live/Dead stain  for 15 min in the dark using fluorescence microscopy .After 24 h of bacterial growth and subsequent centrifugation, supernatants were separated from bacterial pellets by simply transferring to new tubes. In order to remove remaining bacteria, supernatants were filtered using 0.22 μm filters (Millipore). eDNA in filtered supernatants was quantified using the double stranded DNA fluorescent dye assay (dsDNABR) from Qubit, Invitrogen using their standard protocol. To quantify DNA the Qubit 2.0 fluorometer was calibrated using the standards  supplied by Qubit Invitrogen. After calibration, the total amount of dseDNA present in the supernatants was quantified directly by mixing 20 μl aliquots of supernatants with 180 μl dsDNABR assay buffer-fluorescent dye mixture in Qubit assay tubes, followed by vortexing and incubating for 2 mins at room temperature. The intensity of the fluorescent dye after binding with eDNA in the supernatant was measured by using the Qubit 2.0 fluorometer .2+ ) , in the presence and absence of eDNA, bacterial cells were first diluted to OD  = 0.5±0.05/ml in PBS. The percentage reduction in optical density (OD) resulting from bacterial cells settling due to aggregation in the presence and absence of eDNA and as a function of added Ca2+ concentration was monitored after 90 min incubation in 1 ml plastic cuvettes  at 600 nm using a spectrophotometer (Bio-Rad Smart Spec™ 3000). PBS without bacteria was used as a blank. For additional control experiments, bacterial cells were also treated with heat inactivated DNase I (heating DNase I for 30 min at 75°C) and subjected to the aggregation assay. Aggregation was quantified according to equation 1: X = 100× (OD1–OD2)/OD1 To study the aggregation pattern of all bacterial strains with different concentrations of added Ca2+ were studied using fluorescence microscopy. This was done by suspending bacterial cells (with or without DNase I treatment) in PBS (OD  = 0.5±0.05/ml) as mentioned previously and then mixed (by vortexing) with added Ca2+ (0 and 1000 μM). A 1 ml aliquot of bacterial cells were immediately added to 6 well polystyrene plates and incubated for 90 min at room temperature in static condition. After 90 min the well plate's surfaces were gently washed with PBS. The adhered bacteria to the well plate's surface were imaged after staining with the Live/Dead stain as described above.Initial adhesion and aggregation of planktonic bacterial cells in the presence and absence of naturally occurring eDNA and added Ca2+ (1000 μM), chromosomal DNA was extracted from one day old cultures of P. aeruginosa using standard protocols 2+ was calculated after 90 min using equation 1.To quantify bacterial aggregation in the presence of exogenous DNA and CaTo quantify biofilm biomass, untreated and DNase I treated bacterial cells suspended in PBS (OD  = 0.5±0.05/ml) were incubated for 90 min in 96 well microtiter plates at 37°C. After 90 min of incubation wells were washed with PBS three times in order to remove any unattached bacterial cells. Subsequently, 200 μl of LB medium was added to the wells and incubated for 24 h at 37°C in order to initiate biofilm formation. After 24 h of biofilm growth, the LB medium was gently removed by pipetting and subsequently rinsed with PBS three times. The total biofilm biomass attached to the wells was quantified using O'Toole's crystal violet assay at 550 nm To establish the amount of eDNA released during biofilm growth, the biofilm biomass grown over 0, 16 and 24 h was suspended in 1 ml of PBS. The biomass suspended in PBS was then filtered with 0.22 μm filters to remove any bacterial cells and subsequently eDNA concentration present in filtered PBS (supernatants) was quantified as above.P. aeruginosa cultures were incubated for 90 min in the presence and absence of eDNA either as is (with no added Ca2+) or with addition of 1000 μM Ca2+. Bacterial lawns were prepared by depositing bacteria from suspension on a 0.2 μm nitrocellulose filter (Millipore) using negative pressure For contact angle measurements LW) and acid-base (γAB) surface free energy components using the LW-AB approach −) and electron–accepting (γ+) parameter To conduct surface thermodynamical analysis, contact angles were converted into Lifshitz-Van der Waals  or milliQ water as a negative control was mixed with CaCl2  and divided into 25 injections, with an interval of 240 sec after each injection. All measurements were performed at 25°C, with a reference power of 5 µcal/sec and stirring speed of 800 rpm. Data was analysed using Origin 7.0 software . Gibbs free energy changes (ΔG) were determined using the relationship: ΔG  =  ΔH-TΔS where, ΔH is the enthalpy change, T is temperature (Kelvin) and ΔS is the change in entropy.Isothermal Titration Calorimetry (iTC) was used to measure the binding affinity (Ka), enthalpy changes (ΔH), and binding stoichiometry (n) of the interaction between Cap<0.05.The student's t-test was used for statistical analysis and differences were considered significant if P. aeruginosa supernatant has the highest amount of eDNA (6.5 μg/ml) followed by E. coli (4.5 μg/ml) and A. hydrophilla (2.5 μg/ml). All Gram-positive  strains showed significantly less eDNA in its supernatants averaging only 2 μg/ml. The optical density of bacterial cultures  measured at various time intervals  did not show any significant difference  and absence of eDNA. In the absence of eDNA and added Ca2+ all three Gram-negative bacterial strains  responded with a significant decreases in settling. In contrast, the Gram-positive bacteria  showed no significant difference in aggregation. However, in the absence of eDNA, addition of Ca2+ (10–1000 μM) resulted in a significant reduction in aggregation for P. aeruginosa, E. coli, S. aureus and E. faecalis but not for S. epidermidis. At biologically relevant concentrations, Ca2+ (1000 μM) resulted in a significant increase in aggregation of A. hydrophila regardless of the presence of eDNA, however in presence of eDNA the aggregation was significantly higher than in its absence. Out of the six strains considered the aggregation of S. epidermidis was unique in that it was not influenced by the removal of eDNA.The settling of Gram-negative bacteria under gravity as a consequence of aggregation was influenced more by the presence of eDNA than aggregation of Gram-positive bacteria . UntreatP. aeruginosa, A. hydrophila and E. coli) and two out of three Gram-positive bacteria tested  regardless of the Ca2+ concentration. Further, addition of Ca2+, especially at 1000 μM, aggregation increased significantly, from 31% at 0 μM to 40% at 1000 μM for P. aeruginosa, 26% at 0 μM to 32% at 1000 μM Ca2+ for A. hydrophila, from 6% at 0 μM to 12% at 1000 μM Ca2+ for S. aureus and from 18% at 0 μM to 24% at 1000 μM Ca2+ for E. faecalis (The settling (aggregation) assay revealed that the presence of eDNA (including results of heat inactivated DNase I) promoted aggregation for all three Gram-negative bacterial strains used in this study  S. aureus and E. faecalis showed more adhesion and aggregation in comparison to the presence of eDNA alone. Interestingly, removal of eDNA by DNase I treatment significantly decreased S. aureus and E. faecalis adhesion and aggregation, regardless of added Ca2+ (1000 μM)  .2+ and exogenous DNA on bacterial aggregation was further investigated by incubating DNase I pre-treated bacterial strains with and without addition of exogenous DNA (1 μg/ml) and Ca2+  showed significant increases in bacterial aggregation in addition of both DNA and Ca2+ (1000 μM) in comparison to aggregation in addition of Ca2+ alone. Further, A. hydrophilla, S. aureus and E. faecalis showed significant increases in aggregation when both DNA and Ca2+ were added in comparison to aggregation observed in the addition of DNA alone . However, addition of Ca2+ did not influence the contact angle values aka hydrophobicity of P. aeruginosa cell surface regardless of the presence or absence of eDNA. Hydrophobicity determines surface energies . The surface energy components: Lifshitz-Van der Waals  which is generally attractive (due to dipole interactions between molecules or between parts of same molecules) values about 35 mJ/m2 were not affected by the presence or absence of eDNA or added Ca2+. However, the electron donating and electron accepting parameters of the acid-base component  that form the basis for the surface hydrophobicity of bacterial cells was significantly different in the absence of eDNA. Especially, in presence of eDNA the electron donating group has surface energies about half the value (averaging 25 mJ/m2) than in absence of eDNA, regardless of added Ca2+. This result indicates that in the presence of eDNA the bacterial cell surface has low surface energies but strong hydrophobic forces that promote aggregation.P. aeruginosa at close approach in the presence and absence of eDNA and added Ca2+. Lifshitz-Van der Waals aggregation energies were negative averaging −3 mJ/m2 regardless of the presence of eDNA or Ca2+ (2+ to 3 mJ/m2 at 1000 μM Ca2+ and much higher (approximately 40 mJ/m2) in the absence of eDNA regardless of addition of Ca2+ (2+ to 0.5 mJ/m2 at 1000 μM Ca2+) in the presence of eDNA, whereas in the absence of eDNA the total aggregation energies remained high (approximately 35 mJ/m2) regardless of addition of Ca2+ (P. aeruginosa is a consequence of coordination between hydrophobic forces and acid-base interactions driven by the presence of specific surface molecules (eDNA). or Ca2+ . However of Ca2+ . The tot of Ca2+ . In brie2+ was introduced exogenously. In the absence of DNA the Kcal/mol of Ca2+ injection remained static regardless of the number of injections. The results indicate that approximately 19 moles of Ca2+ interacts with 1 mole of DNA. The change in Gibbs free energy (ΔG) for the interaction is −8.25 Kcal/mol, composed of change in enthalpy (ΔH) with a value of −7.509 Kcal/mol and change in entropy (ΔS) at almost zero  at constant temperature (298 Kelvin).2+ further increased biofilm biomass in A. hydrophila and E. faecalis over 24 h. In the absence of eDNA Ca2+ does not enhance biofilm biomass in any of the strains used in this study. Further, 2+ via electrostatic interactions 2+ are primarily known for stabilizing bacterial cell walls and promoting ionic cross bridging among bacterial cells. In this study it is shown that Ca2+ binding with eDNA facilitates bacterial cell-to-cell aggregation and subsequent biofilm formation.Recent discoveries reported that eDNA bind with various biopolymers  and metabolites (phenazines) produced by bacteria P. aeruginosa in comparison to all other bacterial strains tested is likely due to a combination of the active release of eDNA via membrane vesicles/blebs P. aeruginosa cell growth/density (OD) were observed in comparison to other bacterial strains and none of the strains used in this study are known to possess lytic proteins/enzymes. In contrast, microscopy imaging revealed an increase in pyocyanin mediated cell lysis and ultimately increased release of eDNA in P. aeruginosa cultures Pseudomonas species cannot be ruled out. Variations in the concentration of eDNA between strains likely explain the differences in aggregation and biofilm formation behaviour between the bacteria used in this study. Gram-negative bacterial strains showed significant decreases in aggregation when eDNA was removed through DNase I treatment. In the presence of biologically relevant Ca2+ concentrations Gram-positive bacteria showed significant increases in aggregation. Interestingly, under conditions when both eDNA and Ca2+ are present an increase in bacterial aggregation was observed for 4 out of 6 bacterial strains and this increase is likely due to cationic bridging interactions mediated by Ca2+2+ promotes aggregation or clustering of bacteria. Increased bacterial adhesion to any kind of substratum surfaces by eDNA is due to strong interaction/adhesion forces and mature bond formation between bacterial cell surfaces in the presence of eDNA and substratum surfaces triggered by physico-chemical forces The significant increase in eDNA recorded in the supernatant of 2+ there are other influential factors that promote bacterial aggregation such as acid-base interactions. To investigate how acid-base interactions and cationic bridging complement each other in promoting bacterial aggregation we chose P. aeruginosa as model organism because of its naturally high eDNA content in its supernatant. In the presence of eDNA P. aeruginosa showed significant increases in cell surface hydrophobicity and DNA being a hydrophilic molecule likely interacts with hydrophilic (polar) sites on the P. aeruginosa cell surface, leaving hydrophobic (non-polar) sites unattended, consequently promoting hydrophobicity Apart from cationic bridging interactions mediated by Ca2+ did not influence acid-base interactions energies regardless of the presence of eDNA, however, Ca2+ influenced bacterial aggregation. This indicates that Ca2+ binding with eDNA promotes cationic bridging only. Negative ΔG values in iTC data confirmed the interaction between DNA and Ca2+ and revealed that it is thermodynamically favourable and the binding process is spontaneous and exothermic (heat released during interaction) owing to its overall highly negative enthalpy. A previous calorimetric study reported that the presence of polypeptides (antigen I/II) on streptococcal cell surfaces promotes highly negative enthalpy associated with high heat release when binding with salivary protein S. aureus strain with fibronectin binding protein on its cell surface binds to fibronectin in comparison to its mutant strain deficient in fibronectin binding protein 2+ and promote aggregation.Interestingly, incubation of bacterial cells with Ca2+ on bacterial cell surface decreases electrostatic repulsion and therefore facilitates adhesion and aggregation 2+ bacteria adhered and aggregated more. At the bacterial cell surface, specific interactions could result from the strong affinity of Ca2+ at multiple functional sites. Ca2+ binds to several constituents of matrix including lipopolysaccharides 2+ (via electrostatic interactions) affecting aggregation in a variety of bacterial strains. Studies comparing other biopolymers and eDNA in facilitating matrix integrity and aggregation through Ca2+ binding have not been undertaken. The results of the current study support the assertion that eDNA plays a central role in promoting Ca2+ regulated ionic cross-bridging and subsequent aggregation in most of the bacterial strains used in this study. S. epidermidis aggregation is not influenced by removal of eDNA by DNase I treatment is a clear indication that eDNA did not influence cell-to-cell interaction or matrix integrity. This is not surprising because it is well documented that in many S. epidermidis strains matrix formation, bacterial interactions and biofilm formation is primarily dependent on polysaccharides S. epidermidis, which is well known for causing biomaterial-associated infection in humans produces different types of extracellular polysaccharides that are responsible for various roles ranging from bacterial adhesion to biofilm formation and promoting virulence Another study suggests that since most cells are negatively charged, including bacteria, adsorption of Ca2+ is known to increase the production of extracellular components such as proteins P. aeruginosa biofilm 2+-eDNA-polysaccharides matrix. In contrast, Ca2+ does not influence release of eDNA under planktonic or biofilm growth conditions  cell density (OD) in planktonic condition in LB medium measured at various time intervals  (A). Comparison of live and dead cells in planktonic culture of P. aeruginosa and S. aureus grown in LB medium for 24 h at 37°C in a static incubator using fluorescence microscopy  (B).(TIF)Click here for additional data file.Figure S22+ concentration.Quantification of eDNA concentration as a function of added Ca eDNA quantified in planktonic culture of P. aeruginosa grown in LB medium for 24 h at 37°C in a static incubator in presence of added Ca2+ .(TIF)Click here for additional data file.


Changes in Ca2+ concentrations via CaBPs can disturb Ca2+ homeostasis. Brain damage can be induced by the prolonged electromagnetic field (EMF) exposure with loss of interacellular Ca2+ balance. The present study investigated the radioprotective effect of ginseng in regard to CaBPs immunoreactivity (IR) in the hippocampus through immunohistochemistry after one-month exposure at 1.6 SAR value by comparing sham control with exposed and ginseng-treated exposed groups separately. Loss of dendritic arborization was noted with the CaBPs in the Cornu Ammonis areas as well as a decrease of staining intensity of the granule cells in the dentate gyrus after exposure while no loss was observed in the ginseng-treated group. A significant difference in the relative mean density was noted between control and exposed groups but was nonsignificant in the ginseng-treated group. Decrease in CaBP IR with changes in the neuronal staining as observed in the exposed group would affect the hippocampal trisynaptic circuit by alteration of the Ca2+ concentration which could be prevented by ginseng. Hence, ginseng could contribute as a radioprotective agent against EMF exposure, contributing to the maintenance of Ca2+ homeostasis by preventing impairment of intracellular Ca2+ levels in the hippocampus.Calcium binding proteins (CaBPs) such as calbindin D28-k, parvalbumin, and calretinin are able to bind Ca Ginseng, the root of Panax ginseng C.A. Meyer, is a widely used herbal medicine with numerous efficacious effects but possessing very low rate of side effects, thereby becoming one of the top-selling natural remedies plus the most popular dietary supplement . GinsengRapid advancement in the field of telecommunications has led to dramatic increase in the use of mobile phone technology, generating interest in the biological effects and possible health outcomes of electric and magnetic fields. The extensive use of mobile phones raises questions of their possible biological effects  due to E2+) entry into neurons regulates normal neuronal development, metabolism, and ageing and is involved in the control of synaptic transmission and its long-term modulation [2+ levels leading to neuronal degeneration [2+ efflux from the brain and isolated neurons [2+ homeostasis in the brain is regulated by influx and efflux systems but is also affected by calcium binding proteins (CaBPs). Alteration of intracellular signaling pathway via changes in ionic distribution or membrane fluidity could be influenced by EMF . Physioldulation  with eleneration . Radiofr neurons –24. Ca2+2+ plays a complex role as an important moderator of a number of vital physiological processes like neuronal excitability, axonal transport, synthesis and release of neurotransmitters, membrane permeability, and enzyme activities [2+ might play a role in neuron survival as well as programmed cell death and pathological neuronal degeneration with disturbance in Ca2+ regulation leading to lethal effects [2+concentration is crucial for neuronal function and survival, which can be achieved by an active uptake mechanism of the cell's internal membrane structures such as mitochondria and endoplasmic reticulum as well as CaBPs. Considering the importance of Ca2+ homeostasis in numerous processes for cell viability, including neurons, and modulation of intracellular Ca2+ concentration, CaBPs such as calbindin D28-k (CB), parvalbumin (PV), and calretinin (CR), belonging to the EF-hand type, could play a crucial role in the regulation of Ca2+ homeostasis by buffering the intracellular Ca2+concentration and transporting Ca2+ [2+ buffer has been postulated for CaBPs, the impairment of which leads to the failure of buffering of the intracellular Ca2+ and neuronal death [2+ homeostasis and is related with both neuronal and behavioral deficits [2+ homeostasis could contribute to impaired memory processes of the hippocampus. Characterization of CaBPs is important as each neuron may be associated with particular functional properties, loss of which could lead to different manifestations.Cativities . Neurona effects . Therefoing Ca2+ . A neuroal death  among whal death , and CR al death  are consdeficits . Hence, Considering the reported effect of purified ginseng components as radioprotective agents in irradiated rodents , 32 and P. ginseng plants at 90°C to 100°C for 3 hr and subsequent drying at 50°C to 80°C. RG was extracted seven times with distilled water at 85°C for 8 hr followed by cooling. RG contained 0.52 mg/g of major ginsenoside-Rg1, 4.03 mg/g of -Rb1, 2.89 mg/g of -Rg3(s), 1.18 mg/g of -Re, 1.98 mg/g of -Rc, 1.97 mg/g of -Rb2, 1.51 mg/g of -Rd, and other minor ginsenosides. Red Ginseng extracts (RG) manufactured by Korea Ginseng Corporation  were used in all experiments. RG was made by steaming fresh roots of 6-year-old n = 30) were used for the experiment. Upon arrival, animals were randomized and housed six per cage under the condition of 20 to 25°C. Food and water were supplied ad libitum. Mice were acclimated for one week. All animal procedures were performed according to the National Institutes of Health guidelines of animal research and were approved by Dankook University Institutional Animal Care and Use Committee (DUIACUC), which adheres to the guidelines issued by the Institution of Laboratory of Animal Resources (ILAR). Before conducting the study, mice were categorized into three groups (n = 10): (A) sham control (SC), (B) exposed (E1.6), and (C) exposed treated with 30 mg/kg of RG (G1.6).Six-week-old 20–30 g ICR (Orient Bio Inc.) male mice (3) on 835 MHz CDMA frequency. Waves were generated and amplified in an electronic unit and eventually were radiated by a pyramidal rectangular horn antenna connected by a waveguide to coaxial transition. A standard mouse cage of 22 inches was used for the apparatus. Output powers of horn antenna of the exposure apparatus are 2.5 W for SAR 1.6 W/kg and 6.3 W for SAR 4.0 W/kg. Electric field intensities due to SAR values can be calculated, and power value was obtained by a computer simulation with HFSS (High Frequency Structure Simulator) manufactured by Ansoft Co. . 5 cylinder-shaped models of mice were used for simulation. The simulation variable was both the mice location and the distance form horn aperture for freely moving mice. Power was obtained by averaging the simulated peak electric field intensities on each mouse body. The wave exposure from horn antenna to the mouse cage was provided by wave absorption material (TDK ceramic absorber) mimicking the radiation exposure in the open environment, which limits the influence the number of mice might have on exposure. The exposure apparatus provides an automatic light system and air conditioning system with a water feeder, with no restriction in movement during exposure eliminating stress during exposure.The exposure system (Wave Exposer V20) has been described in detail . BrieflyFor the RF exposure experiment, the entire body of mice was exposed to 835 MHz radiation for one month with average SAR of 1.6 W/kg by using Wave Exposer V20 instrument. The RG extract solution was prepared in a solvent consisting of 0.9% NaCl and 4% Tween 80 and was given orally before exposure according to individual body weight. The exposure was conducted for five hours per day for 30 consecutive days for each group. Three hours after the final exposure in the 30th day, animals were anesthetized with diethyl ether, and their brains were collected using perfusion and fixation procedure with phosphate buffer saline and 4% paraformaldehyde solution.μm, was cut with freezing, sliding microtome and collected in wells. Three separate experiments with all three groups simultaneously of immunohistochemistry were performed with the free floating method. Polyclonal anti-rabbit CB , polyclonal anti-rabbit PV , and polyclonal anti-goat CR  were applied to brain sections at dilution ratios of 1 : 5,000, 1 : 10,000, and 1 : 15,000, respectively, in phosphate buffer saline based blocking buffer containing 1% bovine serum albumin, 0.3% Triton X-100, and 1% normal horse serum. Sections were incubated for 48 hours at 4°C. After three washes with phosphate buffer saline, sections were incubated with the biotinylated secondary antibodies at a dilution ratio of 1 : 250 for 1.5 hours at room temperature. Following additional washes, sections were mounted on gelatin coated slides, dehydrated in ethanol, cleared in xylene, and cover-slipped with DPX. Brains were removed from the skull and were postfixed in paraformaldehyde for 24 hours. Then brain tissues were cryoprotected after soaking in a series of sucrose solution  at 4°C until they sank. Serial coronal section, 40 −2). Mathematically, Mean Density = Immunoreactivity/Area (mm−2). Data are expressed as mean ± SD. Comparison of the mean density of the different subfields of the hippocampus  between the sham group and exposed groups was done individually by unpaired Student's t-test. Differences were considered significant at P < 0.05. Analysis was performed under Olympus BX 51 microscope, and pictures of the sections were taken by a microscope digital camera system . The NIH image program (Scion Image) was used to determine staining densities. The sum of the gray values of all pixels in a selected region was divided by the total number of pixels in the selected region to determine the mean density of immunoreactivity per unit area  of all three groups displayed the presence of CB IR very distinctly. Granular layer (GL) was highly stained as compared with molecular layer (ML) and polymorphic layer (PL) in all the three groups Figures . PerceivWide distribution of PV IR was noted in the hippocampal region throughout the CA1 and CA3 areas as well as in the dentate gyrus. SO of CA1 area displayed consisted of various multipolar and bipolar cells, which were also seen in the SP Figures . The SP PV IR was detected in all three layers of the dentate gyrus as well as in the mossy fibers of all the three groups to a varying degree Figures . The graLayer specific alterations of CR IR were observed in the CA areas as well as the DG. CA1 and CA3 areas showed CR IR in the subgranular layer along with scattered interneurons. Strongly labeled scattered bipolar, multipolar, and pyramidal neurons were noted in the SL, SR, and SO of the CA1 areas Figures . NumerouIntense CR IR was noted in the neuropil of inner molecular layer (IML) with moderate staining in the other layers. Strongly immunoreactive infragranular neurons were observed in the SC  and G1.6Image analysis assessment of the mean density distribution of CB IR in all subfields of the hippocampus  displayeP < 0.01), CA3 (P < 0.001), ML (P < 0.0001), GL (P < 0.0001), and PL (P < 0.01) as compared with SC (P < 0.01). No significant difference was noted in the CA1, CA3, ML, and PL areas of G1.6 when compared with SC , CA3 area (P < 0.05), and GL (P < 0.001) of the E1.6 when compared with SC (P < 0.01) while the CA1, CA3, GL, and PL areas did not show any significant difference between the SC and G1.6 groups (Significant difference was observed in the CA1 area ( with SC . Similar6 groups .P < 0.0001), CA3 (P < 0.0001), OML (P < 0.001), GL (P < 0.001), and PL (P < 0.0001)  . No sign 0.0001) . In the present study, we evaluated the CaBP IR in the hippocampal subfields to examine the radioprotective effects of the RG extract against RF exposure at 835 MHz at SAR 1.6 W/kg for one month at 5 hr/day. The study provided results showing the protective effects of RG against EMF exposure, demonstrating that RG could be useful as a radioprotective agent in the CNS.2+ homeostasis, and its specific distribution pattern in the CNS suggests its involvement in important neuronal activities. CB is associated with regulation of intracellular Ca2+and is implicated as a neuronal population marker [2, contributing to the maintenance of synaptic properties [2+ [2+ buffering capacity which could influence diverse functions, such as segregating signaling pathways by limiting calcium diffusions [2+ is an important component of normal cellular functions and mediates most of the physiological effects triggered by EMF and the ions that are liberated from their intracellular stores. Interactions of Ca2+ at the cell membrane have been identified as the first link in the bioeffects from RFR. Release of the neurotransmitter that transfers signals between neurons requires a prerequisite of programmed flow of Ca2+ ions through the membranes. Disruption leading to Ca2+ leakage would increase the background concentration making the cells hypersensitive, with the transmission of spurious signals which would cloud normal mental activity, trigger random thoughts and loss of concentration [2+ buffering capacity, leading to cell death.CaBPs like CB, PV, and CR have an important role in maintaining intracellular Can marker . PV is koperties , while Cties [2+ . Alteratffusions . Ca2+ isntration . Hence, 2+ channels [2+ influx by RG has been noted in the hippocampal neurons subjected to oxygen/glucose deprivation [2+ influx through both NMDA receptor channels and L-type voltage-dependent Ca2+ channels, with the resultant reduction of intracellular free Ca2+ [2+ influx by RG has been reported in a glutamate (0.5 mM) toxicity model of cultured hippocampal neurons of rats [RG has been used in oriental medicine for thousands of years with ginsenosides as its main bioactive component plays an important role in CNS , 3. Ginschannels . Inhibitrivation . RG has ree Ca2+ . Inhibit of rats . Similar of rats –43. SimiWith the ever-increasing growth of mobile communication, an increase in EMF density has occurred which can influence neuronal function, neurotransmitter release, neuronal survival, and learning and memory . The pre2+ buffering capability which might ultimately lead to cell death, while loss of neurons in the CA regions and dentate gyrus further strengthens the reasoning behind the increased Ca2+ influx. In the same model the ginseng-administered group did not show any loss of CaBP IR or neuronal loss due to RF exposure. Various studies have also noted the beneficial effects of ginseng in the hippocampus [2+, and the inhibition of homocysteine-induced elevation of intracellular free Ca2+ has been noted as well. Memory test could be helpful in further consolidating the present findings. Ginseng is also known to have anti-inflammatory and antiapoptosis activities which might be the reason it did not affect CaBP IR. A TUNEL experiment might be of help to prove this point. Another possible explanation could be the promotion of cell genesis by RG, which is also strengthened by our findings (data not shown) that are similar to the findings that noted increased brain-derived neurotrophic factor (BDNF) expression in the hippocampus [2+ homeostasis in the hippocampus.The hippocampus is an important area of interest as it is the primary target for research in the molecular and cellular mechanism of memory and information storage through its synaptic connections . The prepocampus –52, whicpocampus , which rpocampus . BDNF hapocampus . It alsopocampus  and bothpocampus . Increas2+, the lack of which might cause severe damage leading to tissue degeneration. However, the proper mechanism is yet to be unveiled. Further studies with protein assay test, western blots, and electrophysiological recording might help to shed more light on it. In order to thoroughly understand the beneficial effect of RG against RF exposure, much research is still needed to further consolidate RG as a universally accepted radioprotective agent.In summary, the present study showed the protective effect of RG against RF exposure by maintaining the CaBP IR involved in the buffering of Ca


The regimen of cytarabine, aclarubicin and G-CSF (CAG) has been widely used in China and Japan for treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We searched literature on CAG between 1995 and 2010 and performed a meta-analysis to determine its overall efficacy using a random-effects or fixed-effects model. Thirty five trials with a total of 1029 AML (n = 814) and MDS (n = 215) patients were included for analysis. The CR rate of AML (57.9%) was significantly higher than that of MDS (45.7%) (p < 0.01). No difference in CR was noted between the new (56.7%) and relapsed/refractory AML (60.1%) (p > 0.05). The CR rate was also significantly higher in patients with favorable (64.5%) and intermediate (69.6%) karyotypes than those with unfavorable one (29.5%) (p < 0.05). Remarkably, the CR rate of CAG was significantly higher than those of non-CAG regimens (odds ratio 2.43). CAG regimen was well tolerated, with cardiotoxicity in 2.3% and early death in 5.2% of the cases. In conclusion, CAG regimen was an effective and safe regimen for the treatment of AML, and may be more effective than non-CAG regimens. Randomized controlled trials are strongly recommended to evaluate its efficacy and safety in comparison with the current standard treatment. Howevia (AML) -6. Novelia (AML) -10.2, SQ Q12 hr on days 1-14, aclarubicin 7 mg/m2, QD on days 1-8, or 14 mg/m2, IV QD on days 1-4, and G-CSF 200 μg/m2, SQ QD on days 1-14. The rationale for the regimen includes: (1) G-CSF priming has been found to preferentially potentiate Ara-C and anthracycline- mediated cytotoxicity on AML cells and AML progenitor cells (CFU-AML), presumably by enhancing G0 resting AML cells into the cell cycle [Aclarubicin is an oligosaccharide anthracycline, and an antineoplastic antibiotic. It can intercalate into DNA and interact with topoisomerase I and II, thereby inhibiting DNA replication and DNA repair . This agll cycle ; (2) proll cycle ; (3) Aclll cycle ; (4) CAGll cycle . Since tCAG, chemotherapy, leukemia, and MDS. An independent search using the citation database Wanfang Data (http://www.wanfangdata.com) was also performed to identify those publications in Chinese only.The databases of PubMed, Wanfang Data, as well as American Society of Hematology (ASH) and American Society of Clinical Oncology (ASCO) annual meeting abstracts were searched for articles published in English, Chinese and Japanese languages between January 1995 and December 2010. Eligible studies were relevant clinical trials on AML and MDS patients treated with CAG regimen. Key words used were The publications identified were carefully screened. Preclinical studies, case reports and reviews were excluded. Several reports had duplicate or overlapping information. Only the latest updated reports were included for meta-analysis. For two studies by Qian's group ,20, it wi.e. young AML: age < = 60; Elderly AML: age >60.We extracted details on study characteristics, patient characteristics, treatment information, results and follow-up from the selected trials. Two investigators reviewed the data independently (GW and DL). The primary end point of the meta-analysis was CR rate. AML CR was defined using the criteria developed by an International Working Group . The criAll statistical analyses were performed using version 2 of the Comprehensive MetaAnalysis program . The CR rates of patients treated with CAG regimen were directly extracted from individual studies. For subgroup analysis of patients with newly diagnosed, refractory/relapsed AML, and MDS or AML transformed from MDS (MDS/t-AML) patients, numbers of patients in CR were extracted from individual studies and CR rates were recalculated from the derived data. For studies with a control group, the odds ratio (OR) of CR rates was also calculated. For the meta-analysis, both fixed-effects and random-effects models were considered. For each meta-analysis, the Cochrane's Q statistic was first calculated to assess the heterogeneity of the included studies. For p values less than 0.1, the assumption of homogeneity was deemed invalid, and the random-effects model was used only after substantial efforts were made to explore the possible reasons for the heterogeneity. Otherwise, data were assessed using both fixed-effects and random-effects models. We used the Begg and Egger tests to evaluate the presence of publication bias regarding the primary end point CR rates. A two-tailed p value of less than 0.05 was deemed statistically significant. All the statistical analyses were done by GW and DL.Using the key words, our search yielded a total of 135 studies on CAG. Fifty seven studies were irrelevant to CAG regimen trials, and were excluded. Another 32 studies were eliminated due to inadequate information, duplicate and/or overlap reporting. Nine case reports and 2 studies focus on other kinds of leukemia were also excluded. A total of 35 clinical studies were eligible for inclusion and were used for final meta-analysis .2 32.695, indicating the CR event rates were highly variable. Therefore, the CR event rates were calculated using the random-effects model  than in MDS (45.7%) was statistically significant (p = 0.004) Figure . This isAmong AML patients, the median OS was 15 months (range 9-28 months). The number of CAG cycles was specified in 17 studies involving 397 patients, of whom 144 (36.2%) were induced twice with CAG. Of the patients who received one cycle, 46.60% (185/397) went into CR, another 8.31% (33/397) of the patients achieved CR after re-induction with CAG.2 42.396, indicating the CR event rates were highly variable. Therefore, the CR event rates were calculated using the random-effects model  AML from 15 studies. Four of these studies enrolled both new and R/R AML   Tables . Among A2 + Ara-C 100 mg-200 mg/m2) (57%-65%) [CAG regimen was originally designed in Japan for the treatment of relapsed AML patients in 1995. It quickly became popularized in China for the treatment of high-risk AML and MDS patients due to relatively mild toxicity. Most of the studies were single-center non-randomized trials with small sample size. CAG has not been systematically compared with other induction regimens. In this meta-analysis, 1029 patients with AML and MDS were treated with CAG regimen. Two findings on CR rate for AML are quite intriguing. Number one, there was no significant differences in CR rates between new 56.7%) and relapsed/refractory (60.1%) AML. This is surprising since the CR rate is generally lower for R/R than new AML. One possibility is that this novel CAG regimen can overcome the drug resistance of the R/R AML clone and lead to high CR rate. Alternatively, it may be that the first-line induction therapy was substandard in these patients from diverse institutions with different supportive care standards. Therefore, the relapsed and refractory AML was still sensitive to alternative chemotherapy. The CR rate of CAG (56.7%) for new AML appears to be slightly lower than the CR rate of standard "3+7" regimen (daunorubicin 45 mg/m% and rel57%-65%) ,31 . The non-CAG regimens included anthracyclines plus Ara-C in 5 studies (AA regimens), and homoharringtonine plus Ara-C (HA regimen) in two other studies. HA is only used in China . When CAIn this meta-analysis, the CR rate of CAG regimen for high-risk MDS/t-AML patients was 45.7% . Aza-001 trial, a prospective randomized study, compared azacitidine with conventional care regimen  in higher-risk MDS patients . The CR There have not been many reports focusing on toxicities of AML inductions. In the report of "3+7" regimen on 326 new AML patients from the Cancer and Leukemia Group B 8321 study, the overall induction mortality was 15% . Data frThis study has severe limitations. First of all, this study is based on 35 trials which were conducted in China and Japan, where significant variations in supportive care may have existed. In addition, the studies were between 1995 and 2010. Newer molecular prognostic markers, such as FLT3 and NPM1, were not routinely tested in most of the studies. The standard of supportive care for AML has changed dramatically over the decade. The toxicity data from this analysis may therefore overestimate the adverse events of the regimens under current health care system. Majority of the studies were small (only 9 of the 35 studies had 50 or more subjects). Although all the studies used similar CAG regimens, the dosage of aclarubicin was variable. Due to the small sample sizes and ambiguity in reporting the AML status in the 7 studies, it was difficult to ascertain whether the variation of the aclarubicin dosage could have played any role in the outcomes among the different groups of AML (new vs R/R) and MDS/t-AML. There was no randomized study among the 35 reports. CAG was compared with non-CAG regimens from historical controls in the 7 reports. The non-CAG regimens also varied. Two of the seven reports did not include anthracyclines. It is therefore possible that the superiority of CAG over non-CAG regimen may have been overestimated, even when the more stringent fixed-effects model was used. Estimation of CR event rates using random-effects model may minimize the inherent variances. Finally, CAG regimen is used exclusively in China and Japan. It is not clear whether similar outcome is reproducible in the Western countries.In conclusion, CAG regimen was effective and safe for the treatment of AML and MDS patients. Its activity may vary among these patients with significantly higher CR rates observed in AML than MDS. It was as active in new AML as in relapsed and refractory AML. CAG regimen may be more effective than non-CAG regimen with the CR rate of CAG regimen significantly higher than those of non-CAG induction regimens in AML patients. This regimen was well tolerated with low cardiotoxicity and ED rate. We strongly recommend that CAG regimen be compared with standard anthracycline plus Ara-C in a prospective randomized study, particularly in high-risk and elderly AML and MDS patients.The authors declare that they have no competing interests.GW and DL participated in concept design, data collection and analysis, drafting and critically revising the manuscript. All authors are involved in reviewing and revising the manuscript. All authors have read and approved the final manuscript.