Using individually purified MSC-like precursor cell subsets, we and other researchers have been able to investigate the differential phenotypes and regenerative capacities of these contributing cellular constituents in the MSC pool. In this review, we will discuss the identification and characterization of perivascular MSC precursors, including pericytes and adventitial cells, and focus on their cellular kinetics: cell adhesion, migration, engraftment, homing, and intercellular cross-talk during tissue repair and regeneration.Mesenchymal stem/stromal cells (MSCs) and MSC-like multipotent stem/progenitor cells have been widely investigated for regenerative medicine and deemed promising in clinical applications. In order to further improve MSC-based stem cell therapeutics, it is important to understand the cellular kinetics and functional roles of MSCs in the dynamic regenerative processes. However, due to the heterogeneous nature of typical MSC cultures, their native identity and anatomical localization in the body have remained unclear, making it difficult to decipher the existence of distinct cell subsets within the MSC entity. Recent studies have shown that several blood-vessel-derived precursor cell populations, purified by flow cytometry from multiple human organs, give rise to  The availability of mesenchymal stem/stromal cells (MSCs) and MSC-like multipotent stem/progenitor cells marked a major milestone in stem cell therapies , 2. For in vivo [Nevertheless, owing to the nature of MSC isolation by plastic adherence in tissue culture, the native identity and anatomical localization of MSCs have remained unclear for years . Recentlin vivo , 17. Incin vivo –21. Follin vivo . These hin vivo , adventiin vivo , and myoin vivo , can be In this review, we will discuss the identification and characterization of perivascular MSC precursors  from multiple organs and focus on their cellular kinetics during regenerative events, including cell adhesion, migration, engraftment, homing, and intercellular cross-talk.MSCs and MSC-like stem/progenitor cells have been found in nearly all organs in the human body. Despite slight differences in phenotypes and cellular functions, MSCs and MSC-like cells from various ontogenies share basic features in general, including selective plastic adherence, expression of typical MSC surface markers, and mesenchymal multipotency such as osteogenesis, chondrogenesis, and adipogenesis. Some of the most common MSCs and MSC-like multilineage cells are briefly introduced here. α, and HLA-DR1 [in vitro, BM-MSCs typically exist at a very low frequency within the BM mononucleated cell population  but can be efficiently expanded in culture, making them one of the most investigated autologous stem/progenitor cell populations. Interestingly, multipotent BM-MSC clones retain approximately twofold higher CD146 expression level than unipotent clones [Bone marrow (BM) harbors multiple types of stem/progenitor cells, including hematopoietic stem cells (HSCs), endothelial progenitor cells (EPCs), and BM-MSCs , 27. As  HLA-DR1 . Estimatt clones . in vitro using the same criteria as bona fide BM-MSCs [in situ. This misperception was accentuated in light of the recent characterization of CD34-negative PCs as a source of MSCs in a variety of mesodermal tissues, including fat [in vitro [in vitro [ in vivo [The stromal vascular fraction (SVF) of adipose can be isolated via enzymatic digestion of intact fat tissue or lipoaspirate, followed by the depletion of mature adipocytes through centrifugation. The SVF embodies a broad and heterogeneous cellular compartment, including vascular cells , hematopoietic cells (resident and circulating cells), and stromal fibroblasts. In 1976, human adipogenic progenitors (aka preadipocytes) were successfully isolated by two independent groups from the adipose SVF by selective adherence to culture plastics , 31. The BM-MSCs , includi BM-MSCs , althoug BM-MSCs . Unlike  BM-MSCs , a well- BM-MSCs  and endo BM-MSCs  cell linding fat . While tding fat , the CD3in vitro , 39, 40.in vitro , 42. Botin vitro  and may  in vivo . Stem/progenitor cells isolated from disposable perinatal tissues, including amnion/amniotic fluid, umbilical cord blood, placental tissue, umbilical cord blood vessels, and the Wharton's jelly, have been deemed promising for clinical applications because of the minimal safety and ethical concerns , 46. MSCtunica intima, tunica media, and tunica adventitia [in vivo [The similarities between MSCs derived from many different tissues aroused the idea that a common reservoir of MSCs may exist in the body. The blood vessel, which typically consists of three structural layers: ventitia , is distventitia . Recentlventitia , 50. New[in vivo –21. the intima of microvessels and capillaries, are traditionally regarded as a structural component of blood vessels, regulating vascular contractility, stability, and integrity [tunica adventitia, the outermost layer of large blood vessels, has long been considered as a structural bystander, consisting of loosely structured collagen-rich extracellular matrix (ECM), which embeds stromal cells/fibroblasts, the vasa vasorum, and perivascular nerves [tunica adventitia was recently reevaluated due to a number of studies reporting its active role in vascular remodeling, immune response mediation, cell trafficking, and atherosclerosis [in situ and purified these cells to homogeneity based on their unique expressions of cell surface antigens. Details of the isolation and characterization of PCs and ACs will be described in the following sections.Microvascular pericytes (PCs), a set of perivascular mural cells surrounding ntegrity , 52. Intntegrity , 53–55. ntegrity . On the r nerves . The impclerosis –59. In aclerosis –62. Receclerosis , 63–65. tunicae media and adventitia, the subendothelial zone of tunica intima has previously been suggested as one of the sources of EPCs [in vitro [Unlike the  of EPCs , 67. Apa of EPCs . MECs, pin vitro . Furtherin vitro , 68, 69.While placenta and umbilical cord are often discarded at birth, these extraembryonic tissues contain large numbers of stem/progenitor cells, making them attractive sources of donor cells for regenerative medicine. We and others have isolated multipotent PCs (CD146+/CD34−/CD45−/CD56−) from these tissues and utilized them toward multiple tissue repair/regeneration, including skeletal muscle , lung 7, dermal α-SMA) but not hematovascular markers  and were competent for trilineage mesenchymal differentiation [N = 3, 19 to 21 weeks of gestation) and 2.1 ± 0.43%  of PCs (CD146+/CD34−/CD45−/CD56−), respectively  and MSC  surface antigens and were deprived of EC antigens  [β, NG2, CD90, and calponin), including 65 transcripts that are highly expressed in undifferentiated MSCs, and lacked endothelial/hematopoietic cell marker expression  [The native expression of CD146 by mesenchymogenic PCs in many tissues including bone marrow, fetal and term placentas has been reported , 70. Usiand vWF) . Consistnd CD45) . αSMA) but did not express the established EC markers: CD144, CD34, CD31, and Ulex europaeus agglutinin (UEA-1) receptor. Using FACS, Montemurro et al. isolated a population of PCs (CD146+/NG2+/PDGFRβ+) from umbilical cords of preterm newborns [αSMA, and PDGFRβ as well as retained their multipotency to differentiate towards different cell types, including osteogenic, adipogenic, and myogenic cell lineages [Human umbilical cord (HUC) has been known as an abundant source of ECs as well as MSCs derived from the Wharton's jelly. Recently some of us demonstrated that human full-term UCs and, at a higher frequency, fetal (preterm) UCs contain perivascular cells that exhibit features of MSCs. These perivascular smooth muscle-like cells present in the HUC co-expressed CD146 and alpha-smooth muscle actin , and α-smooth muscle actin (α-SMA), with the absence of EC markers: CD31, CD34, CD144, and vWF as well as the hematopoietic cell marker CD45 and myogenic cell marker CD56. Muscle PCs can be efficiently expanded in culture, at the clonal level, while maintaining robust mesodermal developmental potentials. Freshly isolated and long-term cultured muscle PCs both displayed robust myogenic capacity in vitro and in vivo. Moreover, muscle PCs natively and in culture expressed classic MSC markers: CD44, CD73, CD90, and CD105, indicating their developmental status as MSC ancestors [Skeletal muscle has been shown to harbor several adult stem/progenitor cell populations in mammals including humans, in addition to the typical muscle stem cells, that is, satellite cells –83. Many and fat , 84–86. via FACS . Similarncestors . vasa vasorum of large blood vessels such as the vena saphena [αSMA, CD146, NG2, PDGFRβ) [∼50%) of CD34+/CD146− progenitor cells in the nonhematopoietic adipose SVF [γ) reporter mouse model, Tang et al. demonstrated that adipogenic progenitors emerge from CD34+ cells which later adopt a perivascular niche and express PC markers  [αSMA, CD146, NG2, PDGFRβ) in vitro, following treatment with angiotensin II or angiopoietin-2 [Vasculogenic CD34+/CD31− cell populations have been described in the adventitial  saphena  and the  saphena , but mic saphena . Apart f PDGFRβ) , 39, 42.pose SVF , 89 and pose SVF  do not p PDGFRβ) . Similaroietin-2 .  bona fide pericyte features, including angiogenic/vasculogenic capacity and expression of CD146, NG2, and PDGFRβ but not αSMA, CD56, CD34, or EC markers [in vivo similar to their adult counterparts [While developmentally mesenchymogenic PCs may arise from transient CD34+ cell population(s), the persistence of such CD34+ precursors in the adult and their ontological relationship to the bulk of CD34+ ACs in human fat will require further investigation. Indeed, rare CD34+ mesenchymogenic cells have been reported in fetal , 90, 91  markers . These hterparts . Yet, thα, PDGFRβ) without requirement of blood vessel remodeling growth factors in contrast to CD34+CD146− cells [β), adventitial (CD34), or MSC  cell subsets. A number of studies have employed preliminary sorting strategies relying on single markers, such as CD146 [A rare CD34+/CD146+/CD31−/CD45− population of adipose PCs has also been characterized in the SVF , 98–103 6− cells . Though as CD146 , 105 or as CD146 , 107, whin vivo and in culture [in vivo but require prudent interpretations in culture due to its instability.Recently, using a combination of above-mentioned positive and negative selection antigens, we performed advanced flow cytometry analyses and FACS in the adipose SVF in order to identify and simultaneously purify these MSC precursor subpopulations , 39, 101 culture , 24, 101 culture , the CD3in vivo action of stem/progenitor cells. Since cell adhesion and migration capacities are tightly correlated with in vivo cell trafficking and homing, these parameters represent potential predictors for the clinical outcome of stem cell-treated patients and require further investigation [In view of future stem cell-based approaches and therapies, it is crucial to identify predictive parameters that allow the researchers and clinicians to foresee the tigation –110. Herβ+/CD144− PCs exhibited more robust adherence to extracellular matrix substrates  and greater migratory capacity than the CD146− population. Enhanced adherence and migratory capacities may result from high expression levels of alpha and beta subunits of integrin and matrix metalloproteinase (MMP)-2, respectively [in vitro [Anatomically, PCs closely surround ECs populating the vascular intima with specific adhesion and migration properties that allow them to regulate the blood vessel stability/integrity as well as the proliferation and motility of adjacent ECs . Up to 1ectively . On the in vitro . Moreovein vitro , 115.β signaling pathways has also been detected during wound healing and tumor vascular remodeling [β pathways may occur during pathologic conditions , resulting in PC apoptosis and augmented permeability of the vascular wall [in vivo [PC recruitment and migration occur frequently in response to pathophysiological events such as wound healing, inflammation, or angiogenesis. During vascular development, ECs release PDGF-BB to recruit PCs and stabilize the newly formed blood vessels , 117. Inmodeling , 118. Inlar wall , 119. Up[in vivo , 120. A [in vivo , 115. Thin vitro model of tissue damage, some of us previously mimicked the ability of HUCPCs to migrate towards the injury site in vivo and predicted their capacity to secrete cytokines and trophic factors [Using an  factors . Envisio factors . Dye-lab factors . KGF app factors , 122. Si factors , 124.in vitro expansion requirements [β1, and TNFα [β receptor 2, TNF receptor superfamily member 1A, and PDGF receptors α and β [The abundance of mesenchymogenic progenitors in the SVF of adipose tissue   providesirements , 127. ASirements , 129, liirements , 131, olirements , hypoxiairements , allergiirements , inflammirements , sciaticirements , cranialirements , and musirements , 139. Thand TNFα . These s α and β –142.in vitro migration of vascular ECs, fibroblasts, and keratinocytes [β, SDF-1, and VEGF [ASCs have been proposed to affect various neighboring cells within the subcutaneous tissue via paracrine signals during active remodeling processes such as wound healing –145. In inocytes . These dand VEGF . Similarly to BM-MSCs , 151, AS2). This activation of ERK signaling and associated integrins occurred without any detectable alteration on the cell phenotypes or differentiation potentials [α) and Akt phosphorylation [2 hypoxia, the paracrine function of PCs remained highly active when compared to 21% O2 normoxic culture, with increased expression of VEGF-A, PDGF-B, and TGFβ1 and decreased expression of angiopoietin-1, bFGF, EGF, HGF, and MCP-1, and similar levels of leukemia inhibitory factor (LIF), cyclooxygenase-2 , heme oxygenase-1 (HMOX-1), IL-6, HIF-1α, and MMP-2 [Hypoxia has been shown to promote proliferation and migration of both PCs and MSCs , 161. A tentials , 162. A tentials , which ctentials . Culturetentials . The acttentials , 166, anrylation . Additiond MMP-2 . Understnd MMP-2 . Perivascular MSC precursors have recently been demonstrated as efficient regenerative/supportive units for tissue repair and regeneration. In particular, human muscle PCs and saphenous vein-derived ACs exhibited superior angiogenic, paracrine, and cardioprotective capacities and augmented functional recovery in murine myocardial infarction and hind-limb ischemia models when compared to myoblasts or unfractionated MSCs , 169. AdWhen intramyocardially transplanted into a mouse model of acute myocardial infarction (AMI), purified human muscle PCs contributed to cardiac functional and anatomic recovery after infarction, presumably through multiple cardioprotective and regenerative mechanisms: reversal of ventricular remodeling, reduction of cardiac fibrosis, diminution of chronic inflammation, promotion of host angiogenesis, and small-scale myocardial regenerative events . The engSimilarly, Katare et al. reported that transplantation of human saphenous vein-derived ACs (hSV-ACs), a putative PC progenitor population, promoted functional improvement in a mouse model of MI, primarily through angiocrine activities and neovascularization via both donor and recipient cells as well as other cardioprotective mechanisms including improved myocardial blood flow, attenuated vascular permeability, and reduction of myocardial remodeling, cardiomyocyte apoptosis, and interstitial fibrosis . hSV-ACsμm to 2 mm) but also in far more distant regions (up to 2 cm), suggesting active migration of outgrown human myogenic precursors over long distances. Similarly, freshly isolated placental PCs possessed high migratory activity and actively contributed to host skeletal muscle regeneration [As mentioned previously, we have demonstrated that intramuscular injection of freshly sorted or cultured PCs derived from human adipose or skeletal muscle regenerated human myofibers efficiently in the mouse dystrophic or injured muscle . In anotneration . in vitro toward alveolar type II cells damaged by bleomycin, with an elevated secretion of KGF and VEGF [in vivo therapeutic potential of HUCPCs [As mentioned previously, PCs isolated from umbilical cords migrated efficiently and VEGF . Using af HUCPCs . To examα. Accordingly, both donor and host stem cell homing can be further enhanced by MSCs genetically modified to overexpress SDF-1 [To investigate their skeletal regenerative capacity, human PCs and ACs purified from lipoaspirate SVF have been seeded onto osteoinductive scaffolds and implanted into animal models of ectopic bone formation or critical-sized calvarial bone injury, respectively , 89, 176ss SDF-1 . in vitro data highlighting EC influence on mesenchymal differentiation potential of PCs points to a possible role of ECs as gatekeepers within the context of an adult stem cell niche.PCs are ubiquitously present in microvasculature where they extend primary cytoplasmic processes along the abluminal surface of the endothelial tube. They are enveloped in a basement membrane that is continuous with the EC basement membrane to which both cells contribute , 182. Thcorpus luteum indicate that PCs are also capable of guiding sprouting processes by migrating ahead of ECs and expressing VEGF [β, angiopoietins and Tie receptors, sphingosine-1-phosphate signaling, TGF-β signaling, Notch and Wnt [in vitro through Matrigel culture. Human muscle PCs alone can form network structures in Matrigel culture that were morphologically similar to networks formed by ECs but at an accelerated fashion  stimulate ECs, which in turn secrete proteases that degrade basement membrane and allow EC invasion. An endothelial column, guided by a migrating EC at the very tip, then moves toward a VEGF gradient . Studiesing VEGF –200. Eme and Wnt , 202. It Figures . Cocultu Figures . Blocki in situ but environmentally downregulated. Studies using unfractionated SVF have demonstrated poor and unreliable tissue formation [in vitro [γ is a prime inducer of adipogenesis that inhibits osteogenesis, highlighting the mutual exclusivity of these lineages [A growing number of studies demonstrate that tissue resident stem cells reside in vascular niches, including neural, hematopoietic, and MSCs , 204–206ormation  or lowerormation , lendingormation , 210. Inin vitro . The secin vitro  with stulineages . It is tIn this review, we described the identification and characterization of perivascular MSC precursors with regard to their adhesion, migration, engraftment/homing, and intercellular cross-talk in culture and in experimental animal models. Although PCs and ACs both exhibit multilineage mesenchymogenic capacities and are derived from adjacent perivascular structural layers, further investigations are required to clarify their developmental relationship as well as the involvement of an ontogenic intermediate. Through the understanding of their unique cellular kinetics and regenerative potential, we will be able to define the pathophysiological role and therapeutic value of the individual blood-vessel-derived MSC precursor population under a particular pathological circumstance. Ultimately, through the purification and/or recombination of these distinct subsets of MSC precursors, it is feasible to further enhance stem cell therapy by eliminating cells with none or limited regenerative potentials in a specific disorder, creating a customized therapeutic modality for the personalized medicine.


AbstractArctoseiinae of the family Ascidae, and describe a new genus, Maxiniagen. n., based on a new species, Maxinia arctomontanasp. n., whose adults display a combination of attri-butes uniquely different from other genera of the subfamily. The geographical range of Maxinia arctomontana is limited by arctic and mountain landscapes of Siberia. This description provides furtherdata on the arctic distribution and morphological diversity of the subfamily Arctoseiinae, which is unusually well represented in that region . Conceptual problems with the genus Iphidonopsis Gwiazdowicz, 2004 are reviewed, and a new combination, Iphidonopsis magnanalis  comb. n., is presented for Iphidozercon magnanalis Ma & Yin, 1999 from China. The genus Diseius Lindquist & Evans, 1965 is provisionally moved from the family Ascidae to the Digamasellidae. A new key to the genera of Arctoseiinae is presented.We redefine the subfamily  Arctoseiinae at present numbers ca 90 species , among which approximately one-third  inhabit Arctic landscapes  and ventrianal  shields respectively;lSt/wSt, lVA/wVAthe ratio of the lengths of the movable cheliceral digit and the dorsal shield, %;lCh/lDthe ratio of the lengths of the movable cheliceral digit and the corniculus.lCh/lCoIn the male description, the features common with the female are omitted.PageBreakHolotype and most of paratypes are deposited in Zoological Institute, Russian Academy of Sciences (ZIRAS), St.-Petersburg; part of paratype series  in Canadian National Collection of Insects and Arachnids, Ottawa (CNC).Arctoseiinae was first proposed by Gamasina. It was based on a reduced chaetotaxy of the genu and tibia of legs II to IV, and “the nature of the chaetotaxy of the opisthonotal region of the dorsal shield in the female.” A formal and more detailed description of the subfamily, including a key to its genera was presented by Ascidae:The S setae, S2 usually not added in change from larva to protonymph. Marginal (r-R) series of setae lacking r6 in podonotal region and often R6 posteriorly; marginal R setae on lateral soft cuticle on female, but variably on dorsal shield margin on male. Submarginal (UR) setae absent. Female sternal shield with third pair of lyrifissures on its posterior margin; fourth pair of sternal setae on soft cuticle. Sternal setae st5 usually on soft cuticle flanking female epigynal shield. Male sternitigenital shield free from, but sometimes abutting ventrianal shield, and often not fully integrated with endopodal strips alongside coxae III–IV. Maximum number of setae on genua I–II–III–IV, respectively, 12-10-8-7; on tibiae, 12-9-7-7; in change from protonymph to deutonymph, seta pd-3 not added to genu and tibia I, pl-2 not added to genu and tibia II, and al-2 not added to genua and tibiae III–IV.Dorsal shield of adults entire, with or without lateral incisions; opisthonotal region usually with maximum of four pairs of lateral Arctoseiinae include the following:Plesiomorphic attributes of the ad-3 added to genua I–II and tibia I, ad-2 added to tibia II, and al-2 added to tibia II.Gnathotectum basically bi- or tri-ramous, each process simple or denticulate; cheli- ceral movable digit bidentate in female, unidentate in male, without pointed process on mid-ventral surface, and with fringed arthrodial envelope basally; cheliceral fixed digit with small, setiform pilus dentilis, with usually few teeth restricted to apical half of masticatory surface, and without hyaline serrate rim near base on paraxial surface. Tarsus I usually with a conspicuously lanceolate-tipped seta dorsodistally. In change from protonymph to deutonymph, seta urn:lsid:zoobank.org:act:271F12C1-9B82-44F6-BD00-B29F44B05C39http://species-id.net/wiki/MaxiniaMaxinia arctomontana new species. Monotypic. Genus based on adult female and male material representing one newly described species.Maxinia are immediately distinguished from those of other arctoseiinae genera by having a broad, robust dorsal shield; by the female having an PageBreakapomorphically expansive ventrianal shield that encompasses the metapodal plates anterolaterally and includes setae JV1 anteriorly; and by the male having an expansive sternitigenital shield that is fully consolidated with the endopodal strips beside coxae III–IV and abuts an expansive ventrianal shield. In addition, apomorphically, the subcapitulum has a narrow deutosternal groove, and the opisthogaster lacks a setal pair ZV1. None of the setae on tarsi II to IV are conspicuously differentiated by thinness, elongation, or curved shape. As in Arctoseius Thor, 1930 and Iphidozercon Berlese, 1903, genu III carries only seven setae (lacking pv-1) on adults; however, adults of those genera have less robust, parallel-sided dorsal shields, retain opisthogastric setae ZV1, and their females have free metapodal plates and an anal shield. As in Zerconopsis Hull, 1918, adults have robustly sclerotized dorsal and ventral shields. However, adults of Zerconopsis have two to several pairs of dorsal shield setae (at least s4 and Z5) uniquely paddle-like in form, retain opisthogastric setae ZV1, eight setae (including pv-1) on genu III, and the female ventrianal shield is never expansive enough to include the metapodal plates and setae JV1; also, their tarsi II to IV have dorsal proximal setae ad-2, pd-2 conspicuously elongated and curved.Adults of Idiosomal dorsum. Dorsal shield relatively broad, entire, without lateral incisions, well sclerotized; surrounding soft integument smoothly striate; all dorsal shield setae simple, undifferentiated in form; opisthonotal region lacking setae S2 , and without markedly elongated setae apically, but with seven finger-shaped sensillae of different lengths. Tarsi II–IV with apical setal processes inconspicuous, shorter than ambulacrum (to base of claws), and with acutely triangular apical process ventrally. Complement of setae on segments of legs I–II–III–IV typical for Arctoseiinae as presented by pd-3 not added to genu and tibia I, pv-1 but not pl-2 added to genu II, and al-2 not added to genua and tibiae III–IV (this combination of hypotrichy is apomorphic for subfamily). Leg setae collectively smooth; none of setae on tarsi II–IV conspicuously differentiated by thinness, elongation, or curved shape. Legs of male without dimorphically modified setae.swelling , with paThe name of the genus is tribute to the first author’s spouse, Maxine Lindquist. Together with him for 55 years, she supported his acarological endeavors, accompanied him in field work throughout North America, and hosted many visiting colleagues at home. The name is feminine in gender.Arctoseiinae, adults of Maxinia resemble those of the genus Neojordensia Evans, 1957, Ascinae, e.g., the relatively broad, well sclerotized dorsal shield, expansive venPageBreaktrianal shield encompassing the metapodal plates and insertions of setae JV1, absence of setae ZV1, expansive male sternitigenital shield, and the narrow rows of deutosternal denticles. However, adults of Neojordensia present dorsal shield setation and leg chaetotactic attributes typical of the subfamily Ascinae, and are characterized by different distinctive attributes, including absence of paravertical setae z1, absences of setae av-2 on genu I and ad-2 on tibia II, gnathotectum with convex, smooth anterior margin, female with most or all of r-R marginal series of setae on margins of dorsal shield, female sternal region with setae st1 on separate presternal plates, and female epigynal shield apomorphically widened to include the paragenital poroids as well as setae st5 .Holotype, female: EAST SIBERIA, YAKUTIYA, Suntar-Khayata Range, upper reaches of Kyubyume R., Rhododendron aureum, 29.VII 2002, O.L. Makarova (CNC); MAGADAN REGION, 8 females, 2 males, 3 protonymphs, 1 larva, Olskoye Plateau, upper reaches of Ola R., 1149 m a.s.l., snowbed, litter under Rhododendron redowskianum, 10.VIII 2011, O.L. Makarova (ZIRAS).Paratypes: 16 females, 3 males with same data as for holotype (ZIRAS); 6 females, 1 male, the same district, 1600 m a.s.l., snowbed, litter under Pinus pumila, 26.VII 1992, M.B. Potapov; 1 female, Khalerchinskaya Tundra, 69°24'N, 158°37'E, VIII 1991, O.V. Starikova; 4 females, mouth of Kolyma R., 69°32'N, 160°44'E, Pokhodskaya Yedoma, dwarf shrub  thicket, 18–19.VII 1994, A.B. Babenko; 2 females, delta of Yana R., Shirokostan Peninsula, vicinity of Ledyanoye Lake, Dryas community on south slope of valley, 4–6.VIII 1994, A.B. Babenko; 2 females, the same district, date and collector, forb-grassy meadow; MAGADAN REGION, 6 females, 1 male, upper reaches of Kolyma R., Aborigen Mt., Betula exilis thicket within icing in valley, litter, 23.VIII 2006, A.A. Alfimov; TAYMYR PENINSULA, 1 female, vicinity of Pyasino Lake, Nyapan’ Upland, Dryas-community on hill, 10.VII 1999, O.L. Makarova; 1 female, vicinity of Ragozinka R., 72°57'N, 80°56'E, “lemming hay” on slope, 10.VII 1983, A.B. Babenko; 1 female, mouth of Tareya R., hummocky tundra, 28.VII 2010, O.L. Makarova; 3 females, 3 protonymphs, 72°50'N, 101°15'E, bank of Zakharova Rassokha R., mossy tundra, 3.VII 2011, A.V. Barkalov; YAMAL PENINSULA, 2 females, Seyakha Lake, tundra, litter, 9.VII 1986, V.I. Bulavintsev; 1 female, Yakhadyyakha R., 72°53'N, 70°56'E, lemming hill with Poa arctica, 21.VIII 1994, A.B. Babenko; VAIGATCH ISLAND, 1 female, VII 1984, V.I. Bulavintsev; SOUTH SIBERIA, 5PageBreak females, Rudnyi Altai Mts., Ivanovskyi Belok Mt., 3.VII 1983, I.P. Vtorov; 1 female, West Sayan Mts., Alashskoye Upland, 2200 m a.s.l., Dryas-community, 20.VII 2001, S.K. Stebaeva; 2 females, the same region, vicinity of Sut-Khol Lake, Larix-Vaccinium vitis-idaea-mosses-community, 27.VII 2001, S.K. Stebaeva.Other material: YAKUTIYA, 3 females, vicinity of Ust-Nera settlement, 1400 m a.s.l., litter under Middle-sized dark-yellow or brownish mites with rather broad, somewhat pyri-form idiosoma . IdiosomFemale.Idiosomal dorsum. Dorsal shield 448–520 × 296–356, moderately broad, lD/wD ca 1.34–1.62, its maximal width at level of setae S1 . Opisthonotal region with 14 pairs of setae . Among podonotal setae z1, s1, s2 distinguished by much shorter length (7–11), length of j1,2 18–26, other setae 22–36. On opisthonotal region setae J1-4 (20–28) shorter than others (30–44), and J5 clearly shortest (11–15). Dorsal shield with 4 pairs of cuPageBreakPageBreaktaneous glands gdj4, gdz6, gdZ3 and gdZ4. All marginal setae on soft cuticle (r (their length 16–24), 6 setae in series R (22–38); marginal poroid Rp in usual position between setae R3 and R4.setae S1 . Podonot cuticle ; 4 setaeIdiosomal venter. Base of tritosternum narrow (18–22 × 11–14); laciniae with sparse large barbs, free for nearly entire length, fused basal area broadest, with short spicules  and lyrifissures iv1-3; rarely, vestiges of gland gv1 present, off posterior margin of sternal shield. Setae st4 (22–28) on soft cuticle. Free endopodal fragments between coxae II–III well sclerotized with angular inner margin. Endopodal strips between coxae III and IV free, well developed  and paragenital poroids iv5 remain on soft cuticle. Two openings of gland gv2 medially to coxa IV and on posterior margin of exocoxal strip behind coxa IV usually poorly visible  opisthogastric setae, plus the circumanal setae; setae similar in moderate length (22–30); paranal setae (20–26) inserted at level of anterior margin of anus, and nearly as long as postanal seta (24–32); opening of gland gv3 inconspicuous. Setae ZV1 absent, and ZV4, JV5 on soft cuticle; JV5 rather long (32–40). Exopodal strip usually fragmented alongside coxae II–III, mostly contiguous with peritrematal shield but ending freely from it, with extension that abuts or merged with endopodal extensions between coxae I–II ; groove width 8–9. Hypostomatic pair hp3 (39–44) longer than other subcapitular setae (22–30); all setae simple, attenuate. Corniculi of moderate length and width, 30–36 × 11–13. Internal malae slightly longer than corniculi, gradually tapering to tip, with lateral margins fimbriated basally. Chelicera not large (lCh/lD 9.5–12.1 %), one and a half longer than corniculus (lCh/lCo 1.48–1.75). Fixed digit of chela ending in apical hook, masticatory surface with one subapical tooth and pilus dentilis in antaxial position and four denticles in paraxial position. Movable digit (44–55) slightly shorter than fixed one, bidentate. Palp length 156–175; internal seta of trochanter (28–32) longer than external seta (19–26); palp with typically specialized setae on femur  and genu  thick with oblique tip; palp tarsus without macroseta ; leg I shorter than dorsal shield. Length of tarsi I 92–106, II 80–90, III 84–89, IV 102–124. Leg chaetome as described for genus. Setae of legs simple, generally of moderate length; tarsi II–IV each with dorso-proximal setae ad-2, pd-2 not elongated or curved, and with al-1, pl-1 not thinner or more elongated than adjacent setae. Ambulacrum I on pedicellate base, claws I (8–10) smaller than claws II–IV (12–15). Tarsus I distally with 7 rod-like solenidia, 5 of them inserted apically. Ambulacra of legs II–IV (length 24–30) with moderately long paradactyli (9–11) extending clearly beyond apices of claws. Tarsi II–IV with apical setae ad-1 and pd-1 shorter (8–12) than claws (av-1 and pv-1 slightly weaker and shorter (18–23) than lateral setae al-1 and pl-1 (20–27).an claws . Four suMale.Idiosomal dorsum. Dorsal shield 396–424 × 236–276, narrower than in female (lD/wD ca 1.50–1.75), fully reticulated, with all r-R marginal setae (10 pairs) on shield, such that podonotal region with 22 pairs of setae, including r2-5, and opisthonotal region with 20 pairs of setae, including R1-6 . Relative lengths of setae as on female.Idiosomal venter. Tritosternum base (12–16 × 10–12) shorter than in female  longer than setae st4,5 (20–24), and with lyrifissures iv1,2; lyrifissures iv3 and vestiges of glands gv1 not discernible. Ventrianal shield abutting but free from sternitigenital and peritrematal shields, fully reticulated, expansive, slightly wider (194–224) than long (176–202), yet narrower than in female, lVA/wVA 0.82–0.98, consolidated with metapodal platelet sigillae laterally; anterior margin straight or slightly concave, posterior margin and cribrum formed as in female. Ventrianal shield with setation and form, placement, and lengths of setae as in female, except JV1 more removed from anterior margin and poroids iv5 present on shield. Setae ZV1 absent, ZV4 and JV5 on soft cuticle.n female . Prestern female ; length PageBreakPageBreakGnathosoma. Apices of gnathotectum similar to female but sometimes variously shaped in different ways (lCh/lD 9.1–10.7 %) longer than corniculus . Fixed digit with dentition similar to female .of setae  and no lIdiosomal venter. Tritosternum as in adults  metapodal platelets, 4 pairs of setae and 5 pairs of poroids. Peritremes extending to posterior margins of coxae III.n adults . SternalGnathosoma. Gnathosomal structures in general as in adults, tectum as in Legs. Leg chaetotaxy typical for protonymphs of Ascidae , transversally oval, wider than long, with circumanal setae large, paranals (54) longer than postanal (36), and with gland opening gv3 nearly posterior to paranal setae; anal valves with tiny euanal setae; cribrum undeveloped.n adults . IntercoGnathosoma. Hypostome, gnathotectum  communities.Recorded from dry and humid tundra sites, meadows including zoogenic ones (e.g. bird of prey outlooks) and shrub .The species is rather stable morphologically. The variability concerns mainly the form of gnathotectum , 26–28, IphidonopsisGenus Iphidonopsis Gwiazdowicz, 2004 is treated in the original sense  comb. n. Subsequently, Iphidonopsis to accommodate other species  and thereby distinguished his genus from Arctoseius Thor, 1930, primarily on the dorsal shield of adults having lateral incisions in Arctoseius, and these lacking in Iphidonopsis. However, not only is the lack of such incisions a plesiomorphic character state, as presented in some other genera of Arctoseiinae, but the presence of lateral incisions varies greatly among species, and is even subject to geographical variability and sexual dimorphism among some species, in Arctoseius. So, Arctoseius pannonicus Willmann, 1949 from Corsica and Spain, although shallow ones are present in other parts of Europe. Observations by Arctoseius venustulus  by Seiulus minutus has been previously designated as type-species of the genus Arctoseiopsis by Iphidonopsis would render this genus a junior synonym of Arctoseiopsis. With added phylogenetic perspective, we continue to follow the previous concept of Arctoseius embraces Arctoseiopsis and includes the species Arctoseiopsis minutus, Arctoseiopsis pulvisculus, and Arctoseiopsis venustulus. Adult females of Iphidonopsis are somewhat similar to those of Arctoseius and Iphidozercon in retaining what may be considered to be an apomorphically reduced, deutonymphal form of anal shield. However, as indicated in the following key, the genus Iphidonopsis shares with Zerconopsis and Xenoseius Lindquist & Evans, 1965 the apomorphic attribute of having dorsoproximal setae ad-2 and pd-2 of tarsi II to IV elongated, curved, somewhat whip-like, while retaining the plesiomorphic attribute of genu III having eight setae, including pv-1. In contrast, Arctoseius, Iphidozercon and Maxinia are plesiomorphic in having setae ad-2 and pd-2 of tarsi II to IV unmodified, but sharing the apomorphic attribute of genu III having seven setae, in absence of pv-1.In the following key, al sense  to incluDiseius Lindquist & Evans, 1965 from the Arctoseiinae and from the following key to its genera. Based exclusively on gnathosomatic and idiosomatic attributes, the type-species of this genus, Iphidozercon ulmi Hirschmann, 1962, has been assigned to various genera as conceived by different authors’ concepts of the family Ascidae, i.e., to the genus Iphidozercon  sensu Leioseius (Leioseius) (including Gamasellodes but excluding Arctoseius and Iphidozercon) by Leioseius (including Gamasellodes but excluding Arctoseius and Iphidozercon) by PageBreak species was assigned to a separate, new genus and placed in the tribe Ascini of the subfamily Ascinae (separate from the subfamily Arctoseiinae), by Diseius ulmi resembles that of genera of Arctoseiinae, but Diseius was excluded from the Arctoseiinae based primarily on the adult female having a fully divided dorsal shield, and having sternal setae st5 inserted on the edges of the epigynal shield, rather than on soft cuticle. However, new observations of female Diseius by one of us (EEL) revealed the unanticipated absence of setae al-3 and pl-3 on basitarsi III–IV, and also of pd-3 on basitarsus IV. The chaetotaxy of tarsi II to IV is extremely constant throughout the families of free-living Gamasina  Chant, sensu Diseius ulmi with scolytid beetles is in common with that of a variety of digamasellids. As adult females of Diseius ulmi are highly neotenous in both leg and dorsal idiosomal setation, the similarity of leg chaetotaxy on genua and tibiae II–IV with Arctoseiinae is thought to be convergent, and our new observations persuade us to place Diseius tentatively in the Digamasellidae instead of the Ascidae, until such time as additional attributes become available from description of the female spermathecal system and of attributes of the adult male.Provisionally, we exclude the monotypic genus Gamasina . Losses acaridae . Also, i


AB103 is a novel CD28 antagonist peptide currently in clinical development that modulates CD28 signaling in T cells, without affecting the normal humoral immune response. In experimental models of Gram-positive, Gram-negative and polymicrobial sepsis, AB103 demonstrated significant activity, increasing overall survival.The AB103 activity and mode of action (MOA) were evaluated in a murine model of cecal ligation and puncture (CLP). AB103 (5 mg/kg) was administered to mice  at various times points following CLP (2 to 24 hours), together with moxifloxacin.90, given at 12 hours) rescued only 25% (P < 0.05) of the animals. In a separate set of experiments investigating the MOA, AB103 administration  was associated with: a reduction in Th-1 cytokine levels in peritoneum  and plasma (IL-3 and IL-6); a reduction in splenocyte proliferation, stimulated ex vivo with anti-CD3 and anti-CD28 antibodies; a reduction in neutrophil recruitment to the spleen, liver and kidney, as determined by MPO activity; and a reduced bacterial load in peritoneum, blood and tissues .A single dose of AB103, given at 12 or 24 hours post CLP, rescued 100% and 62.2% of the animals (respectively) from sepsis-induced mortality, whereas moxifloxacin alone (LDThese data demonstrate that attenuation of CD28 signaling is a viable therapeutic approach to the treatment of sepsis. Due to its robust activity and good safety profile in humans already established in a phase 1 study, AB103 should be clinically evaluated in sepsis patients.


Male infertility refers to the inability of a male to achieve a pregnancy in a fertile female . GeneticA total of 500 infertile male patients attending private infertility clinics of Guwahati, Assam, were selected for the study. Blood and semen samples were collected from the patients. Genomic DNA was isolated from both type of samples and PCR amplification was carried out using specific primer sets. The genes and sequence-tagged site (STS) markers included in the study are: DBY, USP9Y, PRY-2, RBMY, BPY-2, XKRY, CDY-1, CSPG4LY, DAZ, sY84, sY254, sY127, SY145, sY152 and sY153. All genes and STS were amplified efficiently in samples from 100 fertile men tested, but failed to be amplified in samples from fertile women. In order to study the role of mtDNA in sperm motility, semen DNA from 50 patients including 10 Asthenospermic, 20 Asthenoteratozoospermic and 20 oligoasthenoteratozoospermic patients and 20 fertile men were studied using specific primers for four mitochondrial genes, namely ND2, ND4, ATPase6 and ATPase8 followed by DNA sequencing.Among the 500 patients included in this study, 130 26%) were azoospermic, 185 (37 %) were oligozoospermic and 185 (37 %) were asthenozoospermic. Analysis of PCR amplified products showed that the frequency of Yq microdeletion in semen samples was 20.8% (104/500) but 18.6% (93/500) in blood samples, both of which lies in the range ( 0%-55%) as stated by Kihaile et.al [6% were aThe study reveals that the frequency of Yq microdeletion is higher in semen samples than blood samples, possibly because most of the genes studied are testis-specific in nature. This is in accordance with the previous studies ,4. In bo


Objective. To evaluate the efficacy and safety of docetaxel plus oxaliplatin and capecitabine (DOX) in the first line treatment of advanced gastric adenocarcinoma. Methods. A total of 37 patients were enrolled into this study, and they received DOX regimen ; treatment was repeated every 3 weeks. Results. All 37 patients were assessable for evaluation. The numbers of patients with complete response (CR), partial responses (PR), stable disease (SD), and progressive disease (PD) were 1, 10, 23, and 3, respectively. The objective response rate (ORR) was 29.7%, with the disease control rate (DCR) of 91.9%. Median progression-free survival (mPFS) and overall survival (mOS) were 197 days and 364 days, respectively. The most common grade 3/4 toxicities were hematological toxicities. The most common grade 3/4 nonhematological toxicities were fatigue, nausea, vomiting, anorexia, diarrhea, and hand-foot syndrome. Conclusion. The DOX regimen demonstrated a promising efficacy as the first line regimen in treating advanced gastric cancer patients with good performance status, the toxicities were tolerated and controllable. Large-scale clinical observation is necessary to get further evidence. Gastric cancer is the second most common cause of cancer-related deaths worldwide. The outcome of advanced gastric cancer patients is poor, and the median survival time of untreated patients is less than 6 months . A numbeDuring the past decade, several new agents have emerged, including taxanes , topoisomerase-I inhibitor irinotecan, and third-generation platinum derivative oxaliplatin, which provided more effective and better tolerated regimens in treating this life-threatening disease. Although taxanes share a number of pharmacologic characteristics and similar mechanisms of action (tubulin stabilization and cell cycle arrest), they are distinctly different. Docetaxel demonstrated greater affinity for tubulin, longer plasma half-life, and intracellular retention time than paclitaxel. In addition, the taxanes exhibit different profiles of drug resistance –10. DoceOral fluoropyrimidine agents, such as capecitabine and S-1, may provide an alternative to standard infusional 5-FU. Cunningham et al.  addresseBased upon the results of V-325 and REAL-2 studies, we design this single arm phase II study to evaluate the safety and efficacy of DOX  regimen in treating unresectable locally advanced or metastatic gastric cancer.The main eligibility criteria included (1) age 18–75 yrs, (2) histologically proven gastric adenocarcinoma, (3) measurable and/or evaluable targeted lesion according to RECIST criteria (version 1.0), (4) good performance status with the Eastern Cooperative Oncology Group (ECOG) score of 0–2, (5) no prior chemotherapy, and (6) adequate hepatic, renal, and hematological function.Exclusion criteria included (1) concomitant cancers , (2) neuropathy, brain or leptomeningeal involvement, (3) uncontrolled significant comorbid conditions, (4) patient could not comprehend the purpose of the study and could not comply with the protocol, and (5) women who were pregnant or breastfeeding. The Ethics Committee at Ruijin Hospital, affiliated to Shanghai Jiaotong University School of Medicine, approved the study and written informed consent was obtained.2 was give intravenous infusion for 2 hours on day 1; oxaliplatin 130 mg/m2 was also intravenous infusion for 2 hours on day 1, and capecitabine 1000 mg/m2 were orally intaked twice daily on d1–14. The regimen was repeated every 3 weeks. The dosage of docetaxel was referred from TAX 325 trial, the dosages of oxaliplatin and capecitabine were referred from REAL-2 and ML17032 trials. Only those who completed at least 2 cycles were assessed efficacy. Patients who finished all 8 cycles of treatment without disease progression were turned into follow-up period. Docetaxel 75 mg/mBefore entering the study, all patients received physical examination, full blood count, and serum chemistry analyses. Chest X-ray, ECG, upper gastrointestinal endoscopies, abdominal computer tomographic (CT) scans, and other appropriate procedures were also performed as needed. During treatment, full blood count and serum chemistry analyses were conducted before each cycle, and X-ray and CT scans were conducted every two cycles. After the treatment completed, patients were receiving these evaluations every 2 months. PFS  was defined from the day of enrollment to first evidence of disease progression or death occurring within 12 weeks of the last assessable tumor assessment. OS  was defined from the date of enrollment to death from any cause. DCR (disease control rate) was defined as follows: complete response (CR) + partial response (PR) + stable disease (SD)/all enrolled patients. Responses were evaluated according to RECIST criteria (version 1.0). Toxicities were graded according to the National Cancer Institute of Canada Common Toxicity Criteria (version 1.0). The primary endpoint of this study was response rates (RR). The secondary endpoints were PFS, OS, DCR, and the toxicity profile. Patients were considered assessable for response if they received at least two cycles of chemotherapy. Safety analyses included all treated patients and involved the analysis of treatment-emergent adverse events. The statistical analysis was carried out using SPSS software . Descriptive statistics were used for safety evaluation. ORR and DCR and their two-sided 95% confidence interval (CI) were calculated. PFS and OS were estimated using Kaplan-Meier method and their medians along with two-sided 95% CIs were calculated.A total of 37 late stage gastric cancer patients were enrolled from April 2007 to April 2013. The demographics and baseline characteristics of these patients are presented in The median number of treatment cycles was six, ranging from one to eight with the total cycles of 216. According to the RECIST criteria (version 1.0), CR was observed in one patient, while PR was observed in 10 patients, SD was in 23 patients, and PD in 3 patients. The ORR was 29.7% , while DCR was 91.9% . Median progression-free survival (mPFS) and overall survival (mOS) time were 197 days  and 364 days , respectively .The most common grade 3/4 toxicities were hematological toxicities. The percentages of patients with grade 3/4 leucopenia, neutropenia, and febrile neutropenia were 37.8%, 37.8%, and 10.8%, respectively. The most common grade 3/4 nonhematological toxicities were fatigue, nausea, vomiting, anorexia, diarrhea, and hand-foot syndrome, with the percentages of 27.0%, 5.4%, 5.4%, 8.1%, 2.7%, and 8.1%, accordingly. No severe liver or renal dysfunction or chemotherapy-related death was observed. Only one patient developed grade 2 peripheral neuropathy .Patients who developed grade 4 hematological toxicities or grade 3 nonhematological toxicities received a 20% dosage reduction. Among all enrolled patients, 13 had one time dose reduction , and other 3 patients got one dose reduction of capecitabine owing to grade 3 hand-foot syndrome. The results of this trial showed that triplet regimen with novel agents demonstrated obvious survival benefit as the first-line treatment of late stage gastric adenocarcinoma. The progression-free survival time was 197 days, and the overall survival time was 364 days, which was significantly longer than those in REAL-2 and V-325 trials . The rea2 (day 1) plus cisplatin 75 mg/m2 (day 1), and fluorouracil 750 mg/m2/d continuous infusion (days 1 to 5) [2); cisplatin (60 mg/m2) was given intravenously with hydration in the ECF and ECX groups, and oxaliplatin (130 mg/m2) was administered intravenously during a 2-hour period in the EOF and EOX groups [2 day 1) plus capecitabine  or 5-FU  [The results of V325 and REAL-2 trials, which indicated high efficacy and well tolerability of triplet regimen as the first line treatment of advanced gastric cancer, were referred to in deciding the dose of each chemotherapy agent. In V-325 trial, patients in DCF group were assigned to receive once every 3 weeks docetaxel 75 mg/m 1 to 5) . In REALX groups . In addiliplatin 0 mg/m2 wSimilar as those in the other trials, the majority of dose-related grade 3-4 toxicities of docetaxel observed in this trial were hematological toxicities. Of all these 37 patients, the percentage of patients who experienced grade 3/4 leucopenia, neutropenia, and febrile neutropenia were 37.8%, 37.8%, and 10.8%, respectively. With G-CSF support, the hematological toxicities were well controlled and tolerable. No dose cumulative toxicities and chemotherapy-related deaths were observed in this study.Oxaliplatin is the third-generation platinum-based drug, and has been widely used in the treatment of colorectal carcinoma. Compared with cisplatin, the advantages of oxaliplatin were less renal toxicity and sensorineural hearing loss, as well as no need for hydration. The results of REAL-2 trial demonstrated that oxaliplatin was more noninferior than cisplatin as the first line treatment of metastatic gastric cancer, while combining with fluoropyrimidine and anthracyclin. The results of our study also confirmed the efficacy of this agent as the first line treatment of late-stage gastric cancer, with low incidence of neurotoxicity. Either bolus injection or continuous infusion through a central venous access device (CVAD) was used as intravenous administrative routes for fluorouracil. Bolus injection might lead to more toxicities, especially heart toxicity. Yet, continuous infusion was associated with an increased risk of infection and thrombosis as well as the implantation of inconvenient delivery system. Capecitabine is an oral thymidine phosphorylase (TP)-activated fluoropyrimidine. It is an orally administered systemic prodrug of 5′-deoxy-5-fluorouridine (5′-DFUR), which is converted to 5-fluorouracil. It was rationally designed to generate 5-FU preferentially at the tumor site, and it was activated in tumor tissue by a three-step enzymatic conversion culminating with TP. This high tumor selectivity is achieved through exploitation of the significantly higher activity of TP in tumor tissue compared with healthy tissue. Capecitabine mimics continuous infusion 5-FU and potentially offers a more convenient alternative to intravenous infusion. Some cytotoxic agents, such as taxanes, oxaliplatin, and cyclophosphamide, are known to up-regulate TP activity in tumor cells, offering potential synergistic action. Several powerful clinical trials, such as REAL-2, ML17032, and ToGA, have elucidated the efficacy of capecitabine in chemonaïve gastric cancer patients .P = 0.04). Progression-free survival was also significantly longer in doublet group  [The SPIRITS trial evaluated S-1 plus cisplatin versus S-1 alone in the first-line treatment of advanced gastric cancer. A total of 148 patients were assigned to S-1 plus cisplatin group and 150 patients were assigned to S-1 monotherapy group. Median overall survival was significantly longer in S-1 plus cisplatin group than that in S-1 group  . All theThe results of AVAGAST  and REALThe results of our study demonstrated a promising good response, survival benefit, and well tolerance of triplet regimen, including docetaxel, oxaliplatin, and oral fluoropyrimidine capecitabine, in the first line treatment of late stage gastric cancer patients with good performance status. The shortage of this study is single arm, limited patient number, and lack of biomarker exploration. The precise efficacy of this regimen, and the subgroup of gastric cancer patients who benefit more from this regimen need to be investigated by a large-scale study.


Background. Progressive diseases including cancer, metabolic, and cardiovascular disorders are marked by platelet activation and chronic inflammation. Studies suggest that dietary flavonoids such as quercetin possess antioxidant, anti-inflammatory, and antiplatelet properties, which could prevent various chronic diseases including atherosclerosis and thrombosis. However, the mechanism and the signaling pathway that links quercetin's antiplatelet activity with its anti-inflammatory property is limited and thus further exploration is required. The aim of this paper was to examine the link between antiplatelet and anti-inflammatory roles of quercetin in agonist-induced platelet activation. Methods. Quercetin effects on agonist-activated platelet-aggregation, granule-secretion, [Ca2+]i, and glycoprotein-IIb/IIIa activation were examined. Its effects on PI3K/Akt, VASP, and MAPK phosphorylations were also studied on collaged-activated platelets. Results. Quercetin dose dependently suppressed collagen, thrombin, or ADP-induced platelet aggregation. It significantly inhibited collagen-induced ATP release, P-selectin expression, [Ca2+]i mobilization, integrin-αIIbβ3 activation, and augmented cAMP and VASP levels. Moreover, quercetin attenuated PI3K, Akt, ERK2, JNK1, and p38 MAPK activations, which were supported by platelet-aggregation inhibition with the respective kinase inhibitors. Conclusion. Quercetin-mediated antiplatelet activity involves PI3K/Akt inactivation, cAMP elevation, and VASP stimulation that, in turn, suppresses MAPK phosphorylations. This result suggests quercetin may have a potential to treat cardiovascular diseases involving aberrant platelet activation and inflammation. Our results also showed that quercetin inhibited collagen-induced PI3K and Akt phosphorylations downstream of collagen receptor. This effect was supported by suppressive effect of PI3K-inhibitors (wortmannin or LY294002) to platelet activation via inhibition of [Ca2+]i mobilization and αIIbβ3 activation. An enhanced integrin αIIbβ3 receptor binding to fibrinogen is particularly considered to be the final common pathway for platelet aggregation [γ activations and [Ca2+]i mobilization in platelets i mobilization, which is an essential factor for platelet aggregation [αIIbβ3 activation and platelet aggregation [2+] mobilization. Recent reports indicated that Akt activation decreased cAMP levels through increment of PDE activity i mobilization, αIIbβ3, PI3K/Akt and MAP kinases activations, and an enhanced cAMP-dependent VASP phosphorylation in platelet aggregation reflects the potential use of the compound as a candidate dual antiplatelet, anti-inflammatory agent. Extensive studies, using various experimental setups, have indicated the role of ERK2, JNK1, and p38 in platelet granule secretion and aggregation , 46. UsiIIbβ3 . Thus, t2+]i mobilization, integrin activation, ATP release, and P-selectin expression during platelet aggregation, in combination with its anti-inflammatory effects, suggests that quercetin could be considered as an antiatherothrombosis and anti-inflammatory agent. Given the observed effects of quercetin on platelet signaling and functional responses, it will be important to identify the specific active metabolite that is responsible for the observed effects that link PI3K/Akt and MAPK inhibition and cAMP-dependent VASP activation. This will enable a more detailed mode of action at the molecular level to be determined, and the therapeutic potential of quercetin supplementation to be assessed.In conclusion, this study suggests that the inhibitory property of quercetin in platelet aggregation may involve (i) inhibition of PI3K/Akt signaling, (ii) induction of cAMP-mediated VASP phosphorylation, and (iii) inhibition of the ERK2, p38, and JNK1 MAP kinase phosphorylations in activated platelets. Thus, the ability of quercetin to inhibit [Ca


In vitro experiments showed that Mfn2 overexpression suppressed gastric cancer cell proliferation and colony formation, weakened the invasion and migratory ability of cancer cells by downregulating MMP-2 and MMP-9, halted the cell cycle and induced apoptosis. Western blotting indicated the likely involvement of P21 and PI3K/Akt signaling. Therefore, Mfn2 is a potential anti-tumor gene and a potential therapeutic target for treating gastric cancer. The progress of gastric cancer may be delayed by controlling Mfn2 expression.Mitofusin-2 (Mfn2) is a mitochondrial outer membrane protein involved in mitochondrial fusion. Its mutation can cause Charcot-Marie-Tooth disease. Recent studies of Mfn2 in cancer research have not included gastric cancer. We confirmed that Mfn2 expression was lower in tumor tissue than in normal gastric mucosal tissue and that it was negatively correlated with tumor size, indicating an anti-tumor role for Mfn2.  Gastric cancer is one of the leading causes of cancer-related mortality worldwide. It has been estimated that a total of 989,600 new gastric cancer cases and 738,000 deaths occurred in 2008 . Countri2+ uptake [Mitofusin-2 (Mfn2), a potential target molecule, has been extensively studied in Charcot-Marie-Tooth (CMT) disease . Point mutations in Mfn2 have been found in several types of CMT disease and are likely to be the fourth most common cause of CMT . Mfn2 is+ uptake . Previou+ uptake . Recentl+ uptake –9.in vitro using gastric cancer cell lines.However, the mechanism underlying the anti-tumor effect of Mfn2 is unclear, and Mfn2 has never been studied in gastric cancer. In the current study, we examined the expression of Mfn2 in tissues from 90 gastric cancer cases and evaluated its prognostic significance in gastric cancer. Furthermore, we explored the cellular function and mechanism of Mfn2 p < 0.001). By Western blot analysis, the expression of Mfn2 in normal mucosal tissue was higher than that in tumor tissue for 15 patients, whereas the other five patients showed no obvious difference.We analyzed the expression of Mfn2 in normal mucosal tissue and tumor tissue in gastric cancer patients using three different methods and obtained consistent results. We analyzed 30 pairs of samples by QRT-PCR , 20 pairiii, MGC803, SGC7901, MKN28 and MKN45) by Western blotting at first. We selected AGS and SGC7901 as our main object of study, due to their low expression of Mfn2. Because Mfn2 is a mitochondrial outer membrane protein, we observed mitochondrial gathering around the nucleus using fluorescence microscopy after gastric cancer cells were transfected with Mfn2-YFP . The proliferation curve indicated that cellular proliferation was suppressed by Mfn2. To confirm this, we performed a colony-forming assay (p < 0.05). There was no difference between the negative control and blank (p > 0.05).In cell proliferation assay , the absng assay . After sp < 0.05, compared with both the negative control and blank). There was no significant difference between the negative control and blank. To explore the mechanism underlying this change, we analyzed proteins associated with the cell cycle, including cyclinA, cyclinB, cyclinD, cyclinE, CDK2, CDK4, CDK6, P53 and P21. We didn’t acquire significant change of cyclin A, B, D, E and CDK2, 4, 6 (data not shown). However, we found that P53 and its downstream protein, P21 [To further explore how Mfn2 weakens proliferation, we stained cells with PI and analyzed the cell cycle by flow cytometry . Mfn2 siein, P21 , were inein, P21 ,12. Thisp < 0.05, compared with both the negative control and blank; p > 0.05). Moreover, we observed a visible difference in Hoechst 33342/PI staining  compared with the negative control or blank, whereas there was no significant difference between the negative control and blank. We also observed the downregulation of MMP-2 and MMP-9 by Western blotting (A Transwell assay  was condblotting . Low-levp < 0.05), indicating that the lower the expression of Mfn2, the more the tumor growth was out of control. No significant difference was found between Mfn2 expression and gender, age, adjacent organ invasion, lymphovascular invasion, differentiation degree, T stage, lymph node metastasis, distant metastasis or TNM  stage  stage .p < 0.05), while the Mfn2 expression showed no significance on overall survival  , lymphovascular invasion , distant metastasis  and TNM stage III + IV  as independent factors associated with worst overall survival . However, Mfn2 expression still showed no significance on overall survival in multivariate analysis.For the 90 patients, the one-year, two-year and three-year overall survival were 82.2%, 77.8% and 67.8%, respectively. In univariate analysis, the variables of age, tumor size, adjacent organ invasion, lymphovascular invasion, differentiation degree, T stage, distant metastasis and TNM stage acquired significant prognostic values on overall survival (OS)  . In ordesurvival , while tGiven recent advancements in medicine, especially the improved early diagnosis rate, the morbidity and mortality of gastric cancer have declined in many advanced countries. However, these advances have not changed the treatment of gastric cancer by much. Surgery remains the most efficient therapy, but a high survival rate cannot be attained with advanced stage patients, and chemical therapy and radiotherapy lack specificity. Recently, with the development of molecule-targeted therapy, scientists have focused increasingly on gastric cancer treatment. Many relevant target molecules or pathways have been identified, including epidermal growth factor receptor, vascular endothelial growth factor receptor, fibroblast growth factor receptor, insulin-like growth factor receptor, the PI3k/Akt/mTor pathway and the c-Met pathway ; howeverCaenorhabditis elegans, fruit flies and mammals [Mfn2, which controls mitochondrial fusion, is a highly conserved GTPase . Similar mammals . Mfn2 po mammals ,19.Previous studies have demonstrated that the mutation of Mfn2 can cause CMT neuropathy type 2A ,22. AddiIn the current study, we demonstrated that Mfn2 suppressed the proliferation of SGC7901 gastric cancer cells using both a CCK-8 cell proliferation assay and colony-forming assay. Moreover, we confirmed that Mfn2 expression was higher in normal gastric mucosa than in tumor tissue, as shown by QRT-PCR, Western blotting and immunohistochemical analysis. The expression of Mfn2 in tumor tissue was negatively related to tumor size. Taken together, we have reason to consider Mfn2 to be a potential anti-tumor gene. Meanwhile, we did not observe a significant difference between the expression of Mfn2 and TNM stage or clinical prognosis, though the one-year, two-year and three-year overall survival of the Mfn2 low-expression group was lower than the moderate and high-expression group. However, the findings may have been affected by limited sample size or follow-up time.We found that Mfn2 caused a cell cycle arrest in the G0/G1 phase. The cell cycle consists of four distinct phases . Each phase is regulated by its relevant cyclins, CDKs and CDK inhibitors (CDKIs). The p21 (Ras) signature motif in the N-terminal region of Mfn2 is important for its regulation of the cell cycle. A yeast two-hybrid assay confirmed that Mfn2 interacts with a Ras analog . This waIn addition to blocking the cell cycle, we demonstrated that Mfn2 induced apoptosis. Karbowski  implicatin vivo invasive and angiogenic phenotype of malignant keratinocytes. In our study, the expression of MMP-2 and, especially, MMP-9 was significantly reduced. As a result, the invasion ability of AGS cells was suppressed by Mfn2 in a transwell assay. We further discovered that PI3K and phospho-Akt were reduced following transfection with Mfn2. These findings indicate that the downregulation of phospho-Akt may have diminished the expression of MMP-2 and MMP-9, thereby reducing the invasion ability of gastric cancer cells.Akt is also associated with cell invasion and migratory abilities. Kim  demonstrBetween February 2009 and December 2009, a total of 90 patients diagnosed with gastric cancer, none of whom received radiotherapy or chemotherapy before surgery, at the Department of Gastrointestinal Surgery, First Affiliated Hospital, Medical College, Zhejiang University , were enrolled in this study. Paired tumor tissue and normal mucosal tissue samples were obtained from each patient during surgery. The specimens were stored at −80 °C until use and were embedded in paraffin. Prior to their participation in the study, informed consent was obtained from all patients and appropriate permission was granted by the ethical committee of the First Affiliated Hospital, Medical College, Zhejiang University.2.Plasmid Mfn2-YFP, containing the complete Mfn2 ORF (2282 bp) was a gift from Richard Youle (Addgene plasmid 28010). Empty YFP-N1 vector was used as a negative control. The human gastric cancer cell lines, SGC7901 and AGS, were obtained from the Chinese Academy of Medical Sciences and Shanghai Institute for Biological Sciences, respectively. Cells were cultured in RPMI-1640 medium  with 10% fetal bovine serum (FBS)  and maintained in an incubator at 37 °C with 5% COImmunohistochemical analysis was carried out using paired paraffin-embedded cancer tissues and normal mucosal samples. Briefly, serial 4 μm sections were deparaffinized and rehydrated, then subjected to heat-induced epitope retrieval in a microwave oven. Next, the sections were treated with 3% hydrogen peroxide to quench endogenous peroxidase activity, followed by incubation with 5% FBS to block nonspecific binding. After blocking, the sections were incubated overnight at 4 °C with primary monoclonal antibodies ). The next day, the sections were incubated with HRP-conjugated secondary antibodies  and visualized using 3,3′-diaminobenzidine , followed by counterstaining with hematoxylin. For the negative control, the primary antibody was replaced with phosphate-buffered saline (PBS).The stained slides were scored independently by two observers in a blinded manner under microscope . As the expression of Mfn2 was universal, the scores were determined by grading the staining intensity as follows: low expression ; moderate expression ; high expression .Total protein was extracted from tissues or cells (48 h after transfection) using RAPI lysis buffer  containing 1 mM PMSF. SDS-PAGE was used to separate the extracted proteins, followed by transfer to PVDF membranes. After blocking with 5% non-fat milk, the membranes were incubated overnight on ice with primary antibodies against Mfn2 (1:1000), matrix metalloproteinase (MMP)-2 (1:1000), MMP-9 (1:1000), P53 (1:1000), P21 (1:200), cyclin-dependent kinase (CDK)2, CDK4, CDK6 , PI3K (1:1000), cyclinB, cyclinD, cyclinE  (Abcam), cyclinA , Akt  and phospho-Akt . The next day, the membranes were washed with TBST and incubated with HRP-conjugated anti-rabbit and anti-mouse secondary antibodies for 2 h at room temperature. Next, a membrane-enhanced chemiluminescence reagent ECL kit  was used to detect the immunoreactive bands on the blots. β-Actin  was used as an internal control.We performed QRT-PCR  to compare the mRNA expression of Mfn2 in 30 paired tumor tissue and normal mucosal tissue samples from gastric cancer patients (selected at random). The primers used were 5′-CCCCCTTGTCTTTATGCTGATGTT-3′ and 5′-TTTTGGGAGAGGTGTTGCTTATTTC-3′.The plasmid was transfected into cells using X-tremeGENE HP DNA Transfection Reagent , according to the manufacturer’s instructions. The efficiency of transfection was tested by flow cytometry and Western blot analysis.2; detection: 450 nm, reference: 630 nm). A colony-forming assay was included to further test cell viability. After transfection, the cells were digested and counted using an automated cell counter . Briefly, 1000 viable cells were seeded in 6-well plates and cultured at 37 °C with 5% CO2 for 10–14 days. Next, the colonies were stained with crystal violet and counted. All experiments were repeated three times.SGC7901 cells were seeded into 96-well plates at a density of 2000 cells per well, grown at 37 °C overnight and then transfected with Mfn2-YFP  or YFP-N1 (negative control) or treated with transfection reagent alone (blank). Cell proliferation was tested using a CCK-8 Kit  every 24 h after transfection for 7 days . Before transfection, we synchronized the cell cycle by serum-free medium. At 48 h after transfection, the cells were digested and washed with PBS, then fixed in 70% alcohol overnight. The next day, the cells were washed and incubated with RNase A at 37 °C for 30 min and then incubated with propidium iodide (PI) at 4 °C for another 30 min. The samples were examined by flow cytometry .Cell apoptosis was analyzed using a Hoechst 33342/PI Detection Kit . We stained cells after transfection in 6-well plates with Hoechst 33342 and PI successively and then acquired images under a fluorescence microscope . Cells were also digested and tested by flow cytometry using an Annexin V-APC/PI Apoptosis Analysis Kit . All experiments were repeated three times.5 cells were seeded with serum-free RPMI-1640 into each transwell, with 10% FBS RPMI-1640 in a 24-well plate. After 48 h, the transwell membrane was washed, fixed and stained. Finally, images were acquired using a BX41 microscope . All experiments were repeated three times.A transwell  assay was used to estimate the influence of Mfn2 on migration and invasion by gastric cancer cells. Because the invasion ability of SGC7901 cells was weak, we selected AGS cells for our experiment and we used BD Matrigel™ Matrix  to coat 8.0 μm transwells. After transfection, 3 × 10t-test was used to analyze numerical variables. The Wilcoxon signed-rank test or Chi-square test was used to analyze our immunohistochemical results and correlations with the clinicopathological factors. The follow-up period commenced at the start of surgery. Overall survival was calculated from the date of surgery until death or last contact. Potential prognostic factors, including gender, age, tumor size, adjacent organs invasion, lymphovascular invasion, differentiation degree, TNM stage and Mfn2 expression were entered into a univariate analysis using the Kaplan-Meier product-limit method and compared using the log-rank test. Prognostic factors with p-values of <0.10 in the univariate analysis were enrolled into Cox proportional hazard regression models with the forward likelihood method to determine which variables were associated independently with overall survival. Statistical analyses were performed using SPSS software . A value of p < 0.05 was considered to indicate statistical significance. ImageJ software was used to quantify the results of Western blotting. GraphPad Prism 5.0 software  was used to create artwork.The results are expressed as the mean ± SD. A two-tailed, paired Student’s In summary, we demonstrated that the expression of Mfn2 was lower in tumor tissue than in normal mucosal tissue in gastric cancer patients and that it was negatively correlated with tumor size. We also confirmed that Mfn2 suppressed cell proliferation, arrested the cell cycle in the G0/G1 phase, induced apoptosis and weakened the invasion ability of gastric cancer cells. Therefore, Mfn2 is a potential anti-tumor gene and a potential therapeutic target for the treatment of gastric cancer. If we could control the expression of Mfn2, it is possible that the progression of gastric cancer could be delayed.


Mantle cell lymphoma (MCL) is a distinct non-Hodgkin’s lymphoma type that commonly affects extra nodal sites. The most often affected sites are bone marrow, gastrointestinal tract and Waldeyer’s ring, being the skin rarely involved. We report a case of 56 year-old man with MCL, exhibiting multiple large maculopapular skin rashes and skin ulcers. Histopathological examination had not shown direct infiltration by any atypical cells. He had significant improvement of skin lesions with combination chemotherapy and debridement. Awareness of skin manifestations of MCL is crucial for dermatologists and haematologists to establish the early diagnosis and timely administration of appropriate treatment. The bone marrow examination showed 45% immature lymphoid cells, negative for MPO, SB, AP, NSE & PAS on cytochemistry. The bone marrow biopsy showed infiltration by atypical lymphoid cells. Immunophenotyping by flow cytometry of bone marrow showed positivity for co-expression of CD19/5, CD19, FMC7, Kappa clonality, CD25, HLADR, CD22, CD20, CD79b, SIgM and negative for co-expression of CD23/5, CD10, CD23, CD11C, CD103, CD2, CD3, CD34, SIgG, TdT. The axillary lymph node biopsy was suggestive of blastoid variant of mantle cell lymphoma, which showed immunopositivity for CD 20, CD43, Cyclin D1, BCL-2, BCL-6 and immunonegative for CD5, CD3, CD10, CD23. The skin biopsy from the left arm maculopapular lesion  showed congested vessels along with extravasations of RBC in the dermis without any atypical lymphoid cells  is a malignant tumor derived from B cells in the mantle zone of lymphoid follicles characterized by specific pathologic, immunophenotypic and molecular genetics features, and usually takes an aggressive clinical course, defined in the World Health Organization (WHO) classification.CCND-1 gene on chromosome 11 with the immunoglobulin heavy chain gene on chromosome 14, resulting in an over expression of cyclin D1 protein, that leads to a positive signal for transition to the S phase.10Immunophenotypically, the tumor cells are positive for B-cell markers CD79a, CD19, CD20 and CD22 as well as CD5, and are usually negative for CD10 and CD23.MCL frequently involves extra nodal organs, particularly the bone marrow, gastrointestinal tract and Waldeyer’s ring; thus it has been considered in the past as an extracutaneous lymphoma. However, MCL rarely affects the skin. In two large series of 121 and 59 cases of MCL, only 3 and 2 patients respectively had skin lesions, respectively.12In most of the reported cases of skin manifestations of MCL, skin lesion has shown infiltration by atypical lymphoid cells, while a reactive cutaneous eruption, simulating insect bites, has been only incidentally described in association with MCL.


Pseudomonas aurantiaca PB-St2 exhibits antifungal activity and represents a biocontrol agent to suppress red rot disease of sugar cane. Here, we report the completely sequenced 6.6-Mb genome of P. aurantiaca PB-St2. The sequence contains a repertoire of biosynthetic genes for secondary metabolites that putatively contribute to its antagonistic activity and its plant-microbe interactions.The endophytic bacterium  Pseudomonas aurantiaca PB-St2   ( PB-St2)  is a membacteria , 4 and ehybrids) . Strain hybrids) , 4, 6.P. aurantiaca PB-St2 was sequenced using a combination of next-generation sequencing platforms. Genomic DNA was first subjected to 50-cycle paired-end sequencing with an Illumina GAIIx . The de novo assembly of 4,870,658 reads (45-fold median coverage) was performed using the CLC Genomics Workbench 4.0, yielding 555 contigs and a total assembled length of 6,685,786 bp. To improve the quality of the sequence, the genome was additionally sequenced using the PacBio RS technology . The data collected from the PacBio RS instrument were processed and filtered , and the results of both sequencing platforms were subsequently used to perform a de novo hybrid assembly. The contigs were linked and placed into superscaffolds based on the alignment of the PacBio continuous long reads (CLR). Alignment was performed with BLASR (P. chlororaphis strains GP72 (The genome of iaaMH)  (N-acetylglutaminylglutamine amide (NAGGN) and trehalose    was readiaaMH) , the sidiaaMH) , 17, theiaaMH) , an inco absent) , 20, an rehalose , 22, homR, hdtS) , 24, twoR, hdtS) , and genR, hdtS)  and AprAR, hdtS) , 28, werThe genome sequence of PB-St2 not only revealed a true capacity to produce a multitude of secondary metabolites and exoenzymes but also provides a foundation for further understanding of the lifestyle, antagonistic properties, and respective modes of action of this strain and for biosafety assessments when introduced into agricultural environments.AYUD00000000. The version described in this paper is version AYUD01000000.This whole-genome sequencing project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Pharmaceutical preparations of HA are used in clinical practice for wound healing, joint pain, kerato-conjunctivitis, asthma, mouth care, oesophageal-reflux, and gastritis. Moreover, it is used as a filler to counteract ageing and facial lipoatrophy. Our study aims at investigating the the MTT test was used to rule out the potential toxic effects of HA on the different cell lines used in the antiviral assays. The antiviral activity of HA against Coxsackievirus B5, Herpes Simplex Virus-1, Mumps Virus, Adenovirus-5, Influenza Virus A/H1N1, Human Herpesvirus-6, Porcine Parvovirus, Porcine Reproductive and Respiratory Syndrome Virus was assessed by virus yield assays.the most effective inhibition was observed against Coxsackievirus B5, with 3Log reduction of the virus yield at 4 mg/ml, and a reduction of 3.5Log and 2Log, at 2 mg/ml and 1 mg/ml, respectively: the selectivity index was 16. Mumps virus was highly inhibited too showing a reduction of 1.7Log at 1 mg/ml and 1Log at 4 mg/ml and 2 mg/ml (selectivity index = 12). The selectivity index for Influenza Virus was 12 with the highest inhibition (1Log) observed at 4 mg/ml. Herpes Simplex Virus-1 and Porcine Parvovirus were mildly inhibited, whereas no antiviral activity was observed with respect to Adenovirus-5, Human Herpesvirus-6, Porcine Reproductive and Respiratory Syndrome Virus. No HA virucidal activity was ever observed against any of the viruses tested. Kinetic experiments showed that both Coxsackievirus B5 and Herpes simplex virus-1 replication were consistently inhibited, not influenced by the time of HA addition, during the virus replication cycle.the spectrum of the antiviral activity exhibited by HA against both RNA and DNA viruses, known to have different structures (with or without envelope) and replication strategies, suggests a non specific mechanism of action, probably involving cell membrane-virus interaction steps. The results of the kinetic experiments support this hypothesis. Hyaluronic acid (HA) is a non-sulphated glycosaminoglycan which consists of alternately repeating D-glucuronic acid and N-acetylglucosamine units. A huge variety of HAs, with different molecular weights, has been described, probably retaining distinct physicochemical and biological properties. HA is naturally present throughout all mammalian systems, especially synovial fluid, vitreous humour serum and many connective tissues . MoreoveInitially introduced in clinical practice as wound healing promoter, HA is currently used in many medical and cosmetic fields. Some examples of HA applications include eye drops for kerato-conjunctivitis, intra-articular injections for osteoarthritic joint pain, irrigations for bladder and vaginal chronic inflammatory disorders, tracheobronchial aerosolization for asthma, oral solutions for mouth care or for oesophageal-reflux and gastritis. Besides, HA is commonly used for cosmetic interventions, as a filler to counteract ageing and facial lipoatrophy, especially in HIV patients .in vitro. In particular, the replication of Herpes Simplex Virus type 2 [There is evidence showing the ability of HA to interfere with viral replication s type 2 , Respiras type 2  and retrs type 2  is inhibs type 2 . Such liin vitro effects of a high molecular weight HA against a wide group of viruses covering a large spectrum of structural features and replication strategies: ADV-5, Coxsackievirus B5 (COXB5), Herpes Simplex Virus type 1 (HSV-1), Human Herpesvirus-6 (HHV-6), Influenza Virus A/H1N1, Mumps Virus (MV), Porcine Parvovirus (PPV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). We observed an antiviral activity against COXB5, HSV-1, MV, PPV and Influenza Virus encouraging the use of such compound as a medical tool in specific clinical circumstances.In this study we investigated the A high molecular HA (1.800 KD) in powder  was used. It was dissolved in Minimum Essential Medium (EMEM) at 8 mg/ml solution and sterilized by filtration through 0.45 μm filters.The following cell lines were used to cultivate the different viruses: two monkey kidney lines, VERO cells for ADV-5, COXB5, HSV-1, and MV and MARC145 cells for PRRSV; the human T-leukaemia lymphoblast line JJHAN for HHV-6; the canine kidney line MDCK for Influenza Virus; the pig cell line PK15 for PPV. VERO, MARC145, PK15 and MDCK cells were cultured in EMEM added with 10% (growth medium) or 5% (maintenance medium) foetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 μg/ml); RPMI 1640 medium supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml) was used for JJHAN cells. All the cell lines were incubated at 37°C with 5% CO2.The viral strains of HSV-1, ADV-5, COXB5 and MV were clinical isolates, laboratory adapted through serial passages (>50) on VERO cells. The Influenza Virus strain used was the highly neurotropic cell culture adapted WSN33 strain (A/H1N1). For HHV-6, the U1102 strain (variant A) was employed, whereas the two swine viruses tested were reference strains: NADL-2 for PPV and the ATCC strain (Cat. N° VR-2402) for PRRSV. Batches of each virus were prepared, titrated on the suitable cell line and kept frozen at -80°C until they were used for the experiments.The MTT test  was usedTwenty-four hour growth VERO and MDCK cells in 96 well plates were exposed for 1 h to HA (4 mg/ml); then a cell lysis solution Triton X-100 was added to each well at a final concentration of 0.1% and 0.5%. After 5' for 0.1% concentration and 15' for 0,5% concentration, the lysis solution was removed and replaced with fresh medium. The cell viability was measured by MTT test and the survival of HA treated cells was compared with that one of HA untreated cultures. In each assay, cells, not exposed to lysis solutions, were used as controls (100% viability). Three experiments were carried out, each one with samples in triplicate.The antiviral activity was ascertained by means of virus yield assays. Twenty-four hour growth cell cultures were infected with the different viruses at the following multiplicity of infection values: for COXB5, ADV-5 and MV 0.1Tissue Culture Infectious Dose 50% (TCID50)/cell; for HSV-1, 0.1 Plaque Forming Unit (PFU)/cell; for WSN33 virus, 0.1 PFU/cell; for PPV, 0.1 TCID50/cell; for PRRSV, 0.1 TCID50/cell; for HHV-6, 0.1 TCID50/cell. After 1 hr adsorption at 37°C, the inoculum was removed, the plates washed with PBS and the different dilutions of HA, in maintenance medium, added to the cell cultures (each HA dilution was tested in triplicate). After 24 hr incubation for COXB5, ADV-5, MV and HSV-1, 48 hr for WSN33 and PRRSV, 72 hr for PPV and 6 days for HHV-6 the plates were frozen and thawed three times and the viral yield was titrated by end-point titration for ADV, COXB5, MV, PPV, PRRSV, and HHV-6 and by plaque assay for HSV-1 and WSN33. As far as end-point titration is concerned, 10-fold dilutions of each cell lysate were seeded on the 24 hr growth cells in a 96-well culture plate. After 3 days (6 days for HHV-6), the viral titre of each sample, expressed as TCID50/ml, was read taking into account the final dilution still showing the typical viral cytopathic effect and the results were elaborated using the Reed and Muench formula [Time course experiments were carried out with COXB5 and HSV-1 within a single replication cycle. HA (2 mg/ml) was added, at different time points, within 7 hr for COXB5 and 18-20 h for HSV-1 according to each viral replication cycle. In parallel wells, HA was added together with the viral inoculum (t = 0). The virus yield was assessed by end point titration (for COXB5) or plaque assay (for HSV-1). For each virus, 3 experiments were carried out, each in duplicate.The different viral inocula were exposed to HA at a final concentration of 4 mg/ml, for 30' at room temperature and then their residual infectivity was titrated on the suitable cells, as described above. A viral inoculum, treated with medium without HA was used as control for each virus. Two experiments for each virus were performed, each in duplicate.Initially, in order to rule out any direct cytotoxic effect of HA, dose-dependent experiments were performed exposing the five cell lines, employed in the experiments, to HA for different times  followed by MTT assay. We found modest cytotoxicity only at the highest HA concentration (4 mg/ml) which caused a OD reduction of about 20% in 4 of the 5 tested cell lines  were carried out. We found that both cell lines pre-treated for 1 hr with HA at 4 mg/ml were significantly more resistant to lysis than the untreated controls. In particular, for VERO cells in the HA pre-treated groups, the cell viability was close to 100% with the mild treatment (0.1% for 5') and 24.4% with the stronger one (0.5% for 15'), whereas in parallel groups, not pre-exposed to HA before lysis, the cell viability was reduced to 68.1% and 15.2%, respectively  after HA treatment, indicating no virucidal activity by HA against any of the tested viruses (data not shown).This paper provides a new information on the biological properties of HA, describing its antiviral in vitro activity against 5 viruses, with different structural and biological features.in vivo and, though to a lesser extent, also in vitro. The different cell system we used may account for the lack of replication enhancement observed in our experiments.HA is a non-sulfated glycosaminoglycan widely present in the extracellular matrix of soft tissues and in several biologic fluids . ExperimOur findings provide evidence on the effects of HA on a variety of RNA and DNA viruses, with or without the lipidic envelope, characterized by very different replication strategies. Therefore, we can speculate that HA antiviral mechanism(s) probably involves general/non-specific host cell-virus interaction at membrane level, such as virus entry or release, rather than restricted, virus-specific events occurring inside the cell. This speculation is supported by the kinetic results showing that both COXB5 and HSV-1 are growth inhibited irrespective of the time of HA addition during the virus replication cycle. In line with this hypothesis, we may suggest that HA, known to have a heavy ionic charge, may alter the electrostatic interactions between virus and cell receptors and/or other cell membrane components, thus in turn affecting virus entry and/or exit. Literature data substantiate this hypothesis. Dengue virus uses heparan-sulphate (HS) as a cell receptor: different types of HA, interacting with the virus envelope glycoprotein, responsible for virus attachment to HS, are able to inhibit virus attachment and entry . HSV-1 rin vivo.In conclusions, our study provides a wide spectrum demonstration of the antiviral activity by HA, opening new perspectives in prophylaxis and therapy of some viral diseases. The hypothesis of specifically counteracting cell-virus attachment and viral release, by local administration of HA, is very appealing especially for oral, genital and ano-rectal anatomical areas, where compound(s) can be administered as creams, gels or wash solutions. Many HA-based commercial products already available for topic use have HA concentrations much higher or equal to those we found active against different viruses: so it can be hypothesized that effective concentrations can be locally reached. The inhibitory activity observed against HSV-1 is particularly interesting since HA is the basic component of mouthwash solutions and ophthalmic drops, used as artificial tears. According to our findings, HA may be also considered/included as an antiviral agent in the treatment of HSV-1-associated stomatitis and kerato-conjunctivitis in this kind of preparations. Moreover, since Enteroviruses are often responsible for a childhood form of vesicular stomatitis as well as respiratory diseases, the present findings on Coxsackievirus inhibition may open new perspectives for oral administration of HA as natural atoxic medical treatment of newborns and babies. Similarly, the anti-influenza virus activity observed might be exploited in nasal sprays to locally reduce viral replication. Moreover, HA has been demonstrated to have pro-inflammatory activity. Low weight HA fragmentation products and, in the presence of IFNγ, even high molecular weight HA molecules can also activate innate immunity mechanisms through Toll-like Receptor 4 and CD44 ,22. ThisIn conclusion, our findings strongly support the use of this safe glycosaminoglycan in clinical practice as a potential antiviral compound, both for disease prevention and treatment. Further clinical trials on this topic are required to better understand the antiviral activity of this compound.ADV-5: Adenovirus-5; COXB5: Coxsackievirus B5; EMEM: Eagle Minimum Essential Medium; FBS: Foetal Bovine Serum; HHV-6: Human Herpes Virus-6; HSV-1: Herpes Simplex Virus-1; HS: heparan sulphate; MV: Mumps Virus; PFU: Plaque Forming Unit; PPV: Porcine Parvovirus; PRRSV: Porcine Reproductive and Respiratory Syndrome Virus; OD: Optical Density; SI: selectivity index; TCID50: Tissue Culture Infectious Dose 50The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.The authors hereby certify that all work contained in this paper is original work of CC, AC, MS, BC, RN, AA, EB, TI and BP. The authors claim full responsibility for the contents of the article. The authors contributed equally to this work. This article was not supported by grants.Coxsackievirus B5 - 16.1Mumps virus - 12.1Influenza A/H1N1/WSN33 - 11.9Herpes Simplex Virus-1 - 4.8Porcine Parvo - 3.6Selectivity indexes were calculated for those viruses inhibited by HA as the ratio between the toxic dose 50 and the inhibiting dose 50.