After sacrifice, the liver was perfused with 5 mL phosphate-buffe

After sacrifice, the liver was perfused with 5 mL phosphate-buffered saline (PBS) through the portal vein and homogenized. Total liver cells were then resuspended in perfusate buffer containing Hank’s balanced salt solution (HBSS; Ca2+ and Mg2+), collagenase (0.01%), and DNase I (0.001%). After filtration through a 70-μm cell strainer, pelleted cells were resuspended in RPMI and layered with 24% OptiPrep. Subsequent to centrifugation, mononuclear cells (MNCs) were isolated at the 40/60% interface. Cells were washed once with a perfusate

buffer containing HBSS (free Ca2+ and Mg2+), bovine serum albumin (0.25%), and DNase I (0.001%) and supplemented with complete culture media (RPMI; fetal bovine serum [10%], penicillin [100 U/mL], streptomycin [100 μg/mL], selleck kinase inhibitor and L-glutamine [200 mM]). Cell types were determined using microscopy. KC-derived ROS were assayed using the Total ROS Detection Kit (ENZO-51011; Enzo Life Sciences, Inc., Farmingdale, NY). In brief, cell preparations were stimulated using lipopolysaccharide (LPS) MDV3100 (Escherichia coli 0111:B4; Sigma-Aldrich, St. Louis, MO) and incubated for 30 minutes at 37°C. Samples were then washed and cells were resuspended in ROS detection solution, incubated with TruStain FcX (antimouse CD16/32; BioLegend, Inc., San Diego, CA), and stained with F4/80 antibody (Ab; AbD Serotec, Oxford, UK).

Subsequent flow cytometric analysis (FCA) is detailed below. Microspheres MCE (Fluoresbrite YG Microspheres, 1.00 μm; Polysciences, Inc., Warrington, PA) were incubated with a total MNC suspension for 20 minutes at 37°C. Reaction was stopped by the addition of 2 mL of ice-cold PBS. Cell preparations were then washed and incubated with TruStain FcX (antimouse CD16/32; BioLegend)

and stained with F4/80 Ab. Subsequent FCA is detailed below. Cell preparations were stained with CD3-fluorescein isothiocyanate/NK1.1-PerCp and F4/80 clone BM8-PerCP-Cy5.5 Abs (AbD Serotec) for identification of NKTs and KCs, respectively. Cells were incubated at 4°C for 20 minutes, followed by the addition of 1 mL fluorescence-activated cell sorting (FACS) buffer (BioLegend) and centrifugation. Cells were resuspended in a final volume of 100 μL of FACS buffer and analyzed by FCA (BD LSR II; BD Biosceinces, San Jose, CA). Quantification of data was performed using FlowJo 5.6.1. Offspring liver sections, at 3 and 12 months of age, were formalin (10%) fixed and paraffin embedded before sectioning. All sections were then stained with hematoxylin and eosin (H&E) and Masson’s trichrome to assess steatosis, inflammation, and fibrosis. Brunt-Kleiner’s NAS was used to semiquantitatively assess degree of injury by an expert liver pathologist blinded to the identity of the groups.

After sacrifice, the liver was perfused with 5 mL phosphate-buffe

After sacrifice, the liver was perfused with 5 mL phosphate-buffered saline (PBS) through the portal vein and homogenized. Total liver cells were then resuspended in perfusate buffer containing Hank’s balanced salt solution (HBSS; Ca2+ and Mg2+), collagenase (0.01%), and DNase I (0.001%). After filtration through a 70-μm cell strainer, pelleted cells were resuspended in RPMI and layered with 24% OptiPrep. Subsequent to centrifugation, mononuclear cells (MNCs) were isolated at the 40/60% interface. Cells were washed once with a perfusate

buffer containing HBSS (free Ca2+ and Mg2+), bovine serum albumin (0.25%), and DNase I (0.001%) and supplemented with complete culture media (RPMI; fetal bovine serum [10%], penicillin [100 U/mL], streptomycin [100 μg/mL], AZD0530 and L-glutamine [200 mM]). Cell types were determined using microscopy. KC-derived ROS were assayed using the Total ROS Detection Kit (ENZO-51011; Enzo Life Sciences, Inc., Farmingdale, NY). In brief, cell preparations were stimulated using lipopolysaccharide (LPS) AP24534 solubility dmso (Escherichia coli 0111:B4; Sigma-Aldrich, St. Louis, MO) and incubated for 30 minutes at 37°C. Samples were then washed and cells were resuspended in ROS detection solution, incubated with TruStain FcX (antimouse CD16/32; BioLegend, Inc., San Diego, CA), and stained with F4/80 antibody (Ab; AbD Serotec, Oxford, UK).

Subsequent flow cytometric analysis (FCA) is detailed below. Microspheres medchemexpress (Fluoresbrite YG Microspheres, 1.00 μm; Polysciences, Inc., Warrington, PA) were incubated with a total MNC suspension for 20 minutes at 37°C. Reaction was stopped by the addition of 2 mL of ice-cold PBS. Cell preparations were then washed and incubated with TruStain FcX (antimouse CD16/32; BioLegend)

and stained with F4/80 Ab. Subsequent FCA is detailed below. Cell preparations were stained with CD3-fluorescein isothiocyanate/NK1.1-PerCp and F4/80 clone BM8-PerCP-Cy5.5 Abs (AbD Serotec) for identification of NKTs and KCs, respectively. Cells were incubated at 4°C for 20 minutes, followed by the addition of 1 mL fluorescence-activated cell sorting (FACS) buffer (BioLegend) and centrifugation. Cells were resuspended in a final volume of 100 μL of FACS buffer and analyzed by FCA (BD LSR II; BD Biosceinces, San Jose, CA). Quantification of data was performed using FlowJo 5.6.1. Offspring liver sections, at 3 and 12 months of age, were formalin (10%) fixed and paraffin embedded before sectioning. All sections were then stained with hematoxylin and eosin (H&E) and Masson’s trichrome to assess steatosis, inflammation, and fibrosis. Brunt-Kleiner’s NAS was used to semiquantitatively assess degree of injury by an expert liver pathologist blinded to the identity of the groups.

The rate was higher in stage of CKD 3–5, and preventive medicatio

The rate was higher in stage of CKD 3–5, and preventive medication should be used. Key Word(s): 1. low dose aspirin; 2. UGIB; 3. CKD; Presenting Author: SATOSHI SUGITA Additional Authors: MASAFUMI INOMATA, TOMONORI AKAGI, KENTARO NAKAJIMA, YOSHITAKE UEDA, MANABU TOJIGAMORI, HIDEFUMI SHIROSHITA, TSUYOSHI ETOH, NORIO SHIRAISHI, SEIGO KITANO Corresponding

Author: SATOSHI SUGITA Affiliations: Department of Gastroenterological Surgery, Oita University Faculty of Medicine Objective: Fluorouracil-based chemoradiotherapy (CRT) is regarded as a standard perioperative treatment in locally Selleckchem GPCR Compound Library advanced rectal cancer. We investigated the efficacy and safety of substituting fluorouracil with the oral prodrug TS-1. Methods: A multi-institutional (17 specialized centers), interventional phase II trial, was conducted from April 2009 to August 2011.This study is registered with UMIN-CTR, number C003396. For inclusion, patients must fulfill the following requirements before neoadjuvant CRT: (i) histologically proven rectal carcinoma; (ii) tumor located in the rectum (upper,lower); (iii) cancer

classified as T3-4, N0–3 and M0; Two cycles of neoadjuvant CRT with TS-1 (100 mg/m2 on days 1–5, 8–12, 22–26, and 29–33) is administered, and irradiation (total 45Gy/25fr, 1.8Gy/day, on days 1–5, 8–12, 15–19, 22–26, and 29–33) is performed. Total mesorectal excision with D3 lymphadenectomy is performed during the 4th and 8th week after the end of the neoadjuvant CRT. The primary endpoint is rate of complete treatment of neoadjuvant CRT. Secondary endpoints are response rate of neoadjuvant CRT, short-term clinical outcomes, INCB024360 in vitro rate of curative resection, and pathological response (grade2/3). Results: This

trial included 37 patients. A complete treatment of neoadjuvant CRT was found in 86.5% of patients (95%CI;75.5~97.5%), and an adverse event (grade 3/4) occurred in 4 patients (11.1%). Response rate (PR/CR;RECIST 1.0) was 56.8% (95%CI; 40.8~72.7%), and pathologic response rate (grade2/3) was 48.6% (95%CI; 32.5~64.8%). The median operating time was 448.5 min (IQR 340.5–505.5), 上海皓元医药股份有限公司 and median blood loss was 422.5 mL (IQR 182.5–1125). Grade 3–4 postoperative complications occurred in 6 (16.7%) patients. The most common grade 3 or 4 postoperative complication was anastomotic leakage (2 [5.6%]). Conclusion: Our prospective phase-II study demonstrated that a neoadjuvant-synchronus TS-1 + RT for locally advanced rectal cancer is feasible in terms of pathological response and adverse events. Key Word(s): 1. Rectum; 2. chemoradiotherapy; Presenting Author: VARUT LOHSIRIWAT Corresponding Author: VARUT LOHSIRIWAT Affiliations: Mahidol University Objective: To evaluate the clinical outcomes of enhanced recovery program (ERP) after colorectal surgery performed by a consultant colorectal surgeon in a University Hospital in Thailand.

1)7 The type I collagenase, matrix metalloproteinase (MMP)-13 (b

1).7 The type I collagenase, matrix metalloproteinase (MMP)-13 (but not MMP-2, MMP-8, or MMP-14), was identified as a major macrophage-derived matrix degrading enzyme in this resolving process (Fig. 1).8 The investigators did not, however, investigate the expression of CD11c or MHC II in the cells they ablated. In studies published in this current addition of Hepatology, Jiao et al.9 now report a critical role for DCs in resolution of fibrosis following CCl4-mediated liver fibrosis and identify MMP-9 as a key effector enzyme in DCs (Fig. 1). MMP-9

is a gelatinase that cleaves type IV collagen and Bortezomib research buy elastin, also constituents of pathological matrix, and is widely reported as a major effector molecule in macrophages.10 In order to draw these conclusions the investigators used a different transgenic mouse to ablate myeloid cells, CD11c-DTR (DTR under regulation of the CD11c promoter),11 and they focused on the resolution phase after CCl4-mediated fibrosis during which accumulated scar tissue is resorbed and remodeled. To the uninitiated, these investigations might seem to have uncovered a completely novel pathway of resolution

of fibrosis. However, we now appreciate that CD11c and CD11b are poor discriminators of cells with DC functions and macrophage functions (Table 2). Moreover, in peripheral organs 3-Methyladenine mouse such as the liver it seems that the vast majority of myeloid cells express both CD11b and CD11c to varying degrees.6, 9, 12 Therefore, it may simply be that the investigators have unwittingly ablated the same cells in the liver that the studies from 2005 ablated and have chosen to call them DCs, whereas the investigators in 2005 chose to call them macrophages. In keeping with that line of thought, the processes of digesting, phagocytosing, and clearance of matrix and its constituents are widely recognized as macrophage-type functions rather than DC-type functions

in many organs (Table 1), and MMP-9 is widely reported in the literature as a macrophage effector, not a DC effector molecule.10 As such, therefore, the cells they have identified are not DCs, rather they are CD11c-DTR-sensitive macrophages. However, if we step back from the controversies and problems with nomenclature medchemexpress of peripheral organ tissue effector cells, the investigators have identified a CD11b+, CD11chighCD11c-DTR-sensitive subpopulation of liver inflammatory myeloid cells (IMCs) that specifically are responsible for resolution of scarring, in part by producing MMP-9 (Fig. 1). They clearly identify this subpopulation in the resolving liver among other myeloid leukocytes that are presumably either not directly contributing to scar resolution or are contributing to scar resolution by other mechanisms (it is not possible to determine to what extent fibrosis-resolution is halted by ablation of this subpopulation).

1)7 The type I collagenase, matrix metalloproteinase (MMP)-13 (b

1).7 The type I collagenase, matrix metalloproteinase (MMP)-13 (but not MMP-2, MMP-8, or MMP-14), was identified as a major macrophage-derived matrix degrading enzyme in this resolving process (Fig. 1).8 The investigators did not, however, investigate the expression of CD11c or MHC II in the cells they ablated. In studies published in this current addition of Hepatology, Jiao et al.9 now report a critical role for DCs in resolution of fibrosis following CCl4-mediated liver fibrosis and identify MMP-9 as a key effector enzyme in DCs (Fig. 1). MMP-9

is a gelatinase that cleaves type IV collagen and Cisplatin ic50 elastin, also constituents of pathological matrix, and is widely reported as a major effector molecule in macrophages.10 In order to draw these conclusions the investigators used a different transgenic mouse to ablate myeloid cells, CD11c-DTR (DTR under regulation of the CD11c promoter),11 and they focused on the resolution phase after CCl4-mediated fibrosis during which accumulated scar tissue is resorbed and remodeled. To the uninitiated, these investigations might seem to have uncovered a completely novel pathway of resolution

of fibrosis. However, we now appreciate that CD11c and CD11b are poor discriminators of cells with DC functions and macrophage functions (Table 2). Moreover, in peripheral organs Stem Cells antagonist such as the liver it seems that the vast majority of myeloid cells express both CD11b and CD11c to varying degrees.6, 9, 12 Therefore, it may simply be that the investigators have unwittingly ablated the same cells in the liver that the studies from 2005 ablated and have chosen to call them DCs, whereas the investigators in 2005 chose to call them macrophages. In keeping with that line of thought, the processes of digesting, phagocytosing, and clearance of matrix and its constituents are widely recognized as macrophage-type functions rather than DC-type functions

in many organs (Table 1), and MMP-9 is widely reported in the literature as a macrophage effector, not a DC effector molecule.10 As such, therefore, the cells they have identified are not DCs, rather they are CD11c-DTR-sensitive macrophages. However, if we step back from the controversies and problems with nomenclature MCE公司 of peripheral organ tissue effector cells, the investigators have identified a CD11b+, CD11chighCD11c-DTR-sensitive subpopulation of liver inflammatory myeloid cells (IMCs) that specifically are responsible for resolution of scarring, in part by producing MMP-9 (Fig. 1). They clearly identify this subpopulation in the resolving liver among other myeloid leukocytes that are presumably either not directly contributing to scar resolution or are contributing to scar resolution by other mechanisms (it is not possible to determine to what extent fibrosis-resolution is halted by ablation of this subpopulation).

Anders, MD, PhD Hepatology Associates Course Hepatology Associate

Anders, MD, PhD Hepatology Associates Course Hepatology Associates Course Sunday, November 3 8:00 AM -1:30 PM Room 147 COURSE DIRECTORS: Linda M. Stadheim, RN Mary Panther, RN 5.5 CME Credits / 5 Contact Hours This one day course aims to provide basic and advanced up-to-date knowledge

for the management of patients with liver disease. Learning Objectives: Explain the current nonalcoholic steatohepatitis (NASH) practice guidelines and appropriate, patient specific strategies to treat NASH & nonalcoholic fatty liver disease (NAFLD) www.selleckchem.com/B-Raf.html Identify patient selection indication for liver biopsy or a non-invasive alternative to stage fibrosis Describe controversial decisions with transplant patient selections and orgean allocations including split vs full liver transplant Discuss benefits and risks of Coffee, CAM and Cannabis in the liver patient Examine complex Hepatitis C (HCV) treatment populations and identify appropriate strategies to Depsipeptide mw manage difficult HCV treatment side effects 8:00 – 8:05 AM Opening Remarks

Session I MODERATORS: Donald Gardenier, DNP Linda M. Stadheim, RN 8:05 – 8:45 AM NASH & NAFLD: Cutting Through the Fat Andrea A. Gossard, NP 8:45 – 8:55 AM Awarded Poster Presentation Geri Hirsch, MSN, RN-NP and Gail Butt, MD 8:55 – 9:30 AM Biopsy versus Non-invasive Approaches to Assessing Fibrosis R. Todd Stravitz, MD 9:30 – 9:40 AM Awarded Poster Presentation Amy Nelson, BSN, RN, ACRN 9:40 – 10:20 medchemexpress AM Liver Transplantation Controversies Jacqueline Laurin, MD 10:20 – 10:30 AM Discussion 10:30 – 11:00 AM Break and Brunch Session II MODERATORS: Mary Panther, RN Dustin C. Latimer, PA-C 11:00 – 11:45 AM HCV Triple Therapy Lessons Learned Antonio J. Sanchez, MD 11:45 AM – 12:30 PM Point-Counterpoint: Hepatitis C Treatment Douglas R. LaBrecque, MD and Paul Y. Kwo, MD 12:30 – 1:00 PM Coffee, CAM and Cannabis: Stirring the Pot Kiran Bambha, MD 1:00 – 1:30 PM Discussion and Closing Thomas E. Starzl Transplant Surgery State-of-the-Art Lecture Sunday, November 3 9:30 – 10:00 AM Hall

E/General Session Regenerative Medicine: New Approaches to Healthcare SPEAKER: Anthony Atala, MD MODERATOR: Kenneth D. Chavin, MD, PhD Patients with diseased or injured organs may be treated with transplanted organs. There is a severe shortage of donor organs which is worsening yearly due to the aging population. Regenerative medicine and tissue engineering apply the principles of cell transplantation, material sciences, and bioengineering to construct biological substitutes that may restore and maintain normal function in diseased and injured tissues. Stem cells may offer a potentially limitless source of cells for tissue engineering applications and are opening new options for therapy. Recent advances that have occurred in regenerative medicine will be reviewed and applications of these new technologies that may offer novel therapies for patients with end-stage tissue and organ failure will be described.

The duration of each step was determined experimentally using spe

The duration of each step was determined experimentally using specific controls (Supporting Fig. 2). The results clearly show that a decrease in HCVcc infection was only observed when EGCG was present during virus infection (Fig. 3A, second, third, fourth, and sixth bars in the bar-graph), and that there was no effect of EGCG if added as a pretreatment of the cells (Fig. 3A, first bar) or postinfection (Fig. 3A, fifth bar). These results suggest that EGCG inhibits an early step of the HCV life cycle, most likely the entry step. To confirm the effect of EGCG on HCV entry, HCVpp harboring E1 and E2 of different genotypes were produced. HCVpp

infectivity was reduced by approximately 10-fold with a concentration of 50 μM, confirming the effect of EGCG on HCV entry, whatever the genotype used (Fig. selleckchem 3B). However, MG-132 clinical trial some differences between genotypes could be observed at a lower EGCG concentration (5 μM). In contrast, vesicular stomatitis virus (VSV)pp entry was much less inhibited. These results suggest that the antiviral activity of EGCG is directed against HCV envelope glycoproteins and is genotype independent. Together, these data indicate that

EGCG inhibits HCV entry in a genotype-independent manner. Although the above data indicate that EGCG has a strong effect on HCV entry, we cannot exclude additional effects on other steps of the HCV life cycle. To analyze the effect of EGCG on HCV genome replication, Huh-7 cells were electroporated with in vitro transcribed assembly-defective JFH1-ΔE1/E2-Luc RNA, to bypass the entry step, and avoid any interference with late steps of the HCV life cycle. EGCG had no major effect on HCV replication, even after a longer period of treatment (96 hours postelectroporation) (Fig. 4A). In contrast, IFN-α, at 2 IU/mL, approximately twice the IC50 calculated for HCVcc in Huh-7 cells (1.15 IU/ml), 28 induced 1 log10 decrease of luciferase activity. To determine whether EGCG could have any effect on HCV assembly or secretion, intra- and extracellular core protein was quantified in infected

cells treated postinfection with 50 μM of EGCG for 70 hours. The amount medchemexpress of core in the culture supernatant reflects the quantity of secreted viral particles. A slight, but not significant (P = 0.10), decrease in intracellular core was observed in the presence of EGCG (Fig. 4B). This cannot be explained by a decrease in RNA replication, because it has been shown above that EGCG has no effect on HCV replication (Fig. 4A). However, the quantification of extracellular core showed a small, but not significant (P = 0.10), increase of secreted core in the presence of EGCG, as compared to the nontreated control (Fig. 4B), showing that EGCG does not impair viral secretion. Similar experiments were performed with JFH1-ΔE1/E2 to avoid reinfection of the cells and to quantify the levels of extracellular core resulting from cell lysis.

Use of these agents has been studied across a variety of indicati

Use of these agents has been studied across a variety of indications and populations, and at different stages in the disease course. Failure to respond or loss of response can result from different causes, and can be medically managed in many cases. More research on the pleiotropic

Fer-1 research buy effects, safety of biological agents and biomarkers in the prediction of response will provide a sounder basis for individually directing therapy. Adverse events such as opportunistic infection and malignancy can occur, and screening prior to therapy and discussion on risk-benefit of the various management options are important. Cost of these medications especially with maintenance therapy remains an important issue in many Asia-Pacific countries. New and more specific agents will better target therapy and minimize adverse events. Biological agents” describe a class of substances manufactured from a living organism

or by recombinant technology that includes peptides, monoclonal antibodies, fusion proteins and antisense oligonucleotides that bind to nucleic acids.1 In inflammatory bowel diseases (IBD), this group of drugs manipulates key molecules that are involved in the induction and maintenance of intestinal inflammation. They are expensive and risk adverse effects, but this is counterpoised by their superiority to conventional immunosuppressive agents in their efficacy to decrease refractory inflammation, induce mucosal healing, and reduce rates of surgical intervention and long-term complications. Romidepsin concentration Molecules MCE and targets.  An evolving understanding of the pathogenesis of IBD has identified several immunomodulatory therapeutic targets. In Crohn’s disease (CD), T helper 1 (Th1) and the distinctively different T helper 17 (Th17) cells mediate proinflammatory cytokines.2 Both Th1 and Th17 pathways lead to increases in circulatory and tissue

tumor necrosis factor (TNF)-α, the target of anti-TNF-α therapy in both CD and ulcerative colitis (UC).1 Three anti-TNF agents are used in the treatment of IBD. Infliximab is a murine-human chimeric monoclonal antibody, adalimumab is a humanized monoclonal antibody, and certolizumab pegol is a pegylated monoclonal antigen binding fragment (Fab’) to TNF-α. Other anti-TNF-α biological agents currently in clinical trial include golimumab, and this may offer an additional therapeutic option. The alternative Th17 pathway involves interleukin (IL)-23, liberating IL-17A, IL-17F, IL-22 and TNF-α. IL-23 receptor polymorphism is known to protect against the development of CD, and other polymorphisms of the pathway are linked to the development of inflammatory diseases such as ankylosing spondylitis and Grave’s ophthalmopathy.3 Therapeutic neutralization of human IL-12/23 using ustekinumab and briakinumab, approved or are under trial for psoriasis, are currently under evaluation for IBD.

[109] They are also low in abundance and difficult to detect[109

[109] They are also low in abundance and difficult to detect.[109] The pure arithmetic and subtlety of protein transformations and interactions has led to many semiofficial subcategories of proteomics that constrict their profiling domains to specific molecule classes, cellular functions, or PTMs, etc.[110-112] Simply put, the scale of the proteome is staggering. The constitution SCH727965 mw of the metabolome meanwhile continues to be deliberated. From an original study of glucose processing in

E. coli under specific growth rates, the “metabolome” could be defined as “total complement of metabolites in a cell,” with an emphasis on representing the global metabolic processes of a cell by low-molecular weight compounds.[113] This definition was further expounded by successive metabolome studies, perpetuating a correlation between small molecule size (a metabolite weighs less than 15 kilodaltons[114]), with biochemical finality (“metabolites serve as direct signatures of biochemical activity”[21])

because a small molecule must be the result of many enzymatic processes from a gene-transcribed protein origin. While this may be true, it is inadequate in describing the important functional roles of metabolites in biology. The specific physiological functions of metabolites are appreciated and scrutinized in detail in metabolomic studies,[21, 115] but the widely accepted GPCR Compound Library definition of the metabolome, “the comprehensive study of naturally occurring small molecules,”[116] or some variation thereof,[21] does not generally take this into account. Metabolites may be end points of metabolism but they are not end points of physiological process, acting as catalysts, signaling molecules, and nutrients, among other

roles.[115, 117] The metabolome can perhaps be more comprehensively described as the study of the complete expression and biological function of molecules less than 25 kilodaltons within a given cell. This higher molecular mass inclusion takes MCE into account the capability of NMR spectroscopy and MS in reliably resolving higher mass molecules in metabolomic studies than what some would consider a metabolite.[118] Molecule size runs along a continuum, and those not chemically or proportionally considered a metabolite or a protein may be important. This aspect is further discussed in this review. Fundamentally, reevaluations of what constitutes the proteome and metabolome illustrate the immense complexity of human biology and how “omics” have allowed us to understand this in a more complete way. The next challenge is in maneuvering this new expanse of information to unravel the mechanisms behind complex diseases such as the IBDs and build functional tools for their treatment and management. Some of the principles behind the novel ways in which the proteomic and metabolomic toolboxes are being used to these effects are discussed.

ostenfeldii and A peruvianum Phylogenetic analysis of rDNA sequ

ostenfeldii and A. peruvianum. Phylogenetic analysis of rDNA sequences from the A. ostenfeldii XL184 cell line or A. peruvianum cultures examined in this study revealed a complex genetic structure, consisting of six distinct, but closely related groups. A detailed qualitative and quantitative analyses of isolates belonging to four of these groups showed that the diagnostic morphological characters (shape differences in the 1′,

s.a. and 6″ plates) used to define the original species were more variable than previously assumed, exhibiting extensive intra- and inter-strain variability. Instead of the morphological features being consistently associated with a given group, as would be expected if A. ostenfeldii and A. peruvianum were distinct species, each group examined contained strains morphologically click here identified as either A. ostenfeldii or

A. peruvianum. In group 1, for instance, Baltic A. ostenfeldii and North American A. peruvianum strains (as identified by Kremp et al. 2009, Borkman et al. 2012, Tomas et al. 2012) form a monophyletic subgroup in the phylogenetic tree (Fig. 1). Two nearly identical sequences were obtained from A. ostenfeldii (AOKAL0909) and A. peruvianum (e.g., AP0905). Also, strains from the type localities of A. ostenfeldii and A. peruvianum were closely nested in the same phylogenetic group, group 6. Strain IMPLBA033, which represents the type location of the species in Callao, Peru (Balech and de Mendiola 1977) and which is morphologically in accordance with the A. peruvianum description, appears as the immediate neighbor of AONOR4, an A. ostenfeldii strain isolated from the location of the A. ostenfeldii redescription in Norway (Balech and Tangen 1985). The strain AOIS4 from the Iceland where Paulsen first found the species, was nested in group 5. AONOR4 in contrast, more closely resembles the description of A. peruvianum than the type described from the same location. Thus, though MCE the A. ostenfeldii and A. peruvianum

morphotypes as originally described appear distinct, their often nearly identical rDNA sequences indicate they represent the extreme ends in a continuum of A. ostenfeldii morphotypes. Consistent with this conclusion, the isolates examined in this study often showed a combination of the type A. ostenfeldii and A. peruvianum morphologies. Morphological characters were generally not consistently distributed. AONOR4 has some features that are typical for A. peruvianum such as small cell size and a predominantly A-shaped s.a. plate, which is not in accordance with what Balech and Tangen (1985) observed in field samples, collected from the same location. Cells of the Peruvian strain, on the other hand, were not particularly small as originally reported in the species description. The most inconsistent character, considered diagnostic in the original description, is the curved right anterior margin of the 1′ plate of A. peruvianum.