Proliferative activity of blood microvascular endothelium of intestinal mesentery during rat postnatal ontogenesis

The study of mitotic activity of mesenteric microvascular endothelium cells (EC) in 4-, 12-, and 30-day old rats has been carried out using following parameters: number of labeled nuclei per vessel, or per 100 microm of vascular length, or per 1 mm2 of endothelial surface area, as well as shares of labeled EC and of vessels with labeled EC, have been estimated. The highest density of labeled nuclei was revealed in the pericapillary vessels in all rats. Its values were significantly higher in 12-day-old rats and were the lowest in 30-day-old ones.     

A staining procedure for identifying viable cell hybrids constructed by somatic cell fusion, cybridization, or nuclear transplantation

A general procedure for identifying viable hybrid cells was developed. One cell type was labeled by a brief incubation in the Kodak laser dye rhodamine 123, which accumulates in the mitochondria; a second cell type was labeled by a brief incubation in the Hoechst fluorochrome 33258, which binds to chromatin. The substances which are eventually lost from the organelles, appeared to be nontoxic; the plating efficiencies of numerous cell lines tested was unaffected. Either whole cells or cytoplasts labeled with rhodomine 123 were fused, using inactivated Sendai virus, to whole cells or karyoplasts labeled with Hoechst 33258. When living cells were illuminated with ultraviolet light, individual whole cell hybrids, cybrids or cytoplasmic- nuclear hybrid cells could be rapidly identified by the appropriate staining pattern.     

Use of tritium labeled amino acid conjugates of prostaglandins and thromboxanes as labeled ligands in prostanoid radioimmunoassay

Conjugates of prostaglandins and thromboxanes with tritium labeled amino acids were prepared and employed as labeled ligands in prostaglandin and thromboxane radioimmunoassays. Assays for PGF2 alpha, 15-keto-13, 14-dihydro-PGF2 alpha, TXB2 and 15-keto-13,14-dihydro-TXB2 were evaluated in comparative studies using either these heterologous ligands or the corresponding homologous tritiated eicosanoid as tracers. Binding properties for the respective antibodies were found to be similar using either tracer. Three biological studies were also conducted, viz. study of the release of TXB2 during collagen induced platelet aggregation, of 15-keto-13,14-dihydro-TXB2 during guinea pig pulmonary anaphylaxis, and of PGF2 alpha (measured as 15-keto-13,14-dihydro-PGF2 alpha in peripheral plasma) during bovine luteolysis. The analyses gave comparable results using either the heterologous or the homologous assay. Thus, this type of labeled prostanoid conjugates may serve as a convenient alternative to homologous tracers in radioimmunoassay. Heterologous tracers may even in certain cases provide the only simple solution to the problem of preparing a labeled ligand of high specific activity.     

Intragranular colocalization of arginine vasopressin and methionine-enkephalin-octapeptide in CRF-axons in the rat median eminence

Summary Ultrastructural appearances of axonal terminals containing corticoliberin (CRF) were examined in the rat median eminence prepared by a freeze-drying procedure. Immunolabeling was performed by using 5-, 8-, or 15-nm gold-antibody complexes for CRF, arginine vasopressin (VP) and methionine-enkephalin-octapeptide (Enk-8), singly or in combination. In intact animals, the CRF-containing secretory granules were only slightly labeled with goldanti-VP or -Enk-8. In adrenalectomized rats, granules within single axons appeared to be labeled with all the immunogold complexes. This intragranular colocalization of the three antigens was confirmed by using three neighboring sections of the same axon terminals which were stained separately with each one of the antibodies and visualized with the avidin-biotin-peroxidase complex method. The granules labeled for CRF had decreased 9 days after adrenalectomy but had increased again by day 21, while those labeled for VP steadily increased after adrenalectomy. However, this did not correspond with the appearances of cell bodies in the paraventricular nucleus; the cell bodies labeled for both CRF and VP steadily increased in number and in stainability. By contrast, Enk-8 immunoreactivity in the axonal terminals and cell bodies was not affected by adrenalectomy. These findings suggest that although the three peptides could be released simultaneously from the axonal terminals, VP may play some special role in the expression of CRF activity.     

Long-term tracking of lymphocytes in vivo: the migration of PKH-labeled lymphocytes

Studies of in vivo cell migration using cell markers such as 51Cr, 111In, FITC, or XRITC have been limited to short time periods due to the elution, toxicity, or rapid loss of label detectability. We have labeled sheep lymphocytes in vitro with PKH-2, a new fluorescent cell membrane label, and, after their intravenous injection back into donor sheep, have been able to detect them in efferent lymph, using flow cytometry, for longer than 38 days. The PKH-2-labeled lymphocytes migrated with similar kinetics, efficiency, and tissue specificity as lymphocytes labeled with cell markers used previously. PKH-2-labeled cells mediated graft versus host reactions indistinguishable from those mediated by unlabeled cells, and cell surface antigens were equally detectable on the surface of labeled and unlabeled lymphocytes. According to the slow, consistent loss of fluorescence intensity of the labeled cells in vivo, we predict that labeled lymphocytes could remain detectable by flow cytometry for greater than 7 weeks with the labeling protocol used in these experiments.     

Nonradioactive in Situ Hybridization Histochemistry in Leukemic and Nonleukemic Culture

Technical limitations are associated with conducting successful in situ hybridization. In this study, three cell types including a tumor neuroblastoma cell line (Neuro-2a), an oligodendrocyte primary culture, and a nonneuronal acute lymphoblastic leukemia cell line (Reh) were used to conduct successful nonradioactive in situ hybridization. Two cDNA probes were used. A 1 kb probe was used to identify the expression of proteolipid protein (PLP) mRNA in a primary culture of oligodendrocytes. A 760 bp cDNA was used to identify the expression of ubiquitin C-terminal hydrolase (UCH-L1) mRNA in Neuro-2a and Reh cells. The probes were labeled with digoxigenin-11-dUTP, denatured, and hybridized with cells fixed on coverslips. The efficiency of the labeling was tested using dot blot analysis by comparing the intensity of our labeled probes with known concentration of the probe labeled by the provider. The nonspecific signals were washed off, followed by detection of a signal specific to the gene. The specificity of the probes was determined by treating the cells with RNase A, hybridizing with bacterial Dig-labeled cDNA (pBR322) and hybridizing the tissues in the absence of labeled probe. During the labeling step, we found that addition of co-precipitants, such as tRNA or glycogen, during precipitation of the labeled probe followed by overnight incubation at -20 C is essential for good recovery of labeled cDNA. Dissolving the labeled probe in a buffer solution containing sodium dodecyl sulfate improves the quantity of the labeling. At the cellular level, prehybridization treatments optimize the permeability of the cell and allow efficient penetration of the labeled probe. Fixing with paraformaldehyde or an ethanol-acetic acid mixture can preserve the structure of cultured cells. To increase the signal to noise ratio, cells were treated with 0.2 N HC1 followed by extensive washes using a solution with a high salt concentration and containing dextran sulfate. This treatment significantly improves the signal and reduces the background in cell cultures, but not in tissue sections. The ability to reuse the labeled probe-hybridization mixture is another advantage for using nonradioactive in situ hybridization.     

DNA sequence analysis. Terminal sequences of bacteriophage phi80.

Sequences of the cohesive ends and the 3'-terminal regions of phi80 DNA have been determined. Sequences of the cohesive ends were obtained through the use of two standard methods. The first method involved the incorporation of all four labeled deoxyribonucleotides into the phi80 cohesive ends using DNA polymerase I. The DNA was then partially digested with micrococcal nuclease or pancreatic DNase. The products were separated by two-dimensional electrophoresis and characterized by composition, 3'-terminal, and nearest neighbor analyses. The second method involved partial incorporation using one, two, or three labeled deoxyribonucleotides followed by similar analyses. Sequences of the double-stranded regions adjacent to the cohesive ends were determined by three new methods. These methods were: (a) the DNA was specifically labeled at the 3' terminus and then partially degraded. Labeled oligonucleotide products were sequenced by their mobilities on various separation systems. (b) The cohesive ends were enlarged by limited degradation with exonuclease III. After this treatment, the DNA was partially repaired with labeled nucleotides, digested, and the products were analyzed. (c) A synthetic ologonucleotide primer was bound to phi80 DNA which had been repaired with DNA polymerase I, and then partially digested with lambda-exonuclease. The primer was extended into the region of interest by partial repair with labeled nucleotides. The extended primer was isolated and analyzed.     

Localization of a phytohormone using immunocytochemistry

The localization of cytokinins in corn root tips was investigated using antibodies or antibody fragments directed against dihydrozeatin riboside and labeled with rhodamine or colloidal gold. Roots were sectioned at -30 degrees to -40 degrees for immunofluorescence or freeze-substituted in ethanol or acetone and embedded in plastic for electron microscopy. Meristematic cells surrounding the quiescent center as well as root cap cells were specifically labeled using direct immunofluorescence techniques, whereas cells of the quiescent center did not bind label. Tissue sections treated with colloidal gold-labeled antibody fragments had gold particles widely distributed in the cytoplasm. The results show that the quiescent center is not the major site of cytokinin localization in root tips.     

Short-wave ultraviolet irradiation increases photo-affinity labeling of benzodiazepine sites

Photoaffinity labeling of benzodiazepine sites with [3H]-flunitrazepam was examined using either long-wave (366 nm) or short-wave (254 nm) ultraviolet irradiation. A multiple exposure protocol was employed so that the time course of the process could be determined as well as the fraction of total sites labeled. At 366 nm, approximately 20% of the total sites present were labeled and the remainder showed reduced affinity for flunitrazepam, in agreement with published reports. When membranes from either cortex or cerebellum were irradiated at 254 nm, however, the fraction of sites labeled increased above 40%. The change in the ratio of labeled sites to those showing reduced affinity, as well as differences in the time courses of the two phenomena, are taken as evidence that the labeling and affinity change are independent processes rather than two effects of a single event. Inhibition of labeling by clonazepam and Ro15-1788 indicated the "central" nature of the labeled sites.     

Cellular organization of normal mouse liver: a histological,quantitative immunocytochemical,and fine structural analysis

The cellular organization of normal mouse liver was studied using light and electron microscopy and quantitative immunocytochemical techniques. The general histological organization of the mouse liver is similar to livers of other mammalian species, with a lobular organization based on the distributions of portal areas and central venules. The parenchymal hepatocytes were detected with immunocytochemical techniques to recognize albumin or biotin containing cells. The macrophage Kupffer cells were identified with F4-80 immunocytochemistry, Ito stellate cells were identified with GFAP immunocytochemistry, and endothelial cells were labeled with the CD-34 antibody. Kupffer cells were labeled with intravascularly administered fluorescently labeled latex microspheres of both large (0.5 μm) and small (0.03 μm) diameters, while endothelial cells were labeled only with small diameter microspheres. Neither hepatocytes nor Ito stellate cells were labeled by intravascularly administered latex microspheres. The principal fine structural features of hepatocytes and non-parenchymal cells of mouse liver are similar to those reported for rat. Counts of immunocytochemically labeled cells with stained nuclei indicated that hepatocytes constituted approximately 52% of all labeled cells, Kupffer cells about 18%, Ito cells about 8%, and endothelial cells about 22% of all labeled cells. Approximately, 35% of the hepatocytes contained two nuclei; none of the Kupffer or Ito cells were double nucleated. The presence of canaliculi and a bile duct system appear similar to that reported for other species. The cellular organization of the mouse liver is quite similar to that of other mammalian species, confirming that the mouse presents a useful animal model for studies of liver structure and function.     

Protein disulfide isomerase and sulfhydryl-dependent pathways in platelet activation

The inhibition of blood platelet aggregation and secretion was studied using covalent thiol reagents, maleimides, or mercuribenzoates, or using inhibitors of protein disulfide isomerase (PDI), bacitracin or antibodies to PDI. As expected, both types of inhibitors were effective against stimulation by normal physiologic stimuli. On the other hand, when stimulation was initiated with the peptide LSARLAF, that specifically activates the integrin alphaIIbbeta3 (the fibrinogen receptor), the PDI inhibitors were without effect. LSARLAF-induced aggregation was, however, inhibited by the sulfhydryl reagents. To further investigate the role of sulfhydryl-containing proteins and alphaIIbbeta3, platelets were labeled with membrane-impermeant sulfhydryl reagents. Nine bands were found labeled on gel electrophoresis. Two of the labeled bands were identified as alphaIIb and beta3. The conclusions are that while PDI is required for platelet aggregation and secretion, an additional sulfhydryl-dependent step or protein is also required. This latter reaction occurs at the level of alphaIIbbeta3. In distinction to most literature reports, at least a subpopulation of alphaIIbbeta3 contains free sulfhydryl groups, consistent with the possibility that it is a substrate for PDI or part of the sulfhydryl-dependent response.     

Preparation of specifically labeled C-(lignin)- and C-(cellulose)-lignocelluloses and their decomposition by the microflora of soil

Microbial decomposition of lignocellulose in soil was studied using radioisotope techniques. Natural lignocelluloses containing C in either their lignin or cellulose (glucan) components were prepared by feeding plants l-[U-C]phenylalanine or d-[U-C]glucose, respectively, through their cut stems. Detailed chemical and chromatographic characterization of labeled lignocelluloses from three hardwood and three softwood species showed that those labeled by the [C]glucose incorporation method contained specifically labeled cellulosic components, whereas those labeled by the [C]phenylalanine incorporation method contained specifically labeled lignin components. Microbial degradation of these differentially labeled lignocelluloses was followed by monitoring CO(2) evolution from selected soil samples incubated with known amounts of radiolabeled lignocelluloses. The lignin components of the six woods were shown to be decomposed in soil 4 to 10 times more slowly than their cellulosic components. These rates of mineralization were comparable to the generalized patterns previously reported in the literature. The present technique, however, was thought to be simpler, more sensitive, and less prone to interference than methods previously available.     

利用荧光蛋白标记西瓜枯萎病菌的过氧化物酶体及细胞核

西瓜枯萎病是一种世界范围的西瓜毁灭性病害,其病原菌为尖孢镰刀菌西瓜专化型(Fusarium oxysporum f.sp.niveum,FON)。研究病原菌生长发育和侵染的机制是解决病害的根本途径。利用荧光蛋白对细胞或细胞器进行标记,是病原菌研究中的重要方法。该研究利用绿色荧光蛋白和红色荧光蛋白对FON的细胞核和过氧化物酶体进行了荧光标记。通过农杆菌介导转化(Agrobacterium tumefaciens-mediated transformation,AtMT),该文将3种不同的荧光定位载体分别导入FON,获得了细胞核红色荧光标记的转化子(潮霉素抗性,含mCherry-H2B融合蛋白),以及过氧化物酶体绿色(潮霉素抗性,含GFP-PTS1融合蛋白)和红色(潮霉素抗性,含DsRED-PTS1融合蛋白)荧光标记的转化子各1种。在标记细胞核的菌株中,菌丝、孢子都可见明亮、圆形的红色荧光点,荧光点与DAPI染色标记的细胞核区域完全重合。在过氧化物酶体标记的菌株中,菌丝、孢子中可见明亮的红色或绿色荧光成小点状分布,符合过氧化物酶体的分布特征,而且在脂类物质诱导的条件下,荧光点的数量明显增加。此外,该文还利用细胞壁荧光染色剂卡氏白对3种荧光蛋白标记菌株进行染色。结果显示,卡氏白染色产生的蓝色荧光与红、绿荧光蛋白的荧光在FON中互不干扰。转化子继代培养和初步分析表明,其表型与野生型无差异,菌株继代后荧光表达稳定、定位明显。该结果为进一步研究FON细胞器动态、生长发育与致病分子机制提供了方法和工具。     

Comparative studies using 125I- and 111In-labeled monoclonal antibodies

HDP-1 monoclonal antibody was labeled with ¹¹¹In using deferoxamine, diethylenetriaminepentaacetic acid or 1-(para-bromoacetamidobenzyl)-EDTA as chelating agents or with ¹²⁵I. The in vitro binding capacity and stability of the labeled molecules were evaluated using affinity chromatography. The biodistribution and imaging capabilities were compared using an animal model system that does not involve the use of tumors. Similar studies were done using the corresponding labeled F(ab′)₂ and Fab′ fragments. All labeled molecules, except those treated with deferoxamine, were stable in vitro. When tested in vivo, all retained their capacity to localize in the target tissue (lung). The lung %ID/g levels for the ¹¹¹In-labeled molecules were, however, slightly lower than those observed for the corresponding ¹²⁵I-labeled molecules. High uptake was also observed in the liver or kidneys when the ¹¹¹In-labeled molecules were used; no such results were obtained with the ¹²⁵I-labeled molecules. More work appears to be necessary before the use of bifunctional chelates becomes the optimal method for radiolabeling monoclonal antibodies for use in tumor imaging.     

An autoradiographic study of the role of satellite cells and myonuclei during myogenesis in vitro

Satellite cells and myonuclei of neonatal rat muscles were differentially labeled with 3H-thymidine according to the procedure of Moss and Leblond (1971). Minced muscles fragments containing either labeled satellite cells or labeled myonuclei were cultured until multinucleated myotubes grew out from the explants. Reutilzation of isotope released from degenerating nuclei was competitively inhibited by using a culture medium containing excess (0.32-0.41 mM) cold thymidine. after an 8-10 day growth period, the explants were fixed and prepared for autoradiographic (ARG) examination to determine whether labeled satellite cells or myonuclei had contributed to the myonuclear population of the developing myotubes. Counts were made of the number of labeled myotubes in the explants and compared with the number of labeled satellite cells and myonuclei in samples of the original muscle tissues fixed at the time of explantation. The original muscles showed a mean satellite cell labeling index of 51.7% and gave rise to myotubes with a mean labeling incidence of 40%. In contrast, myonuclear labeling in the original muscle tissues showed no correlation with subsequent myotube labeling. Only 3.4% myotube labeling was found in explants in which over 30% of the original tissue myonuclei had been labeled. Under conditions controlled for isotope reutilization, these observations confirm results of in vivo ARG studies indicating that satellite cells are the only significant source of regenerating myoblasts in injured muscle tissue.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either ¹²⁵I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10⁷ binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

Comparative Study of 125I- and [3H]Acetate-Labeled Antibodies in Detecting Iridescent Viruses

Radioimmunoassays for detecting cell-associated or released virus are described using either (125)I- or [(3)H]acetate-labeled antibodies. In the first assay system, antigen-antibody complexes were separated from free antibody by centrifugation. Sensitivities of 0.1 mug of iridescent virus could be achieved with either (125)I- or [(3)H]acetate-labeled antibody. In the second assay, the antigen was fixed to cover-slip cell cultures, and then reacted with labeled antibody, unbound radioactivity being removed by repeated washing. Nonspecific binding with this method was 0.5 to 1% of the total radioactivity added and sensitivities of 0.1 or 10 mug were achieved with (125)I and [(3)H]acetate, respectively. Immunoglobulins were labeled at the rate of 1 in 300 for (125)I and 1 in 200 with [(3)H]acetate although there was a 400-fold greater isotopic abundance of (125)I relative to (3)H. The possibility of preparing labeled protein of high specific activity using carrier-free [2-(3)H]iodoacetic acid is discussed.     

Identification of porcine oocyte proteins that are associated with somatic cell nuclei after co-incubation

Relatively little is known with respect to the oocyte proteins that are involved in nuclear reprogramming of somatic cells in mammals. The aim of the present study was to use a cell-free incubation system between porcine oocyte proteins and somatic cell nuclei and to identify oocyte proteins that remain associated with these somatic cell nuclei. In two separate experiments, porcine oocytes were either labeled with biotin to label total proteins at the germinal vesicle stage or metaphase II stage or they were labeled with 0.1 mM (35)S-methionine either during the first 6 h or 22-28 h of in vitro maturation to characterize protein synthesis during two distinct phases. To determine which oocyte proteins associate with somatic nuclei, labeled proteins were incubated in a collecting buffer and energy-regenerating system with isolated ovarian epithelial-like cell nuclei. After incubation, the nuclei were subjected to a novel affinity-binding system to recover biotin-labeled oocyte proteins or two-dimensional SDS-PAGE for separation and visualization of radiolabeled proteins. Proteins of interest were sent for identification using either matrix-assisted laser desorption/ionization time of flight or liquid chromatography-tandem mass spectrometry. Of the proteins that remain associated with isolated nuclei after incubation, 4 were identified using the affinity-binding system and 24 were identified using mass spectrometry and the two-dimensional gel interface. This study has identified porcine oocyte proteins that associate with somatic cell nuclei in a cell-free system using proteomics techniques, providing a novel way to identify oocyte proteins potentially functionally involved in nuclear reprogramming.     

The use of the fluorescence photobleaching recovery technique to study the self-assembly of tubulin

Fluorescently labeled microtubule-associated proteins or poly-L-lysine (13,000 MW) were prepared by reaction with fluorescein isothiocyanate. The labeled compounds were used as probes of the assembly of calf brain tubulin using fluorescence photobleaching recovery techniques which allow measurement of the diffusion coefficient and percentage mobility of the fluorescent probe. When unfractionated tubulin (defined as material containing tubulin and microtubule-associated proteins) was polymerized at room temperature or 37 degrees C, either probe could be incorporated into microtubules, since the observed diffusion coefficient (approximately 1.7 X 10(-8) cm2/s) was much slower than that for either probe free in solution. The microtubules formed in the presence of labeled microtubule-associated proteins were free to diffuse while those formed in the presence of labeled polylysine were partially immobilized. Thus the fluorescence photobleaching recovery technique can be used to measure crosslinking of microtubules as well as assembly or interactions with other structures. When unfractionated tubulin was incubated with labeled polylysine in the presence of Ca2+ at room temperature, the observed diffusion coefficient (approximately 5.1 X 10(-8) cm2/s) probably represents the formation of rings of tubulin. The effect of mild and vigorous shearing, of cholchicine, and of different Mg2+ concentrations on the properties of the system were examined.     

Intracerebral estrogen provision increases cytogenesis and neurogenesis in the injured zebra finch brain

To determine whether or not local, injury‐induced aromatization and/or estrogen provision can affect cyto‐ or neuro‐genesis following mechanical brain damage, two groups of adult male zebra finches sustained bilateral penetrating brain injuries. The first received contralateral injections of vehicle or the aromatase inhibitor fadrozole. The second group received contalateral injections of fadrozole, or fadrozole with 17β‐estradiol. Subsequent to injury, birds were injected with the thymidine analog 5‐bromo‐2′‐deoxyuridine (BrdU). Two weeks following injury, the birds were perfused, and coronal sections were labeled using antibodies against BrdU and the neuronal proteins HuC/HuD. In a double blind fashion, BrdU positive cells and BrdU/Hu double‐labeled cells in the subventricular zone (SVZ) and at the injury site (INJ) were imaged and sampled. The average numbers of cells per image were compared across brain regions and treatments using repeated measures ANOVAs and, where applicable, post‐hoc, pairwise comparisons. Fadrozole administration had no detectable effect on cytogenesis or neurogenesis, however, fadrozole coupled with estradiol significantly increased both measures. The dorsal SVZ had the greatest proportion of new cells that differentiated into neurons, though the highest numbers of BrdU labeled and BrdU, Hu double‐labeled cells were detected at the INJ. In the adult zebra finch brain, local estradiol provision can increase cytogenesis and neurogenesis, however, whether or not endogenous glial aromatization is sufficient to similarly affect these processes remains to be seen. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 170‐181, 2011