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Based on our previous observation that heparin-induced thrombocytopenia (HIT) sera can generate platelet microparticles from washed platelets in a heparin-dependent fashion, we developed a test for HIT using flow-cytometry to measure heparin-dependent platelet microparticle formation. During the developmental phase of the assay the optimal physical conditions for microparticle generation were defined. 133 sera were then evaluated using the microparticle assay and the serotonin release assay to determine the threshold for defining a positive result that gave optimal sensitivity and specificity. The microparticle assay was then prospectively evaluated against the serotonin release assay in 202 sera referred to our laboratory for HIT testing. Overall agreement between the two assays was 96% (Cohen's kappa = 0.91). When the clinical data were reviewed on patients whose sera gave discrepant results between the two assays, no case of HIT was detected by one assay and missed by the other. The platelet microparticle assay is as accurate as the serotonin release assay and may be a useful non-radioactive test for HIT.

The production of amino acids and their condensation to polypeptides under plausibly prebiotic conditions have long been known. But despite the central importance of molecular self-replication in the origin of life, the feasibility of peptide self-replication has not been established experimentally. Here we report an example of a self-replicating peptide. We show that a 32-residue alpha-helical peptide based on the leucine-zipper domain of the yeast transcription factor GCN4 can act autocatalytically in templating its own synthesis by accelerating the thioester-promoted amide-bond condensation of 15- and 17-residue fragments in neutral, dilute aqueous solutions. The self-replication process displays parabolic growth pattern with the initial rates of product formation correlating with the square-foot of initial template concentration.

Purpose: To describe a unique case of chronic intraocular inflammation secondary to scleral buckle infection with Mycobacterium chelonae that was successfully treated with buckle explantation. Methods: Case report. Results: A 59-year-old male with a history of retinal detachment repair at the age of 41 presented with chronic, recurrent intraocular inflammation responsive to topical corticosteroids. Conjunctival erosion with exposure of the scleral buckle occurred five months after initial presentation. The scleral buckle was removed and cultured. After three weeks of postoperative topical tobramycin and dexamethasone treatment, the patient has remained symptom-free without medications. The explanted material grew acid-fast bacilli later identified as M. chelonae. Conclusions: This case describes a new finding of chronic intraocular inflammation associated with a scleral buckle infected with M. chelonae and the successful resolution of extraocular infection and intraocular inflammation after buckle removal.

This is a study of a particular technique of administering intramuscular injections and the rationale nurses give for employing such a technique. Instead of mixing the two medications which are compatible, registered nurses in the study used the 'two syringes and one needle' technique when two medications were prescribed. The reasons for such practice are unclear. Some nurses argue that this method prevents the occurrence of incompatibility when medications are mixed in a syringe. The study indicates that such a technique is little more than a ritualistic practice, one based on tradition, which passes on from one nurse to another and from one generation of nurses to the next. Rationales for such a technique given by registered nurses in the study are found to be contradictory and have no sound or scientific base.

End of life care is often overlooked in busy day-to-day medical practice. Physicians need to recognize that death is inevitable for many medical conditions despite aggressive treatment. Optimal end of life care begins with an honest discussion of disease progression and prognosis. By coordinating the care with the family and a hospice program, terminally ill patients can achieve relief of pain and other unwanted symptoms, leading to a good quality of life during their remaining days.

Facile fabrication of building blocks with precisely controlled dimensions is imperative in the development of functional devices and materials. We demonstrate the assembly of nanoscale viral building blocks of controlled lengths using a biologically motivated strategy. To achieve this we exploit the simple self-assembly mechanism of Tobacco mosaic virus (TMV), whose length is solely governed by the length of its genomic mRNA. We synthesize viral mRNA of desired lengths using simple molecular biology techniques, and in vitro assemble the mRNA with viral coat proteins to yield viral building blocks of controlled lengths. The results indicate that the assembly of the viral building blocks is consistent and reproducible, and can be readily extended to assemble building blocks with genetically modified coat proteins (TMV1cys). Additionally, we confirm the potential utility of the TMV1cys viral building blocks with controlled dimensions via covalent and quantitative conjugation of fluorescent markers. We envision that our biologically inspired assembly strategy to design and construct viral building blocks of controlled dimensions could be employed to fabricate well-controlled nanoarchitectures and hybrid nanomaterials for a wide variety of applications including nanoelectronics and nanocatalysis.

Experimental autoimmune encephalomyelitis (EAE) is a widely used model of multiple sclerosis. In NOD mice, EAE develops as a relapsing–remitting disease that transitions to a chronic progressive disease, making the NOD model the only mouse model that recapitulates the full clinical disease course observed in most multiple sclerosis patients. We have generated a TCR transgenic mouse that expresses the α- and β-chains of a myelin oligodendrocyte glycoprotein (MOG) 35–55–reactive TCR (1C6) on the NOD background. 1C6 TCR transgenic mice spontaneously generate both CD4+ and CD8+ T cells that recognize MOG and produce proinflammatory cytokines, allowing for the first time to our knowledge the simultaneous examination of myelin-reactive CD4+ and CD8+ T cells in the same host. 1C6 CD8+ T cells alone can induce optic neuritis and mild EAE with delayed onset; however, 1C6 CD4+ T cells alone induce severe EAE and predominate in driving disease when both cell types are present. When 1C6 mice are crossed with mice bearing an IgH specific for MOG, the mice develop spontaneous EAE with high incidence, but surprisingly the disease pattern does not resemble the neuromyelitis optica-like disease observed in mice bearing CD4+ T cells and B cells reactive to MOG on the C57BL/6 background. Collectively, our data show that although myelin-reactive CD8+ T cells contribute to disease, disease is primarily driven by myelin-reactive CD4+ T cells and that the coexistence of myelin-reactive T and B cells does not necessarily result in a distinct pathological phenotype.

Comparability studies lie at the heart of assessments that evaluate differences amongst manufacturing processes and stability studies of protein therapeutics. Low resolution chromatographic and electrophoretic methods facilitate quantitation, but do not always yield detailed insight into the effect of the manufacturing change or environmental stress. Conversely, mass spectrometry (MS) can provide high resolution information on the molecule, but conventional methods are not very quantitative. This gap can be reconciled by use of a stable isotope-tagged reference standard (SITRS), a version of the analyte protein that is uniformly labeled with 13C6-arginine and 13C6-lysine. The SITRS serves as an internal control that is trypsin-digested and analyzed by liquid chromatography (LC)-MS with the analyte sample. The ratio of the ion intensities of each unlabeled and labeled peptide pair is then compared to that of other sample(s). A comparison of these ratios provides a readily accessible way to spot even minute differences among samples. In a study of a monoclonal antibody (mAb) spiked with varying amounts of the same antibody bearing point mutations, peptides containing the mutations were readily identified and quantified at concentrations as low as 2% relative to unmodified peptides. The method was robust, reproducible and produced a linear response for every peptide that was monitored. The method was also successfully used to distinguish between two batches of a mAb that were produced in two different cell lines while two batches produced from the same cell line were found to be highly comparable. Finally, the use of the SITRS method in the comparison of two stressed mAb samples enabled the identification of sites susceptible to deamidation and oxidation, as well as their quantitation. The experimental results indicate that use of a SITRS in a peptide mapping experiment with MS detection enables sensitive and quantitative comparability studies of proteins at high resolution.

Type 1 diabetes (T1D) is an autoimmune disease that shows familial aggregation in humans and likely has genetic determinants. Disease linkage studies have revealed many susceptibility loci for T1D in mice and humans. The mouse T1D susceptibility locus insulin-dependent diabetes susceptibility 3 (Idd3), which has a homologous genetic interval in humans, encodes cytokine genes Il2 and Il21 and regulates diabetes and other autoimmune diseases; however, the cellular and molecular mechanisms of this regulation are still being elucidated. Here we show that T cells from NOD mice produce more Il21 and less Il2 and exhibit enhanced Th17 cell generation compared with T cells from NOD.Idd3 congenic mice, which carry the protective Idd3 allele from a diabetes-resistant mouse strain. Further, APCs from NOD and NOD.Idd3 mice played a central role in this differential Th17 cell development, and IL-21 signaling in APCs was pivotal to this process. Specifically, NOD-derived APCs showed increased production of pro-Th17 mediators and dysregulation of the retinoic acid (RA) signaling pathway compared with APCs from NOD.Idd3 and NOD.Il21r-deficient mice. These data suggest that the protective effect of the Idd3 locus is due, in part, to differential RA signaling in APCs and that IL-21 likely plays a role in this process. Thus, we believe APCs provide a new candidate for therapeutic intervention in autoimmune diseases.

The INK4a proteins p16INK4a and p19ARF regulate cell cycle arrest and senescence. However, the role of these proteins in controlling these processes in the normal kidney and following injury is unknown. We performed unilateral ureteral obstruction (UUO) to induce fibrosis in 2- to 3-mo-old wild-type (WT) C57/B6 and INK4a knockout mice. By quantitative RT-PCR, p16INK4a levels were increased sixfold in WT mice 7 days after UUO and p19ARF remained undetectable. Kidney sections were examined to determine levels and localization of p16INK4a, apoptosis, fibrosis, and senescent cells. INK4a knockout mice displayed mesangial cell proliferation, increased matrix deposition, and myofibroblast differentiation under normal conditions. Following UUO, INK4a knockout mice displayed 10-fold increased tubular and interstitial cell proliferation, 75% decreased collecting duct apoptosis, 2-fold greater collagen and fibronectin deposition, and no cell senescence by senescence-associated β-galactosidase staining compared with WT mice. Both INK4a knockout mesangial cells and kidney lysates from knockout mice following injury showed elevated levels of IL-6 by ELISA compared with WT samples. INK4a knockout epithelial cell cultures displayed increased mesenchymal cell markers when exposed to transforming growth factor-β. These results confirm that p16INK4a controls cell proliferation and matrix production and mitigates fibrosis following injury and suggest that the mechanism involves a role in limiting inflammation and cell proliferation.

An ancient landslide was re-activated in 1998 in a hillside-graded area of San Juan Capistrano, California. The landslide occurred within a park site and threatened an adjacent residential development. The ensuing litigation led to the design and construction of a retention system with high-capacity tieback anchors in 2000. This system consisted of five levels of lateral support, with each level containing up to three rows of tiebacks and continuous walers. A total of 864 tieback anchors were installed, ranging in design load capacity from 300 to 365 kips. In order to account for the continuing slide movement that was expected to occur during the ongoing construction activities, the anchors were locked off at or above 50 percent of the design capacity. Post-construction monitoring has indicated that the measured loads in about 60 percent of the anchors continued to increase over a period of several years, up to 38 percent higher than the design capacity in some anchors. The anchor loads have now decreased to an equilibrium state at generally less than, or equal to, the design capacity (with few exceptions). This design approach has proven successful in resisting the active slide movement within the mitigated area.

The clinical value of antibiotic prophylaxis in preventing Lyme disease remains uncertain, owing to a meta-analysis lacking sufficient power to demonstrate efficacy and a more recent trial showing effectiveness but lacking precision. Our objective was to update our prior meta-analysis on antibiotic prophylaxis for the prevention of Lyme disease, to obtain a more precise estimate of treatment effect.Clinical trials were identified by searching MEDLINE, Embase, the Cochrane Library and trial registries, and by an assessment of the bibliographies of retrieved articles and reviews. Trials were selected if their patients were randomly allocated to a treatment or placebo group within 72 h following an Ixodes tick bite and had no clinical evidence of Lyme disease at enrollment. Details of the trial design, patient characteristics, interventions and outcomes were extracted from each article. Study quality was assessed using the Jadad scale.Four placebo-controlled clinical trials were included for review. Among 1082 randomized subjects, the risk of Lyme disease in the placebo group was 2.2% [95% confidence interval (CI), 1.2%-3.9%] compared with 0.2% (95% CI, 0.0%-1.0%) in the antibiotic-treated group. Antibiotic prophylaxis significantly reduced the odds of developing Lyme disease compared with placebo (pooled odds ratio=0.084; 95% CI, 0.0020-0.57; P=0.0037).The available evidence to date supports the use of antibiotic prophylaxis for the prevention of Lyme disease in endemic areas following an Ixodes tick bite. Pooled data from four placebo-controlled trials suggests that one case of Lyme disease is prevented for about every 50 patients who are treated with antibiotics.

Tim-3 (T cell immunoglobulin, mucin domain)-3 is a membrane protein expressed at late stages of interferon-gamma secreting CD4+ T helper type 1 (Th1) cell differentiation and constitutively on dendritic cells (DCs). Ligation of Tim-3 on Th1 cells terminates Th1 immune responses. In addition, Tim-3 plays a role in tolerance induction, though the mechanism by which this is accomplished has yet to be elucidated. While it is clear that Tim-3 plays an important role in the immune system, little is known regarding the molecular pathways that regulate Tim-3 expression. Here we have examined the role of Th1-associated transcription factors in regulating Tim-3 expression. Our experiments reveal that Tim-3 expression is regulated by the Th1-specific transcription factor T-bet. This introduces a novel paradigm into the generation of a Th1 response, whereby a transcription factor responsible for effector Th1 cell differentiation also increases the expression of a specific counter-regulatory molecule to ensure appropriate termination of pro-inflammatory Th1 immune responses.

A series of 5-alkyltryptamines (6) and the corresponding conformationally constrained analogues (8) have been synthesized. The structure activity relationships (SAR) at the 5-position of the indole skeleton and the ethylamine side chain have been studied. Functional activities were assessed using isolated rabbit saphenous vein. Potent, selective ligands were found (6e, Ki 2.5 nM, 5-HT1B/5-HT1D 125-fold) that have potential for treating acute migraine.

The Idd3 genetic interval confers protection against multiple autoimmune diseases, including type 1 diabetes and experimental autoimmune encephalomyelitis. The favored candidate gene in this interval is Il2, which is polymorphic between susceptible and resistant strains of mice. IL-2 regulates the growth/death of effector T cells as well as the generation/maintenance of regulatory T cells (Tregs), and recent studies have shown that NOD.Idd3 Tregs are more suppressive than their NOD counterparts. We have further dissected the mechanisms underlying the differential suppression by NOD and NOD.Idd3 Tregs and find that it is determined by CD11b+CD11c− APCs. Thus, contrary to what might be expected, our data suggest that the differential suppressive activity of NOD and NOD.Idd3 Tregs is not due to an effect of the Idd3 genetic interval on T cells but rather is due to differences in the APC compartment.

Efficient methods to model and optimise the design of open pit mines have been known for many years. Although the underground mine design problem is conceptually more difficult it has a similar potential for optimisation. Recent research demonstrates some useful progress in this topic. Here we provide an overview of some of this research.

Despite advances in the safety, processing, and storage of conventional 22°C liquid-stored allogeneic platelet concentrates (see Chapter 69), there are still significant limitations to standard platelet concentrates (Table 70-1). These limitations have led to efforts to minimize the exposure of recipients to allogeneic blood products and to develop safe and effective platelet products and substitutes with longer shelflives. This chapter reviews advances in the development of new platelet products, synthetic platelet substitutes, new storage methods, and novel pathogen inactivation technology for platelet concentrates.

Dem Entstehen von Fehlern (der Produktion von mutierten Peptiden) wirkt dieses einfache nichtlineare chemische Netzwerk durch verstarkte Produktion der nativen Sequenz entgegen. Das Netzwerk reagiert auf diese spontan auftretenden Fehler, indem die mutierten Peptide T9A und T26A als Katalysatoren fur die Kondensation der Peptidfragmente E und N, bei der die native selbstreplizierende Sequenz T gebildet wird, herangezogen werden. Dieser Mechanismus der dynamischen Fehlerkorrektur (siehe Bild) konnte fur die Stabilisierung von genomischer Information von Bedeutung sein.

Synthesis and in vitro cytotoxicity assays of new anthranilamide MDR modulators have been performed to assess their inhibition potency of the P-glycoprotein (P-gp) transporter. The aromatic spacer group between nitrogen atoms (N1 and N2) in the known inhibitor XR9576 was replaced with a flexible alkyl chain of 2 to 6 carbon atoms in length. 6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline and their open-chain N-methylhomoveratrylamine counterparts were shown to be potent P-gp inhibitors. The maximal inhibition was obtained when using an ethyl or propyl spacer. Several compounds were more potent than verapamil and intrinsically less cytotoxic than XR9576. In addition, in vitro metabolism studies of 23a with a subset of human CYP-450 isoforms revealed that, unlike XR9576, 23a inhibited CYP3A4, an enzyme that colocalizes with P-gp in the intestine and contributes to tumor cell chemoresistance by enhancing the biodisposition of anticancer drugs such as paclitaxel toward metabolism. In this context, 22a might be a suitable candidate for further drug development.

Since the introduction of highly active antiretroviral therapy (HAART), the frequency of opportunistic disorders (ODs) of the gastrointestinal (GI) tract in human immunodeficiency virus (HIV)-infected patients has fallen dramatically. We have found, however, that despite the use of HAART, HIV-infected patients can still present with GI ODs. To evaluate the prevalence of GI ODs in HIV-infected patients on HAART who were undergoing endoscopic evaluation for GI-related symptoms. From January 1996 through February 2002, all HIV-infected patients undergoing GI endoscopy were prospectively identified; mucosal biopsies were obtained in a standardized fashion and histologic specimens were examined by a single GI pathologist. All the patients on HAART presenting with a GI OD are described. Results showed that 294 patients (88% men; mean age, 36.5 +/- 10 years; median CD4 lymphocyte count, 64 cells/microl; range, 1-884; median viral RNA level, 40,357 copies/ml; range, 0-7,721,715) underwent 401 upper and/or lower endoscopies during the study period. The use of HAART increased from 10% in early 1996 to 85% in 2002. Nevertheless we found that 27 patients (9%) presented with a GI OD despite HAART. Forty percent of the patients with a GI OD were noncompliant with HAART. We conclude that GI ODs can occur despite HAART and normal CD4 counts. The prevalence of GI ODs in HIV-infected patients taking HAART is 9%. The reasons for this are multifactorial and likely include noncompliance with medications, viral resistance to the drugs, and decreased drug bioavailability. Although the use of HAART has led to a decreased incidence of GI ODs in AIDS, the gastroenterologist evaluating these patients should not discard the possibility that the GI symptoms in HIV-infected patients taking HAART may be secondary to an OD, even when the CD4 count is normal and the viral load is low.

Background The aim of this study was a detailed endoscopic-pathologic assessment of patients with various forms of GI strongyloidiasis. Methods Six patients with a diagnosis of GI strongyloidiasis who underwent endoscopic evaluation during a 3-year period (January 1998-January 2001) were included. Published information was reviewed in detail, focusing on the endoscopic features and the diagnostic approach to this parasitosis. Observations Strongyloidiasis has a broad range of endoscopic features. In the duodenum, the findings included edema, brown discoloration of the mucosa, erythematous spots, subepithelial hemorrhages, and megaduodenum. In the colon, the findings included loss of vascular pattern, edema, aphthous ulcers, erosions, serpiginous ulcerations, and xanthoma-like lesions, and, in the stomach, thickened folds and mucosal erosions. A histopathologic diagnosis of strongyloidiasis was made in all cases. Conclusions Strongyloidiasis can involve any segment of the GI tract. EGD with procurement of biopsy specimens from the duodenum was the most accurate method of diagnosis in this case series.

Adipocyte complement-related protein of 30 kDa (Acrp30)/adiponectin is an adipocyte-derived hormone that affects lipid and glucose metabolism in muscle and liver, but its physical and biochemical properties are poorly characterized. Here we have used several approaches to show that Acrp30 expressed in and purified from Escherichia coli and human embryonic kidney 293T cells forms trimers and hexamers; 293T cells also produce a higher molecular weight species. Similar Acrp30 oligomers were found in mouse serum as well as in 3T3-L1 adipocyte-conditioned medium, although in different proportions. In parallel, we assessed whether Acrp30 is a signaling molecule by searching for promoter or enhancer elements that respond to Acrp30 or its isolated trimeric globular C-terminal domain, gAcrp30. Acrp30 addition to C2C12 myocytes or myotubes led to activation of NF-kappa B transcription factor in a manner dependent upon phosphorylation and degradation of I kappa B-alpha. Importantly, only hexameric and larger isoforms of Acrp30 activated NF-kappa B; trimeric Acrp30 or gAcrp30 could not activate NF-kappa B. Our data indicate that oligomerization of Acrp30 is important for at least some of its biological activities, and changes in the relative abundance of each oligomeric isoform in plasma may regulate Acrp30 activity.

Estrogen-triggered nuclear transcription factor, estrogen receptor (ER) is expressed in about 70% of breast cancer. ER mediates various cellular signaling events including the genomic regulation of target genes that affect reproduction, development, and general metabolism. The antiestrogen tamoxifen is currently administered to patients in order to induce regression of the tumor growth. However, upon continued administration, breast cancer patients develop resistance to tamoxifen. In addition, calcium binding proteins such as, calmodulin (CaM) which are significantly overexpressed in cancer cells, can activate transcription of target genes by directly binding to ER in lieu of estrogen. Calmodulin antagonists (w7 and melatonin) have been shown to significantly inhibit human estrogen receptor alpha (hERalpha) transcriptional activity. In search of a novel anti-cancer agent, we generated a library of interaction profiles of human ER-EF hand protein complexes and identified S100B and S100P (high sequence and structure homology with CaM) as potential targets. Furthermore, S100P is shown to mediate tamoxifen-acquired resistance and cell migration capacity in MCF-7 breast cancer cells. Our objective is to develop peptidomimetics and small molecules to block ER-S100 interactions using an integrated proteomics-assisted drug discovery approach. Briefly, using the fold information and contact regions obtained from the FT-ICR mass spectrometry, 3D structures of ER-S100 protein complexes were reconstructed. These structures of ER best represent the conformational state sensed by the specific interacting partner. ER-S100 complex is distinct from CaM. The lack of the connective peptidic region between the EF hand pairs in S100 attributed to less interaction coverage than CaM. The identified ER conformation-sensing regions of the interacting EF hand proteins were used to develop a library of top peptidomimetics and potential small molecule sensors targeting the interfaces of ER-S100 interactions. We have evaluated their ability to block specific ER-S100 interactions in a high throughput fluorescent polarization based screen, to inhibit cancer cell proliferation and ER-mediated transcription of target genes. We believe that the ensemble of ER-EF hand protein complexes generated by our integrated proteomics-assisted protein interaction profiling will shed light on the lingering issue of hormone independent activation of ER at the molecular level and novel therapeutics targeting ER-S100 interactions. Citation Format: David H. Lee, Bethany K. Asare, Matthew L. Hudson, Rajendram V. Rajnarayanan. Discovery at the interface: novel anticancer agents targeting human estrogen receptor/S100 interactions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4173. doi:10.1158/1538-7445.AM2014-4173

We report the case of a highly sensitized 37 year old male who suffered an acute humoral rejection due to donor specific anti-HLA DP (DP) following a second kidney transplant. A previous living donor kidney graft failed in 2005 due to noncompliance with immunosuppression and he returned to hemodialysis. Past history included two blood transfusions. On evaluation for retransplant in 2010, his cPRA using microarray single antigen beads (One Lambda) was 100% including anti-DP1 (MFI 4221). On 2/17/14, he received a deceased donor kidney (KDPI 17%). T and B cell crossmatches by CDC-AHG/CDC long incubation and by the rosette method (Life Technologies) were compatible. There were no donor specific antibodies (DSA) to HLA A B Cw DR or DQ. DP typing of deceased donors was not performed. Retrospective DP typing indicated that the donor was DP1. The clinical staff was advised. He received Thymoglobulin induction and was placed on Mycophenolate Mofetil (MMF), prednisone and Tacrolimus. By post op day (POD) 10, his creatinine decreased to 1.6 mg/dL. On POD 18, his creatinine increased to 2.5 mg/dL. Biopsy results showed acute glomerulitis and acute tubular necrosis. C4d staining was positive. A biopsy day serum showed anti-DP1 (MFI 7627) by microarray single antigen beads and (MFI 26038) using the C1q binding assay (One Lambda). C1q testing of archived crossmatch serum did not show anti-DP1. After treatment with plasmapheresis and 1 g/kg IVIg, his creatinine stabilized at 1.8–2 mg/dL. He is currently maintained on MMF, Tacrolimus and prednisone. A survey of 1069 renal transplant candidates revealed that 13% had DP antibodies. Two of three transplanted patients with DP antibodies retrospectively determined to be DSA had humoral rejections. Another patient with no pre-transplant DSA received a crossmatch compatible kidney and had a rejection due to de novo anti-DP. These 3 cases were regrafts. Our experience is consistent with recent literature. With increasing evidence that DP antibodies can cause graft rejection and with technology available to perform HLA DP typing and antibody monitoring, the transplant community should take action to recognize the potential risk of DP antibodies in renal transplant, require DP typing of all donors prior to transplant and consider incompatible donor DP antigens unacceptable for regrafts.

A 33-residue de novo designed peptide ligase is reported which catalyzes the template-directed condensation of suitably activated short peptides with catalytic efficiencies in excess of 10(5) ([k(cat)/K(m)]/k(uncat)). The ligase peptide, derived from natural and designed alpha-helical coiled-coil proteins, presents a surface for substrate assembly via formation of a hydrophobic core at the peptide interface. Charged residues flanking the core provide additional binding specificity through electrostatic complementarity. Addition of the template to an equimolar fragment solution results in up to 4100-fold increases in initial reaction rates. Dramatic decreases in efficiency upon mutation of charged residues or increase in ionic strength establishes the importance of electrostatic recognition to ligase efficiency. Although most of the increase in reaction efficiency is due to entropic gain from binding of substrates in close proximity, mechanistic studies with altered substrates demonstrate that the system is highly sensitive to precise ordering at the point of ligation. Taken together these results represent the first example of a peptide catalyst with designed substrate binding sites which can significantly accelerate a bimolecular reaction and support the general viability of alpha-helical protein assemblies in artificial enzyme design.

A series of pyrrolo[3,2,1-ij]quinoline derivatives was synthesized, evaluated for their activity against the 5-HT2c and 5-HT2a, receptors and found to be agonists at 5-HT2c with selectivity over 5-HT2a.

The transition from inanimate to animate chemistry is thought to involve self-organised networks of molecular species whose collective emergent property gives rise to the overall characteristics of living systems. In the past, simple autocatalytic networks have been constructed that display basic forms of cooperative behaviour. These include reciprocal catalysis, autocratic, and hypercyclic networks. The design and emergent properties of these novel molecular networks are reviewed here.

ESPITE CONSIDERABLE progress in assessing in vitro platelet function, in vivo function is relatively poorly understood, because few methods exist to study platelets after transfusion. Thus the in vivo efficacy of the various hemostatic products, developed for the prevention or treatment of thrombocytopenic bleeding, has been difficult to evaluate. This has sometimes led to confusion about optimal conditions for the preparation and storage of human platelets for transfusion.~ This is true not only for random donor platelet concentrates prepared by standard and plateletapheresis methods, but also for platelet products that have been cryopreserved, lyophilized, or prepared and stored in other ways. In addition, various putative platelet substitutes, which represent an emerging group of hemostatic agents, require not only toxicity testing but also test systems to evaluate in vivo efficacy. A requirement for hemostatic products is that they prevent or stop bleeding in thrombocytopenic humans, with minimal toxicity. Contemporary human platelet concentrates must therefore assume the gold standard position against which other hemostatic preparations can be measured. How hemostatic efficacy can be measured has been problematic, and a surrogate in vivo measure for the correction of thrombocytopenic bleeding in humans has been difficult to define. 1 Many of the available in vitro tests of platelet function have been validated against in vivo survival of the transfused product. Survival for many years has represented the in vivo 'measure of platelet function. However, a new platelet product that has equal in vivo survival to a conventional platelet concentrate does not imply that the two necessarily have equal hemostatic function. In the same way, two platelet products that have equivalent in vivo hemostatic efficacy may not have equivalent in vivo survival. Thus, the ability of in vitro tests to predict in vivo hemostatic function is wanting.

Estrogen Receptor (ER) belongs to the nuclear receptor super family of ligand-triggered transcription factors. ER is present in about 70% of breast cancers and plays a critical role in the biology of breast carcinoma and improves prognosis. Estrogen binds to ER and mediates a cascade of cellular signaling events. It has been shown that several proteins including calcium binding proteins with EF-hand motifs, which control cellular calcium and calcium mediated signaling pathways, including calmodulin (CaM) could bind to unliganded ER and activate ER. CaM has four EF-hand motifs that change conformation upon binding Ca2+ ions. Ca2+-CaM complex binds to the target proteins and initiates various signaling cascades. Each EF-hand motif contains two alpha helices connected by a 12-residue loop. Both X-ray and NMR studies show that the alpha helices of the EF-hand motifs change their position relative to each other, forming an almost perpendicular conformation. This allows calmodulin to increase its binding affinity for a number of target proteins. CaM forms a compact globular conformation by bending its central helix upon binding its target peptides. S100, a soluble protein that is recently reported to interact with ER has only two EF-hand motifs. S100 protein is overexpressed in several cancers and shares a high degree of sequence homology with calmodulin. Methods: Protein-protein interactions of CaM, S100 with ER alpha were simulated using HADDOCK and HEX. Docking results were analyzed in UCSF Chimera and integrated with data from in vitro proteomics experiments to determine the conformations of ER bound EF-hand protein complexes. Proteomic experiments consisted of chemical crosslinking of the corresponding protein complexes followed by tryptic digestion, analyzing the resulting peptide peaks using FTICR- Mass Spectrometry. Results/Discussion: Using the fold information and contact regions obtained from the Mass spectrometry, 3D structures of ER-EF hand protein complexes were reconstructed. These structures of ER best represent the conformational state sensed by the specific interacting partner. ER-S100 complex is distinct from CaM. The lack of the connective peptidic region between the EF-hand pairs in S100 attributed to less interaction coverage indicative of only inducing a partial agonist-like conformation. We believe that the ensemble of ER-EF hand protein complexes generated by our integrated proteomics-assisted protein interaction profiling will shed light on the lingering issue of hormone independent activation of ER at the molecular level. Acknowledgements: This research was supported via funds from the New York State, National Institutes of Health (5P20MD002725), through the NCMHD -RIMI program (RVR) Citation Format: David H. Lee, Bethany K. Asare, Suchitra Singh, Rajendram V. Rajnarayanan. Comparative analyses of human estrogen receptor- EF-hand protein complexes: molecular basis for hormone independent activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2891. doi:10.1158/1538-7445.AM2013-2891

Purpose: Although the literature on strongyloidiasis is extensive, there is a remarkable paucity of information about the endoscopic findings of gastrointestinal (GI) strongyloidiasis, with most information coming from sporadic case reports. The aim of this study was to present a detailed endoscopic-pathologic study of patients with various forms of GI strongyloidiasis.

An approach to generate mimics of phosphorylated serine proteins chemically through site-specific sulfonation of cysteine is presented. This chemical modification is reversible in the presence of reducing agent and therefore is analogous to the kinase/phosphatase system used in nature.

The swelling properties of the anterior and posterior stroma of bovine and rabbit corneas were examined by three different methods. The results show that the posterior stroma can swell more than the anterior stroma. Also, at a given swelling pressure, the posterior stroma is more hydrated than the anterior stroma. In a third experiment it is demonstrated that the posterior stroma is also more hydrated in the living eye. It is concluded that the stroma is not a homogeneous structure. The superficial stroma has different physiological properties from the deep stroma. These differences must be taken into account in theoretical models of corneal deturgescence.

Methods were devised using information from an objective analysis to determine the assimilation level of cloud motion vectors. Rawinsonde data and SMS 2 satellite-derived cloud motion vectors were assimilated using the NCAR Limited Area Multivariate Statistical Objective Analysis, with persistence as the first guess. The geographical area included the northwestern Pacific Ocean, Central America and most of North America; a 6 h time interval between analyses and a three-day period were used. The four experiments varied the method of cloud-wind vertical placement, and included wind insertion 1) at the level nearest the cloud-top temperature; 2) at all levels within the cloud layer as defined by the cloud-top temperature and a physical thickness; 3) at the level nearest the cloud base; and 4) at the wind-fit level, at which the insertion wind most agrees with the first guess wind. Insertion at the wind-fit level had characleristics and produced results which were unreasonable. Other level determinat...