Development of a RapidFire mass spectrometry assay and a fluorescence assay for the discovery of kynurenine aminotransferase II inhibitors to treat central nervous system disorders

Kynurenine aminotransferases convert kynurenine to kynurenic acid and play an important role in the tryptophan degradation pathway. Kynurenic acid levels in brain have been hypothesized to be linked to a number of central nervous system (CNS) disorders. Kynurenine aminotransferase II (KATII) has proven to be a key modulator of kynurenic acid levels in brain and, thus, is an attractive target to treat CNS diseases. A sensitive, high-throughput, label-free RapidFire mass spectrometry assay has been developed for human KATII. Unlike other assays, this method is directly applicable to KATII enzymes from different animal species, which allows us to select proper animal model(s) to evaluate human KATII inhibitors. We also established a coupled fluorescence assay for human KATII. The short assay time and kinetic capability of the fluorescence assay provide a useful tool for orthogonal inhibitor validation and mechanistic studies.     

When alcohol is the answer: Trapping,identifying and quantifying simple alkylating species in aqueous environments

Alkylating agents are a significant class of environmental carcinogens as well as commonly used anticancer therapeutics. Traditional alkylating activity assays have utilized the colorimetric reagent 4-(4-nitrobenzyl)pyridine (4NBP). However, 4NBP based assays have a relatively low sensitivity towards harder, more oxophilic alkylating species and are not well suited for the identification of the trapped alkyl moiety due to adduct instability. Herein we describe a method using water as the trapping agent which permits the trapping of simple alkylating electrophiles with a comparatively wide range of softness/hardness and permits the identification of donated simple alkyl moieties.     

Inflammatory Breast Cancer: A Distinct Clinicopathological Entity Transcending Histological Distinction

Introduction

Although well recognized in breast oncology literature, histologic subtypes have not been previously described in inflammatory breast cancer (IBC). The purpose of this study was to describe lobular subtype in IBC and assess the impact of histology on patient outcomes.

Methods

We performed a retrospective analysis of 659 IBC patients at MD Anderson Cancer Center between January 1984 and December 2009. Patients with Invasive Lobular, Mixed Invasive Ductal and Lobular, or Invasive Ducal Carcinomas (ILC, MIC, IDC, respectively) comprise the subject of this report. Patient characteristics and survival estimates were compared by using chi-square test and Kaplan-Meier method with log-rank statistic. Cox proportional hazards models were fit to determine association of histology with outcomes after adjustment for other characteristics.

Results

A total of 30, 37, and 592 patients were seen to have invasive lobular, mixed, or ductal histology, respectively. Grade 3 tumors were more common in the ductal group (78%) than in the lobular (60%) or mixed (61%) group (P = 0.01). The 3-year overall survival rates were 68%, 64%, and 62% in the lobular, mixed, and ductal groups, respectively (P = 0.68). After adjustment, histology did not have a significant effect on death in the lobular group (HR = 0.70, 95% confidence interval [CI]: 0.26–1.94; P = 0.50) or mixed group (HR = 0.53, 95% CI: 0.25–1.13; P = 0.10) compared with the ductal group.

Conclusion

In this cohort of IBC patients, lobular histology was seen in 4.5% cases. Histology does not appear to have a significant effect on survival outcomes in IBC patients, unlike in patients with non-inflammatory breast cancer (n-IBC), indicating the distinct biological behavior of the IBC phenotype.     

Modulation of Macrophage Polarization and HMGB1-TLR2/TLR4 Cascade Plays a Crucial Role for Cardiac Remodeling in Senescence-Accelerated Prone Mice

The aim of this study was to investigate the role of macrophage polarization in aging heart. Macrophage differentiation is pathogenically linked to many inflammatory and immune disorders. It is often preceded by myocardial inflammation, which is characterized by increased cardiac damage and pro-inflammatory cytokine levels. Therefore, we investigated the hypothesis that senescence accelerated-prone (SAMP8) mice cardiac tissue would develop macrophage polarization compared with senescence-resistant control (SAMR1) mice. Both SAMP8 and SAMR1 mice were sacrificed when they became six month old. We evaluated, histo-pathological changes and modifications in protein expression by Western blotting and immuno-histochemical staining for M1 and M2 macrophage markers, high mobility group protein (HMG)B1 and its cascade proteins, pro-inflammatory factors and inflammatory cytokines in cardiac tissue. We observed significant upregulation of HMGB1, toll-like receptor (TLR)2, TLR4, nuclear factor (NF)κB p65, tumor necrosis factor (TNF)α, cyclooxygenase (COX)2, interferon (IFN)γ, interleukin (IL)-1β, IL-6 and M1 like macrophage specific marker cluster of differentiation (CD)68 expressions in SAMP8 heart. In contrast, M2 macrophage specific marker CD36, and IL-10 expressions were down-regulated in SAMP8 mice. The results from the study demonstrated that, HMGB1-TLR2/TLR4 signaling cascade and induction of phenotypic switching to M1 macrophage polarization in SAMP8 mice heart would be one of the possible reasons behind the cardiac dysfunction and thus it could become an important therapeutic target to improve the age related cardiac dysfunction.     

Next generation sequencing of Apis mellifera syriaca identifies genes for Varroa resistance and beneficial bee keeping traits

Apis mellifera syriaca exhibits a high degree of tolerance to pests and pathogens including varroa mites. This native honey bee subspecies of Jordan expresses behavioral adaptations to high temperature and dry seasons typical of the region. However, persistent honey bee imports of commercial breeder lines are endangering local honey bee population. This study reports the use of next‐generation sequencing (NGS) technology to study the A. m. syriaca genome and to identify genetic factors possibly contributing toward mite resistance and other favorable traits. We obtained a total of 46.2 million raw reads by applying the NGS to sequence A. m. syriaca and used extensive bioinformatics approach to identify several candidate genes for Varroa mite resistance, behavioral and immune responses characteristic for these bees. As a part of characterizing the functional regulation of molecular genetic pathway, we have mapped the pathway genes potentially involved using information from Drosophila melanogaster and present possible functional changes implicated in responses to Varroa destructor mite infestation toward this. We performed in‐depth functional annotation methods to identify ～600 candidates that are relevant, genes involved in pathways such as microbial recognition and phagocytosis, peptidoglycan recognition protein family, Gram negative binding protein family, phagocytosis receptors, serpins, Toll signaling pathway, Imd pathway, Tnf, JAK‐STAT and MAPK pathway, heamatopioesis and cellular response pathways, antiviral, RNAi pathway, stress factors, etc. were selected. Finally, we have cataloged function‐specific polymorphisms between A. mellifera and A. m. syriaca that could give better understanding of varroa mite resistance mechanisms and assist in breeding. We have identified immune related embryonic development (Cactus, Relish, dorsal, Ank2, baz), Varroa hygiene (NorpA2, Zasp, LanA, gasp, impl3) and Varroa resistance (Pug, pcmt, elk, elf3‐s10, Dscam2, Dhc64C, gro, futsch) functional variations genes between A. mellifera and A. m. syriaca that could be used to develop an effective molecular tool for bee conservation and breeding programs to improve locally adapted strains such as syriaca and utilize their advantageous traits for the benefit of apiculture industry.     

Seasonal variation in the plankton of Porto Novo Waters (India)

Summary The seasonal variations of marine (collected at the ten fathom line-Station A) and estuarine (collected at the Mouth of the Vellar estuary-Station I) plankton of Porto Novo (India) are described, covering the period 1960–63. The biological, temporal and numerical variations of phytoplankton and zooplankton are described for the two stations. The succession of species was more or less similar at both stations, but certain forms like colonial Radiolaria, Scyphomedusae, Anthozoa, trochophore of Sipunculids, Stomatopod larvae and Protochordate larvae were relatively more confined to Station A, and crustacean larvae other than Stomatopods Actinotrocha larva, fish eggs and larvae were relatively more confined to Station I.The Diatom main peak was about June–July at Station A and about May–July at Station I. The Diatom peak was generally heralded with the appearance of Coscinodiscus. Noticluca usually showed three spurts- mainly two distict ones about May–June and August. The third development was only during certain years and not so intensive. Copepods showed two peaks generally during June–July after few days of the Diatom bloom, and during October–November following the first monsoon rains.The peaks of Diatoms, Peridinians, Noctiluca, Molluscan larvae and Tunicate larvae were more marked at Station A than at Station I. A rich and varied zooplankton was observed about February at Station A. The appearance of salps at Station A seems in a way to be related to salinity, when the salinity values were between 28.89–34.55, the salps appearing more with a fall in salinity. Similarly at Station I, Creseis acicula, Prawn larvae and fish larvae seem to be associated with a sudden fall in temperature and salinity when the monsoon rains start, at Station I.The numerical relationship of plankton at Station A was generally inverse between phyto- and zooplankton and this was generally parallel at Station I.

---

Sumario La variación estacional del plancton marino (collectado en la ten fathom lineestacción A) y estuarino (Mouth of the Vellar estuary) és descrita para el período 1960–63. Las variaciones biológicas, temporales y numéricas del fitoplancton y del zooplancton son descritas para las dos estacciones. La succesión de especies fué más o menos comparable en las dos estacciones; algunas formas como colonial radiolaria, scyphomedusae, anthozoa, trochophore y sipunculids, stomatopod larvae, y protochordate larvae fuéron confinadas a la estacción A, y otras como crustacean larvae, Actinotrocha larva, peces eggs y larvae confinades a la estacción I.El maximum para las diatomeas ocurrió en Junio–Julio en la estacción A,y Mayo–Junio en la estación I. Las diatomeas están representadas principalmente por el génere Coscinodiscus antes del maximum. El maximum para Noctiluca fue observado en Mayo–Junio y en Agosto. Los copepodes presentaran dos períodos de maximum Junio–Julio despues del maximum de las diatomeas y en Octubre–Noviembre. El maximum de las diatomeas, peridianians, Noctiluca, molluscan larvae y tunicate larvae en la estacción A fué mas distincto que en la estacción en el estuario. El zooplancton se mostró rico y variado en Febrero en la estacción A.La presencia de salpas en la estacción A parace ser relacionada en ciertos aspectos con la diminuición de la salinidad cuando esta fué 28.89–34.55. Por la misma forma la presencia de Creseis acicula, prawn larvae, peces eggs y larvae parace ser relacionada con diminuición de salinidad y temperatura del agua durante Octobre–Noviembre–Deciembre en la estacción I. Las relaciones numéricas del plancton indican que en la estacción A fueran contrarias y en la estacción I paralelas.

     

Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design

A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, K_D, in the μM range or lower). Amide hydrogen deuterium Exchange mass spectrometry (HDXMS) is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (K_D ~ nM) and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD).