CoMFA and HQSAR of acylhydrazide cruzain inhibitors

An approach combining CoMFA and HQSAR methods was used to describe QSAR models for a series of cruzain inhibitors having the acylhydrazide framework. A CoMFA study using two alignment orientations (I and II), three different probe atoms and changes of the lattice spacing (1 and 2 A) was performed. Alignment II and an sp3 probe carbon atom yielded good cross-validation (q2=0.688) employing lattice spacing of 1 A. The best HQSAR model was generated using atoms, bond, and connectivity as fragment distinction and fragment size default (4-5) showing similar cross-validated value of CoMFA (q2=0.689). Based upon the information derived from CoMFA and HQSAR, we have identified some key features that may be used to design new acylhydrazide derivatives that may be more potent cruzain inhibitors.     

An unusual allosteric mobility of the C-terminal helix of a high-affinity alphaL integrin I domain variant bound to ICAM-5

Integrins are cell surface receptors that transduce signals bidirectionally across the plasma membrane. The key event of integrin signaling is the allosteric regulation between its ligand-binding site and the C-terminal helix (alpha7) of integrin's inserted (I) domain. A significant axial movement of the alpha7 helix is associated with the open, active conformation of integrins. We describe the crystal structure of an engineered high-affinity I domain from the integrin alpha(L)beta(2) (LFA-1) alpha subunit in complex with the N-terminal two domains of ICAM-5, an adhesion molecule expressed in telencephalic neurons. The finding that the alpha7 helix swings out and inserts into a neighboring I domain in an upside-down orientation in the crystals implies an intrinsically unusual mobility of this helix. This remarkable feature allows the alpha7 helix to trigger integrin's large-scale conformational changes with little energy penalty. It serves as a mechanistic example of how a weakly bound adhesion molecule works in signaling.     

Flexibility of the MHC class II peptide binding cleft in the bound, partially filled, and empty states: a molecular dynamics simulation study

Major histocompatibility (MHC) Class II cell surface proteins present antigenic peptides to the immune system. Class II structures in complex with peptides but not in the absence of peptide are known. Comparative molecular dynamics (MD) simulations of a Class II protein (HLA-DR3) with and without CLIP (invariant chain-associated protein) peptide were performed starting from the CLIP-bound crystal structure. Depending on the protonation of acidic residues in the P6 peptide-binding pocket the simulations stayed overall close to the start structure. The simulations without CLIP showed larger conformational fluctuations especially of alpha-helices flanking the binding cleft. Largest fluctuations without CLIP were observed in a helical segment near the peptide C-terminus binding region matching a segment recognized by antibodies specific for empty Class II proteins. Simulations on a Val86Tyr mutation that fills the peptide N-terminus binding P1 pocket or of a complex with a CLIP fragment (dipeptide) bound to P1 showed an unexpected long range effect. In both simulations the mobility not only of P1 but also of the entire binding cleft was reduced compared to simulations without CLIP. It correlates with the experimental finding that the CLIP fragment binding to P1 is sufficient to prevent antibody recognition specific for the empty form at a site distant from P1. The results suggest a mechanism how a local binding event of small peptides or of an exchange factor near P1 may promote peptide binding and exchange through a long range stabilization of the whole binding cleft in a receptive (near bound) conformation.     

4-Amino-6-arylamino-pyrimidine-5-carbaldehyde hydrazones as potent ErbB-2/EGFR dual kinase inhibitors

Members of a novel class of 4-amino-6-arylamino-pyrimidine-5-carbaldehyde hydrazones were identified as potent dual ErbB-2/EGFR kinase inhibitors using concept-guided design approach. These compounds inhibited the growth of ErbB-2 over-expressing human tumor cell lines (BT474, N87, and SK-BR-3) in vitro. Compound 15 emerged as a key lead and showed significant ability to inhibit growth factor-induced receptor phosphorylation in SK-BR-3 cells (IC(50)=54 nM) and cellular proliferation in vitro (IC(50)=14, 58, and 58 nM for BT474, N87, and SK-BR-3 respectively). The X-ray co-crystal structure of EGFR with a close analog (17) was determined and validated our design rationale.     

Effect of Nitrogen Fertilization and Residual Nitrogen on Biomass Yield of Switchgrass

Switchgrass, Panicum virgatum L., grown for biomass has been extensively researched where the annual precipitation >760 mm and the climate varies from humid to moist-subhumid. Research is needed for areas that receive <700 mm of precipitation, where the climate varies from dry-subhumid to semiarid. The objectives were to determine (1) the effect of nitrogen fertilization on biomass production, (2) the effect of residual nitrogen on biomass production, (3) the nitrogen yield from harvested biomass, and (4) the concentration of soil organic carbon (SOC) from switchgrass plots. Plots were fertilized annually with nitrogen at the rates of 0, 40, 80, and 120 kg ha^?1 from 2008 to 2011 and unfertilized from 2012 to 2015. The biomass yield varied with N rate × production year interactions (P < 0.05), and biomass yield as a function of N rate was either linear or curvilinear depending upon production year. When fertilized, the biomass yield averaged 4.4, 9.4, 11.6, and 13.2 ± 0.4 Mg ha^?1 for the 0, 40, 80, and 120 kg ha^?1 N rates, respectively. Residual nitrogen sustained high biomass yields for 1 year after fertilization ceased. The nitrogen harvested in biomass varied with N rate × production year interactions (P < 0.05), and the harvested nitrogen yield as a function of N rate was linear each year. Fertilization increased the concentration of SOC an average of 1.0 ± 0.2 mg g^?1 of soil. The data suggest that producers could occasionally skip a year of nitrogen fertilization without detrimentally impacting the production of switchgrass biomass.     

The Quantitative Methods Boot Camp: Teaching Quantitative Thinking and Computing Skills to Graduate Students in the Life Sciences

The past decade has seen a rapid increase in the ability of biologists to collect large amounts of data. It is therefore vital that research biologists acquire the necessary skills during their training to visualize, analyze, and interpret such data. To begin to meet this need, we have developed a “boot camp” in quantitative methods for biology graduate students at Harvard Medical School. The goal of this short, intensive course is to enable students to use computational tools to visualize and analyze data, to strengthen their computational thinking skills, and to simulate and thus extend their intuition about the behavior of complex biological systems. The boot camp teaches basic programming using biological examples from statistics, image processing, and data analysis. This integrative approach to teaching programming and quantitative reasoning motivates students’ engagement by demonstrating the relevance of these skills to their work in life science laboratories. Students also have the opportunity to analyze their own data or explore a topic of interest in more detail. The class is taught with a mixture of short lectures, Socratic discussion, and in-class exercises. Students spend approximately 40% of their class time working through both short and long problems. A high instructor-to-student ratio allows students to get assistance or additional challenges when needed, thus enhancing the experience for students at all levels of mastery. Data collected from end-of-course surveys from the last five offerings of the course (between 2012 and 2014) show that students report high learning gains and feel that the course prepares them for solving quantitative and computational problems they will encounter in their research. We outline our course here which, together with the course materials freely available online under a Creative Commons License, should help to facilitate similar efforts by others.This is part of the PLOS Computational Biology Education collection.     

Endothelial cells proactively form microvilli-like membrane projections upon intercellular adhesion molecule 1 engagement of leukocyte LFA-1

Specific leukocyte/endothelial interactions are critical for immunity and inflammation, yet the molecular details of this interaction interface remain poorly understood. Thus, we investigated, with confocal microscopy, the distribution dynamics of the central adhesion molecules ICAM-1 and LFA-1 in this context. Monolayers of activated HUVECs stained with fluorescent anti-ICAM-1 Fabs or Chinese hamster ovary-K1 cells expressing ICAM-1-green fluorescent protein were allowed to bind LFA-1-bearing monocytes, neutrophils, or K562 LFA-1 transfectants. ICAM-1 was rapidly relocalized to newly formed microvilli-like membrane projections in response to binding LFA-1 on leukocytes. These ICAM-1-enriched projections encircled the leukocytes extending up their sides and clustered LFA-1 underneath into linear tracks. Projections formed independently of VCAM-1/very late Ag 4 interactions, shear, and proactive contributions from the LFA-1-bearing cells. In the ICAM-1-bearing endothelial cells, projections were enriched in actin but not microtubules, required intracellular calcium, and intact microfilament and microtubule cytoskeletons and were independent of Rho/Rho kinase signaling. Disruption of these projections with cytochalasin D, colchicine, or BAPTA-AM had no affect on firm adhesion. These data show that in response to LFA-1 engagement the endothelium proactively forms an ICAM-1-enriched cup-like structure that surrounds adherent leukocytes but is not important for firm adhesion. This finding leaves open a possible role in leukocyte transendothelial migration, which would be consistent with the geometry and kinetics of formation of the cup-like structure.     

Introduction to the background, principles, and state of the art in suicide gene therapy

Gene therapy is defined as a technology that aims to modify the genetic component of cells to gain therapeutic benefits. Suicide gene therapy (or gene-directed enzyme prodrug therapy [GDEPT]) is a two-step treatment for cancer (especially, solid tumors). In the first step, a gene for a foreign enzyme is delivered to the tumor by a vector. Following the expression of the foreign enzyme, a prodrug is administered during the second step, which is selectively activated in the tumor. This article discusses the principles and the theoretical background of GDEPT. A special emphasis is put on enzyme/prodrug systems developed for GDEPT, the design of prodrugs and the kinetic of their activation, the types and the mechanisms of bystander effect and its immunological implications. The possible strategies to improve GDEPT are also discussed.     

Correlation between troglitazone cytotoxicity and drug metabolic enzyme activities in cryopreserved human hepatocytes

Troglitazone (TRO) was developed for the treatment of type II diabetes. It was withdrawn from use due to idiosyncratic liver damage and failure. The mechanism of toxicity is still not determined, moreover, it is still not clear whether toxicity is due to the parent compound or its metabolite(s). The cytotoxicity of TRO was evaluated in human hepatocytes using previously cryopreserved hepatocyte suspensions from 27 human donors. Cellular adenosine triphosphate content was used as a viability endpoint. To investigate the role of xenobiotic metabolism in TRO toxicity, the correlation between the drug metabolism activities of the hepatocytes from each donor to EC(50) values TRO cytotoxicity. The activities examined were cytochrome P450 (CYP) isoform activities (CYP2A6, CYP2D6, CYP2C19, CYP1A2, CYP2E1, CYP3A4 and CYP2C9) and phase 2 conjugation enzyme activities (phenol sulfotransferase (PST) and glucuronyl transferase (UGT)). Taken individually, none of the phase 1 or 2 enzyme activities correlated to the EC(50). However, when three enzyme activities ((CYP3A4 x UGT)/PST) were taken into account, a correlation was made (r(2)=0.53). Based on the correlation, we hypothesize that TRO and TRO sulfate are direct acting toxicants, whereas CYP3A4 oxidation and glucuronidation are detoxification pathways.     

The lateral organ boundaries gene defines a novel,plant-specific gene family

The LATERAL ORGAN BOUNDARIES (LOB) gene in Arabidopsis defines a new conserved protein domain. LOB is expressed in a band of cells at the adaxial base of all lateral organs formed from the shoot apical meristem and at the base of lateral roots. LOB encodes a predicted protein that does not have recognizable functional motifs, but that contains a conserved domain (the LOB domain) that is present in 42 other Arabidopsis proteins and in proteins from a variety of other plant species. Proteins showing similarity to the LOB domain were not found outside of plant databases, indicating that this unique protein may play a role in plant-specific processes. Genes encoding LOB domain proteins are expressed in a variety of temporal- and tissue-specific patterns, suggesting that they may function in diverse processes. Loss-of-function LOB mutants have no detectable phenotype under standard growth conditions, suggesting that LOB is functionally redundant or required during growth under specific environmental conditions. Ectopic expression of LOB leads to alterations in the size and shape of leaves and floral organs and causes male and female sterility. The expression of LOB at the base of lateral organs suggests a potential role for LOB in lateral organ development.