A Markov decision process approach to temporal modulation of dose fractions in radiation therapy planning.

The current state of the art in cancer treatment by radiation optimizes beam intensity spatially such that tumors receive high dose radiation whereas damage to nearby healthy tissues is minimized. It is common practice to deliver the radiation over several weeks, where the daily dose is a small constant fraction of the total planned. Such a 'fractionation schedule' is based on traditional models of radiobiological response where normal tissue cells possess the ability to repair sublethal damage done by radiation. This capability is significantly less prominent in tumors. Recent advances in quantitative functional imaging and biological markers are providing new opportunities to measure patient response to radiation over the treatment course. This opens the door for designing fractionation schedules that take into account the patient's cumulative response to radiation up to a particular treatment day in determining the fraction on that day. We propose a novel approach that, for the first time, mathematically explores the benefits of such fractionation schemes. This is achieved by building a stylistic Markov decision process (MDP) model, which incorporates some key features of the problem through intuitive choices of state and action spaces, as well as transition probability and reward functions. The structure of optimal policies for this MDP model is explored through several simple numerical examples.

Mu-opioid receptor activation induces transcriptional plasticity in the central extended amygdala.

Addiction develops from the gradual adaptation of the brain to chronic drug exposure, and involves genetic reprogramming of neuronal function. The central extended amygdala (EAc) is a network formed by the central amygdala and the bed nucleus of the stria terminalis. This key site controls drug craving and seeking behaviors, and has not been investigated at the gene regulation level. We used Affymetrix microarrays to analyze transcriptional activity in the murine EAc, with a focus on mu-opioid receptor-associated events because these receptors mediate drug reward and dependence. We identified 132 genes whose expression is regulated by a chronic escalating morphine regimen in the EAc from wild-type but not mu-opioid receptor knockout mice. These modifications are mostly EAc-specific. Gene ontology analysis reveals an overrepresentation of neurogenesis, cell growth and signaling protein categories. A separate quantitative PCR analysis of genes in the last of these groups confirms the dysregulation of both orphan (Gpr88) and known (DrD1A, Adora2A, Cnr1, Grm5, Gpr6) G protein-coupled receptors, scaffolding (PSD95, Homer1) and signaling (Sgk, Cap1) proteins, and neuropeptides (CCK, galanin). These transcriptional modifications do not occur following a single morphine injection, and hence result from long-term adaptation to excessive mu receptor activation. Proteins encoded by these genes are classically associated with spine modules function in other brain areas, and therefore our data suggest a remodeling of EAc circuits at sites where glutamatergic and monoaminergic afferences interact. Together, mu receptor-dependent genes identified in this study potentially contribute to drug-induced neural plasticity, and provide a unique molecular repertoire towards understanding drug craving and relapse.

Transcriptome analysis identifies genes with enriched expression in the mouse central extended amygdala.

The central extended amygdala (EAc) is an ensemble of highly interconnected limbic structures of the anterior brain, and forms a cellular continuum including the bed nucleus of the stria terminalis (BNST), the central nucleus of the amygdala (CeA) and the nucleus accumbens shell (AcbSh). This neural network is a key site for interactions between brain reward and stress systems, and has been implicated in several aspects of drug abuse. In order to increase our understanding of EAc function at the molecular level, we undertook a genome-wide screen (Affymetrix) to identify genes whose expression is enriched in the mouse EAc. We focused on the less-well known BNST-CeA areas of the EAc, and identified 121 genes that exhibit more than twofold higher expression level in the EAc compared with whole brain. Among these, 43 genes have never been described to be expressed in the EAc. We mapped these genes throughout the brain, using non-radioactive in situ hybridization, and identified eight genes with a unique and distinct rostro-caudal expression pattern along AcbSh, BNST and CeA. Q-PCR analysis performed in brain and peripheral organ tissues indicated that, with the exception of one (Spata13), all these genes are predominantly expressed in brain. These genes encode signaling proteins (Adora2, GPR88, Arpp21 and Rem2), a transcription factor (Limh6) or proteins of unknown function (Rik130, Spata13 and Wfs1). The identification of genes with enriched expression expands our knowledge of EAc at a molecular level, and provides useful information to toward genetic manipulations within the EAc.

Gene expression is altered in the lateral hypothalamus upon activation of the mu opioid receptor.

The lateral hypothalamus (LH) is a brain structure that controls hedonic properties of both natural rewards and drugs of abuse. Mu opioid receptors are known to mediate drug reward, but whether overstimulation of these receptors impacts on LH function has not been studied. Here we have used a genome-wide microarray approach to identify LH responses to chronic mu opioid receptor activation at the transcriptional level. We have subjected wild-type and mu opioid receptor knockout mice to an escalating morphine regimen, which produces severe physical dependence in wild-type but not mutant animals. We have analyzed gene profiles in LH samples using the 430A.2 Affymetrix array and identified a set of 25 genes whose expression is altered by morphine in wild-type mice only. The regulation was confirmed for a subset of these genes using real-time quantitative PCR on samples from independent treatments. Altered expression of aquaporin 4, apolipoprotein D, and prostaglandin synthase is indicative of modified LH physiology. The regulation of two signaling genes (the serum glucocorticoid kinase and the regulator of G protein signaling 4) suggests that neurotransmission is altered in LH circuitry. Finally, the downregulation of apelin may indicate a potential role for this neuropeptide in opioid signaling and hedonic homeostasis. Altogether, our study shows that chronic mu opioid receptor stimulation induces gene expression plasticity in the LH and provides a unique collection of mu opioid receptor-dependent genes that potentially contribute to alter reward processes in addictive diseases.

Identification of novel striatal genes by expression profiling in adult mouse brain.

Large-scale transcriptome analysis in the brain is a powerful approach to identify novel genes of potential interest toward understanding cerebral organization and function. We utilized the microarray technology to measure expression levels of about 24,000 genes and expressed sequence tags in mouse hippocampus, frontal cortex and striatum. Using expression profile obtained from whole brain as a reference, we categorized the genes into groups of genes either enriched in, or restricted to, one of the three areas of interest. We found enriched genes for each target area. Further, we identified 14 genes in the category of genes restricted to the striatum, among which were the orphan G protein-coupled receptor GPR88 and retinoic acid receptor-beta. These two genes were already reported to be selectively expressed in the striatum, thus validating our experimental approach. We selected 6 striatal-restricted genes, as well as 10 striatal-enriched candidates, that were previously undescribed. We analyzed their expression by in situ hybridization analysis in the brain, and quantitative RT-PCR in both brain and peripheral organs. Two of these unknown genes displayed a notable expression pattern. The striatal-restricted gene H3076B11 shows uniform expression throughout and uniquely in the striatum, representing a genuine striatal marker. The striatal-enriched gene 4833421E05Rik is preferentially expressed in the rostral striatum, and is also abundant in kidney, liver and lung. These two genes may contribute to some of the many striatal-controlled behaviors, including initiation of movement, habit formation, or reward and motivation.

Characterization of antibody-binding sites on proteins: development of a knowledgebase and its applications in improving epitope prediction.

Immunoinformatics provides tools for reverse vaccinology and encompasses development of knowledge bases and algorithms for prediction of epitopes. AgAbDb, a database archiving molecular interactions of antigen-antibody co-crystal structures, has been developed (http://202.41.70.51:8080/agabdb2/). Analyses of antibody-binding sites on proteins helped to fine-tune the parameters for prediction of sequential and conformational B-cell epitopes.

Family planning and voluntary organizations.

PIP: The government of India's family planning program has expanded both in concept and scope. Voluntary organizations have contributed considerably by motivating family planning acceptors and by successfully trying out several alternative approaches. The intention of this discussion is to describe the activities of a selective few family planning organizations that are distinctive in approach and have had a catalytic effect on social change. The descriptions are presented in a somewhat historical perspective. The All India Women's Conference (AIWC) was formally registered as a society in 1930. Initially it was concerned with the education of women, but it soon recognized the need to deal with "repeated and quick pregnancies." In 1946 under a mobile van scheme, "SKIPPO," medical services and later family planning services were taken to rural areas of India. The SKIPPO scheme brought motivation, information and family planning services to rural areas and exhibited and established the need for rapport and continuity of social workers with the rural clientele. The primary contribution of the AIWC has been linking family planning to development through information dissemination and personalizing communication strategies. The Family Planning Association of India (FPAI) began in 1949 as the Family Planning Committee. As of March 1980 FPAI had 109 rural and 59 urban centers. FPAI has over a period of time worked to break new ground and has extended its guidance to the industrial sector. The FPAI does extensive work through its network of rural and urban centers, but work in the rural areas has been taken up more comprehensively and purposefully by 2 other organzations, i.e., the Gandhigram Institute of Rural Health and Family Welfare Trust and the Comprehensive Rural Health Project at Jamkhed. The Family Planning Foundation, founded in 1972, is a funding organization. Through action demonstration projects, the organization worked to fund projects in the following areas: biomedical; information; education; communication, health, and social sciences; and other fields relating to family planning. Currently, the Foundation is funding 50 projects in various fields and its experimental work has received serious consideration at various levels. The wide range of incentives in India at this time basically focus on sterilization. Voluntary organizations have long been active in family planning, but the government sought active collaboration with them in April 1976. Since that time, collaborations have slowly and steadily increased.^ieng

Site-directed mutagenesis of catalytic residues of influenza virus neuraminidase as an aid to drug design.

The neuraminidase of influenza virus is a surface glycoprotein that catalyzes the hydrolysis of glycosidic linkages between terminal sialic acids and adjacent sugar moieties. Neuraminidase function is critical for the spread of virus to new cells, and if the enzyme activity is inhibited, then virus infection is abrogated. The neuraminidase active site is conserved in all influenza type-A and type-B isolates, which makes it an excellent target for drug design. To determine the potential for resistance to develop against neuraminidase inhibitors, we have constructed mutations in seven of the conserved active-site residues of a type B (B/Lee/40) neuraminidase and analyzed the effect of the altered side chains on enzyme activity. There is a reduction in k(cat) in all our mutants. A transition-state analogue inhibitor shows variation in Ki with the mutant neuraminidases, allowing interpretation of the effects of mutation in terms of transition-state binding and product release. The results show that Tyr409 is the most critical residue for enzyme activity, but that Asp149, Arg223, Glu275 and Arg374 also play important roles in enzyme catalysis. Based on the pH profile of neuraminidase activity of the D149E mutant protein, we conclude that Asp149 is not a proton donor, but is involved in stabilizing the transition state. If designed inhibitors are targeted to these residues where mutations are highly deleterious, particularly Tyr409, Glu275 and Asp149, the virus is unlikely to generate resistance to the drug.

Influenza type B neuraminidase can replace the function of type A neuraminidase.

Influenza A and B viruses do not form reassortants with each other, presumably due to selection at either the RNA or protein level. Although differences in the promoter sequences of type A and B viruses have been studied, selection at the protein level has not been addressed. In this paper we describe experiments to determine whether differences in structure and/or function of the neuraminidase (NA) protein preclude formation of A/B NA reassortants. Influenza type A (N9) NA or B/Lee/40 NA expressed from plasmids can support multicycle growth of a NA-deficient type A virus (NWS-Mvi), indicating that their function in tissue culture is similar. To determine whether the type A or B NA supplied in trans can be incorporated into the virion of NWS-Mvi, the virus grown in NA-expressing cells was purified by sucrose gradient centrifugation. In each case there was a peak of NA activity coincident with the virus peak, indicating that some NA protein is packaged into the virion. The experiments suggest that, in spite of large sequence differences, the functions of the head, stalk, signal-anchor, and cytoplasmic domains of type A and B NAs are similar in tissue culture. Thus, lack of formation of A/B NA reassortant viruses is not due to restriction at the protein level.

Cardiotonic activity of the water soluble forskolin derivative 8,13-epoxy-6 beta-(piperidinoacetoxy)- 1 alpha, 7 beta, 9 alpha-trihydroxy-labd- 14en-11-one.

8,13-Epoxy-6 beta-(piperidinoacetoxy)-1 alpha,7 beta, 9 alpha-trihydroxy-labd -14en-11-one (HL 706, CAS 114376-11-3) is a water soluble derivative of forskolin with positive inotropic and vasodilating properties. In both in vitro and in vivo preparations, it exhibited significant positive inotropic activity with concomitant increase in heart rate and decrease in mean blood pressure. Though its potency is lower than that of forskolin, its duration of action is more prolonged. In conscious dog experiments, HL 706, administered orally, also showed a dose related increase in LV dP/dtmax. A significant reversal of cardiac failure was attained in anaesthetised dogs subjected to propranolol induced cardiac insufficiency. HL 706, like forskolin increased cAMP by virtue of its adenylate cyclase stimulant activity. Through increase in cAMP it also exhibited non-specific smooth muscle relaxant activity in isolated vascular and ileal preparations. Its therapeutic ratio is quite favourable.

Hydroxyacyl derivatives of forskolin--their positive inotropic activity.

Using appropriate protection and deprotection sequence novel hydroxyacyl chains of the type CO(CH2)nOH are synthesized and are utilized to develop new analogues of forskolin. Several compounds showed good positive inotropic activity. Compound 12 is almost 10 times more active than forskolin (EC50 = 0.002 microgram/ml).].

In search of novel water soluble forskolin analogues for positive inotropic activity.

Using the novel lead from hydroxy acetyl substituted forskolin analogues, such as 7 beta-hydroxyacetyl-7 beta-deacetyl forskolin or 6 beta-hydroxyacetyl forskolin, a number of water soluble omega-amino acyl derivatives were synthesized. Two such compounds 6 and 18 showed better in vitro activity but failed to show in vivo activity.

Generation and characterization of a mutant of influenza A virus selected with the neuraminidase inhibitor BCX-140.

Influenza neuraminidase (NA) plays an important role in viral replication, and characterization of viruses resistant to NA inhibitors will help elucidate the role of active-site residues. This information will assist in designing better inhibitors targeted to essential active-site residues that cannot generate drug-resistant mutations. In the present study we used the benzoic acid-based inhibitor BCX-140 to select and characterize resistant viruses. BCX-140 binds to the NA active site in an orientation that is opposite that of a sialic acid-based compound, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GANA). Thus, the guanidino group of BCX-140 binds to Glu-276, whereas in GANA the guanidino group binds to Glu-119. We passaged influenza A/Singapore/1/57 (H2N2) in Madin-Darby canine kidney cells in the presence of BCX-140, and virus resistant to this inhibitor was selected after six passages. The NA of this mutant was still sensitive to inhibition by BCX-140. However, the mutant virus was resistant to BCX-140 in plaque and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Sequence analysis of hemagglutinin (HA) and NA genes revealed changes in both, although none were in the active site of the NA. Depending on the method of selection of the resistant virus, two types of changes associated with the sialic acid binding site were seen in the HA. One is a change in HA1 of Ala-133 to Thr, a residue close to the binding site, while the other change was Arg-132 of HA1 to Gln, which in HA1 of serotype H3 is a sialic acid contact (Asn-137). Binding studies revealed that both types of resistant viruses had reduced receptor binding affinity compared to that of the wild type. Thus, resistance to BCX-140 was generated by modifying the HA. NA active-site residue 276 may be essential for activity, and thus, it cannot be changed to generate resistance. However, drug-induced changes in the HA can result in a virus that is less dependent on NA activity for growth in cells and, hence, resistant to NA inhibitors.

Editing of the mitochondrial small subunit rRNA in Physarum polycephalum.

Post-transcriptional insertion, substitution or deletion of nucleotides in RNA (RNA editing) has been observed in RNAs from a number of organisms but always in messenger RNA or transfer RNA. We report here that the 17S rRNA of the mitochondrial ribosome of Physarum polycephalum is edited at 40 sites with single cytidine insertions. The locations of the editing sites are fairly evenly distributed throughout the RNA and do not correspond to any obvious feature of the primary sequence or secondary structure. In addition to these cytidine editing sites are editing sites in which a nucleotide other than cytidine is inserted. At two sites a uridine is inserted and at two sites adenosine residues are inserted. This is the first report of mixed nucleotide insertional editing. These results imply that the editing mechanism in Physarum may be different from those proposed for the kinetoplastid protozoa.

HL 752: a potent and long-acting antispasmodic agent.

Ester analogues of methyl-2-(4-(2-piperidinoethoxy)benzoyl)-benzoate hydrochloride (pitofenone) (2) were prepared with an aim to find a more potent and metabolically stable antispasmodic compound. The compounds were evaluated for their in vitro and in vivo antispasmodic activity, and stability to in vitro enzymatic hydrolysis. Of the compounds synthesised, HL 752 (21) showed the most potent and long-lasting antispasmodic activity and was selected as the candidate for clinical development.