X-ray crystallography: Assessment and validation of protein-small molecule complexes for drug discovery

INTRODUCTION: Crystallography is the key initial component for structure-based and fragment-based drug design and can often generate leads that can be developed into high potency drugs. Therefore, huge sums of money are committed based on the outcome of crystallography experiments and their interpretation. AREAS COVERED: This review discusses how to evaluate the correctness of an X-ray structure, focusing on the validation of small molecule-protein complexes. Various types of inaccuracies found within the PDB are identified and the ramifications of these errors are discussed. The reader will gain an understanding of the key parameters that need to be inspected before a structure can be used in drug discovery efforts, as well as an appreciation of the difficulties of correctly interpreting electron density for small molecules. The reader will also be introduced to methods for validating small molecules within the context of a macromolecular structure. EXPERT OPINION: One of the reasons that ligand identification and positioning, within a macromolecular crystal structure, is so difficult is that the quality of small molecules widely varies in the PDB. For this reason, the PDB can not always be considered a reliable repository of structural information pertaining to small molecules, and this makes the derivation of general principles that govern small molecule-protein interactions more difficult.     

Proximal row carpectomy in treatment of post traumatic changes]

In surgical approaches in disease of proximal row of the carpus, authors review the method of proximal row carpectomy. This procedure is among limited number of alternatives for surgical treatment of posttraumatic changes of the carpus. Recalling literature upon this topic. The authors describe their clinical case.     

Medulloblastoma: histological evaluation and prognosis

Eighty-four cases of medulloblastoma were examined immunohistochemically and 12 by electron microscopy to assess differentiation in these tumors. Based upon the largest series of medulloblastoma studied to date, we demonstrated glial fibrillary acidic protein (GFAP) positivity, in 25% (21/84) of these tumors showing glial differentiation. GFAP-positive cells were seen more frequently in the desmoplastic variant of medulloblastoma (7/10). Under electron microscopy, the major part of the 12 tumors studied appeared primitive and undifferentiated. In 7 cases, groups of cells were found with primitive neuronal and/or glial features. GFAP positivity was confirmed at light microscopy level in all cases where cells showed glial differentiation in the form of glial-like filaments in cytoplasma. However, a follow-up questionnaire study of those patients who had received only surgical treatment revealed no difference in mean survival time between GFAP-positive and GFAP-negative medulloblastoma.Supported by the Governmental Grant PR-06 for Fighting Cancer     

Depressive symptoms and taste reactivity in humans

Animal studies suggest that induction of depression-like states may alter preference for sweet tastants. A major goal of the present study was to search for correlations between depressive symptoms measured by the Beck Depression Inventory (BDI) and taste responses to sweet and bitter substances. Thirty-three nonclinical volunteers rated intensity and pleasantness of chocolate and vanilla milk as well as of sucrose- and quinine-soaked filter paper disks. Reactivity to citric acid (sour) and sodium chloride (salty) was also tested with the paper disk methodology. Taste detection thresholds were assessed by means of electrogustometry. A weak inverse relationship was found between the BDI scores (range: 3-33) and rated intensity of paper disks soaked in 60% sucrose. No correlations were found between depressive symptoms and intensity, pleasantness or identification of the other samples. Similarly, there was no relationship between the BDI scores and responses to chocolate and vanilla milk. BDI scores were not associated with electrogustometric thresholds. These data suggest that depressive symptoms may not influence taste reactivity in nonclinical population.     

NAHR-mediated copy-number variants in a clinical population: Mechanistic insights into both genomic disorders and Mendelizing traits

We delineated and analyzed directly oriented paralogous low-copy repeats (DP-LCRs) in the most recent version of the human haploid reference genome. The computationally defined DP-LCRs were cross-referenced with our chromosomal microarray analysis (CMA) database of 25,144 patients subjected to genome-wide assays. This computationally guided approach to the empirically derived large data set allowed us to investigate genomic rearrangement relative frequencies and identify new loci for recurrent nonallelic homologous recombination (NAHR)-mediated copy-number variants (CNVs). The most commonly observed recurrent CNVs were NPHP1 duplications (233), CHRNA7 duplications (175), and 22q11.21 deletions (DiGeorge/velocardiofacial syndrome, 166). In the ∼25% of CMA cases for which parental studies were available, we identified 190 de novo recurrent CNVs. In this group, the most frequently observed events were deletions of 22q11.21 (48), 16p11.2 (autism, 34), and 7q11.23 (Williams-Beuren syndrome, 11). Several features of DP-LCRs, including length, distance between NAHR substrate elements, DNA sequence identity (fraction matching), GC content, and concentration of the homologous recombination (HR) hot spot motif 5′-CCNCCNTNNCCNC-3′, correlate with the frequencies of the recurrent CNVs events. Four novel adjacent DP-LCR-flanked and NAHR-prone regions, involving 2q12.2q13, were elucidated in association with novel genomic disorders. Our study quantitates genome architectural features responsible for NAHR-mediated genomic instability and further elucidates the role of NAHR in human disease.Copy-number variants (CNVs) are an important cause of multiple genomic disorders (Stankiewicz and Lupski 2010; Girirajan et al. 2011). One major mechanism responsible for CNV formation is nonallelic homologous recombination (NAHR) (Stankiewicz and Lupski 2002), which occurs between two paralogous low-copy repeats (LCRs) or segmental duplications (Bailey et al. 2002). Utilizing directly oriented paralogous LCR (DP-LCR) copies in cis as recombination substrates for ectopic crossovers, NAHR can lead to recurrent genomic deletions and reciprocal duplications. Recent evidence suggests a greater than twofold genome-wide enrichment for CNVs between DP-LCRs (Li et al. 2012). NAHR events in trans between LCRs on nonhomologous chromosomes can cause recurrent constitutional translocations (Giglio et al. 2002; Ou et al. 2011). For LCRs in inverted orientation, Dittwald et al. (2013) showed that 12.0% of the human genome is potentially susceptible to NAHR-mediated inversions between inverse paralogous LCRs, with 942 genes (99 of which are on the X chromosome) predicted to be disrupted secondary to such an inversion. Locus-specific studies have shown that LCR size is correlated with NAHR frequency, suggesting that ectopic synapsis precedes ectopic crossing-over (Liu et al. 2012).To date, ∼40 nonoverlapping genomic loci with deletion and/or reciprocal duplication associated with known syndromes have been identified as genomic disorders (Lupski 1998, 2009; Mefford 2009; Liu et al. 2012; Vissers and Stankiewicz 2012). Bioinformatic analyses have revealed many more regions of genomic instability in the human genome that are potentially prone to recurrent DNA rearrangements via NAHR; some of them may be pathogenic, but their phenotypic consequences remain to be elucidated.Using genome-wide bioinformatic analyses in the human genome build hg16 (July 2003), Sharp et al. (2005) predicted 130 genomic intervals flanked by DP-LCRs >10 kb in size, of >95% DNA sequence identity, with the distance between the DP-LCRs ranging from 0.05–10 Mb. Using the same parameters for bioinformatic analyses of the genome build hg19 (February 2009), Liu et al. (2012) identified 608 intervals that collapsed into 89 regions prone to DP-LCR/NAHR. Most of the differences between these data sets result from the different DP-LCRs identified in these genome builds as well as various methods for collapsing the overlapping regions.Here, we constructed bioinformatically a new genome-wide map of the DP-LCR-flanked regions in human genome build hg19 using a concept of LCR clusters. We then queried and cross-referenced our database of 25,144 high-resolution genomic analyses performed on patients referred for chromosomal microarray analysis (CMA) (Cheung et al. 2005). This approach enabled us to determine the relative frequencies in this clinical population of known recurrent genomic disorders and also to quantitate genome-wide genomic architectural features that are associated with individual locus events, to gain insights into the parameters rendering genomic instability. The frequency for ascertaining these genomic disorders varies dramatically and, as predicted previously, may reflect genome architecture and mechanism. We report the computationally determined genomic features that correlate with the empirically observed frequency of de novo recurrent rearrangements and further test, on a genome-wide scale, the “ectopic synapsis precedes ectopic crossing-over” hypothesis.     

Association of human leukocyte antigen ancestral haplotype 8.1 with adverse outcome of non-Hodgkin's lymphoma

Previous reports have implicated the tumor necrosis factor (TNF-308) locus to non-Hodgkin's lymphoma (NHL) outcome. The purpose of the study was to examine other chromosome components of the HLA 8.1 ancestral haplotype (AH) and their relation to the clinical course of NHL. HLA class I, II, TNF-308, and lymphotoxin alpha (LTA+252) alleles were analyzed in 154 newly diagnosed NHL patients. Three locus haplotypes were inferred from the unphased genotypes by a Bayesian implementation of the expectation maximization (EM) algorithm using the PHASE 2.1 program. TNF-308A was the only allele associated with fever, poor performance status, elevated beta2-microglobulin, TNF and its p75 receptor plasma levels. Although TNF-308A was in strong linkage disequilibrium with the remaining alleles of 8.1 AH, only HLA-A*01 and HLA-B*08 showed association with prognostic variables. A part of 8.1 AH (A*01-B*08-TNF-308A) was predictive for shorter freedom from progression and overall survival (RR=2.47, P=0.041; RR=3.15; P=0.0049), an association that was stronger than TNF-308A alone and independent from International Prognostic Index (RR=1.55, P<0.001; RR=2.36; P<0.0001). A*01-B*08-TNF-308A fragment of 8.1 AH remained an independent predictive factor in a multivariate model. We conclude that 8.1 AH is an important contributor to NHL outcome. In contrast to A*01-B*08-TNF(-308A, the remaining alleles (Cw*07, DRB1*03, LTA+252G) associated with the 8.1 AH seem to be its passive components.     

Living-donor kidney transplantation: the Freiburg experience

Background and aim The objective of this study was to determine outcome after living-donor kidney transplantation in a single-center institution in Germany. Materials and methods From 1976 to May 2005, a total of 298 living-donor kidney transplants were performed at the University of Freiburg. Most recipients (78.8%) were placed on cyclosporine, mycophenolate mofetil, and corticosteroids maintenance immunosuppression. Cox proportional hazard model was applied to analyze predictors for patient and graft survival. Mean follow-up was 5.3 years. Results According to Kaplan–Meier calculation, 1-, 5-, and 10-year patient survival was 98.6, 92.7, and 86.8%, respectively. Kidney function rate was 95.5, 82.8, and 67.9%, respectively. A 5-year graft function rate continued to increase from 79.5% in patients transplanted before 1996 to 83.6% in patients transplanted thereafter. In a Cox regression model recipient age above 50 years, duration of dialysis above 2 years and preexisting type 1 diabetes mellitus were associated with a decreased patient survival. Graft survival was mostly influenced by the type of immunosuppression and preexisting hypertension of the recipient. Conclusions Our results demonstrate that living-donor kidney transplantation is a highly effective therapy for patients with end stage renal failure. Updates in immunosuppression, recipient selection, and operative technique may have contributed to the improved graft survival over the past three decades.     

Dopamine receptor supersensitivity: Development, mechanisms, presentation, and clinical applicability

The process of receptor supersensitivity (RSS) has a long history and is an epiphenomenon of neuronal denervation. Dopamine (DA) RSS (DARSS) similarly occurs after DA-denervation, and this process is invoked in neuropsychiatric and neurodegenerative disorders. From studies largely over the past 25 years, much has been learned regarding DARSS. For example, overt D₁ DARSS occurs after perinatal destruction of nigrostriatal DA fibers. However, following perinatal destruction of DA innervation, the mostprominent behavioral effects of a D₁ agonist are observed after a series of D₁ agonist treatments- a process known aspriming of D₁DA receptors. Moreover, perinatal lesioning of DA fibers produces prominent serotonin (5-HT) RSS, and in fact 5-HT RSS appears to modulate D₁ DA RSS. In rodents, receptor supersensitization by these means appears to be irreversible. In contrast to the observedD ₁ DARSS, D₂ DARSS apparently does not occur after perinatal DA denervation. Also, while repeated D₁ agonist treatment of intact rats has no observable effect, repeated D₂ agonist treatments, during or after the ontogenetic phase, produces prominent life-long D₂ RSS. The process may have an association with substance abuse. Therefore, production of D₁ and D₂ DARSS occurs by different means and under different circumstances, and in association with perhaps different neuronal phenotypes, and with greater incidence in either intact (D₂) or DA-lesioned counterparts (D₁). The physiological consequence of RSS are multiple.