Design and Analysis of Multiple Events Case–Control Studies

Summary In case–control research where there are multiple case groups, standard analyses fail to make use of all available information. Multiple events case–control (MECC) studies provide a new approach to sampling from a cohort and are useful when it is desired to study multiple types of events in the cohort. In this design, subjects in the cohort who develop any event of interest are sampled, as well as a fraction of the remaining subjects. We show that a simple case–control analysis of data arising from MECC studies is biased and develop three general estimating‐equation‐based approaches to analyzing data from these studies. We conduct simulation studies to compare the efficiency of the various MECC analyses with each other and with the corresponding conventional analyses. It is shown that the gain in efficiency by using the new design is substantial in many situations. We demonstrate the application of our approach to a nested case–control study of the effect of oral sodium phosphate use on chronic kidney injury with multiple case definitions.     

A population-based study of soft tissue sarcoma incidence and survival in Australia: An analysis of 26,970 cases

BackgroundSoft tissue sarcomas (STS) are rare, often fatal tumors, but little is known of the epidemiology and survival in the Australian population. This study aims to provide the first epidemiological analysis of incidence and survival rates of STS in the Australian population.MethodsA retrospective population-based observational study was conducted between 1982 and 2009 of all patients with a diagnosis of STS using the Australian Institute of Health and Welfare (AIHW) Australian Cancer Database. Incidence rates per 100,000; incidence rate ratios, age-standardized incidence rates, prevalence and incidence rates of subtypes of STS, median, one-year and 5-year survival rates were examined.ResultsA total of 26,970 patients were identified. Between 1982 and 2009 STS incidence rates significantly increased from 3.99 [95% CI 3.68–4.32] to 6.12 [95% CI 5.80–6.46] per 100,000 Australian population, with a peak incident rate ratio (IRR) of 1.59 [95% CI 1.51–1.69] (p < 0.0001) in 2001. Median age at diagnosis increased from 58 to 63 years. Incidence rates were stable across all 10-year age cohorts, except for people aged over 70 where it increased. Overall, age-standardized incidence rates increased from 4.70 [95% CI 4.42–5.00] in 1982 to 5.87 [95% CI 5.63–6.11] per 100 000 Australians in 2009. Leiomyosarcoma (20.43%), malignant fibrous histiocytoma (16.14%), and soft tissue tumors/sarcomas, not otherwise specified (10.18%) were the most common STS subtypes. Median survival from diagnosis increased from 5.80 years [95% CI 5.06–6.54] in 1985–1989 cohort to 8.18 years [95% CI 7.54–8.81] in the 2000–2004 cohort (log-rank test p < 0.0001).ConclusionThe incidence of STS is increasing in Australia, most noticeably in those aged over 70 years, with a small but statistically significant increase in overall survival rates.     

Weighted Likelihood Method for Grouped Survival Data in Case–Cohort Studies with Application to HIV Vaccine Trials

Summary Grouped failure time data arise often in HIV studies. In a recent preventive HIV vaccine efficacy trial, immune responses generated by the vaccine were measured from a case–cohort sample of vaccine recipients, who were subsequently evaluated for the study endpoint of HIV infection at prespecified follow‐up visits. Gilbert et al. (2005, Journal of Infectious Diseases 191 , 666–677) and Forthal et al. (2007, Journal of Immunology 178, 6596–6603) analyzed the association between the immune responses and HIV incidence with a Cox proportional hazards model, treating the HIV infection diagnosis time as a right‐censored random variable. The data, however, are of the form of grouped failure time data with case–cohort covariate sampling, and we propose an inverse selection probability‐weighted likelihood method for fitting the Cox model to these data. The method allows covariates to be time dependent, and uses multiple imputation to accommodate covariate data that are missing at random. We establish asymptotic properties of the proposed estimators, and present simulation results showing their good finite sample performance. We apply the method to the HIV vaccine trial data, showing that higher antibody levels are associated with a lower hazard of HIV infection.     

The validity of self-reported cancer in a population-based cohort compared to that in formally registered sources

BackgroundSelf-reported cancer has been validated with heterogeneous results across populations. The aim was to assess the validity of self-reported cancer in the Lifelines population-based cohort and to search for explanations for not reporting cancer.MethodsData from adult participants (n = 152,780) from Lifelines was linked to the Dutch-Nationwide pathology databank (PALGA), which has nearly 100% coverage of cancer diagnoses in the Netherlands and is considered as the gold standard for ascertainment of cancer diagnosis in this study. Sensitivity and positive predictive value (PPV) for self-reported cancers -reported as hand-written free text- were described. Logistic regressions analyses were performed to evaluate whether socio-demographic factors were associated with the presence of self-reported cancer when there was a diagnosis in PALGA.Results6611 (4.50%) participants had at least one self-reported diagnosis of cancer, where 9960 (6.97%) participants had at least one cancer diagnosis in PALGA. The sensitivity of self-reported cancer was 64.68% [95%CI:63.71–65.66], and 70.18% [95%CI:68.83–71.56] after excluding skin and cervical cancers. Skin and cervical cancers represented 61.24% of non-self-reported cancers. The overall PPV was 97.45% [95%CI:97.45–97.81], and 97.33% [95%CI:96.72–97.82] after the exclusion of skin and cervical cancers. Participants who did not self-report their cancer were more likely to be male, had longer time since diagnosis and lower educational level.ConclusionOverall, the reports of cancer in Lifelines have a high positive predictive value and moderate sensitivity. One third of the cancers were not reported, mainly skin and cervical cancers. Male participants, those with a lower educational level and those with longer time since diagnosis were less likely to self-report a diagnosed cancer.     

A Frailty‐Model‐Based Approach to Estimating the Age‐Dependent Penetrance Function of Candidate Genes Using Population‐Based Case‐Control Study Designs: An Application to Data on the BRCA1 Gene

Summary The population‐based case–control study design is perhaps one of, if not the most, commonly used designs for investigating the genetic and environmental contributions to disease risk in epidemiological studies. Ages at onset and disease status of family members are routinely and systematically collected from the participants in this design. Considering age at onset in relatives as an outcome, this article is focused on using the family history information to obtain the hazard function, i.e., age‐dependent penetrance function, of candidate genes from case–control studies. A frailty‐model‐based approach is proposed to accommodate the shared risk among family members that is not accounted for by observed risk factors. This approach is further extended to accommodate missing genotypes in family members and a two‐phase case–control sampling design. Simulation results show that the proposed method performs well in realistic settings. Finally, a population‐based two‐phase case–control breast cancer study of the BRCA1 gene is used to illustrate the method.     

Ferredoxin:thioredoxin Reductase: Disulfide Reduction Catalyzed via Novel Site-specific [4Fe–4S] Cluster Chemistry

Thioredoxin-mediated light regulation in plant chloroplasts involves a unique class of disulfide reductases that catalyze disulfide reduction in two one-electron steps using a [2Fe–2S] ferredoxin as the electron donor and an active site comprising a [4Fe–4S] cluster and a redox-active disulfide. This review summarizes structural and spectroscopic studies of ferredoxin:thioredoxin reductase (FTR) and a chemically modified form, termed NEM–FTR, which provides a stable analog of the one-electron reduced catalytic intermediate. Detailed spectroscopic characterization of FTR and NEM–FTR using absorption, EPR, electron–nuclear double resonance, variable-temperature magnetic circular dichroism, resonance Raman and Mössbauer spectroscopies indicate that the one-electron reduced catalytic intermediate involves two-electron disulfide reduction coupled with one-electron cluster oxidation of a [4Fe–4S]²⁺ cluster to yield a unique type of S= 1/2 [4Fe–4S]³⁺ cluster with two cysteine residues ligated at a specific Fe site. The results provide the basis for a novel mechanism for disulfide cleavage in two one-electron steps involving site-specific [4Fe–4S] cluster chemistry. A similar mechanism is proposed for direct [4Fe–4S]-mediated cleavage of the CoM–S–S–CoB heterodisulfide in methanogenic archaea by heterodisulfide reductases.     

Rare case of XX/XY mosaicism and trisomy 13 in early prenatal diagnosis

Coexistence of XX/XY sex mosaicism and autosomal trisomy in prenatal diagnosis is particularly rare. Herein, we report the first, to our knowledge, case of a fetus with cyclopia, ambiguous genitalia and a 47,XX,+13,inv9[47]/47,XY,+13[13] karyotype detected at 13 weeks of gestation after chorionic villus sampling. Molecular analysis after prenatal diagnosis suggests that this is a case of sex mosaicism coexisting with trisomy 13, rather than chimera.     

Polyelectrolytic aspects of the thermodynamics of conformational transition: κ‐carrageenan in formamide

A recently published method for the determination of the enthalpy and entropy changes of nonionic origin upon conformational transition of linear biopolyelectrolytes in solution [J. C. Benegas, A. Cesàro, R. Rizzo and S. Paoletti (1998) Biopolymers, Vol. 45, pp. 203–216] has been extended from the case of aqueous salt solutions to that of the organic solvent formamide (FA). The calculation have been applied to the case of the intramolecular transition of the K⁺ salt form of the sulfated polysaccharide κ‐carrageenan. The method proved to be effective in providing the desired data in FA, as it has been previously been successful for the water cases. The comparison between the predicted enthalpy change of transition and the microcalorimetric experimental one turned out to be excellent, thereby ensuring on the validity of the approach. © 1999 John Wiley & Sons, Inc. Biopoly 49: 127–130, 1999     

Diagnostic validation of selected serological tests for detecting scrub typhus

Clinical diagnosis of scrub typhus is often difficult because the symptoms are very similar to those of other febrile illness such as dengue, leptospirosis, malaria and other viral hemorrhagic fevers. Though better diagnostic tests are available for rickettsial diseases and scrub typhus elsewhere, the Weil–Felix test is still commonly used in India, mainly because microimmunofluorescence assays (M‐IFA) were not available in India till recently and relevant staff had insufficient training. The present study was performed to investigate the performance of M‐IFA, IgM ELISA, and Weil–Felix test on 546 non‐repeated serum samples from subjects suspected of having scrub typhus. One hundred and forty‐three of these 546 samples were positive by M‐IFA; these cases were also confirmed clinically to have scrub typhus based on their dramatic responses to doxycycline therapy. IgM ELISA was positive in 122 of the 143 M‐IFA positive cases and the Weil–Felix test in 96. Though the Weil–Felix test is a heterophile agglutination test, it was found in this study to have good specificity but far too little sensitivity to use as a routine diagnostic test. IgM ELISA can be a good substitute for M‐IFA. Incorporation of multiple prototype antigens on M‐IFA slides is likely one of the reasons for its superior performance. As newer and better diagnostic assays become available for scrub typhus diagnosis in developed countries, it will be imperative to also use such tests in other endemic countries to prevent over‐ or under‐diagnosis of scrub typhus.     

Synthesis and application of a targeting diagnosis system via quantum dots coated by amphiphilic polymer for the detection of liver cancer cells

Water‐soluble quantum dots (QDs) for liver cancer diagnosis were prepared using QDs with oleylamine ligand coated with poly(aspartate)–graft–poly(ethylene glycol)–dodecylamine (PASP–Na–g–PEG–DDA). Dynamic light scattering and transmission electron microscopy imaging showed that the novel QDs have an ellipsoidal morphology with a size of ~ 45 nm which could be used for biomedical application. Furthermore, the PASP–Na–g–PEG–DDA was then modified with anti‐(vascular endothelial growth factor) (VEGF antibody), and a 1‐(4,5‐dimethylthiazol‐2‐yl)‐3,5‐diphenylformazan (MTT) assay showed that the novel anti‐VEGF‐targeting QDs in vitro had low toxicity. Confocal laser scanning microscopy observations revealed an intracellular (HepG2) distribution of the novel anti‐VEGF‐targeting QDs and the targeting efficiency of anti‐VEGF. These novel QDs could be used as a probe for liver cancer cell imaging because of anti‐VEGF targeting. Copyright © 2014 John Wiley & Sons, Ltd.     

Secreted phosphoprotein‐24 kDa (Spp24) attenuates BMP‐2‐stimulated Smad 1/5 phosphorylation and alkaline phosphatase induction and was purified in a protective complex with alpha2‐Macroglobulins From Serum

Secreted phosphoprotein‐24 kDa (Spp24) binds cytokines of the bone morphogenetic protein/transforming growth factor‐β (BMP/TGFβ) superfamily and is one of the most abundant serum phosphoproteins synthesized by the liver. Little is known about how Spp24 binding affects BMP signal transduction and osteoblastic differentiation or how this labile protein is transported from the liver to remote tissues, such as bone. When Spp24 was administered to W‐20‐17 mesenchymal stem cells with rhBMP‐2, short‐term Smad1/5 phosphorylation was inhibited, intermediate‐term alkaline phosphatase (ALP) induction was blunted, and long‐term mineralization was unaffected. This supports the hypothesis that Spp24 proteolysis restricts the duration of its regulatory effects, but offers no insight into how Spp24 is transported intact from the liver to bone. When Spp24 was immunopurified from serum and subjected to native PAGE and Western blotting, a high molecular weight band of >500 kDa was found. Under reducing SDS–PAGE, a 24 kDa band corresponding to monomeric Spp24 was liberated, suggesting that Spp24 is bound to a complex linked by disulfide bonds. However, such a complex cannot be disrupted by 60 mM EDTA under non‐reducing condition or in purification buffers containing 600 mM NaCl and 0.1% Tween‐20 at pH 2.7–8.5. LC–MS/MS analysis of affinity‐purified, non‐reducing SDS–PAGE separated, and trypsin digested bands showed that the Spp24 was present in a complex with three α₂‐macroglobulins (α₂‐macroglobulin [α₂M], pregnancy zone protein [PZP] and complement C3 [C3]), as well as ceruloplasmin and the protease inhibitor anti‐thrombin III (Serpin C1), which may protect Spp24 from proteolysis. J. Cell. Biochem. 114: 378–387, 2013. © 2012 Wiley Periodicals, Inc.     

Verification of a proteomic biomarker panel to diagnose minor stroke and transient ischaemic attack: phase 1 of SpecTRA,a large scale translational study

Objective: To derive a plasma biomarker protein panel from a list of 141 candidate proteins which can differentiate transient ischaemic attack (TIA)/minor stroke from non-cerebrovascular (mimic) conditions in emergency department (ED) settings.

Design: Prospective clinical study (#NCT03050099) with up to three timed blood draws no more than 36?h following symptom onset. Plasma samples analysed by multiple reaction monitoring-mass spectrometry (MRM-MS).

Participants: Totally 545 participants suspected of TIA enrolled in the EDs of two urban medical centres.

Outcomes: 90-day, neurologist-adjudicated diagnosis of TIA informed by clinical and radiological investigations.

Results: The final protein panel consists of 16 proteins whose patterns show differential abundance between TIA and mimic patients. Nine of the proteins were significant univariate predictors of TIA [odds ratio (95% confidence interval)]: L-selectin [0.726 (0.596–0.883)]; Insulin-like growth factor-binding protein 3 [0.727 (0.594–0.889)]; Coagulation factor X [0.740 (0.603–0.908)]; Serum paraoxonase/lactonase 3 [0.763 (0.630–0.924)]; Thrombospondin-1 [1.313 (1.081–1.595)]; Hyaluronan-binding protein 2 [0.776 (0.637–0.945)]; Heparin cofactor 2 [0.775 (0.634–0.947)]; Apolipoprotein B-100 [1.249 (1.037–1.503)]; and von Willebrand factor [1.256 (1.034–1.527)]. The scientific plausibility of the panel proteins is discussed.

Conclusions: Our panel has the potential to assist ED physicians in distinguishing TIA from mimic patients.     

Prevalence,comorbidities and mortality of toxic shock syndrome in children and adults in the USA

Toxic Shock Syndrome (TSS), a superantigen‐mediated illness, is characterized by rash, hypotension and multi‐organ dysfunction. Predictors of TSS and related morbidity and mortality are poorly defined. In this study, data on 61,959,084 hospitalizations from the 2003–2012 Nationwide Inpatient Sample, a 20% stratified sample of US hospitalizations, were analyzed and ICD‐9‐CM coding used to identify 4491 hospitalizations with a diagnosis of TSS. Incidence, in‐hospital mortality rate, comorbidities, length of stay and costs of care attributable to TSS were determined. In multivariate survey logistic regression models, TSS was associated with female sex (adjusted odds ratio [95% confidence interval], 1.54 [1.48–1.60]), younger age (0–17 years, 2.17 [2.06–2.29]; 40–59: 0.53 [0.50–0.56]; 60–79: 0.28 [0.26–0.30]; 80+: 0.13 [0.11–0.14] compared with 18–39) and race/ethnicity (black, 0.63 [0.59–0.67]; Hispanic: 0.60 [0.56–0.64]; Asian, 1.11 [1.00–1.11]; and other, 0.83 [0.75–0.92] compared with white). Patients with TSS had a three‐fold greater cost of care (mean: $36,656 ± 942) and length of stay (LOS) (mean: 10.65 ± 0.23 days) than patients without TSS. Shared predictors of increased LOS and costs in patients with TSS were male sex; age 40–79 years; Black, Hispanic, Asian and other race/ethnicity; and more than one chronic condition. Predictors of in‐hospital mortality included respiratory failure (13.66 [11.37–16.43]), liver disease/failure (3.36 [2.59–4.34]), chickenpox (91.26 [27.74–300.25]), coagulopathy (2.14 [1.85–2.48]), and higher age. In conclusion, there are significant racial/ethnic, socioeconomic, and comorbid disparities in the incidence and mortality of TSS in adults and children in the USA.

     

Human α1β3γ2L gamma‐aminobutyric acid type A receptors: High‐level production and purification in a functional state

Gamma‐aminobutyric acid type A receptors (GABA_ARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA_ARs determine their function and pharmacological profile. GABA_ARs are heteropentamers of subunits, and (α1)₂(β3)₂(γ2L)₁ is a common subtype. Biochemical and biophysical studies of GABA_ARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABA_AR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)₃GK–1D4 GABA_AR. These cells achieved expression levels of 70–90 pmol [³H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [³H]flunitrazepam to [³H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABA_ARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ～30%. Typical purifications yielded 1.0–1.5 nmoles of [³H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [³H]muscimol binding were maintained in the purified state.     

The translocation of transportin–cargo complexes through nuclear pores is independent of both Ran and energy

Active transport between nucleus and cytoplasm proceeds through nuclear pore complexes (NPCs) and is mediated largely by shuttling transport receptors that use direct RanGTP binding to coordinate loading and unloading of cargo [1], [2], [3], [4]. Import receptors such as importin β or transportin bind their substrates at low RanGTP levels in the cytoplasm and release them upon encountering RanGTP in the nucleus, where a high RanGTP concentration is predicted. This substrate release is, in the case of import by the importin α/β heterodimer, coupled directly to importin β release from the NPCs. If the importin β –RanGTP interaction is prevented, import intermediates arrest at the nuclear side of the NPCs [5], [6]. This arrest makes it difficult to probe directly the Ran and energy requirements of the actual translocation from the cytoplasmic to the nuclear side of the NPC, which immediately precedes substrate release. Here, we have shown that in the case of transportin, dissociation of transportin–substrate complexes is uncoupled from transportin release from NPCs. This allowed us to dissect the requirements of translocation through the NPC, substrate release and transportin recycling. Surprisingly, translocation of transportin–substrate complexes into the nucleus requires neither Ran nor nucleoside triphosphates (NTPs). It is only nuclear RanGTP, not GTP hydrolysis, that is needed for dissociation of transportin–substrate complexes and for re-export of transportin to the cytoplasm. GTP hydrolysis is apparently required only to restore the import competence of the re-exported transportin and, thus, for multiple rounds of transportin-dependent import. In addition, we provide evidence that at least one type of substrate can also complete NPC passage mediated by importin β independently of Ran and energy.     

A Semiparametric Missing‐Data‐Induced Intensity Method for Missing Covariate Data in Individually Matched Case–Control Studies

Summary In individually matched case–control studies, when some covariates are incomplete, an analysis based on the complete data may result in a large loss of information both in the missing and completely observed variables. This usually results in a bias and loss of efficiency. In this article, we propose a new method for handling the problem of missing covariate data based on a missing‐data‐induced intensity approach when the missingness mechanism does not depend on case–control status and show that this leads to a generalization of the missing indicator method. We derive the asymptotic properties of the estimates from the proposed method and, using an extensive simulation study, assess the finite sample performance in terms of bias, efficiency, and 95% confidence coverage under several missing data scenarios. We also make comparisons with complete‐case analysis (CCA) and some missing data methods that have been proposed previously. Our results indicate that, under the assumption of predictable missingness, the suggested method provides valid estimation of parameters, is more efficient than CCA, and is competitive with other, more complex methods of analysis. A case–control study of multiple myeloma risk and a polymorphism in the receptor Inter‐Leukin‐6 (IL‐6‐α) is used to illustrate our findings.     

Dual effects of [Tyr6]‐γ2‐MSH(6–12) on pain perception and in vivo hyperalgesic activity of its analogues

[Tyr⁶]‐γ2‐MSH(6–12) with a short effecting time of about 20 min is one of the most potent rMrgC receptor agonists. To possibly increase its potency and metabolic stability, a series of analogues were prepared by replacing the Tyr⁶ residue with the non‐canonical amino acids 3‐(1‐naphtyl)‐L ‐alanine, 4‐fluoro‐L ‐phenylalanine, 4‐methoxy‐L ‐phenylalanine and 3‐nitro‐L ‐tyrosine. Dose‐dependent nociceptive assays performed in conscious rats by intrathecal injection of the MSH peptides showed [Tyr⁶]‐γ2‐MSH(6–12) hyperalgesic effects at low doses (5–20 nmol) and analgesia at high doses (100–200 nmol). This analgesic activity is fully reversed by the kyotorphin receptor‐specific antagonist Leu–Arg. For the two analogues containing in position 6, 4‐fluoro‐L ‐phenylalanine and 3‐nitro‐L ‐tyrosine, a hyperalgesic activity was not observed, while the 3‐(1‐naphtyl)‐L ‐alanine analogue at 10 nmol dose was found to induce hyperalgesia at a potency very similar to γ2‐MSH(6–12), but with longer duration of the effect. Finally, the 4‐methoxy‐L ‐phenylalanine analogue (0.5 nmol) showed greatly improved hyperalgesic activity and prolonged effects compared to the parent [Tyr⁶]‐γ2‐MSH(6–12) compound. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Studies of conformational isomerism in α-melanocyte stimulating hormone by design of cyclic analogues

Results of energy calculations for α-MSH (α-melanocyte stimulating hormone, Ac-Ser¹-Tyr²-Ser³-Met⁴-Glu⁵-His⁶-Phe⁷-Arg⁸-Trp⁹-Gly¹⁰-Lys¹¹-Pro¹²-Val¹³-NH₂) and [D -Phe⁷]α-MSH were used for design of cyclic peptides with the general aim to stabilize different conformational isomers of the parent compound. The minimal structural modifications of the conformationally flexible Gly¹⁰ residue, as substitutions for L -Ala, D -Ala, or Aib (replacing of hydrogen atoms by methyl groups), were applied to obtain octa- and heptapeptide analogues of α-MSH(4–11) and α-MSH(5–11), which were cyclized by lactam bridges between the side chains in positions 5 and 11. Some of these analogues, namely those with substitutions of the Gly¹⁰ residue with L -Ala or Aib, showed biological activity potencies on frog skin comparable to the potency of the parent tridecapeptide hormone. Additional energy calculations for designed cyclic analogues were used for further refinement of the model for the biologically active conformations of the His-Phe-Arg-Trp “message” sequence within the sequences of α-MSH and [D -Phe⁷]α-MSH. In such conformations the aromatic moieties of the side chains of the His⁶, L/D -Phe⁷, and Trp⁹ residues form a continuous hydrophobic “surface,” presumably interacting with a complementary receptor site. This feature is characteristic for low-energy conformers of active cyclic analogues, but it is absent in the case of inactive analogues. This particular spatial arrangement of functional groups involved in the message sequence is very close for α-MSH and [D -Phe⁷]α-MSH, as well as for biologically active cyclic analogues despite differences of dihedral angle values for corresponding low-energy conformations. © 1998 John Wiley & Sons, Inc. Biopoly 46: 155–167, 1998     

A molecular dynamics simulation of the flavin mononucleotide–RNA aptamer complex

We report on an unrestrained molecular dynamics simulation of the flavin mononucleotide (FMN)–RNA aptamer. The simulated average structure maintains both cross‐strand and intermolecular FMN–RNA nuclear Overhauser effects from the nmr experiments and has all qualitative features of the nmr structure including the G10–U12–A25 base triple and the A13–G24, A8–G28, and G9–G27 mismatches. However, the relative orientation of the hairpin loop to the remaining part of the molecule differs from the nmr structure. The simulation predicts that the flexible phosphoglycerol part of FMN moves toward G27 and forms hydrogen bonds. There are structurally long‐lived water molecules in the FMN binding pocket forming hydrogen bonds within FMN and between FMN and RNA. In addition, long‐lived water is found bridging primarily RNA backbone atoms. A general feature of the environment of long‐lived “structural” water is at least two and in most cases three or four potential acceptor atoms. The 2′‐OH group of RNA usually acts as an acceptor in interactions with the solvent. There are almost no intrastrand O2′H(n)⋮O4′(n + 1) hydrogen bonds within the RNA backbone. In the standard case the preferred orientation of the 2′‐OH hydrogen atoms is approximately toward O3′ of the same nucleotide. However, a relatively large number of conformations with the backbone torsional angle γ in the trans orientation is found. A survey of all experimental RNA x‐ray structures shows that this backbone conformation occurs but is less frequent than found in the simulation. Experimental nmr RNA aptamer structures have a higher fraction of this conformation as compared to the x‐ray structures. The backbone conformation of nucleotide n + 1 with the torsional angle γ in the trans orientation leads to a relatively short distance between 2′‐OH(n) and O5′(n + 1), enabling hydrogen‐bond formation. In this case the preferred orientation of the 2′‐OH hydrogen atom is approximately toward O5′(n + 1). We find two relatively short and dynamically stable types of backbone–backbone next‐neighbor contacts, namely C2′(H)(n)⋮O4′(n + 1) and C5′(H)(n + 1)⋮O2′(n). These interactions may affect both backbone rigidity and thermodynamic stability of RNA helical structures. © 1999 John Wiley & Sons, Inc. Biopoly 50: 287–302, 1999