LC-MS/MS screening method for designer amphetamines, tryptamines, and piperazines in serum

Since the late 1990s and early 2000s, derivatives of well-known designer drugs as well as new psychoactive compounds have been sold on the illicit drug market and have led to intoxications and fatalities. The LC-MS/MS screening method presented covers 31 new designer drugs as well as cathinone, methcathinone, phencyclidine, and ketamine which were included to complete the screening spectrum. All but the last two are modified molecular structures of amphetamine, tryptamine, or piperazine. Among the amphetamine derivatives are cathinone, methcathinone, 3,4-DMA, 2,5-DMA, DOB, DOET, DOM, ethylamphetamine, MDDMA, 4-MTA, PMA, PMMA, 3,4,5-TMA, TMA-6 and members of the 2C group: 2C-B, 2C-D, 2C-H, 2C-I, 2C-P, 2C-T-2, 2C-T-4, and 2C-T-7. AMT, DPT, DiPT, MiPT, DMT, and 5MeO-DMT are contained in the tryptamine group, BZP, MDBP, TFMPP, mCPP, and MeOPP in the piperazine group. Using an Applied Biosystems LC-MS/MS API 365 TurboIonSpray it is possible to identify all 35 substances. After addition of internal standards and mixed-mode solid-phase extraction the analytes are separated using a Synergi Polar RP column and gradient elution with 1 mM ammonium formate and methanol/0.1% formic acid as mobile phases A and B. Data acquisition is performed in MRM mode with positive electro spray ionization. The assay is selective for all tested substances. Limits of detection were determined by analyzing S/N-ratios and are between 1.0 and 5.0 ng/mL. Matrix effects lie between 65% and 118%, extraction efficiencies range from 72% to 90%.

The binding mode of side chain- and C3-modified epothilones to tubulin

The tubulin-binding mode of C3- and C15-modified analogues of epothilone A (Epo A) was determined by NMR spectroscopy and computational methods and compared with the existing structural models of tubulin-bound natural Epo A. Only minor differences were observed in the conformation of the macrocycle between Epo A and the C3-modified analogues investigated. In particular, 3-deoxy- (compound 2) and 3-deoxy-2,3-didehydro-Epo A (3) were found to adopt similar conformations in the tubulin-binding cleft as Epo A, thus indicating that the 3-OH group is not essential for epothilones to assume their bioactive conformation. None of the available models of the tubulin-epothilone complex is able to fully recapitulate the differences in tubulin-polymerizing activity and microtubule-binding affinity between C20-modified epothilones 6 (C20-propyl), 7 (C20-butyl), and 8 (C20-hydroxypropyl). Based on the results of transferred NOE experiments in the presence of tubulin, the isomeric C15 quinoline-based Epo B analogues 4 and 5 show very similar orientations of the side chain, irrespective of the position of the nitrogen atom in the quinoline ring. The quinoline side chain stacks on the imidazole moiety of beta-His227 with equal efficiency in both cases, thus suggesting that the aromatic side chain moiety in epothilones contributes to tubulin binding through strong van der Waals interactions with the protein rather than hydrogen bonding involving the heteroaromatic nitrogen atom. These conclusions are in line with existing tubulin polymerization and microtubule-binding data for 4, 5, and Epo B.

Carbohydrate counting of food

Carbohydrate counting is a principal strategy in nutritional management of type 1 diabetes. The Nutri-Learn buffet (NLB) is a new computer-based tool for patient instruction in carbohydrate counting. It is based on food dummies made of plastic equipped with a microchip containing relevant food content data. The tool enables the dietician to assess the patient's food counting abilities and the patient to learn in a hands-on interactive manner to estimate food contents such as carbohydrate content.

Effects of implant design on marginal bone changes around early loaded, chemically modified, sandblasted Acid-etched-surfaced implants: a histologic analysis in dogs

A minimal marginal bone loss around implants during early healing has been considered acceptable. However, the preservation of the marginal bone is related to soft tissue stability and esthetics. Implant designs and surfaces were evaluated to determine their impact on the behavior of the crestal bone. The purpose of this study is to evaluate histologic marginal bone level changes around early loaded, chemically modified, sandblasted acid-etched-surfaced implants with a machined collar (MC) or no MC (NMC).

Predictive ability of a modified Örebro Musculoskeletal Pain Questionnaire in an acute/subacute low back pain working population

The original 'Örebro Musculoskeletal Pain Questionnaire' (original-ÖMPQ) has been shown to have limitations in practicality, factor structure, face and content validity. This study addressed these concerns by modifying its content producing the 'Örebro Musculoskeletal Screening Questionnaire' (ÖMSQ). The ÖMSQ and original-ÖMPQ were tested concurrently in acute/subacute low back pain working populations (pilot n = 44, main n = 106). The ÖMSQ showed improved face and content validity, which broadened potential application, and improved practicality with two-thirds less missing responses. High reliability (0.975, p < 0.05, ICC: 2.1), criterion validity (Spearman's r = 0.97) and internal consistency (α = 0.84) were achieved, as were predictive ability cut-off scores from ROC curves (112-120 ÖMSQ-points), statistically different ÖMSQ scores (p < 0.001) for each outcome trait, and a strong correlation with recovery time (Spearman's, r = 0.71). The six-component factor structure reflected the constructs originally proposed. The ÖMSQ can be substituted for the original-ÖMPQ in this population. Further research will assess its applicability in broader populations.

Evolution of bombesin conjugates for targeted PET imaging of tumors

Bombesin receptors are under intense investigation as molecular targets since they are overexpressed in several prevalent solid tumors. We rationally designed and synthesized a series of modified bombesin (BN) peptide analogs to study the influence of charge and spacers at the N-terminus, as well as amino acid substitutions, on both receptor binding affinity and pharmacokinetics. This enabled development of a novel (64/67)Cu-labeled BN peptide for PET imaging and targeted radiotherapy of BN receptor-positive tumors. Our results show that N-terminally positively charged peptide ligands had significantly higher affinity to human gastrin releasing peptide receptor (GRPr) than negatively charged or uncharged ligands (IC(50): 3.2±0.5 vs 26.3±3.5 vs 41.5±2.5 nM). The replacement of Nle(14) by Met, and deletion of D-Tyr(6), further resulted in 8-fold higher affinity. Contrary to significant changes to human GRPr binding, modifications at the N-terminal and at the 6(th), 11(th), and 14(th) position of BN induced only slight influences on affinity to mouse GRPr. [Cu(II)]-CPTA-[βAla(11)] BN(7-14) ([Cu(II)]-BZH7) showed the highest internalization rate into PC-3 cells with relatively slow efflux because of its subnanomolar affinity to GRPr. Interestingly, [(64/67)Cu]-BZH7 also displayed similar affinities to the other 2 human BN receptor subtypes. In vivo studies showed that [(64/67)Cu]-BZH7 had a high accumulation in PC-3 xenografts and allowed for clear-cut visualization of the tumor in PET imaging. In addition, a CPTA-glycine derivative, forming a hippurane-type spacer, enhanced kidney clearance of the radiotracer. These data indicate that the species variation of BN receptor plays an important role in screening radiolabeled BN. As well, the positive charge from the metallated complex at the N-terminal significantly increases affinity to human GRPr. Application of these observations enabled the novel ligand [(64/67)Cu]-BZH7 to clearly visualize PC-3 tumors in vivo. This study provides a strong starting point for optimizing radiopeptides for targeting carcinomas that express any of the BN receptor subtypes.

Monoclonal antibody directed against Neospora caninum tachyzoite carbohydrate epitope reacts specifically with apical complex-associated sialylated beta tubulin

Monoclonal antibodies (mabs) were generated against whole sonicated Neospora caninum tachyzoites as immunogen. Initial ELISA screening of the reactivity of hybridoma culture supernatants using the same antigen and antigen treated with sodium periodate prior to antibody binding resulted in the identification of 8 supernatants with reactivity against putative carbohydrate epitopes. Following immunoblotting, mab6D12 (IgG1), binding a 52/48-kDa doublet, and mab6C6 (IgM), binding a 190/180-kDa doublet, were selected for further studies. Immunofluorescence of tachyzoite-infected cultures localized the corresponding epitopes not to the surface, but to interior epitopes at the apical part of N. caninum tachyzoites. During in vitro tachyzoite to bradyzoite stage conversion, mab6C6 labeling translocated toward the cyst periphery, while for mab6D12 no changes in localization were noted. Upon extraction of tachyzoites with the nonionic detergent Triton-X-100, the 52-kDa band recognized by mab6D12 was present exclusively in the insoluble, cytoskeletal fraction of both N. caninum and Toxoplasma gondii tachyzoites. Tandem mass spectrometry analysis identified this protein as N. caninum beta tubulin. The 48-kDa band labeled by mab6D12 was a Vero cell protein contamination. The protein(s) reacting with mab6C6 could not be conclusively identified by mass spectrometry. Immunofluorescence consistently failed to label T. gondii tachyzoites, indicating that beta tubulin in T. gondii and N. caninum could be differentially modified or that the reactive epitope in T. gondii is masked. Immunogold TEM of isolated apical cytoskeletal preparations and dual immunofluorescence with antibody to tubulin confirmed that mab6D12 binds to the anterior part of apical complex-associated microtubules. The sodium periodate sensitivity of the beta tubulin associated epitope was confirmed by immunoblotting and ELISA, and treatment of N. caninum cytoskeletal proteins with sialidase prior to mab6D12 labeling resulted in a profound loss of antibody binding, suggesting that mab6D12 reacts with sialylated beta tubulin.

A modified calculation of ankle-brachial pressure index is far more sensitive in the detection of peripheral arterial disease

BACKGROUND: Ankle-brachial pressure index (ABI) is a simple, inexpensive, and useful tool in the detection of peripheral arterial occlusive disease (PAD). The current guidelines published by the American Heart Association define ABI as the quotient of the higher of the systolic blood pressures (SBPs) of the two ankle arteries of that limb (either the anterior tibial artery or the posterior tibial artery) and the higher of the two brachial SBPs of the upper limbs. We hypothesized that considering the lower of the two ankle arterial SBPs of a side as the numerator and the higher of the brachial SBPs as the denominator would increase its diagnostic yield. METHODS: The former method of eliciting ABI was termed as high ankle pressure (HAP) and the latter low ankle pressure (LAP). ABI was assessed in 216 subjects and calculated according to the HAP and the LAP method. ABI findings were confirmed by arterial duplex ultrasonography. A significant arterial stenosis was assumed if ABI was <0.9. RESULTS: LAP had a sensitivity of 0.89 and a specificity of 0.93. The HAP method had a sensitivity of 0.68 and a specificity of 0.99. McNemar's test to compare the results of both methods demonstrated a two-tailed P < .0001, indicating a highly significant difference between both measurement methods. CONCLUSIONS: LAP is the superior method of calculating ABI to identify PAD. This result is of great interest for epidemiologic studies applying ABI measurements to detect PAD and assessing patients' cardiovascular risk.

Exercise training in normobaric hypoxia in endurance runners. III. Muscular adjustments of selected gene transcripts

We hypothesized that specific muscular transcript level adaptations participate in the improvement of endurance performances following intermittent hypoxia training in endurance-trained subjects. Fifteen male high-level, long-distance runners integrated a modified living low-training high program comprising two weekly controlled training sessions performed at the second ventilatory threshold for 6 wk into their normal training schedule. The athletes were randomly assigned to either a normoxic (Nor) (inspired O2 fraction = 20.9%, n = 6) or a hypoxic group exercising under normobaric hypoxia (Hyp) (inspired O2 fraction = 14.5%, n = 9). Oxygen uptake and speed at second ventilatory threshold, maximal oxygen uptake (VO2 max), and time to exhaustion (Tlim) at constant load at VO2 max velocity in normoxia and muscular levels of selected mRNAs in biopsies were determined before and after training. VO2 max (+5%) and Tlim (+35%) increased specifically in the Hyp group. At the molecular level, mRNA concentrations of the hypoxia-inducible factor 1alpha (+104%), glucose transporter-4 (+32%), phosphofructokinase (+32%), peroxisome proliferator-activated receptor gamma coactivator 1alpha (+60%), citrate synthase (+28%), cytochrome oxidase 1 (+74%) and 4 (+36%), carbonic anhydrase-3 (+74%), and manganese superoxide dismutase (+44%) were significantly augmented in muscle after exercise training in Hyp only. Significant correlations were noted between muscular mRNA levels of monocarboxylate transporter-1, carbonic anhydrase-3, glucose transporter-4, and Tlim only in the group of athletes who trained in hypoxia (P < 0.05). Accordingly, the addition of short hypoxic stress to the regular endurance training protocol induces transcriptional adaptations in skeletal muscle of athletic subjects. Expressional adaptations involving redox regulation and glucose uptake are being recognized as a potential molecular pathway, resulting in improved endurance performance in hypoxia-trained subjects.

Biomechanical evaluation of the interfacial strength of a chemically modified sandblasted and acid-etched titanium surface

The functional capacity of osseointegrated dental implants to bear load is largely dependent on the quality of the interface between the bone and implant. Sandblasted and acid-etched (SLA) surfaces have been previously shown to enhance bone apposition. In this study, the SLA has been compared with a chemically modified SLA (modSLA) surface. The increased wettability of the modSLA surface in a protein solution was verified by dynamic contact angle analysis. Using a well-established animal model with a split-mouth experimental design, implant removal torque testing was performed to determine the biomechanical properties of the bone-implant interface. All implants had an identical cylindrical shape with a standard thread configuration. Removal torque testing was performed after 2, 4, and 8 weeks of bone healing (n = 9 animals per healing period, three implants per surface type per animal) to evaluate the interfacial shear strength of each surface type. Results showed that the modSLA surface was more effective in enhancing the interfacial shear strength of implants in comparison with the conventional SLA surface during early stages of bone healing. Removal torque values of the modSLA-surfaced implants were 8-21% higher than those of the SLA implants (p = 0.003). The mean removal torque values for the modSLA implants were 1.485 N m at 2 weeks, 1.709 N m at 4 weeks, and 1.345 N m at 8 weeks; and correspondingly, 1.231 N m, 1.585 N m, and 1.143 N m for the SLA implants. The bone-implant interfacial stiffness calculated from the torque-rotation curve was on average 9-14% higher for the modSLA implants when compared with the SLA implants (p = 0.038). It can be concluded that the modSLA surface achieves a better bone anchorage during early stages of bone healing than the SLA surface; chemical modification of the standard SLA surface likely enhances bone apposition and this has a beneficial effect on the interfacial shear strength.

Toward stable N4-modified neurotensins for NTS1-receptor-targeted tumor imaging with 99mTc

A series of Gly-neurotensin(8-13) analogues modified at the N-terminus by acyclic tetraamines (Demotensin 1-4) were obtained by solid-phase peptide synthesis techniques. Strategic replacement of amino acids and/or reduction of sensitive peptide bonds were performed to enhance conjugate resistance against proteolytic enzymes. During 99mTc-labeling, single species radiopeptides, [99mTc]Demotensin 1-4, were easily obtained in high yields and typical specific activities of 1 Ci/micromol. Peptide conjugates displayed a high affinity binding to the human neurotensin subtype 1 receptor (NTS1-R) expressed in colon adenocarcinoma HT-29 or WiDr cells and/or in human tumor sections. [99mTc]Demotensin 1-4 internalized very rapidly in HT-29 or WiDr cells by a NTS1-R-mediated process. [99mTc]Demotensin 3 and 4, which remained stable during 1 h incubation in murine plasma, were selectively studied in nude mice bearing human HT-29 and WiDr xenografts. After injection, [99mTc]Demotensin 3 and 4 effectively and specifically localized in the experimental tumors and were rapidly excreted via the kidneys into the urine, exhibiting overall biodistribution patterns favorable for NTS1-R-targeted tumor imaging in man.

Inhibition of HIV-1 multiplication by a modified U7 snRNA inducing Tat and Rev exon skipping

The HIV-1 regulatory proteins Tat and Rev are encoded by multiply spliced mRNAs that differ by the use of alternative 3' splice sites at the beginning of the internal exon. If these internal exons are skipped, the expression of these genes, and hence HIV-1 multiplication, should be inhibited. We have previously developed a strategy, based on antisense derivatives of U7 small nuclear RNA, that allows us to induce the skipping of an internal exon in virtually any gene. Here, we have successfully applied this approach to induce a partial skipping of the Tat, Rev (and Nef) internal exons. Three functional U7 constructs were subcloned into a lentiviral vector. Two of them strongly reduced the efficiency of lentiviral particle production compared to vectors carrying either no U7 insert or unrelated U7 cassettes. This defect could be partly or fully compensated by coexpressing Rev from an unspliced mRNA in the producing cell line. Upon stable transduction into CEM-SS or CEM T-lymphocytes, the most efficient of these constructs inhibits HIV-1 multiplication. Although the inhibition is not complete, it is more efficient in combination with another mechanism inhibiting HIV multiplication. Therefore, this new approach targeting HIV-1 regulatory genes at the level of pre-mRNA splicing, in combination with other antiviral strategies, may be a useful new tool in the fight against HIV/AIDS. Copyright (c) 2007 John Wiley & Sons, Ltd

Two-step postmortem angiography with a modified heart-lung machine: preliminary results

OBJECTIVE: The purpose of this study was to adapt and improve a minimally invasive two-step postmortem angiographic technique for use on human cadavers. Detailed mapping of the entire vascular system is almost impossible with conventional autopsy tools. The technique described should be valuable in the diagnosis of vascular abnormalities. MATERIALS AND METHODS: Postmortem perfusion with an oily liquid is established with a circulation machine. An oily contrast agent is introduced as a bolus injection, and radiographic imaging is performed. In this pilot study, the upper or lower extremities of four human cadavers were perfused. In two cases, the vascular system of a lower extremity was visualized with anterograde perfusion of the arteries. In the other two cases, in which the suspected cause of death was drug intoxication, the veins of an upper extremity were visualized with retrograde perfusion of the venous system. RESULTS: In each case, the vascular system was visualized up to the level of the small supplying and draining vessels. In three of the four cases, vascular abnormalities were found. In one instance, a venous injection mark engendered by the self-administration of drugs was rendered visible by exudation of the contrast agent. In the other two cases, occlusion of the arteries and veins was apparent. CONCLUSION: The method described is readily applicable to human cadavers. After establishment of postmortem perfusion with paraffin oil and injection of the oily contrast agent, the vascular system can be investigated in detail and vascular abnormalities rendered visible.