Assessment of aldehyde dehydrogenase in viable cells

Cytosolic aldehyde dehydrogenase (ALDH), an enzyme responsible for oxidizing intracellular aldehydes, has an important role in ethanol, vitamin A, and cyclophosphamide metabolism. High expression of this enzyme in primitive stem cells from multiple tissues, including bone marrow and intestine, appears to be an important mechanism by which these cells are resistant to cyclophosphamide. However, although hematopoietic stem cells (HSC) express high levels of cytosolic ALDH, isolating viable HSC by their ALDH expression has not been possible because ALDH is an intracellular protein. We found that a fluorescent aldehyde, dansyl aminoacetaldehyde (DAAA), could be used in flow cytometry experiments to isolate viable mouse and human cells based on their ALDH content. The level of dansyl fluorescence exhibited by cells after incubation with DAAA paralleled cytosolic ALDH levels determined by Western blotting and the sensitivity of the cells to cyclophosphamide. Moreover, DAAA appeared to be a more sensitive means of assessing cytosolic ALDH levels than Western blotting. Bone marrow progenitors treated with DAAA proliferated normally. Furthermore, marrow cells expressing high levels of dansyl fluorescence after incubation with DAAA were enriched for hematopoietic progenitors. The ability to isolate viable cells that express high levels of cytosolic ALDH could be an important component of methodology for identifying and purifying HSC and for studying cyclophosphamide-resistant tumor cell populations.     

Accuracy of supplementary serologic testing for human T-lymphotropic virus types I and II in US blood donors. Retrovirus Epidemiology Donor Study

Blood donations in the United States have been screened for antibody to human T-lymphotropic virus type I (HTLV-I) by HTLV-I enzyme immunoassay (EIA) since November 1988. Specimens repeatedly found to be reactive by EIA undergo confirmation by supplementary serologic tests. We assessed the accuracy of blood center testing of 994 HTLV-I EIA repeat-reactive specimens in five US blood centers between November 1988 and December 1991. Of 410 confirmed HTLV-I/II donations, 407 (99.3%) were infected with HTLV-I/II, as determined by polymerase chain reaction (PCR) (403 cases) and by repeat serologic testing (4 cases). The three false- positive results occurred in the first year of testing. Of 425 HTLV- indeterminate specimens, 6 (1.4%) were found to be infected by PCR (5 with HTLV-II and 1 with HTLV-I). None of 159 confirmatory test-negative donations was PCR positive. Of HTLV-I/II-seropositive specimens, 80.2% to 95.4% could be typed as HTLV-I or HTLV-II by type-specific serologic assays. These results support recommendations that HTLV-I/II- seropositive donors should be advised that they are infected with HTLV- I, HTLV-II, or HTLV-I/II (depending on results of type-specific assays). HTLV-indeterminate donors should be advised that their results only rarely indicate HTLV infection. HTLV confirmatory test-negative donors should be reassured that they are not infected with HTLV-I or HTLV-II.     

Ultra-high-resolution paleoenvironmental records via direct laser-based analysis of lipid biomarkers in sediment core samples

Marine microorganisms adapt to their habitat by structural modification of their membrane lipids. This concept is the basis of numerous molecular proxies used for paleoenvironmental reconstruction. Archaeal tetraether lipids from ubiquitous marine planktonic archaea are particularly abundant, well preserved in the sedimentary record and used in several molecular proxies. We here introduce the direct, extraction-free analysis of these compounds in intact sediment core sections using laser desorption ionization (LDI) coupled to Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). LDI FTICR-MS can detect the target lipids in single submillimeter-sized spots on sediment sections, equivalent to a sample mass in the nanogram range, and could thus pave the way for biomarker-based reconstruction of past environments and ecosystems at subannual to decadal resolution. We demonstrate that ratios of selected archaeal tetraethers acquired by LDI FTICR-MS are highly correlated with values obtained by conventional liquid chromatography/MS protocols. The ratio of the major archaeal lipids, caldarchaeol and crenarchaeol, analyzed in a 6.2-cm intact section of Mediterranean sapropel S1 at 250-µm resolution (∼4-y temporal resolution), provides an unprecedented view of the fine-scale patchiness of sedimentary biomarker distributions and the processes involved in proxy signal formation. Temporal variations of this lipid ratio indicate a strong influence of the ∼200-y de Vries solar cycle on reconstructed sea surface temperatures with possible amplitudes of several degrees, and suggest signal amplification by a complex interplay of ecological and environmental factors. Laser-based biomarker analysis of geological samples has the potential to revolutionize molecular stratigraphic studies of paleoenvironments.Microbial lipids in aquatic sediments reflect phylogeny, environmental conditions, and biogeochemistry of the water column in which they were produced. After sedimentation and because of the persistence of lipid structures in the sedimentary record, the information archived in these lipids remains available on geological time scales. Retrieval of this information from sediments and rocks has increasingly contributed to our understanding of past environments, microbial communities, and biogeochemical processes (e.g., refs. 1–5). Merged with the concept of molecular stratigraphy (2, 6), i.e., the analysis of selected biomarkers in solvent extracts of sediment samples in a stratigraphic framework, unique information can be gleaned regarding the temporal changes of past ecological and environmental conditions in aquatic systems. Because of analytical requirements, the temporal resolution is typically limited by centimeter-scale-sized samples, which, dependent on the depositional setting, tend to integrate time periods of decades to millennia, even in high-sedimentation settings such as the Santa Barbara Basin (e.g., refs. 4, 7, 8). In consequence, our knowledge of the temporal changes of the environmental and ecological history recorded by lipid biomarkers is rather coarse and based on long-term averages of their distributions in the sedimentary record.In this study we seek to extend this approach to ultra-high-resolution molecular stratigraphy. We focused on archaeal glycerol dialkyl glycerol tetraethers (GDGTs), which are produced by planktonic archaeal communities and are ubiquitous and persistent components in marine sediments (e.g., refs. 9, 10) with widely recognized potential for paleoenvironmental studies. The number of cycloalkyl rings in GDGTs (Fig. S1) appears highly sensitive to ecological and environmental factors such as phylogeny (ref. 5 and references therein), temperature (ref. 11 and references therein), or pH (12, 13). This sensitivity of GDGT composition to environmental and ecological factors has driven the development of multiple molecular proxies. Adaptation to temperature has been demonstrated in thermophilic cultures of archaea (e.g., ref. 14) and serves as concept for the reconstruction of paleo sea surface temperatures (SST) in sediments using the TEX₈₆ (tetraether index of 86 carbon atoms) (3), which is based on fossil lipids of planktonic archaea. This proxy has been used to characterize oscillations of SST in different geological episodes (e.g., refs. 8, 15, 16). Complications could arise from additional GDGT sources, e.g., allochthonous inputs of GDGT-bearing soils, lateral transport, in situ production, and/or recycling by sedimentary archaea (cf. refs. 5, 17). Relative GDGT distributions required for proxy calculations are obtained by HPLC atmospheric pressure chemical ionization mass spectrometry (HPLC/APCI-MS) of solvent extracts from gram-sized sediment samples (18, 19).To enhance temporal resolution of GDGT-based proxies, we explored the utility of laser desorption ionization coupled to Fourier transform ion cyclotron resonance mass spectrometry (LDI FTICR-MS). In LDI, the impact of a pulsed laser beam on the sample leads to desorption, vaporization and ionization of the analytes, forming a cloud of charged molecules. An additional matrix is frequently applied to facilitate the generation of ions (matrix assisted laser desorption ionization [MALDI]). A complete understanding of underlying ionization mechanisms has not been achieved yet (20). LDI or MALDI are best known in the field of proteins or peptides and have also been successfully used in lipid research (21). The fact that LDI analysis can generate ions directly from the sample placed on a sample holder without time-consuming wet-chemical pretreatment while producing mass spectra from submillimeter-sized spots (as small as 10 µm) makes this technique particularly attractive for molecular stratigraphy. LDI may allow direct analysis of nanogram-sized samples on the surface of cut, intact sediment cores at ultra-high spatial resolution, equivalent to temporal resolution on the order of months to decades.We aimed to develop a technique that takes advantage of both the exquisite sensitivity and unequivocal molecular information of ultra-high-resolution mass spectrometry by FTICR-MS and the high spatial resolution of LDI within an intact and unaltered sedimentological context. The key steps of validation and implementation included (Fig. S2) (i) the detection of archaeal GDGTs, (ii) verification of results obtained by LDI FTICR-MS by parallel analysis using established HPLC/APCI-MS methods, and (iii) the examination of the first continuous high-resolution GDGT profile from a sediment core section of the eastern Mediterranean sapropel S1, which was deposited under anoxic conditions during the Holocene climatic optimum (HCO; refs. 22, 23). The data thus may provide an unprecedented view of the dynamic variations of both SST and ecological and environmental factors during sapropel formation.     

Electrochemical functionalization of gold and silicon surfaces by a maleimide group as a biosensor for immunological application

In the present study we investigated the preparation of biofunctionalized surfaces using the direct electrochemical grafting of maleimidophenyl molecules with subsequent covalent immobilization of specific peptide to detect target antibody, thereby extending the application of the biosensing systems towards immunodiagnostics. Para-maleimidophenyl (p-MP) functional groups were electrochemically grafted on gold and silicon surfaces from solutions of the corresponding diazonium salt. A specially synthesized peptide modified with cysteine (Cys-peptide) was then immobilized on the p-MP grafted substrates by cross-linking between the maleimide groups and the sulfhydryl group of the cysteine residues. Accordingly, the Cys-peptide worked as an antigen that was able to bind specifically the target antibody (anti-GST antibody), while it was non-sensitive to a negative contrast antibody (i.e. anti-Flag β). The immobilization of both specific and non-specific antibodies on the Cys-peptide-modified surfaces was monitored by infrared spectroscopic ellipsometry, a quartz crystal microbalance integrated in flow injection analysis system and potentiometric response. The results obtained clearly demonstrated that the direct modification of a surface with maleimidophenyl provides a very simple and reliable way of preparing biofunctionalized surfaces suitable for the construction of immunological biosensors.     

股骨近端锁定加压钢板治疗股骨转子间骨折

目的 探讨股骨近端锁定加压钢板（LCP）内固定治疗股骨转子间骨折的临床疗效.方法 采用股骨近端LCP治疗23例股骨转子间骨折的患者.随访观察骨折愈合时间,按Harris评分标准评价疗效.结果 23例均获随访,时间6~12（9±1.4）个月.骨折愈合时间14~20（17±1.7）周.髋关节Harris评分：优13例,良9例,一般1例.结论 股骨近端LCP内固定治疗股骨转子间骨折创伤小、出血少、对骨膜影响小,符合解剖形态,临床疗效满意.     

Association of CHRM2 with IQ: Converging Evidence for a Gene Influencing Intelligence

The cholinergic neurotransmitter system is thought to be involved in many aspects of memory, attention, and higher cognition. In the Collaborative Study on the Genetics of Alcoholism (COGA) sample, we have previously reported linkage and association to the cholinergic muscarinic 2 receptor gene (CHRM2) on chromosome 7 with evoked EEG oscillations (Jones et al. 2004), providing evidence that this gene may be involved in human brain dynamics and cognition. In addition, a small number of genetic markers were genotyped in CHRM2 in the Minnesota Twin and Family Study (Comings et al. 2003) and a Dutch family study (Gosso et al. 2006, in press) and both research groups found evidence that this gene may be involved in intelligence. In the COGA sample, we have extensively genotyped SNPs within and flanking the CHRM2 gene. We find evidence of association with multiple SNPs across CHRM2 and Performance IQ, as measured by the Wechsler Adult Intelligence Scale-Revised (WAIS-R). These results remain significant after taking into account alcohol dependence and depression diagnoses in the sample. Edited by Danielle Posthuma Henri Begleiter—Deceased     

CT evaluation of an anterior urethral tear

A retrograde urethrogram is usually performed to evaluate the urethra in patients with suspected urethral injuries. A computed tomography (CT) scan is performed after the retrograde urethrogram to evaluate for further intrabdominal injuries. We present a case in which a CT scan performed after a retrograde urethrogram in a trauma patient identified a urethral tear.     

Mefloquine and psychotomimetics share neurotransmitter receptor and transporter interactions in vitro

Rationale

Mefloquine is used for the prevention and treatment of chloroquine-resistant malaria, but its use is associated with nightmares, hallucinations, and exacerbation of symptoms of post-traumatic stress disorder. We hypothesized that potential mechanisms of action for the adverse psychotropic effects of mefloquine resemble those of other known psychotomimetics.

Objectives

Using in vitro radioligand binding and functional assays, we examined the interaction of (+)- and (?)-mefloquine enantiomers, the non-psychotomimetic anti-malarial agent, chloroquine, and several hallucinogens and psychostimulants with recombinant human neurotransmitter receptors and transporters.

Results

Hallucinogens and mefloquine bound stereoselectively and with relatively high affinity (K _i?=?0.71–341 nM) to serotonin (5-HT) _2A but not 5-HT_1A or 5-HT_2C receptors. Mefloquine but not chloroquine was a partial 5-HT_2A agonist and a full 5-HT_2C agonist, stimulating inositol phosphate accumulation, with similar potency and efficacy as the hallucinogen dimethyltryptamine (DMT). 5-HT receptor antagonists blocked mefloquine’s effects. Mefloquine had low or no affinity for dopamine D₁, D₂, D₃, and D_4.4 receptors, or dopamine and norepinephrine transporters. However, mefloquine was a very low potency antagonist at the D₃ receptor and mefloquine but not chloroquine or hallucinogens blocked [³H]5-HT uptake by the 5-HT transporter.

Conclusions

Mefloquine, but not chloroquine, shares an in vitro receptor interaction profile with some hallucinogens and this neurochemistry may be relevant to the adverse neuropsychiatric effects associated with mefloquine use by a small percentage of patients. Additionally, evaluating interactions with this panel of receptors and transporters may be useful for characterizing effects of other psychotropic drugs and for avoiding psychotomimetic effects for new pharmacotherapies, including antimalarial quinolines.     

Nonclinical safety of mavrilimumab,an anti-GMCSF receptor alpha monoclonal antibody,in cynomolgus monkeys: Relevance for human safety

Mavrilimumab (CAM-3001) is an investigational human IgG4 monoclonal antibody (MAb) targeting GM-CSF receptor alpha which is currently being developed for the treatment of RA. GM-CSF plays a central role in the pathogenesis of rheumatoid arthritis (RA) through the activation, differentiation, and survival of macrophages and neutrophils. To support clinical development, the nonclinical safety of mavrilimumab was evaluated in several studies with cynomolgus monkeys as the pharmacologically relevant species. Comprehensive toxicity parameters were assessed in each study, and treatment duration ranged from 4 to 26 weeks. Mavrilimumab has an acceptable safety profile in monkeys with no changes in any parameters other than microscopic findings in lung. In several studies, minimal accumulation of foamy alveolar macrophages was observed. This finding was only seen in studies of at least 11 weeks duration, was reversible following a dose-free recovery period and was considered non-adverse. At higher dose levels (≥ 30 mg/kg/week), in a 26-week repeat-IV dose study, the presence of lung foreign material, cholesterol clefts, and granulomatous inflammation was also observed in a few animals and was considered adverse. The dose- and time-related accumulation of foamy macrophages in lung following exposure to mavrilimumab observed in several NHP studies was expected based upon the known role of GM-CSFRα signaling in the function of alveolar macrophages. Overall, a clean no-observed-adverse-effect-level (NOAEL) without any effects in lung was established and provided adequate clinical safety margins. In clinical studies in RA patients, mavrilimumab has demonstrated good clinical activity with adequate safety to support further clinical development. A Phase 2b study of mavrilimumab in subjects with RA is in progress.