Use of a Mycobacterium tuberculosis H37Rv bacterial artificial chromosome library for genome mapping, sequencing, and comparative genomics

The bacterial artificial chromosome (BAC) cloning system is capable of stably propagating large, complex DNA inserts in Escherichia coli. As part of the Mycobacterium tuberculosis H37Rv genome sequencing project, a BAC library was constructed in the pBeloBAC11 vector and used for genome mapping, confirmation of sequence assembly, and sequencing. The library contains about 5,000 BAC clones, with inserts ranging in size from 25 to 104 kb, representing theoretically a 70-fold coverage of the M. tuberculosis genome (4.4 Mb). A total of 840 sequences from the T7 and SP6 termini of 420 BACs were determined and compared to those of a partial genomic database. These sequences showed excellent correlation between the estimated sizes and positions of the BAC clones and the sizes and positions of previously sequenced cosmids and the resulting contigs. Many BAC clones represent linking clones between sequenced cosmids, allowing full coverage of the H37Rv chromosome, and they are now being shotgun sequenced in the framework of the H37Rv sequencing project. Also, no chimeric, deleted, or rearranged BAC clones were detected, which was of major importance for the correct mapping and assembly of the H37Rv sequence. The minimal overlapping set contains 68 unique BAC clones and spans the whole H37Rv chromosome with the exception of a single gap of approximately 150 kb. As a postgenomic application, the canonical BAC set was used in a comparative study to reveal chromosomal polymorphisms between M. tuberculosis, M. bovis, and M. bovis BCG Pasteur, and a novel 12.7-kb segment present in M. tuberculosis but absent from M. bovis and M. bovis BCG was characterized. This region contains a set of genes whose products show low similarity to proteins involved in polysaccharide biosynthesis. The H37Rv BAC library therefore provides us with a powerful tool both for the generation and confirmation of sequence data as well as for comparative genomics and other postgenomic applications. It represents a major resource for present and future M. tuberculosis research projects.     

Application of a spectroscopic method to estimate the olive oil oxidative status

A rapid Fourier transformed infrared (FTIR) attenuated total reflectance (ATR) spectroscopic method coupled with partial least squares (PLS), was developed to estimate the oxidation degree of extra virgin olive oil (EVOO). The reference values of EVOO oxidation for the FTIR calibration were obtained by the specific absorptions at 232 and 270 nm, due to the presence of conjugated diene (CD) and conjugated triene (CT) groups, as monitored by the UV spectrophotometric determination. Specific washing procedures were applied to the EVOO to obtain EVOOP and EVOOTP samples, without phenolic compounds and without tocopherols and phenols, respectively. To obtain different oxidation degrees covering wide CD and CT ranges, EVOO, EVOOP, and EVOOTP samples were subjected to a forced oxidation at 60°C for 20 days and aliquots of the oils were daily analyzed. Regression of the FTIR/PLS‐predicted CD and CT of individual oxidized oils EVOO, EVOOP, EVOOTP, and all combined oils (EVOOALL) against UV–Visible reference values demonstrated the good quality of the models in terms of R² and RMSECV values. The results of the study indicated that a strong correlation existed between FTIR and UV–Visible peak intensities. Practical applications: The FTIR‐ATR method coupled with PLS elaboration was developed and applied to predict the oxidation degree of EVOO samples with considerable advantages in terms of simplicity, analysis time, and solvent consumption as compared to the standard method. Moreover, suitable adjustments of the equipment could permit a rapid control at‐line in oil sector.     

Antigenic implications of human immunodeficiency virus type 1 envelope quaternary structure: oligomer-specific and -sensitive monoclonal antibodies

A majority of monoclonal antibodies (mAbs) raised against soluble oligomeric human immunodeficiency virus type 1 isolate IIIB (HIV-1IIIB) envelope (env) glycoprotein reacted with conformational epitopes within the gp120 or gp41 subunits. Of 35 mAbs directed against gp41, 21 preferentially reacted with oligomeric env. A subset of these mAbs reacted only with env oligomers (oligomer-specific mAbs). In contrast, only 1 of 27 mAbs directed against the gp120 subunit reacted more strongly with env oligomers than with monomers, and none were oligomer-specific. However, 50% of anti-gp120 mAbs preferentially recognized monomeric env, suggesting that some epitopes in gp120 are partially masked or altered by intersubunit contacts in the native env oligomer. Two mAbs to oligomer-dependent epitopes in gp41 neutralized HIV-1IIIB and HIV-1SF2, and binding of these mAbs to env was blocked by preincubation with HIV-1-positive human serum. Thus, immunization with soluble, oligomeric env elicits antibodies to conserved, conformational epitopes including a newly defined class of neutralizing antibodies that bind to oligomer-specific epitopes in gp41, and may also minimize the production of antibodies that preferentially react with monomeric env protein.     

Spectrum of activity of soluble intercellular adhesion molecule-1 against rhinovirus reference strains and field isolates

The antiviral potency of soluble intercellular adhesion molecule-1 (ICAM-1; the major receptor for human rhinoviruses) was determined for a subset of American Type Culture Collection reference serotypes and field isolates of rhinovirus. The results indicate that soluble ICAM-1 exhibits a broad spectrum of activity against rhinoviruses and that field isolates have a sensitivity indistinguishable from that of laboratory strains.     

Immunogenetics of human IgE

The purpose of this study was to see whether and how the number of suicide attempters with alcohol problems and their drinking habits differ between the Nordic areas under study. Problem-drinkers were defined as persons who themselves felt that they had an alcohol problem. The analyses were based on data collected at five Nordic research centers participating in the WHO/Euro Multicentre Study on Parasuicide, namely: Helsinki (Finland); Ume? and Stockholm (Sweden); S?r-Tr?ndelag (Norway); and Odense (Denmark). The results showed that the frequency of problem-drinking among suicide attempters differed markedly between the areas under study; the Finnish male and the Danish female suicide attempters included the highest proportions of self-identified problem-drinkers. The pattern of drinking among the suicide attempters also differed between the areas. The analyses indicate that the point when alcohol becomes a problem to somebody, especially to a degree that it increases the risk of suicidal behavior, not only depends on how much and how often the person drinks alcohol; the prevailing drinking pattern, the attitudes towards drinking alcohol, and the level of social control are also important factors to take into consideration when relations between alcohol and suicidal behavior are under study.     

Ictal and interictal technetium-99m-bicisate brain SPECT in children with refractory epilepsy

Identification of epileptogenic foci in patients with refractory epilepsy remains a significant diagnostic challenge. Magnetic resonance imaging studies frequently fail to reveal an anatomic origin for the seizures, and scalp electroencephalography is often limited to identification of the involved hemisphere. Functional imaging modalities such as PET and SPECT are more promising tools for this application because they reflect the functional pathology associated with the seizure. These changes are more pronounced ictally, but until recently, no radiopharmaceutical was available that could be used routinely for ictal SPECT. The present study was therefore undertaken to determine whether 99mTc-bicisate could be used in ictal SPECT in pediatric patients with refractory epilepsy, to compare the patterns of ictal and interictal blood flow in these patients and to compare the localization information provided by ictal SPECT with that available from other techniques. METHODS: Technetium-99m-bicisate/SPECT was compared prospectively with scalp EEG for its ability to identify a possible seizure focus in pediatric patients with refractory epilepsy. Ictal and interictal SPECT studies were performed in 10 patients (3-19 yr old, mean age 10.9 +/- 4.3 yr; 7 female, 3 male) in whom MRI scans revealed no lesions that might be responsible for the seizures. RESULTS: Ictal SPECT was performed in all patients, and all ictal studies revealed focal perfusion abnormalities. By comparison, four of the interictal SPECT studies showed regional hypoperfusion that corresponded to the regions of hyperperfusion in the ictal studies, and three showed regional hyperperfusion corresponding to the hyperperfused regions in the ictal studies. Three interictal studies revealed no abnormal perfusion. Scalp EEG provided localization information in five patients. CONCLUSION: These initial results suggest that ictal SPECT with 99mTc-bicisate is a more promising tool for the identification of epileptogenic foci than interictal SPECT or scalp EEG in patients without focal abnormalities on MRI.