Chemical images: technical approaches and issues.

Chemical mapping techniques using Raman microscopy are introduced, and using an example of a pharmaceutical tablet, the practical aspects of data collection and processing to produce a chemical image of the sample are detailed. Issues related to data processing, instrument standards, chemical image reportable errors, and the interpretation of chemical images are presented to encourage debate, develop solutions, and promote use in other challenging scientific applications. applications.     

Physicians' strike and general mortality: Croatia's experience of 2003

The aim of the study was to establish whether the physicians' strike, which took place in Croatia in 2003, had an impact on the mortality of the population. Mortality data from the National Bureau of Statistics relating to the strike period (15 January - 14 February 2003) were selected and compared with the previous and subsequent periods of the same duration in 2001, 2002 and 2004. Of the 52,575 deaths in 2003, Croatia recorded 4,682 (8.9%, 95% Confidence interval 8.4-9.4) in the strike period from the 15th of January to the 14th of February 2003 or 1.1 deaths per 1000. No deviations of the 15th of January to the 14th of February period's share of the death total in relation to other observation periods were noted. It is impossible to associate the strike based on the figures shown in this paper with either an increase or decrease in population mortality.     

KATP channel knockout worsens myocardial calcium stress load in vivo and impairs recovery in stunned heart

Gene knockout of the KCNJ11-encoded Kir6.2 ATP-sensitive K(+) (K(ATP)) channel implicates this stress-response element in the safeguard of cardiac homeostasis under imposed demand. K(ATP) channels are abundant in ventricular sarcolemma, where subunit expression appears to vary between the sexes. A limitation, however, in establishing the full significance of K(ATP) channels in the intact organism has been the inability to monitor in vivo the contribution of the channel to intracellular calcium handling and the superimposed effect of sex that ultimately defines heart function. Here, in vivo manganese-enhanced cardiac magnetic resonance imaging revealed, under dobutamine stress, a significantly greater accumulation of calcium in both male and female K(ATP) channel knockout (Kir6.2-KO) mice compared with sex- and age-matched wild-type (WT) counterparts, with greatest calcium load in Kir6.2-KO females. This translated, poststress, into a sustained contracture manifested by reduced end-diastolic volumes in K(ATP) channel-deficient mice. In response to ischemia-induced stunning, male and female Kir6.2-KO hearts demonstrated accelerated time to contracture and increased peak contracture compared with WT. The outcome on reperfusion, in both male and female Kir6.2-KO hearts, was a transient reduction in systolic performance, measured as rate-pressure product compared with WT, with protracted increase in left ventricular end-diastolic pressure, exaggerated in female knockout hearts, despite comparable leakage of creatine kinase across groups. Kir6.2-KO hearts were rescued from diastolic dysfunction by agents that target alternative pathways of calcium handling. Thus K(ATP) channel deficit confers a greater susceptibility to calcium overload in vivo, accentuated in female hearts, impairing contractile recovery under various conditions of high metabolic demand.     

Effect of maturation on the relationship between physical performance and body size

The purpose of the study was to explore the effect of maturation on the body size-physical performance relationship. Based on our previous study that evaluated only muscle strength, we hypothesized that the physical performance tested on pubescent subjects would be related to body size at a higher rate than predicted by standard scaling theory applied on pre- and postpubescent subjects. Six age groups of highly selected and trained junior soccer players (N = 478; age 12-17 years) presumably including prepubescent, pubescent, and postpubescent subjects were evaluated. They were tested using various standard tests of maximum physical performance including muscle strength of 3 leg muscle groups, 2 jumps, sit-ups, hand and foot tapping, and agility. The results revealed a steeper increase of the tested performance with an increase in body size for the pubescent age than for the pre- and postpubescent age. The observed phenomenon was interpreted by different level of maturation of the subjects tested within the same pubescent age group. We conclude that maturation alters the effect of body size on various physical performances and, therefore, this phenomenon needs to be taken into account when interpreting the data from the physical performance tests applied on pubescent subjects.     

DnaB helicase dynamics in bacterial DNA replication resolved by single-molecule studies

In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments have significantly changed the classical model of highly stable replication machines by showing that components exchange with free molecules from the environment. However, due to technical limitations, accurate assessments of DnaB stability in the context of replication are lacking. Using in vitro fluorescence single-molecule imaging, we visualise DnaB loaded on forked DNA templates. That these helicases are highly stable at replication forks, indicated by their observed dwell time of ∼30 min. Addition of the remaining replication factors results in a single DnaB helicase integrated as part of an active replisome. In contrast to the dynamic behaviour of other replisome components, DnaB is maintained within the replisome for the entirety of the replication process. Interestingly, we observe a transient interaction of additional helicases with the replication fork. This interaction is dependent on the τ subunit of the clamp-loader complex. Collectively, our single-molecule observations solidify the role of the DnaB helicase as the stable anchor of the replisome, but also reveal its capacity for dynamic interactions.     

Contrasting roles for CD4 vs. CD8 T-cells in a murine model of virally induced "T1 black hole" formation

MRI is sensitive to tissue pathology in multiple sclerosis (MS); however, most lesional MRI findings have limited correlation with disability. Chronic T1 hypointense lesions or "T1 black holes" (T1BH), observed in a subset of MS patients and thought to represent axonal damage, show moderate to strong correlation with disability. The pathogenesis of T1BH remains unclear. We previously reported the first and as of yet only model of T1BH formation in the Theiler's murine encephalitis virus induced model of acute CNS neuroinflammation induced injury, where CD8 T-cells are critical mediators of axonal damage and related T1BH formation. The purpose of this study was to further analyze the role of CD8 and CD4 T-cells through adoptive transfer experiments and to determine if the relevant CD8 T-cells are classic epitope specific lymphocytes or different subsets. C57BL/6 mice were used as donors and RAG-1 deficient mice as hosts in our adoptive transfer experiments. In vivo 3-dimensional MRI images were acquired using a 7 Tesla small animal MRI system. For image analysis, we used semi-automated methods in Analyze 9.1; transfer efficiency was monitored using FACS of brain infiltrating lymphocytes. Using a peptide depletion method, we demonstrated that the majority of CD8 T-cells are classic epitope specific cytotoxic cells. CD8 T-cell transfer successfully restored the immune system's capability to mediate T1BH formation in animals that lack adaptive immune system, whereas CD4 T-cell transfer results in an attenuated phenotype with significantly less T1BH formation. These findings demonstrate contrasting roles for these cell types, with additional evidence for a direct pathogenic role of CD8 T-cells in our model of T1 black hole formation.     

Intercondylar notch width and inner angle of lateral femoral condyle as the risk factors for anterior cruciate ligament injury in female handball players in Herzegovina

The principal purpose of this prospective study was to examine intercondylar notch size and the value of inner angle of lateral femoral condyle as the risk factors for noncontact anterior cruciate ligament ACL injury and than to correlate them to the physical values of the athletes such as body mass index (BMI), hight, wight, etc. There are indentified two type of risk factors, external include shoes-surface interaction, type of playing surface, weather conditions and internal include anatomic, neuromuscular, biomechanical and hormonal factors that may predispose female athlets to noncontact injury of ACL. Among anatomic factors, intercondylar notch stenosis and larger inner angle of lateral condyle of femur as the factors which can cause impigement of ACL, were related to an increased risk of injury of ACL. In this study were included 51 female athlete. In the study group there were 24 female handball players with ACL tear and in control group there were 27 female handball players without any type of injury of the knee, who are practicing handball on a daily basis for at least for two years. In the first step, were gathered clinical data performed by orthopaedic surgeon. In the second step, the femoral notch width and the inner angle of lateral condyle of femur were measured on coronal MR-images. Study has shown that value of inner angle of lateral condyle of femur was significantly higher in athletes with ACL tear compared to those without. Value of width of intercondylar notch was statisticaly smaller in athletes with ACL tear, compared to those without. In the conclusion the inner angle of lateral femoral condyle is better predicting factor for ACL tear in young female handball players compared to intercondylar notch width.     

Methylenetetrahydrofolate reductase (MTHFR-677 and MTHFR-1298) genotypes and haplotypes and plasma homocysteine levels in patients with occlusive artery disease and deep venous thrombosis

The aim was to investigate different genotypes and haplotypes of methylenetetrahydrofolate reductase (MTHFR-677, -1298) and plasma concentration of total homocysteine (tHcy) in Macedonian patients with occlusive artery disease (OAD) and deep venous thrombosis (DVT). Investigated groups consists of 80 healthy, 74 patients with OAD, and 63 patients with DVT. Plasma tHcy was measured with Microplate Enzyme Immunoassay. Identification of MTHFR genotypes and haplotypes was done with CVD StripAssay. The probability level (P-value) was evaluated by the Student's t-test. Plasma concentration of tHcy in CC and CT genotypes of MTHFR C677T was significantly increased in patients with OAD and in patients with DVT. Plasma concentration of tHcy in AC genotype of MTHFR A1298C was increased in patients with OAD and in patients with DVT. Plasma concentration of tHcy was significantly increased in AA genotype of patients with OAD, but not in patients with DVT. We found a significant increase of plasma tHcy in patients with OAD in comparison with healthy respondents for normal:heterozygote (CC:AC), heterozygote:normal (CT:AA), and heterozygote:heterozygote (CT:AC) haplotypes. Plasma concentration of tHcy in patients with DVT in comparison with healthy respondents was significantly increased for normal:normal (CC:AA), normal heterozygote (CC:AC), and heterozygote:heterozygote (CT:AC) haplotypes. We conclude that MTHFR C677T and MTHFR A1289C genotypes and haplotypes are connected with tHcy plasma levels in Macedonian patients with OAD and DVT.     

Structural dependencies of protein backbone 2JNC' couplings

Protein folding can introduce strain in peptide covalent geometry, including deviations from planarity that are difficult to detect, especially for a protein in solution. We have found dependencies in protein backbone (2)J(NC') couplings on the planarity and the relative orientation of the sequential peptide planes. These dependences were observed in experimental (2)J(NC') couplings from seven proteins, and also were supported by DFT calculations for a model tripeptide. Findings indicate that elevated (2)J(NC') couplings may serve as reporters of structural strain in the protein backbone imposed by protein folds. Such information, supplemented with the H-bond strengths derived from (h3)J(NC') couplings, provides useful insight into the overall energy profile of the protein backbone in solution.