Species determination and characterization of developmental stages of ticks by whole-animal matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of crude bacterial samples has been introduced as a very cost-efficient and rapid, yet highly informative tool to identify and classify bacteria. The potential of this approach to characterize whole animals, so far preferentially insects, is only evolving. Here, a simple protocol was developed to perform MALDI-MS analysis on extracts from whole ticks of 7 species and 4 developmental stages. Using commercially available software designed for the identification of bacteria, a reference database of spectra was constructed that allowed the species determination of ticks using larvae, nymphs, or adult individuals as starting material. Cluster analysis on the basis of MALDI mass spectra indicated that the primary determinant for the mass spectra was the species, followed by the developmental stages, which formed distinct clusters within the given species. With certain limitations, species identification was also possible using body parts and engorged animals. Spectra of developing Ixodes ricinus eggs showed dramatic changes with time, suggesting that, beyond its usefulness for species determination, MALDI-typing may have applications in developmental biology.     

Scanning Electron Microscopic Examination of the Extracellular Matrix in the Decellularized Mouse and Human Cochlea

Decellularized tissues have been used to investigate the extracellular matrix (ECM) in a number of different tissues and species. Santi and Johnson JARO 14:3-15 (2013) first described the decellularized inner ear in the mouse, rat, and human using scanning thin-sheet laser imaging microscopy (sTSLIM). The purpose of the present investigation is to examine decellularized cochleas in the mouse and human at higher resolution using scanning electron microscopy (SEM). Fresh cochleas were harvested and decellularized using detergent extraction methods. Following decellularization, the ECM of the bone, basilar membrane, spiral limbus, and ligament remained, and all of the cells were removed from the cochlea. A number of similarities and differences in the ECM of the mouse and human were observed. A novel, spirally directed structure was present on the basilar membrane and is located at the border between Hensen and Boettcher cells. These septa-like structures formed a single row in the mouse and multiple rows in the human. The basal lamina of the stria vascularis capillaries was present and appeared thicker in the human compared with the mouse. In the mouse, numerous openings beneath the spiral prominence that previously housed the root processes of the external sulcus cells were observed but in the human there was only a single row of openings. These and other anatomical differences in the ECM between the mouse and human may reflect functional differences and/or be due to aging; however, decellularized cochleas provide a new way to examine the cochlear ECM and reveal new observations.     

Mechanism of death in avalanche victims

Summary The autopsies of 12 victims from two snow avalanches in North-Norway are reported. Supportive evidence from non-autopsied and surviving victims is included. Consistent autopsy findings were prominent lung oedema, moderate cerebral oedema, extreme contraction of the left ventricle, petechiae in the superior vena cava drainage area, and acute congestion in lungs and kidneys. In four cases in whom no resuscitation was attempted, aortic oxygen pressure was in the range expected in pure asphyxial-type deaths in one and in pure cardiac-type deaths in three. No air pocket was seen in front of the mouth and nose in any of the fatal cases. Three fatal cases had fractures. It is concluded that the immediate cause of death in most cases was general body compression with acute respiratory and circulatory failure.     

An ultrastructural study of the hypertrophied human papillary muscle cell with special emphasis on specific staining patterns,mitochondrial projections and association between mitochondria and SR

Summary Biopsy material of the hypertrophied human papillary muscle has been processed according to various electron microscopical techniques in order to study the mitochondrial ultrastructure and the association between mitochondria and sarcoplasmic reticulum (SR).En bloc staining with a Cu-Pb citrate solution resulted in specifically contrasted mitochondrial and sarcotubular membranes, characterized by numerous, discrete, electron-dense particles. The differences in staining patterns between the perinuclear mitochondria and their subsarcolemmal and interfibrillar counterparts suggest differences in chemical properties and/or metabolic activities. The selectively contrasted mitochondrial particles may represent a conglomorate of extrinisic and intrinisic respiratory enzymes and other membrane-associated proteins, while the majority of the electron-dense particles of the sarcotubular membrane may represent positively stained Ca²⁺-pumps. Ultrastructural findings in the present study strongly indicate that the slender mitochondrial projections represent an initial stage in a process leading to the formation of large and pleomorphic mitochondria. Intimate contact between adjacent mitochondria as well as between mitochondria and SR are documented. In the contact regions some of the specifically contrasted particles of the adjacent membranes had fused with each other. It is suggested that these particles represent membrane-bound transport proteins providing a system for interorganelle exchanges of metabolites and/or ions.     

Event-Related Potentials to Time-Deviant and Pitch-Deviant Tones

Twelve subjects were run in a paradigm designed to compare event-related brain potentials (ERPs) elicited by infrequent pitch-deviant and time-deviant tones under different attentional conditions. Tones with a pitch of 500 Hz or 1000 Hz were presented at regular interstimulus intervals of 800 ms. Changes in pitch or a shortening of interstimulus interval to 400 ms each occurred with 10% probability. Three different tasks (reading or reaction time to pitch or to timing deviances) were assigned on separate runs. N150 to deviant tones was not influenced by task. Its amplitude, peak latency, and frontally maximum distribution did not differ between pitch and timing deviance, but to pitch deviance it had an earlier onset. P350 amplitude to both types of deviant tones was larger than to standard tones when subjects pressed to time-deviant tones; only P350 to pitch-deviant tones was larger than to standards when subjects pressed to pitch-deviant tones. P350 latency was longer to timing deviance regardless of task. Our results support the view that negativities generated by mismatches in expected timing and pitch are qualitatively the same. ERP differences between these two types of deviance were probably due to differences in stimulus salience or discriminability.     

Polyhalogenated dibenzo-p-dioxins and dibenzofurans and the immune system. 1. Effects on peripheral lymphocyte subpopulations of a non-human primate (Callithrix jacchus) after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Monoclonal antibodies were used to analyse the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on peripheral lymphocytes from marmosets (Callithrix jacchus) following injections of single low doses of TCDD. A reduction of the percentage and the total number of lymphocytes among the white blood cells was seen 3 weeks after a single administration of 300 ng TCDD/kg body wt, but not following 167 ng TCDD/kg or less. After treatment of marmosets with the single subcutaneous dose of 10 ng TCDD/kg body wt, we observed an average decrease of about 20% in the percentage of CD4⁺ cells in the venous blood of treated animals. This change was not seen at the time of maximum absorption (2 weeks after the injection), but was demonstrable after 4 weeks and reached its maximum 15 weeks after the injection. The time course of the changes suggests an indirect effect (possibly via the thymus). Due to the considerable inter- and intra-individual variability in the number of peripheral lymphocytes the effect was less convincing on the basis of the total CD4⁺ cells per microliter blood. There was a significant concomitant increase in the percentage of cells with the CD8⁺ marker. A closer analysis of the lymphocyte subpopulation involved revealed a predominant effect on the cells with the CD4CDw29 (leu 3a⁺4B4⁺) surface marker (helperinducer cells). Subsequent to a dose of 10 ng TCDD/kg body wt the percentage of these cells was reduced by 50% or more when compared with the (24) controls. On an absolute basis (number of cells/l blood) these CD4⁺CDw29⁺ cells were clearly reduced in only two out of four marmosets. There was no significant effect on the percentage of the CD4⁺CD45R⁺ (leu3a⁺2H4⁺) subpopulation (suppressor-inducer cells). The ratios: CD4⁺CDw29⁺/CD4⁺CD45R⁺, or CD4⁺CDw29⁺/CD8⁺ appear to be convenient measures for monitoring the effect described. Since the difference between the percentage of CD2⁺ cells minus the sum of CD4⁺ plus CD8⁺ cells was found to be increased following rather high doses (167 ng TCDD/kg body wt or more), this suggests that immature cells (CD2⁺ CD4-CD8-) are released into the periphery as a result of the exposure to TCDD. Furthermore, a decrease in the percentage of CD20⁺ (B₁ ⁺) cells (about 50% when compared with controls) was observed in the treated animals following a single dose of 10 ng TCDD/kg body wt or higher. Subsequent to a dose of 10 ng TCDD/kg body wt the percentage of CD56⁺ (NKH1⁺) cells seemed to be slightly increased. The dose-response for the effects observed was rather poor. At present it cannot be decided whether the changes have to be considered as adverse health effects. All of the animals involved were apparently healthy and did not exhibit, e.g. any infections. However, the deviations described certainly represent biologicaleffects induced at very low dose levels. From the data available up till now, a single dose of 10 ng TCDD/kg body wt seems to be the lowest-observed-effect-level (LOEL) in the marmoset for all the effects studied. More extensive experiments within the dose range between 1 and 10 ng TCDD/kg body wt are under way in our laboratory. When peripheral lymphocytes of TCDD-treated animals were incubated in vitro with a mitogen (PWM = poke weed mitogen) an exaggeration of thedecrease in the percent of cells with the CD4 surface marker and a concomitant increase in the percentage of CD8⁺ cells was observed. This seems to be an especially sensitive experimental approach for demonstrating biological effects (but not necessarily adverse health effects) of this class of substances. Such an effect was demonstrated and found to be statistically significant already 2 weeks after a single injection of 10 ng TCDD/kg body wt in comparison to vehicle-treated controls.     

Transfer of various polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs and PCDFs) via placenta and through milk in a marmoset monkey

A defined mixture of polychlorinated dibenzop-dioxins and dibenzofurans (PCDDs and PCDFs) was subcutaneously administered to a pregnant marmoset monkey (Callithrix jacchus) 11 weeks prior to delivery. Transfer of PCDDs and PCDFs via placenta and mother's milk was investigated by measurement of concentrations in a newborn 1 day after birth and in an infant of the same litter after a lactation period of 33 days. Furthermore, comparative measurements were performed in different tissues of the mother at the end of the lactation period, and in addition, in two groups of four adult monkeys each 1 and 6 weeks after treatment. Deposition of the PCDDs and PCDFs into fetal liver was very low for most of the 2,3,7,8-substituted congeners. Highest deposition was observed for 2,3,7,8-T4CDD and 1,2,3,7,8-P5CDD. For all other compounds concentrations in the hepatic tissue of newborn shortly after birth were lower than one tenth of corresponding concentrations in adults. Especially for PCDFs, prenatal deposition in fetal liver was extremely low. Fetal liver is apparently largely unable to accumulate PCDDs/PCDFs. In contrast to liver, concentrations of 2,3,7,8-substituted PCDDs/PCDFs in adipose tissue of the newborn were at least one third of the levels in adults. However, concentrations of OCDD and OCDF were about three times higher in the newborn than in adult adipose tissue. Transfer of some of the 2,3,7,8-substituted PCDDs and PCDFs to the off-spring via mother's milk was considerable, leading to hepatic concentrations in the suckled infant at the end of the 33-day nursing period well above corresponding concentrations in the dam. When hepatic concentrations in the infant and dam were compared 2- to 4-fold higher concentrations were found in the infant's liver for 2,3,7,8-T4CDD/F and for 1,2,3,7,8-P5CDD. In the case of the 2,3,7,8-substituted H6CDDs, P5CDFs, and most of the H6CDFs, hepatic concentrations in the infant and dam were in the same range at the end of the suckling period. In contrast to this, less than one tenth the concentration of OCDD was found in the infant's liver when compared with adult liver. A corresponding phenomenon was observed for PCDFs. At the maximum absorption, 1 week after injection, for almost all 2,3,7,8-substituted congeners highest concentrations were measured in hepatic tissue of adult monkeys. This is especially true for those substances with six and more chlorine atoms. Besides adipose tissue, comparatively high levels were found in thymus and also in lung tissue. The limited data available point to a long persistence of some of the 2,3,7,8-substituted congeners in the thymus. Especially low concentrations of all the congeners were measured in the brain and testes.The term deposition is used throughout this paper to designate the presence of PCDDs/PCDFs in a given tissue. This term does not imply any mechanism (storage, active binding, etc.).     

Are occupational factors important determinants of socioeconomic inequalities in musculoskeletal pain?

OBJECTIVES: The aim of this study was to quantify socioeconomic inequalities in low-back pain, neck-shoulder pain, and arm pain in the general working population in Oslo and to examine the impact of job characteristics on these inequalities. METHODS: All economically active 30-, 40-, and 45-year-old persons who attended the Oslo health study in 2000-2001 and answered questions on physical job demands, job autonomy, and musculoskeletal pain were included (N=7293). Occupational class was used as an indicator of socioeconomic status. The lower occupational classes were compared with higher grade professionals, and prevalences, prevalence ratios, prevalence differences, and population attributable fractions were calculated. RESULTS: There were marked, stepwise socioeconomic gradients for musculoskeletal pain, steeper for the men than for the women. The relative differences (prevalence ratios) were larger for low-back pain and arm pain than for neck-shoulder pain. The absolute differences (prevalence differences) were the largest for low-back pain. Physical job demands explained a substantial proportion of the absolute occupational class inequalities in low-back pain, while job autonomy was more important in explaining the inequalities in neck-shoulder pain and arm pain. The estimated population attributable fractions supported the impact of job characteristics at the working population level, especially for low-back pain. CONCLUSIONS: In this cross-sectional study, physical job demands and job autonomy explained a substantial proportion of occupational class inequalities in self-reported musculoskeletal pain in the working population in Oslo. This finding indicates that the workplace may be an important arena for preventive efforts to reduce socioeconomic inequalities in musculoskeletal pain.     

Vascular endothelial growth factor gene-activated matrix (VEGF165-GAM) enhances osteogenesis and angiogenesis in large segmental bone defects.

Healing of fractures is dependent on vascularization of bone, which is in turn promoted by VEGF. It was shown that 0.1 and 1 mg of pVEGF165-GAM led to a significant increase in vascularization and bone regeneration in defects that would otherwise have led to atrophic nonunions. INTRODUCTION: One reason for lack of bone healing in nonunions is the absence of vascularization. In skeletogenesis, which is tightly linked to angiogenesis, vascular endothelial growth factor (VEGF) promotes the vascularization of the growth plate and transformation of cartilage to bone. We postulate that a gene-activated matrix (GAM), created with a plasmid coding for human VEGF165, coated on a collagen sponge could efficiently accelerate bone healing in large segmental defects. MATERIALS AND METHODS: Sixty New Zealand white rabbits received a 15-mm critical size defect on one radius, which was filled with either 0.1 or 1 mg plasmid-DNA as GAM. Radiographs were obtained every 3 weeks. After 6 or 12 weeks, animals were killed. New bone was measured by microCT scans. Vascularity was measured using anti-CD31 staining of endothelial cells in 18 regions of interest per implant. RESULTS: Scaffold and control plasmid showed no defect healing, whereas most of the animals in the VEGF groups showed partial or total bone regeneration. Significantly more bone was found in the VEGF groups, with no significant differences between the 0.1- and 1-mg groups. Immunohistochemical staining of endothelial cells revealed that the VEGF groups showed two to three times the number of vessels and a significantly larger endothelial area after 6 weeks. Twelve weeks after surgery, the amount of vascularization decreased, whereas more new bone was detectable. CONCLUSIONS: The rabbit critical size defect was appropriate in size to produce atrophic nonunions. We showed that angiogenesis and osteogenesis can be promoted by a VEGF165-GAM that is an appropriate tool to induce bone healing in atrophic nonunions.