Vladimir Zivkovic

Micro-structural basis for particular vulnerability of the superolateral neck trabecular bone in the postmenopausal women with hip fractures

In this study we analyzed the trabecular bone micro-architecture in the inferomedial and superolateral subregions of the femoral neck in a group with hip fractures and a control group of elderly women, with aim to clarify the micro-structural basis of bone fragility. Proximal femora from 29 Caucasian female cadavers were collected at Institute of Forensic Medicine in Belgrade (15 women with hip fracture: age 79.5±8.5 yrs.; and 14 women without hip fractures: age 74.1±9.3 yrs.). The femoral neck section was scanned in dry conditions using a micro-computed tomography (Scanco μCT 40), at 70 kV, 114 μA, 300 ms integration time, 36 μm resolution, isotropic, 1024×1024 pixels per slice, automatically evaluating trabecular micro-architecture using the built-in program of the micro-CT with direct 3D morphometry. The samples were foam padded to avoid any movement artifacts during scanning. Analysis of the neck section in the fracture group compared to the control cases demonstrated significantly lower bone volume fraction (mean: 6.3% vs. 11.2%, p=0.002), lower connectivity density (0.33/mm(3) vs. 0.74/mm(3), p=0.019) and higher trabecular separation (0.87 mm vs. 0.83 mm, p=0.030). Division into the superolateral and inferomedial regions of interest revealed that the superolateral neck displayed even more differences in micro-architectural properties between the fracture and non-fracture groups. Namely, while in the inferomedial neck only bone volume fraction and degree of anisotropy displayed significant inter-group variability (lower BV/TV with higher degree of anisotropy in the fracture group), in the superolateral neck almost all parameters were different between the fracture cases and the controls, where the fracture group showed a lower trabecular bone volume fraction (3.6% vs. 8.2%, p=0.001), lower connectivity (0.21 vs. 0.63/mm(3), p=0.008), more rod like trabecular structure (SMI: 2.94 vs. 2.62, p=0.049), higher separation and the thinned trabeculae (Tb.Sp: 0.89 vs. 0.85 mm, p=0.013; Tb.Th: 0.17 vs. 0.20 mm, p=0.05). In addition, after adjusting for the effects of BV/TV, the majority of differences disappeared, demonstrating that the bone loss manifests itself via the changes in micro-architectural parameters: trabecular thinning, rising the spacing between individual trabeculae, reducing trabecular connectivity and accentuating trabecular perforations leading to predominance of rod-like trabecular elements. Preferential impairment of the superolateral neck trabecular structure and organization in women with hip fracture reveals the region-dependent micro-structural basis of bone fragility in elderly women.

Age-related deterioration in trabecular bone mechanical properties at material level: Nanoindentation study of the femoral neck in women by using AFM

Despite general belief that the mechanical properties of bone material contribute to whole bone strength, it is still obscure what the age effects are on mechanical behavior of the bone material, particularly in the case of the femoral neck trabeculae. In this study, atomic force microscopy was used for imaging and measuring the size of mineral grains, as well as nano-scale mechanical characterization (nanoindentation) of the bone mineralized matrix of trabeculae, with the aim to explore the age effects on bone elasticity and give new insight into age-related bone fragility. The bone samples in this study comprised trabecular bone specimens of the femoral neck region, collected from eight skeletal healthy women (five young adults: 27-38yrs., three elderly: 83-94yrs.) at autopsy. Bone trabeculae in the elderly displayed a higher modulus and nanohardness, signifying a decreased amount of energy that can be accommodated by the bone tissue during loading. Regression analysis revealed that nearly 65% of variability in the bone matrix elastic modulus can be statistically explained by the changes in size of the matrix mineral grains. This study revealed that the bone trabeculae of elderly women express less elastic behavior at the material level, which makes them more vulnerable to unusual impact loads originating from a fall. The observed age-related structural and mechanical alteration at the bone material level adds new evidence for understanding why hip fractures are more frequent in elderly women.

Nano-structural, compositional and micro-architectural signs of cortical bone fragility at the superolateral femoral neck in elderly hip fracture patients vs. healthy aged controls.

To unravel the origins of decreased bone strength in the superolateral femoral neck, we assessed bone structural features across multiple length scales at this cortical fracture initiating region in postmenopausal women with hip fracture and in aged-matched controls. Our combined methodological approach encompassed atomic force microscopy (AFM) characterization of cortical bone nano-structure, assessment of mineral content/distribution via quantitative backscattered electron imaging (qBEI), measurement of bone material properties by reference point indentation, as well as evaluation of cortical micro-architecture and osteocyte lacunar density. Our findings revealed a wide range of differences between the fracture group and the controls, suggesting a number of detrimental changes at various levels of cortical bone hierarchical organization that may render bone fragile. Namely, mineral crystals at external cortical bone surfaces of the fracture group were larger (65.22nm±41.21nm vs. 36.75nm±18.49nm, p<0.001), and a shift to a higher mineral content and more homogenous mineralization profile as revealed via qBEI were found in the bone matrix of the fracture group. Fracture cases showed nearly 35% higher cortical porosity and showed significantly reduced osteocyte lacunar density compared to controls (226±27 vs. 247±32#/mm(2), p=0.05). Along with increased crystal size, a shift towards higher mineralization and a tendency to increased cortical porosity and reduced osteocyte lacunar number delineate that cortical bone of the superolateral femoral neck bears distinct signs of fragility at various levels of its structural organization. These results contribute to the understanding of hierarchical bone structure changes in age-related fragility.

Enhanced trabecular micro-architecture of the femoral neck in hip osteoarthritis vs. healthy controls: a micro-computer tomography study in postmenopausal women

PurposeA controversial relationship between osteoarthritis (OA) and bone fragility has been attracting considerable attention. However, despite interest in the effects of OA on femoral neck fracture risk and numerous studies analysing the changes in the arthritic femoral head, there is insufficient data about femoral neck 3D bone micro-architecture in individuals with hip osteoarthritis. We compared trabecular micro-architecture of the femoral neck between postmenopausal women with coxarthrosis and controls to explore whether coxarthrosis may indicate reduced bone fragility from the trabecular micro-architectural perspective.MethodsThe study sample included nine women with hip osteoarthritis and 13 age-matched controls. The femoral neck sections were scanned using micro-computed tomography, evaluating the cancellous bone from the superolateral and inferomedial neck subregions.ResultsOsteoarthritic subjects demonstrated a general trend of improved trabecular micro-architecture in both analysed subregions when compared with age-matched controls. In particular, several architectural properties that are important predictors of cancellous bone strength showed significantly better values in the OA group, even after adjusting for bone volume fraction. Namely, the OA group expressed higher trabecular connectivity (p = 0.008), lower SMI indicating more plate-like structure (p = 0.005), and reduced anisotropy (p = 0.006) particularly in the inferomedial neck. Osteoarthritic cases also trended towards higher BV/TV, particularly in the superolateral neck. All micro-architectural parameters displayed significant regional heterogeneity (p ≤ 0.01), with the inferomedial neck region showing more favourable values than the superolateral region.ConclusionsEnhanced trabecular micro-architecture of the femoral neck in postmenopausal osteoarthritic subjects suggests reduced cancellous bone fragility in comparison with their age-matched healthy controls.

[Suicidal asphyxiation by propane-butane mixture inside a plastic bag: case report].

INTRODUCTION Volatiles are easily accessible and widely used in a form of liquid petroleum gas. Death as a consequence of inhalation of volatiles can be either accidental or suicidal. CASE OUTLINE We present a 62-year-old men who committed suicide by placing a plastic bag over the head and inhaling propane-butane mixture from a domestic gas tank. A rubber hose was attached to the tank valve and connected with the plastic bag. The body of the deceased showed signs of advanced postmortal changes. A suicidal note was found at the scene. CONCLUSION Propane-butane mixture, i.e. liquified petroleum gas leads to the depletion of oxygen in the air consequently causing hypoxia and anoxia, and therefore, unconsciousness and eventual death. The mechanisms of death in cases of volatile inhalation include cardiac arrhythmias, reflex cardiac vagal inhibition, and/or central nervous system depression. Similar mechanisms occur in cases of asphyxiation with a plastic bag. The reconstruction of the event in this case was based, not so much on autopsy findings (because of significant putrefaction changes), but on the police investigation and traces found at the scene.

Expression of guanylyl cyclase-B (GC-B/NPR2) receptors in normal human fetal pituitaries and human pituitary adenomas implicates a role for C-type natriuretic peptide.

C-type natriuretic peptide (CNP/Nppc) is expressed at high levels in the anterior pituitary of rats and mice and activates guanylyl cyclase B receptors (GC-B/Npr2) to regulate hormone secretion. Mutations in NPR2/Npr2 can cause achondroplasia, GH deficiency, and female infertility, yet the normal expression profile within the anterior pituitary remains to be established in humans. The current study examined the expression profile and transcriptional regulation of NPR2 and GC-B protein in normal human fetal pituitaries, normal adult pituitaries, and human pituitary adenomas using RT-PCR and immunohistochemistry. Transcriptional regulation of human NPR2 promoter constructs was characterized in anterior pituitary cell lines of gonadotroph, somatolactotroph, and corticotroph origin. NPR2 was detected in all human fetal and adult pituitary samples regardless of age or sex, as well as in all adenoma samples examined regardless of tumor origin. GC-B immunoreactivity was variable in normal pituitary, gonadotrophinomas, and somatotrophinomas. Maximal transcriptional regulation of the NPR2 promoter mapped to a region within -214 bp upstream of the start site in all anterior pituitary cell lines examined. Electrophoretic mobility shift assays revealed that this region contains Sp1/Sp3 response elements. These data are the first to show NPR2 expression in normal human fetal and adult pituitaries and adenomatous pituitary tissue and suggest a role for these receptors in both pituitary development and oncogenesis, introducing a new target to manipulate these processes in pituitary adenomas.

Atomic force microscopy characterization of the external cortical bone surface in young and elderly women: potential nanostructural traces of periosteal bone apposition during aging.

On the basis of the suggestion that bone nanostructure bears “tissue age” information and may reflect surface deposition/modification processes, we performed nanoscale characterization of the external cortical bone surface at the femoral neck in women using atomic force microscopy (AFM). The specific aims were to assess age-related differences in bone nanostructure and explore the existence of nanostructural traces of potential bone apposition at this surface. Our findings revealed that the external cortical surface represents a continuous phase composed of densely packed mineral grains. Although the grains varied in size and shape, there was a domination of small grains indicative of freshly deposited bone (mean grain size: young, 35 nm; old, 37 nm; p > 0.05). Advanced quantitative analysis of surface morphological patterns revealed comparable roughness and complexity of the surface, suggesting a similar rate of mineral particle deposition at the surface in both groups. Calcium/phosphorus ratio, a measure of bone tissue age, was within the same range in both groups. In summary, our AFM analyses showed consistent nanostructural and compositional bone features, suggesting existence of new bone at the periosteal bone surface in both young and elderly women. Considering observed age-related increase in the neck diameter, AFM findings may support the theory of continuous bone apposition at the periosteal surface.

HIV-related risk behaviors among Roma youth in Serbia: results of two community-based surveys.

PURPOSE The Roma constitute a large ethnic minority in Serbia, and are one of the poorest and most marginalized groups in Europe. Roma youth may be at high risk for hepatitis C, HIV, and other sexually transmitted infections, but little is known about the prevalence of these infectious diseases, HIV-related knowledge, and risky sexual behaviors in this vulnerable population. METHODS We used a respondent-driven sampling to conduct biobehavioral surveys of Roma youth (aged 15-24 years) in Belgrade and Kragujevac, and to document HIV-related knowledge and risky sexual behaviors, health-seeking behaviors, and seroprevalence of HIV, hepatitis C virus (HCV), and syphilis. RESULTS Four hundred eleven Roma youth participated in this study. One participant had HIV, four had HCV, and none had syphilis. Risky sexual behaviors were highly prevalent, especially among male subjects: 36.2% (Belgrade) and 45.1% (Kragujevac) had sexual debut before the age of 15 years; 53.9% (Belgrade) and 61.1% (Kragujevac) had more than one sexual partner in the past year; 11.5% (Belgrade) and 4.6% (Kragujevac) reported engaging in commercial sex; and 4.0% (Belgrade) and 3.2% (Kragujevac) reported having anal sex with other men. Among female subjects aged <25 years, 33.5% (Belgrade) and 25.7% (Kragujevac) reported having an abortion. One-quarter of all participants answered all five HIV knowledge questions correctly. CONCLUSIONS Fortunately, the current prevalence of HIV, HCV, and syphilis is low; however, the high prevalence of reported risky behaviors suggests that Roma youth in Serbia are at high risk of contracting sexually transmitted infections.

Systematic Investigation of Expression of G2/M Transition Genes Reveals CDC25 Alteration in Nonfunctioning Pituitary Adenomas

Dysregulation of G1/S checkpoint of cell cycle has been reported in pituitary adenomas. In addition, our previous finding showing that deregulation of Wee1 kinase by microRNAs together with other studies demonstrating alteration of G2/M transition in nonfunctioning pituitary adenomas (NFPAs) suggest that G2/M transition may also be important in pituitary tumorigenesis. To systematically study the expression of members of the G2/M transition in NFPAs and to investigate potential microRNA (miRNA) involvement. Totally, 80 NFPA and 14 normal pituitary (NP) tissues were examined. Expression of 46 genes encoding members of the G2/M transition was profiled on 34 NFPA and 10 NP samples on TaqMan Low Density Array. Expression of CDC25A and two miRNAs targeting CDC25A were validated by individual quantitative real time PCR using TaqMan assays. Protein expression of CDC25A, CDC25C, CDK1 and phospho-CDK1 (Tyr-15) was investigated on tissue microarray and immunohistochemistry. Several genes’ expression alteration were observed in NFPA compared to normal tissues by transcription profiling. On protein level CDC25A and both the total and the phospho-CDK1 were overexpressed in adenoma tissues. CDC25A correlated with nuclear localized CDK1 (nCDK1) and with tumor size and nCDK1 with Ki-67 index. Comparing primary vs. recurrent adenomas we found that Ki-67 proliferation index was higher and phospho-CDK1 (inactive form) was downregulated in recurrent tumors compared to primary adenomas. Investigating the potential causes behind CDC25A overexpression we could not find copy number variation at the coding region nor expression alteration of CDC25A regulating transcription factors however CDC25A targeting miRNAs were downregulated in NFPA and negatively correlated with CDC25A expression. Our results suggest that among alterations of G2/M transition of the cell cycle, overexpression of the CDK1 and CDC25A may have a role in the pathogenesis of the NFPA and that CDC25A is potentially regulated by miRNAs.

Fatal Subarachnoid Hemorrhage During Sexual Anal Self-Stimulation in a Female: A Case Report

Sudden death after sexual activity is a well-known entity, but it is rare in females. Herein we present a case of fatal, massive subarachnoid hemorrhage from a ruptured berry-shaped aneurysm, during sexual anal self-stimulation. A 39-year-old woman was found dead on a couch, with the wooden handle of a spring twirl whisk inserted inside her anus and rectum as a dildo-like object. External examination was unremarkable with no signs of injuries. Intracranial examination showed a massive subarachnoid hemorrhage, up to 6 mm in thickness, especially around the brainstem and the inferior side of the brain. After removing the blood clots, the saccular aneurysm was found at the site of the bifurcation of the left internal carotid. In this case study, we underline the utility of forensic autopsy, as well as death scene investigation, in reconstructing the mechanism of death, as well as the dynamics of the event.