The Effect of Low-Intensity Pulsed Ultrasound on Bone Regeneration and the Expression of Osterix and Cyclooxygenase-2 during Critical-Size Bone Defect Repair.

Low-intensity pulsed ultrasound (LIPUS) is a form of ultrasound that utilizes low-intensity pulsed waves. Its effect on bones that heal by intramembranous ossification has not been sufficiently investigated. In this study, we examined LIPUS and the autologous bone, to determine their effect on the healing of the critical-size bone defect (CSBD) of the rat calvaria. The bone samples underwent histological, histomorphometric and immunohistochemical analyses. Both LIPUS and autologous bone promoted osteogenesis, leading to almost complete closure of the bone defect. On day 30, the bone volume was the highest in the autologous bone group (20.35%), followed by the LIPUS group (19.12%), and the lowest value was in the control group (5.11%). The autologous bone group exhibited the highest intensities of COX-2 (167.7 ± 1.1) and Osx (177.1 ± 0.9) expression on day 30. In the LIPUS group, the highest intensity of COX-2 expression was found on day 7 (169.7 ±1.6) and day 15 (92.7 ± 2.2), while the highest Osx expression was on day 7 (131.9 ± 0.9). In conclusion, this study suggests that LIPUS could represent a viable alternative to autologous bone grafts in repairing bone defects that are ossified by intramembranous ossification.

Deposition of heavy metals in biological tissues of workers in metal workshops.

Welding and cutting of metals produce large amounts of particulate matter (PM), which poses a significant health risk to exposed workers. Appropriate biological markers to estimate exposure are of great interest for occupational health and safety. Here, hair and nail samples from metal workers were analyzed, which appear to be more suitable than blood or urine samples for assessing long-term exposure. Four workshops working with steel components were included in the study. The hair and nail samples were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) to measure the concentrations of 12 elements. At the workplaces, the concentrations of 15 elements in particulate matter were determined using X-ray fluorescence (XRF) and particle-induced X-ray emission (PIXE) techniques. The hair and nail samples of the workers contained significantly higher metal concentrations than the analytical results of a nonexposed control group. The most significant difference between the groups was found for Ti, Mn, Fe, and Co.

Trends in Professional Radiation Exposures of Medical Staff Covered by Personal Dose Monitoring at a Rijeka Clinical Hospital Centre (2000 to 2015).

ABSTRACT: The increase in the number of radiological procedures observed in recent years also means greater exposure to ionizing radiation for the medical staff performing these procedures. This is most pronounced in the teams that perform interventional and invasive radiological procedures. The aim of this study was to examine the impact of increasing numbers of radiological procedures on the effective dose received by medical staff and to determine which professions received the highest exposure to ionizing radiation. Data for effective doses of 326 employees of Rijeka Clinical Hospital Centre covered by personal dosimetry in the period from 2000 to 2015 have been analyzed. Employees were divided by sex, by departments in which they were employed, and according to their professions. The analysis has shown that the exposure level of workers working in the ionizing radiation zone is typically well below the dose limits. During 2015, most employees (over 96.3%) received an annual effective dose of less than 0.1 mSv. Only three persons received an annual dose higher than 0.5 mSv, and one person received an annual dose of 6.9 mSv. Comparison of the radiation exposure doses of medical workers of different professions has shown that the highest dose of radiation is received by cardiologists involved in interventional cardiology procedures. Therefore, the additional need is to take care of their protection, check the manner of their work, and ensure more even workload of cardiologists participating in procedures that involve higher exposure to ionizing radiation.

The Correlation between Enrolment Criteria and Students' Success in the Study of Medicine: a Five-Year Retrospective Study.

OBJECTIVE: To investigate whether the students' study-success correlates with the achievements evaluated in the enrolment process based on the State Graduation Exam. METHODS: The retrospective study included a total of 637 students enrolled in the study of Medicine at the Faculty of Medicine from the academic year 2010-11 to 2014-15. We analysed the structure of students enrolled on the basis of the State Graduation Exam, examined their success in two courses of the study programme and then correlated this study-success with individual admission parameters. In particular, we investigated whether the success in the study was influenced by the changes made in the enrolment requirements during this period. Pearson's correlation coefficient was used to measure strenght of correlation between two variables. RESULTS: The changes in the admission parameters, i.e., the inclusion of STEM subjects as obligatory enrolment requirements, made in 2012-13 enabled a better selection of students with high average secondary education grades and with significantly better results in Biology, Physics and particularly in Chemistry. This resulted in a higher success in the study, especially in the last two new-criteria generations. The Pearson correlation coefficient between students' success in Physiology and Pathophysiology II and the achievements valuated in enrolment procedure as total cumulative score were statistically significant (r=0.27, P<0.001). Total points achieved in enrolment procedure was also correlated with students' success in Medical Physics and Biophysics and correlation was even stronger (r=0.52, P<0.001). CONCLUSION: Our results proved that a well-designed combination of the relevant admission parameters is a crucial prerequisite for a better success in the study.

Matrix metalloproteinases MMP-1, MMP-2, and MMP-13 are overexpressed in primary nodular melanoma.

BACKGROUND: The spread and invasion of malignant melanoma cells involve degradation and reorganization of the extracellular matrix by the activation of several matrix metalloproteinases (MMPs). This study analyzed the expression of MMP-1, MMP-2, and MMP-13 proteins in primary nodular melanoma (NM) and dysplastic nevi (DN) as a significant risk factor for melanoma development. The secondary goal was to analyze the correlation of MMPs protein expression in NM with tumor invasion, BRAF V600 mutation status, and overall survival. METHODS: Immunohistochemistry for MMP-1, MMP-2, and MMP-13 was performed on nodular melanoma (n = 52) and dysplastic nevi (n = 28) on tissue microarray (TMA). BRAF V600 mutation analysis on NM samples was performed by the Sanger sequencing method. RESULTS: A high level of MMPs expression in NM samples (>30%) compared with DN (<8%) was statistically significant (P < 0.001). BRAF V600 mutations were detected in 15 of 39 (38.5%) NM samples. This study revealed an interesting finding that MMP-1 and MMP-13 protein expression in the BRAF V600 mutated melanomas were significantly lower than in the BRAF V600 wild type (P < 0.05). CONCLUSION: Cox analysis revealed that Clark categories, Breslow thickness, and MMP-1 high protein expression are predictive factors for shorter overall survival (P < 0.05).

Determination of physiotherapy ultrasound beam quality parameters from images derived using thermochromic material.

The evaluation of the performance of nine physiotherapy ultrasound transducers used clinically was performed in the hospital environment using an acoustically absorbing thermocromic tile developed at the National Physical Laboratory (UK). The method consists of exposing an acoustic absorber tile, part of which contains a thermochromic pigment, to the ultrasonic beam, thereby forming an image of the intensity profile of the transducer. Images acquired using thermochromic materials were postprocessed in order to estimate effective radiating area (ERA) and beam nonuniformity ratio (BNR) for ultrasound transducers operating within the frequency range from 1.0 to 3.3 MHz, and nominal applied intensities in the range of 1-2W/cm2. Results of our measurements have shown that thermocromic tile can be used for quality control of ultrasound transducers in the hospital environment. Experimental results show that proposed method can be used to distinguish highly non - uniform ultrasound beams with high value of BNR. Influence of exposure duration on obtained ERA and BNR values was also analysed. Our results show that values for ERA increase with insonation time, while BNR values decrease. In order to compare our results with theory we have estimated temperature rise in thermochromic material experimentally and compare it with theoretical prediction.

Optimization of Ultrasonic-Assisted Extraction of Major Phenolic Compounds from Olive Leaves (Olea europaea L.) Using Response Surface Methodology.

The ultrasound-assisted extraction (UAE) of oleuropein (OLE), verbascoside (VER), and luteolin-4'-O-glucoside (L4OG), as the major phenolics from olive leaves, was optimized using response surface methodology (RSM). A Box⁻Behnken design (BBD) was used to monitor the effect of different modes of ultrasound operation (pulsed and continuous), liquid⁻solid (L⁻S) ratio, and sonication time on each phenolic yield. The yield of UAE and conventional solid extraction (CSE) was determined after performing ultrahigh-performance liquid chromatography with a diode-array detector (UHPLC-DAD) analysis on the extracts. The results suggested that, under optimal conditions, the concentrations of OLE, VER, and L4OG were 13.386, 0.363, and 0.527 mg/g of dry powdered olive leaves (DPOL), respectively. Verification of experiments was carried out under the modified optimal conditions and the relative errors between the predicted and experimental values were dependent on the examined phenolic compound (OLE 8.63%, VER 11.3%, and L4OG 22.48%). In comparison with CSE, UAE improved the yields of OLE, VER, and L4OG (32.6%, 41.8%, and 47.5%, respectively, after 1 min) at a temperature of 60 °C, an L⁻S ratio of 15 (v/w), and in the continuous mode of UAE. We demonstrated that the UAE technique is an efficient method for enhancing yields of OLE, VER, and L4OG in olive-leaf extracts, while the chosen model was adequate to optimize the extraction of major phenolic compounds from olive leaves.

Diagnostic value of tumour markers in pleural effusions.

INTRODUCTION: We investigated whether tumour markers carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 125 (CA-125), and cytokeratin 19 fragment (CYFRA 21-1) in pleural effusions and serum can be used to distinguish pleural effusion aetiology. MATERIALS AND METHODS: During the first thoracentesis, we measured pleural fluid and serum tumour marker concentrations and calculated the pleural fluid/serum ratio for patients diagnosed with pleural effusion, using electrochemiluminescence immunoassays. Receiver operating characteristic (ROC) analysis was carried out and the Hanley and McNeil method was used to test the significance of the difference between the areas under ROC curves (AUCs). In order to detect which tumour marker best discriminates between malignant and non-malignant pleural effusions and to establish the predictive value of those markers, discriminant function analysis (DFA) and logistic regression analysis were utilized. RESULTS: Serum tumour markers CYFRA 21-1 and NSE as well as pleural NSE were good predictors of pleural effusion malignancy and their combined model was found statistically significant (Chi-square = 28.415, P < 0.001). Respective ROC analysis showed significant discrimination value of the combination of these three markers (AUC = 0.79). CONCLUSIONS: Serum markers showed superiority to pleural fluid markers in determining pleural fluid aetiology. Serum CYFRA 21-1 and NSE concentrations as well as pleural fluid NSE values had the highest clinical value in differentiating between malignant and non-malignant pleural effusions. The combination of these three markers produced a significant model to resolve pleural effusion aetiology.

Quantitative testing of physiotherapy ultrasound beam patterns within a clinical environment using a thermochromic tile.

The implementation of the non-standardized method developed at the National Physical Laboratory (UK) supporting the quality assurance of therapeutic ultrasonic beam parameters within a clinical environment is presented. The method consists of exposing an acoustic absorber tile, part of which contains a thermochromic pigment, to the ultrasonic beam, thereby forming an image of the two-dimensional intensity profile of the transducer. Nine different physiotherapy ultrasound treatment heads currently used clinically were tested using this method. Thermochromic images were postprocessed in order to estimate the Effective Radiating Area (ERA) for treatment heads operating within the frequency range from 1 MHz to 3.2 MHz, and nominal applied intensities in the range of 1-3 W/cm(2). Experimental results and comparisons with manufacturer specified values of ERA are presented. Differences in the experimentally derived results and the manufacturer values are typically well within 25%. The root-mean squared difference calculated over the nine treatment heads is 15.1%, with the thermochromic material tended to underestimate the ERA.

Exploiting thermochromic materials for the rapid quality assurance of physiotherapy ultrasound treatment heads.

Significant nonuniformities in the acoustic intensity distribution generated by physiotherapy ultrasound treatment heads are not uncommon, potentially leading to significant localised temperature rises and tissue damage. An acoustic absorber tile containing a thermochromic pigment has been developed to provide rapid quality assurance of physiotherapy ultrasound treatment heads by virtue of a thermochromic colour change, indicating the time-averaged intensity distributions generated by these devices. As a bench-top device, the use of the tile is designed to mimic the nature of the physiotherapeutic application, requiring minimal training. Two designs where thermochromic pigments are added to the various polymeric layers of the tile are presented. Testing has been conducted with two physiotherapy treatment heads of differing performance, one of them notably exhibiting a strong "hot-spot" in localised acoustic time-averaged intensity. Findings show good qualitative agreement with classical hydrophone scans. Techniques are explored for the correction of nonlinearities in the thermochromic relationship, to enhance the accuracy of quantitative assessment.

Craniofacial morphologic and anthropometric features of Croatian schizophrenia patients and non-psychiatric controls--a pilot study.

AIM: to evaluate differences in craniofacial morphologic features and several anthropometric measures between schizophrenia patients and non-psychiatric controls, and to find the best-fit model to differentiate between two groups. METHODS: 40 morphologic features of the head and face, and 5 craniofacial anthropometric measures were evaluated using the Lane Dysmorphology Scale in 58 patients and 46 controls. Total MPA score and subscores for different craniofacial regions were calculated. Individual items were examined using logistic regression analyses to define a model that can discriminate between patient vs. control status. RESULTS: total MPA score, and several subscores (general asymmetry, nasal, lip, ear and tongue) were significantly higher in the patient group. Patients were distinguished by significantly higher measures of maxillary and mandibular facial arcs, general and subtle facial asymmetries presented as deviation of facial landmarks from the vertical facial midline and horizontals, more variable vermilion of the upper lip, tongue surface, frenulum and anterior hair margin, and more adherent and underdeveloped earlobes. A final regression model including maxillary are, facial asymmetry, and adherent earlobes as independent predictors proved useful to efficiently recognize schizophrenia patients (specificity and positive prediction value of 100% when all the three items were present in an individual) or to exclude risk for schizophrenia (sensitivity and negative prediction value of 96.6% and 84.6%, respectively, in cases no one of the three items was present). CONCLUSIONS: schizophrenia patients evidenced significantly more craniofacial dysmorphology than controls. The model revealed in the study needs to be verified in larger samples and other populations.

Progress in developing a thermal method for measuring the output power of medical ultrasound transducers that exploits the pyroelectric effect.

Progress in developing a new measurement method for ultrasound output power is described. It is a thermal-based technique with the acoustic power generated by a transducer being absorbed within a specially developed polyurethane rubber material, whose high absorption coefficient ensures energy deposition within a few mm of the ultrasonic wave entering the material. The rate of change of temperature at the absorber surface is monitored using the pyroelectric voltage generated from electrodes disposed either side of a 60 mm diameter, 0.061 mm thick membrane of the piezoelectric polymer polyvinylidene fluoride (pvdf) bonded to the absorber. The change in the pyroelectric output voltage generated by the sensor when the transducer is switched ON and OFF is proportional to the delivered ultrasound power. The sensitivity of the device is defined as the magnitude of these switch voltages to a unit input stimulus of power (watt). Three important aspects of the performance of the pyroelectric sensor have been studied. Firstly, measurements have revealed that the temperature dependent sensitivity increases over the range from approximately 20°C to 30°C at a rate of +1.6% °C(-1). Studies point to the key role that the properties of both the absorbing backing layer and pvdf membrane play in controlling the sensor response. Secondly, the high sensitivity of the technique has been demonstrated using an NPL Pulsed Checksource, a 3.5 MHz focused transducer delivering a nominal acoustic power level of 4 mW. Finally, proof-of-concept of a new type of acoustic sensor responding to time-averaged intensity has been demonstrated, through fabrication of an absorber-backed hydrophone of nominal active element diameter 0.4 mm. A preliminary study using such a device to resolve the spatial distribution of acoustic intensity within plane-piston and focused 3.5 MHz acoustic fields has been completed. Derived beam profiles are compared to conventional techniques that depend on deriving intensity from acoustic pressure measurements made using the sensor as a calibrated hydrophone.

Dosimetric verification of compensated beams using radiographic film.

INTRODUCTION: External photon beam modulation using compensators in order to achieve a desired dose distribution when brachytherapy treatment is followed by external beam radiation is a well-established technique. A compensator modulates the central part of the beam, and the dose beneath the thickest part of the compensator is delivered mostly by scattered, low energy photons. A two-dimensional detector with a good spatial resolution is needed for the verification of those beams. In this work, the influence of different types of detectors on the measured modulated dose distributions was examined. MATERIALS AND METHODS: Dosimetric verification was performed using X-Omat V, Eastman Kodak radiographic films at different depths in a solid water phantom. The film measurements were compared with those made by ionization chambers. Photon beams were also modelled using EGSnrc Monte Carlo algorithm to explain the measured results. RESULTS: Monte Carlo calculated over-response of the film under the thickest part of the compensator was over 15%, which was confirmed by measurements. The magnitude of over-response could be associated with changes in the spectra of photon energy in the beam. CONCLUSIONS: The radiographic film can be used for the dosimetry of compensated high energy photon beams, with limitations in volumes where photon spectra are hardly degraded.

Radiation therapy photon beams dose conformation according to dose distribution around intracavitary-applied brachytherapy sources.

Intracavitary application of brachytherapy sources followed by external beam radiation is essential for the local treatment of carcinoma of the cervix. Due to very high doses to the central portion of the target volume delivered by brachytherapy sources, this part of the target volume must be shielded while being irradiated by photon beams. Several shielding techniques are available, from rectangular block and standard cervix wedge to more precise, customized step wedge filters. Because the calculation of a step wedge filter's shape was usually based on effective attenuation coefficient, an approach that accounts, in a more precise way, for the scattered radiation, is suggested. The method was verified under simulated clinical conditions using film dosimetry. Measured data for various compensators were compared to the numerically determined sum of the dose distribution around brachytherapy sources and one of compensated beam. Improvements in total dose distribution are demonstrated, using our method. Agreement between calculation and measurements were within 3%. Sensitivity of the method on sources displacement during treatment has also been investigated.

Expression of Bcl-2 family proteins in psoriasis.

AIM: To elucidate the mechanisms involved in apoptosis of psoriatic keratinocytes by examining the expression of pro-apoptotic (Bak, Bax) and anti-apoptotic (Bcl-2, Bcl-X) Bcl-2 family of proteins, as well as the expression of p53 and Ki-67 proteins in normal skin, and uninvolved and involved psoriatic skin. METHODS: A total of 90 skin samples (30 cases of involved and uninvolved psoriatic skin and normal skin) were examined immunohistochemically to determine the protein expression of p53, Ki-67, Bcl-2, Bcl-X, Bax, and Bak. The results were quantified and expressed as a percentage of positive keratinocytes. RESULTS: There was a significant increase in Ki-67 (17.05 vs 3.65; P<0.001), Bcl-X (40.21 vs 13.97; P<0.001), Bak (89.46 vs 73.36; P<0.001), and Bax (50.00 vs 29.25; P<0.001) expression and a decrease in Bcl-2 (3.23 vs 6.25; P=0.008) expression in involved psoriatic skin, as well as an increase in Bcl-X (25.13 vs 13.97; P<0.001) expression in uninvolved psoriatic skin, when compared to normal skin. Samples with higher percentage of Ki-67 positive cells showed a higher percentage of p53 positive cells (correlation coefficient r=0.75 in involved psoriatic samples, P<0.001; r=0.88 in uninvolved psoriatic samples, P<0.001; and r=0.85 in normal skin samples, P<0.001). Samples with higher percentage of p53 positive cells expressed pro-apoptotic Bak and Bax in higher percentage of cells; the correlation coefficients were r=0.74 and r=0.68 in involved psoriatic samples (P<0.001 for both), r=0.75 and r=0.69 in uninvolved psoriatic samples (P<0.001, for both), and r=0.87 and r=0.70 in normal skin samples (P<0.001, for both). CONCLUSION: Increased expression of Bcl-X protein was associated with psoriatic epidermal hyperplasia. Strong Bax and Bak expression in involved psoriatic skin are probably inhibitory mechanisms counteracting intensive proliferation.

Expression of cell cycle and apoptosis regulatory proteins in keratoacanthoma and squamous cell carcinoma.

Some authors view keratoacanthoma (KA) as a variant of squamous cell carcinoma (SCC), while others consider it a separate entity that must be distinguished from SCC. Involution displayed by KA is an important difference between these two entities. It has been suggested that apoptosis plays a role in the involution process of KA, although the exact trigger for it remains unclear. A hundred and fifty specimens were included in this study, 30 cases for each of the following groups: normal skin (NS), proliferative keratoacanthoma (pKA), regressing keratoacanthoma (rKA), well-differentiated squamous cell carcinoma (wdSCC), and poorly differentiated squamous cell carcinoma (pdSCC). They were immunohistochemically examined for the expression of p53, Ki-67, bak, and bcl-2. Significantly higher p53 and Ki-67 expressions were observed in all tumor lesions examined as compared with NS. There was higher bak expression in KAs compared to NS and a significant reduction of bak expression in pdSCC together with a significant reduction of bak expression in SCCs compared to pKA. Bcl-2 expression was similar in NS and SCCs, but was lower in rKA. We found a significant positive correlation between p53 and Ki-67, p53 and Bak in NS and examined skin tumors. Lower bcl-2 expression in conjunction with higher bak expression in rKA suggests a possible role of these apoptosis-regulating proteins in tumor regression. In contrast to this finding, a steady level of bcl-2 expression in pdSCC combined with lower bak expression levels and a high proliferation rate could contribute to progression and aggressiveness in these tumors. Bak and p53 expression is a sun-related and age-dependent process in NS and skin tumors.

The role of perforin-mediated apoptosis in lichen planus lesions.

Lichen planus is recognized as a T-cell-mediated disease. Histologically, it is characterized by the formation of colloid bodies representing apoptotic keratinocytes. The apoptotic process mediated by CD8(+) cytotoxic T lymphocytes (CTLs) and NK cells mainly involves two distinct pathways: the perforin/granzyme pathway and the Fas/FasL pathway. So far, little is known regarding the role of perforin-mediated apoptosis in lichen planus. In the present study, the expression and distribution of perforin, T and NK cell subsets in the epidermis and dermis of lesional and nonlesional lichen planus skin were studied. Skin biopsy specimens from lesional and nonlesional skin of ten patients with lichen planus and eight healthy persons were analysed by immunohistochemistry. Significant accumulation of T cells, particularly of CD4(+) and CD8(+) subsets, was found in both epidermis and dermis of lichen planus lesions compared with nonlesional and healthy skin. There were no significant differences in the incidence of NK cells (CD16(+) and CD56(+)) between lesional, nonlesional and healthy skin. Perforin expression was significantly upregulated in the epidermis of lichen planus lesions. In conclusion, accumulation of perforin(+) cells in the epidermis of lichen planus lesions suggest a potential role of perforin in the apoptosis of basal keratinocytes.

The quantitative analysis of the vascularization following two basic auditory canal skin incisions.

Three groups of nine patients each were analyzed. The first two groups consisted of those that underwent tympanoplastic due to chronic inflammation of middle ear. Two different standard auditory canal skin incisions were applied, i.e. tympanomeatal flap (TMF) or vascular strip (VS). The third control group consisted of non-operated patients. All the operated patients were subjected to a quantitative analysis of the auditory canal revascularization by means of the Weibel stereological test method, i.e. the B 100 double network system. The density of capillaries, arterioles, venulolymphatic spaces and a total volume density of all vascular elements of the auditory canal skin were measured. The obtained results of vascularization were compared with those of the target control group. It was found out that there were no significant differences in vascularization of auditory canal skin between TMF and VS patients from one side and the control group on the other side.

Inflamed urachal cyst containing calculi in an adult.

The urachus is an embryonic structure that persists after birth in some individuals and can cause various problems. We report a case of an inflamed urachal cyst filled with a thick yellow fluid and several calculi in a woman with a 1-month history of dysuria. Physical examination revealed a fist-sized tumor located infraumbically in the midline. The patient's erythrocyte sedimentation rate was elevated; the results of all other routine laboratory studies were normal. Sonography showed a regularly shaped, ovoid, hypoechoic cystic area in the abdominal wall measuring 8 x 4 x 3 cm and containing several hyperechoic masses associated with acoustic shadowing. The wall of the cyst was inhomogeneous, and a thin hypoechoic linear tract linked the superior aspect of the mass to the umbilicus. The results of excretory urography, voiding cystography, and cystoscopy excluded an abnormality of the urinary system. A urachal cyst was diagnosed, and the mass was surgically removed. The surgical specimen was sent for histopathologic analysis, which confirmed the diagnosis.