Direct aperture optimization for online adaptive radiation therapy

This paper is the first investigation of using direct aperture optimization (DAO) for online adaptive radiation therapy (ART). A geometrical model representing the anatomy of a typical prostate case was created. To simulate interfractional deformations, four different anatomical deformations were created by systematically deforming the original anatomy by various amounts (0.25, 0.50, 0.75, and 1.00 cm). We describe a series of techniques where the original treatment plan was adapted in order to correct for the deterioration of dose distribution quality caused by the anatomical deformations. We found that the average time needed to adapt the original plan to arrive at a clinically acceptable plan is roughly half of the time needed for a complete plan regeneration, for all four anatomical deformations. Furthermore, through modification of the DAO algorithm the optimization search space was reduced and the plan adaptation was significantly accelerated. For the first anatomical deformation (0.25 cm), the plan adaptation was six times more efficient than the complete plan regeneration. For the 0.50 and 0.75 cm deformations, the optimization efficiency was increased by a factor of roughly 3 compared to the complete plan regeneration. However, for the anatomical deformation of 1.00 cm, the reduction of the optimization search space during plan adaptation did not result in any efficiency improvement over the original (nonmodified) plan adaptation. The anatomical deformation of 1.00 cm demonstrates the limit of this approach. We propose an innovative approach to online ART in which the plan adaptation and radiation delivery are merged together and performed concurrently-adaptive radiation delivery (ARD). A fundamental advantage of ARD is the fact that radiation delivery can start almost immediately after image acquisition and evaluation. Most of the original plan adaptation is done during the radiation delivery, so the time spent adapting the original plan does not increase the overall time the patient has to spend on the treatment couch. As a consequence, the effective time allotted for plan adaptation is drastically reduced. For the 0.25, 0.5, and 0.75 cm anatomical deformations, the treatment time was increased by only 2, 4, and 6 s, respectively, as compared to no plan adaptation. For the anatomical deformation of 1.0 cm the time increase was substantially larger. The anatomical deformation of 1.0 cm represents an extreme case, which is rarely observed for the prostate, and again demonstrates the limit of this approach. ARD shows great potential for an online adaptive method with minimal extension of treatment time.     

D-Optimal Design in the Development of Rheologically Improved In Situ Forming Ophthalmic Gel

In situ forming ophthalmic gels need to be fine tuned considering all the biopharmaceutical challenges of the front of the eye in order to increase drug residence time at the application site resulting in its improved bioavailability and efficacy. The aim of this study was to develop in situ forming ophthalmic poloxamer P407/poloxamer P188/chitosan gel fine tuned in terms of polymer content, temperature of gelation, and viscosity. Minimizing the total polymer content while retaining the advantageous rheological properties has been achieved by means of D-optimal statistical design. The optimal in situ forming gel was selected based on minimal polymer content (P407, P188, and chitosan concentration of 14.2%, 1.7%, and 0.25% w/w, respectively), favorable rheological characteristics, and in vitro resistance to tear dilution. The optimal in situ forming gel was proved to be robust against entrapment of active pharmaceutical ingredients making it a suitable platform for ophthalmic delivery of active pharmaceutical ingredients with diverse physicochemical properties.     

Effects of high fat diet,ovariectomy, and physical activity on leptin receptor expression in rat brain and white fat tissue

Aim

To evaluate in a rat animal model whether ovariectomy, high fat diet (HFD), and physical activity in the form of running affect leptin receptor (Ob-R) distribution in the brain and white fat tissue compared to sham (Sh) surgery, standard diet (StD), and sedentary conditions.

Methods

The study included 48 female laboratory Wistar rats (4 weeks old). Following eight weeks of feeding with standard or HFD, rats were subjected to either OVX or Sh surgery. After surgery, all animals continued StD or HFD for the next 10 weeks. During these 10 weeks, ovariectomy and Sh groups were subjected to physical activity or sedentary conditions. Free-floating immunohistochemistry and Western blot methods were carried out to detect Ob-R in the brain and adipose tissue.

Results

StD-ovariectomy-sedentary group had a greater number of Ob-R positive neurons in lateral hypothalamic nuclei than StD-Sh-sedentary group. There was no difference in Ob-R positive neurons in arcuatus nuclei between all groups. Ob-R distribution in the barrel cortex was higher in HFD group than in StD group. Ob-R presence in perirenal and subcutaneous fat was decreased in StD-ovariectomy group.

Conclusion

HFD and ovariectomy increased Ob-R distribution in lateral hypothalamic nuclei, but there was no effect on arcuatus nuclei. Our results are first to suggest that HFD, ovariectomy, and physical activity affect Ob-R distribution in the barrel cortex, which might be correlated with the role of Ob-R in election of food in rats.Obesity is one of the leading health issues worldwide, associated with an increased risk of morbidity and mortality (1). In 1997, the World Health Organization (WHO) formally recognized obesity as a global epidemic (2). Increase in body fat stores and obesity is caused by an imbalance between energy intake and energy expenditure (3,4). Since childhood obesity is a predictor of an increased death rate, the “obesity epidemic” may reverse the current declining rate of death from cardiovascular diseases (5). Factors that contribute to obesity can be environmental (6), social (7), behavioral (8), psychological (9), and genetic (10,11).Women generally have more body fat than men (12). Nevertheless similar odds ratios were recorded in women and men for the association of abdominal obesity with acute myocardial infarction (13). Weight gain is common after menopause, indicating an association between hormones and fat stores (14). A large scale observational study found that both the body mass index and the level of physical activity were independent predictors of mortality and that a higher level of physical activity did not eliminate the risk associated with adiposity. At the same time, women who were both lean and physically active had the lowest mortality (15). In animal studies menopause can be induced by ovariectomy (OVX) (16).Obesity can also be called a disorder of appetite and it is controlled by complex homeostatic mechanisms involving the hypothalamus and brainstem (17). Many gut peptides like cholecystokinin, ghrelin, glucagon-like peptide-1 (GLP-1), and -2 and peptide YY (PYY) act on the brain to control eating behavior (18). There are two different system for controlling feeding behavior: short-term and long-term (19). Short-term regulation involves neural signals from the GI tract and its hormones, like insulin, glucagon, and ghrelin (20). A hormone that functions mainly within long-term regulation is leptin (16 kD), a hormonal product of the obesity (ob) gen, primarily secreted by adipocytes (21) and released in the brain. It generates a feeling of satisfaction and acts like an appetite-suppressing agent. Circulating leptin levels are lower in ovariectomized rats (22).Food intake is regulated via neural circuits located in the hypothalamus (23). Leptin acts via its leptin or Ob receptors (Ob-R) and is primarily expressed in hypothalamic neurons (19) especially in arcuate, ventromedial, and dorsomedial nuclei (24). Leptin is transported across the blood-brain barrier (BBB) by a saturable transporter (25). Ob-R is also detected in nonhypothalamic areas in the mice and in human brain neocortex, cerebellum, entorinal cortex, amygdale, and rostral medulla (26). Adipocytes, endothelial cells, and macrophages also have leptin receptor at its surface, which suggests autocrine and paracrine action for leptin in human adipose tissue (27). Association between the expression of Ob-R in target tissues and physiological and hormonal controlled processes is still unclear. Leptin receptors mRNA is found in each of the major components of the CNS “feeding” circuitry – the brainstem, hypothalamus, and is distributed reward centers (Allan brain) (28). Therefore, the aim of the current study was to evaluate whether HFD affects Ob-R distribution compared with StD specifically in the barrel field and piriform cortex compared to standard feeding centers in the hypothalamus. We supposed that the combination of OVX and HFD is interesting for further research on selected brain regions, which might be alleviated by physical activity. We also supposed that changes in Ob-R level in white fat tissue would correlate with the changes in brain regions.     

Single acute stress-induced progesterone and ovariectomy alter cardiomyocyte contractile function in female rats

Aim

To assess how ovarian-derived sex hormones (in particular progesterone) modify the effects of single acute stress on the mechanical and biochemical properties of left ventricular cardiomyocytes in the rat.

Methods

Non-ovariectomized (control, n = 8) and ovariectomized (OVX, n = 8) female rats were kept under normal conditions or were exposed to stress (control-S, n = 8 and OVX-S, n = 8). Serum progesterone levels were measured using a chemiluminescent immunoassay. Left ventricular myocardial samples were used for isometric force measurements and protein analysis. Ca²⁺-dependent active force (F_active), Ca²⁺-independent passive force (F_passive), and Ca²⁺-sensitivity of force production were determined in single, mechanically isolated, permeabilized cardiomyocytes. Stress- and ovariectomy-induced alterations in myofilament proteins (myosin-binding protein C [MyBP-C], troponin I [TnI], and titin) were analyzed by sodium dodecyl sulfate gel electrophoresis using protein and phosphoprotein stainings.

Results

Serum progesterone levels were significantly increased in stressed rats (control-S, 35.6 ± 4.8 ng/mL and OVX-S, 21.9 ± 4.0 ng/mL) compared to control (10 ± 2.9 ng/mL) and OVX (2.8 ± 0.5 ng/mL) groups. F_active was higher in the OVX groups (OVX, 25.9 ± 3.4 kN/m² and OVX-S, 26.3 ± 3.0 kN/m²) than in control groups (control, 16.4 ± 1.2 kN/m² and control-S, 14.4 ± 0.9 kN/m²). Regarding the potential molecular mechanisms, F_active correlated with MyBP-C phosphorylation, while myofilament Ca²⁺-sensitivity inversely correlated with serum progesterone levels when the mean values were plotted for all animal groups. F_passive was unaffected by any treatment.Conclusion Stress increases ovary-independent synthesis and release of progesterone, which may regulate Ca²⁺-sensitivity of force production in left ventricular cardiomyocytes. Stress and female hormones differently alter Ca²⁺-dependent cardiomyocyte contractile force production, which may have pathophysiological importance during stress conditions affecting postmenopausal women.The relation between stress, gender, and cardiovascular diseases is well established (1-4). Some of the known risk factors for cardiovascular disease such as smoking, unhealthy diet, and behavioral and psychosocial stress have deleterious effects on the cardiovascular system via activation of the sympathetic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis (5-8). Acute restraint stress is a preferred and widely used method to induce physical stress in animal models (9). Moreover, restraint and immobilization are important as models for psychological stress, which was shown to adversely affect ovarian function (10) and to play a pivotal role in the pathomechmanism of Takotsubo (stress) cardiomyopathy in postmenopausal women (11).Gender is a very important factor in the development of cardiovascular diseases. Premenopausal women have better lipid profile, endothelial function (12), and a lower risk to develop coronary artery disease and myocardial infarction (MI) than men. These advantages of female gender, however, are abolished after menopause, which is associated with increased prevalence of left ventricular (LV) hypertrophy, decreased LV ejection fraction, and LV contractility (13). One of the explanations for the distinct myocardial responses is the cardioprotective effect of female sex hormones (eg, estrogens) (14,15).Progesterone performs several actions on the heart: it exerts an antiarrhythmic effect by accelerating cardiac repolarization (16) and has a preventive role in ischemia-reperfusion injury via reducing inflammatory response (17). It has been shown to inhibit cardiomyocyte apoptosis (18), induce vasodilation, and reduce blood pressure via increasing nitric oxide (NO) levels in normotensive and hypertensive patients (19). Importantly, progesterone is produced by the both ovaries and the adrenal gland: Moreover, the adrenal progesterone content is similar or even larger than that in the ovaries (20). Adrenal progesterone production and secretion increase along with corticosterone regardless of gender and estradiol under stress conditions (21). Progesterone, being an indirect precursor of cortisol (22), increases in response to adrenocorticotrophic hormone (ACTH) stimulation (23).In the heart, there are multiple estrogen hormone receptor types (24). The expression of aromatase in the heart suggests that estrogen may be synthesized also within the cardiomyocyte to exert autocrine/paracrine actions (25). Myocyte contractility seems to be modulated by systemic estrogen levels and altered in cardiomyocytes derived from ovariectomized (OVX) rats (26). In particular, myofilament Ca²⁺-sensitivity is increased in isolated myofibrillar preparations from OVX rats, and restored to the basal levels with estrogen supplementation (27,28).Activation of the sympathetic nervous system plays a central role in the regulation of cardiomyocyte contractile function and myofilament Ca²⁺-sensitivity through beta-adrenergic receptor stimulation, activating the protein kinase A (PKA). PKA-mediated phosphorylation of Ca²⁺-handling and myofilament proteins (myosin binding protein-C [MyBP-C], troponin I [TnI], titin) were shown to alter cardiomyocyte contractile function (29,30). It has been suggested that female cardiomyocytes operate at lower levels of intracellular Ca²⁺ than those of males, particularly under inotropic conditions (31). This difference in Ca²⁺ homeostasis may be related to the fact that estrogen suppresses the L-type Ca²⁺ current (32,33) and may reduce the amount of Ca²⁺ released from the sarcoplasmic reticulum (SR) (34), which was shown to be larger in myocytes from OVX rats (35). Not only cardiomyocyte contraction, but relaxation may also be affected by estrogen via altered Ca²⁺ re-uptake into the SR and modified Ca²⁺ efflux via increased sarcolemmal Na⁺/Ca²⁺ exchange (36). Interestingly, despite similar SR Ca²⁺ content in males and females (37), studies using OVX models report conflicting results concerning changes in the expression and activity of the SR Ca²⁺-ATPase and its regulator protein phospholamban (38-41). Much less is known about the possible effect of progesterone on cardiomyocyte contractile function. We hypothesized that progesterone affected force production of single isolated cardiomyocytes. Therefore, in the present study we aimed to investigate how sex hormones (particularly progesterone) and single acute restraint stress altered cardiomyocyte contractile function and to identify the consequent posttranslational myofilament protein modifications in OVX rats.     

Dental and Skeletal Age in Patients with Palatally Displaced Canines

ObjectiveTo determine potential associations between dental and skeletal maturation and palatally displaced canines (PDC) considering gender and chronological age.Material and methodsThis study included pretreatment panoramic and lateral cephalometric radiographs of 43 subjects with PDCs and 203 randomly selected orthodontic subjects with normally erupted canines. Both groups were non syndromic patients. Chronological age of subjects was rounded and noted in years with decimal points and compared with chronological age according to Demirjian''s dental age assessment. Skeletal maturation was determined by cervical vertebrae changes on cephalometric radiographs.ResultsFemale subjects with PDC were more affected by left side canine displacement than males (p=0.027) with five times higher odds ratio (OR = 4.9; 95% CIL=1.2-19.7). A comparison of chronologic and skeletal age indicated that PDC subjects were skeletally younger than unaffected groups with statistically significant differences at the age of 10, 12 and 13. (p=0.05).ConclusionYoung subjects with PDCs showed skeletal maturation delay compared to control group, indicating that skeletal maturation assessment could be one of unexplored predicting factors of a PDC, especially at the age between 10 and 13 years in both genders. Subjects with PDC showed intensive growth spurt after the age of 12 years in females, and after the age of 13 in males. Dental maturation delay showed no statistical significance in PDC prediction.     

Epidemiological and clinical aspects of congenital heart disease in children in Tuzla Canton,Bosnia-Herzegovina

Congenital heart disease (CHD) is among the most frequent of all congenital anomalies. The purpose of this study was to present the results of an initial registration of children with CHD from January 1994 to December 1999 in Tuzla Canton, Bosnia-Herzegovina. The population studied consisted of all 39,699 live-born children in this area. Diagnosis of CHD was made by clinical findings, electrocardiography, chest X-ray, echocardiography, catheterisation or autopsy. In the 6-year period, 243 children were found to have CHD, i.e. a prevalence of 6.12 per 1000 live-born. Critical CHD was present in 58 of them, or 1.46 children per 1000 live-born. The average age at diagnosis was 1.47 years. There were 132 boys (54.3%) and 111 girls. The most frequent anomaly was ventricular septum defect with a prevalence of 2.49 per 1000 live-born, representing 40.7 % of the total anomalies. Of the total group, 46 (18.9%) had extracardiac anomalies related to syndromes. Cardiac surgery was indicated in 98 patients (40.3%) but could only be carried out in 42 (17.3%). A total of 63 (25.9%) patients died, 54 of whom within the 1st month of life. CONCLUSION: congenital heart disease is a very significant health problem in Tuzla Canton. It requires urgent measures in terms of organisation of early diagnosis and proper management.     

Loss of heterozygosity of DPC4 tumor suppressor gene in human sporadic colon cancer

We investigated the prevalence of DPC4 loss of heterozygosity in sporadic colorectal cancer. Thirty-six cases of human sporadic colon carcinoma and corresponding normal tissue samples were examined to evaluate loss of heterozygosity at the DPC4 tumor suppressor locus using variable nucleotide tandem repeat (VNTR) analysis and three polymorphic markers. From 36 analyzed samples 35 (97%) were heterozygous or informative. Loss of heterozygosity at the DPC4 locus was detected in 18 (51%) of informative tumor DNAs. The DPC4 LOH was more frequent in smaller tumors (<5 cm) than in larger ones. There was no correlation between DPC4 LOH and age or sex of patients. There was a negative correlation between DPC4 LOH and histological grade or Dukes' stage of tumors, but without statistic significance. Observed results are in agreement with the view that malignant progression is consequence of many genetic changes. It can be concluded that inactivation of the DPC4 gene plays a role in a multistep process of outgrowth and progression of colon cancer.     

Prevalence of G-6-PD deficiency in the Croatian Adriatic Coast population

BACKGROUND: The objective of this study was to determine the prevalence of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency among the population of the Croatian Adriatic Coast, part of the Mediterranean basin. METHODS: The fluorescent spot test was used to screen 2,726 randomly selected high school students in the Croatian Adriatic coastal area. Fluorescence readings were performed at the beginning and at 3, 6, 10, and 25 min of incubation. Results were classified into the following three groups: bright fluorescence (BF), weak fluorescence (WF), and no fluorescence (NF). All NF and WF samples at 3 min were quantitatively measured using the spectrophotometric method. RESULTS: Twelve persons, 10 boys and 2 girls, were found to be deficient in G-6-PD, rendering a 0.44% prevalence of G-6-PD deficiency. All NF samples at fluorescent spot test were G-6-PD-deficient. WF at 3 min of the incubation period was present in 33 (1.2%) subjects, and only 2 (6%) were true positive. Fluorescence reading at 10 min of incubation omits five (41%) of the G-6-PD deficient samples. CONCLUSIONS: Prevalence of G-6-PD deficiency in the Croatian Adriatic coastal population is 0.44%. Fluorescent spot test for moderate enzyme deficiency is reliable in early fluorescence reading.