Polymorphisms in toll-like receptor 3 and 4 genes as prognostic and outcome biomarkers in melanoma patients.

Melanoma is one of the most aggressive tumors, and in the setting of rising incidence and mortality, there is an urgent need to identify new prognostic markers. Toll-like receptors (TLRs), are aberrantly expressed in numerous cancers, including melanoma. TLR signaling provides a microenvironment that is involved in antitumor immune response, chronic inflammation, cancer cell proliferation and evasion of immune destruction. In the present study, we investigated whether single nucleotide polymorphisms (SNPs) in TLR3 and TLR4 genes are associated with clinicopathologic features, progression and survival of melanoma patients. The study was conducted on 120 melanoma patients. DNA extracted from peripheral blood was genotyped for TLR3 polymorphisms rs5743312 and rs3775291 (L412F) and TLR4 polymorphisms rs4986790 (D299G) and rs4986791 (T399I), by TaqMan Real-Time PCR Assays. Kaplan-Meier survival curves were compared by the log-rank test. TLR3 polymorphism L412F was associated with a higher mitotic index ( P = 0.035). TLR4 D299G and T399I polymorphisms were associated with indicators of melanoma severity, nodal metastases ( P = 0.005 and P = 0.007, respectively) and advanced stage III ( P = 0.005 and P = 0.004, respectively). Cox regression analysis showed that the presence of tumor-infiltrating lymphocytes (TILs) predicted better overall survival (HR = 0.318; P = 0.004). TLR4 T399I polymorphism was significantly associated with worse survival, P = 0.025. The overall survival rates were significantly lower for patients carrying variant allele T of TLR4 T399I SNP (TC and TT genotypes combined) ( P = 0.008, log-rank test), compared to wild-type genotype CC. Our findings indicate that TLR4 polymorphisms T399I (rs4986791) and D299G (rs4986790) could be potential prognostic and survival markers for melanoma patients.

Does Shoot Age Influence Biological and Chemical Properties in Black Currant (Ribes nigrum L.) Cultivars?

The aim of this study was to examine the influence of shoot age on the biological and chemical properties of 13 black currant cultivars with different origins and ripening times. Phenological observations together with examined pomological and chemical characteristics were studied in two consecutive years at the experimental field near Belgrade, Serbia. The total content of phenols was estimated spectrophotometrically by the Folin-Ciocalteu method, while quantitative analysis of anthocyanin and flavonols aglycones was performed using a high-performance liquid chromatographic (HPLC) method. Principal component analysis was performed to establish differences in biological and chemical properties of black currants. Three-year-old shoots had an earlier start of all examined phenological stages, better generative potential, higher yields, while clusters and berries from 2-year-old shoots had significantly higher values for physical properties, total phenols, anthocyanin and flavanols aglycones and antiradical capacity. Late ripening cultivars had higher contents of all chemical compounds. The berries on 2-year-old shoots had total phenolics that ranged between 123.0 ('Titania') and 298.3 mg/100 g fresh weight (FW) ('Ometa'), while total anthocyanins ranged between 398.5 ('Ojebyn') and 1160.8 mg/kg FW ('Ometa'). According to the obtained results, cultivars 'Ometa', 'Ben Lomond', 'Tsema' and 'Malling Juel' can be recommended as the most promising for growing in the continental climate because they stood out with higher generative potential and yield, physical traits of cluster and berry, higher level of primary and secondary metabolites and DPPH activity in their berries.

GLI-1 polymorphisms of Hedgehog pathway as novel risk and prognostic biomarkers in melanoma patients.

In adult organisms, deregulation of the sonic hedgehog (SHH) signaling pathway is significantly correlated with different malignancies. Currently, data associating genetic polymorphisms in the SHH pathway with melanoma are scarce and largely unknown. The objective of our study was to elucidate an association between gene polymorphisms in the SHH pathway and prognosis of melanoma skin cancer patients. The current study investigated the association of PTCH1 (rs357564), SMO (rs2228617) and GLI1 (rs2228224, rs2228226), polymorphisms with melanoma predisposition and prognosis. Single-nucleotide polymorphisms were assessed by TaqMan SNP Genotyping Assays. The study involved 93 melanoma patients and 97 individuals in the control group. Melanoma patients with the variant mutant genotype GG of GLI1 rs2228226 polymorphism had poorer overall survival and recurrence-free survival (P = 0.0001 and P = 0.037, respectively). The multivariate analysis revealed that disease progression [hazard ratio (HR) = 14.434, P = 0.0001] and the GLI1 rs2228226 polymorphism (HR = 4.161, P = 0.006) persisted as independent prognostic factors. Mutated allele carriers (combined heterozygous and mutated genotypes) for GLI1 rs2228224 G and GLI1 rs2228226 G allele significantly increased melanoma risk [odds ratio (OR) = 2.261, P = 0.007; OR = 2.176, P = 0.010]. Our study demonstrated that genetic variants in GLI1, downstream member of the HH signaling pathway, are the risk factors for melanoma susceptibility and it can be a novel marker for melanoma prognosis. As a crucial SHH signaling member, GLI1 can also be regarded as a novel drug target for anti-cancer treatment in melanoma.

Biological and nutritional properties of blackcurrant berries (Ribes nigrum L.) under conditions of shading nets.

BACKGROUND: Changes of environmental factors, created under the influence of various shading nets, could significantly affect the biological characteristics of plants grown in such conditions as well as biosynthesis of primary metabolites and ascorbic acid. Five blackcurrant cultivars - 'Ben Sarek', 'Ben Nevis', 'Ben Lomond', 'Ometa' and 'Cacanska Crna' - were cultivated in the shade of two green polyethylene nets and exposed to direct sunlight during two experimental seasons. RESULTS: In the control treatment, all cultivars contained the highest amounts of soluble solid content and number of flower buds per shoot in both years of cultivation. The bushes exposed to direct sunlight had the highest sunburn damage to the berries and leaves, and loss of yield. The greatest yield loss caused by berry damage in the present study during the experimental period was in cultivar 'Ben Sarek': 9.0% in 2010 and 15.4% in 2011. Growing in the shade of light-green net contributed the highest content of ascorbic acid. Control bushes and those shaded by light-green net had significantly higher radical scavenging activity, with values between 1.15 and 1.22 mg mL(-1). CONCLUSION: Plants shaded by nets in both years of cultivation had lower damage to leaves and berries and percentage of loss of yield, and usage of the net would be economically advantageous to growers. Fruit of blackcurrant cultivars grown in shading conditions still represent a good source of valuable nutritive and biologically active compounds.

Surface enlargement of a new arterialised venous flap by the surgical delay method.

BACKGROUND/AIM: The delay method is a surgical, pharmacological and combined method that includes two or more time separated phases, which gives bigger flap surface. In our research we explored the possibility of flap surface enlargement in a new arterialised venous flap (AVF) on an experimental rabbit ear model by the delay surgical method. The aim of this research was to establish vitality surface of our AVF and to maintain the difference in flap vital surface between AVF flaps, with or without performing the delay surgery method. METHODS: We used both ears of "Big Chinchilla" rabbits in 10 experimental male animals, divided into two groups, average weight 3-3.5 kg, and average age 8-10 months. In the first (experimental) group, a venous flap was arterialised by our method. In the second (control) group, the venous flap was arterialised 14 days after the delay surgical method. AVF surface was measured on the 1 and 14 days by the method of trapezoid rule. RESULTS: Vital surface on our AVF experimental model was bigger than 87% of elevated flap surface after the delay surgical method. Vital surface on AVF without delay on our experimental model was bigger than 30% of elevated flap surface (p < 0.001). CONCLUSION: Analysis of previous experimental models on the rabbit ear, non-delayed and delayed (to enlarge flap surface) led us to conclusion that previously created experimental models of non-delayed AVF are hemodynamically negative. Our experimental non-delay AVF model is hemodynamically more positive than previously created models of non-delay AVF and provides better conditions for AVF survival and enlargement of vital flap surface of elevated flap. On the other hand, surgical delay method significantly enlarges vital surface of AFF.

Clinical aspects of reconstruction of the lower third of the leg with fasciocutaneous flap based on peroneal artery perforators.

BACKGROUND/AIM: Fasciocutaneous flaps are tissue flaps that include the skin, subcutaneous tissue and underlying fascia, and are based on the septocutaneous system of blood vessels. They have a number of characteristics that give them an advantage in many cases, especially in treatment of lower extremity defects. The aim of this study was to clinically analyse the outcomes of patients surgically treated with a. peroneae perforator-based reverse flaps of the lower leg. METHODS: This prospective study was conducted on a group of 22 patients with tissue defects in the lower third of the lower leg and foot. All the 22 patients underwent standard diagnostic procedures and the preoperative preparation for spinal anesthesia. During the surgery, the location of the recipient site with a future flap and an accompanying vascular stem was marked. Having prepared the recipient site in accordance with the surgical principles, the lobe was prepared, rotated and positioned in the defect region. RESULTS: The proportion of male to female patients in our study group was 19 (86%) to 3 (14%), respectively. The maximal defect frequency (14 or 63%) was found in the age group of 30-50 years, i.e. working-age people. Trauma in 16 (72%) of patients was the most common reason for surgical treatment. The most common defects our patients had were found in the distal third of the leg (5, 22%), medial malleolus (4, 18%), the dorsum of the foot (3, 13%) and the foot (4, 18%). The number of lobes (18, 81%) applied in the course of our clinical study was statistically more significant (p < 0.05) than the number of lobes applied along with the skin autografts (4, 19%). During the final follow-up of the applied lobes, 6 (27.2%) of early and 2 (9%) of late complications were observed, making a total of 36.2% of complications occurring in the applied lobes, of which only 1 was lost. CONCLUSION: Clinical application of a. suralis superficialis mediani-based reverse flap is justified by a high percentage (95.50%) of preserved lobes. Functional recovery of Achilles tendon injuries treated with the fasciocutaneous flap appeared to be effective. The results obtained during the clinical investigation can be very helpful in using this flap more optimally and more often in a wide range of different reconstructive surgical procedures in the field of plastic surgery.

Biochemical properties of the fresh and frozen black currants and juices.

Thirteen black currant varieties (Ribes nigrum L.) cultured in Serbia were characterized for their pomological properties and chemical composition (total phenolics, total anthocyanins, anthocyanin aglycones, sugars, and vitamin C). The average amount of vitamin C varied from 122.4 to 193.2 mg/100 g fresh weight (FW), while concentration of invert sugars ranged from 6.3% to 11.1%. The highest amounts of total phenolics and anthocyanins were detected in variety Ometa (278.9 mg of gallic acid equivalents per 100 g of FW [mg GAE/100 g FW] and 135.4 mg/100 g, respectively). Quantitative analyses of anthocyanin aglycones in berries were performed using high-performance liquid chromatography, and delphinidin was found to be dominant compound in 11 varieties. Total phenolics and anthocyanins contents decreased during the processing of berry fruits to juices, and the reduction of anthocyanins was more pronounced, 12%-80%. The radical scavenging activity of black currant juices was investigated by 1,1-diphenyl-2-picrylhydrazyl, and the IC(50) value ranged from 1.9 to 4.0 mg/mL. Our results also showed that freezing as a way of preservation and storage could save important phytochemicals and health benefits of berries and berry juices. The amount of total phenolics in berries increased during 1 year of storage by 46.09%-171.76% and in juices by even 107.58%, while the amount of total anthocyanins in berries and juices decrease by 5.63%-52.76% and 13.04%-36.82%, respectively.

Surgical anatomy and histology of the levator palpebrae superioris muscle for blepharoptosis correction.

BACKGROUND/AIM: The detailed knowledge of the architecture of the upper eyelid is very important in numerous upper eyelid corrective surgeries. The article deals with the detailed anatomy of the major components of the upper lid, which are commonly seen in surgical practice. METHODS: This study was conducted on 19 human cadavers (12 adults and 7 infants) without pathologic changes in the orbital region and eyelids. Anatomic microdissection of the contents of the orbita was performed bilaterally on 12 orbits from 6 unfixed cadavers (3 male and 3 female). Micromorphologic investigations of the orbital tissue were performed on 8 en bloc excised and formalin-fixed orbits of infant cadavers. Specimens were fixed according to the Duvernoy method. An intra-arterial injection of 5% mixture of melt formalin and black ink was administered into the carotid arterial system. Using routine fixation, decalcination, dehydration, illumination, impregnation and molding procedures in paraplast, specimens were prepared for cross-sections. RESULTS: The measurement of the muscle length and diameter in situ in 6 nonfixed cadavers (12 orbits) showed an average length of the levator palpbrae superioris (LPS) muscle body of the 42.0 +/- 1.41 mm on the right, and 40.3 +/- 1.63 mm on the left side. In all the cases, the LPS had blood supply from 4 different arterial systems: the lacrimal, supratrochlear, and supraorbital artery and muscle branches of the ophthalmic artery. The LPS muscle in all the specimens was supplied by the superior medial branch of the oculomotor nerve. The connective tissue associated with the LPS muscle contains two transverse ligaments: the superior (Whitnall's) and intermuscular transverse ligaments (ITL). The orbital septum in all the specimens originated from the arcus marginalis of the frontal bone, and consisted of two layers--the superficial and the inner layer. In addition, a detailed histological analysis revealed that the upper eyelid's crease was formed by the conjoined fascia including the fascia of the orbicularis muscle, the superficial layer of the orbital septum, and the aponeurosis of the LPS muscle, as well as the pretarsal fascia. CONCLUSION: The conducted study provided a valuable morphological basis for biomechanical and clinical considerations regarding blepharoptosis surgery.

Epidemiology of nosocomial colonization/infection caused by Acinetobacter spp. in patients of six surgical clinics in war and peacetime.

BACKGROUND/AIM: Acinetobacter spp. has emerged as nosocomial pathogen during the past few decades in hospitals all over the world, but it has increasingly been implicated as a serious nosocomial pathogen in military hospitals. The aim of this study was to analyse and compare the surveillance data on Acinetobacter nosocomial colonization/infection (NCI) collected during the wartime with the data collected in peacetime. METHODS: We conducted a prospective study of incidence of Acinetobacter spp. colonization/infection. Also, the two nested case-control studies were conducted. The patients with nosocomial infection (cases) were compared with those with nosocomial colonization (controls) during the two different periods, wartime and peacetime. The patients with NCI by Acinetobacter spp. were identified by the case-based surveillance. The surveillance covered all the patients in 6 surgical clinics. RESULTS: During the study periods a total of 166 patients had cultures that grew Acinetobacter spp. and the pooled rates of Acinetobacter spp. colonization and infection were significantly higher in wartime. When patients with NCI in wartime were compared with those with NCI in peacetime significant differences were observed. In the war year, the patients were more significantly males (p < 0.000). In a period of peace, most of the colonization/infections were reported from patients with certain chronic diseases (p = 0.020) and the survival of patients was more significant (p = 0.049). During the peacetime, proportions of Acinetobacter isolates resistent to ciprofloksacin, imipenem and meropenem were significantly higher (p < 0.001). CONCLUSION: This study provides additional important information about the risk factors of nosocomial Acinetobacter spp. infections in a large cohort of surgical patients. This is also the first study that directly examines epidemiological differences between NCI caused by Acinetobacter spp. during the war and peace period.

Biochemical properties of red currant varieties in relation to storage.

Eleven red currant varieties (Ribes rubrum L.) cultured in Serbia were evaluated for some of their biochemical properties such as total phenolics, anthocyanins, ascorbic acid, invert sugar, soluble solid content, and acidity. The average amount of ascorbic acid varied from 50.5-71.6 mg/100 g FW, while concentration of invert sugars ranged from 6.0%-9.0%. The highest amounts of total phenolics and anthocyanins were detected in variety Redpoll (153.4 mg GAE/100 g FW and 19.3 mg/100 g, respectively). Red currants were processed to juice, and the phenolic and anthocyanin contents changed as a result of processing. Berries and juices were long-term stored at -18 °C and changes in phytochemicals were monitored. In berries, storage caused the decrease of ascorbic acid content up to 49%, and a general reduction of total phenolics was also noticed. In juices, total phenolics content increased after one year of storage. In both berries and juices total anthocyanins increased during storage by up to 85% and 50%, respectively. This study demonstrates that certain varieties, namely Redpoll, Jonkheer and London Market are good source of phytochemicals, retaining the nutritional value during processing and storage.

Surgical site infections in a tertiary health care center: prospective cohort study.

PURPOSE: To investigate the incidence and risk factors associated with the development of surgical site infections (SSIs) using the National Nosocomial Infection Surveillance (NNIS). METHODS: A prospective cohort study was conducted at a tertiary health care center. Infection control personnel collected general and health care related data about patients. The NNIS risk index was calculated on the basis of data relating to the operation: wound contamination class, duration of surgery, and the American Society of Anesthesiologists (ASA) score. RESULTS: A total of 5109 surgical procedures were included in the study. The overall cumulative incidence rate was 6.3%. The incidence of SSIs was 2.3% (63.5% of operative procedures), 8.3% (29.7%), 34.6% (6.2%), and 43.3% (0.6%), in patients with 0, 1, 2, and 3 risk index, respectively. The length of hospital stay (OR: 1.0; 95% CI: 1.053-1.075), preoperative length of stay (odds ratio [OR]: 1.9; 95% confidence interval [CI]: 1.953-1.981), antibiotic prophylaxis (OR: 2.5; 95% CI: 1.421-4.628), drainage (OR: 1.7; 95% CI: 1.360-2.353), ASA score (OR: 1.5; 95% CI: 1.235-1.946), class of wound contamination (OR: 2.0; 95% CI: 1.745-2.003), and NNIS risk index (OR: 1.3; 95% CI: 1.063-1.7) were independently associated with an increased risk for SSIs. Staphylococcus aureus was the most frequently isolated microorganism, 64% of them being methicillin-resistant. CONCLUSION: The aim of this study was to investigate the most important risk factors associated with the development of surgical site infections (SSIs). Therefore, greater attention has been given to adherence to recommendations for the prevention and control of SSIs as well as to antibiotic prophylaxis protocols.

Principles of surgical treatment of congenital, developmental and acquired female breast asymmetries.

BACKGROUND/AIM: There is a natural asymmetry in normal female brests. When the difference in the shape, size or position of the breast and nipple-areola complex is visible, surgical correction is the only treatment option and presents one of the greatest challenges for a plastic surgeon. Based on the Nahai classification presented in details, the aim of the study was to present the possibilities of plastic surgery to correct primary (congenital), secondary (developmental) and tertiary (acquired) brest asymmetries. METHODS: We conducted a retrospective analysis of female breast asymmetry surgeries performed in the Clinic for Plastic Surgery and Burns, Military Medical Academy (MMA), Belgrade over the last seven years (January 2002 - January 2009). RESULTS: During the above mentioned period, 82 female patients, 18 - 65 years of age, underwent surgery for breast asymmetry. The most frequent asymmetries were developmental, "pubertal" (n = 43); acquired asymmetries as a consequence of tumor surgery were found in the other 22 patients, while 7 patients were diagnosed with primary asymmetries such as congenital chest-wall asymmetry (Sy. Poland), accessory and tuberous breasts. All patients underwent preoperative ultrasound examination, while hormone status was determined in those with developmental, "pubertal" asymmetries. The selection of surgical procedure for correction of breast asymmetry depended upon clinical examination findings and patient's wish relating to the shape and size of the breasts. The most of breast asymmetries were corrected by a combination of surgical procedures including primary and secondary reconstruction, reduction, suspension or augmentation mammoplasty. Having combined different surgical procedures, we managed to achive satisfactory results. The hypertrophic scar formation after reduction mamoplasty was seen in some cases, however, they caused no significant patient's discomfort. CONCLUSION: Application of plastic, reconstructive and aesthetic surgical principles can considerably contribute to achieving excellent results in corrective surgery for breast asymmetries. In addition to most suitable breast asymmetry surgical procedures choice, motivation of a patient is also very important for achieving satisfactory results.

[Fasciocutaneous perforators of the lower leg--anatomic study and clinical significance].

BACKGROUND/AIM: Soft tissue defects of the lower leg, especially in its distal third, still remain a challenging problem in reconstructive surgery. The aim of this study was to examine septocutaneous system of lower leg vascularization, quantitatively and qualitatively, which is the basis of fasciocutaneous and neurocutaneous flaps. METHODS: Septocutaneous systems a. tibialis posterior and a. peroneae were examined by anatomical suprafascial lower leg microdissection of 40 fresh cadavers. Septocutaneous perforators located intermusculary, in deep crural fascia duplicature, were followed from its origin (at main arterial trunks of the lower leg) till the point they reach the deep crural fascia. The number and localization of emergence of the septocutaneous perforators were examined for both above mentioned arterial trunks, and the obtained data were documented by photographs, tables and graphics. Statistical analysis (especially cluster analysis), was used for establishing reliable septocutaneous perforators levels. RESULTS: Septocutaneous perforator systems of the lower leg have constant and reliable pattern of existance. Lower leg length was 36 cm in average, (between 33 and 43 cm). Lower leg was divided in 10 equal segments, 3.6 cm each. A. tibialis posterior and a. peronea had 5 septocutaneous perforators. Cluster analysis of a. tibialis posterior perforators (with diameter > or = 0.5 mm), discovered 5 reliable levels of septocutaneous perforators. These levels are at 3.6-10.8 cm; 14.4--21.6 cm and 25.2-28.8 cm. For a. peronea reliable perforators were found at 3.6-10.8 cm, 14.4--18 cm and 21.6-25.2 cm. Posterior tibial artery perforators had the greatest diameter (from 0.5-1.8 mm; mean value 1.14 mm and SD = 0.26 mm). A mean diameter for peroneal artery perforators was 0.9 mm. Conclusion. Existance of reliable levels of septocutaneous perforators of the lower leg enables and makes reconstruction of the soft tissue defects of the lower leg, especially its distal third and foot, much easier.