Effect of ClAlPcS(2) photodynamic and sonodynamic therapy on HeLa cells.

Photodynamic therapy (PDT) uses photosensitive substance to provoke a cytotoxic reaction causing a cell damage or cell death. The substances, photosensitizers, are usually derivates of porphyrine or phtalocyanine. Photosensitizers must be activated by light in order to produce reactive oxygen species, mainly singlet oxygen. Sonodynamic therapy (SDT) utilizes ultrasound to enhance a cytotoxic effects of compounds called sonosensitizers. In this study we investigated photodynamic and sonodynamic effect of chloraluminium phtalocyanine disulfonate (ClAlPcS(2)) on HeLa cells. DNA damage, cell viability and reactive oxygen species (ROS) production were assessed to find whether the combination of PDT and SDT inflicts HeLa cells more than PDT alone. We found that the combined therapy increases DNA fragmentation, enhances ROS production and decreases cell survival. Our results indicate that ClAlPcS(2) can act as a sonosentitiser and combined with PDT causes more irreversible changes to the cells resulting in cell death than PDT alone.

Intravitreal anti-vascular endothelial growth factor combined with half-fluence photodynamic therapy for choroidal neovascularization in chronic central serous chorioretinopathy.

PurposeTo evaluate the results of indocyanine green angiography (ICGA)-guided verteporfin photodynamic therapy (PDT) with half-fluence rate combined with intravitreal application of anti-VEGF in treating choroidal neovascularization (CNV) in chronic central serous chorioretinopathy (CSCR).Patients and methodsIn this retrospective cohort study 17 consecutive patients with secondary CNV due to chronic CSCR had their diagnosis verified with fluorescein angiography (FA) and ICGA at baseline. All eyes received either intravitreal ranibizumab (IVR) or bevacizumab (IVB). On the consecutive day following the initial IVR/IVB treatment, ICGA-guided verteporfin (6 mg/m(2)) PDT with half-fluence rate (25 J/cm(2)) was performed on every patient. IVR or IVB was rescheduled on a pro re nata regimen. Main outcome measures were changes in visual acuity (VA) according to the ETDRS letter score and changes in the central foveal thickness (CFT).ResultsBest-corrected VA at baseline was 65.6 letters (±6.7; n=17) according to the ETDRS letter score. At 12 months, mean ETDRS letter score improved to 71.2 letters (P=0.34). CFT was 309 µm and decreased to 216 µm at month 12 control (P=0.0004). Nine eyes (52.9%) received additional treatment with IVR/IVB due to recurrence of subretinal fluid, with an overall mean number of IVR/IVB treatment of 1.8±3.6 per patient with no systemic side effects during 12 months' follow-up.ConclusionsIVR or IVB combined with ICGA-guided half-fluence PDT with verteporfin is effective in treating CNV in chronic CSCR, with choroidal hyperpermeability in ICGA, resulting in stable vision and significant reduction of CFT.

Deubiquitylating enzyme USP9x regulates radiosensitivity in glioblastoma cells by Mcl-1-dependent and -independent mechanisms.

Glioblastoma is a very aggressive form of brain tumor with limited therapeutic options. Usually, glioblastoma is treated with ionizing radiation (IR) and chemotherapy after surgical removal. However, radiotherapy is frequently unsuccessful, among others owing to resistance mechanisms the tumor cells have developed. Antiapoptotic B-cell leukemia (Bcl)-2 family members can contribute to radioresistance by interfering with apoptosis induction in response to IR. Bcl-2 and the closely related Bcl-xL and Mcl-1 are often overexpressed in glioblastoma cells. In contrast to Bcl-2 and Bcl-xL, Mcl-1 is a short-lived protein whose stability is closely regulated by ubiquitylation-dependent proteasomal degradation. Although ubiquitin ligases facilitate degradation, the deubiquitylating enzyme ubiquitin-specific protease 9x (USP9x) interferes with degradation by removing polyubiquitin chains from Mcl-1, thereby stabilizing this protein. Thus, an inability to downregulate Mcl-1 by enhanced USP9x activity might contribute to radioresistance. Here we analyzed the impact of USP9x on Mcl-1 levels and radiosensitivity in glioblastoma cells. Correlating Mcl-1 and USP9x expressions were significantly higher in human glioblastoma than in astrocytoma. Downregulation of Mcl-1 correlated with apoptosis induction in established glioblastoma cell lines. Although Mcl-1 knockdown by siRNA increased apoptosis induction after irradiation in all glioblastoma cell lines, USP9x knockdown significantly improved radiation-induced apoptosis in one of four cell lines and slightly increased apoptosis in another cell line. In the latter two cell lines, USP9x knockdown also increased radiation-induced clonogenic death. The massive downregulation of Mcl-1 and apoptosis induction in A172 cells transfected with USP9x siRNA shows that the deubiquitinase regulates cell survival by regulating Mcl-1 levels. In contrast, USP9x regulated radiosensitivity in Ln229 cells without affecting Mcl-1 levels. We conclude that USP9x can control survival and radiosensitivity in glioblastoma cells by Mcl-1-dependent and Mcl-1-independent mechanisms.

How prepared are medical students to diagnose and manage common ocular conditions.

It is essential that primary care physicians have a solid fund of knowledge of the diagnosis and management of common eye conditions as well as ocular emergencies, as management of these diseases commonly involves appropriate referral to an ophthalmologist. Thus, it is crucial to receive comprehensive clinical knowledge of ophthalmic disease in the primary care setting during medical school. This study investigated how well prepared medical students are to diagnose and manage common ocular conditions. The study used scores from a standardized 12-question quiz administered to fourth-year medical students (N = 97; 88% response rate) and second-year medical students (N = 97; 97% response rate). The quiz comprising diagnosis and referral management questions covered the most frequently tested ophthalmology topics on board exams and assessed students' ability to recognize when referral to an ophthalmologist is appropriate. Fourth-year medical students had quiz scores ranging from 0%-94.5% with an average score of 68.7%. Second-year students had quiz scores ranging from 27.2%-86.4%, with an average score of 63.8%. Passing rate was 70%. Student's t-test showed fourth-year students had a significantly higher quiz average (P = 0.003). In general, both classes performed better on diagnostic questions (fourth-year, 73.7%; second year, 65.8%) rather than on management questions (fourth-year, 64.8%; second year, 61.8%). Both second-year and fourth-year students on average fell short on passing the ophthalmology proficiency quiz, and in general students were more adept at diagnosing rather than managing ocular conditions and emergencies.

Nonperturbative shell-model interactions from the in-medium similarity renormalization group.

We present the first ab initio construction of valence-space Hamiltonians for medium-mass nuclei based on chiral two- and three-nucleon interactions using the in-medium similarity renormalization group. When applied to the oxygen isotopes, we find experimental ground-state energies are well reproduced, including the flat trend beyond the drip line at (24)O. Similarly, natural-parity spectra in (21,22,23,24)O are in agreement with experiment, and we present predictions for excited states in (25,26)O. The results exhibit a weak dependence on the harmonic-oscillator basis parameter and reproduce spectroscopy within the standard sd valence space.

Strengthening student communication through pediatric simulated patient encounters.

As medical students enter the role of physician, clinical outcomes not only rely on their mastery of clinical knowledge, but also on the effectiveness in which they can communicate with patients and family members. While students typically have numerous opportunities to practice clinical communication with adult patients, such practice in pediatric settings is limited. This study examines if simulated patient (SP) encounters strengthen third-year medical students' communication skills during the pediatrics clerkship. During 2011-2013, three SP encounters (comprising 3 pediatric scenarios) were incorporated into a pediatrics clerkship at one United States medical school to give students a safe venue to practice advanced communication with observation and direct feedback. Third-year medical students engaged in the scenarios and received both written and oral feedback from an evaluator observing the encounter. With IRB approval, students' self-perceived confidence and abilities at performing the advanced communication skills were measured using an eightitem, Likert scale questionnaire administered pre and post the SP encounter. Pre- and post-questionnaires (n=215; response rate, 96%) analyzed using a Wilcoxon-matched pairs signed-rank test demonstrated statistically significant increases in students' perception of their confidence and abilities regarding their performance (P<0.05; Bonferroni correction, P<0.006). There was an increases in student confidence and self-perceived ability in: first, communicating with children and family members of young patients; second, managing confrontational situations involving parents; third, performing a thorough psychosocial history with an adolescent; and fourth, using Evidence Based Medicine to motivate parents.

Electronic screening-enhanced hole pairing in two-leg spin ladders studied by high-resolution resonant inelastic x-ray scattering at Cu M edges.

We study the electronic screening mechanisms of the effective Coulomb on-site repulsion in hole-doped Sr(14)Cu(24)O(41) compared to undoped La(6)Ca(8)Cu(24)O(41) using polarization dependent high-resolution resonant inelastic x-ray scattering at Cu M edges. By measuring the energy of the effective Coulomb on-site repulsion and the spin excitations, we estimate superexchange and hopping matrix element energies along rungs and legs, respectively. Interestingly, hole doping locally screens the Coulomb on-site repulsion reducing it by as much as 25%. We suggest that the increased ratio of the electronic kinetic to the electronic correlation energy contributes to the local superexchange mediated pairing between holes.

Development of monoclonal antibodies to pre-haptoglobin 2 and their use in an enzyme-linked immunosorbent assay (ELISA).

Haptoglobins (HPs) are alpha 2-globulin proteins that bind free hemoglobin in plasma to prevent oxidative damage. HPs are produced as preproteins that are proteolytically cleaved in the ER into alpha and beta chains prior to forming mature, functional tetramers. Two alleles exist in humans (HP1 and HP2), therefore three genotypes are present in the population, i.e., HP1-1, HP2-1, and HP2-2. A biochemical role for nascent haptoglobin 2 (pre-haptoglobin 2 or pre-HP2) as the only known modulator of intestinal permeability has been established. In addition, elevated levels of serum pre-HP2 have been detected in multiple conditions including celiac disease and type I diabetes, which are believed to result in part through dysregulation of the intestinal barrier. In this study, we report the development of a monoclonal antibody that is specific for pre-HP2 with a binding affinity in the nanomolar range. Additional antibodies with specificities for preHP but not mature haptoglobin were also characterized. A sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated. The ELISA showed high specificity for pre-HP2 even in the presence of excess pre-HP1 or mature haptoglobins, and has excellent linearity and inter- and intra-assay reproducibility with a working range from 3.1ng/mL to 200ng/mL. Testing of sera from 76 healthy patients revealed a non-Gaussian distribution of pre-HP2 levels with a mean concentration of 221.2ng/mL (95% CI: 106.5-335.9ng/mL) and a median value of 23.9ng/mL. Compared to current approaches, this ELISA offers a validated, monoclonal-based method with high sensitivity and specificity for measuring pre-HP2 in human serum.

Palliative and end-of-life care training during the surgical clerkship.

BACKGROUND: In 2000, the Liaison Committee on Medical Education required that all medical schools provide experiential training in end-of-life care. To adhere to this mandate and advance the professional development of medical students, experiential training in communication skills at the end-of-life was introduced into the third-year surgical clerkship curriculum at Wright State University Boonshoft School of Medicine. MATERIALS AND METHODS: In the 2007-08 academic year, 97 third-year medical students completed six standardized end-of-life care patient scenarios commonly encountered during the third-year surgical clerkship. Goals and objectives were outlined for each scenario, and attending surgeons graded student performances and provided formative feedback. RESULTS: All 97 students, 57.7% female and average age 25.6 ± 2.04 y, had passing scores on the scenarios: (1) Adult Hospice, (2) Pediatric Hospice, (3) Do Not Resuscitate, (4) Dyspnea Management/Informed Consent, (5) Treatment Goals and Prognosis, and (6) Family Conference. Scenario scores did not differ by gender or age, but students completing the clerkship in the first half of the year scored higher on total score for the six scenarios (92.8% ± 4.8% versus 90.5% ± 5.0%, P = 0.024). CONCLUSIONS: Early training in end-of-life communication is feasible during the surgical clerkship in the third-year of medical school. Of all the scenarios, "Conducting a Family Conference" proved to be the most challenging.

Ab Initio calculations of even oxygen isotopes with chiral two-plus-three-nucleon interactions.

We formulate the in-medium similarity renormalization group (IM-SRG) for open-shell nuclei using a multireference formalism based on a generalized Wick theorem introduced in quantum chemistry. The resulting multireference IM-SRG (MR-IM-SRG) is used to perform the first ab initio study of all even oxygen isotopes with chiral nucleon-nucleon and three-nucleon interactions, from the proton to the neutron drip lines. We obtain an excellent reproduction of experimental ground-state energies with quantified uncertainties, which is validated by results from the importance-truncated no-core shell model and the coupled cluster method. The agreement between conceptually different many-body approaches and experiment highlights the predictive power of current chiral two- and three-nucleon interactions, and establishes the MR-IM-SRG as a promising new tool for ab initio calculations of medium-mass nuclei far from shell closures.

Behavioral alterations and changes in Ca/calmodulin kinase II levels in the striatum of connexin36 deficient mice.

Gap junctions (GJ) are intercellular channels which directly connect the cytoplasm of adjacent cells. GJ allow direct cell-to-cell communication via the diffusion of ions, metabolites and second messengers such as IP(3). The connexin36 (Cx36) protein has been detected in GJ between interneurons of the hippocampus, cerebral cortex, striatum, amygdala, the inferior olive, cerebellum and other brain structures, such as the olfactory bulb. Cx36 knockout (Cx36 KO) mice display changes in synchronous network oscillations in the hippocampus, neocortex and inferior olive and exhibit impaired spatial alternation and one-trial object recognition in a Y-maze. Here, we further characterized the behavioral changes induced by Cx36 deficiency in the mouse by using different behavioral measures and experimental procedures. Additionally, we examined the effects of Cx36 deficiency on acetylcholine esterase (AChE) activity and calcium calmodulin kinase II alpha (CaMKII) protein levels in the striatum. The homozygous Cx36 KO mice displayed increased locomotion and running speed in the open-field, reduced object exploration and impaired one-trial object-place recognition. Furthermore, they exhibited more anxiety-like behavior as compared to the heterozygous controls in the light-dark box. Homozygous Cx36 KO mice exhibited reduced CaMKII levels in the striatum as compared to the heterozygous mice. AChE activity in the striatum was not significantly different between groups. The present results suggest that Cx36 deficiency in the mouse leads to reduced CaMKII levels in the striatum and behavioral changes in open-field activity, anxiety-related behavior in the light-dark box and one-trial object-place recognition.

In vitro cytotoxicity and phototoxicity study of cosmetics colorants.

The aim of the work was early identification of preventable risk factors connected with the consumers usage of products of everyday use, such as cosmetics, toys and children products, and other materials intended for contact with human skin. The risk factor is represented by substances with irritation potential and subsequent possible sensitisation, resulting in negative impact on human physical and psychical health with social and societal consequences. The legislation for cosmetics, chemical substances and other products requires for hazard identification the application of alternative toxicological methods in vitro without the use of animals. For this reason we used a battery of alternative assays in vitro, based on cell cultures. Progressive methods of molecular biology, based on fluorimetry and fluorescence, were employed for identification of early morphological and functional changes on cellular level. Four colorants frequently used in cosmetics (P-WS Caramel, Chlorophyllin, Unicert Red K 7054-J and Unicert Red K 7008-J) were tested on cell line NIH3T3 (mouse fibroblast cell) and 3T3 Balb/c with/without UV irradiation (dose 5 J cm(-2)). Fluorescence methods for the study of cell damage using fluorescence probes offer results for the evaluation of cytotoxicity and cell viability of adherent cells. We detected intracellular production of ROS investigated by molecular probe CM-H(2)DCFDA, which is primarily sensitive to the increased production of hydrogen peroxide or its downstream products. Toxic effects on the cellular level were identified by viability tests using Neutral Red uptake and MTT assay, where the live cells reduce yellow soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to insoluble formazan crystals. The reaction was investigated on mitochondrial membrane of living cells and the type of cell death was determined using Apoptosis detection kit. Cytotoxicity tests revealed health risks of using Chlorophyllin and Unicert Red K 7054-J.

Phototoxic effect of TPPS4 and MgTPPS4 on DNA fragmentation of HeLa cells.

Photodynamic therapy (PDT) is an alternative method of tumour treatment. It is based on a photochemical reaction of a photosensitizer, irradiation, and O(2) which converts to cytotoxic (1)O(2) and other forms of reactive oxygen species (ROS). The comet assay (also called single-cell gel electrophoresis, SCGE) is a sensitive, simple and quantitative technique for detection of DNA damage. In our study we investigated the phototoxicity of the two porphyrin photosensitizers, TPPS4 and MgTPPS4, on HeLa cells. Three different radiation doses and six different concentrations of the photosensitizers were used. Our results show that the DNA of the cells treated with the TPPS(4) and MgTPPS(4) at the concentrations higher than 5 µM was highly fragmented indicating a strong phototoxic effect resulting in a cell apoptosis. On the base of our results we can hypothesize that even the irradiation dose of 1 J cm(-2) is sufficient enough to provoke the DNA fragmentation.

Modeling the effect of facial topographies upon the visual field of humans and primates.

PURPOSE: To establish a virtual device that can predict the effect of facial features on the visual field of humans and primates. METHODS: Virtual masks were obtained from human subjects, and macaque, chimpanzee and baboon taxidermic specimens, and aligned with upright head orientation at the center of a virtual perimeter-like dome (radius = 50 m) developed with Cinema 4D. Virtual searchlights positioned at the masks' pupils were then allowed to 'paint' facial elements obstructing their path, and demarcate the unobstructed rays at the perimetric surface and on a virtual ground floor related to eye level. Searchlight positions along the human mask's pupillary axis were identified by maximum congruence to Goldmann visual field limits. RESULTS: The human contours largely concur with large-stimulus isopters displaying the limiting role of the nasal ridge, and the relatively extended ventral and temporal limits. In contrast, the facial design of chimpanzees and baboons obstructs significant portions of the ventral foreground (>2 m cf < 0.5 m in human), while there appear to be larger binocular overlaps (125 degrees in chimp cf 90 degrees in human). CONCLUSIONS: The model provides information on anatomical constraints for monocular and binocular visual field extensions including projection of the ventral field on a virtual floor.

Two-component dynamics of the order parameter of high temperature Bi2Sr2CaCu2O8+delta superconductors revealed by time-resolved Raman scattering.

We study the dynamics of the superconducting order parameter in the high-Tc cuprate Bi2Sr2CaCu2O8+delta by employing a novel time-resolved pump-probe Raman experiment. We find two different coupling mechanisms that contribute equally to the pair-breaking peak. One coupling sets in very fast at 2 ps and relaxes slowly, while the other one is delayed and sets in roughly at 5 ps and relaxes fast. A model that couples holes through phonons is able to reproduce one part of the condensate dynamics; thus, we argue that hole-spin interactions are of importance as well.

Systemic bevacizumab (Avastin) therapy for exudative neovascular age-related macular degeneration. The BEAT-AMD-Study.

BACKGROUND: Double-blinded, randomised, prospective, pilot-study to determine the effect of systemic bevacizumab therapy. METHODS: Subjects with fibrovascular pigment epithelial detachment, subfoveal choroidal neovascularisation extending under the geometric centre of the foveal avascular zone and/or macular thickness of at least 300 microm in both eyes were included. Sixteen eyes were included and randomised equally to receive either three infusions of 5 mg/kg Avastin or 100 ml of 0.9% sodium chloride every 2 weeks. The main outcome measure was the lesion size. The follow-up time was 24 weeks. RESULTS: Throughout the 24-week follow-up, the lesion size and macular thickness decreased in the Avastin group by 0.5 (SD 0.08) mm and 103.6 (14.9) microl respectively. In both groups, visual acuity remained stable in seven eyes and decreased in one eye. At the end of follow-up, 50% of the eyes in the Avastin group became fibrotic, 37.5% remained unchanged, and 12.5% developed a subretinal bleeding. There was a treatable rise in blood pressure after Avastin treatment. CONCLUSION: Systemic Avastin could be offered to patients with exudative age-related macular degeneration in both eyes and/or patients who refuse intravitreal injections if blood pressure is normal and there is no history of thrombosis.

Complement factor H gene polymorphisms and Chlamydia pneumoniae infection in age-related macular degeneration.

PURPOSE: To investigate the association of the complement factor H gene (CFH)Y402H polymorphism and age-related macular degeneration (AMD) in the Austrian population (Caucasoid descent), and to determine whether there is an association between exposure to Chlamydia pneumoniae-responsible for up to 20% of community-acquired pneumoniae-and the AMD-associated CFHrisk polymorphism. METHODS: Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism analysis in 75 unrelated AMD patients and compared with 75 healthy, age-matched control subjects. C. pneumoniaeserum IgG was tested by ELISA (R&D) in both groups. The association between the CFHY402H genetic polymorphism and the disease was examined by chi (2)-test and logistic regression. RESULTS: CFH Y402H genotypefrequencies differed significantly between AMD patients and healthy controls (1277 TT, 22.7%; 1277 TC, 53.3%; and 1277 CC, 22.7% in the AMD group; 1277 TT, 48.0%; 1277 TC, 38.7%; and 1277 CC, 13.3% in the control group) showing a P-value <0.005 (OR:2.920/3.811).No association was found between a positive C. pneumoniae titre and AMD (P=0.192), nor was any association found between C. pneumoniae and the CFH Y402H polymorphism. CONCLUSIONS: Our data confirm that the CFHY402H polymorphism is a risk factor for AMD in the Austrian population with a higher frequency of the Y402 polymorphism in AMD patients. No association between preceding C. pneumoniaeinfection and diagnosed AMD was found.

[Long-term results of scleral buckling using a microsurgical approach]. / Langzeitresultate nach mikrochirurgischer skleraeindellender Ablatiochirurgie.

BACKGROUND: The purpose of this study is to report the long-term results of a refined scleral buckling technique for the treatment of retinal detachments. PATIENTS/MATERIALS AND METHODS: We retrospectively reviewed the charts of 152 consecutive cases (129 patients), operated in our clinic by one surgeon. Indirect ophthalmoscopy was used for the identification and treatment of retinal breaks. All other steps of the operation were performed under the operating microscope. The main outcome measures were the retinal redetachment rate, intraoperative and postoperative complication rates as well as the functional outcome. RESULTS: Reattachment was achieved in 130 eyes (85.5 %) after the first procedure and in 151 eyes (99.3 %) after reoperation. No intraoperative complications occurred in our series. Eighty-one eyes (53.3 %) showed a significant improvement in visual acuity after surgery, whereas 16 eyes (10.5 %) showed a significant deterioration in visual acuity. At the last follow-up visit, 101 cases (66.4 %) showed a visual acuity of 0.5 or better, 68 cases (44.7 %) had a visual acuity of 0.8 or better and in 7 cases (4.6 %) the visual acuity was lower than 0.1. CONCLUSIONS: Adding microsurgical techniques to scleral buckle surgery may help to minimise the incidence of intraoperative and postoperative complications resulting in a favourable functional outcome.

23-Gauge versus 20-gauge system for pars plana vitrectomy: a prospective randomised clinical trial.

AIM: To compare the sutureless 23-gauge system with a standard 20-gauge system in pars plana vitrectomy. METHODS: 60 patients in two randomised groups were included in this prospective clinical trial. Pars plana vitrectomy with either 23- or 20-gauge instruments was performed. The main outcome measures were postoperative conjunctival injection and pain. Secondary outcome parameters were time of surgery, intraocular pressure, visual acuity and complications. RESULTS: Conjunctival injection (p = 0.0003) and postoperative pain (p = 0.01) were significantly reduced following 23-gauge vitrectomy compared with the 20-gauge procedure. Opening (p = 0.006) and closure times (p<0.00001) were significantly shorter, and vitrectomy time (p = 0.001) significantly longer in the 23-gauge system compared with 20-gauge vitrectomy. However, retinal manipulation and overall surgery times did not differ significantly between both groups. The same applies for eye pressure, distance and reading acuity. Regarding complications, two choroidal haemorrhages and one flat serous choroidal detachment occurred in the 23-gauge group. CONCLUSION: The 23-gauge system for pars plana vitrectomy offers significantly higher patient comfort during the early postoperative period. Time of surgery is almost equal--a shorter time for wound closure is neutralised by a longer vitrectomy time in the 23-gauge group.