APC activation is therefore a necessary prerequisite for an efficient adaptive immune response. DCs not only provide antigen and co-stimulation to naïve T cells, but also contribute to the initial commitment of naïve T helper cells into Th1, Th2 or other subsets. This directs the efficient induction of T helper cells selleck chemical with appropriate cytokine profiles early during infections, without the need for direct contact between antigen-specific T cells and pathogens. Undigested pathogen-derived antigens are also drained by the lymph and transported to the B cell-rich area of the lymph node, where they are exposed to BCR-expressing cells. An

adaptive immune response is therefore initiated in a draining lymph node by the concerted action of innate immune cells and free antigens. These activate T and B lymphocytes, respectively, to proliferate and differentiate into effector and memory cells. The type of communication employed by the immune system represents a unique approach to multi-system signalling and communication over distances. As well as employing the soluble mediators – proinflammatory messengers, chemokines and soluble danger signals – the immune system uses migratory APCs to physically transport messages from the periphery to the induction sites of adaptive immune response, eg in lymph nodes. Notably, by selectively migrating in response to infectious/cell-damaging events, DCs act as filters

for the adaptive immune response, helping T and B cells to ignore innocuous foreign antigens. Thus, the innate immune response plays an important role in selecting antigens that represent a real SB431542 concentration threat to the organism that requires an adequate adaptive response. The response to pathogens in humans takes place over a large anatomical distance and in distinct phases, which are summarised in Figure 2.9. The innate immune response is initiated at the site of challenge when a foreign entity triggers a defensive response, which is mediated by chemical signals. These signals attract responding innate immune cells (monocytes, DCs etc) which travel to the site and engulf fragments of the pathogen. The monocytes and DCs then leave

the site via lymphatic vessels and begin to mature and Dolutegravir differentiate, while travelling to the local draining lymph nodes. Differentiation gives rise to APCs that interact with naïve T cells at the lymph nodes and bear receptors for the antigenic peptides expressed on the surface of the APC. Molecular, antigenic and cytokine signals combine to direct the differentiation and activation of CD4+ T cells into distinct effector subtypes. This is the induction phase of the adaptive immune response. A sub-population of CD4+ T cells differentiates into memory cells, which are capable of responding rapidly on repeat exposure to the same antigen. CD8+ T cells also receive antigenic and cytokine stimulation from APCs and undergo differentiation either into memory-type cells or armed effector cytotoxic cells.

Because the subtests designed to probe the central executive and phonological loop depend heavily ABT-199 concentration on language, it is possible that the observed working memory deficits in the participants with SLI might be due to their language problems rather than to working memory deficits per se. Therefore we performed additional analyses in which we covaried out a measure of language abilities. We computed a single composite variable of language by submitting the four measures of language (expressive and receptive lexical and grammatical abilities; see Table 2) to a principal components analysis, and extracted

a single factor. This approach aims to create a composite variable that maximizes the shared variance of all four language measures, and minimizes the variability that is unique to a single measure or is shared only between two or three of them. The four measures accounted for PLX3397 cost 67.7% of the variance in the language factor. The factor loadings were as follows: Expressive Vocabulary = .853, Receptive Vocabulary = .832, Expressive Language = .769 and Receptive Grammar = .834. The MANCOVAs with the language factor included as covariate yielded significant multivariate group effects both for the central executive (p < .001) and phonological loop subtests (p < .001), although with a reduction of effect sizes in both cases ( Table 3, Covariates: Language

Factor). The post-hoc univariate tests controlling for language abilities revealed significant differences on all the central executive and eltoprazine phonological loop subtests except the Word List Matching subtest, mostly with medium (partial η2 ≥ .059) or large effect sizes ( Table 4, under “Covariate: Language Factor”). The next set of analyses tested SLI-TD group differences on the CMS, to examine declarative memory for verbal and visual information. Results from between-subjects MANOVAs revealed a significant multivariate group effect for the subtests probing verbal information (p < .001), with a large effect size, but not for the subtests of visual information (p = .350),

which yielded a small effect size ( Table 3, Covariates: None). The post-hoc univariate tests ( Table 5, under “No covariates”) yielded significant group differences, with medium to large effect sizes, on all measures designed to assess verbal aspects of declarative memory. In contrast, small effect sizes were found on all visual subtests, only one of which showed a significant group difference. Many of the subtests from the CMS require children to temporarily store information, and thus the observed group differences could in part be explained by working memory deficits rather than problems with declarative memory itself. Group differences on the CMS were therefore examined while controlling for working memory.

At Day 4, the shape of the latebra in the center of the yolk changes significantly from a spherical region to a flat horizontal

star-like structure (Fig. 1E). The latebra becomes smaller and less distinct after ABT-888 order Day 6 (Fig. 1G). Segmentation and 3D surface rendering of the embryo, yolk, albumen, EEFs and latebra during first 120 h of development were carried out for three eggs in the longitudinal study. This allowed the 3D changes in the shape, location and volume of the various components during embryonic development to be visualized (Fig. 3) and quantified (Fig. 4) (Supplementary Data Table S1). At Day 0 (Fig. 3A), about 70% of the egg is albumen and the rest is yolk. Over a 120-h period, the mean total volume of fluids in the egg decreases by 5.8% from 9.41 to 8.86 ml. This is due to water loss by evaporation through the shell, even though HSP inhibitor the incubators are humidified to help reduce water loss. The volume of yolk in the quail egg is about 3 ml and this does not significantly change during early embryonic development. In contrast, dramatic changes in the aqueous regions are detected after 48 h of incubation. An aqueous region becomes visible above the yolk, which includes SEF (Fig. 1C). From Day 0 to Day 5, the volume of albumen decreases by 67.6% from 6.37 to 2.06 ml. Much of the

reduction in the volume of albumen is due to movement of water to SEFs and the other to EEFs; by Day 5, the volume of EEF has increased to 3.51 ml. The EEFs consist

of fluid lying under the embryo in the sub-germinal space and fluids within extra-embryonic cavities enclosed by the chorion and amnion. The SEF is less dense than the albumen [24] and lies within the yolk sac above the yolk. The amniotic fluid around the embryo and the fluid in the allantois can be distinguished in high-resolution images of the embryo (annotated in Fig. 2H). By Day 7, the image intensity of the allantois fluid is lower (darker image) than that of the amniotic fluid. The drop in image intensity in allantois arises from an increase in biomolecules including paramagnetic iron which decrease the water’s transverse relaxation rate. The embryo first becomes visible in the MR images of quail eggs on Day 3 (Fig. 1D). The sagittal crown-rump length of the Day 3 embryo is around 4 mm (Fig. 5A). Some anatomical structures can be made out and the image digitally segmented Aurora Kinase to produce 3D representations (Fig. 5B) of features of the vessels (red), spine (white), brain (light blue) and eyes (cream). Sagittal crown-rump length increases to 7 mm (Day 4), 10 mm (Day 5) and 14 mm (Day 6), respectively (Fig. 2). The volume of the embryo at Day 3 is about 0.02 ml which increases to 0.038 ml at Day 4 and 0.105 ml at Day 5; thus the volume of the embryo nearly trebles between Day 4 and Day 5. The Day 3 embryo lies close to the top of the egg as the embryo is slightly less dense compared to the other aqueous fluids (Fig.

Results depicted in Fig. 4 indicate that complex I inhibition by Ebs, (PhSe)2 and (PhTe)2 was not modified by the addition of SOD (Fig. 4A), CAT (Fig. 4B) or SOD + CAT (Fig. PARP inhibitor 4C). In order to test the hypothesis that organochalcogens-induced complex I inhibition is mediated by oxidation of thiol groups, we investigated the efficacy of GSH

to reverse the organochalcogens-induced inhibition of complex I. Fig. 5 shows that GSH (500 μM) completely reversed the organochalcogens-induced complex I inhibition in hepatic (Fig. 5A) and in renal (Fig. 5B) membranes. In order to check the inhibitory effect of different organochalcogens in mitochondria complex II activity, we carried out experiments at two different conditions. In brief, in condition 1 the membranes were incubated with the organocompounds (at different concentrations) in the presence of succinate

5 mM for 10 min. The reaction was stopped 3 min after MTT by addition of ethanol. In condition 2, the mitochondrial membranes see more were incubated with various concentrations of organocompounds in the absence of succinate for 10 min. Succinate (5 mM) and MTT were then added and the reaction stopped after 3 min by the addition of ethanol. Statistical analysis indicates that Ebs and (PhTe)2 significantly inhibited both hepatic and renal complex II activity in both conditions (Fig. 6). In contrast, (PhSe)2 did not change the mitochondrial complex II activity from liver (Fig. 6A and B), but inhibited renal complex II activity under condition 6-phosphogluconolactonase 1 (Fig. 6C), without inhibiting it under experimental condition 2 (Fig. 6D). The IC50 (μM) values for inhibition by organochalcogens of mitochondrial complex II activity, in both conditions, are showed in Table 1. Malonate (8 mM) caused a significant inhibition of the mitochondrial complex II activity that varied from 40% to 70% inhibition (see Fig. 6A–D). GSH (500 μM) completely reversed the organochalcogens-induced complex II inhibition both in hepatic (Fig.

7A) and renal (Fig. 7B) membranes. Ebs and (PhTe)2 inhibited the mitochondrial complexes II–III activity from liver (Fig. 8A) and kidney (Fig. 8B). (PhSe)2 did not inhibit hepatic complexes II–III activity (Fig. 8A), but significantly inhibited renal complexes II–III activity (Fig. 8B). The IC50 (μM) values for inhibition by organochalcogens of mitochondrial complexes II–III activity are showed in Table 1. Statistical analysis revealed that Ebs did not modify the hepatic (Fig. 9A) or renal (Fig. 9B) complex IV activity. (PhSe)2 slightly inhibited complex IV activity from liver and kidney (Fig. 9A and B), whereas (PhTe)2 did not change the renal complex IV activity (Fig. 9B); but it inhibited hepatic complex IV activity at 50 μM (Fig. 9A). The IC50 (μM) value for inhibition by (PhSe)2 of mitochondrial complex IV activity is showed in Table 1.

5–7 pmol L− 1 d− 1 in snow, and the corresponding numbers for CH2ClI were 0.1–0.9 pmol L− 1 d− 1 in ice and 0.1–1 pmol L− 1 d− 1 in snow (n = 7). Again, these values are comparable to release rates from the Arctic Ocean (Karlsson et al.) in snow for CHBr3, although the maximum release rate for CH2ClI

was 10 times lower. Atmospheric selleck chemicals halocarbons that have been naturally produced could have two sources: sea ice/snow and surface sea water. To establish which of the two that was most important, saturation anomalies were calculated for the systems sea ice/air and surface water/air. The saturation anomalies, SA (%), for CHBr3 and CH2ClI were determined by the equation: equation(2) SA=Cw−Ca/HCa/H×100%where Cw = concentration in brine or sea water, Ca = concentration in air, and H = temperature dependent Henry’s law constants, determined by Moore et al. (Moore et al., 1995). They stated that they are valid in the salinity

range 30 ± 5. The brine salinity in this study varied between 30 and 36, and no correction for ionic strength was therefore needed. CHBr3 was found to be both over- and under-saturated in brine at different stations, with SA varying between − 61 and 97% (Fig. 5a, Table 5). Highest over-saturation coincided with elevated CHBr3 concentrations in air. Production time studies also showed that all halocarbons were released from sea ice as well as from snow (see supplemental material). CH2ClI was over-saturated in brine at all stations, varying between 91 and 22, 000% (Fig. 5b, Table 5). CHBr3 was LDN-193189 cost under-saturated in surface waters throughout the Amundsen Sea, with saturation anomalies ranging between − 83 and − 8% (Fig. 5a, Table 6), with the highest undersaturation in the surface water (Ice station 4, − 83%) coinciding with highest Alanine-glyoxylate transaminase oversaturation in brine (97%). This implies that sea water was not the dominating source of CHBr3 in air; conversely, it implies that a sea-ice environment may be a major contributor to the atmosphere. As can be seen in Fig. 5, the variation in saturation anomalies mostly depends on the concentration of the halocarbons in air. In earlier work by Carpenter et al. (2007),

a mean mixing ratio of the halocarbons in air was used to calculate the saturation anomaly. Their approach of using mean mixing ratios results in a smoothed distribution, whereas our data accounts for spatial and temporal variations. The calculated saturation anomaly for CH2ClI in the surface water suggested that CH2ClI was oversaturated in the Amundsen Sea, varying between 9 and 1200% (Fig. 5b, Table 6), although it was lower when compared to the saturation anomaly in brine. One explanation for this difference in the saturation anomalies between CHBr3 and CH2ClI is the different atmospheric half-lives, where the half-life for CH2ClI is as short as 0.1 day compared to the CHBr3 half-life of 26 days (Law et al., 2007) . CH2ClI therefore quickly degrades in air when released from the sea ice or surface water (i.e.

More oxidation was caused by freeze–thawing 10 times over 14 d at −20 °C (Fig. 5d), or leaving the peptide at −20 °C

over 80 d (Fig. 5e), or leaving the peptide at 4 °C for 37 months (Fig. 5f). Storing peptide III_24 in N2-saturated solution with repeated freeze–thawing over 14 d slowed oxidation four-fold (data not shown). Long-term storage of other Toolkit peptides resulted in variable polymerization. Just 12% of 37 month-old III-04 had formed helical polymers while 44% of 41-month old II-56 was polymeric, similar to the level shown for III-24 in Fig. 5f. A sample of III-24 (Tm 51 °C at 2.5 mg mL−1) stored for 48 months at 4 °C was 47% triple-helical when analyzed by gel filtration at 40 °C. However, after 10 min reduction with 2 mM TCEP, the proportion of triple-helical peptide was 18%. Helicity was ∼75% if gel filtration was run at 10 °C, regardless of the presence of TCEP. Peptides were heat-denatured after storage Fasudil at 4 °C for 9 months or longer. They were analyzed by gel filtration at 60 °C, and by MALDI and electrospray mass spectrometry immediately after heating to 60 °C. Their cysteine thiol content was determined using Ellman’s reagent. This allowed Compound C in vitro us to characterize the peptide polymer mixture (Suppl. Figs. S2–S5, Tables S1 and S2, Sections 3.8, 4.4, 4.5). Briefly, >90% of

cysteine in peptides aged for 9 months or more is oxidized, and cross-linked such that 5–13% of the peptide is monomeric (mostly cyclic), 7–50% is dimeric, correlating with peptide stability and purity, where CRPcys has less dimer than the other peptides, and the remainder is polymeric. Positive controls using

fresh peptide were ∼95% reduced as expected. Gel filtration revealed that, in the presence of 2 mM TCEP, peptide III-24 at 2.5 mg mL−1 Myosin was almost free of any component bigger than a single helix, no matter what temperature (4–50 °C) was maintained before loading onto the column (see, for example, Fig. 5a). To confirm this, we undertook DLS experiments under reducing conditions in neutral buffer. There was no evidence of any species larger than around 16.5 kDa, equivalent to a single helix. We could not resolve peptide monomer from helix, so mass and Stokes Radius shown in Table 2 represent average values, decreasing with increasing temperature due to helix denaturation. Stokes Radius correlated well with values obtained from gel filtration, and are as expected for rod-like molecules of this mass. We evaluated the coating of biotinylated peptides with or without cysteine to 96-well plates, detected as described in Section 2. We could detect coating of the plastic by cysteine-containing biotinylated peptide (B-GFOGERcys), but biotinylated peptides lacking cysteine-adhered poorly (B-GFOGER) or not at all (B-CRP) (Fig. 6a). Additionally, all peptides containing motifs that bind integrin α2β1 or GpVI and terminal cysteine supported platelet adhesion (CRPcys, GFOGERcys, B-GFOGERcys, Fig. 6b).

Arterial compliance was characterized by cerebral pulse transit time derived from phase difference analysis between ECG and TCD signals. Sleep time was dichotomized into periods with high density of consecutive respiratory events vs. periods with low density of consecutive respiratory events. TCD measurements of CBF velocity showed a regular, undulating pattern with flow minima immediately before apneas or hypopneas and maxima closely after their termination, reciprocally to peripheral O2 saturation.

CBF velocity reactivity was significantly diminished in consecutive respiratory events compared to non-consecutive respiratory event periods. The authors discussed severe disturbances of cerebrovascular reactivity in OSAS patients and interpreted their data as a sign of loss of vasoreactivity and increase of arterial stiffness. The combined long-term recordings of intracranial High Content Screening Talazoparib cell line flow patterns

and polysomnography constitute an important method for evaluating dynamic aspects of brain function and cerebral perfusion during sleep. Numerous studies concerning this scientific field using this technique have contributed to a better understanding of the physiology of the normal sleep and the pathophysiology of sleep disorders as well as that of nocturnal stroke. “
“The mechanism of cerebral autoregulation (CA) minimizes fluctuations of cerebral blood flow (CBF) during changes of cerebral perfusion pressure (CPP). Pressure triggered dilatation or constriction of small artery vessels may control cerebral blood flow resistance and prevent the brain from ischemia during decrease as well as from hyperemia during increase of CPP. This so-called cerebrovascular pressure reactivity (CVR) is a pre-condition of a working CA. While cerebral autoregulation is characterized by its regulating effect on cerebral blood flow, CVR describes the state of its underlying mechanism. Since CA may be affected in patients with severe brain injuries [1] and [2] its monitoring

provides important information for clinical treatment. Various monitoring methods are based on the concept of dynamic CA [3] which not NADPH-cytochrome-c2 reductase only describes a steady-state relationship between CPP and CBF [1] but also assesses the flow dynamics during rapid pressure changes. During monitoring these pressure changes may either be induced under controlled conditions [4] and [5] or due to spontaneous oscillations of ABP or CPP [6] and [7]. In recent publications the question whether CA was symmetric, i.e. whether CA response was equally effective during increase and decrease of pressure challenge, was subject to investigation and partly contradictive results. For the first time Aaslid reported a stronger response of dynamic autoregulation during increasing ABP compared to decreasing ABP [8]. This effect was demonstrated in 14 patients with traumatic brain injuries (TBI) during cyclic changes of ABP which have been induced by sequentially repeated leg cuff tests.

All patients were operated more than five years after the diagnosis. None of the patients with essential tremor had a preexisting cerebellar injury on the basis of history, neurological examination, and brain imaging as reviewed with a neuroradiologist. None of the patients with cerebellar tremor had any family or personal history of essential tremor or any symptoms of tremor before their cerebellar injury. The control group consisted of patients

with neuropathic lower extremity pain. None of them had a personal or family history of tremor. They had no cerebral or cerebellar pathology based on detailed neurological examination and MRI imaging. Their electrophysiological recordings were therefore suitable selleck chemicals to use a control for comparison of firing rates and other parameters with the tremor patients. The protocol was reviewed and approved annually by the Institutional Review Board of the Johns Hopkins University. All

patients signed an informed consent for these studies. Details of the methods used in this study have been previously described (Hua and Lenz, 2005). Thalamic exploration was performed as a stereotactic procedure using the Leksell frame in patients who were off tremor medications for at least 18 h. First, the frame coordinates of the anterior (AC) and posterior commissures (PC, Fig. 1A) were measured by magnetic resonance imaging (MRI) or computed tomography. These coordinates were used to estimate the nuclear locations. selleck chemicals llc Physiological corroboration of nuclear location was then performed under local anesthesia without sedation (i.e. subject fully conscious) by single unit recording and microstimulation through a microelectrode. We used a platinum–iridium electrode etched to

a tip of 3–4 mm and coated with solder glass to give an impedance of approximately 2.5 MΩ, which was reduced to approximately 5 MΩ by microstimulation (50 µA) in the brain. The electrode was advanced toward the target as localized by pre-operative imaging. The signals recorded on magnetic tape (Model 4000, Vetter Corp., Rebersberg, PA, USA) or electronically (Cambridge Electronic Design, CED PIK-5 1401 interface) during the procedure included: the foot pedal indicating events during the examination, the microelectrode signal, electromyogram (EMG) for wrist flexors and extensors plus elbow flexors and extensors in the contralateral upper limb, the audio channel describing instructions to the patient as well as technical details of the procedure. The physiological exploration with the microelectrode involved both the recording of neuronal activity and stimulating at microampere current levels. When a neuron was isolated, spontaneous activity was recorded. The activity of the isolated neuron was then studied to identify neurons responding to cutaneous stimuli such as light touch, tapping or pressure to skin.

One way to increase WG intake on a broad level is by making changes in regulations for federally funded meal and food supplement programs. The fourth School Nutrition Dietary Assessment Study

conducted in 2009 to 2010 indicated that average National School Lunch Program (NSLP) lunches only provided 6% to 10% of recommended daily amounts of WG [35] for children/adolescents. The new school meal regulations requiring that whole grain–rich foods be served in the NSLP [36] may result in an increase in the daily amount of WG consumed over time among those who participate in the NSLP. Evidence for a potential increase in WG can be drawn from improvements in the availability and intake of WG foods for women and children participating in the selleck BGB324 in vivo Special Supplemental Nutrition Program for Women, Infants, and Children after new regulations were established

to increase WG foods in Women, Infants, and Children food packages [37], [38] and [39]. Ready-to-eat cereals are an important source of many vitamins and minerals, especially for children. On average, RTE cereals contribute 20% of folic acid and iron and more than 10% of B vitamins, vitamin A, and zinc while contributing less than 4% of calories and total sugar in the diets of children 2–18 years of age [40]. In the current study, cooked and RTE cereals made substantial contributions to total dietary fiber, making up about 20% of the total dietary fiber intake for adults and children/adolescents. Several previous studies have shown that intake of RTE cereals among children and adolescents is related to greater total dietary fiber intake [41], [42] and [43]. Analysis of secondary data from the National Growth and Health Study showed that as children

age through adolescence, more frequent RTE cereal consumption was related to higher fiber intakes [42]. Cross-sectional data from a national Australian sample of 12- to 16-year-old boys showed that those consuming RTE cereals of all types had a higher total dietary fiber intake compared with those not eating RTE cereal [43]. Data from School Nutrition Dietary Assessment Study III (2004-2005) showed that RTE cereal consumption among Paclitaxel concentration school-aged children participating in the School Breakfast Program was related to higher WG intake [41]. Previous studies have not examined the contribution of different types of RTE cereals to fiber intake as in the current study. Whole grain and non-WG RTE cereals with no added bran provided the most total dietary fiber among all children and adolescents. The relationship between the total dietary fiber content of RTE WG cereals and top fiber sources was also examined by Williams and Felt-Gunderson [44] for adults completing a 14-day eating frequency diary.

Briefly, blood samples were drawn by antecubital venipuncture while the individuals, who had not been fasting prior to any invasive procedure, were seated. The samples were collected in an 8.5-cc

Serum Separator Vacutainer Tube (BD Diagnostics, Plymouth, UK) and maximally within 4 h at room temperature were centrifuged at 1000 × g for 10 min. Serum samples were then distributed into sterile 500-μL barcode labeled polypropylene aliquots (TrakMate; Matrix TechCorp.) and stored at −80 °C. All serum samples were thawed on ice once and randomly placed in barcode labeled BYL719 solubility dmso racks in an 8-channel Hamilton STAR® pipetting robot (Hamilton) for automated aliquotting into 60-μL daughter tubes. The aliquots were stored in 96-tubes racks at −80 °C until further sample processing. Samples from the calibration and the validation set were distributed over three 96-tubes racks as following: one full 96-tube rack for both the calibration and validation set and one partially filled 96-tube rack with 63 samples from the calibration set and 18 samples from the validation set. Identical

processing steps were followed for the two sample sets. The isolation of peptides from human serum was performed using RPC18-functionalized MBs as previously described [27]. In short, RPC18-MBs were first activated by a three-step washing with a 0.1% TFA solution. Then, for each sample 5 μL of serum was added to the activated beads and incubated for 5 min at room selleck chemicals llc temperature. The beads were washed again three times with 0.1% TFA and peptides were eluted with a 1:1 mixture of water and acetonitrile. Two microliters of each

(stabilized) eluate were mixed with 10 μL of an α-cyano-4-hydroxycinnamic acid MALDI matrix solution in a 384-well PCR plate. Then, 1 μL of this mixture was spotted in quadruplicate onto a 600 μm Anchor-Chip™ MALDI-target plate (Bruker Daltonics). The so-called next RPC18 eluates from the calibration and the validation set were spotted onto three 384-spots MALDI-target plate as following: 96 eluates from the calibration set and 96 eluates from the validation set were spotted in quadruplicate onto two distinct MALDI-target plates; the remaining eluates from the two sets were spotted in quadruplicate onto the same MALDI-target plate. This SPE- and MALDI-spotting procedure requires approximately 3 h per plate of 96 samples. MALDI-FTICR experiments were performed on a Bruker 15 tesla solariX™ FTICR mass spectrometer equipped with a novel CombiSource (Bruker Daltonics). The MALDI-FTICR system was controlled by Compass solariXcontrol software and equipped with a Bruker Smartbeam-II™ laser system that operated at a frequency of 200 Hz. The ‘medium’ predefined shot pattern was used for the irradiation.