Use of bioengineered human commensal gut bacteria-derived microvesicles for mucosal plague vaccine delivery and immunization.

Plague caused by the Gram-negative bacterium, Yersinia pestis, is still endemic in parts of the world today. Protection against pneumonic plague is essential to prevent the development and spread of epidemics. Despite this, there are currently no licensed plague vaccines in the western world. Here we describe the means of delivering biologically active plague vaccine antigens directly to mucosal sites of plague infection using highly stable microvesicles (outer membrane vesicles; OMVs) that are naturally produced by the abundant and harmless human commensal gut bacterium Bacteroides thetaiotaomicron (Bt). Bt was engineered to express major plague protective antigens in its OMVs, specifically Fraction 1 (F1) in the outer membrane and LcrV (V antigen) in the lumen, for targeted delivery to the gastrointestinal (GI) and respiratory tracts in a non-human primate (NHP) host. Our key findings were that Bt OMVs stably expresses F1 and V plague antigens, particularly the V antigen, in the correct, immunogenic form. When delivered intranasally V-OMVs elicited substantive and specific immune and antibody responses, both in the serum [immunoglobulin (Ig)G] and in the upper and lower respiratory tract (IgA); this included the generation of serum antibodies able to kill plague bacteria. Our results also showed that Bt OMV-based vaccines had many desirable characteristics, including: biosafety and an absence of any adverse effects, pathology or gross alteration of resident microbial communities (microbiotas); high stability and thermo-tolerance; needle-free delivery; intrinsic adjuvanticity; the ability to stimulate both humoral and cell-mediated immune responses; and targeting of primary sites of plague infection.

Status of Women in Academic Anesthesiology: A 10-Year Update.

BACKGROUND: Gender inequity is still prevalent in today's medical workforce. Previous studies have investigated the status of women in academic anesthesiology. The objective of this study is to provide a current update on the status of women in academic anesthesiology. We hypothesized that while the number of women in academic anesthesiology has increased in the past 10 years, major gender disparities continue to persist, most notably in leadership roles. METHODS: Medical student, resident, and faculty data were obtained from the Association of American Medical Colleges. The number of women in anesthesiology at the resident and faculty level, the distribution of faculty academic rank, and the number of women chairpersons were compared across the period from 2006 to 2016. The gender distribution of major anesthesiology journal editorial boards and data on anesthesiology research grant awards, among other leadership roles, were collected from websites and compared to data from 2005 and 2006. RESULTS: The number (%) of women anesthesiology residents/faculty has increased from 1570 (32%)/1783 (29%) in 2006 to 2145 (35%)/2945 (36%) in 2016 (P = .004 and P < .001, respectively). Since 2006, the odds that an anesthesiology faculty member was a woman increased approximately 2% per year, with an estimated odds ratio of 1.02 (95% confidence interval, 1.014-1.025; P < .001). In 2015, the percentage of women anesthesiology full professors (7.4%) was less than men full professors (17.3%) (difference, -9.9%; 95% confidence interval of the difference, -8.5% to -11.3%; P < .001). The percentage of women anesthesiology department chairs remained unchanged from 2006 to 2016 (12.7% vs 14.0%) (P = .75). To date, neither Anesthesia & Analgesia nor Anesthesiology has had a woman Editor-in-Chief. The percentage of major research grant awards to women has increased significantly from 21.1% in 1997-2007 to 31.5% in 2007-2016 (P = .02). CONCLUSIONS: Gender disparities continue to exist at the upper levels of leadership in academic anesthesiology, most importantly in the roles of full professor, department chair, and journal editors. However, there are some indications that women may be on the path to leadership parity, most notably, the growth of women in anesthesiology residencies and faculty positions and increases in major research grants awarded to women.

Review of triazine antiprotozoal drugs used in veterinary medicine.

Triazines are relatively new antiprotozoal drugs that have successfully controlled coccidiosis and equine protozoal myeloencephalitis. These drugs have favorably treated other protozoal diseases such as neosporosis and toxoplasmosis. In this article, we discuss the pharmacological characteristics of five triazines, toltrazuril, ponazuril, clazuril, diclazuril, and nitromezuril which are used in veterinary medicine to control protozoal diseases which include coccidiosis, equine protozoal myeloencephalitis, neosporosis, and toxoplasmosis.

Defects in chondrocyte maturation and secondary ossification in mouse knee joint epiphyses due to Snorc deficiency.

OBJECTIVE: The role of Snorc, a novel cartilage specific transmembrane proteoglycan, was studied during skeletal development using two Snorc knockout mouse models. Hypothesizing that Snorc, like the other transmembrane proteoglycans, may be a coreceptor, we also studied its interaction with growth factors. METHODS: Skeletal development was studied in wild type (WT) and Snorc knockout mice during postnatal development by whole mount staining, X-ray imaging, histomorphometry, immunohistochemistry and qRT-PCR. Snorc promoter activity was studied by applying the LacZ reporter expressed by the targeting construct. Slot blot binding and cell proliferation assays were used to study the interaction of Snorc with several growth factors. RESULTS: Snorc expression was localized in the knee epiphyses especially to the prehypertrophic chondrocytes delineating the cartilage canals and secondary ossification center (SOC). Snorc was demonstrated to have a glycosaminoglycan independent affinity to FGF2 and it inhibited FGF2 dependent cell growth of C3H101/2 cells. In Snorc deficient mice, SOCs in knee epiphyses were smaller, and growth plate (GP) maturation was disturbed, but total bone length was not affected. Central proliferative and hypertrophic zones were enlarged with higher extracellular matrix (ECM) volume and rounded chondrocyte morphology at postnatal days P10 and P22. Increased levels of Ihh and Col10a1, and reduced Mmp13 mRNA expression were observed at P10. CONCLUSIONS: These findings suggest a role of Snorc in regulation of chondrocyte maturation and postnatal endochondral ossification. The interaction identified between recombinant Snorc core protein and FGF2 suggest functions related to FGF signaling.

Large real-time holographic 3D displays: enabling components and results.

A holographic 3D display with 300 mm×200 mm active area was built. The display includes a spatial light modulator that modulates amplitude and phase of light and thus enables holographic reconstruction with high efficiency. Furthermore, holographic optical elements in photopolymer films and laser light sources are used. The requirements on these optical components are discussed. Photographs taken at the display demonstrate that a 3D scene is reconstructed in depth, thus enabling selective accommodation of the observer's eye lenses and natural depth perception. The results demonstrate the advantages of SeeReal's holographic 3D display solution.

Vaccination mitigates the impact of PRRSv infection on the pharmacokinetics of ceftiofur crystalline-free acid in pigs.

The pharmacokinetics of intramuscularly administered ceftiofur crystalline-free acid (CCFA) were determined in pigs that were clinically healthy (n = 8), vaccinated with a Porcine reproductive and respiratory syndrome modified live virus (PRRS MLV) (n = 10), challenged with wild-type porcine reproductive and respiratory syndrome virus (PRRSv) VR-2385 (n = 10), or vaccinated with PRRS MLV and later challenged with wild-type PRRSv VR-2385 (n = 10). Animals were given a single dose of CCFA intramuscularly at 5 mg/kg body weight. Blood was collected at 0 (pretreatment), 0.25, 0.5, 1, 6, 12, 24, 48, 96, 144, 192, and 240 h postinjection. Plasma was analyzed using liquid chromatography-mass spectrometry. Plasma concentration-time curves for each group were evaluated with noncompartmental modeling. When compared to control animals, those receiving the PRRSv wild-type challenge only had a lower AUC0-last , higher Cl/F, and higher Vz/F. The PRRSv wild-type challenge only group had the longest T1/2λ . The Cmax did not differ among all four treatments. Control animals had no statistically significant differences from animals vaccinated with PRRS MLV alone or animals vaccinated with PRRS MLV and later challenged with wild-type PRRSv. Our results suggest that PRRSv wild-type infection has the potential to alter CCFA pharmacokinetics and PRRS MLV vaccination may attenuate those changes.

Fc-gamma receptors are not involved in cartilage damage during experimental osteoarthritis.

OBJECTIVE: Fc-gamma receptors (FcγRs) have been shown to play a crucial role in cartilage degradation during experimental arthritis. Although most of their effect on cartilage degradation has been attributed to their potential to promote inflammation in the presence of immunoglobulins, activating FcγRs promote cartilage degeneration in antigen-induced arthritis (AIA) independently of the level of inflammation. This prompted us to investigate, whether FcγRs may also play a role in osteoarthritis (OA)-related cartilage degradation. METHODS: FcγR expression was measured by RT-PCR and FACS in murine cartilage tissue and chondrocytes. Experimental OA was induced by destabilisation of the medial meniscus (DMM) in WT mice and animals lacking either activating (Fc receptor γ-chain-deficient) or inhibitory (FcγRIIB-deficient) FcγRs. Cartilage damage was investigated histologically 8 weeks post-surgery by assessing proteoglycan loss and structural damage according to OARSI recommendations. Osteophyte size was measured to investigate alterations in bone turnover. RESULTS: Expression analyses revealed significant levels for all four types of murine FcγRs in mouse chondrocytes and cartilage tissue from newborn and 8-week-old mice. Surprisingly, yet, ablation of either activating or inhibitory FcγRs did not affect cartilage damage or bone turnover during DMM-induced OA in mice. CONCLUSION: While FcγRs appear to have a crucial role in cartilage degradation during inflammatory arthritis our data indicate that FcγRs do not influence cartilage destruction in experimental OA. This indicates that a certain threshold of inflammation is a prerequisite for FcγR-induced cartilage destruction in arthritis.

Origin and genome evolution of polyploid green toads in Central Asia: evidence from microsatellite markers.

Polyploidization, which is expected to trigger major genomic reorganizations, occurs much less commonly in animals than in plants, possibly because of constraints imposed by sex-determination systems. We investigated the origins and consequences of allopolyploidization in Palearctic green toads (Bufo viridis subgroup) from Central Asia, with three ploidy levels and different modes of genome transmission (sexual versus clonal), to (i) establish a topology for the reticulate phylogeny in a species-rich radiation involving several closely related lineages and (ii) explore processes of genomic reorganization that may follow polyploidization. Sibship analyses based on 30 cross-amplifying microsatellite markers substantiated the maternal origins and revealed the paternal origins and relationships of subgenomes in allopolyploids. Analyses of the synteny of linkage groups identified three markers affected by translocation events, which occurred only within the paternally inherited subgenomes of allopolyploid toads and exclusively affected the linkage group that determines sex in several diploid species of the green toad radiation. Recombination rates did not differ between diploid and polyploid toad species, and were overall much reduced in males, independent of linkage group and ploidy levels. Clonally transmitted subgenomes in allotriploid toads provided support for strong genetic drift, presumably resulting from recombination arrest. The Palearctic green toad radiation seems to offer unique opportunities to investigate the consequences of polyploidization and clonal transmission on the dynamics of genomes in vertebrates.

The effects of firocoxib on cautery disbudding pain and stress responses in preweaned dairy calves.

Perioperative analgesic effects of oral firocoxib following cautery disbudding were investigated in preweaned calves. Twenty Holstein calves approximately 4 to 6wk old received a single oral dose of firocoxib, a nonsteroidal antiinflammatory, at 0.5mg/kg (n=10) or placebo (n=10) in a randomized controlled clinical trial. Responses, including ocular temperature determined by infrared thermography, pressure algometry measuring mechanical nociception threshold, and heart rate, were evaluated at 2, 4, 7, 8, and 24h after cornual nerve block and cautery disbudding. Blood samples were collected over 96h and analyzed for plasma cortisol and substance P concentrations by RIA. Additionally, ex vivo prostaglandin E2 concentrations were determined over a 72-h study period using an enzyme immunoassay. Data were analyzed using a linear mixed effects model with repeated measures. An inhibition of ex vivo prostaglandin E2 synthesis was observed from 12 to 48h following disbudding in calves treated with firocoxib. Cautery disbudding was associated with an increased nociception for the duration of sampling (24h). During the initial 24-h period following disbudding, no difference in response between treatment groups was noted. Following 24h, mean cortisol concentrations diverged between the 2 study groups with placebo-treated calves having increased cortisol concentrations at approximately 48h after disbudding. Furthermore, the overall integrated cortisol response as calculated as area under the effect curve tended to be reduced in firocoxib-treated calves. The prolonged effects of cautery dehorning require further investigation. Moreover, the effect of firocoxib on cortisol reduction observed in this study requires additional exploration.

Impact of an experimental PRRSV and Streptococcus suis coinfection on the pharmacokinetics of ceftiofur hydrochloride after intramuscular injection in pigs.

This study determined the impact of porcine reproductive and respiratory syndrome virus (PRRSV) and Streptococcus suis coinfection on the pharmacokinetic (PK) profile of ceftiofur hydrochloride in pigs after intramuscular (i.m.) injection. Eighteen clinically normal crossbred gilts were assigned by weight into a challenge group (10 pigs) and control group (eight pigs). Pigs in both groups received a single i.m. injection of ceftiofur hydrochloride (Excenel RTU Sterile Suspension; Zoetis) at a 5 mg/kg BW dose. Serial blood samples were collected to characterize the plasma concentration curve. After a 10 days drug washout period, the challenge group was inoculated with 2 mL of PRRSV isolate VR-2385 (10(5.75) 50% tissue culture infective doses per mL) intranasally and 8 days later inoculated S. suis. When clinical disease was evident, the second PK assessment began in both challenge and control groups. Coinfected pigs demonstrated lower values of AUC and CMAX , but higher values of Cl/F and Vz/F indicating drug kinetics were altered by infection. The data from this study have implications on ceftiofur treatment regimens in diseased pigs.

Anti-inflammatory and anti-bacterial properties of tetramethylhexadecenyl succinyl cysteine (TSC): a skin-protecting cosmetic functional ingredient.

BACKGROUND: The skin is the first line of defence against exposure to microbial, physical, environmental and chemical insults. In mobilizing a protective response, several different cell types located in our skin release and respond to pro-inflammatory cytokines ensuring skin homeostasis and health. However, chronic activation of this response eventually causes damage resulting in premature ageing. Diosodium tetramethylhexadecenyl succinyl cysteine (TSC or SIG1273), an isoprenylcysteine small molecule, down modulates these inflammatory signalling pathways in various cell types (keratinocytes, peripheral blood mononuclear cells (PBMCs) and endothelial cells) and possesses anti-bacterial properties. Thus, TSC represents a novel cosmetic functional ingredient that provides a broad spectrum of benefits for the skin. OBJECTIVE: To assess the anti-inflammatory properties of TSC in several cutaneous cell types and further investigate its anti-microbial activity. METHODS: Cultured normal human epidermal keratinocytes were exposed to chemical irritant phorbol 12-myrisate 13-acetate (TPA) or ultraviolet-B light (UVB) to induce pro-inflammatory cytokine (IL-6, IL-8 and TNF-α) production. T-cell receptor (TCR) activation of PBMCs and nickel (Ni(2+) ) treatments of human dermal microvascular endothelial cells (HDMECs) were performed resulting in IL-4, IL-6, IL-8 and IL-17 production. Streptococcus pyogenes were cultured to determine minimal inhibitory concentration values. RESULTS: In vitro studies demonstrate TSC blocks TPA and UVB-induced cytokine production in cultured keratinocytes. Similarly, TSC inhibits overproduction of IL-4 and IL-17 in T-cell receptor (TCR)-activated PBMCs as well as nickel induction of IL-6 and IL-8 in HDMECs. Lastly, TSC demonstrated anti-microbial properties, inhibiting cell growth of S. pyogenes. CONCLUSIONS: Tetramethylhexadecenyl succinyl cysteine represents a novel cosmetic functional ingredient that provides a dual modulating benefit of skin protection to individuals by reducing inflammation in keratinocytes, endothelial and mononuclear cell types and S. pyogenes counts.

RECIPE COMPLETION USING MACHINE LEARNING TECHNIQUES.

Completing a recipe is a non-trivial task, as the success of ingredient combinations depends on a multitude of factors such as taste, smell, texture, etc. The aim of our work is to build a model that adds one or more ingredients to a given number of ingredients. The idea is based on leftover ingredients in a fridge. A person could list the available ingredients in his or her fridge and the model would suggest some additional ingredients to create a full recipe.

Inferring the degree of incipient speciation in secondary contact zones of closely related lineages of Palearctic green toads (Bufo viridis subgroup).

Reproductive isolation between lineages is expected to accumulate with divergence time, but the time taken to speciate may strongly vary between different groups of organisms. In anuran amphibians, laboratory crosses can still produce viable hybrid offspring >20 My after separation, but the speed of speciation in closely related anuran lineages under natural conditions is poorly studied. Palearctic green toads (Bufo viridis subgroup) offer an excellent system to address this question, comprising several lineages that arose at different times and form secondary contact zones. Using mitochondrial and nuclear markers, we previously demonstrated that in Sicily, B. siculus and B. balearicus developed advanced reproductive isolation after Plio-Pleistocene divergence (2.6 My, 3.3-1.9), with limited historic mtDNA introgression, scarce nuclear admixture, but low, if any, current gene flow. Here, we study genetic interactions between younger lineages of early Pleistocene divergence (1.9 My, 2.5-1.3) in northeastern Italy (B. balearicus, B. viridis). We find significantly more, asymmetric nuclear and wider, differential mtDNA introgression. The population structure seems to be molded by geographic distance and barriers (rivers), much more than by intrinsic genomic incompatibilities. These differences of hybridization between zones may be partly explained by differences in the duration of previous isolation. Scattered research on other anurans suggests that wide hybrid zones with strong introgression may develop when secondary contacts occur <2 My after divergence, whereas narrower zones with restricted gene flow form when divergence exceeds 3 My. Our study strengthens support for this rule of thumb by comparing lineages with different divergence times within the same radiation.

Sex comparisons of strength and coactivation following ten weeks of deadlift training.

OBJECTIVES: To examine the effects of a ten week deadlift training program on peak torque and agonist-antagonist coactivation. METHODS: Fifty-four untrained subjects (mean age=23 years) participated in this investigation, and were randomly assigned to a training (males, n=17; females, n=17) or control (males, n=9; females, n=11) group. The subjects in the training group performed deadlifts twice per week. Isometric peak torque for the leg extensors and flexors and surface electromyographic (EMG) amplitude for the superficial quadriceps and biceps femoris muscles were assessed. RESULTS: Deadlift training increased leg extension peak torque for the males (P=.008, Cohen's d=0.43) and females (P=.003, d=0.48). Leg flexion peak torque improved for the females (P=.001, d=0.45). Increased EMG amplitude for the superficial quadriceps femoris muscles when they served as agonists was demonstrated for the females (P=.010, d=0.40), but not the males (P=.059, d=0.20). For both sexes, the effect sizes for the decline in biceps femoris coactivation were large. CONCLUSION: Deadlift training elicited improvements in strength and agonist-antagonist coactivation in untrained subjects, and particularly, novice females.

The impact of 3 strategies for incorporating polled genetics into a dairy cattle breeding program on the overall herd genetic merit.

Dehorning in cattle has been associated with behavioral, physiological, and neuroendocrine responses indicative of pain. Unaddressed, the pain associated with a routine production procedure could contribute to a negative public perception of livestock production practices. Alternative considerations of dehorning include the selection of polled cattle within herds, thereby avoiding pain and production loss. As polledness results from an autosomal dominant pattern of inheritance, genetic selection for polled cattle could reduce the prevalence of the horned trait. Herein we discuss 3 strategies to incorporate polled genetics into a cow herd and the estimated impact on the overall genetic merit of the herd. Furthermore, the availability and genetic merit of polled artificial insemination bulls in the United States is summarized. Both Holstein and Jersey dairy bulls registered with the National Association of Animal Breeders from December 2010 through April 2013 were queried. Polled bulls were identified as either being homozygous (PP) or heterozygous (Pp) and the average net merit (NM) predicted transmitting ability (PTA) of each sire group was calculated. The percentage of polled calves born each year over a 10-yr period was calculated for the following 3 scenarios: (A) various percentages of horned cows were randomly mated to Pp bulls, (B) various percentages of horned cows were preferentially mated to Pp bulls, and (C) horned cows were selectively mated to PP bulls, heterozygous cows to Pp bulls, and homozygous polled cows to horned bulls. Additionally, the change in NM PTA of the cow herd was calculated over the same period. The highest percentage of polled animals (87%) was achieved in scenario C. An evaluation of the herd NM PTA highlights the trade-offs associated with increasing polled genetics. Given the current genetic merit of horned and polled bulls, increasing the percentage of polled calves will decrease the NM PTA in Holstein, but may have minimal impact in Jersey herds. Decisions regarding selective breeding to increase polled genetics will need to be evaluated in the context of production objectives, cost of dehorning, and impact on overall genetic merit.

Pharmacokinetics and effect of intravenous nalbuphine in weaned Holstein calves after surgical castration.

The objective of this study was to investigate the pharmacokinetics and effect of nalbuphine administered intravenously to calves immediately prior to surgical castration. Ten healthy calves were randomly assigned to two treatments (n = 5): (i) 0.9% sodium chloride (CONT) placebo, (ii) nalbuphine hydrochloride (NAL) (0.4 mg/kg). Blood samples collected over 10 h postcastration were analyzed for nalbuphine and cortisol concentrations. Additionally, heart rate, respiratory rate, rectal temperature, and step count was compared between groups using a random-effects mixed model. Changes in behavior and attitude were assessed using a six-point ordinal scoring system and compared using chi-square analysis. Plasma NAL concentrations were only detectable for 3 h postadministration (T½ = 0.68 h; Range: 0.53-0.79 h). There was no effect of NAL treatment prior to castration on cortisol concentrations (P = 0.99), heart rate (P = 0.73), respiratory rate (P = 0.59), rectal temperature (P = 0.22), and step count (P = 0.08) but fewer calves showed signs of head shaking, kicking, and tail flicking in the NAL group compared with the CONT group (P = 0.036). Therefore, we conclude that a single intravenous injection of nalbuphine at 0.4 mg/kg reduced some pain-related behaviors but did not significantly eliminate the physiological signs of distress in calves after surgical castration.

Pharmacokinetics of firocoxib in preweaned calves after oral and intravenous administration.

The objective of this study was to determine the pharmacokinetics of intravenous and oral firocoxib in 10 healthy preweaned calves. Firocoxib (0.5 mg/kg) was initially administered i.v. to calves, and following a 14-day washout period, animals received firocoxib orally prior to cautery dehorning. Firocoxib concentrations were determined by liquid chromatography-tandem mass spectrometry. Changes in hematology and plasma chemistry were determined using automated methods. Computer software was used to estimate pharmacokinetic parameters best described with a two-compartment model for i.v. administration and a one-compartment model for p.o. administration. Following i.v. dosing, the geometric mean (range) T1/2K10 and T1/2ß were 6.7 (4.6-9.7) and 37.2 (23.5-160.4) h, respectively, Vss was 3.10 (2.10-7.22) L/kg, and CL was 121.7 (100.1-156.7) mL/h/kg. Following oral administration, geometric mean (range) Cmax was 127.9 (102.5-151.3) ng/mL, Tmax was 4.0 (2.6-5.6) h, and T1/2K10 was 18.8 (14.2-25.5) h. Bioavailability of oral firocoxib was calculated using the AUC derived from both study populations to be 98.4% (83.1-117.6%). No adverse clinical effects were evident following firocoxib administration. Pharmacokinetic analysis of i.v. and p.o. firocoxib indicates high bioavailability and a prolonged terminal half-life in preweaned calves.

Low rates of X-Y recombination, not turnovers, account for homomorphic sex chromosomes in several diploid species of Palearctic green toads (Bufo viridis subgroup).

Contrasting with birds and mammals, most ectothermic vertebrates present homomorphic sex chromosomes, which might be due either to a high turnover rate or to occasional X-Y recombination. We tested these two hypotheses in a group of Palearctic green toads that diverged some 3.3 million years ago. Using sibship analyses of sex-linked markers, we show that all four species investigated share the same pair of sex chromosomes and a pattern of male heterogamety with drastically reduced X-Y recombination in males. Phylogenetic analyses of sex-linked sequences show that X and Y alleles cluster by species, not by gametolog. We conclude that X-Y homomorphy and fine-scale sequence similarity in these species do not stem from recent sex-chromosome turnovers, but from occasional X-Y recombination.

Requirements for dose calculation on an active scanned proton beamline for small, shallow fields.

INTRODUCTION: A growing interest in using proton pencil beam scanning in combination with collimators for the treatment of small, shallow targets, such as ocular melanoma or pre-clinical research emerged recently. This study aims at demonstrating that the dose of a synchrotron-based PBS system with a dedicated small, shallow field nozzle can be accurately predicted by a commercial treatment planning system (TPS) following appropriate tuning of both, nozzle and TPS. MATERIALS: A removable extension to the clinical nozzle was developed to modify the beam shape passively. Five circular apertures with diameters between 5 to 34mm, mounted 72cm downstream of a range shifter were used. For each collimator treatment plans with spread-out Bragg peaks (SOBP) with a modulation of 3 to 30mm were measured and calculated with GATE/Geant4 and the research TPS RayStation (RS11B-R). The dose grid, multiple coulomb scattering and block discretization resolution were varied to find the optimal balance between accuracy and performance. RESULTS: For SOBPs deeper than 10mm, the dose in the target agreed within 1% between RS11B-R, GATE/Geant4 and measurements for aperture diameters between 8 to 34mm, but deviated up to 5% for smaller apertures. A plastic taper was introduced reducing scatter contributions to the patient (from the pipe) and improving the dose calculation accuracy of the TPS to a 5% level in the entrance region for large apertures. CONCLUSION: The commercial TPS and GATE/Geant4 can accurately calculate the dose for shallow, small proton fields using a collimator and pencil beam scanning.