Dosimetric and Monte Carlo verification of jaws-only IMRT plans calculated by the Collapsed Cone Convolution algorithm for head and neck cancers

AimThe aim of this study is to verify the Prowess Panther jaws-only intensity modulated radiation therapy (JO-IMRT) treatment planning (TP) by comparing the TP dose distributions for head-and-neck (H&N) cancer with the ones simulated by Monte Carlo (MC).BackgroundTo date, dose distributions planned using JO-IMRT for H&N patients were found superior to the corresponding three-dimensional conformal radiotherapy (3D-CRT) plans. Dosimetry of the JO-IMRT plans were also experimentally verified using an ionization chamber, MapCHECK 2, and Octavius 4D and good agreements were shown.Materials and methodsDose distributions of 15 JO-IMRT plans of nasopharyngeal patients were recalculated using the EGSnrc Monte Carlo code. The clinical photon beams were simulated using the BEAMnrc. The absorbed dose to patients treated by fixed-field IMRT was computed using the DOSXYZnrc. The simulated dose distributions were then compared with the ones calculated by the Collapsed Cone Convolution (CCC) algorithm on the TPS, using the relative dose error comparison and the gamma index using global methods implemented in PTW-VeriSoft with 3%/3 mm, 2%/2 mm, 1%/1 mm criteria.ResultsThere is a good agreement between the MC and TPS dose. The average gamma passing rates were 93.3 ± 3.1%, 92.8 ± 3.2%, 92.4 ± 3.4% based on the 3%/3 mm, 2%/2 mm, 1%/1 mm criteria, respectively.ConclusionsAccording to the results, it is concluded that the CCC algorithm was adequate for most of the IMRT H&N cases where the target was not immediately adjacent to the critical structures.     

Ex Vivo Assessment of Contractility,Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca²⁺ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca²⁺ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.     

Phosphatidylinositol 4-Phosphate 5-Kinase α Facilitates Toll-like Receptor 4-mediated Microglial Inflammation through Regulation of the Toll/Interleukin-1 Receptor Domain-containing Adaptor Protein (TIRAP) Location

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP₂), generated by PI 4-phosphate 5-kinase (PIP5K), regulates many critical cellular events. PIP₂ is also known to mediate plasma membrane localization of the Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), required for the MyD88-dependent Toll-like receptor (TLR) 4 signaling pathway. Microglia are the primary immune competent cells in brain tissue, and TLR4 is important for microglial activation. However, a functional role for PIP5K and PIP₂ in TLR4-dependent microglial activation remains unclear. Here, we knocked down PIP5Kα, a PIP5K isoform, in a BV2 microglial cell line using stable expression of lentiviral shRNA constructs or siRNA transfection. PIP5Kα knockdown significantly suppressed induction of inflammatory mediators, including IL-6, IL-1β, and nitric oxide, by lipopolysaccharide. PIP5Kα knockdown also attenuated signaling events downstream of TLR4 activation, including p38 MAPK and JNK phosphorylation, NF-κB p65 nuclear translocation, and IκB-α degradation. Complementation of the PIP5Kα knockdown cells with wild type but not kinase-dead PIP5Kα effectively restored the LPS-mediated inflammatory response. We found that PIP5Kα and TIRAP colocalized at the cell surface and interacted with each other, whereas kinase-dead PIP5Kα rendered TIRAP soluble. Furthermore, in LPS-stimulated control cells, plasma membrane PIP₂ increased and subsequently declined, and TIRAP underwent bi-directional translocation between the membrane and cytosol, which temporally correlated with the changes in PIP₂. In contrast, PIP5Kα knockdown that reduced PIP₂ levels disrupted TIRAP membrane targeting by LPS. Together, our results suggest that PIP5Kα promotes TLR4-associated microglial inflammation by mediating PIP₂-dependent recruitment of TIRAP to the plasma membrane.     

Adenoviral overexpression of the glutamylcysteine ligase catalytic subunit protects pancreatic islets against oxidative stress

The catalytic subunit of glutamylcysteine ligase (GCLC) primarily regulates de novo synthesis of glutathione (GSH) in mammalian cells and is central to the antioxidant capacity of the cell. However, GCLC expression in pancreatic islets has not been previously examined. We designed experiments to ascertain whether GCLC is normally expressed in islets and whether it is up-regulated by interleukin-1 beta (IL-1 beta). GCLC expression levels were intermediate compared with other metabolic tissues (kidney, liver, muscle, fat, and lung). IL-1 beta up-regulated GCLC expression (10 ng/ml IL-1 beta, 3.76 +/- 0.86; 100 ng/ml IL-1 beta, 4.22 +/- 0.68-fold control) via the p38 form of mitogen-activated protein kinase and NF kappa B and also increased reactive oxygen species levels (10 ng/ml IL-1 beta, 5.41 +/- 1.8-fold control). This was accompanied by an increase in intraislet GSH/GSSG ratio (control, 7.1 +/- 0.1; 10 ng/ml IL-1 beta, 8.0 +/- 0.5; 100 ng/ml IL-1 beta, 8.2 +/- 0.5-fold control; p < 0.05). To determine whether overexpression of GCLC increases the antioxidant capacity of the islet and prevents the adverse effects of IL-1 beta on glucose-induced insulin secretion, islets were infected with an adenovirus encoding GCLC. IL-1 beta significantly decreased glucose-stimulated insulin secretion (control, 123.8 +/- 17.7; IL-1 beta, 40.2 +/- 3.9 microunits/ml insulin/islet). GCLC overexpression increased intraislet GSH levels and partially prevented the decrease in glucose-stimulated insulin secretion caused by IL-1 beta. These data provide the first report of GCLC expression in the islet and demonstrate that adenoviral overexpression of GCLC increases intracellular GSH levels and protects the beta cell from the adverse effects of IL-1 beta.     

Biodegradation of Acetochlor and 2-methyl-6-ethylaniline by Bacillus subtilis and Pseudomonasfluorescens

Microbiology - Two bacterial strains, Pseudomonas fluorescens KT3 and Bacillus subtilis 2M6E, isolated from soil utilized acetochlor and 2-methyl-6-ethylaniline (2M6E) as carbon and nitrogen...     

Restored immune cell functions upon clearance of senescence in the irradiated splenic environment

Some studies show eliminating senescent cells rejuvenate aged mice and attenuate deleterious effects of chemotherapy. Nevertheless, it remains unclear whether senescence affects immune cell function. We provide evidence that exposure of mice to ionizing radiation (IR) promotes the senescent‐associated secretory phenotype (SASP) and expression of p16^INK4a in splenic cell populations. We observe splenic T cells exhibit a reduced proliferative response when cultured with allogenic cells in vitro and following viral infection in vivo. Using p16‐3MR mice that allow elimination of p16^INK4a‐positive cells with exposure to ganciclovir, we show that impaired T‐cell proliferation is partially reversed, mechanistically dependent on p16^INK4a expression and the SASP. Moreover, we found macrophages isolated from irradiated spleens to have a reduced phagocytosis activity in vitro, a defect also restored by the elimination of p16^INK4a expression. Our results provide molecular insight on how senescence‐inducing IR promotes loss of immune cell fitness, which suggest senolytic drugs may improve immune cell function in aged and patients undergoing cancer treatment.     

Intra- and interlaboratory performance of antibiotic disk-diffusion-susceptibility testing of bacterial control strains of relevance for monitoring aquaculture environments

In the course of an international research project on hazard analysis of antimicrobial resistance in SE Asian aquaculture environments, 2 European Union and 3 SE Asian laboratories attempted to harmonize a procedure for antimicrobial agent susceptibility testing based on disk diffusion (DD). For this purpose, a selected panel of 10 bacterial control strains of relevance for monitoring warm-water aquaculture environments was sent by the central laboratory to the other participating laboratories. In each laboratory, 10 independently replicated DD determinations of each control strain to 6 antibiotics were performed using Iso-Sensitest Agar (ISA) according to a standard operating procedure (SOP); in total, this study thus yielded 300 data sets for all 5 laboratories. At the end of the study, strain authenticity of subcultures of the control strains used by the respective participating laboratories was verified by the central laboratory. Based on the arithmetic mean of 10 inhibition-zone diameter measurements and standard deviation (SD), intralaboratory SD variations ranged from 0 to 2 mm when 79% of the recorded data sets were considered. In 8% of the data sets, the SD value exceeded 4 mm, which in most cases could be attributed to the fact that the data points for a given strain-disk combination were not normally distributed in one of the laboratories. At the interlaboratory level, 81% of the SD values based on global averaging of 50 data points per strain-disk combination were situated in the 0 to 5 mm range. Comparison with a minimal data set from literature of DD testing performed with Mueller-Hinton (MH) medium indicated that the use of either ISA or MH medium in DD testing has a limited impact on the method's precision among different laboratories. In conclusion, the current study has provided a validated SOP to promote the coordination and harmonization of DD-susceptibility methodologies for aquaculture-associated organisms at an international level. As one of the main action items for the future, new interpretive breakpoints should be specifically designed and validated for aquaculture drugs and organisms.     

Evidence for population genetic structure in two exploited Mekong River fishes across a natural riverine barrier

Impacts of urban development on aquatic populations are often complex and difficult to ascertain, but population genetic analysis has allowed researchers to monitor and estimate gene flow in the context of existing and future hydroelectric projects. The Lower Mekong Basin is undergoing rapid hydroelectric development with around 50 completed and under-construction dams and 95 planned dams. The authors investigated the baseline genetic diversity of two exploited migratory fishes, the mud carp Henicorhynchus lobatus (five locations), and the rat-faced pangasiid catfish, Helicophagus leptorhynchus (two locations), in the Lower Mekong Basin using the genomic double digest restriction site-associated DNA (ddRAD) sequencing method. In both species, fish sampled upstream of Khone Falls were differentiated from those collected at other sites, and N_e estimates at the site above the falls were lower than those at other sites. This was the first study to utilize thousands of RAD-generated single nucleotide polymorphisms to indicate that the Mekong's Khone Falls are a potential barrier to gene flow for these two moderately migratory species. The recent completion of the Don Sahong dam across one of the only channels for migratory fishes through Khone Falls may further exacerbate signatures of isolation and continue to disrupt the migration patterns of regionally vital food fishes. In addition, H. lobatus populations downstream of Khone Falls, including the 3S Basin and Tonle Sap system, displayed robust connectivity. Potential obstruction of migration pathways between these river systems resulting from future dam construction may limit dispersal, which has led to elevated inbreeding rates and even local extirpation in other fragmented riverine species.     

Autoproteolysis and Intramolecular Dissociation of Yersinia YscU Precedes Secretion of Its C-Terminal Polypeptide YscUCC

Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscU_CC. Here we show that depletion of calcium induces intramolecular dissociation of YscU_CC from YscU followed by secretion of the YscU_CC polypeptide. Thus, YscU_CC behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscU_CC in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscU_CC dissociation for Yop secretion. We propose that YscU_CC orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms.     

Antenatal antioxidant prevents adult hypertension, vascular dysfunction, and microvascular rarefaction associated with in utero exposure to a low-protein diet

Developmental programming of hypertension is associated with vascular dysfunction characterized by impaired vasodilatation to nitric oxide, exaggerated vasoconstriction to ANG II, and microvascular rarefaction appearing in the neonatal period. Hypertensive adults have indices of increased oxidative stress, and newborns that were nutrient depleted during fetal life have decreased antioxidant defenses and increased susceptibility to oxidant injury. To test the hypothesis that oxidative stress participates in early life programming of hypertension, vascular dysfunction, and microvascular rarefaction associated with maternal protein deprivation, pregnant rats were fed a normal, low protein (LP), or LP plus lazaroid (lipid peroxidation inhibitor) isocaloric diet from the day of conception until delivery. Lazaroid administered along with the LP diet prevented blood pressure elevation, enhanced vasomotor response to ANG II, impaired vasodilatation to sodium nitroprusside, and microvascular rarefaction in adult offspring. Liver total glutathione was significantly decreased in LP fetuses, and kidney eight-isoprostaglandin F2alpha (8-isoPGF(2alpha)) levels were significantly increased in adult LP offspring; these modifications were prevented by lazaroid. Renal nitrotyrosine abundance and blood levels of 1,4-dihydroxynonene and 4-hydroxynonenal-protein adducts were not modified by antenatal diet exposure. This study shows in adult offspring of LP-fed dams prevention of hypertension, vascular dysfunction, microvascular rarefaction, and of an increase in indices of oxidative stress by the administration of lazaroid during gestation. Lazaroid also prevented the decrease in antioxidant glutathione levels in fetuses, suggesting an antenatal mild oxidative stress in offspring of LP-fed dams. These studies support the concept that perinatal oxidative insult can lead to permanent alterations in the cardiovascular system development.