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Abstract Background To analyze the accuracy and inter-observer variability of image-guidance (IG) using 3D or 4D cone-beam CT (CBCT) technology in stereotactic body radiotherapy (SBRT) for lung tumors. Materials and methods Twenty-one consecutive patients treated with image-guided SBRT for primary and secondary lung tumors were basis for this study. A respiration correlated 4D-CT and planning contours served as reference for all IG techniques. Three IG techniques were performed independently by three radiation oncologists (ROs) and three radiotherapy technicians (RTTs). Image-guidance using respiration correlated 4D-CBCT (IG-4D) with automatic registration of the planning 4D-CT and the verification 4D-CBCT was considered gold-standard. Results were compared with two IG techniques using 3D-CBCT: 1) manual registration of the planning internal target volume (ITV) contour and the motion blurred tumor in the 3D-CBCT (IG-ITV); 2) automatic registration of the planning reference CT image and the verification 3D-CBCT (IG-3D). Image quality of 3D-CBCT and 4D-CBCT images was scored on a scale of 1–3, with 1 being best and 3 being worst quality for visual verification of the IGRT results. Results Image quality was scored significantly worse for 3D-CBCT compared to 4D-CBCT: the worst score of 3 was given in 19 % and 7.1 % observations, respectively. Significant differences in target localization were observed between 4D-CBCT and 3D-CBCT based IG: compared to the reference of IG-4D, tumor positions differed by 1.9 mm ± 0.9 mm (3D vector) on average using IG-ITV and by 3.6 mm ± 3.2 mm using IG-3D; results of IG-ITV were significantly closer to the reference IG-4D compared to IG-3D. Differences between the 4D-CBCT and 3D-CBCT techniques increased significantly with larger motion amplitude of the tumor; analogously, differences increased with worse 3D-CBCT image quality scores. Inter-observer variability was largest in SI direction and was significantly larger in IG using 3D-CBCT compared to 4D-CBCT: 0.6 mm versus 1.5 mm (one standard deviation). Inter-observer variability was not different between the three ROs compared to the three RTTs. Conclusions Respiration correlated 4D-CBCT improves the accuracy of image-guidance by more precise target localization in the presence of breathing induced target motion and by reduced inter-observer variability.

Abstract Background Incontinence associated dermatitis (IAD) is an inflammatory skin disease mainly triggered by prolonged skin contact with urine, feces but also liberal detergent use when cleansing the skin. To minimize the epidermal barrier challenge we optimized the design of adult incontinence briefs. In the fluid absorption area we interposed a special type of acidic, curled-type of cellulose between the top sheet in contact with the skin and the absorption core beneath containing the polyacrylate superabsorber. The intention was to minimize disturbance of the already weak acid mantle of aged skin. We also employed air-permeable side panels to minimize skin occlusion and swelling of the stratum corneum. Methods The surface pH of diapers was measured after repeated wetting with a urine substitute fluid at the level of the top sheet. Occlusive effects and hydration of the stratum corneum were measured after a 4 hour application of different side panel materials by corneometry on human volunteers. Finally, we evaluated skin symptoms in 12 patients with preexisting IAD for 21 days following the institutional switch to the optimized diaper design. Local skin care protocols remained in place unchanged. Results The improved design created a surface pH of 4.6 which was stable even after repeated wetting throughout a 5 hour period. The "standard design" briefs had values of 7.1, which is alkaline compared to the acidic surface of normal skin. Side panels made from non-woven material with an air-permeability of more than 1200 l/m2/s avoided excessive hydration of the stratum corneum when compared to the commonly employed air-impermeable plastic films. Resolution of pre-existing IAD skin lesions was noted in 8 out of 12 patients after the switch to the optimized brief design. Conclusions An improved design of adult-type briefs can create an acidic pH on the surface and breathable side panels avoid over-hydration of the stratum corneum and occlusion. This may support the epidermal barrier function and may help to reduce the occurrence of IAD.

Salivary calcium plays an important role in the pathogenesis of dental caries and the bio-mineralization of dental enamel and exposed dentin. The cellular and molecular basis of calcium secretion by the human salivary glands is, however, poorly understood. Recently a transcellular transport of calcium by the acinus cells has been proposed. In this paper we looked for evidence for paracellular calcium transport by investigating the presence and cellular localization of paracellin-1 (claudin-16) that has been implied in paracellular magnesium and calcium transport in the kidney. At the mRNA level, using RT-PCR with primers of appropriate sequence, paracellin-1 mRNA could be found in human Glandula parotis, Glandula submandibularis, Glandula labialis and Glandula sublingualis samples. In addition, a splice variant was detected in three out of 15 glands consisting of exons one and five of the paracellin gene. In immunohistochemical studies paracellin-1 colocalised in the salivary excretory ducts with the tight junction proteins ZO-1 and occludin suggesting a potential role in paracellular calcium and magnesium transport. In the acini no such colocalisation was observed; paracellin was instead detected at the basal poles of the cells, between cells of the same acinus as well as between cells of neighboring acini. At this location paracellin-1 might act as selectivity filter for the paracellular movement of ions and water during stimulated secretion. Thus, both in the ducts and in the acini a paracellular transport of calcium appears possible. Whether it occurs at all and the extent to which it contributes to the overall salivary calcium secretion remains, however, to be determined.

Salivary calcium plays a vital role in bio-mineralization of dental enamel and exposed dentin. In order to elucidate the yet unknown cellular and molecular mechanisms of calcium secretion in human salivary glands the presence of various relevant plasma membrane transport systems for calcium were investigated. Using an RT-PCR approach, expression of the epithelial calcium channel (CaT-Like), the calcium binding protein (calbindin-2), the endoplasmic reticulum pumps (SERCA-2 and -3), and the plasma membrane calcium ATPases (PMCA-1, -2, and -4), were found in parotid and submandibular glands. Immunohistochemistry revealed that CaT-Like is located in the basolateral plasma membrane of acinar cells; while calbindin-2, SERCA-2 and SERCA-3 were found inside the acinar cells; and PMCA-2 was found in the apical membrane and in the secretory canaliculi between the cells. Based on these findings, we propose the following model of calcium secretion in human salivary glands: (1) calcium enters the acinar cell at the basolateral side via calcium channel CaT-Like (calcium influx); (2) intracellular calcium is taken up into the endoplasmic reticulum by SERCA-2 and possibly SERCA3 or bound to calbindin-2 (intracellular calcium pool); and (3) calcium is secreted by PMCAs at the apical plasma membrane (calcium efflux).

Objective: In order to elucidate the cellular and molecular mechanisms of phosphate secretion by human salivary glands, the expression and intracellular distribution of sodium-phosphate cotransporters was investigated., Design: Total RNA was extracted from 33 parotid gland (PG) and 35 submandibular gland (SMG) samples and RT-PCR was performed using gene specific primers for all known sodium-phosphate cotransporters. An antibody was raised against an NPT2b epitope and the cellular and intracellular distribution was investigated by immunohistochemistry., Results: No mRNA for the type I cotransporter NPT1 was found. Out of the type II phosphate cotransporters only message for NPT2b but not for NPT2a or NPT2c could be detected in about the same number of samples (76% in PG versus 69% in SMG). Type III cotransporter mRNA was also found in both glands, PIT1 gave positive results for 93% of PG samples compared to 69% of SMG samples. For PIT2 also, a higher expression was found in PG than in SMG, although the difference was smaller (79% versus 51%). Immunostaining for NPT2b was found both in the acini and in the ducts, with a stronger reaction in the latter. In acinar cells, NPT2b was restricted to the basal-lateral plasma membrane, in duct cells, a broad band of reactivity was located in the apical part of the cell., Conclusions: These findings suggest a secondary active secretion of phosphate into the primary saliva. Ductal cells appear to be able to reabsorb phosphate, thereby modifying the phosphate concentration in the final saliva.

AREA (NIT2) is a general transcription factor involved in derepression of numerous genes responsible for nitrogen utilization in Gibberella fujikuroi and many other fungi. We have previously shown that the deletion of areA-GF resulted in mutants with significantly reduced gibberellin (GA) production. Here we demonstrate that the expression level of six of the seven GA biosynthesis genes is drastically reduced in mutants lacking areA. Furthermore, we show that, despite the fact that GAs are nitrogen-free diterpenoid compounds, which are not obviously involved in nitrogen metabolism, AREA binds directly to the promoters of the six N-regulated genes. The binding of AREA was analysed in more detail using the promoter of one of the GA-biosynthesis genes encoding the ent-kaurene oxidase (P450-4). Deletion/mutation analysis of the P450-4 promoter fused to the Escherichia coli uidA gene, which encodes beta-glucuronidase, allowed the in vivo identification of functional GATA motifs. We have also analysed the nmr gene of G. fujikuroi (nmr-GF) which has high similarity to the Neurospora crassa nmr-1 and Aspergillus nidulans nmrA genes, both involved in nitrogen metabolite repression. In contrast to our expectation, deletion of nmr-GF did not result in significant derepression of the GA biosynthesis genes in the presence of ammonium, glutamine or glutamate. Overexpression of the nmr-GF gene fused to the strong promoter of the G. fujikuroi glutamine synthetase (gs) gene revealed only a very slight repression of the nitrate reductase (niaD) gene, resulting in weak resistance to chlorate. Surprisingly, this effect was only observed in the presence of high amounts of glutamate; cultivation on ammonium failed to induce any resistance to chlorate. Despite the limited effect of gene replacement and overexpression of nmr-GF on the nitrogen metabolism of G. fujikuroi itself, the gene fully restored nitrogen metabolite repression in A. nidulans and N. crassa nmr mutants. Therefore, we postulate that, in contrast to A. nidulans and N. crassa, NMR does not function independently as the main modulator of AREA in G. fujikuroi.

Farnesylpyrophosphate synthase (FPPS) is a key enzyme in isoprenoid biosynthesis. Several classes of essential metabolites, including sterols, quinones, carotenoids and gibberellins, are terpenoids with high biological activity. The structural gene for FPP synthase was isolated from two ascomycete fungi, Neurospora crassa and Gibberella fujikuroi. A comparative analysis of the nucleotide sequences of both FPPS genes revealed the presence of introns at the same positions at the 5' end of the coding regions. Furthermore, the most conserved region of the gene was isolated from two other plant pathogenic fungi, Sphaceloma manihoticola and Claviceps purpurea, by PCR. Sequence analysis showed a high degree of similarity between the deduced proteins of all known FPP synthase genes. In contrast to animals, all analyzed fungi contain a single copy of the gene, although FPP is the precursor for essential sterol and quinone biosynthesis and secondary metabolites, such as gibberellins, as well. Transcription analysis in different light regimes has shown that the FPPS genes in G. fujikuroi and N. crassa are not regulated by light induction.