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Abstract Background The aim of environmental risk assessment (ERA) for pesticides is to protect ecosystems by ensuring that specific protection goals (SPGs) are met. The ERA follows a prospective tiered approach, starting with the most conservative and simple step in risk assessment (RA) (so-called tier 1) using the lowest available appropriate endpoint derived from ecotoxicological tests. In 2015, for the tier 1 RA of aquatic primary producers, the recommendation was changed from using the lowest of the 50% inhibition (EC50) values based on biomass (area under the curve—EbC50), increase in biomass (yield- EyC50) or growth rate (ErC50) to only using the growth rate inhibition endpoint (ErC50) because it is independent of the test design and thus more robust. This study examines the implications of this such on the level of conservatism provided by the tier 1 RA and evaluates whether it ensures a suitable minimum protection level. Results Our analysis shows that replacing the lowest endpoint with the growth rate inhibition endpoint while maintaining the assessment factor (AF) of 10 significantly reduces the conservatism in the tier 1 RA. Comparing protection levels achieved with different endpoints reveals that the current assessment is less protective. To maintain the previous level of protection, and since the protection goals have not changed, we recommend to multiply the default AF of 10 by an extra factor of minimum 2.4 in the tier 1 RA based on ErC50. Independently of the endpoint selected in tier 1 RA, several issues in the general RA of pesticides contribute to uncertainties when assessing the protection levels, e.g., lack of appropriate comparison of the higher tier experimental studies (i.e., best achievable approximation of field situation, so-called surrogate reference tier) with field conditions or the regulatory framework's failure to consider realistic conditions in agricultural landscapes with multiple stressors and pesticide mixtures. Conclusions We advise to consider adjusting the risk assessment in order to reach at least the previous protection level for aquatic primary producers. Indeed continuing using an endpoint with a higher value and without adjustment of the assessment factor is likely to jeopardize the need of halting biodiversity loss in surface waters.

A growing body of scientific literature stresses the need to advance current environmental risk assessment (ERA) methodologies and associated regulatory frameworks to better address the landscape-scale and long-term impact of pesticide use on biodiversity and the ecosystem. Moreover, more collaborative and integrative approaches are needed to meet sustainability goals. The One Health approach is increasingly applied by the European Food Safety Authority (EFSA) to support the transition towards safer, healthier and more sustainable food. To this end, EFSA commissioned the development of a roadmap for action to establish a European Partnership for next-generation, systems-based Environmental Risk Assessment (PERA). Here, we summarise the main conclusions and recommendations reported in the 2022 PERA Roadmap. This roadmap highlights that fragmentation of data, knowledge and expertise across regulatory sectors results in suboptimal processes and hinders the implementation of integrative ERA approaches needed to better protect the environment. To advance ERA, we revisited the underlying assumptions of the current ERA paradigm; that chemical risks are generally assessed and managed in isolation with a substance-by-substance, realistic worst-case and tiered approach. We suggest optimising the use of the vast amount of information and expertise available with pesticides as a pilot area. It is recommended to as soon as possible adopt a systems-based approach, i.e. within the current regulatory framework, to spark a step-wise transition towards an ERA framed at a system level of ecological and societal relevance. Tangible systems-based and integrative steps are available. For instance, the rich sources of existing data for prospective and retrospective ERA of pesticides could be used to reality-benchmark existing and new ERA methods. To achieve these goals, collaboration among stakeholders across scientific disciplines and regulatory sectors must be strengthened., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Soluble factors released from irradiated human mesenchymal stromal cells (MSC) may induce genetic instability in human CD34+ cells, potentially mediating hematologic disorders. Recently, we identified four key proteins in the secretome of X-ray-irradiated MSC, among them three endoplasmic reticulum proteins, the 78 kDa glucose-related protein (GRP78), calreticulin (CALR), and protein disulfide-isomerase A3 (PDIA3), as well as the glycolytic enzyme glucose-6-phosphate isomerase (GPI). Here, we demonstrate that exposition of CD34+ cells to recombinant GRP78, CALR, PDIA3 and GPI induces substantial genetic instability. Increased numbers of γH2AX foci (p < 0.0001), centrosome anomalies (p = 0.1000) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells upon incubation with these factors. Specifically, γH2AX foci were found to be induced 4−5-fold in response to any individual of the four factors, and centrosome anomalies by 3−4 fold compared to control medium, which contained none of the recombinant proteins. Aberrant metaphases, not seen in the context of control medium, were detected to a similar extent than centrosome anomalies across the four factors. Notably, the strongest effects were observed when all four factors were collectively provided. In summary, our data suggest that specific components of the secretome from irradiated MSC act as mediators of genetic instability in CD34+ cells, thereby possibly contributing to the pathogenesis of radiation-induced hematologic disorders beyond direct radiation-evoked DNA strand breaks.

Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.

Genotoxic bystander signals released from irradiated human mesenchymal stromal cells (MSC) may induce radiation-induced bystander effects (RIBEs) in human hematopoietic stem and progenitor cells (HSPC), potentially causing leukemic transformation. Although the source of bystander signals is evident, the identification and characterization of these signals is challenging. Here, RIBEs were analyzed in human CD34+ cells cultured in distinct molecular size fractions of medium, conditioned by 2 Gy irradiated human MSC. Specifically, γH2AX foci (as a marker of DNA double-strand breaks) and chromosomal instability were evaluated in CD34+ cells grown in approximate (I) < 10 kDa, (II) 10-100 kDa and (III) > 100 kDa fractions of MSC conditioned medium and un-/fractionated control medium, respectively. Hitherto, significantly increased numbers of γH2AX foci ( p = 0.0286) and aberrant metaphases ( p = 0.0022) were detected in CD34+ cells grown in the (II) 10-100 kDa fraction (0.67 ± 0.10 γH2AX foci per CD34+ cell ∨ 3.8 ± 0.3 aberrant metaphases per CD34+ cell sample; mean ± SEM) when compared to (I) < 10 kDa (0.19 ± 0.01 ∨ 0.3 ± 0.2) or (III) > 100 kDa fractions (0.23 ± 0.04 ∨ 0.4 ± 0.4) or un-/fractionated control medium (0.12 ± 0.01 ∨ 0.1 ± 0.1). Furthermore, RIBEs disappeared after heat inactivation of medium at 75 °C. Taken together, our data suggest that RIBEs are mainly mediated by the heat-sensitive (II) 10-100 kDa fraction of MSC conditioned medium. We postulate proteins as RIBE mediators and in-depth proteome analyses to identify key bystander signals, which define targets for the development of next-generation anti-leukemic drugs.

There are mainly two options for the dealcoholization of beer: evaporation of ethanol by heat treatment, whereby desired aroma-active compounds are also removed, and stopped fermentation that leads to beers still containing high amounts of unfermented sugar in parallel with lower amounts of aroma-active fermentation products. Thus, dry-hopping could be an opportunity to compensate for these aroma deficiencies. Therefore, following the sensomics approach, odorants were characterized in dry-hopped (Hallertauer Mandarina Bavaria, Hallertauer Cascade, or Hallertauer Mittelfrüh) top- and bottom-fermented alcohol-free beers either after thermal dealcoholization or stopped fermentation. Twenty-three odorants were quantitated via stable isotope dilution analysis, and odor activity values (OAVs; ratio of concentration to odor threshold) were calculated. Thermally dealcoholized samples showed high losses (up to 100%) of key odorants like 3-methyl-1-butanol or 3-methylbutyl acetate. During stopped fermentation, aroma compounds like ethyl butanoate or 2-phenylethanol were formed in relevant concentrations, leading to OAVs ≥ 1, but the amounts were significantly lower compared to beers with normal alcohol contents. For hop-derived odorants (linalool, geraniol, myrcene, and esters), transfer rates between 20 and 90% were found, leading to OAVs ≥ 1 in beer. Furthermore, hop addition apparently induced the formation of ethyl esters of hop-derived monocarboxylic acids.

DNA damage and alterations in the DNA damage response (DDR) are critical sources of genetic instability that might be involved in BCR-ABL1 kinase-mediated blastic transformation of chronic myeloid leukemia (CML). Here, increased DNA damage is detected by γH2AX foci analysis in peripheral blood mononuclear cells (PBMCs) of de novo untreated chronic phase (CP)-CML patients ( n = 5; 2.5 γH2AX foci per PBMC ± 0.5) and blast phase (BP)-CML patients ( n = 3; 4.4 γH2AX foci per PBMC ± 0.7) as well as CP-CML patients with loss of major molecular response (MMR) ( n = 5; 1.8 γH2AX foci per PBMC ± 0.4) when compared to DNA damage in PBMC of healthy donors ( n = 8; 1.0 γH2AX foci per PBMC ± 0.1) and CP-CML patients in deep molecular response or MMR ( n = 26; 1.0 γH2AX foci per PBMC ± 0.1). Progressive activation of erroneous non-homologous end joining (NHEJ) repair mechanisms during blastic transformation in CML is indicated by abundant co-localization of γH2AX/53BP1 foci, while a decline of the DDR is suggested by defective expression of (p-)ATM and (p-)CHK2. In summary, our data provide evidence for the accumulation of DNA damage in the course of CML and suggest ongoing DNA damage, erroneous NHEJ repair mechanisms, and alterations in the DDR as critical mediators of blastic transformation in CML., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The use of hops in beer brewing is mainly based on its content of bitter acids and aroma compounds. Due to the loss of volatile odorants during wort boiling, the so-called dry hopping is a possibility to intensify the hoppy aroma in the final beer. To clarify the potential of different hop varieties for aroma modulation of beer via dry hopping, key aroma compounds of three different hop varieties were characterized using the sensomics approach. Aroma extract dilution analysis revealed 41 aroma-active compounds, of which 39 were identified via gas chromatography-olfactometry and gas chromatography-mass spectrometry. The highest flavor dilution factor was determined for myrcene with a geranium-like odor. Fourteen substances were quantitated by stable isotope dilution analysis and further two odorants via the internal standard method; all of them revealed odor activity values (OAVs; ratio of concentration to odor threshold) ≥1. Linalool, 3-methylbutanoic acid, myrcene, and dimethyl trisulfide showed the highest OAVs (>1000) in all analyzed hop varieties. For validation of the analytical data, reconstitution models were prepared by adding all quantitated aroma compounds with OAVs ≥ 1 in their naturally occurring concentrations to cellulose as matrix. All three recombinates showed a very high similarity to the aroma profile of the respective hop sample, confirming the correct identification and quantitation of all key aroma compounds.

Malignant hematopoietic cells of myelodysplastic syndromes (MDS)/chronic myelomonocytic leukemias (CMML) and acute myeloid leukemias (AML) may be vulnerable to inhibition of poly(ADP ribose) polymerase 1/2 (PARP1/2) and apurinic/apyrimidinic endonuclease 1 (APE1). PARP1/2 and APE1 are critical enzymes involved in single-strand break repair and base excision repair, respectively. Here, we investigated the cytotoxic efficacy of talazoparib and APE1 inhibitor III, inhibitors of PARP1/2 and APE1, in primary CD34+ MDS/CMML cell samples ( n = 8; 4 MDS and 4 CMML) and in primary CD34+ or CD34- AML cell samples ( n = 18) in comparison to healthy CD34+ donor cell samples ( n = 8). Strikingly, talazoparib and APE1 inhibitor III demonstrated critical antileukemic efficacy in selected MDS/CMML and AML cell samples. Low doses of talazoparib and APE1 inhibitor III further increased the cytotoxic efficacy of decitabine in MDS/CMML and AML cells. Moreover, low doses of APE1 inhibitor III increased the cytotoxic efficacy of talazoparib in MDS/CMML and AML cells. In summary, talazoparib and APE1 inhibitor III demonstrated substantial antileukemic efficacy as single agents, in combination with decitabine, and combined with each other. Hence, our findings support further investigation of these agents in sophisticated clinical trials.

Accumulation of DNA damage and alteration of the DNA damage response (DDR) are critical features of genetic instability that is presumed to be implicated in the pathogenesis of monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL). Here, we show increased numbers of γH2AX foci, a marker of DNA double-strand breaks (DSB), in CD19+ cells of CLL patients as compared to CD19+ cells of MBL patients and healthy individuals. Furthermore, numerous γH2AX/53BP1 foci in CLL cells suggest activation of error-prone non-homologous end-joining repair mechanisms. Signatures of DDR proteins further indicate alterations of the DDR in CLL in contrast to a largely regular activation in MBL and healthy controls. In summary, our results provide evidence for the stepwise accumulation of DNA damage in the progression of MBL towards CLL and suggest increased DNA damage, error-prone DNA repair and altered DDR signaling to be critical mechanisms of clonal evolution in MBL and CLL.

Little is known about the ecotoxicity of heterocyclic aromatic hydrocarbons (NSO-HETs) to aquatic organisms. In the environment, NSO-HETs have been shown to occur in a strong association with their unsubstituted carbocyclic analogues, the polycyclic aromatic hydrocarbons (PAH), for which much more information is available. The present study addressed this issue by investigating the toxicity of four selected NSO-HETs in green algae (Desmodesmus subspicatus), daphnids (Daphnia magna) and fish embryos (Danio rerio). The four high molecular weight NSO-HETs dibenz[a,j]acridine (DBA), 7H-dibenzo[c,g]carbazole (DBC), benzo[b]naphtho[2,1-d]thiophene (BNT) and benzo[b]naphtho[1,2-d]furan (BNF) were selected, based on the results of a previous research project, indicating a lack of toxicity data and a high potential for persistence and bioaccumulation. The solubilities of the NSO-HETs in the test media were determined and turned out to be comparatively low (2.7-317 µg/L) increasing in the following order: DBA < BNT « DBC « BNF. Exposure concentrations during the toxicity tests were quantified with GC-MS and decreased strongly possibly due to sorption or metabolising during the test periods (48-96 h). Therefore, the estimated effect concentrations were related to the mean measured concentrations, as endpoints related to nominal concentrations would have underestimated the toxicity many times over. Within the range of the substance solubilities, BNF affected all test organisms with fish embryos being the most sensitive (fish: EC 50 6.7 µg/L, algae: EC 10 17.8 µg/L, daphnids: EC 50 55.8 µg/L). DBC affected daphnids (EC 50 2.5 µg/L,) and algae (EC 10 3.1 µg/L), but not fish embryos. The lowest toxicity endpoint was observed for BNT affecting only algae (NOEC 0.556 µg/L) and neither daphnids nor fish embryos. DBA did not show any effects on the tested organisms in the range of the water solubility. However, we would expect effects in long-term toxicity studies to fish and aquatic invertebrates for all substances at lower concentrations, which needs further investigation. All four NSO-HETs were identified in mussels (Mytilus edulis) from the German coasts, in green kale (Brassica oleracea var. acephala) and in freshwater harbor sediment in concentrations between 0.07 and 2 µg/kg, highlighting their relevance as environmental contaminants. There is a need to regulate the four NSO-HETs within the REACH regulation due to their intrinsic properties and their environmental relevance. However, acquisition of additional experimental data appears to be pivotal for a regulation under REACH., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

In low risk MDS, increased apoptosis of erythroid progenitors mediated via CD95 (Fas) activation has been described to result in peripheral cytopenia. Blockade of the CD95 system can improve erythropoiesis in MDS. Asunercept (APG101) is a fusion protein consisting of the extracellular domain of human CD95 and the Fc domain of human IgG1 blocking the interaction between CD95 and its ligand. Here we report on results from a phase I study in 20 transfusion-dependent low and intermediate risk MDS patients treated with intravenous asunercept (EudraCT 2012-003027-37). Primary objectives were safety and tolerability as well as pharmacodynamic effects. Secondary objectives were hematologic improvement, incidence and time to leukemic progression as well as overall survival. Frequency and severity of adverse events were in range of what could be expected in a patient cohort comprising of elderly MDS patients. Two patients experienced a serious adverse event with a suspected relationship to asunercept. The incidence of disease progression was low. In the 20 patients a decrease of the transfusion need from a mean of 10,8 (±5,1) pRBCs during the 12 weeks treatment phase to a mean of 10,0 (±4,2) pRBCs at the end of the study was observed. In conclusion, asunercept was well tolerated and showed efficacy in transfusion-dependent low and intermediate risk MDS patients. Further clinical investigation is warranted, particularly in combination with erythropoiesis stimulating agents (ESAs)., (Copyright © 2018. Published by Elsevier Ltd.)

Background: Short-chain PFASs (per- and polyfluoroalkyl substances) are widely used as alternatives to long-chain PFASs. Long-chain PFASs become gradually regulated under REACH (EC No. 1907/2006) and other international regulations, due to having persistent, bioaccumulative and toxic properties and/or being toxic for reproduction. The increasingly used short-chain PFASs are assumed to have a lower bioaccumulation potential. Nonetheless, they have other properties of concern and are already widely distributed in the environment, also in remote regions. The REACH Regulation does not directly address these emerging properties of concern, complicating the implementation of regulatory measures. Therefore, this study illustrates these environmental concerns and provides a strategy for a regulation of short-chain PFASs within REACH., Results: Short-chain PFASs have a high mobility in soil and water, and final degradation products are extremely persistent. This results in a fast distribution to water resources, and consequently, also to a contamination of drinking water resources. Once emitted, short-chain PFASs remain in the environment. A lack of appropriate water treatment technologies results in everlasting background concentrations in the environment, and thus, organisms are permanently and poorly reversibly exposed. Considering such permanent exposure, it is very difficult to estimate long-term adverse effects in organisms. Short-chain PFASs enrich in edible parts of plants and the accumulation in food chains is unknown. Regarding these concerns and uncertainties, especially with respect to the precautionary principle, short-chain PFASs are of equivalent concern to PBT substances. Therefore, they should be identified as substances of very high concern (SVHC) under REACH. The SVHC identification should be followed by a restriction under REACH, which is the most efficient way to minimize the environmental and human exposure of short-chain PFASs in the European Union., Conclusion: Due to an increasing use of short-chain PFASs, an effective regulation is urgently needed. The concerns of short-chain PFASs do not match the "classical" concerns as defined under REACH, but are not of minor concern. Therefore, it is of advantage to clearly define the concerns of short-chain PFASs. This might facilitate the following restriction process under REACH.

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of γH2AX and 53BP1, the combined analysis of γH2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on γH2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the γH2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of γH2AX and 53BP1.

Objective: To step-wise evaluate image quality of sinogram-affirmed iterative reconstruction (SAFIRE) in reduced-dose (RD) thoracoabdominal computed tomography (CT) compared to full-dose (FD) and RD filtered back projection (FBP) in a longitudinal study., Materials and Methods: 122 patients were included in this prospective study. 49 patients (14 men: mean age ± SD, 56±0.4 years; 35 women: 58±1.3 years) completed FD, RD1 (80%-dose) and RD2 (60%-dose) thoracoabdominal CT. Each CT dataset was reconstructed with FBP and SAFIRE. For quantitative image analysis image noise was measured in defined tissue regions. Qualitative image evaluation was performed according to the European Guidelines on Quality criteria for CT. Additionally artifacts, lesion conspicuity, and edge sharpness were assessed., Results: Compared to FD-FBP noise in soft tissue increased by 12% in RD1-FBP and 27% in RD2-FBP reconstructions, whereas SAFIRE lead to a decrease of 28% (RD1) and 17% (RD2), respectively (all p <0.001). Visually sharp reproduction, lesion conspicuity, edge sharpness of pathologic findings, and overall image quality did not differ statistically significant between FD-FBP and RD-SAFIRE datasets. Image quality decreased in RD1- and RD2-FBP compared to FD-FBP, reaching statistically significance in RD2 datasets (p <0.001). In RD1- and RD2-FBP (p <0.001) streak artifacts were noted., Conclusion: Using SAFIRE the reference mAs in thoracoabdominal CT can be reduced by at least 30% in clinical routine without loss of image quality or diagnostic information.

Increased DNA damage and alteration of the DNA damage response (DDR) are critical features of genetic instability presumably implicated in pathogenesis of myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). We used immunofluorescence staining of γH2AX and 53BP1 for analyzing DNA double-strand breaks (DSB) in MDS and AML cell lines, in CD34+ selected cells of normal and MDS bone marrow (including three cases of chronic myelomonocytic leukemias) and in blasts of AML bone marrow. In addition, we screened for activation of the DDR by immunoblotting of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53. As compared to γH2AX foci levels in normal bone marrow samples (0.2 focus per CD34+ cell±0.0; mean±standard error of mean), increased levels of γH2AX foci were detected in 16/16 MDS bone marrow samples (2.8 foci per CD34+ cell±0.5), 18/18 AML bone marrow samples (5.5 foci per blast±0.5), 1/1 MDS cell line (6.4 foci per cell) and 6/6 AML cell lines (12.0 foci per cell±0.6). γH2AX and 53BP1 co-localized in all tested samples forming diffuse, clustered and marginal patterns. Further, DDR proteins were expressed heterogeneously suggesting impairment of the DDR. In summary, our results provide evidence for a continuous increase of DSB across the spectrum from MDS to AML in conjunction with an impaired DDR. Co-localization of γH2AX and 53BP1 indicates promotion of (in)effective nonhomologous end-joining repair mechanisms at sites of DSB. Moreover, γH2AX/53BP1 foci distribution presumably reveals a non-random spatial organization of the genome in MDS and AML., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: Computed tomography (CT) scans are an important source of ionizing irradiation (IR) in medicine that can induce a variety of DNA damage in human tissues. With technological improvements CT scans at reduced absorbed doses became feasible presumably lowering genotoxic side effects., Materials and Methods: For measuring DNA damage we performed γH2AX foci microscopy in peripheral blood mononuclear cells (PBMC) after exposure to reduced and conventional absorbed radiation doses using 3rd generation dual-source CT (DSCT) technology., Results: CT scans performed at reduced absorbed doses of 3 mGy induced significant lower levels (p < 0.0001) of DNA damage (0.05 focus per cell ± 0.01 [mean ± standard error of mean]) at 5 min after IR compared to conventional absorbed doses of 15 mGy (0.30 focus per cell ± 0.03). With ongoing DNA repair background γH2AX foci levels (0.05 focus per cell) were approached at 24 h after CT with both protocols., Conclusion: Our results provide evidence that reduced absorbed doses mediated by adjusted tube current in 3rd generation DSCT induce lower levels of DNA damage in PBMC compared to conventional absorbed doses suggesting a lower genotoxic risk for state-of-the-art tube current reduced CT protocols.

The passive elasticity of the sarcomere in striated muscle is determined by large modular proteins, such as titin in vertebrates. In insects, the function of titin is divided between two shorter proteins, projectin and sallimus (Sls), which are the products of different genes. The Drosophila sallimus (sls) gene codes for a protein of 2 MDa. The N-terminal half of the protein is largely made up of immunoglobulin (Ig) domains and unique sequence; the C-terminal half has two stretches of sequence similar to the elastic PEVK region of titin, and at the end of the molecule there is a region of tandem Ig and fibronectin domains. We have investigated splicing pathways of the sls gene and identified isoforms expressed in different muscle types, and at different stages of Drosophila development. The 5' half of sls codes for zormin and kettin; both proteins contain Ig domains and can be expressed as separate isoforms, or as larger proteins linked to sequence downstream. There are multiple splicing pathways between the kettin region of sls and sequence coding for the two PEVK regions. All the resulting protein isoforms have sequence derived from the 3' end of the sls gene. Splicing of exons varies at different stages of development. Kettin RNA is predominant in the embryo, and longer transcripts are expressed in larva, pupa and adult. Sls isoforms in the indirect flight muscle (IFM) are zormin, kettin and Sls(700), in which sequence derived from the end of the gene is spliced to kettin RNA. Zormin is in both M-line and Z-disc. Kettin and Sls(700) extend from the Z-disc to the ends of the thick filaments, though, Sls(700) is only in the myofibril core. These shorter isoforms would contribute to the high stiffness of IFM. Other muscles in the thorax and legs have longer Sls isoforms with varying amounts of PEVK sequence; all span the I-band to the ends of the thick filaments. In muscles with longer I-bands, the proportion of PEVK sequence would determine the extensibility of the sarcomere. Alternative Sls isoforms could regulate the stiffness of the many fibre types in Drosophila muscles.

Thick filaments in striated muscle are myosin polymers with a length and diameter that depend on the fibre type. In invertebrates, the length of the thick filaments varies widely in different muscles and additional proteins control filament assembly. Thick filaments in asynchronous insect flight muscle have an extremely regular structure, which is likely to be essential for the oscillatory contraction of these muscles. The factors controlling the assembly of thick filaments in insect flight muscle are not known. We previously identified a thick filament core protein, zeelin 1, in Lethocerus flight and non-flight muscles. This has been sequenced, and the corresponding proteins in Drosophila and Anopheles have been identified. The protein has been re-named myofilin. Zeelin 2, which is on the outside of Lethocerus flight muscle thick filaments, has been sequenced and because of the similarity to Drosophila flightin, is re-named flightin. In Drosophila flight muscle, myofilin has a molecular weight of 20 kDa and is one of five isoforms produced from a single gene. In situ hybridisation of Drosophila embryos showed that myofilin RNA is first expressed late in embryogenesis at stage 15, a little later than myosin. Antibody to myofilin labelled the entire A-band, except for the H-zone, in cryosections of flight and non-flight muscle. The periodicity of myofilin in Drosophila flight muscle thick filaments was found to be 30 nm by measuring the spacing of gold particles in labelled cryosections; this is about twice the 14.5 nm spacing of myosin molecules. The molar ratio of myofilin to myosin in indirect flight muscle is 1:2, which is the same as that of flightin. We propose a model for the association of these proteins in thick filaments, which is consistent with the periodicity and stoichiometry. Myofilin is probably needed for filament assembly in all muscles, and flightin for stability of flight muscle thick filaments in adult flies.

In a search for the pathophysiologic mechanisms, we estimated isoprenoid synthesis and concentration, cellular growth, and the activity of the LDL receptor pathway in fibroblasts from patients with mevalonate kinase deficiency (MKD), a severe multisystemic disorder of cholesterol and non-sterol isoprenoid biosynthesis. In response to different concentrations of LDL and non-lipoprotein-bound cholesterol, MKD cells partially counteracted their enzyme defect by increased activities of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (results from earlier studies) and the LDL receptor pathway, responses similar to the pharmacologic effects seen upon administration of HMG-CoA reductase inhibitors. Rates of N-linked protein glycosylation, estimated as the amount of [14C]galactose-labeled macromolecules secreted into cell culture medium, were significantly decreased in MKD fibroblasts in comparison with control cells which may indicate alterations in the dolichol or dolichol phosphate pool. In response to exogenous cholesterol, the major feedback inhibitor of isoprenoid biosynthesis, growth velocities of MKD fibroblasts declined in comparison with control cells, further suggesting an impairment of non-sterol isoprenoid biosynthesis in MKD. Our results suggest an imbalance in the multilevel regulation of the biosynthesis of cholesterol and non-sterol isoprenoids in MKD, representing an additional causative factor responsible for the pre- and postnatal pathology of MKD.

It could be ascertained that the quantitative and qualitative identification of bacterial antigens and antibodies can be improved and completed, respectively, by DCIE in selected indications given. The diffusion counter immunoelectrophoresis has several advantages (easy handling, saving of time, easy evaluability). Our own tests confirmed the possibility of developing DCIE as a screening method to identify diphtheria and tetanus antibodies.

The retinal guanine nucleotide-binding protein, transducin (TD), was subjected to chromatography on Blue Sepharose (BLS). A simple two-step protocol was developed, allowing the resolution of the alpha-subunit and the beta gamma-complex of the protein extracted from bovine retina by the use of a poorly hydrolysable GTP analogue. If TD was applied to BLS in a divalent cation-containing buffer, the beta gamma-complex did not bind to the resin, whereas the alpha-subunit was retained; elution of the latter was achieved by removing the divalent cation from the buffer. Binding of the alpha-subunit to BLS was not affected by nucleotides or by ADP ribosylation catalysed by bacterial toxins. However, adsorption of the alpha-subunit by BLS or by a strong cation exchanger (Mono S) depended strictly on divalent cations. In contrast to previous reports, the data suggest the formation of a complex between a sulphonyl residue of Cibacron Blue, a divalent metal ion, and the alpha-subunit as the relevant binding mechanism causing adsorption of the alpha-subunit to BLS.