Aerobic Methanotrophy and Co-occurrence Networks of a Tropical Rainforest and Oil Palm Plantations in Malaysia.

Oil palm (OP) plantations are gradually replacing tropical rainforest in Malaysia, one of the largest palm oil producers globally. Conversion of lands to OP plantations has been associated with compositional shifts of the microbial community, with consequences on the greenhouse gas (GHG) emissions. While the impact of the change in land use has recently been investigated for microorganisms involved in N2O emission, the response of the aerobic methanotrophs to OP agriculture remains to be determined. Here, we monitored the bacterial community composition, focusing on the aerobic methanotrophs, in OP agricultural soils since 2012, 2006, and 1993, as well as in a tropical rainforest, in 2019 and 2020. High-affinity methane uptake was confirmed, showing significantly lower rates in the OP plantations than in the tropical rainforest, but values increased with continuous OP agriculture. The bacterial, including the methanotrophic community composition, was modified with ongoing OP agriculture. The methanotrophic community composition was predominantly composed of unclassified methanotrophs, with the canonical (Methylocystis) and putative methanotrophs thought to catalyze high-affinity methane oxidation present at higher relative abundance in the oldest OP plantation. Results suggest that the methanotrophic community was relatively more stable within each site, exhibiting less temporal variations than the total bacterial community. Uncharacteristically, a 16S rRNA gene-based co-occurrence network analysis revealed a more complex and connected community in the OP agricultural soil, which may influence the resilience of the bacterial community to disturbances. Overall, we provide a first insight into the ecology and role of the aerobic methanotrophs as a methane sink in OP agricultural soils.

A hydrogen bond network in the active site of Anabaena ferredoxin-NADP(+) reductase modulates its catalytic efficiency.

Ferredoxin-nicotinamide-adenine dinucleotide phosphate (NADP(+)) reductase (FNR) catalyses the production of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) in photosynthetic organisms, where its flavin adenine dinucleotide (FAD) cofactor takes two electrons from two reduced ferredoxin (Fd) molecules in two sequential steps, and transfers them to NADP(+) in a single hydride transfer (HT) step. Despite the good knowledge of this catalytic machinery, additional roles can still be envisaged for already reported key residues, and new features are added to residues not previously identified as having a particular role in the mechanism. Here, we analyse for the first time the role of Ser59 in Anabaena FNR, a residue suggested by recent theoretical simulations as putatively involved in competent binding of the coenzyme in the active site by cooperating with Ser80. We show that Ser59 indirectly modulates the geometry of the active site, the interaction with substrates and the electronic properties of the isoalloxazine ring, and in consequence the electron transfer (ET) and HT processes. Additionally, we revise the role of Tyr79 and Ser80, previously investigated in homologous enzymes from plants. Our results probe that the active site of FNR is tuned by a H-bond network that involves the side-chains of these residues and that results to critical optimal substrate binding, exchange of electrons and, particularly, competent disposition of the C4n (hydride acceptor/donor) of the nicotinamide moiety of the coenzyme during the reversible HT event.

Modification of the pheophytin redox potential in Thermosynechococcus elongatus Photosystem II with PsbA3 as D1.

In Photosystem II (PSII) of the cyanobacterium Thermosynechococcus elongatus, glutamate 130 in the high-light variant of the D1-subunit (PsbA3) was changed to glutamine in a strain lacking the two other genes for D1, psbA1 and psbA2. The resulting PSII (PsbA3/Glu130Gln) was compared with those from the "native" high-light (PsbA3-PSII) and low-light (PsbA1-PSII) variants, which differ by 21 amino acid including Glu130Gln. H-bonding from D1-Glu130Gln to the primary electron acceptor, PheophytinD1 (PheoD1), is known to affect the Em of the PheoD1/PheoD1(-) couple. The Gln130 mutation here had little effect on water splitting, charge accumulation and photosensitivity but did slow down S2QA(-) charge recombination and up-shift the thermoluminescence while increasing its yield. These changes were consistent with a ≈-30mV shift of the PheoD1/PheoD1(-)Em, similar to earlier single site-mutation results from other species and double the ≈-17mV shift seen for PsbA1-PSII versus PsbA3-PSII. This is attributed to the influence of the other 20 amino-acids that differ in PsbA3. A computational model for simulating S2QA(-) recombination matched the experimental trend: the S2QA(-) recombination rate in PsbA1-PSII differed only slightly from that in PsbA3-PSII, while in Glu130-PsbA3-PSII there was a more pronounced slowdown of the radical pair decay. The simulation predicted a major effect of the PheoD1/PheoD1(-) potential on (1)O2 yield (~60% in PsbA1-PSII, ~20% in PsbA3-PSII and ~7% in Gln130-PsbA3-PSII), reflecting differential sensitivities to high light.

External loops at the ferredoxin-NADP(+) reductase protein-partner binding cavity contribute to substrates allocation.

Ferredoxin-NADP(+) reductase (FNR) is the structural prototype of a family of FAD-containing reductases that catalyze electron transfer between low potential proteins and NAD(P)(+)/H, and that display a two-domain arrangement with an open cavity at their interface. The inner part of this cavity accommodates the reacting atoms during catalysis. Loops at its edge are highly conserved among plastidic FNRs, suggesting that they might contribute to both flavin stabilization and competent disposition of substrates. Here we pay attention to two of these loops in Anabaena FNR. The first is a sheet-loop-sheet motif, loop102-114, that allocates the FAD adenosine. It was thought to determine the extended FAD conformation, and, indirectly, to modulate isoalloxazine electronic properties, partners binding, catalytic efficiency and even coenzyme specificity. The second, loop261-269, contains key residues for the allocation of partners and coenzyme, including two glutamates, Glu267 and Glu268, proposed as candidates to facilitate the key displacement of the C-terminal tyrosine (Tyr303) from its stacking against the isoalloxazine ring during the catalytic cycle. Our data indicate that the main function of loop102-114 is to provide the inter-domain cavity with flexibility to accommodate protein partners and to guide the coenzyme to the catalytic site, while the extended conformation of FAD must be induced by other protein determinants. Glu267 and Glu268 appear to assist the conformational changes that occur in the loop261-269 during productive coenzyme binding, but their contribution to Tyr303 displacement is minor than expected. Additionally, loop261-269 appears a determinant to ensure reversibility in photosynthetic FNRs.

Isolated noncatalytic and catalytic subunits of F1-ATPase exhibit similar, albeit not identical, energetic strategies for recognizing adenosine nucleotides.

The function of F1-ATPase relies critically on the intrinsic ability of its catalytic and noncatalytic subunits to interact with nucleotides. Therefore, the study of isolated subunits represents an opportunity to dissect elementary energetic contributions that drive the enzyme's rotary mechanism. In this study we have calorimetrically characterized the association of adenosine nucleotides to the isolated noncatalytic α-subunit. The resulting recognition behavior was compared with that previously reported for the isolated catalytic ß-subunit (N.O. Pulido, G. Salcedo, G. Pérez-Hernández, C. José-Núñez, A. Velázquez-Campoy, E. García-Hernández, Energetic effects of magnesium in the recognition of adenosine nucleotides by the F1-ATPase ß subunit, Biochemistry 49 (2010) 5258-5268). The two subunits exhibit nucleotide-binding thermodynamic signatures similar to each other, characterized by enthalpically-driven affinities in the µM range. Nevertheless, contrary to the catalytic subunit that recognizes MgATP and MgADP with comparable strength, the noncatalytic subunit much prefers the triphosphate nucleotide. Besides, the α-subunit depends more on Mg(II) for stabilizing the interaction with ATP, while both subunits are rather metal-independent for ADP recognition. These binding behaviors are discussed in terms of the properties that the two subunits exhibit in the whole enzyme.

Molecular mechanisms of superoxide production by complex III: a bacterial versus human mitochondrial comparative case study.

In this mini review, we briefly survey the molecular processes that lead to reactive oxygen species (ROS) production by the respiratory complex III (CIII or cytochrome bc1). In particular, we discuss the "forward" and "reverse" electron transfer pathways that lead to superoxide generation at the quinol oxidation (Qo) site of CIII, and the components that affect these reactions. We then describe and compare the properties of a bacterial (Rhodobacter capsulatus) mutant enzyme producing ROS with its mitochondrial (human cybrids) counterpart associated with a disease. The mutation under study is located at a highly conserved tyrosine residue of cytochrome b (Y302C in R. capsulatus and Y278C in human mitochondria) that is at the heart of the quinol oxidation (Qo) site of CIII. Similarities of the major findings of bacterial and human mitochondrial cases, including decreased catalytic activity of CIII, enhanced ROS production and ensuing cellular responses and damages, are remarkable. This case illustrates the usefulness of undertaking parallel and complementary studies using biologically different yet evolutionarily related systems, such as α-proteobacteria and human mitochondria. It progresses our understanding of CIII mechanism of function and ROS production, and underlines the possible importance of supra-molecular organization of bacterial and mitochondrial respiratory chains (i.e., respirasomes) and their potential disease-associated protective roles. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

The mechanism of ubihydroquinone oxidation at the Qo-site of the cytochrome bc1 complex.

1. Recent results suggest that the major flux is carried by a monomeric function, not by an intermonomer electron flow. 2. The bifurcated reaction at the Qo-site involves sequential partial processes, - a rate limiting first electron transfer generating a semiquinone (SQ) intermediate, and a rapid second electron transfer in which the SQ is oxidized by the low potential chain. 3. The rate constant for the first step in a strongly endergonic, proton-first-then-electron mechanism, is given by a Marcus-Brønsted treatment in which a rapid electron transfer is convoluted with a weak occupancy of the proton configuration needed for electron transfer. 4. A rapid second electron transfer pulls the overall reaction over. Mutation of Glu-295 of cyt b shows it to be a key player. 5. In more crippled mutants, electron transfer is severely inhibited and the bell-shaped pH dependence of wildtype is replaced by a dependence on a single pK at ~8.5 favoring electron transfer. Loss of a pK ~6.5 is explained by a change in the rate limiting step from the first to the second electron transfer; the pK ~8.5 may reflect dissociation of QH. 6. A rate constant (<10(3)s(-1)) for oxidation of SQ in the distal domain by heme bL has been determined, which precludes mechanisms for normal flux in which SQ is constrained there. 7. Glu-295 catalyzes proton exit through H(+) transfer from QH, and rotational displacement to deliver the H(+) to exit channel(s). This opens a volume into which Q(-) can move closer to the heme to speed electron transfer. 8. A kinetic model accounts well for the observations, but leaves open the question of gating mechanisms. For the first step we suggest a molecular "escapement"; for the second a molecular ballet choreographed through coulombic interactions. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

The Tll0287 protein is a hemoprotein associated with the PsbA2-Photosystem II complex in Thermosynechococcus elongatus.

Cyanobacteria have multiple psbA genes encoding PsbA, the D1 reaction center protein of the Photosystem II (PSII) complex. The thermophilic cyanobacterium Thermosynechococcus elongatus has three psbA genes differently expressed depending on the environmental conditions. Among the 344 residues of PsbA, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2 and 27 between PsbA2 and PsbA3. In this study, we found a new hemoprotein that is expressed when the T. elongatus genome has only the psbA2 gene for D1. This hemoprotein was found in both the non-membrane proteins and associated to the purified PsbA2-PSII core complex. This protein could be removed by the washing of PSII with Tris-washing or CaCl2-washing. From MALDI-TOF/TOF spectrometry, N-terminal sequencing and MALDI-MS/MS analysis upon tryptic digestion, the new hemoprotein was identified to be the tll0287 gene product with a molecular mass close to 19kDa. Until now, tll0287 was registered as a gene encoding a hypothetical protein with an unknown function. From the amino acid sequence and the EPR spectrum the 5th and 6th axial ligands of the heme iron are the His145 and likely either the Tyr93, Tyr159 or Tyr165, respectively. From EPR, the heme containing Tll0287 protein associated to PsbA2-PSII corresponds to approximately 25% of the Cytc550 content whereas, from SDS page analysis, the total amount of Tll0287 with and without the heme seems almost in a stoichiometric amount with PsbA2-PSII. Homologous genes to tll0287 are found in several cyanobacteria. Possible roles for Tll0287 are suggested.

Evidence for a role of CETP in HDL remodeling and cholesterol efflux: role of cysteine 13 of CETP.

Cholesteryl ester transfer protein (CETP), a key regulator of high-density lipoprotein (HDL) metabolism, induces HDL remodeling by transferring lipids between apolipoprotein B-containing lipoproteins and HDL, and/or by promoting lipid transfer between HDL subparticles. In this study, we investigated the mechanism as to how CETP induces the generation of lipid-poor particles (pre-ß-HDL) from HDL, which increases ATP-binding cassette transporter 1-mediated cholesterol efflux. This CETP-dependent HDL remodeling is enhanced by the CETP modulator dalcetrapib both in plasma and isolated HDL. The interaction of dalcetrapib with cysteine 13 of CETP is required, since this effect was abolished when using mutant CETP in which cysteine 13 was substituted for a serine residue. Other thiol-containing compounds were identified as CETP modulators interacting with cysteine 13 of CETP. In order to mimic dalcetrapib-bound CETP, mutant CETP proteins were prepared by replacing cysteine 13 with the bulky amino acid tyrosine or tryptophan. The resultant mutants showed virtually no CETP-dependent lipid transfer activity but demonstrated preserved CETP-dependent pre-ß-HDL generation. Overall, these data demonstrate that the two functions of CETP i.e., cholesteryl ester transfer and HDL remodeling can be uncoupled by interaction of thiol-containing compounds with cysteine 13 of CETP or by introducing large amino acid residues in place of cysteine 13.

Relationship Between Alexithymia, Smartphone Addiction, and Psychological Distress Among University Students: A Multi-country Study.

Objectives: Increasing dependence on smartphones results in the appearance of psychological problems, especially among young people. This study aims to determine the rates of alexithymia and its relationship with smartphone addiction and psychological distress in university students. Methods: A total of 2616 students (mean age = 22.5±3.5 years; 73.1% female) from universities in Egypt, Oman, and Pakistan were included in a cross-sectional and comparative study conducted through a web survey during the COVID-19 pandemic from October to December 2021. The following scales were used: Toronto Alexithymia Scale (TAS-20), Depression Anxiety Stress Scale (DASS-21), and Smartphone Addiction Scale-Short Version (SAS-SV). The survey also included questions related to sociodemographic and smartphone usage patterns.

Adipose transplant for inborn errors of branched chain amino acid metabolism in mice.

Liver transplantation appears to be quite beneficial for treatment of maple syrup urine disease (MSUD, an inherited disorder of branched chain amino acid metabolism); however, there is a limited availability of donor livers worldwide and the first year costs of liver transplants are quite high. Recent studies have suggested that intact adipose tissue, already widely used in reconstructive surgery, may have an underappreciated high capacity for branched chain amino acid (BCAA) metabolism. Here we examined the potential for adipose tissue transplant to lower circulating BCAAs in two models of defective BCAA metabolism, BCATm and PP2Cm [branched chain keto acid dehydrogenase complex (BCKDC) phosphatase] knockout (KO) mice. After 1-2g fat transplant, BCATm and PP2Cm KO mice gained or maintained body weight 3weeks after surgery and consumed similar or more food/BCAAs the week before phlebotomy. Transplant of fat into the abdominal cavity led to a sterile inflammatory response and nonviable transplanted tissue. However when 1-2g of fat was transplanted subcutaneously into the back, either as small (0.1-0.3g) or finely minced pieces introduced with an 18-ga. needle, plasma BCAAs decreased compared to Sham operated mice. In two studies on BCATm KO mice and one study on PP2Cm KO mice, fat transplant led to 52-81% reductions in plasma BCAAs compared to baseline plasma BCAA concentrations of untreated WT type siblings. In PP2Cm KO mice, individual BCAAs in plasma were also significantly reduced by fat transplant, as were the alloisoleucine/Phe ratios. Therefore, subcutaneous fat transplantation may have merit as an adjunct to dietary treatment of MSUD. Additional studies are needed to further refine this approach.

Reference range and short- and long-term biological variation of interleukin (IL)-6, IL-17A and tissue necrosis factor-alpha using high sensitivity assays.

Interleukin (IL)-6, and tissue necrosis factor alpha (TNF-α) are established biomarkers for clinical practice and use in clinical trials of patients with cardiovascular disease. IL-17A may be an emerging marker for atherosclerosis disease progression. We measured IL-6, TNF-α, and IL-17A using a high sensitivity immunoassay (Erenna, Singulex, Inc.) to determine the reference range and to calculate the weekly (25 subjects over 6weeks) and monthly (17 subjects over 9months) biological variation (BV) from apparently healthy subjects and those attending a cardiovascular disease clinic. As a validation for the experimental and statistical approach taken, the weekly BV for high sensitivity C-reactive protein was also determined and result compared to previous reports. The upper 95th percentile reference limit for IL-6, TNF-α, and IL17-A was 4.45, 2.53, and 1.93pg/mL, respectively. The intra-individual variability ranged from 21% to 57% and the inter-individual variation ranged from 22% to 53%. The corresponding index of individuality was 0.65-1.6 and reference change values from 63% to 161%. The BV for IL-6 and TNF-α are similar to previous reports, documenting their diagnostic utility in clinical practice. Up until now, the biological variation of IL-17A and upper reference limit has not been previously reported, thereby limiting the use of this marker in clinical trials.

The effect of syndecan-4 and glypican-1 expression on age-related changes in myogenic satellite cell proliferation, differentiation, and fibroblast growth factor 2 responsiveness.

Satellite cells are multipotential stem cells responsible for muscle growth and regeneration. Satellite cell proliferation, differentiation, and responsiveness to fibroblast growth factor 2 (FGF2) is, in part, regulated by the heparan sulfate proteoglycans syndecan-4 and glypican-1. Syndecan-4 and glypican-1 expression declines with satellite cell age and may be associated with decreased satellite cell activity. The objective of the current study was to determine if overexpression of syndecan-4 and glypican-1 would increase proliferation, differentiation and FGF2 responsiveness in satellite cells isolated from pectoralis major muscle from 16-wk-old turkeys. Overexpression of syndecan-4 and glypican-1 did not have a significant effect on proliferation and differentiation in 1d, 7 wk, and 16 wk satellite cells, and did not affect FGF2 responsiveness during proliferation. Expression of syndecan-4 and glypican-1 increased differentiation at 48 h in 1d, 7 wk, and 16 wk cells treated with FGF2. Expression of myogenic regulatory factors MyoD, myogenin, and MRF4 was affected by the overexpression of syndecan-4 and glypican-1. However, changes in myogenic regulatory factor expression did not have a significant effect on proliferation or differentiation. These data demonstrate that syndecan-4 and glypican-1 are likely not directly associated with the age related decrease in satellite cell activity.

The use and the cost of outpatient diagnostic procedures for cardiovascular diseases in Isfahan province: A utilization study.

BACKGROUND: Cardiovascular diseases (CVDs) are among the most important causes of premature death, disability, disease burden, and increasing the cost of healthcare worldwide. Having an overview of service utilization can help policymakers to plan more effective use of those services and to cut costs. Thus, this study aims to determine the amount of use as well as the cost of various outpatient diagnostic procedures for CVDs in Isfahan province of Iran from 2011 to 2017. MATERIALS AND METHODS: This descriptive study used insurance claim data (time period: 2011-2017) from Health Insurance Organization in Isfahan province to determine the amount of use and the cost of various outpatient diagnostic procedures for CVDs. Afterward, based on these data, the use and the cost of various outpatient diagnostic procedures for CVDs were estimated for the total population of Isfahan province. The list of outpatient diagnostic procedures for CVDs was carefully chosen according to experts' opinions. RESULTS: The use and the cost of outpatient diagnostic procedures for CVDs have drastically increased in the study period (2011-2017). Since 2011, the number of procedures and their related costs have increased 6.6 and 30.76 times (11.74 times, adjusted with PPP conversion factor), respectively. Per capita use (per thousand people) was 18.75 in 2011, reaching 116.51 in 2017. Per capita cost (per thousand people) was 1,887,660 IRR (355 PPP$) in 2011, reaching 54,660,365 IRR (3920 PPP$) in 2017. The highest cost and use were related to echocardiography and electrocardiography, respectively. A notable increase has been observed in the share of radionuclide myocardial perfusion scan and analysis of pacemakers and ICDs of the total cost. CONCLUSIONS: The use of outpatient diagnostic procedures for CVDs has drastically increased during the studied period. Consequently, the cost borne by the health system and the patients have notably increased. This may be because of the increase in the incidence and prevalence of CVDs during the study period. Greater access to related health services can be mentioned as another reason for this increase. Further research is needed to explain all potential reasons and their importance, which can provoke a suitable health policy reaction.

Characterization of the novel heterozygous SCN5A genetic variant Y739D associated with Brugada syndrome.

Genetic variants in SCN5A gene were identified in patients with various arrhythmogenic conditions including Brugada syndrome. Despite significant progress of last decades in studying the molecular mechanism of arrhythmia-associated SCN5A mutations, the understanding of relationship between genetics, electrophysiological consequences and clinical phenotype is lacking. We have found a novel genetic variant Y739D in the SCN5A-encoded sodium channel Nav1.5 of a male patient with Brugada syndrome (BrS). The objective of the study was to characterize the biophysical properties of Nav1.5-Y739D and provide possible explanation of the phenotype observed in the patient. The WT and Y739D channels were heterologously expressed in the HEK-293T cells and the whole-cell sodium currents were recorded. Substitution Y739D reduced the sodium current density by 47 ± 2% at -20 mV, positively shifted voltage-dependent activation, accelerated both fast and slow inactivation, and decelerated recovery from the slow inactivation. The Y739D loss-of-function phenotype likely causes the BrS manifestation. In the hNav1.5 homology models, which are based on the cryo-EM structure of rat Nav1.5 channel, Y739 in the extracellular loop IIS1-S2 forms H-bonds with K1381 and E1435 and pi-cation contacts with K1397 (all in loop IIIS5-P1). In contrast, Y739D accepts H-bonds from K1397 and Y1434. Substantially different contacts of Y739 and Y739D with loop IIIS5-P1 would differently transmit allosteric signals from VSD-II to the fast-inactivation gate at the N-end of helix IIIS5 and slow-inactivation gate at the C-end of helix IIIP1. This may underlie the atomic mechanism of the Y739D channel dysfunction.

Evaluation of image-guided and surface-guided radiotherapy for breast cancer patients treated in deep inspiration breath-hold: A single institution experience.

INTRODUCTION: Nowadays, deep inspiratory breath-hold is a common technique to reduce heart dose in left-sided breast radiotherapy. This study evaluates the evolution of the breath-hold technique in our institute, from portal imaging during dose delivery to continuous monitoring with surface-guided radiotherapy (SGRT). MATERIALS AND METHODS: Setup data and portal imaging results were analyzed for 98 patients treated before 2014, and SGRT data for 228 patients treated between 2018 and 2020. For the pre-SGRT group, systematic and random setup errors were calculated for different correction protocols. Residual errors and reproducibility of breath-holds were evaluated for both groups. The benefit of using SGRT for initial positioning was evaluated for another cohort of 47 patients. RESULTS: Online correction reduced the population mean error from 3.9 mm (no corrections) to 1.4 mm. Despite online setup correction, deviations greater than 3 mm were observed in about 10% and 20% of the treatment beams in ventral-dorsal and cranial-caudal directions, respectively. However, these percentages were much smaller than with offline protocols or no corrections. Mean absolute differences between breath-holds within a fraction were smaller in the SGRT-group (1.69 mm) than in the pre-SGRT-group (2.10 mm), and further improved with addition of visual feedback (1.30 mm). SGRT for positioning did not improve setup accuracy, but slightly reduced the time for imaging and setup correction, allowing completion within 3.5 min for 95% of fractions. CONCLUSION: For accurate radiotherapy breast treatments using deep inspiration breath-hold, daily imaging and correction is required. SGRT provides accurate information on patient positioning during treatment and improves patient compliance with visual feedback.

In vivo and in silico studies of Dennettia tripetala essential oil reveal the potential harmful effects of habitual consumption of the plant seed.

Dennettia tripetala G. Baker (Annonaceae), is a plant with nutritional, social economy, and medicinal values. Its rising medicinal profile makes this plant a prospect in drug discovery. However, the reported strong addictive potential among habitual consumers makes the need to establish its safety imperative. In this report, we evaluated the safety profile of the essential oil of the seed of D. tripetala (EODS) in nulliparous female Wistar rats using in vivo single and repeated dose toxicity profiling, as well as in silico toxicity profiling of its known seed oil derived phytoconstituents. Our results showed consistent significant dose-dependent alterations in relative body weights, organ-body and organ-brain weight ratios, haematological and biochemical indices, as well as liver and kidney histoarchitectures, following single and repeated oral administrations. Significant alterations in liver and kidney histoarchitectures were consistent with the observed significant increase in AST/ALT ratio, suggesting deleterious effects of EODS on the kidney and liver. However, the lack of alterations in the histoarchitectures of the hippocampus and hypothalamus suggests that the brain may not have been adversely affected. Also, the in silico analysis suggests that hepatotoxic effects of EODS may be linked to Benzylnitrile, Humulene, Linalool, (Z)-ß-Ocimene. In addition, the failure of ß-Phenylnitroethane, the most abundant phytoconstituent of EODS, to pass phases I and II in silico toxicity screening, and the presence of Caryophyllene oxide, a known toxic compound, coupled with the predicted binding of both to DNA and protein, low LD50 and high percent mortality at 250 mg/kg of repeated doses, further confirmed the potentially toxic nature of EODS. We concluded that based on our in vivo and in silico observations, there is an urgent need for public education to regulate the excessive consumption of the seeds of D. tripetala.

Inter-clinician delineation variation for a new highly-conformal flank target volume in children with renal tumors: A SIOP-Renal Tumor Study Group international multicenter exercise.

BACKGROUND AND PURPOSE: Recently, the SIOP-RTSG developed a highly-conformal flank target volume definition for children with renal tumors. The aims of this study were to evaluate the inter-clinician delineation variation of this new target volume definition in an international multicenter setting and to explore the necessity of quality assurance. MATERIALS AND METHODS: Six pediatric renal cancer cases were transferred to ten radiation oncologists from seven European countries ('participants'). These participants delineated the pre- and postoperative Gross Tumor Volume (GTVpre/post), and Clinical Target Volume (CTV) during two test phases (case 1-2 and 3-4), followed by guideline refinement and a quality assurance phase (case 5-6). Reference target volumes (TVref) were established by three experienced radiation oncologists. The Dice Similarity Coefficient between the reference and participants (DSCref/part) was calculated per case. Delineations of case 5-6 were graded by four independent reviewers as 'per protocol' (0-4 mm), 'minor deviation' (5-9 mm) or 'major deviation' (≥10 mm) from the delineation guideline using 18 standardized criteria. Also, a major deviation resulting in underestimation of the CTVref was regarded as an unacceptable variation. RESULTS: A total of 57/60 delineation sets were completed. The median DSCref/part for the CTV was 0.55 without improvement after sequential cases (case 3-4 vs. case 5-6: p = 0.15). For case 5-6, a major deviation was found for 5/18, 12/17, 18/18 and 4/9 collected delineations of the GTVpre, GTVpost, CTV-T and CTV-N, respectively. An unacceptable variation from the CTVref was found for 7/9 participants for case 5 and 6/9 participants for case 6. CONCLUSION: This international multicenter delineation exercise demonstrates that the new consensus for highly-conformal postoperative flank target volume delineation leads to geometrical variation among participants. Moreover, standardized review showed an unacceptable delineation variation in the majority of the participants. These findings strongly suggest the need for additional training and centralized pre-treatment review when this target volume delineation approach is implemented on a larger scale.

Improved osteosarcoma survival with addition of mifamurtide to conventional chemotherapy - Observational prospective single institution analysis.

PURPOSE: Conventional osteosarcoma is an orphan disease. Current treatment approaches include combining a three drug chemotherapy schedule and surgery. The 3- and 5-year event-free survival (EFS) in localized disease is roughly 65 and 60%, respectively. The registration study of mifamurtide reported survival benefit, but some methodological controversies have been insufficient for FDA market authorization in contrast to EMA. METHODS: prospective single centre survival analysis of a mifamurtide addition to conventional therapy in 23 patients over a 5.5 year enrolment period is reported and compared to a historical control of 26 patient with localized disease. Bias arising from observational methodology was addressed using Landmark analysis and time-dependent Cox models. Blood count dynamics were analysed during the treatment. RESULTS: The adverse event profile was as expected with no dose limiting toxicities. There were no local relapses observed, one patient died in the first complete remission due to doxorubicin cardiotoxicity, one patient had pulmonary metastatic relapse. The observed 3- and 5-year EFS was 87.4% (CI 72.4-100%) and 87.4% (CI 72.4-100%), progression free survival (PFS) was 92.9% (CI 80.3-100%) and 92.9% (CI 80.3-100%), overall survival was 94.1% (CI 83.6-100) and 80.7% (CI 58.3-100), respectively. Comparison to the historical control showed statistically significant better PFS for mifamurtide patients (Landmark analysis; p = 0.044). Risk of progression was 5-times lower for the mifamurtide group (Cox model; HR 0.21, p = 0.136). Only subtle differences in lymphocyte counts were observed across treatment. CONCLUSION: the PFS benefit of mifamurtide is reported herein. The addition of mifamurtide could be considered as a best treatment option for localized osteosarcoma.

Bioassay-guided isolation and evaluation of anti-osteoporotic polysaccharides from Morinda officinalis.

ETHNOPHARMACOLOGICAL RELEVANCE: Morinda officinalis is a well-known Chinese tonic herb that has shown clinical efficacy in the treatment of bone disease. However, its anti-osteoporotic potential and the M. officinalis polysaccharides (MOPs) responsible for activity require further investigation. AIM OF THE STUDY: This study aimed to investigate the anti-osteoporotic effects of different MOP fractions in ovariectomized (OVX) rats, and to identify the osteoprotective components by bioassay-guided isolation. MATERIALS AND METHODS: MOPs were prepared by hot water and alkali extraction, separated into three fractions (MO50, MO70, and MOB) and evaluated in the classic OVX rat model and in MC3T3-E1 cells for anti-osteoporotic activity. RESULTS: Administration of MOPs (400 mg/kg/day) provided significant protection against ovariectomy-induced bone loss and biomechanical dysfunction in rats. Treated animals exhibited reduced deterioration of trabecular microarchitecture and lower levels of bone turnover markers. Bioactivity-guided fractionation led to the isolation of two inulin-type fructans from MO50, MOW50-1 and MOP50-2, with potential anti-osteoporotic activities. These consisted of (2 → 1)-linked ß-D-fructosyl residues with degrees of polymerization (DP) of 7 and 13, respectively. Furthermore, MOW50-1 promoted osteogenic differentiation of MC3T3-E1 cells by increasing alkaline phosphatase activity. CONCLUSIONS: These data suggest very strongly that MOPs, especially MO50 and MOW50-1, may play important roles in the prevention and treatment of osteoporosis.