Subphysiological Concentrations Research Articles

Comparison of the developmental competence of in vitro-produced mouse embryos cultured under 5 versus 2% O2 with in vivo-derived blastocysts.

The prevalence of infertility in Canada has substantially increased over 30 years, and plateaued success rates of culture systems warrant further optimization for transfer outcomes. In clinical programs, embryos commonly undergo extended culture under 5% O2 until the blastocyst stage. The aim of this study is to characterize the developmental competence and stress-related responses of embryos cultured under 5 versus 2% O2 in comparison to in vivo-derived blastocysts. We hypothesized 2% O2 compromises developmental competence through altered embryonic stress responses and induction of apoptosis-related genes relative to those cultured under 5% O2 and in vivo-derived blastocysts. Quantitative measures of development and relative expressions of a cohort of stress-related genes in CD1 mouse zygotes cultured to blastocysts under 5 or 2% O2 were compared to in vivo-derived embryos. Apoptotic responses were evaluated using an immunofluorescence assay for Caspase-3. The mean percentage of blastocysts developed, and total cell number of embryos derived in vivo or cultured under 5% O2 was significantly higher than those cultured under 2% O2. Blastocyst expansion was greatest in embryos cultured under 5% O2. Stress response genes were significantly upregulated in embryos cultured under 2% O2, and expression of antioxidant-related genes was significantly lower in cultured versus in vivo-derived embryos. Caspase-3 immunofluorescence was significantly higher in cultured embryos versus in vivo-derived embryos. We inferred that 5% O2 systems better approximate physiologic oxygen availability for culture of mouse embryos, warranting re-evaluation of culturing embryos under threshold or sub-physiologic oxygen concentrations during clinical IVF programs.

Phage activity against Staphylococcus aureus is impaired in plasma and synovial fluid

S. aureus is a pathogen that frequently causes severe morbidity and phage therapy is being discussed as an alternative to antibiotics for the treatment of S. aureus infections. In this in vitro and animal study, we demonstrated that the activity of anti-staphylococcal phages is severely impaired in 0.5% plasma or synovial fluid. Despite phage replication in these matrices, lysis of the bacteria was slower than phage propagation, and no reduction of the bacterial population was observed. The inhibition of the phages associated with a reduction in phage adsorption, quantified to 99% at 10% plasma. S. aureus is known to bind multiple coagulation factors, resulting in the formation of aggregates and blood clots that might protect the bacterium from the phages. Here, we show that purified fibrinogen at a sub-physiological concentration of 0.4 mg/ml is sufficient to impair phage activity. In contrast, dissolution of the clots by tissue plasminogen activator (tPA) partially restored phage activity. Consistent with these in vitro findings, phage treatment did not reduce bacterial burdens in a neutropenic mouse S. aureus thigh infection model. In summary, phage treatment of S. aureus infections inside the body may be fundamentally challenging, and more investigation is needed prior to proceeding to in-human trials.

Low progesterone concentration in early pregnancy is detrimental to conceptus development and pregnancy outcome in horses

High progesterone concentrations in the early luteal phase support pregnancy, whereas subphysiological progesterone concentrations delay embryonic development at least until placentation. In this study, fetal growth and development of pregnancy was investigated in pregnancies with prostaglandin F2α-induced low progesterone concentrations (PGF) in the early luteal phase and control pregnancies (CON) in the same mares (n = 12). Mares were inseminated and in PGF pregnancies received the prostaglandin F2α analogue cloprostenol (62.5 µg) on days 0–3 after ovulation to induce subphysiological progesterone concentrations; CON pregnancies remained untreated. Mares were assigned to PGF or CON treatments in alternating order and received the opposite treatment in the following year. Blood was collected and conceptus size determined repeatedly by transrectal (≤day 101) and transabdominal (>day 101) ultrasonography. After birth, foals were weighed, measured and submitted to a clinical examination. Treatment PGF resulted in fewer pregnancies than CON treatment. All foals born from CON pregnancies were healthy and mature, whereas 4/7 PGF pregnancies were either lost (one embryonic death, one abortion) or resulted in the birth of compromised foals (P = 0.018). Size of the conceptus (e.g., diameter day 49: PGF 6.6 ± 0.7, CON 7.7 ± 0.7 cm, P = 0.006) and embryo proper (e.g., crown rump length day 54; PGF 4.4 ± 0.8, CON 5.8 ± 0.6 cm, P = 0.015) differed between treatments. These size differences decreased over time and at birth PGF foals did not differ significantly from CON foals. In conclusion, reduced progesterone concentration in the early luteal phase leads to delayed conceptus growth beyond placentation and increased pregnancy loss.

Effects of sub-physiological plasma progesterone concentration in the early luteal phase on embryonic and fetal development, pregnancy outcome and foal characteristics

High progesterone concentration in the early luteal phase supports pregnancy and postovulatory progestin treatment stimulates conceptus growth (Willmann et al., Theriogenology 2011;75,421) whereas reduced progesterone concentration changes histotroph composition (Beyer et al., Theriogenology 2019;125:236). Embryos obtained on day 34 from mares with reduced progesterone concentration were less developed than embryos from control pregnancies (Okada et al., Reprod Fert Develop 2020;32:1156). Sub-physiological progesterone concentrations in early pregnancy thus delay embryonic development at least until placentation. In this study, subsequent fetal growth, pregnancy loss and foal characteristics were investigated in pregnancies with reduced progesterone concentrations in the early luteal phase (PGF; n=7) and control pregnancies (CON; n=7) in the same mares. Estrous mares were inseminated every 48h until ovulation (day 0). Group PGF received 62.5µg of the prostaglandin F2α analogue cloprostenol daily on days 0-3 to induce sub-physiological progesterone concentrations whereas CON pregnancies remained untreated. Mares were assigned to PGF and CON treatments in alternating order and received the opposite treatment in the following year. Blood was obtained and conceptus size determined by transrectal (≤day 100) and transabdominal (>day 100) ultrasonography at regular intervals. IgG concentration was determined in colostrum at foaling and in foal plasma at 18-22 hrs after birth. Foals were weighed,measured, submitted to a clinical examination and the neutrophil / lymphocyte (N / L) ratio was determined as an indicator of maturity. Time from birth to first standing and first suckling was recorded. Data were analyzed with the SPSS statistics software, using two-way repeated measures ANOVA and χ2 analysis. Progesterone concentration was reduced in the first two weeks of PGF pregnancies (p<0.001; day 7 CON 35±3, PGF 11±2 ng/ml). All foals from CON pregnancies were healthy and mature, whereas 3/7 PGF pregnancies were lost (one embryonic death, one abortion, one non-viable foal, p=0.05). Size of the conceptus (day 49: PGF 6.6±07, CON 7.7±07 cm, p=0.006), embryo proper (day 54; PGF 4.4±0.8, CON 5.8±0.6 cm, p=0.015) and fetal orbita diameter (day 73 PGF 0.7±0.1, CON 1.0±0.1 cm, p=0.007) were smaller in PGF than in CON pregnancies. Size differences between PGF and CON pregnancies decreased over time and were no longer present at foaling. The viable PGF foals also did not differ in N / L ratio and time to standing and first suckling from CON foals In conclusion, reduced plasma progesterone concentration in the early luteal phase delays fetal growth beyond placentation and increases pregnancy loss. When viable foals are born, however, they are not compromised. Results indicate that pregnancies with sub-physiological progesterone concentrations will benefit from early progestogen substitution.

Stress Hormone Corticosterone Controls Metabolic Mitochondrial Performance and Inflammatory Signaling of In Vitro Cultured Sertoli Cells.

Stress, as a physiological response, is a major factor that affects several processes, including reproductive functions. The main hormonal players of stress are cortisol (humans) and corticosterone (rodents). Sertoli cells (SCs), as key contributors for the testicular homeostasis maintenance, are extensively challenged by different hormones, with glucocorticoid corticosterone being the signaling modulator that may impact these cells at different levels. We aimed to characterize how corticosterone modulates SCs energy balance, putting the mitochondrial performance and signaling output in perspective as the cells can disperse to the surroundings. TM4 mouse SCs were cultured in the absence and presence of corticosterone (in nM: 20, 200, and 2000). Cells were assessed for extracellular metabolic fluxes, mitochondrial performance (cell respirometry, mitochondrial potential, and mitochondrial complex expressions and activities), and the expression of androgen and corticosteroid receptors, as well as interleukine-6 (IL-6) and glutathione content. Corticosterone presented a biphasic impact on the extracellular fluxes of metabolites. Low sub-physiological corticosterone stimulated the glycolytic activity of SCs. Still, no alterations were perceived for lactate and alanine production. However, the lactate/alanine ratio was decreased in a dose-dependent mode, opposite to the mitochondrial complex II activity rise and concurrent with the decrease of IL-6 expression levels. Our results suggest that corticosterone finely tuned the energetic profile of mouse SCs, with sub-physiological concentrations promoting glycolytic expenditure, without translating into cell redox power and mitochondrial respiratory chain performance. Corticosterone deeply impacted the expression of the pro-inflammatory IL-6, which may alter cell-to-cell communication in the testis, in the last instance and impact of the spermatogenic performance.

Artifactual FA dimers mimic FAHFA signals in untargeted metabolomics pipelines

FA esters of hydroxy FAs (FAHFAs) are lipokines with extensive structural and regional isomeric diversity that impact multiple physiological functions, including insulin sensitivity and glucose homeostasis. Because of their low molar abundance, FAHFAs are typically quantified using highly sensitive LC-MS/MS methods. Numerous relevant MS databases house in silico-spectra that allow identification and speciation of FAHFAs. These provisional chemical feature assignments provide a useful starting point but could lead to misidentification. To address this possibility, we analyzed human serum with a commonly applied high-resolution LC-MS untargeted metabolomics platform. We found that many chemical features are putatively assigned to the FAHFA lipid class based on exact mass and fragmentation patterns matching spectral databases. Careful validation using authentic standards revealed that many investigated signals provisionally assigned as FAHFAs are in fact FA dimers formed in the LC-MS pipeline. These isobaric FA dimers differ structurally only by the presence of an olefinic bond. Furthermore, stable isotope-labeled oleic acid spiked into human serum at subphysiological concentrations showed concentration-dependent formation of a diverse repertoire of FA dimers that analytically mimicked FAHFAs. Conversely, validated FAHFA species did not form spontaneously in the LC-MS pipeline. Together, these findings underscore that FAHFAs are endogenous lipid species. However, nonbiological FA dimers forming in the setting of high concentrations of FFAs can be misidentified as FAHFAs. Based on these results, we assembled a FA dimer database to identify nonbiological FA dimers in untargeted metabolomics datasets.

Autoreactive antibodies control blood glucose by regulating insulin homeostasis

Homeostasis of metabolism by hormone production is crucial for maintaining physiological integrity, as disbalance can cause severe metabolic disorders such as diabetes mellitus. Here, we show that antibody-deficient mice and immunodeficiency patients have subphysiological blood glucose concentrations. Restoring blood glucose physiology required total IgG injections and insulin-specific IgG antibodies detected in total IgG preparations and in the serum of healthy individuals. In addition to the insulin-neutralizing anti-insulin IgG, we identified two fractions of anti-insulin IgM in the serum of healthy individuals. These autoreactive IgM fractions differ in their affinity to insulin. Interestingly, the low-affinity IgM fraction (anti-insulin IgMlow) neutralizes insulin and leads to increased blood glucose, whereas the high-affinity IgM fraction (anti-insulin IgMhigh) protects insulin from neutralization by anti-insulin IgG, thereby preventing blood glucose dysregulation. To demonstrate that anti-insulin IgMhigh acts as a protector of insulin and counteracts insulin neutralization by anti-insulin IgG, we expressed the variable regions of a high-affinity anti-insulin antibody as IgG and IgM. Remarkably, the recombinant anti-insulin IgMhigh normalized insulin function and prevented IgG-mediated insulin neutralization. These results suggest that autoreactive antibodies recognizing insulin are key regulators of blood glucose and metabolism, as they control the concentration of insulin in the blood. Moreover, our data suggest that preventing autoimmune damage and maintaining physiological homeostasis requires adaptive tolerance mechanisms generating high-affinity autoreactive IgM antibodies during memory responses.

Robust thrombolytic and anti-inflammatory action of a constitutively active ADAMTS13 variant in murine stroke models

AbstractAdvances in our understanding of ADAMTS13 structure, and the conformation changes required for full activity, have rejuvenated the possibility of its use as a thrombolytic therapy. We have tested a novel Ala1144Val ADAMTS13 variant (constitutively active [ca] ADAMTS13) that exhibits constitutive activity, characterized using in vitro assays of ADAMTS13 activity, and greatly enhanced thrombolytic activity in 2 murine models of ischemic stroke, the distal FeCl3 middle cerebral artery occlusion (MCAo) model and transient middle cerebral artery occlusion (tMCAO) with systemic inflammation and ischemia/reperfusion injury. The primary measure of efficacy in both models was restoration of regional cerebral blood flow (rCBF) to the MCA territory, which was determined using laser speckle contrast imaging. The caADAMTS13 variant exhibited a constitutively active conformation and a fivefold enhanced activity against fluorescence resonance energy transfer substrate von Willebrand factor 73 (FRETS-VWF73) compared with wild-type (wt) ADAMTS13. Moreover, caADAMTS13 inhibited VWF-mediated platelet capture at subphysiological concentrations and enhanced t-PA/plasmin lysis of fibrin(ogen), neither of which were observed with wtADAMTS13. Significant restoration of rCBF and reduced lesion volume was observed in animals treated with caADAMTS13. When administered 1 hour after FeCl3 MCAo, the caADAMTS13 variant significantly reduced residual VWF and fibrin deposits in the MCA, platelet aggregate formation, and neutrophil recruitment. When administered 4 hours after reperfusion in the tMCAo model, the caADAMTS13 variant induced a significant dissolution of platelet aggregates and a reduction in the resulting tissue hypoperfusion. The caADAMTS13 variant represents a potentially viable therapeutic option for the treatment of acute ischemic stroke, among other thrombotic indications, due to its enhanced in vitro and in vivo activities that result from its constitutively active conformation.

Network Pharmacology of Red Ginseng (Part I): Effects of Ginsenoside Rg5 at Physiological and Sub-Physiological Concentrations.

Numerous in vitro studies on isolated cells have been conducted to uncover the molecular mechanisms of action of Panax ginseng Meyer root extracts and purified ginsenosides. However, the concentrations of ginsenosides and the extracts used in these studies were much higher than those detected in pharmacokinetic studies in humans and animals orally administered with ginseng preparations at therapeutic doses. Our study aimed to assess: (a) the effects of ginsenoside Rg5, the major “rare” ginsenoside of Red Ginseng, on gene expression in the murine neuronal cell line HT22 in a wide range of concentrations, from 10−4 to 10−18 M, and (b) the effects of differentially expressed genes on cellular and physiological functions in organismal disorders and diseases. Gene expression profiling was performed by transcriptome-wide mRNA microarray analyses in HT22 cells after treatment with ginsenoside Rg5. Ginsenoside Rg5 exhibits soft-acting effects on gene expression of neuronal cells in a wide range of physiological concentrations and strong reversal impact at high (toxic) concentration: significant up- or downregulation of expression of about 300 genes at concentrations from 10−6 M to 10−18 M, and dramatically increased both the number of differentially expressed target genes (up to 1670) and the extent of their expression (fold changes compared to unexposed cells) at a toxic concentration of 10−4 M. Network pharmacology analyses of genes’ expression profiles using ingenuity pathway analysis (IPA) software showed that at low physiological concentrations, ginsenoside Rg5 has the potential to activate the biosynthesis of cholesterol and to exhibit predictable effects in senescence, neuroinflammation, apoptosis, and immune response, suggesting soft-acting, beneficial effects on organismal death, movement disorders, and cancer.

Novel Peptide-drug Conjugate Melflufen Efficiently Eradicates Bortezomib-resistant Multiple Myeloma Cells Including Tumor-initiating Myeloma Progenitor Cells.

Introduction of the proteasome inhibitor bortezomib has dramatically improved clinical outcomes in multiple myeloma. However, most patients become refractory to bortezomib-based therapies. On the molecular level, development of resistance to bortezomib in myeloma cells is accompanied by complex metabolic changes resulting in increased protein folding capacity, and less dependency on the proteasome. In this study, we show that aminopeptidase B, encoded by the RNPEP gene, is upregulated in bortezomib-resistant myeloma cell lines, and in a murine in vivo model. Moreover, increased RNPEP expression is associated with shorter survival in multiple myeloma patients previously treated with bortezomib-containing regimens. Additionally, expression is increased in plasma cell precursors, a B-lymphoid compartment previously associated with myeloma stem cells. We hypothesized that increased aminopeptidase B expression in aggressive myeloma clones may be used therapeutically toward elimination of the cells via the use of a novel peptide-drug conjugate, melphalan flufenamide (melflufen). Melflufen, a substrate of aminopeptidase B, efficiently eliminates bortezomib-resistant myeloma cells in vitro and in vivo, and completely suppresses clonogenic myeloma growth in vitro at subphysiological concentrations. Thus, melflufen represents a novel treatment option that is able to eradicate drug-resistant myeloma clones characterized by elevated aminopeptidase B expression.

KRAS-Mutated Myeloma Cells Exploit Autophagy and Macropinocytosis to Tolerate Glutamine-Deprived Conditions

Introduction: Sustained cancer cell growth requires up-regulation of nutrient acquisition mechanisms. Novel approaches can identify “tumor fuel”, the way it is obtained and then altered for cellular usage. Autophagy is a conserved catabolic process that is thought to replenish nutrient pool and thereby starvation survival by providing bioenergetic and biosynthetic substrates. Some KRAS-mutated cancers show heightened dependency to glutamine as a source of energy. Since a large proportion of myeloma tumors are KRAS-mutated, we investigated in the present study the contribution of macropinocytosis and autophagy as the cellular mechanisms involved in protein uptake and degradation, respectively, to growth and survival of myeloma cells under glutamine-deprived condition.Methods: Macropinosomes were visualized using confocal microscopy in KRAS-mutated and WT human myeloma cells utilising TMR-dextran and 5-[N-ethyl-N-isopropyl] amiloride (EIPA) as a marker and specific inhibitor of macropinocytosis, respectively. Internalization and degradation of labelled albumin and its co-localization with TMR-dextran were evaluated in parallel. Bafilomycin was employed to evaluate the dependency of protein degradation on autophagy. Cell sensitivity to glutamine deprivation was examined with an ATP-based viability test. To evaluate the possible role of macropinocytosis and autophagy at glutamine deprivation conditions, the effect of albumin supplementation on cell growth and viability was studied in the presence or absence of EIPA or chloroquine. To test whether the degradation of macropinocytosed albumin results in the production of intracellular glutamine, the intracellular concentrations of glutamine was directly measured.Results: KRAS-mutated KMS28-PE, KMS18 and MM1S cells showed higher levels of EIPA-sensitive macropinocytosis compared to WT-KRAS KMS34, KMS12-BM and TK1 cells. Macropinocytosis led to the internalization of albumin which subsequently underwent bafilomycin-sensitive proteolytic degradation in KRAS-mutated cells. The viability of WT-KRAS cells was reduced in sub-physiological concentrations of glutamine when compared with KRAS-mutated cells that not only remained viable but could continue to slowly proliferate over a period of 7 days (p˂0.05). The compromised growth rate of KRAS-mutated cells at very low concentrations of glutamine was rescued by albumin and this effect was abrogated by EIPA or chloroquine (p˂0.05). The KRAS-mutated cells demonstrated an EIPA-sensitive increase in intracellular concentration of glutamine following albumin supplementation (p˂0.05) that was abrogated following the treatment with chloroquine. In WT cells, in contrast, the level of intracellular glutamine remained almost unchanged following albumin supplementation.Conclusions: Autophagy and macropinocytosis maintain cellular growth and survival in the context of nutrient deprivation by provision of glutamine, and potentially other amino acids, in RAS-mutated myeloma cancer. These data suggest that novel anti-cancer strategies interrupting these cellular mechanisms may represent a promising therapeutic approach to myeloma. DisclosuresSpencer:Janssen: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding.

A Novel A\u03b240 Assembly at Physiological Concentration

Aggregates of amyloid-β (Aβ) are characteristic of Alzheimer’s disease, but there is no consensus as to either the nature of the toxic molecular complex or the mechanism by which toxic aggregates are produced. We report on a novel feature of amyloid-lipid interactions where discontinuities in the lipid continuum can serve as catalytic centers for a previously unseen microscale aggregation phenomenon. We show that specific lipid membrane conditions rapidly produce long contours of lipid-bound peptide, even at sub-physiological concentrations of Aβ. Using single molecule fluorescence, time-lapse TIRF microscopy and AFM imaging we characterize this phenomenon and identify some exceptional properties of the aggregation pathway which make it a likely contributor to early oligomer and fibril formation, and thus a potential critical mechanism in the etiology of AD. We infer that these amyloidogenic events occur only at areas of high membrane curvature, which suggests a range of possible mechanisms by which accumulated physiological changes may lead to their inception. The speed of the formation is in hours to days, even at 1 nM peptide concentrations. Lipid features of this type may act like an assembly line for monomeric and small oligomeric subunits of Aβ to increase their aggregation states. We conclude that under lipid environmental conditions, where catalytic centers of the observed type are common, key pathological features of AD may arise on a very short timescale under physiological concentration.

Low plasma progestin concentration during the early postovulatory phase impairs equine conceptus development in the late preimplantation phase

In horses, low postovulatory progestin concentrations delay downregulation of endometrial progesterone receptors, impairing histotroph composition. The aim of this study was to evaluate conceptus development until placentation in mares under experimentally reduced progestin concentrations in the early postovulatory phase. Eleven mares were inseminated until ovulation and received the prostaglandin F2α agonist cloprostenol (125 µg) once daily for 4 days after ovulation (treatment) or were left untreated (control). Conceptus growth and development was analysed daily from Day 10 after ovulation by ultrasound. On Day 34 conceptuses were recovered and underwent microcomputed tomography. Statistical comparison was performed by non-parametric Wilcoxon test or Chi2. First visualisation of embryonic vesicle, embryo proper and heartbeat did not differ. Conceptus fixation in the uterus occurred earlier in controls than in conceptuses recovered from treatment pregnancies (P < 0.05). Embryos from treatment pregnancies were smaller (P < 0.05) and lighter (P < 0.05) than controls. In the lung, the mean number of bronchi was lower in embryos from treatment pregnancies than from control pregnancies (P < 0.05). Persistence of communication between the cardiac ventricles was observed more often in embryos from treatment than control pregnancies (P < 0.05). In conclusion, subphysiological progestin concentrations in the early postovulatory period impaired conceptus development before placentation in horses.

61 Low plasma progestin concentration during the early luteal phase impairs equine conceptus development until placentation

During the early luteal phase, low progesterone concentrations delay downregulation of endometrial progesterone receptors. This contributes to impaired histotroph production at Day 14 of pregnancy (Beyer et al. 2019 Theriogenology 125, 236-241). Until the beginning of placentation (i.e. Day 37 of pregnancy), nutritional supply of the equine conceptus depends on histotrophy alone. The aim of the present study was to analyse development of the equine conceptus under reduced plasma progesterone concentrations until shortly before placentation. Fertile Haflinger mares (n=11; 4-11 years old) were examined daily by transrectal ultrasonography, and when in oestrus, they were inseminated every 48h until spontaneous ovulation. Mares were randomly assigned to either the treatment group and received the prostaglandin F2α agonist cloprostenol (PGF2α; 125μg) once daily for 3 days after ovulation (Beyer et al. 2019) or the control group and left untreated. After conceptus collection on Day 34, mares were allowed one oestrous cycle for recovery and subsequently assigned to the opposite treatment, thus serving as their own controls. From Day 10 after ovulation, conceptus development including size, uterine fixation, and heartbeat detection was evaluated daily by transrectal ultrasonography. On Day 34, conceptus and fetal membranes were recovered transcervically. The recovered material was weighed, measured, and stored for further analysis. Conceptuses underwent microcomputed tomography that was evaluated by Amira (Thermo Fisher Scientific). Statistical comparison for differences between control and treatment pregnancies was performed by nonparametric Wilcoxon test or chi-square analysis. The day of first visualisation of the conceptus and of the embryo proper and heartbeat did not differ between treatments. Uterine fixation occurred on Day 19.3±0.5 in conceptuses from PGF2α-treated pregnancies but on Day 16.7±0.4 in controls (P&amp;lt;0.05). At 34 days of gestation, the conceptuses from PGF2α-treated mares were smaller (P&amp;lt;0.05) than control conceptuses when measured with ultrasound callipers in utero (maximal length: PGF2α 17.4±0.3mm, control 19.0±0.4mm). Conceptus weight determined after recovery was less in treated pregnancies (PGF2α 2.5±0.2 g; control 3.5±0.3 g; P&amp;lt;0.05). Microcomputed tomography analysis of selected inner organs showed some differences in development. Seven complete pairs of undestroyed conceptuses were available. Mean numbers of 33.4±3.2 and 18.8±7.5 bronchi were detected in control and PGF2α conceptuses, respectively (P&amp;lt;0.05). Heart volume did not differ, but communication between ventricles was detected in only 1/7 control but 5/7 PGF2α conceptuses (P&amp;lt;0.05). The footpad was present in 6/7 control and 0/7 PGF2α conceptuses (P&amp;lt;0.001). In conclusion, subphysiological progesterone concentration during the early luteal phase delays development of the equine conceptus before placentation. The condition may contribute to early conceptus loss in horses, which occurs in up to 20% of pregnancies.

Ile73Asn mutation in protein C introduces a new N-linked glycosylation site on the first EGF-domain of protein C and causes thrombosis

Activated protein C exerts its anticoagulant activity by protein S-dependent inactivation of factors Va and VIIIa by limited proteolysis. We identified a venous thrombosis patient who has plasma protein C antigen level of 63% and activity levels of 44% and 23%, as monitored by chromogenic and clotting assays. Genetic analysis revealed the proband carries compound heterozygous mutations (c.344T>A, p.I73N and c.1181G>A, p.R352Q) in PROC. We individually expressed protein C mutations and discovered that thrombin-thrombomodulin activates both variants normally and the resulting activated protein C mutants exhibit normal amidolytic and proteolytic activities. However, while protein S-dependent catalytic activity of activated protein C-R352Q toward factor Va was normal, it was significantly impaired for activated protein C-I73N. These results suggest that the Ile to Asn substitution impairs interaction of activated protein C-I73N with protein S. This conclusion was supported by a normal anticoagulant activity for activated protein C-I73N in protein S-deficient but not in normal plasma. Further analysis revealed Ile to Asn substitution introduces a new glycosylation site on first EGF-like domain of protein C, thereby adversely affecting interaction of activated protein C with protein S. Activated protein C-R352Q only exhibited reduced activity in sub-physiological concentrations of Na+ and Ca2+, suggesting that this residue contributes to metal ion-binding affinity of the protease, with no apparent adverse effect on its function in the presence of physiological levels of metal ions. These results provide insight into the mechanism by which I73N/R352Q mutations in activated protein C cause thrombosis in proband carrying this compound heterozygous mutation.

Effect of Cu(ii) on in vitro glycation of human serum albumin by methylglyoxal: a LC-MS-based proteomic approach

It has been reported that glycation of human serum albumin (HSA) changes its capability for copper binding whereas the increase of free copper might have an impact on protein glycation - a key process in diabetes progression. In this work, proteomic analysis of non-glycated HSA and HSA glycated with methylglyoxal (MGo) in the absence or in the presence of Cu(ii) (0.1; 1.0; 5.0 mg Cu L-1) has been undertaken. Trypsin hydrolysates were subjected to capillary HPLC-ESI-QTOF-MS and MS/MS. Raw data were analyzed using two proteomic platforms: MaxQuant () and ProteinScape (Bruker). Considering seven MGo-derived modifications, the sequence coverage was 98% for non-modified HSA and ≥93% for HSA incubated with MGo or MGo + Cu(ii). Peptide mapping yielded 76 identical peptides in all samples though important differences were found between non-modified HSA and protein glycated with or without Cu(ii). Overall, 46 peptides with residues from 1 to 3 modified were detected/sequenced; the MGo-derived modifications found were: hydroimidazolone, argpyrimidine, Nε-carboxyethyl-lysine and S-carboxyethyl-cysteine; 39 modified sites were identified (22 on arginine, 12 on lysine, and 5 on cysteine) and among them, 27 were common for ProteinScape and MaxQuant. The count of the modified peptides and the comparative analysis of their abundance in different samples indicated that Cu(ii) at physiological and sub-physiological concentrations inhibited HSA glycation as compared to the glycation of the Cu-devoid protein; at higher concentrations (5 mg Cu L-1), this inhibitory effect tends to be inverted. The results obtained suggest that increased protein glycation might be associated with Cu-deficiency and with excessive Cu(ii) concentrations, calling for more detailed studies performed on real-world samples with a strict control of copper concentration.

Prospective association of sex steroid concentrations with periodontal progression and incident tooth loss.

The aim of this study was to determine the prospective association between sex steroid concentrations with periodontal progression and incident tooth loss in men and women. We used data from 1465 women and 1838 men (age 20-81 years) with completed five-year-follow-up from the Study-of-Health-in-Pomerania, a population-based longitudinal cohort. Serum levels of total testosterone (TT) and other sex steroids were measured. Mean clinical attachment loss (CAL) and the number of teeth were assessed. Generalized regression models were implemented for cross-sectional and longitudinal analyses, adjusting for age, education, smoking, waist circumference, diabetes, physical activity, blood sampling time and time between baseline and follow-up. Fully adjusted models revealed no consistent associations between TT and mean CAL, neither in cross-sectional [men: ß = -0.0004 (-0.023;0.022), p = 0.97; women: ß = -0.033 (-0.057; -0.009), p = 0.006] nor in longitudinal analyses [men: ß = -0.033 (-0.100;0.034), p = 0.33; women: ß = -0.023 (-0.086;0.040), p = 0.47]. For tooth loss, neither cross-sectional nor longitudinal associations with any of the sex steroid concentrations were found. No consistent associations of sexual steroids with periodontal progression or tooth loss were found. Further cohort studies are necessary to evaluate possible associations between endocrinological parameters, like supra- or subphysiologic testosterone concentrations, and periodontal progression or tooth loss.

PWE-350 Resilience and protection against the dose-related toxicity of hydrogen sulphide

Introduction Hydrogen sulphide (H2S) is thought to have a biphasic effect on cells leading to cellular proliferation at “low” doses (micromolar) and cell death in “high” doses (millimolar). It is unclear how this is related to colonic physiology and in-vivo concentrations. H2S is cytotoxic by inhibiting cytochrome c, while genotoxicity may be mediated by free radicals of uncertain origin. We tested the effect of sub-physiological and physiological concentration of H2S on epithelial cell lines to identify proliferative/cytotoxic effects and whether a free radical scavenger could be protective. Method Cell lines were seeded at a concentration of 2.5 × 10 4 per well and cultured until 70–80% confluency. NaHS was dissolved in distilled water at 0, 100 μM, 500 μM, 1 mM, 2.5 mM and 5 mM and added to the culture. The number of surviving cells was counted after 24 h. In the protection experiments 1mM Tiron, a free radical scavenger, dissolved in distilled water was added concurrently. Results H2S proved considerably cytotoxic to CACO2 cells at even physiological doses (100 μM, 76.7% death), while at the higher end of the claimed physiological range (2.5 mM) 98.8% cell death was noted). Similar findings occurred with HCT116, though increased survival was noted with HT29 cells compared to the other colorectal cell lines (100 µM 43.9% death). Tiron was protective in all cell lines, especially in the CACO2 cells, with 100 μM + 1 mM Tiron mitigating cell death at 20% and 2.5 mM at 68.8%. P-values were significant at p Conclusion H2S is cytotoxic to cell lines, especially colonic lines, at estimated physiological doses. Tiron, a free-radical scavenger, increases cell survival after H2S exposure. This implicates excess free radicals in cytotoxicity. HT29 cells, with intact mismatch repair, were more resilient than other colorectal lines with mismatch deficiency. Contrary to some published research, this experiment suggests that H2S is cytotoxic, even at physiological levels, via a free radical mechanism, rather than protective against such insults. Colonic H2S may potentiate the risk of colorectal cancer. Steps to reduce its production or inhibit its action may reduce the risk of progression to cancer and reduce the chance of recurrence after surgery. Disclosure of interest None Declared. References Szabo C, et al . Tumor-derived hydrogen sulfide, produced by cystathionine-beta-synthase, stimulates bioenergetics, cell proliferation, and angiogenesis in colon cancer. Proc Natl Acad Sci USA. 2013;110(30):12474–9 Deplancke B, Gaskins HR. Hydrogen sulfide induces serum-independent cell cycle entry in nontransformed rat intestinal epithelial cells. FASEB J. 2003;17(10):1310–2

Imaging tumor metabolism using positron emission tomography.

Positron emission tomography (PET) is an extraordinarily sensitive clinical imaging modality for interrogating tumor metabolism. Radiolabeled PET substrates can be traced at subphysiological concentrations, allowing noninvasive imaging of metabolism and intratumoral heterogeneity in systems ranging from advanced cancer models to patients in the clinic. There are a wide range of novel and more established PET radiotracers, which can be used to investigate various aspects of the tumor, including carbohydrate, amino acid, and fatty acid metabolism. In this review, we briefly discuss the more established metabolic tracers and describe recent work on the development of new tracers. Some of the unanswered questions in tumor metabolism are considered alongside new technical developments, such as combined PET/magnetic resonance imaging scanners, which could provide new imaging solutions to some of the outstanding diagnostic challenges facing modern cancer medicine.

Spectroscopic Study of Full-Length Recombinant Proteoglycan 4 (rhPRG4): Self-Assembly and Interactions with Hyaluronan

Proteoglycan 4 (PRG4) is a mucin-like glycoprotein present in synovial fluid (SF) and at the surface of articular cartilage. It functions as a critical cartilage boundary lubricant, both alone in a dose-dependent manner and synergistically with the repeating disaccharide hyaluronan (HA). The mechanism of the functional interaction with HA remains to be elucidated, but appears to be non-covalent in nature and be dependent on PRG4 structure and assembly. Furthermore, concentration of both PRG4 and HA, which can vary in disease, may affect the interaction as well. Recently, full-length recombinant human PRG4 (rhPRG4) has become available. The objective of this study was to characterize aggregation of rhPRG4 and investigate its interaction with HA using spectroscopic techniques.Spectroscopic techniques including uv absorbance, fluorescence, and light scattering, were employed to characterize the aggregation of rhPRG4 and interaction with HA over a range of physiological concentrations. Various dilutions of rhPRG4 in different buffers and their mixtures with different concentrations of HA, with and without other physiological molecules, were examined. rhPRG4 was provided by Lubris.UV absorbance and fluorescence spectroscopic data demonstrated concentration based aggregation and suggested a non-covalent linear aggregation of rhPRG4 molecules with increasing concentration. However, multiple species were also observed even at sub-physiological concentrations. Dynamic as well as multi-angle light scattering techniques suggested presence of species with various and large (∼MDa) molecular weights. These techniques also suggested the subtle interaction of PRG4 with HA can be influenced by sample preparation, storage conditions, and presence of other molecules.These results contribute to the understanding of rhPRG4's aggregation and interaction with HA, which can have functional consequences and therefore provides the framework for the development of potential new rhPRG4+HA containing biotherapeutics for the treatment of osteoarthritis and other conditions.