Bacillus methanolicus is critical to understand the origin of their efficient methylotrophy. This knowledge will then be leveraged to transform such natural strains into new cell factories or to design methylotrophic capability in other strains already used by the industry.Methanol is inexpensive, is easy to transport, and can be produced both from renewable and from fossil resources without mobilizing arable lands. As such, it is regarded as a potential carbon source to transition toward a greener industrial chemistry. Metabolic engineering of bacteria and yeast able to efficiently consume methanol is expected to provide cell factories that will transform methanol into higher-value chemicals in the so-called methanol economy. Toward that goal, the study of natural methylotrophs such as  Bacillus methanolicus MGA3 is a thermotolerant and relatively fast-growing methylotroph able to secrete large quantities of glutamate and lysine. These natural characteristics make B. methanolicus a good candidate to become a new industrial chassis organism, especially in a methanol-based economy. Intriguingly, the only substrates known to support B. methanolicus growth as sole sources of carbon and energy are methanol, mannitol, and, to a lesser extent, glucose and arabitol. Because fluxomics provides the most direct readout of the cellular phenotype, we hypothesized that comparing methylotrophic and nonmethylotrophic metabolic states at the flux level would yield new insights into MGA3 metabolism. In this study, we designed and performed a 13C metabolic flux analysis (13C-MFA) of the facultative methylotroph B. methanolicus MGA3 growing on methanol, mannitol, and arabitol to compare the associated metabolic states. On methanol, results showed a greater flux in the ribulose monophosphate (RuMP) pathway than in the tricarboxylic acid (TCA) cycle, thus validating previous findings on the methylotrophy of B. methanolicus. New insights related to the utilization of cyclic RuMP versus linear dissimilation pathways and between the RuMP variants were generated. Importantly, we demonstrated that the linear detoxification pathways and the malic enzyme shared with the pentose phosphate pathway have an important role in cofactor regeneration. Finally, we identified, for the first time, the metabolic pathway used to assimilate arabitol. Overall, those data provide a better understanding of this strain under various environmental conditions.IMPORTANCE Methanol is inexpensive, is easy to transport, and can be produced both from renewable and from fossil resources without mobilizing arable lands. As such, it is regarded as a potential carbon source to transition toward a greener industrial chemistry. Metabolic engineering of bacteria and yeast able to efficiently consume methanol is expected to provide cell factories that will transform methanol into higher-value chemicals in the so-called methanol economy. Toward that goal, the study of natural methylotrophs such as Bacillus methanolicus is critical to understand the origin of their efficient methylotrophy. This knowledge will then be leveraged to transform such natural strains into new cell factories or to design methylotrophic capability in other strains already used by the industry. It has successfully been used to identify new pathways  and/or nuclear magnetic resonance (NMR). With NMR, carbon isotopomers are determined; hence, positional information is provided. With MS, isotopologues are identified. A metabolic model is then used to fit these measurements to theoretical data that are simulated by optimizing reaction flux values from the metabolic model. Assuming mass balance, if the measurements are coherent and the topology of the metabolic model is correct, the experimental and simulated data will converge, yielding the estimated reaction fluxes. Finally, 13C-MFA provides a flux map: a predicted snapshot of the metabolic fluxes through the organism of interest during the experiment, i.e., its metabolic state. 13C-MFA flux maps are conceptually similar to those from flux balance analysis (FBA), a purely in silico method that, from a metabolic model , computes the optimal reaction flux distribution to maximize an objective defined in terms of metabolite production, often designed to model cell growth. Importantly, 13C-MFA flux maps are estimates based on experimental data, whereas FBA flux maps are purely in silico predictions, which can be confirmed by gene deletion analysis or 13C-MFA, for example.In the last decade, pathways , improvepathways , and, mopathways  such as pathways –8. BriefBacillus methanolicus MGA3 is a Gram-positive bacterium that was first isolated in the 1990s from freshwater marsh soil samples after an enrichment culture on methanol at 55°C  and mannitol (C6H14O6) are the best-known carbon sources for this strain (5H12O5) is significantly slower  and two putative arabitol phosphate dehydrogenases (AtlD and AtlF) H-dependent reduction. A number of potential sugars  and sugar alcohols  were also tested as the substrates, but no significant activity  was detected. The kinetic parameters measured for AtlD and AtlF reduction using either Xyl5P or Ribu5P as the substrate are summarized in mK of 0.07 ± 0.03 mM for AtlD with Xyl5P as the substrate and NADH as cofactor was 2.5 times lower than the value obtained for AtlF (0.18 ± 0.05 mM). With Ribu5P as the substrate, the mK for AtlD was 17 times lower (1.21 ± 0.42 mM) (Vmax for AtlD with Xyl5P as the substrate and NADH as cofactor was 2.7 times higher than with Ribu5P as the substrate (0.49 ± 0.06 U mg−1) and 12 times higher than for AtlF with Xyl5P (0.11 ± 0.01 U mg−1) (mK (0.01 ± 0.01 versus 0.11 ± 0.09 mM). The affinity and NADPH-dependent activity (0.24 ± 0.06 U mg−1) could only be determined with AtlD and Xyl5P, as no significant activity was detected for any of the other reactions. Liquid chromatography-mass spectrometry (LC-MS) analyses were performed to confirm the formation of arabitol phosphate in the enzyme reactions catalyzed by AtlD. Although arabitol 1-phosphate and arabitol 5-phosphate could not be distinguished, arabitol phosphate was clearly produced with both Xyl5P and Ribu5P as the substrates (see The operon responsible for arabitol assimilation in nd AtlF) , which and AtlF) , 32 (seend AtlF)  Fig. 1)B. methan0.42 mM) , and for U mg−1) . NADH waates see .10.1128/mSystems.00745-20.5TEXT S1Text S1, PDF file, 0.5 MB.Phylogeny of AtlD and AtlF. Download Copyright © 2020 Delépine et al.2020Delépine et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro and that AtlD and AtlF both prefer Xyl5P. This suggests that arabitol is mainly assimilated through the arabitol 1-phosphate pathway. However, assimilation via arabitol 5-phosphate cannot be excluded, since AtlD can also use Ribu5P, albeit with reduced efficiency, as shown both by the kinetic parameters , were inspected manually and then used with additional NMR and physiological data to fit a model of B. methanolicus’s central metabolism  tabolism .Some isotopologues of 6-phosphogluconate (Gnt6P) (M + 2 and M + 3) could not be quantified, and the flux through the oxidative pentose phosphate pathway (PPP) could not be accurately resolved. Hence, to exploit these partial data and estimate the flux through the oxidative PPP, we applied the ScalaFlux approach . Using sB. methanolicus was grown in batches on three different carbon sources. For each cultivation, the growth, consumption, and production rates were determined. Results are given in −1) is slightly higher than reported in a previous proteomic study of B. methanolicus MGA3 (0.40 h−1) (B. methanolicus PB1 (0.32 h−1)   or for t.32 h−1) . Biomassd values .B. methanolicus MGA3 is known to overproduce glutamate at up to 50 g/liter  may operate in vivo. To confirm whether either or both arabitol assimilation pathways are operative in B. methanolicus, as suggested for E. avium   via PTS-mediated uptake and phosphorylation to arabitol 1-phosphate followed by oxidation to Xyl5P with AtlD as the major dehydrogenase  . Intereslation”) . This inrogenase .B. methanolicus and rule out the hypothesis of additional catabolism through arabitol 5-phosphate and Ribu5P derived from our enzymatic analyses  cycle that condenses formaldehyde (For) and ribulose 5-P (Ribu5P) into hexulose 6-P (Hex6P)  . The reg (Hex6P) . Interes (Hex6P) . The uti (Hex6P) , 45—is oet al.’s  study ofntration , and we ntration .1 acceptor under those conditions. As expected, the nonoxidative PPP was more oriented toward carbon assimilation on arabitol than on mannitol. On arabitol, the ribulose-phosphate 3-epimerase’s flux (rpe) was in the opposite direction, and the ribose-5-phosphate isomerase’s absolute flux (rpi) flux was almost 2 times higher , whereas arabitol is converted to Xyl5P as discussed above. However, on those carbon sources, the nonoxidative PPP was operating in the opposite direction than when on methanol , which is higher  and C ins higher . Simulats higher . Howevers higher  and the s higher .glpXP) on the pBM19 plasmid, whereas there is only one transaldolase gene (taC) in the chromosome. Proteomic  and ta (associated with taC) carried a comparable flux; thus, both variants may be active  cycle and glyoxylate shunt , the reported expression levels suggest that this pathway does not carry a high flux under mannitol or under methanol growth conditions  was observed on methanol than on mannitol and arabitol    and C.Overall, as reported before, the TCA cycle is mainly active under nonmethylotrophic conditions, and the glyoxylate shunt was inactive under our conditions. The novelty is the noncyclic operation of the TCA cycle under methylotrophic conditions.B. methanolicus balances its redox and energy needs, we used the estimated fluxes from the 13C-MFA to infer production and consumption rates of ATP, NADH, and NADPH, as shown in 13C-MFA is constrained by carbon balance and the distribution of the isotopic tracer, but unlike flux balance analysis, it is typically not constrained by cofactor balance. We used the estimated fluxes from the MFA and the expected stoichiometry of the associated reactions to assess the absolute rates of cofactor production and consumption. In the absence of specific measurements, we used the biomass requirements of Bacillus subtilis  than on mannitol and arabitol  because of the conversion of methanol into formaldehyde by methanol dehydrogenase . This suarabitol . It was Overall, nonmethylotrophic growth on mannitol and on arabitol share the same features despite their associated different growth rates (with the notable exception of the contribution of the oxidative PPP for NADPH production). On methanol, however, we observed more differences in the origin of cofactor production, which clearly highlight a different metabolic state. Importantly, the linear detoxification pathways share with the PPP and the malic enzyme an important role in cofactor regeneration.B. methanolicus MGA3 provides three snapshots of its metabolic states for growth on methanol, mannitol, or arabitol. Isotopic data are consistent with prior knowledge of MGA3 methylotrophy, showing greater flux in the RuMP cycle than in the TCA cycle. The 13C-MFA provided new insights related to the utilization of cyclic RuMP versus linear dissimilation pathways and between the RuMP cycle variants; finally, the characterization of the arabitol assimilation pathway was completed using enzymatic data. In future studies, these validated flux maps will be used as references for constraint-based modeling to validate genome-scale model predictions. Overall, the information provided in this work and previous omics studies on B. methanolicus metabolism can be used to improve design strategies for new strains . For instance, the comparison between methanol and arabitol might help identify key control steps in the metabolic network that allows shifting from a cyclic  to a linear  mode of operation. The experimental path outlined here likely leads to B. methanolicus becoming a viable alternative to existing cell factories.In summary, this MFA of B. methanolicus wild-type MGA3 (ATCC 53907) strain was used for metabolic flux analyses. Strains used for cloning and expression are described below in “Strains and culture conditions” and are listed in The (i) Culture conditions and parameters. For the carbon source methanol, two batch cultures were performed in 0.5-liter bioreactors  with a working volume of 0.40 liters coupled to a Dycor ProLine Process mass spectrometer . The culture medium per liter was 3.48 g Na2HPO4·12H2O, 0.606 g KH2PO4, 2.5 g NH4Cl, 0.048 g yeast extract, 1 ml of 1 M MgSO4 solution, 1 ml of trace salt solution, 1 ml of vitamin solution, 0.05 ml Antifoam 204, and 150 mM methanol. The trace salt solution per liter was 5.56 g FeSO4·7H2O, 0.027 g CuCl2·2H2O, 7.35 CaCl2·2H2O, 0.040 g CoCl2·6H2O, 9.90 g MnCl2·4H2O, 0.288 g ZnSO4·7H2O, and 0.031 g H3BO3. The vitamin solution per liter was 0.10 g d-biotin, 0.10 g thiamine-HCl, 0.10 g riboflavin, 0.10 g pyridoxine-HCl, 0.10 g pantothenate, 0.10 g nicotinamide, 0.02 g p-aminobenzoic acid, 0.01 g folic acid, 0.01 g vitamin B12, and 0.01 g lipoic acid. The precultures were grown in two 0.5-liter shake flasks containing 150 ml of the preculture medium and inoculated with cryostock of B. methanolicus wild-type MGA3 cells. The cultures were grown overnight at 50°C under shaking at 200 rpm and used to inoculate the reactors. The aeration rate of 1 vvm (volume of gas per volume of liquid per minute) was controlled by a mass flow meter , and partial O2 pressure (pO2) was maintained at >25% throughout all cultures. Temperature, pH, and stirring speed were maintained at 50°C, pH 6.8 (with KOH 1 M), and 800 rpm, respectively. The N2, O2, Argon, CO2, 13C-CO2, and methanol concentrations in the bioreactors off-gas were measured on-line with the mass spectrometer.13C-methanol  was added to the cultures at an optical density of 600 nm (OD600) of 2.5. Growth curves are available in To perform the pulse of tracer, 100 mM (ii) Quantification of cells and supernatant NMR analysis. For determination of the dry weight of cells, a conversion factor of 0.389 g/liter (dry weight) of cells per OD600 unit was used. Supernatant samples were taken to analyze substrate methanol consumption as well as by-product formation by subtracting 1 ml of culture and centrifuging it at 13,000 × g for 60 s. Thereafter, supernatant was collected and stored until analysis at −20°C. Supernatant analysis was performed by 1D 1H NMR at 292°K, using a 30° pulse and a relaxation delay of 20 s, with an Avance 800 MHz spectrometer . Deuterated trimethylsilyl propionate (TSP-d4) was used as an internal standard for quantification.(iii) Sampling and MS analysis of intra- and extracellular pool sizes. When the cultures reached an OD600 of 2, metabolome samples were collected using the optimized method described in reference Escherichia coli, cultivated on 99% [13C6]glucose  as an internal standard  and extracellular (B) pools of key metabolites of the central metabolism of Copyright © 2020 Delépine et al.2020Delépine et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00745-20.2FIG S2m/z values for arabitol 5-phosphate (https://doi.org/10.1073/pnas.1423570112). Download FIG S2, TIF file, 0.1 MB.Detection of formed arabitol phosphate in AtlD-catalyzed enzyme reactions using LC-MS. Chromatograms of detected pentitol phosphates (top) and their respective LC-MS spectra (bottom) of enzyme reactions using Xyl5P (A) and Ribu5P (B) as the substrates. The peaks for the given compounds were identified by characteristic mass spectra and retention time using standards for Xyl5P and Ribu5P or by comparison to previously reported Copyright © 2020 Delépine et al.2020Delépine et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the (iv) Sampling and MS analysis of labeled metabolites. Label enrichments in the intracellular metabolites were followed after performing a pulse of 100 mM 13C-methanol at an OD600 of 2.5. Whole broth  and culture filtrate  were sampled to indirectly track label incorporation in the intracellular metabolites. Specifically, 13 WB and 3 CF samples were collected in 3.5 min from each bioreactor. Exact sampling times can be seen in d-glycerate (G3P) plus phosphoglycerate (PGA), citrate (Cit), aconitate (Aco), fumarate (Fum), malate , and succinate (Suc). After manual peak integration, the raw peak areas were corrected for the contribution of all naturally abundant isotopes using IsoCor software  Mass balance. Experimental data consistency of the measured rates was verified using standard data reconciliation procedures under the elemental mass balance constraints  Culture conditions. For the carbon sources mannitol and arabitol, the culture medium composition remained unchanged. The experiments were performed in 500-ml baffled shake flasks using 40 ml of medium, and cells were grown at 50°C and 200 rpm. Precultures contained yeast extract and 15 mM unlabeled mannitol or arabitol. They were inoculated as stated previously and grown overnight. The precultures were used to inoculate triplicate main cultures to an OD600 of 0.05 after centrifugation and resuspension in medium without yeast extract. The medium to perform the 13C MFA contained 15 mM [1-13C]mannitol, 15 mM [5-13C]arabitol, or 15 mM a mixture of 10% [1-13C]arabitol and 90% [2-13C]arabitol . An experimentally determined conversion factor of 0.22 g/liter (dry weight) of cells per OD600 unit was used. Growth curves are available in 13C-isotopologues.(ii) Measurements of proteinogenic amino acids  Mannitol (or arabitol) cultures were sampled around 10 h (or 30 h) once they reached an OD600 of 1.3. The pellets obtained from the cellular extract were hydrolyzed 15 h at 110°C with 500 μl 6 N HCl. Samples were evaporated and washed twice with 500 μl of ultrapure water, evaporated to dryness, resuspended , and diluted  for the mass spectrometry analysis.2O and solvent B was 0.1% formic acid in acetonitrile at a flow rate of 250 μl/min. The gradient was adapted from the method used in reference Amino acids were separated on a pentafluorophenyl (PFP) column . Solvent A was 0.1% formic acid in Hm/z) in full-scan mode, with the following source parameters: capillary temperature, 275°C; source heater temperature, 250°C; sheath gas flow rate, 45 AU (arbitrary unit); auxiliary gas flow rate, 20 AU; S-lens radio frequency (RF) level, 40%; source voltage, 5 kV. Isotopic clusters were determined by extracting the exact mass of all isotopologues, with a tolerance of 5 ppm. Experimental carbon isotopologue distributions (CIDs) of alanine, glycine, valine, serine, threonine, phenylalanine, aspartate, glutamate, histidine, isoleucine, leucine, lysine, arginine, tyrosine, proline, and methionine were obtained after correction of raw MS data for naturally occurring isotopes other than carbon, using IsoCor (High-resolution experiments were performed with an Ultimate 3000 high-performance liquid chromatography (HPLC) system  coupled to an LTQ Orbitrap Velos mass spectrometer  equipped with a heated electrospray ionization probe. MS analyses were performed in positive Fourier transform MS (FTMS) mode at a resolution of 60,000 (at 400 g IsoCor . However(iii) NMR measurements. Concentrations in supernatants were measured by 1D 1H NMR at 290°K using a 30° angle pulse, and a presaturation of water signal was applied during a relaxation delay of 8 s. TSP-d4 was used as an internal standard for calibration and quantification.13C-isotopologues MS analysis, which were redried and suspended in 200 μl D2O (0.1% DCl). The positional isotopomer distribution of alanine C-2 and C-3 was extracted from the analysis of 13C-13C couplings in two-dimensional (2D) 1H-13C heteronuclear single quantum coherence (HSQC) experiments  (The measurement of isotopomers and specific enrichments of targeted biomass components were performed using the same samples used for proteinogenic amino acids and eriments . The carF-TOCSY) .1H spectra using TopSpin 3.5 .NMR spectra were collected on an Avance III 800 MHz spectrometer  equipped with a 5-mm z-gradient QPCI cryoprobe. Every acquisition, 1D, 2D, and absolute quantifications were performed on 1D (i) Metabolic flux analysis software. All simulations needed for metabolic flux analysis (MFA) on methanol, mannitol, and arabitol were performed with influx_si software v4.4.4  Metabolic models. We designed the models to cover central carbon metabolism and biomass needs of B. methanolicus. influx_si uses a nonstandard legacy file format (FTBL) to encode the metabolic network and the associated atom-atom transitions. This format centralizes the metabolic network with all biological measurements, i.e., metabolite pool sizes, fluxes, and carbon isotopologue distribution. Consequently, we developed distinct model files for each carbon source. Models share the same nomenclature and the same general topology between them, which is displayed in 2 pool was explicitly modeled within the system to allow for reincorporation of tracer via CO2. Amino acid synthesis pathways were modeled as part of biomass needs along with important precursors. The biomass equation was borrowed from a Bacillus subtilis genome-scale model  were also exploited. No acetate production was observed. Because some isotopologues of Gnt6P (M + 2 and M + 3) could not be quantified, these isotopic measurements could not be included in the influx_si model; hence, the flux through the oxidative PPP could not be accurately resolved. To exploit these partial data and estimate the flux through the oxidative PPP, we applied the ScalaFlux approach  and isotopologue profiles  through the influx_si optimization process. Only the measurement of the COapproach , which a(iii) Quality checks and flux conversion. Experimental data were fitted to our models as described above. For each culture replicate, we performed a Monte Carlo sensitivity analysis (n = 100) on the fit to assess its robustness to small variations around the fitted values. We also performed a chi-squared goodness-of-fit statistical test to ensure that simulated data for each biological replicate were significantly close to experimental data. All tests were significant with a significance level (α) of 0.05 (https://fairdomhub.org/data_files/3269?version=2). For convenience, we provide figures of measured versus simulated data points , mannitol (B), and arabitol (C) conditions. Each data point refers to the proportion of one of the isotopologues from the carbon isotopologue distribution (CID) of a metabolite used to constrain the models, i.e., either metabolites from central metabolism (A) or proteinogenic amino acids (B and C) as described in Materials and Methods. On the Copyright © 2020 Delépine et al.2020Delépine et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the On mannitol and arabitol, the precision of the fluxes through malate dehydrogenase , malic enzyme , and pyruvate carboxylase  was not satisfactory. We thus applied a flux minimization analysis  includedSince both the substrate uptake rates see  and the (i) Strains and culture conditions. In this study, Escherichia coli DH5α (E. coli BL21(DE3) (E. coli strains were routinely cultivated at 37°C and 180 rpm in lysogeny broth (LB) or on LB agar plates supplemented with 100 μg ml−1 ampicillin and 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) when relevant.oli DH5α  was usedL21(DE3) . A summa(ii) Recombinant DNA work. Molecular cloning was performed as previously described  with primers P192, P193, P194, and P195 (escribed  using prand P195 . All clo(iii) Overproduction and purification of AtlD and AtlF. Plasmids for protein production using E. coli BL21(DE3) were constructed on the basis of pET16b  and are presented in atlD or atlF gene was PCR amplified from B. methanolicus MGA3 genomic DNA using the primers P192 and P193 or P194 and P195, respectively  Arabitol phosphate dehydrogenase enzymatic assays. Determination of purified AtlD and AtlF activities in the reductive reaction using Xyl5P or Ribu5P as the substrate were performed as previously described (escribed . The assB. methanolicus MGA3 genome NZ_CP007739.1. https://fairdomhub.org/data_files/3269?version=2.Gene loci mentioned throughout the text are from NCBI annotation of


Apparent digestibility, total digestible nutrient, digestible crude protein, dry matter intake, daily weight gain (DWG), price of DWG, daily profit, and economics of feed efficiency were higher (P < 0.05) for the additives-treated DPLS relative to the control, with T2 enhancing these parameters compared with T3. With exception of ruminal pH, which was reduced, concentrations of ruminal NH3-N and total volatile fatty acids (VFA) increased 4 h post feeding. However, ruminal NH3-N and total VFA were greater (P < 0.05) for the additives-treated DPLS, with T2 producing higher values than T3. Ruminal pH and feed cost/kg LW gain were lower for T2 relative to other treatments. Blood constituents were within the normal ranges for lambs, though slightly altered by treatments. Whereas serum total protein, albumin, and globulin were affected (P < 0.05) in this rank order, T1 < T3 < T2, other serum parameters were not affected. Relative feed cost and relative daily profit were lower and higher respectively for T2 than for T3. It is concluded that additives-treated DPLS is nutritionally superior to untreated DPLS as a roughage source in total mixed rations fed to growing lambs. However, for improved performance of the lambs and economic benefits, EM1-treated DPLS is recommended.Eighteen 4-month-old lambs, with a mean live weight (LW) of 19.47 ± 0.20 kg, were used to evaluate the nutritive value of date palm leaves (DPL) ensiled with different additives in a completely randomized design. Lambs were stratified into three groups of 6 lambs each and fed a control diet comprising 60% concentrate feed mixture (CFM) and 40% DPL silage (T1). In other treatments, the DPL silage (DPLS) of the control treatment was replaced with EM Date fruit production yields several crop residues, including date palm fronds or leaves (DPL), petioles, racemes (without the dates), and pedicels. These by-products are usually used as feed for animals during winter, though they can be used year round , containing a combination of 70 to 80 different types of beneficial microorganisms. The principal organisms are photosynthetic bacteria (phototrophic bacteria), lactic acid bacteria, yeasts, actinomycetes and fermenting fungi  or 40% El-Mofeed additive-treated DPLS (T3). Animals were randomly assigned to one of three experimental treatments and fed ad libitum, twice daily at 08:00 h and 15:00 h, to provide allowance for orts collection. The CFM and DPLS were sampled daily, composited weekly, oven dried at 60 °C for 48 h, and stored pending chemical analyses. The chemical composition of component of the diets is shown in Table The study was carried out at Sakha Experimental Station, Animal Production Research Institute, Agriculture Research Centre, Egypt. Eighteen crossbred ram lambs (½ Finnish Landrace × ½ Rahmani) at 4 months of age (19.47 ± 0.20 kg LW) were used in a completely randomized design. They were weighed, divided into three homogenous groups (6 lambs/group), and housed individually in a 120-day feeding trial. Lambs were fed a control diet (T1) comprising 60% DM concentrate feed mixture (CFM) and 40% DM DPL to meet their nutrient requirements according to NRC . In the Feed intake was measured daily throughout the experimental period, while nutrient digestibility coefficients were determined using six animals for each ration at the end of the experimental period. Faeces were collected daily and oven dried at 60 °C for 48 h. The oven-dried daily faecal samples of each ram were bulked, thoroughly mixed, subsampled, and kept in a refrigerator for chemical analysis. Samples of feed, orts, and faeces were ground to pass a 1-mm screen using a Wiley mill  and analysed for dry matter , ash (method ID 942.05), EE (method ID 956.02), and crude protein  according to the methods of AOAC . Fibre f3-N) concentration was measured according to Conway and O'Mally . Ammonia nitrogen . The following mathematical model was used:ij = μ + Ti + eijYYij is the observation on the experimental unit, μ is the overall mean, Ti is the effect due to treatment, and eij is the experimental error.where P < 0.05.Duncan’s multiple range test was used to separate significant treatment means, and significance was declared if 1 and El-Mofeed increased DPLS CP by 74.3 and 33.3% but decreased NDF, ADF, and ADL by 9.0 and 5.3%, 12.4 and 9.6%, and 31.2 and 23.2%, respectively. The chemical composition of the untreated DPLS is consistent with the earlier reports  improved anaerobic fermentation efficiency and thus nutritive value of DPLS compared to DPLS treated with El-Mofeed .Additive treatments of DPL decreased the DM, OM, fibre fractions, and NFC but increased CP Table . EM1 andP < 0.05) the nutrient digestibility, digestible crude protein (DCP), and total digestible nutrient (TDN), with T2 (EM1-treated DPLS) producing better (P < 0.05) results compared with El-Mofeed-treated DPLS (T3)  nutrient digestibility and nutritive value (TDN and DCP).Additive treatments T2 and T3) increased ( and T3 i3) Table . Moreove3) Table . The imp3) Table . The che3) Table . Both TDy et al.  earlier f et al.  and Mahrf et al.  found thP < 0.05) (Table P < 0.05) ruminal NH3-N values at 0 and 4 h post feeding followed by T2 and T3. The ruminal NH3-N concentrations for all treatments at 0 and 4 h post feeding were greater than the minimal level of 5 g NH3-N/L required for optimum rumen microbes growth and activity  increased the concentration of VFA. Increased VFA for the treatments may be due to improved nutrient digestibility and ruminal fermentation  Table . Sheep fn et al.  reportedP < 0.05)  Table . Increas5) Table . Moreove5) Table  and Ayli5) Table  reported5) Table  who foun5) Table .Table 4BP < 0.05) for lambs fed additives-treated DPLS diets compared with control, but T2 improved these parameters relative to T3. Feed conversion was lowest for T2 followed by T3 and T1  in T1, price of daily gain, daily profit, and economics of feed efficiency increased in T2 and T3 compared to T1, with T2 having greater values than T3 (Table P < 0.05) in T2 and T3, indicating that it was significantly cheaper and more economical to produce 1 kg of LW with additives-treated DPLS than for the control untreated DPLS. This is in agreement with the previous studies where processed cassava peels reduced feed cost/kg LW gain relative to the unprocessed peels in rabbit diets (Olafadehan 1-treated conocarpus at 15 or 30% compared to those fed the control diet. Relative daily profit was 14.2% higher in T2 than in T3, indicating superior nutritive and economic benefit of EM1-treated DPLS relative to El-Mofeed-treated DPLS in the lambs’ diets. Olafadehan et al. (Piliostigma thonningii foliage in partial replacement of concentrate to superior quality of the foliage relative to the locally produced concentrate.Total body gain, daily gain, and feed intake were higher P < 0.0 for lambT1 Table  who attrn et al.  attributn et al.  and Mahrn et al.  who repon et al.  indicaten et al.  attribut1- and El-Mofeed-treated DPLS in lambs’ total mixed rations improves voluntary intake, ruminal fermentation, nutrient digestibility, nutritive value, economic benefit, and growth performance. However, EM1 additive improved the quality of DPLS and performance of lambs compared with El-Mofeed additive.It can be concluded that incorporation of EM


The camels sampled from the central desert of Iran exhibited higher relatedness than Sudanese and lower than Arabian Peninsula dromedaries. Recent Ne of Iran’s camels was estimated to be 89. Predicted Tajima’s D (1.28) suggested a bottleneck or balancing selection in dromedary camels in the central desert of Iran. A general decrease in effective and census population size poses a threat for Iran’s dromedaries. This report is the first SNP calling report on nearly the chromosome level and a first step towards understanding genomic diversity, population structure and demography in Iranian dromedaries.The development of camel husbandry for good production in a desert climate is very important, thus we need to understand the genetic basis of camels and give attention to genomic analysis. We assessed genome-wide diversity, linkage disequilibrium (LD), effective population size (Ne) and relatedness in 96 dromedaries originating from five different regions of the central desert of Iran using genotyping-by-sequencing (GBS). A total of 14,522 Single Nucleotide Polymorphisms (SNPs) with an average minor allele frequency (MAF) of 0.19 passed quality control and filtering steps. The average observed heterozygosity in the population was estimated at 0.25 ± 0.03. The mean of LD at distances shorter than 40 kb was low ( Camels have a unique morphology and physiology as they are capable of providing vital products in a desert climate, even under harsh conditions. Camelini (one- and two-humped camels) and Lamini (New World camels) are two tribes of the Camelidae family . Camelidn = 41), Bahabad (n = 8), Khatam (n = 17), Mehriz (n = 8) and Saghand (n = 22)) ) .The blood samples were gathered with EDTA tubes, and genomic DNA was extracted using the modified salt-out method. In this method, optimization includes utilization of separate buffers instead of buffy coat isolation, chloroform for DNA phase isolation and extraction of purified DNA, and sodium acetate for more concentrated DNA . The extThe sequence reads were mapped to the dromedary reference genome assembly on chromosome level  by usinFST among populations were calculated by using the StAMPP package in R [r2 (Equation (1)) [ABP, abP, AbP, and aBP are the haplotype frequencies and AP, aP, BP, and bP are the allele frequencies, respectively. The LD decay plot was created in popLDecay [Quality control (QC) steps, linkage disequilibrium (LD), genome-wide diversity (observed and expected heterozygosity), as well as admixture analyses were performed using TASSEL V5.0 . Variantage in R . To inveion (1)) :(1)r2=(PopLDecay .Ne) was predicted using the pairwise LD [r2, Ne, and c (Equation (1) [r2 was estimated following Equation (3) [c is the recombination rate, n is the number of individuals sampled; β = 2 when the gametic phase is known and β = 1 if instead the phase is not known. Ne during t generations ago was predicted (Equation (4)):TN is the effective size t generations ago, computed as t = (2ƒ(tc)) − 1 [tc is the recombination rate defined for a specific physical distance between markers and optimally adjusted with the mapping functions mentioned above; α = {1, 2, 2.2} is a correction for the mutation rates [Effective population size (rwise LD . The SNerwise LD  based ontion (1) ). The cotion (3) :(2)E(r2)ct)) − 1 ; ct is ton rates .ijk is the difference between sequence i and j.Tajimas’s D was estimated to investigate demography or signals of selection (Equation (5)) .Tajima_A total of 14,522 SNPs resulted after filtering and were used for final analysis. The largest gap between SNPs  was located on chromosome 11. We note that this is the first SNPs-based report at chromosome-level . Before r2 = 0.089 ± 0.234) was observed at distances shorter than 40 kb, which decreased to 0.019 ± 0.034 in 40–60 kb. The LD decay trend is shown in r2 > 0.20 related to 0–40 kb intervals (Linkage disequilibrium (LD) was estimated for each pairwise combination of SNPs . Average LD   .jk = −0.011 ± 0.113).Because of a limited number of bull camels and half-sib calves within herds, there were node clusters visible, although the median of relatedness among camels was very low  was estimated as 89 to 487, respectively . The treCamelus bactrianus breeds in East Asia by using the GBS technique [S across different studies can be due to different techniques or the heterozygosity levels in the other camel groups.Guo et al., (2020) identified 2,433,732 variants in 366 camels, including the seven domestic echnique . Bahbahaechnique . Al Abrijk = − 0.007 ± 0.018) and lower ones than individuals from the Arabian Peninsula (median Ajk = 0.347 ± 0.093) [Heterozygosity of the Iranian central desert dromedaries was predicted as low (0.25 ± 0.30). Arabian Peninsula and Sudanese dromedaries’ ectively . The cam± 0.093) . Predict± 0.093)  and consr2 (LD) was 0.155, 0.156, and 0.128 across all chromosomes for three sheep breeds [r2 = 0.6 in 5 kb and up to 20 kb in Thoroughbred horses. The average r2 > 0.2 at 1.0–1.5 Mb intervals was predicted in two pig populations [Until recently, LD patterns of many livestock species have been reported. For example, the amounts of LD at 0–30 kb distances in Angus, Charolais, and C beef cattle were 0.29, 0.22, and 0.21, respectively . The meap breeds . Corbin p breeds  reportedulations . The meaPrincipal component analysis suggested that Sudanese camel populations are almost homogeneous . On the The trend of Ne shows a bottleneck around 2000–6000 years ago in Iranian central desert dromedary camels. This is consistent with a decline in Ne in Old World camels around 4000–5000 years ago and might be related to the domestication scenarios of dromedary and Bactrian camels [Commercial SNP arrays have been developed for many livestock animals but no SNPchip is available for dromedaries. GBS based on whole-genome sequencing has solved the problem of ascertainment bias  as the e


However, in both cases, absolute ratios varied greatly among different BAs. The average hepatic reuptake of BAs agonistic for FXR/TGR5 was similar to non-agonists. However, as the sum of non-agonist BAs in vena portae was 2–3-fold higher than the sum agonist (p < 0.05), the peripheral BA pool was composed mostly of non-agonist BAs. We conclude that hepatic BA reuptake varies substantially by type and does not favor FXR/TGR5 BAs agonists.Farnesoid X receptor (FXR) and Takeda G-protein coupled receptor 5 (TGR5) are the two known bile acid (BA) sensitive receptors and are expressed in the intestine and liver as well as in extra-enterohepatic tissues. The physiological effects of extra-enterohepatic FXR/TRG5 remain unclear. Further, the extent BAs escape liver reabsorption and how they interact with extra-enterohepatic FXR/TGR5 is understudied. We investigated if hepatic BA reuptake differed between BAs agonistic for FXR and TGR5 compared to non-agonists in the rat. Blood was collected from the portal vein and inferior caval vein from anesthetized rats before and 5, 20, 30, and 40 min post stimulation with sulfated cholecystokinin-8. Plasma concentrations of 20 different BAs were assessed by liquid chromatography coupled to mass spectrometry. Total portal vein BA AUC was 3–4 times greater than in the vena cava inferior (2.7 ± 0.6 vs. 0.7 ± 0.2 mM x min,  Bile acids (BAs) are amphipathic, steroidal fat emulsifiers secreted from the liver and released from the gallbladder into the intestinal lumen to facilitate lipid absorption . Bile acFXR and TGR5 are also found in non-enterohepatic tissues. TGR5 expression is high in the brain, spinal cord, smooth muscle tissue ,15,16,17p < 0.001, p = 0.13) to increase around 40 min post CCK stimulation in vena portae, whereas the concentrations in vena cava inferior did not change (p = 0.47). Total BA AUC values in vena portae were 4 times higher than total BA AUC in vena cava inferior  and 19.1 ± 6.56 µM in vena cava inferior . The concentrations of conjugated total BAs at 0 min were 22.5 ± 9.32 µM in vena portae and 1.43 ± 0.57 in vena cava inferior, but neither was significantly different at 40 min (p > 0.90). Total AUC for conjugated BAs was approximately 10-fold higher in vena portae than in vena cava inferior  but did not affect TCA, CDCA, or TCDCA concentrations (p = 0.13–0.99), which in case of CDCA and TCDCA remained low throughout the experiment (p = 0.71–0.99) at time 40 min versus 0 min, except for CDCA which was mildly lower  (Cholic acid (CA) and tauro (T) cholic acid concentrations were threefold higher in vena portae compared to chenodeoxy cholic acid (CDCA) and TCDCA. CCK increased the concentration of CA in vena portae by approximately 50% (periment . The fra < 0.01) . Concentp < 0.05, compared to respective basal levels), (whereas TDCA concentrations remained near the quantification limit (p < 0.05) and tended to be lower in vena cava inferior (3–5-fold) for TDCA (p = 0.11), UDCA (p = 0.08), and TUDCA (p = 0.11) (p = 0.23–0.99) , and TUDCA were generally low (<2.5 µM) in vena portae at 0 min, expect for UDCA which was 7.02 ± 3.28 µM . In venaon limit . AUCs we = 0.11) . Fractio23–0.99) .p < 0.01) , but varied among BAs (Murine CA (MCA) alpha and MCA beta concentration in vena cava inferior was at time 0 min 1.95 ± 0.87 and 2.65 ± 0.94 µM, respectively. Taurine-conjugated forms of these were in vena cava inferior at same time point 2.01 ± 0.82 µM and 5.29 ± 2.60 µM, respectively. MCA alpha and TMCA beta concentrations did not change over the time course of the study, but MCA beta and TMCA alpha were 2–3-fold higher at time point 40 min ( < 0.01) . Correspmong BAs . Glycinep < 0.01. n = 8, p > 0.05, n = 8).It is well established that the liver retains a large proportion of BAs from the portal vein . We asseCompared to the gastrointestinal tract and liver, extra-enterohepatic FXR and TGR5 regulation has been studied to a limited extent with focus mainly on distribution of tissue expression, not on ligand concentration. TGR5 expression is high in the brain, spinal cord, smooth muscle tissue, and adipocytes ,15,16,1750 values of 0.4–10 µM in the following hierarchy of potencies: lithocholic acid (LCA) > DCA > CDCA, independent of conjugation, whereas neither CA nor UDCA (both conjugated and unconjugated isoforms) activated the receptor at concentrations up to 10 µM [50 values in the range of 10–100 µM [+ taurocholate cotransporting protein) and non-conjugated BAs (organic anion transporting protein family) into the hepatocytes [p-value = 0.200) nor their corresponding conjugated forms . However, since our study was not designed and powered to address this aspect, future work with expanded sample sizes is needed to appropriately address this possible explanation. However, as activation of FXR/TGR5 differs among BAs and is unaffected by conjugation, total concentrations of conjugated and unconjugated BAs provides little information with regard to potential extra-enterohepatic FXR and TGR5 activation. We therefore quantified the concentrations of unconjugated and conjugated CA, CDCA, DCA, UDCA, MCA alpha, and MCA. In the portal plasma, CA and TCA predominated and composed approximately 40% of the total BA concentration, whereas CDCA and DCA and their taurine-conjugated counterparts each accounted for only 2%–7%. These ratios are in agreement with another study in rat [We and others  have shoto 10 µM ,23. Huma0–100 µM ,25,26. I0–100 µM ,25,26. I0–100 µM ,29,30. H0–100 µM ,32,33. I0–100 µM ,31,32. Watocytes . Furthery in rat . In humay in rat . This spy in rat . Indeed,50 values for these agonists on FXR/TRG5 are 1–10 µM, suggesting BAs could not activate either receptor in this study.Of note, concentrations of different BA specimens in the periphery varied greatly and differed by almost 50-fold between the most (UDCA) and the least abundant (TDCA) BA. Moreover, the ratio difference between portal and peripheral concentrations for the quantified BAs varied widely, ranging from 0.07 µM (TCA) to 0.46 µM . However, when grouped into agonist for FXR/TGR5  and poor-agonists/non-agonists , average ratios between concentrations in vena portae and vena cava inferior were similar between groups, suggesting hepatic reuptake of BAs is regulated by hepatic BA delivery rather than ability to activate FXR/TGR5. As non-agonists accounted for more than 2/3 of the total BA pool in the portal vein, peripheral concentrations of non-agonist BAs dominated and the average concentration of agonist BAs amounted to around 1 µM at baseline and peaked at approximately 1.3 µM. As a reference, ECA general observation in our study is that concentrations of BA as well as the transhepatic BA ratios differed significantly between rats. Although we were unable to narrow down the underlying reason for this pronounced animal-to-animal variation, other studies also showed great individual variation in monkeys and humans ,33,37,38A weakness of our study is that the injected CCK only resulted in moderately increased BA secretion, and it remains to be investigated whether stronger stimuli  would have resulted in different or similar hepatic reuptake of the investigated BAs and extra-enterohepatic plasma concentrations that were more within respective EC50s for FXR/TGR5 activation.Animal studies were conducted with permission from the Danish Animal Experiments Inspectorate (2013-15-2934-00833) and approved by the local ethical committee . Animal studies were conducted in accordance with the EU Directive 2010/63/EU and guidelines of Danish legislation governing animal experimentation (1987) and the NIH (publication number 85-23) and were designed to minimize pain or discomfort to the animals.g, 15 min, 4 °C) within half an hour of collection. Supernatant was transferred to fresh centrifuge tubes and immediately stored at −20 °C until analysis as described below. After collection of the zero min sample, sulfated CCK-8  was injected at a dose of 20 nmol/kg body weight (~260–550 µL depending of body weight). CCK-8, diluted in isotonic saline supplemented with 10% (w/v) human serum albumin  was injected intravenously through the needle inserted in the inferior caval vein to stimulate bile acid release into the intestine. In total ~6 mL blood was withdrawn from each animal, corresponding to ~18% of the theoretical total blood volume of 33.2 mL (estimated by: blood volume (mL) = 0.06 × body weight (g) + 0.77, as described previously [Male Wistar rats were obtained from Janvier  and housed in pairs under standard conditions with ad libitum access to chow and water and a 12:12 h light and dark cycle. Rats were allowed to acclimatize for at least one week before the study. Studies were carried out between 10 am and 1 pm. Rats were allowed free access to chow and water in the hours leading up to the experiment, but as the dark period ended at 6 am, the rats can in our experience be considered to be semi-fasted at study initiation. Rats (mean weight ± standard error of the mean (SEM) = 492 ± 34 g) were anesthetized with a subcutaneous injection of Hypnorm/midazolam (0.079 mg fentanyl citrate + 2.5 mg fluanisone + 1.25 mg midazolam/mL: 0.3 mL/100 g body weight). The abdominal cavity was opened and a needle inserted into the inferior caval vein (vena cava inferior) concurrent with insertion of a non-obstructing plastic catheter into the portal vein. Blood (600 µL/time point) was withdrawn simultaneously from the two veins into EDTA coated syringes . The samples were immediately transferred into 1.5 mL EDTA-coated tubes . To prevent clot formation and replace fluid lost from blood collection, the needle and catheter were both flushed with 400 µL isotonic saline (room temperature) immediately after sample collections. Samples were drawn at the time points −5, 0, 20, and 40 min and transferred onto ice and centrifuged  and dried via speed vacuum. Quality controls were composed in triplicate at three different concentrations  in the presence of liquid chromatograph mass spectrometry grade water or human plasma. The quality controls as well as a standard curve were processed in the same manner as samples. A blank was composed in the presence and absence of human plasma. Quality controls were interspersed at even intervals throughout the queue. Samples were analyzed in a random order to control for systematic error. Metabolites were separated, detected, and quantified as described [10 µL of plasma was extracted with 80 µL of 87.5% methanol containing 12 pmol of internal standard. Extracts were clarified by centrifugation .Statistical methods of this study were reviewed by Prof. Jens Juul Holst. Data are presented as mean concentrations ± SEM. Values below the limit of quantification were imputed using the lowest standard concentration. BAs whose concentrations were below the limit of quantification in >50% of samples were excluded from analyses. These BAs were glycochenodeoxycholic acid, glycodeoxycholic acid, glycoursodeoxycholic acid, lithocholic acid, muricholic acid gamma, taurolithocholic acid, and tauromuricholic acid gamma Graphs were constructed in GraphPad Prism 6  and collected and prepared in Adobe Illustrator . Test for statistical significance was performed in GraphPad Prism. Significance was assessed at individual time points against own baseline  by two-way ANOVA for repeated measurements followed by Tukey’s multiple comparison test. Significance amongst relative level of BAs was tested by two-way ANOVA followed by Tukey’s multiple comparison test, whereas significance between respective AUC’s in v. portae and v. cava inferior was tested by paired We find that total BA concentrations in the vena cava inferior were 4–5 times lower than the total concentration in vena portae. Conjugated BA hepatic reuptake was on average 3–4 times more efficient than the reuptake of unconjugated BAs, but reuptake varied greatly between individual BAs. Collectively, BAs that are agonist for FXR/TGR5 were reabsorbed by the liver to the same extent as non-agonists. The concentration of non-agonists was 2–3-fold higher in vena portae than agonists, suggesting regulation of extra-enterohepatic FXR/TGR5 activity is mediated by hepatic delivery of BAs rather than by specific/differential hepatic reuptake of agonistic BAs towards FXR/TGR5.


Osteogenic cells were seeded on bio-inert glass slides with or without the presence of Hst1 in dose-dependent or time-course assays. 1 scrambled and 6 truncated Hst1 variants were also evaluated. Cell spreading was analyzed using a well-established point-counting method. Fluorescent microscopy was adopted to examine the cellular uptake of fluorescently labeled Hst1 (F-Hst1) and also the cell spreading on sandblasted and acid etched titanium surfaces. Signaling inhibitors, such as U0126, SB203580, and pertussis toxin (PTx) were used to identify the potential role of extracellular-signal-regulated kinase, p38 and G protein-coupled receptor pathways, respectively. After 60 min incubation, Hst1 significantly promoted the spreading of osteogenic cells with an optimal concentration of 10 μM, while truncated and scrambled Hst1 did not. F-Hst1 was taken up and localized in the vicinity of the nuclei. U0126 and SB2030580, but not PTx, inhibited the effect of Hst1. 10 μM Hst1 significantly promoted the spreading of osteogenic cells on both bio-inert substrates and titanium SLA surfaces, which involved ERK and p38 signaling. Human salivary histatin-1 might be a promising peptide to enhance bone healing and implant osteointegration in clinic.Promoting cell spreading is crucial to enhance bone healing and implant osteointegration. In this study, we investigated the stimulatory effect of human salivary histatin-1 (Hst-1) on the spreading of osteogenic cells  Large-volume bone defects (LVBD) can result from various diseases, such as congenital malformation, trauma, infection, inflammation and cancer . Due to in vitro ], was cultured in alpha-Minimum Essential Medium (α-MEM) . All media were supplemented with 10% fetal bovine serum , 10 units/mL penicillin and 10 μg/mL streptomycin . Cells were cultured at 37°C in a moist atmosphere at 5% COAll peptides  were manN = 6 wells per group. Cells were serum deprived for 24 h, detached using 0.05% trypsin (Gibco), and suspended in culture medium containing 2% FBS to inactivate the trypsin and centrifuged at 200 g for 5 min at room temperature. Next, cells were re-suspended in their prescribed medium without serum and counted using a hemocytometer. Cells were seeded on glass coverslips  in 12-wells suspension cell culture plates  at a density of 6 × 104 cells/well, treated with 0–20 μM of the Hst1 or 10 μM truncated Hst1 or scrambled Hst1. Cells were imaged every 20 min during a 3 h period using an EVOS-FL microscope (Thermo Fisher Scientific) equipped with a LPlanFL PH2 20x using the phase contrast setting or the Cy5 light cube . For each well, 6 images were taken randomly with about 90–120 cells per image. Thereafter, we randomly selected 3 images for further analysis by measuring the surface area of cells’ filopodia and lamellipodia  after which serum-free medium was added. Two micrometer F-Hst1 was added and after a 1 h period, the cells were washed four times with DPBS containing 0.9 mM Ca2+ and 0.5 mM Mg2+. As a negative control, incubations without F-Hst1 were included. The cells were studied by the EVOS-FL microscope with a 20x objective with a phase contrast setting and a “Cy-5 light cube” with a 628/40 excitation filter and a 692/40 nm emission filter. Digital photographs were recorded by a computer integrated in the microscope.To analyze the uptake and localization of fluorescently labeled Hst1 (F-Hst1) by osteogenic cells, cells from a semi-confluent cell culture were transferred into a 48-wells plate at a density of about 3.5 × 10TM live cell stain  and PKH67GL  respectively, following manufacturer’s instructions.To more precisely observe the uptake and localization of F-Hst1, the cells were further studied using a LEICA TCS SP8 confocal laser scanning microscopy (CLSM) system as previously described . Before in vitro . Finally, the discs were cleaned ultrasonically in dH2O for 15 min three times and dried overnight in oven. These discs were thereafter autoclaved for sterilization and kept in sterile condition. Cell spreading on titanium SLA surface was performed as described in the section of cell spreading assay. 1.5 h after seeding, cells on titanium discs were fixed, dehydrated and stained with DAPI and FITC-Phalloidin to stain nuclei and cell skeleton respectively. Fluorescent micrographs were randomly taken using a fluorescent microscope (Olympus BX61) with Excitation/Emission wavelengths (nm) of 358/461 and of 496/516, respectively. On the micrographs, spreading surface of each cell was estimated using the abovementioned point-counting method. More than 30 cells per group were calculated.Commercially titanium  foil was cut into round discs (5 mm in diameter). To make SLA surface, the titanium discs were first sandblasted with large corundum grits. Thereafter, the discs were cleaned ultrasonically in alcohol and dHt-test and one- way ANOVA with Bonferroni’s post hoc test for multiple comparisons. For the data from different groups at different time points in post hoc test for multiple comparisons. Results were reported as mean ± SD. A value of P < 0.05 was considered as statistical significance. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.All experiments were carried out at least three times and in quadruple. Data were plotted using Graphpad Prism , analyzed using Student’s We first evaluated the dose-dependent effect of Hst1 on cell spreading. The presence of Hst1 promoted the spreading of osteogenic cells with larger lamellipodia and more filopodia in comparison with the control (no Hst1) . Comparein vitro wound-healing properties of Hst1 (amino acids 12–38) appeared comparable to those of the parent peptide Hst1 , p38 MAPK inhibitor (SB203580) and PTx (an inhibitor of GPCR) on the Hst1-induced cell spreading. U0126 (10 μM) or SB203580 (10 μM) completely abolished the stimulating effects of Hst1 on the spreading of osteogenic cells . In cont2) was significantly higher than that without Hst1 (537.38 ± 108.19 μm2)  .The adhesion, spreading and migration of osteogenic cells on medical devices are indispensable for effective bone formation of LVBD and osteointegration of implants . In thisIt is well established that cell-substrate contact area can determine cell activities, such as proliferation, quiescence or apoptosis . When plHst1 has already been shown to be efficacious in promoting the adhesion, spreading and migration of various epithelial cells from skin , mucosalin vitro 60 min post incubation. The time-dependent course of Hst1’s effect was consistent with the previous findings on epithelial cells that the surface of fibroblasts increased remarkably after 60 min treated with Hst1 when compared to the control   . However β or γ) . Further β or γ) . Such a  β or γ) . Furtherin vitro osteogenic differentiation and in vivo osteogenesis, thereby contributing to osteoinductivity—another important property to heal LVBD and facilitate osteointegration. Hitherto, this issue is rarely reported. In our recent study, we show that Hst1 alone does not promote ALP activity . Therefore, although ERK and p38 signaling are involved in Hst1’s effects on cell adhesion and spreading, they don’t directly lead to osteogenic differentiation at least in MC3T3-E1 pre-osteoblasts. All these data suggest that Hst1 does not enhance osteogenic differentiation, thus bearing no osteoinductivity. On the other hand, in our recent in vivo study, Hst1 significantly promotes angiogenesis and osteogenesis under the stimulation of BMP2 in an ectopic bone induction model (On the other hand, ERK1/2 and p38 are proved to be involved in osteogenic differentiation . Under tactivity . Additioon model . Such anon model  that favon model . HoweverAll datasets presented in this study are included in the article/GW, HL, and EV: conceptualization. WS, GW, and DM: investigation. GW and JB: resources. WS and GW: formal analysis. KN and FB: data curation. WS, FB, and GW: writing—original draft preparation. PS, WS, DM, FB, JB, KN, HL, GW, and EV: editing. WS, GW, PS, HL, and EV: funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To investigate the clinical and genetic characteristics of patients with glycogen storage disease type Ib (GSD Ib).SLC37A4 gene were detected, of which c. 572C > T was the common variant and c. 680G > A was a novel variant. The 3 cases of GSD Ib were mainly affected by liver enlargement, growth retardation, etc., and all had a history of repeated infections. At the onset, patients mainly manifested as mildly elevated alanine-aminotransferase (ALT), accompanied by decreased absolute neutrophil count (ANC), hypertriglyceridemia, and metabolic disorders . After long-term treatment by oral uncooked cornstarch, the abnormal liver enzymes gradually returned to normal, and metabolic abnormalities were basically controlled most of the time. With increasing age, ANC of 2 patients decreased progressively, whereas the times of infections was reduced.This report retrospectively analyzed the clinical data of 3 patients with GSD Ib admitted into our hospital, and summarized their onset characteristics, clinical manifestations, related examinations and treatment as well as mutational spectrum. After gene sequencing, the diagnosis of GSD Ib was confirmed in all 3 patients. Five variants of SLC37A4 variant. The possibility of GSD type Ib should be kept on alert when a patient suffers recurrent infections, accompanied by hepatomegaly, elevated liver enzymes, hypoglycemia, dyslipidemia, and metabolic disorders.We reported 3 cases with GSD Ib and a novel  G6PC gene (OMIM #613742) or SLC37A4 gene (OMIM # 602671), and the incidence is about 1:100,000 [G6PC gene variant causes the deficiency of glucose-6-phosphatase alpha (G6Pase-α) activity, which leads to GSD type Ia (OMIM#232200), accounting for about 80% of GSDI patients, whereas the SLC37A4 gene variant causes the deficiency of glucose-6-phosphate transporter protein (G6PT), which underlies GSD type Ib (OMIM# 232220), accounting for about 20% of GSDI cases [Glycogen storage disease type I (GSDI) is a group of autosomal recessive inherited metabolic disorders with varying clinical severity caused by variants in the e OMIM #63742 or Se OMIM #63742 or SSLC37A4 gene is highly expressed in hematopoietic progenitor cells, its defect has a significant effect on myeloid progenitor cells [Both type Ia and Ib are characterized by hepatomegaly and metabolic abnormalities such as hypoglycemia, hyperlipemia, lactic acidosis, and hyperuricemia. The or cells , 5.SLC37A4 variant, one of which had novel variant.We report 3 additional cases of GSD Ib with The clinical features of the 3 patients were summarized in Tables This female patient was admitted to hospital because of “abdominal distension for over 1 year” at the age of 15 months. At admission, her height SDS was − 2.26 and weight was 9 kg (10th–25th centile). There were abnormalities in absolute neutrophil count (ANC 1.07 × 10^9/L), liver function , blood lipid (TG 8.84 mmol/L), fasting glucose (3.7 mmol/L), lactic acid (8.78 mmol/L), and blood gases (BE − 8.5). The liver ultrasound suggested hepatomegaly. She underwent liver biopsy and showed glycogen storage as confirmed by periodic acid-Schiff staining (PAS). She was also tested for glucose response to epinephrine stimulation after overnight fasting and the result was positive. Combined with the above results, she was clinically diagnosed as GSD. She was given oral uncooked cornstarch four daily doses of 1.0–2.0 g/kg (3am–9am–3pm–9pm). According to current guidelines, follow-up is recommended every 3–6 months (adjusted by disease changes and their ages). However, the patient did not take oral uncooked cornstarch regularly as prescribed. At the age of 12 years, she was finally diagnosed with GSD type 1b through genetic testing. She had an infection frequency of about 0.36 times/year before the diagnosis of genetic classification and was hospitalized for “repeated vomiting and diarrhea” 3 times in the past year, and had secondary “inflammatory bowel disease (IBD) and pancreatitis” during the last hospitalization . She received G-CSF treatment irregularly. At the last visit, the liver enzymes gradually returned to normal, triglycerides and lactic acid were decreased than before, but did not fall to normal levels. Her pyruvic acid and uric acid increased, and fasting hypoglycemia and metabolic acidosis have not been significantly improved.At the age of 3 years and a half, this male patient was admitted to hospital because of “hepatomegaly for over 3 years, upper respiratory tract infection for 1 week”. At admission, his height SDS was − 2.97 and weight was 13 kg (10th–25th centile). There were abnormalities in ANC 0.95 × 10^9 /L, liver function , TG 5.53 mmol/L, fasting glucose (3.8 mmol/L), lactic acid (10.75 mmol/L), and blood gases (BE − 10.9). The liver ultrasound also suggested hepatomegaly. He also underwent liver biopsy and showed glycogen storage. He was tested for glucose response to epinephrine stimulation after overnight fasting and the result was positive, too. According to all the results, he was clinically diagnosed as GSD. He took oral uncooked cornstarch regularly. At 13 years old, he was diagnosed with GSD type 1b through genetic testing. At the last visit, the liver enzymes gradually returned to normal, triglycerides, lactic acid, pyruvic acid, and uric acid were decreased than before, but did not fall to normal levels. His fasting hypoglycemia and metabolic acidosis have been significantly improved. He had a history of oral ulcers. As the treatment time extended, the number of infections was significantly reduced, only presented with minor infections, such as upper respiratory tract infection or oral ulcers that did not require hospitalization.This female patient was admitted to hospital because of “short stature, hepatomegaly for 6 months” at the age of 3 years old. At admission, her height SDS was − 1.48 and weight was 15 kg (50th–75th centile). There were abnormalities in ANC (0.82 × 10^9 /L), TG (3.92 mmol/L), fasting glucose (3.48 mmol/L), lactic acid (9.61 mmol/L), and uric acid (519 μmol/L). The liver ultrasound suggested hepatomegaly. She also underwent liver biopsy and was tested for glucose response to epinephrine stimulation after overnight fasting and the results were positive. Therefore, she was clinically diagnosed as GSD. She was given oral uncooked cornstarch regularly. At the age of 10 years and a half, she was finally diagnosed with GSD type 1b through genetic testing. At the last visit, her triglycerides, lactic acid, pyruvic acid and uric acid were decreased than before. Her fasting hypoglycemia and metabolic acidosis have been significantly improved. She had a history of upper respiratory tract infections, about once a year.GYS1, GYS2, G6PC, SLC37A4, GAA, AGL, GBE1, PYGM, PYGL, PFKM, PHKA2, PHKB, PHKG2, PHKA1, PGAM2, LDHA, ALDOA, ENO3, PGM1, GYG1, PRKAG2) was used for GSDs. We fragmented the genomic DNA which extracted from the sample, and the DNA probes were designed to tile along the exon regions and exon–intron boundaries of the target genes. After enrichment of DNA fragments, Illumina HiSeq X ten sequencer was used for high-throughput sequencing of the captured exon region. Sanger sequencing was finally used to verify co-segregation in the family. Suspected candidate variants were screened by comprehensively considering the genetic pattern and the clinical characteristics of the disease. The pathogenicity of variants was predicted according to the 2015-ACMG Standards and Guidelines.After obtaining the written informed consents, gene sequencing (Beijing MyGenostics Inc.) was performed on the probands and their parents. Our genetic testing strategy is a GSD panel based on target gene capture technology , 20 GSD SLC37A4 gene were detected in 3 patients  Type Ib is a group of inherited metabolic disorders caused by variants in the d Serbia .SLC37A4 gene is located on chromosome 11q23, consists of 9 exons, spans approximately 5.3 kb of genomic DNA, and is expressed ubiquitously in liver, kidney, intestine, blood and skeletal muscle [SLC37A4 gene encodes G6PT, G6PT transports G6P from the cytoplasm to the lumen of the endoplasmic reticulum and delivers it to the catalytic site of G6Pase-α or G6Pase-β. G6Pase-α and G6Pase-β are G6P hydrolases in the endoplasmic reticulum membrane, which in turn hydrolyze G6P to glucose and inorganic phosphate [SLC37A4 gene can cause G6PT deficiency or dysfunction, failing to complete the transport of G6P and resulting in disturbed glucose homeostasis, and then leading to hyperlipidemia, lactic acidosis, hyperuricemia, and other metabolic abnormalities. Whereas G6Pase-β couples functionally with G6PT to maintain neutrophil function and homeostasis. Consequently, G6PT is essential to maintain both interprandial glucose homeostasis and myeloid cell energy homeostasis [The human hosphate . Among tSLC37A4 gene that have been identified, including missense variants, nonsense variants, frameshift variants, splice site variants, and deletion variants, etc. There are ethnic variability in variant types and proportions [SLC37A4 gene were detected in 3 patients. Among them, c.680G > A is a novel variant, the c.572C > T (p.Pro191Leu) variant is the most common and only reported in the Chinese population [So far, there are 115 pathogenic variants in the portions , 12. Preportions –16. In opulation –19. We sSLC37A4 gene variants is both a metabolic and an immune disorder [The typical clinical manifestations of GSD Ib are similar to those of type Ia, including impaired glucose homeostasis such as liver enlargement and growth retardation. By contrast, neutropenia and neutrophil dysfunction are major clinical phenotypes of patients with GSD Ib. GSD Ib patients are often accompanied by neutropenia and are prone to frequent infectious diseases, such as recurrent upper respiratory tract infections, oral ulcers, enterocolitis and inflammatory bowel disease (IBD). There are also reports of GSD Ib combined with Crohn's disease . The exadisorder .A decreased number of neutrophils in peripheral blood is an important feature that distinguishes GSD Ib from GSD Ia. It is worth mentioning that not all patients diagnosed with GSD Ib based on metabolic phenotypes and genetic testing develop neutropenia, which may be related to the residual transport activity of G6PT . AccordiThe current treatment of GSD Ib is mainly symptomatic. As a serious metabolic and immune multisystem disorder, if not actively treated, it may cause the patient to be fatal in adolescence. Clinically, diet therapy  can maintain the patient's glucose stability and reduce the early symptoms of the disease , 21. ThoThe 3 patients in our study all had typical clinical manifestations such as elevated liver enzymes, fasting hypoglycemia, hyperlipidemia, hyperlactacidemia, lactic acidosis, and decreased neutrophil count, accompanied by hepatomegaly, growth retardation, and repeated infections. With increasing age, ANC of the 3 patients decreased progressively. Among them, the condition of patient 1 was poorly controlled, G-CSF was used irregularly, and the effect was not good, with secondary IBD and frequent hospitalizations due to infection. The other 2 patients were effectively controlled with age, and the number of infections was significantly decreased. Therefore, the clinical manifestations of patients with GSD Ib have certain heterogeneity.SLC37A4 variant. When recurrent upper respiratory tract infections or digestive tract symptoms are accompanied by hypoglycemia, dyslipidemia, metabolic disorders, elevated liver enzymes and/or neutropenia clinically, the possibility of GSD Ib should be vigilant.In summary, GSD Ib patients have various gene variant types and different clinical symptoms. We reported 3 cases with GSD Ib and a novel


NFKBIE) and 24% (SF3B1). Mutations beyond Sanger resolution (<12% VAF) were observed in all genes, with KRAS mutations principally composed of these low VAF variants. Firstly, employing orthogonal approaches to confirm <12% VAF TP53 mutations, we assessed the clinical impact of TP53 clonal architecture. Whilst ≥ 12% VAF TP53mut cases were associated with reduced PFS and OS, we could not demonstrate a difference between <12% VAF TP53 mutations and either wild type or ≥12% VAF TP53mut cases. Secondly, we identified biallelic BIRC3 lesions (mutation and deletion) as an independent marker of inferior PFS and OS. Finally, we observed that mutated MAPK-ERK genes were independent markers of poor OS in multivariate survival analysis. In conclusion, our study supports using targeted resequencing of expanded gene panels to elucidate the prognostic impact of gene mutations.Despite advances in chronic lymphocytic leukaemia (CLL) treatment, globally chemotherapy remains a central treatment modality, with chemotherapy trials representing an invaluable resource to explore disease-related/genetic features contributing to long-term outcomes. In 499 LRF CLL4 cases, a trial with >12 years follow-up, we employed targeted resequencing of 22 genes, identifying 623 mutations. After background mutation rate correction, 11/22 genes were recurrently mutated at frequencies between 3.6% ( TP53 ), 35 medium- (P < 0.01) and 76 low-confidence associations (P < 0.05). Significant associations between mutations were found in only 10/171 possible associations, such as NOTCH1 + 3′UTR with BIRC3 (P = 0.02) and FBXW7 (P = 0.01), as well as BRAF with TP53 (P = 0.03)  Fig. .n = 288), including <12% VAFs (n = 98), and showed an agreement bias of only 5% , those at larger subclonal or clonal levels , and those concomitant with deletion events  , while ATM, MYD88, NOTCH1, SF3B1, TP53 and XPO1 mutations were found to be contain a significant majority of ≥12% VAF mutations. KRAS mutations were more likely to be composed of low VAF variants, with a mean VAF of 10.7%   or negative (<12% VAF) by accounting for the impact of tumour purity on VAF. Initially, we studied 233 patients with tumour purity derived from CD5/CD19 flow cytometry. Raw VAFs were compared with purity-adjusted VAFs across all variants  Fig. . SAMHD1 5]) Fig. .Fig. 2CLTP53 (with/without del(17p), termed ‘TP53ab’) and EGR2 were associated with reduced PFS , EGR2, RPS15, NFKBIE, BRAF, KRAS and NRAS were associated with reduced OS  cases, having no significant impact on OS in this subgroup of patients  were confirmed using orthogonal approaches (Table TP53 mutated cases were enriched for BRAF and FBXW7 mutations (Table TP53 mutations could be further subdivided into those with < 12% VAF (n = 16) or ≥12% VAF (n = 43), with no difference in the site or type of TP53 mutation between subgroups , isolated TP53 mutations (n = 27) or both (n = 23). Five TP53 mutated cases lacked FISH data. cohorts . We obsents Fig. ; all of es Table . These <ns Table . TP53 muups Fig. . After iTP53mut cases. Both <12% VAF and ≥12% VAF TP53mut groups had increased mutation/CNA frequency in comparison to TP53wt cases (both P < 0.001)  Fig. . To furtns Table  and S14.TP53mut subgroups in pairwise KM analysis. ≥12% VAF TP53mut were associated with reduced PFS and OS compared with cases with wild-type TP53 . In contrast, we could not demonstrate a significant difference between the <12% VAF TP53mut cases and either the wild type or ≥12% VAF TP53mut patients for PFS or OS   Fig. . These o96) Fig. , confirmATM nor BIRC3 mutations, regardless of their VAF , using shallow WGS  were then categorised into five distinct subgroups: sole 11q deletion (n = 71), biallelic ATM abnormalities (abs) (n = 12), biallelic BIRC3 abs (n = 9), sole ATM mutations (n = 24) and sole BIRC3 mutations (n = 19).Although neither AF Figs.  and S17,AF Figs.  and S13,ly shown  , as well as biallelic ATM  and biallelic BIRC3 , were associated with a significantly reduced survival.After omitting ten cases with co-existing TP53ab , we condATM vs. 11q = 1 year vs. 1.4 years, P = 0.336; biallelic BIRC3: 1 year vs. 1.4 years, P = 0.178); however cases with biallelic BIRC3 abs had a significantly reduced OS, whilst cases with biallelic ATM abs did not significantly differ in median survival times compared with sole 11q deleted cases . This suggests that biallelic loss of BIRC3 represents the subgroup of 11q deleted CLL with the worst outcome following initial treatment with chemotherapy.The outcome of cases with biallelic abs was then compared with those with del(11q) only. There were no significant differences in PFS , KRAS (34/29) and NRAS (11/10), were principally composed of specific hotspot variants   only harboured a mutation in one of these genes , KRAS , and NRAS  , and were negatively associated with long-term survival  , and significantly less likely to harbour del(13q) as a sole aberration  Fig. , and thenes Fig. . Interesnts Fig. . In univ01) Fig. . StratifVAF Fig. . Taken t03) Fig. , with onATM and BIRC3 as they emerged from our stratified 11q deletion analysis, and short telomeres based on our previous paper on the topic [P value < 0.05. For PFS, the final model was constructed from 225 patients and 210 events (274 were excluded due to missing data) and showed that TP53ab , biallelic BIRC3 , short telomeres , sole 11q deletion , and increased prolymphocytes  were independent markers of PFS. For OS, the final model was constructed from 391 patients and 323 events (108 observations were excluded due to missing data). TP53ab , biallelic BIRC3 , mutations in EGR2 , MAPK-ERK genes , SF3B1 , as well as IGHV-U genes  and Binet stage B&C , were all observed as independent markers of OS. This data extends on our univariate survival analysis, showing that cases with biallelic BIRC3 deletions exhibit reduced PFS and OS, and that mutations in the MAPK-ERK pathway lead to reduced OS.Finally, we constructed comprehensive multivariate Cox Proportional Hazards models for PFS and OS Table , which ihe topic . A backwTP53ab on PFS and OS in MVA, SF3B1, EGR2 [RPS15 [NFKBIE [SF3B1 and EGR2 mutations retained as independent markers of OS in MVA.We report targeted resequencing analysis of 22 genes known to be recurrently mutated in CLL in the UK CLL4 clinical trial. CLL4 represents an ideal candidate for such an analysis, with expansive clinical and biological description , 40, 41 B1, EGR2 , 26, RPS2 [RPS15  and NFKB [NFKBIE , 28, 42 MAPK-ERK mutations represent a biologically distinct subgroup, where MAPK-ERK mutations are frequently mutually exclusive, are enriched for trisomy 12, unmutated IGHV genes and other adverse biological markers , and are linked to inferior time to first treatment in retrospective cohorts [MAPK-ERK genes, BRAF, KRAS and NRAS (collectively representing 12.2% of patients) are also independently associated with short OS in a cohort of patients requiring treatment. Vendramini et al. showed a similar frequency of mutations in these genes (14%) [MAPK-ERK pathway [BRAF mutations accelerated disease progression in Eµ-TCL1 mice [BRAF has been implicated in venetoclax resistance [KRAS mutated cases associated with poor response to chemoimmunotherapy [The literature suggests that patients with  cohorts , 43–45. es (14%) , while G pathway , (2) preCL1 mice , (4) mutsistance , and (5)otherapy  and lenaotherapy .TP53ab using FISH and Sanger sequencing has known prognostic value [TP53 mutations that present at low VAFs, below the detection limit of conventional Sanger sequencing may also be positively selected by chemotherapy, and also predict inferior survival, at least in retrospective, institutional cohorts [TP53 Network of ERIC provide expansive guidelines on the most suitable approach for TP53 mutational analysis, but also conclude that the clinical importance of low-level TP53 clones remains an unresolved issue, requiring validation in clinical trials [TP53 mutations, but we could not demonstrate inferior survival associated with cases harbouring <12% VAF TP53 mutations, the inference perhaps is that these cases represent an intermediate-risk group. Given the unexpected nature of this finding, we also conducted stratified 17p deleted survival analysis, identifying the same result for <12% VAF TP53 mutations without 17p deletion. Furthermore, we proceeded to show that our observation was not associated with any differences in the type of TP53 mutation, their co-existence with other more clonal prognostically-important gene mutations or biological features, nor the enrichment of any specific treatment. As a consequence, we feel that our observation is technically sound, and warrants confirmation in further studies.Screening for ic value , 20, 31,ic value . TP53 mu cohorts . The TP5l trials . We demoBIRC3 and ATM genes, both mapping to the long arm of chromosome 11. The ATM gene is mutated in 30–40% of 11q deleted patients [ATM, driving an impaired DNA damage response [ATM mutations is controversial in unselected cohorts [ATM mutations based on their putative pathogenicity, several are reported in both somatic (i.e. COSMIC) and germline  databases, lending uncertainty to their prognostic impact. The sequencing of matched germ-line material would provide additional clarity, but was not possible due to the historical nature of CLL4. Preliminary studies support a pathogenetic role of BIRC3 [BIRC3 mutations were not linked to inferior PFS or TTFT, respectively. Another comparator would be the RESONATE2 trial, which compared first line treatment with Ibrutinib vs. chlorambucil [BIRC3 cases is equally good under modern small molecule inhibition. In our previous CLL4 analysis, we demonstrated that BIRC3 dysfunction (defined as deletion AND/OR mutations of BIRC3) did not impact survival in 11q deleted CLL, while biallelic ATM lesions remained informative [BIRC3 mutations. Our current study, therefore aimed to expand the analysis with a larger patient cohort with significantly improved technology. This approach permitted the identification of a meaningful number of cases with loss and mutation of BIRC3. As neither ATM nor BIRC3 mutations were linked to survival in univariate analysis, we performed a stratified analysis in 11q deleted cases. In so doing, we show that biallelic BIRC3 cases have a further reduction in survival in comparison to sole 11q deleted cases and were found to be independent prognostic markers for PFS and OS in MVA. Finally, ATM and BIRC3 mutated cases without 11q deletion have a similar survival to wild-type cases.There remains disagreement regarding the relative clinical significance of deletion and mutation of the patients , where iresponse . The pro cohorts , with thof BIRC3 , 39, morof BIRC3  and the of BIRC3 , BIRC3 mrambucil . The 24 ormative . HoweverSF3B1, EGR2 and the MAPK-ERK genes. Our analysis suggests that <12% VAF TP53 mutations are an intermediate survival group. Finally, we show that biallelic BIRC3 aberrations identify a novel patient subgroup with poor survival, inferior to those with 11q deletions alone. Taken together, we demonstrate that a more expansive genomic screening approach provides additional clinical information, thereby helping to establish the precise importance of genetic alterations in the context of other established and emerging biomarkers. Furthermore, our work will facilitate the development of international standards for the detection and interpretation of somatic mutations in CLL.In conclusion, our study makes three main contributions to the field. We show an expansive analysis of the impact of clinico-biological disease features on the clinical importance of important gene mutations, including Supplementary MaterialSupplemental Table S3Supplemental Table S4Supplemental Table S6


However, this parameter is difficult to assess after blastocyst expansion. In this study, we used mechanical hatching to confirm the presence of cellular fragments in the perivitelline space of bovine IVP blastocysts. We further looked for associations between possible apoptosis within extruded cells/ cellular fragments and the quality of bovine blastocysts using quantitative RT-PCR and immunofluorescence. Surprisingly, more than 42% of expanded blastocysts had cellular fragments in the perivitelline space; however, more than 37% of extruded cells were TUNEL negative. We observed no significant difference in embryo quality between expanded blastocysts with and without cellular fragments in the perivitelline space. Overall, our data suggest that embryos extrude abnormal cells to maintain their developmental potential. The presence of fragmented cells is not an indicator of embryo quality.The presence of cellular fragments in the perivitelline space is a commonly used parameter to determine quality before transfer of  The total cell number and the ratio of inner cell mass (ICM) to trophectoderm cells are directly correlated with blastocyst quality   for further development at 38.5°C, in a humidified atmosphere containing 5% CO2 and 7% O2. On day 5, embryos were transferred to fresh SOF and cultured until day 9 , unless otherwise stated. Cattle ovaries were obtained from a local slaughterhouse within 2 h after slaughter. Cumulus-oocyte complexes (COCs) were aspirated from 2-8 mm follicles using a winged infusion set (18G) connected to a vacuum aspiration system. COCs were isolated from the aspirated follicular fluid using a stereomicroscope. Groups of 40–60 oocytes were matured and fertilized 3, 0.05 mg/mLNaH2PO4 (Merck), 0.22% (v/v) of a 60% sodium lactate solution, 2.38 mg/mL HEPES, 0.20% (v/v) phenol red, 0.39 mg/mL CaCl2·2H2O, 0.10 mg/ mL MgCl2·6H2O (Merck), 0.11 mg/mL sodium pyruvate, 100 U/mL penicillin-Streptomycin  and 6.00 mg/mL bovine serum albumin fraction V (BSA) , set at an osmolality of 280 ± 2 osmol/kg and pH 7.3 ± 0.05. Embryos were mechanically separated from their zonae pellucida using tungsten needles in washing medium under a stereomicroscope. Isolated embryos and zonae pellucidae were rinsed in PBS before collection for quantitative reverse transcription PCR and immunofluorescence.Day 7–9 expanded blastocysts were collected and placed in a washing medium composed of 6.66 mg/mL NaCl , 0.24 mg/mL KCl (Merck), 0.17 mg/mL NaHCOGroups of 47–55 zonae pellucidae containing obvious cell clumps or cellular fragments, and groups of 21–32 embryos were collected and stored in 100 μL RLT buffer  at −80°C until RNA isolation. Total RNA extraction and genomic DNA digestion was performed using the RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized directly after RNA extraction. The mixture used for reverse transcription (RT) was made up of 10 μL of the RNA sample and 4 μL of 5 × RT buffer  in a total volume of 20 μL containing 10 mM DTT (Invitrogen), 0.5 mM dNTP , 8 units RNAse inhibitor (Promega) and 150 units Superscript III reverse transcriptase (Invitrogen). Minus-RT controls were prepared from 5 μL RNA sample using the same reagents as above, without the reverse transcriptase. After incubation at 70°C for 5 min, the mixtures were cooled down on ice for 5 min, followed by 1 h at 50°C, and 5 min at 80°C. cDNA samples were stored at −20°C.http://www.ncbi.nlm.nih.gov/tools/primer-blast) according to Bos taurus mRNA . The quantitative reverse transcription PCR (qRT-PCR) mixture contained 10 μL iQ SYBR Green supermix , 9 μL RNAse-free and DNAse-free water (Invitrogen) and 1 μL cDNA with a final primer concentration of 500 nM. The specificity and annealing temperature of primers  were designed on Primer-Blast  for 15 min and then stored in 1% PFA at 4°C until further use. After washing twice in PBST , samples were blocked in PBS with 5% goat serum and 0.3% Triton X-100 for 60 min. Following three washes in PBST, samples were incubated with mouse monoclonal antibody against CDX2  and Alexa Fluor™ 568 Phalloidin  in dilution buffer  overnight. After three washes in PBST, secondary antibody incubation was performed with goat anti mouse Alexa488 (Invitrogen) for at least 1 h in the dark. Subsequently, nuclei were stained with DAPI (Sigma Aldrich) for 20 min in the dark. After three washes in PBST, samples were mounted with Vectashield  on slides and stored at 4°C in the dark before imaging. Fluorescent images were obtained using a confocal laser scanning microscope  with a Z-stack projection and were further analyzed using IMARIS software .Apoptotic cells were identified by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) based on the Click-iT Plus TUNEL assay  with a few modifications. After fixation, samples were washed twice in PBST and incubated with 100 μL of TdT reaction buffer at 37°C for 10 min, followed by incubation with TdT reaction mixture for 60 min at 37°C. After two washes in Milli Q water, samples were incubated with 3% BSA and 0.1% Triton X-100 in PBS for 5 min at room temperature. Following two washes in PBST, samples were incubated with 100 μL of the Click-iT Plus TUNEL reaction cocktail for 30 min at 37°C in the dark.After three washes in PBST, immunofluorescence and imaging were performed as described above.https://www.graphpad.com/scientific-software/prism/). Pools of embryos or zonae with cellular fragments from three biological replicates were analyzed for gene expression. Individual embryos or zonae with cellular fragments from three biological replicates were analyzed after immunostaining. Differences between two groups were analyzed by two-tailed unpaired Student's t-tests, and differences between multiple groups were examined by one-way ANOVA, followed by a post-hoc Tukey test. A linear mixed model [SPSS ] was used to investigate differences in the total cell number, CDX2 positive cell number and TUNEL positive cell number per blastocyst and cellular fragment comparison. The “individual embryo” was set as “subject,” different parameters as “fixed effect” and “run of IVF” as “random effect” because of the variability between batches of ovaries. Statistical significance was set at P < 0.05.Statistical analysis was performed using Excel and GraphPad Prism 8 (n = 140), 8 (n = 155) and 9 (n = 148) blastocysts , and is closely related to apoptotic processes  and extruded cells (n = 151), we next performed qRT-PCR to determine expression of genes in the caspase and Bcl-2 families . In contrast to expectations, the expression of caspase family genes was not statistically different between extruded cells and blastocysts (P > 0.17 for CASPASE 3 expression and P > 0.11 for CASPASE 9 expression). In addition, the expression levels of BAX (pro-apoptotic regulator) and BCL2L1 (anti-apoptotic regulator) were similar in blastocysts and extruded cells , we performed TUNEL staining combined with immunofluorescence staining for CDX2 (trophectoderm biomarker) and DAPI staining to label all nuclei  and apoptosis regulator genes in day 8 blastocysts. In agreement with the TUNEL/combined immunofluorescence results, there were no significant differences in the levels of CDX2, SOX2, CASPASE 3, CASPASE 9, BAX, and BCL2L1 expression between blastocysts with (n = 64) and without (n = 66) cellular fragmentation  and BCL2L1 (anti-apoptotic) expression between day 9 blastocysts and day 9 zonae pellucidae with cellular fragments, or day 8 blastocysts with and without cellular fragments.The technique of qRT-PCR does not allow determination of caspase protein activity, is partly dependent on post-translational modification (Parrish et al., Generally, the presence of cellular fragments in the perivitelline space is considered as an indicator of poor embryo quality (Jurisicova et al., To conclude, the presence of cellular fragments in the perivitelline space is common in expanded blastocysts. A limitation of this study is that even blastocysts with good morphology will not necessarily implant and give rise to healthy offspring. Blastocyst morphology is however a criterion used in practice to select or prioritize embryos for transfer. Although the most convincing demonstration of bovine blastocyst quality, i.e., birth of a healthy calf, has not been examined, we conclude that there is no correlation between the presence of cellular fragments in the perivitelline space and blastocyst quality.The original contributions presented in the study are included in the article/BY, HT, TS, and BR conceived and designed the experiments. BY, HT, and BR performed the experiments and collected the data. BY analyzed the data. BR contributed the reagents, materials, and analysis tools. BY, TS, and BR wrote the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.