Peptidase M20 Domain Containing 1 (PM20D1). We observed that PM20D1 DNA methylation, RNA expression, and genetic background are correlated and, in turn, associated with AD. We provided mechanistic insights for these correlations and had shown that by genetically increasing and decreasing PM20D1 levels, AD-related pathologies were decreased and accelerated, respectively. However, since the PM20D1 QTL region encompasses also other genes, namely Nuclear Casein Kinase and Cyclin Dependent Kinase Substrate 1 (NUCKS1); RAB7, member RAS oncogene family-like 1 (RAB7L1); and Solute Carrier Family 41 Member 1 (SLC41A1), we investigated whether these genes might also contribute to the described AD association.Alzheimer’s disease (AD) is a complex disorder caused by a combination of genetic and non-genetic risk factors. In addition, an increasing evidence suggests that epigenetic mechanisms also accompany AD. Genetic and epigenetic factors are not independent, but multiple loci show genetic-epigenetic interactions, the so-called quantitative trait loci (QTLs). Recently, we identified the first QTL association with AD, namely . Using this approach, we show that only PM20D1 DNA methylation and expression are significantly correlated with the AD-risk associated background. We find that the expression of SLC41A1 and PM20D1—but not NUCKS1 and RAB7L1—is increased in mouse models and human samples of AD, respectively. However, SLC41A1 and PM20D1 are differentially regulated by AD-related stressors, with only PM20D1 being upregulated by amyloid-β and reactive oxygen species, and with only PM20D1 being neuroprotective when overexpressed in cell and primary cultures.Here, we report a comprehensive analysis of these QTL genes using a repertoire of in silico methods as well as in vivo and in vitro experimental approaches. First, we analyzed publicly available databases to pinpoint the major QTL correlations. Then, we validated these correlations using a well-characterized set of samples and locus-specific approaches—i.e., Sanger sequencing for the genotype, cloning/sequencing and pyrosequencing for the DNA methylation, and allele-specific and real-time PCR for the RNA expression. Finally, we defined the functional relevance of the observed alterations in the context of AD in vitroPM20D1 as the most likely gene responsible of the previously reported PM20D1 QTL association with AD.Our findings reinforce  Alzheimer’s disease (AD) is the most common neurodegenerative disorder in western societies. It is characterized by a progressive decline in mental abilities, neuronal loss, and the accumulation of two types of protein aggregates, amyloid plaques and neurofibrillary tangles . The cauThe genetic component of AD has been generally interrogated by genome-wide association studies (GWAS), which have identified an important number of risk loci associated with AD –7, but aPredicted Metalloproteinase 20D1 (PM20D1) [PM20D1 expression, that PM20D1 expression is upregulated in symptomatic APP/PS1 AD mice and human AD samples, and that genetic manipulation of PM20D1 levels can modify the progression of the disease in APP/PS1 mice: When PM20D1 was overexpressed, disease progression was delayed; when PM20D1 was decreased, disease progression was accelerated.Recently, we reported the first mQTL association with AD, which converges on the gene (PM20D1) . We havePM20D1 expression QTL (eQTL) region, showing that the expression of neighboring genes to PM20D1 also correlate with several SNPs in that region, which include Nuclear Casein Kinase And Cyclin Dependent Kinase Substrate 1 (NUCKS1); RAB7, member RAS oncogene family-like 1 (RAB7L1); and Solute Carrier Family 41 Member 1 (SLC41A1), plus PM20D1 [PM20D1 [At the same time, two high-throughput studies have expanded the s PM20D1 , 13. The [PM20D1 , we cannPM20D1 eQTL genes, curated evidence for an AD association centered on PM20D1, and further support for the protective role of PM20D1 in AD.In the present study, we combine a series of in silico methods with in vivo and vitro experiments to provide a comprehensive analysis of p values were directly reported. mQTL SNPs were then investigated in our own postmortem human brain cohort of samples from the IDIBELL Biobank . DNA was isolated by phenol-chloroform extraction from gray matter of 18 control  and 21 Alzheimer’s disease frontal cortex samples . Genotypes were obtained by Sanger sequencing using primers listed in Additional file Hannon et al.’s mQTL database was interrogated for mQTLs—SNPs and CpGs—in each of the potential PM20D1 QTL region genes . When sip values were directly reported. Only previously annotated genes were considered for LIBD prefrontal (PFC) and hippocampal (Hip) datasets—i.e., Type = “Gene,” Class = “InEns”; and Type = all, Class = “InGen,” respectively. eQTL SNPs were then investigated in our cohort of samples. RNA purification was performed using TRIzol (Invitrogen), reverse-transcribed using the Thermoscript RT-PCR system (Invitrogen), and tested using StepOnePlus Real-Time PCR System (Applied Biosystems) and SYBR Green PCR MasterMaster Mix (Applied Biosystems). Three housekeeping genes were used for normalizing PCR signals. Primers for real-time PCR are listed in Additional file GTEX  and LIBDhttp://www.genenetwork.org) was also analyzed for the PM20D1 QTL region genes in the BxD mice population. BxD mice derive from multiple intercrosses of the C57BL/6 J (B) and DBA/2 J (D) progenitor mice, later inbred to fix the generated genetic variation [http://www.genenetwork.org/)—which constitutes a well-established genetic reference for the analysis of QTLs [The GeneNetwork database (ariation . To date of QTLs . The eQT of QTLs . Images PM20D1 QTL region gene expression was investigated in the aforementioned human brain cohort and five 12-month-old APP/PSEN1  and five2. SH-SY5Y cells were treated with 0.2‰ hydrogen peroxide (Merck) and with synthetic amyloid-β (1–42)-derived diffusible ligands (ADDLs) (Abcam) during 6 h and 24 h. Cell viability was measured using Alamarblue cell viability assay (Invitrogen) according to the manufacturer’s instructions. Primary hippocampal neuron-glia co-cultures derived from P0 wild-type mice were cultured in media consisting of Neurobasal (Invitrogen), B27 supplement (Invitrogen), l-glutamine (Invitrogen), and penicillin/streptomycin (Invitrogen) (0.2 ml per well) on 96-well plates (2.5 × 104 cells per well) coated with Cultrex poly-l-lysine (Trevigen). Cells were infected at DIV6 with 20 × 103 (200 ng/well) viral particles containing either a GFP (mock) or a PM20D1/SLC41A1 version of the pLVX-IRES-ZsGreen1vector (Promega). At DIV 14–17, cytotoxicity was assessed using the CytoTox96 non-radioactive cytotoxicity assay (PROMEGA) according to the manufacturer’s instructions.SH-SY5Y neuroblastoma cells (ATCC) were cultured in DMEM supplemented with 20% FBS, 100 μ/ml penicillin, and 100 mg/ml streptomycin at 37 °C in a humidified atmosphere of 5% COp values smaller than 0.05 were considered statistically significant and provided in the figures as follows: *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.0001.The analyses were performed using Prism 6.0 (GraphPad). Correlations were calculated using Pearson’s correlation coefficients, and comparisons using one-way ANOVA with post hoc Holm-Sidak’s multiple comparison tests. PM20D1 DNA methylation is strongly correlated with the rs708727-rs960603 haplotype, and both, methylation and haplotype, are associated with AD [PM20D1 promoter show strong correlations with rs1172198, rs708727, rs823082, rs823088, rs1361754, and rs960603 mQTL SNPs [NUCKS1, RAB7L1, and SLC41A1 [NUCKS1, RAB7L1, and SLC41A1 in the cerebellum; for PM20D1 in the hippocampus; and for NUCKS1, RAB7L1, SLC41A1, and PM20D1 in other non-brain tissues  [RAB7L1, SLC41A1, and PM20D1 in the dorsolateral prefrontal cortex, and for RAB7L1 and PM20D1 in the hippocampus [ with AD   . In addiNUCKS1, RAB7L1, and SLC41A1 DNA methylation levels and the genetic background (Fig. NUCKS1, RAB7L1, and SLC41A1 promoter regions remained largely unmethylated, independently of the genetic background (Fig. PM20D1 DNA methylation was strongly correlated with the genetic background (Fig. SLC41A1, i.e., values ranging from 7 to 9% of DNA methylation [PM20D1 arises as the only gene in this QTL region that truly qualifies as mQTL.Hence, we have expanded our previous analysis and measured the levels of DNA methylation of these genes in a genetically well-characterized human cohort of brain samples by locus-specific bisulfite sequencing and pyrosequencing Fig. b, c. We hylation  (Table 1PM20D1 QTL genes by allele-specific and real-time PCR (Fig. NUCKS1, RAB7L1, and SLC41A1 were found to be expressed from both chromosomes (Fig. SLC41A1 was observed (Fig. PM20D1 were significantly correlated with the genetic background, and PM20D1 was mainly expressed from non-methylated chromosomes (Fig. Similar to DNA methylation, we analyzed the RNA expression levels of the PM20D1 QTL region, we took advantage of the BxD mouse population [PM20D1 QTL locus. Supporting our previous results, similar relationships were also observed in the BxD population, i.e., no effect of the genetic background for Nucks1, Rab7l1, and Slc41a1, but a strong correlation for Pm20d1 (Fig. PM20D1 in the region under investigation.To further explore the pulation , since mpulation , includiPM20D1 is upregulated in human and mouse samples of AD [SLC41A1 and PM20D1 eQTL low expression carriers seem to be associated with the same AD-risk haplotype (e.g., rs708727 AA carriers, Table PM20D1 QTL genes in APP/PS1 mice and AD human postmortem samples, which were stratified by the rs708727 genotype. No significant expression differences were observed for NUCKS1 or RAB7L1 in the frontal cortex of APP/PS1 mice at symptomatic stages (Fig. SLC41A1 levels were significantly increased in both APP/PS1 mice (Fig. PM20D1 (Fig. SLC41A1 might have similar neuroprotective functions as PM20D1 or, alternatively, whether it participates of AD pathology.Genes in close proximity tend to share common regulatory elements and to correlate in expression , 20. PM2es of AD , which sges Fig. a, nor inges Fig. b. Converice Fig. a and hum0D1 Fig. b, which PM20D1 QTL genes in AD, we next assayed their expression upon Aβ and ROS exposure, two pathogenic hallmarks of AD [NUCKS1 or RAB7L1 genes (Fig. PM20D1 (Fig. SLC41A1 expression, albeit in opposite direction (Fig. PM20D1 and SLC41A1 was expected to be positively correlated since both eQTLs are associated with the same AD-risk haplotype—i.e., they share common regulatory elements—and both genes are increased in mouse and human samples of AD. However, our results suggest that PM20D1 and SLC41A1 are regulated by different mechanisms since only PM20D1 is upregulated by AD-related stressors—i.e., Aβ and ROS.To further disentangle the potential influence of nes Fig. a, b. Con0D1 Fig. a, b, whiSLC41A1, alongside with PM20D1 [SLC41A1 expression, together with PM20D1, which seems to indicate that the epigenetic association with AD and the upregulation of SLC41A1 in AD are the consequence of different mechanisms.Lastly, to investigate whether h PM20D1 , might ah PM20D1 , PM20D1 h PM20D1 . Counterh PM20D1  is assocIn sum, these lines of evidence corroborate our previous results on PM20D1 and suggest that PM20D1—which is upregulated and protective in response to AD-related stressors—is the main gene responsible of the AD-risk haplotype, while the evidence for SLC41A1 is less consistent and in a different direction.PM20D1 as a risk factor for AD [PM20D1 DNA methylation and RNA expression were correlated with the genetic background, which, in turn, was associated with AD. Moreover, we demonstrated that by genetically increasing and decreasing PM20D1 expression, AD-related pathologies were decreased and increased, respectively. Recently, the PM20D1 QTL region has been expanded to other genes, including NUCKS1, RAB7L1, and SLC41A1 genes [PM20D1 and, thereby, potentially contribute to our AD-associated risk haplotype.Our previous study has identified r for AD . We founA1 genes , 13 (datPM20D1 QTL genes, at the DNA methylation, RNA expression, and functional level, using APP/PS1, human postmortem AD samples, as well as mouse in vivo and in vitro experiments. We found no significant correlations between the genetic background and the DNA methylation and/or RNA expression levels of other PM20D1 QTL genes, except for the previously reported PM20D1 itself (Fig. PM20D1, reported significant RNA expression correlations with the genetic background for NUCKS1, RAB7L1, and SLC41A1 (Table PM20D1 DNA methylation, and for rs708727 and PM20D1 RNA expression in all datasets containing PM20D1, which pinpoints PM20D1 as the major QTL in the region (Tables PM20D1 is not found in the cerebellum GTEX dataset. Consequently, whether PM20D1 is cell-type and/or tissue-specifically regulated will surely be a matter of future investigations.In order to test this hypothesis, we have performed a comprehensive analysis of PM20D1 QTL genes either, except for the previously reported PM20D1 and the newly reported SLC41A1, which were both increased in mouse and human samples of AD (Fig. PM20D1 expression, but not SLC41A1, which was either not affected or downregulated, respectively. In line with these results, only PM20D1 overexpression was found to protect against ROS-mediated cytotoxicity (Fig. PM20D1 overexpression and depletion have been shown to be well tolerated [N-acyl amino acid production, which affects thermogenesis regulation [SLC41A1 have been found to be detrimental. SLC41A1 overexpression reduced cell survival in multiple cell lines [PM20D1 and SLC41A1 are expressed by astrocytes [Between control and AD samples, we observed no significant differences between the levels of RNA expression of  AD Fig. a, b. Howolerated , 23. Botolerated  and PM20gulation , 23. On ll lines , while mll lines , which strocytes  and mightrocytes , 26. HowInterestingly, PM20D1 has previously been shown to activate mitochondrial uncoupling , which pPM20D1 is the most likely responsible candidate of the previously reported QTL association with AD, and reinforce its protective role in AD. How precisely PM20D1 exerts its protective functions now requires further investigations.In sum, our results further support that Additional file 1: Table S1. List of primers used for the different techniques.


Vision suddenly has a new set of identities.When children grow up, the first word or first step in walking is always a significant event. These events rarely coincide neatly with calendar dates. So it is with the birth and growth of a new journal. Vision can now be found on PubMed at https://www.ncbi.nlm.nih.gov/pmc/journals/3742/. This is a major step forward for the new journal in expanding its visibility and its credibility as a permanent new feature in the research space.The main open source for paper-scanning and literature searches for current and future readers of this journal would most likely be PubMed. This database is well known as the standard open-source database in medical sciences. We are very pleased to be able to announce that Vision remain in the public domain in perpetuity.The indexing on PubMed will include all the papers published to date, thus increasing their visibility. We will also now be present on Scopus, as of October 2019, and are pleased to have this recognition. Both of these advances are backed by the policy of our publisher, MDPI, who have arranged for all of the articles published in their journals to be deposited in the Swiss National Library electronic archiving system. These archives would survive any change of ownership, and this arrangement effectively ensures that all papers published in We are very grateful for the efforts of the current editors and referees in helping us get to this new status. Nonetheless, we can only be a viable journal if research groups send their papers forward. For those who have been involved in the early stages of this new journal, we believe that the current news will be especially welcome.First Basel Sustainable Publishing Forum. This is an important effort for the future of scientific publishing. We wish our umbrella organization well in taking forward these efforts and encourage them to go further.I would also like to draw your attention to some other, recent activity of our publishers, MDPI. They have been a major sponsor of the An additional scientific development that we greatly encourage at the moment is further collections of Special Issues, that is to say focused collections of papers covering specific topics in visual science. These Special Issues provide a common reference point for researchers specializing in those topics and provide new generations of students and researchers with a compact way of accessing particular research themes. Thanks to electronic publishing, the size and format of Special Issues have become much more flexible and the journal has a policy of publishing individual component articles as soon as they become accepted for publication. If you have proposals or ideas for a Special Issue, I invite you to write directly to the journal offices, who will bring forward the proposal for prompt appraisal by the editorial board.Vision is administratively in good hands for its next phase of development.In terms of in-house management, the journal office in Wuhan continues to work efficiently on behalf of the scientific community. I am confident in stating that I feel that 15 December 2019Andrew J. Parker


Starch has a complex molecular structure, with properties dependent on the relative chain lengths and branching structure of its constituent molecules, which varies due to polymorphisms in starch biosynthetic genes, as well as environmental factors. Here we present the application of ultra-high performance size exclusion chromatography to the separation of starch chains from plant seeds. Several methods, have been used to analyse chain length distributions in starch, all with limitations in terms of analysis time, sample preparation and molecular weight range. Here we demonstrate that chain length distributions can be obtained with dramatically reduced analysis time using ultra-high performance size exclusion chromatography. The method may also show improvements in resolution of some fine structural features. Understanding links between starch fine structure and biosynthetic genes will allow bioengineering of starches with tailored properties. This technique may have application to the size separation and resolution of a range of biopolymers of value to the food, drink and pharmaceutical industries. Amylopectin is a far more complex molecule formed from short chains of α–1 → 4- linked anhydroglucose residues interspersed with α–1 → 6 branch points. These two polymers make up the complex, hierarchical structure of the starch granule. The chain lengths, degree of branching and position of the branches within the molecule are all dictated by a complex system of biosynthetic enzymes including elongating starch synthases, starch branching enzymes, debranching enzymes and amylolytic enzymes. Plants possess several isoforms of each of these biosynthetic and degradative enzymes, which can play subtly different roles and act in concert to generate the final complex structure of the starch molecule.Starch is one of the most important biopolymers on earth, forming the main source of energy in the human diet, as well as being an important feedstock for a range of industrial processes, from the food industry to biofuels and paper manufacture. Starch comprises two distinct biopolymers, amylose and amylopectin. Amylose is an essentially linear molecule formed from α–1 → 4- linked anhydroglucose residues with a molecular weight of 10The most widely measured structural feature of starch polymers is the chain length distribution (CLD), which is the fundamental structural level of starch which determines a range of functionalities such as crystallinity, texture properties and digestion . To obta4)  , HPLC-ba4) . HPLC-SE4)  In theorsing AF4 .Over the last two decades, the field of liquid chromatography has been revolutionised by the introduction of Ultra-high Perfomance Liquid Chromatography (UPLC), which utilises very high pressures of 40 MPa and above, and column packing materials with a particle size of sub–2 μm . The use2Hordeum vulgare) (cv. Tipple) was provided as a gift from Phil Howell . The grain sample was ground to a flour in an IKA MF 10 mill to pass a 150 μm mesh and starch was extracted from the flour following the method of Pisum sativum) plants were grown at the John Innes Centre (UK), using two smooth-seeded pea cultivars, BC1/19RR (derived from introgression of the wild-type SbeI (starch-branching enzyme I) allele into the wrinkled-seeded genotype, JI 430) and cv. Cameor. Pea seeds were manually dehulled and then ground in a coffee grinder to pass a 150 μm mesh. From all three samples the starch was then solubilised and debranched as described in Wu, Li, and Gilbert (2014). Pullulan standards with peak molecular weights ranging from 342 to 708,000 Da (PSS-pulkit) were obtained from Kromatek (UK). All samples and standards were dissolved in DMSO containing 0.5% (w/v) LiBr prior to analysis at a concentration of 1 mg/mL. All samples and standards were analysed using a Waters ACQUITY Advanced Polymer Chromatography system fitted with 3 columns in series; XT-450 Å, XT-125 Å and XT-45 Å and a differential refractive index (DRI) detector. The flow rate was set to 0.3 mL/min and the column temperature was 90 °C.Three different starches were used in this study. Barley (h) for the linear glucans, based on Mark-Houwink parameters of K = 0.0002427 dL g−1 and α = 0.6804 for pullulan in DMSO at 90 °C. Using this calibration, DRI elution profiles of the debranched starch samples were converted to SEC weight distributions expressed as w(logVh) using the relationship:el(Vh) is obtained from the calibration curve of the pullulan standards (de(X)N) can be obtained from the corresponding weight distribution obtained from the DRI signal through the relationship:Pullulan standards were used for calibration, using the method described by tandards . CLDs we3DRI responses for each of the standards and the associated standard curve are shown in With the small particle size, narrow bore columns and elevated pressures used in UPLC-SEC, there is increased likelihood of both column overloading and shear scission of polymers occurring due to the elevated pressure in the column. To identify if either of these effects were giving rise to artefacts in our results we re-analysed Tipple at three different injection concentrations  and at three different flow rates .The effect of flow rate on possible shear scission was also investigated by running injections at a range of flow rates. The results are shown in 44 or FACE with no limitations on the molecular weight range that can be analysed, no requirement for derivatization, and advantages of speed. The UPLC-SEC method presented here opens up the possibilities to carry out high-throughput phenotypic screening of starch structures, using germplasm collections to identify biodiversity, to correlate phenotypes with genomic and genetic data linked to variation in the underlying genes, and to facilitate breeding programmes aimed at specific food and feed uses. The method can also be applied to other biopolymers in applications where rapid determination of molecular weight distribution is beneficial. In conclusion, we present a rapid, flexible method for biopolymer molecular weight characterisation, particularly applicable to starch CLD, which overcomes many of the technical limitations of previous methods.The results presented in this paper demonstrate that UPLC-SEC can be successfully applied for the rapid and efficient separation of biopolymers. Despite the very high pressures, and consequently shear rates, involved, CLDs produced by the system match very well with the molecular weights and distributions observed in a conventional HPLC-SEC set-up. The resolution obtained in terms of structural features within both the amylose and amylopectin regions is comparable or superior to existing approaches such as HPAEC-PAD, AF


Arabidopsis hypocotyls as an example. During the study, the formation of a layer that covers the surface of the graft union was observed. So, this study also aimed to describe the histological and cellular changes that accompany autografting of Arabidopsis hypocotyls and to perform preliminary chemical and structural analyses of extracellular material that seals the graft union.Grafting is a technique widely used in horticulture. The processes involved in grafting are diverse, and the technique is commonly employed in studies focusing on the mechanisms that regulate cell differentiation or response of plants to abiotic stress. Information on the changes in the composition of the cell wall that occur during the grafting process is scarce. Therefore, this study was carried out for analyzing the composition of the cell wall using During grafting, polyphenolic and lipid compounds were detected, along with extracellular deposition of carbohydrate/protein material. The spatiotemporal changes observed in the structure of the extracellular material included the formation of a fibrillar network, polymerization of the fibrillar network into a membranous layer, and the presence of bead-like structures on the surface of cells in established graft union. These bead-like structures appeared either “closed” or “open”. Only three cell wall epitopes, namely: LM19 , JIM11, and JIM20 (extensins), were detected abundantly on the cut surfaces that made the adhesion plane, as well as in the structure that covered the graft union and in the bead-like structures, during the subsequent stages of regeneration.Arabidopsis grafting. The results showed that unmethyl-esterified homogalacturonan and extensins are together involved in the adhesion of scion and stock, as well as taking part in sealing the graft union. The extracellular material is of importance not only due to the potential pectin–extensin interaction but also due to its origin. The findings presented here implicate a need for studies with biochemical approach for a detailed analysis of the composition and structure of the extracellular material.To the best of our knowledge, this is the first report on the composition and structure of the extracellular material that gets deposited on the surface of graft union during The online version of this article (10.1186/s12870-019-1748-4) contains supplementary material, which is available to authorized users. Grafting is a technique in which either organs from different plants (heterografting) or from the same plant (autografting) are joined so as to continue their growth together. The upper part of the combined plant is called the scion, while the lower part is called the rootstock. In addition, the isolated organ fragments , or evenGrafting leads to the development of a stable union zone in which a structural and functional connection between the vascular system and other tissues of the scion and rootstock is established . A stablBefore the above-described cell events occur, an adhesion between the scion and the stock develops and stabilizes , 11. DurPectins comprise a heterogeneous group of polysaccharides composed mainly of galacturonic acid residues . Within Arabinogalactan proteins (AGPs), a class of highly glycosylated proteins –31, haveArabidopsis hypocotyl, we observed the formation of a layer covering the surface of the graft union. As this phenomenon has not been described so far, we focused on the exterior area of a graft union instead of the adhesion zone, which has been the subject of numerous studies. The aims of this study were 1) to describe the histological and cellular changes that occur during the process of regeneration in autografted Arabidopsis hypocotyls and 2) to perform preliminary chemical and structural analyses of the material that extracellularly deposits and finally seals the graft union.Understanding the roles of the cell wall components in plant cell differentiation processes is crucial for enhancing the knowledge base and for their commercial applications. While studying the changes in cell wall composition that occur during grafting of the Arabidopsis hypocotyls during grafting based on the occurrence of dominant cellular events  analysis showed that extracellular deposition of material accompanied the formation of graft union during all the stages of regeneration. During the second time frame (4–5 dag), material that was deposited on the surface of the callus cells appeared in two structural forms: fibrillar and membranous Fig. a and b. At the beginning of the regeneration process , the callus cells in the graft union zone had a smooth surface with no visible bead-like structures; however, strands of fibrillar material were associated with some of these cells Fig. a. In theArabidopsis grafting, monoclonal antibodies against cell wall components were utilized. Immunohistochemical analysis showed the abundant presence of two HG epitopes – LM19 and LM20  – in the walls of the dividing cells near the site of the cut; they were also seen within the maternal tissues of the scion and stock  and LM21 (heteromannan), during grafting process was also studied. LM15 epitope occurred abundantly in the walls of cells in the graft union area, except for endodermal or peripheral cells  and LM20 (scarce), were detected on the surface of graft union  formation of a fibrillar material outside the walls, 2) polymerization leading to the formation of a membranous layer, and 3) the presence of bead-like structures, which appeared either “closed” or “open”. Moreover, the structure of the extracellular material that was observed under SEM resembled an ECMSN as was mentioned earlier , 58.Bead-like structures, or beads, have previously been observed on the surface of the callus cells that emerged during grafting , 77. HowIn the present study, analysis of the sectioned material indicated that grafting process was accompanied by deposition of extracellular material. SEM revealed two forms of the extracellularly deposited material – fibrillar and membranous – as well as the occurrence of bead-like structures on the surface of the graft union cells, long after a connection was established between the scion and stock. The extracellular material subsequently sealed the graft union. Only unesterified HG and extensins were detected abundantly in both the extracellular material and the bead-like structures. Thus, our results contribute to the current state of knowledge regarding plant grafting and introduce a structure, which is interesting in terms of its form and composition, that potentially protects the graft union area and which has not been observed during the grafting of other species before. The results also indicated a possibility of ionic interaction between the two polymers, acidic pectins and basic extensins, due to their chemical nature, which can result in the formation of a network with beneficial properties.Arabidopsis thaliana Col-0  were surface-sterilized with 20% commercial bleach  for 8 min, rinsed five times with sterile distilled water (each time for 5 min), and left for 3 days at 4 °C in darkness. Then, the sterilized seeds were sown on Petri dishes with a medium containing ½ Murashige and Skoog salts , 1% sucrose , and 0.8% agar , pH 5.80–5.84. After seeding, the Petri dishes were sealed with parafilm  and placed vertically in a growth chamber . Following incubation, 4-day-old seedlings (six to seven seedlings per Petri dish) were transferred under sterile conditions onto Petri dishes with a medium containing ½ Murashige and Skoog salts , 1% sucrose , and 1.8% agar , pH 5.80–5.84. Later, another set of Petri dishes were prepared with a medium which was allowed to solidify at an angle of 15°, and grafting procedure was performed according to the method previously described [− 2 s− 1).Seeds of The grafted seedlings were collected at 0–9 dag (60 seedlings on each day) and analyzed using an SZH10 stereomicroscope . The roots, cotyledons, and developing leaves were excised, following which the grafted hypocotyls were fixed and embedded in Steedman’s wax as described previously . Longituv/v) solution, and then in distilled water; each wash for 10 min), and prepared for the histochemical analysis using a 0.05% (w/v) toluidine blue O aqueous solution used for general histological evaluation  and 0.5% (w/v) Sudan III solution used for detection of lipid substances . Toluidine blue O is metachromatic dye that imparts different colors to particular cell components , wherea− groups .w/v) Calcofluor White, a dye that stains cellulose , in PBS for 10 min. All the observations were performed and photographs were taken using a Nikon Eclipse Ni-U microscope equipped with a Nikon Digital DS-Fi1-U3 camera with the corresponding software , at a maximum excitation wavelength of 490 nm (AlexaFluor 488) or 330 nm . The details of the staining procedures are described in previous studies [For the immunolabeling procedure, the sections were dewaxed and rehydrated in a series of ethanol solutions  solution in phosphate-buffered saline (PBS); each wash for 10 min). The primary rat monoclonal antibodies  used in the current study are listed in Table  studies , 82. On For SEM analysis, the roots, cotyledons, and developing leaves were first excised from the grafted seedlings (4 and 9 dag). The remaining hypocotyls with the graft union zone were fixed by immediately placing them in 100% methanol  for 30 min to 1 h, according to a previously described procedure . After fw/v) paraformaldehyde  and 1.25% (v/v) glutaraldehyde  in PBS, pH 7.2, overnight at 4 °C. Following fixation, the samples were washed three times with PBS (each time for 10 min) and placed in a blocking buffer containing 2% bovine serum albumin  in PBS (w/v) for 30 min. After washing, the samples were incubated with primary antibodies  using an epifluorescence and confocal microscope, and were combined using Fiji . The figures (photographs and schemes) were assembled using CorelDrawX7 graphics program.Additional file 1:Figure S1. Immunohistochemistry of grafted hypocotyl sections –homogalacturonan (LM7 and LM8 epitopes). A – lack of fluorescence signal. A′ A, Calcofluor White. B – epitope detected in random sections (arrow) between groups of tracheary elements (asterisk) and other graft union cells. C – epitope present in some locations (arrow) within graft union area. c Calcofluor White. Scale bars: A and A′ = 50 μm; B and C = 10 μm (JPG 2277 kb)Additional file 2:Figure S2. Immunohistochemistry of grafted hypocotyl sections – rhamnogalacturonan I . A and B – epitope detected abundantly in walls of graft union cells (arrows) and in low amount in walls of cortical cells (full arrow), epitope absent from extracellular material on the surface of graft union (arrowheads). A′ A, Calcofluor White. C and D – epitope present in cellular compartments of graft union cells (arrows), no epitope observed in extracellular material on the surface of graft union (arrowheads). C′ C, Calcofluor White. E – epitope detected in walls of some graft union cells (arrows), apart from extracellular material on the surface of graft union (arrowhead). E′ E, Calcofluor White. F – strong fluorescence signal in cell wall of sieve tubes (arrows). G – epitope absent from graft union cells (arrows) and from extracellular material (arrowheads). G′ G, Calcofluor White. c Calcofluor White. Scale bars: A, A′, C, C′, E, E′, G, and G′ = 50 μm; B, D, and F = 10 μm. (JPG 2868 kb)Additional file 3:Figure S3. Immunohistochemistry of grafted hypocotyl sections – extensins (JIM12 and LM1 epitopes) and AGPs . A – epitope present in some of the cortical cells (full arrow) and graft union area (arrowheads), intensive fluorescence signal detected in the outer periclinal cell walls and cuticle of the epidermis (arrow); inset: intensive fluorescence signal detected in the outer periclinal cell walls and cuticle of the epidermis (arrow). B – epitope detected in the cell wall (arrow) and on the outside of the cell (arrowhead). C – epitope present in the cytoplasmic compartments of cortical cells near the graft union area (arrow). D – occurrence of epitope in the cells of the regenerated vascular bundle (arrows), in some endodermal cells (arrowhead), and peripheral cells of the graft union (inset: arrowhead), no fluorescence signal detected on the cell surface (full arrow). E – epitope present in the cytoplasm and/or plasmolemma of the graft union cells located peripherally (arrowheads), no fluorescence signal detected on the cell surface (arrow). F and inset – weak labeling in the cytoplasmic compartments of the peripheral cells (arrowheads), no fluorescence signal detected on the cell surface (arrows). c Calcofluor White, ep epidermis. Scale bars: A, D and D inset, and F = 50 μm; B, C, E, A inset, and F inset = 10 μm (JPG 2588 kb)Additional file 4:Figure S4. Immunohistochemistry of grafted hypocotyl sections –xyloglucan (LM15 epitope), and heteromannan (LM21 epitope). A and B – abundant occurrence of the epitope in the walls of graft union cells (asterisks) except for endodermal or peripheral cells (arrows), no fluorescence signal detected on the graft union surface (full arrow). A′ A, Calcofluor White. C and D – epitope detected in cellular compartments (arrows) and in the cell walls (full arrows) but not on the graft union surface (arrowheads). C′ C, Calcofluor White. c Calcofluor White. Scale bars: A, A′, C, and C′ = 50 μm; B and D = 10 μm (JPG 3122 kb)


Antibody-mediated rejection (AMR) is the main barrier to renal graft survival, and mouse renal AMR models are important to study this process. Current mouse models are established by priming the recipient to donor skin for over 7 days before kidney transplantation. The robustness of AMR in these cases is too strong to mimic clinical AMR and it is unclear why altering the priming times ranging from 7 to 91 days fails to reduce the AMR potency in these models. In the present study, we found that the donor-recipient combination and skin graft size were determinants of donor-specific antibody (DSA) development patterns after skin transplantation. DSA-IgG was sustained for over 100 days after skin challenge, accounting for an identical AMR robustness upon different skin priming times over 7 days. However, decreasing the skin priming time within 7 days attenuated the robustness of subsequent renal allograft AMR in C3H to Balb/c mice. Four-day skin priming guaranteed that recipients develop acute renal AMR mixed with a high ratio of graft-infiltrating macrophages, renal grafts survived for a mean of 6.4 ± 2.1 days, characterized by typical AMR histological changes, such as glomerulitis, peritubular capillary (PTC) dilation, and capillaritis, deposition of IgG and C3d in PTCs, but less prevalence of microthrombus, whereas the cellular rejection histological change of tubulitis was absent to mild. With this scheme, we also found that the renal AMR model can be developed using common mouse strains such as C57BL/6 and Balb/c, with mean prolonged renal graft survival times of 14.4 ± 5.0 days. Finally, we proved that donor-matched skin challenge after kidney transplantation did not strongly affect DSA development and kidney graft outcome. These findings may facilitate an understanding and establishment of mouse renal allograft AMR models and promote AMR-associated studies. Antibody-mediated rejection (AMR) has been recognized as the leading cause of kidney graft failure, which accounts for 30–50% of acute rejection episodes in kidney transplantation and over 60% of death-censored late graft loss –3. The h(−/−) mice, which spontaneously generate donor-specific antibodies (DSAs) after transplantation : anti-C3d; Bethyl Laboratories : anti-IgG; the secondary antibodies were purchased from Beyotime Biotechnology : HRP-labeled anti-anti-C3d and HRP-labeled streptavidin. The antibodies used for flow cytometric analysis were purchased from BioLegend : BV570-CD45, FITC-CD4, APC-F4/80, BV421-B220; eBioscience : eFluor 450-CD8a, Alexa Fluor 700-CD3, PE-CD49b; BD Biosciences: PE-Cy7-CD11b; Abcam : anti-IgG; and Jackson ImmunoResearch Laboratories : anti-IgM.Male C3H (H-2k), C57BL/6 (H-2b), and BALB/C (H-2d) mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and were used at the age of 8–12 weeks. All mice were housed in a specific pathogen free facility at Sun Yat-sen University and all animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals  and according to the Sun Yat-sen University Institutional Ethical Guidelines for animal experiments.2 or 4–5 × 5 mm2 from the tails of donor mice were transplanted onto the dorsal area of recipient mice. Murine kidney transplantation models were established as described previously  or periodic acid-Schiff (PAS) staining. For immunohistochemical staining, the paraffin-embedded kidney tissues were cut into 4-µm sections, deparaffinized in xylene, rehydrated through graded ethanol, then subjected to quenching of endogenous peroxidase activity in 0.3% hydrogen peroxide, antigen retrieval by microwave heating in 10 mM citrate buffer (pH 6.0), and overnight incubation with primary antibodies against C3d or IgG at 4°C followed by detection using HRP-labeled donkey anti-goat secondary antibody or HRP-labeled streptavidin at 37°C for 30 min. After washing, the tissue sections were stained with diaminobenzidine (DAB) solution and counterstained with hematoxylin. The sections were examined for the severity and category of rejection by light microscopy based on the Banff criteria: 0, absent; 1, mild; 2, moderate; 3, prominent .Fresh kidney grafts were milled gently in phosphate-buffered saline supplemented with 1% heat-inactivated fetal bovine serum using a needle of a 10-ml syringe before pressing through a 200-mesh nylon screen. The collected cells were then stained with fluorochrome-conjugated antibodies against CD45, CD3, CD4, CD8a, B220, CD49b, CD11b, F4/80, and acquired on a FACSCalibur flow cytometer with Cell Quest Software (BD Biosciences). Data were analyzed with FlowJo Software .t-test was utilized to compare the differences between two groups. Graft survival among groups was compared using the log-rank test. Data are shown as mean ± SD. Differences with P-values <0.05 were considered statistically significant.Student’s 2, the DSA-IgG levels conformably began to rise on day 7 after transplantation and were increasingly higher from then onward till they reached the peak time in recipient mice from all groups, and were maintained at near peak levels after reaching the highest point within 100 days. The peak times for the DSA-IgG levels in recipients from the above four combinations were posttransplant day 49, 42, 35, and 28, respectively, and the fold increases in the DSA-IgG level at peak times to the baseline levels were 3.82, 2.34, 3.94, and 2.58, respectively. By contrast, the rise in DSA-IgM was extremely slight and exhibited fluctuation within 100 days after skin transplantation in the four different combinations. Compared to the graft size of 8–10 × 10 mm2, the 4–5 × 5 mm2 skin graft displayed greater efficiency at sensitizing recipients to generate circulating DSAs, which manifested as both DSA-IgG and IgM levels that started to rise much earlier, at day 2 after transplantation; however, from postoperative day 7 onward, this discrepancy resulting from a different graft size was gradually leveled out  in all groups  compared to the baseline levels on day 0 before skin transplantation (ST0). By contrast, recipient mice in both the primed-4d and primed-7d groups displayed significantly augmented DSA levels on day KT5 compared to the skin pre-transplant baseline level on day ST0 Figure B. Kidneyi score 0–3) were observed in the renal graft on day KT5. Flow cytometry revealed that macrophages accounted for 58.3 ± 3.8% of the CD45+ immune cells infiltrated in the renal graft, followed by CD8+ T cells (20.5 ± 3.9%), NK cells (11.4 ± 2.4%), CD4+ T cells (10.7 ± 0.7%), and B cells (1.8 ± 0.7%) . The survival time in primed recipients was significantly prolonged compared to that in the former C3H to Balb/c combination . The outcome of DSA levels after kidney transplantation suggested that both the IgG and IgM levels in recipient sera from the primed group were significantly increased on day KT5 vs. ST0, and were higher than the levels on day KT7 in the nonprimed group . Dynamic monitoring of DSA levels over 30 days after kidney transplantation demonstrated that challenge with donor-matched skin grafts after kidney transplantation slightly strengthened both DSA-IgG and IgM development but failed to reach significance  in the skin Tx group .QS and DZ designed the research; DZ, TL, SL, YZ, HZ, JZ, FH, and YD performed the experiments; DZ, TL, YZ, JZ, and YD analyzed the data; DZ, TL, and SL wrote the manuscript. All the authors read and approved the final 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.


Grandparenting can be a rewarding and health-promoting experience for older adults. However, grandparent-caregivers often experience greater stress and poorer health relative to non-caregiving grandparents. Further, little is known about grandparents caring for a child with a rare, chronic illness. This study aimed to extend knowledge of the expectations, roles, and experiences of grandparents providing care to a child affected with an inherited metabolic condition. The sample included 23 grandparent-mother dyads from the Inherited Diseases, Caregiving, and Social Networks Study. The grandparent sub-sample ranged from 49 to 79 years of age (Mage = 64), the majority were female (83%) and married (74%), and almost half (48%) were retired. Social network assessments were analyzed to determine concordance between mother- and grandparent-reports of grandparents’ role in the child’s caregiving network. Fifteen mother-grandparent dyads (65%) agreed on grandparents’ role in the child’s network, with the majority of those (93%) considering the grandparent to be very close and important (versus less close or excluded from the caregiving network). Grandparents whose self-reports of their role in their child’s caregiving networks were consistent with mother-reports appeared more likely to report that they spend enough time caregiving than those whose reports were inconsistent. Content analysis of grandparents’ interviews provided supporting information about the joys and regrets of their grandparenting experience and perspectives on caregiving expectations. This research leverages multi-informant social network and qualitative data to illuminate grandparents’ role in the caregiving networks of chronically ill children and adaptation to non-normative grandparenting experiences.


Current stiffness and displacement stiffness are two important parameters of radial active magnetic bearing (RAMB) that are generally considered as constants in a control system. However, such presumption may lead to the probable degradation of the control performance of the RAMB system when the perspective that the current and displacement stiffness should be variable due to variations in the speed. On this regard, a structure analysis and stiffness measurement method based on non-variable stiffness would not be feasible for RAMB under high-speed conditions. This paper presented an analysis of the dynamic stiffness characteristics of RAMB by means of a dynamic equivalent magnetic circuit method and laid out a comparison between the results of the theoretical analysis and the simulation results of finite element method. In particular, a novel dynamic stiffness measurement method of RAMB under high-rotation frequency was introduced. Results of experiments on the dynamic stiffness of RAMB demonstrated an excellent agreement with the theoretical research and the finite element analysis results, thereby verifying the rationality of the discussed dynamic stiffness characteristics. Practically, the proposed measurement method of RAMB dynamic stiffness provides an accurate analysis for the dynamic stiffness and contributes inspiring research significance for the dynamic properties of RAMB. Bearings are used in molecular pumps to reduce friction and oil contamination. Compared to the traditional ball bearings and oil film bearings, the magnetic bearing is a new type of bearing with special advantages of non-contact characteristics, much less friction, low power consumption, low maintenance cost, dynamical controllability and active control ability of rotor dynamic imbalance, giving it broad prospects in industrial applications6. The magnetic bearing used in molecular pumps can realise oil-free, wear-free and quiet operation and minimal vibration, which are especially suitable conditions for the semiconductor industry, such as in ultra-high vacuum applications9.A molecular pump is kind of high-end scientific instrument, such as cyclotron, laser, mass spectrometer, gyro equipment and so on, typically used in obtaining high vacuum12. However, these bearings lack active controllability and exhibit low damping and stiffness. On the other hand, active magnetic bearings, which generate supporting force by means of electromagnets, a feedback control loop and other elements as sensors and power amplifiers, present extraordinary characteristics in terms of high stiffness, good controllability and damping properties relative to the passive ones16. Consequently, most magnetically suspended molecular pumps adopt active magnetic bearings to achieve ultra-high pumping speed at present.Accordingly, the two most commonly used magnetic bearing types are the passive and active magnetic bearings. On one hand, passive magnetic bearings with low power consumption realise the passive suspension of rotor through the interaction between the stator and rotor permanent magnets or the reluctance suction between the stator and rotor18 of magnetic bearings and is categorised mainly into displacement stiffness and current stiffness. Literature19 asserted that both coefficients of current and displacement stiffness for a power magnetic bearing can be precisely determined via application of an open-loop static levitation force on the rotor. Reference20 put forward a new stiffness measurement method of repulsive passive axial magnetic bearing with Halbach magnetic array, for the single gimbal magnetically suspended control moment gyroscope. Moreover, literature21 proposed a new stiffness measurement method for the magnetically suspended flywheel for determining the current stiffness and displacement stiffness of permanent magnet biased radial magnetic bearings. Further, a detailed stiffness measurement method for a radial hybrid magnetic bearing was described in22. In all these studies, the stiffness of magnetic bearings has been regarded as a constant (not affected by rotation frequency) and the measurement experiments were performed when the rotor was suspended steadily at zero-frequency. For the low speed applications in22, the stiffness at zero speed can be used as a constant in the whole speed range. However, some researchers have found that the stiffness of radial active magnetic bearing (RAMB) would decline in a discernible manner with an increase in rotor frequency and would influence the stability of the radial magnetic bearing system in the process24. The literature presented herein also depict that the frequency response of the current stiffness could be obtained by addition of a balancing mass; nonetheless, the method is complicated and no explicit description of measuring current stiffness is provided in this research. This aside, the frequency response of the displacement stiffness is not analysed nor measured. Therefore, it is necessary implement the meterage of dynamic stiffness of RAMB for the magnetically suspended molecular pump (MSMP).Stiffness is generally known to be one of the most important parameters for the whole control systemIn this paper, an accurate dynamic stiffness measurement method of RAMB is put forward for MSMP. A build-up of the characteristics of the RAMB dynamic stiffness is first established by means of an equivalent magnetic circuit with varying frequency. Diagrams of magnetic line distribution and change trend of the stiffness is then obtained using finite element method (FEM). Finally, the proposed dynamic stiffness measurement method of RAMB is verified by prototyped experiments via comparison of results of the theoretical and FEM analyses.Figure 23. Such phenomenon results in substantial reduction in stiffness compared to the 0 Hz stiffness value that is considered constant in the RAMB system. As such, the traditional magnetic circuit model, which ignores the rotor frequency variation and defaults the control current and magnetic resistances to a constant value, is not suitable for the RAMB stiffness analysis. Through an analysis of the dynamic characteristics when the rotor speed is variable while other parameters remain unchanged, studies in24 have proved that rotor frequency variation and eddy current effect leave a marked impact on magnetic bearing stiffness. Accordingly, these studies established an analytical method for a hybrid radial magnetic bearing system based on an equivalent circuit model whose parameters were frequency-dependent. Although it has been generally recognised that eddy current effects could be mitigated by replacing the solid structure with laminated cores and by designing modifications, the magnetic bearings stiffness was still considerably influenced by eddy currents when the rotation frequency was sufficiently high27. Therefore, it is necessary and is of great significance to carry out a quantitative analysis on the dynamic stiffness of RAMB at different rotation speeds.While the rotor operates at or near the rated speed, eddy currents caused by time-varying control currents will affect the resistance of the rotor and the stator, which is an important factor influencing the stiffnessSpecifically, the rotor speed change will lead to a time-varying control current, which will induce the eddy current effect in the stator core and rotor core, leading to the modification of their magnetic permeability. Based on the dynamic permeability of the stator and rotor, an analysis of accurate quantitative dynamic stiffness characteristics for the varying frequencies can be performed by establishment of an equivalent magnetic circuit.25. In particular, the relative permeability of stator (μrs) could be deduced using Eq. (μnr) can be expressed through Eq.  as the total transfer function of the displacement sensor, PID controller and power amplifier. According to the theory of vibration, the equation of motion for second-order system can be expressed by Eq.  for the phase angle of the transfer function G(s),For the RAMB structure shown in Fig. sing Eq. , which dd by Eq. , where Ding Eqs. , s = 2πfough Eq. , with ϕStiffnessStiffness with air gap length. The variation of air gap length leaves a considerable influence on the magnetic reluctance of the air gap, leading to a corresponding variation in current and displacement stiffness of magnetic bearings. Herein, several air gap values were selected from 0 to 1 mm, for an investigation of the change of current and displacement stiffness at 1000 Hz. Figure Stiffness with pole width of stator. From the dynamic stiffness characteristics obtained in (ained in  and 18)StiffnessStiffness with pole coil turns. As an important parameter of the proposed dynamic stiffness model shown in Eqs. ( in Eqs.  and 18)StiffnessBy the electromagnetic field analysis model in ANSYS software, the 2D-FEM mesh model of RAMB introduced in Fig. 26 indicated that the rotor magnetic induction line would no longer be evenly distributed but would tend to the rotor surface due to the skin effect caused by the eddy currents. For the RAMB structure and FEM simulation conditions described in this paper, the FEM simulation results were consistent with the theoretical analysis as depicted in Fig. Based on Fig. Accordingly, the corresponding values of dynamic stiffness with varying rotor frequencies were calculated herein using 2D FEM and the calculated values were compared with those of the analytical results, as shown in Fig. It is clear from the above discussions that current and displacement stiffness are important parameters for characterising the performance of magnetic bearing and thus, stiffness measurement is a very essential research topic. This, however, most accounts on such a measurement method were limited at 0 Hz and have regarded the measured values as constants, which is obviously not feasible for high-speed conditions. To adequately verify the correctness of the proposed dynamic stiffness on this research as well as obtain the actual stiffness values for RAMB with varying rotation frequencies, a dynamic stiffness measurement method is proposed under this section.f = kx·(h − h0) + ki·i. For this expression, f represents the electromagnetic force of the measured channel, h donates the rotor displacement, h0 donates the magnetic centre position of RAMB, i donates the control current in the RAMB coils and kx and ki donate the measurement dynamic current and displacement stiffness, respectively. As the electromagnetic force of RAMBs in MSMP balances the gravity of the rotor with certain frequency, the electromagnetic force of each RAMB in the measured channel can be calculated with the solution f = f(mg) = G/2, with m indicating the rotor mass and G donating the rotor gravity. Consequently, the control current can be represented as:In general, stiffness is measured on account of Eq. , which ih0 is known, measurement of different displacements near h0 and the corresponding control currents at any rotation speed will result to the finding the current and displacement stiffness at the rotation speed through Eq.  and ij(ω), the current and displacement stiffness of RAMB under different frequency can be obtained via changing the rotor speed and repeating the steps.The secondary stage involves determining the dynamic current and displacement stiffness, which is much different from the literatureg to Eq. , the corugh Eqs.  and 28)22, by adThe feasibility of the proposed dynamic stiffness characteristics and measurement method, the dynamic stiffness test experiment of RAMB was completed in a MSMP platform with a rated speed of 500 Hz (30000 r/min), as illustrated in Fig. Figure During the experiment process, some factors as the interference caused by rotor unbalance will influence the performance of the RAMB system. To better simulate the system, two noise signals including a unit impulse signal and a white noise signal that result in a signal-to-noise ratio of 14 dB, were introduced for the simulation conducted in Matlab/Simulink. Table In the experiment, the magnetic centre was determined according to the primary stage described in the proposed measurement method, with the bias current set at 0.38 A. As a consequence, the control current can be measured when the rotor rotates at the magnetic centre of RAMB with different frequencies and the corresponding dynamic current stiffness under each speed can be obtained through force analysis of the MSMP system and by using Eq. . The rel22, the RAMB current stiffness can be obtained in the measurement test system described herein as 133.6 N/A, with no variation in the rotor frequency. Nonetheless, Fig. By adoption of the measurement method presented in literaturex-axis faces upward in the vertical direction and the magnetic centre of RAMB was set as the coordinate origin. Under such condition, the magnetic force in the RAMB would be zero when the rotor is suspended stably, such that the control current is i0 = 0 with h0 = 0. Consequently, Eq. (In order to obtain a convenient measurement of the dynamic displacement stiffness, the attitude of the prototype of the measured MSMP was adjusted so that the tly, Eq.  can be itly, Eq.  yieldingFinally, the dynamic displacement stiffness can be obtained by the recorded control current values via a series of set equilibrium positions under different rotor frequencies in accordance to Eq. . Figure 22, the RAMB displacement stiffness obtained through the proposed measurement test system was 1.44 × 105 N/m, which is regarded as a constant and is unrelated with the rotor frequency. Nonetheless, it is discerned that the displacement stiffness of RAMB obtained using the proposed measurement method varied with rotor frequency, where the displacement stiffness of RAMB in the rated speed of 500 Hz was 1.34 × 105 N/m. Furthermore, the experimental results were consistent with the analytical data and FEM results and produced low maximum errors of 2.3% at 550 Hz and 1.5% at 550 Hz.By adopting of the measurement method presented in literatureThe experiments were performed on a prototyped MSMP, which suggests that interference from the electric motor was unavoidable. To describe the scenario, the electric motor’s position in Fig. Some ideal hypotheses were aimed to promote calculation convenience in the theoretical analysis part, say, the laminated material of the rotor was linear and the magnetic flux was a spatial function varying with time in the Z-direction.Figures The errors between the measured results and theoretical analysis can be minimise in future works by focusing on the separation design of the electric motor and measured RAMB, for elimination of the influence generated by the motor.The content of this work highlighted the proposal of an accurate model of dynamic stiffness, along with a measurement method of dynamic stiffness for RAMB. It conducted an investigation of dynamic stiffness characteristics influenced by rotor frequency for RAMB by application of the dynamic magnetic circuit method and FEM. Based on the results, dynamic stiffness exhibited remarkable variation with an increase in frequency. The proposed measurement method is a two-stage process. The magnetic centre is first determined so that the dynamic stiffness under a certain frequency can be obtained through measurement of different current values when the rotor is adjusted to different equilibrium positions near the magnetic centre. The method was validated by an experiment on a prototyped MSMP. Upon comparison of the experimental values to the theoretical results and the values obtained by FEM, as the rotor rotates at the same frequency, the maximum error of current stiffness was 5.0% and 3.1%, respectively, while the maximum error of the displacement stiffness was 2.3% and 1.5%.The findings herein have shown great progress in the research of the dynamic properties of RAMB and have as well provided an evaluation standard for dynamic performance improvement and structure optimisation of RAMB.The theoretical analysis for the dynamic stiffness model was based on the RAMB structure in MSMP, as shown in Fig. The parameters in the FEM magnetic bearing model were consistent with the parameters in Table The experimental conditions and control system are shown in Fig.


Idli is mostly prepared through fermentation. It has high palatability, and the rapid and highly digestible nature of the food product results in a higher post-glucose level in diabetic patients. A heat-treated Idli rice sample was prepared by roasting parboiled rice at the temperature range of 155 to 165 °C for 65 to 75 s. Idli/rice-based Dokala made from heat-treated rice is better when compared to untreated rice in terms of its microbiological profile and physiochemical properties. The proximate composition of heat-treated parboiled rice Idli/Rice Dokala showed slightly higher values than the untreated parboiled rice Idli/Rice Dokala, which reveals that it has marginally higher nutritive value. Determination of the Rapidly Available Glucose (RAG) and Slowly Available Glucose (SAG) values, SEM analysis, resistant starch analysis, microbial assay, and in vivo studies were performed to determine the glycemic index (GI) and glycemic load in normal and diabetic persons. Sensory analysis also proved that heat-treated Idli/Rice Dokala is superior to untreated based on the color, flavor, appearance, taste, and texture. The Asian food pattern primarily embraces rice and rice-based products, which mainly comprise 90% starch. Among these various food products,  Diabetes has developed into a chronic disease in humans, affecting approximately 200 million adults globally and almost 15 million Americans in 2000. In 2002, the rate of diabetes increased drastically; also, the treatment and medicine productivity costs were raised. Likewise, the incidence of diabetes among U.S. adults has grown to 61%; previously, it was 5% in 1990, and it continuously increased to 8.1% in 2000 . It has Idli and Dokala are mainly prepared with parboiled rice, which is rich in amylose content, leading to soft and spongy textural characteristics [Famous traditional rice-based Indian foods such as eristics . Fermenteristics . Severaleristics . The RS eristics . It leaderistics . The quaeristics .The complex nature of inter-connections between nutrients and nourishment in the diet and the hazard of diabetes and high glucose levels in the blood has been inspected by numerous researchers. The most prominent research indicates that a lifestyle of consuming a high-fat and high-calorie diet leads to type 2 diabetes, but epidemiologic research information has shown that this is relatively unpredictable . Recent Currently, the emphasis being placed on the glycemic reaction to various carbohydrate-enhanced foods varies from the conventional conviction about basic versus complex carbohydrates in the management of diabetes mellitus . RecentlRice, a staple food in India, is a prime source of carbohydrate, and different varieties are applied as a primary food. However, the GI of rice is higher than that of any other starch-containing food . Rice caRelatively less epidemiologic research-based evidence has shown the correlation of carbohydrates with diabetes to be indirectly proportional to high glucose in the blood ,18,19,20The American Diabetes Association (ADA) endorses the consumption of nutritious food such as fruits, vegetable, cereals, pulses, and dairy products as a healthy food diet for diabetes patients, because lower amounts of carbohydrate lead to lower incidence of diabetes . The ADAThe current research shows that higher-carbohydrate/fiber diets result in a reduction in blood glucose and other beneficial factors towards controlling diabetes, specifically in those with type 2 diabetes mellitus ,32,33. SThe final product of the digestion of starch is glucose, which is classified into Rapidly Available Glucose (RAG), Slowly Available Glucose (SAG), Free Glucose (FG), and Total Glucose (TG) . RAG andResistant starch (RS) is well defined as the starch and products of starch degradation that are not absorbed in the small intestine of healthy individuals . RS resiThe proportion of amylose and amylopectin, physiochemical form, degree of gelatinization, heat treatments, refrigeration, and storage affect the RS quantity in foods ,41,42. RAs recorded, there are four types of RS: RS1 is starch which is physically difficult to reach due to the manifestation of rigid cell walls in grains, kernels, or rhizomes. RS2 is native crystalline starch granules that are resistant to enzymatic hydrolysis; for example, the starch in uncooked potatoes, the green variety of banana, and elevated-amylose corn. RS3 is retrograded starch which is cooked followed by cooling for a few hours or longer. RS4 is the chemically changed starches; these are due to cross-linking with substance reagents, ethers, esters, and so forth .Insulin is considered to be the hormone accountable for the transportation of blood-based glucose from the circulatory system to the muscle and (fat) cells . AvoidinIdli is the most common fermented legume and rice product in South India. Idli is highly digestible and is thus recommended as a food for patients, infants, and older people with impaired digestion [Idli because of its rapid digestibility, which in turn results in a rapid rise in their postprandial blood glucose level. This is detrimental to diabetic patients [Idli made in the traditional method is as follows, making Idli not suitable for the diabetic diet. Fermented foods are often more nutritious than their unfermented counterparts, and they are easily digestible. This is true of naturally fermented legume products; hence, such foods are still produced in India by simple traditional methods . Idli isigestion . Howeverpatients . The GI Idli suitable for diabetic patients, the digestibility must be reduced to give it lower RAG values [In order to make G values . This wiIn food processing, physio-chemical treatments may meaningfully affect starch ingestion. For grain-based foods, modification based on dampness, condition, and so forth is vital . Starch Moisture–heat action has been generally considered a main strategy for altering starch yet heat treatment of starch in a dehydrated environment has been rarely examined. One exemption is pyrodextrin creation by the treatment of starch at high temperatures . DextrinEliasson et al.  reportedThe GI concept was initially developed to categorize diverse sources of sugars and starch-rich foods in the diet based on their influence on postprandial glycemia ,71,72,73An ADA spot report on nourishment concluded that the application of GI and GL would possibly give greater advantage in regulating diabetes based on total carbohydrate . AdditioThe glycemic sugar fraction, which is captured in the small intestine, is defined as the quantity of carbohydrate and starch excluding Resistant Starch (RS). The sugar fraction can be subdivided into Readily Available Glucose (RAG) and Slowly Available Glucose (SAG), and in vitro analysis reflects the likely degree of absorption of glucose in the small intestine. Analytical procedures have been developed to characterize foods and reflect their expected biological uptake. The values of RAG and SAG were determined in hydrolyzed foods by enzymes amyloglucosidase and amylase at 20 minutes and 120 minutes of an in vitro carbohydrate digestion method, respectively, by modifying the method by Eliasson . The RAGIn food processing, physio-chemical treatments may profoundly affect starch indigestibility. For cereal foods, modifications in humidity, thermal conditions, etc., have been verified . Starch Further bacterial fermentation leads to the production of volatile fatty acids. This has been observed in the majority of carbohydrate-enriched foods. Consumption of natural RS by humans has been indicated to result in a reduced glycemic response in healthy individuals , decreasAn important association was detected when the percentage of rapid carbohydrate ingestion and the Lente carbohydrate assimilation percentage were interrelated with the GI of the food consumed, but only when a single rapid carbohydrate absorption rate was considered. These outcomes validate this as a beneficial, simple, and low-cost method to evaluate the biotic response of high-carbohydrate foods ,69. Idli and other rice-based food products. The review showed that a slight amendment to rice treatment can modify the physiochemical properties of rice. This rice provides bulk, that is, to satiety, but most is not absorbed and it does not lead to an increase in blood glucose level. By the additional effect of fiber and further effects as discussed, overall and Low-density lipoprotein (LDL) dietary fat was reduced. The process is economically affordable and not expensive. The only disadvantage is minor differences in taste and the raised fuel rate required for heating the rice. Based on this review, it was concluded that the thermal treatment of rice is a better method for increasing the resistant starch content, which, in turn, may be useful for diabetes patients. Hence, heat treatment of rice is preferred for the preparation of low-GI


Cassava starch was oxidized using the electrolysis system. Sodium chloride was added to this system at various concentrations from 0.5 to 5.0 % (w/v). The whiteness of modified starches proportionally increased based on the NaCl concentration and human eyes could recognize the difference of color. Under treatment, dents occurred on the surface of starch granule. Concentration of carbonyl and carboxyl groups was increased compared to native starch. Based on X-ray diffraction pattern, oxidized starch kept its A-type. Besides, the ratios of alpha-helix/amorphous regions remained indicating oxidation reaction mainly subjected on amorphous region. Intrinsic viscosity was used to indirectly calculate the average molecular weight of sample. Furthermore, results showed that average molecular weight was significantly reduced (from 2.09-fold to 13.22-fold) based on the reacting NaCl concentration. The increase of NaCl content related to the increase of retrogradation of treated starches. At various temperatures (30-95°C), swelling factor and clarity reflected negative and positive correlations to NaCl concentration. Cassava starch is one of major starch sources in Vietnam. However, native starch properties are not fit industrial requirements. Many methods have been used to modify natural starch to improve its functions in other industries. Oxidized starch has been widely used in various food and non-food products.2O2, (NH4)2SO4, NaBrO3, KMnO4, and O3. Except ozone, all others are harmful to environment. The new challenges of starch modification require a safer method for both manufacturer and environment. Electrolysis treatment, an electrochemical method, could be useful for starch oxidation [Main reagents used for starch oxidation are NaOCl, Hxidation . This meUnder electrolysis, the extent of oxidation can be affected by many factors including NaCl concentration, input voltage, input ampere, distance of electrodes, and reaction time –5. On thϕ=12cm). Initial starch slurry was around 6.0. Two titanium electrodes (anode and cathode), which were coated with a mixture of RuO2, IrO2, and TiO2 for prevention of corrosion, were flooded in this suspension. These electrodes have a distance of 10 cm and were supplied by a 10V and 3A DC electric current. The treatment was conducted for 1 h at ambient temperature (30°C) with lightly continuous mixing by a magnetic stirrer. After treatment, the electric current was off and starch suspension was adjusted to pH 7 (by 1M HCl). Starch suspension was washed (three times) with 3-fold volume of distilled water and then centrifuged . The washed starch sediment was dried  to reach the final moisture content . CN0, CN0.5, CN0.75, CN1, CN2, and CN5 were treated starch in 0-5% NaCl solution.Cassava starch (400g) was suspended in 4.0 liters of NaCl solution  in a cylindrical container  following the method of Singhal and Kulkarni . Starch ηi, ml/g) had a relationship to the average molecular weight of starch that was dissolved in an alkaline solution. Starch was dissolved in 1M KOH to make a solution of 1.0 to 5.0 mg/ml. Kinematic viscosity  was measured using an Ostwald viscometer  at 30-70°C. The relative viscosity (ηrel)= η/ηo . The reduced viscosity (ηred)= (ηrel -1)/c (c is the concentration of starch in KOH solution). Intrinsic viscosity (ηi)=limc→0ηred. Based on the Staudinger-Mark-Houwink equation, the average molecular weight (M) of starch was calculated as ηi=KMα  [Intrinsic viscosity ( α=0.89) .−1 to 4500 cm−1 . Starch (2.0 g) and KBr (0.2 g) were mixed well and pressed at 8.0 bar for 10 min before measurement.FTIR spectra were measured following the method of Kizil R.  at the wα radiation of 0.15406 nm . Each scan was performed from 5 to 30° (2θ) [c was the area of crystalline portion and Aa was the area of amorphous portion.XRD was determined using a powder X-ray diffractometer . The operating conditions were 40 kV and 40 mA with Cu-K30° (2θ) . DRC wasColor of sample was measured using a Minolta CR-400 Chroma Colorimeter. The difference of color was calculated following a previous method .Experiments were conducted in triplicate, and the mean value was reported. Data were analyzed using analysis of variance (ANOVA), and the mean separations were determined by Duncan's test (p<0.05). All statistical analyses were carried out using SPSS software .In this study , both caScanning electron micrographs (SEMs) of native and modified starches are shown in −1 can be attributed to O–H and C–H bond stretching, respectively. The peaks of starch at 1650 and 1460 cm-1 were assigned as C–O–C stretching and C–H bending, respectively. The absorbances at 1150 and 1085 cm−1 are both assigned as the coupling of C–O, C–C, and O–H bond stretching, bending, and asymmetric stretching of the C–O–C glycosidic bridge. Absorbance at 1010 cm−1 is assigned to the vibration of C–O–H deformation, and absorbances at 918 and 846 cm−1 are assigned both for C–H bending [−1 has been considered sensitive to ordered  structure and the band at 1022 cm−1 has been associated with amorphous in starch. Thus, the ratio of intensity of 1047/1022 cm−1 could express the degree of order in starch [ bending . The IR n starch . In our ηi) and average molecular weight (M) of starches are shown in η and M. Thus, these results evidently proved the depolymerization of starch under treatment. The oxidative reaction promoted by electrolysis treatment caused partial degradation of glycosidic bonds and lowering of molecular weight that is reflected by the decrease in starch paste viscosity. Compared with hydroxyl groups, the presence of bulky carboxyl groups loosed the granular structure of starch and resulted in the decrease of the viscosity [Kuakpetoon D.S. Wang Y.J.  suggesteiscosity , 24.θ). Actually, the oxidation reaction caused by the electrolysis did not cause any change in X-ray pattern of starch samples [The X-ray diffraction spectra (XRD) and relative crystallinity degree (DRC) of starches are shown in  samples , 25. In  samples  and Kuak samples  reported samples  suggesteUnder treatment, starch samples were brighter  . The dif2, B3 amylopectin side-chains, and amylose molecules resulted in higher retrogradation [The syneresis tendency (retrogradation) of the starches was expressed as liquid liberated from their pastes after three freeze-thaw cycles . The valradation . Obviousradation .The solubility reflected the ratio of molecules leached from the starch granule after swelling, and the swelling factor expresses the ability of starch to hydrate under specific conditions . These pAt around 60°C, swelling factor of starch suddenly increased reflecting the intense hydration of sample . The higBoth the scission of the glycosidic linkages and oxidation of hydroxyl groups to carbonyl and carboxyl groups resulted under electrolysis treatment of granular cassava starch. Both carbonyl and carboxyl groups increased rapidly when increasing NaCl content in electrolysis reaction. These chemical changes led to the decreasing of swelling factor and paste viscosity and the increasing of paste clarity and solubility. Furthermore, oxidized starch enhanced syneresis during freeze-thaw storage. At low concentration of 0.5% NaCl, electrolysis treatment caused the increasing of crystallinity. Moreover, higher concentration of NaCl resulted in the disruption of crystal structure. However, A-type X-ray pattern remained during treatment. Besides, the treatment caused rougher surface and large holes on the surface of starch granules. Thus, with the above-described characteristics, electrolyzed starch can find many different applications in industries.


Exp. 1 and 2); 2) To assess effects of partially replacing corn with glycerin in beef cattle diets on ruminal fermentation pattern (Exp. 3 and 4); and 3) To evaluate the effects of glycerin inclusion on the extension of ruminal DM digestibility of feeds with high (orchard hay) and low (corn) fiber content (Exp. 5). For Exp. 1 and 2, two in vitro systems (24-bottle AnkomRF and 20-serum bottles) were used in four consecutive fermentation batches to evaluate gas production (GP), fermentation profiles, enteric methane (CH4), and carbon dioxide (CO2) of corn, glycerin, and starch. The 24 h total GP, acetate concentration, and acetate: propionate ratio decreased only when glycerin was added to the diet (P < 0.01). The 48-h total GP and metabolizable energy were greatest for corn (P < 0.01), and similar between glycerin and starch. The starch treatment had the lowest total volatile fatty acids concentration (P = 0.01). Glycerin had greatest CH4 production, lag time, and maximum gas volume of the first pool (P < 0.05). However, the maximum gas volume of the second pool was greatest for corn (P < 0.05), and similar between glycerin and starch. The starch treatment had the greatest specific rates of digestion for first and second pools (P < 0.05). Production of CO2 (mL/g) was greater for corn (P < 0.01), but similar for glycerin and starch. For Exp. 3 and 4, the same systems were used to evaluate four different levels of glycerin  replacing corn in beef cattle finishing diets. Glycerin levels did not affect 24 and 48 h total GP, CH4, and CO2 (P > 0.05). The inclusion of glycerin linearly decreased acetate concentration (P = 0.03) and acetate: propionate ratio (P = 0.04). For Exp. 5, two DaisyII incubators were used to evaluate the in vitro dry matter digestibility (IVDMD) of the following treatments: orchard hay; corn; orchard hay + glycerin; and corn + glycerin. Glycerin inclusion decreased orchard hay IVDMD (P < 0.01) but did not affect corn IVDMD (P > 0.05). We concluded that, under these experimental conditions, glycerin has similar energy efficiency when used in replacement of corn and included at up to 300 g/kg in beef cattle diets.Five in vitro experiments were conducted with the following objectives: 1) To evaluate the ruminal fermentation of three different single ingredients: corn, glycerin, and starch ( The expansion of the biodiesel industry has increased glycerin supply  making i4) production , and starch. Ingredients were incubated in four 48-h fermentation incubations to assess the in vitro GP profiles . Steers were maintained on a total mix diet composed of 400 g/kg orchard grass hay, 470 g/kg dry ground corn, 100 g/kg soybean meal, and 30 g/kg mineralized salt [dry matter (DM) basis]. Two h after feeding, 2000 mL of rumen fluid were collected, immediately filtered through 4 layers of cheesecloth and kept into pre-warmed thermal containers and transported to the lab.For °C and purged continuously with nitrogen (N2) for 30 min. Resazurin was used as color indicator to control the buffer pH and N2 saturation . The rumen fluid was mixed with the buffer solution (1:2 v/v) in water bath at 39°C under anaerobic conditions by flushing N2  x 100 / (W2 x DM / 10)) x 10, where: W1 = bag tare weight, W2 = sample weight, W3 = final bag weight after in vitro incubation, C1 = blank bag correction , DM = g/kg dry matter.Corn and orchard hay were individually weighed (0.4 g/bag) into filter bags , which were heat-sealed and placed into the digestion vessels. Each vessel received 6 bags of one of the treatments plus 2 bags with no samples (blanks) and 2000 mL of rumen/buffer solution. Then, jars with glycerin treatments were inoculated with 1.0 g of glycerin. After inoculation, vessels were closed and then placed into the incubator with temperature at 39.5d for DM . The IVDAll the ingredients used in this study were ground through a 2-mm screen  for all incubations and analysis performed. Samples were analyzed for DM (method 934.01), ash (method 938.08), crude protein , and ether extract  according to AOAC . The OM P > 0.05). For Exp. 1 through Exp. 4, data were collected and analyzed according to a randomized complete block design using mixed models methodology with ingredients considered fixed factors , and fermentation batch as random factor (3 degrees of freedom for Exp. 1 through Exp. 4). For Exp. 5, data were collected and analyzed following a replicated 4 x 4 Latin square design. All statistical procedures were carried out using SAS 9.4 for Windows  with significance declared at P ≤ 0.05, through MIXED and GLIMMIX procedures. For Exp. 1 through Exp. 4, experimental units were considered to be the mean of the bottles within ingredients/diets in each fermentation batch (true replicate), whereas for Exp. 5, experimental units were considered to be the jars within incubators in each fermentation batch (true replicate). For total GP over time data, logistic nonlinear functions for two pools and a discrete lag [Exp. 1; corn, glycerin, and starch) as well as diets , in order to compare possible differences in fermentation profiles. Parameters of the nonlinear functions were then compared by means of sum of squares reduction test and differences were declared at P ≤ 0.05.All results were tested for normality  and follrete lag  were adjExp. 1, the first 24 h of total GP (mL/g DM) indicated that corn and starch did not differ, but glycerin had the lowest values . The lag time was longest for glycerin (P < 0.05), while corn and starch did not differ , but smallest second pool . Starch, corn, and glycerin reached the maximum gas volume of the first pool at 600h, 800h, and 1830h after the beginning of the fermentation, respectively. Starch had intermediary values for both pools whereas corn had a smaller V1 but a greater V2 (P < 0.05). Specific rates of digestion for first and second pools (C1 and C2) were faster for starch (P < 0.05) but similar for corn and glycerin. For Exp. 2, glycerin had the greatest CH4 (mL/L and mL/g) production (P < 0.01), but the lowest CO2 (mL/L) production , but similar for glycerin and starch.For t differ . Also, g3-N compared to glycerin and corn (P < 0.01). Starch had lower total VFA concentration compared to glycerin (P = 0.01) although did not differ from corn. Acetate was lowest for glycerin (P < 0.01) whereas propionate was lowest for corn (P < 0.01). Glycerin had the greatest butyrate and valerate concentrations (P < 0.01) although had the lowest (P < 0.01) acetate: propionate ratio, iso-butyrate as well as iso-valerate molar proportions; followed by starch and corn, respectively. For Exp. 2, starch also had lower pH than glycerin and corn (P < 0.01). Tested ingredients had no effect on total VFA (P = 0.50), propionate (P = 0.86), iso-butyrate (P = 0.10), and iso-valerate (P = 0.08) concentrations. Acetate concentration (P < 0.01) and acetate: propionate ratio (P = 0.01) were greater for corn and starch, whereas butyrate and valerate concentrations were greater for glycerin (P = 0.01).The effects of ingredients on ruminal variables are presented in Exp. 3, glycerin levels did not affect total GP, both at 24 h and 48 h incubations . The second pool however, had increased GP as the proportion of glycerin increased, especially when levels went from 100 to 300 g/kg of glycerin inclusion (P < 0.05). For Exp. 4, glycerin inclusion did not affect productions of CH4 in mL/L and mL/g of DM, and CO2 in mL/L and mL/g of DM (P > 0.05), which averaged 6.01 ± 0.97 mL/L, 9.65 ± 3.73 mL/g DM, 30.0 ± 3.31 mL/L, 48.0 ± 9.64 mL/g DM, respectively.For Exp. 3, glycerin levels did not affect pH, NH3-N, and total VFA (P > 0.05), which averaged 5.85 ± 0.21, 28.8 ± 4.33 mL/g DM, and 6.64 ± 1.84 mM/g DM, respectively. The inclusion of glycerin linearly decreased acetate concentration (P = 0.03) and acetate: propionate ratio (P = 0.04). Additionally, increasing levels of glycerin increased valerate concentration (P < 0.01). Also, glycerin inclusion did not affect propionate, butyrate, iso-butyrate, and iso-valerate (P > 0.05), which averaged 27.1 ± 2.09, 23.3 ± 2.41, 2.98 ± 0.36, and 7.27 ± 0.68 mol/100mol, respectively. For Exp. 4, glycerin inclusion also did not affect final pH, and total VFA (P > 0.05), which averaged 5.33 ± 0.13, and 20.5 ± 9.06 mL/g DM respectively. A linear decrease in acetate concentration (P = 0.04) was observed due to inclusion of glycerin in the diets. The inclusion of glycerin linearly increased valerate concentration (P < 0.01). Glycerin levels did not affect the concentrations of propionate, butyrate, iso-butyrate, iso-valerate, as well as acetate: propionate ratio (P > 0.05), which averaged 27.4 ± 3.95 mol/100 mol, 29.0 ± 2.80 mol/100 mol, 2.24 ± 0.34 mol/100 mol, 5.37 ± 0.87 mol/100 mol, and 1.13 ± 0.29, respectively.The effects of glycerin inclusion in beef cattle finishing diets on ruminal variables are presented in P < 0.01; The inclusion of glycerin with orchard hay decreased IVDMD  do not have CP. Furthermore, the ME observed in this study was calculated considering that 1000 g/kg of glycerin was fermented in the rumen. Rémond et al.  observed4 and CO2 production because its fermentation leads to more propionate production than starch [4 formation by changing the overall electron balance in the rumen. Furthermore, there is no CO2 formation in pathways to propionate production (2 (mL/L) production compared to corn and starch. However, the greater CH4 production for glycerin, compared to the other treatments, contradicts our hypothesis. An explanation for this result is the fact that propionate formation from glycerin does not act as a hydrogen sink [2, and CO2 balance of each one. Each mol of glycerin enters the glycolysis pathway as D-glyceraldehyde 3-P, releasing one mol of NADH + H+ and consuming one ATP. Therefore, to produce VFA, glycerin releases more hydrogen than hexoses and contributes more than corn or starch to increases on CH4 production. The greater butyrate concentration when glycerin is fermented also may explain the greater CH4 production for this treatment. Butyrate formation releases more reducing equivalents than propionate, and contributes to the enhancement of methanogenesis  was included at up to 150 g/kg (DM basis) in forage-based diets. However, the in vitro productions of CH4 and CO2 were unaffected by glycerin inclusion using ruminal inoculum from adapted and non-adapted donor animals [4 production was not affected when glycerin was added at up to 210 g/kg of DM in an in vitro system. Avila-Stagno et al. [4 production when lambs were fed diets containing up to 210 g/kg (DM basis) of glycerin. Others have observed a reduction in CH4 and CO2 productions in Nelore steers when crude glycerin was included up to 300 g/kg (DM basis) in the diet [4 production. Nevertheless, the lack of differences in VFA concentration, total GP, and CH4 and CO2 production in this study indicates similar energy efficiency when glycerin replaces corn in beef cattle finishing diets.The lack of effects for ruminal variables may also be the reason for partial dietary replacement of corn with glycerin did not have affect CHoduction . Thus, to et al. , the pre animals . Similar animals  observedo et al.  also notResults from this experiment confirm our hypothesis that glycerin addition would not impact the corn IVDMD. However, adding glycerin reduced the IVDMD of orchard hay. These results suggest suppression in fiber digestibility when glycerin is added in forage feedstuffs. In the study by Roger et al.  an inhibCompared to starch, glycerin had slower rate of degradation but similar 48 h total gas production. Furthermore, glycerin and starch had similar metabolizable energy, which suggests that glycerin may be used as alternative energy source in beef cattle finishing diets.4 production were observed when glycerin was included in beef cattle finishing diets. Therefore, our results suggest that glycerin has similar energy efficiency when used in replacement of corn and included at up to 300 g/kg in beef cattle finishing diets.Despite lower dry matter digestibility when glycerin was added to orchard hay-based diet, no negative effects on ruminal fermentation, total gas production, and enteric CHS1 Table(XLSX)Click here for additional data file.