Fecal microbial transplantation (FMT) has been used with the therapeutic intent to change the functions of the gut microbial community in metabolism and host immunity. For most of these therapies, the recipients are not given antibiotics to eliminate the microbial community prior to transplant with donor fecal microbes resulting in the initial gut microbial community following FMT consisting of a consortium of donor and recipient microbes. The detailed analysis of the fecal samples from these FMT over time provides a unique opportunity to study the changes in the gut microbial strain community that occurs following the introduction of new microbial strains (donor) into an established community (recipient).Bacteroides vulgatus post FMT, we used BLAST+ to analyze the data sets for Bacteroidales-specific antimicrobial proteins (BSAP-3) that have known functions to restrict species specific replication.In this study, we have metagenomic data set consisting of 5 FMT that contained donor, recipient and recipient post FMT taken multiple times for periods up to 535 days after the FMT. We used two established strain tracking methods, Window-based Single Nucleotide Variant (SNV) Similarity (WSS) and StrainPhlAn, to determine the presence of donor and recipient microbial strains following FMT. To assess recombination between donor and recipient strains of Alistipes onderdonkii, Alistipes shahii, Alistipes putredinis, and Parabacteroides merdae, all had patterns post FMT consisting of either dominant donor or recipient microbial strains in the feces. In contrast, the analysis of Bacteroides spp. in five FMT pairs revealed inter-individual oscillation over time with the appearance of either donor or recipient fecal strain dominance. In some instances, B. vulgatus and B. uniformis were also identified after FMT that were not related to either the donor or recipient. Finally, in one of the FMT, we identified a distinct B. vulgatus strain post-FMT that matched the pre-FMT strain but was BSAP-3 positive, suggesting a possible recombination event between the donor and recipient strains.We found that The complex oscillating patterns of the appearance of fecal dominant donor, recipient or unrelated strains following extended times post FMT provide new insights into the dynamics of the microbial community interactions with the recipients following FMT. The result from our analysis has implications for the use of FMT to predictably change the biological functions of the gut community in metabolism and host immunity. Clostridium difficile [C. difficile patients that have had suppressive antibiotics that FMT can result in the stable long-term colonization of donor microbial strains for up to two years post FMT, the longest time examined [Numerous studies have shown fecal microbial transplant (FMT) to be highly effective for the treatment of patients with recurrent ifficile –3. For tifficile –7. To beifficile –11. We, examined ,8.FMT has been also used with therapeutic intentions to change the biological functions of the gut microbial community in chronic diseases that impact metabolism and host immunity –15. The In the current study, we have further investigated the patterns of change in the diversity of donor and recipient microbial strains in recipients following FMT not pre treated with antibiotics using a data set from a recent study that used FMT to enhance immunotherapy using anti-PD-1 . In contWe obtained the publicly available original sequence files from the NCBI BioProject database under accession number PRJNA672867. All participants’ samples were collected, sequenced, and deposited by Davar et al. . The seqWe used publicly available data set, Davar et al. , to perfhttps://github.com/lh3/seqtk). The number of subsampled reads for all samples were shown in A total of 1,398,692,232 metagenomic sequencing reads were downloaded from Davar et al. . Due to Taxonomic profiling for all samples was conducted using MetaPhlAn2 ,21, howeBacteroides uniformis for two selected donor-recipient pairs  and for Bacteroides vulgatus for three selected donor-recipient pairs  using StrainPhlAn with default parameters and with the options ‘-relaxed_parameter3, -marker_in_clade 0.1’ [B. uniformis and B. vulgatus were visualized using the neighbor-joining method along with the Maximum Composite Likelihood [Our WSS strain-tracking analysis was conducted for all donor-recipient pairs and the full details of the WSS methods can be found in our previously published papers ,22–26. Wade 0.1’ . As a coade 0.1’ . The reskelihood  in MEGA kelihood ,29.The details of the analysis for the genes encoding BSAP-3 can be found in Koo and Morrow . BrieflyIn a previous study, we have developed a microbial strain tracking analysis called Window-based Single Nucleotide Variant (SNV) Similarity (WSS) program to assess the relatedness of the microbes from two separate fecal samples . Using pAlistipes spp., Bacteroides spp., or Parabacteroides merdae for the days analyzed post FMT.Our first analysis used WSS strain tracking to track donor or recipient strains in the feces following two independent FMT . A consiAlistipes spp.. In contrast, for the Bacteroides spp., we found the presence of both donor and recipient strains after FMT. For example, for B. vulgatus we found that the recipient was fecal dominant for the first 28 days after the FMT and then the donor microbes were dominant from days 34 to 56. Interestingly, we found for both B. uniformis and B. vulgatus that the donor sample had a greater relative abundance than the recipient (We next examined a third FMT (donor 18–0014 and recipient 19–0024) in which samples were collected for 118 days post FMT . Similarecipient . Howeverecipient .B. uniformis from days 20, 41, 48, and 69 and B. vulgatus from days 69, 77, 83, 92, and 113 all had WSS scores higher than the starting WSS score of donor compared to the recipient pre FMT [B. uniformis, we found two distinct clusters on the phylogenetic tree one for donor related microbes (blue boxes) and another one for recipient microbes (purple boxes)  . ImportaB. vulgatus where we found many of the samples with high WSS scores related to donor and recipient where the donor and recipient were in separate clusters on the phylogenetic tree  was then subjected to strain tracking analysis. This FMT was unique in that it had numerous samples that were collected and sequenced for up to 535 days post FMT . In commpost-FMT .B. uniformis, we found that early after FMT, up to approximately 383 days, a pattern of either donor or recipient dominant fecal strains. However, after day 383, we noted several instances where the WSS scores were higher, but below the cutoff value, when compared to both donor and recipient . In fact, at some times, the WSS scores were both above the cutoff value indicating relatedness, to both the donor and recipient . We used StrainPhlAn to further characterize the relationship of the B. uniformis strains  and the recipient (purple (note: only one sample was related to recipient R-D29)) clustered differently. (e.g. R-D 535) and another one related to the recipient (e.g. R-D514)  and the recipient (purple) were located in distinct clusters on the phylogenetic tree. Importantly, the samples with WSS scores above the cutoff for both donor and recipient were, for the most part, located in branches positioned in between the donor and recipient. A similar result was found for ecipient . Analysierently. . Within  R-D514) . CollectB. vulgatus at days 36, 42, 47, 56, 63, 69, 75, 90 and at two later times, days 342 and 383 had a WSS score of 80–86 compared to the donor and a WSS score of 1–6 compared to the recipient  that have known functions to restrict species specific replication of Bacteroides vulgatus. From this analysis, we found that the gene encoding BSAP-3 in the individuals B. vulgatus were either present or absent [In a recent study, we have characterized the HMP data set for the presence of genes encoding r absent . To furte.g., Alistipes spp., P. merdae). In contrast, for Bacteroides spp. we detected the coexistence of donor and recipient strains for extensive times post FMT. For two FMT pairs , fecal dominant recombinant strains of donor-recipient were evident post FMT. In addition, FMT pair , strain-tracking analysis revealed the presence of a new strain that was most probably a minor strain in the donor feces that emerged following FMT. We also provide evidence for recombination between the donor and recipient in one FMT. Collectively, our results provide a new perspective into the complexity of the microbial strain community in the gut ecosystem following FMT.In this study, we have analyzed an extensive time series data set that contains samples for some recipients up to 535 days post FMT. For some donor-recipient post FMT pairs, we found complete fecal dominance of certain donor or recipient microbes  and possessed the capacity to initially outcompete both the donor and recipient for fecal dominance. Interestingly, the fecal dominance followed by re-appearance with the same WSS score  after time supports that this strain effectively colonized the recipient post FMT. The oscillating patterns of fecal dominance of this strain after FMT could reflect differences in the recipient from the donor with respect to the availability of nutrients (e.g. diet) or microbe-microbe and host-microbe interactions in the new ecosystem that transiently favors this strain over the original donor and recipient [Finally, from analysis of FMT pairs donor 18–0002 & recipient 18–0018, we found that ecipient .Bacteroides spp., the mixed donor and recipient microbe communities (and in some instances even new communities of Bacteroides spp. recombination), could have a wider impact by altering the interaction between other microbes in the gut ecosystem needed for functions of the gut community in metabolism, colonization resistance and the immune system [Our time series analysis shows that in FMT where there was no pre-conditioning to reduce or eliminate the recipient microbe community that an individual specific oscillating pattern of donor, recipient and new microbial communities post FMT. The results from our study then provide an important, new framework in which to understand the lack of predictability of FMT to change the gut microbial community function to correspond to that of the donor ,39. Althe system –14. The e system ,8. In ade system .S1 TableThe original sequence files were sequenced and deposited by Davar et al. in the BioProject database under accession number PRJNA672867. The table represents the sample information with sequence read information used for the analysis in this study.(XLSX)Click here for additional data file.S2 TableMicrobial community composition was profiled at the species level for (A) donor 18–0002, recipient 18–0018, (B) donor 18–0014, recipient 19–0024, (C) donor 18–0031, recipient 19–0013, (D) donor 18–0006, recipient 19–0002, and (E) donor 18–0005, recipient 18–0007 from Davar data set. The number shown in the table shows the relative abundances obtained using MetaPhlAn2.(XLSX)Click here for additional data file.


Arabidopsis thaliana mutants that lack SUS activity in mesophyll cells (quadruple sus1234) or have no SUS activity (sextuple sus123456) have wild-type levels of ADPG and starch, while ADPG is 20 times lower in the pgm and adg1 mutants that are blocked in the consensus chloroplastic pathway of starch synthesis. We conclude that the ADPG needed for starch synthesis in leaves is synthesized primarily by ADPG pyrophosphorylase in the chloroplasts.Many plants accumulate transitory starch reserves in their leaves during the day to buffer their carbohydrate supply against fluctuating light conditions, and to provide carbon and energy for survival at night. It is universally accepted that transitory starch is synthesized from ADP-glucose (ADPG) in the chloroplasts. However, the consensus that ADPG is made in the chloroplasts by ADPG pyrophosphorylase has been challenged by a controversial proposal that ADPG is made primarily in the cytosol, probably by sucrose synthase (SUS), and then imported into the chloroplasts. To resolve this long-standing controversy, we critically re-examined the experimental evidence that appears to conflict with the consensus pathway. We show that when precautions are taken to avoid artefactual changes during leaf sampling,  Arabidopsis leaves, resolving a 20-year-old controversy about one of the most important pathways of photosynthetic metabolism.Mutant analysis shows that sucrose synthase makes no substantial contribution to transitory starch synthesis in  It is universally accepted that starch is synthesized from ADP-glucose (ADPG) and that in leaves starch is synthesized in the chloroplasts, primarily in mesophyll cells. However, there is disagreement about the subcellular compartmentation and pathway of ADPG synthesis. Studies on isolated chloroplasts4 and starch-deficient mutants of Arabidopsis thaliana11 led to a consensus view that ADPG is produced in the chloroplasts by ADPG pyrophosphorylase (AGPase), with its glucose 1-phosphate (Glc1P) substrate being derived from fructose 6-phosphate (Fru6P) that is withdrawn from the Calvin-Benson cycle in the light, and converted sequentially to glucose 6-phosphate (Glc6P) and then Glc1P by the plastidial phosphoglucose isomerase (PGI) and phosphoglucomutase (PGM), respectively. This pathway is consistent with the near absence of starch in source leaves of pgm and adg1 null mutants that lack plastidial PGM or AGPase, respectively7. ADPG and starch synthesis are thought to be restricted to the chloroplasts in leaves of all species and to the amyloplasts in non-photosynthetic tissues , except for the endosperm of cereals and other Poaceae13. Here the majority of ADPG is synthesized by a cytosolic AGPase and imported into the amyloplasts via BRITTLE1 (BT1)-type ADPG transporters, probably in exchange for AMP19. In barley (Hordeum vulgare) and rice (Oryza sativa), alternative splicing of the AGPS small subunit gene in different tissues leads to expression of a chloroplast-targeted AGPS polypeptide in leaves, while the AGPS polypeptide expressed in the endosperm has no plastidial transit peptide and is located in the cytosol21. In maize (Zea mays) endosperm, the cytosolic AGPase derives from gene duplication and neo-functionalization during tetraploidization22.Many plants accumulate starch reserves in their leaves during the day, which are remobilized at night to provide carbon and energy for survival at night29. In addition to uridine diphosphate (UDP), the preferred substrate, SUS can use ADP as an alternative substrate, producing ADPG instead of UDPG as a product of sucrose cleavage30. The proposed SUS-mediated pathway of ADPG synthesis was based on two lines of evidence. First, if the consensus pathway operates, levels of ADPG and starch would be expected to be much lower in leaves lacking PGM or AGPase (the pgm and adg1 (aps1) mutants, respectively) than in wild-type (WT) leaves. This was claimed not to be the case: ADPG levels in pgm and adg1 mutants were reported to be similar to those in WT plants29. Second, in an attempt to discover the effects of reductions in ADPG levels on starch synthesis in either the chloroplast or the cytosol on starch synthesis, Baroja-Fernández et al.24 expressed an adenosine diphosphate sugar pyrophosphatase (ASPP) from Escherichia coli in one or the other of these compartments in WT plants. This enzyme cleaved ADPG into Glc1P and AMP, but could also hydrolyze other ADP-sugars, with ADP-ribose being the preferred substrate31. Expression of ASPP in chloroplasts had little effect on ADPG levels, but reduced starch accumulation by 50%. Expression in the cytosol reduced ADPG levels by 30% and starch levels by 20%24. From these observations, it was argued that a pool of ADPG located in the cytosol is essential for normal rates of starch synthesis. As AGPase is strictly chloroplastic in Arabidopsis leaves33, Baroja-Fernández and colleagues proposed that SUS synthesizes ADPG in the cytosol, using ADP as the glucosyl acceptor in the sucrose-cleavage reaction, followed by transfer of ADPG into the chloroplast via a hypothetical transporter in the chloroplast envelope23. However, it remains possible that the reduction in starch levels in plants with cytosolic ASPP is a pleiotropic effect, brought about by ASPP-mediated changes in ADP-sugars other than ADPG.This consensus has been challenged by a proposal that ADPG is synthesized primarily by sucrose synthase (SUS) in the cytosol, and then imported into the chloroplasts for starch synthesis Fig. 23–29. In34 and Neuhaus et al.35. One of their major criticisms was that the SUS pathway, as originally proposed, did not explain the starch-deficient phenotypes of the pgm and adg1 mutants. In response to this criticism, it was proposed that a futile cycle of starch synthesis and degradation operates inside the chloroplast during the day, in parallel with synthesis and import of cytosolic ADPG. In the proposed futile cycle, starch is degraded hydrolytically to glucose, which is then converted via plastidial hexokinase, PGM and plastidial AGPase to ADPG for resynthesis of starch27. According to this scheme, loss of either plastidial PGM or AGPase would prevent recycling of the products of starch degradation and thus prevent starch accumulation. Such futile cycling would need to operate under all conditions and throughout the photoperiod to explain the starch-deficient phenotypes of the pgm and adg1 mutants.The proposed SUS pathway for ADPG synthesis has been critically discussed by Okita13C-labelling studies have shown that there is some turnover of starch in Arabidopsis leaves in the light, but this is limited to certain times of day and prevalent only in long-day conditions36. Second, Streb et al.37 introgressed the pgm mutation into null mutants for key steps in the major pathway of starch degradation in Arabidopsis leaves38: glucan, water dikinase , phosphoglucan phosphatase (SEX4), isoamylase3 (ISA3) debranching enzyme and the maltose transporter (MEX1). All of the resulting double mutants were as starch-deficient as pgm, consistent with a requirement for plastidial PGM in the primary pathway of starch synthesis. Third, even if the proposed futile cycling of starch and glucose salvage pathways were operating, the SUS pathway does not readily explain the starch-deficient phenotype of Arabidopsis plastidial pgi mutants9. Proponents of the SUS pathway argue that plastidial pgi mutants are starch-deficient because of a defect in the synthesis of isoprenoid-derived phytohormones  that perturbs their growth and seed production, rather than a simple metabolic block in the flow of carbon from Fru6P, an intermediate of the Calvin-Benson cycle, to ADPG39. Fourth, the SUS pathway also does not explain why triose-phosphate translocator (tpt) mutants, which are unable to export triose-phosphates for sucrose biosynthesis, accumulate more starch than WT plants in the light41. Likewise, mutants defective in the pathway of sucrose synthesis usually have elevated starch45. If ADPG were synthesized primarily from sucrose via SUS, all of these mutants would be expected to have less starch than WT. Furthermore, f2kp mutants that lack fructose-2,6-bisphosphate have enhanced rates of sucrose synthesis but less starch than WT plants46, not more as would be expected if ADPG and starch were derived from sucrose. Fifth, there is no experimental evidence that Arabidopsis leaf chloroplasts have the capacity to import ADPG from the cytosol. On the contrary, eudicot forms of the plastidial BRITTLE1 adenylate transporter, including the Arabidopsis orthologue AtBT1, have been shown to transport AMP, ADP and ATP but not ADPG, and Arabidopsisbt1 mutants have near-WT levels of starch47. These observations argue against import of ADPG via AtBT1 from the cytosol making a meaningful contribution to starch synthesis in Arabidopsis leaves.The proposal that futile cycling of starch occurs continuously in the light is called into question by several observations. First, recent sus1 sus2 sus3 sus4 (sus1234) mutant. This lacks all four of the SUS isoforms that are expressed in mesophyll cells and has little or no soluble SUS activity in the leaves, yet has WT levels of starch49. The main proponents of the SUS pathway confirmed the WT starch levels in the sus1234 mutant, but argued that the result was inconclusive because, unlike Barratt et al.48, they observed high soluble SUS activity in the sus1234 mutant. They suggested that SUS activity had been underestimated by Barratt et al.48 and that Arabidopsis might have other, cryptic forms of SUS50.A direct indication that SUS is not required for starch synthesis in mesophyll cells came from a study of a quadruple 48 also questioned whether WT Arabidopsis leaves have sufficient SUS activity to account for their rates of starch synthesis. This was based on a previous report that SUS activity in WT leaves (0.023 µmol min−1 g−1FW)51 was lower than the rates of starch synthesis typically observed under standard laboratory conditions (up to 0.1 µmol [Glc] min−1 g−1 FW). Bieniewska et al.51 measured SUS activity at 25 °C in the direction of sucrose synthesis by the production of [14C]sucrose from UDP-[14C]glucose and fructose. Baroja-Fernández et al.25 contended that SUS activity had been underestimated due to instability of the UDP-glucose substrate under the assay conditions. They reported an activity of 0.185 µmol min−1 g−1 FW in WT leaves, measured at 37 °C in the sucrose cleavage direction using a high-performance liquid chromatography (HPLC)-based assay for UDP-glucose and ADPG, which would be sufficient to mediate observed rates of starch synthesis. The basis for these claims was in turn questioned by studies showing that UDP-glucose is more stable at moderately alkaline pH than claimed by Baroja- Fernández et al.52. These conflicting reports leave open the question of whether there is sufficient SUS activity to sustain observed rates of starch synthesis via the proposed cytosolic pathway of ADPG synthesis.Barratt et al.Arabidopsis rosettes showed that ADPG co-localized with the chloroplastic markers, with 100% of the total ADPG being assigned to the chloroplastic fraction in a two-compartment model (chloroplast vs cytosol)53. When WT Arabidopsis rosettes were labelled with 13CO2 under steady-state photosynthetic conditions, the pool of ADPG was labelled more rapidly than sucrose, and had kinetics similar to those of Calvin-Benson cycle intermediates53. These labelling patterns are consistent with direct synthesis of ADPG from intermediates of the Calvin-Benson cycle in the chloroplasts, rather than via sucrose in the cytosol.In addition to the genetic evidence favouring the consensus pathway, there are biochemical data that conflict with the proposed SUS pathway. Non-aqueous fractionation of Arabidopsis mutants that completely lack SUS activity and report that these sextuple (sus1 sus2 sus3 sus4 sus5 sus6) mutants have near wild-type levels of ADPG and starch, and only mild growth phenotypes under standard laboratory growth conditions.Transitory starch is a major end product of photosynthesis, therefore uncertainty about its synthesis is a major obstacle to understanding the regulation of photosynthetic carbon metabolism and how the fixed carbon is allocated between storage and export for growth. To resolve the long-standing controversy surrounding the pathway of starch synthesis in leaves, we critically re-examine the main lines of evidence that ADPG is synthesized by SUS in the cytosol for transitory starch synthesis in the chloroplasts. Furthermore, we generated 29 claiming support for the SUS pathway. However, plant extracts contain many other types of nucleotide that absorb in the UV range54, and it is difficult to achieve baseline separation of ADPG from other UV-absorbing compounds, potentially leading to overestimation of ADPG. Likewise, HPLC-PAD, as used in Baroja-Fernández et al.25, cannot distinguish ADPG from any co-eluting molecules, so is also prone to overestimation of ADPG. HPLC coupled to tandem mass spectrometry (LC-MS/MS) offers much greater specificity and sensitivity than HPLC-UV or HPLC-PAD55, and has become the method of choice for assaying ADPG.HPLC with UV detection at 260 nm (HPLC-UV) or pulsed amperometric detection (HPLC-PAD) has been widely used to assay ADPG, including all but the most recent studyArabidopsis leaves are generally low (<5 nmol g−1 FW), with an estimated turnover time of <1 s in illuminated leaves56, suggesting that leaf sampling in the light must be done with great care to avoid artefactual decreases in ADPG content below the steady-state level before the tissue is quenched. To assess the sensitivity of ADPG to unintentional shading during leaf sampling, rosettes were harvested from WT, pgm and adg1Arabidopsis plants in the light either by pouring liquid nitrogen directly onto the plants, or by cutting the hypocotyl from below and then quickly transferring the cut rosette into liquid nitrogen while in the growth chamber, taking care not to shade the leaves at any time during the procedure. In parallel, rosettes were harvested from plants that had been deliberately shaded by transfer from 160 µE m−2 s−1 to <10 µE m−2 s−1 for 2–3 s. This brief shading treatment led to a large (94%) fall in rosette ADPG levels (measured by LC–MS/MS) compared with rosettes that had been harvested in the light without shading  conditions, and rosettes were harvested at the end of the night and at intervals during the day for metabolite analysis. There were no significant differences in Glc6P and Glc1P between WT and sus1234 plants  4  onwards  in all genotypes in the dark  in the two genotypes and remaining fairly constant throughout the day with no significant differences between WT and sus1234 plants  conditions36.WT, rds Fig. . Glc1P w WT Fig. . ADPG leark Fig. . Upon ilnts Fig. . In contely Fig. . The sus WT Fig. . The lag13CO2 under ambient CO2 conditions to compare the labelling kinetics of ADPG with UDPG. The latter nucleotide-sugar is the substrate for sucrose biosynthesis and potential product of sucrose cleavage by SUS in the cytosol. ADPG was labelled much more rapidly and more completely than UDPG, with very similar labelling kinetics to Calvin-Benson cycle intermediates  mutants by CRISPR/Cas9-mediated gene editing of the SUS5 and SUS6 genes in the sus1234 mutant background. The CRISPR /Cas9 (CRISPR-associated protein 9)-generated mutations in the SUS5 (sus5-1 and sus5-2) and SUS6 (sus6) genes gave rise to premature stop codons  lacked functional copies of all six ArabidopsisSUS genes.Based on localization of the SUS1–SUS4 isoformssus1234 and the two sextuple sus123456 mutants. Developing siliques were chosen initially because all six SUS genes are expressed in siliques and/or seeds, as visualized using the Plant eFP browser , sus1234516  and sus1234526  mutants , sus1234516  and sus1234526  mutants  conditions and rosettes were harvested just before dawn and at intervals during the day for metabolite analysis. In WT Col-0 plants, Glc6P, Glc1P and ADPG increased upon illumination 56. We used LC–MS/MS56 to assay ADPG with greater sensitivity and specificity than the HPLC-UV/HPLC-PAD methods that were used for most previous studies. We found that shading of the leaves of WT plants for just a few seconds leads to a rapid decrease in measurable ADPG content, down to levels that resemble those found in the starch-deficient pgm and adg1 mutants. Consequently, unless extreme care is taken during sample collection, ADPG levels are likely to be underestimated and therefore unrepresentative of the levels during steady-state photosynthesis. This issue may well have affected values presented in earlier papers, including some of our own.According to the consensus pathway, the starch-deficient pgm and adg1 mutants were <6% and <4% of WT, respectively, but the same as WT in the sus1234 mutant . In earlier work that employed the HPLC-UV assay for ADPG, these researchers reported that adg1 leaves also had ADPG levels comparable with those of WT leaves28. One possible explanation for this discrepancy between our results and those of Bahaji and colleagues is that their plants were inadvertently shaded during harvest, potentially leading to large losses of ADPG before quenching. Consistent with this idea is the fact that their reported values for ADPG in WT leaves (0.13 nmol g−1 FW)29 are substantially lower than the range we observed in WT leaves  Columbia-0 wild-type and the pgm5, adg17 and sus123448 mutant germplasm were from in-house collections.sus123456 mutants were generated by gene editing of the SUS5 and SUS6 genes in the sus123448 background using CRISPR/Cas9 and two pairs of guide RNA (gRNA) targets: SUS5—GAAATGACATCTGGATCGTT and TGTAGAACTTGGTGAATCTC; SUS6—GGTAAGGGTAGATATCGAAT andSextuple 63. The construct was introduced into the sus1234 quadruple mutant by Agrobacterium-mediated transformation using the floral-dip method64 to generate sextuple sus123456 mutants. The CRISPR/Cas9 construct was subsequently eliminated by crossing the sextuple sus123456 lines with the quadruple sus1234 parent. Progeny were screened by PCR to identify lines that were homozygous for the T-DNA insertions in SUS1–SUS4 and for gene-edited mutant alleles of SUS5 and SUS6  and day/night temperatures of 20 °C/18 °C. For the 13CO2-labelling experiment, plants were grown with a 12 h photoperiod (120 µE m−2 s−1 irradiance). Seedlings were also grown on agar plates containing 0.5× Murashige–Skoog medium.Plants were grown in a 1:1 mixture of compost and vermiculite in 6-cm-diameter pots with a 16 h photoperiod  for 5 min, an aliquot (180 µl) of the supernatant was desalted by centrifugation through a MicroSpin column  equilibrated with extraction buffer. Soluble SUS activity was determined in the sucrose cleavage direction by measuring the UDP-dependent production of UDPG from sucrose, with enzymatic determination of UDPG as previously described61. Reaction mixtures (225 µl) contained: 20 mM PIPES-KOH (pH 6.5), 3 mM MgCl2, 100 mM sucrose and 2 mM UDP, with UDP being omitted from blank reactions. Reactions were started by addition of 25 µl of desalted extract and incubation at 30 °C for 25 min. The reaction was stopped by addition of 250 µl 50 mM Tricine-KOH (pH 8.3) and heating at 100 °C for 2 min. UDPG was measured enzymatically by coupling to reduction of NADP+ and monitoring the increase in absorbance at 340 nm. The initial reaction mixture (500 µl) contained: 50 mM Tricine-HCl (pH 8.0), 4 mM MgCl2, 12.5 mM NADP+, 0.3 U glucose-6-phosphate dehydrogenase , 0.4 U phosphoglucomutase  and 400 µl of the SUS assay reaction. When the A340 was stable, UDPG was determined by addition of sodium pyrophosphate  and 0.2 U UDP-glucose pyrophosphorylase . All coupling enzymes were from Sigma-Aldrich.Finely ground frozen leaf tissue (20 mg) was homogenized in 1 ml of ice-cold extraction buffer as previously described55 and measured by ion-pair reverse phase LC–MS/MS56 or anion-exchange LC–MS/MS55 (with modifications as previously described66). Soluble sugars were measured in ethanolic extracts and starch was measured in the ethanol-insoluble residue as previously described67.Rosettes were rapidly quenched in the light by either pouring liquid nitrogen directly onto the rosettes, or by cutting the hypocotyl from below and rapidly transferring the cut rosette into liquid nitrogen in the light, taking care not to shade the leaves at any time. Phosphorylated intermediates were extracted using chloroform-methanol as previously describedArabidopsis Col-0 plants were pulse-labelled with 13CO2 (400 µl l−1) at an irradiance of 120 µE m−2 s−1, for 0, 1 or 20 min, and rapidly quenched with liquid nitrogen under ambient illumination as previously described53. Labelling was performed between ZT4 and ZT7. Isotopomers of nucleotide sugars and phosphorylated intermediates were quantified by LC–MS/MS53.Four-week-old WT https://www.microsoft.com) or Sigmaplot for Windows version 14.5 .Statistical analysis was performed using Microsoft Excel 2019 MSO (Further information on research design is available in the Supplementary InformationSupplementary Figs. 1–6 and Tables 2–4.Reporting Summary.Supplementary Table 113C-labelling data from wild-type Arabidopsis Col-0 leaves.


Osteopathic manipulative treatment (OMT) has been found to be effective in the context of premature infants. Nonetheless, no studies have investigated the immediate effects of OMT on heart rate variability (HRV). As altered HRV reflects poor or worsening newborn’s clinical conditions and neurodevelopment, should OMT improve HRV fluctuations, it could become a relevant intervention for improving the care of preterm newborns. Therefore, this study aimed to evaluate whether OMT could affect HRV. The study was carried out at the Buzzi Hospital in Milan. From the neonatal intensive care unit, ninety-six preterm infants  were enrolled and were randomly assigned to one of two treatment groups: OMT or Static Touch. The infants were born at 33.5 weeks (±4.3) and had a mean birth weight of 2067 g (±929). The study had as primary outcome the change in the beat-to-beat variance in heart rate measured through root mean square of consecutive RR interval differences (RMSSD); other metrics were used as secondary and exploratory analyses. Despite the lack of statistically significant results regarding the primary outcomeand some study limitations, compared to static touch, OMT seemed to favor a parasympathetic modulation and improved HRV, which could reflect improvement in newborn’s clinical conditions and development. Worldwide, 1 out of every 10 newborns is affected by prematurity, that is, before the completion of 37 weeks of gestation. Such a preterm birth entails several and serious complications that increase the risk of death: indeed, about one-third of global neonatal mortality is due to prematurity .The neonatal intensive care unit (NICU) represents a stressful environment due to the many procedures—some of which can be quite painful—performed to take care of preterm infants, which are extremely sensitive to various stimuli  due to their delicate conditions ,4. TheseIn NICU, the autonomic nervous system (ANS) is evaluated through the measurement of parameters such as oxygen saturation (SpO2) and heart rate (HR) to monitor and protect newborns’ fragile life . Indeed,Regarding both ANS development and the potential comorbidities due to prematurity, in the last few decades it has been shown that heart rate variability (HRV), the fluctuation of intervals between heartbeats, can be a reliable marker to estimate vagal nerve modulation, ANS maturation, and even the risk of developing life-threatening pathologies such as sepsis. Indeed, two recent reviews highlighted how HRV monitoring could be an effective tool for studying the development, growth, and clinical conditions of fetuses, newborns, and infants ,13. HRV HRV fluctuations emerge from the complex elaboration carried out by the central autonomic network (CAN), a brain network whose role is both to regulate the internal homeostasis and to orchestrate the organism adaptation to stressors ,17. MoreDue to the relationships between HRV and organism’s functioning just outlined, every therapy that can positively affect the newborn’s HRV becomes worth investigating since such intervention could, in the same way, positively affect the newborn’s clinical conditions ,13.In the last decade, many researchers have found that a specific kind of touch, that is, “gentle touch”, can reduce the stress experienced by premature babies. This type of manual contact can directly influence the children’s ANS and stress regulation: indeed, during or after a stressful event, gentle touch can reduce the amount of cortisol produced, dampen the sympathetic tone, and improve the general conditions in preterm infants ,22,23. ILack of maternal touch has instead shown negative consequences on infant’s response during social stress. In very preterm newborns, whereas maternal contact has been shown to buffer the stress response even in the presence of increased serotonin transporter gene (SLC6A4) methylation due to NICU-related stress, when gentle touch was absent, newborns experienced higher levels of negative emotions ,31.To optimally elicit C-tactile fibers is particularly important in infants, in particular if preterm, as they are central to neurodevelopment and growth since gestation  . The stiAmong the possible ways for eliciting C-tactile fibers, we can find osteopathic medicine, a system of manual diagnosis and treatment. Since the osteopathic techniques performed on newborns involve a light amount of force and movement, osteopathic touch shows some similarity with gentle touch and, therefore, could play a fundamental role in the clinical management of the premature newborn . Indeed,As of today, OMT could be recommended as adjuvant therapy within the NICU routine practice. Several studies showed positive influences of OMT in preterm newborns: in particular, a systematic review with meta-analysis showed OMT to be a safety procedure with the potential to reduce the days of prematures’ hospitalization . NeverthIn adults, it is known that OMT may lead to an increase in PNS tone, resulting in an increase in the amplitude of brain waves and a decrease in their frequency, an increase in skin temperature, and a decrease in HR, respiratory rate, muscle tension, and anxiety ,39. MoreBased on these assumptions, in this paper we investigated the biological effects that OMT may induce HRV changes in preterm infants. Specifically, we addressed whether OMT could elicit specific HRV modifications through several metrics that the literature has related to various physiological and pathological conditions ,13. TherWe chose to compare OMT with static touch, a kind of touch that, as a previous study revealed , is veryThis study was a randomized clinical trial with two intervention arms: (1) OMT and (2) Static Touch. Participants were allocated in the two groups based on an allocation ratio of 1:1.ClinicalTrials.gov .The study was approved by the local Research Ethics Committee (38657/2017), and the trial was registered on Whereas the registered study aimed to assess the changes in HR and SpO2, the present paper extends the data analysis by evaluating changes in several HRV metrics.From March 2019 to June 2019, preterm infants were recruited within 1 week of birth from the NICU of the Buzzi Hospital in Milan, Italy. Inclusion criteria were: to be born at Buzzi hospital; preterm birth, in particular, a gestational age (GA) between 28.0 and 36.6 weeks; absence of clinical  or congenital diseases. Before the baby’s enrollment, we obtained written informed consent from parents or legal guardians. During the study period, all participants continued their routine neonatal clinical care.As outlined in a previous publication , we coulA computer generated the randomization sequence, without stratification, in blocks of ten to allocate the enrolled infants in one of the two intervention arms. Randomization was performed and stored by the coordinating center, and the process was overseen by an information technology consultant. Thus, 100 preterm infants were randomly assigned to receive OMT (50) or Static Touch (50). Due to clinical complications, four infants in the Static Touch group were excluded before the intervention: therefore, the Static Touch group included 46 participants.The NICU professionals were not informed about the study’s outcomes, design, or patient allocation. Additionally, the statistician was blinded to the patients’ allocation and had no contact with the patients, osteopaths, or NICU staff.Only osteopaths knew about the patient assignment, but they played no role in the decision-making process regarding patient care.A pulse oximeter was used to monitor HR . The pediatric pulse oximetry probe was wrapped around the infant’s right foot’s dorsal aspect. The physiological signal was digitized and recorded at a sampling rate of 500 Hz using the New Life Box physiological recording system  in conjunction with the Polybench software . HR data was then output as a CSV file with a data point for every 0.5 s of recording.The babies were evaluated and treated during quiet sleep, when the HRV is predominantly affected by the PNS. Nonetheless, due to the babies being preterms, there could be a sympathetic influence even during quiet sleep . This maPreterms were subjected to a single 20 min protocol in which they received either OMT or Static Touch. Osteopaths with extensive experience (at least 5 years of experience in NICU) carried out the interventions.Each protocol session included the following: (a) a 5 min baseline recording prior to the touch, (b) a 10 min touch procedure, and (c) a 5 min post-touch recording. The hands were placed in an incubator throughout the baseline period to match the infant’s skin temperature.The osteopath performed the manual assessment by standing beside the crib: the cranial hand was placed on the baby’s occiput, and the caudal hand on the sacrum. With both hands, the osteopath tried to cover the whole bone surfaces of the cranium and the sacral bone. This manual assessment lasted about a minute and aimed to recognize potential areas of restricted mobility. The assessment focused on the infant’s body’s compliance and homogeneity in response to manual light pressure, whether the body resisted the applied pressure and whether the tissue texture changed. The operator looked for postural asymmetries, deformities, strain patterns, and altered range of motion in several regions of the infant’s body, including fontanelles abnormalities, condylar compression, sacral torsion or flexion, intraosseous lesions, sacroiliac compression, pubic dysfunction, altered rib mobility or diaphragm functionality, and, for viscera evaluation, altered range of motion of tissues of the anterior area and the referred dermatomeric area of the column .Based on the obtained palpatory findings, the osteopath performed a personalized treatment designed to alleviate the restricted mobility detected in the baby’s body. This second stage lasted about 9 min. The techniques chosen by the osteopath, in particular, indirect techniques such as cranial, functional, and balanced ligamentous tension, have been previously shown to be safe for preterm infants ,49. It iThe researcher who performed the static touch intervention used their dominant hand by placing it on the baby’s back, in particular between the first thoracic and the last lumbar vertebrae. The static touch procedure lasted 10 min and, throughout this period, the researcher maintained their hand in that position with the approximate force (0.3 N) remaining constant. Static touch was not a therapeutic intervention, but a form of non-specific touch that professionals randomly performed when taking care of infants.Contact was always made with bare hands on bare skin. All preterm babies were placed in their cribs on their right side and remained in this position throughout the intervention. The right-sided position was chosen clinically because it was seen by NICU professionals as the most advantageous position to avoid interference from probes or tubes. The pulse oximetry probe was attached to the babies’ right foot, and then they were placed on their right side approximately two minutes prior to the start of the recording.Throughout the session, the fraction of inspired oxygen was kept constant. Additionally, any drug administration occurred at least three hours prior to the start of the experiment/treatment.From HR data, R-R intervals (RRI) were obtained through the formula 60,000/HR and then divided into three study periods, i.e., Baseline, Touch, and Post-Touch. Intervals were then imported in Kubios software to exclude artifacts, whether physiological or technical  and to cIn particular, both linear and non-linear metrics were extracted.Regarding linear metrics, root mean square of consecutive RR differences (RMSSD) and standard deviation of NN intervals (SDNN) were chosen among the time-domain metrics due to their connection with the ANS. Whereas several studies have shown RMSSD to reflect mainly the effects of vagal modulation on HR, SDNN seems to more reflect the global neuroanatomic regulation due to being influenced by both branches of the ANS, although sometimes  SDNN seems to be influenced particularly by the sympathetic division ,51. In fFor the HRV linear analysis, frequency-domain metrics were also extracted: in particular, low-frequency  and high-frequency  bands relative power (%) were obtained from power spectra through Fast Fourier transformation (FFT) of equidistant linear interpolated (4 Hz) tachograms (resampled to 2 Hz). Whereas the HF band reflects vagal modulation similarly to RMSSD and is affected by respiratory sinus-arrhythmia, the LF band reflects baroreceptors activity and is affected by both ANS branches .Considering non-linear analysis, the following metrics were used: sample entropy (SampEn), approximate entropy (ApEn) and detrended fluctuation analysis α1 (DFAα1). SampEn and ApEn are used to detect fetal distress  or, in iThrough the Kubios software, two composite metrics were also computed: the PNS index and the sympathetic nervous system (SNS) index. These metrics, which are derived through the computation of specific metrics as described in The primary outcome measure was the change in RMSSD, due to its correlation with vagal modulation .As secondary and exploratory outcomes, we chose to evaluate the changes in the other metrics reported in t-tests, whereas sex distribution between groups was compared using a Chi-squared test.The general characteristics of the two groups (OMT and Static Touch) were calculated and shown as mean (±standard deviation) for the numerical data and as absolute frequency (percentage) for the categorical data. Then, GA, weight at birth and Baseline HR were compared with independent samples To explore whether the extracted HRV metrics changed between groups over time, we carried out separate repeated-measures analyses of variance (ANOVAs), followed by the subsequent Tukey post hoc tests, with Group (OMT vs. Static Touch) as the between-factor and Period  as the within-factor.Lastly, we carried out a post hoc sample size estimation through power analysis through a Monte Carlo simulation, in order to evaluate the optimal sample size needed to find out a statistically significant difference in the primary outcome between the two groups. For running this simulation, we took the characteristics (mean and standard deviation) of our sample as reference points.Statistical significance was set for p e size estimation through power analysis through a Monte Carlo simulation, in order to evaluate the optimal sample size needed to find out a statistically significant differencand doParallel.Despite HR being lower in the OMT group, every measured outcome did not show statistically significant differences between the two groups at Baseline.Regarding the primary outcome, the OMT group did not show any statistically significant change in RMSSD values compared to the Static Touch group. The within-group analysis also showed a lack of statistically significant changes. Concerning the secondary outcomes, we did not find any statistically significant change in SDNN values either.p < 0.001). Indeed, during both the Touch period and the Post-Touch period, the OMT group showed a higher PNS index . It is worth noting that the within-group analysis showed statistically significant changes between the Touch period and the Baseline  and then no significant change between the Post-Touch and Touch periods for both groups.On the other hand, a significant influence of the osteopathic touch on the PNS index compared to the static touch was observed . Furthermore, whereas the OMT group showed a statistically significant change only between the Touch period and the Baseline , the Static Touch group showed an increase in the SNS index during the Post-Touch period compared to both the Baseline and Touch period .Almost specularly, the OMT group showed a lower SNS index during both the Touch period and the Post-Touch period, compared to the Static Touch group . Despite being non-statistically significant, these two results seemed to remain in the Post-Touch period. No differences were found regarding HF power . The same result, although non-statistically significant, was found in the SampEn analysis. Concerning instead the DFA metrics, the two groups did not differ in DFA1 values . Nonetheless, some interesting results were found. In particular, the two composite metrics aimed to specifically assess PNS and SNS modulation changed differently based on the kind of touch received by newborns: indeed, compared to static touch, OMT induced a parasympathetic modulation as revealed by an increase in the PNS index and a decrease in the SNS index both during and after the intervention. Moreover, concerning the within-group analysis, OMT elicited a parasympathetic modulation during the intervention compared to the Baseline, whereas static touch induced a particularly strong sympathetic activation, especially after the intervention had ended.Regarding the frequency-domain metrics, the decrease in LF power and the lack of HF power change could point to a more balanced sympathovagal regulation . On the As another useful intervention for improving the conditions and development of preterm newborns is skin-to-skin care, it is interesting to compare the present findings with the effect of maternal contact on HRV. On the one hand, higher frequency of maternal contact correlated with higher HF in newborns ; on the Concerning our study, such results might be interesting for two reasons: first, the skin-to-skin reduction in HR decelerations correlated with a decrease in RMSSD, SDNN, LF, and HF, and an increase in LF/HF ,71. AlthThis is the reason why, in recent years, several composite scores have been developed and machine learning algorithms are taking the lead in HRV analysis ,20,60,73This could be the reason why we found significant results through the PNS and SNS indexes, but not through RMSSD and SDNN. In fact, RMSSD is included in the computation carried out to obtain the PNS index together with a non-linear metric such as SD1 . On the The second reason why skin-to-skin results are interesting for the present paper is that our two groups showed a statistically significant difference in HR at Baseline, which is actually a serious limitation of our study since, as a consequence, our groups cannot be considered as homogeneous. Furthermore, as HR is included in the computation of both PNS index and SNS index, this difference could have strongly biased our statistically significant findings. Nonetheless, the OMT group showed a lower HR, that is, a possible higher parasympathetic tone at Baseline. Therefore, although this difference could have biased the change in the PNS index and other metrics, in light of the evidence regarding skin-to-skin care, we might argue that, compared to skin-to-skin contact, OMT has a greater potential of inducing a parasympathetic tone, even in those infants who already show a PNS predominance.Obviously, this is not to say that the faulty randomization does not have consequences: indeed, the higher peak in the SNS index shown by the Static Touch group could have been due to the already higher sympathetic tone of those infants. However, since the osteopathic touch induced a strong decrease in this index despite the lower HR at Baseline, only future studies with bigger samples and more accurate randomization could exactly point out how the heterogeneity between our groups have affected our findings.It is noteworthy that the Monte Carlo simulation we performed showed that the analyzed sample was too low to effectively catch significant differences in the primary outcome (RMSSD) between the two groups. In fact, we would have needed a sample at least double in size compared to the one used. Considering the other findings, it is highly probable that we would need a higher sample size for detecting significant differences even in the other measured outcomes.Interestingly, some findings were retained after the end of the intervention , whereas others became apparent only after the intervention . These results can have a twofold meaning. First, OMT might indeed affect ANS regulation in a permanent manner, thus enabling preterm infants to better regulate their clinical conditions. In fact, OMT seems to have an initial metabolic effect in infants, which is followed by the autonomic regulation effect only after that . Since CAs the second meaning, it could be useful to record whether the effects of OMT, or any other intervention, could last beyond the 5 min post-touch period assessed in the present study. Surely, due to the complexity of the clinical conditions of infants hospitalized in NICU and the many procedures that are administered to them, such long-term recording might prove difficult, both in actually doing and in interpreting. Nonetheless, it could be a source of useful data to understand the effects that interventions such OMT could have on infants’ autonomic regulatory capacities. Indeed, since NICU professionals touch infants hundreds of times per day to effectively take care of them , a manuaConcerning the precise effects of touch on infants, it is crucial to emphasize that there is a dearth of data regarding the effects of static touch and other types of touch on the baby’s state, i.e., quiet sleep, active sleep, or wakefulness. Although the literature indicates that these states involve varying degrees of autonomic functioning, as indicated by differences in HRV metrics , it is uLastly, we could argue that the present study added to the existing literature the fact that OMT indeed has an influence on the PNS, but more complex and accurate measurements are needed to highlight this effect. Despite the important limitations of the present study, in particular randomization and sample size, the analyzed data and the considerations put forth could help future research in defining more sound studies to detect the effects of OMT, or touch in general, on HRV metrics.Should OMT actually induce a parasympathetic modulation on HRV, or a more global HRV rebalancing, it could be speculated that OMT could positively affect CAP activation ,79. AlthThe current study failed to demonstrate a change in RMSSD in preterm infants due to osteopathic touch. The other analyses showed, instead, that the approach used within the OMT was associated with increased PNS index and a decrease in SNS metrics. These findings suggest that a single osteopathic intervention might benefit autonomic effects during the preterm period, but it is necessary to perform more accurate studies with bigger samples, especially since positive results would provide important new insights into optimizing modern perinatal care approaches.


Its chemical modification was confirmed by 1H and 29Si NMR and FT-IR. Interestingly, the POE blends with the m-HCR showed that the melting peak temperature (Tm) was not changed. However, Tm shifted to lower values with increasing m-HCR content after crosslinking. Additionally, the mechanical properties did not significantly differ with increasing m-HCR content. Meanwhile, with increasing m-HCR content in the POE blend, the peel strength increased linearly without sacrificing their transmittance. The test photovoltaic modules comprising the crosslinked POE blend encapsulants showed little difference in the electrical performance after manufacturing. After 1000 h of damp-heat exposure, no significant power loss was observed.In this study, polyolefin elastomer (POE) was blended with a chemically modified hydrocarbon resin ( High-quality and long-durability component materials are very important for photovoltaic (PV) modules to operate for long lifetimes of more than 20 years while reducing the lifespan cost of solar cells ,2,3,4,5.Thus far, ethylene-vinyl acetate copolymer (EVA) is widely applied as an encapsulant material for crystalline silicon-based solar cells because of its specific advantages such as high flexibility, good optical and mechanical properties, and field experience for more than 30 years ,9,10. UnRecently, polyolefin elastomer (POE) has been considered an encapsulant for PV modules because of its high transmittance, persistent bonding, and good creep resistance with small deformation ,23,24,25m-HCR) using γ-methacryloxypropyl trimethoxy silane (MTS) to obtain both the tackifier characteristic and adhesion to glass substrates. Then, the expected polymer blend series, POE/m-HCR with the additives of dicumyl peroxide (DCP), were prepared with a secondary master batch process. Lastly, this work addressed the feasibility of a POE encapsulant material as a design solution for PV modules in crystalline silicon-based solar cells.Therefore, this work focused on developing an adhesion-enhanced PV encapsulant material instead of the classical EVA commonly used for crystalline silicon-based solar cells. For this purpose, we designed and synthesized a chemically modified hydrocarbon resin  was purchased from the Dow Chemical Company . Hydrocarbon resin (HCR)  was generously donated by Kolon Industries . DCP and MTS were purchased from Sigma-Aldrich Inc. . Other organic solvents and chemicals were purchased from Samchun Chemicals Co. . The solar cells used in this study were based on multicrystalline silicon  produced by Hae Sung Solar Co., Ltd. .POE , and then 2 g of MTS and DCP (0.3 g) were added to the mixture. The mixture was stirred for 5 h at 120 °C under Nmpletely . The MTSm-HCR blend samples were mixed uniformly with DCP [by 2 phr (parts per hundred POE resin)]. The m-HCR contents in the POE blends were controlled as 3, 6, 9, and 12 phr, respectively. Melt compounding was conducted for 8 min at 105 °C and a rotation speed of 50 rpm. The uncrosslinked blend film was carefully prepared using a manual hot press under mild conditions (~110 °C), and the obtained film was cured in a manual hot press under 170 °C for 10 min for the instrumental analysis.Using a batch-type internal mixer , neat POE and POE/The gel contents were determined according to ASTM D2765-16. First, the crosslinked samples were placed in folded 120-mesh copper screen cages and, their weights were measured before immersion in xylene. The samples in the cage were extracted under refluxing for 6 h to remove uncrosslinked polymer parts from the blends. The residue specimens were dried at 120 °C to a constant weight and subsequently reweighted. The gel content was calculated using the following formula:The encapsulant laminator curing adopted in this work used an automatic laminating machine  for 18-cell multicrystalline solar cell-based PV modules. The sample was first loaded to the laminator (with a temperature of 140 °C and a pressure of 100 kPa) and kept in there for 300 s. Then, the pressure was sequentially reduced to 90 kPa (30 s) and 55 kPa (30 s) on top of the samples. Thereafter, the pressure was reduced to 25 kPa and kept for 600 s and was finally vented to atmospheric pressure. The stack structure of the PV modules was backsheet/encapsulant/silicon solar cell/encapsulant/glass. The fabricated PV module and its stacking diagram are shown in d1 (CDCl3) as a solvent. Additionally, Fourier transform-infrared (FT-IR) spectra were recorded on a Nicolet iS20 spectrophotometer  in absorbance mode under air conditions. The thermal characterization was studied using a DSC 1 differential scanning calorimeter . The scans were performed at a heating rate of 20 °C/min under a nitrogen gas atmosphere. A tensile test (ASTM D412) was performed using an Instron universal testing machine  equipped with a 1-kN load cell, and measurements were conducted at a constant crosshead speed of 500 mm/min. The tests were evaluated from the averages of at least five parallel tests. The peel strength for the POE—Glass interface was measured according to the ASTM 3330M method with a separation angle of 180° and a separation rate of 150 mm/min at 25 °C. For this test, glass (soda-lime glass) and a compound mode of POE blend film were laminated and cured together in the manual hot press under 150 °C at 0.1 MPa for 10 min. The characteristics of the encapsulated solar cell modules were measured using a Newport solar simulator . The current—Voltage (I–V) characteristics were determined using a digital source meter  under standard testing conditions: irradiance of 1000 W/m2 with an AM 1.5 G spectrum at room temperature. The samples for the damp heat test were placed in the chamber at 85 °C and 85% relative humidity (RH) (85/85 test).Nuclear magnetic resonance (NMR) for chemical structure analysis was performed on an Agilent 400 MHz NMR Magnet spectrometer  using chloroform-m-HCR was obtained through a simple radical grafting reaction using MTS, which could play a role as an adhesion promotor to a glass surface. FT-IR measurements were performed to confirm the chemical structure of m-HCR, and the acquired spectra for the neat HCR and m-HCR are shown in −1 attributed to the C–H stretching  group. From the results of the FT-IR, 1H NMR, and 29Si NMR spectra, we could conclude that the plausible grafting reaction of MTS on the HCR was performed successfully.f m-HCR , an isom-HCR are presented in Tc) of neat POE was detected at 37.0 °C, beginning at 27.3 °C and completing at 45.3 °C  due to the melting of monoclinic crystals [Tm of the POE blend samples showed values similar to that of the neat POE, implying that the uncrosslinked POE blends have no significant impact on the second heating thermogram shape and shifting of the exothermic peak. However, the DSC thermograms of the crosslinked neat POE and their POE blend samples showed lower Tm values than those of the uncrosslinked neat POE and their POE blend samples because of the increased content of shorter crystallizable chain blocks induced by the crosslinking reaction [Tm of the crosslinked POE blend samples increased slightly as the m-HCR content in the blends increased. This increase may be attributed to the reduced crosslinking density of the POE blend samples. Nonetheless, the Tc of the crosslinked samples was not changed. Meanwhile, the glass transition temperature (Tg) of all samples increased with increasing m-HCR content because the Tg of m-HCR is higher than that of the other polyolefins [Tg for all samples indicated that the phase morphology of the POE blend samples with m-HCR was homogeneous.DSC thermograms recorded during the cooling and second heating scan for neat POE and its blends with  45.3 °C . The Tc crystals  measuredreaction ,31. As syolefins . As we cm-HCR content on the gel content of the crosslinked neat POE and its blends are presented in m-HCR content. This decreasing trend was related to the decreasing degree of crosslinking. Therefore, the TMS grafting on the HCR should be a reasonable pathway to incorporating the coupling agent without any disturbance in the curing process.The effects of The UV/visible (UV/vis) transmission spectra of the crosslinked neat POE and its blend samples are presented in m-HCR are shown in m-HCR, the ultimate properties, and tensile modulus values were similar to those of the uncrosslinked neat POE. These results indicated that the mechanical properties of the POE matrix could be sustained with the addition of m-HCR. With crosslinking, the tensile modulus and elongation at break values showed trends similar to those of the uncrosslinked POE blends. However, the tensile strength values slightly decreased with increasing m-HCR content because of the decreased degree of crosslinking of the POE blends.The tensile properties of the crosslinked neat POE and its POE blends with The adhesion strength between the glass and the encapsulant film was measured with a universal testing machine using the 180° peel test according to ASTM 3330M. Peel adhesion strength is a very important factor in evaluating the bonding of different interfaces in the PV module to ensure its longevity. In commercial encapsulant materials, the profile of peel testing fluctuates because of the uneven peeling path between the encapsulant and glass surface . Therefom-HCR chain as grafted moiety in the POE blends with m-HCR, there was no need for additional grafting during lamination. With increasing m-HCR contents in the POE blends, the adhesion strengths increased linearly, indicating that the entire amount of the silane moiety added to the POE matrix promoted adhesion. These results showed that the improved adhesion strength can help prevent moisture diffusion and reduce the opportunities for interfacial debonding/delamination, hence improving the reliability of PV modules.According to previous reports ,37,38, fI–V curves of the PV modules encapsulated by the POE blends with m-HCR are shown in m-HCR are better candidates than the classical EVA encapsulant for module encapsulant applications.The damp-heat test is an accelerated test conducted at high levels of temperature and humidity and is an important test for screening applied materials to certify the longevity of PV modules for actual applications ,40. Thesm-HCR), which was prepared by radical reaction of MTS on the HCR. Using the MTS-grafted HCR (m-HCR), POE blend films were prepared by hot-pressing the blends after melt-mixing. The simple addition of m-HCR in the POE blends shifted their recrystallization temperature (Tc) to lower temperatures. However, Tc did not change even with increasing m-HCR content after crosslinking. Indeed, the Tg of all samples increased with increasing m-HCR content and appeared as a single value, which indicated that the POE blend samples with m-HCR were homogeneous in the blends. Even with increasing m-HCR content in the POE blends, the transmittance values were almost similar to those of EVA (90%). The mechanical properties of the POE blends could be sustained even with the addition of m-HCR. After crosslinking, the tensile modulus and elongation at break values showed trends similar to those of the uncrosslinked POE blends, but the tensile strength values slightly decreased with increasing m-HCR content. Additionally, with increasing m-HCR content in the POE blend, the peel strength increased linearly. This means that the modified polyolefin elastomer can effectively be used to adhere to glass in PV modules. The damp-heat test results of the PV modules with the POE blends showed no significant power loss. This implies that the performance of PV modules encapsulated with the POE blends with m-HCR could be sustained. Regarding this result, it can be inferred that this POE blend system can play the role of weather-resistant encapsulant for PV modules. Finally, the POE blends with m-HCR could provide a feasible solution for the development of high-quality and long-durability component materials for PV modules.In this study, a polyolefin encapsulant material designed for photovoltaic modules had been successfully constructed by compounding polyolefin elastomer (POE) with chemically modified hydrocarbon resin (


Aesthetic restorations should be able to mimic the natural colour depth of teeth, affected by several factors including material properties. There is a lack of information regarding the effect of cement shade and material thickness on the final colour of ultratranslucent multilayered zirconia veneers.  This study evaluated the effect of ceramic thickness and resin cement shade on the final colour of different layers of ultratranslucent multilayered (UTML) zirconia veneers. n = 9). Each adhesive resin cement (Panavia V5) was applied between the ceramic samples and composite substrate. The colour values were measured using a spectrophotometer in baseline and after resin cement application according to the CIELab system. For all samples, ΔE00 values were obtained. Data were evaluated with SPSS 25 using the three-way ANOVA test (p < 0.05).  This in vitro study produced 90 rectangular-shaped specimens with nonsintered Katana UTML monolithic zirconia , shade A1 blocks. Ceramic samples were prepared in two groups of 0.7 mm and 0.5 mm thicknesses, 45 of each . Specimens of each thickness were further divided into 5 groups: universal, clear, brown, white, and opaque (p < 0.001). The results showed lower ΔE00 values with thicker ceramic veneers. Tukey test results showed that the opaque and brown shade had a significantly greater ΔE00 values comparing to universal (p = 0.004), clear, and white shades (p < 0.001).  The factors of cement shade, ceramic thickness, and ceramic layers have statistically significant effect on ΔE00 values ( The colour change was greater in lower ceramic thickness. Different shades of resin cement and layers of UTML zirconia differently affected the final colour. Different material selection indications exist including direct versus indirect restorative combinations. Guidance is necessary to help dental professionals make the right decisions between available options to replicate morphology, function, and aesthetics .For the patients' satisfaction with aesthetic effects in anterior teeth, restorative materials whose optical characteristics mimic the natural teeth are necessary . CeramicThe increasing application of zirconia was witnessed due to its unique biocompatibility and biochemical properties . The maiRecently, to further improve the aesthetic properties of dental restorations, ultratranslucent multilayered zirconia systems have been introduced. Their microstructure and composition were modified to increase translucency. The aim of multilayered zirconia design is to mimic the shade gradient of natural teeth: where the incisal area of a crown is most translucent, increasing in chroma and opacity towards the gingival area. According to their unique properties, different grades of such zirconia are advocated for various indirect dental restorative applications. Thus, since its first introduction to the dental market, this multilayered monolithic zirconia system has drawn tremendous attention from clinicians and researchers –9.This product represents a remarkable improvement in minimally invasive monolithic CAD/CAM restorations. Ultratranslucent multilayered zirconia (UTML) can be utilized for veneer with a minimum thickness of 0.4 mm and needs simple adhesive cementation . Two cliThe mechanical and optical properties of 5Y-ZP (Katana UTML) are between those of 3Y-TZP and lithium disilicate, and the bond strength was comparable to those of lithium disilicate. The use of zirconia for full-arch framework would achieve similar mechanical results for implants and peri-implant tissues, in addition to similar vertical fitting . HoweverAn aesthetic restoration should fulfil the optimum shade matching with natural teeth to be clinically successful. Although the ceramic systems promote shade and translucency of restorations, a perfect aesthetic outcome remains unpredictable .Ceramic restorations can be aesthetically influenced by several factors such as underlying tooth colour, resin cement, structure and thickness of ceramic material, and preparation design , 16. PreAdditionally, adhesive cement and ceramic thicknesses have a major influence on the final shade of restoration in respect to the ceramic material and structure. The cement influences approximately ten to fifteen percent of the ultimate optical characteristics of all ceramic restorations , 16. AltFew studies evaluated the zirconia veneers, and there is little information regarding multilayered ultratranslucent zirconia. The final shade of a ceramic restoration made from monochromatic blocks would be similar for each layer after adhesive cementation due to its homogeneous structure, but multilayered blocks have variable colours and translucencies in each layer. Considering the gap of information regarding the possible effect of each layer on the final optical colour of ceramic restoration after the adhesive cementation in multilayered ultratranslucent zirconia, this in vitro study was aimed at investigating the effect of various cement shades and ceramic thickness on the final colour of CAD/CAM ultratranslucent multilayered zirconia ceramic veneers in different layers. The null hypothesis was that the ceramic thickness and cement shade would have no significant effect on the final colour of each layer of a CAD/CAM multilayered ultratranslucent zirconia ceramic.In this in vitro study, 90 specimens were produced with ultratranslucent multilayered monolithic nonsintered zirconia, shade A1 blocks . Rectangular-shaped ceramic samples were prepared in two thicknesses (0.7 mm and 0.5 mm), 45 of each group  utilizing CAD/CAM milling . Sintering was carried out in accordance with the manufacturer's instruction, and the surface of specimens was smoothed and polished with 1200-grit silicon carbide abrasive paper  Figure .Ninety composite resin specimens  were fabricated using rubber mold (8 × 11 × 5 mm) to make the background uniform .n = 9), and dual cure adhesive resin cement  in five shades of clear, universal, white, brown, and opaque was applied between the treated surface of ceramic specimen and composite resin substrate. Compressive pressure of 250 gr was applied for 10 s in order to obtain similar thickness of resin cement using universal test machine  2. The cement was irradiated with a light polymerization device  for 40 s in each layer of incisal, body, and cervical  was performed. After abrasion, ceramic surface was treated by a single bottle MDP-based adhesive primer . To simulate the clinical scenario for cementation, tooth primer was applied to the composite resin substrate surface  in accordance with manufacturer's instruction . 45 speccervical .To measure the baseline colour values, ceramic specimens were placed on a white background . The colAfter cementation process, colour measurement was performed for each cemented specimen in the same manner.One trained operator performed all spectrophotometer assessments. The instrument was calibrated in accordance with the manufacturer's instructions before and after each measurement. Measurements were made at 3 different points of the cervical, middle, and incisal layers using “tooth area” mode simultaneously, and the mean value was obtained for each portion both in baseline and after cementation. The difference in colour was calculated for each specimen using the ΔE00 (CIE DE2000) formula as follow:Η/ΚHSH)), ΔE00 = √((ΔL/KLSL)2 + (ΔC/KcSc)2 + (ΔH/KHSH)2 + Rt(Δc/KcSc) (ΔΔL∗ = L0 (baseline)–L1 (resin cemented), Δa∗ = a0 (baseline)–a1 (resin cemented), Δb∗ = b0 (baseline)–b1 (resin cemented).In this study, the threshold of ΔE00 < 2.25 was set as clinically acceptable colour change .p = 0.05.Data were presented as means ± SD. To analyse the effect of adhesive resin cement shade and zirconia ceramic thickness on ΔE00 values of different layers, three-way ANOVA and Tukey post hoc test was used. Kolmogorov-Smirnov test was used to assess the normality of ΔE00 values. All statistical analyses were conducted using SPSS for Windows 25.0 (SPSS). Statistical significance was preset at Mean ± SD of ΔE00 (CIE DE2000) values for all study groups are presented in p < 0.001). Moreover, ceramic thickness and cement shade interaction was statistically significant (p < 0.001).Based on the results of three-way ANOVA, ΔE00 values were significantly influenced by resin cement shade, ceramic thickness, and different layers (p < 0.001). According to the Tukey test, brown and opaque shades had significantly greater ΔE00 values comparing to universal , clear, and white shades (p < 0.001).For all shades (except white shade), ΔE00 values of 0.7 mm thickness were lesser than those of 0.5 mm thickness (p < 0.001). ΔE00 values were higher for incisal (3.74 ± 1.24) comparing to the other two layers, followed by the body layer (3.18 ± 1.21), with the lowest values for the cervical layer (2.65 ± 1.15) regardless of specimen's thicknesses.The obtained results represented a significant difference between ΔE00 values of different layers of ceramic  .L values decreased, and “a” increased. Although it is a translucent cement (11.2%), increasing “a” coordinate due to pigmentation lowers its translucency (L) and high ΔE00 values are observed. Chen et al. also demonstrated a significant increase in “a” value in brown colour [Barath et al. revealed that the shade of luting agent has a considerable effect on the final colour of translucent all-ceramic restorations . Since Un colour . Universn colour .L∗ value and brightness and causes less chroma [The results showed that ΔE00 values of opaque cement were significantly greater. Niu et al. and Chang et al. revealed that the opaque shade remarkably enhances the s chroma , 34. Coms chroma .Different studies showed that using opaque cements improves the final colour of restorations when the underlying structure shade is darker , 35, 36.Bayindir et al. showed that clear and opaque shades decrease the translucency parameter of translucent zirconia after cementation. However, TP values of the specimens of all thicknesses cemented with transparent shade were higher than those of cemented with opaque shade .Based on the results, the interaction of cement shade and ceramic thickness was significant. Xing et al. revealed that the effect of luting agent shade on the final colour of laminate veneers was associated with ceramic thickness .This study's results showed that various layers of multilayer ceramic significantly affected the ΔE00. The incisal layer showed the highest ΔE00 values due to lower colour pigment ingredients in this area compared to the body and cervical layers .L increased from DEL to ENL, while “a” and “b” values diminished across the same layer sequence [UTML zirconia used in the present study has 43% of light transmittance across layers. Four layers of DEL, FTL, STL, and ENL are presented in this zirconia ceramic. Harada et al. indicated that UTML has higher transmittance (Tt%) when compared with other types of zirconia . Moreovesequence . Pigmentsequence . Kolakarsequence .As previously investigated, different ceramic systems show different optical properties. Masking properties of different ceramics for veneer restorations depends on the translucency and thickness of material , 39. TheOne of the limitations of this study was that one shade of background composite was used. As it was mentioned, opaque resin cements can improve the colour in darker tooth shades and the underlying tooth structure may affect the final colour of restoration; therefore, further studies are needed to evaluate the effects of a wider range of underlying shades on the colour of ultratranslucent monolithic zirconia restorations. Since the samples tested in this study were rectangular-shaped rather than ceramic veneer restorations, the clinical conditions are not completely simulated, and it is suggested to conduct a clinical study to completely assess the final shade of ceramic veneers.In short, considering the limits of this investigation, the following conclusions were drawn: Firstly, the colour change was greater in lower ceramic thickness. Secondly, various shades of cement considerably affect the final colour of ceramic veneers. Finally, the layers of UTML zirconia differently affected the final colour.


Deutsche Zeitschrift für die gesamte gerichtliche Medizin . With the exception of a war-related interregnum from 1944 to 1948, the broadly scoped journal was published with this title until 1969. In 1970, responding to changes in the designation commonly used to describe the specialty and institutes of legal medicine, it became the Zeitschrift für Rechtsmedizin . In renaming it the International Journal of Legal Medicine in 1991, the publishing house and editor were following the growing trend towards academic internationalisation. Under the editorship of Bernd Brinkmann, International Journal of Legal Medicine has become the world’s leading journal of legal medicine and has maintained this top position ever since.100 years ago, Berlin publisher Julius Springer released the first volume of the The 135 volumes published to date reflect advances in legal medicine over the past 100 years. On the occasion of the anniversary, the amazing development of the journal will be honored in a four-part series of articles. Part 1 covers the period from 1922 to 1944, Part 2 from 1948 to 1969, Part 3 from 1970 to 1990, and Part 4 from 1991 to 2022. The articles analyze the major areas of focus of the journal during the respective period and place it in the context of the development of our discipline.Part 1 of the article series appears in this issue. We wish you interesting reading.


Starch and flour from various plants have been widely used for sundry applications, especially in the food and chemical industries. However, native starch and flour have several weaknesses, especially in functional, pasting, and physicochemical properties. The quality of native starch and flour can be improved by a modification process. The type of modification that is safe, easy, and efficient is physical modification using hydrothermal treatment techniques, including heat moisture treatment (HMT) and annealing (ANN). This review discusses the hydrothermal modifications of starch and flour, especially from various tubers and cereals. The discussion is mainly on its effect on five parameters, namely functional properties, morphology, pasting properties, crystallinity, and thermal properties. Modification of HMT and ANN, in general, can improve the functional properties, causing cracking of the granule surface, stable viscosity to heat, increasing crystallinity, and increasing gelatinization temperature. However, some modifications of starch and flour by HMT and ANN had no effect on several parameters or even had the opposite effect. The summary of the various studies reviewed can be a reference for the development of hydrothermal-modified starch and flour applications for various industries. Natural flours and starches from various plants generally have several weaknesses in their characteristics, which limit their application in various industries ,2,3. TheOther starch treatments have several advantages but also some disadvantages. Chemical modification can produce changes in starch molecules quickly and significantly but has the potential to leave chemical residues and is not environmentally friendly. Enzymatic modifications also result in rapid, specific, and significant changes in starch molecules, but controlling the process is relatively difficult ,12,13,14w/w) or moderate (40% w/w) water content at temperatures above the glass transition temperature (Tg) and below the initial gelatinization temperature (To), i.e., ±40–75 °C for a certain period [g and above the starch gelatinization temperature, namely ± 84–120 °C for 15 min–16 h [Hydrothermal modifications (ANN and HMT) are the type of physical modification that has the principle of using specific humidity, heat, and pressure levels ,28,29,30n period ,33,34,35min–16 h ,36,37.The positive impact of hydrothermal modification is an opportunity for the development of starch and flour-based products, especially for various food and chemical industries ,39,40. TThis review was carried out by collecting various articles using the Scopus, Web of Science, and Google search engines, using keywords of hydrothermal treatment, heat moisture treatment, annealing, from August 2021 to October 2022. The articles obtained were selected based on their suitability and were not included in the predatory journals.The functional properties of ANN and HMT hydrothermal-modified starch and flour are quite diverse, and they can be in the form of increasing or decreasing the parameter values. However, in general, both experienced a decrease in swelling power (SP) and solubility. The decrease in swelling power and solubility was related to the changes in the overall arrangement of starch granules. There was an improvement in the structure of amylopectin, amylose, and amylopectin levels in starch granules where swelling power increased with high amylose content and the intensity of intra- and interchain interactions ,42,43. TGenerally, HMT has a greater effect on the solubility and crystallinity of flour than starch. Sun et al.  reportedHydrothermal treatments (HMT and ANN) generally cause a decrease in swelling power and solubility in line with water absorption capacity and inversely with an increase in gel hardness. The less water absorbed by the starch molecules, the greater the starch gel hardness due to structural damage and cross-link interactions between starch chains , making the gel stiffer ,22,65. IThe decrease in swelling power and solubility values was shown by the modification of HMT ,55,61,62Changes in functional properties can also be affected by the applied cycle of hydrothermal treatments. As the modification cycle increases, the level of interaction between the starch chains also increases ,75. ThisThe decrease in swelling power and solubility was shown by ANN modifications and cyclical ANN. The modification of ANN made the crystalline structure stronger as the hydration level of the amorphous portion of the starch decreased ,32. ThisChanges in functional characteristics can also be affected by the applied cycle. Cyclic ANN modifications in sweet potato starch  and red Changes in the functional properties of starch will be indirectly related and reflected in the characteristics of the granules that can be identified through microscopic observations, which are known as the morphological properties of starch.Morphological properties of ANN- and HMT-modified starch granules showed a change, but some studies were not significant. Changes are seen in the size and irregularity of the starch granule surface: texture and the presence of holes, fissures, cavities, indentations, or cracks ,78,79. TChanges that occur in granule morphology tend to have a more significant effect on HMT-modified starch than ANN, which is proven to be dominant in combination modification ,83. ThisHMT modifications have different effects on starch and flour granules for each type of starch and treatment. Several factors affect the morphology of starch granules during HMT, including internal factors (amylose and amylopectin composition), as well as conditions before and during modification  ,88. ThesAggregation and agglomeration processes take place along with swelling and partial gelatinization during modification. These events cause morphological changes in the form of indentations, cracks, holes/gaps/cavities, irregularities, and changes in the size/shape of starch granules, so that the amorphous part of the starch granules becomes more compact ,79,89. HThe agglomeration process can also continue along with HMT modification cycles and times which cause disruption of starch granules . This isHMT modification also causes a decrease in brightness intensity; the starch hilum becomes darker, the edges of the starch granules become more opaque, and the growth rings are more difficult to detect. These changes also indicated a decrease in the starch crystallinity index. This decrease occurs due to the destruction of amylose-amylose and amylose-amylopectin chains during cyclical and continuous modifications. . This reAlmost the same thing happened to the modification of starch and flour by ANN. ANN modifications in potato, barley, lentil, oat, and wheat starch did not show significant morphological changes, but other high-amylose starches showed significant changes ,46,90. TThe effect of ANN modification on the morphological characteristics of starch can also be seen from its birefringence pattern. The relative ANN modification maintains the birefringence pattern. The results of the study that did not show changes in the birefringence pattern were found in ANN modifications for corn starch, lentils, and peas , cyclicaHowever, if other studies found different birefringence patterns, this indicates that the changes that occur in the starch granules are not always the same due to various factors, both internal and external, during different processes ,87,88. CThe pasting properties of various ANN- and HMT-modified starches showed very different results on the same parameters, either with or without the application of certain cycles. In general, hydrothermal modification causes a decrease in peak viscosity (PV) and breakdown (BD) ,91,92. TViscosity changes that occur are more significant in HMT-modified starch than in ANN ,40,82,86Apart from the changes in pasting properties, HMT and ANN showed better efficiency when applied with certain cycles rather than singly. Treatment cycles (a certain number of cycles) were more effective than single or continuous treatment in modifying starch ,56,57,99Changes in the pasting properties of starch due to modification are strongly influenced by the process of perfecting the crystalline part that occurs. This event can be studied further through an approach to starch crystallinity, which can be analyzed using X-ray diffraction (XRD) ,100.Starch crystallinity can be seen from the relative crystallinity (RC) index with an RC value of 15–45%. Starch crystallinity does not always show the same results because there are varying influencing factors, including the nature of the natural polymorph of the material, which includes amylose/amylopectin ratio, amylopectin chain length and branching, the mole percentage of amylopectin fraction/degree of polymerization (DP), crystal size, and interactions, and a constituent double helix (density and orientation) ,101,102.The degree of crystallinity greatly determines the type of starch crystallinity, which is divided into three types, namely types A, B, and C. The type of starch crystallinity , as shown by X-ray diffraction patterns, can be seen in ANN > RCNatural/HMT/Combination. Optimum ANN modification can produce an orderly and perfect structure. The choice of the type of modification affected the crystallinity of starch more significantly than the choice of treatment (cycles/continuous), although cycles/continuous treatments still had an effect on starch [Starch crystallinity index may increase, decrease, or change insignificantly with the main characteristic of hydrothermally modified starch, namely the type of crystallinity that is relatively fixed or transformed into type A ,106,107.n starch ,108. ChaThermal properties in this study focused on three parameters, namely gelatinization temperature, gelatinization temperature range (GTR), and heating enthalpy (ΔH). The characteristics of the three parameters from each study are not necessarily the same, even though the modified method used is the same. However, in general, ANN and HMT have similar thermal properties in the form of increasing gelatinization temperature ,109,110.The heating enthalpy parameter (ΔH) of ANN-modified starch could be an increase, decrease, or insignificant change, while HMT-modified starch generally decreased ,113,114.Likewise, the gelatinization temperature range (GTR) of ANN- and HMT-modified starch showed different results. The GTR of ANN-modified starch showed a decrease, while the HMT modification caused an increase in GTR. GTR is a parameter of the homogeneity of the starch granule structure. A decrease in GTR indicates a more homogeneous starch granule structure, and more even crystal melting occurs, while an increase in GTR indicates a more heterogeneous starch granule structure. The increase in GTR is also in line with the increase in gelatinization temperature. Both indicate an increase in the need for heat/temperature energy for the melting process ,106,108.Hydrothermal-treated starch and flour (ANN and HMT) can be applied to a variety of food products, especially those that require a stable product when exposed to heating/cooling and a strong texture. HMT- and ANN-modified starches can be used in noodles, baking products, frozen foods, canned foods, edible films, and other various products ,115,116.Hydrothermal treatments (ANN and HMT) in general have a significant effect on the physical and functional properties of starch, granule morphology, pasting properties, crystallinity, and the thermal properties of starch and flour. However, several studies have produced various characteristics depending on the source of starch/flour and the conditions of the modification process. The combination of hydrothermal modification with ANN and treatment cycles can increase the effectiveness and quality of modified starch products so that they are potentially suitable for application in various industries in the development of flour and starch from various plant sources.Hydrothermal-modified starch and flour have been widely used for various applications in the food, chemical, and pharmaceutical fields. However, modification of starch through hydrothermal treatments still has several weaknesses, such as instability in some physical properties, the appearance of cracks or wrinkles on the surface of the starch granules, and a darker color. Therefore, several additional treatments are needed to improve these characteristics. Further studies are needed to combine with other treatments, especially environmentally friendly ones, to improve the characteristics. These treatments include oxidation with ozonation, the use of vacuum pressure to reduce heat, cavitation energy through ultrasonication, irradiation with ultraviolet (UV) light, sublimation with freeze drying, and other treatments. Oxidation by ozonation can improve color brightness and solubility; vacuum pressure can improve process efficiency; ultrasonication can increase the porosity of the granule surface and improve process efficiency; UV light can increase solubility; and sublimation can increase the porosity of the granules and increase the solubility. In addition, the effects of several hydrothermal treatments can also produce porous starch granules, so that they have the potential to be developed into porous starch which can be used as an adsorbent, encapsulant, and fast-cooking starch.