The main challenge faced by cows during the first three weeks after calving is the sudden increase in nutrient demand for milk production while dry matter intake and nutrient supply lag. Although metabolic hormone changes are a normal adaptive process in high yielding cows, failure to adapt to this challenge can lead to disease states and affect production and reproductive efficiency. Therefore, a smooth transition from prepartum to postpartum period for optimal dairy cow health and performance. Whether rumen-protected niacin supplementation could be a useful additive was controversial. This work tested whether the widespread use of rumen-protected niacin in multiparous Holstein dairy cows could be justified.n = 12, parity = 3.5 ± 0.5, body weights = 740 ± 28 kg) were divided into two groups supplemented with either 0 (CON) or 20 g/d RPN (RPN). Our results showed that RPN supplementation increased DMI and milk production of cows during the first three weeks after calving (p < 0.05). The concentrations of neuropeptide Y and orexin A were significantly higher in RPN group than that in the CON group during postpartum period (p < 0.05). The apparent total-tract digestibility of nutrients was similar between the CON and RPN groups at 2 weeks after calving (p > 0.05). The 16S rRNA gene sequencing analysis showed that RPN had no impact on the alpha and beta diversity, although 4 genera were changed in cow feces at 14 days after calving. Overall, 20 g/d RPN added to the diet could improve DMI and milk yield up to two weeks after calving with little influence on feed digestibility.Extensive studies about rumen-protected niacin (RPN) supplementation on dairy cows in early-lactation have been done, but the effects of RPN on changes in dry matter intake (DMI), milk production, feed digestibility, and fecal bacterial community were conflicting. The aim of this study was to investigate them affected by RPN in postpartum cows. Multiparous Holstein dairy cows ( As for farmed animals, the health and biological functioning of dairy cattle are often prioritized. Pregnancy and postpartum are recognized as inducing remarkable physiological and metabolic adaptations in dairy cows essential for a good reproductive performance and to ensure the suitable development of the fetus and to provide adequate substrates that are needed in utero and following birth ,2,3. DesNiacin or nicotinic acid (NA) is a B vitamin and precursor of nicotinamide adenine dinucleotide phosphate and nicotinamide adenine dinucleotide, which are key coenzymes in many enzymatic reactions in intermediary metabolism, including lipid and glucose metabolism ,10. In dFeed intake in dairy cows may be controlled by many hormones such as neuropeptide Y (NPY) ,21 and oThroughout the experiment, all animals involved in this study were managed according to the herd standard protocol at the Sunlon Livestock Jinyindao Farm . All animal protocols were reviewed and approved by China Agricultural University, Institutional Animal Care and Use Committee .p > 0.10) among groups at the beginning of the study. The cows were dried off on d 60 before calving and housed in an individual pen that includes comfortable bedding and dry lot. During the trial, the total mixed ration (TMR) of the dry cow and lactating cow was provided twice daily at 7:30 and 14:00. During the close-up period, the prepartum TMR was provided once daily at 14:00. The RPN  included 55% of pure niacin, and the 12-h ruminal stability was 96%. RPN supplementation was from 49 days before to 21 days after calving. In each day morning before feeding, cows in the RPN group were fed a mixture of 20 g of RPN and 1 kg of fresh TMR, where a small amount of molasses was used to combine them together. For the CON group, cattle were fed a mixture of 1 kg of fresh TMR and a small amount of molasses. The composition of the basal diet and the proportion of nutrients are provided in A total of 12 multiparous Holstein dairy cows were divided into two groups with diet supplemented with either 0 (CON) or 20 g/day RPN (RPN), each group contained three 3rd parity and three 4th parity cows, respectively. The expected calving date (difference less than two weeks) and cumulative milk yield  of the cows enrolled in the trial were similar. The body weight , ages (57.83 ± 6.74 and 57.0 ± 6.84 months in CON and RPN group respectively), and body condition score were not different (g at 4 °C for 15 min) and stored at −20 °C for further analysis of NPY, OXA, NEFA, and BHBA. Blood samples from all time points would be uniformly tested once the animal experiment was done. Faecal samples were collected three times a day, eight hours apart on days 13, 14, and 15. All the samples from each cow were mixed with tartaric acid and homogenized to create a single bulk sample, dried at 65 °C to a constant weight in an air-forced oven, and ground for further chemical analysis. To coincide with sampling times for feed digestibility, 2 g faecal samples were sealed in 2 mL conical tubes at two weeks after calving (morning of day 14) and frozen in liquid nitrogen, then stored at −80 °C until genomic DNA was extracted. However, one of the 2 g faecal samples in the CON group lost during the transfer of samples from farm to laboratory.All the samples were taken individually from each cow besides TMR samples. Samples of TMR were collected twice a week during the experiment to monitor the dry matter (DM) content. Daily feed intake was monitored by Roughage Intake Control System , a system that can identify the cattle ID before opening the trough and measure feed intake by collecting the feed weight before and after eating. Milk production was recorded every day after milking. Milk samples were collected and mixed with potassium dichromate stored at 4 °C for no more than 24 h on days 7, 14, and 21 after calving. Blood samples were collected from the coccygeal vein into 5 mL evacuated serum tubes  on days 1, 3, 7, 14, and 21 at 6:00 a.m. The sera were obtained by centrifuging , ether extract (EE), ash, calcium, and phosphorus in basal diets and feces were analyzed according to the methods established by the Association of Official Analytical Chemists (AOAC) . NeutralProtein and fat in milk were analyzed in a DHI Testing Center  by using an automated near-infrared milk analyzer . Blood serum NEFA (No. E030-1-1) and BHBA (No. A042-1-1) were analyzed on a Hitachi 7600 automated biochemistry analyzer  using kits from Nanjing Jiancheng Bioengineering Institute . The ELISA method was used to determine the NPY  and OXA  kits from Shanghai Horabio Biotechnology Co., Ltd. .The main protocol of faecal bacterial DNA extraction has followed the procedure described by Hao et al. . 16S rRN16S rRNA microbiota data analysis has followed the pipeline published in the author’s study . Brieflyp ≤ 0.05. The weekly means were calculated from daily records of DMI and milk production prior to statistical analyses. Analyses of variance of the data were performed using PROC MIXED of SAS 9.2 . The model included the fixed effects of treat, time, treat × time interaction, and the random effect of individual animals. Effects of treatment on DMI, NPY, OXA, NEFA, BHBA, and milk production for each time point were tested using one-way ANOVA. For all variables, significant treatment and interaction effects were noted at p < 0.05). DMI, the concentration of NPY and OXA, milk production, and milk protein yield were higher in RPN than that in CON (p ≤ 0.001) during the first three weeks after calving. Yet, the concentration of the milk protein and fat, production of energy-corrected milk and milk fat were not changed significantly by RPN supplement. The changes of sera NEFA were affected by not only treatment but also the time points. The concentration of BHBA in blood was significantly increased by treatment. However, it was affected by the interaction of RPN and time. As shown in p < 0.05) and kept increasing until the third week (p = 0.068). Milk yield increased significantly in RPN group during the second and third weeks after calving (p < 0.01) though no affected in the first week (p = 0.317). The concentration of NPY in blood was significantly higher on days 1, 3, and 14 in RPN compared with CON. The concentration of OXA was significantly higher on day 3 in RPN than that in CON (p < 0.05). The concentration of NEFA in blood was significantly higher on days 3 and 14 after calving. The effects of the RPN on the concentration of BHBA were not significant on days 3, 7, 14, and 21, although BHBA was significantly higher on the first day after calving in RPN group (p < 0.05).As shown in p > 0.05), alpha and beta diversity of the faecal microbial community (p > 0.05) were observed in multiparous Holstein dairy cow at the second week after calving. The abundances of most genera were not significantly different .No effects of RPN on the apparent total-tract digestibility of NDF, ADF, EE, CP, OM, and DM  resulted in linear decreases in DMI . Rumen-pChanges in milk production and DMI were consistent in our study (improved 5.32 and 3.22 kg/d respectively). However, other studies found that diet supplemented with a low dosage of RPN (12 g/d) ,40 or noRuminococcus was a fiber utilizer [Bacteroides was a polysaccharides utilizer [Ruminococcus and Bacteroides between the two groups in our study. These findings were consistent with no difference in NDF and ADF digestibility. Genera of Eubacterium_nodatum_group, Dorea, and Escherichia-Shigella were conventional in cattle feces and reported in many studies [Eubacterium_nodatum_group and Escherichia-Shigella [Eubacterium_nodatum_group could utilize lysine and arginine to produce acetate and butyrate [Escherichia-Shigella was the dominant genus in the uterus of healthy cows compared with the cows with metritis [Eubacterium_nodatum_group and Escherichia-Shigella in this study were very low . Therefore, changes in their relative abundance may not be of biological relevance. Dorea is a gram-positive bacterial genus from the family Lachnospiraceae [Dorea in the feces of dairy cows increased when probiotics administration improved the host health [Dorea in feces. These indicated that host healthy conditions benefit the growth of Dorea. Mycoplasma was isolated from bovine feces as early as 1975 [Mycoplasma genus are pathogenic in cattle [Gut microbes are closely related to host health . Feces autilizer  and Bactutilizer . However studies ,56,59,60Shigella . Genus obutyrate , which wbutyrate . Howevermetritis . Moreovepiraceae . The abut health  and the t health . Similar as 1975 . Severaln cattle ,68. The Our work provides a reference for the use of RPN during the transition period to improve DMI and milk production performance in multiparous Holstein dairy cows in early lactation. Dietary supplementation with 20 g/d RPN could improve DMI via increased intake-related hormones, NPY and OXA, but had little influence on feed digestibility and faecal bacterial community. Dietary supplementation with 20 g/d RPN could also significantly improve milk production without significant impact on ECM or negative impacts on serum NEFA and BHBA. Further research is needed to determine whether RPN has a cumulative effect on multiparous Holstein dairy cows in subsequent transition periods. Moreover, further research is deserving whether the above-mentioned effects are physiological or pharmacological.


Atta are considered important pests toagriculture in the Americas, although Atta species are alsoimportant contributors to ecosystem functions in the various habitats in whichthey occur. The aim of this study was to assemble four complete mitochondrialgenomes of the genus Atta, construct the phylogenomic tree, andanalyze the gene content, order, and organization. The mitogenomes of A.colombica, A. opaciceps, A.texana, and A. sexdens rubropilosa comprise18,392, 19,257, 19,709, and 19,748 bp, respectively. The four Atta mitogenomesshowed the charactistics typical of those of insects, with 13 protein-codinggenes, 22 tRNAs, and 2 rRNAs, with genes displayed in the conventional order.Analysis for intergenic spacer regions showed that Attaintergenic spacers are larger than those of the outgroups. Phylogenomic analysesusing partial cytochrome oxidase I gene sequences showed similar topologies toprevious phylogenetic analyses, with high clade support values. We conclude thatAtta mitogenomes are characterized by high conservation ingene order and have giant intergenic spacers in the genusAtta.The ants of the genus  Atta are leafcutters belonging to the tribe Attini(Hymenoptera: Formicidae: Myrmicinae) and are considered important pests to agriculturein the Americas, although Atta species are also important contributorsto ecosystem functions in the various habitats in which they occur. The specieswidespread in Brazil are A. bisphaerica Forel, 1908, A.capiguara Gonçalves, 1944, A. cephalotes Lineu, 1758,A. goiana Gonçalves, 1942, A. laevigata F. Smith,1858, A. opaciceps Borgmeier, 1939, A. robustaBorgmeier, 1939, A. sexdens piriventris Santschi, 1919, A.sexdens rubropilosa Forel, 1908, A. sexdens sexdens Lineu,1758, A. silvai Gonçalves, 1982, and A. vollenweideriForel, 1939.The ants of the genus A. texana, A.mexicana, and A. insularis in the Archeatta clade; (2)A. colombica and A. cephalotes in the Atta s. str.clade; (3) A. opaciceps, A. laevigata, A.capiguara, A. bisphaerica, A.vollenweideri Forel 1939 and A. saltensis in the Epiattaclade, and (4) A. sexdens and A. robusta in theNeoatta clade .Phylogenetic analyses using gene fragments of cytochrome oxidase I, tRNA leucine, andcytochrome oxidase II revealed four clades: (1) Attawere assembled. Mitogenomes were utilized for phylogenomic analyses, gene content, andorder for exploring the evolution of the genus Atta. For development ofmitogenomes, reads of A. opaciceps were sequenced, and reads ofA. colombica, A. texana, and A. sexdensrubropilosa were downloaded from the NCBI database and utilized to assemblethe complete mitogenomes.In this study, four complete mitochondrial genomes of the genus A. opaciceps, the biological sample was collected in the state ofAlagoas, Brazil, and DNA extraction was performed using the cetyltrimethylammoniumbromide (CTAB) extraction method (For n method . The quaA. colombica (SRR3187022 and SRR3168931), A. texana(SRR5438011), and A. sexdens rubropilosa (SRR5651498), short reads wereobtained from public data in NCBI, from which the SRA files were unpacked into FASTQusing the FASTQ-DUMP tool executable from the SRA Toolkit. FASTQ files were thenfiltered with a minimum quality of 10, converted into FASTA files, and utilized forgenome assembly. Thirty million reads of A. colombica, 24 million readsof A. opaciceps, 3.3 million reads of A. texana, and4.2 million reads for A sexdens rubropilosa were used.For A. laevigata as reference, using the softwareGeneious R9 (http://www.geneious.com). The draft mitogenomes were checked using contigsfrom the de novo assembling generated by Ray software  method based on the GTR+I+G nucleotidesubstitution model .The mitogenomes of ectively . After oAtta mitogenomes showed the typical characteristics of thosefor insects, with 13 protein-coding genes, 22 tRNAs, and 2 rRNAs, as well as thenoncoding region . The mitogenome arrangement for the genusAtta was identical, wherein the protein-coding genes and the rRNAsdisplayed the same order and orientation. Additional tRNAs were observed between ATP8and COX2 in A. cephalotes .The four g region , with thA. sexdensrubropilosa) to 82.5% (A. texana). For the coding region,the lowest A + T content was in COXI, COX3, and ATP6, whereas the highest A + T contentwas between ND5 and ND3. The A + T contents between the coding and noncoding regionswere different, ranging from 77.6%  to 78.5% (A.texana), whereas the noncoding regions showed an A + T content ranging from84.1% (A. sexdens rubropilosa) to 90.3% (A. texana).The four genomes of the genus Atta revealed 60 SSRs, which were evenlydistributed, and the di-nucleotide motifs were more abundant, except for A.cephalotes for which the tetra-nucleotide motifs were more abundant.The A + T contents of mitogenomes were high, ranging from 72.7%  regions showed thatoutgroup , rangingoutgroup . The lar spacers . For theoutgroup .A. texana as the basal clade and theother species as the derived clade . When using only coding regions, A.colombica and A. cephalotes species formed one group andA. laevigata and A. opaciceps formed another. In the PCA for complete mitogenomes, the firstprincipal component separated A. laevigata, A.opaciceps, and A. sexdens rubropilosa from the otherspecies, and the second principal component separated A. colombica andA. cephalotes from A. texana.The phylogenetic analysis showed ed clade . Thetoped clade  and codied clade showed nAtta mitogenomes arecharacterized by a high frequency of A + T. The same result was reported previously forA. laevigata by Atta mitogenomes, like in the ancestral insectmitochondrial genome (Atta mitogenomes were larger, suggesting aphylogenetic signal. The size variation is influenced by expansions in intergenicspaces, confirmed by the larger intergenic spacers found in Attamitogenomes. Expansions in the intergenic spaces do not affect gene functions, and thuscan be considered selectively neutral. Intergenic spaces from other insect mitogenomeshave been reported to range from 216 bp in Naupactus xanthographus(Protaetia brevitarsis (Attamitogenomes are characterized by larger intergenic spacers.The nucleotide compositions in all analyzed Atta, Atta species.Mitogenomes have an impact on insect genetics, as they are widely utilized forphylogenetic studies. Regarding the genus A. texana in the basalclade, A. cephalotes and A. colombica in the secondclade, A. laevigata and A. opaciceps in the thirdclade, and A. sexdens rubropilosa in the fourth clade. However,bootstrap values using mitogenomes were larger than those for partial COI-tRNA-COII,indicating more robust phylogenetic inference with mitogenomes. We conclude thatAtta mitogenomes are characterized by high conservation in geneorder and organization and by giant intergenic spacers.Topologies using complete mitogenomes were similar to the phylogeny generated withpartial COI-tRNA-COII sequences (


Xanthosoma sagittifolium) is a tuber indigenous to Indonesia, which has starch as the major storage carbohydrate.Low glycemic foods are beneficial for people with type II diabetes. At the same time, sustained glucose release is also beneficial for people suffering from glycogen storage diseases. Taro . Samples were taken from the dialysate, which reflected glucose absorbed in the blood stream.This was investigated in a validated, dynamic computer-controlled Native taro starch showed a ~1.5-fold reduced digestibility compared to glucose and a ~ 1.35-fold compared to wheat starch. In addition, digestion of native taro starch was moved towards the ileum, and later in time compared to glucose and wheat. With modified taro starch, these effects were not observed.In conclusion, native taro starch showed a lower glycemic load than wheat starch and modified taro starch and could be used as a substitute for refined foods by diabetics and people suffering from other glucose metabolic diseases. Type II diabetic people have high blood glucose levels, caused by dietary ingestion of simple sugars and starch, the latter of which is digested by pancreatic amylase into glucose.Starch is frequently ingested from many staple foods, however not every starch is equally digestible, and hence the rise in blood glucose levels is starch-source dependent.Digestion of foods can be tested in a validated, sophisticated, dynamic computer-controlled in vitro model that closely mimics the stomach and small intestine of humans (TIM-1).Here, we show that in TIM-1 native starch from the taro-tuber is slowly digested (slower than wheat starch) and leads to sustained (low) blood glucose levels.Therefore, this taro-starch could be used as a substitute for refined foods by diabetics and people suffering from other glucose metabolic diseases.(Xanthosoma sagittifolium) tuber, which belongs to the monocotyledonous Araceae family (the aroids), is one of the many underused indigenous tubers of Indonesia. Indonesia has the highest taro diversity in the world, and taro can be found in areas in Borneo, Java, Sumatra and Sulawesi  carbohydrate in taro is starch. In the gastrointestinal tract (GIT), starch is degraded to glucose, maltose, maltotriose and dextrins by pancreatic amylase. Maltose, maltotriose and the dextrins are further degraded to glucose by the brush-border enzymes maltase and iso-maltase of the epithelial lining , 7. The in vitro models are very helpful. However, simple batch incubations usually do not predict the actual digestibility in vivo. Therefore, we have used here a validated, dynamic, computer-controlled in vitro model of the stomach and small intestine  with the aim to determine the glycemic load and speed of digestion of taro starch (native and after modification) compared to a free glucose solution and wheat starch. This model has been extensively validated and has been used before to determine starch digestion . Modified taro starch was manufactured by autoclave cooling according to a modification of the method of Zhao and Lin . In briein vitro digestion was performed for 6 h at 37°C. A mixture of 20 g of test-product (glucose or starch), electrolyte solution, water, pepsin , lipase  and gastric start-residue were introduced into the gastric compartment. Computer-controlled peristaltic valve pumps controlled mixing through peristalsis and meal transit through the individual compartments . Glucose was subsequently determined enzymatically using the Hexokinase/Glucose-6-phosphatedehydrogenase assay as described before . Starch Every hour samples were taken from the jejunal dialysate , ileal dAlthough glucose in solution does not require any digestion, from past experience with the model and from clinical data, we know that not all of it is absorbed. Figure 3 shows that approximately 90% is absorbed in the cumulative fractions Ja+Ia, which means that 10% is present in the Ie fractions plus the residue. Compared to glucose, wheat starch, a highly digestible starch, follows more or less the same kinetics, but the absorbed glucose is slightly lower [81% (vs. 90% for glucose)], with 72% thereof absorbed in the jejunum and 28% in the ileum. This means that, compared to glucose, absorption is shifted a little to the ileum. Due to the lower absorption, the amount in the ileal efflux (and residue) is higher (19% vs. 10% for glucose). Also, in the first 3 h, 89% of the absorbed glucose (Ja+Ia-1 to Ja+Ia-3) becomes available for absorption, indicating that absorption is slightly slower than for free glucose, but that digestion of the wheat starch is rather efficient, since it results in very similar amounts of absorbed glucose.Digestion of native taro starch in contrast was much lower and much slower. Overall, 59% of the ingested glucose was absorbed . Also, of the absorbed glucose, 62% was absorbed in the jejunum and 38% in the ileum, showing delayed absorption. Moreover, in the first 3 h, 62% of the absorbed glucose was absorbed, leaving a significant fraction to be absorbed in the last 3 h of the experiment. This can also be observed in Lastly, we modified the taro starch according to a protocol by Zhao and Lin . They shThe same model has been used for digestibility of other starch sources. For instance, maize starch was shown to be 86% digestible with a comparable protocol , while sin vitro model used here. It could be an excellent replacement for refined foods for people with type II diabetes, which are also on the rise in Indonesia, and would lead to sustained glucose release for individuals suffering from glycogen storage disease. The RS that makes it to the colon would be fermented by the gut microbiota and lead to increases in the health-beneficial microbial metabolite butyrate. This has already been shown by us in a rat model for diabetes , and thus use of the indigenous tuber in the Indonesian diet holds great promise for health.In conclusion, native taro starch is an excellent source of RS, and would lead to low and slow accumulation of glucose in blood, based on the validated The authors declare no potential conflicts of interest. The study was funded by the Bina Nusantara Foundation with a 2019 Eminent Research Grant of Bina Nusantara University. The study was also partly funded by the Centre for Healthy Eating & Food Innovation (HEFI) of Maastricht University – campus Venlo. This research has been made possible with the support of the Dutch Province of Limburg with a grant to HEFI.


This study analyzed changes in the starch structures and in vitro digestion profiles of a specific maize cultivar, Jike 728 (JK728), in Jilin, China, after 0–5 days of germination. The total starch, amylose, and amylopectin contents decreased significantly during germination. The average molecular weight of the starch compounds also decreased significantly during germination. The proportion of amylopectin with a degree of polymerization (DP) of 13–24 significantly decreased, while the relative abundance of amylopectin with DP values of 6–12, 25–36, and 37–60 significantly increased. The X‐ray diffraction (XRD) patterns of all samples were characteristic of A‐type starch, and the starch relative crystallinity decreased over time. The proportions of slowly digestible starch and resistant starch decreased significantly, while the proportion of rapidly digestible starch increased significantly during germination. Germination is an easy, inexpensive, and low‐carbon processing method. This study indicates that germination is an effective way to control the physical properties and digestibility of starch in maize. The changes observed in the physical properties and digestibility of maize starch after germination provide scientists with a platform to understand starch modification mechanisms that might have potential applications on an industrial scale. The total starch, amylose, and amylopectin concentrations decreased significantly during maize germination (0–5 days). The proportions of amylopectin with a DP of 13–24 significantly declined and those with a DP of 6–12, DP of 25–36, and DP of 37–60 significantly rose during maize germination (0–5 days). The XRD patterns of all starch samples were characteristic of A‐type starch, and the starch relative crystallinity was reduced during maize germination (0–5 days). The SDS and RS levels significantly declined, and the RDS levels significantly rose during maize germination (0–5 days). Maize is rich in nutrients and is composed of 8% protein, 0.8% fat, 79.2% carbohydrates, and 14.4% dietary fiber. Maize is also rich in vitamins and mineral elements. Furthermore, maize is an important feed source in the animal husbandry and aquaculture sectors and is an indispensable raw material in relation to food, healthcare, and chemical industries. There is a high starch content (70%) in maize. Thus, maize starch plays an important role in the maize processing industry.The corn plant maize (Starch is the main carbohydrate in the human diet and serves as the main source of human energy. This polysaccharide can be categorized into three types based upon the characteristics of digestion: rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS)  are decomposed and nutrients  are released, thereby causing changes to kernel composition . Maize sticks were collected manually when the moisture content was 35% ± 3%, and the maize seeds were subsequently stripped . The resultant maize was stored at a temperature of 20 ± 2°C and a relative humidity of 55% ± 5%. These conditions ensured that there was no invasion of insects and mold during storage.Jike 728 (jk728), a common maize in Northeast China, was harvested in Jinzhou, Liaoyuan, China . The resultant liquids were subsequently centrifuged in an Allegra X‐30R high‐speed freezing centrifuge  at 4,000 g for 10 min. The supernatant was discarded, the upper layer of soft yellow starch was scraped off, and the precipitates were washed three times with deionized water. Next, the centrifugation step was repeated, the supernatant was discarded, and the soft yellow starch was removed. The lower layer of starch was oven‐dried at 40°C for 24 hr, crushed, and filtered (100 mesh). The resultant starch powder was stored in a dryer until further required.A method published (with minor modifications) by Lin and Chang  was util2.4The total starch contents of the maize flour were measured using an enzymatic method  for observation with a 10 kV electron beam. The morphological changes in the starch granules were observed, and representative photographs were taken.In accordance with a method published by You et al.  —Dawn DSP‐F multiangle laser scattering meter (Wyatt Technology Co.)—Optilab T‐rEx differential refractive index detector (Wyatt Technology Co.) system (HPSEC‐MALLS‐RI). The chromatogram conditions were as follows: mobile phase was 90% DMSO solution containing 50 mmol/L LiBr, flow velocity was 0.5 ml/min, and column temperature was 60°C.In accordance with a previously reported method  distribution was analyzed using high‐performance size‐exclusion chromatography (HPSEC)  coupled to a refractive index detection (HPSEC‐RI) system. In accordance with a previously reported method  and then treated in a boiling water bath for 20 min. After cooling to 37°C, 5 µl of isoamylase  was added, and the mixture was incubated at 37°C for 24 hr. Isoamylase was subsequently inactivated in a boiling water bath for 10 min. After cooling to 4°C, the resultant solution was centrifuged at 3,000 g for 10 min. Next, the supernatant was degassed and filtered through a 0.22 μm cellulose ester microporous membrane. The supernatant was subsequently analyzed by HPSEC‐RI. The chromatogram conditions were as follows: A Shodex SB‐803  column (Shodex) and a Shodex SB‐802.5  column (Shodex) were connected according to the separation ranges from large to small. A Shodex SB‐G (Shodex) protection column was connected in front of the analytical column. The column temperature was set at 40°C. The mobile phase was 0.1 mol/L Tris, 0.1 mol/L NaCl, and pH 7.40, and the flow rate was 0.5 ml/min. The Shodex RI‐101 differential refractive index detector (Shodex) was set at 37°C. Samples were fed via a trace flat injector at a concentration of 10 g/L and dosage of 20 µl. The normalization analysis and peak area calculation were carried out on Origin 9.0 . The peak area ratio of the amylopectin branch chain (fraction) is its DP.The 2.8The relative crystallinity of the maize starch was detected on a D8 Avance XRD meter (Bruker Corp.) using CuKa rays, a tube voltage of 35 kV, a tube ampage of 20 mA, a scanning range of 5–40°, and a scanning rate of 0.02°/s using a method previously published by Zhang et al.  with min2.9g for 10 min. The supernatant was then added to 1.0 ml of amyloglucosidase .Starch digestibility was measured using an in vitro simulated enzyme hydrolysis method . The starch contents were calculated as follows:Then, 0.3 g of starch was placed into a screw‐cap centrifuge tube. Next, 7.5 ml of 0.1 mol/L sodium acetate buffer (pH 5.2) was added, and the mixture was placed into a water bath at 95°C for 30 min to facilitate gelatinization. Five glass beads and 0.75 ml of the enzyme mixture were added, and the mixture was shaken at 200 2.10p < .05).The experiments were repeated at least three times, and the results were expressed in the form of mean ± standard deviation. The statistical software SPSS 19.0  was used to analyze the experimental data, and Duncan's Multiple Range test in ANOVA was used to analyze the difference (33.1Changes to the starch composition during maize germination are listed in Table 3.2SEM images of maize starch during germination. The native maize starch and germinated maize starch granules are both irregular polygons. The images on day 0 show that the raw maize starch granules contained pore‐like structures (A), or "holes." These structures are also found in broomcorn starch, millet starch, wheat starch, and barley starch  and number‐average molecular weights (Mn) of maize starch both decreased significantly during germination. These results are consistent with those published by Xu et al.  increased from 2.20 ± 0.16 to 3.14 ± 0.36 after germination, indicating that the starch granules became nonuniform after germination.The molecular weights of maize starch during germination are shown in Table u et al.  and You u et al. . This ph3.4Table 3.5JK728 exhibited strong diffraction peaks at 15.550, 17.510, 18.450, and 23.646 before germination Figure . Followi3.6SEM (Figure Changes to starch following in vitro digestion during maize germination are listed in Figure M Figure  analysisM Figure , a factoM Figure . As the M Figure , indicat4During 0–5 days of germination, the relative abundance of amylopectin branch chain segments with a DP of 13–24 significantly decreased, whereas the relative abundance of amylopectin branch chain segments with a DP of 6–12, 25–36, and 37–60 significantly increased over time. The XRD patterns of all the starch samples assessed are characteristic of A‐type starch, and the relative crystallinity was reduced with time. The in vitro digestion analysis showed that the SDS and RS levels both significantly decreased, while the RDS levels significantly increased. This study indicates that germination is an effective way to improve the RDS content of maize starches.The authors declare no conflict of interest.This study does not involve any human or mammalian testing.


In situ hybridization chain reaction (HCR) is a method to visualize specific mRNAs in diverse organisms by applying a HCR that is an isothermal enzyme-free nucleotide polymerization method using hairpin DNAs. Although in situ HCR is a versatile method, this method is not widely used by researchers because of their higher cost than conventional in situ hybridization (ISH). Here, we redesigned hairpin DNAs so that their lengths were half the length of commonly used hairpin DNAs. We also optimized the conjugated fluorophores and linkers. Modified in situ HCR showed sufficient fluorescent signals to detect various mRNAs such as Penk, Oxtr, Vglut2, Drd1, Drd2, and Moxd1 in mouse neural tissues with a high signal-to-noise ratio. The sensitivity of modified in situ HCR in detecting the Oxtr mRNA was better than that of fluorescent ISH using tyramide signal amplification. Notably, the modified in situ HCR does not require proteinase K treatment so that it enables the preservation of morphological structures and antigenicity. The modified in situ HCR simultaneously detected the distributions of c-Fos immunoreactivity and Vglut2 mRNA, and detected multiple mRNAs with a high signal-noise ratio at subcellular resolution in mouse brains. These results suggest that the modified in situ HCR using short hairpin DNAs is cost-effective and useful for the visualization of multiple mRNAs and proteins.The visualization of multiple gene expressions in well-preserved tissues is crucial for the elucidation of physiological and pathological processes.  In situ hybridization (ISH) is a commonly used technique for detecting specific mRNAs in cells, tissues or whole bodies  is an isothermal enzyme-free polymerization method that uses two different hairpin nucleotides: H1 and H2 . The haiIn situ HCR is a method that applies the HCR technique to the visualization of a specific nucleotide in cells, tissues, and whole-mount samples using an RNA probe that has a sequence enabling the hybridization to a target mRNA and a sequence to work as an initiator of HCR -long DNA hairpins costs more than conventional ISH methods. The cost of oligonucleotides increases in proportion to their length, and the yield of full-length oligos decreases as their lengths increase. Thus, shorter DNA hairpins are favorable as long as they progress HCR.Despite the technical advantages of in situ HCR with split probes. We also simplified the in situ HCR protocol by removing proteinase K treatment. Our modified in situ HCR protocol is sensitive to a low abundance mRNA, is easier to handle and enables better antigenicity preservation for immunohistochemistry at a decreased cost.In this study, we optimized and shortened DNA hairpins and initiators for All nonlabeled oligo DNAs were synthesized as standard desalted oligos (Integrated DNA Technologies). We modified and optimized the design of DNA probes using short hairpin DNAs from previous studies . ssH-labeling of the 5′ end of DNAs enables subsequent coupling to a fluorophore .Nonlabeled DNA hairpins were used only for All hairpin DNAs were snap-cooled (heated to 95°C for 2 min and cooled to room temperature for 30 min) to form a hairpin structure before use. HCR was performed in 5× standard saline citrate (SSC) with 0.1% Tween 20 (5× SSCT). Each microtube was prepared by adding 2 μl of 3 μM hairpin DNAs, 6 μl of 0, 0.005, 0.02 or 0.1 μM initiator DNA, and an appropriate amount of 20× SSC with 10% Tween 20 to bring the reaction volume to 12 μl. Two hours after mixing the DNAs, the samples were supplemented with prestained loading dye (Bio-craft) and loaded into native 1% agarose gels. The gels were electrophoresed in sodium borate buffer (10 mM NaOH and 35 mM boric acid) at 135 V for 30 min and then were imaged. The intensity of each band was analyzed by ImageJ to quantify the amount of HCR products.We determined that a pair of hairpins showed strong amplification when HCR products exceeded 10 kb with 1/200 of the initiator-to-short hairpin ratio. We also judged that initiator-independent HCR (leakage) was low when a very weak signal of the HCR product was observed after 2 h of HCR without initiator nucleotides. Also, HCR efficiency was evaluated based on the amount of unreacted hairpin see .All animal procedures were conducted following the Guidelines for Animal Experiments of Toho University and were approved by the Institutional Animal Care and Use Committee of Toho University (Approved protocol ID #19-51-405). Breeding pairs of C57BL/6J mice were obtained from Japan SLC and CLEA Japan. Mice were raised in our breeding colony under controlled conditions . Male mice (20–30 weeks old) were used except for c-Fos staining after maternal behavior. Female mice (20–30 weeks old) were sacrificed 2 h after pup presentation for c-Fos immunohistochemistry  for 20 min. After washing, the sections were prehybridized for 10 min at 37°C in a hybridization buffer containing 30% formamide, 10% dextran sulfate, 5× SSC, 10 mM citric acid, 0.1% Tween 20, 50 μg/ml heparin, 1× Denhardt’s solution as described , which was followed by overnight incubation with rabbit anti-c-Fos antibody  in 0.4% Block Ace/PBST at 4°C. After washing three times with PBST, sections were incubated with an Alexa488-conjugated donkey anti-rabbit goat antibody  with Hoechst 33342 (1 μg/ml) for an hour at room temperature. The sections were mounted on the slide glass and cover-slipped with mounting media containing antifade .in situ HCR and enzyme-based ISH, we also performed chromogenic ISH and fluorescent ISH using tyramide signal amplification. The cDNA fragment of oxytocin receptor (Oxtr) mRNA  was amplified, inserted into the pGEM-T plasmid , which was used into DH5α E. coli. After confirmation that the DNA sequence was correct, template cDNA was produced using polymerase chain reaction with specific primers (5′-ATTTAGGTGACACTATAG-3′) and (5′-TAATACGACTCACTATAGGG-3′). The probe was transcribed by SP6 RNA polymerase  in the presence of digoxigenin-labeled UTP , which was followed by precipitation with LiCl with ethanol. The riboprobe was digested by alkaline hydrolysis to reduce the average size to 500 bases. Although hydrolyzed riboprobes sometimes increase nonspecific hybridization, our ISH protocol using a stringent wash, RNase A treatment, and non-excessive hydrolysis suppresses the background.To compare the sensitivity between in situ HCR. After acetylation, sections were washed with PBST, which was followed by incubation at 57°C overnight in a hybridization mixture containing 1 μg/ml riboprobe, 50% deionized formamide, 5× SSC (pH 7.0), 5 mM EDTA (pH 8.0), 0.2 mg/ml yeast tRNA, 0.2% Tween-20, 0.2% sodium dodecyl sulfate, 10% dextran sulfate, and 0.1 mg/ml heparin. After hybridization, the sections were washed twice with 2× SSC containing 50% formamide at 57°C for 10 min, incubated with RNAse A solution (20 μg/ml) at 37°C for 30 min, rinsed twice with 2× SSC and 0.2× SSC at 37°C (10 min each), and incubated in an alkaline phosphatase-conjugated or peroxidase-conjugated anti-digoxigenin antiserum  for 2 h at room temperature. Then the alkaline phosphatase-labeled sections were washed with 100 mM Tris-HCl (pH 8.0) and 150 mM NaCl and were incubated with BCIP/NBT (Roche) in 100 mM Tris-HCl (pH 9.5), 150 mM NaCl, 1 mM MgCl2 and 10% polyvinyl alcohol for 3 days at room temperature. The sections were dehydrated by treatment with methanol, ethanol, and xylene, and were mounted with Marinol . The peroxidase-labeled sections were washed and immersed in 0.1 M boric buffer (pH 8.5) containing 10 μM Alexa568-labeled tyramide, 10% dextran sulfate, 0.05 mg/ml iodophenol and 0.003% H2O2 for 30 min. After washing, the sections were mounted as they were in the in situ HCR experiments.The prehybridization procedure was identical to that of Fluorescent photomicrographs were obtained using a Nikon Eclipse Ni microscope equipped with the A1R confocal detection system under 20×, 40× and 100× objective lenses . The photomicrographs for the bright field observation were taken by a Nikon AZ-100 microscope equipped with a digital camera (Sony α7s). Images were analyzed using ImageJ software . Quantification of the fluorescent photographs was performed at the same threshold and adjustment of contrast.To find the shortest sequences of DNA hairpins that reliably proceed with the HCR, we systematically examined hairpin DNAs with different lengths for their HCR efficiency based on the appearance of HCR products larger than 10 kb and the intensity of the unreacted hairpin DNA band. Because the commonly used DNA hairpins have toehold and loop domains of equal length and are 72-nt long containing 12-nt toehold, 24-nt stem and 12-nt loop domains .Since insufficient stability of hairpin DNAs leads to initiator-independent HCR, we optimized the sequence of the stem domains of short hairpin pair based on the abundance of initiator-independent HCR. Through this optimization using short hairpins with different nucleotide contents, we noticed that short hairpin pair with a similar number of G and C on one strand of the stem domain tended to result in frequent initiator-independent HCR . In contAlso, we noticed that the shift of the transition site between the stem and loop domains by one or two nucleotides sometimes decreased initiator-independent HCR . For exaWe examined which fluorophore to use affects HCR efficiency in microtubes. The conjugation of ATTO390, FAM, ATTO488, Alexa488, ATTO550, and Alexa647  to 42-ntin situ HCR for mouse brain using the fluorophore-labeled short hairpin DNAs described above and the reagents and buffers with reaction conditions reported in previous studies  mRNA than 36-nt hairpin DNA (in situ HCR to detect mRNAs in this study. Penk mRNA-positive cells were abundantly detected in the striatum after HCR amplification initiated by five split-initiator probe sets for Penk mRNA (Penk mRNA. The incubation of split-initiator probes with one of the hairpin pairs (H1) or the incubation of hairpin pairs  without split-initiator probes did not generate any signals, indicating the in situ HCR specific to Penk mRNA .Consistent with itro HCR , ssH-lin ATTO550 . The use6 linker . PAGE puin situ HCR using short hairpins with other ISH methods by detecting Oxtr mRNA, which we selected as an example of very low abundance mRNAs. After hybridization of 10 split-initiator probe sets for Oxtr mRNA, in situ HCR detected Oxtr mRNA signals in the rhomboid nucleus of the bed nucleus of the stria terminalis (BNST) and the magnocellular nucleus (MN) and weak signals in the principal nucleus of the BNST and the medial preoptic area  2 in the MPOA , Drd2 and Penk mRNAs were simultaneously detected in the mouse striatum using the corresponding probes (five probe sets for each mRNA) and hairpin DNA pairs conjugated to different fluorophores  -stem (12 nt)—A—loop (7 nt)—T—stem (12-nt); A and T are transition bases], which often decreased initiator-independent HCR but did not exhibit a decrease in HCR efficiency. Many designed hairpins showed a certain level of initiator-independent HCR, polymerization without an initiator. Many factors cause initiator-independent HCR  was used to link hairpin DNAs with ATTO550 succinimidyl ester, HCR did not form long products . In cont in situ . Also, t in situ . We founin situ HCR  using alkaline phosphatase and BCIP/NBT after a long chromogenic reaction which is thought to be most sensitive among enzyme-based conventional ISH . Also, as the oligos get longer, mis-synthesized oligos increase exponentially, which further makes short hairpins cost-effective.In summary, our short hairpin All data are available from the corresponding authors upon reasonable request.The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Toho University.YT: conceptualization, methodology and formal analysis. YT and HF: writing, funding acquisition and supervision.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Periostin, a recently found matricellular protein, has been implicated in neointima formation after balloon injury. However, the relationship between periostin and hyperplastic intima formation after PTFE graft implantation is unclear. Under mixed anesthesia, PTFE grafts were implanted between the canine carotid artery and jugular vein, and PTFE graft samples were harvested 1, 2, and 4 months after implantation. Intima formation started on the luminal surface of PTFE grafts at the venous anastomotic region 1 month after implantation. Thereafter, the increase in intimal volume was not only observed in the venous and arterial anastomotic regions, but also in the middle region of the PTFE grafts. In accordance with the increased intimal formation, time-dependent increases in mRNA expressions of periostin and transforming growth factor beta 1 (TGF-β1), as well as a strong positive correlation between periostin and TGF-β1, were observed. These findings suggest that periostin may play a very important role in the pathogenesis of hemodialysis vascular access stenosis through the acceleration of intimal formation. Thus, periostin may be a very important therapeutic target for the treatment of vascular access graft dysfunction in hemodialysis patients. If kidney function drops to less than 85 to 90 percent of the normal condition in patients with end-stage renal disease (ESRD), these patients will need to have hemodialysis treatment for their whole life unless they are able to obtain a kidney transplant. Advances in hemodialysis techniques have been remarkable, and the survival time on hemodialysis can even reach several decades . Of courIn the clinical setting, a native arteriovenous (AV) fistula in hemodialysis patients as a vascular access route is the first-choice vascular access route due to a lower incidence of complications and better long-term patency . HoweverWith respect to PTFE vascular access graft dysfunction, it has been recognized that the stenosis caused by intimal hyperplasia on the venous side is the main hemodynamic cause . AlthougRecently, periostin, a newly identified extracellular matrix (ECM) protein, was reported to be important in the promotion of several types of vascular remodeling, such as that involving the carotid artery intima in rats , atherosAs can be seen in At 1 month after PTFE graft implantation A, periosLike the periostin immunostaining, TGF-β1-positive staining was also found in the intima of both arterial and venous regions, as well as at the tissues surrounding the PTFE graft 1 month after PTFE graft implantation A. A simiα-SMA. These loci were matched to the yellow frames of Azan–Mallory staining, as shown in α-SMA-positive cells, and the immunostaining for vimentin in the serial section showed that most of these α-SMA-positive cells were overlapped with the vimentin-positive cells. On the other hand, as can be seen in the high magnification views of vimentin and α-SMA immunostaining, although vimentin-positive staining was found in all cellular components at these venous regions , some cellular components in the border zone between the intima and the PTFE material wall, as well as in the splits of the PTFE material wall, were negative for α-SMA , some cellular components in the border zone between the intima and the PTFE material wall, as well as in the splits of PTFE material wall, were negative for α-SMA  phosphorylation, indicating that these integrins act as periostin receptors to modulate cell migration. In the present study, periostin expression determined by RT-PCR showed that mRNA periostin expression could be observed as early as 1 month after PTFE graft implantation at the three examined regions and was further increased 4 months after PTFE graft implantation . Anesthesia was induced in the dogs by intramuscular (IM) injection of medetomidine (0.02 mg/kg) and midazolam (0.3 mg/kg), followed by an intravenous (IV) injection of pentobarbital sodium (10 mg/kg) 10 min later. To avoid blood clotting during and after the surgery, heparin (100 U/kg) was subcutaneously injected after the anesthesia procedure. Under sterile conditions, the PTFE grafts with an internal diameter of 0.6 cm and length of 7 to 8 cm  were implanted between the carotid artery and jugular vein with arterial-graft and venous-graft side-to-end anastomoses. Finally, the skin was closed in layers, and the animals were allowed to recover. All animals received antibiotic therapy through an IM injection of cefminox sodium (0.5 g). Graft patency was monitored weekly by auscultation and palpation. The experimental procedures for animals were conducted in accordance with the guidelines of Osaka Medical College for medical experiments, and they were approved by the ethics committee (29003).n = 6), 2 (n = 6), and 4 (n = 6) months post-implantation; their grafts, together with the connected native arteries and veins, were then carefully removed. All the samples were cut into three equal parts (average length: 2.3–2.6 cm): arterial anastomotic, middle, and venous anastomotic regions, as described previously [The animals were sacrificed at 1 , a rabbit anti-human TGF-β1 antibody , a mouse anti-human vimentin antibody , and a mouse anti alpha-smooth muscle actin antibody , according to the protocol described elsewhere ,17,18. ITo extract the RNA of the PTFE grafts, 10 sheets of the 20-μm-thick paraffin sections were collected from the respective Carnoy-fixed, paraffin-embedded tissue blocks using a microtome . Total RNA was extracted in accordance with the protocol provided in the total RNA isolation kit .Total RNA (1 μg) was transcribed into cDNA with SuperScript VILO . Then, mRNA levels were measured by RT-PCR on a Stratagene Mx3000P  using Taq-Man fluorogenic probes. RT-PCR primers and probes for periostin, TGF-β1, collagen I, and GAPDH were designed by Roche Diagnostics . The primers were as follows: 5′-ttcctgattctgccaaacaa-3′ (forward) and 5′- gtccgtgaaagtggtttgct-3′ (reverse) for periostin, 5′-cattaacgggttcagttccag-3′ (forward) and 5′-agcaggaagggtcggttc-3′ (reverse) for TGF-β1, 5′-gtctgcccggtgaaagag-3′ (forward) and 5′-cagtagcaccatcgtttcca-3′ (reverse) for collagen I, and 5′-aatgtatcagttgtggatctgacc-3′ (forward) and 5′-gcttcactaccttcttgatgtcg-3′ (reverse) for GAPDH. The probes were as follows: 5′- ctggctgg-3′ for periostin, 5′-ggccacca-3′ for TGF-β1, 5′-cctggagc-3′ for collagen I, and 5′-cctggaga-3′ for GAPDH.The mRNA levels of periostin, TGF-β1, and collagen I were normalized to that of GAPDH.The Azan–Mallory-stained sections obtained from three different regions were used to determine the intimal areas using a computerized morphometry system  3.07, Nikon, Tokyo, Japan). First, the intimal areas covering the grafts were measured. To normalize the observed intimal areas, the lengths of the prosthetic vessels were also measured. The degree of neointima formation in each graft was expressed as the respective area divided by the length of the graft.p < 0.05 was considered significant.All numerical data shown in the text are expressed as means ± standard error of the mean (S.E.M.). Significant differences among the mean values of multiple groups were evaluated by one-way analysis of variance (ANOVA) followed by a post hoc analysis (Fisher’s test). Pearson’s correlation coefficient was measured to test the linear relationship between two variables using linear regression analysis. The present study showed that periostin expression in the hyperplastic intima, as well as in the tissues surrounding the PTFE graft, was significantly increased after artificial PTFE graft implantation, and a close correlation between the expressions of periostin and TGF-β1 was found, independent of time point and region. These findings suggest that periostin may play a very important role in the pathogenesis of hemodialysis vascular access stenosis through the acceleration of intima formation; thus, periostin may be a therapeutic target for the treatment of vascular access graft dysfunction in hemodialysis patients.


Biological Cybernetics. The journal was founded under the German title Kybernetik in 1961 opened the journal to new applications in biology, beyond contributions to the field of computational neuroscience. Looking at the articles in this issue, most papers are still devoted to classical problems dealing with, for instance, neuromechanical models, neural network models with plasticity, and models of perceptual inference and motor control. There is one contribution on sensitivity minimization and biological homeostasis that considers as example applications glycolytic oscillations and bacterial chemotaxis—great examples of regulatory systems beyond neuroscience of which we would like to see more in future articles.Last year, we also started to offer new and original forms of contributions to junior scientists. This initiative has met with enthusiasm, and we publish in this issue the first paper by a junior scientist; more are coming! We see this development as not only an early opportunity to involve young researchers into the writing of journal articles, an essential form of scientific communication, but also into the responsibilities and skills associated with peer review. We hope many graduate students and postdoctoral fellows will contribute as authors or reviewers and help make this initiative a success.0, or statistical quantities describing the variability of model outcomes, to journalists and other nonspecialists. We hope that with our journal’s review and prospect formats, and, last but not least, with the journal club format of the junior scientist corner, we can modestly contribute to making research results in the field of biological cybernetics more accessible to a broader scientific audience and, by that, indirectly eventually also to the public. In the long run, explanation and education is the only way to defend scientific reasoning, be it in epidemiology, in computational biology, or in neuroscience.We cannot welcome the reader to 2021 without reflecting on the COVID-19 pandemic, which has taken already the lives of two millions and transformed life for billions more. As a journal that often communicates the results of mathematical modelling of biological systems, we have been encouraged by the extent to which mathematical modelling in biology and medicine has attracted public attention. Despite isolated controversies arising naturally from the sensitivity of model predictions to modelling assumptions and reliable data, the importance of modelling and its predictive power have become widely appreciated by the general public. At the same time, growing public awareness of modelling as an intellectual enterprise highlights the difficulty and the importance of clearly communicating quantitative concepts, such as the basic reproduction number RIn this issue, we also say farewell to our longest-serving board member, Horace Barlow, who passed away last June at the age of 98. Amazingly, he has been with the journal's editorial board since its foundation in 1961 (cf. Fig. We close by wishing all of our readers, authors, editors and reviewers a healthy and successful year 2021.


Journal of Patient-Reported Outcomes (JPRO) is an international, open access, multi-disciplinary journal publishing original research and review articles, brief communications, commentaries, editorials, and reviews of recent books and software advances in the field of patient-reported outcomes. The JPRO is an official journal of the International Society for Quality of Life Research (ISOQOL). The first JPRO articles were published on September 12, 2017.The Quality of Life Research” and was the “beginning of a beautiful journal” (https://jpro.springeropen.com/articles/10.1186/s41687-017-0009-2). The success of the JPRO is due to their extensive knowledge, hard work and dedication. The journal’s success also reflects the diligent contributions made by reviewers and the quality of the science in the published manuscripts.Dennis Revicki and David Feeny were the founding Co-Editors-in-Chief. Dennis served until the start of 2020 and David finished at the end of 2020. They noted at the onset of the JPRO that it was “intended to complement and extend ISOQOL’s existing journal, Quality of Life Research (2009–2017) and David served as ISOQOL president (2004–2005). We are grateful for the giant steps forward they made with the JPRO.Dennis and David are luminaries in the field of patient-reported outcomes. Both are former ISOQOL President’s Award recipients (Dennis in 2007 and David in 2010). In addition, Dennis is a former co-Editor-in-Chief of We hope to continue the upward trajectory of the journal and obtain the coveted impact factor soon. We encourage those doing patient-reported outcomes research to submit manuscripts and to serve as peer reviewers for the JPRO. We can only move forward with your help.Sincerely,Ron D. Hays and Chih-Hung ChangJPRO Co-Editors-in-Chief


In this paper, a piezoelectric tactile sensor for detecting tissue stiffness in robot-assisted minimally invasive surgery (RMIS) is proposed. It can detect the stiffness not only when the probe is normal to the tissue surface, but also when there is a contact angle between the probe and normal direction. It solves the problem that existing sensors can only detect in the normal direction to ensure accuracy when the degree of freedom (DOF) of surgical instruments is limited. The proposed senor can distinguish samples with different stiffness and recognize lump from normal tissue effectively when the contact angle varies within . These are achieved by establishing a new detection model and sensor optimization. It deduces the influence of contact angle on stiffness detection by sensor parameters design and optimization. The detection performance of the sensor is confirmed by simulation and experiment. Five samples with different stiffness  are used. Through blind recognition test in simulation, the recognition rate is 100% when the contact angle is randomly selected within 30°, 94.1% within 45°, which is 38.7% higher than the unoptimized sensor. Through blind classification test and automatic k-means clustering in experiment, the correct rate is 92% when the contact angle is randomly selected within 45°. We can get the proposed sensor can easily recognize samples with different stiffness with high accuracy which has broad application prospects in the medical field. With the development of medical treatment and the advancement of robot technology, robot-assisted minimally invasive interventional surgery (RMIS) is becoming popular. It has the advantages of small trauma, quick recovery and high accuracy. Compared with traditional surgery, doctors cannot touch tissue directly in RMIS, which may cause misdiagnosis or wrong resection. Since the lumps are often harder than the surrounding normal tissues, doctors can usually directly distinguish the lump by the difference in stiffness felt. ,2 In ordThere are many kinds of tactile sensors that can be used to detect force and stiffness. C. H. Chuang  designs So far, most tactile sensors in literature can detect the stiffness of tissue only when the contact is in the normal direction. However, most MIS procedures are performed in a confined space such as the transurethral resection of bladder tumor (TURBt) and uterine fibroid embolization (UFE). The limited degree of freedom (DOF) of the instrument causes the tactile sensor to become unable to be placed in the normal direction to contact the tissue surface, which may lead to lump undetectable. The method of achieving stiffness detection and lump identification under multiple constraints is particularly important. However, the use of existing tactile sensors is mostly limited by the contact angle between the probe and normal direction of tissue surface. The stiffness palpation method proposed in  can distThis paper proposes a piezoelectric tactile sensor that can not only detect the stiffness when the probe is normal to the tissue surface, but also when there is a contact angle relative to the normal direction. The detection model with a contact angle is proposed in α, a stiffness detection method is proposed. Based on that, parameters of the sensor are designed and optimized to minimalize the influence of angle change on detection.A piezoelectric sensor is preliminarily designed, and the sensing principle explains how the sensor works. In order to know the relationship between stiffness and contact angle α, as shown in This paper proposes a piezoelectric bimorph tactile sensor, and the structure is shown in Due to the inverse piezoelectric effect, applying an AC voltage in the polarization direction of the PZT Zx=rxAfter the sensor touches the tested tissue, the impedance of the system changes. The resonance frequency of the system shifts due to the change which can be measured.The frequency shift after the sensor touches the tissue can be expressed as ,19:(2)Δff and the characteristics of the tested tissue:Substituting Equation (1) into (2) finally obtains the relationship between the frequency shift ΔThe contact between the probe of the sensor and the tested tissue can be expressed as static contact and dynamic contact. Static contact means that a small force makes the probe contact the tissue at a certain indentation depth; dynamic contact means that an AC voltage is applied to the sensor which causes a small vibration between probe and tissue. However, for measuring the stiffness of object with a large anisotropy factor and Poisson’s ratio, the contact between the sensor and tissue can be approximated by static contact . BiologiNF, the sensor has a normal relative displacement Δ to the surface of the tissue, which is called indentation depth. Since the probe is spherical with a radius of R, the contour of the contact part can be approximated as a circle with a diameter of 2a, a≤ R. From the geometric relationship of contact, we can get:As shown in α, the probe produces a displacement of Δ in this direction. The contour of the contact part can still be approximately circular, with a diameter of 2a’, as shown in When the sensor’s DOF is limited, it cannot detect tissue in the normal direction but at angle α (α < 90°), the smaller the contact area S.It can be seen from Equation (6) that if the depth Δ is maintained at a constant value, the larger the angle Substituting Equation (6) into (3):α, the larger the angle α, the smaller the frequency shift Δf. However, the change of Δf can be reduced.It can be seen from the Equation (7) that the stiffness of the tested tissue is proportional to the frequency shift. When the sensor presses the tissue at a contact angle f is not only related to the stiffness and the angle during detection, but also related to the parameters of the sensor itself: the length L of PZT and the radius R of the probe. Through the design and optimization of these two items, the influence of α on Δf can be reduced. In order to prevent the slip phenomenon, we only discuss the angle in the range of α ∈ . Taking the angle α in Equation (7) as the independent variable, the schematic image of Δf-α function is shown in The sensor’s Δf0°and Δf45° is close to 0 (D1 is close to 0), to ensure that the angle change has the smallest impact on the detection result. (2) When tissues with different stiffness are tested, the difference between Δif and Δjf should be larger than D1 to ensure that they can be reliably distinguished, as shown in D2 in i and j represent samples with different stiffness. Stiffness of sample i is larger than sample j.To optimize the performance of the sensor, two goals of sensor parameters optimization are: (1) As shown in D1 ≥ 0, get R ≥ 0.42Δ. In Equation (9), select the extreme case  for discussion, set α = 0°, x(i)E = 1.2x(j)E, the solution is R < 2.5Δ. In order to ensure the contact surface is approximately circular, control R≥ Δ. Therefore, take R = Δ. For the L, two sets of parameters are selected for comparison in simulation.Solve Equation (8) In order to test the sensor performance on distinguishing different stiffness samples with arbitrary contact angle, effect of optimization and blind recognition ability, simulation is designed.L = 35 mm is preliminarily chosen, and probe tip radius and indentation depth R = Δ = 0.5 mm are selected. The materials of sensor are shown in A model of the sensor and the tested samples was established, and the model was simulated in Ansys Workbench. Sensor structure is shown in Five samples with different stiffness are set, including the normal tissue with the smallest stiffness (sample 1) and lumps  , and theα is set to 0°, 15°, 30° and 45°, respectively. The contact with the sample is set as frictional contact, and the friction coefficient is 0.1 ,  and  to detect the stiffness value of each point of each sample. The stiffness value of each sample at the boundary of the interval is used as the recognition range of the sample surface point. That is, when sample 1 is tested, if the stiffness value of a certain point is within the recognition range of sample 1, this point will be judged as sample 1. If it is also in the range of sample 2 at the same time, it may be misjudged as sample 2. The boundaries of the stiffness range of the five samples are obtained by simulation. The results of normal detection and random angle detection are shown in In order to prove the ability of the optimized sensor to recognize samples with different stiffness when the contact angle is from 0° to 45°, five samples (the same as mentioned before) are used as the tested objects, comparing the blind recognition effect of the optimized sensor , far better than the recognition rate before optimization.Based on simulation, an experiment is designed, a piezoelectric sensor is fabricated. Five soft silica gel samples with different stiffness are made. The stiffness of samples consistent with the simulation. The actual stiffness measured by the 00-type hardness tester is shown in As shown in α) to facilitate analysis.The resonance frequency of the un-load sensor is 137.6 Hz, which is approximately the same as the simulation result. It can be seen from In order to prove the sensor’s ability to blindly recognize samples with different stiffness, the labels of the five samples are torn off and the order was disturbed. The proposed sensor is used to randomly detect a sample with a contact angle within 45°. Each random detection process includes randomly selecting a sample of unknown stiffness, randomly selecting a point on this sample (the sample stiffness is uniform), and testing the stiffness of this point at a random contact angle. After each test is completed, each sample is shuffled for the next test, repeating 50 times. These 50 stiffness test results (expressed in frequency shift) are imported into k-means clustering algorithm for automatic classification. The classification result is shown in From In this paper, a piezoelectric tactile sensor for detecting tissue stiffness in RMIS is proposed, whose detection principle is based on the resonant frequency change of the system after making contact with the tissue. The stiffness can be detected without causing damage to the tissue. The outstanding feature of the sensor is that it can detect the stiffness and distinguish different stiffness samples not only in the normal direction, but also when there is a contact angle between probe and normal direction of tissue surface, solving the problem that when the DOF of surgical instruments is limited, the sensor cannot detect in normal direction. For the traditional tactile sensor, when it deviates from the normal direction, the stiffness cannot be detected, or the accuracy is greatly reduced. The proposed sensor solves this problem well.Through theoretical analysis, the principle of sensing and the stiffness detection method with a contact angle are obtained. The sensor system’s resonance frequency shift is proportional to the stiffness of the tested tissue and is negatively related to the contact angle. The parameters of the sensor are optimized to minimalize the influence of angle on stiffness. In order to test the optimized sensor performance, a simulation and an experiment are designed. Normal tissue sample and lump samples are set. When the angle changes from 0° to 45°, samples with different stiffness can be clearly distinguished. The sample recognition rate of the optimized sensor is 100% when the contact angle is selected randomly within 30°  in simulation, 94.1% within 45° (92% in experiment), which is 38.7% higher than the unoptimized sensor. It can be seen that the proposed sensor is hardly affected by angle when detecting stiffness. Additionally, it has abilities to distinguish different stiffness, distinguish lump from normal tissue, and recognize different stiffness samples with high precision. It signifies that the stiffness detection is no longer restricted by space and DOF, and that the sensor can detect tumors in any posture with high accuracy.Although the sensor has been well verified in this study, there are still some areas that need to be improved. The proposed sensor is sensitive within the stiffness range of samples used in tests, but the sensitivity can be further analyzed with a larger stiffness range with continuous values. For the contact angle aspect, subsequent study will focus on effectively compensating the influence of the contact angle on the sensor’s detection to obtain more accurate stiffness results. For sensor products, an isolation coating will be used for insulation and protection. Additionally, to further test the performance of the sensor, the sensor will be attached to the end of the surgical robot and a porcine bladder will be used. At the same time, the sensor will be further studied how to be more compact to meet the needs of more minimally invasive surgery.For the proposed piezoelectric sensor, we establish a new detection model with contact angle to optimize the sensor’s parameters. It makes the sensor insensitive to angle during detection, so as to meet the requirement of detecting stiffness in any posture. At the same time, the accuracy of stiffness identification is verified, proving that it has the potential to be used in interventional surgery with limited space.