Strombidium sp., Strombidium cf. sulcatum, and Halteria grandinella) in two dominant ciliate lineages. Comparative and phylogenetic analyses of newly sequenced and previously published ciliate mitogenomes were performed and revealed a number of important insights. We found that the mitogenomes of these three species are linear molecules capped with telomeric repeats that differ greatly among known species. The genomes studied here are highly syntenic, but larger in size and more gene-rich than those of other groups. They also all share an AT-rich tandem repeat region which may serve as the replication origin and modulate initiation of bidirectional transcription. More generally we identified a split version of ccmf, a cytochrome c maturation-related gene that might be a derived character uniting taxa in the subclasses Hypotrichia and Euplotia. Finally, our mitogenome comparisons and phylogenetic analyses support to reclassify Halteria grandinella from the subclass Oligotrichia to the subclass Hypotrichia. These results add to the growing literature on the unique features of ciliate mitogenomes, shedding light on the diversity and evolution of their linear molecular architecture.Determination and comparisons of complete mitochondrial genomes (mitogenomes) are important to understand the origin and evolution of mitochondria. Mitogenomes of unicellular protists are particularly informative in this regard because they are gene-rich and display high structural diversity. Ciliates are a highly diverse assemblage of protists and their mitogenomes  were amongst the first from protists to be characterized and have provided important insights into mitogenome evolution. Here, we report novel mitogenome sequences from three representatives ( Mitochondria are double-membraned semiautonomous organelles that contain their own genomes (mitogenomes) that originated from an endosymbiotic α-proteobacterium that evolved prior to the last eukaryotic common ancestor ,2,3. AltTetrahymena and Paramecium were the first confirmed to be linear double-stranded DNA molecules and to have their telomeric sequences identified, which provided novel insights into the replication of linear mitochondrial DNA [Mitogenomes usually evolve more rapidly than their nuclear counterparts in most species ,7. Additrial DNA ,26,27.Tetrahymena pyriformis, T. thermophila, T. malaccensis, T. paravorax, T. pigmentosa, T. rostrata, Ichthyophthirius multifiliis, Uronema marinum, Paramecium caudatum, P. aurelia, P. tetraurelia, P. sexaurelia, P. multimicronucleatum, P. biaurelia, P. octaurelia, P. novaurelia, P. decaurelia, P. dodecaurelia, P. quadecaurelia, and P. jenningsi) [Oxytricha trifallax, Laurentiella strenua, Stylonychia lemnae, Paraurostyla sp., Urostyla grandis, Pseudourostyla cristata, Euplotes minuta, and E. crassus) [Stentor coeruleus and Gruberia lanceolata) [Nyctotherus ovalis, Metopus contortus, Metopus es, Metopid sp., Heterometopus sp., Parablepharisma sp., and Muranothrix gubernata) [N. ovalis ~81.51% for I. multifiliis) and relatively large gene complements (20–30 protein-coding genes) [In the years since those seminal discoveries, more ciliate mitogenomes have been characterized, including 20 mitogenomes from species in the class Oligohymenophorea (nningsi) ,34,35,36crassus) ,38,39, tceolata) , as wellbernata) ,42,43. Cg genes) ,44. Mitog genes) .Strombidium sp. and Strombidium cf. sulcatum) and a representative species of Halteriidae  whose phylogenetic position is controversial . lar data . We annoStrombidium sp.: one contig; Halteria grandinella: one contig; Strombidium cf. sulcatum: 9 contigs) are recovered from the assembled mitochondrial or genomic data by BLASTN searches. They are assembled into linear molecules for the mitogenomes of Strombidium sp., S. cf. sulcatum, and H. grandinella with coverage of 194.2, 492.2–564.8, and 40.2 respectively  (tRNA_Phe (trnF) and tRNA_Tyr (trnY) in each genome. However, the central repeats differ from one another in terms of length and sequence. In Strombidium sp., the central repeat region is 170 bp long, composed of 11 repeats of the unit ATA ATA TAA TAA TAT. This region is 142 bp long in S. cf. sulcatum, composed of two repeats of the unit ATA AAT TTA ATT TTA and an irregular sequence for the rest. In H. grandinella, this region is 168 bp long and composed of nine repeats of the unit TAT ACA TAT AAT ATA TA  . A centrT ATA TA . DivergiT ATA TA .Strombidium sp., 94.89% of the mitogenome is coding sequence, including 29 known protein genes, 8 tRNA genes, 2 rRNA genes, and 12 open reading frames (ORFs) with unknown function. The mitogenome of S. cf. sulcatum also contains 29 known protein genes, but instead has 9 tRNA genes, 2 rRNA genes, and 11 ORFs, with the coding region comprising 95.09%. The proportion of coding sequence is 90.65% in the H. grandinella mitogenome, the lowest among the three taxa, which comprises 29 known protein genes, 9 tRNA genes, 2 rRNA genes, and 9 ORFs , S. cf. sulcatum: seven cases , H. grandinella: fifteen cases ) , and rps3  are separated into two parts for all the three mitogenomes, while the ccmf (cytochrome c maturation-related gene) is split only for H. grandinella. Two duplicated genes of nad1_a and tRNA_Met (trnM) are found in S. cf. sulcatum mitogenomes (For d 9 ORFs . The mit 39 bp)) . Three gogenomes . No intrOxytricha trifallax and Laurentiella strenua are complete, with both ends capped by telomeres  among the available mitogenomes in ciliates. Within the class of Spirotrichea, the A+T content of the mitogenome in Paraurostyla sp. is the highest (80.59%), while that of Urostyla grandis is the lowest , the tandem repeat region shows similar stem-loop structure: the sun-shape structure containing 4–17 helices with similar length for P. cristata, H. grandinella, U. grandis, O. trifallax, E. crassus, Strombidium sp., and Stylonychia lemnae, but with divergent length for the remaining three species. , cytochrome c reductase and oxidase genes , ATP synthase gene (atp9), ribosomal protein genes , and cytochrome c maturation-related gene (ccmf). Nevertheless, rps2 and rps8 are not detected in the mitogenome of U. grandis. In addition, the ccmf gene is split into ccmf_i and ccmf_ii in the mitogenomes of all Spirotrichea species apart from two oligotrichous species, which will be discussed in detail below  of S. lemnae and L. strenua, trnM of S. cf. sulcatum and O. trifallax, tRNA_Leu (trnL), tRNA_Lys (trnK), and tRNA_Ser (trnS) of S. lemnae, and tRNA_His (trnH) of U. grandis (tRNA_Glu (trnE), trnF, trnY, and tRNA_Trp (trnW) are present in all these eleven mitogenomes, while trnS is only detected in S. lemnae (tRNA_Cys (trnC) is detected in the mitogenome of O. trifallax  in mitogenomes of two Strombidium species and six ORFs  in two Euplotes mitogenomes are unique and no homolog is found in mitogenomes of other species  and one inversion (nad6). The mitogenomes of S. cf. sulcatum and H. grandinella are largely collinear, except that two terminal genes of nad6 and rpl14 in S. cf. sulcatum are moved to the position between cox1 and cox2 in H. grandinella. The gene orders in H. grandinella and O. trifallax mitogenomes are identical. The mitogenome synteny is also significant among the hypotrichs, except for the inversion of rps2 in Paraurostyla sp. and the gene duplication of rnl in S. lemnae, L. strenua, and Paraurostyla sp. The two Euplotes mitogenomes are collinear and are largely collinear with those of the hypotrichs in the core region, from nad3 to rnl, except for the inversion of nad5, ccmf, and cob. Besides, the gene order of tRNA, tRNA_Gln (trnQ)-trnL-trnE-trnF-trnY-trnW, is conserved in all the species except for the missing trnQ in E. crassus and trnL in U. grandis, E. minuta, and E. crassus.Gene order comparison shows that the gene synteny is highly conserved among mitogenomes within the class Spirotrichea . For theTetrahymena and Paramecium), with the exception of one large inversion and translocation from cob to rnl [Similar to the extensive collinearity within the class Spirotrichea, the mitogenomes are largely collinear within the class Oligohymenophorea  based on the mitochondrial data (ML/BI: 100/0.77), which is consistent with previous studies [α-tubulin, and actin I genes), H. grandinella was always clustered within core hypotrichs [H. grandinella and Hypotrichia [H. grandinella nests within hypotrichs, while the subclass Oligotrichia, represented by two Strombidium species because of a limitation of data, forms a monophyletic clade that is sister to hypotrichs  test. However, the clustering of H. grandinella and Strombidium taxa is rejected on the basis of mitochondrial data in the present work (p = 0.0024).The phylogenetic trees based on mitochondrial proteins reveal a concordant topology with that inferred from nuclear small subunit ribosomal DNA (nSSU-rDNA) data, thus only the topology based on mitochondrial protein with support values generated from both maximum likelihood (ML) and Bayesian inference (BI) analyses is presented . Accordi studies ,49. Accopotrichs ,51,52. Rotrichia ,48. In opotrichs . In ordeEuplotes mitogenomes, the mitogenomes of the species in Spirotrichea are generally both gene-rich and relatively large. For example, Spirotrichea mitogenomes are generally 50–70 kb, containing about 30 known protein genes and 7–12 tRNA genes, whereas the Oligohymenophorea mitogenomes are generally 40–50 kb, with about 20 known protein genes and 3–8 tRNA genes.The main features of the 34 mitogenomes in ciliates, including all the published mitogenomes of aerobic ciliates and a representative hydrogenosomal genome of anaerobic ciliates, are also compared and mapped on the phylogenetic trees . Except ccmf gene. According to previous studies, the ccmf gene is suspected to be split into two parts in the mitogenomes of O. trifallax, L. strenua, U. grandis, and P. cristata [ccmf could also be a split gene for E. minuta and E. crassus, but have three parts . Nevertheless, we found that the split of ccmf_i for E. crassus results from the insertion of an adenine nucleotide based on the comparison of our mitogenomes of E. crassus and E. vannus (unpublished) with the published data. Similarly, many insertions and deletions of ccmf_ii of E. minuta were detected. Although the insertion and deletion might come from the interspecific divergence, we think the additional split of ccmf_ii of E. minuta might not be a true split but instead is the product of sequencing errors. This speculation is consistent with the length, transmembrane (TM) profile, and sequence alignment of ccmf proteins as discussed below  based on a prediction with the NCBI Open Reading Frame Finder and SmartBLAST, with the best BLAST hit generally coming from one of the ciliate mitogenomes. Secondly, each of the two hypothetical split parts (ccmf_i and ccmf_ii) possess similar lengths among Hypotrichia and Euplotia mitogenomes, respectively: the lengths of ccmf_i are 543 amino acids for H. grandinella, 480–548 (avg. 524) amino acids for Hypotrichia, and 405 amino acids for Euplotia; the lengths of ccmf_ii are 648 amino acids for H. grandinella, 219–645 (avg. 547) amino acids for Hypotrichia, and 440–445 (avg. 442) amino acids for Euplotia. Thirdly, ccmf is a mitochondrial integral membrane protein containing multiple transmembrane helices [H. grandinella, Hypotrichia, and Euplotia . Besides, the split phenomenon was not detected based on the ccmf sequence of two Strombidium species, nearby the conserved split positions of Hypotrichia and Euplotia ccmf proteins  (repeat sequences with opposite directions at each end of the linear molecule), telomeric repeats , and central repeats , though some mitogenomes may contain one, two, or all of them. TIR is a common characteristic of linear mitogenomes from diverse eukaryotes as reviewed in  and is p–3100 bp . Notably strenua . These sStrombidium species). The telomeric repeats are also found in Tetrahymena mitogenomes (31–53 bp), but even different at each end in the T. pigmentosa mitogenome [O. trifallax, which supports the previous proposal of the telomere transfer between mitogenomes and linear plasmids [According to Nosek and Tomáška , we spectogenome . It was togenome . A lineaplasmids .Tetrahymena mitogenomes (Cl.: Oligohymenophorea) also contain a conserved putative GC-box control region located in the highly AT-rich stretch, which is speculated to be significant to bidirectional transcription [Rhodomonas salina [Hemiselmis andersenii [Chondrus crispus [Porphyra purpurea [An AT-rich central tandem repeat is discovered in all the available Spirotrichea mitogenomes, where a bidirectional transcription initiates from this region. The repeat region comprises a palindromic sequence and could be predicted to form stable stem-loop structures . Similarcription . Except s salina  and Hemidersenii ), plants crispus  and Porppurpurea ) and anipurpurea  and lagopurpurea ). Accordpurpurea ,58,61. Apurpurea ,61. All Paramecium mitogenomes, the possession of covalently-closed single-stranded hairpins (consisting of direct tandem repeats) at one end and an open structure at the opposite side was found. The hairpin structure was demonstrated to be associated with a replication origin [Paramecium mitogenomes were published [Paramecium mitogenomes: with covalently-closed sun-shape central repeat at one end and telomeric repeats at the other end  which are not detected in H. grandinella. The foregoing findings demonstrate that Halteria has a closer relationship with hypotrichs than oligotrichs.Except for the phylogenetic analyses, the characteristics of the mitogenome also support the close relationship of rifallax . Furtheron-split . It is nH. grandinella might be a “oligotrich-like” hypotrich. Both our mitogenome information and phylogenetic analyses support this hypothesis, though limited mitochondrial data in the subclass Oligotrichia is available now. Besides, as discussed in Wang et al. 2019 [H. grandinella shared more nuclear orthologous genes with oxytrichids than with oligotrichs. Considering that Halteria is the type genus of the family Halteriidae, we agree with Lynn [According to Lynn and Kolisko  and Wangal. 2019 , H. granith Lynn  that HalHalteria grandinella was collected from the pond of the Baihuayuan Park in Qingdao , China. Several cells were isolated by glass micropipette under microscope and washed 4–5 times, and then were cultured in filtered and autoclaved in situ water at room temperature, with Escherichia coli as food source. Strombidium sp. was kindly supplied by Dr. Sheng-Fang Tsai of the Institute of Environmental Biology and Fisheries Science in National Taiwan Ocean University. Strombidium cf. sulcatum was sampled from Daya Bay in Guangdong , China. Both Strombidium species were cultured in filtered and autoclaved marine water at room temperature with E. coli as food source. All of the three morphospecies were identified by live microscopic observation and silver impregnations [gnations .H. grandinella and Strombidium sp., cells were starved and treated with antibiotic-antimycotic  for about 24 h before mitochondria isolation. Samples were collected by centrifugation at 200× g for 3 min. The total numbers of cells were approximately 3 × 104 for H. grandinella and 5 × 104 for Strombidium sp. Mitochondria were isolated using a Qproteome Mitochondria Isolation Kit , following the manufacturer’s protocol without washing with 0.9% sodium chloride solution. Subsequently, the DNA extraction was performed by DNeasy Blood & Tissue Kit . Finally, the mitochondrial DNA was amplified using a REPLI-g® Mitochondrial DNA Kit  with 10 µL template DNA.For the cultures of S. cf. sulcatum, cells were starved and treated with the antibiotic-antimycotic as described above. After that, cells were harvested by filtering through a 5 µm membrane . Subsequently, cells (ca. 2 × 105) were lysed using Proteinase K solution from DNeasy Blood & Tissue Kit  and a lysis buffer  at 56 °C in a water bath for 3 h. Genomic DNA was then extracted by Phenol-Chloroform method. RNA was eliminated with RNase A .For the culture of ® Ultra™ DNA Library Prep Kit  according to the manufacturer’s instructions: mitochondrial DNA of H. grandinella, mitochondrial DNA of Strombidium sp., and genomic DNA of S. cf. sulcatum. Paired-end sequencing (150 bp reads length) was performed using an Illumina HiSeq4000 sequencer . Sequencing adapters and low-quality reads  were removed by FASTX-Toolkit with the parameters of -q 5 -p 0.5 [Three illumina libraries (350 bp) were constructed using NEBNext5 -p 0.5 .H. grandinella and Strombidium sp. as well as the whole genome of S. cf. sulcatum, were assembled using SPAdes v.3.11 with default parameters varying k-values: 21, 33, 55, and 77 [−5) using eight mitogenomes downloaded from the National Center for Biotechnology Information (NCBI) as a reference database, including Oxytricha trifallax (acc. no. JN383843), Tetrahymena pyriformis (acc. no. AF160864), Tetrahymena thermophila (acc. no. AF396436), Paramecium aurelia (acc. no. NC001324), Paramecium caudatum (acc. no. FN424190), Euplotes minuta (acc. no. GQ903130), Euplotes crassus (acc. no. GQ903131), and Nyctotherus ovalis (acc. no. GU057832).Mitogenomes of , and 77 ,67. AfteH. grandinella and Strombidium sp., one resulting contig  possessing high similarity with the reference data was found in each species . For S. cf. sulcatum, three contigs were recovered (similarity: >72.82%). These three contigs were subsequently used as references for the second search in the genomic data with BLASTN  and another six contigs were obtained. Nine mitochondrial contigs of S. cf. sulcatum with sizes ranging from about 1.2 kb to 19 kb were assembled into two scaffolds by SeqMan v.7.1.0 (DNAStar). Polymerase chain reactions (PCRs) were performed to confirm the junctions of these contigs and link the two scaffolds together into a linear mitogenome , with primers designed according to the nine mitochondrial contigs (For togenome . The PCR contigs . PCR pro contigs .https://www.ncbi.nlm.nih.gov/orffinder (accessed on 23 February 2021)) and annotated by searching against the NCBI non-redundant protein sequences (nr) database with SmartBLAST or BLASTP. The ribosomal RNA (rRNA) genes were confirmed by BLASTN searches against the NCBI nucleotide collection (nr/nt) database. The transfer RNA (tRNA) genes were also predicted using tRNAscan-SE v.2.0 with default mode and other mitogenomes as reference sources [http://www.unafold.org/mfold/applications/rna-folding-form.php (accessed on 23 February 2021)) with default parameters. RnaViz v.2.0 [Three mitogenomes were annotated firstly using both MITOS and MFannot with genetic code 4 of “the mold, protozoan, and coelenterate mitochondrial code and the mycoplasma/spiroplasma code” ,72. Then sources . Mitogen sources . The seciz v.2.0  was usediz v.2.0 .Stentor coeruleus and Gruberia lanceolata were selected as the outgroup taxa. The fourteen datasets were aligned using the MAFFT algorithm on the GUIDANCE2 Server (http://guidance.tau.ac.il/ver2/ (accessed on 23 February 2021)) and trimmed by trimAl v.1.4 with default parameters [http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 23 February 2021)).Fourteen conserved mitochondrial protein sequences were used to construct phylogenetic trees. Newly characterized sequences were combined with relevant orthologs of the other 31 ciliates downloaded from the GenBank database, resulting in fourteen mitochondrial datasets . Stentorrameters . Subsequrameters . The finrameters  with therameters  under thH. grandinella and the subclass Oligotrichia, the constrained ML tree was generated with a constraint block by enforcing the monophyly of H. grandinella and two Strombidium species. Subsequently, the approximately unbiased (AU) test [To test the monophyly of AU) test  was perfAU) test .


JNCI Cancer Spectrum! Launched in 2017 by the nonprofit organization Oxford University Press, the Journal is fully open access with a reasonable publication fee for authors, consistent with calls from international organizations for free and wide dissemination of research findings not only to researchers but also to patients. The editors of the Journal are committed to provide rapid and rigorous peer review of submitted manuscripts and publish accepted papers online in a timely fashion so important cancer research can quickly start to make an impact.Welcome to the fifth year of Journal of the National Cancer Institute (JNCI), JNCI Cancer Spectrum seeks to publish high-quality original research as well as timely reviews and commentaries. The Journal publishes a broad spectrum of cancer research that has direct relevance to cancer patient care and/or health policy, ranging from population health and epidemiology to clinical (both prospective and retrospective) and translational research. The editors encourage direct manuscript submissions to the Journal, which also provides a seamless transfer process for manuscripts that are deemed of high quality but not of sufficient publication priority for JNCI. In addition, we encourage submission of well-conducted studies that either report a novel finding or confirm or validate previously reported findings as well as clinical trials with either “positive” or “negative” results.Along with our sister journal, Under Dr Pamela Goodwin’s leadership as the inaugural Editor-in-Chief, the Journal published its first issue in September 2017. It is currently published 6 times per year. The Journal has experienced tremendous growth in the past 4 years by all metrics, including submissions, published articles, and citations . In 2020raison d'être.As Dr Goodwin completed her term as Editor-in-Chief at the end of 2020, the Journal is primed for continued and increasing success. I have been working with Pam since the Journal’s inception, initially as an Associate Editor and then as Deputy Editor. I want to thank Oxford University Press for giving me the honor and opportunity to lead this journal in its next phase; Pam for her strong leadership over the past 4 years; and the hard work of the Journal’s esteemed and international group of staff, editors, and reviewers—who together have built this journal from its beginning. Most of all, I want to thank you, the authors—because you are the foundation of the Journal—and the readers, the Journal’s I invite you to submit your manuscript to the Journal and welcome your feedback and input for the Journal as it continues to grow in its exciting next phase.


Biomechanical deviations at individual joints are often identified by gait analysis of patients with cerebral palsy (CP). Analysis of the control of joint and leg stiffness of the locomotor system during gait in children with spastic diplegic CP has been used to reveal their control strategy, but the differences between before and after surgery remain unknown. The current study aimed to bridge the gap by comparing the leg stiffness—both skeletal and muscular components—and associated joint stiffness during gait in 12 healthy controls and 12 children with spastic diplegic CP before and after tendon release surgery (TRS). Each subject walked at a self-selected pace on a 10-meter walkway while their kinematic and forceplate data were measured to calculate the stiffness-related variables during loading response, mid-stance, terminal stance, and pre-swing. The CP group altered the stiffness of the lower limb joints and decreased the demand on the muscular components while maintaining an unaltered leg stiffness during stance phase after the TRS. The TRS surgery improved the joint and leg stiffness control during gait, although residual deficits and associated deviations still remained. It is suggested that the stiffness-related variables be included in future clinical gait analysis for a more complete assessment of gait in children with CP. Spastic diplegic cerebral palsy (CP) accounts for 22% of all types of cerebral palsy , with imThe efficacy of tendon release on CP during level walking has been predominantly evaluated using functional scores and/or clinical evaluation ,10, suchLeg and joint stiffness during gait has been used for the assessment of pathology and of the efficacy of treatment because of its critical role in modulating the kinematics and kinetics of the locomotor system –21. Chanversus healthy controls. It was hypothesized that, when compared to healthy controls, the CP group would have decreased leg stiffness but with increased muscular contributions and joints stiffness during gait, and that after TRS the CP group would increase the leg stiffness with reduced muscular contributions and joint stiffness when compared to those before TRS.The purpose of this study was to compare the leg and joint stiffness, the contributions of skeletal and muscular components, and the associated joint kinematics and kinetics in children with spastic diplegic CP during level walking before and after TRS The work was approved by the Institutional Review Board of National Taiwan University Hospital, Taiwan, R.O.C (Permit Number: 201605138RIND). All participants were informed of the procedure and they provided written informed consent prior to the study. This observational, cohort study was conducted in the National Taiwan University Hospital Gait Laboratory.a priori power analysis for a two-group independent sample t-test for the comparison of leg stiffness between diplegic CP and healthy children based on pilot results using GPOWER [d = 0.88) at a significance level of 0.05.A convenience sample of twelve children with spastic diplegic cerebral palsy (CP)  was recruited from the outpatient orthopedic clinics at National Taiwan University Hospital during August 2016 to July 2019, and 12 healthy controls  were recruited from the local community to match the patient group for sex, age, height and weight during the same time period . Informeg GPOWER  determinIn a hospital gait laboratory, each subject walked at a self-selected pace on a 10-meter walkway while the motions of the body segments were measured at 200 Hz via 39 retro-reflective markers placed on specific anatomical landmarks  using anThe measured GRF and marker data were low-pass filtered using a fourth-order Butterworth filter with cut-off frequencies of 25 Hz and 10 Hz, respectively . With thEach of the lower limbs was modeled as a non-linear spring which connected the center of pressure (COP) of the GRF and the hip joint center . The varThe leg stiffness, the skeletal and muscular components and their ratios, as well as the angles, moments and stiffness at the hip, knee and ankle in the sagittal plane, were calculated for each of the lower limbs. Time-averaged values of the variables were then calculated over the sub-phases of the stance phase of each limb, i.e., loading response , mid-stance (MS), terminal stance (TS) and pre-swing . For each subject of both groups, the calculated variables from both limbs were averaged for subsequent statistical analysis.t-test, while within-group comparisons of the CP group were performed using a paired t-test. All significance levels were set at α = 0.05. All the statistical analyses were performed using SPSS version 20.0 .For each of the calculated variables, the time-averaged values of each sub-phase of the CP group were compared with those of the Control group for both Pre-OP and Post-OP using an independent Compared to Control, both Pre-OP and Post-OP of the CP group showed significantly decreased gait speed and stride length but increased step width, while only the Pre-OP showed decreased cadence and increased stride time (p<0.05) . No signCompared to Control, the CP group showed significantly increased hip and knee flexion in both Pre-OP and Post-OP throughout the stance phase, and increased plantar flexion during MS and TS, but increased ankle plantarflexion only in the Pre-OP during LR and PS (p<0.05)  Table 3Table 3. When compared to Control, both Pre-OP and Post-OP of the diplegic CP group showed significantly decreased leg stiffness during LR and PS with increased MC/SC ratio during LR and decreased SC during PS, while the Pre-OP also showed decreased leg stiffness during MS and an increased MC/SC ratio during MS to TS (p<0.05) . CompareFor the stiffness of individual joints, both Pre-OP and Post-OP of the CP group showed significantly increased joint stiffness at the hip during TS, at the knee during PS, at the ankle during LR to MS, and decreased joint stiffness at the knee during MS, when compared to Control. The Pre-OP also showed increased hip joint stiffness during LR to MS and increased knee joint stiffness during LR, while the Post-OP showed increased hip and ankle joint stiffness during PS . CompareThe current study aimed to quantify and compare the leg stiffness and the associated skeletal and muscular components, as well as lower limb joint stiffness during the stance phase of gait between healthy controls and children with diplegic CP before and after tendon release surgery. Before TRS, the patients with diplegic CP walked with decreased leg stiffness but with increased muscular contributions and joint stiffness when compared to healthy controls. After TRS, the CP group kept the leg stiffness unaltered with significantly decreased muscular contributions  and decreased joint stiffness at the hip and knee. This was accompanied with postural adjustments, including decreased knee flexion and decreased ankle plantarflexion during stance, for a more extended posture with reduced extensor moments at the knee and a more stable base of support (better foot contact).The TRS appeared to improve the lower limb joint kinematics and kinetics of the diplegic CP group to be closer to those of the healthy controls, except for hip flexion and ankle plantarflexor moments which remained unchanged. The improvement included reduced hip extensor moments during LR, reduced knee flexion angles during most of the stance phase, and reduced knee extensor moments and ankle plantarflexion angles during TS and PS when compared to Pre-OP. These results suggest that release of the tight tendons of the gastrocnemius and hamstrings helped improve the motions at the ankle and knee, and thus the dynamic alignment of the lower limb segments, contributing to the reduction of the extensor moments at the knee and hip. The underlying strategy of such kinematic and kinetic changes could be revealed through further analysis of the stiffness-related variables.During loading response, or during the initial double-limb support phase, the CP group showed significantly reduced leg stiffness with increased muscular contribution, as well as increased joint stiffness at the hip, knee and ankle before TRS when compared to the Controls. It has been suggested that the reduced leg stiffness in the CP group was as a result of the more flexed posture of the lower limbs during body weight-transfer, as was the increased effort of the muscles . The TRSDuring mid-stance, similar to loading response, the CP group also showed significantly reduced leg stiffness with increased muscular contribution, as well as increased joint stiffness when compared to the Controls. These parameters are also related to the more flexed posture of the stance limb . The TRSDuring pre-swing, or the terminal double-limb support, when the body weight was transferred to the contralateral limb, the CP group showed significantly reduced leg stiffness with reduced skeletal contribution, as well as increased knee joint stiffness when compared to the Controls. After the TRS, the leg stiffness was not altered but the knee joint stiffness and muscular contribution to the leg stiffness were significantly reduced, while the hip and ankle joints’ stiffness were increased. These changes showed the redistribution of the stiffness between the lower limb joints following TRS. The stiffer ankle and hip with a more flexible knee in the trailing limb were helpful for pushing the body forward and for the transfer of the body weight to the leading stance limb, as well as for the subsequent forward movement of the trailing limb. While the TRS helped improve the weight transfer performance during the pre-swing phase, it did not bring the leg and joint stiffness variables to normal values. Other surgical or rehabilitative intervention may be needed for further improvement of the remaining deviations in children with diplegic CP.The current study showed that the TRS altered the stiffness control of the lower limb joints during walking while retaining an unaltered whole leg stiffness in children with diplegic CP. Such a change of control strategy was helpful for reducing the demand on the muscles in maintaining the same leg stiffness and body posture against collapse. However, residual deficits and associated deviations in leg and joint stiffness still remained. Rehabilitative training such as muscle strengthening after TRS may be needed for further improving the leg and joint stiffness control. The current study was the first attempt to use leg and joint stiffness to evaluate the efficacy of TRS in diplegic CP. The current patients were limited to GMFCS grade II; for children with different GMFCS grades, further studies will be needed. Another limitation was that the subjects in this study were all male patients. Further study would be needed to test whether the current results would apply to female patients. The current results encourage future application of such analysis in the assessment of the efficacy of orthopaedic and rehabilitative treatment in patients with walking impairments.The control strategy of body support during gait in children with diplegic CP after TRS was revealed through the analysis of leg and joint stiffness, the contributions of skeletal and muscular components, and the associated joint kinematics and kinetics. The CP group altered the stiffness of the lower limb joints and decreased the demand on the muscular components while maintaining an unaltered leg stiffness during stance phase after the TRS. The TRS surgery improved the joint and leg stiffness control during gait although residual deficits and associated deviations still remained. The analysis of leg stiffness and related variables in clinical gait analysis could provide more information on treatment effects in children with CP.S1 File(XLSX)Click here for additional data file.


Prosthetic graft infection (PGI) is associated with low patient survival rates. The effectiveness of ultrasound debridement in chronic wound healing has been previously reported; however, data on the use of ultrasound technology and its effect on the treatment of PGI are still lacking. We report a case in which PGI in the groin was managed by graft removal using ultrasound debridement.A 70-year-old man was diagnosed with chronic limb-threatening ischemia and underwent a femoral-femoral bypass with a polytetrafluoroethylene graft. Eight months postoperatively, he developed an infection at the femoral incision site. Graft removal was performed using ultrasound debridement. The estimated blood loss was 10 mL. The wound healed, and the patient has remained in good health for 2 years postoperatively.When the ultrasonic probe is applied to the wound, ultrasonic energy penetrates into the tissue, and a fibrinolytic action removes necrotic or infected tissue without removing healthy tissue, thereby minimizing bleeding. Using this technique, we were able to perform effective debridement at not only the wound but also the anastomosis.It is our opinion that this technique can be used to achieve adequate debridement with little bleeding during graft removal and may provide a new option for the treatment of PGI. •Prosthetic graft infection (PGI) is associated with low patient survival rates.•We present a case in which PGI in the groin was managed by graft removal using ultrasound debridement (UD).•Ultrasonic energy penetrates into the tissue and cleaves dead tissue without removing healthy tissue.•UD can be used to achieve adequate debridement during graft removal and may provide a new option for the treatment of PGI. Traditionally, the treatment of PGI has included removing the infected prosthesis, administering antibiotics, and performing an extra-anatomic bypass. The effectiveness of ultrasound debridement in chronic wound healing has been previously reported . However2The graft removal and debridement were performed with an ultrasonically activated scalpel . Treatment with ceftriaxone, vancomycin, and local drainage was initiated. Despite continuous antibiotic treatment, CT showed that an air bubble remained 1 week later. Therefore, we performed a graft removal.A 70-year-old man presented to our hospital with rest pain and severe claudication in the right leg. He was diagnosed with chronic limb-threatening ischemia with an occluded right common iliac artery using computed tomography (CT). He initially underwent an endovascular revascularization a few years prior in a different hospital, but it failed. Therefore, we underwent a femoral-femoral crossover bypass with a polytetrafluoroethylene graft. He recovered well from this procedure and was discharged to return home 10 days postoperatively. Duplex scans were performed at follow-ups every 3 months, which indicated no significant problems. However, 8 months after the crossover surgery, he presented to our hospital with claudication in the right leg and an infected abscess in contact with the graft at the right femoral incision site . CT reveMH and HI performed the procedure . A bilateral groin and midline incision was made while the patient was under general anesthesia and in the supine position. The infected prosthetic graft was occluded by thrombosis, and it was extremely incorporated with surrounding tissues; therefore, extensive manipulation and debridement were required to remove the graft. The prosthetic graft was partially removed by ligating 3 cm from the left femoral anastomosis without disrupting the anastomosis because we did not find any significant infection in that region. The graft was removed completely from the right side of the groin, and a midline incision was made with an ultrasonically activated scalpel. The surrounding tissues received focused treatment (with the hook moving over the surface) until the infected areas and fibrin were removed; notably, the tissue appeared macroscopically normal upon the surgeon's inspection. The wall of the right common femoral artery was debrided ultrasonically  and sutuThe postoperative period of the patient was uneventful. The patient continued antibiotics until postoperative day 7 and was discharged on postoperative day 11. The wound was fully healed 1 month later. Claudication remained in the right leg; however, as of 2 years postoperatively, no significant problems have occurred.4Peripheral graft infections occur in approximately 4% of cases and may lead to limb loss ,4. When Ultrasound treatment for cutting and coagulating tissues was originally developed for use in abdominal surgery, particularly with regard to laparoscopy ,7. CurreApplying this ultrasound debridement technique on the anastomosis is the most important and effective component of this procedure. The main bleeding point during the removal of an infected graft is usually at or near the anastomosis because collateral vessels often appear near the anastomosis and because the walls of the vessels are fragile because of the infection. Ultrasonic energy can be used to induce coagulation in small collateral vessels and remove the infected tissue without disrupting healthy tissue, thereby minimizing bleeding. In addition, because, in our opinion, the ultrasound debridement completely removed the infected tissue, we were able to simply close the wound without inserting a surgical drain or performing negative pressure wound therapy.Conversely, the main disadvantage of this technique is the high cost of using this equipment. The cost of using an ultrasonically activated scalpel is approximately $900. The Harmonic Scalpel is relatively expensive, and there are additional costs for disposable single-use shears, cleaning of the shears, and sterilization of the shears and hand piece. However, this procedure can be performed on an outpatient basis and may reduce direct staff costs . WithoutA second disadvantage of ultrasound debridement is that the prolonged debridement of the wall of the graft can impact the impermeability of the graft or cause considerable damage to the graft. Thus, the safety and effectiveness of debridement or of the removal of vein grafts using this procedure are unknown. Careful manipulation of the probe is recommended during the procedure.Because this report involved a single case, no definite conclusions can be made regarding the suitability of the approach for other patients with PGI. A larger series of patients, including an analysis of the functional long-term outcomes of affected patients, is needed to confirm the findings of the present case.We described a case in which PGI in the groin was managed by ultrasound debridement with graft removal. It is our opinion that this technique can be used to achieve adequate debridement with little bleeding during graft removal and may provide a new option for the treatment of PGI.Not commissioned, externally peer-reviewed.


Brain structures are diverse among species despite the essential molecular machinery of neurogenesis being common. Recent studies have indicated that differences in the mechanical properties of tissue may result in the dynamic deformation of brain structure, such as folding. However, little is known about the correlation between mechanical properties and species-specific brain structures. To address this point, a comparative analysis of mechanical properties using several animals is required. For a systematic measurement of the brain stiffness of remotely maintained animals, we developed a novel strategy of tissue-stiffness measurement using glyoxal as a fixative combined with atomic force microscopy. A comparison of embryonic and juvenile mouse and songbird brain tissue revealed that glyoxal fixation can maintain brain structure as well as paraformaldehyde (PFA) fixation. Notably, brain tissue fixed by glyoxal remained much softer than PFA-fixed brains, and it can maintain the relative stiffness profiles of various brain regions. Based on this method, we found that the homologous brain regions between mice and songbirds exhibited different stiffness patterns. We also measured brain stiffness in other amniotes  following glyoxal fixation. We found stage-dependent and species-specific stiffness in pallia among amniotes. The embryonic chick and matured turtle pallia showed gradually increasing stiffness along the apico-basal tissue axis, the lowest region at the most apical region, while the ferret pallium exhibited a catenary pattern, that is, higher in the ventricular zone, the inner subventricular zone, and the cortical plate and the lowest in the outer subventricular zone. These results indicate that species-specific microenvironments with distinct mechanical properties emerging during development might contribute to the formation of brain structures with unique morphology. Although the vast majority of molecular machinery to generate neurons from progenitors are commonly conserved in amniotes , the aliin vivo. However, there are practical difficulties in handling several kinds of living animals, such as breeding and shipping. Furthermore, stiffness measurements should be performed under identical experimental conditions and an identical AFM system to minimize deviations. Therefore, we examined whether fixed tissues could substitute for living tissues for stiffness measurements. For fixatives, we chose 4% paraformaldehyde (PFA), a common fixative, and 3% glyoxal, a novel fixative. Recent studies have demonstrated the powerful ability of glyoxal to preserve tissue and cellular structures  were raised in KBRI. Juvenile birds  were used in this study. Other brains  were obtained according to the guidelines of each institute .Animal protocols for mice and songbirds, including breeding and experiments, were approved by and performed according to guidelines of the Committee of Korea Brain Research Institute (KBRI). Pregnant ICR mice were purchased from Core Tech and bred in KBRI. The noon on which the virginal plug was detected was defined as embryonic day 0.5 (E0.5). The day of birth was defined as postnatal day 0 (P0). E16.5 embryos and 4 weeks juvenile mice were used in this study. Songbirds , 3.13 ml of glyoxal , and 0.3 ml of acetic acid  were mixed well by vortex. After adjusting to pH 4.0 with 1 N of NaOH, the solution was filled up to 40 ml with ddH2O. The final concentration of glyoxal was 3%. Both fixative solutions were prepared on the day of the experiment and kept cool until use.Paraformaldehyde  was dissolved in PBS (pH 7.4) at a final concentration of 4%. The glyoxal fixative solution was prepared according to published protocol . BrieflyImmersion fixation was applied in the embryonic stage for the mice and chicks, at 4 months and 2.5 years for the turtles, and at E35 and P0 for ferrets. Mouse embryos were taken from uteruses following the cervical dislocation of the mothers and kept in ice-cold PBS. Chick embryos were taken from fertilized eggs and fixed at 7 and 10 days of incubation at 37°C  . These sThe transcardial perfusion was applied to juvenile animals. ICR mice were deeply anesthetized with intraperitoneal injections of pentobarbital  and then perfused transcardially with either 4% PFA or 3% glyoxal (pH 4.0) fixative followed by PBS. The songbirds were deeply anesthetized with isoflurane  and then perfused like the mice. The brains were dissected out and post-fixed with fixative with the same perfusion overnight at 4°C and then kept in PBS at 4°C until vibratome sectioning.All procedures were performed in ice-cold media according to a previous publication with slight modifications . BrieflyTo obtain the acute slices of juvenile mouse and songbird brains, artificial cerebrospinal fluid containing sucrose (slicing ACSF) was used to dissect and make vibratome sections. Brains were immediately dissected into ice-cold slicing ACSF and glued directly on the stage with cyanoacrylate glue. The brains were cut into 300-μm-thick slices in ice-cold slicing ACSF using a vibratome. Acute slices were incubated in slicing ACSF for 45 min and then transferred to measurement ACSF. The ACSF composition was described in a previous study . SupplemThe measurement method was slightly modified from our previous publication  to optimTo confirm the measured regions, the acute slices were immediately fixed with 4% PFA for 1 h at room temperature after measurement for DAPI staining. For immunohistochemistry, the adjacent cryosections of stiffness measured slices were incubated in 0.5% Triton-X 100/PBS for permeabilization for 10 min and then a 2% BSA/0.1% Triton-X-100 solution for 2 h for blocking followed by washing with PBS. Subsequently, the cryosections were incubated with primary antibodies for overnight at 20°C and then incubated with secondary antibodies for 2 h at room temperature followed by washing with PBS. The primary and secondary antibodies used in this study were rabbit anti-Tbr1 , rat anti-Ctip2 , and mouse anti-Satb2 . The secondary antibodies were Alexa 488-, 555-, and 647-conjugated . DAPI was used to counter stain nuclei. Stained samples were mounted with PermaFluro (Thermo Fischer Scientific) and then observed using an upright confocal laser microscopy  and Panoramic Scan II (3DHISTECH).t-test was applied to compare two conditions, and one-way analysis of variance (ANOVA) and the Tukey post hoc test were applied to compare more than three conditions. Differences were considered significant at ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ****P < 0.0001. Error bars in graphs are represented as the mean ± SEM.All statistical analyses were performed using Prism 8 (GraphPad). A two-tailed unpaired in situ. For this purpose, we chose 4% PFA and 3% glyoxal solutions as fixatives. PFA is common in histological studies, and glyoxal is a small dialdehyde molecule that is reported to provide better morphological preservation and strong fixation of both proteins and RNAs at cellular resolution because of its rapid penetration , and proliferative region, including SVZ and VZ (SVZ+VZ), based on the phase-contrast images . SubsequWe further compared the stiffness tendencies of PFA- and glyoxal-fixed brains and acutely prepared living brains using 4-week-old juvenile mice. We made coronal slices, including the hippocampus, from each condition  and measSubsequently, we investigated the effect of fixative solutions on tissue stiffness using other species. For this purpose, we measured stiffness in juvenile songbird brains (30–40 dph). Juvenile songbirds can begin to feed themselves around this age, and this developmental period is considered relevant to the weaning stage of mice around 4 weeks after birth. We prepared coronal slices containing pallium in the dorsal part and striatum (Str) in the ventral part of the brains and measured the stiffness in both regions . As withThe mammalian cerebral cortex consists of six layers, whereas the avian telencephalon consists of neuronal nuclei. Despite this difference in brain structure, mammalian and avian brains share representative neuronal markers, such as Satb2, Ctip2, and Tbr1 , althougAs reported previously , corticaThe pallium of songbirds consists of the hyperpallium (HP), mesopallium (M), nidopallium (N), and entopallium (E) . The sonFinally, we applied our glyoxal brain-fixation method to comparatively analyze the stiffness of various species using chicks (E7 and E10), turtles (4 months and 2.5 years), and ferrets (E35 and P0). These brains were fixed by glyoxal immediately after dissection and shipped overseas. Subsequently, slices were made to measure their stiffness in one place using the same conditions and an AFM system. We prepared coronal brain slices of each animal and then measured the stiffness in the pallium along the apico-basal axis . The embThe highlighted findings of this study are as follows: (1) Glyoxal-fixed brains can fairly maintain the relative stiffness of living tissue, and (2) brains fixed with glyoxal exhibit species-specific stiffness profiles.in situ hybridization and next-generation sequencing analysis (Regarding (1), glyoxal has several advantages in fixation. It was reported several decades ago as an alternative fixative to formalin . Its simanalysis . Importaanalysis , a coloranalysis , 6. ThisThe stiffness in the fixed tissue was higher than that of living tissue irrespective of brain size and fixation method, that is, PFA or glyoxal fixation. Remarkably, however, brain tissue fixed with glyoxal remained much softer than PFA and maintained relative stiffness like living conditions in most cases . Since gRegarding (2), we examined the stiffness profiles of homologous structures across species  and stagFirst, we compared the stiffness of the homologous structure shared by the mouse NCx and songbird HP to investigate whether the pallial cytoarchitecture affected the tissue stiffness . In the Second, we applied glyoxal fixation to several amniotes to investigate the stiffness of their pallia exhibiting different morphologies. We chose representative species based on phylogenetic and histological reasons . We succWe found stage-dependent and species-specific stiffness in pallia among amniotes. For instance, a monotonous pattern of stiffness was observed in chicks at E7 . This teIn comparing the matured pallia, gradually increasing stiffness along the apico-basal axis was observed in the 2.5 years turtle DC, which was derived from the dorsal pallium, a homologous region of the mammalian NCx . This grIn conclusion, glyoxal fixation can be applicable to the study of the mechanical properties of the brain in combination with AFM. Our findings based on our method strongly suggest that species-specific microenvironments might exist in the brain and that distinct mechanical properties could provide different cues to neural cells to form diverse brain structures as a result of migration during development. To identify the interactions between cells and microenvironments, further systematic analysis is required. Therefore, we propose a novel research model for brain development based on the mechanical properties of microenvironments . CombiniThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Committee of Korea Brain Research Institute.MI and YK designed the research concept and wrote the manuscript. MI prepared the mouse brains, measured the stiffness, and analyzed the results. TN prepared the turtle and chick brains and wrote the manuscript. TS and FM prepared the ferret brains. SK prepared the songbird brains. All the authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: This study aimed to assess the association between the percentage of glomerulosclerosis (GS) in procurement allograft biopsies from high-risk deceased donor and graft outcomes in kidney transplant recipients. Methods: The UNOS database was used to identify deceased-donor kidneys with a kidney donor profile index (KDPI) score > 85% from 2005 to 2014. Deceased donor kidneys were categorized based on the percentage of GS: 0–10%, 11–20%, >20% and no biopsy performed. The outcome included death-censored graft survival, patient survival, rate of delayed graft function, and 1-year acute rejection. Results: Of 22,006 kidneys, 91.2% were biopsied showing 0–10% GS (58.0%), 11–20% GS (13.5%), >20% GS (19.7%); 8.8% were not biopsied. The rate of kidney discard was 48.5%; 33.6% in 0–10% GS, 68.9% in 11–20% GS, and 77.4% in >20% GS. 49.8% of kidneys were discarded in those that were not biopsied. Death-censored graft survival at 5 years was 75.8% for 0–10% GS, 70.9% for >10% GS, and 74.8% for the no biopsy group. Among kidneys with >10% GS, there was no significant difference in death-censored graft survival between 11–20% GS and >20% GS. Recipients with >10% GS had an increased risk of graft failure , compared with 0–10% GS. There was no significant difference in patient survival, acute rejection at 1-year, and delayed graft function between 0% and 10% GS and >10% GS. Conclusion: In >85% KDPI kidneys, our study suggested that discard rates increased with higher percentages of GS, and GS >10% is an independent prognostic factor for graft failure. Due to organ shortage, future studies are needed to identify strategies to use these marginal kidneys safely and improve outcomes. In the United States, more than 90,000 patients with end-stage kidney disease (ESKD) are currently waiting for a kidney transplant ,2. A sigThe shortage of deceased donor organs continues to be a problem in kidney transplantation despite the implementation of expanded criteria donor (ECD) programs in 2002 to increase the use of organs from donors with ≥60 years or comorbidities . In 2013Despite conflicting evidence regarding the prognostic capability of histologic findings for differentiating donor kidneys at greater risk of inferior outcomes ,18,19, tThe aim of this study is to explore the association between the percentage of GS and graft outcomes in kidney transplant recipients who received KDPI >85% kidneys between 1 January 2005 to 2 December 2014 using the Organ Procurement and Transplantation Network (OPTN)/UNOS database.We used the OPTN/UNOS database to identify deceased-donor kidneys recovered from January 1, 2005 to December 2, 2014 . The study was exempt from the institutional review board due to the publicly available nature of the de-identified database of the OPTN/UNOS database. All data used in the analysis were provided by UNOS through the Standard Transplant Analysis and Research (STAR) database. The database is a de-identified, patient-level data source that contains donor, waitlist, and transplant recipient variables derived from UNet forms for any transplant in the United States after October 1, 1987. KDPI (reference year of 2017) was calculated based on donor factors to summarize the likelihood of graft failure after deceased donor kidney transplant. Higher KDPI scores are associated with shorter estimated graft function. Although the KDPI was not formally introduced into allocation policy until implementation of the new kidney allocation system (KAS) on December 2014, the OPTN/UNOS database has KDPI values for 99% of all deceased donor recipients who underwent kidney transplantation during the study period. To assess the predictive value of procurement biopsy GS percentage in high-risk deceased donors, we only included deceased-donor kidneys with a KDPI score > 85%. We excluded recovered kidneys for dual-kidney transplant and kidneys from donors with body weight < 20 kg. Subsequently, we assess the post-transplant outcomes based on GS percentage in deceased-donor kidney transplant recipients who received kidney with KDPI > 85%. We excluded patients undergoing kidney re-transplants or multi-organ transplant from the analysis. We categorized deceased donor kidneys into four groups based on the percentage of GS: 0–10%, 11–20%, >20% and no biopsy performed. We investigated the kidney discard rates and post-transplant deceased donor allograft outcomes based on GS groups. The primary outcome was death-censored graft survival. Death-censored graft survival began at kidney transplant, was followed until graft failure, defined as the requirement of renal replacement therapy and/or kidney re-transplant, and was censored at death or the end of study (6 September 2018), whichever was earlier. The secondary outcomes were patient survival, rate of delayed graft function, and 1-year acute rejection. Delayed graft function was defined as a requirement of dialysis within the first week of transplantation. As there was no statistical difference in any post-transplant outcomes between 11–20% and >20% GS , we combDonor-related factors included donor age, sex, race, diabetes mellitus, hypertension, body mass index, the last serum creatinine before kidney procurement, donation after cardiac death, hepatitis C virus (HCV) antibody status, cause of death, and machine perfusion. Recipient-related factors included recipient age, sex, race, body mass index, diabetes mellitus, preemptive transplant, dialysis duration, and panel reactive antibody. Transplant-related factors included HLA-DR mismatch, cold ischemic time, transplant period, and induction therapy.t-test or ANOVA, as appropriate. Categorical variables were compared between GS groups using the Chi-squared test. Patient survival and death-censored graft survival outcomes were estimated using the Kaplan–Meier method with significance tested using the log-rank test. The associations of the GS percentage group with death-censored graft failure and patient mortality was assessed using Cox proportional hazards analysis. The proportional hazards assumption was tested using Schoenfeld residuals (p = 0.29). Because the OPTN/UNOS database did not specify the date of occurrence, the associations of the GS percentage group with delayed graft function and 1-year acute rejection were assessed using logistic regression analysis. Multivariable analysis was performed to adjust for covariates associated with outcomes of interest with p < 0.05 in univariate analysis. All p-values were two-tailed, and p-values of <0.05 were considered significant. Stata version 13  was used for all statistical analyses.Baseline characteristics were described using mean ± standard deviation (SD) for continuous variables or frequencies with percentage for categorical variables. Continuous variables were compared between GS groups using the student’s During the study period, 25,154 kidneys were recovered from deceased donors with KDPI > 85%. A total of 3014 kidneys recovered for dual-kidney transplant and 134 kidneys from donors with a body weight < 20 kg were excluded. A total of 22,006 kidneys with KDPI > 85% were included in the kidney procurement cohort. Of these kidneys, 58.0% had 0–10% GS, 13.5% had 11–20% GS, 19.7% had >20% GS, and 8.8% had no kidney biopsy performed . Overallp = 0.70). The donor, recipient, and transplant-related characteristics stratified by percent of GS are shown in In this cohort of 22,006 deceased donor kidneys with KDPI > 85%, 10,662 kidneys were discarded. After excluding 1032 recipients with prior kidney transplants or undergoing multi-organ transplant, a total of 10,312 recipients with donor KDPI > 85% were included in the post-transplant outcome analysis. Of these patients, 75.6% had 0–10% GS, 11.9% had 11–20% GS, 4.9% had >20% GS, and 7.6% had no kidney biopsy performed . The medp < 0.001).The median (IQR) follow-up was 4.87  years after kidney transplant. During follow-up, 3015 (29.2%) patients had allograft failure, and 4433 (43.0%) patients died. A total of 1443 (14.0%) patients had acute rejection within one year, and 3436 (33.3%) patients had delayed graft function. p < 0.001). After adjusting for baseline donor, recipient, and transplant-related factors, kidneys with >10% GS remained significantly associated with a 27% higher risk of graft failure compared to kidneys with 0–10% GS  (p = 0.40), rate of acute rejection at 1-year , and rate of delayed graft function  between 0–10% GS and >10% GS  . Of note< 0.001) . There w >10% GS . p < 0.001), as shown in We examined the graft outcomes of >85% KDPI kidney with a low degree of GS, compared with 71–85% KDPI kidneys. The death-censored graft survival at 5 years in >85% KDPI kidneys with 0–10% GS was inferior to in 71–85% KPDI kidneys , as shown in Kidneys donors with no biopsy performed were younger, more were female, and had a greater prevalence of positive hepatitis C antibody, but had a lower prevalence of diabetes, hypertension, body weight, donation after cardiac death, use of machine perfusion, and expanded criteria donation when compared with kidneys with 0–10% GS . Graft sOver 700,000 patients in the United States have ESKD, with the United States having the second-highest incidence rate of treated ESKD in the world . DespiteOur study showed that procurement biopsies are becoming increasingly common in marginal deceased donors in the United States. Ninety-one percent of KDPI >85% kidneys were biopsied on procurement during this study period compared to 85% between 2000 and 2003 in the United States . The utiUsing the UNOS database, we demonstrated that GS > 10% is an independent prognostic factor for graft failure in >85% KDPI kidneys, with an adjusted 1.28-fold increased risk of graft failure at 5 years when compared to kidneys with 0–10% GS. The findings of our study suggest that the use of GS percentage in procurement biopsy of >85% KDPI kidneys may improve risk stratification for recipient allograft survival. While GS > 10% was associated with a higher risk of graft failure in >85% KDPI kidneys, we did not find a difference in death-censored graft survival between allografts with 11–20% GS and >20% GS. This may suggest that >10% GS in procurement biopsies can potentially be utilized as a cutoff for risk prediction in clinical practice. Given that the presence of GS > 10% in >85% KDPI kidneys had no significant impact on delayed graft function rate, acute rejection, or patient survival, the underlying explanation for higher graft failure in GS > 10% kidneys is likely due to the progressive kidney aging process in a kidney with less residual function. As the phenotype of GS is associated with podocyte detachment and a reduced number of functioning and viable glomeruli, this leads to increasing ESKD prevalence ,37. It h2 [This data should not discourage the use of >85% KDPI kidneys with >10% GS. There is an organ shortage with a growing number of individuals who develop ESKD every year  combined2 ; furtherAlthough our study aimed to assess the impact of GS degree on >85% KDPI graft outcomes, the findings of our study cannot be generalized to lower KDPI kidneys. We did compare graft outcomes between KDPI >85% kidneys with 0–10% GS to the overall KDPI 71–85% kidneys. This demonstrated that graft outcomes of KDPI >85% kidneys with 0–10% GS were inferior to KDPI 71–85% kidneys, suggesting a stronger impact of KDPI-related factors on graft outcomes over the percentage of GS on procurement biopsies. As KDPI is comprised of several clinically important donor characteristics that impact outcomes , it is hAlthough our study using the UNOS database is among the largest cohorts investigating procurement biopsies with KDPI > 85%, there are some major limitations. First, there is a lack of uniform criteria for procuring, processing and interpreting procurement graft biopsies ,54. CoreIn conclusion, we demonstrated that procurement biopsies for >85% KDPI kidneys are very commonly obtained in the United States, at a rate of 91.2%. A higher percentage of GS in >85% KDPI kidney biopsies are associated with an increased discard rate. Among KDPI >85% kidneys, GS >10% is an independent risk factor for allograft failure. However, graft survival from 0–10% GS kidneys is still inferior to kidneys with KDPI 71–85%, suggesting a stronger impact of KDPI on graft outcomes. Instead of discarding kidneys, future studies are needed to identify strategies to optimally utilize these “marginal” kidneys safely.


Cryoconite holes are miniature freshwater aquatic ecosystems that harbor a relatively diverse microbial community. This microbial community can withstand the extreme conditions of the supraglacial environment, including fluctuating temperatures, extreme and varying geochemical conditions and limited nutrients. We analyzed the physiological capabilities of microbial isolates from cryoconite holes from Antarctica, Greenland, and Svalbard in selected environmental conditions: extreme pH, salinity, freeze-thaw and limited carbon sources, to identify their physiological limits. The results suggest that heterotrophic microorganisms in cryoconite holes are well adapted to fast-changing environmental conditions, by surviving multiple freeze-thaw cycles, a wide range of salinity and pH conditions and scavenging a variety of organic substrates. Under oxic and anoxic conditions, the communities grew well in temperatures up to 30°C, although in anoxic conditions the community was more successful at colder temperatures (0.2°C). The most abundant cultivable microorganisms were facultative anaerobic bacteria and yeasts. They grew in salinities up to 10% and in pH ranging from 4 to 10.5 (Antarctica), 2.5 to 10 , and 3 to 10 (Greenland). Their growth was sustained on at least 58 single carbon sources and there was no decrease in viability for some isolates after up to 100 consecutive freeze-thaw cycles. The elevated viability of the anaerobic community in the lowest temperatures indicates they might be key players in winter conditions or in early melt seasons, when the oxygen is potentially depleted due to limited flow of meltwater. Consequently, facultative anaerobic heterotrophs are likely important players in the reactivation of the community after the polar night. This detailed physiological investigation shows that despite inhabiting a freshwater environment, cryoconite microorganisms are able to withstand conditions not typically encountered in freshwater environments , making them physiologically more similar to arid soil communities. The results also point to a possible resilience of the most abundant microorganisms of cryoconite holes in the face of rapid change regardless of the location. Ice sheets and glaciers are the biggest freshwater ecosystem on the planet , which iRates of microbial activity in cryoconite sediment are similar to those found in temperate freshwater sediments . Yet micMetagenomic and molecular studies of Alpine cryoconite hole communities have attempted to characterize the mechanisms of adaptation to these extreme stressors. At Rotmoosferner, Austria, it was demonstrated that microbial community members not only have a large array of stress response genes, but that they also have significant genetic potential for effective nutrient and organic carbon scavenging/recycling . UtilizaMuch of the research on polar cryoconite holes has been focused on geochemistry, net ecosystem productivity and carbon cycling , whilst n = 3) were collected from ice-lidded cryoconite holes on Canada Glacier in McMurdo Dry Valleys  in the Austral summer of 2005/2006. Svalbard samples (n = 3) were collected from Midtre Lovénbreen glacier, approximately 4 km from the United Kingdom Arctic Research Station in Ny-Ålesund  in the melt season of 2016. The Greenland study site (n = 3), “Black and Bloom” , with three samples from each location analyzed. Antarctic samples  was diluted 1 to 10 in substrate free medium, then fixed in 1.6% formaldehyde and stained with acridine orange. Total cell counts were determined in three technical replicates with a Zeiss Axioskop microscope after staining.The total cell count of each of the sediment sample was performed using the epifluorescence microscopy following method of n = 3 for Greenland and Svalbard, n = 2 for Antarctica). Following initial dilution of ∼1 g of the sediment in the 9 ml of water, a series of eight 10-fold dilutions in three replicates was prepared on a 96-well plate (–1), MgCl2 ⋅ 6H2O (0.25 g l–1), CaCl2 ⋅ 2H2O (0.1 g l–1), KCl (0.1 g l–1), NH4Cl (0.1 g l–1), KH2PO4 (0.1 g l–1), casamino acids (0.25 g l–1), and yeast extract (0.05 g l–1). The medium was supplemented with 1 ml l–1 of the trace element solution SL 10, 0.2 ml l–1 of a selenite and tungstate solution (–1) and the pH was adjusted to 7.2 with 1 M NaOH prior to autoclaving. After autoclaving, the medium was supplemented with 2 ml l–1 of vitamin solution (–1). For anaerobic microorganisms, a bicarbonate-buffered fermenter medium was used, containing: MgCl2 ⋅ 6H2O (0.1 g l–1), CaCl2 ⋅ 2H2O (0.35 g l–1), KCl (0.1 g l–1), NH4Cl (0.1 g l–1), KH2PO4 (0.1 g l–1), casamino acids (0.25 g l–1), vitamin solution (2 ml l–1), glycine betaine (0.5 mM), sodium acetate (0.5 mM), TCA mixture . Overalrium sp. , whereasCellulomonas strains can have differences in their physiology (Flavobacterium genus, which is also widespread in cryoconite holes, where two distinct species had 98.9% 16S rRNA gene sequence similarity (Some closely related isolates assigned to the same OTU showed striking differences in their metabolic capabilities. Without further studies it is impossible to pinpoint the source of the differences, but possible reasons include the initial incubation temperature, fitness of the culture and the metabolic impact of genome rearrangement in stressed and starving microorganisms. Such differences are quite common, for example ysiology , even whmilarity . The resmilarity . Isolatemilarity . Finallymilarity  and barrmilarity .Microorganisms inhabiting cryoconite holes are exposed to a wide range of extreme conditions, and this study demonstrates their broad tolerance. The generally oxygen-rich cryoconite holes harbor an active, culturable anaerobic community. Anaerobic cultivability is better in the coldest conditions tested, which suggests their adaptation to and dominance of the beginning or end of the melt season, when anoxic conditions are likely to occur. Apart from anoxia, cultured microorganisms can withstand a wide range of other physical stresses, including extreme pH and salinity. As pH tolerance broadly reflected the values found in the samples locations, this may indicate that extreme salinities could also be found in cryoconite holes in certain conditions, for example, during freeze-thaw cycles, or that the microbial community is seeded from nearby saline habitats, such as arid soils or melt ponds. Cryoconite microorganisms use a wide range of substrates and as a community are effective in scavenging limited carbon sources in cryoconite holes. Their metabolic capabilities seem to depend not only on the genetic affiliation, but also, perhaps, fitness of the culture and phenotypic differences between closely related species. Such phenotypic differences are especially likely, as the bacteria with a high 16S rRNA gene similarity show differences in their physiology. Antarctic isolates showed greater resistance to freezing and thawing cycles and greater use of variable carbon sources when compared to Arctic ones, suggesting they might be adapted to harsher conditions of Antarctic cryoconite holes. However, we found some of the same genera in both Arctic and Antarctic cryoconite holes, similar total cell numbers and the same range of salinities withstood, demonstrating that cryoconite hole microorganisms from both poles also share some physiological traits.The datasets generated for this study can be found in the Genbank MT430950, MT432272–MT432304, MT473233, and MT473713–MT473721.EP, EB, HS, MT, and AA contributed to the conception and design of the study. EP, HS, GW, CW, and AS performed the research. EP, EB, and HS analyzed the data. All authors contributed to the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The essence of the improvement in the toughness of the PLA/PSt/PEG/CA blend was the esterification reaction among CA, PEG, and starch. During the melt-blending process, the CA with abundant carboxyl groups reacted in the amorphous region of the starch. The shape and crystal form of the starch did not change, but the surface activity of the starch improved and consequently increased the adhesion between starch and PLA. As a plasticizer for PLA and starch, PEG effectively enhanced the mobility of the molecular chains. After PEG was dispersed, it participated in the esterification reaction of CA and starch at the interface and formed a branched/crosslinked copolymer that was embedded in the interface of PLA and starch. This copolymer further improved the compatibility of the PLA/starch blends. PEGs with small molecules and CA were used as compatibilizers to reduce the effect on PLA biodegradability. The esterification reaction on the starch surface improved the compatibilization and toughness of the PLA/starch blend materials and broadens their application prospects in the fields of medicine and high-fill packaging.In this study, poly(lactic acid) (PLA)/starch blends were prepared through reactive melt blending by using PLA and starch as raw materials and vegetable oil polyols, polyethylene glycol (PEG), and citric acid (CA) as additives. The effects of CA and PEG on the toughness of PLA/starch blends were analyzed using a mechanical performance test, scanning electron microscope analysis, differential scanning calorimetry, Fourier-transform infrared spectroscopy, X-ray diffraction, rheological analysis, and hydrophilicity test. Results showed that the elongation at break and impact strength of the PLA/premixed starch (PSt)/PEG/CA blend were 140.51% and 3.56 kJ·m With the increasing tension in petroleum resources and environmental issues, several bio-based degradable resources have been drawing wide attention in recent years. Poly(lactic acid) (PLA), which is the most promising bio-based degradable material, can be obtained through the polymerization  of lactiStarch contains numerous hydroxyl groups and has high polarity. However, when starch and PLA are blended, using the former as a filler cannot improve and even reduces the toughness of the PLA/starch blends. The layered microstructure along the flow direction is formed in the PLA/starch blends through the pressure-induced flow (PIF) process proposed by Zhang et al. . During In this study, PLA/premixed starch (PSt)/PEG/citric acid (CA) blends were prepared through reactive blending to examine the effects of CA and PEG on the toughness of PLA/PSt  blends. CA with high carboxyl underwent an esterification reaction with starch and consequently enhanced the interface adhesion between starch and PLA. As a plasticizer of PLA and starch, PEG effectively dispersed in the matrix and enhanced the mobility of the molecular chains. When the PEG that entered the matrix participated in the esterification reaction between CA and starch, a branched/crosslinked polyester was easily formed at the interface. This polyester further improved the compatibility of the PLA/starch blend materials. This PLA/starch composite material uses biodegradable raw materials, and at the same time, a highly filled PLA/starch composite material with excellent performance is obtained through reactive blending. Therefore, this composite material has broad application prospects in the medical field and packaging field.3 and 160 °C, respectively) was purchased from NatureWorks LLC . The corn starch (St) was provided by Wuhan Huali Environmental Protection Technology Co., Ltd . The citric acid (CA) monohydrate and PEG 1000  were purchased from Sinopharm Chemical Reagent Co., Ltd . The vegetable oil polyol (VOP) was provided by Dongguan Aoda Environmental New Materials Co., Ltd .The PLA  was used to test the samples at a tensile speed of 5 mm/min to obtain the stress–strain curve, tensile strength, and elongation at break. The 4 mm-thick samples were cut into a standard spline in accordance with ISO 179-1, and the impact energy was measured through the Charpy impact test  with a range of 0.5 J. The impact strength of the sample was obtained using data calculation. The data of the samples in every test were measured on at least five splines.A rotating plate rheometer  was used to obtain the linear rheological behavior of the mixture with plate–plate geometry. The sample is a 1 mm-thick disc with a diameter of 25 mm. The set temperature was 170 °C, the frequency range was 0.1–100 Hz, and the strain was 0.1%.cX represents the crystallinity of PLA, A DSC  was used for crystallization performance analysis of the PLA/PSt/PEG/CA blend. The sample was heated from 30 to 200 °C at a rate of 10 °C/min and maintained at 200 °C for 3 min to eliminate the previous thermal history and then cooled to 30 °C at a rate of 10 °C/min. The sample was heated again from 30 to 200 °C at the same increase rate. The second heating curve was recorded, and the crystallinity of PLA was calculated as (1)Xc=ΔHThe morphology of the impact fracture surface was examined using a SEM  at an acceleration voltage of 10 kV. The 4 mm-thick sample was broken at room temperature, and the impact fracture surface was washed with ethanol.The XRD analyzer from Panalytical B.V.  was used to test the pretreated powder samples with the target of Cu, test conditions: voltage = 40 kV, current = 30 mA, start angle = 5°, end angle = 40°, and scan step width = 0.02°.2Cl2), washed with ethanol, and dried to obtain powder samples. The powders were mixed with potassium bromide at a weight ratio of 1:100 and subsequently compressed. Measurements were performed using an FTIR spectrometer  with a resolution of 4 cm−1 and scan number of 32.The blend was dissolved in dichloromethane . The blends were cut into small pieces with sizes of 10 × 10 × 2 mm and placed in a vacuum oven at 50 °C for 48 h. The initial weight mmined as .(2)Wate−2, respectively, which reflects the brittleness of PLA. Compared with pure PLA, the elongation at break of PLA/PEG/CA blends increased slightly, while the tensile strength decreased, which may be due to the ability of low-molecular-weight PEG to plasticize PLA [The mechanical properties of pure PLA and PLA/PSt/PEG/CA blends with different PSt mass ratios are shown in cize PLA . There icize PLA , and thecize PLA . When thTo explore the contribution of each component of the PLA/PSt/PEG/CA blend to the mechanical properties of the blend, several blends with different components were fabricated. The test results are summarized in By testing the rheological properties of the blend, it is helpful to analyze the changes in the compatibility of the blend components. Generally, after PLA is blended with poorly compatible materials such as polyamide11 , starch The behavior of the storage and loss moduli as a function of frequency is shown in g and Tc, which may be due to the VOP entering the PLA matrix during the blending process destroying the crystallization of PLA. Secondly, the VOP attached to the surface of the starch blocks the interface between PLA and starch, inhibiting the heterogeneous nucleation [g, Tc, and Tm of PLA/PSt/PEG blends further decreased, which was mainly due to the plasticization of PLA by PEG. PEG can enter between PLA macromolecules during melt mixing and establish physical interactions such as hydrogen bonding or dipole-dipole interactions in between atoms. As a result, some of the rigid homogeneous PLA-PLA interactions were replaced by heterogeneous PLA-PEG interactions. This phenomenon enhanced the mobility of PLA and decreased the energy consumption during glass transition (decreased Tg) [c and Tm of the PLA/PSt/CA blend increased. This may be because the surface polarity of the starch changed after CA was introduced into the blend. The reason for the change may be that CA participated in the esterification reaction and formed a branched/crosslinked copolymer on the surface of the starch, which enhanced the interaction between the components of the blend [g will increase as the interaction force between the components of the blend increases. However, the Tg of PLA/PSt/CA blends only increased slightly, which may be because CA tends to change the surface polarity of starch rather than acting on PLA. Moreover, the depolymerization of PLA by CA will destroy the crystallization of PLA and reduce Xc. Comparing PLA/PSt blends with PLA/PSt/CA blends, the result is that CA can slightly increase the Tg of the blends. However, when the PLA/PSt/PEG blend is compared with the PLA/PSt/PEG/CA blend, the result is that CA slightly reduces the Tg of the blend. This shows that when CA and PEG are contained in the blend, PEG may also participate in the esterification reaction in the melt-blending process. Some interesting facts have also been discovered from the perspective of crystallinity changes in g of the blend to decrease. The change of the interface structure between PLA and starch indicates that the compatibility of the two is increased. At this time, PSt can be regarded as a heterogeneous nucleating agent of PLA and promotes the crystallization of the PLA matrix. The increase in effective nucleation sites in the blend enhances the interfacial adhesion between PLA and starch and allows the blend exhibit excellent toughness in the macroscopic view, and this has been confirmed from the mechanical performance test results.PLA is a typical semi-crystalline polymer  whose mecleation  of PLA based Tg) . Comparehe blend . UsuallyOn the basis of the above analysis, CA is an important factor in toughening PLA/PSt/PEG/CA blends. To observe the effect of CA and PEG on the blend, a separate dispersed phase of PLA/PSt/PEG/CA blends was obtained by dissolving and washing. The surface morphology of the dried dispersed phase was observed using SEM. The morphologies of several samples examined under 5k× magnification are shown in The XRD patterns of pure starch and pretreated PLA/PSt/CA and PLA/PSt/PEG/CA powder samples are shown in −1 can only be detected in VOP and PSt. No C=O absorption peak is observed in the FTIR spectra of the PLA/PSt and PLA/PSt/PEG powder samples after pretreatment. This result indicates that the PLA and VOP in the blend are completely removed, and PEG may display a plasticizing effect. However, C=O absorption peaks are detected in the PLA/PSt/CA and PLA/PSt/PEG/CA powder samples after pretreatment, which means that ester groups are still present in the powder samples at this time. CA participates in the esterification reaction during the blending process, and a branched/crosslinked copolymer forms. This branched/crosslinked copolymer is coated on the surface of the starch to prevent the removal of the ester group during the blending process. Another obvious change in peak position occurs near 2863 cm−1, which can be attributed to the C–H symmetric stretching vibration absorption peak in VOP and PSt. The respective absorption peaks of pretreated PLA/PSt and PLA/PSt/PEG powder samples at 1743 and 2863 cm−1 disappear, implying that no chemical changes occur during the blending process. Starch only acts as a large-sized filler, and PEG acts as a plasticizer. The immiscibility between PLA and starch decreases the macroscopic tensile toughness of the blend. The esterification reaction might occur in the PLA/PSt/CA and PLA/PSt/PEG/CA powder samples after pretreatment, which might improve the surface activity of starch and increase the compatibility between PLA and starch. Furthermore, the blend shows a significant increase in tensile and impact toughness.Chemical reactions may affect the surface morphology of starch, and this effect will influence the compatibility of the components of the blend. The dispersed phase was extracted from several blends, and FTIR spectroscopy was performed to analyze the changes in the compatibility among the components. The FTIR test results of several samples are presented in 2O molecules to form hydrogen bonds. From the results shown in The water absorption rates of several blends are shown in In view of the change of the hydrophilicity of the blends, the dynamic contact angles of several blends were analyzed. As shown in In addition, low-molecular-weight PEG acts as a plasticizer to PLA and exerts a certain plasticizing effect on starch. This feature of PEG allows the branched/crosslinked copolymer with PEG chains that formed after the esterification reaction to penetrate the interface between PLA and starch and act as a bridge between the two to increase their interaction force. Meanwhile, the branched/crosslinked polyester forms a highly stable coating on the starch surface, which facilitates the compatibilization between PLA and starch.2. The addition of a small amount of CA did not change the crystal form of starch, but CA participated in the esterification reaction during the blending process. The generated crosslinked/branched polyester adhered to the surface of the starch granules and improved the surface activity of starch and the compatibility between starch and PLA. The particle size and shape of the starch in the blend did not significantly change, which indicated that the esterification reaction only occurred in the amorphous region of starch. The decrease in the hydrophilicity and the increase in the tensile toughness of the PLA/PSt/PEG/CA blend compared with that of PLA/PSt/CA suggested that PEG may also participate in the esterification reaction during melt blending.In this study, the compatibilization between PLA and starch was improved through reactive blending, and the toughness of the PLA/PSt/PEG/CA blend was improved to a certain extent. The elongation at break of the blend with a PSt content of 30 wt% reached a maximum value of 140.51%, and the impact strength increased to 3.56 kJ/mThe findings suggest that PEG, which acts as a plasticizer for PLA and starch, can lubricate the system. Moreover, when PEG is dispersed in the PLA and starch, the branched/crosslinked polyester containing PEG chains, which is formed by the esterification reaction, is easily embedded in the interface between PLA and starch. This polyester enhances the interaction between PLA and starch. These results indicate that PEG can form a highly stable crosslinked/branched polyester on the starch surface after participating in the esterification reaction to further improve the compatibility between PLA and starch. Furthermore, the decrease in the water absorption can help enhance the stability of the blend material in a natural environment.


In this study, starch extracted from lily bulbs were modified using an ultra‐high pressure (UHP) treatment at six different pressure levels . The effects of UHP treatment on the physicochemical and morphological properties of lily starch were investigated. The morphological observation revealed that UHP treatment led to particle expansion and aggregation. Compared with the native and lily starch treated at 100–500 MPa, the lily starch treated at 600 MPa exhibited almost completely disrupted morphology and a larger particle size, indicating nearly complete gelatinization of the starch. The relative crystallinity of the UHP‐treated starch remarkably reduced. Gelatinization temperatures via differential scanning calorimetry decreased with increasing pressure. The rapid viscoanalyzer results revealed that the lily starch treated with UHP at 600 MPa showed low values of peak viscosity, trough viscosity, breakdown, final viscosity, and setback. These results indicated that UHP was an effective physical modification method for lily starch, UHP treatment  caused nearly complete gelatinization of lily starch, and lily starch modified using UHP might expand the application of lily in the food field. In this study, starch extracted from lily bulbs were modified using an ultra‐high pressure (UHP) treatment at six different pressure levels . The results indicated that UHP was an effective physical modification method for lily starch, UHP treatment  caused complete gelatinisation of lily starch, and lily starch modified using UHP might expand the application of lily in the food field. It is regarded as a green and environment‐friendly physical modification method and can be used for starch modification. In previous reports, the effects of UHP on different kinds of starch have been studied. Li et al.  found thHowever, to the best of our knowledge, no report is available on the physicochemical and morphological properties of lily starch treated with UHP. In this study, lily starch was treated at six UHP levels. The surface morphology, particle size, X‐ray patterns, thermal properties, Fourier‐transform infrared (FTIR) spectrum, and pasting properties of native lily and UHP‐treated starch were investigated. The experimental results can provide references for the application of lily starch.22.1Lilium brownii var. viridulum Baker) used in this work were cultivated in Longhui County, Hunan Province, China. After harvesting, the bulbs were transported to the laboratory immediately. Bulbs with physical damage and pests were selected out. Undamaged lily bulbs were peeled and washed with tap water, and then, the washed lily scales were placed into a wall breaker to homogenate for 2 min .The fresh bulbs . The extracted starch was prepared into 15% (w/w) starch–water suspension and divided into seven equal parts. The suspension was packed into polythene bags, shaken thoroughly to move the bubbles of the vacuum bag, and sealed with a vacuum packer. The sealed samples were transferred into a pressure chamber and subjected to different pressure levels  at room temperature for 30 min. After UHP treatment, the samples were vacuum‐filtered, and the supernatant was removed after centrifugation with 5,000 × 2.4−4 MPa). Scanning electron micrographs were taken at 500 magnifications.Scanning electron microscopy (SEM) of lily starch samples was obtained on a scanning electron microscope  following the method of Ovando‐Martínez et al. . Approxi2.5Polarized and normal light microscopic images were recorded on a polarized light microscope  by using the method of Guo, Zeng, Zhang, et al. . A starc2.6D), surface particle size ), D10, D50, and D90 were recorded. D presents the particle diameter of volume, D presents the particle diameter of surface, and D10, D50, and D90 represent the corresponding particle sizes which are smaller than 10%, 50%, and 90% of the sample particles, respectively.The particle size parameters of starch were measured using a laser diffraction particle size analyzer , as described by Li et al. . In deta2.7λ = 1.5418 nm), X‐ray tube 40 kV, and voltage of 40 mA, the diffraction scanning angle was from 5° to 45° (2θ), the step size was 0.015°, and the scanning speed was 8°/min. The relative crystallinity of lily starch was calculated using MDI Jade software.X‐ray diffractograms of lily starch samples were obtained using an X‐ray diffractometer  in accordance with the method of Ahmed et al.  with som2.8−1.The FTIR spectra of starch samples were recorded on an FTIR spectrophotometer  at room temperature, as described by Rafiq et al. . Starch 2.9To), peak temperature (Tp) and conclusion temperature (Tc), the gelatinization temperature range (ΔTr), and the gelatinization enthalpy (ΔH), were recorded.The thermal properties of the lily starch samples were measured using a differential scanning calorimeter  in accordance with the method of Li et al. . Each st2.10The test was performed as described by Zhang, Ma, et al.  with som2.11p < .05). ORIGIN 7.5 was also used for statistical analysis.All the tests were performed in triplicate. Data were expressed as mean ± standard deviations. Statistical analysis was conducted using SPSS20.0 for Windows, and data were analyzed using ANOVA with Duncan's multiple range tests , D, D10, D50, and D90 for 0–500 MPa high‐pressure treatment were 31.24–32.25 μm, 16.72–17.68 μm, 15.58–15.95 μm, 29.57–30.36 μm, and 50.59–52.96 μm, respectively. Such parameters for 600 MPa treatment were 80.40, 429.45, 789.24, 137.79, and 439.98 μm, respectively. No obvious difference existed in the size of starch granules treated with 0–500 MPa, but the size of starch granules treated with 600 MPa increased significantly; this result is consistent with the observation results of SEM and PNM. The gelation of starch granules resulted in the expansion and aggregation of starch granules, which led to an increase in starch particle size. A similar phenomenon has been observed for quinoa starch , B‐type , and C‐type  (Liu, Guo, et al., In accordance with the X‐ray diffraction pattern, the starch can be divided into three categories: A‐type (mainly exists in corn starch, with a strong diffraction peak at 23.5Tr and ∆H for 0–500 MPa high‐pressure treatment were 7.05°C–7.69°C and 11.62–12.16 J/g, respectively. The parameters for 600 MPa treatment were not detected. The UHP treatment at 500 MPa caused decreased onset temperature and enthalpy of gelatinization compared with the treatment at 400 and 600 MPa. This result suggested that the samples treated at 500 MPa had better crystal structure than those treated at 400 and 600 MPa. The starch crystal structure might have undergone an annealing stage during UHP treatment. Consequently, the crystalline region was further strengthened, while the amorphous region was disrupted. Two types of phase transitions, namely, helix–helix dissociation and helix–coil transition, occurred in starch granules under different treatment conditions (Liu, et al., Figure 3.6−1 observed in the lily starch samples corresponded to the crystallinity of the starches (Remya et al., −1 was caused by the C–H bending or stretching vibration of carbohydrates (Zheng & Li, –1 region indicated the breakage of C–H bonds after the 600 MPa treatment. The sharp band at 1,649 cm–1 corresponded to the molecules absorbed in the amorphous region and the stretching vibration of the amide C = O band; the peak at 2,100 cm−1 originated from the free moisture content (Dankar et al., −1 increased with UHP treatment at 0–500 MPa, thereby indicating that the amount of free water in the above starches increased. The peak was relatively gentle at 600 MPa, indicating that the content of free water in starch decreased significantly. The peak at 2,368 cm−1 was due to C–H stretching associated with ring methane hydrogen atoms (Rafiq et al., −1 was related to the C–H stretching and bending vibration of methyl and methylene groups of polysaccharides (Wang, Xie, et al., –1 region were related to the absorption of hydrogen‐bonded O–H groups in starch (Kizil et al., Fourier‐transform infrared spectroscopy of native and UHP‐treated starches is presented in Figure 3.7Variations in pasting properties of the native and UHP‐treated samples are shown in Table 4In this article, lily starch was physically modified using UHP. UHP treatment showed an evident effect on the granule structural, PSD, and physicochemical properties of lily starch. The smooth lily starch surface became rougher and more agglomerate than before after UHP treatment. Lily starch treated at 600 MPa had the largest particle size and a distinct gelatinized structure. XRD results showed a decreased relative crystallinity after UHP treatment. The value of gelatinization temperatures gradually increased with increasing pressure. The lily starch treated at 600 MPa exhibited lower PV, TV, BD, FV, and SB compared with that treated at 0–500 MPa. The UHP treatment at 600 MPa for 30 min caused nearly complete gelatinization of lily starch. This experiment has a certain reference value for the nonheat treatment of lily starch. However, the exact mechanism of various parameter changes caused by UHP should be studied in the future.The authors declare no conflict of interest.