NPHP1 and a heterozygous PKD1 mutation. We suggest that the PKD1 mutation may have epistatically ameliorated NPHP disease progression and that the screening of larger cohorts for similar possible epistatic effects is needed.We report a patient with adult‐onset nephronophthisis (NPHP) that was identified a homozygous full gene deletion of  In fact, NPHP is considered to be a ciliopathy, since it is caused by structural and functional damage of primary cilia, basal body, and centrosome functions.Nephronophthisis (NPHP) is a progressive renal disease that onsets during childhood or adolescence and presents with a range of clinical characteristics including polydipsia,polyuria, and secondary enuresis, that cause patients to progress to end‐stage renal disease (ESRD).NPHP1‐associated juvenile NPHP progress to ESRD by an average age of 18 years, a recent study reported that some do not exhibit ESRD until they reach adulthood.NPHP patients are classified as either infantile, juvenile, or adolescent‐type NPHP according to the age of onset. Specifically, infantile NPHP onsets between 0 and 3 years, whereas the average age of onset for juvenile and adolescent‐type NPHP is 13 and 19 years, respectively.PKD1 and PKD2, which encode polycystin1 (PC1) and polycystin 2 (PC2) proteins, respectively, that are expressed in the cilia of renal tubular cells. PC1 normally functions to sense the flow of urine, and then transmits a signal to PC2, which is an intracellular calcium release channel. The resulting PC2‐mediated influx of calcium regulates the renal tubular diameter. Impairment of these functions by ADPKD‐causative mutations leads to cyst formation.Autosomal dominant polycystic kidney disease (ADPKD) has similarly been shown to be a ciliopathy that is characterized by the progressive development and accumulation of numerous renal cysts, eventually leading to ESRD.NPHP1 and a heterozygous PKD1 substitution.Herein, we report a patient with adult‐onset NPHP‐induced ESRD that was identified to harbor a homozygous full gene deletion of 2The patient was a 34‐year‐old Japanese man, whose family history included a father with hypertension, and a mother that suffered a subarachnoid hemorrhage, but which did not include either a consanguineous marriage or any incidence of renal cysts. At the age of 31, the patient presented with mild albuminuria, and a serum creatinine (Cr) level of 2.98 mg/dL. At the age of 34, the patient was admitted to hospital with a creatinine level of 8.2 mg/dL.NPHP1 (NM_000272.3:g110879716‐110962709) was resultantly identified, as well as heterozygous substitutions in PKD1 (NM_0001009944.2:c.6395T>G(p.Phe2132Cys)) ), and INPP5E (NM_019892.4:c.1652C>T(p.Thr551Met)).Upon admission, the patient's height, weight, and blood pressure were 179 cm, 58 kg, and 126/80 mm Hg, respectively. The patient exhibited anemia (Hb 8.2 g/dL), azotemia (Cr 8.38 mg/dL), hyperphosphatemia, metabolic acidosis, and secondary hyperparathyroidism; thus, he was diagnosed with ESRD. An abdominal ultrasonography revealed that, although the size of both kidneys appeared to be normal, the patient's renal parenchyma showed increased brightness. We performed a percutaneous renal biopsy; histologically, six of 16 glomeruli displayed global sclerosis, along with mild cellular infiltration, conspicuous interstitial fibrosis, renal tubular atrophy, and cystoid irregular dilation   identified in this patient was previously reported in the PKD Mutation Database as a “likely pathogenic mutation”.BBS1 substitution  (rs750668484 in NCBI (https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=750668484) has been previously reported as a heterozygous mutation , its pathological significance is not yet clear.INPP5E substitution (p.Thr551Met) is to date considered to be a variant of uncertain significance . Each of these genetic mutations may have modulated the clinical course of the patient's NPHP via incurred epistatic effects, as has been previously proposed for many other hereditary disorders and ciliopathies. Indeed, while autosomal recessive disorders develop as a result of homozygous or compound heterozygous mutations, their phenotypes are often modified by the presence of a third gene mutation. For example, a family patient of ciliopathic Bardet‐Biedl syndrome (BBS) was previously reported, in which affected members harbored causative mutations in BBS1, BBS2, and/or BBS6, as well as additional heterozygous mutations in the other BBS genes, which is considered to enhance the disease phenotype.MGC1203, which is a gene shown to be related to BBS4, similarly enhance the BBS phenotype.NPHP genes have been shown to modulate the disease phenotype.Nphp1‐knockout (Nphp‐/‐) with heterozygous Ah1‐knockout mice (Ah1+/−) (the causative gene in Joubert syndrome) has been shown to enhance the development of retinal degenerative lesions.The genetic analyses in our adult‐onset patient revealed a homozygous full gene deletion ofion p.Phe32Cys , ES and SU: performed genetic analysis and wrote “Genetic Assessments” part of the manuscript. ST: performed histopathological evaluations and wrote “Pathological Assessments” part of the manuscript. TA, SM, HK and KN: helped to evaluate and edit the manuscript. TM(Mochizuki): supervised development of work, helped in data interpretation and manuscript evaluation, revised the manuscript, and finally approved the version to be published.


Candidatus Micrarchaeota” are widely distributed in acidic environments; however, their cultivability and our understanding of their interactions with potential hosts are very limited. Their habitats were so far attributed with acidic sites, soils, peats, freshwater systems, and hypersaline mats. Using cultivation and culture-independent approaches , we surveyed the occurrence of these archaea in geothermal areas on Kamchatka Peninsula and Kunashir Island and assessed their taxonomic diversity in relation with another type of low-pH environment, acid mine drainage stream . We detected “Ca. Micrarchaeota” in thermophilic heterotrophic enrichment cultures of Kunashir and Kamchatka that appeared as two different phylotypes, namely “Ca. Mancarchaeum acidiphilum”-, and ARMAN-2-related, alongside their potential hosts, Cuniculiplasma spp. and other Thermoplasmatales archaea without defined taxonomic position. These clusters of “Ca. Micrarchaeota” together with three other groups were also present in mesophilic acid mine drainage community. Present work expands our knowledge on the diversity of “Ca. Micrarchaeota” in thermophilic and mesophilic acidic environments, suggests cultivability patterns of acidophilic archaea and establishes potential links between low-abundance species of thermophilic “Ca. Micrarchaeota” and certain Thermoplasmatales, such as Cuniculiplasma spp. in situ. “ Ca. Micrarchaeota” and “Ca. Parvarchaeota” [Archaeal lineages represented in relatively low abundance in communities are still largely underexplored, which leaves many gaps in our knowledge regarding their distribution patterns, specific roles in the environment and interconnection with other community members. One example of these organisms, often referred to as microbial “dark matter”, is a group of Archaeal Richmond Mine Acidophilic Nanoorganisms (ARMAN), firstly detected in acid mine drainage systems of Iron Mountain  and later confirmed to be widely distributed in numerous low pH settings as well as in areas with neutral pH values ,5,6,7,8.chaeota” . Ca. Micrarchaeota” were shown to cluster with “Ca. Diapherotrites” and “Ca. Parvarchaeota” to be associated with Nanoarchaeota [Nanoarchaeota and Candidate phyla “Aenigmarchaeota”, “Altiarchaeota”, “Diapherotrites”, “Huberarchaeota”, “Nanohaloarchaeota”, “Pacearchaeota”, and “Woesearchaeota” [Nanoarchaeota and “Ca. Micrarchaeota”. Cultured thermophilic associations between Nanoarchaeota and Crenarchaeota described and studied so far, are noteworthy to mention. The very first example of a co-culture was Nanoarchaeum equitans and Ignicoccus hospitalis [Nanoobsidianus stetteri and Sulfolobales Acd1 [Candidatus Nanopusillus acidilobi” and Acidilobus sp. 7a [Candidatus Nanoclepta minutus” and Zestosphaera tikiterensis (representing a new member of the family Desulfurococacceae) [Ca. Micrarchaeota” two cultured associations were documented: “Ca. Mancarchaeum acidiphilum” Mia14 with Cuniculuplasma divulgatum PM4 [Ca. Micrarchaeota”, ARMAN-1-related organism in a co-culture with consortium of two archaea  together with a fungus [Ca. Micrarchaeota” in metagenomic data occurs at a global scale [Based on the phylogenetic analysis of three concatenated protein sets, these groups are placed divergently . “Ca. Mirchaeota . Both grchaeota” ,11. Thischaeota” . Howeverspitalis , followeles Acd1  a “Candis sp. 7a , and fincacceae) . For mesatum PM4  and “Ca.a fungus . Neverthal scale .Ca. Micrarchaeota” [Ca. Micrarchaeota” in environmental microbiomes was shown to constitute <1% in 17 of 25 genomes reconstructed from metagenomic data, although an abundance of one assembly variant represented by FK_Sed_bin12_4  was as high as 5.3%–21.3% in certain subsamples [Ca. Micrarchaeota”/ARMAN-2 reads [Ca. Micrarchaeota” in comparison to “Ca. Parvarchaeota” possess wider distribution patterns since signatures of this group were detected not in acidic settings only, but in soils, peats, freshwater systems, and hypersaline mats. It may imply that “Ca. Micrarchaeota” demonstrates enhanced resistance to selective pressure, which enables their spreading and genome stability. Altogether, connection with Thermoplasmatales was predicted for 16 “Ca. Micrarchaeota” and for 13 “Ca. Parvarchaeota” species [Estimation of phylogenetic diversity for “chaeota”  based onchaeota”  suggestebsamples . Two aci-2 reads  that “Ca species .Ca. Micrarchaeota”, based on the aerobic respiratory chain, fermentation potential and presence of cytochrome bd-II encoding genes [Analysis of 39 ARMAN-related genomes with different levels of completeness from various emplacements pointed at a certain variability in genome sizes 0.64–1.08 Mb) and predicted their potential involvement in carbon, nitrogen and iron cycles [ Mb and png genes . To understand further patterns of distribution and phylogenetic variance of these archaea in particular environments, we focused our study on two types of acidic environments. Ca. Micrarchaeota” -related sequences in acidic geothermal areas via establishment of enrichment cultures with samples from terrestrial hydrothermal vents of Kamchatka Peninsula and Kunashir Islands (Russian Far East) and their consequent molecular study. We also address here the issue of relations between “Ca. Micrarchaeota” and Thermoplasmatales archaea, abundant in these niches and recovered in a number of enrichment cultures experiments. We surveyed the presence of “Euryarchaeota accounted for 64%, “Ca. Micrarchaeota” (DPANN group) for 3% and “Ca. Parvarchaeota” for 0.2%, with the rest of the archaea making up 0.5 % of total prokaryotic reads [Ca. Micrarchaeota” to represent 0.3%–0.4% and 1.7%–1.4% of total reads in these microenvironments, respectively [Ca. Micrarchaeota” at a local scale of Parys Mt acid mine drainage site. Diversity, heterogeneity, and similarity among “Ca. Micrarchaeota” variants in these two particular low-pH places differ in nature and genesis of acidity and will be discussed here.Another site used in this work was an extremely acidic low/moderate temperature environment: a shallow stream located in mine-impacted area of Parys Mountain , which was in focus of extensive studies for several years ,18,19,20Samples of water/sediment from geothermal areas of Kunashir Island (Russia) were taken in July 2014 and were described to have further characteristics. KY1: A thermal vent in a solfataric area, with temporary coverage by lake water with parameters in a sampling spot of pH 2.1 and 58 °C . KY2: a stream in a Northwest part of a fumarolic field, the sampling spot was located 3–5 m below fumaroles after junction of high-temperature acidic stream and cold neutral stream. The characteristics of the site were pH 2.3, 54 °C . Additionally, KM3 enrichment was established from Kamchatka Peninsula geothermal site (Russia), characterised by pH 2.5 and 44 °C (54°30′4.61″ N 160°00′0.17″ E). These samples were used for the establishment of enrichment cultures and their consequent study via 16S rRNA gene clonal libraries and metabarcoding experiments. Samples of the top 1–3 cm water-saturated sediments of acidic stream and water and sediment as separate fractions located in Parys Mt  have been taken in October of 2014 and in October 2016, respectively. Characteristics of the site were reported previously [−1 were added to the medium. Additionally, the Medium 9K with supplements was used, as it was discussed previously [For enrichment cultures, the modified Medium 88 (DSMZ) was used . Mixtureeviously . The temCa. Micrarchaeota” SSU rRNA gene amplification, designed pairs of primers MIAF (GCTTGGCGAATAAGTGCTGGGC) and MIAR (ATCTTGCGACCGTACTCCCCAG); and also ARM-MIA Fwd3 (GCGTACGGCTCAGTAACACGTAG) and ARM-MIA Rev (TTGAGGTGATCTATCCGCAGG) were used. The annealing temperature used in the PCR program with ARM/MIA primers was 60 °C. Generation of SSU rRNA gene clone libraries was done with TOPO TA 2.1 Cloning Kit for sequencing , according to the protocol of the supplier.The DNA from all samples was isolated using MoBio Soil DNA isolation kit (QIAGEN). Selective targeting of archaea from enrichment cultures was done with specific primers. The primers used for archaeal amplification were archaeal Forward A23 (TCYGGTTGATCCTGCC) and Reverse universal R1492 (TACGGYTACCTTGTTACGACTT). For ARMAN and “The procedures for preparation of V3-V4 rRNA gene amplicon libraries as well as the metabarcoding analysis pipeline were described previously . Processblastn algorithm [Thermoplasmatales and “Ca. Micrarchaeota” were developed using a total number of 62 and 87 sequences, respectively. First, sequences of clones were matched against the NCBInr database using lgorithm . ReferenMafft v. 7 [UGENE [Trimal [Both sets of sequences were aligned using fft v. 7  with def7 [UGENE  and fina [Trimal , removinR environment using ape [phangorn [Final phylogenetic trees were obtained using GTR as an evolution model by maximum likelihood method. Bootstrap was calculated using 1000 replicates. Generation of the final phylogenetic trees and final visualization have been both performed using scripts under sing ape  and phanphangorn  packages. PCR amplification with Archaea-specific oligonucleotides revealed the presence of various Thermoplasmatales archaea and “Ca. Micrarchaeota” in heterotrophic enrichment cultures. A number of 16S rRNA gene amplicon libraries were established during the first year of cultivation to monitor the content of archaeal counterparts of three enrichment cultures  enrichment cultures with samples from geothermal terrestrial hot springs from Kunashir and Kamchatka was revealed to be 50 °Ccultures . The stuAcidiplasma spp. [Thermoplasmatales or “Ca. Micrarchaeota” were detected. In relation to this, both iron oxidation capability in these particular “Ca. Micrarchaeota”-related organisms and their interactions with Acidiplasma spp. are very unlikely. Additionally, the reason for the lack of “Ca. Micrarchaeota” in iron-containing enrichment cultures might be that these certain conditions were favourable for Acidiplasma aeolicum-like archaea, but not other Thermoplasmata, on which “Ca. Micrarchaeota” might be metabolically dependent.All enrichment cultures established in iron (II)-containing 9K medium (pH 1.7) showed the presence of sma spp.  only; noThermoplasmatales, namely Acidiplasma spp. and organisms distantly related to Thermogymnomonas and Cuniculiplasma (91%–93% 16S rRNA gene sequence identity). From all established heterotrophic variants, KY1, KY2 and KM3 enrichments showed the presence of “Ca. Micrarchaeota”, related to ARMAN-2 and Mia14-like containing clusters. Classifying the clones into OPUs , based on clones with ~99% of identity percentage among them, and checking the frequency of each OPU on each sample, we have noticed that OPUs 5 (Cuniculiplasma-related) and 8 (ARMAN-2 related) are clearly pronounced in KM3 and KY2 communities, respectively, whereas OPUs 1 (Cuniculiplasma-related) and 6 (Acidiplasma-related) are more present in the KY1 variant , showed the presence of  variant .Ca. Micrarchaeota”. We have observed that KY1 and KM3 enrichment cultures were similar one with another and with Mia14-like archaea (100% 16S rRNA gene sequence identity). Organisms taxonomically similar to Mia14 and belonging most likely to the same genus, were also detected in metagenomic data in geothermal areas in China  and in different acid mine drainage systems . AccordiThe clustering of KY2 and one of the sequences from KM3 with ARMAN-2 affiliated to genus 12/Family 2 according to  classifiThermoplasmatales archaea and “Ca. Micrarchaeota” in enrichment cultures. The KY1 enrichment culture was composed by Acidiplasma spp. and Cuniculiplasma spp. (97% sequence identity to C. divulgatum) archaea and Mia14-like “Ca. Micrarchaeota”. Another Kunashir Island enrichment KY2 showed the presence of three clades Thermoplasmatales. One variant showed sequence identity to Acidiplasma aeolicum of 99%, others were only distantly related to Thermogymnomonas acidicola and Cuniculiplasma divulgatum (89%–92%), and finally, the third group was represented by organisms with 97% SSU rRNA gene sequence identity to C. divulgatum. “Ca. Mancarchaeum acidiphilim” and ARMAN-2-related clades of “Ca. Micrarchaeota” were found in KY2 enrichment. The assessment of the content of Kamchatka enrichment culture (KM3) showed the presence of Cuniculiplasma spp. (with identity to C. divulgatum 97%), Mia14- and ARMAN-2-related clades of “Ca. Micrarchaeota” (We observed certain co-occurrence patterns between chaeota” . Ca. Micrarchaeota” after 2 years of cultivation in minor numbers (0.3% reads), assessed by the DNA barcoding technique in a KY2 variant only. Initially, it was the most diverse enrichment culture, exemplified by three different Thermoplasmatales species. Among archaea in this particular variant, we identified sequences related to Acidiplasma aeolicum, 100% identity (35.8% reads) and sequences only distantly related to Acidiplasmaaeolicum (81% identity), sequences of the last lineage were not detectable earlier by clonal libraries approach. Other Thermoplasmatales archaea constituted 63.8% of all reads with Cuniculiplasma spp. in the variant KY2.2 A. Another parallel enrichment culture a KY2.2 B showed the presence of 4% reads of Acidiplasma aeolicum (95–100% identity) and other Thermoplasmatales in a number of 96.1% reads with 92% sequence identity with Cuniculiplasma divulgatum. The content of enrichment cultures determined the presence of “Acidiplasma aeolicum (100% sequence identity), and 12% of reads to Cuniculiplasma divulgatum (97%). In KY2.2.B 94% reads were related to Acidiplasma aeolicum and 5% to Cuniculiplasma divulgatum with the same levels of identities of 16S rRNA gene sequence. The reason for the disappearance of “Ca. Micrarchaeota” from enrichment cultures after 3 years is not entirely clear since the possible host, Cuniculiplasma sp., which is able to provide essential metabolic precursors or other biomolecules was still present in the culture although in relatively smaller numbers being significantly outcompeted by Acidiplasma spp. It could be that for some reasons, higher densities of host cells are needed to support the life of “Ca. Micrarchaeota”. We cannot also exclude the possible involvement of Acidiplasma species into the interaction with “Ca. Micrarchaeota”. However, this needs further experimental confirmation.Furthermore, after almost 3 years of cultivation, only two organisms persisted in a KY2.2.A: 87% of all reads were affiliated with Ca. Micrarchaeota” in samples from geothermal sites of Kunashir and Kamchatka was intriguing. The occurrence of Thermoplasmatales was rather expected, since these organisms are known inhabitants of Kamchatka hot springs and were detected to make up to 39% of all archaea in groundwater microbiome  in the East Thermal Field of Uzon Caldera [Acidiplasma representatives, which probably cannot interact with “Ca. Micrarchaeota” poses the question of who the “Ca. Micrarchaeota” actual host is, since the temperatures of incubation of Kamchatka and Kunashir enrichments (45 and 50 °C) seem to be too high for Cuniculiplasma divulgatum representatives.The detection of “ Caldera . HoweverThermoplasmatales, namely Thermoplasmatales group A10, were previously found in quite significant numbers  in Kamchatka hot springs Kaskadny and Arkashin Shurf characterized by moderate acidity and temperatures [Nanoarchaeota in Kamchatka hot springs was also confirmed, yet no information about “Ca. Micrarchaeota” was presented [Ca. Micrarchaeota”, in microbiomes.eratures . Moreoveresented . These rCa. Micrarchaeota”-related groups in Parys Mt environment. Altogether, we detected a number of variants with a variety of affiliations to families and genera proposed by phylogenetic analysis [Our results of the analysis of Parys Mt AMD 16S rRNA gene sequences have revealed significant diversity of “analysis .Ca. Micrarchaeota”-like variants and belongs to genera 3–4 (Family 1), according to the classification of [The first cluster represented by numerous PM clones showed relatively low sequence identity to other “ation of . The neaThe single PM clone 18 with sequence ID MH463124 was phylogenetically located outside this cluster  and was Ca. Micrarchaeota”-related sequences. One clone 23 (MH463107) was shown to be distantly related to other “Ca. Micrarchaeota”  was derived from ARMAN-2–related organisms, Kunashir (KY1) and Kamchatka (KM3) variants and many other sequences from Fankou AMD sediment, Tengchong geothermal area  and in genomic data from acidic stream of the Rincon de la Vieja Volcano National Park (Costa Rica). Additionally, similar sequences were identified in metagenomic data from acidic biofilm (KC127696) Harz Mountains, Germany [The Family 2 proposed by  includeschaeota” , another Germany . AnotherCa. Micrarchaeota” was represented by three groups, categorised as ARMAN-1-3, together with “Ca. Parvarchaeota” lineages, with ARMAN-2 group being the most abundant in seven biofilms studied [Ca. Micraerchaeota” represented by two clusters related to ARMAN-1 and 2 lineages [Interestingly, in Iron Mountain AMD site  “lineages . These dCa. Micrarchaeota” in Parys Mt environment may rely on interactions with different archaea. Firstly, the dependency of “Ca. Micrarchaeota” on symbiotic interactions with Cuniculiplasma and Cuniculiplasmataceae, as previously reported [Thermoplasmatales in this ecological niche  one couCa. Micrarchaeota” genomes that lack the genes for amino acids and nucleotides synthesis [Nanoarchaeota [Cuniculiplasma spp. could be a host for different ARMAN organisms (Mia14- and ARMAN-1-related species) [Ca. Mancarchaeum acidiphilum Mia14” metagenomic reads together with ARMAN-1-related “A-DKE” sequences were detected, favouring heterotrophic, aerobic and mesophilic cultivation conditions [Ca. Micrarchaeota” spp., two Thermoplasmatales archaea, one of which was Cuniculiplasma divulgatum, and a fungus. CARD-FISH experiments suggested localisation of ARMAN cells with both Thermoplasmatales organisms [Ca. Micrarchaeota” in the ecosystem. The dependency of ARMAN archaea on multiple hosts was proposed from analysis of 39 “ynthesis . The prorchaeota . Our prespecies) . “Ca. Manditions . Additionditions  reportedrganisms . To sum Ca. Micrarchaeota” in geothermal areas of Kunashir and Kamchatka. The content of moderately thermophilic heterotrophic enrichment cultures suggests Cuniculiplasma spp., Acidiplasma spp. and other Thermoplasmatales without defined taxonomic positions being potential hosts for “Ca. Micrarchaeota”. The assessment of the diversity of two types of acidic sites, thermal springs, and the AMD stream revealed significant variety among “Ca. Micrarchaeota”. Two phylotypes found to be widely represented in geothermal areas, namely “Ca. Mancarchaeum acidiphilum” and ARMAN-2-related, were detected also in AMD ecosystems, confirming ubiquity and abundance for these archaeal groups. Five different clusters of sequences were observed for Parys Mt “Ca. Micrarchaeota” lineages with 88% 16S rRNA gene sequence identities between the most distant groups. The present study is a step forward towards understanding of diversity and cultivability of low-abundance microbial species in thermal and low/moderate temperature acidic environments. We also think that these results expand our knowledge on the cultivability patterns of acidophilic archaea of the order Thermoplasmatales from geothermal acidic placements and emphasise the remarkable co-occurrence of “Ca. Micrarchaeota” and certain Thermoplasmatales species, which points at their interactions in situ.We revealed the presence of “


High-intensity interval training (HIIT) is frequently employed to improve the endurance of various types of athletes. To determine whether youth soccer players may benefit from the intermittent load and time efficiency of HIIT, we performed a meta-analysis of the relevant scientific literature.Our primary objective was to compare changes in various physiological parameters related to the performance of youth soccer players in response to running-based HIIT to the effects of other common training protocols . A secondary objective was to compare specifically running-based HIIT to a soccer-specific form of HIIT known as small-sided games (SSG) in this same respect, since this latter type of training is being discussed extensively by coaches.dppc2) and associated 95% confidence intervals for the comparison of the responses to HIIT and other interventions were calculated.A systematic search of the PubMed, SPORTDiscus, and Web of Science databases was performed in August of 2017 and updated during the review process in December of 2018. The criteria for inclusion of articles for analysis were as follows: (1) comparison of HIIT to SSG or some other training protocol employing a pre-post design, (2) involvement of healthy young athletes (≤ 18 years old), and (3) assessment of variables related to endurance or soccer performance. Hedges’ g effect size ), shuttle runs (expressed as the distance covered or time to exhaustion), and time-trials, as well as submaximal variables such as running economy and running velocity at the lactate threshold. HIIT induced a moderate improvement in soccer-related tests involving technical exercises with the soccer ball and other game-specific parameters . Neuromuscular parameters were largely unaffected by HIIT or SSG.Nine studies, involving 232 young soccer players (mean age 16.2 ± 1.6 years), were examined. Endurance training in the form of HIIT or SSG produced similar positive effects on most parameters assessed, including peak oxygen uptake and maximal running performance during incremental running (expressed as The present meta-analysis indicates that HIIT and SSG have equally beneficial impacts on variables related to the endurance and soccer-specific performance of youth soccer players, but little influence on neuromuscular performance. Various reports on responses of both youth and adults to high-intensity interval training (HIIT) have been published, but no systematic comparison of the effects of HIIT and other training regimes on parameters related to the performance of youth soccer players has yet been published.The calculated effect sizes indicate that HIIT has more beneficial effects than various other exercise programs on all of the parameters examined, with the exceptions of sprinting and jumping performance and repeated sprint ability.Small-sided games (SSG), a sport-specific form of HIIT, exerted similar effects on parameters related to soccer performance .HIIT and SSG both enhance the key performance of youth soccer players in a time-efficient manner, leaving more time for training sport-specific skills such as technique and tactics.Youth soccer is characterized by constant changes in the intensity of a variety of activities, including standing, walking, running, and sprinting with frequent changes in direction, as well as jumping, often with involvement of the ball and/or opponents . This va2peak), individual anaerobic threshold, and/or maximal velocity in youth soccer players [peak) alternating with periods of low-intensity activity [2peak [High-volume or continuous low-intensity training has been employed successfully to improve certain aspects of endurance performance, such as peak oxygen uptake  the overall responses of youth soccer players to HIIT in comparison to other endurance protocols with respect to endurance and other important determinants of performance, such as the ability to sprint and jump; and (2) whether SSG, a sport-specific form of HIIT, results in improvement of these same parameters comparable to those achieved with running-based HIIT.Our aim here was to systematically compare the effects of HIIT on variables related to the performance by youth soccer players as reported in numerous publications during the past decade to those of other training regimes (in particular SSG). Such analysis should aid in designing HIIT and/or SSG to improve the endurance of youth soccer players most effectively.This systematic analysis of peer-reviewed investigations on the effects of HIIT and SSG on several parameters related to performance in youth soccer players was conducted in accordance with established guidelines . A comprThe search was limited to original research articles written in English and published in peer-reviewed journals. The screening and selection process is illustrated in Fig. In addition, the references cited by the articles retrieved were examined for potential relevance. Data was extracted from the studies included by one of the authors and this extraction checked independently by a second author.peak [The inclusion criteria were as follows: (1) involvement of endurance training in the form of HIIT ; (2) inStudies on patients with specific conditions  were excluded, as were conference abstracts, dissertations, theses, and articles that had not undergone peer review.To ensure the methodological quality of the articles selected, the criteria of the Physiotherapy Evidence Database (PEDro) scale were applied, with one point for each criterion fulfilled and a maximal possible score of 10 . The quaThe findings on HIIT were compared to those with (1) all of the other interventions, (2) all of the other interventions except SSG, and (3) SSG alone.pooled, as recommended by Carlson and Schmidt [n is the number of participants in the experimental (nexp) and control groups (ncont) and SDexp and SDcont the corresponding pretest standard deviations.Effect sizes (ES) were calculated as the mean difference between the experimental and control groups, divided by SD Schmidt .\documenIn addition, in light of the tendency for findings with small samples to be positively biased, leading to overestimation, a correction factor (CP) was employed:dppc2) is optimal in the case of studies involving pre- and post-testing and a control group [This approach to the calculation of ES  calculated.The ES values obtained were classified as trivial (< 0.10), small (0.10–0.30), moderate (0.30–0.50), or large (> 0.50) . HeterogAll statistical analyses were carried out in version 11.5.1.0 of the Med-Calc software .Nine studies were included , 13–18 years of age (mean age: 16.2 ± 1.6 years), of whom 50% performed intense interval training and the other 50% control programs of exercise, i.e., either SSG, some form of low-intensity high-volume exercise, or soccer-specific drills .2peak of the subjects was 57.4 ± 1.7 (range 55.1–60.2) mL·kg−1·min−1. HRmax was determined with incremental treadmill tests [All of these participants played regularly for a club, in most cases professional. Four studies , 27, 28 ll tests –13, 28, ll tests , or the The mean duration of the interventions was 6.4 ± 2.0 weeks (range 4–10), with an average of 2.9 ± 0.9 sessions per week (range 2–4.5).max), with 3-min intervals of rest [The HIIT protocols employed differed with respect to intensity, duration, and work-to-rest ratio Table . Notably of rest , 13, 28.max [In most cases, the target intensity was > 90% HRmax , 28, 30,max , 29. Twomax , 30. Themax , 29 or 4max , 27, 28,max . The recIn four cases, HIIT was compared to SSG , 27–29. dppc2 0.45 ± 0.46; range 0.08–1.11). Among the four analyses of peak or maximal oxygen uptake, one effect was trivial positive [In comparison to all other interventions, HIIT induced a moderate mean positive effect on peak oxygen uptake dppc2 0.4dppc2 0.75 ± 0.50; range 0.40–1.11). In the two studies analyzed in this respect, one reported a moderate positive [In comparison to all interventions except SSG, HIIT resulted in a large mean positive effect on maximal or peak oxygen uptake dppc2 0.7dppc2 0.15 ± 0.10; range 0.08–0.22), as shown by two studies, one demonstrating a trivial positive effect [In comparison to SSG, HIIT exerted a small positive effect on maximal or peak oxygen uptake dppc2 0.1dppc2 0.30 ± 0.58; range − 0.78–1.34) [Vmax) [I2 value of 14% indicated low heterogeneity.As assessed in six studies, HIIT induced a moderate mean positive effect on parameters related to maximal running performance (mean 78–1.34) , 29, 30,) [Vmax) , 29, dif) [Vmax) , 30, or ) [Vmax)  dppc2 0.55 ± 0.70; range 0.01–1.34)  in comparison to SSG, as determined by incremental running tests [Three studies , 27, 29 ng tests  Fig. 4)dppc2 0.1dppc2 1.00 ± 2.29; range − 0.40–4.42). One of these four studies indicated a moderate negative [With respect to running performance at the lactate threshold, reported in four cases, HIIT exerted a large positive mean effect in comparison to all other interventions dppc2 1.0negative  or definnegative , 28.dppc2 2.01 ± 3.41; range − 0.40–4.42) [As shown in two reports, in comparison to all other interventions except SSG, HIIT had a large positive effect on running performance at the lactate threshold (mean 40–4.42)  Fig. 3)dppc2 2.0dppc2 − 0.01 ± 0.02; range − 0.03–0.00)  Fig. .dppc2 0.53 ± 0.74; range 0.00–1.64). Two trivial positive [I2 value of 43%.In comparison to all other interventions, HIIT induced a large positive mean effect on running economy  [One article reported that in comparison to all control interventions except SSG, HIIT produced a large positive mean effect on running economy (mean 10–1.64)  Fig. 3)dppc2 0.8dppc2 0.05 ± 0.05; range 0.00–0.10) [One comparison of HIIT to SSG revealed a trivial positive mean effect of the former on running economy (mean 00–0.10)  Fig. 4)dppc2 0.0dppc2 0.10 ± 0.12; range − 0.01–0.35) over the various distances  examined in five different studies. Trivial effects were found for 5 m [I2 value of 12% indicated low heterogeneity.HIIT exerted a small positive effect on linear sprinting performance . One small positive effect was found for 10 m [In comparison to all other interventions except SSG, HIIT exerted a trivial positive effect on sprinting performance , with one trivial and one moderate positive effect for 5 m [In comparison to SSG, HIIT produced a small positive effect on sprint performance . Small negative effects were found in three studies [I2 = 0%).In comparison to all other interventions, HIIT had a trivial positive effect on jumping performance  , based on two small negative effects on the vertical jump [In comparison to all other interventions except SSG, HIIT showed a trivial negative effect on jumping performance , as a result of two small positive effects, one on the countermovement jump [In comparison to SSG, HIIT had a trivial positive effect on jumping performance dppc2 0.0dppc2 − 0.05)  Figs. .dppc2 0.59) [One publication showed that in comparison to SSG, HIIT exerted a large positive effect on change-of-direction performance c2 0.59)   [I2 of 8% indicated a low degree of heterogeneity.The effect of HIIT on variables related to soccer performance was moderately positive in comparison to that of all other interventions mean 21–1.15) , 13, 28 1–1.15 , 21–1.15) , 13, 28,dppc2 0.47 ± 0.39; range − 0.09–1.15) [Comparison of HIIT to all other interventions except SSG revealed a moderate positive effect of the former on soccer-related performance, including kicking velocity , the Lou09–1.15) , 13 , as assessed by the Ekblom’s test [In comparison to SSG, HIIT exhibited a small negative effect on soccer-related performance dppc2 − 0The average durations of one session of HIIT and of all the other programs were 33 ± 14 min and 41 ± 15 min, respectively—a difference that is noteworthy, even if not statistically significant. Considered separately, in the two studies involving high-volume endurance exercise as the control training , the aveThe present meta-analysis compares the effects of HIIT on youth soccer players to those of alternative training regimes, including SSG.(i)In comparison to all other interventions, HIIT induces moderate-to-large positive effects on maximal or peak oxygen uptake, variables related to running performance , change-of-direction performance, and soccer-related performance tests (ii)In comparison to all other interventions except SSG, HIIT demonstrates moderate-to-large effects on maximal or peak oxygen uptake, variables related to running performance, and, again, soccer-related performance(iii)In comparison to SSG, HIIT exerts a large effect on change-of-direction ability(iv)In comparison to all other interventions including or excluding SSG, as well as to SSG alone, HIIT has little or no impact on sprint running performance, jumping performance, or repeated sprint abilityThe overall findings were as follows:2peak to a greater extent than other training strategies [max and intensities as high as 90–95% of HRmax can be reached [Although endurance performance is unquestionably a key determinant of the success of advanced youth soccer players , 32, numrategies . Further reached .2peak for HIIT, in agreement with previous findings [In this regards, HIIT and SSG improve endurance parameters  to a similar extent, with slightly higher values of VOfindings . Howeverfindings , improvifindings . Unfortufindings , as wellfindings , 39. AltThe present analysis indicates that improvements in neuromuscular parameters  with HIIT and SSG were trivial or, at best, small. In most cases, HIIT involved 4-min intervals at 90–95% of peak heart rate, but not sprinting for only a few seconds at 100% intensity , and theThe single investigation that examined repeated sprints  observedIn practice, coaches may focus on the effects of various training regimes on performance in connection with soccer-related tests. The medium-positive effect of HIIT on such performance compared to all other interventions can be attributed to the large positive effects on the number of sprints, total distance covered, and number of involvements with the ball, all of which require the endurance that can be achieved with HIIT. The negative effect of HIIT in the Ekblom’s test in comparison to SSG Fig.  may be eHIIT and SSG provide similar benefits with respect to most of the parameters analyzed here Fig. . Moreove2peak than SSG, thereby developing adequate aerobic endurance. During a season, running-based protocols that do not involve ball drills often appear impractical. From this perspective, SSG may provide a viable alternative for maintaining endurance and skills such as ball handling and tactical thinking.From a practical point of view, the choice of a HIIT or SSG protocol for training youth soccer players depends to a large extent on the time-point during the season and the coach’s overall strategy. HIIT induces somewhat more pronounced increases in VOThe following limitations of the present analysis need to be considered: (1) Only nine studies met our criteria for inclusion, and in some cases, no more than one ES could be calculated for each parameter; 2) for some parameters, there was considerable heterogeneity (as indicated by the 95% CI), especially for running performance at the lactate threshold; (3) we categorized each report on the basis of what the authors stated. For instance, in one of the studies  involvin for someOn the basis of the present analysis, we conclude that HIIT in general and the more sport-specific SSG both result in similar improvements in maximal or peak oxygen uptake, variables related to running performance , and soccer-related performance tests in youth players. In contrast, repeated sprint ability and sprinting and jumping performance are virtually unaffected by either HIIT or SSG. Change of direction performance was improved to a greater extent by HIIT than SSG, but this difference requires further investigation. Thus, HIIT and SSG both offer an effective approach to simultaneously improve certain physiological characteristics and the sport-specific skills of youth soccer players.


OsNUC1-S’s function was investigated using transgenic rice lines overexpressing OsNUC1-S. Under non-stress conditions, the transgenic lines showed a lower yield, but higher net photosynthesis rates, stomatal conductance, and transpiration rates than wild type only in the second leaves, while in the flag leaves, these parameters were similar among the lines. However, under salt-stress conditions at the booting stage, the higher yields in transgenic lines were detected. Moreover, the gas exchange parameters of the transgenic lines were higher in both flag and second leaves, suggesting a role for OsNUC1-S overexpression in photosynthesis adaptation under salt-stress conditions. Moreover, the overexpression lines could maintain light-saturation points under salt-stress conditions, while a decrease in the light-saturation point owing to salt stress was found in wild type. Based on a transcriptome comparison between wild type and a transgenic line, after 3 and 9 days of salt stress, the significantly differentially expressed genes were enriched in the metabolic process of nucleic acid and macromolecule, photosynthesis, water transport, and cellular homeostasis processes, leading to the better performance of photosynthetic processes under salt-stress conditions at the booting stage.Rice nucleolin (OsNUC1), consisting of two isoforms, OsNUC1-L and OsNUC1-S, is a multifunctional protein involved in salt-stress tolerance. Here,  Oryza sativa L.) is a staple food and a main source of energy for humans, especially in Asia. There are several biotic or abiotic stresses that limit rice growth and yield, such as soil salinity, drought, and soil nutrition [a, chlorophyll b, and carotenoids, because salt stress increases enzyme activities involved in chlorophyll degradation, which leads to a decrease in chlorophyll levels [Rice  and a shorter  form. OsNUC1-S lacks an N-terminal region, but the cDNA sequences of the other regions are the same as the longer form. In 2013, Sripinyowanich and colleagues studied OsNUC1-S overexpression in Arabidopsis and found that this form promoted salt tolerance by increasing the number of lateral roots and enhancing root growth. In addition, the overexpression of this gene in rice leads to shoot fresh weight increasing when seedlings are grown under salt-stress conditions [OsNUC1-L form resulted in an increase in salt tolerance in both rice and Arabidopsis by enhancing photosynthesis [In rice, two forms of Nucleolin1 were found, a longer  and the transgenic lines, when grown in the control and salt stress condition at reproductive stage. Significantly higher OsNUC1 gene expression could be detected in transgenic lines when compared to WT, especially in the salt stress condition , and seed number per plant of the transgenic rice, TOSL1, TOSL2, TOSL3, and WT, were evaluated as shown in OsNUC1-S caused the reduction in % fertility, leading to the reduction in seed number per plant. However, under salt stress, the transgenic lines tended to have the higher tiller number per plant, panicle number per plant, panicle length, % fertility, and seed number per plant, except the TOSL2 line that showed similar % fertility and seed number per plant to WT. The reduction percentage of seeds/plants in WT was 64%, while the transgenic lines had the seeds/plant reduction of 17%, 47%, and 27%. These data suggested that there should be some changes in metabolisms in the transgenic lines due to OsNUC1-S over-expression.To investigate if the overexpression of OsNUC1-S over-expressing lines under control and salt stress conditions. Under the control condition, only second leaves of transgenic rice overexpressing OSNUC1-S showed higher net photosynthetic rates (PN) and stomatal conductance levels (gs), while the flag leaves had similar levels, except TOSL2, which had a lower PN than the other lines. These results may reflect positional effects of the transformation. After 9 days of salt stress, the overexpression of OsNUC1-S increased the PN and gs of all the transgenic lines   E,F, but nditions G,H.−2·s−1, except TOSL2, which had a lower PN than other lines. The light-saturation points were approximately 1000 µmol·m−2·s−1 in all lines   B.N values of the WT’s second leaves started to decline when the light intensity was greater than 800 µmol·m−2·s−1; however, for the transgenic lines, the light-saturation point was 1000 µmol·m−2·s−1  was found in WT after 9 days of salt treatment, while in all the transgenic lines, a reduced effect was found  between WT and TOSL3, when grown under control conditions, the DEGs were highly enriched in the cellular macromolecule metabolic processes, including the macromolecule biosynthetic process. Genes involved in transmembrane transport, regulation of cellular process, and the pigment metabolic process were also found.Because of the effects of OsNUC1-S’s role in the enhancement of macromolecule production for grain filling in the flag leaves. Interestingly, the molecular function enrichment was found for only linoleate 13S-lipoxygenase activity. This enzyme is involved in plant growth and development [For the cellular component enrichment, a plasma membrane and chloroplast envelope were reported. This supported elopment , and alselopment ,17  (LOC_Os03g39610 (CAB) (LOC_Os04g33830 (PSAO) (LOC_Os01g64960 (PsbS1)  A, LOC_Os10 (CAB) B, and LO0 (PSAO) C, were c0 (PSAO) . The com (PsbS1) D showed  (PsbS1) D. The inOsNUC1-S, one of the mRNA splice forms, lacks an N-terminal region that contains several acidic stretches and a nuclear localization signal. However, based on the localization study [OsNUC1-S did not change the photosynthetic activity levels in flag leaves when plants were grown under control conditions  or transgenic seeds were germinated for 7 days. Then, seedlings were transferred to pots containing 5 kg of soil. When plants were at the booting stage, 150 mM NaCl solution was added to cause salt stress at 8–10 ds·m−1. All photosynthetic parameters were measured after 9 days salt treatment using a Gas Analysis System  with a 1200 µmol·m−2·s−1 light intensity and 380 µmol mol−1 carbon dioxide (CO2) concentration.The experiment was conducted in a planting house for transgenic plants in the Botany Department, Faculty of Science, Chulalongkorn University in Thailand. Three independent transgenic rice lines expressing lleagues  in a “Niv/Fm) using Pocket PEA . In total, 30 min was used for dark adaptation, and then a 1-s saturation flash was used to measure the potential maximum photochemical efficiency of PSII.For chlorophyll fluorescence parameters, either the flag leaf or second leaf was used to determine maximum quantum yield of photosystem II (PSII) photochemistry . A Dynabeads mRNA purification kit  and a KAPA Stranded mRNA-Seq Kit were used for mRNA isolation and cDNA libraries’ preparation, respectively. Fragment sizes of ~300 bp were selected and connected with adaptors. Then, the fragments were enriched by PCR for 12 cycles. All libraries were sequenced using the Genome Analyzer . Adaptors were subsequently removed from all short-sequence reads before grouping, following the protocol of Missirian et al. [LOC_Os01g58470, LOC_Os01g64960, LOC_Os03g39610, and LOC_Os04g33830 predicted to localize in plastids were selected. The primers for detection of the gene expression are shown in OsEF-1∝ gene expression was used as an internal control. For q-RT-PCR, briefly, 1 μL of RNA extraction from either WT’s or transgenic rice’s flag leaves was used to synthesize the cDNA with an AccuPower® RT PreMix . Gene expression of the target genes was detected by quantitative PCR using Luna® Universal qPCR Master MiX . The thermal cycle was performed at 95 °C for 60 s, then 40 cycles of 95 °C for 15 s, 58.5 °C for 30 s, followed by 95 °C for 30 s, and the extension was done at 70 °C for 5 s. The relative expression of interesting genes was calculated using the Pfaffl method [For the transcriptomic analysis, flag leaves of WT and TOSL3, the transgenic line with n et al. . All then et al. . The genl method  with thep-value < 0.5 and log2fold change less than −1 or more than 1 were classified into GO terms using the ClueGO tool [Genes that had a eGO tool . They weeGO tool  pathway a, chlorophyll b, and total carotenoid contents were studied according to the method of Lichtenthaler [Chlorophyll enthaler . Briefly−1. Seeds were harvested when they were fully developed and then desiccated. The numbers of panicles per plant and seeds per panicle, as well as the fertility rates (%), were determined.After a 9 days salt-stress treatment, plants were recovered by the addition of water until soil salinity was below 2 ds·mp-value < 0.05 using SPSS 21.0 statistical software. All the results are presented as mean ± standard error of the mean.A randomized complete block design with four replications was used for the experimental plots. Analysis of variance was performed to detect the differences among means and a Tukey’s range test was used to detect significant differences between each mean at OsNUC1-S in the salt tolerance of rice during the reproductive stage was suggested to be due to the enhancement of photosynthetic processes in both flag and second leaves through the modification of gene expression levels in water transport, photosynthesis, cellular homeostasis, and carotenoid biosynthesis. These could help maintain the grain yield after a salt stress (Based on the experimental results, a role of t stress .


In past, several biomarkers, based on concentration of proteins involved in type-2/intrinsic/mitochondrial apoptotic pathway, have been identified for prognosis of colorectal cancer patients. Recently, a prognostic tool DR_MOMP has been developed that can discriminate high and low risk CRC patients with reasonably high accuracy . This prognostic tool showed an accuracy of 59.7% when used to predict favorable/unfavorable survival outcomes. In this study, we developed knowledge based models for predicting risk scores of CRC patients. Models were trained and evaluated on 134 stage III CRC patients. Firstly, we developed multiple linear regression based models using different techniques and achieved a maximum HR value of 6.34 with p-value = 0.0032 for a model developed using LassoLars technique. Secondly, models were developed using a parameter optimization technique and achieved a maximum HR value of 38.13 with p-value 0.0006. We also predicted favorable/unfavorable survival outcomes and achieved maximum prediction accuracy value of 71.64%. A further enhancement in the performance was observed if clinical factors are added to this model. Addition of age as a variable to the model improved the HR to 40.11 with p-value as 0.0003 and also boosted the accuracy to 73.13%. The performance of our models were evaluated using five-fold cross-validation technique. For providing service to the community we also developed a web server ‘CRCRpred’, to predict risk scores of CRC patients, which is freely available at  Colorectal cancer (CRC) or large bowel cancer is one of the prevalent and fatal cancers with about 95 percent of them as adeno-carcinomas . It is tMitochondrial or apoptotic type 2 signaling pathway is a conserved pathway in many organisms, which controls the lifespans of cells in different tissues and leads to cellular death or apoptosis as a result of genotoxic and other stresses . Alteratη. The authors in this study demonstrated that η can be used to classify a group of 26 CRC patients into responders (favorable outcomes) and non-responders (unfavorable outcomes) to chemotherapy  and X is a n × 5 dataset matrix where n rows signify patients in the dataset and 5 columns have the protein expression values s.t for a patient j and at W = Wi:A column vector βi and estimate HR values on the complete dataset using mean and median cutoffs. Algorithm is given in the β* as Risk Score (RS), for reasons that will become clear later.The dataset corresponding to 134 stage III CRC patients was uniformly divided into 5 subsets. A parallel parameter (W) search upto one decimal digit precision was performed iteratively, in order to maximize the objective ‘Hazard Ratio’ at mean and median cutoffs, on five training sets (each train set ∼ 4 out of 5 subsets). Running five search algorithms parallely with reduced yet homogeneous data saved computation time. The top parameter set obtained for each case  was then used to evaluate Following the univariate analysis presented in , we straIn order to find a relationship between level of protein concentration and survival time, we developed multiple linear regression models. The protein concentrations were used as independent/input variables and overall survival time (OS) was used as output/target/dependent variable. This implementation was done using Python’s sklearn package. After model fits and test validations, predicted OS from different methods was used to stratify high and low risk patients. Results are provided in It was observed that LassoLars (LassoLarsCV from sklearn.linear model) based model performed better than other models and achieved a maximum HR value of 6.34 with p-value = 0.0032 for predicted OS at median cutoff. KM plot for this is shown in One of the limitations of multiple linear regression models is that they are extensions of linear regression models where they evaluate relationship between a dependent and each independent variable. In addition, it is assumed that these protein concentrations (independent variables) have no relationship with each other. Thus, there is a need to develop a model which can handle non-linear data and correlated variables. In this study, we used a simple parameter optimization technique, where all possible weights were tried in an iterative manner to obtain best weights. By means of this method, Risk Score (RS) is predicted that is derived from anti and pro-apoptotic protein levels.β*. These results also correlate with isolated studies on BclXL and Mcl1 which showed their relevance as prognostic markers in the past [In parameter optimization technique, parameters or weights are optimized using iterative techniques which increases the possibility of over optimization. In order to avoid any over optimization, we used the concept of five-fold cross validation . Resultsthe past , 17. Howzη [zη. Results are summarized in The CRC stage III cohort dataset contains 95 favorable patients i.e patients that were alive during the 5 years study period and 39 unfavourable patients consisting of cases of recurrences/deaths. A comparison between a recently established mathematical model based prognostic marker, zη , and RS β1, β2, β3, β4, β5 were also significant in a multivariate setting (results in β*) performs better than them. These results strengthen the conclusion that RS is a significantly improved prognostic marker over previously established histo-pathological biomarkers.A multivariate analysis using cox proportional hazard models, was performed to see the association of other pathological features present in the dataset with the mortality risk of patients. RS was estimated for CRC stage III cohort dataset, to stratify high risk (RS < 0 or < 0.266) and low risk (RS ≥ 0 or ≥ 0.266) patients. Results are reported in Other factors like age , gender β* and allotted optimized weights through an iterative selection as earlier, to see whether such hybrid combinations could further add value to the current protein based RS. These hybrid combinations can be represented as follows:Clinical factors were added to Combi corresponds to a specific combination of i features with β* and wi are the corresponding optimized weights. The results corresponding to single-feature combinations of features whose information was available for all 134 patients are shown in Combi values in β* with age shows the most significant improvement and uses the involvement of least number of clinical variables. Adding more features to this sum didn’t change the performance. We call the z-normalized version of this combination as Hybrid Risk Score (RSH). Patients with RSH <mean(RSH) are at 40 times more risk than patients with RSH >mean(RSH) with HR value of 40.11 and p-value of 0.0003. KM plot corresponding to stratification of patients by RSH is given in Where, https://webs.iiitd.edu.in/raghava/crcrpred, in order to provide service to the community. This web-server implements the current study in order to predict high and low risk patients given the nanomolar concentration of required Bcl2 family protein(s). Following are the two prediction modules with their brief descriptions:We also developed a web-server CRCRpred, which is freely available at Often the user will not possess the concentrations of all required Bcl2 family proteins, mainly because protein level quantification is a challenging task in itself. Keeping this in mind we provide this module to the user where, with the limited knowledge of concentration(s) of one or more than one proteins, the risk can be predicted. The prediction here is a protein wise prediction. Input concentration from the user is fed into a linear regression model and risk percentage is estimated. This regression model comprises of fitting bin-wise mean protein concentrations with probability of high risk patients in that bin. High risk and low risk patient stratification was done on the basis of median survival time in CRC dataset.This module computes the Risk Score (RS) of a patient, given the concentrations of all five proteins by estimation of RS for the given patient. The patient is classified into high/low risk category based on the cutoff, RS = 0. The distance from cutoff point is displayed as percentage risk to the user along with the risk grade.Colorectal cancer is a mortal disease, with incidences all over the world, and requires better clinical strategies. Recent experiments have suggested a crucial role of apoptotic type 2 pathway in tumor progression and development, as a result of which many new drugs are targeted on the proteins involved in this pathway. For e.g. several BH3 mimetics, which are small molecules that target and inhibit anti-apoptotic Bcl2 proteins, such as ABT-737, ABT-263 (Navitoclax), ABT-199 (Venetoclax), WEHI-539, A-1155463 etc. have been introduced in the market while several others are in clinical trials –24. Thusβ* value obtained for each patient. Patients with RS<0 (mean cut-off) or RS<0.266 (median cut-off) are classified as high risk patients, indicating that a higher anti-apoptotic concentration increases the risk associated with CRC. This has a direct correlation with cell death mechanism related to type 2 apoptotic pathway, where a higher anti-apoptotic concentration as compared to pro-apoptotic concentration has been shown to be the reason for evasion of apoptosis by the tumor cell [H which uses protein concentrations and age of a patient, which is readily available, to predict high/low risk class.To tackle this, we first took at the total pro- and anti- apoptotic protein concentration, since both of these are upregulated in the event of cellular stress conditions such as tumor , and strmor cell . RS alsomor cell , 26–29. mor cell –21. A prhttps://webs.iiitd.edu.in/raghava/crcrpred was also developed. CRCRpred is built on a responsive template, compatible for desktop, tablet, and smartphone. These templates are dynamic that fit content based on screen size of the device. This web server can be used to distinguish high risk CRC patients from low risk CRC patients given the protein concentration of one or more apoptotic proteins  involved in the process of Mitochondrial Outer Membrane Permeabilization (MOMP), which is defunct in the case of cancer. This risk estimation is based on statistical and survival analysis on a recent CRC dataset and risk score (RS) obtained from the same can be a promising prognostic tool in clinical/research domain.To provide valuable help to scientific community, a web server, CRCRpred, S1 Fileβ1 (i) Patients with β1 < mean(β1) are classified as High Risk as compared to patients with β1 ≥ mean(β1). (ii) Patients with β1 < median(β1) are classified as High Risk as compared to patients with β1 ≥ median(β1). Fig B, Stratification of high and low risk CRC patients based on OS using β2 (i) Patients with β2 < mean(β2) are classified as High Risk as compared to patients with β2 ≥ mean(β2). (ii) Patients with β2 < median(β2) are classified as High Risk as compared to patients with β2 ≥ median(β2). Fig C, Stratification of high and low risk CRC patients based on OS using β3(i) Patients with β3 < mean(β3)re classified as High Risk as compared to patients with β3 ≥ mean(β3). (ii) Patients with β3 < median(β3) are classified as High Risk as compared to patients with β3 ≥ median(β3). Fig D, Stratification of high and low risk CRC patients based on OS using β4(i) Patients with β4 < mean(β4)re classified as High Risk as compared to patients with β4 ≥ mean(β4). (ii) Patients with β4 < median(β4) are classified as High Risk as compared to patients with β4 ≥ median(β4). Fig E, Stratification of high and low risk CRC patients based on OS using β5(i) Patients with β5 < mean(β5)re classified as High Risk as compared to patients with β5 ≥ mean(β5). (ii) Patients with β5 < median(β5) are classified as High Risk as compared to patients with β5 ≥ median(β5). Fig F, Multivariate COX-PH hazard analysis for risk estimation in the CRC patients based on β1 (i) At mean cutoff. and (ii) At median cutoff. Here right tumor location—hepatic flexure, caecum, ascending, traverse; left tumor location—splenic flexure, descending, sigmoid and rectal—rectum, rectosigmoid. Fig G, Multivariate COX-PH hazard analysis for risk estimation in the CRC patients based on β2 (i) At mean cutoff. and (ii) At median cutoff. Here right tumor location—hepatic flexure, caecum, ascending, traverse; left tumor location—splenic flexure, descending, sigmoid and rectal—rectum, rectosigmoid. Fig H, Multivariate COX-PH hazard analysis for risk estimation in the CRC patients based on β3 (i) At mean cutoff. and (ii) At median cutoff. Here right tumor location—hepatic flexure, caecum, ascending, traverse; left tumor location—splenic flexure, descending, sigmoid and rectal—rectum, rectosigmoid. Fig I, Multivariate COX-PH hazard analysis for risk estimation in the CRC patients based on β4 (i) At mean cutoff. and (ii) At median cutoff. Here right tumor location—hepatic flexure, caecum, ascending, traverse; left tumor location—splenic flexure, descending, sigmoid and rectal—rectum, rectosigmoid. Fig J, Multivariate COX-PH hazard analysis for risk estimation in the CRC patients based on β5 (i) At mean cutoff. and (ii) At median cutoff. Here right tumor location—hepatic flexure, caecum, ascending, traverse; left tumor location—splenic flexure, descending, sigmoid and rectal—rectum, rectosigmoid. Fig K, Kaplan Meier survival curves for risk estimation in the sub-populations of CRC stage III patient cohort based on RS using the mean (RS = 0) cutoff, show significant differences in high/low risk groups. (i) Samples with patient age less than 60y were stratified  in high/low risk groups. (ii) Female patients were stratified  within high/low risk groups. (iii) Patient with no lymphovascular invasion were stratified  in high/low risk groups. (iv) Patient samples with N-stage 2 or higher were stratified  in high/low risk groups. Fig L, Kaplan Meier survival curves for risk estimation in the sub-populations of CRC stage III patient cohort based on RS using the mean (RS = 0) cutoff, show significant differences in high/low risk groups. (i) Samples with tumor location on left side were stratified  in high/low risk groups. (ii)Samples with tumor location on rectal side were stratified  in high/low risk groups. (iii) Patient with T stage 2 or higher were stratified  in high/low risk groups.Fig A, Stratification of high and low risk CRC patients based on OS using (DOCX)Click here for additional data file.S2 Fileβ values for specified weight parameter W, for each patient. Table G, Risk Score (RS) values . Table H, Estimated Combi values for each single-clinical feature combination. Hybrid Risk Score (RSH) values  are provided in the last column.Table A, Clinical information about the patients used in this study as adapted from Lindner AU, et al. Gut. 2017. OS predicted from multiple regression models are also provided. Tables B-F, Estimated (XLSX)Click here for additional data file.


Maternal smoking and/or exposure to environmental tobacco smoke continue to be significant factors in fetal and childhood morbidity and are a serious public health issue worldwide. Nicotine passes through the placenta easily with minimal biotransformation, entering fetal circulation, where it results in many harmful effects on the developing offspring, especially on the developing respiratory system.  Recently, in a rat model, electroacupuncture (EA) at maternal acupoints ST 36 has been shown to block perinatal nicotine-induced pulmonary damage; however, the underlying mechanism and the specificity of ST 36 acupoints for this effect are unknown. Here, we tested the hypothesis that compared with EA at ST 36, EA at LU 5 acupoints, which are on lung-specific meridian, will be equally or more effective in preventing perinatal nicotine-induced pulmonary changes.  Twenty-four pregnant rat dams were randomly divided into 4 groups: saline (“S”), nicotine (“N”), nicotine + ST 36 (N + ST 36), and nicotine + LU 5 (N + LU 5) groups. Nicotine  and EA  were administered from embryonic day 6 to postnatal day 21 once daily. The “S” group was injected saline. As needed, using ELISA, western analysis, q-RT-PCR, lung histopathology, maternal and offspring hypothalamic pituitary adrenal axes, offspring key lung developmental markers, and lung morphometry were determined. γ and an increase of β-catenin and glucocorticoid receptor expression in lung tissue and corticosterone in the serum of offspring rats. Electroacupuncture at ST 36 normalized all of these changes, whereas EA at LU 5 had no obvious effect.  With nicotine exposure, alveolar count decreased, but mean linear intercept and septal thickness increased. It also led to a decrease in pulmonary function and PPAR Electroacupuncture applied to ST 36 acupoints provided effective protection against perinatal nicotine-induced lung changes, whereas EA applied at LU 5 acupoints was ineffective, suggesting mechanistic specificity and HPA axis' involvement in mediating EA at ST 36 acupoints' effects in mitigating perinatal nicotine-induced pulmonary phenotype. This opens the possibility that other acupoints, besides ST 36, can have similar or even more robust beneficial effects on the developing lung against the harmful effect of perinatal nicotine exposure. The approach proposed by us is simple, cheap, quick, easy to administer, and is devoid of any significant side effects. The concept that smoking is harmful to health has been deeply rooted among the populace, but tobacco smoking continues to hurt, particularly, the most vulnerable members of the society, i.e., fetuses and newborns. Maternal smoking and/or exposure to environmental tobacco smoke continue to be significant factors in fetal and childhood morbidity and are a serious public health problem worldwide. According to the World Health Organization, more than 1 billion people around the world smoke, and 5 million die from cigarettes each year. In China, the current smoking rate for adults, aged 15 and over, is 27.7, percent and the total number of smokers is more than 300 million . Even though the rate of active smoking during pregnancy in China is relatively low, the exposure rate to secondhand smoke during pregnancy is alarmingly high (>50%) , 5.There are hundreds of toxicants in tobacco smoke, among which nicotine is the main chemical with known toxicity for the developing lungs and brain , 7. NicoDespite aforementioned concerns, at present, other than smoking cessation and avoiding cigarette smoke exposure altogether, there is no effective preventive or therapeutic option. Unfortunately, due to aggressive advertising strategies from the tobacco industry, especially towards the youth, and the highly addictive property of nicotine and negative reinforcement of withdrawal symptoms , 19, it While pharmacological interventions remain far-fetched, interestingly, perinatal electroacupuncture (EA) offers a promising, practical approach that can be, safely and relatively easily, translated to clinical practice. Using a rat model, we recently showed that EA at ST 36 acupoints (Zusanli) effectively blocked perinatal nicotine-induced pulmonary phenotype in the exposed offspring , 21. HowZusanli (ST 36) is a commonly used acupuncture modality, utilized mainly, for general well-being and gastrointestinal ailments, although it has been also shown to be effective against respiratory conditions such as allergic asthma , chronicNicotine bitartrate (99% purity) and pentobarbital (2%) were purchased from Sigma-Aldrich, USA. HAN's acupoint stimulation equipment (LH202H) was obtained from Beijing Huawei Industrial Development Co., Ltd., and Huatuo brand aseptic acupuncture needles (0.20 mm × 13 mm) were acquired from Suzhou Medical supplies Factory Co., Ltd. Pulmonary function studies were performed using AniRes2005 animal lung function analysis system.Twenty-four specific pathogen-free (SPF) Sprague Dawley female  and eight male  rats were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (certificate no. SCXK (Beijing) 2006-0009). The experiments were performed in the barrier animal room of the SPF Animal Research Center of Beijing University of Chinese Medicine. Before the experiment, the animals were allowed to acclimatize for 72 h at room temperature (23 ± 1)°C in 55 ± 5% relative humidity and 12 : 12 h light:dark cycle. All animal procedures were conducted in accordance with the Guidelines for the Animal Care and Use advocated by the U. S. National Institutes of Health, as well as the “3R” principle of animal experimentation with reduction, substitution, and optimization. The animal protocol passed the Ethics Committee of Beijing University of Chinese Medicine.µL volumes was administered to dams subcutaneously from embryonic day (E) 6 to postnatal day (PND) 21 except on the day of delivery. The “S” group was administered the same amount of saline subcutaneously as the “N” group from E6 to PND 21. The N + ST 36 group, in addition to nicotine, was administered acupuncture at acupoints ST 36, which are located in the posterolateral aspect of the knee joint, 5 mm below the fibulae capitulum  were inserted perpendicularly 7 mm into ST 36 or LU 5 acupoints (connected to the EA negative electrode) and 2 mm subcutaneously below ST 36 or LU 5 (connected to the EA positive electrode). The EA parameters were set as follows: Sparse-dense wave frequency, 2/15 Hz; intensity, 1 mA; and duration, 20 minutes, administered by the same operator, once a day between 10 AM–12 noon every day.For pulmonary function studies, on PND 21, after instrument calibration for airway pressure and lung volume, rat pups were weighed and anesthetized using 2% pentobarbital sodium (55 mg/kg), followed by tracheostomy, cannulation, and ventilation for plethysmography. After respiratory stability was achieved, using AniRes2005 software, airway resistance (RL), peak inspiratory flow (PIF), peak expiratory flow (PEF), forced vital capacity (FVC), and dynamic pulmonary compliance (Cdyn) were derived.μm thickness hematoxylin and eosin-stained tissue slices from different experimental groups. Using Image-Pro Plus 6.0 software, alveolar count, mean linear intercept, and septal thickness were determined as described by us previously [At sacrifice, the lungs were collected and instilled with ice-cold 4% paraformaldehyde (PFA) dissolved in 1 × PBS. These were kept immersed in 4% PFA for 4-5 hours and then moved to 30% sucrose dissolved in PBS. When settled to the bottom of the container, the lungs were removed, and the left lung was paraffin-embedded. An investigator unaware of experimental groups performed lung morphometry on 5 eviously .γ and β-catenin, serum Cort, and pituitary GR, ACTH, and adrenal MC2R levels were determined using ELISA according to manufacturers' instructions. As needed, the lung tissue, pituitary, and adrenal glands were ground, lysed, and centrifuged. ELISA kits used were PPARγ from LifeSpan BioSciences Co., Ltd. (LS-F15392); β-catenin from Enzo Life Sciences Co., Ltd. (ADI-900-135); ACTH, MC2R, and GR from WUHAN CUSABIO Co., Ltd. .At the end of the experimental period (PND 21), dams and pups were euthanized , and blood was collected via cardiac puncture. Serum was separated by centrifugation, and samples were frozen at −80°C until processing. Offspring lungs and maternal pituitary and adrenal glands were collected and immediately flash-frozen in liquid nitrogen. Lung PPARγ and β-catenin were obtained from Invitrogen (USA) and amplified by the real-time fluorescence quantitative PCR . The reaction was carried out by activating the DNA polymerase at 95°C for 10 minutes, and then up to 40 PCR denaturation cycles at 95°C for 15 s and annealing at 60°C for 1 minute were performed. The relative quantitative value of each gene was calculated using the 2ΔΔCT method based on CT values.RNA extraction was performed using Qiagen RNAeasy Mini kit (category no. 74106) obtained from Life Technologies. The extracted RNA was qualitatively analyzed using Agilent nucleic acid and protein analyzer  and quantified using Nanodrop micronucleic acid protein analyzer  and stored at −80°C until needed for assay. Reverse transcription of RNA to cDNA was performed using SuperScript VILOTM cDNA Synthesis Kit (category no. 11754) for qRT-PCR System  and the PCR automatic analyzer . Real-time PCR was performed using the upstream and downstream primers, obtained from Sangon Biotech Co., Ltd., Shanghai, China . SYBR GrP < 0.05 indicated statistically significant difference.All data were analyzed using the statistical software (IBM SPSS 20.0). The data are expressed as mean ± SD. The differences between the groups were compared by one-way analysis of variance, followed by the Tukey test. P < 0.01, P < 0.05, and P < 0.01, respectively), while the PIF and RL increased significantly (P < 0.01). Compared with the “N” group, the PEF, FVC, and Cdyn in the N + ST 36 group increased , and the PIF and RL decreased significantly (P < 0.01). Furthermore, compared with the “N” group, while the PEF, FVC, and Cdyn in the N + LU 5 group showed an upward and the RL, a downward trend; these changes did not reach statistical significance (P > 0.05)  .γ protein level decreased, and β-catenin protein increased significantly in the “N” (P < 0.05). Compared with the “N” group, in the N + ST 36 group, PPARγ protein level increased, and β-catenin levels decreased significantly (P < 0.05). However, in the N + LU 5 group, while there was a trend towards normalization, the changes did not statistical significance (P > 0.05)  .γ and β-catenin expression were in-line with the ELISA data outlined above, i.e., compared with the “S” group, “N” group showed a significant decrease in PPARγ and a significant increase in β-catenin expression (P < 0.05). While these changes were corrected in the N + ST 36 group, there was no statistically significant effect in the LU 5 group (P > 0.05), even though there was a trend towards normalization . Compared with the “N” group, pituitary ACTH, adrenal MC2R, and serum Cort levels in N + ST 36 group were significantly lower  and not statistically different from the “S″ group; however, compared with the “N” group, there was no significant change in the levels of all these biomarkers in the N + LU 5 group (P > 0.05)  .P < 0.01, P < 0.05, respectively). The GR and Cort levels in the N + ST 36 group were significantly lower compared with the “N” group  and were not statistically different from the “S” group (P > 0.05). In contrast, in the N + LU 5 group, while there was a downward trend, these levels were not statistically different compared with the “N” group (P > 0.05)  .P < 0.01 for both). Compared with the “N” group, these changes improved significantly in the N + ST 36 group (P < 0.05) and were not statistically different from the “S” group (P > 0.05). In contrast, in the N + LU 5 group, while there was an improving trend in these parameters, these did not reach statistical significance vs. the “N” group (P > 0.05). Note that lung morphometry data outlined here have been published previously [Compared with the “S” group, the mean linear intercept increased and alveolar count decreased significantly in the “N” group (eviously .γ downregulation and β-catenin upregulation), lung morphometry , and pulmonary function . In contrast, EA at LU 5 acupoints had no significant effect on either maternal/fetal HPA axis, or lung molecular, structural, and functional phenotypes.Our data demonstrate that EA at ST 36 blocked the perinatal nicotine-induced changes in maternal  and offspring (serum Cort level and lung GR expression) HPA axes. It also blocked perinatal nicotine-induced changes in key lung developmental signaling pathways (PPARγ and inhibit Wnt signaling in offspring lung, and preserve lung development and function in offspring perinatally exposed to nicotine [As outlined above, at present, there is no effective pharmacologic intervention against the harmful effects of perinatal smoke/nicotine exposure on the developing lung; however, our recent work has suggested acupuncture to be a promising, potential approach. Recently, we demonstrated that EA at ST 36 could regulate maternal HPA axis, promote PPARnicotine , 21. Allγ signaling is vital for lung lipid differentiation programming, which is essential for normal lung development and homeostasis [γ signaling in turn interacts with Wnt signaling, which is involved in many key developmental biological processes involved in lung development and maturation [γ and Wnt signaling is critically important during lung development [β-HSD-2 expression, it results in fetal exposure to a high glucocorticoid environment. This high fetal glucocorticoid environment potentially disrupts fetal neuroendocrine environment, inhibiting fetal HPA axis, intrauterine growth retardation [γ and β-catenin expressions [PPAReostasis . PPARγ sturation –31. A baelopment , a balanelopment –36. Addielopment . The matardation , and eliressions –41. In pIn the theory of traditional Chinese medicine, the main effects of acupoints' stimulation are directly related to the meridians and position to which they belong. The acupoints on the meridians can treat the diseases of the Zang-fu organs, which the meridians belong to, and the diseases of the Zang-fu organs and tissues, through which the meridians travel, as well as the disease of the adjacent areas. In addition, some acupoints have special therapeutic effects. The therapeutic effects of meridian acupoints are specific, an important factor affecting the beneficial effects of acupuncture. There are differences in functional effects between acupoints and nonacupoints, different acupoints of the same meridian, and different acupoints of different meridians. However, the specificity is relative, as one meridian point can treat multiple viscera and multisystem diseases, different meridian points can treat the same meridian or the same visceral diseases, and different meridian points have different effects on the same disease . The twoIt is also important to highlight that in our previous studies, we did not observe any significant effects of EA at sham acupoints on lung morphometry and lung maturation supporting specific mechanisms involved in mediating ST 36 acupoints' effects on the developing lung. However, since we applied EA during both antenatal and postnatal periods, it is difficult to distinguish between the effects for each period, and to determine if postnatal intervention has any effect.γ and increase in Wnt signaling, and provides a more homeostatic milieu for offspring lung development. In contrast, even though LU 5 is on the lung meridian and has been used to treat many lung diseases, it did not prove to be useful against perinatal nicotine's exposure on the developing lungs. It reinforces that the beneficial effects of EA at ST 36 on the developing lung are via its effect on maternal/offspring HPA axis, rather than on offspring lung directly. This opens the possibility that other acupoints besides ST 36 having similar or even more robust beneficial effects on the developing lung against the harmful effect of perinatal nicotine exposure. The approach proposed by us is simple, cheap, quick, easy to administer, and is devoid of any significant side effects.To sum up, comparing the effects of EA at ST 36 and LU 5 acupoints on lung development of the perinatally nicotine exposed offspring, we conclude that the mechanism underlying the beneficial effects of EA at ST 36 acupoints is via modulation of maternal/offspring HPA axis. This likely helps in avoiding offspring exposure to excessive maternal glucocorticoid environment induced by nicotine exposure, which, in turn, blocks the perinatal nicotine-induced decrease in alveolar PPAR


We demonstrate that a generalized anomalous diffusion (AD) model, which uses a simple power law to relate the mean square displacement to time, more accurately captures individual cell migration paths across a range of engineered 2D and 3D environments than does the more commonly used PRW model. We used the AD model parameters to distinguish cell movement profiles on substrates with different chemokinetic factors, geometries (2D vs 3D), substrate adhesivities, and compliances. Although the two models performed with equal precision for superdiffusive cells, we suggest a simple AD model, in lieu of PRW, to describe cell trajectories in populations with a significant subdiffusive fraction, such as cells in confined, 3D environments.Appropriately chosen descriptive models of cell migration in biomaterials will allow researchers to characterize and ultimately predict the movement of cells in engineered systems for a variety of applications in tissue engineering. The persistent random walk (PRW) model accurately describes cell migration on two-dimensional (2D) substrates. However, this model inherently cannot describe subdiffusive cell movement, i.e., migration paths in which the root mean square displacement increases more slowly than the square root of the time interval. Subdiffusivity is a common characteristic of cells moving in confined environments, such as three-dimensional (3D) porous scaffolds, hydrogel networks, and  Cell migration is integral to a variety of physiological processes including organ development, tissue morphogenesis, wound healing, and immune response. A greater understanding of the motility effects of environmental cues can inform the design of biotechnologies such as movement-directing scaffolds. Research into the relationship between cell migration and cues from the cellular microenvironment increasingly takes advantage of the capability to manipulate properties such as the extracellular matrix (ECM) compliancein vivo conditions.P < 50 min) motile cells in 3D collagen gels have been found to be poorly fitted by the PRW model (R2 < 0.6).Descriptive  models of migration dynamics facilitate analysis of microenvironment dependence in part by assigning parameters to characterize cells, individually and in aggregate. One of the most commonly used models for describing individual cell migration in 2D is the persistent random walk (PRW) model,τ, in anomalous diffusion, the mean squared displacement grows as a power, α < 2, by definition lending this model the flexibility to describe both sub- and superdiffusive motion. Variants of anomalous diffusion, in which α may be constant or τ-dependent, accurately describe a variety of physical and biological phenomena;individual cell trajectories to the best of our knowledge.No simple descriptive model is commonly used to capture a broad range of types of cell motion, from highly constrained to ballistic. We therefore adapted the anomalous diffusion (AD) model for individual and aggregate cell migration. In contrast to normal (free) diffusion, in which the mean squared displacement grows linearly with the time interval α more clearly differentiated subdiffusive cells from each other than did the PRW parameter P (persistence time). The AD parameters as well as the PRW parameters were found to predictably vary with geometry, elastic modulus, ECM composition, and ECM ligand density. Therefore, we suggest the AD model is a more robust model of individual cell movement, particularly in constrained, 3D environments.Given that many cells migrating in 3D are subdiffusive, we undertook to systematically characterize the trajectories of individual cells (and aggregate sample-wide migration) under various extracellular conditions using the AD model. We found that PRW and AD gave similar correlation coefficients for superdiffusive cells, but that the AD model was better at describing subdiffusive cells. The AD parameter 1 integrin (red)  or  at observation times t = 0, Δt, 2Δt, … tmax, where Δt is the time between image acquisitions, tmax is the last acquisition time, and n ranges from 1 to N. Cells that contacted other cells, underwent division or apoptosis, or were not fully in frame for the entire 12- or 18-h observation period were excluded from all calculations.On coverslips, cells were seeded at 4000 cells per cmτ = 0.25 h, 0.50 h, 0.75 h, …, by averaging the square displacements over all available time intervals of length τ within the cell's trajectory  and 8 h (32 lowest data points) on the resulting τ–MSD data. This experiment was done with the pooled data from 2 biological replicates, each with 2 technical replicates.Fitting of τ of 4 h were fitted to the persistent random walk (PRW) equation used by DunnS is root mean square speed over all available Δt-sized intervals (15 min) and P is persistence time, a characteristic parameter describing the average length of time over which cells resist major changes in direction of movement. For individual cells, the persistence is nP and the root mean square speed is calculated asPagg andP (nP for cells and Pagg for conditions) between 0 and tmax were obtained using the Matlab (Mathworks) function lsqcurvefit, which attempts to minimize the residual sum of squares (RSS), defined asIndividual and aggregate MSD data up to a maximum S and P are allowed to vary. So a 2-parameter PRW fit was performed to obtain additional, arguably more “fair” values of S, P, and R2PRW. Generally, the use of the 2-parameter fit did not significantly increase R2PRW but sometimes drastically altered the best-fit parameters (see Table S2). Unless otherwise indicated, reported P values are from the 1-parameter fit.Whereas the PRW model has only 1 fitted parameter (P), the AD model has 2. Since additional “degrees of freedom” can be expected to increase the correlation coefficient, some comparisons of the accuracy of the AD and PRW model might be reversed in favor of PRW if both τ of 4 hours, was fitted to the equation using lsqcurvefit and Microsoft Excel's slope and intercept functions. AD parameters αn and Γn, for individual-cell MSD data, or αagg and Γagg, for aggregate MSD data, were simultaneously determined. Best-fit α was restricted to between 0 and 2 and best-fit Γ restricted to between 0 and 10 000.The anomalous diffusion (AD) model equationFor AD model fitting of MSD vs. τ data , RSS, TSS, and COD were defined, respectively, asn and nP were not weighted differently depending on the strength of the fit, i.e., R2 value, when calculating condition-wide averages. Individual cells were considered subdiffusive for descriptive purposes if 0 ≤ αn < 1 and superdiffusive if 1 < αn ≤ 2.A suite of purpose-built Matlab programs was used to process and analyse the migration data at the cell and aggregate level, including model fittings, and report, classify, and compare the results, and produce analytical plots such as Γ-binned-by-α plots and histograms. The MSD calculations and PRW model fittings were confirmed using the Visual Basic program DiPer.Ethics approval is not required for this study. Conditioned medium from patient cells described in Fig. supplementary material for complete data for cell motility parameters and model fitting data.See


A novel methodology towards fabrication of multilayer organic devices, employing electrochemical polymer growth to form PEDOT and PEDTT layers, is successfully demonstrated. Moreover, careful control of the electrochemical conditions allows the degree of doping to be effectively altered for one of the polymer layers. Raman spectroscopy confirmed the formation and doped states of the PEDOT/PEDTT bilayer. The electrochemical deposition of a bilayer containing a de-doped PEDTT layer on top of doped PEDOT is analogous to a solution-processed organic semiconductor layer deposited on top of a PEDOT:PSS layer without the acidic PSS polymer. However, the poor solubility of electrochemically deposited PEDTT  raises the possibility of depositing a subsequent layer via solution-processing. N-methylpyrrole) to develop symmetric supercapacitors [Fabrication of multilayer organic electronic devices has been extensively researched in the past 20 years, resulting in numerous processes and techniques . Recent pacitors . However3CN  and 3,4-ethylenedithiothiophene (EDTT), were either purchased commercially or prepared according to literature procedures . Both weed PEDTT  whilst ted PEDTT . The abs−4 M monomer solution) were grown on ITO and analysed by Raman spectroscopy, alongside doped and de-doped mono-layers of PEDOT and PEDTT for comparison. −1, the Raman spectra are dominated by medium to weak bands which can be predominantly assigned to out-of-plane deformations [−1 and 991 cm−1, assigned to dioxyethylene ring deformations, and at 1099 cm−1, assigned to C–O–C deformation, are absent in PEDTT , 1413 cm−1 (symmetric Cα=Cβ stretching mode), 1371 cm−1 (Cβ=Cβ stretching vibration), and 1255 cm−1 (inter-ring Cα=Cα’ stretching) are present [−1 shifts to 1521 cm−1 and increases in relative intensity, while the νsym(C=C) band shifts from 1413 cm−1 to 1426 cm−1. Additionally, the broad band at 1255 cm−1 resolves into two distinct features (at 1227 and 1271 cm−1) [−1 is observed. Similar changes were ascribed by Garreau et al. [On the contrary, the Raman features appearing above 1150 cmspectrum , Raman f−1 and 1321 cm−1, can be ascribed to νs(Cα=Cβ) and νs(Cβ=Cβ) vibrations, respectively. However, the out-of-plane bands appearing in the region below 1150 cm−1 are less sensitive to the de-doping process, but the C=C bands show important changes, such as the marked downshift of the Cβ=Cβ band from 1321 cm−1 to 1312 cm−1, and the relative intensity increase of the Cα=Cβ band at ≈1396 cm−1. In particular, Kocharova et al. [α=Cβ band in polythiophene structures to the higher localised charge at the Cα=Cβ linkage as a consequence of the positively charged doping of the material.By analogy with the band assignment for PEDOT, the Raman features in the PEDTT spectrum  at 1396 a et al.  associat−1, can be recognised in the bilayer spectrum. The appearance of signals arising from the underlying PEDOT layer, although distant from the focal point of the laser, is likely the result of a partial de-doping of the underlying PEDOT layer. Thus, the much higher Raman scattering efficiency of PEDOT in the neutral state, as compared to PEDTT (Figure S5, In summary, we have shown a novel processing methodology for the fabrication of multilayer organic electronic devices that utilises electrochemical polymerisation to form the first two layers. Successful PEDOT/PEDTT bilayer formation has been confirmed by Raman spectroscopy. Electrodeposition of the bilayer has advantages over traditional processing methods including avoiding the acidity of PSS which is detrimental to the lifetime of devices containing PEDOT:PSS  and the File 1General experimental and additional spectra.