The widespread and indiscriminate use of antimicrobials in food animals is a key contributor to antimicrobial resistance and antimicrobial residue, which have become a growing public and animal health concern in developing countries such as Bangladesh. This study was aimed to assess the knowledge, attitude, and practices (KAP) of large-animal farmers towards antimicrobial use (AMU), antimicrobial resistance (AMR), and antimicrobial residue (AR) with their correlation. A cross-sectional survey was conducted with a structured and pretested questionnaire in the Mymensingh division of Bangladesh. A total of 212 large-animal farmers  were surveyed. Results showed that most of the farmers are male (85.8%) and belong to the 18–30 age group (37.3%). About 20.3% had no formal education, and nearly half of the participants (48.1%) received training regarding antibiotic use and resistance. Penicillin is the most common class of antibiotic used (61.8%) in the study area, followed by other antimicrobials. Only 37.7% of the farmers used antimicrobials on the recommendation of their veterinarian. Overall, 41.5%, 42.5%, and 21.7% of farmers possess adequate knowledge and a satisfactory attitude and perform desirable practices, respectively. Farmers in the 31–40 age group have adequate knowledge, attitude, and ability to implement desired practices compared to farmers in the 18–30 age group. Farmers having a graduate or post-graduate degree scored better in relation to knowledge, attitude, and practice than other farmers. Analysis revealed that farmers who received training on AMU and AMR had 10.014 times , 9.409 times , and 25.994 times  better knowledge, attitude, and performance, respectively, compared to their counterparts. A significant proportion of farmers (97.2%) dispose of leftover antibiotics inappropriately. The findings of the present study will be used to intervene in the education and training of the farmers, which will help to limit the indiscriminate and irrational use of antimicrobials, leading to reducing the chances of developing AMR. Antimicrobials are the drugs of choice for the treatment of infections in both humans and animals  and exhiIrrational AMU in animal production is thought to be a key contributor to antimicrobial resistance (AMR) despite the fact that the development of AMR is a complex multifactorial process ,6. AMU iCurrently, antimicrobial resistance has become a major public and animal health problem that threatens the effective prevention and treatment of a wide range of infections caused by bacteria . AMR enhInappropriate use of antimicrobials and non-observance of withdrawal periods may result in the deposition of antibiotic residue in animal tissue ,15. AntiAlthough AMR is a global health concern, people in low- and middle-income countries bear the brunt of the consequences . BangladIn accordance with the Global Action Plan (GAP) guidelines of the WHO, Bangladesh has developed and adopted a National Action Plan (NAP) for the prevention and control of AMR in human, animal, and environmental sectors for the period of 2017–2022 . NoticeaWe conducted 212 interviews for the current investigation in all four districts of Mymensingh Division, namely, Mymensingh, Sherpur, Jamalpur, and Netrokona.The characteristics of the study interviewees are shown in Several antimicrobials were used on large-animal farms in the study area. Antimicrobials were used on the farm either alone or in combination with other antimicrobials. In the study, the most common class of antimicrobials used in large-animal farms were penicillin (61.8%), oxytetracycline (55.7%), sulfa drug (55.7%), and streptomycin (54.7%) followed by ciprofloxacin (51.9%), gentamicin (43.1%), and ceftriaxone (34.9%) .As shown in We asked nine questions to the large-animal farmers to assess their attitudes towards AMU and AMR. The results are shown in We asked 10 questions to assess respondents’ practices regarding AMU and AMR. The results are shown in p = 0.003), level of education (p < 0.001), training program regarding antimicrobial use and resistance from any institutions (p < 0.001), and their farm population size (p < 0.001) were the significant factors influencing the knowledge theme. The analysis revealed that a farmer’s sex (p < 0.001), level of education (p < 0.001), training received (p < 0.001), type of farm the farmer owned (p = 0.028), and farm population size (p < 0.001) were the significant factors affecting their attitudes. The analysis also revealed that a farmer’s age (p = 0.044), sex (p < 0.001), education (p < 0.001), training (p < 0.001), and farm population size were the significant factors influencing their practice  compared with female farmers. They further revealed that the respondents who belonged to the age group 31–40 had a ‘better’ attitude  compared with the farmers who were in the 18–30 age group. Furthermore, farmers educated at PSC, JSC, SSC, HSC, graduate, and masters levels were found to be 1.25 times , 3.646 times , 3.603 times , 6.836 times , 10.208 times , and 39.379 times  more likely to have better attitudes regarding AMU and AMR, respectively, compared to illiterate farmers. Further, the farmers who received training regarding antibiotic use and resistance from any institution had 9.844 times the odds of having better attitudes on AMU and AMR compared with their counterparts . Regarding a farmer’s farm population, farmers who had 6–10, 11–20, and >20 animals on their farms had 2.313 times , 5.946 times , and 55.5 times  the odds of having better attitudes regarding AMU and AMR than those who had 3–5 animals on their farms. There was no significant variation found for the other variables. The analysis of AMU and AMR practices showed that males had 1.127 times the odds of having a ‘better’ practice towards AMU and AMR  compared with female farmers. They further revealed that the respondents who belonged to the age group 31–40, 41–50 years, and >50 years had a ‘better’ practice  compared with the farmers who were in the 18–30 age group. Furthermore, farmers educated at PSC, JSC, SSC, HSC, graduate and masters levels were found to be 2.562 times , 3.237 , 5.979 times , 8.483 , 20.5 times , and 47.833 times  more likely to have better practices regarding AMU and AMR, respectively, compared to illiterate farmers. Further, the farmers who received training regarding antibiotic use and resistance from any institution had 25.994 times the odds of having better practices regarding AMU and AMR compared with their counterparts . Regarding a farmer’s farm population, farmers who had 6–10, 11–20, and > 20 animals on their farms had 1.75 times , 14.086 times , and 48.375 times  the odds of having better practices regarding AMU and AMR than those who had 3–5 animals on their farms.There was no significant variation found for the other variables in the present study.p ≤ 0.001). Knowledge–attitudes, knowledge–practices, and attitudes–practices all showed a reasonable correlation [According to Spearman’s rank-order correlation, each pair of respondents’ knowledge, attitude, and practice scores had a positive relationship  with his/her knowledge, attitudes, and practices regarding AMU and AMR [To combat against AMR, human behavior and educational level are crucial . In addi and AMR . The res and AMR . Farmers and AMR . In this study, farmers with inadequate knowledge scores were linked to a higher number of respondents who had no training (51.5%). The farmers who received training regarding antimicrobial use and resistance from any institution had 10.014 times the odds of having adequate knowledge of AMU and AMR, 9.844 times the odds of having desirable attitudes, and 25.994 times the odds of having appropriate practices compared with their counterparts. According to a study in Cameroon, farmers with lower knowledge scores were more likely to be untrained in poultry farming . A previFarmers having larger population size farms are more likely to have a favorable attitude to limit AMU than their smaller counterparts ,58,59. AThis study has some limitations due to the nature of gathering data on human behavior through survey approaches. In this survey, a KAP questionnaire was used to collect data. A small number of the respondents were selected from each of the four districts in Mymensingh, Bangladesh, which may not reflect the actual status of KAP for large-animal farmers. Furthermore, because participants self-reported their attitudes and past behaviors, there is a chance of incorrect recall and social desirability or confirmation bias, which could skew the results. Another limitation of the study is KAP survey techniques may mistakenly lead participants to provide responses that they foresee the researcher viewing as appropriate or desirable. The cause-and-effect relationship between the predictor variables and the dependent binary variables  of large-animal farmers may be influenced by the nature of this cross-sectional survey. The number of questions was also reduced in order to cut down on the time it took to complete the survey.The study was conducted in all four districts  in Mymensingh division of Bangladesh . A farmer was described as someone who daily spends time on feeding, watering, rearing, or caring for animals on the farm, having direct or indirect contact with a farm, and obviously with an age ≥18 years. Farmers were selected on a random basis from a list obtained from the corresponding Upazilla Livestock Offices. From each upazilla, a total of 12 farmers were surveyed, covering 6 dairy farmers, 3 goat farmers, 2 beef fattening farmers, and 1 sheep farmer. However, buffalo farmers (n = 20) from 6 buffalo-concentrated upazillas  of four districts were selected. The Raosoft sample volume calculation method  was usedA structured questionnaire was developed based on previous studies ,43,44,65Both English and Bangla versions of the questionnaire were developed to collect information from the farmers . ParaphrData from the interviews were entered and cross-checked in a paper-based questionnaire. The information was then transferred to an MS Excel spreadsheet  for cleaning, processing, and analysis. We gathered information on various knowledge, attitudes, and practices related to AMU and AMR using a variety of closed-ended and open-ended questions. The correct (adequate/desirable/appropriate) response (‘yes’) was assigned a value of 1, while the incorrect (inadequate/undesirable/inappropriate) response (‘no’) was assigned a value of 0. The aggregate of each participant’s responses for that particular segment was tallied to see how they did overall in the knowledge, attitude, and practice areas. The data were examined using IBM SPSS Statistics . The data were coded, and the internal consistency of the themes was assessed using Cronbach’s alpha test, with acceptable values of 0.814 for knowledge, 0.76 for attitude, and 0.71 for practice . The internal consistency of the data was good by the scale of Cronbach’s coefficient alpha value .p-value of less than 0.20 was considered. Cramer’s phi-prime statistic was used to determine if explanatory variables were collinear. If Cramer’s phi-prime statistic was greater than 0.70, a pair of variables was called collinear [The demographic and socioeconomic aspects affecting large-animal farmers’ management, knowledge, attitudes, and practices regarding AMU and AMR were studied individually. Age, sex, education, training, and a farmer’s farm size and farm type were all transformed into categorical variables before being analyzed. The association between a large-animal farmer’s knowledge, attitude, and practices with his/her demographic features was assessed using the Pearson chi-square test. The answers from the respondents regarding knowledge, attitude, and practices were categorized as “adequate” versus “inadequate”, “desirable” versus “undesirable”, and “satisfactory” versus “unsatisfactory”, respectively. For multiple logistic regression analysis, any explanatory variable linked with management, knowledge, attitude, and practices with a ollinear .To explore the effects of demographic characteristics linked with AMU, AMR, and antimicrobial residues on the farmers’ knowledge, attitudes, behaviors, and farm management, we used the enter method for multiple logistic regression models. Variables such as sex, age, education, training, and farm size were categorized. The levels were for sex “male and female”; “illiterate, PSC, JSC, SSC, HSC, graduate and masters” for education; receiving “training or not” for training; and “the number of animals in a farm” for farm size. Multicollinearity among these potential explanatory variables was assessed by pair-wise Pearson correlation tests in IBM SPSS Statistics . A pair of explanatory variables was considered collinear if Cramer’s phi-prime statistic was >0.70 . The finThe anticipated survey data uncovered the perspective of large-animal farmers in Mymensingh division of Bangladesh on knowledge, attitudes, and practices regarding AMU, AMR, and antibiotic residues. The findings of the study suggest that a significant number of large-animal farm owners/workers have inadequate knowledge, undesirable attitudes, and inappropriate antibiotic use practices towards AMU, AMR, and antibiotic residues. The present study data suggested that socio-demographic factors such as sex, age, training, farm population size, and particularly level of education have a significant impact on KAP towards AMU, AMR, and antibiotic residues. The findings of this study provided baseline evidence concerning the KAP of large-animal farmers, which would definitely help respective authorities to focus future interventions on Bangladesh smallholder livestock farming systems to reduce antimicrobial use and resistance. Regulation and control of the usage of veterinary medication and enacting strong antibiotic prescription legislation in Bangladesh to minimize widespread antimicrobial use are strongly recommended. Finally, it is recommended that AMU policies be developed and enforced in a way that is both practical and inclusive.


Z. mays subsp. parviglumis, and 5 partial genomes of Z. mays subsp. mexicana. Using these genomes, plus four other maize genomes and two outgroups from the NCBI database, we reconstructed the phylogenetic relationship of Z. mays. Our results suggest a polyphyletic origin of purple maize in South America and agree with a complex scenario of domestication with recurrent gene flow from wild relatives. Additionally, we identify 18 plastid positions that can be used as high-confidence genetic markers for further studies. Altogether, these plastid genomes constitute a valuable resource to study the evolution and domestication of Z. mays in South America.Peru is an important center of diversity for maize; its different cultivars have been adapted to distinct altitudes and water availability and possess an array of kernel colors , which are highly appreciated by local populations. Specifically, Peruvian purple maize is a collection of native landraces selected and maintained by indigenous cultures due to its intense purple color in the seed, bract, and cob. This color is produced by anthocyanin pigments, which have gained interest due to their potential use in the food, agriculture, and pharmaceutical industry. It is generally accepted that the Peruvian purple maize originated from a single ancestral landrace ‘Kculli’, but it is not well understood. To study the origin of the Peruvian purple maize, we assembled the plastid genomes of the new cultivar ‘INIA 601’ with a high concentration of anthocyanins, comparing them with 27 cultivars/landraces of South America, 9  Zea mays L. subsp. mays) has been particularly important in the Peruvian diet [Peruvian agrobiodiversity is well recognized as a source of globally important crops for food and agriculture, such as potato, quinoa, maize, and cotton, among others; and the traditional practices applied by farmers are known to favor the conservation of genetic resources . Among tian diet . To dateian diet , and 131ian diet . In Peruian diet , and morian diet . Peruviaian diet ,8.Despite their phenotypic diversity, studies based on morphological typing and archaeological evidence suggest that all purple maize landraces in Peru originated from the ancient race called ‘Kculli’ , or ‘KulZea mays subsp. mays cv. ‘INIA 601’). This cultivar was bred from two purple maize landraces, ‘Morado de Caraz’ and ‘Negro de Parubamba’, by the recurrent selection of half siblings over six generations. The selection criteria were based on the anthocyanin concentration, total dry mass, and total yield. A recent comparison between improved cultivars and landraces showed that cv. ‘INIA 601’ produces higher yields and higher concentration of anthocyanins than other improved purple maize landraces and cultivars [Purple maize cultivars have been developed by breeding programs since 1990 . Notablyultivars ,11.Zea species, the subspecie Zea mays subsp. huehuetenangensis (Iltis & Doebley) Doebley, 1990 [Tripsacum dactyloides (L.) L [Sorgum bicolor (L.) Moench [Z. mays are still missing, namely Zea mays subsp. parviglumis Iltis & Doebley, 1980 and Zea mays subsp. mexicana (Schrad.) Iltis, 1971, both of which have contributed enormously to the extant diverse maize germplasm through hybridization and recombination [Z. mays subsp. parviglumis, and 5 Z. mays subsp. mexicana. The parental individuals of cv. ‘INIA 601’ date back to 1990 [Plastid genes and genomes have been widely used in several species to resolve phylogenetic relationships among different taxa ,16,17,18ey, 1990 , and sevey, 1990 ,21, ‘B37ey, 1990 , and ‘Zhey, 1990 . Additios (L.) L  and Sorgbination . Further to 1990  and wereTo assemble the plastid genome of the cv. ‘INIA 601’, we used PacBio long reads. The initial assembly produced the expected plastid genome size (140 kbp) separated into two contigs. One contig  contained the long single copy region (LSC), and the other contig  was formed by the small single copy region flanked by both inverted repeats . Close inspection of the assembly graph showed that both contigs were constructed out of three connected edges in the graph, typical of a circular molecule with an unresolved repeat. While both single copy (SSC and LSC) edges had an approximate depth of ~20×, the inverted repeat (IR) edge had nearly twice the depth (38×), which is consistent with a collapsed duplicated sequence. To reconstruct the complete circular plastid genome, raw reads were aligned with the assembly graph. Of these, 20 reads spanned the IR edge, 9 of which supported the canonical structural configuration (<IR2<LSC>IR1>SSC), and 11 reads supported the non-canonical structural configuration (<IR2>LSC>IR1>SSC). Upon separation of isoform specific reads, two sets of reads were assembled and resulted in correctly circularized assemblies of a similar size, but with different orientations of their LSC and SSC. The non-canonical isoform was ~10 bp shorter than its canonical counterpart, but both isoforms contained the same number of genes  .Z. mays subsp. mays) and the teosintes , we used a total of 46 plastid genomes, from which we assembled the complete genomes of 27 Z. mays subsp. mays and 9 Z. mays subsp. parviglumis, and partial genomes of 5 Z. mays subsp. mexicana; additionally, we recovered four other genomes of Z. mays subsp. mays from the NCBI database . The plastid genome sizes vary between 140,369 bp (Z. mays subsp. parviglumis—PC_I53_ID1_1) and 140,539 (Z. mays subsp. mexicana—RIMME0026)  . InteresRNAs see . When we . IntereZ. mays individuals, we aligned the full genomes’ sequences using MAFFT v7.453 [In order to analyze the sequence variation among the 46 T v7.453 . The resT v7.453  and the T v7.453 , we obseZea nicaraguensis Iltis & B.F.Benz, 2000 and Zea perennis (Hitchc.) Reeves & Mangelsd., 1942). In this phylogeny  and the fertile inbred lines A188, and other cultivated individuals from Peru and Brazil. Moreover, we can observe that the purple maize individuals do not cluster together, the landrace ‘Kculli’ belongs to the first group, while cv. ‘INIA 601’ belongs to the second group. The cv. ‘INIA 601’ is the sister lineage of the landrace ‘Iqueño’ in the group that includes the other teosintes. These results indicate a polyphyletic origin of the purple maize. The second group of Z. mays  were separated from the reads that linked them in opposite directions (< > or > <). The removal of either set from the assembly dataset resulted in a single circular contig with different LSC and SSC orientations. These results suggest that the fragmentation of plastid genome assemblies using long reads is caused by the inability of the assembler to separate conflicting reads that support different co-existing structural configurations of the genome. However, using assembly graphs and graph mapping, we accurately separated reads supporting different structural variations and reconstructed the two isoforms separately. Structural heteroplasmy has been previously reported in both land plants  and algaZ. mays subsp. mexicana ‘TIL11’, a teosinte inbred line [In maize, an early study of cytoplasmic male sterility used restriction patterns of the cosmid library to demonstrate that recombination between the inverted repeats caused the inversion of the SSC region and, consequently, the co-existence of two structural isoforms . Anotherred line . In our red line ,35. Furtred line .Z. mays and two genetic groups for South American landraces [Z. mays clusters [Z. mays individuals into two groups: I) a group formed by some Z. mays subsp. mays, and II) a second group that clusters the four subspecies . This division agrees with the hypothesis that two waves of semi-domesticated maize dispersed southward out of the center of domestication [Z. mays subsp. parviglumis) was reported [Z. mays subsp. mexicana and maize have been observed [Previous studies using nuclear and plastid genetic markers support a monophyletic origin of andraces ,36,37. Handraces ,38. For clusters . Neverthtication ,39. Morereported . AdditioA single origin of the Peruvian purple maize has been proposed . In partCertified seeds from the cv. ‘INIA 601’ were provided by the Maize Breeding Program from the ‘Instituto Nacional de Innovación Agraria’ (INIA-Peru). Seeds were germinated in vitro, and plantlets were transferred to pots with substrate and grown under greenhouse conditions .v/v) were performed. Approximately 400 µL of supernatant were recovered, and DNA was precipitated with cold isopropanol. Additional washes with 70% ethanol were done after DNA pellet recovery. DNA purity was evaluated using the spectrophotometer Nanodrop 8000 , and the concentration of dsDNA was determined using a QubitTM Fluorometer dsDNA BR Assay Kit (Thermo Fisher Scientific). DNA integrity was examined by agarose gel electrophoresis. Afterwards, DNA integrity was also estimated using a Femto Pulsed field gel electrophoresis (Agilent) at the University of Washington, and the adequate fraction was selected for downstream sequencing on the Pacific Biosciences Sequel II platform using the Blue pippin DNA size selection system. One lane in 1 SMRT cell was run in CLR mode, and 15 h movies were produced.Leaves from a single 25-days-old plant were collected, and DNA was extracted following a previously described method  with minLong reads were mapped to the reference maize plastid genome  using minimap2 v2.24 . The assPaired-end Illumina reads were downloaded from NCBI SRA  and 4 genomes obtained from the NCBI database (3 Z. mays subsp. mays and 1 Z. mays subsp. huehuetenangensis)  See . AdditioT v7.453 , and theT v7.453  with theT v7.453 , and theT v7.453 .Z. mays subsp. parviglumis, Z. mays subsp. mexicana, and other South and North American maize landraces. We used these genomes to assess the origin and domestication of purple maize. Our results suggest a polyphyletic origin of purple maize in South America and agree with a complex scenario of domestication with recurrent gene flow from wild relatives. We also identify 18 plastid positions that can be used as high-confidence genetic markers.In this paper, we report the first complete plastid genome of a purple maize and use publicly available data to reconstruct additional plastid genomes for


The control of infectious diseases has always been a top medical priority. For years during the so-called antibiotic era, we enjoyed prolonged life expectancy and the benefits of superior pathogen control. The devastating failure of the medical system, agriculture and pharmaceutical companies and the general population to appreciate and safeguard these benefits is now leading us into a grim post-antibiotic era. Antimicrobial resistance (AMR) refers to microorganisms becoming resistant to antibiotics that were designed and expected to kill them. Prior to the COVID-19 pandemic, AMR was recognised by the World Health Organization as the central priority area with growing public awareness of the threat AMR now presents. The Review on Antimicrobial Resistance, a project commissioned by the UK government, predicted that the death toll of AMR could be one person every 3 seconds, amounting to 10 million deaths per year by 2050. This review aims to raise awareness of the evergrowing extensiveness of antimicrobial resistance and identify major sources of this adversity, focusing on agriculture’s role in this problem and its solutions.Widespread development of antibiotic resistance is a major global health risk.• Antibiotic resistance is abundant in agricultural produce, soil, food, water, air and probiotics.• New approaches are being developed to control and reduce antimicrobial resistance.•  The UK review of antimicrobial resistance (AMR) presented now outdated 2014 data on AMR consequences for human life. They estimated that 700,000 people die every year from AMR. This prediction was recently updated with the data from 2019 presenting that the deaths of 4.95 million people were related to drug-resistant bacterial infections and 1.27 million deaths were directly attributable to AMR  because the ban on antibiotic use for growth-promoting purposes resulted in an increase in disease outbreaks since the antibiotics also kept pathogens in check while improving growth performance measures. This dire need induced the industry to invest in research on alternatives to antibiotics, and the market is now flooding with antimicrobial products that have no overlap with antibiotics. Among the most successful and widely used are phytogens, plant antimicrobial products whose antibacterial properties can successfully provide growth-promoting and pathogen controlling effects  are abundant in raw and treated sewage  were not designed to remove or reduce AMR contamination  in chicken. VRE was then found in processed meat and farmers and abattoir staff slaughtering the VRE + animals, some of whom had to be hospitalised with the VRE infection  (reviewed in Apreja et al. Cyanobacteria are among the most interesting: they use light as an energy source and CO2 as a carbon source, also helping in carbon sequestration during the bioremediation process. Additionally, they are hardy, capable of growing under extreme conditions and capable of fixing up atmospheric nitrogen. Successful examples of algal bioremediation of antibiotics are well presented and reviewed in the literature (Grimes et al. Phytoremediation is the removal of antibiotics using algae. Mycoremediation and phytoremediation are up-and-coming remediation techniques using fungi and plants to remove antibiotics from the environment. We previously discussed how plants accumulate antibiotics from the soil; although this is an issue for vegetable and other edible plant production, it is excellent in bioremediation.One of the superior technologies capable of removing antibiotics, AMR and ARGs, is aerobic and anaerobic digestion using carefully selected microbial inoculums and optimised digestion conditions (reviewed in Waseem et al. There is no doubt that AMR is one of the major global health issues. AMR is a natural phenomenon that existed for as long as microorganisms. The preservation of intestinal content in naturally and deliberately mummified human remains confirmed the presence of resistance genes to most known antibiotics, including those most recently discovered (reviewed in Santiago-Rodriguez et al.


Nanoformulations of KS-58 with CrEL dramatically improved its water solubility and increased its stability. Weekly intravenous administration of KS-58 nanoparticles (NPs) suppressed the growth of CT26 and PANC-1 cell-derived tumors in vivo, and fluorescent bioimaging indicated that the NP-encapsulated near-infrared fluorescent probe indocyanine green selectively accumulated in the tumor and was safely excreted through the kidneys following intravenous injection. Histopathological analysis of CT26 tumors and Western blotting of PANC-1 tumors revealed that KS-58 NPs reduced ERK phosphorylation, a downstream signal of K-Ras(G12D). Our results suggest that KS-58 NPs represent a novel therapeutic agent for treating colorectal and pancreatic cancers.Single amino acid mutations of Ras occur in 30% of human cancers. In particular, K-Ras(G12D) has been detected in the majority of intractable colorectal and pancreatic cancers. Although efforts to target K-Ras(G12D) are currently underway, no effective drugs are available. We previously found that the K-Ras(G12D)-inhibitory bicyclic peptide KS-58 exhibits antitumor activity against syngeneic colon and orthotopic grafted pancreatic tumors; however, pristine KS-58 is difficult to handle because of low water solubility and it requires frequent administration to obtain sufficient antitumor activity. In this study, we used a nanoformulation of KS-58 prepared with the highly biocompatible surfactant Cremophor In 2021, a covalent small molecule drug was identified that targeted K-Ras(G12C), a mutated form of Ras primarily found in lung cancer3. A number of preclinical and human clinical studies are currently underway to evaluate the anti-cancer activity of K-Ras(G12C) inhibitor in combination with other chemotherapeutic drugs6; however, the development of drugs against K-Ras(G12D) in colorectal and pancreatic cancers, which are known as intractable cancers, have not been as successful7. A major reason is that covalent small molecule drug design is difficult to apply to K-Ras(G12D)8. By structurally modifying the K-Ras(G12C) inhibitor, a non-covalent K-Ras(G12D) inhibitor MRTX1133 was developed in late 2021, but twice-daily intraperitoneal administration was required to yield anti-cancer activity in vivo9.Since the discovery of single amino acid mutations of Ras in human cancers, 30 years of research has been focused on developing strategies to ameliorate the effects of mutated Ras12. We also identified a K-Ras(G12D)-inhibitory bicyclic peptide “KS-58”, which is a derivative of KRpep-2d, and reported its in vitro and in vivo growth inhibition activities against CT26  and PANC-1 (human pancreatic cancer cell line) cells both expressing K-Ras(G12D)14. However, there are several challenges associated with the development of KS-58. One is its poor water solubility and the other is the need for frequent dosing. With respect to the latter, the in vivo antitumor activity of KS-58 requires intravenous administration once every 2 days at a dose of 40 mg/kg because of its poor pharmacokinetics. The elimination half-life (t1/2) of KS-58 in the blood is approximately 30 min14. Therefore, it is necessary to reduce the frequency of administration from once every 2 days to once a week to reduce the burden on patients.We attempted to synthesize a K-Ras(G12D) inhibitor using cyclic peptides. In 2017, the first K-Ras(G12D)-selective inhibitory peptide “KRpep-2d” was reported16 or polyethylene glycol18 have been shown to improve the pharmacokinetics of various peptides. Such conjugations can reduce excretion from the kidneys and prolong t1/2 in the blood; however, these strategies have been applied mostly to peptides with extracellular targets. Moreover, peptide drug conjugations exhibit reduced intracellular penetration with increasing molecular weight. Therefore, we evaluated nanoformulations to further improve the solubility, t1/2 in the blood, and the intracellular penetration of the peptide drugs19. Nanoparticles (NPs) in the range of 10–100 nm in diameter have been considered effective drug carriers because they are able to accumulate in solid cancer tissues owing to the enhanced permeability and retention (EPR) effect20.Several strategies using chemical conjugations with albumin-binding tags® EL (CrEL), a biocompatible surfactant approved as a pharmaceutical additive21. KS-58 was stably encapsulated into CrEL NPs. The resulting KS-58 NPs exhibited significant antitumor activity against CT26 colorectal cancer cell-derived syngeneic tumors and PANC-1 pancreatic cancer cell-derived orthotopic tumors following weekly intravenous (i.v.) administration  purification, a structural assignment was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Cremophor® EL (CrEL) (09727-14) was purchased from Nacalai Tesque, Inc. . KS-58 (200 mg) was dissolved in DMSO (2 mL) at a concentration of 100 mg/mL. Then, 10 × D-PBS  (3.5 mL) was mixed with purified water (3.5 mL) for the preparation of a 50% 10 × D-PBS solution in a centrifuge tube (15 mL size). CrEL (1 mL) was added to the 50% 10 × D-PBS solution and mixed well by a vortex mixer. The peptide solution was added dropwise to the CrEL/10 × D-PBS solution, which was being agitated by a vortex mixer at 1,000 rpm. The peptide/CrEL/10 × D-PBS solution was converted to a suspension by repeated vortex agitation and sonication at 50 °C. After repeated chilling on ice, sonication under cold water, and agitation by vortexing, the solution became a transparent KS-58 /DMSO (20%)/CrEL (10%)/10 × D-PBS (35%) solution and was stored at 4 °C until use. DMSO without KS-58 was used for the preparation of control NPs.A K-Ras(G12D)-inhibitory peptide KS-58 (1333.6 g/mol) was synthesized by SCRUM, Inc.  as previously reportedNegative staining was used to observe the morphology and structure of NPs using high-resolution TEM  at an acceleration voltage of 100 kV. The sample on a TEM grid was tightly washed to remove impurities before the observation. The particle size of the NPs was measured in a disposable cuvette by dynamic light scattering (DLS) with a Zetasizer Nano ZSP .The CT26.WT (CRL-2638) mouse colorectal cancer and PANC-1 (ATCC CRL-1469) human pancreatic cancer cell lines were purchased from the American Type Cell Collection . The cells were cultured in medium supplemented with 10% fetal bovine serum according to the manufacturer’s recommended protocol.The effect of KS-58 on the proliferation of CT26 and PANC-1 cells was determined following a 72-h exposure to KS-58 with or without nanoformulation. Cells were seeded at 1000 cells/well in 96-well tissue culture plates and allowed to adhere overnight. KS-58 was dissolved in DMSO and diluted with each growth medium with a final DMSO concentration of 0.5%. KS-58 NPs were diluted with each growth media to a final DMSO concentration of 0.04% and a final CrEL concentration of 0.02%. The medium in the wells was removed and replaced with diluted sample solution and the cells were incubated for 3 days. During the incubation, medium containing the samples was replaced each day. The relative cell numbers were estimated using the CellTiter-Glo assay kit  according to the manufacturer’s instructions. Luminescence was detected using a SpectraMax i3x . The cell proliferation rate (%) was calculated based on cell numbers at day 0 as 0% and at day 3 and treatment with DMSO  or control NPs  was designated as 100%.CT26 cells were seeded in imaging dishes  and allowed to adhere overnight. The attached cells were treated with PBS containing indocyanine green (ICG) and CrEL  for 24 h at 37 °C. After washing with PBS, the cells were observed using a fluorescence microscopy system (IX73) and cellSens V3.1 software  equipped with a mirror unit  and an objective  at 20 °C.3; BALB/cCrSIc; Japan SLC) were injected i.v. with 250 μL PBS containing ICG and CrEL . The mice were euthanized and the major organs, including the heart, liver, spleen, and kidneys, in addition to the tumors, were imaged using an in vivo fluorescence imaging system  with a 3-s exposure time and an ICG filter  at 4, 6, 8, and 12 h post injection. The fluorescence images were acquired and analyzed using CleVue software.To monitor the chronological changes in fluorescence intensity resulting from the tumor-targeting effects of NPs, CT26 tumor-bearing mice  (No. 04-002). Female BALB/cCrSlc mice  were obtained from Japan SLC . CT26 cell-derived mouse tumors were generated by injecting 100 μL Matrigel culture medium  containing 1 × 106 cells into the dorsal right side of the mice. After approximately 1 week, when the tumor volume had reached ∼ 50 mm3, the mice (n = 4 in each group) were intravenously injected with D-PBS (vehicle group), D-PBS containing KS-58 NPs (KS-58 concentration = 20 or 40 mg/kg), or D-PBS containing control NPs once a week. Tumor formation and overall health (viability and body weight) were monitored on the indicated days. Tumor volume was calculated by the formula V = L × W2/2, where L and W denote the length and width of the tumor, respectively. When the tumor volume reached > 3000 mm3, the mice were euthanized according to the JAIST Institutional Animal Care and Use Committee guidelines. After the mice were euthanized, the liver, kidney, spleen, and CT26 tumors were harvested for immunohistochemical (IHC) staining.Experimental procedures that involved animals and their care were conducted in compliance with the Guide for the Care and Use of Laboratory Animals6 cells/50 µL of PANC-1 cells were injected into the pancreas of BALB/cAJcl-nu/nu male mice . The mice (n = 8 in each group) were intravenously injected with D-PBS containing KS-58 NPs (KS-58 concentration = 20 or 40 mg/kg), or D-PBS containing control NPs (sham and vehicle groups) once a week for 6 weeks. After the mice were euthanized, the pancreas, liver, and kidneys were collected and their weights were measured.The animal experiments using mouse xenograft models were carried out at UNITECH Co. Ltd. in accordance with the guidelines of the Animal Care and Use Committee of UNITECH . To establish orthotropic xenograft tumor models, 2.5 × 10®   were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (Tunel) and phospho-p44/42 MAPK antibody, respectively.IHC analysis of liver, kidney, spleen, and CT26 tumors collected from allograft model mice was performed by the Biopathology Institute Co., Ltd.  using standard protocols. Briefly, primary tumors were surgically removed, fixed in 10% formalin, processed for paraffin embedding, and cut into 3–4-μm sections. After incubation with primary antibody, the sections were stained with hematoxylin and eosin and examined by light microscopy (IX73). Anti-digoxigenin-peroxidase   and Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP14. Experiments were conducted at UNITECH. Briefly, frozen PNAC-1 tumors excised were homogenized in cold RIPA buffer  supplemented with cOmplete mini EDTA-free Protease Inhibitor Cocktail  and PhosSTOP using a BioMasher II disposable homogenizer . The supernatant containing target proteins was collected and total protein concentration was measured. Supernatant proteins (30 μg per gel lane) were separated by SDS-PAGE using Laemmli mini gels  and electrophoretically transferred onto polyvinylidene difluoride membranes. Membranes were blocked in TBS containing 0.1% Tween 20 (TBST) plus 3% BSA and then incubated with Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® in TBST plus 1% BSA. Blotted membranes were then incubated with horseradish peroxidase (HRP)-conjugated mouse anti-rabbit IgG  in TBST plus 1% BSA. The reactions were detected with ImmunoStar Zeta (FUJIFILM Wako) and Hyperfilm ECL . After detection, membrane was incubated with stripping buffer  and then washed five times with TBST for measurement of GAPDH as described above using anti-GAPDH antibody (1000-fold dilution) . Protein bands were quantified using ImageJ . Full length blots are shown in Supplementary Fig. Western blotting was conducted according to previously reported methodsp < 0.05 were considered statistically significant.Multiple group comparisons were made using a one-way or two-way analysis of variance followed by Dunnett's test. All statistical analyses were performed using GraphPad Prism 6 software . Values of As KS-58 is a hydrophobic bicyclic peptide Fig. , we hypoTEM analysis of KS-58/CrEL revealed the formation of unique spherical nanoparticles with a diameter of 10 nm Fig. B. ParticCell growth suppression activity of KS-58 with or without nanoformulation was evaluated in CT26 and PANC-1 cells Fig. A. This m26. When the particle size is large, they cannot sufficiently penetrate into tumor tissue and they accumulate in the liver, potentially causing hepatotoxicity. However, when the particle size is too small, they are eliminated from the body more rapidly by renal clearance and cannot sufficiently infiltrate the tumor tissue. Near-infrared fluorescent dye ICG was used to identify the tumor location in CT26 tumor-bearing mice following intravenous injection. Strong ICG-mediated fluorescence was observed using an in vivo fluorescent bioimaging system  suppressed CT26 tumors growth to 35% relative to the vehicle group. In the present study, KS-58 NPs  suppressed CT26 tumor growth to 26% relative to the vehicle group.The anti-cancer effect of KS-58 NPs on subcutaneously implanted CT26 cell-derived tumors was evaluated Fig. . The tumWe found that KS-58 NPs inhibited K-Ras(G12D) activity by reducing ERK phosphorylation (pERK), which is a Ras-mediated downstream signaling event, as determined by immunohistochemical (IHC) staining of CT26 tumor tissue sections Fig. B. KS-58 29. One of the reasons is that angiogenesis occurs in subcutaneously implanted tumors, which facilitates the EPR effect; however, tumors formed in the pancreas are covered with stroma and have few blood vessels making it difficult to demonstrate an EPR effect29. KS-58 NPs showed a clear pharmacological effect not only in subcutaneous tumor-bearing mice  are early eliminated by renal clearance30. Poon et al. reported that nanoparticle formulations with a particle size of 5.5 nm or larger delay elimination from the kidneys31. The mean tumor volumes of the KS-58 NPs (20 mg/kg) and KS-58 NPs (40 mg/kg) groups were 68 ± 15% and 50 ± 14%, respectively, compared with that of the Sham and control NPs groups, which were set at 0% and 100%, respectively  suppressed PANC-1 tumors growth to 65% relative to the vehicle group. In the present study, KS-58 NPs  suppressed PANC-1 tumors growth to 50% relative to the control NPs group.Finally, the antitumor activity of KS-58 NPs in PANC-1 cell-derived tumors implanted into the pancreas of mice was evaluated .Supplementary Figures.


Phytate has been classified as an anti-nutrient, but there are no adverse effects from the consumption of a balanced diet with 1 to 2 g of daily phytate  as a calcium magnesium salt, the form naturally present in grains. Furthermore, recent research has shown that phytate consumption may prevent pathological calcifications, such as kidney stones and cardiovascular calcifications. However, many endogenous and exogenous enzymes can hydrolyze phytate to lower inositol phosphates (InsPs) that also have biological activity. We performed a controlled hydrolysis of phytate and identified the products (InsPs) using tandem mass spectrometry (MS/MS). The total level of all InsPs was measured using a non-specific methodology. In addition, we evaluated the effects of the InsP6 hydrolysates on calcium oxalate crystallization using scanning electron microscopy and measuring the time needed for the induction of crystallization. Our results indicate that InsP6 and its hydrolysis products functioned as effective inhibitors of calcium oxalate crystallization. Thus, even though InsP6 is hydrolyzed after consumption, the enzymatic products also have the potential to reduce pathological calcifications. Finally, although it is useful to measure the overall level of InsPs in biological fluids, such as urine, there is a need to develop simple analytical methods to quantify the level of individual InsPs. Mellanby performed the first studies of the possible health effects of phytate during the 1940s by administering sodium phytate to dogs [Researchers first identified inositol-hexaphosphate , it is thought that there are no specific transporters for phytate, as its absorption is produced by paracellular transport.Recent studies reported that the intake of InsPioxidant , protectioxidant , preventioxidant  and previoxidant ,11. Thusioxidant . InsP6 h6 can be difficult because it does not have any characteristic spectral properties and because of its capacity to form metal complexes, allowing it to bind to multiple metal surfaces and molecules. A further complication is that the body has enzymes that hydrolyze phytate (InsP6) to other inositol phosphates (InsP5 to InsP1) that may also have biological activity. This topic has not been thoroughly studied and has led to various controversies. However, the development of new analytical methodologies that incorporate tandem mass spectrometry (MS/MS) [The analytical determination of InsP (MS/MS) ,14 has m6 reduced the development of pathological calcifications, such as cardiovascular calcifications, soft tissue calcifications and kidney stones [6 leads to the formation of less phosphorylated InsPs, it is also necessary to assess the activity of these derivatives as crystallization inhibitors. Polyphosphates with two phosphate groups, such as pyrophosphate and bisphosphonates, are potent inhibitors of the crystallization of calcium salts  in blood and urine [Studies of experimental animals and humans have demonstrated that the consumption of InsPy stones . Since tnd urine ,16. Renand urine .6 hydrolysates on inhibiting the crystallization of calcium oxalate (CaOx).In the present study, we performed a controlled hydrolysis of phytate and evaluated the hydrolysis process using MS/MS and the total pool of inositol phosphates (InsPs) using a non-specific methodology. We also evaluated for the first time the effects of the different InsPA phytate stock solution (1.12 mM) was prepared from phytic acid sodium salt hydrate  and was adjusted to a pH of 2 using HCl (0.5 M). Duplicate aliquots (5 mL) were kept in a dry bath at T = 97 °C for 6, 9, 16, 24, 48 or 72 h for hydrolysis.Diluted samples (1:1000) were injected into a Q Exactive Orbitrap high-resolution mass spectrometer equipped with a heated electrospray ionization (HESI) probe , which was operated in a negative ionization mode. The temperature of the ion transfer capillary was set to 320 °C, the spray voltage was set to 2.9 kV in negative mode and the S-lens RF level was 50 AU. Direct injection in the full scan acquisition mode over a range of 150 to 700 m/z was performed with a resolution of 140,000.The effects of phytate and its hydrolysis products mixture on CaOx crystallization in synthetic urine were assessed using a kinetic turbidimetric system. This system consisted of a spectrometer equipped with a fiber-optic light-guide measuring cell . Crystallization was assessed at a constant temperature (37 °C) with magnetic stirring (300 rpm).In these experiments, the synthetic urine solution, obtained by mixing equal volumes of Solution A and Solution B , was pre6 and its hydrolysis products mixture were examined using scanning electron microscopy  coupled with RX energy dispersive microanalysis .The morphological and structural characteristics of the CaOx crystals that formed in synthetic urine in the absence and presence of InsPThe nonspecific quantification of InsPs, before and after the hydrolysis process, was performed by purification using AG 1-X8 resin and by the formation of InsPs complexes with Al(III), in which InsPs displaced the aluminum-xylenol orange dye, as previously described .The amount of inorganic phosphate that was liberated during phytate hydrolysis was determined using ammonium molybdate and ascorbic acid (a reducing agent), according to the phosphomolybdate–ascorbic acid method . The abs6) solution prior to hydrolysis  led to changes in the levels of the six different InsPs  crystals were predominant. This product is kinetically favored because the thermodynamically stable form is calcium oxalate monohydrate (COM) [6 and samples subjected to hydrolysis for 6, 9, 16 or 24 h, indicating significant inhibition of crystallization of all these samples. However, COT crystals formed in the presence of samples that were hydrolyzed for 48 h or 72 h.We used SEM to observe the different types of CaOx crystals that formed in synthetic urine in the presence of the different InsP hydrolysates . In the te (COM) . Notably6 was the greatest (6) and no detectable free phosphate ion. The 6 h hydrolyzed sample had an InsPs level of 0.71 mM and a free phosphate level of 0.45 mM. As the hydrolysis time increased further, the level of InsPs decreased, and the level of the phosphate ionss increased. The 48 and 72 h hydrolyzed samples had detectable InsPs by the non-specific method (0.16 and 0.10 mM respectively), despite the absence of InsP6 and a very low amount of InsP5 in the hydrolyzed mixtures  can give rise to a diversity of InsPs in the blood, tissues and urine, as documented in previous studies [6 is associated with different health benefits [4 and InsP5 inhibit the activation of the AKT protein [Although the alkaline phosphatases of human mucosal cells apparently do not significantly degrade phytate during intestinal transit, there are other phytases of plant origin that accompany some foods and microbial phytases that can degrade dietary phytate . Further6 . Therefo studies ,23. The benefits . In partbenefits  and on t protein . The les protein , similar6 was a potent inhibitor of the in vitro crystallization of hydroxyapatite [6 inhibited the in vitro calcification of rat cartilage and that the parenteral injection of partially hydrolyzed InsP6 prevented aortic calcification in rats treated with high doses of vitamin D [6 had the greatest capacity to inhibit CaOx crystallization, but that mixtures of InsP5, InsP4 and InsP3 also functioned as important crystallization inhibitors. Our results confirm that a mixture of InsPs inhibits CaOx crystallization, even when InsP6 is not abundant in these mixtures. Therefore, the inhibition of CaOx crystallization in vivo after the consumption of InsP6 is likely attributable to InsP6 and other hydrolyzed InsPs. Thus, it is very important to consider that the products resulting from InsP6 dephosphorylation are also potent inhibitors of calcium oxalate crystallization.A 1972 publication reported that partially hydrolyzed InsPyapatite . Anotheritamin D . However6 ingestion is their lack of analytical determination of the different InsPs in biological fluids and tissues. InsP6 lacks spectral characteristics that allow easy identification and does not have properties that enable the development of simple analytical methods. An added difficulty is that it is necessary to consider that numerous InsPs products result from InsP6 dephosphorylation , and these products have similar chemical and biological properties. The most common analytical methods used to quantify InsP6 are based on the non-specific evaluation of total inorganic phosphate or complex formation by phosphate groups [6 when other InsPs are present [6 decreased because of hydrolysis, the overall amount of InsPs also decreased, and the phosphate ion concentration increased, as can be seen in 6, and yet the total amount of InsPs was only reduced by about 50% (from 0.82 to 0.39 mM). Only after 72 h of hydrolysis, when the level of total InsPs was very low (with InsP2 being the predominant species), the non-specific method indicated a greatly reduced level of total InsPs (0.10 mM).A limitation of many in vivo studies that examined the beneficial effects of InsPe groups ,20, but  present ,23. In t present  to quant6 was eliminated from the diet, the nonspecific determination of InsPs in the urine indicated extremely low levels [6 was re-administered. Other research has shown that experimental animals that were given diets that did not contain InsP6 only excreted small amounts of InsP2 in their urine [It is important to note that, in animal experiments in which InsPw levels , and their urine . Therefo6 functioned as inhibitors of CaOx crystallization. Thus, even though InsP6 is enzymatically hydrolyzed after consumption, its hydrolysis products also inhibit crystallization. This may explain the positive effects of the consumption of phytate on pathological calcifications. Although the determination of total InsPs level can be useful, it is necessary to develop sensitive and specific analytical methods that can quantify each of the hydrolyzed InsPs.Our in vitro experiments indicated that the hydrolysis products of InsP


Kaposi’s sarcoma is an angioproliferative malignancy due to human herpesvirus-8 and is associated with immunosuppression. Although most cases are cutaneous and resolve with treatment of the underlying condition, few cases present with organ involvement and have a fulminant course. We present a case of a 24-year-old sexually active man who presented with fulminant visceral Kaposi’s sarcoma, without cutaneous involvement. He presented with anasarca, high fever, hypoalbuminemia, and anemia on day five of antiretroviral therapy (ART). There was clinical improvement after the first dose of liposomal doxorubicin. However, given that he developed refractory pancytopenia, with disease relapse by the third week, he received a second dose of doxorubicin, with no clinical improvement, and the patient died with multi-organ dysfunction on day 22 of presentation. The main treatment is liposomal doxorubicin with ART, and the disease is typically associated with a poor prognosis. Kaposi's sarcoma (KS) is an angioproliferative tumor associated with the presence of herpesvirus-8 (HHV-8) in more than 90% of the lesions. The mode(s) of transmission of HHV-8 remains unclear, but epidemiologic and virologic data suggest that saliva is a source of the infectious virus and may be an important route of transmission as well as sexual contact . KS was A 24-year-old, sexually active homosexual male, presented with recurrent episodes of odynophagia, fever, enlarged tonsils, and oral thrush. Initial symptoms had started approximately seven months previously when the patient was advised to have an HIV serology performed. He was lost to follow-up during that period until the current presentation. Two weeks before admission he was diagnosed with HIV infection with a viral load of 190,087 copies/ml; the CD4 count was 232 cells/mcl and so started on antiretroviral therapy (dolutegravir/lamivudine/tenofovir) and Bactrim for pneumocystis prophylaxis.During the current presentation, the patient reported high fever, odynophagia, sore throat, and weight loss of approximately 10 kg over the past six months. A physical examination revealed a fever of 38.5 degrees Celsius, blood pressure 120/70 mm Hg, heart rate 110/min, oxygen saturation (SpO2) 98% on room air, pale skin, anicteric sclera, poorly injected conjunctiva, oral thrush, and bilaterally enlarged tonsils, more prominent on the left side with some purple discoloration, as well as axillary and inguinal lymph nodes (LNs). The skin showed no abnormal lesions.The workup showed the following: low hemoglobin level 9.2 g/dL, mean corpuscular volume 90 fl, white blood cells 5 200/mL , platelets 179 000/mL, and normal liver function tests. Cervical, chest, and abdominopelvic chest computed tomography (CT) showed the following: mediastinal and axillary LNs, heterogeneous multi-lobulated lesion partially compressing the pharynx, hepatosplenomegaly, and retroperitoneal, iliac, inguinal, and mesenteric LNs with peritoneal fluid. A tonsillectomy was performed, as well as an excisional LN biopsy in the right inguinal and the left axillary area, and the patient was discharged home pending the results.Preliminary results excluded mycobacterial infection, by a negative polymerase chain reaction (PCR) for mycobacterium, and excluded other infections by negative culture and gram stain and were in favor of a lymphoproliferative disorder, pending immunostaining. The patient was started on antiretroviral therapy (ART) (dolutegravir/lamivudine/tenofovir). Five days later, he started to complain of high fever, lethargy, and diffuse lower limb edema. At re-evaluation on day five, the patient looked ill and pale, with diffuse upper and lower limbs edema and a purple lesion at the right conjunctiva associated with eyelid edema, as well as new purple lesions in the retro pharynx and gingiva, and hypertrophy of a purple mass at the site of the tonsillectomy. The ART was stopped for possible immune reconstitution inflammatory syndrome (IRIS). The final pathology report of tonsils showed reactive lymphoid hyperplasia. Left axillary and right inguinal LN biopsies showed proliferation of small vessels associated with spindle cells involving the nodal parenchyma and the capsule diagnostic of nodal KS in a background of HIV lymphadenopathy Figure .Clostridium difficile toxin A and B were negative. Fecal occult blood was positive, and stools showed a focal aggregate of inflammatory cells. A repeated chest, abdomen, and pelvis CT  immunoglobulin (Ig)G was positive, so the patient started on ganciclovir. The patient reported diarrhea in which T Figure  was perfMycobacterium tuberculosis (MTB) PCR was negative. Urine, sputum, and blood cultures were taken and the patient was put on wide-spectrum antibiotics such as meropenem 1g intravenous every eight hours and vancomycin 1g intravenous every 12 hours along with antifungal therapy, and micafungin 100mg intravenous daily. After five days of presentation, all cultures were negative so superimposed infection was eliminated. There was no clinical improvement with increased anasarca and a persistent high-grade fever; a diagnosis of aggressive visceral KS was made. We reinitiated ART and administered the first dose of liposomal doxorubicin. The patient improved after one week, the lesions disappeared, and there was no more edema. The patient then developed pancytopenia after 15 days of presentation, with hemoglobin 5.3 g/dL, white blood cells 2,800/mL , and platelets 32,000/mL. A bone marrow biopsy showed hypercellularity (80%) with few interstitial reactive CD20+ B lymphocytes and numerous reactive CD3+ T lymphocytes, suggestive of an acute inflammatory process.Serum taken for CMV showed a high viral load of 625 IU/ml, and the patient was started on therapeutic ganciclovir 5mg/kg intravenous every 12 hours. Vitamin B12 and folate were low at <150 pg/ml and 3.43 ng/ml, respectively; thus, cobalamin and folate were given. We administered a second dose of liposomal doxorubicin; however, the patient remained pancytopenic and continued to deteriorate with the appearance of pulmonary edema and pleural effusion, kidney failure, anemia, and coagulopathy. He died within three weeks of initiating highly active antiretroviral therapy (HAART). Until the death, no skin lesions were identified.KS is an angioproliferative tumor of lymphatic endothelium-derived cells infected with HHV-8, typically occurring in patients with immunodeficiency. It has variable presentations that range from indolent, isolated dermatologic manifestations to fulminant disease with visceral involvement . AlthougAlthough four KS variants have been described in the medical literature, all these forms likely represent different manifestations of the same pathological process, given that over 95% of KS lesions, regardless of their source or clinical subtype, have been found to be infected with HHV-8 [Classic KS has a male predominance, with a male to female ratio of 17:1. It occurs primarily in the elderly of Eastern European and Mediterranean descent, who are at greater risk for secondary malignancies , and it Visceral KS can appear in any organ, but it mainly involves the LNs, lungs, and gastrointestinal tract. Although 33-77% of patients with cutaneous KS have been found to have visceral lesions on autopsy, such lesions in the absence of skin manifestations are rare. The most frequent site involved in the gastrointestinal tract is the small intestine, followed by the colon and stomach . GastroiThe first description of PLE due to gastrointestinal KS in the medical literature was in 1987. The importance of investigating hypoalbuminemia in patients with AIDS was discussed in 1989 by Bouhnik et al , becauseGiardia, Cryptosporidium, and CMV infection [In patients with HIV, diarrhea can have several etiologies. Gastrointestinal KS is highly associated with protein-losing enteropathy where fecal α1-antitrypsin >0.3 mg/g, wet stool have a sensitivity and specificity of 94% and 76%, respectively . Therefonfection .Oropharyngeal KS, particularly isolated KS of the tonsils, is rare with only a few cases reported in the literature. One of the earliest cases reports of KS located in the upper pole of the left tonsil was by Raikundalia in 1973, in a 38-year-old woman . The oroFor a definitive diagnosis of KS, a biopsy or excision of the suspicious areas must be performed to look for the characteristic spindle cell proliferation in the dermis; these cells are currently believed to originate from lymphatic endothelium . ImmunohThe aim of treatment is usually palliative and primarily targets symptom improvement and prevention of progression. Regarding treatment options, therapy is started according to the severity of the disease on the initial presentation. If the disease is localized or minimally disseminated cutaneous KS, it usually responds to ART plus either cryotherapy, radiotherapy, or surgical resection of the lesion. ART causes resolution of KS lesions by suppressing HIV viral replication and increasing CD4 cell counts, without acting directly on HHV-8. If the disease is disseminated with visceral involvement, a combination of systemic chemotherapy with ART therapy is typically required . LiposomIn most prospective studies, patients with life-threatening or extensive KS received chemotherapy alone or adjuvant when the KS failed to respond to ART alone. The KS lesions of such patients regressed less often, and more slowly, than the lesions of patients with limited disease who received only ART. Chemotherapy could often be withdrawn when KS lesions regressed with the combination of ART and systemic treatment.Mycobacterium avium-intracellulare infection, and Pneumocystis jirovecii pneumonia.Although antiretroviral therapy is important for AIDS-associated KS improvement and resolution, in disseminated cases, a small proportion of patients exhibit deterioration in their condition following ART initiation. This condition is known as IRIS, and it is of varying severity and even fatality . In the A prospective study in Mozambique showed the presence of four independent factor predictors of KS-IRIS: hematocrit below 30%, KS pretreatment, high plasma viral HIV load ( ≥50 copies/ml), and detection of HHV-8 DNA in plasma . AnotherIRIS can be defined as a group of inflammatory disorders with progressive deterioration in clinical status after the recovery of the immune system, leading to worsening infections in patients infected with HIV after the initiation of ART . KS-IRISIn general, IRIS treatment is a challenge that depends on the clinical severity and disease agent because this entity is not well recognized and its appropriate management is still unclear. In moderate to severe IRIS cases, discontinuation of ART with corticosteroid use leads to decreased inflammation in varying degrees. However, the use of corticosteroids to treat KS-IRIS is not routinely indicated because it can decrease cellular immunity, permitting HHV-8 replication and tumor growth . Thus, wOne study showed that the use of chemotherapy in combination with continuing ART can lead to the resolution of KS-IRIS. This is highlighted in the brief report by Connick et al. in which the patient presented with facial KS and significant neck edema and lymphadenopathy while on HAART . During Visceral KS remains a severe disease. Primary therapy is liposomal doxorubicin with ART, and it is usually associated with a poor prognosis. It is now appreciated that HHV8 can cause several diseases, many of which had not been previously recognized. This case emphasizes the need to recognize and strongly consider visceral KS as a possible cause in any people living with HIV/AIDS with low CD4 counts. CT scan of the abdomen and pelvis can play an important role in the recognition of visceral lesions and lymph nodes and the diagnosis of KS. This case reflects the complexity of HIV and reinforces the need for greater awareness in screening regardless of whether a clear risk behavior has been identified.


Aim: As the most common cardiomyopathy, dilated cardiomyopathy (DCM) often leads to progressive heart failure and sudden cardiac death. This study was designed to investigate the molecular subgroups of DCM.Methods: Three datasets of DCM were downloaded from GEO database . After log2-transformation and background correction with “limma” package in R software, the three datasets were merged into a metadata cohort. The consensus clustering was conducted by the “Consensus Cluster Plus” package to uncover the molecular subgroups of DCM. Moreover, clinical characteristics of different molecular subgroups were compared in detail. We also adopted Weighted gene co-expression network analysis (WGCNA) analysis based on subgroup‐specific signatures of gene expression profiles to further explore the specific gene modules of each molecular subgroup and its biological function. Two machine learning methods of LASSO regression algorithm and SVM-RFE algorithm was used to screen out the genetic biomarkers, of which the discriminative ability of molecular subgroups was evaluated by receiver operating characteristic (ROC) curve.Results: Based on the gene expression profiles, heart tissue samples from patients with DCM were clustered into three molecular subgroups. No statistical difference was found in age, body mass index (BMI) and left ventricular internal diameter at end-diastole (LVIDD) among three molecular subgroups. However, the results of left ventricular ejection fraction (LVEF) statistics showed that patients from subgroup 2 had a worse condition than the other group. We found that some of the gene modules  in WGCNA analysis were significantly related to cardiac function, and each molecular subgroup had its specific gene modules functions in modulating occurrence and progression of DCM. LASSO regression algorithm and SVM-RFE algorithm was used to further screen out genetic biomarkers of molecular subgroup 2, including TCEAL4, ISG15, RWDD1, ALG5, MRPL20, JTB and LITAF. The results of ROC curves showed that all of the genetic biomarkers had favorable discriminative effectiveness.Conclusion: Patients from different molecular subgroups have their unique gene expression patterns and different clinical characteristics. More personalized treatment under the guidance of gene expression patterns should be realized. Dilated cardiomyopathy (DCM) is the most common type of cardiomyopathy and a leading cause of death in the cardiovascular field, which is characterized by enlargement of the ventricle and reduced cardiac function . DCM canCTGF, IGFBP3, SMAD7, INSR, CTGF, IGFBP3) significantly related to DCM by establishing protein-protein interaction (PPI) network  using weighted gene co-expression network analysis (WGCNA) method, and identified gene modules that are related to the progression of DCM  of DCM and uncover the specific biological functions of DEGs, which plays a crucial role in developing clinical therapeutic measures and new drugs . Xiao et network . In addin of DCM .Molecular classification was first proposed in various cancer researches to reveal the heterogeneity between patients with the same tumor, shifting tumor classification from traditional morphology to molecular features-based molecular typing. Considering patients in different molecular subgroups often have different clinical manifestations and prognosis, molecular classification is helpful in judging prognosis and guiding treatment of diseases . In recehttp://www.ncbi.nlm.nih.gov/geo/)  (Three gene expression datasets of DCM were downloaded from GEO database (ov/geo/)  via the ct.org/) , includict.org/) , GSE7996ct.org/) , and GSEct.org/) . GSE1780ct.org/) . GSE7996ct.org/) . GSE3585ct.org/) . The detlimma” package to conduct log2-transformation and background correction, and merged three datasets into a metadata cohort for further analysis  and draw PCA-plot based on the top two principal components in PCA  value was adopted to determine the discriminative power.We created receiver operating characteristic (ROC) curve by the “The detailed characteristics of the datasets included in the analysis, including GSE17800, GSE79962 and GSE3585, was shown in After the batch effect was successfully removed, the merged dataset was employed to conduct molecular subgroup analysis by consensus clustering. The cluster consensus score of each subgroup was higher than 0.7 only in the three categories . In addiDCM cases in subgroup 1, subgroup 2, and subgroup 3 had different gene expression patterns. To further investigate the clinical characteristics of three groups, the age, BMI, LVEF, and LVIDD were analyzed in detail in DCM cases from GSE17800 dataset. We found that patients in subgroup 2 had lower LVEF than patients in subgroup 1 and subgroup 3 with statistical difference . Howeverp < 0.05, absolute difference of mean > 0.2)  . We alson > 0.2) . AccordiTCEAL4, ISG16, RWDD1, ALG5, MRPL20, JTB and LITAF, were finally selected as biomarkers  in ISG15, and AUC of 0.939 (95% CI 0.850–0.996) in RWDD1, AUC of 0.955 (95% CI 0.888–1.000) in ALG5, AUC of 0.874 (95% CI 0.701–1.000) in MRPL20, AUC of 0.966 (95% CI 0.917–0.998) in JTB and AUC of 0.953 (95% CI 0.888–0.996) in LITAF  in in LITAF . The expIn this study, three gene expression profiles of heart tissue samples from patients with DCM and control individuals from GEO database were analyzed in detail. For the first time, we merge the three datasets as a metadata cohort and successfully clustered the DCM cases into three molecular subgroups according to the gene expression profile of DCM. The consensus clustering process based on CDF score and cluster consensus score guaranteed that our molecular subgroup classification was robust. Furthermore, significant correlation between clinical conditions and molecular subgroups was observed. Patients in subgroup 2 had lower LVEF comparing with the other two subgroups. In addition, molecular subgroups-specific functional modules and pathways were also analyzed through WGCNA method. These results taken together showed that the molecular classification of DCM was associated with clinical features of patients with DCM and patients in different molecular subgroups should receive personalized treatment.Molecular subgroup classification based on gene expression patterns has provided great help for clinical diagnosis and treatment, especially in the field of cancer research. In particular, patients in subgroup 2 tended to have a more serious condition than patients from subgroup 1 and subgroup 3. The results of age, BMI, and LVIDd statistics showed that there was no significant difference among three groups. Therefore, DCM patients should be distinguished by the molecular classification and receive more personalized treatment.TCEAL4, ISG15, RWDD1, ALG5, MRPL20, JTB, and LITAF, based on two machine learning methods of LASSO regression and SVM-RFE algorithm. However, the accuracy of its predictions requires further validation in a larger population and roles of the biomarkers in DCM still need to further investigate. A limitation of this study should be noted. The development of DCM is a complex process, although a total of 56 participants were included, the input data might still be insufficient to identify and validate biomarkers. In addition, the 56 participants included in the study came from various regions with different genetic variation, diet, physical activity and so on. Therefore, the conclusions in the present study still need more external validations.Compared to previous studies, the functional modules and pathways identified by WGCNA method were also connected with specific molecular subgroup of DCM . We founIn conclusion, our results showed that, through molecular classification, more detailed disease characteristics and its relationship with clinical features of patients with DCM should be noticed. In addition, patients in different molecular subgroups should receive a more personalized treatment. Similar to molecular classification in cancer, more populations are needed to conduct further validation, moreover, future research in DCM should also introduce multi-omics data to reveal more precise molecular subgroups of DCM.


Antimicrobial resistance (AMR) is currently one of the most concerning threats in public health. The efforts to tackle the problem require a global One Health approach, using multidisciplinary approaches and a thorough understanding of the topic both by the general public and the experts. Currently, the lack of a shared mental model of the problem, the absence of a sense of responsibility amongst the different actors and a deficient education on the topic burden the efforts to slow down the emergency and spread of antimicrobial resistant infections. We here propose a multidisciplinary approach to tackle the AMR problem, taking into consideration not only the input from the biological and medical sciences but also the input from the social sciences. Specifically, we suggest strategies from education and psychology to increase awareness about antimicrobial resistance and to implement more effective interventions. Finally, we advocate for a comprehensive and a solidaristic model as the only solution for a problem which knows no borders. As such, political will and international cooperation will be key to achieve the desired change in antibiotic resistance trend. Antimicrobial resistance (AMR) is widely considered to be the next global pandemic that humanity is going to face. In fact, many consider this pandemic to be already on-going, with the world-wide number of estimated deaths by AMR elevating to almost 5 M in 2019  estimates that almost 9 million nosocomial infections  occur each year in European hospitals alone, with However, AMR is far from being exclusively a healthcare-related problem. The WHO’s list of main drivers for AMR include the misuse and overuse of antimicrobials in both the clinical and farm context, the lack of access to clear water, bad practises in the sanitation and hygiene of both humans and animals, poor access to early diagnostics, a lack of legislation on the matter and last, but definitely not least, a generalised lack of awareness and knowledge on the subject. The AMR crisis is therefore a great example of a problem that should be approached under the holistic perspective of “One Health.”One Health is the understanding that in today’s globalised world, there is no real distinction between human health, animal health and environmental health. A formal definition of One Health, as provided by the One Health Initiative Task Force of the American Veterinary Medical Association, would be “the collaborative efforts of multiple disciplines working locally, nationally, and globally, to attain optimal health for people, animals and our environment.”For years, the approach to fighting multidrug resistant organisms (MDROs) has been an almost exclusively scientific one. As AMR started spreading, a huge effort was placed in the search for new antibiotics and the development of new therapies. Although great advances are being made in these domains, it is clear that, given the planetary scale of the problem, a solely wet-lab approach no longer suffices. We believe that such a complex threat requires a multidisciplinary approach, and that the contribution of both biological and social sciences is necessary to address it.In fact, to fully understand the scale of the AMR problem, one has to take into consideration social disciplines such as economy and politics. On first sight, the link between these disciplines and AMR might seem implausible. However, simple decisions such as, for example, the choice of antibiotic to treat an infection are often made on the basis of cost-effectiveness, which is above all, an economical decision. Given that treatment guidelines are usually set by national public health bodies, this often creates a uniform national strategy against infection, meaning that the same antibiotic will be often used throughout an entire territory Wagner . This crIt is clear that promoting collaboration between radically different disciplines is an essential step in the fight against AMR Fig. . One firHaving an accurate and shared mental model of AMR drivers and the different factors that play a role in the problem, in addition to the diverse knowledge areas that can contribute to preserving the usefulness of antibiotics, would promote conversations and facilitate the necessary investment from the different concerned parties. Additionally, the COVID-19 pandemic has highlighted the urgency of developing global policies and legal frameworks to deal with health crises that in a global society know no borders. The same will be true for the AMR pandemic in the near future.The COVID-19 crisis has also highlighted that a poorly scientifically educated population will be less likely to understand the logic behind health policies, and will therefore be less likely to abide by them. Taking this in consideration, the data on the general public’s understanding of the use of antibiotics and AMR paints a dark picture. Several studies  reveal that all groups tended to believe that the main drivers of AMR came from the other groups , values  and worldviews, these are all factors that influence antibiotic prescribing behaviour and/or use, and they are bound to be very different amongst different people.Efforts have been made in the direction of providing a suitable framework that accounts for the complexity of antibiotic prescription and use. One example of such frameworks that has been proposed is the behaviour change wheel .Conversely, it is essential to specifically train healthcare workers on the topic of AMR. In fact, several institutions have started offering multidisciplinary programs to train physicians on the use of antibiotics, using teams comprising pharmacists, epidemiologists and microbiologists .In the USA, the CDC started the “Be Antibiotics Aware” campaign (Use and | CDC https://www.saveourantibiotics.org).Similarly to human health, animal health, especially that of livestock, also plays a crucial role in the spread of AMR. In fact, the overuse of antibiotics in farms far surpasses the overuse in human health, with roughly two-thirds of the antibiotic consumption being destined at livestock  is a European commission-funded effort that provides teaching resources on the use of antibiotics, hygiene and the spread of infection. The program offers several activities tailored for two age groups (9–11 and 12–15 years old), all covering information on how the immune system fights infection, antibiotic use and the importance of vaccination. MicroMundo , launched by Alberta Health Services and the British Columbia Centre for Disease Control; the debate kit launched by the Spanish National Plan for AMR (https://resistenciaantibioticos.es/en/node/508); or the ReAct Campaign, a set of international independent networks to educate on the nature of AMR and its drivers (https://www.reactgroup.org/). Smaller initiatives have also taken place, such as the “Bugs in Bangkok” board game, which explores the complexity of AMR in great detail (https://bugsinbangkok.wordpress.com/).In the previous sections, we have discussed several existing educational campaigns against AMR. Few of these campaigns, however, have targeted the school educational system. Studies have shown that early childhood is an optimal stage for learning , Technology, Engineering and Maths. The urgency for improving STEM skills is shared by educators, policy developers and industry/business organisations alike, and has its roots mostly on the growing demand for STEM specialists in the labour market and the already existing shortage in the current STEM workforce (Caprile et al. Because of this growing concern for strongly educating future generations, STEM education has been systematically reinforced in the different educational systems. An example of this is the Evolving STEM initiative, aiming at improving the understanding of children of evolution (Cooper et al. In order to do so, several facets will have to be evaluated. The first thing that needs discussing is how the topic of AMR will be introduced within the umbrella of STEM integration. The concept of STEM integration defends the idea that regardless of how different STEM fields are between them, the set of skills required for understanding them are very similar English . BecauseSomething else that needs to be assessed are the psychological and sociological determinants of effective education, which would allow us to tailor AMR education to the different publics. Meaningful learning, i.e. completely understanding the learnt information and connecting it with previously known knowledge, requires fitting the new information with one’s existing understanding of the world (Paolo et al. It has been argued that STEM education, given that it finds itself in the border between science sociology and sociology of education, might suffer from the sociological determinants of both the education and the sciences fields (Xie et al. It would also be interesting to include gender mainstreaming in AMR education, as AMR will affect women and men differently (Schröder et al. Many other questions will have to be addressed as well, such as at what age to start introducing the topic, or how to better assess the effect of all the different initiatives and campaigns. Psychology approaches could inform the best ways to maximise the effectiveness of interventions. For example, evaluation of policies including qualitative data to understand how individual factors such as values impact the implementation of guidelines could help in the adoption of policy measures Redding . TailoriDrawing upon research on other areas could also inform on how to best tackle the AMR issue. For example, AMR shares several characteristics with climate change, such as its perception of being a slowly emerging problem. Therefore, lessons learnt from public awareness campaigns regarding climate change could be applied to combat AMR.All in all, there is no doubt of the urgency of better educating the population and future generations on AMR and how to prevent or slow down this global problem. To effectively tackle AMR, and in line with Broom et al. (Broom et al. There are several initiatives that could be launched, both at local, national and worldwide levels. Regardless of the scale, one thing to keep in mind is to always tailor the content so that it is relevant to the target public. This is crucial to promote interest and assure that the public has the needed prior knowledge to actively learn about the topic.Several factors will contribute to the success of any designed campaign. First, the more aligned the campaign is with the hegemonic values of the community, the more effective it will be. To overcome this difficulty, different platforms and styles should be used to reach different target audiences. Second, all different groups, the whole society, should be targeted, in order to reinforce a true change and create a new social norm that is able to endure and create a behavioural change overtime.Finally, a real political and societal commitment is key, with the will to invest in strategies that will have an impact and a benefit in the long term, and not only focussing on short-term economic goals in healthcare.Finally, educational changes alone will not suffice. It is crucial that policy plays a crucial role in the fight against AMR, by regulating and directing the measures that are to be taken in order to tackle the problem. In economics, situations in which individuals acting independently according to their own self-interest lead to a depletion in a resource at a community level are referred to as “the tragedy of the commons.” The depletion of the resource is caused by an uncoordinated action at a community level. The tragedy of the commons is usually linked to environmental science and sustainability and it applies perfectly to the AMR problem as well Cully . To prev


Carbon fiber insole (CFI), which is lightweight and stiff to reduce energy loss and help wearers perform better in sports, has recently been introduced. However, reports are scarce on the effects of CFI on sports performance, muscle activation, and wearing comfort. This study investigated the acute effects of CFI on sports performance, lower extremity muscle activity, and subjective comfort. Thirty young healthy males with shoe sizes between 260 and 270 mm performed various sports tasks  and treadmill runs with wearable sensors under two experimental insole conditions . The results showed that, compared to the benchmark insole, CFI significantly improved sports performance in terms of power generation (~1.5%) and agility (~1%). However, it activated more of the Tibialis Anterior (~0.7%) and Gastrocnemius Medialis (~0.8%) muscles, and was perceived to be stiffer and less comfortable. These findings suggested that CFI could improve sports performance, but could cause more lower extremity muscle activation and subjective discomfort. Wearing the proper athletic footwear is essential for an athlete to improve comfort, prevent injury, and most importantly, enhance athletic performance . As a reFootwear could play an important role in inducing localized pain, muscular activation and fatigue, and wearing discomfort, all of which can limit sports performance. Effects on muscular fatigue and wearing discomfort have been reported according to the different features and characteristics of the insole, such as material, thickness, and wedge for arch support ,11,12. ESeveral studies have explored the role of carbon plates embedded in shoes on sports performance and injury. They have reported that stiffer shoes improve running economy, speed, agility, and jump performance ,22,23,24Therefore, the purpose of this study is twofold: (1) to investigate whether CFI can improve sports performance; (2) to check if CFI affects muscular activation and wearing comfort during treadmill running. It is hypothesized that the use of CFI will improve sports performance, increase muscular activation, and decrease wearing comfort.Thirty healthy young males with shoe sizes between 260 and 270 mm participated in the experiment. All participants were free from any type of back or lower limb pain for at least 6 months, and they were able to complete all of the required tasks. In this study, males with shoe sizes outside of 260 and 270 mm, back or lower limb injuries, heart/lung disease or diabetes, and difficulty in jumping or running were excluded from the experiment. A within-subject design was used to investigate the influence of CFI on sports performance, muscle activation, and subjective comfort. Two experimental insoles were tested in random order: benchmark commercial insole (COM) and CFI . The expIn this study, three aspects of sports performance were examined: the power generation test was composed of a standing long jump (L-jump) and a vertical jump (V-jump); agility was assessed using a 5-10-5-m agility drill (Agility); and speed was assessed using 50-m sprint (Sprint). A standardized procedure was followed to evaluate each sports performance in terms of power generation ,22,25, aThe effect of CFI on muscle usage was investigated by measuring muscular activation while running on a treadmill . The activation of lower extremity muscles was recorded with a sampling frequency of 1000 Hz using surface electromyography sensors .The experiment consisted of two phases, which were conducted outdoors and indoors, respectively. In the outdoor phase, all participants were asked to perform a stretching and dynamic warm-up for at least 10 min. Considering most of the participants had no experience wearing CFI, they were given sufficient time to familiarize themselves with wearing CFI before a series of sports performance tests. After warm-up and familiarization, participants performed the outdoor experimental task in L-jump, Agility, and Sprint sequence. For the convenience of the experimental setup and test, we conducted the experimental tasks in a consistent sequence instead of a randomized sequence. The sequence of experimental tasks followed the guidelines of the National Strength and Conditioning Association (NSCA) ,30. The After finishing all outdoor experimental tasks, the last sports performance test, V-jump, was performed indoors due to the need for an optical motion capture system to accurately measure vertical jump heights. A reflective marker was attached to the tibialis anterior without interfering during the V-jump. The V-jump consisted of a counter-movement jump with an arm swinging to vertically jump as far as possible. Participants stood upright with a marked point on the floor, then performed the maximum effort V-jump. For a successful trial, participants were required to extend their knees and ankles after toe-off, which was visually monitored by the experimenter. Performance was assessed as the height from the reference to the maximum location of the marker, and the reference was defined as the average location of the marker for standing posture for two seconds before the jump. The participants completed three successful trials for each task in each insole condition. If one record deviated from the other two records by more than 10%, the test was considered invalid and the participant was asked to perform the test again to ensure the validity and accuracy of the jump height.Before the treadmill running task, participants were given at least 10 min to relieve muscular fatigue. Then, the participants’ skin was prepared by removing excessive hair and cleaning with alcohol. Next, four EMG sensors were attached to the skin using adhesive tape and firmly fixed with the strap to minimize potential noise from any detachment or tremble. As shown in Participants were instructed to walk at 3 km/h for at least 30 s to familiarize themselves with the task of running on the treadmill. Afterward, they jogged at 6 km/h for at least 30 s and ran at a speed of 10 km/h for 5 min. Participants then ran or walked at their preferred speeds for at least one minute to cool down. After completing a trial run with each insole, participants gave their subjective evaluation of perceived insole stiffness, energy support, overall comfort, and fatigue through a 9-point rating scale. Participants were given at least ten minutes to rest between trials to avoid the fatigue effect.Dependent variables were performance measures for each sports task , muscular activation of four muscles, and subjective ratings after treadmill running. The average value of three successful trials during sports was used to determine the effect of CFI. The raw coordinates of a reflective marker during the V-jump were filtered with a cutoff frequency of 10 Hz to smooth the motion trajectory. The EMG data during 5-min running at 10 km/h were rectified, normalized, and smoothed using the root mean square (RMS) filter to perform a linear envelope. For EMG signals, raw signals were filtered at 20–450 Hz to minimize signal noise and smoothed by the RMS filter with a window length of 50 ms.t-test  or a one-sample Wilcoxon signed-rank test  was conducted to statistically evaluate the effects from two different insoles, with the level of significance set to α = 0.05 [All data were checked with the Shapiro-Wilk test and box-plot to identify the deviations from normality and detect outliers ,35. A paα = 0.05 ,37,38. Iα = 0.05 ,40. Excet = −2.009, p = 0.054) than that of COM wearers (44.97 cm). In addition, CFI wearers had significantly greater  L-jump distance than COM wearers (211.27 vs. 208.19 cm). For agility, CFI wearers displayed marginally more agile performance than COM wearers . In Sprint, however, there was no significant difference . Detailed statistical analysis results are summarized in Parametric comparison results among sports performance from two different insoles  are presented in t-test revealed significant differences between COM and CFI on activation of TA and GM. However, there was no significant difference between the two different insoles in muscular activation of RF  and BF . When the participants wore CFI, their TA muscle activation was significantly higher  than while wearing COM. In addition, muscular activation of GM while wearing CFI was marginally higher  than when wearing COM. Summary results for comparing muscular activation from two different insoles are summarized in p = 0.389). When the participants wore CFI, they felt that it was significantly stiffer , provided greater energy support , but resulted in less comfort  than when they wore COM. Detailed statistical analysis results are described in This study aimed to examine the acute effect of CFI on sports performance, lower extremity muscle activity, and subjective comfort. CFI significantly improved sports performance in terms of power generation (~1.5%) and agility (~1%) compared to COM. However, it induced more of TA (~0.7%) and GM (~0.8%) muscles, and was perceived as stiffer and less comfortable while running on a treadmill.As expected, CFI significantly improved sports performance in terms of power generation and agility compared to COM. In terms of power generation, the average distance of the L-jump and height of the V-jump were improved by about 1.5%. A possible reason for this improvement may be related to energy loss in the MTP joint. While jumping, it has been shown that the MTP joint does not extend until take-off . Since tThere was a marginal improvement (~1%) in agility while wearing CFI. This finding is in line with a previous study that reported that shoes with stiffer plates enhanced agility performance . Even thFor sprint running, the effect of footwear stiffness on sprint performance is still being examined among researchers, but the evidence is conflicting. While wearing stiffer shoes, a decrease in sprint time was reported in the previous study . ConversEven though the effect of CFI on sports performance has not been examined pertaining to elite athletes, it is possible that athletes could receive more support from CFI. When wearing CFI, sports performance could be improved by adding extra gear to the forefoot . Adding Although sports performance can be improved while wearing CFI, when running on a treadmill, muscular activation was significantly higher. When the participants wore CFI, the activation of TA was significantly higher, by 0.7%, with a moderate effect size  Table A2Comfort is an essential factor in designing footwear, since uncomfortable footwear can alter gait and lower extremity muscle activity during running, which could affect sports performance as well as the risk of injury ,57. In aEven though our study showed statistically significant differences between CFI and COM in terms of sports performance and muscle activation, as well as subjective comfort, the effect size of the different insoles was small to moderate. This should be understandable, since sports performance is determined by a combination of human characteristics ,60, equiThis study has some limitations. First, although wearing the stiffer CFI improved athletic performance, the optimal stiffness could not be investigated in this study as only two different insoles were available. Second, the duration of the treadmill running  may be somewhat short compared to some previous studies ,70. ThusThis study investigated the acute effects of CFI on sports performance, lower extremity muscular activation, and subjective comfort. The results showed that, compared to the benchmark insole, CFI significantly improved sports performance in terms of power generation and agility. However, it activated more of the Tibialis Anterior and Gastrocnemius Medialis muscles and was perceived to be stiffer and less comfortable. These findings suggested that CFI can improve sports performance, but it may lead to more muscular activation and subjective discomfort.