Enterocytozoon hepatopenaei (EHP) in cultured Pacific whiteleg shrimp (Litopenaeus vannamei) with a growth disorder in Korea. The histopathology, electron microscopy and DNA phylogeny indicated that the EHP is a unique strain first recorded in Korea. This finding highlights the need for closer monitoring and surveillance to control EHP in aquaculture to prevent disastrous economic losses.We here report the first detection of Litopenaeus vannamei) are major cultivated invertebrates worldwide. However, shrimp productivity faces a variety of challenges, mainly related to outbreaks of lethal or growth retardation-related diseases. In particular, hepatopancreatic microsporidiosis caused by the microsporidian parasite Enterocytozoon hepatopenaei (EHP) is an important disease associated with growth retardation in shrimp. Here, we report the detection of EHP through histopathological, molecular and electron microscopy methods in the hepatopancreas of Pacific whiteleg shrimp with growth disorder in a South Korean farm. Phylogenetic analysis showed a clade distinct from the previously reported EHP strains isolated in Thailand, India, China and Vietnam. An EHP infection was not associated with inflammatory responses such as hemocyte infiltration. Although EHP infection has been reported worldwide, this is the first report in the shrimp aquaculture in Korea. Therefore, an EHP infection should be managed and monitored regularly for effective disease control and prevention.The consumption of cultured crustaceans has been steadily increasing, and Pacific whiteleg shrimp ( Litopenaeus vannamei) is the most widely farmed species for human consumption [Enterocytozoon hepatopenaei (EHP). HPM was first reported as a growth disorder symptom in the Black tiger shrimp (Penaeus monodon) in Thailand in 2004 [Litopenaeus stylirostris) have been reported as the main hosts infected with EHP [The Pacific whiteleg shrimp  spore phase in the lumen of the digestive tubule, and numerous intracellular spore-forming stages that colonize the digestive epithelial cells of the shrimp hepatopancreas . AlthougAlthough there have been reports of EHP detection in shrimp imported to Korea, there has been no report on pathogen isolation or associated disease occurrence in domestic shrimp farms . In ApriIn April 2020, a significant growth delay occurred in a Pacific whiteleg shrimp farm in Ganghwa-gun, Incheon, Korea. Observation of the field conditions did not reveal any white feces in the culture seawater, which is a common clinical sign of severe infection with EHP ; howeverThe hepatopancreas extracted from the 20 shrimp were pooled into four samples of five shrimp each for PCR analysis. DNA was extracted from sampled shrimp using a 5-min DNA/RNA extraction kit , according to the manufacturer’s instructions. A nested PCR amplification of the small subunit ribosomal RNA (ssu rRNA) gene of the EHP was performed in two rounds of nested PCR, according to a previous study . For theTo measure the prevalence of EHP infection at the shrimp farm, the nested PCR results were input into the prevalence package in R software with the test sensitivity set to 95–100% and the test specificity set to 90–95% ,20.3) of the hepatopancreas were fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, embedded with an epoxy-embedding kit , dehydrated through a graded acetone series, sectioned at 70–90 nm and stained with uranyl acetate and lead citrate. The sections were examined using a Libra120 transmission electron microscope.Histopathological and ultrastructural microscopic analyses were performed on the extracted hepatopancreas samples from Pacific whiteleg shrimp exhibiting delayed growth. For light microscopic observation, the hepatopancreas was fixed in a 10% neutral buffered formalin, dehydrated, embedded in paraffin wax, sectioned at 4 μm and stained with hematoxylin and eosin (H & E) and Giemsa solution. For ultramicroscopic observation, small pieces , electron-dense exospores (12.0 nm) and nuclei .Histopathological and molecular biological analyses of Pacific whiteleg shrimp with growth disorders revealed the presence of an EHP infection in a shrimp farm in Ganghwa-gun, Korea. Phylogenetic analysis indicated that the obtained isolate from this farm can be considered a new strain, distinct from previously reported EHP in other countries, which is classified as an independent clade. This is the first case of EHP infection detected in Pacific whiteleg shrimp in Korea.Numerous EHPs, ranging from 1.4 to 1.7 μm in size, were observed both inside and outside the epithelial cells of the digestive tubule with H & E and Giemsa staining. In contrast to serious inflammation signs, such as granuloma formation and hemocyte infiltration, that commonly accompany the bacterial and fungal infections of shrimp ,22,23,24EHP is considered a wasting disease that continuously causes disturbances in nutrient absorption and storage without inflammatory changes. Recently, a proteomic study identified biomarkers linked to a growth hormone disorder in shrimp with growth retardation ; changesArtemia salina [Changes in environmental factors such as ammonia and nitrites can promote the development of HPM. The safe concentrations of total ammonia nitrogen and nitrite for shrimp culture are known to be less than 0.834 and 0.328 mg/L, respectively ; feedinga salina ; and hora salina . We estia salina , regulara salina  and routa salina .


The appropriate solvent is screened to dissolve FD. The electrospinnability of FD/UHMWPEO aqueous solutions is greatly dependent on the weight ratios of FD/UHMWPEO. The incorporation of UHMWPEO significantly improves the electrospinnability of solution and thermo-stability of nanofibers. Also, it is found that there is good miscibility or no phase separation in FD/UHMWPEO solutions. In vitro biological experiments show that the chitosan-modified FD/UHMWPEO nanofibers greatly facilitate the adhesion of endothelial cells and inhibit the attachment of monocytes. Thus, the designed FD-based nanofibers are promising bio-scaffolds in building tissue-engineered blood vessels.Sulfated polysaccharide fucoidan (FD) is widely applied in biomedical applications owing to its outstanding bioactivities. In addition to the biochemical features, the architecture of biomaterials plays a critical role in tissue repair and regeneration. Particularly, nanofibers have elicited great interest due to their extracellular matrix-like structure, high specific surface area, and favorable biological properties. Herein, chitosan-modified FD/ultra-high molecular weight polyethylene oxide (UHMWPEO) nanofibers are developed  In the past few decades, marine polysaccharides have gained increasing attention in the area of diversified biomedical applications owing to their inherent (bio)physicochemical features, such as biocompatibility, biodegradability, favorable bioactive, biomechanical properties, and structural functionalities . Particuvia electrospinning  are the predominant cell type and generate a continuous inner monolayer of blood vessels, which are responsible for regulating inflammation and vascular homeostasis in healthy blood vessels . Also, t−1), chitosan , and acetic acid  were supplied by Shanghai Macklin Biochemical Co., Ltd. . Both the human umbilical vein endothelial cells (HUVECs) and human acute monocytic leukemia cells (THP-1) were bought from the Shanghai Institutes for Biological Sciences . Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12, RPMI 1640 media, and fetal bovine serum were supplied by Biological Industries (Israel). FITC phalloidin and DAPI were provided by Solarbio . Cell Counting Kit-8 was purchased from Absin Bioscience Inc. (China). Carboxyfluorescein diacetate succinimidyl ester was provided by MedChemExpress . Other chemical reagents were of analytical grade and used without further purification. Ultrapure water used in all experiments was obtained with a Milli-Q apparatus .Fucoidan  was provided by Qingdao Bright Moon Seaweed Group Co., Ltd. . UHMWPEO . The mixed solutions were stirred for ∼6 h at room temperature prior to processing to ensure thorough mixing. The solution was loaded into a 20 ml plastic syringe attached with a 25-gauge blunt-ended needle as the spinneret which was charged at a high electric potential of 10–15 kV by a high voltage power supply . The solution feeding rate (0.3–1 ml/h) was precisely controlled by a syringe pump . The FD-based nanofibers were collected onto an aluminum foil-covered collector placed 15 cm away from the needle tip. Electrospinning processes were performed on a horizontal electrospinning setup at 20–25°C with an ambient humidity of 30–35%.FD-based nanofibers prepared from FD/PEO (90/10) were particularly selected for modification with CS. First, 1% CS was dissolved in an aqueous mixed solvent system consisting of 30, 60, and 90 wt% HAc, respectively. The FD-based nanofibers were immersed in the CS/HAc solution for ∼60 s. All modified samples were dried for 2–3 days in a vacuum oven  at 35°C to remove any potential residual solvent.The morphological structure of the prepared nanofibers was observed using a scanning electron microscope (SEM)  operated at an acceleration voltage of 8–10 kV. Prior to observation, samples were sputter-coated with gold for 120 s to increase the electronic conductivity. The mean diameter of nanofibers was identified by randomly detecting at least 50 fibers from various SEM images for each type of sample using Image J software.The rheometer  equipped with a parallel plate (20 mm) was used to measure the viscous property of FD/PEO aqueous solutions.−1 at a scanning resolution of 2 cm−1 during 32 scans.A Nicolet iN10 FTIR spectrometer  was used to characterize Fourier transform-infrared (FTIR) spectra of the samples over the range of 500–4,000 cmλ = 1.5418 Å).X-ray diffraction (XRD) spectroscopy was performed by DX2700  to measure the crystal structures of nanofiber samples. The samples were tested between 10 and 80° (2θ) at a scanning rate of 0.05° (2θ) per min operating with voltage 40 kV and current 30 mA equipped with Cu Kα radiation  at a scan range from 0 to 800°C with continuous nitrogen flow.Differential scanning calorimetry  was used to measure the thermal properties of the electrospun FD-based nanofibers. A nitrogen atmosphere (flow rate = 50 ml/min) was used throughout. All samples were first quenched to -80°C with liquid nitrogen and then heated at a rate of 10°C/min to 180°C.2 at 37°C. THP-1 cells were cultured in RPMI 1640 media  supplemented with 10% FBS in a humidified 37°C and 5% CO2 incubator. THP-1 cells were used in the following experiments.HUVECs (passage: 3–5) were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12  supplemented with 10% fetal bovine serum  and 1% Penicillin-Streptomycin Liquid  in a humidified incubator of 5% CO4 cells/well for cell adhesion. All plates were stored in an incubator at 37°C and 5% CO2 for 24 h. Then, HUVECs were fixated by 4% paraformaldehyde  for 20 min. Subsequently, the cell membrane was permeabilized with 0.5% Triton X-100 (Sigma) solution for 3 min. Finally, the cells were stained by FITC phalloidin and DAPI for 30 and 10 min, respectively . The images were captured by Fluorescence Microscopy .All substrates (Ø14 mm) were immersed into 75% ethanol for 2 min and then irradiated with UV for 1 h, placed in 24-wells, and washed by PBS. After that, HUVECs were incubated on the substrates in 24-well plates at a density of 3 × 104 cells/well for forming cell monolayers. After 1 day, THP-1 cells (1.5 × 105 cells/well) stained by Carboxyfluorescein diacetate succinimidyl ester  were seeded onto HUVEC monolayer, and co-cultured for 4 h. Afterward, each well was washed with PBS three times and counted the number of THP-1 adhered by HUVECs using the Fluorescence Microscopy .HUVECs were seeded onto the sterilized substrate (Ø14 mm) in 24-well plates at a density of 5 × 10p < 0.05 was considered to be statistically significant.All data were expressed as mean ± SD. Statistical analysis was performed using Origin 9.0. All the data were analyzed using one-way analysis of variance (ANOVA) with Tukey’s test to determine differences between groups. A value of via electrospinning. Also, water-based electrospinning, also named “green electrospinning,” has several advantages of being environmentally friendly, non-toxic, and non-flammable. It was reported that organic solvents remaining in the fibers had a negative effect on cellular adhesion and proliferation both in vitro and in vivo  at room temperature was used in the following experiment. However, when 10% w/v FD aqueous solution was used for electrospinning, only droplets were formed as shown in As reported, the electrospinnability of naturally derived polymer solutions can be greatly improved by introducing a small amount of UHMWPEO . As showQuantification shows that the fiber diameter first decreased and then increased with increasing the amount of PEO . The sizIt was well-demonstrated that the diameter of nanofibers can affect the drug release, modulate cell adhesion, migration, proliferation, differentiation, siRNA uptake, and gene silencing, as well as even tissue repair and regeneration . As depi−1, which is attributed to—CH2 stretching , 1,642 cm−1 (C=O stretching), 1,232 cm−1 (S=O bending), and 833 cm−1 (C-O-S bending). The absorption band associated with C-O-C disappeared in FD/PEO nanofibers, probably because C-O-C is a proton acceptor and may form hydrogen bonding with the OH group in FD molecules . Representative SEM images of FD/PEO nanofibers before and after modification are shown in 2O = 30 wt%, the integrity of the fiber structure was retained  and actin cytoskeleton (green). More adhered cells were found on the CS-modified FD/UHMWPEO nanofibers compared to the CS/UHMWPEO nanofibers and FD/CS/UHMWPEO nonfibrous films, indicating that FD and fiber structure could greatly promote cell adhesion. This result suggests that the CS-modified FD/UHMWPEO nanofibers possessed excellent cytocompatibility as a bio-scaffold for blood vessel tissue engineering.HUVECs were selected because they are the main cell type and play a critical role in the function of the blood vessel . Cell atThe adhesion and migration of monocytes to endothelial cells is a process of the inflammatory response, which is mediated by specific molecules on endothelial cells and monocytes . THP-1 cIn summary, chitosan-modified FD/UHMWPEO nanofibers were fabricated using green electrospinning. Water was screened and used as a solvent to dissolve FD. The defect-free nanofibers with an average diameter (560 ± 88 nm) were prepared in the electrospinning of the FD/UHMWPEO (90:10) solution. The addition of UHMWPEO greatly improved the electrospinnability of the solution and thermo-stability of nanofibers. Cellular experiments demonstrated that the chitosan-modified FD/UHMWPEO nanofibers facilitate HUVEC adhesion and suppressed the attachment of monocytes. Thus, the developed FD-based nanofibers display great potential for vascular tissue engineering.


SARS-CoV-2, the virus that causes COVID-19, can spread rapidly in prisons and can be introduced by staff members and newly transferred incarcerated persons , all units have shared lavatories and common areas, including one 150-person dormitory-style unit with conjoined sleeping and common areas. Before the outbreak, prison A implemented multiple mitigation measures, including mandatory mask wearing for staff members and incarcerated persons.After September 14 test results identified 86 cases of COVID-19 among incarcerated persons, the facility implemented a modified lockdown, restricting movement of incarcerated persons across units and shutting down common areas; however, staff members continued to rotate throughout the facility because of staffing shortages and scheduling policies. By September 22, the facility was unable to medically isolate or quarantine incarcerated persons because of limited space. On September 28, the Wisconsin DHS and Wisconsin Department of Corrections contacted CDC to request assistance in investigating the outbreak.A COVID-19 case was defined as a positive SARS-CoV-2 test resulthttps://stacks.cdc.gov/view/cdc/104507). Twenty-two days later (September 23), cases were identified across all 15 units, with infections in three units progressing from zero cases to attack rates ranging from 66.2% to 84.6% during that period. During August 14–October 22, a total of 869 (79.4%) of 1,095 incarcerated persons  and 69 (22.6%) of 305 staff members  received positive SARS-CoV-2 test results  nasal swab specimens from incarcerated persons sent to the Wisconsin State Laboratory of Hygiene for testing were retained for WGS, and 172 (42%) of these, representing 20% of cases among incarcerated persons across 13 of 15 units, were successfully sequenced. These included specimens from cases identified from intake testing (12 of 12), symptomatic testing (11 of 22), and mass or targeted testing on September 14 (66 of 86), October 6–7 (60 of 153), and October 19 (23 of 34). No specimens from staff members were available for sequencing because the testing laboratory had discarded them.Sequences were compared with those from specimens obtained from persons in the central intake facility, the location of persons with the initial cases before their transfer to prison A, and others across Wisconsin . SequencOn October 16, CDC and Wisconsin DHS recommended that prison A house incarcerated persons with active infection separately from susceptible incarcerated personsInvestigation findings demonstrate that SARS-CoV-2 spread rapidly in prison A, infecting 79% of incarcerated persons and 23% of staff members in 2 months. Factors that likely facilitated transmission include the described intake quarantine procedures, crowded housing units, and movement of incarcerated persons and staff members among units.WGS detected one cluster of cases originating in a group of newly transferred incarcerated persons, with all subsequent cases clustering closely together. CDC’s interim guidance recommendation is to quarantine incarcerated persons at intake and immediately isolate symptomatic persons and persons who receive positive test results  (Because of ongoing within-facility movement of staff members and incarcerated persons, staff members and incarcerated persons both might have contributed to the prolonged outbreak. Assignment of staff members to specific units and routine testing of staff members might reduce within-facility and community transmission (The findings in this report are subject to at least four limitations. First, sociodemographic or clinical comparisons between persons who did and did not have COVID-19 could not be made because data were available only for infected persons. Second, cases in staff members were likely underreported because facilitywide testing and test result reporting were not mandated. Third, maximum capacity was used to calculate the percentage of new cases detected by unit because the number of persons in each unit over time was unavailable. Finally, only 20% of specimens from incarcerated persons with COVID-19 and no specimens from staff cases were sequenced, limiting understanding of staff member and prison-to-community transmission.SARS-CoV-2 transmission is difficult to control in congregate living facilities (SARS-CoV-2 can spread rapidly in prisons and can be introduced by staff members and newly transferred persons.After early detection of SARS-CoV-2 in six newly transferred persons during intake quarantine in a Wisconsin prison, 79.4% of incarcerated persons and 22.6% of staff members contracted SARS-CoV-2 during August 14–October 22, 2020. Whole genome sequencing from 172 incarcerated persons with COVID-19 determined that all specimens clustered in the same lineage.Insufficient quarantine after intake can lead to rapid, widespread SARS-CoV-2 transmission, even after early detection of initial cases. Understanding and addressing the challenges faced by correctional facilities to implement medical isolation and quarantine can help reduce and prevent outbreaks.


Gynecological Teaching Associates (GTAs) and Male Urogenital Teaching Associates (MUTAs) instruct healthcare professional learners to perform accurate and respectful breast, speculum, bimanual vaginal, rectal, urogenital, and prostate examinations. During such sessions, the GTA/MUTA uses their own body to instruct while providing real-time feedback. While GTAs/MUTAs fall under the broader umbrella of Standardized Patient methodology, the specificity of their role indicates need for establishment of Standards of Best Practice (SOBP) for GTA/MUTA programs. On behalf of the Association of Standardized Patient Educators (ASPE), the Delphi process was utilized to reach international consensus identifying the Practices that comprise the ASPE GTA/MUTA SOBP. The original ASPE SOBP was used as the foundation for the iterative series of three surveys. Results were presented at the ASPE 2019 conference for additional feedback. Fifteen participants from four countries completed the Delphi process. Four of the original ASPE SOBP Domains were validated for GTA/MUTA programs: Safe Work Environment, Instructional Session Development, Training GTAs/MUTAs, and Program Management. Principles and Practices were shaped, and in some instances created, to best fit the distinct needs of GTA/MUTA programs. The ASPE GTA/MUTA SOBP apply to programs that engage GTAs/MUTAs in formative instructional sessions with learners. Programs that incorporate GTAs/MUTAs in simulation roles or in summative assessment are encouraged to reference the ASPE SOBP in conjunction with this document. The SOBP are aspirational and should be used to shape Practices within the program’s local context. The ASPE GTA/MUTA SOBP will continue to evolve as our knowledge-base and practice develop.The online version contains supplementary material available at 10.1186/s41077-021-00162-4. The Association of Standardized Patient Educators (ASPE) published the ASPE Standards of Best Practice to identify guidelines for individuals and programs working with standardized patients (SPs). The ASPE SOBP identifies SPs as “a person trained to portray a patient in realistic and repeatable ways” . StandarHistorically, learners practiced pelvic examinations on plastic models, sex workers, anesthetized patients, and/or patients in clinic possibly alongside lectures, videos, and/or observation . In 1972Terminology and application of methodology varies among institutions and locations . As with broader SP methodology , 17, thiConsistent with the ASPE SOBP, the ASPE GTA/MUTA SOBP “will provide a clear and definitive guide for all programs that” incorporate GTA/MUTA instructional sessions, with careful attention to making “these guidelines comprehensive and flexible enough to address the diversity of varying contexts” of GTA/MUTA programs . The oveBecause these standards are aspirational in nature, it is expected that institutions will be at varying degrees of implementation. The ASPE GTA/MUTA SOBP specifically addresses instructional sessions, and the ASPE SOBP should be referenced when GTAs/MUTAs are in the role of a SP or are otherwise incorporated into simulation activities and assessments. Following the ASPE GTA/MUTA SOBP will help  assure tGynecological Teaching Associate (GTA): “a female specifically trained to teach, assess, and provide feedback to learners about accurate pelvic, rectal and/or breast examination techniques. They also address the communication skills needed to provide a comfortable exam in a standardized manner, while using their own bodies [to instruct] in a supportive, non-threatening environment (ASPE)”  in a supportive, non-threatening environment (ASPE)” [ (ASPE)” .SP Educator (SPE): “used to refer to those who work to develop expertise in SP methodology and are responsible for training and/or administering [GTA/MUTA instructional sessions]. Some may be trainers who exclusively work with SPs[/GTAs/MUTAs], while some may be faculty or healthcare professionals who work with SPs[/GTAs/MUTAs] as part of their clinical and/or academic roles” [c roles” . The rolInstructional session: a period of time during which an instructor teaches learners relevant clinical content and safe assessment techniques. It is anticipated that a learner has received at least minimal preparation prior to an instructional session and the GTA/MUTA builds their knowledge, competence, and confidence through hands-on practice with real-time feedback.Learner: an individual who will be instructed by a GTA/MUTA. Learners are typically health professional students, practicing health professionals, or GTA/MUTA trainees.Participant: a person actively engaged in an instructional session including the GTA/MUTA, and learner(s).Sensitive examinations: physical assessment of the breast, vulva, vagina, uterus, adnexa, penis, scrotum/scrotal contents, inguinal region, rectum, and/or prostate.Stakeholder: an individual impacted by GTA/MUTA sessions, which may include GTAs, MUTAs, staff, faculty, learners, and patients.A scoping review was undertaken separately to identify the implementation and utilization characteristics of GTA and MUTA programs . ArticleASPE developed a subcommittee with the goal of using a modified Delphi process to reach international consensus identifying the Practices that comprise the ASPE GTA/MUTA SOBP. Subcommittee members were selected based on demonstration of leadership within the ASPE GTA/MUTA SIG or active ASPE participation with experience in a GTA/MUTA program. The Eastern Michigan University granted Exempt status, UHRSC-FY18–19-65.The Delphi Process was originally developed in the 1950’s as a method of predicting future trends by building consensus among experts. “Delphi may be characterized as a method for structuring a group communication process so that the process is effective in allowing a group of individuals, as a whole, to deal with a complex problem” . Impleme○ Where GTAs and MUTAs instruct independently (as opposed to acting as models)GTA or MUTA (instructing learners), and/orGTA/MUTA educator/trainer (instructing GTAs/MUTAs), and/orGTA/MUTA program administrator.○ In one or more of the following roles:Must have at least 3 years of experience within a GTA and/or MUTA programFluent in English (written)Currently affiliated with GTA/MUTA organization or networkNo conflict of interestSubcommittee members (the authors of this publication) identified individuals with expertise in GTA/MUTA methodology throughout the world using the following resources: known ASPE members/contacts, authors who published on GTA/MUTA methodology, and GTA/MUTA programs with an online presence. Understanding that ASPE is an international organization with a large membership base from the United States, fifteen individuals from Australia, Belgium, New Zealand, Pakistan, Sweden, The Netherlands, the United Kingdom, and Canada were specifically invited to enhance international participation. Email invitations were additionally sent to the ASPE List-serve and the ASPE GTA/MUTA SIG List-serve. The invitation addressed the goals, the modified Delphi process, anticipated timeline, and inclusion criteria as listed:While the SOBP are directed at the program level , GTAs/MUTAs were included to represent their distinct and valuable perspective. Interested individuals emailed the Subcommittee to express interest and verify how they meet the inclusion criteria. To promote diversity, no more than one individual was to be confirmed to participate from each individual program. To ensure all stakeholders were represented, a balance of GTAs/MUTAs, GTA/MUTA educators/trainers, and GTA/MUTA administrators were selected.There is no standardized number of panelists to include in a Delphi process, but rather researchers must identify the number that best fits the breadth and depth of their topic . The intUsing the ASPE SOBP as a framework, panelists completed three iterative rounds of surveys within Survey Monkey, Inc. . The iniAfter the initial survey, the Subcommittee consolidated the qualitative responses into the list of Practices. Identical items were removed and revisions were made only for clarity and grammar, but not for content. The consolidated list was combined with the ASPE SOBP for the second survey. The second and third surveys included only the rating of the Practices on a 5-point scale  and rationale for their rating.Rationale provided by panelists was summarized and provided alongside the mean and standard deviation at the introduction to the second and third surveys . RationaEach survey was available for 2 weeks. Reminder emails were sent to panelists 10 days after each survey was released. Only panelists that completed the prior survey round were invited to participate in the following survey round. An email was sent to individuals that dropped out to explore changes that would have encouraged participation.After each survey, the Subcommittee conducted descriptive statistics using SPSS  on each Upon completion, the authors presented the ASPE GTA/MUTA SOBP at the ASPE 2019 Annual Conference to obtain feedback. All conference attendees were invited to participate in a workshop that included presentation of the survey results and the opportunity to provide feedback. Workshop attendees were divided into small groups to provide written feedback. Feedback was evaluated and incorporated into the final document. The ASPE Executive Committee approved the final document.Twenty-one of the 26 interested individuals were selected for participation Tables  and 2. WThe Practices included within this document met statistical consensus for applicability to GTA/MUTA programs during the modified Delphi process; minor alterations were made to consensus-supported Practices to enhance clarity based on feedback as detailed above. Lack of inclusion of a Domain, Principle, or Practice included within the original ASPE SOBP does not negate the relevance of that item, but rather does  identifyPhysical and psychological safety is critical in all working and learning environments, but it is especially critical when instructing learners to conduct sensitive examinations as GTAs/MUTAs do. This is an unique context for interaction with another person’s body and it may trigger a variety of responses for both the GTA/MUTA –34; and/“For the community of SP educators, there are three distinct principles related to creating a safe work environment: safe work practices, confidentiality, and respect” .PrincipWhile the ASPE SOBP  Domain 2Many GTA/MUTA programs exist within broader SP programs or simulation centers which have a defined learning theory, feedback style, debriefing style, etc. To the extent possible, those interaction patterns should be extended to GTA/MUTA instructional sessions to provide clear support and intent for the sessions –44. “TheAs highlighted in the ASPE SOBP, the development of materials for instructional sessions is an iterative process of creation and refinement . This isTraining may be accomplished in a variety of ways, depending on the program goals. Training curriculum should be designed in a manner consistent with the learning theories and norms of the broader SP program or simulation center, as appropriate. The objectives of the instructional session will determine the extent of the training necessary regarding knowledge of physical examination techniques and effective provision of feedback to facilitate learner confidence and competence.GTAs/MUTAs that will provide feedback on physical examination techniques should receive training that includes a physical exam from a provider or other trained individual so that they can characterize the sensations and learn to guide learners to that goal . GTAs/MUThe second primary component of GTA/MUTA training involves effective and expected provision of feedback. Provision of summative feedback is clearer if the instructor understands the expected outcome: either the learner completed the task appropriately or did not. Provision of formative feedback, however, involves clarification of goals, engaging the learner in self-assessment and dialog, providing instruction on how to modify the performance, and facilitating “opportunities to close the gap between current and desired performance” all while supporting the learner’s self-confidence The majority of GTA/MUTA programs utilize GTAs/MUTAs for formative instructional sessions. If GTAs/MUTAs will be involved in summative evaluation and/or simulation, the ASPE SOBP  should bMany GTA/MUTA programs exist within broader SP and/or simulation programs but regardless of their institutional affiliation or location the broad program should follow standard business processes and procedures. This includes “creating financial management, business, and strategic plans” for the broad organization and (as applicable) specific programs . The purThe ASPE SOBP  should bOverall, the Practices within the first 4 Domains that were omitted reflected components related to SP simulation as opposed to GTA/MUTA instructional sessions. This indicates that the consensus building process was effective. Only one panelist was lost to attrition once the survey process began, indicating that the surveys were not too complex or time-consuming.Far more individuals expressed interest and were invited to participate than completed the process. Perspectives from 4 countries were ultimately represented although we aimed to have more global representation of panelists. While we can expect cultural differences reflected among GTA/MUTA programs in varying locations, a recent Scoping Review did not demonstrate consistent geographic variance in program specifics . AlthougThree members of the research team  also completed the surveys for the Delphi process. They did not have access to participant responses prior to completing the surveys themselves and so were unable to impart bias. The protocol defined that all feedback and responses would be generalized from survey responses, so there was no opportunity for the researchers to impart their own individual bias. Other panelists did not know that these individuals were also participating as panelists.The Practices within Domain 1, Safe Work Environment, reached consensus with ease. Panelists were very agreeable that safety is critical, however safety is infrequently addressed within the literature on GTA/MUTA programs. Further research should explore how SP educators perceive safety and how GTAs/MUTAs experience safety within an instructional session and simulations. The research supporting Domains 2 , 3 (Training GTAs/MUTAs), and 4 (Program Management) is more robust, however the majority of publications are case reports detailing an individual program. Further research is indicated to demonstrate the components of effective instructional session development, strategies to optimize GTA/MUTA training, and analysis of how and whether GTA/MUTA program management is distinct from broader SP program management.Per Lewis et al. , SP educThere were comments within the survey responses expressing concern about the use of gendered terminology in relation to GTA/MUTA sessions. The Association of American Medical Colleges’  work towThe ASPE GTA/MUTA SOBP demonstrates expert consensus regarding aspirational standards that all GTA/MUTA programs should aspire to uphold. These standards were designed to ensure safety, promote educational effectiveness, and reduce institutional risk. ASPE will periodically engage in rigorous methodology to ensure the GTA/MUTA SOBP continue to reflect expert consensus and growth of the roles of the SP educator and GTA/MUTA.Additional file 1. ASPE GTA MUTA SOBP Survey Results


In Japan, there is concern regarding the relation between the inappropriate use of antibiotics and antibiotic resistance (AMR). Increased bacterial resistance is due in part to the inappropriate use of antimicrobial agents. The support of the pharmacist becomes important, and there is growing interest in antimicrobial stewardship to promote the appropriate and safe use of antimicrobials needed for the optimal selection of drugs, doses, durations of therapy, therapeutic drug monitoring (TDM), and implementations of cost containment strategies in Japan. Pharmacists should strive to disseminate the concept of “choosing wisely” in relation to all medicines, implement further interventions, and put them into practice. In this article, we present data for antimicrobial stewardship and Japan’s AMR action plan, focusing on how pharmacists should be involved in enabling physicians to choose antimicrobials wisely. Furthermore, a significant reduction was also found in the total DDDs of the antibiotics used. In addition, the implementation of antimicrobial stewardship reduced the unnecessary antimicrobial prescription rate for acute respiratory tract infections and acute diarrhea (The spread of drug-resistant bacteria poses a serious threat to human beings worldwide . At the diarrhea  6]..p < 0.01Staphylococcus aureus (MRSA), candidiasis, and extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, which are particular problems in hospitals, and we provide suggestions on how pharmacists should practice to enable physicians to choose antimicrobials wisely.Infectious disease (ID) pharmacists have to extend their support for the appropriate use of antimicrobials prescribed by attending physicians to not only hospitalized patients but also outpatients. “Choosing wisely”, the United States-based health education campaign, warns against the unnecessary use of antimicrobials . PharmacIn Japan, the most common AMR organism is MRSA . MRSA isStaphylococcus aureus isolates from hospitalized patients, but, in recent years, there has been a decreasing trend . Th. Th61]. AMR Alliance Japan, a policy proposal announced in July 2019, proposes that items will be added, changed, or emphasized in the Japanese government’s next National Action Plan on Antimicrobial Resistance to be implemented in 2021 . The effHamada et al.  used a pIn our latest study, Hanai et al.  conducteAs a recent topic, azole-resistant Aspergillus species ,76 and aIn this article, AMR Alliance Japan also suggests that efforts should be made to promote an increase in the amount of therapeutic drug monitoring (TDM) in the Japanese government’s next NAP on AMR . Since tWe reported on recent AMR strategies and studies conducted by Japanese pharmacists in the context of the COVID-19 pandemic. Pharmacists are an integral part of the antimicrobial stewardship team and must be actively involved in antimicrobial management. In Japan, the titles of board-certified infection control pharmacy specialist and infectious disease chemotherapy pharmacist exist. However, the staffing constraints that allow pharmacists to complete such programs in some hospitals in Japan and the shortage of ID-trained pharmacists are barriers to the implementation of the program and present challenges. Japanese pharmacists are working to reduce AMR and change the Japanese system while accumulating achievements one step at a time. Japanese pharmacists strive to properly utilize TDM as a tool to improve the adequacy of antimicrobial use. Further validation is needed in the future to discuss the use of a proactive TDM, defining the criteria that could identify the patients, the disease, or the conditions that are more often associated with pharmacokinetic variability. It should be noted that this review does not include a wider approach. A wider approach to antimicrobial stewardship that includes other professionals working in the ICU, infectious disease wards, cardio-surgery units, etc., for whom the possibility of spreading knowledge about the risk of multi-resistant strains and protocols to reduce the risk is of fundamental importance . Pharmac


On this basis, a method for the rapid detection of F− ions by host molecular tweezer 7d was established, and the naked-eye detection of F− was realized through the unique yellow color of the complex solution. According to the determination of F− ions in real food samples, it was proved that the established method had good application prospects in F− ion detection.Chiral cholesteric molecular tweezer 7d was synthesized and its influences on changes in the ultraviolet (UV) and fluorescence spectra of various anions were investigated. The results displayed that molecular tweezer 7d selectively recognized F Fluorine is an indispensable trace element for human growth and development [− ions are ingested by the human body, they may induce cell apoptosis, decrease the immune function of the body [− ion content in daily drinking water is 1.5 mg/L [− ion concentration should not exceed 1.0 mg/L to prevent F− ion poisoning [− ion detection is of great significance to human health and chemical environment.It is well known that anions are abundant in our daily life and play an irreplaceable role, especially in biological processes, environmental detection, and clinical medicine fields ,2,3. To elopment , and plathe body , and evethe body ,7, thus 1.5 mg/L . In Chinoisoning . Hence, − ion identification, including gas chromatography [19F− nuclear magnetic resonance analysis [− ion detection with high sensitivity and selectivity. At present, a large number of studies have reported that the photochemical response recognition technique can promote rapid and efficient selectivity between the guest and receptor, promoting the realization of qualitative and quantitative detection of specific ions [−, Cl−, Br−, I−, Ac−, and H2PO4−, and that the host and guest could form a 1:1 type supramolecular complex by multiple hydrogen bonds. Chahar et al. [− ions with high sensitivity and selectivity.In recent years, many traditional detection methods have been developed for Ftography , the iontography , 19F− nuanalysis , and higanalysis . Howeverfic ions . Generalfic ions ,16, thiofic ions ,18, pyrific ions , and indfic ions ,22 are cfic ions . Liu et fic ions  designedr et al.  performe− ions by chiral molecular tweezer was established and applied to F− detection in food samples. It is expected that the presented cholesteric compounds can provide a new method for the rapid determination of F− ions.In this study, on the basis of our previous work , we synt− ions significantly change UV–vis absorption spectrum. The intensities of the absorption peaks at 255 and 288 nm decrease significantly, and a new absorption peak appears at 350 nm. In contrast, the addition of other anions does not cause significant changes in UV–vis absorption spectrum of host molecular tweezer 7d solution. It should be noted that the solution changes from colorless to distinctly yellow after the interaction with F− ions, while other anions show no color change  solution in the presence of other anions was studied. As shown in − ions by the host molecular tweezer 7d. Thus, the host molecular tweezer 7d shows good anti-interference performance in the selective recognition of F− ions, and has wide environmental adaptability and practicability.In order to further evaluate the selectivity of molecular tweezer 7d to F− ions, the fluorescence intensity at 273 nm was measured after adding molecular tweezer 7d into F− ionic solution . Hence, the host molecular tweezer 7d and F− ions generate a 1:1 type complex. According to the intercept 1/a and slope 1/a•Ka of the straight line, the association constant Ka of the complex was calculated as 1719 L/mol. In addition, by plotting the graph of UV–vis absorbance value versus F− ion concentration during the complex formation process  (210 μM) [− ions  . Compare [− ions , the hos− ions [− ions is 0.5, the complex of 7d + F− ions has the highest absorbance value. Hence, the chiral molecular tweezer 7d complexes with F− ions following a molar ratio of 1:1.The continuous variation method with a constant molar ratio (Job test) is another effective method for determining the coordination ratio of host molecular tweezer 7d to F− ions . As show− ions. In the absence of F− ions , accompanied with significant broadening and weakening of the signal peak. Moreover, the shape of the peak at 7.64 ppm changes significantly, which decomposed from the original triple peak to a dual peak at 7.67 ppm and a single peak at 7.57 ppm. This may be due to the interaction between the active hydrogen in the host molecular tweezer 7d and highly electronegative F− ions, which form hydrogen bonds (N-H... F). As a result, the charge of the benzene rings increases, indirectly leading to the change of aromatic ring hydrogen signal [− ions, promoting changes in the optical properties, thus achieving F− ion detection.It is difficult to accurately describe the recognition mechanism of the chiral molecular tweezer 7d for F spectra ,39. Thern signal . Therefo− ions was optimized using DFT/B3LYP/6-311G method in the Gaussian 09 quantum chemistry software. − ions, the energy-minimized conformation of the host molecular tweezer 7d is a tweezer shape, which can provide enough space for the embedment of F− ions. When F− ions interact with the host molecular tweezer 7d, the conformation of the aromatic rings on the host arm changes. Thus the guest F− ions can enter the cracked cavities of the molecular tweezer 7d more conveniently and form a stable complex. F− ions usually have a high electron density and are easy to attract hydrogen atoms. In the presence of the electron-attracting amide group N-H, F− ions act as good hydrogen bond donors. The electron density of LUMO orbital of the host molecular tweezer 7d is mainly distributed on the benzene ring groups, while the electron density of HOMO orbital is mainly distributed on the -SCH3 groups. After the addition of F− ions, the electron density on LUMO orbital of the host molecular tweezer 7d and F− complex is mainly distributed on the benzene ring structure units; in contrast, the electron density on HOMO orbital is mainly distributed on the benzene rings and amide N-H groups, and slightly distributed on the methoxy groups linked to the benzene rings. This electron cloud distribution is advantageous to the transfer of intramolecular charge between the aromatic rings and hydrogen bonds, thus leading to changes of UV–vis absorption spectra. According to theoretical calculations, the length of the N-H bond in the host molecular tweezer 7d before the addition of F− ions is 1.016 Å; after the addition of F− ions, the bond length increases (1.042 Å), the distance of H…F is 1.555 Å, and the included angle of N—H…F is 174.3°, indicating that strong intermolecular hydrogen bonds occur between F− ions and the host molecular tweezer 7d  to the food samples, UV–vis absorbance values were measured. The results are listed in − ion selective electrode method in the Chinese national standard GB/T 5009.18, i.e., “Determination of Fluorine in Food”, the relative error between our test results is less than 5% (n = 3). Hence, our proposed method can accurately and quantitatively (16–192 μmol/L) detect F− ions in food samples, and has high potential value in real life.As listed Ribes stenocarpum Maxim were picked from Menyuan, Qinghai province.All chemicals were purchased from Sigma and Aladdin reagent company without further purification except stated otherwise. All the organic solvents were of analytical grade. The anionic solution is prepared from their various tetrabutylammonium (TBA) salts. Different food samples, namely, milk powder, wolfberry, quinoa, and wheat flour, were bought from the local market , The 1H-NMR spectra were recorded with the AVANCE NEO nuclear magnetic resonance spectrometer . The microscopic images were observed with the JSM-7900F field-emission scanning electron microscope .Chemical reactions were conducted by MCR-3 microwave chemical reactor ; Ultraviolet spectra were measured by UV-2600 Shimadzu ultraviolet–visible ; The 2 in the presence of L-methionine. Finally, the cholesteric chiral molecular tweezers were synthesized by microwave irradiation with methyl deoxycholate as a spacer, and arylhydrazine and amino acid as arms. The mass, hydrogen, and carbon spectra of the compound are consistent with those reported in reference [Methyl deoxycholate  was prepeference . The syn−4 mol·L−1) and different anions of tetrabutylammonium salt  (0.01 mol·L−1) were prepared separately at room temperature [DMSO solutions containing host molecular tweezer 7d  was added to 0.5 mL of the host molecular tweezer 7d solution of 2 × 10−4 mol·L−1 (dissolved in DMSO). The mixed solution was corrected to a constant volume of 5 mL. The solution colors, fluorescence emission spectra, and UV–vis absorption spectra for different anions were observed.In a typical run, 0.5 mL DMSO solution containing a certain anion of tetrabutylammonium salt (F− ions and other anions with a molar quantity of 50 times that of the host molecular tweezer 7d were successively added into the host molecular tweezer 7d solution (20 μmol·L−1), and the UV–vis absorption spectrum of the mixed solution at 288 nm were measured.The DMSO solutions containing F− ions, 0.5 mL DMSO solution containing 2 × 10−4 mol/L the molecular tweezer 7d was added to 0.5 mL F− ionic solution, and the total volume reached 5 mL by adding DMSO, then the fluorescence intensity at 273 nm was measured every 20 seconds until the fluorescence was stable. The slit width of excitation and emission were both 5 nm.To determine the fluorescence response time of compound 7d to F− ions was added dropwise to the host molecular tweezer 7d solution of 5.0 × 10−4 mol/L, and the UV–vis absorption spectra of the complex solution with different F− ion concentrations (16–192 μmol/L) were determined. The association constant of the host molecular tweezer 7d with F− ions was calculated based on the absorbance values of the complex solutions at 288 nm. Moreover, the standard curve equation was obtained by plotting the graph of F− ions concentration versus the UV–vis absorbance value during the formation of complex. Then, according to the formula LOD = 3 σ/ K, the detection limit of F− ions by the host molecular tweezer 7d could be calculated. Here, in the formula LOD = 3 σ/k, σ is the standard deviation, and k is the slope of linear fitting.The DMSO solution containing F− was kept at 1 × 10−5 mol/L, and a series of mixed solutions with different molar ratios (host molecular tweezer 7d to F−) of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 were prepared successively. The absorbance values of these mixed solutions were measured to obtain the Job point diagram of the complex, and thus the complexing ratio of the complex was determined.The total concentration of the host and guest F− ions more intuitively, a field emission scanning electron microscope (FESEM) was used to conduct microstructure observation and energy spectrum imaging analysis on the host molecular tweezer 7d and its complexes with F− ions.In order to observe the interaction between the host molecular tweezer 7d and F− ions in DMSO-d6 were quantitatively added to obtain the mixed solution. Changes in 1H spectra after the addition of F− ions into the host molecular tweezer 7d solution were observed, and the combining sites of the host molecular tweezer 7d and F− ions and the possible driving force for recognition were determined according to the chemical shift changes of protons on different functional groups. Gaussian 09 software was used for density functional theory (DFT) optimization at B3LYP/6-311G(d) level, as well as simulating the structure of the host molecular tweezer 7d and the molecular structure of the host molecular tweezer 7d and F− ions.According to the composition ratio of the complex, the host molecular tweezer 7d and FRibes stenocarpum Maxim, 4 g quinoa, 4 g milk powder, 4 g flour, and 4 g Chinese wolfberry were weighed respectively and added into a nitrifying tube. Then, 0.2 g copper sulfate, 6 g potassium sulfate, and 10 mL sulfuric acid were added into the nitrifying tube successively. After digestion for 40–50 min at 400 °C, the mixture was centrifuged to obtain the supernatant [−4 mol·L−1) was added to 0.5 mL supernatant sample solution, and the mixed solution was corrected to a constant volume of 5 mL. The absorbance value of the sample was measured using a UV-spectrophotometer.Total of 4 g ernatant . Then, 0− ions. The addition of F− ions promoted regular changes of the UV spectrum of the molecular tweezer 7d, and other anions had little interference. The molecular tweezer 7d had a high sensitivity and selectivity towards F− ions. The association constant was 1.72 × 103 L/mol, and the detection limit was 5.14 μmol/L, which was more sensitive than that in many reported methods.In summary, the chiral molecular tweezer 7d was synthesized via the deoxycholic acid. UV spectrophotometry found that the molecular tweezer 7d had selective recognition ability towards F− ions altered the color of the host molecular tweezer 7d solution from colorless to yellow, while the addition of other anions had no effect. Based on this, the rapid naked-eye detection of F− ions was achieved. Furthermore, the complexing process between the host molecular tweezer 7d and F− ions was explored by Job experiment, where the host 7d and F− ions formed a complex in 1:1 ratio.In DMSO solution, addition of F− ions, the NMR spectra and microstructure changes of the molecular tweezer 7d before and after addition of F− ions were studied. It was found that F− ions readily entered the cracked cavities of the molecular tweezer 7d and formed a stable complex. The 12α-CONH and -PhCONH of the host molecular tweezer 7d were the main binding sites, and F− ions were recognized by the intermolecular hydrogen bonding with hydrogen atoms at the amide N-H groups.In order to further clarify the recognition mechanism of the molecular tweezer 7d to F− ions in actual food samples. Based on the determination of F− ions in quinoa, Ribes stenocarpum Maxim, Chinese wolfberry, milk powder, and wheat flour, it was confirmed that a detection method for F− ions in actual food samples in less than an hour was possible. Compared with other reported methods, our method is more rapid and efficient, and the required instruments and equipment are of low cost with simple operation process. It has high selectivity for F− ions, and the recognition results are reliable. Therefore, based on our method, it is expected to develop F− rapid detection kit for its detection in food, environment, and other fields.The molecular tweezer 7d was applied to the detection of F


First, the activity of sEH on [3H]-trans-diphenyl-propene oxide (tDPPO) was assessed ex vivo using tissue homogenates . Second, the concentrations of sEH substrates and metabolites in plasma, milk, and urine were determined as an index of in vivo sEH activity. Oxidant status was assessed in serum and milk. Data were analyzed by non-parametric methods. Metabolism of tDPPO was greater in mammary tissues from cows with coliform mastitis compared to controls. In contrast, ratios of sEH substrates and metabolites predicted lower sEH activity in cows with coliform mastitis than controls. Milk oxidant status showed greater prooxidant levels in coliform mastitis cows. Cows with coliform mastitis exhibit increased sEH activity in mammary tissue; at the same time, milk oxidant status is increased. Future studies should characterize sEH activity and oxidant status patterns and explore therapies targeting sEH during coliform mastitis.Bovine coliform mastitis presents treatment challenges because of systemic inflammation and oxidative stress. Soluble epoxide hydrolase (sEH) is a promising therapeutic target in conditions characterized by inflammation and oxidative stress but has not been evaluated in cattle. We compared sEH activity and oxidant status in healthy Holstein dairy cows to those with systemic coliform mastitis ( Bovine mastitis continues to substantially hamper dairy producers economically due to decreased milk production, poor milk quality, and increased veterinary and labor costs associated with infection . In addiThe oxygenation of polyunsaturated fatty acid (PUFA) during acute coliform mastitis results in the formation of potent inflammatory mediators, known as oxylipids, that contribute to ROS production and oxidative stress ,7. The oThe epoxygenation of PUFA by the CYP450 pathway generates oxylipids known as epoxy fatty acids that regulate inflammation and mediate oxidative stress . Epoxy fn = 5/group) were enrolled in this case-control study. Systemic clinical disease was judged by the presence of clinical signs, including hyperthermia, tachycardia, tachypnea, episcleral injection, moderate to systemic dehydration, in addition to local mammary gland inflammation. Systemic coliform mastitis was based on at least 2 clinical signs and a positive culture of Escherichia coli (E. coli) in milk from infected mammary glands. Escherichia coli was identified based on typical colony characteristics and growth on MacConkey agar which is selective for coliform bacteria based on National Mastitis Guidelines -trans-diphenyl-propene oxide (t-DPPO) as substrate [final) of 50 µM. The mixture was incubated at 37 °C for 7 min for liver and kidney or 30 min for mammary gland tissue. The reaction was quenched by adding 60 µL of methanol and 200 µL of isooctane, extracting the remaining epoxide from the aqueous phase. Extractions of the stopped reaction with 1-hexanol were performed in parallel to assess the possible presence of glutathione transferase activity which could also transform the substrate [Frozen tissue samples were thawed on ice. After thawing on ice, the samples (approximately 5 g each) were mixed with 20 mL of chilled sodium phosphate buffer (20 mM pH 7.4) containing 5 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. The mixtures were homogenized with a polytron , 3-time 30 s pulse at 11,200× ubstrate . Brieflyubstrate . The actSample processing: Plasma and urine samples previously stored at −80 °C were thawed on ice. One mL of both 4% formic acid, 1 mL plasma sample, 20 µL of AOR agent, and 15 µL of internal oxylipid standards  were combined and mixed in glass tubes. Samples were then passed through SPE columns  that had been preconditioned with 12 mL of methanol followed by 12 mL of water. Samples, transferred to the glycerol-containing glass tube, were dried in a Speed Vac  at 45 °C heat for 105 min and at least 2 h run-time or until dried. Samples were left to cool to room temperature before the residues were resuspended in 150 µL of a 2:1 methanol and water mixture and set on ice. Samples were transferred into tubes with 0.2 µm bio-inert filters  and centrifuged at 14,000× g for 30 s to remove residual particulate material. Samples were transferred to chromatography vials with a 150 µL insert and stored at −20 °C until mass spectrometric analysis within 1 week. Milk samples were processed differently with a hydrolyzation step included to improve detection and quantification of oxylipids. Milk samples (0.5 mL) were mixed with 20 µL of AOR and 355 µL of 6 M potassium hydroxide and vortexed briefly to mix. The mixture was then incubated in a water bath at 45 °C for 45 min. After cooling to room temperature, sample mixtures were combined with 1.5 mL of 1% formic acid in acetonitrile, vortexed briefly, and centrifuged at 7359× g for 10 min at 4 °C. After centrifugation, the supernatant was carefully removed and transferred to a 50 mL conical tube containing 20 mL of HPLC grade water, and 15 µL of internal standards were added. Samples were then passed through SPE columns  that had been preconditioned with 12 mL of methanol followed by 12 mL of water. Columns were washed with 10 mL of 5% methanol and eluted into 16 mm × 100 mm glass tubes with 10 µL of 20% glycerol added. Elution was achieved by adding 6 mL of a 9:1 acetonitrile: methanol mixture and dried in a SpeedVac. Dried samples were processed and stored at −80 °C until mass spectrometric analysis like plasma samples.LC-MS/MS: The LC-MS/MS analyses of all plasma and milk samples were performed using a previously reported method [d method . The quad method . Ratios d method .The 10% neutral-buffered formalin-fixed tissues  were dehydrated, cleared, and infiltrated with paraffin on the VIP 2000 . Sections were placed on charged slides and dried at 56 °C overnight. Subsequently, samples were de-paraffinized, hydrated with distilled water, and followed by Tris-buffered saline (TBS) pH 7.4  for 5 min for pH adjustment. Heat-Induced Epitope Retrieval in Tris-EDTA pH 9.0 was performed using Dako Pascal Micro-Processor controlled pressure cooker  for 30 s at 125 °C and 10 s at 90 °C. Endogenous peroxidase was blocked in a 3% hydrogen peroxide/methanol bath followed by running tap water and distilled water rinses, followed by TBS + Tween 20  for 5 min. Following pretreatment, standard micro-polymer staining was performed at room temperature on the Biocare intelliPATH™ automated stainer . All staining steps followed by rinses in TBS auto wash buffer . Non-specific protein blocked using IP FLX Background Punisher  for 5 min; Rabbit Polyclonal anti-EPHX2 diluted 1:500 (LS-B6940)  in standard antibody diluent  and incubated for 1 h; followed by Rabbit on Farma HRP-Polymer™ detection  for 30 min. The reaction was developed with Betazoid DAB  for 5 min, followed by enhancement with DAB Sparkle  for 1 min. Counterstained with CATHE Hematoxylin diluted 1:5  for 5 min, dehydrated, cleared, and mounted with synthetic mounting media. Brightfield images were collected from 10 randomly selected fields on a Nikon Ni Eclipse upright microscope  using a 20× Plan Apo (NA 0.75) objective and configured with a D2-U3 Nikon Digital Sight color camera . The images were captured and analyzed using Nikon Elements AR software .p ≤ 0.05.Statistical analyses were performed using the SAS software . Data were checked for normality by the modified Levene test. Most of the variables were not normally distributed. Consequently, all data were analyzed using the non-parametric tests for unpaired samples using the Wilcoxon rank-sum procedure. Variables compared were sEH activity variables , oxidant status variables , mRNA expression , tissue IHC staining intensity (sEH), and the CBC and serum biochemistry parameters. Statistical significance was declared at p = 0.008). Serum biochemistry abnormalities were mostly detected in cows with coliform mastitis compared to control cows  metabolism of tDPPO, indicating greater sEH activity than control cows (p = 0.008) than from control cows, while metabolism by kidney tissues was not different (p = 0.310) between the groups  than controls (p = 0.03) in plasma from cows with mastitis compared to controls  in milk from cows with mastitis compared to healthy controls (p = 0.04) in cows with mastitis compared to control (p < 0.05) for the 11,12-EET/11,12-DHET pair relative to the healthy control cows (p = 0.0002) in cows with mastitis compared to healthy cows . The serum OSi in cows with coliform mastitis was lower than in serum from healthy control cows, suggesting a lower prooxidant status.  was measured, it is interesting to note the greater prooxidant status in milk and the greater mammary sEH activity in cows with coliform mastitis. Other studies linked mammary tissue gene expression changes to changes in oxidant status parameters in milk . Conversp > 0.05) in the IHC sEH staining intensity in tissues between cows with mastitis and controls (The mRNA expression agrees with observations in acute inflammatory murine models where in vivo depression of CYP450 isoforms and sEH gene expression was found in the various organs, including the liver and kidneys . We examcontrols , suggestThe specific relationship between oxidant status and sEH activity during coliform mastitis cannot be determined from our study because samples were collected only at the terminal stages of clinical disease. Our findings may not reflect cows in the early stages of the clinical disease when interventions may be beneficial. The decreased plasma OSi in cows with coliform mastitis differs from sepsis studies in humans that showed negative outcomes associated with progressive prooxidant production . SkeletaIn summary, we report, for the first time, a greater sEH activity in the mammary gland tissues during systemic bovine coliform mastitis at the same time when prooxidant production is increased in the mammary gland based on milk samples. The decreased hepatic and renal sEH activity in the cows of this study may suggest that terminal stages of the disease may not respond to therapy. Characterizing hepatic and renal sEH activities and tissue oxidant status require further investigation to determine optimal intervention strategies. We also report that based on the current methods of quantification and analyses, the oxylipid substrates and hydration products of sEH quantified in milk do not predict the activity of sEH in tissue as evaluated by ex vivo analysis. We, therefore, propose further exploration of the sEH activity patterns and how they correlate with changes in oxidant status to determine if targeting sEH modulates oxidative stress.In conclusion, we found that sEH activity is increased in mammary tissue but decreased in the liver and kidneys in cows at the terminal stages of systemic bovine coliform mastitis. The increased sEH activity in the mammary gland at the same time with the increased prooxidant status in milk during coliform mastitis may suggest a possible relationship between oxidative stress and the sEH activity. This finding may mean that therapies targeting the sEH activity may need to be targeted to the mammary gland, but this needs further exploration.


After these composite nanofibers’ exposure to drug-resistant bacteria, they exhibit dual antibacterial behaviors and efficiently kill the drug-resistant bacteria. These dual antibacterial nanofiber membranes with excellent adhesiveness may benefit the application of wound infection as antibacterial dressing.Bacterial infection especially caused by multidrug-resistant bacteria still endangers human life. Photodynamic therapy (PDT) can effectively kill bacteria, and nanofiber-based PDT can effectively reduce damage to normal tissues. However, current photosensitizers coated on the surfaces of fibers would release to the wound, causing some side effects. And nanofibers prepared by traditional method exhibit poor adhesion on the wound, which severely reduces the PDT effect due to its short-range effect. Herein, core–shell curcumin composite nanofibers are prepared by in situ electrospinning method via a self-made portable electrospinning device. The obtained composite nanofibers show superior adhesiveness on different biological surface than that of traditional preparation method. Upon 808-nm irradiation, these composite nanofibers effectively produced singlet oxygen (The online version contains supplementary material available at 10.1186/s11671-021-03513-2. Altho1O2) –19. Howe1O2) , 21, and1O2) , 23. In Electrospinning is a fast and efficient method to prepare nanofibers, including organic and inorganic nanofibers –28. Duri1O2. The UCNPs@Curcumin composite nanofibers are prepared by in situ electrospinning method via a self-made electrospinning device. The adhesion of the composite nanofibers obtained by this method on different biological surfaces is better than the traditional electrospinning preparation method. Upon 808-nm irradiation, these composite nanofibers can effectively produce 1O2 without curcumin falling off. After these composite nanofibers are contaminated with drug-resistant bacteria of MRSA, they will occur dual antibacterial behaviors that effectively kill the drug-resistant bacteria.In this study, curcumin is used as photosensitizers because of its wide sources from organism extracts. Core–shell nanostructure of UCNPs is used as wavelength transfer station, and it shows high conversion efficiency to produce Thulium chloride, ytterbium chloride, neodymium chloride, and yttrium chloride were purchased from Sigma-Aldrich. Methanol, ethanol, cyclohexane, curcumin, dichloromethane, acetone, polyvinylpyrrolidone (PVP), polycaprolactone (PCL), and polyethyleneimine (PEI) were bought from Sinopharm Chemical Reagents. All materials were used without further purification.4:Yb/Tm@NaYF4:Nd were synthesized using co-precipitate methods [Upconversion nanoparticles (UCNPs) of NaYF methods , 34. Aft4:Yb/Tm@NaYF4:Nd@Curcumin were added into 5 mL of acetone. After 12 h of stirring, a homogeneous precursor solution was obtained for electrospinning. Taking 3 mL of the precursor solution in 5-mL syringe, a self-made handheld electrospinning equipment was used for electrospinning, which consists of a 0.4-mm metal needle in diameter, two alkaline batteries, and a high-voltage converter that can convert 3 V of battery to 10 kV for electrospinning. The electrospinning distance between collector and electrospinning needle was about 10 cm.One gram of PCL, 0.16 g of PVP, and 0.1 g of NaYF1O2 formation. An 9 × 9 mm square of as-prepared nanocomposite fiber membrane with different concentration of UCNPs@Curcumin was added in a quartz cuvette, and then 3 mL of methanol containing 25 μM of SOSG was added. Afterward, the cuvette was irradiated under the 808-nm laser with different irradiation time. The fluorescence spectrophotometer with 504 nm of excitation wavelength was used to measure the fluorescence intensity of this solution, which reflects the singlet oxygen level.Singlet oxygen sensor green (SOSG) was utilized to detect the 6 CFU/mL. In total, 100 μL of bacterial solution was placed in each well of 96-well plate on a sterile ultra-clean table. Then, a piece of circular fiber membrane with 6 mm diameter was added to each well of the 96-well plate. After 20 min of 808-nm laser irradiation, the bacterial solution in the plate was diluted 10 times with sterile water. A 10 μL of diluent was placed in a nutritional agar plate to obtain an evenly coated agar plate. The treated agar plate was cultured in a constant temperature bacterial incubator at 37 °C for 18 h and then took photographs. In the control group, the steps were the same as above, except that 808-nm laser irradiation was not used. Each group was repeated with 5 plates.Drug-resistant bacteria of MRSA and Escherichia coli were used to evaluate the antibacterial ability. Briefly, bacterial strains were cultivated in the tryptic soy broth medium. The culture media containing bacterial strains were incubated at 37 °C for 15 h. After culturing, the concentration of bacterial strain was 1 × 10TEM and SEM images were taken from JEM-2010 and SU-1510 electron microscopes. Fluorescence spectrum was measured on Edinburgh FLS1000 fluorescence spectrophotometer. The absorption spectrum was recorded on Shimadzu UV2550 spectrometer. Fourier transform infrared spectroscopy was taken on Nicolet iS50 spectrometer. The zeta potential was measured with WJL-608 analyzer. The hydrophilicity with sessile drop method was tested by PT-602Atest equipment.4:Yb/Tm@NaYF4:Nd nanoparticles (UCNPs). It demonstrates a uniform size distribution of UCNPs with an average diameter of about 45 nm. The zeta potential of these nanoparticles was further tested to be + 19 mV , which origin from curcumin (green line). Meanwhile, there is a stretching vibration of C–N at 1125 cm−1, which comes from the PEI (blue line). Their molecular structure diagrams are illustrated in appendix , and this small distance was conducive to the generation of highly efficient fluorescence resonance energy transfer (FRET). The FRET method can obtain as high as 44.3% energy transfer efficiency, which can also benefit the following efficient production of 1O2.Figure ficiency . Thus, γ1O2, the SOSG method was used. First, we took nanocomposite fibers with a fixed doping concentration and observed the generation of 1O2 under different irradiation time. As shown in Fig. 1O2. The longer the irradiation time, the more 1O2 was produced. However, it also shows that although the concentration of 1O2 gradually increases with the increase in irradiation time, the rising rate slows down gradually and almost remains constant after 20 min, which is manifested by a dense curve interval. This phenomenon may be due to the fast local oxygen consuming by producing 1O2 with sustained NIR light radiations, resulting in a relatively low oxygen level in local area, and thus decreasing the rising rate of producing 1O2.In order to evaluate the ability of nanocomposite fibers to produce 1O2, Fig. 1O2 was produced. However, the rising rate of 1O2 slowed down when the concentration was larger than 0.20 wt%. These experimental results suggest that there is no need to infinitely increase the irradiation time and doping concentration to produce more 1O2. The optimal choice is 0.20 wt% with 20 min and thus in the following experiments will take this concentration and irradiation time.To observe the influence of doping concentration on producing 1O2 is a process that requires UCNPs@Curcumin nanoparticles in fibers to interact with oxygen in body fluid, thus the contact angle of this fiber membrane was further tested. Figure 1O2. In addition, considering that the short-range effect of PDT and the poor adhesion of the fiber membrane prepared by the traditional electrospinning method to the wound surface , and its structure diagram. Figure S3. Schematic illustration of energy transfer and pathway from NaYF4:Yb/Tm@NaYF4:Nd shell to NaYF4:Yb/Tm core under 808-nm laser excitation. Figure S4. Structural formula of curcumin, oleic acid, and PEI. Figure S5. Tensile test of curcumin composite nanofibers prepared by different method.


Advances in molecular technologies, from genomics and transcriptomics to epigenetics, are providing unprecedented insight into the molecular landscape of pediatric tumors. Multi-omics approaches provide an opportunity to identify a wide spectrum of molecular alterations that account for the initiation of the neoplastic process in children, response to treatment and disease progression. The detection of molecular markers is crucial to assist clinicians in accurate tumor diagnosis, risk stratification, disease subtyping, prediction of treatment response, and surveillance, allowing also for personalized cancer management. This review summarizes the most recent developments in genomics research and their relevance to the field of pediatric oncology with the aim of generating an overview of the most important, from the clinical perspective, molecular markers for pediatric solid tumors. We present an overview of the molecular markers selected based on therapeutic protocols, guidelines from international committees and scientific societies, and published data. Recent decades have witnessed an intensive development of molecular research, which has contributed significantly to a more complete understanding of the molecular basis of childhood cancers. The published studies reveal a high heterogeneity of molecular alterations that account for the initiation of the neoplastic process, response to treatment and disease progression. These findings clearly indicate that the profile of molecular changes occurring in childhood malignancies differs significantly from the one observed in adult cancers. Thus, it is not possible to directly extrapolate the knowledge and experience with molecular markers from adults to the pediatric population. The differences observed pertain not only to the molecular basis, but also to the disease itself, its anatomical site and its histopathological features demanding the development of a different diagnostic and therapeutic approach for this group of patients.Knowledge of specific childhood cancer genetic alterations present in tumor cells (somatic markers) as well as germline alterations is useful throughout the entire diagnostic and therapeutic process. There are genetic markers specific for histological types of cancer which are necessary for making a correct diagnosis (a group of diagnostic markers). Furthermore, there are molecular markers that correlate to the tumor’s grade, allowing us to predict the course of disease and prognosis (a group of prognostic markers) as well as to predict responses to a specific treatment (a group of predictive markers). Most promising are the molecular markers for targeted therapies. Extensive research on the biology of cancers, including their molecular profile, has influenced the current way of practicing medicine in the field of cancer diagnosis and treatment as well as the development of the so-called “personalized or precision medicine”. In recent years, there has been an attempt to change the paradigm of anticancer treatment, which assumes that the occurrence of specific molecular alterations may determine the efficacy of the administered treatments irrespective of the site and histological type of the tumor.In addition to the changes occurring in the neoplasm itself, which are used as targets for therapy, alterations identified in the genetic material from patients’ peripheral blood  are also important. They allow us to determine whether the occurrence of a particular cancer is caused by the presence of a molecular defect. If the identified molecular alteration may have been inherited, assessing its presence in other family members enables us to identify individuals at a higher-than-average risk of developing cancer. These individuals should be screened systematically for early cancer detection. Germline alterations can also constitute prognostic and predictive markers.Central nervous system (CNS) tumors (~20–23% *),Neuroblastoma (8–10% *),Wilms tumors, (7–8% *),Malignant bone tumors, (osteosarcoma and Ewing tumor) (~7% *),Soft tissue sarcomas (~7% *),Germ cell tumors (3–6% *),Hepatoblastoma, rarely hepatocarcinoma (0.5–2% *),Retinoblastoma (2.5–3% *),Other malignant epithelial neoplasms and malignant melanomasOther and unspecified carcinomas.Childhood cancers are rare, but they remain the second-leading cause of death in the pediatric population. Their incidence is 14–15 cases per 100,000 per year. They account for about 1–1.5% of cancers in the general population. Solid tumors account for approximately 60% of all childhood malignancies. Their broad spectrum includes ,3: Centr* of all malignant neoplasms of developmental ageSince the mid-1970s, the cure rates for most pediatric solid tumors have increased by as much as 50% . At presNTRK fusion solid tumors and Crizotinib for children and young adults with relapsed or refractory systemic anaplastic large-cell lymphoma received marketing authorization. Phase 2/3 studies with Crizotinib in children and adolescents with recurrent, progressive, and unresectable inflammatory myofibroblastic tumors are forthcoming. In recent decades personalized treatments based on molecular markers have been developed for adults with cancer. Nevertheless, not enough progress has been made in the development and authorization of targeted therapies for childhood malignancies. Though molecular markers are routinely used in diagnosis, establishing risk groups in pediatric oncology, and novel medicinal products are being developed, with some exceptions, few breakthrough medicines have come to the market for children. Only few such medicinal products received marketing authorization for the treatment of pediatric malignancies. Among them are ABL-class inhibitors for Philadelphia positive acute lymphoblastic leukemia, anti-CD antibodies for B cell lymphomas, and anti-GD2 antibodies for children with high-risk neuroblastoma. Recently, Larotrectinib for children with Moreover, genetic data are lacking on the difficulty to treat refractory or relapsed solid tumors, limiting our knowledge of the molecular composition of such entities. To further improve cure rates in pediatric cancer it is essential to continue research and targeted medicine trials using tumor molecular profiling in children and adolescents. This article describes a spectrum of molecular markers of clinical relevance in pediatric solid tumors. The markers were selected based on therapeutic protocols, guidelines from international committees and scientific societies, and a review of the current literature.Central nervous system (CNS) neoplasms are the most common solid tumors in children and a leading cause of childhood cancer-related deaths. Moreover, out of all survivors of childhood cancer, patients cured from CNS tumors present with the most severe treatment-related health conditions. At present, CNS neoplasms are the greatest challenge for pediatric oncology in its broad diagnostic and therapeutic aspects. Nevertheless, it is molecularly the best-understood group of childhood solid tumors. The results of multi-omics research led, amongst other things, to the definition of molecular subgroups in medulloblastoma, which have solid clinical implications . There are also other CNS tumors with molecularly defined subgroups.The clinical validity of molecular markers in diagnostic management has already been demonstrated in the WHO 2016 classification of central nervous system tumors, and their role was further emphasized in the guidelines prepared by an international consortium cIMPACT-NOW . In the latest WHO 2021 classification of tumors of the CNS, molecular markers are fundamental for making a proper diagnosis .At the same time, there has been an increase in the number of molecularly targeted drugs in cancer. The selection of optimal therapy absolutely requires the assessment of specific molecular alterations .A set of clinically relevant molecular markers for pediatric central nervous system tumors is provided in Neuroblastoma (NBL) is the most frequent pediatric extracranial tumor originating from neural crest progenitor cells. It accounts for approximately 10% of all childhood malignancies and for up to 15% of deaths in children from cancer . It is tMYCN amplification status and the presence of characteristic chromosomal aberrations  and the INRG  .A set of molecular markers clinically relevant for neuroblastoma is provided in clear cell sarcoma of kidney (CCSK),renal cell carcinoma associated with MiTF/TFE translocations,malignant rhabdoid tumor of kidney (MRTK),congenital mesoblastic nephroma (CMN), and others.Wilms tumor  is the most common pediatric renal malignancy, accounting for over 90% of renal tumors. Other less frequently occurring malignancies of the kidney include:The incidence of Wilms tumor is approximately 7 cases per 1 million children younger than 15 years of age, accounting for 5–7 percent of all childhood malignancies. WT can occur in both kidneys , found in 5–8% of cases. The mean age at diagnosis is 44 months in unilateral cases and 31 months for bilateral cases of Wilms tumor. Wilms tumor is rare in patients older than age 15. A total of 1.5% of cases occur in related family members. Approximately 5% of WTs are associated with known constitutional predisposition syndromes.WT1, CTNNB1 or AMER1 (WTX) genes are observed in about one-third of Wilms tumor cases ..DICER1 , in childhood malignancies is much lower than in adults. With respect to germline alterations, the opposite is true. The occurrence of childhood cancers is more often determined by the presence of alterations responsible for genetic syndromes. More than 200 such syndromes have been identified, and this number is steadily growing. The fact that congenital cancers are also diagnosed underscores the significance of germline alterations in the pathogenesis of childhood cancers. It is estimated that approximately 7–8% of hematologic malignancies and solid tumors in children are determined by germline alterations . FurtherTP53 gene. Patients with germline RB1 alterations who underwent radiotherapy have twice the risk of developing secondary cancers compared with patients who did not receive radiotherapy [NF1 gene in patients with low-grade gliomas is associated with a better prognosis; hence, the treatment undertaken may be less aggressive [MLH1, MSH2, MSH6, and PMS2 with brain cancers require more aggressive chemotherapy [SMARCB1 alterations is 6 months [Assessment of the presence of germline alterations is also important in optimizing therapeutic management. If patients present with lesions that result from chromosomal instability or that occur in DNA repair genes, the omittance of radiotherapy or a reduced radiation dose are recommended. One such example is children with choroid plexus carcinoma and germline alterations in otherapy . The pregressive . Howeverotherapy . The pre6 months ,98. TherThe following tables  present The starting point for most oncogenetic tests used is a tumor tissue sample. Formalin fixation and paraffin embedding (FFPE) is the most common form of tissue preservation; however, for NGS-based protocols, tissue preservation by freezing is much more beneficial. In selected tumors , the impression smear of tumor tissue may serve as the material for genetic assay. To identify germline alterations, the patient’s peripheral blood, saliva and/or buccal swab samples are also collected in addition to the tumor tissue.Due to the heterogeneous nature of tumors (particularly neuroblastoma and selected gliomas), it is recommended that at least two specimens be collected from the respective tumor tissue. In cases of tumor recurrence and probable changes in the molecular profile of the relapsed tumor tissue, resampling is indicated.Each tumor tissue specimen for molecular assay should be evaluated for the percentage of tumor cells in the tested specimen. This assessment is routinely performed by a pathologist. If more than one biological sample is available, the most appropriate sample should be selected based on the type of molecular assay planned, the availability of the biological material and the need for it at subsequent stages of diagnostic process.It is also very important to maintain sterility when collecting material for molecular assays. NGS methods can detect mosaic-type alterations at very low levels. In cases of contamination of the specimen with even a very small amount of material from another patient, false results may be obtained.The rapid development of high-throughput next-generation sequencing methods has significantly contributed to the understanding of the molecular profile of the most common pediatric cancers. Today, we know that, on the one hand, these tumors exhibit a great variety of molecular alterations, but, on the other hand, their total number is small compared to the number of somatic alterations detected in tumors occurring in adults. This is reflected very frequently in the low TMB score, which translates into limited applicability of immunotherapy. Effective immunotherapy can also be limited by the suppressive tumor microenvironment with relatively few effector cells. Generally, this type of therapy in pediatric solid tumors still remains in the early stages of development and significant clinical benefit has yet to be demonstrated.In contrast, epigenetic changes occupy a special place in the vast spectrum of molecular alterations that are identified in childhood cancers. It seems that this type of alteration is crucial for the initiation of carcinogenesis-related processes. A confirmation of this assumption is the occurrence of different genome DNA methylation patterns in different tumor subtypes. A unique epigenetic signature that represents both the tumor origin and the presence of acquired oncogenic alterations affecting chromatin state constitutes a very promising diagnostic tool for, among others, central nervous system tumors in children. The classification system developed by German National Cancer Institute (DKFZ) in Heidelberg, based on the methylome pattern  is slowlThere is a growing number of published studies demonstrating the important role of liquid biopsy in the diagnostic and therapeutic management of a number of cancers, including pediatric malignancies. It is a complementary or alternative method to surgical biopsy, as well as a non-invasive, promising tool for early cancer detection, that may also overcome problems of tumor accessibility and heterogeneity of tumor tissue. Various biological fluids, including peripheral blood, urine, cerebrospinal, synovial and ocular space fluids, can be used to obtain such circulating material as tumor cells (CTCs), tumor DNA (cfDNA), RNA (cfRNA), proteins and extracellular vesicles (EVs) for diagnostic assays. Thanks to the advances in technology, it is possible to obtain and analyze such biological material with increased effectiveness. There are data demonstrating the usefulness of liquid biopsy in variety of analysis including small- and large-scale mutation analysis, high throughput sequencing technologies, and analysis of structural or copy number alterations. From a clinical perspective, the results from liquid biopsy can provide reliable data as to the status of the disease and allow us to monitor treatment and to evaluate predictive, prognostic and resistance markers. In some cases, it may be helpful in early detection of recurrence. Thus, liquid biopsy, although still a relatively new method, appears to be a significant application for cancer diagnosis and treatment. The results of published research on neuroblastoma, sarcoma, Wilms tumor, hepatoblastoma and retinoblastoma appear to be very promising . NeverthThe introduction of technologies such as massively parallel DNA sequencing and RNA sequencing, as well as tools for the interpretation of the vast amounts of data obtained with these methods, including bioinformatic or crystallographic methods, creates an opportunity to elucidate the molecular mechanisms of childhood cancers and to develop targeted therapies. Artificial intelligence methods are also becoming increasingly employed to design therapeutic algorithms and identify prognostic and predictive markers . The int