Upon examination, there were no significant findings, specifically no neurological abnormality.We present a 1-year-old boy who presented to the emergency department with a 7-day history of diarrhoea and vomiting. The initial renal function profile demonstrated a urea of 55 mmol lHe was prescribed back-to-back Salbutamol nebulisers, to increase the shift of extracellular potassium into the intracellular space, followed by i.v. calcium gluconate, with some improvement in potassium levels. A further 5 mmol of sodium bicarbonate was given, as well as a stat dose of 1 mg/kg furosemide, and per rectal calcium resonium. He was then commenced on an infusion with 10% dextrose with insulin.He was subsequently found to be in urinary retention and a catheter was inserted, which drained 1700 ml.−1, creatinine 300 micromol/L and potassium 3.0 mEq/L.A subsequent renal function profile, 24 hours after admission, demonstrated improvement with urea 39 mmol l The child had an initial ultrasound examination of the renal tract, demonstrating a heterogeneous prostatic mass, deemed to be causing bladder outlet obstruction .These findings were further reinforced after an MRI abdomen and pelvis with contrast was performed .The histological samples taken from the prostate gland confirmed morphological and immunohistochemical appearances in keeping with rhabdomyosarcoma.The patient underwent an MRI head examination 7 days after admission , at whicFollow-up imaging  2 months1 UE has been well-documented in the adult population,2 but appears to be very rare in the paediatric population3,4. After a detailed literature review, to our knowledge, there are no case reports describing bilateral basal ganglia (BG) involvement in a non-diabetic, paediatric patient with no prior history of renal impairment.UE is an uncommon, acquired toxic metabolic disorder, associated with both acute and chronic renal failure. The cause is attributable to the accumulation of uremic toxins, such as creatinine and guanidine, which have the ability to activate the neurotoxic effect of NMDA receptors.5 The BG require a steady supply of adenosine triphosphate (ATP) to maintain transmembrane ion gradients, vital for their neuro-transmissive role. If the normal process of its generation, via aerobic respiration, is interrupted, as seen in conditions such as acute toxic metabolite accumulation, hypoxia, hypoglycaemia and carbon monoxide (CO) poisoning, then insufficient ATP production occurs via anaerobic mechanisms.5The BG, the primary location of abnormality in this case, consist of the caudate nuclei, putamina and globus pallidi; complex structures that control extrapyramidal motor activity.2 Firstly, the most common has cortical involvement, and is a category of posterior reversible encephalopathy syndrome 6,7 ; the second is rarer and demonstrates bilateral BG involvement2 ; and the final one has white matter involvement, limited to case reports.6 Very rarely, as described in a case report concerning a 14-year-old girl by Jia et al., there can be brainstem involvement.3 The ‘Lentiform Fork Sign ’ is characteristically associated with metabolic acidosis and blood-brain barrier disruption. It is demonstrated as a hyperintense T2 rim delineating the medial  and lateral  boundaries of the putamina bilaterally.2 Restricted diffusion, suggestive of cytotoxic oedema, can occur in UE, but is usually limited to the globus pallidi, as it is more vulnerable to cytotoxic damage than the putamina, due to higher ATP requirements.2 Post-contrast enhancement is also not a typical feature. In most cases, lesions on neuroimaging improve or resolve upon normalisation of uremia.6UE has 3 principal patterns of imaging findings, best demonstrated as increased signal on T2 and FLAIR MRI sequences bilaterally, in keeping with vasogenic oedema from the accumulation of interstitial fluid, following a disruption of autoregulation.8In the acute setting, with bilateral symmetrical BG pathology, as demonstrated in this case, one must also consider causes such as hypoxia, hypoglycaemia, CO poisoning, haemolytic uremic syndrome and osmotic myelinolysis, in the presence of appropriate history and laboratory findings.9 and possibly associated haemorrhage.In hypoxia, the acute deprivation of oxygen means anaerobic metabolism predominates. This results in the formation of lactic acid, which changes the intracellular environment, resulting in diffuse BG injury,10 Interestingly, it has an affinity for globus pallidi injury, although the remaining BG may also be affected. Moreover, there is cerebral white matter injury, which in fact determines the overall prognosis.10Hypoglycaemia, as opposed to hypoxia, does not produce lactic acidosis as there is a different mechanism of injury. However, the endpoint of both processes, namely ATP depletion, is similar. The radiological findings include high T2 signal change bilaterally within the posterior limbs of the internal capsules, BG, cerebral cortex  and hippocampi. CO poisoning impairs oxygen delivery to the BG, predominantly by competitive binding to haemoglobin.11Haemolytic-uremic syndrome is a rare and multisystem disorder, with most patients presenting before the age of 5 years. It is characterised by microthrombosis, acute renal failure, thrombocytopenia and microangiopathic haemolytic anaemia. Vascular occlusion results in impaired delivery of both oxygen and glucose to the BG, leading to decreased ATP production and ultimately BG injury.12 There was no history of rapid sodium correction in this case.Osmotic myelinolysis, which can occur following the rapid correction of sodium concentrations, typically affects the pons. However, the BG can be affected (extrapontine myelinolysis), with symmetrical high T2 signal change affecting the lentiform nuclei bilaterally.13 The mean age of cancer diagnosis, however, is 2.5 years, which is significantly older than our patient.13 Intracranial manifestations of NF1, specific to the BG, include focal areas of signal intensities (FASIs), which are focal areas of high T2 signal with no mass effect or contrast enhancement, representing myelin vacuolisation, which may wax and wane for the first decade of life.14 These lesions favour the globus pallidi15 and are usually not symmetrical, hence differing from the features seen in our case. Reversal of the signal changes in a relatively short time period, as seen in this case, is also unusual in NF1.The key, non-acute differential diagnosis to consider with these imaging findings, is NF1 or von Recklinghausen disease, as there is an association with rhabdomyosarcoma.This case highlights bilateral and symmetrical high putaminal T2 signal change in a 1-year-old boy with acute renal failure and hyperuremia secondary to prostatic rhabdomyosarcoma, resolving upon correction of the abnormal urea levels, thus suggestive of UE. Other acute causes of bilateral BG changes were considered, but these did not correlate with the clinical history or specific laboratory findings. Rhabdomyosarcoma is associated with NF1, but the MRI findings do not support this diagnosis.UE can be associated with the ‘Lentiform Fork Sign’ in MRI imaging.The abnormal MRI findings resolve upon correction of the raised urea levels.It is important to consider other acute causes of bilateral BG radiological abnormalities, including hypoxia, hypoglycemia, CO poisoning and osmotic myelinolysis.


Two-dimensional (2D) materials are widely used in microelectronic devices due to their excellent optical, electrical, and mechanical properties. The performance and reliability of microelectronic devices based 2D materials are affected by heat dissipation performance, which can be evaluated by studying the thermal conductivity of 2D materials. Currently, many theoretical and experimental methods have been developed to characterize the thermal conductivity of 2D materials. In this paper, firstly, typical theoretical methods, such as molecular dynamics, phonon Boltzmann transport equation, and atomic Green’s function method, are introduced and compared. Then, experimental methods, such as suspended micro-bridge, 3ω, time-domain thermal reflectance and Raman methods, are systematically and critically reviewed. In addition, the physical factors affecting the thermal conductivity of 2D materials are discussed. At last, future prospects for both theoretical and experimental thermal conductivity characterization of 2D materials is given. This paper provides an in-depth understanding of the existing thermal conductivity measurement methods of 2D materials, which has guiding significance for the application of 2D materials in micro/nanodevices. The thermal conductivity of 2D materials is of great significance for both basic research ,7,8,9,10Various theoretical calculation methods such as molecular dynamics simulation ,15,16,17The theoretical calculation is an effective way to deeply understand the potential mechanism of phonon transport in 2D materials. Currently, the molecular dynamics (MD) simulation, phonon Boltzmann transport equation (PBTE), and atomistic Green’s functions (AGF) were the 3 mainstream theoretical methods.In MD simulation, the motion of each particle in the dynamic process was described based on Newton’s second law, and the position, velocity, and force of each atom were calculated at each step. The interatomic forces were derived from the potential function, and the commonly used empirical potential functions were Lennard–Jones (LJ) potential for interlayer van der Waals (vdW) interaction, Stillinger–Weber (SW)  potentiat,T is the absolute temperature of the system, V is the volume and In EMD method, the thermal conductivity is expressed as the integration of the heat current autocorrelation function (HCACF) with respect to a given correlation time For the MD method, one advantage is that the simulation is based on a real physical model in space, which makes it convenient to study the effects of physical parameters, such as strain, defect, doping, etc. However, the accuracy of MD simulation is highly dependent on the potential empirical function used, which is usually developed by fitting the existing material properties. Moreover, as the MD method is modeled in real space, the calculation range is limited due to the simulation time, which makes the calculation of thermal conductivity not very accurate. In addition, the phonon scattering rate in MD is related to the Maxwell Boltzmann distribution while ignoring the quantum effect below the Debye temperature. Thus, erroneous results were obtained for the calculated thermal conductivity below the Debye temperature .2 2  . Additio= S, Se)  implemenCompared with the complex measurement device of TDTR, Raman-based methods are simpler and have been widely used in the thermal conductivity measurement of 2D materials. By constructing different heat transfer states in the time and space domain, various Raman-based measurement methods were developed. Among them, the optothermal Raman method based on steady-state heating is the most commonly used.2 sample is suspended on a Si2N4 substrate and heated by a focused laser light. The heat can only diffuse around the sample and eventually to the substrate. As shown in 2 is linearly related to the local temperature change  , which uIn TD-Raman, thermal conductivity of 2D materials is measured through the Raman characterization of transient heat transfer. However, in practice, when the heating time is too short, a long time of Raman signal acquisition is needed, which makes it hard for fast thermal transport characterization produces more environmental interference and affects the measurement accuracy. To solve this problem, Wang’s lab further developed frequency-resolved (FR) Raman technique Frequency-resolved Raman for transient thermal probing and thermal diffusivity measurement . As showosphorus . CompareD). The subsequent process is the heat conduction by phonons, which receives energy from the hot carriers or electron–hole recombination, which mainly happens in the in-plane and depends on the thermal conductivity (κ). The third is the heat conduction from sample to substrate, and this process is dominated by the local thermal resistance (R).Besides the time-domain modulation, the energy transport states can also be modulated in the spatial domain. Based on this, a technique named ET-Raman was developed to measure the in-plane thermal conductivities of supported or suspended 2D materials. For supported 2D samples, both a CW laser and a picosecond laser are used. As shown in P), a parameter named Raman shift power coefficient (RSC) can be obtained and expressed as: D, R, laser absorption coefficient and temperature coefficient of Raman shift. According to the 5 heating states in D and R. D, R). Yuan et al. measured that the in-plane thermal conductivity spans from 31.0 to 76.2 W/(m·K) of 2D few layers MoS2 samples (thickness ranging from 2.4 nm to 37.8 nm) supported on a glass substrate by 5 state picosecond ET-Raman method.By using different laser power  as the sample thickness increased from 13 nm to 107 nm with nanosecond ET-Raman technique.Due to the short pulse interval, the picosecond laser, which would generate heat accumulation in the suspended structure, is replaced by a nanosecond laser. As shown in se laser . The infκ of MoS2 and MoSe2 nanofilms and phonon coupling factors [Since the Raman signal comes from optical phonons (OPs), but the heat transfer in the sample is related to acoustic phonons (APs). Considering the measurement error caused by ignoring the temperature difference between the 2 phonons, Wang et al. developed 6 heating states nanosecond ET-Raman technique by changing the objective lens: 3 steady states and 3 transient states, and realized the measurement of intrinsic  factors . As showIn order to compare the different experimental methods for measuring the thermal conductivity of 2D materials much more conveniently, these methods are summarized in The thermal conductivity of 2D materials is affected by many factors, such as length, thickness, temperature, substrate, strain, and so on. These factors will affect the process of phonon transmission and scattering and further affect the thermal conductivity.Unlike bulk materials, the thermal conductivity of nm-thick 2D materials usually exhibits an abnormal size dependence. In Zhang’s work , the in-λ) is larger than the length (l) of the system, heat transfer is ballistic. Certain phonon modes can transmit from the heat-source to the heat-sink without scattering. When l on small scales. In order to extract the thermal conductivity The factor for the size-dependent thermal conductivity originates in phonon scatterings at the sample boundaries. When the phonon mean free path  analysis, it was found that substrate coupling inhibits the thermal transmission of ZA phonons, resulting in a significant reduction in thermal conductivity [100MoS2 monolayer was 50% higher than that synthesized from naturally abundant isotope mixtures. They attribute this to the former having fewer defects, which reduces phonon-defect scattering [As the main heat carrier in 2D materials, the propagation of phonons can be adjusted by many other factors, such as lattice deformation caused by strain, substrate coupling, isotope-engineering, which leads to the change of thermal conductivity. Zhang et al. reported that a small strain has a positive effect on the heat conduction of monolayer h-BCN . With fuuctivity . In addiattering .In this paper, we systematically introduce the theoretical and experimental methods for the thermal conductivity measurement of 2D materials. The basic principles, advantages, and disadvantages are discussed in detail. Furthermore, some factors  that affect the thermal conductivity of 2D materials are also introduced. Based on the thorough analysis, there are many works to conduct to further develop the theoretical and experimental methods.For the theoretical methods, the accuracy can be further improved by taking more parameters of actual materials into consideration. For example, the growth of 2D materials obtained by chemical vapor deposition (CVD) is controlled by macro physical conditions and parameters, such as partial pressures of each gas in the CVD environment, substrate, defects, furnace configuration, temperature conditions, and gas-phase reactions. One idea is to use growth kinetics and parameter settings to establish the growth model for describing the growth mechanism of the material, which makes the established model consistent with the actual growth model. Netto et al. have reported the continuous growth process of CVD diamond films using time-dependent Monte Carlo algorithm with the chemical reaction mechanism . RecentlFor experimental methods, there is also a lot of work to conduct. For suspended micro bridge devices, the contact thermal resistance is an important factor affecting the accuracy of measurement results, which is quantified in the subsequent development of electron beam self-heating method . Further


MLH1, MSH2, PMS2 or MSH6, and typically include truncating variants, such as frameshift, nonsense or splicing variants. However, a significant number of missense, intronic, or silent variants, or small in-frame insertions/deletions, are detected during genetic screening of the MMR genes. The clinical effects of these variants are often more difficult to predict, and a large fraction of these variants are classified as variants of uncertain significance (VUS). It is pivotal for the clinical management of LS patients to have a clear genetic diagnosis, since patients benefit widely from screening, preventive and personal therapeutic measures. Moreover, in families where a pathogenic variant is identified, testing can be offered to family members, where non-carriers can be spared frequent surveillance, while carriers can be included in cancer surveillance programs. It is therefore important to reclassify VUSs, and, in this regard, functional assays can provide insight into the effect of a variant on the protein or mRNA level. Here, we briefly describe the disorders that are related to MMR deficiency, as well as the structure and function of MSH6. Moreover, we review the functional assays that are used to examine VUS identified in MSH6 and discuss the results obtained in relation to the ACMG/AMP PS3/BS3 criterion. We also provide a compiled list of the MSH6 variants examined by these assays. Finally, we provide a future perspective on high-throughput functional analyses with specific emphasis on the MMR genes.Lynch syndrome (LS) is one of the most common hereditary cancer predisposition syndromes worldwide. Individuals with LS have a high risk of developing colorectal or endometrial cancer, as well as several other cancers. LS is caused by autosomal dominant pathogenic variants in one of the DNA mismatch repair (MMR) genes  The integrity of the human genome is continuously challenged by exogenous and endogenous factors that cause DNA damage. Endogenous factors include errors committed by the replicative polymerases, which misincorporate nucleotides, leading to base-base mismatches, as well as insertion/deletion loops that occur when the polymerase slip at repeat sequences, such as microsatellites. The DNA mismatch repair (MMR) system corrects these mismatches, as well as the insertions/deletions . The corMLH1 and MSH2, whereas most variants in CMMRD are found in PMS2 and, to a lesser extent, in MSH6, followed by MLH1 and MSH2 [Heterozygous pathogenic variants in the MMR genes predispose to Lynch syndrome , also known as hereditary nonpolyposis colorectal cancer (HNPCC), while homozygous or compound heterozygous pathogenic variants lead to constitutional MMR deficiency  syndrome. While LS is one of the most common hereditary cancer predisposition syndromes, with a frequency of 1:300 individuals ,14,15. Tand MSH2 . Since tand MSH2 . In contand MSH2 .MSH6 gene is located on chromosome 2, spanning 24 kb genomic sequence and consists of 10 exons, encoding a 1360 amino acid (aa) protein. The MSH6 protein has the following five functional domains: the mismatch binding, connector, lever, clamp, and ATPase domains. MSH6 is pseudosymmetric with MSH2, which has a similar domain structure [The tructure . Even thtructure . In additructure . Howevertructure ,21. The tructure . Moreovetructure .MSH6 interacts with MSH2 at two regions, aa 326 to 575 and aa 953 to 1360, and form the MutSα heterodimer . The hetThe MutSα complex is responsible for mismatch recognition A. This cMSH6 gene.Genetic testing has evolved from small gene panels based on phenotype to multigene panel testing, exome and whole-genome sequencing on a broader spectrum of cancer patients, which has resulted in an increase in the number of variants that need to be classified. Variants such as frameshifts and nonsense are relatively easy to interpret and are often classified as pathogenic. Unfortunately, a significant number of identified variants are missense, intron or silent variants, or small in-frame insertion/deletions, where the biological effects are more difficult to predict. Several tools can be used in the evaluation of MMR variants, including prediction programs that estimate the effect of a variant on the protein or mRNA level, databases with population frequencies, as well as databases such as the International Society for Gastrointestinal Hereditary Tumours (InSiGHT) and ClinVar . InSiGHTIn 2008, a five-tier variant classification scheme was created, which classified benign variants as class 1, likely benign variants as class 2, variants of uncertain significance (VUS) as class 3, likely pathogenic variants as class 4, and pathogenic variants as class 5 . The schMSH6 gene has been reported to have a higher number of VUS compared to the other MMR genes [MSH6 gene.Unless substantial evidence points towards the variant being either pathogenic or non-pathogenic, the variant is—as stated above—classified as a VUS. Since the cancer risk of a VUS in the MMR genes is unknown, the clinical management of an individual with this type of variant is difficult, and relatives are not offered predictive testing. The MR genes , and curIn the following sections, we describe different functional assays that have been used for the reclassification of VUS identified in MSH6.On the protein level, the key functional assay for variants in MSH6 is to measure the effect on MMR activity. Several, rather similar, in vitro MMR activity assays have been developed through the years, which all include a circular DNA substrate with a base:base mismatch or a small insertion in an endonuclease recognition site, as well as a single-stranded nick to direct the repair process. The MMR substrate is then added to variant MSH6 protein supplemented with wt MSH2, as well as cellular extract from an MSH6-deficient cell line. The correction of the mismatch will re-generate the cleavage site, and the degree of MMR can subsequently be determined by gel electrophoresis and compared to the MMR activity of wt MSH6. In total, we have collected data from nine MSH6 MMR activity publications from three different research groups.The Nyström group used an MMR substrate that was developed from the pGEM phagemid, with a total extract of Sf9 insect cells overexpressing the MSH6 variant as well as wt MSH2 proteins, and supplemented this with nuclear extract from MSH6-deficient HCT15 or LoVo cells. As negative controls, nuclear extract from MMR-proficient TK6 or HeLa cells, as well as the MMR-deficient cell line supplemented with purified wt MSH6, were used. The group analyzed the MMR activity of 16 MSH6 variants . Four ofIn 2012, Geng et al. analyzed the activity of a single MSH6 variant, p.(Thr1219Asp), using an assay based on an MMR substrate that was derived from Wang and Hays ,46, as wIn 2012, Drost et al. published their first assay, in which they measured the activity of MSH6 variants . SimilarMsh6+/− mESCs, where one Msh6 allele was inactivated by insertion of a puromycin resistance gene [The 6-thioguanine (6-TG) selection assay in mESCs was first developed to characterize MSH2 variants , and wasnce gene . The assnce gene .Msh6+/− mESCs. (2) The mESCs are then subjected to selection with 6-TG, a purine analog that, when incorporated, creates lesions that are recognized by the MMR system. However, the MMR machinery cannot repair the lesions, resulting in replication arrest and cell death in MMR-proficient cells, but not in MMR-deficient cells [Msh6 alleles was performed. (4) Finally, 6-TG-resistant mESCs that maintained both Msh6 alleles were sequenced, to confirm the presence of the variant of interest. If no 6-TG-resistant mESC colonies contained the desired variant, the screen was repeated with a sense-oriented LMO.The assay consists of the following four steps: (1) Site-directed mutagenesis is performed with antisense-oriented short single-stranded locked nucleic acid-modified DNA oligonucleotides (LMOs), to introduce variants of interest into nt cells ,56,57. , but previously classified as pathogenic and non-pathogenic, respectively, using the 6-TG selection assay . The assNext, a total of 28 VUS, consisting of 27 missense variants and one intron variant (c.3438+6T>C), selected from the literature, the InSiGHT database or a clinical cohort, were investigated ,54. Of tIn agreement with its role in MMR, MSH6 is shown by immunohistochemistry analysis to be predominantly localized within the nucleus . During To examine whether a variant influences the subcellular location of MSH6, the localization of a fluorescent protein-tagged variant and wild type (wt) MSH6 in transfected cells can be examined by confocal laser scanning microscopy. The variants are then scored as nuclear, cytosolic, or nuclear/cytosolic. This has been carried out for several MLH1 and MSH2 variants ,63. CurrTwo papers, by Cyr and Heinen, and Geng et al., present an array of biochemical assays to evaluate the specific mechanistic malfunctions of MSH6 variants ,65. The Geng et al. studied a single MSH6 variant, p.(Thr1219Asp), together with wt MSH2 purified from co-expressed Sf9 insect cells . They foCyr and Heinen investigated seven MSH6 missense variants ((p.(Ser144Ile), p.(Ser285Ile), p.(Gly566Arg), p.(Asp803Gly), p., p.(Arg976His), and p.(His1248Asp)) . SimilarOverall, Cyr and Heinen found that five of the seven investigated MSH6 variants affect steps in the MMR mechanism . HoweverBased on in silico data, it is estimated that approximately 15%–25% of exonic, pathogenic variants mediate their effect via altered RNA splicing ,67,68. Tde novo splice sites, resulting in partial exon skipping or partial exon inclusion, while other variants result in intron retention [The most common splicing aberration involves disruption of the canonical splice donor or splice acceptor sites. Such variants may result in exon skipping. Variants might also activate cryptic splice sites or create etention ,73.MSH6 transcript isoforms [In silico splicing prediction tools are widely used to predict the effect of a variant on splicing , and areisoforms ,75,85,86Finally, one synonymous variant ) resulted in the creation of a de novo splice donor site, while two truncating variants  and c.3991C>T, p.(Arg1331*)) interestingly resulted in exon skipping, by affecting yet undefined exonic splicing enhancer elements .MSH6 germline variants can be classified by clinical data, such as co-segregation and tumor information (MSH2/MSH6 expression and microsatellite status), together with variant information. However, in many cases, this information is lacking, and additional information is needed before a variant can be classified as benign or pathogenic. Functional assays are therefore used to gather more information regarding specific variants.In this review, we have collected 207 unique MSH6 variants that have been previously examined by functional analysis, including MMR activity, 6-TG selection, nuclear localization, mechanistic and RNA splicing assays. The pathogenic missense variants that are described are scattered throughout the different domains of the MSH6 protein. Most are located in the mismatch binding, the connector, and the ATPase domains . StrikinFor all the assays, it is applicable that there are several points that need to be considered before data are included in the assessment of the pathogenicity of germline variants, including experimental design, replication, controls, and validation. Recommendations for the application of the functional evidence criteria PS3/BS3 using the ACMG/AMP guidelines has recently been published to clarify the variant interpretation process for functional assays .Regarding the experimental design, all the assays model the pathogenesis mechanism. The MMR activity, nuclear localization and splicing assays, as well as the various mechanistic assays, are limited to the examination of one specific MSH6 protein function. This means that a test result that shows an effect on MSH6 function can be used in variant classification, whereas a negative test result cannot automatically be interpreted as benign. In contrast, the 6-TG selection assay is a survival assay, which has the advantage that the output includes all the biological functions of MSH6, as well as effects at the mRNA level. However, the 6-TG selection assay is performed in mouse ESCs, and careful consideration should be given when analyzing the functional effects of human variants in non-human cells.The MMR activity (CIMRA) assay includes internal controls (wt and null variants), and is replicated and calibrated/validated using several known pathogenic and benign variants  to determine the sensitivity and specificity of the assay . MoreoveThe development of the nuclear localization assay as a quantitative functional assay is complex, since the amount of protein overexpression can affect protein mislocalization and misfolding. Moreover, before localization analysis using fusion proteins, it is important to examine the effect of tagged MSH6 proteins at C-terminal or N-terminal ends, since this may affect protein expression levels, cellular localization, and MMR activity, as previously shown for MLH1 and PMS2 .Several MSH6 variants were examined by CIMRA and 6-TG selection functional assays with similar classification. However, currently there is no consensus on whether results from different functional assays could be combined. Therefore, if multiple functional assay results are available for a single variant, evidence from the assay that is the most validated and best measures the disease mechanism should be applied . Regarding splicing assays, these are more difficult to validate, at least when RNA from patient samples is used. However, recommendations for RT-PCR protocols that are used in the clinical testing have previously been developed for BRCA1/BRCA2 variants , and theThe rapid increase in the number of VUS discovered due to novel sequencing techniques calls for development of large-scale functional assays. During the latest years, several studies of cancer pre-disposing genes, including BRCA1, PTEN, and TP53, using saturation mutagenesis, together with a high-throughput functional analysis, have been performed ,94,95,96More recently, MSH2 was examined in a human cell line model, in which deletion of MSH2 was complemented with libraries of variants comprising nearly every possible MSH2 missense allele, covering 94% of the 17,746 possible variants . The celhttps://www.varianteffect.org, accessed on 25 May 2021), has been launched, and we anticipate that this effort may increase information regarding interpretation of variants considerably. If properly validated, we expect that data from these high-throughput assays will be incorporated in variant classification guidelines and clinical decision-making in the near future. The MSH6 variants that were examined by functional analysis, collected in this review, may provide a set of variants for validation of these future large-scale assays.Recently, the atlas of variant effects (AVE) alliance, an international collaborative initiative, whose goal is to resolves issues relating to the use of multiplex assays of variant effect (MAVE) data to interpret human genetic variants (In relation to splicing, we expect that long-read RNA sequencing will be implemented in the genetic diagnostic pipeline within the next few years, enabling delineation of complex structures resulting from aberrant splicing, as well as co-occurrence of splicing events and allele-specific expression analysis, and providing a more comprehensive RNA splicing analysis.


The purpose of this research was to investigate the impact of routine assessment of health-related quality of life (HRQoL) on quality of life and satisfaction with care in patients with head and neck cancer (HNC). A randomized controlled open-label clinical trial with 200 patients with HNC managed in four cancer centers in Eastern France was performed. In the intervention arm (regularly completed HRQoL questionnaires), HRQoL mean change was significantly improved at 2 years from baseline. Compared with the control arm, differences were not statistically significant, but minimal clinically important differences in favor of the intervention arm were found for HRQoL, satisfaction with waiting times, and satisfaction with accessibility. In patients with head and neck cancer undergoing treatments, routine assessment of HRQoL is a simple practice and may have HRQoL and satisfaction benefits.p < 0.0001) and exceeded the minimal clinically important difference (mean change at 2 years from baseline = 10.46). In the two arms, mean differences between arms were not statistically significant, but minimal clinically important differences in favor of the intervention arm were found for EQ VAS (mean change difference (MD) = 5.84), satisfaction with care, in particular waiting times (MD = 10.85) and satisfaction with accessibility (MD = 6.52). Routine assessment of HRQoL improves HRQoL and satisfaction with care for patients with HNC treated with radiotherapy.The impact of routine assessment of health-related quality of life (HRQoL) on satisfaction with care and the HRQoL of patients with head and neck cancer (HNC) treated with radiotherapy was assessed. Patients with HNC were randomly assigned to two arms, with stratification on sex, cancer localization, and stage of the disease. In the intervention arm, the patients completed the EORTC QLQ-C30 and EORTC QLQ-H&N35 questionnaires first before randomization, then before each medical appointment during radiotherapy (7 weeks), and then every 3 months until 1 year and at 2 years thereafter. In the control arm, the EORTC QLQ-C30 and EORTC QLQ-H&N35 questionnaires were completed before randomization and at 1 year and 2 years thereafter. The primary endpoint was mean change in HRQoL at score at 2 years from baseline assessed by EQ VAS from the EuroQol questionnaire. The secondary endpoint was mean change in satisfaction with care at 2 years from baseline assessed by QLQ-SAT32. Two hundred patients with head and neck cancers were involved in this study ), of whom 100 were assigned to the intervention arm and 100 to the control arm. Patients in the intervention arm were reported to have a statistically significant increase in EQ VAS at 2 years ( Head and neck cancers (HNC) represent 4% of all cancers worldwide. The main risk factors for HNC are tobacco smoking, alcohol consumption, and HPV infection ,2,3,4. FNowadays, with the ever-increasing numbers of cancer survivors, special attention to HRQoL is warranted among patients in oncology. Accordingly, HRQoL assessment has become a key endpoint in cancer management and clinical trials. HRQoL has the advantage of taking account of the patient’s perception of his or her disease and treatment, providing additional insights beyond the clinical information. Furthermore, the use of HRQoL data in clinical practice has been shown to help guide the choice of treatment , facilitWe conducted a phase III randomized multicenter study to assess the impact of routine assessment of HRQoL on HRQoL and satisfaction with care in patients with HNC.In this prospective multicenter phase III study, patients with HNC were randomized 1:1 into 2 arms. In the intervention arm, patients were invited to complete the EORTC QLQ-C30 and EORTC QLQ-H&N35 ,26,27 quThe trial complied with the Helsinki Declaration and Good Clinical Practice guideline and was approved by the EST I ethics committee and the Agence Francaise de Securite sanitaire des produits de santé. The trial was registered on the clinical Trial.gov website (NCT 01210872).Patients were recruited in 4 cancer centers in Eastern France: George-François Leclerc Center, Paul Strauss Center, Jean Godinot Institute, and Lorraine Cancerology Institute. The eligibility criteria were: (1) diagnosis of primary nonmetastatic HNC, (2) treatment with radiotherapy delivered by intensity-modulated radiation therapy as the initial treatment, (3) age of over 18 years, (4) ability to read and speak French, and (5) provision of written informed consent to participate. Patients with a history of other cancer, patients with a second primary cancer at the time of diagnosis, and patients with a history of psychiatric disorders were excluded.Randomization was performed using TENALEA  software. Patients were randomly assigned at a 1:1 ratio to 1 of the 2 arms of the study according to a minimization method and with stratification on sex, cancer localization, stage, and center. The Biostatistics Unit of the Georges Francois Leclerc Center generated the random allocation sequence and sent the randomization number and patient assignation to intervention to the study investigators via e-mail. The participants, investigators, and healthcare team could not be blinded to the allocation sequence.2), and clinical data, such as cancer subsite, cancer stage, comorbidities as defined by the Charlson comorbidity index , current alcohol consumption, and treatments, were collected. Follow-up self-report questionnaires were given to the participants by a clinical research technician before each medical appointment with physicians, either in the waiting room or in the patient bedside, or in some cases, the questionnaires were posted to the participants.At the first medical appointment, eligible patients were informed about the study by their oncologist. If interested in participating, they were referred to the research team, who explained the study procedures. After the participants received explanations and provided informed consent, they completed the baseline self-report questionnaires before being randomly allocated to either the intervention or the control arm. The patients’ sociodemographic characteristics, such as age, sex, smoking status, marital status, body mass index  consisted of regular completion of self-report questionnaires at each medical appointment throughout the follow-up period, until the end of the study at 2 years. Accordingly, the patients in the intervention arm regularly completed the intervention questionnaires every week during radiotherapy (which lasted 7 weeks), then every 3 months up to 1 year after radiotherapy, and then 2 years after radiotherapy. A duplicate of their self-report questionnaires, with the corresponding HRQoL scores generated, was made available to the physician prior to the corresponding medical appointment.In the control arm, no HRQoL assessments were performed until 1 year and then at 2 years after radiotherapy . No feedThe EORTC QLQ-C30 questionnaire measures common cancer-related symptoms and is composed of 30 items that generate 15 scales, namely, 5 functional scales , 8 symptom scales , global health status, and financial difficulties . The EORThe primary endpoint was mean change in HRQoL score at 2 years from baseline. The EuroQol questionnaire (EQ-5D) was used to assess the mean change in health global state (EQ VAS) at 2 years from baseline. EQ-5D comprises 2 parts: The first part is the EQ-5D index, which explores 5 dimensions , and each dimension has 3 levels: no problems, some problems, and severe problems. It can be presented as a global health index and produces a composite score between 0 and 1. The second part is a visual analog scale (EQ VAS), which assesses global health state with a score of 100 corresponding to the “best imaginable health state” and a score of 0 for the “worst imaginable health state” ,29.The secondary endpoint is mean change in satisfaction at 2 years from baseline, as assessed using the EORTC QLQ-SAT32 questionnaire . It contn = 200) were required to detect a difference of 10 points in HRQoL scores between the two arms with a significance level of 0.01 and a power of 80%. Continuous variables were described as mean ± standard deviation (SD), or median and range, and categorical variables were described as number and percentage. Between-arm differences in continuous variables were compared using Wilcoxon–Mann–Whitney tests, and a chi-square or Fisher’s test was used to compare categorical variables between arms. For endpoints, the mean change difference between the 2 arms was compared using Mann–Whitney tests. HRQoL and satisfaction scores at baseline and after 2 years within each arm were compared using Wilcoxon signed-rank tests. A negative change score indicated a decline in HRQoL and satisfaction. Following the guidelines of Osoba et al., the minimal clinically important difference (MCID) was defined in these analyses as a mean difference of least 5 points in the mean change in HRQoL and satisfaction scores [p-value of 0.01 was considered significant for endpoints. All analyses were performed using SAS, version 9.4 .Two hundred patients  and were less alcohol drunk .The median age was 58.83 years  in the intervention arm and 56.70 years  in the control arm. Sociodemographic and clinical characteristics were similar between the two arms, except for comorbidities and current alcohol consumption . In the At baseline, there were no statistically significant differences in any dimensions of QLQ-C30 and the QLQ-H&N35 between the two arms .p < 0.0001); moreover, this mean change was clinically significant (p = 0.0698). The comparison of mean change scores between the two arms at 2 years was not statistically significant, but the mean difference (MD) in the mean change at 2 years between the two arms was +5.84 points, which corresponds to a clinically meaningful change  in global self-rated health (The mean EQ VAS scores at baseline were 65.50 (SD = 24.10) and 68.80 (SD = 19.50) in the intervention and control arms, respectively . The meanificant A. In thed health A.Regarding the secondary endpoint, overall satisfaction and all subscales of satisfaction were not statistically significant at any time and between both arms. However, the mean difference at 2 years between the arms in subscale satisfaction with waiting time met the criterion for a clinically meaningful difference (mean increase of 6.25 (SD = 21.81) in the intervention arm vs. mean decrease of 4.60 (SD = 25.07) in the control arm, and mean difference = +10.85) B. Similap = 0.002) (p = 0.42). For satisfaction subscales, the study arms were not statistically associated with the patients’ satisfaction with waiting time (p = 0.34) and also with the patients’ satisfaction with access to the hospital (p = 0.25).In mixed models adjusted, regardless of the study arm, EQ VAS scores increased up to 2 years (= 0.002) . The relp = 0.1552). Additionally, there were no statistically significant differences between the arms concerning the use of concomitant medications, such as antidiarrheal agents, antiemetics, psychotropic drugs, and antibiotics (p > 0.05).During the study, painkillers were the most frequently used concomitant drugs with 82.02% in all the patients . Use of This randomized phase III study was conducted in patients with HNC to assess the impact of the routine use of HRQoL measurement on patients’ HRQoL and satisfaction with care. Assessing HRQoL is important for patients with HNC, for whom treatment is burdensome with many potential side effects ,33. In tIn this study, we found that the routine assessment of HRQoL had an impact on the patients’ HRQoL. In the intervention arm, HRQoL scores increased significantly at 2 years from baseline, and this improvement was clinically meaningful. In the control arm, HRQoL score increased, but the increase was neither statistically nor clinically significant. Moreover, a comparison of the mean change between both arms showed a greater mean difference in the intervention arm, with a clinically meaningful improvement of 5.84 points in favor of the intervention. Our findings are in keeping with the studies by Velikova et al. and Basch et al. ,23, who Assessment of satisfaction with care, which provides feedback from patients and takes into account their expectations and perceptions, showed a high overall satisfaction score at baseline and at 2 years in the intervention arm. With a mean change difference of 4.5 points between arms (very close to the MCID criterion of ≥5 points), overall satisfaction showed a trend towards a clinically meaningful impact in the intervention arm. Moreover, for two subscales of satisfaction with service, namely, satisfaction with waiting times and magnitude in satisfaction with accessibility, we found a clinically meaningful difference in the intervention arm. This result is novel in that few studies have shown positive effects of routine assessment on patient satisfaction ,37. IndeWe expected that HRQoL questionnaire completion may motivate patients to discuss more their health issues at their medical appointments, especially in the intervention arm, where duplicates of their HRQoL questionnaires with quality of life score generated were transmitted prior to their medical appointments. Indeed, no significant difference was found in using concomitant treatments, such painkillers, antiemetics, and antibiotics, between the intervention arm and the control arm. Santana et al.  reportedOur study has some potential limitations. We used a generic measure (EQ-5D) to assess HRQOL as an outcome, rather than a cancer-specific measure, such as the FACT-G , which mThe strengths of our study include the use of validated tools to assess HRQoL and the randomized trial design, which should increase the robustness of our analysis and limit potential bias in the conclusions. A further strongpoint is the follow-up over a period of 2 years. HRQoL has not been widely studied in clinical trials as a primary outcome measure, and therefore, this study with HNC patients could be a starting point for future research to confirm whether the use of routine HRQoL may influence global health state and satisfaction in these patients.The results of the present study show that repeated routine assessment of HRQoL in patients with HNC improves patients’ HRQoL and has a positive impact on some subscales of satisfaction with service. This intervention has the potential to improve clinical practice, and its implementation should be encouraged in routine clinical care with a view to improving a comprehensive patient care.


Polyporus leprieurii var. yasuniensis is a prolific wood-decay fungus inhabiting the forest floor of one of the most biodiverse places on earth, the Yasuní National Park in Ecuador. Basidiocarps and aerial rhizomorphs are commonly found growing on woody debris distributed along the floor of this forest ecosystem. Because of the extraordinary abundance of this fungus in the tropical rainforest, we carried out investigations to better understand the biological and ecological aspects contributing to its prolific distribution. Data on growth inhibition in paired competition studies with sixteen fungal isolates exemplifies defense mechanisms used to defend its territory, including pseudosclerotial plates and the development of a melanized rhizomorphic mat. Results of biomass loss on eleven types of tropical wood in microcosm experiments demonstrated the broad decay capacity of the fungus. In and ex situ observations provided information on how long rhizomorphs can prevail in highly competitive ecosystems as well as stressful conditions in the laboratory. Finally, high concentrations of metal ions occur on rhizomorphs as compared to colonized wood. Sequestration of metal ions from the environment by the melanized rhizomorphs may offer protection against competitors. The development of melanized rhizomorphs is key to find and colonize new substrates and resist changing environmental conditions. Studies of biological diversity provide a better understanding of the ecosystem composition, how ecosystems function, and how organisms interact with other organisms in their surrounding environment. This leads to a deeper understanding of the life history of organisms: lifespan/longevity, relative abundance (rarity/commonness) and the biological traits that make them unique as they function in the ecosystem. This information also contributes to conservation assessments for species and communities.Polyporus s.l. of the Polyporaceae family were among the most common taxa found in this ecosystem, and most of them are closely related to Polyporus leprieurii Mont. [In ecology, a general observation is that most species are rare, and few species are common ,2,3. Thiii Mont. .Polyporus leprieurii Mont. was described and proposed by Camille Montagne in 1840 while describing other taxa from French Guiana [P. hemicapnodes Berk. & Broome, P. pusillus Rostr., P. calyculus Pat., & Gaillard, P. savoyanus Pat., Revue, P. subelegans Murrill, P. tephromelas Mont., and P. atripes Rostr. [P. leprieurii and its known synonyms are still unresolved, and a recent study suggested that P. leprieurii is a species complex that needs further revision to resolve its true taxonomy [P. leprieurii, we refer to these taxa in a broad sense as a part of the “leprieurii” group [h Guiana . Initialh Guiana ,12. New h Guiana  and Núñes Rostr. . The phytaxonomy . In simitaxonomy ,15,16. Ti” group .Polyporus. leprieurii var. yasuniensis   between Basidiocarps and rhizomorphs of the fungi used here were sampled using a random stratified sampling method  to cover a significant proportion of the plot that included all topographical habitats  and to dP. leprieurii var. yasuniensis (CT-16F) were paired with other fungal isolates obtained from decayed wood, fresh basidiocarps, senescent basidiocarps and rhizomorphs of P. leprieurii var. yasuniensis found at the field location in Yasuní National Park  were established using two types of media, malt extract agar (MEA) and malt yeast agar (MYA). Media were prepared following Toapanta-Alban et al. . Assays P. leprieurii var. yasuniensis (CT-16F isolate) or the other fungi was determined by the inhibition growth at 10 weeks and the mycelial response at 28 weeks. In the case that opposite responses were observed among repetitions, success was determined by the majority. Conversely, if there was not a clear sign of which fungus dominated the growth (“winner”), success was noted as equal.The success of P. leprieurii var. yasuniensis for wood degradation  for 60 min cycles at 121 °C. Once cooled, blocks were promptly placed in the microcosms.Wood was obtained at local sawmills in Quito and Quevedo (Ecuador). Wood was cut into 2 × 2 × 2 cmFor soil microcosms, soil mixture was prepared with two parts of soil, two parts of vermiculite, and one part of peat moss thoroughly mixed to remove rocks and big pieces of organic matter. Soil mixture was placed in 16 oz (wide mouth) mason jars using a 50 mL beaker, then 45 mL of deionized water was carefully added, and two wood feeder-strips (1.5 cm wide × 3 cm length) were placed on top of the soil. Jars with the soil mixture, water, and feeder strips were autoclaved twice for 60 min at 121 °C. Once cooled, 5 mm disks of inoculum from two-week-old cultures were placed on each corner of the feeder strips and allowed to grow for 4 weeks. For controls, mock inoculation was performed.Agar microcosms were modified from the methods of Schilling and Jacobson  using MYWhen woodblocks were harvested, mycelium was carefully removed from the surface of blocks and then oven-dried at 100 °C for 48 h. Blocks were cooled in a desiccation chamber, and the weight of each block was recorded as w2. Mass loss was calculated as follows: ((w1 − w2)/w1) × 100, where w1 is initial weight and w2 is final weight. Results are expressed as an average of the percentage of weight loss for each type of wood, incubation time, and microcosm type. At the end of the experiments, soil microcosms that had produced rhizomorphs on feeder strips were kept for longevity and survival tests.Longevity and survival of rhizomorphs was observed and tested in and ex situ. In situ observations were gathered over a period of three years to check for the viability and presence of rhizomorphs on collection sites marked as highly dense. Ex situ observations were gathered in soil microcosms with rhizomorphs produced in the laboratory.Polyporus-like species. One hundred of these were associated with rhizomorphs’ production. Polyporus leprieurii var. yasuniensis was present in forty-six collection sites, demonstrating its high local abundance compared to other taxa producing rhizomorphs, P. taromenane (3 collection sites), and A. yasuniensis (1 collection site) [One hundred nine collection sites resulted from the random stratified sampling method focused on on site) . Local aon site) ,47. Sincon site) , we conson site) . We did P. leprieurii var. yasuniensis varied from 0.5 to 40 m2. Due to a large amount of fine and coarse woody debris colonized by rhizomorphs and basidiocarps, we decided to focus on the number of living plants colonized by rhizomorphs at each collection site; these varied from zero to thirty-one. With this information, and due to a large number of collection sites, we selected the collection sites/individuals with an occupied area greater than 5 m2 for the long-term surveys and focused on changes on the colonized area and number of living plants colonized. We also checked for viability of rhizomorphs and basidiocarps. Another parameter used to select these areas was accessibility, as some of the collection sites were difficult to revisit due to the number of fallen trees, flooded terrain, and wasp nests.The area occupied in each collection site/individual of The occupied area was delimited with a fixed-area plot sampling method  that we Sites were found in all topographical habitats, including, ridges, slopes, and valleys (terra firme and temporary flooded areas). In each site, the occupied area and the number of colonized plants were noted at the beginning and end of surveys. In December 2019, the last visit was carried out to check for the presence of viable rhizomorphs in all sites.Ex situ survival was assessed with rhizomorphs produced in soil microcosms during the decay study .Production of rhizomorphs ex situ was previously observed to occur on old MYA cultures . HoweverFifty-two jars from the decay study were selected for having rhizomorphs growing on feeder strips. Rhizomorphs varied in quantity and size in each jar; thus, jars were randomly selected to form five groups that corresponded to the following treatments .For two groups (1 and 2), sterile deionized water was added to each jar to simulate flooded conditions. No water was added to the three remaining groups. Light exposure was suppressed to one group with water and another without water, by keeping jars inside a black box (1 and 3). One group with water and one with no water had light exposure by keeping jars in clear plastic containers (2 and 4). Groups 1, 2, 3, and 4 were placed on a metal shelf near a window. For group 5, light and moisture content remained unchanged by keeping jars in the same place they were during the wood decay study (with no direct sunlight exposure).In each jar, the number of rhizomorphs was recorded at the beginning and end of the experiment. At the end of the experiment (450 days since microcosms were established), rhizomorph viability was assessed by identifying actively growing tissue . When no active growing tissue was identified, viability was confirmed by isolating rhizomorphs and/or a segment of mycelial mat on MYA. Jars that failed to have active growing tissue and were not successfully isolated were marked as dead.Multi-elemental analysis for Al, B, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, Rb, S, Si, Sr, Ti, V, and Zn was determined in dried rhizomorphs and their wood substrates. This was performed using Inductively Coupled Argon Plasma Spectrometry (ICP-AES) dry-ash method at the Research Analytical Laboratory, University of Minnesota. Rhizomorphs and wood substrates from two sites were thoroughly rinsed with deionized water to remove soil particles and then dried at 50 °C. Then, 500 mg of each sample were used in the analysis with methods previously described in Held et al. . ResultsP. leprieurii var. yasuniensis when combined with other fungi, as well as to identify mycelial responses upon interaction.In vitro dual competition studies served to observe the inhibition growth of P. leprieurii var. yasuniensis resulted in less inhibition growth , mycelial contact was confirmed on all combinations . All resMelanization of mycelium was the response most observed, which was absent only on A6 and A7 (MYA) and A3, A6, and A14 (MEA). This response was determined when light to dark brown-reddish superficial pigmentation was present in the mycelium. The development of mycelial cords, conversely, was the response least observed and was only found on A5 and A14 (MYA) and A12 and A14 (MEA). Furthermore, combinations A5 and A14 (MYA) had evidence of all responses. In these combinations, the antagonist was noted as successful due to the percentage of inhibition growth, 62% and 81%, respectively and mycelial responses, including the development of mycelial cords. .P. leprieurii var. yasuniensis, while antagonists were successful “winners” on five combinations on MYA and six combinations on MEA  and A11, A12, A13, A17, and A18 (MEA). In A4, A8, A10, A11, and A12, response and result of interaction changed upon media type with P. leprieurii var. yasuniensis being more successful on MYA and the antagonist being more successful on MEA , and was only present on balsa wood on agar microcosms at 90 days  Table S.P. leprieurii var. yasuniensis occurred among the different wood types, with balsa, pachaco, melina, guayacan, and laurel woods having greater weight loss and cedro, seike, teka, Colorado, saman, and moral woods having lower weight loss (less than 10%) (Non-inoculated woodblocks in both agar and soil microcosms had little to no weight loss (0% to 1.30% with an average of 0.14% and standard deviation of 0.132) in controls after 30, 60, and 90 days . In treahan 10%)  (Table Shan 10%) .In and ex situ observations demonstrated that rhizomorphs are structures able to persist for relatively long periods under challenging environmental conditions.In situ observations served to detect patterns of growth and dispersal of rhizomorphs within a period of 3  and 4 years (CT-16F) in highly abundant collection sites. Changes were reported in meters to see if the colonized area increased or decreased and by counting living plants colonized by rhizomorphs at the beginning (December 2016) and end of surveys (July 2019). One collection site decreased in colonized area (CT-16F), two increased in area (CTR-2-27 and CTR-2-67) and the last two maintained the same area (CTR-2-12 and CTR-2-31) .In all sites, at the end of surveys, survival was confirmed with fresh basidiocarps and rhizomorphs emerging from old and new substrates (coarse and fine-woody debris). In these densely colonized sites, considerable amounts of coarse and fine-woody debris and dead trees were found fully colonized, except in CTR-2-67, where only coarse and fine-woody debris were present .In this research, we use the term “mother tree” to refer to a whole dead tree either standing or fallen that has a tag number and exhibits the most colonization in each site. For this term, we identify the substrate that appears to be the source of dispersal of rhizomorphs and spores (emerging from basidiocarps) that will serve to colonize new substrates available in each site. “Mother trees” were identified in all sites, except for CTR-2-67 . “MotherColonization of living trees by rhizomorphs was registered at all sites; however, with CTR-2-27 and CTR-2-31, rhizomorphs were observed to remain attached to trees since the first observation. These rhizomorphs stayed anchored to an individual substrate. In some cases, rhizomorphs remained active, while in others, rhizomorphs were attacked by insects and antagonistic fungi.Laboratory investigations allowed us to expose rhizomorphs to different conditions, simulating flooding and droughts with direct, undirect and no light exposure. By the end of the observations, rhizomorphs survived and were viable for at least 330 days in 41 of the 52 jars .The number of rhizomorphs tended to increase for most treatments except in group 2. In all treatments, a melanized mycelial mat was observed covering the substrate (feeder strips) and spread above the soil. Mycelium was found to regrow over older tissue, developing white mycelium that eventually became melanized. Other responses observed in the mycelial mat and rhizomorphs included: the development of a melanized mycelial mat above the water in flooded treatments; upward-oriented growth of rhizomorphs in all jars with no light; attachment of mycelial mat and rhizomorphs on the glass wall; below-ground growth of rhizomorphs; extensive growth of rhizomorphs; and constant primordial development of rhizomorphs  Figure .By the end of the experiments (450 days after microcosms were set up), rhizomorph viability was 79% for all treatments . Most moP. leprieurii var. yasuniensis (two rhizomorphs and two wood substrates) revealed higher concentrations of metal ions on rhizomorphs than in their wood substrates , K (1708 and 1437), Mg (809 and 1333), Al (668 and 297), Fe (399.5 and 90), and Mn (100 and 56). For these metals, concentrations in rhizomorphs were 5-, 5-, 1-, 12-, 15-, and 4-fold times more than in their wood substrate, respectively. The metals with the lowest concentrations were Rb (7.98 and 5.927), Ni (3.88 and 0.950), Ti (3 and 0.747), Cr (1.48 and 0.520), V (1.40 and 0.356), Pb (0.74 and 0.235), Co (0.47 and 0.087), Li (0.24 and 0.100), Mo (0.099), and Be (0.09). These elements were also higher in rhizomorphs than in wood substrates. Only Ni had similar concentrations in rhizomorphs as in the wood substrate .Polyporus leprieurii var. yasuniensis is widespread along all topographical habitats in Yasuní National Park, where it exhibits relatively high local abundance compared to other taxa within the “Melanopus” group [P. leprieurii var. yasuniensis interacts with other fungi in controlled environments, assess its ability to decay tropical woods with different densities, report the longevity of rhizomorphs in and ex situ, and determine the content of metal ions in rhizomorphs and their wood substrates. More importantly, we were also able to observe its dispersal methods and growth patterns in densely colonized sites in the field.s” group . In thisP. leprieurii var. yasuniensis displayed three main responses to gain and protect terrain and prevent inhibition, these were overgrowth, melanization of superficial mycelium, and formation of pseudosclerotial plates. The level of responses during the interaction with the paired fungi varied as P. leprieurii var. yasuniensis was more efficient against other wood-decay fungi such as Marasmius sp. , Tinctoporellus sp. , and Ganoderma sp. (A12). Fast growth melanization of superficial mycelium, overgrowth, and the use of pseudosclerotial plates were key to gain and delimit its territory. However, with some other wood-decay fungi such as Caripia montagnei (A13), Ganoderma weberianum (A18), and Cristataspora coffeata (A18), the inhibition growth and final response were established as equal since both fungi exhibited responses that prevented the competitor of invading the other’s territory by exhibiting similar growth rates and the formation of pseudosclerotial plates and a thick layer of mycelium where contact occurred. Conversely, the inhibition and responses of P. leprieurii var. yasuniensis when paired with fungi isolated from senescent rhizomorphs and those considered plant pathogens, such as Russulales (A3), Ascomycota 1 and 3 , Rhizoctonia sp. (A6), and Ascomycota 2 (A7) resulted in higher inhibition of growth and absence of defense responses that allowed the antagonist to dominate the territory. These plant pathogens and saprophytic fungi demonstrated faster growth to overgrow P. leprieurii var. yasuniensis. Samples A5 and A14 even developed mycelial cords on top of P. leprieurii var. yasuniensis. This response was also observed on the substrate where A14 was isolated, as two fungi were isolated from a piece of wood hosting, Caripia montagnei (A13) and Ascomycota. 3 (A14). During this experiment, rhizomorphs did not develop during the interaction with antagonistic fungi, we believe this could be the result of the competition as the fungus has to direct more energy to defense mechanisms rather than to nutrient exploration.Among wood-decay fungi, competition for territory has been reported as the most common type of interaction . The comP. leprieurii var. yasuniensis was reported to be favored by the yeast extract in MYA, where eventually rhizomorphs can be produced [P. leprieurii var. yasuniensis to competitors was somewhat higher in MEA (where its growth is slower than in MYA) for all combinations except when paired with Cristataspora coffeata (A17), where it was slightly higher in MYA. For five combinations , the “winner” of the interaction was reported as opposite upon the media type, while P. leprieurii var. yasuniensis was successful on MYA, and on MEA the antagonist-resulted “winner” of the interactions. These results suggest that more studies should be carried out to understand how the type of media and nutrient availability can influence competition among these tropical fungi. This could indicate a method of niche specialization among wood-decay fungi.In this competition assay, we decided to use two media types, MYA and MEA, because the growth of produced . Thus, wproduced , while tproduced . The inhTrichoderma spp. [Trichoderma would be effective at inhibiting the growth of P. leprieurii var. yasuniensis, as the genus is widely used in the control of plant pathogens [P. leprieurii var. yasuniensis responds to antibiosis and the role of the melanized tissue in defense mechanisms against compounds secreted by Trichoderma.In our study, inhibition of growth was the result of defense mechanisms exemplifying mycelial gross contact responses that were confirmed by the changes in the morphology of mycelium when contact occurred between the paired fungi. Mycelial gross contact responses are common in dual competition studies ,41,42,43rma spp. . In our athogens ,41,52,55P. leprieurii var. yasuniensis when paired with fungi with similar lifestyles as well as those with different lifestyles. Future studies could take a broader approach to understand its place in wood-decay fungal communities. Another focus could be on identifying the fungi attacking P. leprieurii var. yasuniensis, to predict future threats when fungal communities change. Since this was an exploratory study, more work needs to be conducted to better understand how this fungus interacts with other taxa.Some of the antagonistic fungi were not able to be identified on the genus level because their DNA sequences did not have good matches to well-identified sequences available on NCBI. These fungi have not received much research attention and may represent new species. In addition, for the three taxa Ascomycota 1, 2, and 3 , GenBank accession numbers were not obtained because only partial sequences were obtained for these fungi. However, we could give them some identity and infer their ecology based on the source of isolation, morphology of ascocarps, and other matches with fungal sequences in NCBI. In any case, our main objective was to identify the response of Saprophytic fungi associated with wood degradation play key roles in the decomposition of lignified organic matter and thus have important influences on the global recycling of carbon and other elements including N, P, K, S and minerals. These wood-degrading fungi make those substances available for other organisms such as plants and other fungi to utilize, which is key for healthy ecosystem functioning ,56. DecaP. leprieurii var. yasuniensis to exemplify the different types of substrates the fungi available to colonize and obtain nutrients.This study took place in Yasuní National Park, where the diversity of tree species has been reported to be extraordinarily high ,38,62 asIn both microcosm types used, most degradation was observed on balsa, pachaco, melina, and guayacan woods, which have the lowest reported heart-wood densities , and theP. leprieurii var. yasuniensis is effective at colonizing different substrates, but the decay process seems to be slow, as most decay (30%) was observed at 90 days on balsa, the least dense type of wood. In nature, wood decay is a process involving insects, fungi, and other microorganisms [From the information obtained in this research, we can conclude that rganisms ,65,66. IP. leprieurii var. yasuniensis colonizes wood by forming a melanized rhizomorphic mat that covers the substrate. This seems to be an effective strategy to protect the substrate from other competitors and to ensure that the wood and its nutrients are utilized. This was observed by the amount of decay in woodblocks that was completely covered by the fungus versus the ones that were not totally covered  from networks of anastomosing hyphae when encountering and colonizing new substrates. These structures aid the fungus as it moves across the forest floor and as more complex networks are being formed [P. leprieurii var. yasuniensis (on the highly abundant sites) [Armillaria [on earth ,68,69 ang formed ,70,71,72t sites) , and evemillaria ,68,69, tP. leprieurii var. yasuniensis, especially its longevity and resistance, we studied the prevalence of rhizomorphs and basidiocarps in the field and tested their survival in soil microcosms. Our observations confirmed that similar to Armillaria [P. leprieurii var. yasuniensis are long-lasting structures capable of persisting under challenging environmental conditions such as in excess or limited moisture. In the field, a continuous production of rhizomorphs was observed for over 4 years of observations at the collection site, CT-16F. In 2015, its “mother tree” was standing with basidiocarps and rhizomorphs only present on the root collar. In 2016, this tree fell, resulting in abundant inoculum around the ridge near the base of the tree and around the main stem downhill, with fine- and coarse-woody debris colonized with abundant rhizomorphs and basidiocarps. Further distribution was the result of the colonized wood being dragged downhill by the rain, where new substrates were then colonized. This pattern of distribution was also observed on CTR-2-12, CTR-2-27, and CTR-2-31 collection sites, where “mother trees” fell downhill, dispersing significant amounts of inoculum.To learn about some aspects of the lifestyle of millaria , rhizomoIn the collection sites located on terra firme , rhizomorphs are confronted with seasonal droughts near forest gaps during the dry season, and there is direct exposure to UV light. Fungal melanin has been reported to offer protection against UV exposure as melanin absorbs UV radiation and transforms the energy into harmless amounts of heat ,76,77,78Conversely, we observed rhizomorph prevalence under seasonal flooding in the field (CTR-2-67 collection) and in the laboratory in soil microcosms where water was added (groups 1 and 2). In the field, inoculated wood with rhizomorphs and basidiocarps were found underwater with rhizomorphs maintaining their upward growth to stay above the water level. By the end of the surveys in the field, their prevalence and occupied area confirmed that rhizomorphs were constantly developing at this site despite seasonal flooding. In the laboratory assay, survival in groups 1 and 2 was 82% and 73%, respectively. With water covering the soil and substrate, a melanized rhizomorphic mat was key to keep the fungus alive under these conditions. Melanized mycelial mats provide protection to the fungus in temporary flooded environments. This tissue will eventually develop rhizomorphs seeking to colonize new substrates and escape the harsh flooded conditions. Melanin is a hydrophobic pigment with high molecular weight compounds ,76,77,78P. leprieurii var. yasuniensis has been shown to have a high tolerance to both desiccation and water excess with direct or absent light exposure. The development of rhizomorphs appears to prolong the longevity and prevalence of the fungus by generating asexual organs resistant to changing environmental conditions, and the melanized outer layer provides protection from attack by other organisms. More studies should be conducted to have a better estimate of the age of these individuals in each collection site, including genetic studies to identify how big these individuals are. The tropical rainforests likely harbor massive cord-forming fungi that may rival Armillaria.Rhizomorphs are key to help the fungus find new substrates and move from one substrate to another ,70,71,72Polyporus leprieurii var. yasuniensis exhibits dark brown to reddish pigmentation on its stipe, rhizomorphs, and mycelial mats, and it is also observed on mycelia growing in culture media as a coriaceous tissue covering the culture as well as the pseudosclerotial plates [P. leprieurii var. yasuniensis can also absorb metal ions. Rhizomorphs exhibit much higher concentrations of metal ions as compared to the content in wood substrates. Similar results have been reported for Armillaria rhizomorphs [Phellinus weirii exposed to soil [Armillaria and Phellinus weirii, contact with soil allows for high concentrations to be accumulated. In the Amazon rainforest, the aerial rhizomorphs apparently sequester metal ions from moist above-ground sources where they exist in low concentrations, but over time they can accumulate at high levels.Fungal melanized tissue has been reported to play significant roles in pathogenic fungi ,82,83,84l plates . Becausel plates ,78,92,95 to soil . It is a to soil . It is iCladosporium resinae and Aureobasidium pullulans, due to its role in the biosynthesis of DHN melanin polymerization [Armillaria rhizomorphs than its soil substrate [P. leprieurii var. yasuniensis. The interaction with metals and fungi has been of interest not only because metal toxicity has been used in many fungicides, but also because metals can be directly or indirectly involved in fungal growth and metabolism [P. leprieurii var. yasuniensis.Some metals, such as copper, have been shown to increase the production of extracellular melanin in rization . The prerization ,99. In oubstrate . The chetabolism . Howevertabolism . We beliP. leprieurii var. yasuniensis. This is a core species in the Amazon rainforest, where most species are rare. Its rhizomorphs help this fungus to dominate in this highly diverse ecosystem. They are efficient at finding and colonizing new substrates as well as moving out of harsh conditions (flooded areas). Melanin appears to be a key component for the success of this organism, as it provides mycelial mats and rhizomorphs with a physical barrier to prevent water loss and/or to survive excess water. It is also important as a chemical barrier when competing with other organisms for territory. The highly abundant colonized sites investigated in Yasuní National Park in this work exemplify how this organism is occupying significant areas of forest. Its growth and dispersal dynamics are extraordinary, and we speculate that this could be another massive fungal organism, present in many regions of the Amazon.This work contributes with important new information on key biological and ecological aspects of We want to encourage researchers to continue gathering biological and ecological data of rare and common fungal species to develop a better understanding of their roles in ecosystem functioning and predictions under global warming. In addition, the fungal data obtained could be included in the generation of strong conservation assessments to prioritize regions and ecological communities for fungal preservation.


The test results of 140 milk samples from supermarkets and dairy farms in China showed that cloxacillin, aflatoxin M1, acetamiprid, and fipronil sulfone were found with lower concentrations. Combined with the residue results from the literature, penicillin G and cloxacillin (beta-lactams), enrofloxacin and ciprofloxacin (fluoroquinolones), and sulfamerazine (sulfonamides) were more frequently detected in different countries and need to receive more attention regarding their monitoring and control.A multiclass and multiresidue method for simultaneously screening and confirming veterinary drugs, mycotoxins, and pesticides in bovine milk was developed and validated with ultrahigh-performance liquid chromatography–hybrid quadrupole–linear ion trap mass spectrometry (UHPLC-Qtrap-MS). A total of 209 targeted contaminants were effectively extracted using an optimized QuEChERS method. Quantitative and qualitative confirmation were achieved simultaneously by multiple reaction monitoring–information-dependent acquisition–enhanced product ion (MRM-IDA-EPI) scan mode. The validation results exhibited a good sensitivity with the LOQs of 0.05–5 μg/kg, which was satisfactory for their MRLs in China or EU. The recoveries of in-house spiked samples were in the range of 51.20–129.76% with relative standard deviations (RSD) between replicates ( The quality and safety of bovine milk is an important issue for consumers in the world because it is an important resource of lipids, proteins, amino acids, vitamins, and minerals . HoweverThe residues of these contaminants can cause toxic effects in animals and humans and therefore attract global concern. For example, penicillin can result in allergic reactions in sensitive children or senior citizens; AFM1 and OTA have been defined as potential carcinogens by the International Agency for Research on Cancer (IARC) . Thus, mTo simultaneously analyze and monitor the residues of different contaminants in milk, various methods have been reported using different analytical techniques such as high-performance liquid chromatography (HPLC) ,17,21,22As an effective instrument to obtain more mass spectral information of the targeted contaminants, ultrahigh-performance liquid chromatography–hybrid quadrupole–linear ion trap mass spectrometry (UHPLC-Qtrap-MS) can use multiple reaction monitoring (MRM)–information-dependent acquisition (IDA)–enhanced product ion (EPI) to simultaneously obtain quantitative and qualitative results in one analysis . This inTo detect different types of contaminants in bovine milk more effectively, some more powerful methods need to be developed to simultaneously screen and analyze veterinary drugs, mycotoxins, and pesticides. Thus, this study first employed UHPLC-Qtrap-MS to establish and validate a multiclass and multiresidue method for the simultaneous quantitative and qualitative confirmation of 209 contaminants in bovine milk. Then, the developed method was applied to analyze 140 real milk samples that were collected from supermarkets and dairy farms in China. Finally, the analytical results using LC-MS/MS in the literature since 2014 were collected and comparatively discussed, and the key contaminants that need to be considered in different countries were refined.2O) respectively according to their solubility. The mixture of working standard groups was prepared by diluting the stock solutions with ACN. HPLC- and LC/MS-grade solvents used in the study were purchased from Merck . Acetic acid (AA), formic acid (FA), and ammonium formate (NH4FA) were purchased from Fisher Scientific . Ultrapure water (H2O) was prepared from a Milli-Q system . For the dispersive solid-phase extraction (d-SPE) steps, anhydrous magnesium sulfate (MgSO4), primary secondary amine (PSA), octadecyl silane (C18), and EMR-Lipid (EMR) were obtained from Agilent Technologies , and zirconium dioxide (ZrO2) was purchased from Sinopharm .Veterinary drugs, pesticide, and mycotoxin standards were obtained from Alta Technology  with an available purity > 95%. Individual stock solutions at concentrations near 1000 mg/L were prepared in acetonitrile (ACN), methanol (MeOH), or ACN with 50% water , and collision energy (CE) are listed in Sample analysis was achieved in a UHPLC  coupled to a triple quadrupole linear ion trap mass spectrometer  system. The analytical column setup was a ZORBAX RRHD Eclipse Plus C18  with a flow rate of 0.4 mL/min at 40 °C. The mobile phase A was H2, and each of them was tested pairing with MgSO4. The final steps followed: 5 g milk samples were weighed into 50 mL centrifuge tubes, spiked with different drug standard groups and allowed to stand for 30 min at room temperature. During the extraction process, 10 mL of ACN with 2% FA were added into samples and mixed on an automatic vortexer for 1 min. To stratify the organic phase and water, 2 g NaCl were added to each tube. After 1 min of vortexing and centrifuging (4500 r/min for 5 min), 1 mL extracts (upper layer) were transferred to 5 mL centrifuge tubes containing 50 mg C18, which were shaken for 30 s. The tubes were centrifuged at 4500 r/min for 1 min and filtered with 0.22 μm membranes before injecting into the UHPLC-MS/MS.A modified QuEChERS sample preparation method was used. The sample preparation procedure began with a comparison of extraction solutions by ACN with different concentrations of AA and also FA. Then, the cleanup step was evaluated by different sorbents including PSA, C18, EMR, and ZrOThis study was performed with 140 milk samples collected in China, including 99 samples obtained from dairy farms and 41 samples bought from supermarkets. In the supermarket samples, 12 samples were from domestic production, and 29 samples were from imports, according to their labels, including Australia, New Zealand, Germany, and America. All these samples were prepared and analyzed with the developed method, and the results of contaminants were comparatively analyzed with different sources and positive reports in the previous literature.The normal optimization of the extraction procedure is usually performed by investigating recovery. In this study, the extraction solution was first optimized by the matrix effects (MEs) of 209 chemicals from various concentrations of AA in ACN. MEs are problematic and important in residue analysis when using MS detection. The slopes of calibration curves of matrix-matched standards and solvent-based standards were compared to estimate the ME. The ME values were divided into three parts to describe the effects on analytes. If the ME values fell between 0.8 and 1.2, the matrix did not exert very strong effects on chemicals. When the ME values were higher than 1.2 or lower than 0.8, the MEs were considered enhancement and suppression, respectively. In this study, weak effects on analytes would be the primary condition for choosing the extraction solution. Therefore, the increase in quality of analytes of value between 0.8 and 1.2 reflects the weak effects of the matrices.The milk samples were extracted by ACN with 0%, 0.1%, 0.5%, 1%, 2% and 5% AA. Before the cleaning procedure, extraction solutions were filtered similarly to the matrix solutions to dilute the standards. The matrix curves of six extraction solutions were compared with solvent curves respectively to calculate the ME. 2, and 50 mg of each of them was mixed with 150 mg MgSO4. However, the MgSO4 showed a strong adsorbability for sulfonamides and tetracyclines. Therefore, the next step was the evaluation of every single adsorbent. The range of recovery was separated into four stages, and each stage calculated the percentages of analytes. From 2 outperformed the other two sorbents, because we prefer more chemical recoveries falling in the range of 70% to 120%, although individual compounds were still outside the range. C18 and ZrO2 showed very similar results on recovery. However, ZrO2 had not been used as commonly as C18 in the residual analysis. Therefore, C18 was used in the d-SPE cleanup step. To compare with the classic column SPE method, d-SPE removes the procedures of activating agent and elution, which provided a more efficient cleaning procedure.A d-SPE cleanup step was evaluated after extraction by 2% FA ACN. Extracts (1 mL of the upper layer) were transferred to 5 mL centrifuge tubes with sorbents. The initial cleanup step was tested with four sorbents, PSA, C18, EMR and ZrO4FA and FA would show an impact on sensitivity. Thus, low amounts of NH4FA and FA were added to both aqueous and organic (MeOH) phases. FA  was added to the 5 mmol/L NH4FA-based mobile phase to evaluate the impact on the MS signal. However, it was very difficult to demonstrate a signal increase for every chemical. The result showed that the mobile phase with 0.1% FA and 5 mmol/L NH4FA was the best choice. Under these conditions, most of the chemical signals increased. At the same time, there was a slight diminishing in negative ion data acquisition, but the sensitivity was still strong enough.Mass spectrometric parameters were optimized to maximize sensitivity. To increase the dwell time of each transition, the scheduled MRM model was used, and the chromatogram of each chemical was maximized split on the column to reduce the number of transitions at each time point. Then, the mobile phase compositions were evaluated. We found that NHR2 values were ≥0.99. LODs for every chemical ranged from 0.01 to 1 μg/kg, and LOQs ranged from 0.05 to 5 μg/kg. All the limits were under or equal to the MRL that was based mainly on the regulations from China [The parameters for method validation included linearity, detection limits (LODs), quantification limits (LOQs), precision, and accuracy. Calibration curves were obtained for concentrations of 0.25, 0.5, 2.5, 5, and 12.5 μg/L for veterinary drugs and 0.05, 0.25, 0.5, 2.5, and 5 μg/L for mycotoxins and pesticides. The standards were prepared in ACN and matrix solvent, respectively. Both solvent and matrix curves exhibited excellent linearity over the working range, and om China  and EU [om China  if the cAccuracy was evaluated at spiked concentrations of 0.5, 1, 5, 10, and 25 μg/kg for veterinary drugs, and 0.1, 0.5, 1, 5, and 10 μg/kg for mycotoxins and pesticides in milk. The average recoveries of 209 chemicals ranged from 51.2 to 129.76%. Salbutamol, cefixime, and epi-chlortetracycline showed lower recoveries (between 51.2% and 65.3%) at different spiking levels. Furthermore, because of the lower MRL, aflatoxin M1, diclofenac, and clenbuterol were increased for the testing level. The MRL of aflatoxin M1 is 0.5 μg/kg in China but 0.05 μg/kg in Europe. Therefore, we increased the concentration of 0.05 μg/kg spiked in milk samples. The diclofenac increased the spiking concentration to 0.1 μg/kg, which is the MRL in Europe. Clenbuterol is prohibited in China, but the MRL is 0.05 μg/kg in Europe. Therefore, these concentration levels were also spiked in samples to test recovery. Fortunately, these three analytes had a very perfect sensitivity at the lower concentration, and their recoveries ranged between 110% and 118%. Relative standard deviation (RSD) values were used to express precision. From the results, the RSDs of all analytes were from 0.82% to 19.86%, including salbutamol, cefixime, and epi-chlortetracycline .The real milk samples were collected and analyzed with the developed method, which demonstrated good applicability for the screening, quantitation, and confirmation of three types of contaminants in milk. As Comparatively, veterinary drugs showed a higher detection rate than mycotoxins and pesticides. Sulfamethazine and cloxacillin were often used to prevent bovine mastitis but are prohibited in the lactation stages. Cloxacillin can be more widely applied in China than sulfamethazine, because its detection rate (8.85%) was higher than that of sulfamethazine (0.71%). Additionally, cloxacillin was detected in the samples from the supermarket and the dairy farm simultaneously with three samples exceeding the values of the Chinese MRL (30 μg/kg), indicating that the residues of cloxacillin from the farm may sustainably exist in the final product.The lower concentrations (0.17 and 0.24 μg/kg) of AFM1 were found in milk samples that were under the MRL (0.5 μg/kg) set by China. Both of the two positive samples came from the dairy farm, and the possible source may be the migration and metabolism of AFB1 from the moldy feed in cow breeding that can produce mycotoxins, especially under conditions of higher temperature and humidity.Fipronil sulfone (0.08 μg/kg) is the main metabolite of fipronil that resulted in an outbreak of egg contamination in Europe in 2017 because of the use of fipronil in a disinfectant. Fipronil is a broad-spectrum insecticide, which is used as the active ingredient in field pest control for corn and flea control products. The MRL in milk is 8 μg/kg in the EU, which is the sum of fipronil and the sulfone metabolite. Imidacloprid and acetamiprid are both insecticides that are widely used for pest control in agriculture. The MRLs of imidacloprid and acetamiprid in milk are 100 μg/kg and 200 μg/kg, respectively, in the EU. All the values detected in milk samples were under the MRLs. However, these results indicate that the pesticides may be directly used at dairy farms to control the harmful parasites and flies or migrate from the contaminated forage or feeds made from crops on which the pesticides were used.The higher detection rates of contaminants were observed in milk samples from dairy farms compared with the supermarkets, indicating that the farm is a key control point for pollution prevention and quality control of milk. Additionally, both the domestic and imported products contained the residues of cloxacillin above MRL, suggesting that its contamination is a joint problem in different countries.m/z 50 to 200. The solvent standards of targeted contaminants with different concentrations were analyzed first, and all the standard spectra were imported to the database library and then selected as references. The matching correlation of molecular and fragment ion data from the library is typically expressed as the purity fit value, and 70% had been set by the instrument manufacturer as an appropriate threshold for the positive identification of the analytes in samples [The positive samples were further qualitatively confirmed using UHPLC-Qtrap-MS in the MRM-IDA-EPI mode, which can obtain quantitative and qualitative results based on simultaneous scans of MRMs and second product ions in one injection. Usually, the MRMs together with retention time matching could confirm a finding. However, the effects of the matrix and the structural similarity of compounds could bring interference. Thus, the EPI mass spectra will give further qualitative confirming information. Collision energy had a very strong impact on the product ion information. Thus, a collision energy spread (CES) of 15 V was used to obtain the average fragment ion spectrum with different CEs of 15 V, 30 V, and 45 V. The scan rate of EPI was set on 10,000 Da/s from  samples .The purity fit values were observed at >83% for sulfamethazine, cloxacillin, and aflatoxin M1 and >76% for fipronil sulfone, imidacloprid, and acetamiprid. Typical comparative MRM chromatograms and product ion spectra of aflatoxin M1 in milk and in standard solution are shown in The reported results for veterinary drugs, mycotoxins, and pesticides detected in bovine milk using LC-MS/MS in the literature since 2014 and relevant MRLs are listed in In total, 39 veterinary drugs were found in milk with low concentrations compared to relevant MRLs. Among these drugs, fenbendazole sulfone and desacetyl cephapirin were detected as metabolites of fenbendazole and cephapirin, respectively. The most frequently detected classification was beta-lactams  compared to fluoroquinolones  and sulfonamides . Nine drugs and six drugs were cephalosporins and penicillins (beta-lactams), respectively. Penicillin G and cloxacillin (beta-lactams), enrofloxacin and ciprofloxacin (fluoroquinolones), and sulfamerazine (sulfonamide) showed more frequent detection compared to other drugs, indicating that these drugs may be more widely applied in these countries. Cloxacillin and sulfamethazine were also detected in this study, which further proved their wide application. Penicillin G, cefazolin, ampicillin, and cloxacillin are worthy of attention because they contained high concentrations over the MRL in some milk samples. The main reason may be the nonstandard and illegal uses of these veterinary drugs in cow breeding.Of all targeted contaminants, aflatoxin was the most frequently detected. Especially, AFM1 was detected more frequently than AFB1 because most of the AFB1 would metabolize to AFM1 and AFM2 in bovines, and some of them would migrate into the milk. AFM1 is the main metabolite and was often detected in bovine milk in different countries, including in this study. Additionally, alpha-zearalenol is a metabolite of zearalenone, and residual amounts of both were found in milk. Individual cases of exceeding MRL were observed for AFM1 in the literature . Our detAll pesticides from the list including fipronil, acetamiprid, clothianidin, imidacloprid, thiacloprid, and thiamethoxam are insecticides, and no fungicides and herbicides were found in milk. According to these, it can be suggested that the insecticides were more likely used at dairy farms for hygienic purposes, and no obvious residues of fungicides and herbicides would be produced in milk because of indirect contamination from feed and forage. Both imidacloprid and acetamiprid were found in Switzerland . The resIn conclusion, beta-lactams, fluoroquinolones, sulfonamides, and aflatoxins were the main contaminants in bovine milk according to the present literature. Penicillin G, cloxacillin, enrofloxacin, ciprofloxacin, sulfamerazine, and aflatoxin M1 should be given more attention for monitoring and control. The nonstandard and illegal uses of veterinary drugs and pesticides are the main reason to cause their residues to be observed in milk. Additionally, the dairy farm was the main source for these contaminants and should have strict regulatory management in cow breeding.The present method consists of simple sample preparation and a simultaneous screening and confirmatory analysis with UHPLC-QTRAP-MS using the mode of MRM-IDA-EPI, which shows a good quantitative and qualitative ability at the same time. It is also a highly efficient analytical method with advantages of saving time and costs on sample analysis. The method has been validated for achieving 209 contaminants including veterinary drugs, mycotoxins, and pesticides in bovine milk. After applying it to the milk samples obtained from dairy farms and supermarkets, the low concentration of cloxacillin, aflatoxin M1, acetamiprid, and fipronil sulfone were found. Compared to the results from the literature, some contaminants need to receive more attention regarding their monitoring and control.