Hypoxia is a persistent physiological feature of many different solid tumors and a key driver of malignancy, and in recent years, it has been recognized as an important target for cancer therapy. Hypoxia occurs in the majority of solid tumors due to a poor vascular oxygen supply that is not sufficient to meet the needs of rapidly proliferating cancer cells. A hypoxic tumor microenvironment (TME) can reduce the effectiveness of other tumor therapies, such as radiotherapy, chemotherapy, and immunotherapy. In this review, we discuss the critical role of hypoxia in tumor development, including tumor metabolism, tumor immunity, and tumor angiogenesis. The treatment methods for hypoxic TME are summarized, including hypoxia‐targeted therapy and improving oxygenation by alleviating tumor hypoxia itself. Hyperoxia therapy can be used to improve tissue oxygen partial pressure and relieve tumor hypoxia. We focus on the underlying mechanisms of hyperoxia and their impact on current cancer therapies and discuss the prospects of hyperoxia therapy in cancer treatment. Solid tumors gradually form a hypoxic malignant microenvironment during growth, which promotes tumor proliferation and metastasis by affecting metabolism, angiogenesis, and immune escape, and finally leads to therapeutic resistance. In recent years, hypoxia‐inducible factor inhibitors, direct targeting of hypoxic cells, and respiratory hyperoxia have been widely used to improve the hypoxic microenvironment of tumors and enhance the sensitivity of tumor therapy. The HIF‐1 DNA‐binding activity of cells and the concentrations of HIF‐1α and HIF‐1β proteins increased exponentially with decreasing O2 concentration.Many tumors can survive under hypoxic conditions, mainly due to the expression of HIF in tumor cells.2 concentrations of 21%, 6.25%, 4.85%, 3.46%, 2.11%, 1.57%, 1.00%, and 0%), demonstrating that the presence of VEGF mRNA may be a marker of hypoxia.Von Hippel‒Lindau (VHL) syndrome is a rare autosomal dominant genetic disorder caused by pathogenic variants of the VHL gene. Approximately 70% of patients with VHL develop renal cell cancer (RCC) during their lifetime. The VHL protein acts as an E3 ubiquitin ligase and can ubiquitinate the HIF‐α subunit in an oxygen‐dependent manner, resulting in the proteolysis of HIF. VHL gene mutations lead to the stability of HIF subunits and the constitutive activation of HIF‐mediated transcriptional pathways in patients with VHL, causing the progression of renal cell carcinoma.Rapid uncontrolled growth of tumor cells, inadequate blood supply, and hypoxia are typical microenvironmental features of most solid tumors.2.1The role of oxygen in the metabolic pathway of cells is paramount because cancer cells reside in nutrient‐ and oxygen‐deficient environments due to the rapid growth of tumors.Glutamine is the most abundant amino acid in the blood and is critical for the synthesis of tricarboxylic acid cycle metabolites, nonessential amino acids, and adenosine triphosphate (ATP) energy, on which cancer cells and rapidly proliferating cells depend.+ ratio. Loss of SHMT2 in hypoxic cells increases reactive oxygen species (ROS) levels, resulting in cell death.+ entry into mitochondria.Cell metabolism mainly refers to the metabolism of energy and substances. It is widely known that mitochondria are the energy reservoir in organisms. Many studies have focused on whether HIF regulates metabolic pathways that influence mitochondrial viability.2.2In 1971, Professor Folkman proposed the theory that “tumor growth and metastasis depend on neovascularization”, which provided a new research direction and theoretical rationale for antiangiogenic antitumor drugs.2.3Hypoxia is associated with metastasis; however, how it affects metastasis progression is controversial. Currently, epithelial‐mesenchymal transition (EMT) has mostly been studied in relation to hypoxia and metastasis. EMT is a key process in the metastasis and colonization of cancer cells from the primary tumor to distant organs. Hypoxia plays an important role in triggering EMT by regulating HIFs.2.4Some cancer therapies kill tumor cells by causing DNA damage; however, cancer cells have been shown to activate a variety of repair mechanisms and signaling pathways as a response to overcome DNA damage.2.52 is required for T‐cell activation and the development of effector functions and that HIF‐1α acts as a negative regulator of T‐cell responses.Aside from obtaining an adequate energy supply, tumor occurrence and development must evade various immune surveillance mechanisms of the body.3Since the discovery of the hypoxic tumor environment, sensitization of hypoxic cells in tumors and improvement of the hypoxic tumor environment have been the focus of tumor research.3.1HIFs are significantly upregulated in many cancer types, and the application of HIF inhibitors as anticancer therapies has captured great interest.3.2Gene therapy is a promising strategy for the treatment of various genetic and acquired diseases. However, to minimize the side effects of gene therapy, gene expression must be targeted to specific tissues.3.3Researchers have harnessed the properties of hypoxic cells, targeting them with hypoxia‐activated prodrugs (HAPs) or bioreductive agents.2+.Multifunctional nanoparticles can integrate various key components, such as drugs, genes, and targeting ligands, using unique delivery platforms. Due to the enhanced permeability and retention effect (EPR effect),3.4Treatments targeting key components of tumor hypoxia signaling pathways, including mTOR, are one of the current approaches to cancer therapy.Hypoxic TME is still one of the major causes of resistance to radiotherapy, chemotherapy, and immunotherapy,42); nanomaterials to deliver/produce oxygenTo date, several methods have been proposed to increase the oxygen content of tumor tissue Figure , includi4.12 indefinitely without signs of toxicity.2 > 60%. Sustained hyperoxia (>60% oxygen) normally results in pulmonary and ocular toxicity due to oxidative damage to endothelial and epithelial cells.2 (1 h) alleviates hepatic hypoxia by inhibiting HIF‐2α expression.2) can also reverse the immunosuppressive TME and promote antitumor immunity.For normobaric hyperoxic respiration, in a standard atmosphere with an oxygen content between 20% and 100%, mice were treated with 60% O4.2Hyperbaric hyperoxic respiration treatment involves exposure to a high level of oxygen at a higher atmospheric pressure The oxygen pressure is much higher than at sea level. The pressure at sea level is defined as standard atmospheric (ATM) pressure, a value of 101.325 kPa, which can increase the oxygen tension of tissue or venous blood vessels, likely resulting in increased oxygen concentration. The specified criteria in hyperbaric oxygen treatment (HBOT) stipulate that patients are offered pure oxygen (100%) for 2–3 h under 1.5–2.5 absolute atmospheres (ATA).4.3Various researchers have made substantial efforts devoted to the development of different nanocarriers in the hope of using nanotechnology to deliver oxygen to reverse tumor hypoxia and improve the efficacy of existing treatments.Oxygen‐loaded nanocarriers: Oxygen‐containing nanomaterials can directly deliver oxygen to hypoxic tissues and alleviate hypoxia. Researchers have developed a variety of nanomaterials with excellent oxygen‐carrying capacity, as shown by the following examples.Perfluorinated compounds to deliver oxygen: Fluorine has low polarizability; thus, the low polarizability is converted to low intermolecular van der Waals forces, and van der Waals forces are the intermolecular forces that hold nonpolar molecules together. Accordingly, liquid perfluorocarbons behave close to ideal, gas‐like liquids.2 carrier approved for clinical use by the Food and Drug Administration (FDA) is fluorocarbon‐DA (Fluosol‐DA), but it has been withdrawn from the market due to its cumbersome implementation and limited efficacy.Hemoglobin‐based oxygen carriers to deliver oxygen: Hemoglobin‐based oxygen carriers (HBOCs) were originally developed to provide an alternative to blood transfusions. As the realization that hemoglobin solution is not only a substitute for erythrocytes but also has additional properties, including hemodynamic effects related to its osmotic pressure, nitric oxide scavenging effects, and effects on oxygen delivery and microcirculation, the broader concept of “hemoglobin therapy” was born.Oxygen‐producing nanocarriers: Excess hydrogen peroxide (H2O2) exists in the hypoxic microenvironment of tumors, which greatly limits the efficacy of tumor therapy. Materials such as catalase (CAT)2)2O2 as a raw material to generate abundant oxygen, acting as oxygen generators to improve tumor oxygenation and existing therapeutic efficacy.2O2 and relieve tumor hypoxia by encapsulating CAT in tantalum oxide (TaOx) nanoshells, resulting in TaOx@Cat nanoparticles.2 nanoparticles to improve the efficacy of photodynamic therapy under tumor hypoxia.2 into bionanoreactors provides an attractive nanotechnological route to effectively enhance tumor therapy.2, the delivery of nanomaterials can more specifically target tumor tissues and improve tumor oxygenation.Over the years, nanomaterials have been used as delivery platforms and in vivo diagnostic carriers due to their unique nano properties and physical and chemical properties, and many achievements have been made. Blood vessels in tumor tissues are very different from those in normal tissues and have a higher density. There is a large gap between vascular endothelial cells, and the blood vessels of tumor tissue have an EPR effect; therefore, nanomaterials can target tumor tissue and improve the accumulation of drugs in tumor tissue. Compared with atmospheric hyperoxia and hyperbaric hyperoxia, which improve tumor pO4.443T90  divided weekly, and the animals received 1 or 3–4 divided treatments. Compared with baseline, the tumor PO₂ was significantly increased in both treatment groups.Hyperthermia (HT) is defined as an increase in the body tissue temperature above the normal physiological range, approximately 39–45°C.HT increases the local temperature of the tumor, accelerates the circulation, and increases oxygen saturation, thus increasing the oxygen content of the tumor, increasing the number of cells in the sensitive phase of the tumor, and reducing the number of hypoxic cells in the center of the tumor. No damage to the surrounding normal tissue has been observed. As a cancer treatment method, studies have shown that HT can improve the efficacy of conventional cancer treatments, especially radiotherapy.4.52 + 5% CO2 mixture at standard atmospheric pressure.2 concentrations showed different effects on the time course of tumor oxygenation changes. The perfusion recovered to its starting value immediately after the end of hyperoxia (pure oxygen), whereas with high concentrations of CO2, the perfusion remained elevated for at least 30 min. Higher inhaled CO2 concentrations (2.5% or 5%) prolonged the time to improvement in tumor perfusion compared with pure oxygen respiration.In the 1960s, Dusault explored the use of carbogen respiration in C3H breast tumor xenografts and found improvements. Studies have shown that carbogen improves blood flow and oxygenation more than pure oxygen because respiratory stimulation and vasodilation from carbon dioxide deliver more oxygen to the tumor.4.6The concept that growing tumors have a rich vascular network first emerged through the observations of the well‐known scientist Rudolf Virchow.4.7Inhibition of cellular respiration oxygen consumption provides a different type of intratumoral oxygen supply protocol to conserve endogenous oxygen and overcome hypoxia.5Lung ventilation is the beginning of the respiratory function and the primary factor determining the efficiency of respiratory function.5.12, 6 h/day) inhibits lung cancer metastasis.In 1956, Heston and Pratt began to study the role of oxygen in lung tumors, and the results showed that elevated oxygen uptake increased lung tumor initiation in mice.5.22 and 1% O2 were 2.5 and 4 times higher than those in cells treated with 20% O2, respectively. In contrast, hyperoxia (40% O2) significantly inhibited EPO production.1 protein/mRNA levels were significantly reduced.2, 90 min) had a significant inhibitory effect on tumor growth, and the MAPK pathway was significantly downregulated. An antiangiogenic effect was observed after intermittent HBO treatment.Improved tumor oxygenation also has a suppressive effect on non‐pulmonary tumors. Studies have shown that atmospheric and hyperbaric hyperoxia can reduce the hypoxic area of tumors to varying degrees.The different responses of cancer cells and normal cells to hyperoxia also provide a principle of action for hyperoxia in cancer therapy. Little attention has been given to the differences in the way normal cells and cancer cells respond to changes in oxidative stress. The basic antioxidant defense level is usually abnormal in tumor cells. Glutathione (GSH), an important antioxidant in the body, can scavenge free radicals and prevent the induction of ROS damage.2, 40–64 h) induces cyclin‐dependent kinase 2 activity and acute inhibition of DNA synthesis. The S‐phase block also has different degrees of blockade in the G1 and G2 phases  can lead to cell death with or without loss of mitochondrial function Figure .314, s Figure .316, According to current studies, respiratory hyperoxia has inhibitory effects on lung tumors and other tumor types, with no significant difference in effect. Respiratory hyperoxia can alleviate tumor hypoxia and inhibit tumor progression through various mechanisms Table . Althoug66.1Radiation therapy has been used since the late 19th century as a means of controlling tumor burden, both for curative and palliative purposes. Following the discovery of the German physicist Wilhelm Konrad Roentgen in 1895, X‐rays were used for diagnosis, and less than 3 years later, radiotherapy was used to treat cancer.2) and carbogen respiration (95% O2 + 5% CO2) to improve tumor oxygenation, and the results showed that both carbogen and oxygen inhalation increased tumor PO2 and radiation therapy effectiveness.Radiobiology researchers have improved the efficacy of radiotherapy by reversing tumor hypoxia through various means, including the use of HBO therapy,2 is a powerful radiosensitizer has practical implications for cancer treatment with energetic heavy ions, such as carbon ions.Fractionated radiotherapy is the irradiation mode adopted by most radiotherapy centers.Regarding clinical outcomes, several reports have demonstrated that irradiation occurring immediately after routine fractionated HBO has antitumor activity.In the past, hyperoxia therapy was considered to promote tumor proliferation and metastasis; thus, hyperoxia therapy was contraindicated. However, a large number of basic and clinical studies have found that hyperoxia therapy not only does not promote tumorigenesis but enhances the effect of radiotherapy. Hypoxia is an important reason for the poor effect of tumor radiotherapy. Under hyperoxia, the partial pressure of oxygen in tissues is more than 10 times that under normoxia, which can greatly improve the hypoxic state of tumors and enhance the effect of radiotherapy. On the other hand, when radiotherapy is used to treat malignant tumors, normal tissues will also be killed. Hyperoxia therapy can improve the blood supply to normal tissues to protect and promote the repair of normal tissues. In conclusion, hyperoxia is feasible and effective in the treatment of tumors, especially in adjuvant radiotherapy.6.22) can promote the toxic effects of carboplatin on lung tumors.+‐crosslinked microcapsules. The cytotoxicity of DOX is promoted by increased ROS production.Drug delivery and cellular uptake of drugs in hypoxic regions are known to be affected by hypoxia or associated acidity, resulting in the relative resistance of hypoxic tumor cells to chemotherapy.394 and resistance to the chemotherapeutic drug temozolomide is commonly responsible for most disease recurrences.Several studies on the remission of hypoxic tumors in combination with various chemotherapeutic agents have shown increased mean survival time and/or decreased tumor growth/metastasis.Hypoxia is a broadly studied phenotype at the clinical and molecular levels and a definite focus in biomarker studies; thus, exploring oxygen‐sensitive molecules to identify the degree of tumor hypoxia is a feasible approach. In addition to HIF, PHD, VHL, VEGF, CA9, and the other proteins mentioned above that can sense oxygen concentration, lysine demethylase 3A (KDM3A) and lysine demethylase 5C (KDM5C) should be included. Hypoxia inhibited the activities of KDM3A and KDM5C and reduced ROS production and tumor cell apoptosis.6.3Another antitumor mechanism of hyperoxia may be to promote the revitalization of immunity. High concentrations of oxygen activate the anticancer effects of T cells and NK cells by deactivating the adenosine immunosuppressive microenvironment.Polymorphonuclear neutrophils (PMNs) are thought to play an important role in cancer, but their role in tumors is unclear.2) upregulates MHC expression, confirming that MHC antigen presentation regulation is oxygen‐dependent.2 can not only reduce the secretion of exosomes from MDSCs caused by hypoxia but can also inhibit the upregulation of PD‐L1 protein expression.Enhancing tumor oxygenation as monotherapy can improve the tumor immune microenvironment; therefore, could combining hyperoxia and cancer immunotherapy improve the limitations of existing immunotherapies? Programmed death‐1 (PD‐1) blockade of the immune checkpoint has shown promising antitumor effects in a variety of cancer types.Research has shown that an important component of hyperoxia therapy has been overlooked: the immune response. Normally, tumors grow faster than their blood supply, creating a hypoxic environment that stimulates cancer cells to produce a molecule called adenosine, which encourages antitumor T and NK cells to go into a dormant state. Given that respiratory hyperoxia inhibits the hypoxia‐adenosine pathway and promotes T cell infiltration into the tumor by depleting the dense ECM, we also expect hyperoxia to contribute to adoptive cell therapy, such as therapies that employ endogenous T cells and NK cells or genetically engineered T cells, NK cells and macrophages, by improving cell infiltration and functional maintenance in the malignant TME. Although the current studies have only been carried out in mouse models and this approach has not been tested in humans, they open up the possibility of using oxygen‐assisted immunotherapy to fight tumors.6.4PDT is a new therapeutic modality for the treatment of malignant tumors. It has been approved by the FDA for the palliative treatment of advanced lung and esophageal cancer.2 into perfluorocarbon nanodroplets.Hjelde and colleagues investigated the effect of oxygen tension on photodynamic therapy toxicity in an established cell line.Maier evaluated the use of PDT under HBO versus normobaric conditions in patients with advanced esophageal cancer. All patients underwent photosensitization with hematoporphyrin derivatives, and of these patients, 14 received PDT alone, and 17 received PDT under HBO (2 ATA). Tumor length was reduced in both groups, and the tumor reduction was more pronounced in the PDT/HBO group. The 12‐month survival rate was 28.6% in the PDT group and 41.2% in the PDT/HBO group.2, but the specific mechanism remains to be explored. On the other hand, due to the variety of photosensitizers, the combination of hyperoxia therapy and photosensitizers that can better promote PDT requires more basic and clinical research. Oxygen supplementation can solve the problem of PDT resistance caused by tumor hypoxia, but the clinical application of hyperoxia combined with photodynamic therapy still needs further research.Although hyperoxia can improve the efficacy of PDT treatment, HBO seems to be more effective for PDT. Study results indicate that HBO can promote the efficacy of PDT in addition to increasing blood pO7Cancer cells have a complex relationship with oxygen, and for decades, research has been conducted to investigate whether oxygen helps or harms cancer. Tumors that grow in hypoxic environments are known to be more metastatic and more lethal to patients. Tumors are initially susceptible to chemotherapy and radiotherapy, and advanced cancers maintain growth as cells adapt to a hypoxic microenvironment.Theoretically, the application of hyperoxia therapy in tumor therapy is justified for the following reasons: improving oxygenation can remove hypoxia‐stimulated vascular malformations and degrade the dense ECM of tumor cells, improving drug delivery to hypoxic areas of tumors. The improvement in oxygenation causes cells to enter a proliferative phase, which makes them sensitive to radiation therapy and certain chemotherapeutic agents. Improved oxygenation also recruits immune cells to the tumor or enhances the antitumor ability of immune cells. With these facts in mind, although the antitumor effect of hyperoxia therapy is weak, it may improve the effectiveness of some therapies and be beneficial as an adjuvant in cancer treatment.There are still many difficulties in the clinical application of hyperoxia therapy. Oxygen is breathed into the blood to provide nutrients for cells throughout the body. Cancer cells also need oxygen to survive. As earlier studies have shown, respiratory hyperoxia in the early stages of tumorigenesis accelerates tumor development by promoting angiogenesis.At present, researchers seem to be highly interested in the effect of hyperoxia on immunity. Hyperoxia can recruit immune cells, such as T and NK cells, but its specific mechanism has not been fully studied. Why can hyperoxia recruit immune cells? One of the most important and basic effects of hyperoxia is to activate ROS, which kills tumor cells by damaging mitochondria. What is the connection with immunity? Although a large number of studies have verified the immune activation effect of hyperoxia, research on the antitumor effect of hyperoxia is limited. Therefore, the combination of hyperoxia therapy with existing immunotherapy can open up new treatment pathways and will need further exploration.Y.Z. and J.H.W. conceptualized this review and drafted the paper. Y.Z., K.L. and Q.Y.H. produced the figures and tables. J.H.W., X.S.G. and C.P.J. revised the paper. All authors have read and approved the article.The authors declare that they have no conflict of interest.Not applicable.


Pangasianodon hypophthalmus) (gill and intestine) on a seasonal scale.The study was undertaken to evaluate the influences of some physicochemical parameters and viable bacterial counts in cultured ponds (water and sediment) on the viable counts of the Pangasius catfish  and a heatmap coupled with the clustering analysis.Physicochemical parameters, Except for fluctuations in air and water temperatures, the other two physicochemical parameters were almost stable throughout the study periods. The gills and water counts were relatively lower than those of the intestine and sediment. Pearson’s correlation analysis established no significant correlations between the physicochemical parameters and viable bacterial counts. However, significant positive correlations were detected between the viable counts of water and sediment and between the gill and intestine. The RDA plot showed that, except in spring, the viable counts of a particular sample type were similar among the four locations. The results of permutation test showed that, individually none of the studied physicochemical parameters was significant; however, the seasons significantly affected the viable counts. The number and types of bacteria present in the aquaculture ponds greatly regulate the water quality parameters  and are Pangasianodon hypophthalmus) is one of Bangladesh’s most well-regarded aquaculture species. Production and country-wide availability of their seeds, faster growth, convenient culture technique, higher disease resistance, and better adaptability to a wide range of aquatic habitats made them popular among fish farmers [Pangasius catfish  producing areas such as Shutiakhali , Maskhanda , Shomvuganj , and Khagdhohor  under the Sadar Upazila of Mymensingh district (24°45’14”N and 90°24’11”E), Bangladesh  from three different pond locations were collected in sterile sample bottles (200 ml). Bottom sediment samples were also collected in sterile glass bottles from the three different locations of the selected ponds. The bottles with samples were kept in an icebox until carried to the laboratory for further analysis. amples Pangasius catfishes were harvested using a cast net from the sampling ponds and brought to the laboratory live. All the samplings were done in triplicate from each of the selected ponds and carried to the Fisheries Microbiology Laboratory, Department of Fisheries Technology, Bangladesh Agricultural University, Mymensingh, Bangladesh.viz., ambient temperature, water temperature, water transparency, and pH of the pond waters, were measured during sampling. The ambient and surface water temperatures (°C) were monitored using an alcohol thermometer. The collected water’s pH was measured using a DF Universal pH Test Paper Strip . The transparency (cm) of the water was measured by reading the Secchi depth of the pond water using a Secchi disk.Physicochemical parameters, −1; in the case of water samples) or per gram , was determined. Before plating the collected samples, stock solutions were prepared and further diluted following consecutive decimal and serial dilution techniques. For water, 1 ml of the collected sample (water stock solution) was transferred with a micropipette to test tubes containing 9 ml of sterile physiological saline. For a homogeneous dilution, the tubes were thoroughly shaken using a vortex mixture to achieve a 10−1 dilution of the collected water sample. Following the same protocol of decimal dilution, the 10−2, 10−3, and 10−4 dilutions of the samples were achieved consecutively. For sediment, 1 gm of the wet sample was weighed and homogenized in 200 ml of sterile diluent  to prepare the first stock solution. This solution was then serially diluted following the above protocol to achieve the 10−1 to 10−4 dilutions.Standard plate count, expressed as colony-forming units per millimeter  were then mixed and ground in a sterile mortar, aseptically and carefully. One gram of the ground sample was then suspended in a bottle containing 200 ml of sterile physiological saline and mixed thoroughly (by shaking and vortexing). Following the similar processes mentioned above, several dilutions of 10−3 and 10−4) were then spread plated in duplicate in previously prepared plate count agar  plates. From sample solutions of different dilutions, 0.1 ml samples were taken by a micropipette and transferred aseptically onto the agar plates, which were then spread homogenously and carefully onto the surface using a sterile L-shaped glass rod until the sample was dried up. The plates with the cultures were then turned upside down and kept at 30°C for 48 h.The diluted samples  equipped with a guide plate ruled in square centimeters was used to count the colonies. Plates containing 30–300 colonies were considered to calculate viable bacterial counts by using the following formulae:−1),For water samples (CFU ml= No. of colonies on Petri dish × 10 × dilution factor−1),For sediment samples (CFU gm−1),For fish gill/ intestine samples  with the package “ggplot2” . Statist package  based onviz., ambient temperature, water temperature, water transparency, and pH of the studied Pangasius catfish ponds, were recorded. The average values of these parameters are determined for different seasons and sites and presented in box plots  in different ponds of the studied sites throughout the study periods.6 CFU ml−1, 2.16 × 106 CFU gm−1, 3.2 × 106 CFU gm−1, and 4.39 × 106 CFU gm−1. During this season (spring), the maximum average counts of water, sediment, gill, and intestine were 3.75 × 106 CFU ml−1 in site 2, 6.52 × 106 CFU gm−1 in site 3, 6.37 × 106 CFU gm−1 in site 4, and 8.55 × 106 CFU gm−1 in site 4, respectively  and a weak negative correlation with viable counts of gill and intestine . However, significant positive correlations were found among the viable counts of water and sediment (r = 0.78) and the gill and intestine (r = 0.738). A significant negative correlation was observed between viable counts of pond sediment and fish gill (r = −0.520), while moderate negative correlations were observed between the viable counts of the pond (water and sediment) and fish (gill and intestine)  .RDA was carried out to assess the responses of viable counts of bacteria to the physicochemical parameters of the studied Pangasius catfish ponds. The driving factor(s) that controlled the viable counts of bacteria in the ponds was also evaluated . Except r2 = 0.23 and p = 0.17, based on 1,000 permutations). Ambient temperature  and water temperature  had relatively fewer influences than water pH. Among the studied parameters, water transparency  had the least impact on the viable counts of bacteria  above the 99.9% confidence level, i.e., viable counts fluctuated seasonally. The sampling locations had no significant effects , indicating viable counts were similar in different locations within a season  (gill and intestine) to some physicochemical parameters and viable bacterial counts of cultured ponds (water and sediment) were assessed on a seasonal scale. We hypothesized that the viable counts of the pond (water and sediment) and fish (gill and intestine) would vary according to the seasons and correlate with the physicochemical parameters. We also hypothesized that among different physicochemical parameters, the temperature would act as the most important driving factor to influence viable counts of water and sediment in the ponds and the gills and intestines of the Pangasius catfish.Responses of viable counts of the Pangasius catfish . Competition among the organisms for nutritional purposes is also an essential factor responsible for quantitative and qualitative fluctuations in bacterial flora. The temperature was reported as the most influential diver that affects the microbiomes in aquatic environments ,29 and fThe viable bacterial counts of the Pangasius catfish ponds varied significantly among the seasons. The counts of the ponds (water and sediment) showed a weakly positive correlation with the temperature and a weakly negative correlation with the fish counts (intestine and gill). None of the assessed physicochemical parameters found significant drivers to influence the viable bacterial counts of the Pangasius catfish ponds.


We aim to determine the spectrum of cytogenetic abnormalities and outcomes in unbalanced offspring of asymptomatic constitutional balanced t carriers through a systematic literature review. We also include a case of a constitutional balanced t carrier from our institution. Among the 16 balanced t carriers in our review, 13 were maternal and 3 were paternal. Of the 15 unbalanced translocation cases identified, 13 were live births, one was a missed abortion, and one resulted in pregnancy termination. The spectrum of established syndromes reported among the live births was the following: trisomy 9p syndrome (6/13), dual trisomy 9p and DiGeorge syndrome (3/13), dual 9q subtelomere deletion syndrome and DiGeorge syndrome (1/13), 9q subtelomere deletion syndrome (1/13), and DiGeorge syndrome (1/13). One unbalanced case did not have a reported syndrome. The phenotype of the unbalanced cases included cardiac abnormalities (5/13), neurological findings (7/13), intellectual disability (6/10), urogenital anomalies (3/13), respiratory or immune dysfunction (3/13), and facial or skeletal dysmorphias (13/13). Any constitutional balanced reciprocal t carrier should be counseled regarding the increased risk of having a child with an unbalanced translocation, the spectrum of possible cytogenetic abnormalities, and predicted clinical phenotype for the unbalanced derivative. These translocations result from nonallelic homologous recombination through breaks in adenine and thymine residue-rich (AT-rich) repeats repaired by nonhomologous end-joining . Some baIn our systematic literature review of constitutional reciprocal t carriers, we describe the genetic and phenotypic characteristics of their unbalanced offspring. Our review was inspired by a case at our institution, in which a patient with a history of recurrent pregnancy loss carried a constitutional balanced t. This uncommon translocation, which mimics the somatic balanced translocation seen in CML, AML, and ALL subtypes, has only been reported twice in prior literature . In thisA 31-year-old G3P0020 woman of Indian descent presented for prenatal genetic counseling with her partner at 13w5d gestation. The patient has a past medical history of pulmonic stenosis and hidradenitis suppurativa. Her partner is also of Indian descent and a self-reported heterozygote for Factor V Leiden identified through direct-to-consumer genetic testing. The couple had previously experienced two early first trimester pregnancy losses, and genetic testing was not performed on the couple or the products of conception in either pregnancy loss. The couple underwent a workup for recurrent pregnancy loss at a local reproductive endocrinology and infertility clinic.in situ hybridization (FISH) was performed on both interphase nuclei and metaphase cells , while the patient was found to have an apparently balanced reciprocal translocation between chromosomes 9 and 22  . The brese cells . The int1 fusion , while mCR genes . DespiteBCR-ABL1 transcripts. The absence of transcripts would rule out the possibility of a cryptic translocation that could result in leukemia, though unlikely given the patient’s normal CBC. The patient declined further workup with RT-PCR.Additionally, no evidence of leukemic process was identified on complete blood count (CBC). Further workup with reverse transcription-polymerase chain reaction (RT-PCR) was recommended to quantitatively detect t The patient’s nuchal translucency ultrasound and first trimester sequential screen were normal. After reviewing her karyotype and discussing the increased risk of an unbalanced translocation in her fetus, the patient elected amniocentesis. The results of the amniocentesis revealed a normal male fetal karyotype  with no clinically significant numerical or structural chromosome abnormalities. Chromosome 9 and 22 homologs appeared normal at the 450-band resolution of karyotyping. Additionally, the CMA was consistent with a normal male fetus without clinically significant CNV or copy neutral regions detected at a resolution of 50 Kb or higher . FinallyAn echocardiogram was performed to evaluate the patient’s pulmonic stenosis at 18 weeks gestation, which indicated an increase in trans-pulmonic gradient as compared to her cardiac MRI images from two years prior. This increase, though still in the mild range, was attributed to the high-flow state in pregnancy. A repeat echocardiogram was completed at 30 weeks gestation, which indicated no significant changes since the prior study at 18 weeks.The patient underwent parental CMA to determine the etiology of her pulmonic valve stenosis by evaluating sub-microscopic CNV(s) not detectable by standard karyotyping. Her CMA results were normal, making the translocation an unlikely cause of her cardiac finding. However, the normal result does not exclude the presence of a CNV <50 Kb or the possibility of a monogenic disorder as the cause of the stenosis. The patient declined single gene testing.The remainder of the pregnancy was uneventful except for the identification of a fetal right pelvic kidney with normal renal parenchyma diagnosed at 20 weeks gestation by anatomy ultrasound. The patient had a spontaneous vaginal delivery of a live male infant weighing 3.37 kg at 40w3d. Apgar scores were 8 and 9 at 1 and 5 minutes, respectively. The neonate was discharged on the second day of life with no complications. He is currently in good health at 17 months of age without any health consequences from his right pelvic kidney.The literature review was conducted using PubMed and Scopus from database inception to 10 October 2021. Articles discussing constitutional translocations of chromosomes 9 and 22 were identified without limitations on study date or type. Only literature in English were included . The folA total of 11 studies including 15 cases of unbalanced translocations from a balanced t carrier parent met our inclusion criteria. The phenotypes of the unbalanced cases include an array of features such as cardiac abnormalities, neurological findings, intellectual disability, urogenital abnormalities, respiratory and immune dysfunctions, and facial or skeletal dysmorphias , Table 4Congenital heart disease was found in five of 13 live births. Of these five cases, three passed away from cardiopulmonary complications within six months of birth, one case was eight years old at time of report, and one case did not have a reported clinical outcome. Of these same five cases, three had a dual diagnosis of trisomy 9p syndrome and DiGeorge syndrome (22q11.2 deletion syndrome), one had 9q subtelomere deletion syndrome, and one had a dual diagnosis of 9q subtelomere deletion syndrome and DiGeorge syndrome . All thrSeven of 13 live births presented with neurological findings. Two cases had epilepsy , three hAt least six of the 10 live births (60%) who were able to be cognitively assessed have some degree of intellectual or learning disability. Urogenital findings were reported in three of the 13 cases. Three of the 15 cases had pulmonary or immune dysfunction. One case reported in All unbalanced cases with reported phenotypes had some form of facial or skeletal dysmorphia. Seven of the 13 live births had facial dysmorphia, and four had malformations such as microbrachycephaly, microcephaly, micrognathia, retrognathia, and macroglossia . Six casNine of the 15 balanced carriers (60%) had at least one prior miscarriage . RecurreEight of the 15 unbalanced cases included information on their siblings. Case 1 in The apparently balanced translocations of healthy individuals can remain undetected until pregnancy when patients present with recurrent pregnancy loss or fetal congenital abnormalities . Our patBCR-ABL1, could have led to a misdiagnosis of CML if she had also presented with leukocytosis. Though balanced translocations are common, occurring in at least 1 in 500 individuals, our patient’s constitutional balanced t is rare . Her disBalanced t carriers and their families should be counseled on the fertility implications and the reproductive risks of t to help guide parents in their reproductive decision-making. Firstly, parents with a balanced t have an increased risk of pregnancy loss. Though it is unclear if our patient’s previous pregnancy losses were related to her balanced translocation, we found in our review that most balanced t carriers had at least one miscarriage, many of whom had recurrent pregnancy loss. Translocations of maternal origin carry an increased risk for recurrent pregnancy loss, while translocations of paternal origin increase the risk of male infertility . In a coSecondly, balanced t carriers should be counseled on their risk of having a child with an unbalanced translocation. Based on the breakpoints of our patient’s translocation, she has an increased likelihood of having a child with DiGeorge syndrome, 9q subtelomeric deletion syndrome, dual genetic diagnosis of both syndromes, or other unbalanced genomic outcomes . A prospThis systematic literature review is the only comprehensive assessment of constitutional t carriers and the outcomes and phenotypes of their unbalanced offspring. We also incorporated a case from our institution of a balanced t, an analog of the somatic balanced translocation seen in the Philadelphia chromosome of CML. There are only two other published cases of a patient with balanced t . The rarA limitation of this review is the inclusion of clinical cases of t with various chromosomal breakpoints, each of which are associated with a range of possible phenotypic outcomes. There is a relatively small number of accrued cases and each paper reported their cases with a varying level of detail. Additionally, five of the 15 balanced translocation carriers included in the review did not have the karyotype results for each of their offspring reported, limiting insight into the cytogenetic variability amongst siblings . ReporteOur review provides a cytogenetic breakpoint-based genotype-phenotype correlation for unbalanced cases of parental constitutional reciprocal translocation  carriers. This literature review helps providers give anticipatory guidance to balanced t carriers on potential pregnancy outcomes. Balanced t carriers should be counseled on the increased risk of recurrent pregnancy loss, subfertility, and having a child with an unbalanced translocation that could lead to an array of phenotypic manifestations. Phenotypic findings in unbalanced cases include cardiac abnormalities, neurological findings, intellectual disability, urogenital anomalies, respiratory or immune dysfunction, and facial or skeletal dysmorphias. Genetic counseling is recommended for any balanced translocation carrier or breakpoints regardless of the chromosomes that are involved.


Chronic rhinosinusitis is a highly heterogeneous chronic inflammation of the upper respiratory tract caused by immune dysfunction in human beings. However, the underlying etiology of this disease has not yet been well established. Several trials have revealed that serum vitamin D level abnormality might play a role in the pathophysiology of chronic rhinosinusitis.The aim of present study was to perform a meta-analysis of studies to compare the serum vitamin D levels between patients with chronic rhinosinusitis and healthy controls and to evaluate the potential associations of serum vitamin D level with the occurrence of chronic rhinosinusitis.2 value was used to test for heterogeneity between the included studies.Following the PRISMA guidelines, relevant databases incorporating Pubmed, Web of Science, Embase and Cochrane Library were searched from inception to December 1, 2018. Funnel plot symmetry and Egger’ test were used to detect publication biases. The outcomes were presented as Weighted Mean Difference combined with 95% Confidence Intervals to estimate the difference of serum vitamin D levels between patients with chronic rhinosinusitis and controls. Higgins Ip = 0.000). Subgroups analyses based on study location (USA vs. Non-USA), types of biomarkers (25(OH)D3 vs. 25(OH)D) and the study design methods (retrospective vs. prospective) did not reveal heterogeneity. However, phenotypes of chronic rhinosinusitis, with versus without polyposis might account for some degree of heterogeneity. Meanwhile, a lower serum vitamin D level was observed in chronic rhinosinusitis with nasal polyps patients.Out of 176 studies, only eight studies involving 337 chronic rhinosinusitis patients and 179 healthy controls met the criteria and were included in the meta-analysis. In a pooled analysis of the studies, chronic rhinosinusitis patients showed decreased serum vitamin D status (WMD = −7.80, 95% CI −13.28 to −2.31, Our findings indicate that serum vitamin D level might be associated with patients with chronic rhinosinusitis as we detected a significant association between lower serum vitamin D status and chronic rhinosinusitis, especially in chronic rhinosinusitis with nasal polyps patients. However, further comprehensive studies are warranted in order to draw firm conclusions. Chronic rhinosinusitis (CRS) is a disease of the nasal and paranasal mucosa characterized by the persistent inflammation with distinctive inflammatory cells. Epidemiologic studies have revealed wide variation in the prevalence of CRS among regions globally.The specific pathogenesis of CRS isn’t totally clear. In the past, CRS was considered to be a chronic suppurative inflammation caused by bacterial infection. At present, more and more studies have demonstrated that CRS is a highly heterogeneous chronic inflammation of the upper respiratory tract caused by immune dysfunction in human beings.It is generally considered that vitamin D could maintain the healthy balance of calcium and phosphorus playing an important role in the bone metabolism.9To the best of our knowledge, no systematic evaluation and meta-analysis have been conducted on the relationship between serum vitamin D levels and CRS or healthy controls. Up to now, this is the first meta-analysis to investigate the possible associations between serum vitamin D status and CRS.Our meta-analysis was performed according to the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA).Studies were included in the meta-analysis if they met the requirements below: (1) Studies reporting comparison of serum vitamin D levels between CRS and controls; (2) Articles had a clear definition of cases and controls; (3) Vitamin D measured using a standard method and expressed as, or could be converted to, one international unit (ng/mL);The exclusion criteria studies were: (1) Patients with other risk factors, including asthma, allergic rhinitis, fungal infection, nasal tumor and so on; (2) Animal studies, abstracts, letters, editorials, reviews, expert opinions; (3) Articles did not support sufficient data; (4) The full text was not available in English. The final decision was obtained by consensus.Two independent researchers reviewed all candidate articles for inclusion or exclusion. Any conflicts in the data extraction or quality assessment, were resolved by a third reviewer. The data were extracted and recorded in a data extraction form. The following data were collected: first author, publication year, region or country, study design, number of participants, basic characteristics of the individuals, mean of vitamin D level and their standard deviations, vitamin D cutoff value. If necessary, we would contact the corresponding author of the article to obtain unpublished data. The study would be excluded after two failed attempts.The Newcastle-Ottawa Quality Assessment Scale (NOS), which consists of three parts: selection of study groups (4 scores), comparability of groups (2 scores), and outcome assessment (3 scores), was used to assess the quality of the enrolled studies.2 statistic was used to test for heterogeneity between the included studies.2 < 25% can be interpreted as an indicator of mild heterogeneity; I2 value between 25% and 50%, to moderate heterogeneity while I2 > 50%, to significant heterogeneity. To rule out over representation of results from a single study in the meta-analysis, we performed sensitivity analysis by eliminating each study individually. Funnel plot and Egger’s test were used to assess the potential publication bias. All statistical tests were two-sided, and p-value of <0.05 were considered statistically significant.Extracted data were pooled using the STATA software package . Continuous outcomes were presented as Weighted Mean Difference (WMD) and the 95% Confidence Interval (CI) was used to evaluate the precision and significance of that point of estimate. Forest plot was tested under the fixed or random effects models. If there was considerable heterogeneity across the combined studies, the random effects model was considered. Higgins IThe flow diagram of this study was presented in the 2 = 84.4%, p = 0.000)  .Figure 22 = 97.1%, p = 0.000) showed that there was significant association for the lowest serum vitamin D with CRSwNP and CRSsNP subjects  , while lower vitamin D level was associated with CRS in other Countries   (p = 0.005) but with a high heterogeneity (I2 = 73.5%) and no such phenomenon was seen in prospective ones  , types of biomarker (25(OH)D3 and 25(OH)D), geographic area (USA and non-USA) and study design . In an a= 0.226) . When st= 0.000) . The sub= 0.000) . The sub= 0.000) .Table 2Ap-value for the Egger’s test was 0.916, which suggested that there was no evidence of publication bias in this meta-analysis D3), which would circulate and active to calcitriol (1.25(OH)D3) by 1-α-hydroxylase in peripheral tissues. After binding to the intracellular Vitamin D Receptor (VDR), a number of cell signaling pathways are activated.A total of 516 cases in 8 relevant studies were identified in the current meta-analysis. The results indicated that the low serum vitamin D level was significantly associated with CRS under the random effect model. Low systemic vitamin D level might interfere with natural mechanisms to limit the mucosa inflammation, anti-proliferative and anti-angiogenic properties leading to episodes of CRS.However, there was high heterogeneity in the strength of associations between studies. To further explore the relationship, stratified analyses were performed based to the two different phenotypes of CRS and the finding of the subgroup analysis suggested that lower serum vitamin D was related to both CRSwNP and CRSsNP patients, especially to the CRS with polyps. This result was in line with the retrospective studies, which showed that vitamin D deficiency status was more prevalent in CRSwNP subjects.Including sex, race, season, body mass index and geographic area, these factors were known to randomly influence the serum 25(OH)D. Pinto et al. found that serum levels in African American patients with CRS were significantly lower than those in race- and sex-matched control groups while there was no significant difference between white subjects when using the same matched strategy.To our knowledge, this is the first cumulative analysis comparing serum vitamin D level of CRS with healthy ones. The strengths of the present study are: (1) Our analysis included all available studies that investigated the association between serum vitamin D status and CRS and the searching strategy and analysis were performed in an impartial and systematic manner in line with Cochrane standards. (2) The sensitivity analysis and publication bias detection suggested that the conclusion of this meta-analysis is quite stable. Although our results provide compelling evidence to support the phenomenon that serum vitamin D status might be a risk factor for CRS in the general population, limitations were also found in our study as other observational studies. For instance, there were only 8 studies included, and some of them were not of extreme high quality. The fact that no RCTs trials included might induce an inescapable bias. Most of the included studies were retrospective studies, which implied a case-control design, and therefore there was a selection and recall bias. Second, heterogeneity existed between different studies. Through the subgroups analysis, we noticed that the phenotype of CRS might be the reason of heterogeneity in this meta-analysis. Finally, we eliminate some studies published in non-English language, which might lead to a deficiency of data.In summary, our meta-analysis of 8 studies illustrates the lower serum vitamin D status in CRS patients, which indicates that people might get benefit from appropriate vitamin D supplementation. Therefore, due to the heterogeneity of the subjects, more well-designed prospective RCTs should be carried out to further validate these findings in for the general population in the future.Deep Underground Space Medical Center Research Foundation of Sichuan University (DUGM201804).This work was supported by a Grant of the The authors declare no conflicts of interest.


For occupational exposure in adults, cancer risks were even higher, ranging from 10−3 to 10−5, with the highest values associated with dermal exposure. (4) Conclusions: The study results suggest the presence of potentially toxic elements (PTEs) in cosmetics should be regulated/monitored to protect human health, especially for occupational exposure and use by children.(1) Background: Costume cosmetics, such as face paints and pancakes, are used by adults and children during Halloween, Carnival, or children’s parties. However, the metallic-based pigments used as dyes in these products may contain toxic elements associated with different levels of exposure. Objectives: (a) to determine the Al, As, Ba, Cd, Co, Cr, Cu, Ni, Pb, Sb, Sn, and Sr concentrations in face paints and pancakes; and (b) to estimate cancer and non-cancer risks posed by the concentrations of each element in these products for dermal and ingestion exposure scenarios during children and adult use. (2) Methods: A total of 95 samples of face paints and pancakes (four brands in different textures and colors) were purchased at the largest high-street commercial center in São Paulo city, Brazil. An extraction procedure with nitric acid was carried out using a graphite-covered digester block. Toxic element determinations were performed using an ICP-MS. (3) Results: The non-cancer risks estimated were lower than 1, except for dermal exposure in adults for some target systems. High cancer risk values raise concerns in both groups. The risk for children ranged from 10 Every year the consumption of cosmetics increases worldwide. However, consumers may not completely understand the risks to their health associated when using these products . ConcernThe presence of toxic elements has already been detected and is well-documented in traditional cosmetic products such as lipsticks, eyeshadows, and skin creams ,6,7,8,9.The face paints in liquid, cream, or pancake form are freshly applied to any part of the body, but most commonly to the head and trunk surfaces. These paints are used in occupational activities to convey a character’s personality and enhance the actor’s presence on stage . HoweverThe objective of this study was to determine the concentration of twelve potentially toxic elements  in costume cosmetics (face paints and pancakes) available in high street outlets in São Paulo state, Brazil. Cancer and non-cancer risks posed by the content of each element in cosmetics were determined according to the models proposed by the United States Environmental Protection Agency (USEPA). The risks for face paints and pancakes were estimated considering exposure to the elements determined via dermal and non-dietary ingestion routes.A total of 95 samples of face paints and pancakes were purchased from stores at the largest high street commercial center in the city of São Paulo, Brazil. The manufacturers of all the samples were Brazilian. Face paints of four brands in three different types  and multiple colors  were purchased (n = 90). All face paints available in the stores were purchased, whenever possible, in two different batches of each type and color. Pancakes for professional use were also evaluated, but only one brand was available for purchase in five different colors . −1) was added to each. Nitric acid digestion for metal determinations in cosmetic samples was reported previously by Lim et al. .]. After cooling, the volume increased to 40 mL with deionized water . Deionized water was used for all tests and cleaning. Before the ICP-MS analysis, the samples were filtered using a filter membrane of 0.2 µm (cellulose acetate).All samples were weighed out (150–200 mg in triplicate) and 2 mL of nitric acid  were also analyzed. To verify the accuracy of the procedure, CRM was prepared using the same procedure as for the samples. The CRM used were: NIST 2709 (San Joaquim soil), ERM CC 141 (Loam soil), NIST 1573 (Tomato Leaves), and Agro 1003a (Tomato Leaves). The ICP-MS operating conditions are shown in the Element determinations were performed using an inductively coupled plasma-mass spectrometer . A multi-element standard solution was purchased from PerkinElmer with a concentration of 1000 µg LCR) and non-cancer risk or dermal hazard quotient (HQ) during the period of exposure.During the application of face paints or pancakes to the skin, PTEs can undergo dermal absorption. The possibility of a person developing health problems or cancer due to the use and absorption of these products into the skin was evaluated by calculating the cancer risk (DAD) was calculated according to Equations (1) and (2) for soil contact from USEPA [C is the mean concentrations of PTE determined in the paints (mg kg−1); CF is a conversion factor defined by the USEPA as 10−6 mg kg−1 [AF is the amount of skin adherence of the paints per event [SA); ABS is a fraction of a specific metal absorbed dermally : 0.04; 0.001 for other PTEs) [eventDA is the dose absorbed per event (mg/cm2-event); EF is average exposure frequency considering hours per day and days per year [ED is exposure duration in years [SA is the average surface area that comes into contact with the paint, considering only the head surface for children and both head and trunk surfaces for adults [BW is average body weight [AT is averaging time . The estimated dose by dermal absorption (om USEPA .(1)DAev mg kg−1 ; AF is ter event , obtaineer PTEs) ,18; DAevin years ; SA is tr adults ; BW is ay weight ; and AT A list of all the variables and values used in the equations for dermal exposure is presented in HQ) was calculated as shown in Equation (3) according to the USEPA [The non-cancer risk or dermal hazard quotient (he USEPA .(3)HQ=DHQ calculation are presented in All the values used for oRfD is the oral reference dose (mg kg−1 day−1) from the USEPA/IRIS assessment and was used in HQ calculations for As and Sr. The Minimal Risk Levels (MRL) from the Agency for Toxic Substances and Disease Registry (ATSDR) were used for Al, Co, Cu, and Sn, whose oRfD were not available. In the absence of chronic MRL-oral values, intermediate MRL-oral values were used [absRfD) was used instead of oRfD. The USEPA values were adopted only for elements with recommendations for adjustment of toxicity . The USEPA equation based on gastrointestinal absorbed dose was used to obtain the absRfD (Equation (4)) [giABS is the fraction of contaminant absorbed in the gastrointestinal tract. The ere used . For Ba,ion (4)) .(4)RfDaCR) was calculated for As, Cr(VI), and Pb as shown in Equation (5), according to the USEPA [The dermal cancer risk (he USEPA .(5)CR=DoSF is the oral slope factor (mg kg−1 day−1) from the California Office of Environmental Health Hazard Assessment (OEHHA). This value was used in the equations for As and Pb. For Cr(VI), the USEPA equation based on gastrointestinal absorbed dose (Equation (6)) was used to obtain the absorbed cancer slope factor (absSF) [The  (SFabs) .(6)SFabPossible PTE exposure via incidental ingestion was investigated; therefore, the potential exposure from direct ingestion or ingestion via hand-to-mouth contact was evaluated. The exposure assessment was based on the statistical data provided in the USEPA Exposure Factors Handbook  and on cC is the mean concentration determined for each PTE in the paint (mg kg−1), Mass is the number of grams of product applied per use [SA is the surface area of the hand (cm2). The concentration of the product applied to the skin (appliedC) can be used to calculate the oral intake (µg day−1) from hand-to-mouth contact (HMIntake) using Equation (8), where handSA is the surface area of the hands [handFSA is the fractional surface area of the hand involved in hand-to-mouth contact [Df is the conversion factor for direct hand-to-mouth transfer efficiency (0.24) [t is the duration in hours per day that the cosmetic remains applied [The concentration of product applied per use was calculated using Equation (7), where  per use , and SA he hands ; FSAhand contact ; λD is t contact ; fD is ty 0.24) ; and t i ; and t −1) by body weight (BW) yields oral dose . This oral dose value was used to calculate ingestion cancer risk (CR) and hazard quotient (HQ). The same Equations (3) and (5) presented above were used, replacing the DAD value with the oralD, and using the oRfD and oSF for each element. Finally, dividing oral intake  who uses these products as costume cosmetics, and an adult (>21 years) in an occupational exposure scenario. For both of these situations, estimations were determined for the two biological sexes. Exposure was estimated using the mean concentrations of the detected PTEs. The estimates for children were determined for three different age groups , with summed results representing risk during childhood (age 2 to <11 years).CR) were calculated as the sum of the As, Cr(VI), and Pb values determined, given their potential carcinogenic effects and the fact that carcinogenic slope factors were available for these elements, assuming a linear dose-response relationship [HQ) was estimated for all elements except Pb. The final HQ values were summed by type of effect, i.e., according to the elements that had the same target system in the definition of the RfD or MRL, as shown in LT represents an averaging time equal to a mean lifetime of 70 years.For cumulative carcinogenic risks in case of exposure to multiple carcinogens, the risks of each substance were tallied. Information on non-additive interactions is not readily available and without this specific information, the cancer risk from various chemicals has been conservatively assumed to be additive . Therefotionship . The nonMRL), and reference doses (RfD) for the PTEs evaluated in this study are presented in For Cr(VI) risk estimates, the entire concentration of total chromium determined was considered hexavalent chromium. If the risk was within acceptable limits, this implied the lesser fraction of Cr(VI) would also be within safe limits, avoiding the need for chemical speciation ,30. A liDescriptive statistical treatment of the PTE concentrations was performed, including, arithmetic mean, minimum and maximum, and the 95th percentiles of each element. p < 0.05). All statistical analyses were conducted using R software [The Kruskal–Wallis test was performed to evaluate the statistically significant differences in PTE concentrations among samples of different colors  and types . Dunn’s test of multiple comparisons was performed following a significant Kruskal–Wallis test  and pancakes. Results that were below the limit of detection  were assigned a value equal to the detection limit divided by the square root of 2 (LOD/√2) . Resultsp = 0.01). The highest As mean concentrations were found in lilac, brown, and white paints (p = 0.01). Lilac, blue, and green colors had the highest means for Cu (p < 0.01). The highest means for Ba were in red colors (p < 0.01), whereas for Co, the highest means were in brown and yellow (p = 0.03). For almost all elements determined , the means were higher in the pancakes and liquid samples (p < 0.05). The cream samples and professional pancakes had higher means for Cd, Cr, and Pb (p < 0.0001). On the other hand, the means for Sr were higher in fluorescent and liquid paints. Only Cu did not differ in concentration between the types of costume cosmetics analyzed.The PTEs that showed statistically significant differences between colors occurred with As, Ba, Cd, Co, Cu, and Pb. White, blue, and purple colors had the highest mean for Pb and Cd . In all exposure scenarios, the estimated cancer risks for the use of pancakes were higher than the risks for face paint consumption.−8 for face paints and 1 × 10−7 for pancakes. The accidental ingestion risk exceeded 1 × 10−6 for face paints and 1 × 10−5 for pancakes. For the general population, the tolerable acceptable risk is 1 × 10−6, whereas the USEPA deems a risk of 1 × 10−4 tolerable for specific and justified situations [−6. In this case, the cancer risk values calculated for accidental ingestion by children exceeded this limit.For children, dermal exposure risks exceeded 1 × 10tuations ,34,35,36−3 for pancakes and face paints, while ingestion risk exceeded 1 × 10−5 for face paints and 1 × 10−4 for pancakes in adults. The tolerable risk values in occupational exposure are variable. The National Institute for Occupational Safety and Health of the U.S. (NIOSH) considers a maximum of 1 × 10−4 [−3 to 1 × 10−6. Considering this variability and a tolerable range of 1 × 10−4 to 1 × 10−5, many values estimated in the present study proved high, with the highest values for dermal exposure in adults.Dermal exposure values for adults exceeded 1 × 101 × 10−4 . By cont1 × 10−4 , the risThe non-cancer risks were lower than 1 for dermal exposure in children and ingestion exposure in both child and adult groups. Some values for dermal exposure in adults were greater than 1, where elements with the highest contribution to this total risk were arsenic and hexavalent chromium in pancakes. These reflect values greater than 1 for cardiovascular and dermal effects (100% As contribution) and other effects in pancakes (99% CrVI contribution).HQ) and cancer risk (CR) in child and adult exposure to face paints and pancakes are presented in None of the cancer and non-cancer risk values differed significantly between males and females. The total results for non-cancer  were applied based on EPA recommendations to account for the difference in absorbed dose relative to the administered dose and to avoid overestimation of risks. The EPA recommends adjustment for Ba, Cd, Cr, Ni, and Sb considering their absorption in the gastrointestinal system is low. For the other elements, the absorbed dose is equivalent to the administered dose, and therefore no toxicity adjustment was necessary [The exposure assessments used in this study were selected to conservatively estimate PTE exposures due to costume cosmetic application. As oral and dermal reference doses differ, toxicity factors  in costume eye-shadow and body paints sold in the United States, yet similar Co, Ni, and Pb concentrations  in pancakes; whereas Sb levels were higher in the US products (0.12–6.3 mg kg−1). The authors stated that the cumulative daily dose for all users did not exceed the RfD or MRL for As, Co, Ni, and Sb, and concluded that these concentrations do not pose a health risk to intermittent consumers and children, but occupational exposures may exceed health-based guidance values (1 × 10−4 mg kg−1 day−1). Wang et al. [CR ranged from 1.67 × 10−7 to 9.6 × 10−3. The carcinogenic risk in 25 paint samples ranged from 0.01% to 0.96%, with the highest risk for lifetime exposure to Cr-containing paints (above 1 × 10−4). A few studies determining the concentrations of some elements in costume cosmetics have been conducted. Relative to the levels detected in the present study, Perez et al.  found a 3+, Mn, Fe, Co, Ni, Cu, Zn, Cd, Sb, and Ti, but the cancer risk of dermal exposure to cosmetics in adults exceeded acceptable risk levels (>1 × 10−5). Arshad et al. [−6) and the minimum value in eye pencils (5.29 × 10−15). Conversely, the hazard quotient, hazard index, and cancer risk were below acceptable limits in cosmetic products of different brands in Nigeria, indicating a measure of safety [Other studies have evaluated the health risks of different types of cosmetics  for heavy metal contamination in products not specifically considered costume cosmetics. Lim et al.  found a d et al.  concluded et al.  found thf safety . Kilic ef safety  calculatf safety . Samplesf safety .Some countries have established regulations for the allowable amounts of heavy metals in cosmetics. According to Regulation (EC) No 1223/2009 of the European Union, heavy metals such as lead, cadmium, arsenic, and antimony were part of the list of substances prohibited in cosmetic products. However, the unintended presence of these metals in cosmetics is allowed if technically unavoidable . In the Limits have been defined in legislation for only a few PTEs, while some countries have no established limits. An international agreement on the status and safety requirements of these products and their ingredients is needed . MoreoveThe available literature shows that different elements are present in many types of cosmetics produced worldwide ,42,43,53Potentially toxic elements still exist in cosmetics. Therefore, it follows that the amounts applied to the skin or lips each day may accumulate over time. Moreover, countless cosmetics are on the market and may be used in combination, leading to different exposure patterns and health effects ,38. NotaSpecial attention should be paid to the adverse health effects of cosmetic product consumption, considering the growing use, repeated exposure, and lack of uniform legislation governing the presence of toxic metals. It is paramount to investigate cumulative exposure and child use in a bid to improve draft guidelines on impurities in cosmetics and reflect technically avoidable contamination ,51.−8 to 10−5, proving higher in cases of accidental exposure by ingestion. For adults, cancer risks were even higher, ranging from 10−3 to 10−5, with the highest values associated with dermal exposure. The element contributing most to total risk values was arsenic (approximately 90%) and exposure scenarios for pancakes were associated with higher risk values.In conclusion, the data obtained in this study provides useful information regarding the content of PTEs in face paints and pancakes used as costume cosmetics in Brazil. Concerns about contaminated cosmetics are becoming commonplace in the beauty market, but limits for metal impurities in these products remain rare, and regulation governing levels are lacking in some countries. The non-cancer risks were lower than 1 for all exposure scenarios, except dermal exposure in adults for some target systems . The cancer risk for children ranged from 10These products are applied to children during parties, Halloween, and Carnival as part of the entertainment and celebrations. However, this exposure to chemicals at such a young age during these occasions, which are supposed to be safe and fun, is inappropriate and should be avoided. Further, this investigation of PTEs contained in costume cosmetic products suggests that the presence of these elements in cosmetics needs to be regulated and monitored in all countries to protect human health, especially regarding occupational exposure and child consumption.


Immune-mediated demyelination and neurodegeneration are pathophysiological hallmarks of Multiple Sclerosis (MS) and main drivers of disease related disability. The principal method for evaluating qualitatively demyelinating events in the clinical context is contrast-weighted magnetic resonance imaging (MRI). Moreover, advanced MRI sequences provide reliable quantification of brain myelin offering new opportunities to study tissue pathology in vivo. Towards neurodegenerative aspects of the disease, spinal cord atrophy – besides brain atrophy – is a powerful and validated predictor of disease progression. The etiology of spinal cord volume loss is still a matter of research, as it remains unclear whether the impact of local lesion pathology or the interaction with supra- and infratentorial axonal degeneration and demyelination of the long descending and ascending fiber tracts are the determining factors.Quantitative synthetic MR using a multiecho acquisition of saturation recovery pulse sequence provides fast automatic brain tissue and myelin volumetry based on R1 and R2 relaxation rates and proton density quantification, making it a promising modality for application in the clinical routine. In this cross sectional study a total of 91 MS patients and 31 control subjects were included to investigate group differences of global and regional measures of brain myelin and relaxation rates, in different MS subtypes, using QRAPMASTER sequence and SyMRI postprocessing software. Furthermore, we examined associations between these quantitative brain parameters and spinal cord atrophy to draw conclusions about possible pathophysiological relationships.Intracranial myelin volume fraction of the global brain exhibited statistically significant differences between control subjects (10.4%) and MS patients . In a LASSO regression analysis with total brain lesion load, intracranial myelin volume fraction and brain parenchymal fraction, the intracranial myelin volume fraction was the variable with the highest impact on spinal cord atrophy (standardized coefficient 4.52). Regional supratentorial MRI metrics showed altered average myelin volume fraction, R1, R2 and proton density in MS patients compared to controls most pronounced in PMS. Interestingly, quantitative MRI parameters in supratentorial regions showed strong associations with upper cord atrophy, suggesting an important role of brain diffuse demyelination on spinal cord pathology possibly in the context of global disease activity. R1, R2 or proton density of the thalamus, cerebellum and brainstem correlated with upper cervical cord atrophy, probably reflecting the direct functional connection between these brain structures and the spinal cord as well as the effects of retrograde and anterograde axonal degeneration.By using Synthetic MR-derived myelin volume fraction, we were able to effectively detect significant differences of myelination in relapsing and progressive MS subtypes. Total intracranial brain myelin volume fraction seemed to predict spinal cord volume loss better than brain atrophy or total lesion load. Furthermore, demyelination in highly myelinated supratentorial regions, as an indicator of diffuse disease activity, as well as alterations of relaxation parameters in adjacent infratentorial and midbrain areas were strongly associated with upper cervical cord atrophy. It is wThere have been many endeavors in the last 20 years to succeed accurate quantification of myelin content in the brain tissue by using advanced quantitative MRI methods. Some of the most established MRI methods for myelin imaging are myelin water imaging, T1/T2 ratio and magnetization transfer imaging . DrawbacBesides brain atrophy, upper cervical cord atrophy has been reliably used to monitor disease progression in MS as a measure of neurodegeneration and axonal loss . Spinal The aim of this study was to compare global and regional myelin measures and relaxation rates derived from the QRAPMASTER sequence between MS groups and control subjects and to investigate associations of those parameters in several infra- und supratentorial brain structures with the mean upper cervical cord area (MUCCA) in MS patients. We hypothesized that brain myelin loss may show a closer relation to MUCCA loss than brain atrophy and total brain lesion load. Furthermore, we assumed that associations between cervical cord volume and brain structures will be most pronounced in anatomically connected structures, such as the thalamus, brainstem, cerebellum and the corticospinal and spinocerebellar tracts, and that associations between upper cervical cord volume and brain myelin might differ between progressive and relapsing forms of MS.22.1MRI and clinical data from 49 RRMS, 27 SPMS, 15 PPMS and 31 control subjects (CS) were acquired from November 2018 till December 2019 in the context of routine clinical follow up and were retrospectively reviewed in a cross-sectional manner. Patients had to fulfill definitive diagnosis of MS according to 2017 McDonald criteria  with an 2.23) and conventional contrast weighted imaging including sagittal 3D T1-weighted (T1w) MPRAGE  and sagittal 3D FLAIR  images of the brain. MRI sequences with contrast enhancement using gadolinium were always performed after the acquisition of QRAPMASTER sequence to avoid changes of relaxation rates and possible interference with myelin quantification by virtue of blood brain barrier breakdown and paramagnetic effects of contrast agents  using a 16 channel head/neck matrix coil. All patients underwent quantitative MR imaging using the QRAPMASTER sequence , R2 (s−1) and PD (%) within the global brain were calculated. The brain segmentation into white matter (WM), gray matter (GM) and cerebrospinal fluid (CSF) is based on a tissue look up grid in a three dimensional R1-R2-PD space which has been created on the basis of T1 and T2 relaxometry values in different brain regions of healthy volunteers. Myelin https://www.statistical-modelling.de/lst.html) for Statistical Parametric Mapping . Furthermore, we have thoroughly inspected lesion maps for misclassification and manually edited them using the FSLeyes viewer if necessary.To extract the global brain lesion load we generated binary MS lesion maps based on conventional 3D FLAIR images using the lesion prediction algorithm  as imple2.3.2Four different atlases for brain structures  were used to extract average MVF, R1, R2 and PD fractions in 28 brain regions of interests (ROI). The average MVF (aMVF) represents the mean value of MVF in all ROI-voxels. Since our main interest was to study associations between cervical cord area and quantitative brain measures we focused on brain ROIs which are functionally or structurally connected to the spinal cord . We analhttps://fsl.fmrib.ox.ac.uk/fsl/fslwiki) for image registration among the different MRI sequences and for the extraction of average quantitative MRI values within ROIs (3) which were linearly registered to the resampled 3D T1w images (flirt). The 3D T1w images were then registered to the FSL’s 2 mm MNI template using linear and non-linear registration (flirt + fnirt). The obtained warping fields were inverted in order to register each ROI mask in MNI space to individual 3D T1w space. Likewise, the matrices from the registration of synthetic T1w to 3D T1w images were inverted and applied to ROI masks in 3D T1w space in order to obtain ROI masks in individual synthetic MRI space. Mean values and standard deviations of aMVF, R1, R2, and PD within each ROI were extracted using fslstats.We established an analysis pipeline using FSL software tools  were performed based on the sagittal 3D-T1w image series of the brain. MUCCA was measured using the semi-automated software NeuroQLab  over a predefined cord section with a length of 30 mm (starting from the top of the C1 vertebra) as described in previous works. In brie2.4SPSS software  was used for the statistical analyses. Results were considered statistically significant at p < 0.05. All parameters were tested for normality with a Shapiro – Wilk test.As a preprocessing step, prior to further analyses, we tested all MRI variables in the control subject group for significant associations with age by using linear regression models, in order to detect dependency on physiological aging. Subsequently we applied the inverse transformation parameters for a mean age of 40 years to the MRI variable for both, control subject group and the patient group. This resulted in a complete reduction of age dependency in the control group, and a compensation of physiological aging in the patient group.Group comparisons between CS and the patient subgroups regarding age and disease duration were assessed by univariate ANOVA with Games-Howell correction for multiple comparisons of post-hoc pairwise tests between the subgroups. For group differences of EDSS and global and ROI-derived quantitative MRI parameters  we applied Kruskal Wallis tests and corrected pairwise post- hoc tests for multiple comparisons with the Dunn–Bonferroni method correction. In order to provide an overview of the deviations of aMVF, R1, R2 or PD in the patient subgroups from CS we calculated standardized z-scores (z -scores relative to the control group) and summarized them in bar-plots. The individual z-scores were calculated as.We evaluated associations between MUCCA and global as well as regional synthetic MR derived MVF and relaxometry results using Spearman rank correlation analyses. To account for the risk of false-positive findings due to multiple testing of correlations with MUCCA in 28 different atlas-based regions, we applied a Bonferroni – Holm correction for multiple comparisons.In order to investigate whether iMVF, T2w lesion load or brain atrophy were most related to spinal cord atrophy, we used Least Absolute Shrinkage and Selection Operator (LASSO) regression with MUCCA as dependent variable and iMVF, brain parenchymal fraction and lesion load as independent variables. Lasso is a penalized regression technique that models the joint dependence of multiple variables on a quantitative parameter, and is suitable for variables tending to multicollinearity . The obt33.1Demographic and clinical data as well as global MRI parameters are depicted in ). Therein the rho coefficients for correlation with MUCCA were greatest for iMVF, BPF and WMF. Separate Spearmann correlation analyses for RRMS and PMS patient groups yielded significant correlations of iMVF (ρ = 0.325), BPF (ρ = 0.293) and WMF (ρ = 0.349) with MUCCA only in the RRMS group (data not shown). The PMS group demonstrated no statistically significant results in this analysis.Among all MS patients, MUCCA was significantly associated with total brain lesion load, iMVF, GMF, WMF, and BPF by using Spearman rank correlation analyses , Fig. 4.Fig. 4.We analyzed which of the global MRI features had the highest impact on upper cervical cord atrophy in the entire patient group. By virtue of representing measures of global demyelination (including NAWM), focal lesion pathology, and brain atrophy, we chose iMVF, lesion load and BPF as variables. Potential outliers  were excluded, also in order to succeed the normal distribution of the variable, resulting in 78 patients with all parameters available. A small degree of collinearity between these three parameters was obtained by variance inflation factors (VIF) of 1.8 for iMVF, 2.4 for lesion load and BPF as well as Pearson correlation coefficients of −0.63 for lesion load vs iMVF, −0.73 for lesion load vs BPF and 0.69 for iMVF vs BPF. Thus, we used LASSO regression, which is suitable for multicollinearity problems and we evaluated variable importance by use of standardized coefficients. The highest coefficient was obtained for iMVF (4.52). BPF and lesion load showed much lower coefficients of 0.37 and 0.003, respectively.3.2Median and interquartile ranges of the quantitative MRI parameters  were assessed in all brain ROIs separately for CS and the two patient subgroups. The results are summarized as z-deviations compared to the control group in −1, R2 = 11.0 s−1, PD = 82.6 %). Highest R1 and lowest PD were found in the anterior corona radiata , while the pallidum was the structure with highest R2 in the control group (R2 = 15.6 s−1).In the control group the aMVF within the ROIs ranged between 22.4 %  and 30.8 % (left anterior corona radiata) in the supratentorial regions, between 10.1 % (left putamen) and 20.2 %  in the deep gray matter structures, and between 13.0 % (cerebellum) and 25.2 % (middle cerebellar peduncle) in the infratentorial regions. By comparison, lowest R1 and R2 and highest PD were determined in the cerebellum  demonstrated statistically significant associations with MUCCA in these structures. Brainstem, cerebellum, cerebellar peduncles, corticospinal tract, putamen, thalamus, posterior limb of internal capsule and superior corona radiata did not exhibit any significant associations between aMVF and MUCCA.In contrast to aMVF, relaxation rates – especially R1 – presented significant associations with MUCCA not only in supratentorial but also in infratentorial structures, namely in the cerebellum and in the brainstem. Especially in the right thalamus relaxation rates and proton density correlated well with spinal cord atrophy measures. An additional subgroup analysis (data not shown) regarding RRMS and PMS separately showed no significant correlation between MUCCA and any of the four quantitative MRI parameters in both MS subgroups.4Myelin damage occurs in the brain within inflammatory lesions or as diffuse demyelination of white and grey matter and is one of the major drivers of clinical disability in MS . In this4.1Intracranial MVF, WMF and GMF were reduced in relapsing as well as progressive MS compared to CS, with strongest effects in PMS (table 1). Our iMVF results matched well with a recent validation study using the same method that identified lower myelin fractions in the whole brain and NAWM of relapsing–remitting and progressive MS patients relative to healthy controls . That reIn accordance with the literature, we detected significant spinal cord atrophy in both patient groups compared to CS, which was most pronounced in PMS (table 1) . We deteAiming at the question whether the associations between MUCCA and iMVF were based on affection of structures that are functionally connected with the cervical cord, or whether they are caused by parallel but independent processes, we conducted a regional atlas-based quantification of aMVF, R1, R2 and PD and investigated their ROI-based associations with MUCCA.4.2We focused on brain ROIs which are functionally or structurally connected to the spinal cord, namely the corticospinal tracts and related structures  the brainstem and cerebellum with the superior, inferior and middle cerebellar peduncles, and the corpus callosum as a highly myelinated reference structure.Our aMVF results of the control group in the corpus callosum, internal capsule, corona radiata and posterior thalamic radiation (average MVF in ROIs between 22 % and 31 %) agreed with a recent ROI-based validation study on healthy controls that compared QRAPMASTER based myelin quantification with other MRI based methods . DiscrepWe demonstrated consistent decreases of aMVF, R1, and R2 as well as PD increase compared to CS in most of the selected supratentorial ROIs in RRMS and PMS patients, with the PMS group being more severely affected . The chaThe corticospinal tracts showed most pronounced alterations of aMVF, R1, R2 and PD in the left CST in PMS compared to CS. The right CST was involved to a lesser extent and showed mainly reduced values of R1, R2 and higher PD in PMS compared to CS. Thus, the CST as a connecting structure between supratentorial and infratentorial regions was stronger affected in PMS than in RRMS, which supports the hypothesis that the tissue damage may spread downstream from supratentorial structures via the CST towards the spinal cord during the progression of the disease. Furthermore, a left–right hand side asymmetry of CST affection, especially in SPMS patients, is in line with a previous study that regarded multiparametric MRI indices  . SimilarIn the DGM structures, basal ganglia and thalami , we founWhen interpreting the aMVF in the infratentorial structures, similar issues as in the DGM emerged, related to MVF modelling in GM containing structures. The cerebellum and, to a lesser extent, the brainstem contain substantial amounts of GM, in the cerebellum in the form of the tightly folded cerebellar cortex and the dentate nucleus which make up more than half of the cerebellar volume, and in the brainstem by a number of GM nuclei intermingled with the WM fiber bundles. For the ROI containing the entire cerebellum, the aMVF therefore showed relatively low values (about 13 %), which, however, did not differ between the subgroups. By comparison, the aMVF in the middle cerebellar peduncles, which largely consist of the cerebellar WM fiber trunks, were significantly higher (approx. 25 %). In the brainstem we found an aMVF of about 19 %, which was even increased in the MS groups, significantly in PMS compared to the control group. The SyMRI myelin model may, as explained above, be only partly valid in the brainstem and the cerebellum because of their complex histopathologic structure of grey and white matter, and might not give a complete picture of myelin pathology. Therefore we referred to the relaxation parameters R1, R2 and PD for additional information on pathologic changes presumably including myelin loss in these structures. In the brainstem, the cerebellum and the superior cerebellar peduncles the observed R1 and R2 reductions and PD increases, especially in PMS compared to controls, corresponded to our hypothesis of a higher level of pathologic changes within these structures in the progressive MS forms and that in PMS there is a focus on downstream degradation of the infratentorial structures which include regions that belong to the descending corticospinal pathways. There are few previous studies that have examined relaxation parameters in infratentorial areas of the brain that could be used for direct comparison with our quantitative results. However, in our control group, we found good agreement between our R1 values in the brainstem and superior cerebellar peduncles and a recent study  that exa4.3Associations between MUCCA and any ROI-derived aMVF, R1, R2 or PD were not found in the control group, showing that potential physiological associations between cervical cord volume and brain myelin or relaxation parameters were negligible in our study, and that the associations that we found in the patient groups were most probably related to the disease.We detected correlations between aMVF and MUCCA in supratentorial WM structures  in MS patients. Herein, the anterior and posterior corona radiata contain motoric projection fibers that feed into the corticospinal tracts. The body of the corpus callosum is related with the motoric system by incorporating transversing interhemispheric fibers that originate in the bilateral motoric cortical areas. Demyelination in these structures can be a source of transsynaptic or anterograde Wallerian degeneration affecting the upper spinal cord. Recent studies which have shown that atrophy of the spinal cord in MS is more pronounced in the upper cervical cord than in the lower segments, and is mostly independent of local spinal cord lesions, have supported the hypothesis that cord atrophy might result from remote effects originating in the brain . On the In infratentorial structures and DGM, we observed correlations of MUCCA with relaxation rates and PD but hardly any association with aMVF (table 3). The identification of correlations between MUCCA and myelin in these structures may have been compromised by the effects explained in the previous section, which are related to the high proportion of GM in these ROIs limiting the validity of the SyMRI myelin model, and related to iron deposition the basal ganglia, which have an impact on the R2 relaxation rates.Regarding the thalamus we observed correlations between MUCCA and R1, R2 and PD that were plausible when considering that the thalamus is directly connected to the spinal cord via the spinothalamic tracts, and thus, supports our hypothesis of associations between cord atrophy and pathological changes in functionally connected structures. Both, cervical cord and thalamus, have been shown to be highly sensitive to disease related volume loss and degradation of white matter fibers in MS . Still, Furthermore, MUCCA was correlated with aMVF and proton density of the pallidum, but not with R1 or R2 in this structure. We assume that the large proportion of GM and predominant iron accumulation in the pallidum may mask associations of MUCCA with the relaxation rates.Separate correlation analyses of relapsing and progressive MS did not detect significant associations of MUCCA with regional quantitative MR data in the brain, probably due to limited patient numbers in our subgroups. Still, we can assume that the associations between MUCCA and thalamic and infratentorial relaxation parameters in the entire MS group were probably driven by PMS, because alterations of R1, R2 and PD were strongest in PMS and previous studies have shown that microstructural involvement of the infratentorial brain is pronounced in progressive MS .4.43. Despite fair histological validations of supratentorial myelin estimation the SyMRI myelin model showed limitations in GM rich structures like the basal ganglia, brainstem and cerebellum, as described in the discussion. Furthermore, partial volume effects can have an impact on tissue classification in the SyMRI tissue compartment model at the boundaries between different tissue types or at interfaces between brain and CSF. Particularly in infratentorial structures, pulse and motion artifacts produced from vascular structures (transverse and sigmoid sinuses) or CSF can interfere with the relaxation process (EPI-based acquisitions, such as QRAPMASTER could also influence the estimation of myelin based on the SyMRI tissue model (We acknowledge the limitations of this study and the need to consider several technical and methodological issues. The QRAPMASTER sequence generates axial slices with 4 mm slice thickness and a voxel size of 4 mm process . Furtherue model  With reg5In conclusion, global quantitative MR parameters including myelin volume fraction derived from QRAPMASTER sequence were suitable to detect differences between controls and MS patients as well as between relapsing and progressive MS. With regard to the upper spinal cord, the intracranial myelin volume fraction had the strongest impact on spinal cord volume, suggesting that cervical cord atrophy is influenced stronger by demyelination within the brain, than by neurodegeneration or inflammatory brain lesions. Regional investigation of brain quantitative MR parameters indicated more severe involvement of infratentorial structures in progressive MS disease than in RRMS. Associations between MUCCA and supratentorial myelin loss and thalamic and infratentorial R1, R2 and PD could be demonstrated in MS patients. Thus, quantification of myelin and relaxation parameters by QRAPMASTER sequence is suitable to detect disease specific differences in the brain in relapsing and progressive MS and can also contribute to the clarification of the origin of upper spinal cord atrophy in MS.Theodoros Ladopoulos: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization. Britta Matusche: Methodology, Formal analysis, Writing – original draft, Writing – review & editing, Visualization. Barbara Bellenberg: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization. Florian Heuser: Formal analysis. Ralf Gold: Supervision, Validation, Funding acquisition. Carsten Lukas: Conceptualization, Methodology, Resources, Supervision, Validation. Ruth Schneider: Conceptualization, Methodology, Resources, Supervision, Validation, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.