Apart from developmental disabilities, the prevalence of chronic diseases increases with age especially in those with co-morbidities: vitamin D deficiency plays a major role in it. Whether vitamin D deficiency initiates and/or aggravates chronic diseases or vice versa is unclear. It adversely affects all body systems but can be eliminated using proper doses of vitamin D supplementation and/or safe daily sun exposure. Maintaining the population serum 25(OH)D concentration above 40 ng/mL  ensures a sound immune system, minimizing symptomatic diseases and reducing infections and the prevalence of chronic diseases. This is the most cost-effective way to keep a population healthy and reduce healthcare costs. Vitamin D facilitates physiological functions, overcoming pathologies such as chronic inflammation and oxidative stress and maintaining broader immune functions. These are vital to overcoming chronic diseases and infections. Therefore, in addition to following essential public health and nutritional guidance, maintaining vitamin D sufficiency should be an integral part of better health, preventing acute and chronic diseases and minimize their complications. Those with severe vitamin D deficiency have the highest burdens of co-morbidities and are more vulnerable to developing complications and untimely deaths. Vitamin D adequacy improves innate and adaptive immune systems. It controls excessive inflammation and oxidative stress, generates antimicrobial peptides, and neutralizes antibodies via immune cells. Consequently, vitamin D sufficiency reduces infections and associated complications and deaths. Maintaining vitamin D sufficiency reduces chronic disease burden, illnesses, hospitalizations, and all-cause mortality. Vulnerable communities, such as ethnic minorities living in temperate countries, older people, those with co-morbidities, routine night workers, and institutionalized persons, have the highest prevalence of vitamin D deficiency—they would significantly benefit from vitamin D and targeted micronutrient supplementation. At least now, health departments, authorities, and health insurance companies should start assessing, prioritizing, and encouraging this economical, non-prescription, safe micronutrient to prevent and treat acute and chronic diseases. This approach will significantly reduce morbidity, mortality, and healthcare costs and ensure healthy aging. Vitamin D is not a hormone but is essential for human survival. Compared with white people, darker-skinned people need longer skin exposure to sunlight. However, this is impractical for many because of the sun’s intensity, less time available for exposure, and insufficient UVB rays reaching the surface in northern latitudes . Consequently, darker-skinned people likely have a higher prevalence of hypovitaminosis, lower fertility rates, higher rates of infections and complications, a higher prevalence of chronic diseases , and a shorter life span, especially during the winter ,2. The fSignificant advances have been made over the past two decades related to vitamin D, especially in the immune system and its effects on chronic diseases. Parent vitamin D (D), 25-hydroxyvitamin D [25(OH)D: calcifediol], and 1,25-dihydroxyvitamin D  all have specific roles to play in the physiological activities of vitamin D. For example, the parent vitamin D is important to reach into target cells as a precursor generating calcitriol . VitaminVitamin D  is a fat-soluble secosteroid, the clinical importance of which was understood in 1920. Then, patients with tuberculosis were cared for in solariums or with daily exposure to direct sunlight [2D , which helps patients’ recovery [https://www.ncbi.nlm.nih.gov/books/NBK481857/table/chapter6.t1/: Accessed, 5 July 2023) restrict the penetration of UV rays to the dermis [UVB converts 7-dehydrocholesterol in the skin into pre-vitamin D, which is hydroxylated to form 25(OH)D in the liver, the precursor of 1,25,(OH)recovery ,7. The srecovery , but modrecovery ,10. There dermis ; thus, tEvolutionarily, having a varying degree of melanin pigment in the skin was a healthful compromise for those living in regions closer to the equator , where the sun’s rays are intense. However, when humans migrated from central Africa to the northern areas to access more food, they had lesser exposure to sunlight. To overcome this natural disadvantage, the density of melanin pigment in the skin gradually reduced over many generations—a natural evolutionary survival mechanism. Those who developed a lighter skin color (white-skinned people) had a significant survival advantage—better protection against infections, fewer chronic diseases , and a h3, binds to vitamin D binding protein (VDBP), and releases it to circulation within 24 h [The pre-vitamin D generated in the dermis undergoes thermal isomerization to form vitamin Dhin 24 h . Becausehin 24 h . Circulahin 24 h . In the hin 24 h .The internalization rate of vitamin D into cells is higher than that of 25(OH)D in cells that do not express the megalin–cubilin transport system , such as2D has broad physiological functions [Vitamin D is a secosteroid molecule: fully activated vitamin D, 1,25(OH)unctions ,24. Thesunctions , membranunctions , and repunctions ,27.3 is supposed to be obtained naturally by humans following exposure to UVB rays from sunlight. In the skin, 7-dehydrocholesterol converts to pre-vitamin D3, which isomerizes to form vitamin D3. It binds to VDBP and diffuses via capillaries into the circulation [2 in sun-exposed mushrooms and D3 n fatty fish. After intestinal absorption as chylomicron, vitamin D is incorporated into VDBP via lipoproteins and reaches the bloodstream through the thoracic duct [Vitamin Dculation . There icic duct . These rcic duct . In addicic duct ,32,33 socic duct ,35. Howe2D have different dissociation constants in binding to VDBP, which determine the free (unbound to VDBP) D and 25(OH)D concentration in the circulation, which is approximately 1% of the that of the total vitamin D. The dissociation constant of 25(OH)D is about 10−9 m, while for vitamin D and 1,25(OH)2D, it is approximately 10−7 m [2D, a few hours [2D, then D, and lowest for 25(OH)D.Vitamin D, 25(OH)D, and 1,25(OH)y 10−7 m ,23. Consew hours . Accordi3 could be synthesized in the skin after exposure to sunlight, which takes about 24 h to materialize in the circulation [3 appears in circulation between 12 and 20 h after intestinal absorption and transportation [2 and D3 is approximately 24 h; that of D2 is less than that of D3 [A few thousand International Units (IU) of vitamin Dculation . Ingesteortation ,29,37. Tat of D3 ,38. Therat of D3 .Vitamin D deficiency universally impairs its intended benefits in all body systems. Its deficiency increases the vulnerability to infections, increases generalized inflammation, increases risks for diseases and infections, and worsens chronic diseases ,39,40. C2D. This is due to gastrointestinal malabsorption, increased catabolism, and a significant decrease in renal 1α-hydroxylation by CYP27B1. This results in low circulatory calcitriol that causes hyperphosphatemia and elevated fibroblast growth factor-23 (FGF-23) concentrations [Persons with chronic kidney disease (CKD) have insufficient handling of vitamin D, 25(OH)D, and 1,25(OH)trations ; these itrations ,47. The trations .3) [The abovementioned abnormalities of vitamin D metabolism lead to secondary hyperparathyroidism, which rapidly responds to oral cholecalciferol (D3) . Surviva3) . In cont3) .3 and 25(OH)D3 are in the micromolar range, while 1,25(OH)2D3 is present in the nanomolar range, with a calcitriol concentration of approximately nine-hundred-fold lower {Wimalawansa, 2023 #17062}. Therefore, calculating this ratio does not include calcitriol or 1,24,25-trihydroxyvitamin D concentrations in circulation as they are minuscule (as other uncommon metabolites and epimers of vitamin D), making it easier to calculate. While controversial, a reverse J shape of all-cause mortality has been reported with total serum 25-hydroxyvitamin D concentration [The higher catabolic ratios and thus lower 25(OH)D concentrations are associated with modestly increased all-cause mortality . Circulantration , for whintration ,53.The classical actions of vitamin D involve mineral metabolism—calcium absorption and mineral conservation, skeletal calcification, and musculoskeletal functions ,7. TheseThe tissue transport mechanism for steroids—megalin–cubilin endocytotic system —is essenRespiratory tract illnesses, including colds, influenza, and COVID-19, escalate in the winter. There are specific reasons why countries located far north of the equator in the northern (and southern) hemispheres experience winter-associated viral respiratory cycle that increases in colder months with less sunlight ,58,59. DVitamin D deficiency markedly impairs overall immunity and thus increases the risk of illness, including metabolic disorders and infections. This makes individuals vulnerable to microbial infections ,64, primChildren rely primarily on their innate immune systems to counteract pathogenic microbial invasions. Since they have better innate immunity than the elderly, they are less likely to develop symptomatic COVID-19, complications or die from it unless they have severe hypovitaminosis D . Severe Most extra-musculoskeletal biological activities of calcitriol occur following the generation of calcitriol within peripheral target cells , where it acts as a signaling molecule and a local cytokine. The latter functions include controlling cell proliferation and maturity, preventing cancer cell growth, brain development, respiratory and reproductive functions, and mitochondrial energy generation ,81,82,83https://COVID19criticalcare.com/treatment-protocols/: Accessed 5 July 2023), affirmative Substack articles, and websites like https://COVID19criticalcare.com (Accessed 1 July 2023) [A large data set and emerging data support multiple physiological functions of vitamin D, via calcitriol. These data suggest vitamin D should be used as a preventative and adjunct therapy in several common disorders, including sepsis and COVID-19 infection ,70,90,91ly 2023) .Sufficient calcitriol synthesis within immune cells prevents autoimmune reactions profoundly and controls inflammation and infections ,40. ThesThese antimicrobial peptides stimulate white blood cells, macrophages, and natural killer cells and direct the circulating viruses to macrophages to destroy them . Vitamin3) to prevent musculoskeletal disorders [Over the years, the focus has been on cholecalciferol , or have significant fat malabsorption require several fold-higher doses than those mentioned above. Even with such amounts, a vitamin D-deficient person likely takes several months to increase their serum 25(OH)D to therapeutic levels of over 50 ng/mL . Using tSerum 25(OH)D concentrations are reduced in chronic diseases like metabolic disorders, obesity, cancer, infections, and all-cause mortality ,102,103.Meta-analyses of RCTs concerning vitamin D supplementation reported a significant reduction in the incidence and severity of respiratory tract infections ,108,109—Using higher doses of vitamin D consistently has been reported to have better clinical outcomes than the government-recommended doses of 800 IU/day, which have no tangible effect on any disease other than muscular–skeletal disorders ,112. For3 quarterly [However, studies that used minute doses of vitamin D based on outdated recommendations  ,120, as uarterly  failed tuarterly ,122, anduarterly .Most steroid hormones enter cells via diffusion and endocytosis via the membrane-based megalin–cubilin system as in the kidney and parathyroid gland, muscle, and fat cells . In addiIn addition to diffusion, as illustrated above, VDBP bound D and 25(OH)D enters these cells via endocytosis . Since tWhen vitamin D is taken daily, the circulatory vitamin D concentrations are likely to be higher than 25(OH)D concentrations . Therefo3 enhances epithelial and endothelial stability independently of canonical pathways through calcitriol/CTR-derived genomic outcome [3 supplementation. These rapid membrane-related actions of vitamin D are derived from D3 and its two common metabolites, 25(OH)D and 1,25(OH)2D, at a similar potency.D outcome . The dis3 and its metabolites impairs endothelial barriers, leading to vascular fluid leakage into soft tissues [The deficiency of D tissues . Similar tissues . These n3 after one hour of UVB exposure over a third of the upper body [A century ago, it was established that sunrays (vitamin D) reverse rickets in children and are effective against tuberculosis. In addition, a large body of scientific evidence demonstrates that vitamin D plays a central role in disease prevention (maintaining a disease-free state) and preventing severe symptoms, diseases, complications, and deaths . A few yper body ,130,131.Maintaining a steady state of D and 25(OH)D in circulation is helpful for better physiological functions. Marked fluctuating serum 25, and no more than once a month. Therefore, vitamin D should not be administered at intervals longer than two-week intervals . This wiThe importance of the above is highlighted by six positive respiratory tract infection-related RCTs, most conducted in children ,57,58,59Different dosing schedules have varied effects on serum vitamin D and 25(OH)D concentrations—daily doses maintain a stable circulating concentration . In cont3-supplemented to a lactating mother, vitamin D concentration in her breast milk increased by about 80 IU/L. The recommended average dose of vitamin D3 for pregnant and lactating mothers is 6000 IU/d; this provides the infants with 400–500 IU of vitamin D per day [Vitamin D supplementation and sufficient UV exposure increase maternal circulating 25(OH)D concentration in breast milk ,138. It  per day .The circulating concentration of 25(OH)D in the fetus is approximately 70% of that of the mother; thus, a diffusion of 25(OH)D occurs across the placenta . HoweverCalcitriol is the most active vitamin D metabolite, crucial for combating invading pathogens and preventing autoimmunity, and chronic diseases ,2. ThrouPeripheral target cells, like immune cells, depend on vitamin D and 25(OH)D primarily via diffusion from the circulation to generate higher concentrations of non-hormonal calcitriol. When immune cells detect external threats, like circulating microbes or unfamiliar antigens by pattern recognition receptors, like membrane-bound Toll-like receptors TLR (TLR-4) they send signals to increase the expression of 1α of VDR, thus, increasing in the cytoplasm.Higher nmol range concentrations of calcitriol generated in-tracellularly in response to TLR signaling, provides  intracellular autocrine/intracrine signaling that is crucial for immune functions to overcome threats, like infections. Consequently, there is a hold mechanism, increasing serum, i.e., beyond threat like detecting unfamiliar proteins or antigens in the circulation or local tissues. The sporadic increases in the synthesis of calcitriol and VDR in responses to TLR-4 signaling, ensures the formation of sufficient calcitriol-VDR complexes to modulate transcriptions, and intra-cellular autocrine signaling and genomic modulation, as when needed.Regulates inflammation and oxidative stresses through the abovementioned mechanisms, primarily by suppressing inflammatory cytokines and enhancing the synthesis of anti-inflammatory cytokines. Immunomodulatory effects of vitamin D include activation of immune cells such as T and B cells, macrophage and dendritic cells, and enhanced production of several antimicrobial peptides and neutralizing antibodies ,4,5. FigOnce adequate concentrations are generated within the immune cells, calcitriol activates and bonds to the cytosol’s vitamin D receptors (VDRs) that translocate into the nucleus for its genomic actions. The interaction of calcitriol with its receptor leads to the translocation of the complex to the nucleus, where it binds to the genome and modulates over 1200 genes . In addip = 0.004) compared with those with negative results (24.6 ng/mL), demonstrating a higher vulnerability [Because hypovitaminosis D status does not activate immune cells, it causes relative immune paresis and delayed responses. This increases people’s vulnerability, especially to bacteria (like tuberculosis)  and resprability . This isrability .In addition, there is a strong correlation between severe vitamin D deficiency and cytokine storm—a hyper-inflammatory condition caused by an uncontrolled, overactive immune status . Viral iA balanced diet with adequate micronutrients, such as vitamins D, B2, K2, and C, and magnesium, trace minerals, and antioxidants, will support a more robust immune system. In most countries, some communities have one or more prevailing micronutrient deficiencies that increase vulnerability to various disorders, such as metabolic, infectious, and non-communicable diseases. In addition to nutrient supplements, fortifying foods with vitamin D and other essential micronutrients will enable them to develop a robust immune system, which prevents them from becoming frequently sick with viral infections.Nationwide vitamin D supplementation, at least during epidemics and pandemics, markedly reduces acute and chronic diseases, the need for hospitalization, and their complications and deaths, including SARS-CoV-2 infection. It is common to have vitamin D deficiency in those with co-morbidities and chronic diseases, such as hypertension, diabetes mellitus, obesity, and cancer, and to be more vulnerable to developing complications from infections like COVID-19 . Vitamin2, resveratrol, and magnesium, in combination with essential fatty acids, such as omega-3, would facilitate the maintenance of a robust immune system [Maintaining serum 25(OH)D concentrations above 40 ng/mL 100 nmol/L) is thought to significantly reduce microbial infections, particularly respiratory viral ones, including COVID-19 00 nmol/L. Enrichie system ,167,168 Sustained vitamin D deficiency adversely affects human health, which is cost-effectively prevented with vitamin D supplementation and/or regular safe sun exposure. Maintaining population serum 25(OH)D concentrations above 40 ng/mL ensure a robust immune system. Sustained vitamin D deficiency negatively affects all body systems and increases risks for viral infections, outbreaks, and hospitalization. Thus, government and health administrators should consider nationwide educational campaigns for safe sun exposure, vitamin D supplementation, and targeted food fortification programs to strengthen the population’s immunity and keep them healthy. These acts cost less than 0.01% of one day’s hospitalization and significantly reduce healthcare costs.Health insurance companies have a financial incentive to take proactive actions to keep their clients healthy by maintaining their vitamin D sufficiency. While the efficacy of vaccines and their boosters is wading with emerging Omicron mutant viruses, the effectiveness of vitamin D sufficiency remains solid and unchanged. The key to protecting the vulnerable is maintaining a higher circulatory vitamin D concentration, especially in ethnic minorities, older adults, and institutionalized persons, so they will maintain a robust innate immune system to fight against infections promptly.Thus, government and health administrations should consider nationwide educational campaigns for safe sun exposure and vitamin D supplemental programs to strengthen the population’s immunity and keep them healthy. Sun exposure and/or vitamin D supplementation and targeted food fortification can achieve this cost-effectively. This would have marked beneficial effects on reducing symptomatic diseases and preventing complications associated with and deaths caused by COVID-19. The world did not capitalize on this highly cost-effective opportunity during the COVID-19 pandemic. This will also protect vulnerable populations , such as ethnic minorities with darker skin color, older people, and institutionalized persons.Maintaining population serum 25(OH)D concentrations above 40 ng/mL ensures a robust immune system in communities, curtailing the spread of infections, minimizing symptomatic diseases, and reducing the prevalence of chronic diseases. Vitamin D sufficiency also minimizes acute viral infections and outbreaks and the need for hospitalization, saving healthcare costs and lives. Thus, governments, health insurance companies, and health administrators should consider nationwide educational campaigns for safe sun exposure, vitamin D supplementation, and targeted food fortification programs to strengthen the population’s immunity and keep them healthy. Implementing these is less than one day’s cost of healthcare.While the efficacy of vaccines and their boosters has waded with mutants of Omicron viruses, the effectiveness of vitamin D sufficiency remains strong. Vitamin D has multiple beneficial effects on all body systems: however, it is not a panacea for everything. Apart from preventative use, vitamin D should be used as adjunctive therapy with other primary pharmaceuticals and the best/optimal therapies and approaches, such as using antibiotics in bacterial infections. The key to protecting the vulnerable and reducing chronic disease burden in a country is not by expanding hospitals and health centers and recruiting more healthcare professionals but by educating the public on health preservation and maintaining a higher circulatory vitamin D concentration, especially in vulnerable communities—ethnic minorities, older adults, and institutionalized persons—so that they will have robust immune systems to fight against any infection and minimize chronic diseases.


N-donors and susceptible to further modification for possible antitumor/antiviral applications. Specifically, platinum-based nucleoside derivatives [PtII(dien)(N7-Guo)]2+, [PtII(dien)(N7-dGuo)]2+, and [PtII(dien)(N7-dGTP)] -guanosine-triphosphate) were investigated. These Pt(II) complexes were demonstrated to be stable species suitable for incorporation into RBCs. This result opens avenues for the possible incorporation of other metalated nucleobases analogues, with potential antitumor and/or antiviral activity, into RBCs.The therapeutic advantages of some platinum complexes as major anticancer chemotherapeutic agents and of nucleoside analogue-based compounds as essential antiviral/antitumor drugs are widely recognized. Red blood cells (RBCs) offer a potential new strategy for the targeted release of therapeutic agents due to their biocompatibility, which can protect loaded drugs from inactivation in the blood, thus improving biodistribution. In this study, we evaluated the feasibility of loading model nucleobase-containing Pt(II) complexes into human RBCs that were highly stabilized by four  Research and development on red blood cells (RBCs) as drug delivery systems has increased rapidly in the last twenty years. Due to their long lifespans (approximately 120 days in humans), high surface-to-volume ratios, and non-immunogenicity, RBCs represent a widely used biocompatible method for the delivery of peptides, proteins, small molecules, nucleic acids, antibodies, and a large number of nanomaterials compared to other types of drug delivery systems ,6,7,8,9.Metal-based drugs are a class of compounds that have been extensively studied for their wide range of applications, from anticancer ,40,41 toRecently, we devoted our research to new platinated guanine derivatives in order to gain a deeper understanding of their cellular metabolic mechanisms, and to confirm their potential as antimetabolites ,56,57,58II(dien)(N7-Guo)]2+ (1), [PtII(dien)(N7-dGuo)]2+ (2), and [PtII(dien)(N7-dGTP)] (3) -guanosine-triphosphate). In the following discussion, complexes 1–3 (dGuo)]2+, and [Pt(dien)(dGTP)], respectively. These complexes, despite not being intrinsically active as antitumor or antiviral drugs, are recognized by physiological membrane transporters and DNA polymerases, thus representing suitable models for assessing the stability of RBCs.In this context, the present work aims to study whether RBCs can be used as delivery systems for the model platinated nucleoside analogues: 2+ (1), [PtII(dien)(N7-dGuo)]2+ (2), and [PtII(dien)(N7-dGTP)] (3) were added to 0.5 mL 44% Ht RBCs at a 10 mM final concentration. The samples were incubated at room temperature and with slight stirring at different times . Aliquots of these suspensions were taken to evaluate, through a hemocytometer, some typical biological parameters of human RBCs in comparison to those of the control sample . The results reported in 1–3, at each time point, supernatants obtained as reported in “Materials and Methods” section were used to evaluate a possible release of hemoglobin compared to the control untreated sample. The data reported in 2+, in all cases the overall observed HGB released from treated cells (Guo)]2+ (1), [Pt(dien)(dGuo)]2+ (2), and [Pt(dien)(dGTP)] (3) -guanosine-triphosphate) using the loading protocol reported in “Materials and Methods” section. The osmolarities of RBCs suspensions were evaluated with a Milliosmometer  following the dialysis step and resulting in 110 mOsm for unloaded RBCs; 92 mOsm for (RBCs + 1 sample); 100 mOsm for (RBCs + 2 sample); and 91 mOsm for (RBCs + 3 sample). At the end of the loading procedure, the typical properties of human RBCs were evaluated to verify their cell viability and integrity.After carrying out preliminary tests to evaluate the biocompatibility of these specific compounds with human RBCs, we performed the incorporation of the 2+-, [Pt(dien)(dGuo)]2+-, and [Pt(dien)(dGTP)]-loaded RBCs samples that do not appear to be altered after the incorporation of all platinum derivatives . In fact, it is evident that the basal values of the control RBCs are not significantly affected after cell incorporation of the platinum compounds. Additionally, the percentage of final cell recoveries is very similar to that obtained for the control sample; only the MCV appears to be slightly lower, as found for the loading of other compounds or molecules 2+ (1), [Pt(dien)(dGuo)]2+ (2), and [Pt(dien)(dGTP)] (3) complexes, respectively. At the same loading condition, a higher loading efficiency was found for the nucleoside–platinum derivatives [Pt(dien)(dGuo)]2+ (2), and [Pt(dien)(dGTP)] (3) than for the nucleotide–platinum derivative [Pt(dien)(Guo)]2+ (1).The concentrations of 1.42+, [Pt(dien)(dGuo)]2+, and [Pt(dien)(dGTP)] ides through a biomimetic drug delivery, such as that offered by the most common natural cells, the RBCs. Indeed, erythrocytes are biocompatible, have a large capacity, and can accommodate traditional chemical entities, biologics (proteins), and/or contrasting agents (dyes and nanoparticles). The tested new Pt(II) derivatives were found to be biocompatible and encapsulable in human RBCs, thus supporting the primary aim of the present study. These data could be important in the perspective of using autologous RBCs–platinum compound preparations as therapeutic agents. We have previously reported that drug-carrier RBCs obtained with our procedure can be perfused in the blood system without affecting the other cell components, as their behavior is similar to that of endogenous circulating erythrocytes. After an average survival of about 120 days, RBCs are degraded by the cells of the reticuloendothelial system .2+ (1), [Pt(dien)(dGuo)]2+ (2), and [Pt(dien)(dGTP)] (3) complexes-loaded RBCs are similar to each other and to the control sample. Thus, the incorporation of these Pt(II) complexes with four N-donors, including a nucleos(t)ide, into human RBCs is possible, with a final cell recovery very similar to that of unloaded control cells. These data must be considered as a demonstration of effective biocompatibility. All three compounds have demonstrated that they do not affect the biological properties of RBCs even after the loading procedure. Cell integrity and morphology were also maintained, as shown by TEM analyses.The data reported in this work showed that the hematological values of human [Pt(dien)(Guo)]1–3 for our studies on biocompatibility with erythrocytes, also due to their low reactivity, being coordinatively stable, due to the presence of four N-donors, including a guanine nucleoside derivative, N-bonded to platinum. Biocompatibility with RBCs should be enhanced in these complexes due to the absence of a nucleus or other DNA-containing organelles and even for the well-known reduced guanosine metabolism occurring in RBCs 2+ (1), [PtII(dien)(N7-dGuo)]2+ (2), and [PtII(dien)(N7-dGTP)] (3) complexes -guanosine-triphosphate) were synthesized by a previously reported method Cl complex. The reaction was then monitored using 1H NMR spectroscopy. The D2O solutions containing the final 1–3 species were then lyophilized, to eliminate the deuterated solvent and replace it with H2O. NMR  (1) δ 8.44 , 5.98 , 4.73 , 4.42 , 4.28 , 3.90 , 3.30 , 3.08 , 2.91 ppm ; (2) δ 8.43 , 6.36 , 4.64 , 4.18 , 3.83 , 3.30 , 3.08 , 2.91 , 2.78 , 2.58 ppm ; (3) δ , 6.39 , 4.92 , 4.78 , 4.26 , 3.32 , 3.12 , 2.89 , 2.74 , 2.62 ppm ; δ −11.99 , −12.18 , −24.15 ppm .[PtCl(dien)]Cl was prepared following the synthesis reported by Annibale and co-workers . 2+, [Pt(dien)(dGuo)]2+, or [Pt(dien)(dGTP)] at a final concentration of 9 mM, using a tube with a 12-14 kDa cut-off as reported in ref. 2+ (1), [PtII(dien)(N7-dGuo)]2+ (2), and [PtII(dien)(N7-dGTP)] (3) complexes incorporated into erythrocytes, to be considered for further future delivery studies. The choice of RBCs as a drug delivery system was considered due to their natural role as carriers and components of the blood circulatory system. RBC carrier infusions in humans have been previously reported, proving their safety and efficacy   complexeefficacy ,13,21,241–3 to RBCs allowed us to evaluate the maintenance of normal cell biological features, without a significant effect on the typical hemocytometric parameters. Moreover, the loading of platinum complexes in human RBCs was successfully achieved by using a method of hypotonic dialysis previously reported by Antonelli et al. [1–3) was used during the loading procedure. It was also found that processed erythrocytes substantially maintained their fundamental properties and cell integrity. Moreover, the Annexin V binding assay showed that no significant phosphatidylserine RBCs surface exposure occurred after encapsulation of platinum complexes into cells with our loading method. Good cell integrity was also supported by transmission electron microscopy (TEM), which showed that the use of this type of platinum compounds does not affect the typical blood cells morphology.The obtained results revealed that Pt(II)-nucleoside analogues adducts can be fully compatible with red blood cells. Preliminary tests of adding compounds i et al. . The datN-donors, including an N7-bonded guanosine derivative. The biological interactions of these molecules were experimentally evaluated by us in the past years toward DNA and RNA polymerases and cell and mitochondrial membrane transporters [1–3, though not active as antitumor/antiviral drugs, have been used as specific models for their assessed recognition by transporters and potential incorporation into DNA. We previously reported the potential of this class of molecules in developing new antitumor and antiviral drugs [Due to the great interest in the study of platinum-based drugs and the frequent side effects occurring after treatments, our results could open new perspectives for the study of RBCs as possible new delivery systems for platinum-based drugs. This cell type has previously been evaluated for its suitability as a platinum drug carrier, but it showed decreased compatibility with the utilized platinum drugs ,30,31. Fsporters ,56,57,58al drugs . The plaN-donors in the Pt(II) coordination sphere within human RBCs. Overall, we can conclude that these new nucleobase mimetic molecular species are compatible with red blood cells when used as drug delivery carriers.In conclusion, we have demonstrated the possibility of loading platinated nucleoside analogues with a square-planar coordination geometry and four


Bacillota are widely distributed in various environments, owing to their versatile metabolic capabilities and remarkable adaptation strategies. Recent studies reported that Bacillota species were highly enriched in cold seep sediments, but their metabolic capabilities, ecological functions, and adaption mechanisms in the cold seep habitats remained obscure. In this study, we conducted a systematic analysis of the complete genome of a novel Bacillota bacterium strain M8S5, which we isolated from cold seep sediments of the South China Sea at a depth of 1151 m. Phylogenetically, strain M8S5 was affiliated with the genus Abyssisolibacter within the phylum Bacillota. Metabolically, M8S5 is predicted to utilize various carbon and nitrogen sources, including chitin, cellulose, peptide/oligopeptide, amino acids, ethanolamine, and spermidine/putrescine. The pathways of histidine and proline biosynthesis were largely incomplete in strain M8S5, implying that its survival strictly depends on histidine- and proline-related organic matter enriched in the cold seep ecosystems. On the other hand, strain M8S5 contained the genes encoding a variety of extracellular peptidases, e.g., the S8, S11, and C25 families, suggesting its capabilities for extracellular protein degradation. Moreover, we identified a series of anaerobic respiratory genes, such as glycine reductase genes, in strain M8S5, which may allow it to survive in the anaerobic sediments of cold seep environments. Many genes associated with osmoprotectants , transporters, molecular chaperones, and reactive oxygen species-scavenging proteins as well as spore formation may contribute to its high-pressure and low-temperature adaptations. These findings regarding the versatile metabolic potentials and multiple adaptation strategies of strain M8S5 will expand our understanding of the Bacillota species in cold seep sediments and their potential roles in the biogeochemical cycling of deep marine ecosystems. Cold seeps, which are widely distributed on the seafloor of the world’s oceans, play a crucial role in the global marine geochemical cycling of carbon and sulfur ,2,3. ColBacillota is predominantly low G+C content, gram-positive, rod-shaped, obligate anaerobic bacteria capable of forming spores to resist extreme environmental conditions [Bacillota is one of the most abundant bacterial groups and is widely distributed in a variety of environments, including cold seeps [Bacillota was the dominant bacterial group in the cold seep sediments of the South China Sea (SCS), with up to 37% of the relative abundance in the total bacterial communities [Bacillota play an essential role in the biodegradation of lignocellulosic and carbohydrate polymers [Bacillota) has the capability to utilize various complex carbohydrates, such as starch, cellulose, chitin, and xylan, and the Bacillus sp. of this phylum may play an important role in long-chain alkane biodegradation [Bacillota plays a crucial role in biogeochemical cycles.The phylum nditions ,14,15. Bld seeps , marine ld seeps ,19,20,21ld seeps ,23,24,25ld seeps , freshwald seeps , the humld seeps , plateauld seeps , and theld seeps . Notablypolymers , and petpolymers . Other sradation ,33. TogeAbyssisolibacter belongs to the order Clostridiales and the family Clostridiaceae within the phylum Bacillota. At present, the genus Abyssisolibacter contains only one isolated strain, Abyssisolibacter fermentans MCWD3, which was isolated from deep-sea sediment (depth 2150 m) obtained from the Ulleung Basin, East Sea, Korea [Abyssisolibacter.The genus a, Korea . Strain a, Korea . TherefoAbyssisolibacter sp. M8S5, which was isolated from the cold seep sediments of Site F at a depth of 1151 m. Our analysis of the complete genome sequence of strain M8S5 revealed the phylogeny, metabolic versatility, and multiple adaptive strategies of M8S5 in the high-pressure, low-temperature, and anaerobic cold seep environment.Hitherto, more than 40 cold seep sites have been detected in the South China Sea (SCS), only two of which are active: the Haima cold seep and the Site F (or Formosa Ridge) cold seep ,35,36,37Remotely operated vehicle (ROV) sediment push cores were collected at Site F  at a depth of 1151 m from the cold seep of the South China Sea  in May 22 (200 kPa), and incubated anaerobically at 35 °C. The M89 medium contained (L−1) 700 mL seawater, 300 mL distilled water, 1 g tryptone, 2 g yeast extract, 2 g casamino acid, 2 g 4-aminobutyric acid, 3 g HEPES buffer, 0.25 g l-cysteine hydrochloride dihydrate (as a reducing agent), and 1 mg sodium resazurin (as a redox indicator). The pH of the medium was adjusted to 7.0 with 5 M NaOH at room temperature. After autoclaving, 1 mL of vitamin solution [v/v) DMSO.Once in the lab, a subsample was used to inoculate M89 medium, prepared with a gas phase of Nsolution  and 1 mLsolution  were addThe cells of strain M8S5 were cultured in M89 medium at 35 °C for 5 days. The genomic DNA of strain M8S5 was extracted using the Qiagen DNA extraction kit . The total DNA was used for quality control and was obtained by gel electrophoresis and further quantified by a Qubit fluorometer .The complete genome sequence of strain M8S5 was sequenced using Oxford Nanopore Sequencing Technologies at Nextomics Biosciences Co., Ltd. . Oxford Nanopore Technologies sequencing libraries were prepared using the manufacturer’s ligation sequencing kit (SQK-LSK109), and sequencing was carried out on a PromethION device using flow cells (FLO-PRO002) without fragmentation. Finally, Nanopore reads were assembled using Canu (version 1.7)  in the nGenome annotation and ORF prediction in M8S5 and the other four strains were performed by the NCBI prokaryotic genome annotation pipeline (PGAP) . The whoThe taxonomy annotation of strain M8S5 and the related species was performed using the Genome Taxonomy Database Toolkit   with theThe ribosomal RNAs (rRNAs) of strain M8S5 and the other four strains were predicted using Barrnap version 0.9 with the default parameters . Then, t−10. The proteins employed by strain M8S5 but not by strain MCWD3 were considered to be strain-specific. On the other hand, the proteins shared by strain M8S5 and MCWD3 but absent in the other three species  were considered to be genus-specific.The protein sequence alignment was conducted by BLASTp with the following cut-off values: identity, 50%; query coverage, 50%; E-value, 1 × 10The average nucleotide identity (ANI) is referred to as the mean of the identity values between homologous genomic regions shared by two genomes . The ANIThe average amino acid identity (AAI) can provide better resolution when comparing highly different bacterial species, such as species from different genera ,64. The www.ncbi.nlm.nih.gov/sra, accessed on 1 May 2023) datasets with Integrated Microbial Next Generation Sequencing  [The full-length 16S rRNA gene sequences of strain M8S5 were used to screen for the presence of closely related sequences in all publicly available Short Read Archive  , the meteotides) .Abyssisolibacter fermentans MCWD3 . The G+C content of the M8S5 genome is 30.3%. The genome of strain M8S5 consists of 4376 genes, including 4250 protein-coding genes, 7 rRNA operons , 77 tRNAs, 4 ncRNAs, and 24 pseudogenes , which is slightly smaller than the scaffold genome of udogenes .Abyssisolibacter species contains a high proportion of novel genes and may be valuable for further studies. The top five COG categories in strain M8S5 included translation, ribosomal structure and biogenesis , amino acid transport and metabolism , transcription , general function prediction only , and energy production and conversion   and 1729 (40.8%) protein-coding genes of strains M8S5 and MCWD3, respectively, were assigned to 23 different clusters of orthologous groups (COGs) categories . Such a , 6.68%) . A graphSenegalia massiliensis SIT17, Senegalia massiliensis 583, and Clostridiisalibacter paucivorans DSM 22131) were considered to be genus-specific. As many as 20 genomic islands (GIs) were predicted in strain M8S5, and, in particular, seven of these GIs were related to nitrogenase and endospore germination permease, suggesting that this genome harbors obvious evidence of horizontal gene transfer  . It showns MCWD3 , which wA. fermentans MCWD3  evaluates genome-wide identity between distance species. Our results showed that the AAI values between M8S5 and the related four species ranged from 57.27 to 69.97%, and the best-matched strain was ns MCWD3 . The aves 77.85% , suggestAbyssisolibacter M8S5 is shown in Abyssisolibacter species are widely distributed in various marine environments, including seawater (18 of the sample locations), marine sediments (10), coral reefs (9), sponges (7), and an oyster gut (1) Carbohydrate metabolismscrA) and mannose (manA) were detected in the two isolated Abyssisolibacter strains (M8S5 and MCWD3) but not in the three neighboring species . Moreover, strain M8S5 harbored eight genes encoding fructose transporter, including a strain-specific gene (fruAb) (mapA), trehalose transporter , and arabinogalactan oligomer/maltooligosaccharide transporters (ganOPQ) were found in strain M8S5 but not in strain MCWD3, implying that the utilization of these sugars may contribute to the survival of strain M8S5 to the in situ cold seep environment.Metabolic analysis showed that strain M8S5 has the potential to utilize various kinds of carbohydrates, including chitin, cellulose, trehalose, arabinogalactan oligomer/maltooligosaccharide, maltose, sucrose, mannose, glucose, sugar, cellobiose, and galactitol . Among t (fruAb) . These rAbyssisolibacter strains, suggesting they could alleviate the accumulation of aceyl-coA generated from glycolysis via acetate production and export Amino acid metabolism and extracellular protein degradationRegarding the biosynthesis of amino acids, our genomic survey showed that the strain M8S5 preoccupies the complete pathways for the biosynthesis of 18 kinds of amino acids, whereas the biosynthesis pathways of histidine and proline were largely incomplete . SimilarddpDF), oligopeptide (oppABCDF), arginine/lysine/histidine (artQPR), alanine, proline/glycine (opuA), glutamate/aspartate, and polar amino acid from the extracellular cold seep environments d-amino acid metabolismd-amino acids (DAAs), which are essential components of bacterial cell wall peptidoglycan and are also found in various non-ribosomal peptides produced by microbes [It is well known that bacteria produce diverse microbes ,72,73. Amicrobes .d-amino acid transport and utilization were identified in strain M8S5 (d-methionine transport (metNIQ), d-cysteine desulfhydrase (dcyD), d-proline reductase (prdABDE), and d-alanine hydrolase (fmtA) as well as four racemases of amino acids, which catalyze the conversion between l- and d-amino acids . These pieces of evidence suggest that strain M8S5 might contribute to the degradation of d-amino acid-containing recalcitrant organic polymers in cold seep environments.In this study, 21 genes involved in ain M8S5 , includi(4)Nitrogen metabolismCold seep ecosystems are mainly supported by anaerobic methanotrophy, and the abundant input of methane (a non-nitrogenous carbon source) may drive the cold seep microbial community to nitrogen limitation ,76. In tBacillota, Flavobacterium, and Salmonella) from the environments [eutH) and utilization (encoding by eutABCJLMNPQS) were predicted in strain M8S5 , respectively [eutB and eutC, which catabolize ethanolamine into ammonia and acetaldehyde by an ethanolamine ammonia lyase (Ethanolamine (EA), derived from the membrane phospholipid phosphatidyl-ethanolamine of bacterial cells, can be used as a valuable source of carbon and/or nitrogen by a variety of bacteria  were identified in strain M8S5  in strain M8S5, but these were absent in the four phylogenetically related strains: MCWD3, S. massiliensis SIT17, S. massiliensis 583, and C. paucivorans DSM 22131 , which is the reduction of atmospheric dinitrogen to ammonia, is an important way to obtain a nitrogen source for microorganisms . Previouediments . In our SM 22131 . These f(5)Nucleic acid metabolismFor nucleic acid metabolism, four genes encoding nucleoside transporters were identified in strain M8S5 , and the(6)Glycine reductase in substrate-level phosphorylationBacillota species Eubacterium acidaminophilum) that could reduce glycine (acts as an electron acceptor) to acetyl-phosphate in Stickland reactions, with thioredoxin (acts as an electron donor) transformed to thioredoxin disulfide, which finally generates ATP [Glycine reductase (EC:1.21.4.2) is present in many obligate anaerobic microbes , thioredoxin 1 (trxA), glycine/sarcosine/betaine reductase (grdA), glycine/betaine transporter (betL), glycine/sarcosine/betaine reductase (grdC and grdD), and NADPH-dependent thioredoxin reductase (trxB) (grdX), NADPH-dependent thioredoxin reductase (trxB), thioredoxin 1 (trxA), and glycine reductase   . The othnd grdD) . These gTo uncover how strain M8S5 adapted to the cold seep environments, we analyzed its genome and identified a large number of genes that were potentially involved in high hydrostatic pressure (HHP) and low-temperature adaptation , includiopuABCD) and three BCCT family transporters (treB) encoding trehalose transporter was also detected in strain M8S5  were detected in the genome of strain M8S5 , spore maturation protein (cgeBD and spmAB), sporulation protein , and spore coat protein (cotAEHIJS)  . These fAbyssisolibacter and the phylum Bacillota. Metabolically, strain M8S5 could potentially utilize various kinds of simple and complex polysaccharides, such as chitin, cellulose, and trehalose. Although strain M8S5 lacks genes for synthesizing histidine and proline, it possesses not only the transporter genes of many amino acids and peptides but also diverse genes of extracellular peptidases, implying that it is capable of degrading extracellular proteins in the cold seep. Furthermore, the strain can utilize a variety of organic and inorganic nitrogen sources, such as ethanolamine, spermidine, putrescine, nitrogen, and ammonia. The genes of diverse transporters of osmoprotectants, molecular chaperones, and reactive oxygen species-scavenging proteins as well as spore formation may help it to adapt well to high-pressure and low-temperature cold seep environments. The findings regarding the versatile metabolic potentials and adaptation strategies of strain M8S5 provide new insights into the contribution of Abyssisolibacter species to biogeochemical cycling and expand our understanding of Bacillota species in cold seep habitats.In this study, we report the complete genome sequence of a novel anaerobic bacterial strain, M8S5, which was isolated from the Site F cold seep in the South China Sea at a depth of 1151 m. Phylogenetically, strain M8S5 belongs to the genus


Aflatoxin B1 (AFB1) induces lipid peroxidation and mortality in bovine foetal hepatocyte-derived cells (BFH12), with underlying transcriptional perturbations associated mainly with cancer, cellular damage, inflammation, bioactivation, and detoxification pathways. In this cell line, curcumin and resveratrol have proven to be effective in mitigating AFB1-induced toxicity. In this paper, we preliminarily assessed the potential anti-AFB1 activity of a natural polyphenol, quercetin (QUE), in BFH12 cells. To this end, we primarily measured QUE cytotoxicity using a WST-1 reagent. Then, we pre-treated the cells with QUE and exposed them to AFB1. The protective role of QUE was evaluated by measuring cytotoxicity, transcriptional changes (RNA-sequencing), lipid peroxidation , and targeted post-transcriptional modifications (NQO1 and CYP3A enzymatic activity). The results demonstrated that QUE, like curcumin and resveratrol, reduced AFB1-induced cytotoxicity and lipid peroxidation and caused larger transcriptional variations than AFB1 alone. Most of the differentially expressed genes were involved in lipid homeostasis, inflammatory and immune processes, and carcinogenesis. As for enzymatic activities, QUE significantly reverted CYP3A variations induced by AFB1, but not those of NQO1. This study provides new knowledge about key molecular mechanisms involved in QUE-mediated protection against AFB1 toxicity and encourages in vivo studies to assess QUE’s bioavailability and beneficial effects on aflatoxicosis. The ICp ≤ 0.05. The same statistical approach was adopted for the analytical data, but Tukey’s multiple comparisons test was used instead of Dunnett’s test.The statistical analyses of cytotoxicity, qPCR data, and MDA content, as well as of NQO1 and CYP3A enzyme activities, were performed using a one-way ANOVA followed by Dunnett’s multiple comparisons test, with the level of significance set at


Single cell transcriptomics has emerged as a powerful tool for revealing functional variability within cell populations.The type I interferon (IFN) response is an innate immune program that mediates anti-viral, anti-cancer, auto-immune, auto-inflammatory, and sterile injury responses. Bone marrow derived macrophages (BMDMs) are commonly used to model macrophage type I IFN responses, but the use of bulk measurement techniques obscures underlying cellular heterogeneity. This is particularly important for the IFN response to immune stimulatory double-stranded DNA (dsDNA) because it elicits overlapping direct and indirect responses, the latter of which depend on type I IFN cytokines signaling Here, we use single cell RNA-Seq to examine BMDM heterogeneity at steady state and after immune-stimulatory DNA stimulation, with or without IFNAR-dependent amplification.Ccl5 to be an IFNAR-independent marker gene of direct DNA sensing cells.We find that many macrophages express ISGs after DNA stimulation. We also find that a subset of macrophages express ISGs even if IFNAR is inhibited, suggesting that they are direct responders. Analysis of this subset reveals Our studies provide a method for studying direct responders to IFN-inducing stimuli and demonstrate the importance of characterizing BMDM models of innate immune responses with single cell resolution. As a result, therapies based on inhibition of type I IFN signaling using neutralizing antibodies against the IFN alpha receptor (INFAR) have been developed and were recently FDA approved for treatment of lupus (The type I interferon (IFN) response is an innate immune program that fuels inflammation during diverse pathologic processes including infections, malignancies, sterile tissue injury, autoimmunity, and autoinflammatory diseases –9. In thof lupus .The type I IFN response is shown schematically in Type I IFNs are diffusible extracellular cytokines that spread responses from directly-stimulated cells to neighboring cells by binding to cell surface interferon alpha receptors  Figure 1in vitro model of tissue macrophages, at steady state and after exposure to immune stimulatory dsDNA with or without simultaneous IFNAR Ab treatment or genetic deficiency of IFNAR , a commonly used of IFNAR . This enMcm6 and Mcm5 (S phase), Top2a and Cenpa (G1 phase), Birc5 and Nusap1 (G2 phase) , , and . The final cluster was defined as a monocyte-like BMDM Progenitor characterized by expression of S100a4 and S100a6.To dissect direct and indirect responses to transfected immunogenic DNA stimulation with single cell resolution, we designed the following experiment. We used single cell RNA-Seq to define the heterogeneity of cultured BMDMs (i) at steady state, (ii) in response to dsDNA alone, or (iii) in response to dsDNA + IFNAR Ab inhibition of secondary signaling, or (iv) in dsDNA-treated IFNAR KO . We thenIfit2, Oasl2, Rsad2, Isg15) compared to unstimulated controls as well as compared to dsDNA in the context of IFNAR Ab, thus demonstrating successful inhibition of secondary amplification  have firmly established that dsDNA complexed with a transfection reagent, when delivered to macrophages in culture, induces expression of type I IFNs and ISGs . Howeverfication Table 1.Ccl5, Irf7, Ifit1, Isg15, Rsad2, Oasl2, Bst2, and Ifi204  with the Fabp4+MHCII- macrophages from condition 2 (dsDNA Alone) and reclustered . An alternative explanation for the directly stimulated cells that we observed is that there is a population of cells that are resistant to IFNAR Ab blockade. While this cannot be disproven, it seems unlikely since the resistant population would have to also have to uniquely respond to DNA stimulation with overexpression of Ccl5 as an IFNAR-independent marker of direct DNA-sensing cells.In summary, we have used single cell RNA-Seq to define the heterogeneity of macrophages in culture, both at steady state and in response to immune stimulatory DNA, with and without blockade of secondary IFNAR-dependent amplification, revealing WT mice, stock 000664, and 2 IFNAR -/- mice, stock 028288). All experiments were performed with 10 to 14-week-old animals and were carried out using age and gender matched groups without randomization. All mice were maintained in a pathogen-free environment of UC San Diego.Mouse experiments were approved and conducted under the oversight of University of California San Diego Institutional Animal Care and Use Committee (IACUC #17144) using Adult C57BL/6J (3 In vivogen) was complexed with a Lipofectamine transfection agent (ThermoFisher) at a ratio of 1:1.5 in serum free-media and added to 1 million BMDMs in a 6-well multi-well plate with serum- and mCSF-containing media for each experiment. Two independent experiments were performed, yielding n = 5 for each condition. For inhibition of secondary signaling via the IFNAR receptor, cells were treated with 20μg/ml of MAR1-5A3 IFNAR neutralizing antibody or isotype control (BioXCell).Bone marrow derived macrophages (BMDMs) were isolated by flushing femurs of adult mice and culturing the resulting cells in 10% FBS 1% Pen/Strep-containing DMEM supplemented with 10ng/mL recombinant m-CSF (Peprotech) for 7 days. 5μg of immunogenic HT DNA . Cell suspensions were labeled with DAPI just prior to flow cytometric analysis to allow exclusion of dead cells. Doublets and dead cells were excluded by forward scatter and Dapi. Data was acquired by Sony sorter MA900 at UCSD and analyzed with FlowJo software.Single cell RNA-Seq was performed by microfluidic droplet-based encapsulation, barcoding, and library preparation (10X Genomics) as previously described . Paired FindVariableFeatures method from the Seurat R package (version 3.0) by selecting 3,000 genes with the highest feature variance after variance-stabilizing transformation. Integration of multiple single-cell RNA-seq datasets was performed in Seurat to enable harmonized clustering and downstream comparative analyses across conditions  dimensional reduction was performed. Differentially expressed genes (DEGs) between clusters were determined using a Wilcoxon Rank Sum test. Where specified, subsets of cells were isolated and reclustered to identify new DEGs.After scaling and centering expression values for each variable gene, linear dimensionality reduction was performed on integrated data using principal component analysis (PCA). Clustering was performed using the shared nearest neighbor (SNN) clustering algorithm with the Louvain method for modularity optimization, as implemented in the Seurat 4.ISG Scores were measured as the sum of the raw reads for the ISGS: Ifi204, Isg15, Irf7, Ifit1, Rsad2, Oasl2, Oas3, Gpb5, Bst2, Ifit2, Ifit3, Oas2, Isg20, Cxcl10, Oasl1, Gpb2, Ccl5, Ccl3. Direct Scores were measured as the sum of the raw reads for: Ccl5, Cdk8, Cxcl2, and Cd74. ISG and Direct Scores were normalized to reads per cell and scaled by 10P values are indicated by P values less than 0.05 were considered significant and are indicated by asterisks as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.Statistical analysis was performed using GraphPad Prism software. All data are represented as mean values +/- standard error of mean (S.E.M.) unless indicated otherwise. A statistical method was not used to predetermine sample size. All analyses were unpaired. https://zenodo.org/record/7874451).The single cell RNA-Seq datasets presented in this study have been deposited to the Gene Expression Omnibus under accession no. GSE229311 (GEO). The code used to process this data is publicly available at Zenodo .EM, JY, VN, and KK designed the study, performed analysis, and wrote the initial manuscript. VN conducted experiments. EM, JY, and DC performed analysis. KK directed the project. All authors contributed to the article and approved the submitted version.


Background: Neuroblastoma (NB) is a childhood cancer originating from immature nerve cells in the sympathetic nervous system. Current clinical and molecular subtyping methods for NB have limitations in providing accurate prognostic information and guiding treatment decisions.GPR125, CDK4, and GREB1 emerged as potential therapeutic targets in Cluster 3. CD4K inhibitors showed subtype-specific responses, suggesting tailored treatment strategies. Single-cell analysis highlighted unfavorable clinical features in Cluster 3, including high-risk classification and reduced cytotoxicity. Suppressed interactions between monocytes, macrophages, and regulatory T cells were observed, affecting immune regulation and patient prognosis.Results: To overcome these challenges, we explored the microenvironment of NB based on the knowledge-based functional gene expression signatures (Fges), which revealed heterogeneous subtypes. Consensus clustering of Fges activity scores identified three subtypes  that demonstrated significant differences in prognosis compared to mainstream subtypes. We assessed the immune infiltration, immunogenicity, CD8T cytotoxicity, and tumor purity of these subtypes, uncovering their distinct biological functions. Cluster 1 and Cluster 2 exhibited higher immunoreactivity, while Cluster 3 displayed higher tumor purity and poor prognosis. Gene ontology annotation and pathway analysis identified immune activation in Cluster 1, epithelial-mesenchymal transition (EMT) in Cluster 2, and cell cycle processes in Cluster 3. Notably, the impact of EMT activity on prognosis may vary across NB subtypes. A classification model using XGBoost accurately predicted subtypes in independent NB cohorts, with significant prognostic differences. Conclusion: To summarize, we have identified a new independent prognostic factor in NB that underscores the significant correlation between tumor phenotype and immune contexture. These findings deepen our understanding of NB subtypes and immune cell interactions, paving the way for more effective treatment approaches. Neuroblastoma (NB) is a childhood cancer that originates from the neural crest during embryonic development and primarily occurs in the sympathetic nervous system . Common MYCN amplification, often indicates a highly malignant form with an unfavorable prognosis [PHOX2B and ALK genes, which can be inherited and are frequently accompanied by other syndromes related to neurocognitive development [BDNF and TrkB, correlating with the malignancy and poor prognosis [SETD8, a histone methyltransferase, may be associated with a poor prognosis in MYCN non-amplified NB [BARD1 gene and its isoform protein, BARD1β, are linked to high-risk NB and could potentially serve as therapeutic targets [DDX1 and DDX4 have also been identified in specific NB cases, exhibiting associations with distinct disease characteristics [ARID1A/B have been correlated with drug resistance and elevated mortality rates in NB, indicating the potential pivotal roles of these genes in NB development [Considering the heterogeneous nature of NB, patient-specific staging to address different treatment strategies is clinically relevant. Currently, three primary approaches for staging and classifying NB are commonly employed: The International Neuroblastoma Staging System (INSS), the International Neuroblastoma Risk Group (INRG), and the Children’s Oncology Group (COG) risk stratification , 7. The rognosis , 15. Famelopment , 17. Higrognosis . Alteratified NB . Additio targets . DDX1 aneristics . Neverthelopment . NotablyAlthough the acceptance of genomic analysis in clinical decision-making is on the rise, current classification systems and biomarkers also have limitations in accurately portraying the biological and molecular genetic characteristics of tumors, hindering the development of personalized treatment plans and precise prognostic assessments . TherefoIn our research, based on the knowledge-based functional gene expression signatures (Fges) provided by Bagaev et al. , which rn = 151), with expression data normalized by FPKM (Fragments Per Kilobase of exon model per Million mapped fragments). TARGET-NB cohort data were mainly used to explore TME and as the training data for investigating the potential subtypes of NB. Also, GSE49710 (n = 357) [n = 266) [rma function from “affy” package (version 1.70.0) [In the present study, we obtained transcriptome sequencing expression data and clinical data for NB patients from “Therapeutically Applicable Research to Generate Effective Treatments” (TARGET) database  –34 and Gn = 266) , and cli 1.70.0)  for exprhttps://github.com/shenglinmei/NB.immune.atlas/. This dataset includes 46,134 cells from 17 NB patients and covers ten major immune cell types, including B cells, ILC3, Macrophages, mDC, Monocytes, NK cells, Plasma cells, Cytotoxic T cells (Tcyto), Helper T cells (Th), and Regulatory T (Treg) cells.To investigate the intrinsic molecular mechanisms of heterogeneity in NB subtypes, we further obtained a single-cell dataset provided by Verhoeven et al.  from httAddModuleScore function from the Seurat package.We used the GSVA package (version 1.40.1)  with thehttps://github.com/BostonGene/MFP/blob/master/signatures/gene_signatures.gmt  [p-value less than 0.05.Kaplan-Meier (KM) survival analysis, along with the log-rank test, was used to determine if the subtypes showed a significantly different overall survival (OS). For gene expression data, the optimal cutoff point for group stratification was determined using the n 0.4.9) . StatistFindAllMarkers function from “Seurat” package (version 4.3.0) [p-values below 0.01 and an absolute log2FC greater than 1 were deemed significantly differentially expressed.To identify DEGs across subtypes, we utilized the n 4.3.0) . Specifibitr function. Subsequently, gene ontology (GO) and KEGG pathway enrichment analysis was conducted using the enrichGO function with the “ont = BP” parameter and enrichKEGG function, respectively. GO and KEGG terms that had adjusted p-values below 0.01 were regarded as significantly enriched.To examine the distinct biological processes and pathways that exhibited significant differential expression among subtypes, we employed the “clusterProfiler” package (version 4.0.5)  to conveGZMA and PRF1 markers [The estimation of immune score, and tumor purity of NB patients was conducted by utilizing the “ESTIMATE” package (version 1.0.13) , as spec markers .p-value ≤ 0.01) as the features for the training set. (2) We applied the XGBoost model to the training set using 10-fold cross-validation, as implemented by the “xgb.cv” function from the “xgboost” R package (version 1.6.0.1). The parameters used were “nfold = 10, objective = multi:softprob, max.depth = 10, eval_metric = mlogloss” [Given that the Cluster 3 identified by the TARGET-NB cohort demonstrated the worst prognosis, we introduced the XGBoost model  to distilogloss” . The modlogloss” –34 and Glogloss” ).To assess the drug sensitivity of the Cluster 1&2 and Cluster 3, we utilized the “oncoPredict” package (version 0.2)  with theWe employed CellChat  , suggesting that the subtypes portrayed by MYCN may be more closely linked to the TME of NB  that represented the major functional components of the tumor, as well as immune, stromal, and other cell populations from previous study  Supplem and 2. Wnt of NB . Also, wME of NB . NotablyNB  and Cluster 2 the lowest (n = 40), with a more evenly distributed overall distribution  can reveal molecular subtypes of NB, distinct from those identified previously based on clinical and molecular features, we conducted a consensus clustering analysis on the activity scores of Fges, resulting in the categorization of patients into three subtypes: Cluster 1, Cluster 2, and Cluster 3 . We firsribution . We alsoribution .MYCN, Ploidy, COG, and INSS  immune infiltration, immunogenicity and cytotoxicity scores, while Cluster 3 exhibited significantly lower immune infiltration and the greatest level of tumor purity  and showing good efficacy in clinical practice. For example, Palbociclib [CDK4/6 to D-type cyclin, thereby blocking the G1/S phase transition [Considering the potential clinical application of tivities . This fibociclib , 58 can ansition –61.To determine the sensitivity of NB subtypes Cluster 1&2 and Cluster 3 to Palbociclib and Ribociclib inhibitors, we used OncoPredict tool  to prediGREB1 emerged as the important distinctive gene feature in the C3 subtype, with the highest proportion of positive expression observed in Macrophages  , we obtarophages . Abundanrophages .CD4 in Cluster 3, as well as the CXCL16-CXCR6 axis interaction . In the independent NB cohorts, the subtypes predicted by the classifier showed significant prognostic differences consistent with the expected pattern, which is crucial for investigating the Fges-derived subtypes in more extensive NB cohorts. Specifically, we found tastasis , 56, 66,CXCL16 in monocytes and macrophages within Cluster 3 was suppressed, leading to weakened interactions with Treg cells, thus influencing patient prognosis. Specifically, Tregs, known for their immune regulatory role, typically interact with monocytes to maintain immune balance. However, the diminished interaction between monocytes presenting MHC class II molecules and CD4 molecules in Tregs resulted in reduced suppressive effects by Tregs, leading to weakened immune regulation. Consequently, this could potentially trigger excessive or imbalanced immune responses, causing self-tissue damage or triggering a stronger immune response against tumor antigens. This could potentially result in excessive or imbalanced immune responses, causing damage to self-tissues or generating a stronger immune response against tumor antigens [In a separate single-cell analysis conducted on NB patients, we examined the characteristics and interactions among different subtypes. The study unveiled unfavorable clinical prognostic features associated with the Cluster 3 subtype, including high-risk classification, increased proliferative capacity, and lower cytotoxicity scores. Moreover, compared to Cluster 1&2, the expression of MHC-II and antigens , 68. TheDespite the promising results, our study has some limitations. Firstly, the study was based on a relatively small sample size, and more extensive validation is needed. Secondly, we only investigated two drugs, and more drugs need to be examined in future studies. Thirdly, further investigation is needed to validate the clinical utility of our subtyping approach. Nonetheless, our study highlights the potential of using Fges to derive novel subtypes of NB with significant differences in prognosis and treatment response. In conclusion, as research in NB subtyping and treatment strategies continues to advance, a multidisciplinary approach involving clinicians, researchers, and patients will be pivotal in translating these findings into more effective, personalized, and less burdensome treatment options. The ultimate goal is to enhance patient outcomes and the overall experience of those affected by NB.Supplementary Tables 4, 8 and 9Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 5Supplementary Table 6Supplementary Table 7


Briefings in Bioinformatics, Volume 23, Issue 6, November 2022, bbac422, https://doi.org/10.1093/bib/bbac422This is a correction to: Visanu Wanchai, Piroon Jenjaroenpun, Thongpan Leangapichart, Gerard Arrey, Charles M Burnham, Maria C Tümmler, Jesus Delgado-Calle, Birgitte Regenberg, Intawat Nookaew, CReSIL: accurate identification of extrachromosomal circular DNA from long-read sequences, In the originally published version of this manuscript, there was an error in Panel E of Figure 3 due to a mistake in the script to plot the upset plots. This has been corrected in the original article.In addition the following sentence was also incorrectly given as:“However, we found over 90 000 recurrent eccDNAs between replicates of the ARP1 cell line (∼50%), indicating a cell-specific population of eccDNA that possibly benefits the ARP1 cell line ”This has now been corrected to:“However, we found over 28 000 specifically recurrent eccDNAs between replicates of the ARP1 cell line (∼15%), indicating a cell-specific population of eccDNA that possibly benefits the ARP1 cell line .”These errors have been corrected in the original article.


Myeloid-derived suppressor cells (MDSCs) are a heterogeneous myeloid cell population and serve as a vital contributor to the tumor microenvironment. Reactive oxygen species (ROS) are byproducts of aerobic respiration and are involved in regulating normal biological activities and disease progression. MDSCs can produce ROS to fulfill their immunosuppressive activity and eliminate excessive ROS to survive comfily through the redox system. This review focuses on how MDSCs survive and function in high levels of ROS and summarizes immunotherapy targeting ROS in MDSCs. The distinctive role of ROS in MDSCs will inspire us to widely apply the blocked oxidative stress strategy in targeting MDSC therapy to future clinical therapeutics. MDSCs play a crucial role in tumorigenesis and inhibit antitumor immune responses to promote tumor development , 2. In aThis paper summarizes the distinctive regulation, scavenging and effects of ROS in MDSCs. In addition, we generalize immunotherapy that targets ROS in MDSCs. This will provide novel potential insight for targeting MDSC immunotherapy.2+Gr-1+. According to different epitopes (Ly6G and Ly6C) in Gr-1, mouse MDSCs can be further divided into two subgroups: CD11b+Gr-1+Ly6GhighLy6Clowgranulocyte/polymorphonuclear MDSCs (G-MDSCs/PMN-MDSCs) and CD11b+Gr-1+Ly6GlowLy6Chighmonocytic MDSCs (M-MDSCs)  are a heterogeneous population composed of immature myeloid cells (IMCs). In pathological conditions such as cancer, infectious diseases, trauma, and some autoimmune disorders, IMCs cannot differentiate into mature myeloid cells, which causes the activation and expansion of MDSCs , 5. At pM-MDSCs) . The pheess CD14 . In mostess CD14 , 10. IHCess CD14 .The immunosuppressive activity of MDSCs relies on the expansion and activation of MDSCs. There are a variety of factors accounting for the expansion of the MDSCs, such as cyclooxygenase 2 (COX2), prostaglandin, stem-cell factor (SCF), macrophage CSF (M-CSF), granulocyte/macrophage CSF (GM-CSF), vascular endothelial growth factor (VEGF), TNF-α, polyunsaturated fatty acids, MyD88 and HIF-1α –15. MostSeveral major molecules contribute to MDSC-mediated immune suppression, including arginase 1 (Arg-1), inducible nitric oxide synthase (iNOS), COX2, TGF-β, IL-10 and ROS. Many factors, such as STAT3, C/EBPβ, p50 NF-κB, and IDO1, play a critical role in MDSC function by regulating these functional effector molecules , 19, 20.via the immunosuppressive cytokines IL-10 and TGF-β. MDSCs can also reduce the secretion of IL-6 and TNF-α by macrophages and shape them into the M2-type phenotype, which promotes tumor progression , hydrogen peroxide (H2O2), nitric oxide, peroxynitrite, hypochlorous acid, singlet oxygen and hydroxyl radicals , oxygen-containing derivatives, include a range of species such as superoxide , tumor cells, cancer stem cells (CSCs) and immune cells . The proROS are short-lived, strong-effect and short reaction distance compounds that serve as a double-edged sword that elicits both beneficial and harmful effects in cells. The most common influence is the toxic side effects of ROS. Elevated levels of ROS can damage cells and intracellular components, cause DNA hydroxylation, protein denaturation and tissue damage, and ultimately lead to cell cycle G2/M arrest, apoptosis, senescence and death, and ROS can also participate in mitochondria, death receptors, and endoplasmic reticulum-mediated apoptosis . However4NADPH oxidase (NOX) enzymes and mitochondria are major sources of endogenous ROS. In addition, there are numerous cellular sources of ROS, including xanthine oxidase, cyclooxygenases, cytochrome p450 enzymes, lipoxygenases and the endoplasmic reticulum .Two major sources of ROS in MDSCs are NOX2 and mitochondria. Compared with MDSCs, cancer cells and macrophages also utilize mitochondria and NADPH oxidase to produce ROS. However, T cells express no or very low levels of NADPH oxidase .2.-, particularly those derived from mitochondrial electron transport chain (ETC) complexes. Complex I and III of the ETC generate O2.-, which is rapidly converted to H2O2via mitochondrial SOD2, while the O2.- from the complex can be converted into H2O2 by cytosolic SOD1   . Compararesponse . Rats anresponse .5Many factors can regulate ROS production, such as GM-CSF, interleukin, TGF, TNF, FGF, platelet-derived growth factor, TLR agonists, protease, nucleotide receptors, TCR stimulation and peroxynitrite , 45, 46.Various types of cells and survival environments possess different ROS regulatory mechanisms. In terms of MDSCs, it has been proven that multiple molecules can govern intracellular ROS, such as STAT3, fatty acid transport protein 2 (FATP2) and noncoding RNAs. STAT3 is an important transcription factor related to the expansion, differentiation and function of MDSCs. STAT3 directly increases the expression of p47phox, which belongs to the NOX2 complex, by binding to the promoter region of p47phox. Blocking STAT3 could downregulate the expression of gp91phox and p47phox to decrease ROS production , 47, 48.+ T-cell proliferation and function by generating ROS  can polarize monocytes to MDSCs, which suppress CD8ting ROS . Murine ting ROS .In contrast to MDSCs, other myeloid cells, such as macrophages, can stimulate NADPH oxidase expression and activity to elevate the level of ROS by other disparate factors, such as P2X7, brain-specific angiogenesis inhibitor 1 (BAI1), beryllium, myocardin-related transcription Factor A (MRTF-A) and TLRs –60. Howe6In general, the cell needs an appropriate level of ROS to maintain normal physiological function. Either too few or too many ROS are harmful. Normally, ROS production is controlled in a safe range, and superfluous ROS can be neutralized by the antioxidant system to maintain cell homeostasis. The antioxidant system contains antioxidant enzymes and nonenzymatic molecules. Common antioxidant enzymes include superoxide dismutase (SODs), catalase, peroxidase (PRDXs), peroxiredoxins (Prxs) and glutathione peroxidase (GPXs) . Other nvia antioxidant enzymes and autophagy  and mutations in Nrf2 and P53 transcription factors , HIF-1α, pyruvate dehydrogenase kinase 1 (PDK1) and phosphoenolpyruvate (PEP) , NAD(P)H:quinone oxidoreductase 1 (NQO1), catalase and SOD . Under nWith the exception of Nrf2, the existence of HMGB1 in MDSCs cannot be underestimated. HMGB1, a damage-associated molecular pattern (DAMP) molecule, is a vital driver of MDSC accumulation and immunosuppressive function, as reported in early studies. In the tumor microenvironment, elevated ROS can increase cytoplasmic translocation and release HMGB1 . Subsequ+Gr1+ MDSCs from IDO-KO hosts enhanced ROS generation and downregulated the expression of ROS scavenging genes  into kynurenine (Kyn) . CD11b+Gng genes , 85. Morng genes , 87.In addition, hypoxia plays a crucial role in regulating the function of tumor derived MDSCs. HIF-1α could decrease NOX2 expression and excessive ROS production, which may give rise to the preferable survival of MDSCs in the tumor microenvironment . In turnIn contrast to MDSCs, HSCs and CSCs have low ROS content. Several signaling molecules, such as ataxia telangiectasia mutated (TAM), PI3K/Akt, FoxO3 (FoxO transcription Factors 3), phosphatase and tensin homology (PTEN), p53, Prdm16 (PR domain-containing 16), HIF-1α, p38MAPK, and Nrf2, account for the low ROS level to maintain stemness and quiescence in HSCs . For exaMDSCs can produce ROS by mitochondria and NOX2. MDSCs can take up lipids through FATP2 to promote mitochondrial ROS production. Moreover, the transcription factor STAT3 can increase NOX2 expression to upregulate ROS levels in MDSCs. Then, the elevated ROS level can activate the antioxidant system to eliminate excessive ROS. Nrf2 could be transcriptionally activated to initiate the expression of its downstream antioxidant genes. The high level of ROS can also induce nuclear heterotopic HMGB1 to promote the survival of MDSCs by autophagy. In addition, HIF-1α can activate PDK1 to inhibit mitochondrial ROS production. IDO1 can scavenge ROS with its metabolic characteristics. Another enzyme, CaMKK2, can activate AMPK to decrease ROS production. The glycolytic metabolite PEP could also prevent massive ROS production to keep the ROS level in a suitable range.7via the PI3K/Akt/NF-κB pathway at 3 days after stroke , JAK/STAT and phosphoinositide 3-kinase (PI3K)/AKT , 95. Furr stroke .2O2 reduced the TCRζ chain and IFN-γ secretion of T cells to destroy T-cell function , nitroaspirin and Embelin. On account of its capacity to upregulate several antioxidant genes, including NAD(P)H: quinone oxidoreductase 1 (NQO1), thioredoxin, catalase, superoxide dismutase and heme oxygenase, CDDO-Me could efficiently abrogate the immune suppressive effect of MDSCs and enhance T-cell function by activating the target gene NQO1 to decrease MDSC-mediated ROS production, while CDDO-Me did not affect the NO level in MDSCs . NitroasIn addition, many inhibitors are being exploited to reduce ROS level such as N-acetylcysteine (NAC), L-NIL, histamine dihydrochloride (HDC), celecoxib, alisertib, SAHA and sildenafil. NAC, a well-established antioxidant that had the ability to reduce ROS and increase the extracellular pool of cysteine. Many animal models have verified its antitumor efficacy. NAC could stimulate the degradation of HIF-1 and inhibit its activity by neutralizing ROS . MoreoveAlong with inhibitors that target the effector molecules of the immunosuppressive activity of MDSCs, enzyme inhibitors can achieve similar outcomes. The selective Aurora-A kinase inhibitor alisertib directly weakened the immunosuppressive function of MDSCs by notably downregulating the mRNA expression levels of associated genes, such as NOS2, S100A8, S100A9, CYBB and NCF1, and compromising ROS production by inhibiting the JAK2-STAT3 pathway . PhosphoOut of the ordinary, the histone deacetylase inhibitor SAHA could augment the intracellular ROS to induce apoptosis in MDSCs. That might be a promising and novel MDSCs-targeted therapy . In addiNatural products are increasingly being discovered and researched in tumor therapy. With the deeper comprehension of natural products, many plant extracts have antioxidant impacts. Among them, withaferin A (WA), a component of the root extract of the plant Withania somnifera Dunal (WRE), could decrease ROS production in MDSCs through a STAT3-dependent mechanism . The polTraditional Chinese medicines have made enormous achievements in antioxidant activity. Baicalein is a traditional Chinese herbal medicine. Baicalein prevented the expansion and function of MDSCs in lupus mice, which can be attributed to decreased ROS levels and enhanced Nrf2 activation . Jianpi Ganoderma microsporum that reduces MDSC expansion in bone marrow cells (BMCs) stimulated by S. aureus biofilms, which was attributed to increased cytokine expression and a reduction in ROS levels  derived from collagen XVIII has the potential to target PMN-MDSCs selectively, resulting in obviously reduced ROS production . DoxorubS levels , 141. L-oduction .Based on a previously published review, this paper further updated and listed the new molecules found in recent years that can regulate ROS levels in MDSCs and comprehensively summarized the therapeutic drugs that can target ROS levels in MDSCs. This provides a treatment strategy for cancer immunotherapy.Compared to other myeloid cells, such as macrophages or tumor cells, ROS play an irreplaceable and distinctive role in MDSCs. On the one hand, MDSCs are required to produce ROS to suppress the antitumor immune response. In turn, excessive ROS can be removed to promote MDSC survival comfortably by activating factors, such as Nrf2, HMGB1, HIF-1α, IDO1, CaMKK2 and PEP. On the other hand, an appropriate level of ROS can prevent further differentiation of MDSCs to better maintain their state and nature. However, other myeloid cells, such as macrophages, have the same sources of ROS as MDSCs and regulate intracellular ROS levels by different factors, such as P2X7 and BAI1. Tumor cells can also induce autophagy to scavenge excessive ROS.It is widely believed that ROS can promote the development of tumors, but a large number of studies have shown that ROS can promote tumor cell apoptosis and death. At present, studies are emerging that tend to exploit immunotherapies that utilize the ability of ROS to kill tumor cells. Therapy targeting ROS in MDSCs can be therapeutic by impairing MDSC differentiation and function. How to combine targeted ROS therapy in MDSCs and tumor cells requires further consideration.JH drafted the manuscript. YZ, KZ and KY discussed and revised the manuscript. SW designed the study and revised the manuscript. All authors contributed to the article and approved the submitted version.


Their presence in the environment is ubiquitous and many of them have been proven carcinogenic, teratogenic, and mutagenic. These toxic pollutants can therefore pose a threat to both ecosystem and human health and urges for remediation strategies for PAHs and derivatives from water bodies. Biochar is a carbon-rich material resulting from the pyrolysis of biomass resulting in a very porous matter with high surface area for an enhanced interaction with chemicals. This makes biochar a promising alternative for filtering micropollutants from contaminated aquatic bodies. In this work, a previously developed and validated methodology for the analysis of PAHs, oxy-PAHs and N-PACs in surface water samples was adapted for its utilization in biochar treated stormwater with special emphasis on scaling down the solid-phase extraction as well as including an additional filtering step for the removal of particulate matter in the media.  Specifications tablee.g. petroleum, oil, gas or coal Polycyclic aromatic hydrocarbons (PAHs) are persistent and semi-volatile organic compounds primarily formed due to incomplete combustion of organic material, Due to the hydrophobic nature of PACs, their occurrence in aquatic samples is often at lower concentrations than in soils and sediments Biochar is a carbon-rich material resulting from the pyrolysis of biomass or organic waste, such as woodchips or wastewater sludge ,7. Such In this work, a previously developed and validated methodology for the analysis of PAHs, oxy-PAHs and N-PACs in surface water samples 2H8]-9-fluorenone, [2H10]-acenaphthene, [2H8]-acenaphthylene, [2H10]-anthracene, [2H8]-anthracene-9,10-dione, [2H12]-benz[a]anthracene, [2H12]-benzo[a]pyrene, [2H12]-benzo[b]fluoranthene, [2H12]-benzoperylene, [2H12]-benzo[k]fluoranthene, [2H8]-carbazole, [2H12]-chrysene, [2H14]-dibenzoanthracene, [2H10]-fluoranthene, [2H10]-fluorene, [2H12]-indenopyrene, [2H8]-naphthalene, [2H12]-perylene, [2H10]-phenanthrene, [2H10]-pyrene and [2H7]-quinoline. All reference compounds were purchased from Sigma-Aldrich, CDN isotopes and Cambridge Isotope Laboratories with purities ranging from 95 to 100%. The recovery standards (InjS) were [13C]-PCB-97 and [13C]-PCB-188 .The chemicals assessed in this study comprised a group of 17 PAHs, 10 oxy-PAHs and 4N-PACs. The PAHs analysed were: acenaphthene, acenaphthylene, anthracene, benz[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzoperylene, benzo[k]fluoranthene, chrysene, dibenzoanthracene, fluoranthene, fluorene, indenopyrene, naphthalene, perylene, phenanthrene and pyrene. The oxy-PAHs analysed were: 1-indanone, 2-methylanthracene-9,10-dione, 4H-cyclopenta[def]phenanthrenone, 6H-benzo[cd]pyren-6-one, 7H-benz[de]anthracen-7-one, 9-fluorenone, anthracene-9,10-dione, benzo[a]anthracene-7,12-dione, benzo[a]fluorenone and naphthacene-5,12-dione. Finally, the N-PACs were: acridine, benzo[h]quinoline, carbazole and quinoline. The isotopically labelled internal standards (ILIS) used were: anthracene did not show appropriate recoveries; these compounds were therefore excluded from the extraction methodology. Method detection limits (MDLs) and method quantification limits (MQLs) were in range of those previously proposed by Nguyen et al. Instead of 12 L of raw sample extraction, the sample volume was scaled down to 40 mL, i.e. a reduction in sample size by a factor of 300. The size of the SPE cartridge was also reduced, from 6 g HLB sorbent to 200 mg (reduction factor of 30). Additionally, the conditioning and extraction solvents were also adapted to those recommended for 200 mg cartridges . It shou samples  to be abThe biochar contamination strongly impacted the stormwater matrix. During method development, we observed that the powdery and hydrophobic nature of biochar Different approaches were tested to remove biochar suspended particles with the goal of including as few steps as possible in the sample treatment with the least impact on method efficiency. Decantation, centrifugation, glass-wool column filtration and glass microfibers vacuum-aided filtration were all tested as separate treatment steps. Decantation and centrifugation were tested due to their non-invasive and limited sample manipulation required, while glass-wool and glass microfibers filters were tested as suspended particle filter materials with the expectation of limited impact on analyte loss.2H14]dibenzoanthraceneanthracene was excluded from the methodology.From visual inspection, it was concluded that the first two strategies, decantation and centrifugation, performed poorly on removing suspended particles due to the high hydrophobicity of biochar particles. The glass-wool column filtration helped at removing part of the biochar particles , but only the fraction of larger suspended particles, as the filtered water still showed presence of suspended material. However, glass microfibers disk filters, with a pore size of 1.2 µm, performed well, seemingly removing suspended particles to a satisfying degree. In order to evaluate the removal efficiency of biochar particles hindering extraction of PACs by instantly adsorbing the test compounds, the recovery of ILIS was tested with fortification pre- and post-filtration, respectively. These recoveries were normalized to the recovery of ILIS when extracting spiked Milli-Q water. In the case of spiking pre-filtration, it can clearly be observed that ILIS could not be efficiently recovered from the stormwater, with some cases showing even zero recovery . HoweverThe methodology for extracting PAHs and derivatives has been adapted for the analysis of stormwater treated with biochar containing suspended particles originating from the biochar. In brief, the adaption consisted on the scaling down the SPE as well as the inclusion of an additional filtration step to remove biochar suspended particles. It should be mentioned that the addition of an extra filtration step did not significantly impact financial, timing, or environmental costs. Additionally, it was deemed necessary to exclude three substances  from the methodology. Thus, the new extraction method covers 15 PAHs, 9 oxy-PAHs and 4N-PACs.Alberto Celma: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – original draft. Anna-Karin Dahlberg: Conceptualization, Methodology, Writing – review & editing, Funding acquisition. Karin Wiberg: Conceptualization, Methodology, Writing – review & editing, 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.