Glycolipid surfactants are biocompatible and biodegradable compounds characterized by potential applications in various sectors including pharmaceuticals, cosmetics, agriculture, and food production. A specific overview regarding synthetic methodologies and properties of 6′-lactose-based surfactants is presented herein, particularly all the synthetic approaches to this class of lactose esters, such as enzymatic and traditional organic syntheses. Moreover, detailed descriptions of physicochemical data and biocompatibility properties of these molecules, that is, surface tension, critical micelle concentration, emulsifying ability, foaming, particle size distribution, biocompatibility, and safety, are described. Biological applications with a focus on permeability enhancing, antimicrobial activity, and antibiofilm properties of 6′-lactose-based esters are also reported. In particular, the use of derivatives consisting of fatty acid chains linked to the sugar by an ester function has become increasingly widespread. These molecules, known as sugar-based fatty acid esters or glycolipids, are non-ionic surfactants because of the presence of a hydrophilic polar head  and a hydrophobic tail (saturated or unsaturated carbon chains). These amphiphilic compounds are of great interest for their multiple uses in the cosmetic, food, and pharmaceutical industries. In fact, in comparison with other surfactants, they are biodegradable, non-toxic, non-irritant, and odourless, and possess a safe biocompatibility profile . Thus, tGlycolipids can be easily obtained in large quantities from inexpensive raw materials such as oily by-products and industrial waste, as well as from microorganisms that optimize the fermentation process. In general, the yields of these bioconversion processes are so high that they allow the retention of huge amounts of glycolipids for various applications . They caAmong all sugar-based surfactants, sucrose and glucose esters are the most studied and applied derivatives, while lactose esters have received less attention, despite studies showing their potential .O-esters are considered.In this report, synthetic procedures, physicochemical and biocompatibility properties, and biological applications regarding lactose-6′-O-acyl(aryl)lactose esters are obtained from various aliphatic fatty acids  or aromatic acids and are represented in The known 6′-O-lactose fatty acids esters are preferentially synthesized by enzyme-catalyzed reactions. Lipases are characterized by stability in non-aqueous environments and green properties and are the enzymes most utilized to carry out regioselective acylation reactions. In fact, the stability in various conditions  and the possibility of using an immobilization system involving resins or other approaches, which allow a reuse of the enzyme without significant loss of activity, favor their use  mannitol Papp (apparent permeability coefficient) was observed at concentrations > 1 mM (p < 0.01). This rise is inversely correlated with the decreasing TEER effect. C12-lactose showed a better permeability enhancing effect than C12-trehalose ester and comparable results with the commercially available C12-sucrose ester [50 values were applied and a dose–effect relation for TEER was observed. For C16:1, TEER values sharply decrease, with maximal lowering by 62–68% of the baseline value after 2.5 h. However, C24:1 leads to a gradual decrease in TEER, and a minimal value was observed after 3 h (41–65%). For C18:1, the maximum decrease in TEER was observed after 120 min, with TEER remaining stable from that point and up to 180 min subsequent application of the compound. TEER values at 120 min were roughly 70% (0.06 mg/mL), 55% (0.125 mg/mL), and 40% (0.25 mg/mL) of the baseline value. TEER recovered after 24 h, emphasizing the reversible effect of the lactose-unsaturated fatty acid monoesters on tight junction openings. The effect of C16:1 and C24:1 on the permeability of Fitc-ovalbumin (45 Kda) as model protein across Caco-2 cell monolayer was also studied [Sugar-based esters can be used as permeability enhancers in drug delivery, offering a suitable non-invasive alternative route of administration, such as oral or pulmonary, to others . The permeability enhancing activity of 6′-lactose aliphatic saturated  and unsaTT assay . Therefo mucosae . In factse ester . TEER wase ester , and C18 studied . At 0.2 E. faecalis ATCC 29212, showing the same MIC value as sodium benzoate (64 μg/mL) [Various sugar derivatives have been proposed as safe and efficient preservatives, particularly to replace parabens in the pharmaceutical, cosmetic, and food fields. In this context, the antibacterial activities of various lactose fatty acid esters against several different food-borne and human pathogens, gram positive, gram negative, and fungi, were measured by minimum inhibitory concentration (MIC) values ,20,24,254 μg/mL) . The ant4 μg/mL) .E. coli O157:H7 ATCC 35150 strain with respect to the same reference drug  [S. aureus ATCC 43387 and P. aeruginosa ATCC 9027, for which a higher concentration was measured.Nevertheless, the best antimicrobial effect was observed with 6′-lactose unsaturated fatty acid esters ,18. In pctively) . The antctively) . The fewctively) . Aryl orctively) , except Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, and Staphylococcus enteritidis) [S. aureus, 2× MIC 55%), whereas Bpa was the best, being active after 24 h, 48 h, and 96 h with MIC and 2× MIC. In the last case, the best result was obtained with E. coli after 96 h , but good/consistent antibiofilm properties were shown after 24 h  and 48 h  or versus other bacteria  [A very recent promising tool in the armamentarium of biological applications is the possibility that some surfactants counteract the formation of bacteria biofilms. In fact, two 6′-lactose derivatives, aliphatic C10 and aromatic Bpa, were able to do this during the experiments carried out after a 24 h, 48 h, or 96 h incubation and with selected bacteria (ritidis) . In partMIC 53%) . OverallMIC 53%) .O-lactose monoester derivatives, highlighting their main synthetic procedures, physicochemical and biocompatibility properties, and the most interesting biological applications.Lactose-based surfactants are an emerging broad group of biocompatible and biodegradable compounds with established and potential future applications in the pharmaceutical, biomedical, cosmetic, and food industries. Specifically, this report focuses on 6′-


The application of nanoparticles in consumer products and nanomedicines has increased dramatically in the last decade. Concerns for the nano-safety of susceptible populations are growing. Due to the small size, nanoparticles have the potential to cross the placental barrier and cause toxicity in the fetus. This review aims to identify factors associated with nanoparticle-induced fetotoxicity and the mechanisms involved, providing a better understanding of nanotoxicity at the maternal–fetal interface. The contribution of the physicochemical properties of nanoparticles (NPs), maternal physiological, and pathological conditions to the fetotoxicity is highlighted. The underlying molecular mechanisms, including oxidative stress, DNA damage, apoptosis, and autophagy are summarized. Finally, perspectives and challenges related to nanoparticle-induced fetotoxicity are also discussed. The rapid development of nanotechnology has led to the increased production and use of nanomaterials in industry, biomedicine, and everyday life ,2,3, thuSo far, numerous studies have indisputably demonstrated that maternal exposure to nanomaterials during gestation result in fetotoxicity, including adverse prenatal effects on the fetus ,15,16,17Here, we summarized the progress of NP-induced fetotoxicity based on the categories of NPs to identify crucial factors associated with NP-induced fetotoxicity and the mechanisms involved. We discussed how the physicochemical properties of NPs, administration schedule, gestation stages, and maternal pathophysiological conditions affected NPs’ fetotoxicity, as well as the possible underlying mechanisms, which will be a cornerstone of protecting susceptible populations from NP exposure.In this section, NP-induced fetotoxicity, including detrimental prenatal outcomes, neurodevelopmental toxicity, reproductive toxicity, immune toxicity, and respiratory toxicity, was summarized.Recent epidemiological studies have indicated a significant association between maternal occupational exposure to NPs and being small for gestational age ,40. MateAmbient fine or ultrafine particles. Air pollution is a global threat to health. The potential detrimental effects of ambient (ultra)fine particles on fetal and child development have been evidenced. For example, pregnant C57BL/6J pun/pun female mice were intratracheally instilled with collected ultrafine particles at the accumulated dose of 400 μg/kg every other day during gestational day 7 (GD7)-GD17 to evaluate the fetal development under the adverse intrauterine environment induced by particulate matter (PM) [ter (PM) . This inter (PM) . Howeverter (PM) . Recentlter (PM) .Carbon-based nanomaterials. Carbon-based nanomaterials, such as carbon black (CB), carbon nanotubes (CNTs) and fullerenes are produced and widely used in the industry. Their biosafety has become a major concern. When pregnant C57BL/6BomTac mice were administered with CB NPs (14 nm) via lung exposure, both inhalation exposure  from GD8–GD18  and intratracheal instillation induced maternal inflammation [ammation . Maternaammation . Both inammation . Qi et aammation . Other rammation . The repammation ,73. ThesThe surface modification of NPs could affect nanotoxicity. When pregnant mice were intravenously injected with amino-functionalized polyethylene glycol (PEG)-SWCNTs at a single dose of 0.1, 10, and 30 μg/mouse on GD6 or in multiple administrations of 10 μg/mouse on GD6, GD9, and GD12 , occasional teratogenic effects were observed only at a dose of 30 μg/mouse, possibly due to their transfer to the fetal–placenta unit. Functionalized SWCNTs significantly increased fetal resorptions, morphological abnormalities and skeletal deformities following a single oral exposure of 10 mg/kg on GD9. Ultraoxide-functionalization aggravated the toxicity of SWCNTs considerably with the higher induction of early miscarriages and fetal resorptions compared with pristine or oxidized SWCNTs . The intMetal oxide NPs. Metal oxide NPs, especially zinc oxide NPs (ZnO NPs) and titanium dioxide NPs (TiO2 NPs), have become one of the most promising nanomaterials due to their wide application in sunscreens, food, biomedicine, and environmental remediation. Thus, their potential toxicity after maternal exposure has gained more attention. The oral administration of zinc oxide (ZnO) NPs, once daily by gavage during the organogenesis period, retarded fetal growth, reduced live fetal number, and increased fetal resorption and abnormalities [malities ,49,50,51malities . ZnO NPsmalities . When pr2 NPs during pregnancy induced alterations in gestational parameters, such as increased fetal resorption rate, smaller uteri and fetuses, and skeletal abnormalities in fetuses [2 NPs and SiO2 NPs (70 nm) significantly enhanced the fetal resorption rate, and decreased the fetal weight and uterine weight [2 NPs  during the whole gestation to investigate their adverse effects on the developing fetus [2 NPs transferred through the placenta, accumulated in the fetus and suppressed fetal development. Maternal inhalation exposure to TiO2 NPs (21 nm) during organogenesis decreased pup weight due to reduced placenta efficiency. However, in another study, no alterations in gestational parameters, such as litter size, fetal weight, sex distribution, and implantations, were observed [2 NPs. The maternal–fetal transfer and accumulation of food-grade TiO2 NPs in the human placenta and meconium emphasized the need for the risk assessment of chronic exposure to TiO2-NPs during pregnancy [Maternal exposure to TiO fetuses ,52,53,74e weight , which we weight ,54. Timeregnancy .2 NPs and CdO NPs also led to poor pregnancy outcomes [2 NPs into pregnant mice at a dose of 5 mg/kg daily from GD5–GD7 resulted in aberrations in decidualization, which exhibited “ripple effects” leading to fetal loss, fetal growth retardation, placental dysfunction, and even infertility [3) or daily (230 mg/m3) for 2.5 h/exposure from GD5–GD17, the accumulation of CdO NPs in placenta decreased the fetal length and delayed neonatal growth without direct NP transfer to the fetus [Maternal exposure to other metal oxide NPs during pregnancy such as CeOoutcomes ,55. The ertility . When prMetal NPs. The extensive application and large-scale production of metal NPs, such as Ag NPs and Au NPs, raise safety concerns in the vulnerable stages of life. A single intravenous administration of polyvinylpyrrolidone (PVP) or citrate-coated Ag NPs  in the late stages of pregnancy produced moderate size- and vehicle-dependent alterations in vascular tissue contractility, suggesting possible fetal growth retardation [ardation . Nose-onardation . The intardation . Au NPs ardation .In summary, in utero exposure to NPs induced potential fetal developmental toxicity or teratogenicity, such as fetal growth retardation, decreased litter size, fetal deformities, and fetal resorption. NP-induced embryo–fetal developmental toxicity depended on not only the physiochemical properties of NPs but also the exposure schedule, including administration doses and times, as well as maternal pathophysiological conditions.2 NPs [Maternal exposure to xenobiotics usually causes the most common developmental abnormalities in humans, especially those associated with neurodevelopment in fetus/offspring. Previous studies demonstrated that ultrafine particles/NPs penetrated through the blood–brain barrier, reached the brain, and induced neurodevelopmental toxicity ,77,78,792 NPs ,87,88,892 NPs  induced Ambient fine or ultrafine particles. Epidemiological studies indicated a positive association between air pollution and autism spectrum disorder, attention-deficit hyperactivity disorder, cognitive impairment, and decreased corpus callosum (CC) volumes in children [2.5  altered the neuroimmune phenotype [3 during GD1–GD17 to recognize the critical window of neurodevelopmental disorders. Such exposure induced ventriculomegaly, increased the CC area, and decreased the hippocampal area in both sexes of offspring. Meanwhile, both sexes exhibited CC hypermyelination, increased microglial activation, and decreased total CC microglia. Oxidative stress and neuroinflammation are the main mechanisms of CNS toxicity induced by ambient ultrafine particles during gestation [children ,101,102.children ,77. Devechildren ,91,103. children . In uterhenotype . Gestatihenotype . PregnanCarbon-based nanomaterials. Intratracheal instillation, intranasal instillation, and airway exposure were often adopted to evaluate neurodevelopmental disorders induced by CB NPs. Maternal inhalation exposures to CB NPs triggered habituation pattern and brain blood vessel alteration, dysregulated gene expression in the frontal cortex, and reactive astrogliosis in fetal brains [2), larger-sized MWCNTs moved across the blood–placenta barrier (BPB), restricted the development of fetuses, and induced brain deformity, whereas SWCNT and smaller-sized MWCNTs showed no or less fetotoxicity. Furthermore, p53-/- fetuses showed obvious brain deformity, indicating that CNTs might have a genetic background-dependent toxic effect on the normal development of the embryo [l brains ,82,83,84l brains . After me embryo .Metal oxide NPs and Metal NPs. Gestational exposure to metal oxide led to the accumulation of NPs in the fetal brain, causing neurotoxicity and neurobehaviors associated with enhanced depression-like behaviors, impaired learning and memory, and autism spectrum disorder in offspring [2 NPs during gestation caused their accumulation of fetal brain; hence, they were dangerous to fetal brain development, especially in early pregnancy [2 NPs (6.5 nm) to mice from prenatal day 7 to postnatal day (PND) 21 led to brain retardation, retarded axonal and dendritic outgrowth, and impaired cognitive ability; it was related to the excessive activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK1/2/MAPK) signaling pathway [2 NPs into pregnant mice altered the expression levels of genes related to CNS development and function [ffspring ,87,88,89regnancy . Continu pathway . The subfunction . Exposurfunction . RecentlNP-induced neurotoxicity depends on the physicochemical parameters of NPs, pregnancy stages, and administration procedures. The dissolution of NPs  plays an important role in fetotoxicity after maternal exposure.Regarding the reproductive toxicity, reproductive dysfunction in male offspring after maternal exposure to NPs has been reported .Ambient fine or ultrafine particles. The intratracheal administration of Asian sand dust  on GD8 and GD15 into pregnant ICR mice induced significantly decreased testis and epididymis weight in offspring. Pathological alterations included the vacuolation of seminiferous tubules and cellular adhesion of seminiferous epithelia, which resulted in significantly decreased daily sperm production (DSP) in offspring [ffspring . Gestatiffspring ,108.Carbon-based nanomaterials. Most in vivo findings indicated that in utero exposure to CB NPs and MWCNTs might affect the reproductive system and function in male/female offspring [ffspring ,82,83,84ffspring . Similarffspring . Howeverffspring . Treatmeffspring ,71.Metal oxide NPs. Previous investigations indicated that in utero exposure to ZnO NPs, TiO2 NPs, Fe2O3 NPs, and SiO2 NPs affected male reproductive ability and the reproductive function of offspring [2 NPs into pregnant mice resulted in alterations in spermatogenesis and impairment in the testis of offspring. TiO2 NPs were transferred to the offspring and accumulated in Sertoli cells, Leydig cells, or spermatids of offspring testes, leading to reduced DSP and the number of Sertoli cells, disorganized and disrupted seminiferous tubules, and failed spermiation from Sertoli cells and sperm motility [2 NPs during gestation at the total doses of 0.5, 5, 50, and 500 μg did not affect the male reproductive function in two generations of male offspring mice [2 NPs (250 mg/kg) triggered alterations in spermatogenesis and pathological changes in testis, such as epithelial vacuolization, cellular adhesion of epithelia, and decreased seminiferous tubule diameter [2O3 NPs. Polyethyleneimine Fe2O3 NPs(+) treatment induced morphological alterations in offspring uteri and testis during organogenesis, while poly(acrylic acid) Fe2O3 NPs(−) treatment only induced mild alterations in the offspring uterine cavity [ffspring ,106,107.ing mice . The oradiameter . Similare cavity . The afoThe innate immune system of an organism provides the first line of defense against particulate materials. Pregnant women and developing fetuses are populations susceptible to immunotoxicity induced by xenobiotic pollutants. Developmental immunotoxicity induced by various NPs in mice is summarized in Ambient fine or ultrafine particles. In recent years, detrimental effects on the immune development of the offspring after maternal exposures to PM have received more attention [ttention ,30,109. ttention . The intttention .Carbon-based Nanomaterials and Metal NPs. El-Sayed et al. reported the adverse effects on the immune system of offspring after gestational exposure to CB NPs [Traf6 gene expression in the thymus. Therefore, respiratory exposure to CB NPs in middle and late gestation might cause allergic or inflammatory effects in male offspring mice [+, CD4+, and CD8+ cells, and upregulated Il15 , Ccr7 and Ccl19  gene expression [3) via inhalation from GD3–GD19 [o CB NPs ,111. Afting mice . When exGD3–GD19 . Among 8Epidemiological studies have proved that maternal exposure to ambient PMs during the gestational period is associated with disturbed lung development and increased risk of respiratory diseases after birth. So far, investigations have mostly focused on PMs; only a few have explored metal oxide NPs and metal NPs.Ambient fine or ultrafine particles. The exposure of pregnant C57/BL6 mice to combustion-derived PMs via oropharyngeal aspiration aggravated asthma development in offspring mice [2.5 (3 mg/kg) by oropharyngeal aspiration every other day from GD1–GD17 [ing mice . This aging mice . Yue et GD1–GD17 .Metal NPs and Metal oxide NPs. Maternal exposure to metallic NPs during pregnancy, such as TiO2 NPs, CeO2 NPs, and Ag NPs, might lead to the transfer of NPs from the mother to the fetal lung and induce impairment in lung development and even a lasting effect in adulthood [2 NPs (21 nm) daily for 7 days during pregnancy [2 NPs, CeO2 NPs, and Ag NPs resulted in irreversible impairment in offspring lungs regardless of the chemical properties of NPs with alterations in lung development-related genes and proteins in the fetal and alveolarization stages [dulthood ,113. Forn stages .In summary, the aforementioned results provided a comprehensive understanding on how the key parameters of particles affected NP-induced fetotoxicity, including neurotoxicity, reproductive toxicity, developmental immunotoxicity, and respiratory toxicity, in offspring . HoweverThe placenta is a vital organ connecting the mother and the fetus in mammals. During gestation, the placenta not only is the site for the exchange of substances, metabolism, secretion, and excretion, but also protects the embryo/fetus from the harmful environmental pollutants. Although anatomical differences in the placenta exist among species, the most commonly selected models used to explore transplacental transfer ability and fetotoxicity are human and murine placenta models. Different from human term placenta, the mature murine placenta consists of three trophoblast layers, including the decidua, junctional zone, and labyrinth, constituting the primary barrier ,115,116.2 NPs [2 NPs [2 NPs [2 NPs, Au NPs [Direct particle translocation across the placental barrier has been considered as the most potential mechanisms underlying NP-induced fetotoxicity . Previou2 NPs , TiO2 NPs [2 NPs , ZnO NPss [2 NPs ,48, ZrO2s [2 NPs ,119, CeO, Au NPs ,121,122,, Au NPs ,123 coul, Au NPs ,125.2 NPs [2 NPs coated with -COOH/-OH or QDs coated with -PEG/-SiO2 shell prevented or reduced NP translocation into the fetus [Particle size is a pivotal determinant regulating NP translocation across the placental barrier ,121,126.2 NPs ,127, ZnO2 NPs , and QDs2 NPs ,123, was2 NPs . SiO2 NPhe fetus ,123. Addhe fetus ,129,130.he fetus . Finallyhe fetus ,131.2 (16 nm)-induced maternal–fetal transfer of NPs across the placenta via the upregulation of clathrin- and caveolin-mediated endocytosis [So far, investigations regarding the transfer mechanisms of NPs across the placental barrier have been limited ,121,128.ocytosis ,135. In ocytosis .Maternal NP exposure may induce fetotoxicity by the direct translocation of NPs to the fetus or indirect NP-induced maternal and placental mediators ,38,117. 2 NPs during pregnancy triggered oxidative damage to nucleic acids and lipids, and induced reactive astrocytes via the overexpression of GFAP and Aquaporin 4 (Aqp4) in offspring brain [2 NPs  1h/day from GD8–GD18 induced the deposition of NPs in lungs and long-term maternal lung inflammation, resulting in a sex-specific neurobehavioral alteration in offspring [2 NPs  into pregnant mice from GD13–GD14 upregulated nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome and significantly increased the levels of placental inflammatory cytokines  [2 NPs, SWCNTs, and TiO2 NPs, induced oxidative stress and a high incidence of detrimental developmental outcomes [Reactive oxygen species (ROS) generation and inflammatory responses are generally considered as the main mechanisms in NP-induced fetotoxicity . First, ng brain ,84. Inhaffspring . Second,nd CCL2) . This exnd CCL2) . After pnd CCL2) . Maternaoutcomes ,64,89. T-/- fetuses, which is related to MWCNT 50-induced DNA damage [2 NPs exposure altered the gene expression of the retinoic acid signaling pathway in female rather than male mice [DNA damage is generally thought to be responsible for initiating fetal developmental abnormalities caused by environmental chemicals. Maternal exposure to NPs during pregnancy may cause severe DNA damage, such as DNA strand breaks, DNA deletions, mutations and oxidative DNA adducts. SWCNT-50 can induce p53-dependent fetotoxicity and brain deformity in p53A damage . Maternaale mice ,137. Traale mice ,138. Matale mice .2 NPs, TiO2 NPs, Au NPs, QDs, functionalized CNTs, and carboxylate-modified polystyrene beads during pregnancy induced cell apoptosis in spongiotrophoblasts of the placenta [Maternal exposure to SiOplacenta ,139,140,placenta ,23,92,93placenta ,141,142,2 NPs (<25 nm) via gavage from GD1–GD13 triggered apoptosis, dysregulated vascularization, and proliferation, which significantly impaired placental growth and development [2 NPs suppressed axonal or dendritic outgrowth, through the induction of apoptosis in the offspring hippocampus [2 NPs, ZrO2 NPs, and Au NPs likewise induced pathological and structural alterations in apoptosis and apoptosis-related factors in the placenta following gestational NP exposure [For example, gestational exposure to TiOelopment . In addipocampus ,93,107. pocampus . In anotpocampus . Carboxypocampus . Other nexposure ,65,120.2 nanosheets induced autophagy relying on the variation in surface chemistry and shape [2 NPs induced autophagy and possible placental dysfunction in HTR-8/SVneo cells through the activation of endoplasmic reticulum stress [2 NPs in a human trophoblast model [2 NPs induced excessive autophagy with significant enhancement in expression levels of autophagy-related factors and LC3 II/LC3I, resulting in the severe suppression of dendritic outgrowth in offspring hippocampal neurons [NP-induced autophagy has been widely reported ,145. Fornd shape ,147. Thrnd shape , implantnd shape , placentnd shape ,151, embnd shape ,153, prend shape ,154 and nd shape . Especiand shape  and protnd shape . Howevernd shape . CoCr NPnd shape . TiO2 NPm stress ,160. MiR neurons .The aforementioned studies revealed that NPs might disrupt placental development and embryo–fetal development via autophagy. The placenta acted as a vital support unit for the developing fetus and played a pivotal role in regulating fetal development such as fetal brain . TherefoIn addition, NPs may induce fetotoxicity via interfering with endocrine signaling ,163, vasThe evaluation of NP-induced toxicity across placental barriers has become increasingly important with the increased application of nanomaterials in medical products. Maternal exposure to NPs during pregnancy led to adverse gestational parameters, neurotoxicity, reproductive toxicity, immunotoxicity, and respiratory toxicity in offspring. The characteristics and functionalization of NPs, as well as maternal conditions and exposure routes, play crucial roles in NP-induced fetotoxicity. Oxidative stress and inflammation, DNA damage, apoptosis, and autophagy have been considered as the main mechanisms underlying the NP-induced fetotoxicity. However, the detailed mechanisms underlying the embryo–fetal toxicity need further investigation due to the complexity in the pharmacokinetics of NPs and interactions with physiological systems, in particular during pregnancy.The nano-safety evaluation during pregnancy has been extensively explored. However, more systematic investigations should be conducted on the following issues in the future. First, the NP-induced fetotoxicity focused on the CNS, reproductive, respiratory and immune systems. More studies on other systems, including cardiac function, early placental development, microvascular function, and fetal metabolism should be performed. Second, NP libraries and more effective placental transfer models  should be constructed to systematically elucidate how NP translocation is dependent on the physiochemical properties of NPs and clarify the underlying mechanisms. Third, the exposure route adopted in current investigations, especially intravenous administration, is not the representative route for maternal environmental and occupational exposure. Hence, strategies for exposure assessment in the environment and at the workplace should be elaborated, which is the base point for the establishment of occupational exposure limits for NPs.


For instance, in bacteria, genetic and biochemical studies suggest a close proximity and coordination of action of the RecF, RecR and RecO proteins in order to ensure their RMP function, which is to overcome the single-strand binding protein (SSB) and facilitate the loading of the recombinase RecA onto ssDNA. In contrary to this expectation, using single-molecule fluorescent imaging in living cells, we showed recently that RecO and RecF do not colocalize and moreover harbor different spatiotemporal behavior relative to the replication machinery, suggesting distinct functions. Finally, we address how new biophysics tools could be used to answer outstanding questions about RMP function.This review recapitulates the initial knowledge acquired with genetics and biochemical experiments on Recombination mediator proteins in different domains of life. We further address how recent The recombination mediator proteins (RMPs) are ubiquitous and play a crucial role in genome stability. RMPs facilitate the loading of recombinases like RecA onto single-stranded (ss) DNA coated by single-strand binding proteins like SSB. Despite sharing a common function, RMPs are the products of a convergent evolution and differ in (1) structure, (2) interaction partners and (3) molecular mechanisms. The RMP function is usually realized by a single protein in bacteriophages and eukaryotes, respectively UvsY or Orf, and RAD52 or BRCA2, while in bacteria three proteins RecF, RecO and RecR act cooperatively to displace SSB and load RecA onto a ssDNA region. Proteins working alongside to the RMPs in homologous recombination and DNA repair notably belongs to the RAD52 epistasis group in eukaryote and the RecF epistasis group in bacteria. Although RMPs have been studied for several decades, molecular mechanisms at the single-cell level are still not fully understood. Here, we summarize the current knowledge acquired on RMPs and review the crucial role of biophysical tools to investigate molecular mechanisms at the single-cell level in the physiological context. This chapter summarizes genetic studies which identified and helped to characterize the function of Recombination Mediators Proteins (RMPs) as well as their partners in DNA replication and repair across different kingdoms of life.recF, recO, and recR genes which belong to the same DNA repair pathway , including recF [recO and recR [recF, recO, and recR were grouped with other genes  in the recF epistatic group. Genetic experiments revealing the similarity between SSB overexpression and recF deletion were critical to understand the role of the RecF pathway in SSB removal [recFOR deletion mutants are generally characterized by functional defects related to RecA presynaptic nucleofilament  activities, such as: (1) conjugation defect, (2) UV, Gamma rays and drug sensitivity, (3) delayed SOS induction, (4) translesion synthesis (TLS) defects and (5) replication fork instability [Deinococcus radiodurans naturally lacking the recBC genes, the RecF pathway plays a significant role in double strand break (DSB) repair through extended synthesis-dependent strand annealing process [In bacteria, RMPs are encoded by  pathway . Their ding recF . This geand recR ,3. Due t removal ,5. recFOsuch as:  conjugat defect,  UV, Gammtability ,9,10,11.tability ,13, medi process . recF, recO, and recR gene conservation despite their apparent epistasis [recO or a recO ortholog gene is present in most bacterial species, whereas the recF gene is much less widely distributed [recO and recF in Bacillus subtilis and E. coli [Phylogenetic analysis reveals considerable variability in pistasis . In parttributed ,16. This E. coli ,19,20,21uvsX was identified, then uvsY, and finally uvsW [uvsX, uvsW, and uvsY genes act in the same non-linear DNA repair pathway [uvsX gene encodes a RecA-like recombinase [uvsW and uvsY [In bacteriophage T4 (lytic), identification of DNA recombination genes was carried out using genetic screening of deletion mutants presenting sensitivity to UV, DNA damaging agents, such as ethyl and methyl methanesulfonate (EMS and MMS), hydroxyurea (HU), and furthermore showed HR defects ,25,26,27lly uvsW ,24,26. Slly uvsW ,26,28. Dombinase ,23,29, wand uvsY . Geneticand uvsY . The bacteriophage λ (lysogenic) also encodes its own recombination system called λRed able to repair ss- and dsDNA breaks. The λRed combines an exonuclease (5′ to 3′) Exo, a Gam protein, which prevents the action of the host RecBCD and a single-strand binding protein involved in DNA annealing, called Beta . AdditioSaccharomyces cerevisiae [S. cerevisiae, the RAD52 deletion only presented a subtle HR deficient phenotype in vertebrates, suggesting the existence of another DNA repair mechanism in this phylum. Beyond this, recent work uncovered a new mitosis-specific functions of RAD52 in mammals, showing that RAD52 promotes a break-induced replication-like pathway, dubbed mitotic DNA synthesis, that allows completion of DNA replication during chronic stress [RAD52 was identified as a radiation-sensitive mutation in revisiae . Additiorevisiae . This grrevisiae ,37. Althc stress ,39,40. Tc stress ,42.BRCA2 is another crucial gene for HR in eukaryotes and was first identified in humans [Ustilago maydis and mammalian cells, BRCA2 also acts as RMP during meiosis by facilitating the loading of DMC1 (RecA-like) onto ssDNA [Caenorhabditis elegans only encodes BRC2, mammalian cells encode both and show a different extent in HR phenotypes for the single BRCA2 and double knockdown BRCA2/RAD52 [The breast cancer susceptibility gene n humans . Analysin humans . The intn humans . Togetheto ssDNA ,47. RAD5to ssDNA . RMP conA2/RAD52 ,49. Biochemical and structural studies were critical to understand both common function and specificities of RMPs. Based on their overall structure, RMPs can be grossly classified into two groups, globular RMPs harboring oligonucleotides/oligosaccharides binding (OB) fold domains such as RecO or BRCA2, and RMPs adopting a multimeric ring-like shape such as UvsY or RAD52 . The folD. radiodurans and E. coli either alone or in complex with RecR or SSB [The entire RecO protein structure has been solved for R or SSB ,53,54,55R or SSB . Bulk exR or SSB ,56,57. RR or SSB . D. radiodurans, Pseudomonas aeruginosa, Thermoanaerobacter tengcongensis, and Thermus thermophilus [B. subtilis, the interaction between RecO and RecR is required to displace SSB tetramers from ssDNA [The structure of the bacterial RecR has been solved for mophilus ,60,61,62mophilus ,62. The mophilus ,63,64. Rmophilus . Structumophilus . With thom ssDNA ,66. Whenom ssDNA ,67,68,69D. radiodurans and T. tengcongensis (with and without ATP) [The structure of RecF protein was solved for out ATP) . RecF beout ATP) ,71. RecFout ATP) ,73,74. Iout ATP) ,74. Althout ATP) ,75. Oppoout ATP) . Notablyout ATP) ,68,69,77out ATP) . Consistout ATP) . Importaout ATP) ,68Overall, biochemical and structural studies agree on a consensus model in which RecOR cooperatively act as RMPs . HoweverRMP UvsY in T4 adopts a heptameric open barrel conformation  which wrE. coli SSB was demonstrated by far western-blot [Structurally, RMP λOrf in bacteriophage λ is composed of two domains, (1) a small N-terminal domain and (2) a larger C-terminal domain . λOrf foern-blot . Sequencern-blot . The homologous pairing domain of RAD52 forms an undecamer ring folded into a mushroom-like structure consisting of a “stem” and a “domed cap” . The steIn the case of BRCA2, only the structures of truncated C-terminus (~800 residues) and short N-terminus have been solved. BRCA2 was solved in complex with its various binding partners DSS1, DSS1, and DNA, RAD51 or PALB2 ,87,88,92in vivo and in vitro biophysical tools used to capture population dynamics of biochemical reactions and their intermediates. Genetics, structural and bulk-biochemical studies have provided a wealth of knowledge about the role of RMPs. Nonetheless, these methods come with limitations and may have missed spatiotemporal information of the reaction dynamics. They may have overlooked transient or underrepresented intermediates in the reaction population, or even that biochemical reactions may proceed via different pathways. These limitations can however be addressed by employing biophysical tools. In this chapter, we discuss In vivo biophysical tools provide spatiotemporal information of biochemical reactions. Utilizing microfluidic devices, cells are flattened on a coverslip in the imaging plane , result5 μm2/s, ). These 5 μm2/s, ,101,102)5 μm2/s, , can inf5 μm2/s, ,104, infE. coli cells, we recently investigated the behavior of RMPs RecF and RecO [recF-YPet recO-mKate2. E. coli cells produced ~18 RecF molecules and ~12 RecO molecules per cell, corresponding to ~5 nM and ~4 nM, respectively. Despite similar concentrations, RecF formed foci more frequently (2.2 ± 0.2 RecF-YPet and 0.3 ± 0.1 RecO-YPet foci per cell). Upon UV exposure, cellular intensities stayed constant, giving no increase in protein concentrations, the number of foci per cell however increased  has been used to determine binding affinities by measuring kinetics of mass adsorption or desorption on a chip . At equimolar ratios of RecO and SSB-ssDNA, the authors observed slow displacement of SSB by RecO. From 40 min after RecO addition, ~60–70% of DNA molecules displayed a FRET efficiency of 0.78, suggesting SSB displacement by RecO. DNA-bound RecO could however not be displaced by SSB. Upon immobilization of DNA on coverslips, using FRET traces, the authors found that RecO association with ssDNA is slowed in the presence of SSB . In the process of SSB displacement, traces showed a low FRET intermediate, indicating ssDNA extensions, potentially due to the formation of a RecO–ssDNA–SSB ternary complex. Interestingly, most displacements occurred after this low FRET intermediate state. The authors further discovered that RecO has two main binding states, one at a low FRET where DNA is extended and one at a high FRET where ssDNA ends are close, consistent with ssDNA wrapping around RecO. Mutation of positively charged N-(K35E/R39E) or C-terminal residues (R195E/R196E) impaired the formation of a high FRET state, suggesting that the first binding state involves stretching of ssDNA similarly seen during SSB displacement. These mutants showed a large reduction in SSB displacement and an increase in intermediate state. The authors proposed a sequential mechanism  and Rad51 paralogs, RFS/RIP-1, stimulate nematode Rad51 filament growth . FurtherTaken together, these studies uncovered fundamental principles about the role of RMPs in displacing the single-stranded binding protein, recombinase loading and nucleofilament stabilization across organisms. Employing biophysical tools revealed that the formation of a ternary complex, RMP-single-stranded binding protein-ssDNA, appears to be a conserved mechanism. in vivo assays.Biophysical tools are powerful techniques used to uncover fundamentals of biochemical reactions. Despite the knowledge already gained on RMP’s molecular mechanisms, some questions are still outstanding. In this last chapter, we discuss how future studies could address several open questions using biophysical tools in combination with bulk and D. radiodurans RecF with RecO-like biochemical properties [in vivo pull downs could in the future be helped by mass photometry [In living bacteria, single-molecule fluorescence observations contradict the accepted model of RecF and RecO proteins working in close proximity during DNA repair ,76,131. operties . Identifotometry . New intotometry .The current knowledge of bacterial as well as phagic RMPs combined with future protein designing and biophysical experiments could allow the development of new generations of therapeutics in the fight against multidrug resistant bacteria. Phage therapy has been used by some countries , paving BRCA2-deficient cells being more sensitive to DNA damage, initiating IR and cisplatin treatment. Eventually, based on the finding that BRCA2 acts in both HR and base excision repair, new therapeutics such as PARPi were developed [BRCA2-deficient breast cancer cells [in vivo single-cell imaging could improve the general understanding of RMPs [The development of a new generations of cancer therapeutics were initiated after the observation of eveloped . Similarer cells ,142. Diser cells ,144. Com of RMPs , paving


In this study, recognizing the importance of 1,25VD3 as one of only 2 molecules  photosynthesized for energy during the beginning stage of life on this planet, we explored the functional role of 1,25VD3 in AML metabolism.Acute myeloid leukemia (AML) has the lowest survival rate among the leukemias. Targeting intracellular metabolism and energy production in leukemic cells can be a promising therapeutic strategy for AML. Recently, we presented the successful use of vitamin D  gene therapy to treat AML mouse models in vivoTranscriptome database (RNA-seq) of four different AML cell lines revealed 17,757 genes responding to 1,25VD3-treatment. Moreover, we discovered that fructose-bisphosphatase 1 (FBP1) noticeably stands out as the only gene  with a 250-fold increase in gene expression, which is known to encode the key rate-limiting gluconeogenic enzyme fructose-1,6-bisphosphatase. The significant increased expression of FBP1 gene and proteins induced by 1,25VD3 was confirmed by qPCR, western blot, flow cytometry, immunocytochemistry and functional lactate assay. Additionally, 1,25VD3 was found to regulate different AML metabolic processes including gluconeogenesis, glycolysis, TCA, de novo nucleotide synthesis, etc. In summary, we provided the first evidence that 1,25 VD3-induced FBP1 overexpression might be a novel therapeutic target to block the “Warburg Effect” to reduce energy production in AML blasts.The online version contains supplementary material available at 10.1186/s40364-022-00367-3. To the Editor,Acute myeloid leukemia (AML) is the most common type of leukemia in adults . DespiteVitamin D is known to be the oldest hormone on earth . Some ofFirst, we performed transcriptome analyses (RNA-seq) of 4 different AML-FLT3 cell lines including MV4–11, MOLM-14, MV4–11-midostaurin-resistant cells and MOLM-14-midostaurin-resistant cells which were previously reported . Among mIn addition to the central pathway of metabolizing glucose to pyruvate via glycolysis, AML metabolism involves diverse processes of nucleotides, amino acid, lipids and their end metabolites to perform signaling functions and produce energy to support tumorigenesis . Here, wAdditional file 1.


Cancer development is associated with abnormal proliferation, genetic instability, cell death resistance, metabolic reprogramming, immunity evasion, and metastasis. These alterations are triggered by genetic and epigenetic alterations in genes that control cell homeostasis. Increased reactive oxygen and nitrogen species  induced by different enzymes and reactions with distinct molecules contribute to malignant transformation and tumor progression by modifying DNA, proteins, and lipids, altering their activities. Nitric oxide synthase plays a central role in oncogenic signaling modulation and redox landscape. Overexpression of the three NOS isoforms has been found in innumerous types of cancer contributing to tumor growth and development. Although the main function of NOS is the production of nitric oxide (NO), it can be a source of ROS in some pathological conditions. Decreased tetrahydrobiopterin (BH4) cofactor availability is involved in NOS dysfunction, leading to ROS production and reduced levels of NO. The regulation of NOSs by BH4 in cancer is controversial since BH4 has been reported as a pro-tumoral or an antitumoral molecule. Therefore, in this review, the role of BH4 in the control of NOS activity and its involvement in the capabilities acquired along tumor progression of different cancers was described. Cancers are a set of diseases characterized by genetic instability, abnormal cell proliferation, cell death resistance, metabolic reprogramming, angiogenesis, metastasis capability, and immune response evasion caused by genetic and epigenetic alterations in oncogenes and tumor suppressor genes . These aAmong other cause effects, tobacco, obesity, and related metabolic syndromes, infections, and UV are associated with inflammation, loss of redox homeostasis, and oxidative stress, contributing to cancer development ,8,9,10. NOS1), endothelial NOS (NOS3), and inducible NOS (NOS2), are encoded by different genes localized in distinct chromosomes. All three isoforms are homodimeric and each monomer contains a C-terminal reductase domain and an N-terminal oxygenase domain connected by a calmodulin-binding peptide linker. The binding sites for the cofactors flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide phosphate (NADPH) are found in the reductase domain, which shares homology with cytochrome P450 (CYT P450) reductases while the oxygenase domain contains binding sites for heme, the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) and the substrate L-arginine. The electron transfer occurs from the NADPH cofactor into the oxygenase domain, where the L-arginine hydroxylation and subsequently NO formation takes place. Stable dimerization between the two NOS monomers is required for efficient NOS activity, as electrons flow from the reductase domain of one monomer to the oxidase domain localized in the other monomer (Three NOS (EC 1.14.13.39) isoforms have been described in mammalian cells. NOS isoforms, named neuronal NOS NO is a pleiotropic gaseous molecule with a reactive free radical activity. NO regulates innumerous physiological processes, including vascular  and neurological functions (nervous system development and neurotransmission) . Decreas+ effector cells activation and intratumoral infiltration, attenuating tumor growth . In. Inde no1.1.153) . In the ) and AR . GTPCH1 ) and AR ,48,49. G) and AR ,51.2•− and hydrogen peroxide (H2O2)   47]. Al. Al2•− aDifferent molecules have been described to contribute positively or negatively to tumor progression, which is associated with several factors, including the type of neoplasia and the stage of tumor development, reflecting the cross-talking between altered oncogenic signaling pathways and the interaction with other molecules. The compartmentalization within the cell and the location in the cancer microenvironment in addition to the expression and/or concentration of such factors also cooperate with its role.As discussed below, the involvement of tetrahydrobiopterin as a pro- or anti-tumoral molecule has been demonstrated in different biological processes, including tumor microenvironmental reprogramming, cell growth, metabolism, and metastasis, contributing or impairing cancer development .Proliferation is a biological process controlled by innumerous signaling pathways triggered by ligand-receptor interaction, which in turn, among other processes, regulates cell cycle progression, nutrition status, and genome integrity. In cancer, uncontrolled cell growth is associated with genetic and epigenetic alterations of key genes that maintain cell homeostasis and also by cell metabolism dysregulation ,69.As discussed, NOS coupling does not only depend on the total amount of BH4. The BH4/BH2 ratio and the stoichiometric balance between BH4 and NOS have been shown to determine the functional state of the enzyme ,70. In t2•− and increased cGMP levels in MCF-7 and MDA-231 cancer breast cells, which suggested NOS recoupling. Moreover, L-sepiapterin supplementation of MCF-7 cells induced cGMP-dependent protein kinase (PKG) activation, which in turn promoted β-catenin expression and decreased TCF4 promoter activity [2•− and increases NO amount, we can conclude that BH4 metabolism alteration modulates the role of NF-κB in cancer and probably the activity of other signaling pathways. Finally, L-sepiapterin inhibited clonogenic capability of MCF-7 and MDA-231 cells in vitro which was shown to be dependent on NO and PKG. MDA-231 xenografts tumor growth and progression were also impaired in the presence of L-sepiapterin. Although MCF-7 and MDA-231 breast carcinoma cells express both iNOS and eNOS isoforms, these results suggested that iNOS is uncoupled since the iNOS selective inhibitor 1400 W impaired the L-sepiapterin-induced cGMP levels [Rabender et al. observed decreased BH4/BH2 ratio in human breast cancer cells grown in culture, as xenografts, or in a spontaneous mouse breast cancer model (MMTVneu). Besides that, a low BH4/BH2 ratio was found in human colorectal carcinoma biopsies compared with the adjacent normal colon tissue. Treatment with L-sepiapterin partially reduced Oactivity . The proactivity . The actP levels .2•− [2•− and NO must be in close proximity [2•− [Previously, the same group described that L-sepiapterin impaired chemically induced murine colitis and azoxymethane-induced colorectal cancer . Inflamm2•− . Since proximity , which nity [2•− . Burhanuity [2•− . ROS is ity [2•− , implicaity [2•− .Nos3 expression. Moreover, O2•− was suppressed by L-NAME (N-Nitro-L-arginine methyl ester), a NOS inhibitor [2•− concentration [2•− and lower levels of NO. Treatment with BH4 increased the BH4/BH2 ratio, NO concentration, and decreased O2•−. More importantly, BH4 attenuated cell proliferation and induced apoptosis of metastatic melanoma cells, but not melanocytes, pointing to its use as a promising therapy [Nos3 expression abrogated the increase in O2•− and the reduction of NO. Moreover, cell growth in vitro and in vivo was attenuated . Increased expression of all three isoforms has been shown in melanoma cells and tissues [2•− modulate different signaling pathways in melanoma, provoking different tumor phenotypes. Furthermore, innumerous authors suggested that the imbalance between NO and O2•− results in cancer growth and progression or tumor development impairment.Campos et al. demonstrated for the first time evidence of the association between NOS uncoupling and murine melanocyte malignant transformation. Melanocytes submitted to a chronic stress condition showed increased nhibitor . The samntration . Recentl therapy . The hyp tissues ,40,41. HGCH1 is downregulated in HCC tissues and different cell lines by promoter methylation. Decreased GCH1 expression is significantly associated with a higher Barcelona Clinic Liver Cancer stage and larger tumor size. Moreover, GCH1 is correlated with poor clinical outcomes in HCC patients. GCH1 silencing promotes proliferation in vitro and in vivo through inhibition of oxidative stress-induced AKT/p38 activation, indicating the involvement of uncoupled NOS in HCC carcinogenesis [NOS3 T-786C polymorphism (rs2070744) genotype, resulting in reduced activity of NOS3 gene promoter and NO production, is associated with better clinical outcomes in patients treated with sorafenib [NOS2 and PTGS-2 expression was correlated with poor prognosis in hepatitis C virus–positive HCC patients. However, NOS activity was not analyzed [Recent data showed that GTPCH1/BH4/NO axis impaired hepatocellular carcinoma (HCC) development. ogenesis . Althougogenesis ,83. Gonzorafenib ,85. Rahmanalyzed .On the other hand, Zhang et al. reported an oncogenic role of BH4 in breast cancer development. Increased sepiapterin reductase protein (SR) was found in breast cancer tissues when compared to the adjacent non-tumorigenic tissue and correlated with tumor aggressiveness. SR downregulation inhibited metastatic breast cancer cell proliferation and induced ROS-mediated apoptosis. Although NO amount was not evaluated, decreased BH4 concentration was reported in MDA-MB-231 and MDA-MB-468 breast cancer cells, suggesting the involvement of uncoupled NOS in metastatic breast cancer survival impairment . The redin silico analyses showed that increased GCH1 expression was correlated with higher glioma grade, recurrence, and worse survival. Furthermore, GCH1 overexpression and BH4 levels elevation in GBM cells increase proliferation and decrease survival in an intracranial GBM-mouse model, which is correlated with brain tumor-initiating cells maintenance and ROS suppression [In glioblastoma (GBM), pression . GBM devpression ,92. On tpression ,56,93. Tpression .GCH1 expression and NO amount, which in turn decreased cell growth and increased apoptosis in squamous carcinoma Eca-109 cells [2•− analyses are required.Increased expression of iNOS and eNOS have been related to esophageal squamous cell carcinoma (ESCC). However, neither the regulation of NOS by BH4 nor the mechanism of contribution to the pathogenesis of ESCC have not been investigated. AU-rich element RNA-binding factor 1 (AUF1) is a family of proteins involved in the post-transcriptional regulation process of mRNA and has been related as a promoter or inhibitor of cancer progression ,94,95,9609 cells . Althoug09 cells ,98, NOS de novo BH4 synthesis pathway is associated with cell death triggered by RSL3, a GPX4 inhibitor, in different cancer cell lines. Exogenous BH4 supplementation increased proliferation and restored the resistance of T- and B-cell acute lymphoblastic leukemia and lymphomas to GPX4 inhibitors, indicating BH4 synthesis as a new target therapy in those tumors. The authors showed that ferroptosis impairment was caused by the ability of BH4 to act as a radical-trapping antioxidant in lipid membranes, avoiding lipid peroxidation in accordance with another study using fibroblasts [2•− generation. BH4 is not consumed in the reaction catalyzed by NOS, instead, it is regenerated by the enzyme itself, not resulting in a BH4 decrease [2•− levels were not evaluated to be able to infer about NOS activity. NOS inhibition or eNOS silencing renders leukemic cells more sensitive to RSL3, suggesting the protective function of NO. This data is in agreement with other studies showing that NOS activity is important in leukemia cell survival [In a very elegant approach, Soula et al. showed that BH4 abrogates lipid peroxidation–induced ferroptosis caused by glutathione peroxidase 4 (GPX4) inhibition . Using mroblasts . It was decrease . Moreovesurvival ,55.PTS silencing impaired LTBP nitrosylation and cell proliferation, showing the involvement of the BH4/NO pathway in colorectal cancer progression [In the early stages of colorectal cancers, PTPS was found to be elevated. Under hypoxia, a common characteristic of aggressive cancers, AMPK-induced PTPS phosphorylation, which in turn promotes LTBP (Latent TGF-β binding protein)-PTPS-iNOS interaction leading to LTBP nitrosylation and ubiquitin-dependent degradation. In this condition, TGF-β secretion is increased contributing to colorectal tumor growth. gression . The appgression , contribHeterogeneous cell types, including fibroblasts, endothelial, and immune cells, along with extracellular matrix components comprise the tumor microenvironment (TME). Through the secretion of various factors, cancer cells can reprogram and control the function of the surrounding environment, contributing to biological processes, such as angiogenesis .Angiogenesis is a physiological process consisting of the growth of new blood vessels from pre-existing ones, providing oxygen and nutrient supplies. In cancer, pathological angiogenesis contributes to tumor development, increasing proliferation, growth, and metastasis ,101. AngGCHI expression and L-sepiapterin or BH4 supplementation augmented NO production, cell proliferation, migration, and the capacity of endothelial cells of different species to form capillary-like structures through PI3K/AKT signaling pathway activation [Ser473 and eNOSSei1177 phosphorylation and NO production. Increased cell proliferation, migration, and tube formation required PI3K signaling since LY treatment, a PI3K signaling inhibitor, abrogated it. Furthermore, Ras activation and PI3K/Akt/eNOS up-regulation were impaired by L-NAME, suggesting a positive feedback mechanism. It was shown that NO caused S-nitrosylation (SNO) of p21Ras in Cys118, triggering the activation of ERK/MAPK signaling pathway and the proliferation of innumerous cells as neural stem cells, breast cancer, and endothelial cells [Experiments in vitro demonstrated that increased tivation ,106,108.al cells ,110,111.GCHI expression in vitro impaired angiogenesis through abrogation of AKT activation, eNOS phosphorylation, NO production, and consequently reduction of endothelial cell proliferation, migration, and tubulogenesis [On the other hand, the treatment with 2,4-diamino-6-hydroxypyrimidine (DAHP), a GTPCHI inhibitor, and decreased ogenesis ,62,112. ogenesis .GCH1 was cloned into a plasmid that contains tetracycline responsive element under cytomegalovirus promoter control. In the presence of doxycycline (DOX), GCH1 expression is abrogated. Mice with tumors around 100 mm3 in size (25th day after implantation) were then fed with DOX in drinking water to inhibit GCH1 expression or injected with DAHP (300 mg/kg/day) for 7 days. Although angiogenesis was reduced, as showed by decreased CD34-positive microvessels, tumor growth was not inhibited [Many authors evaluate the participation of BH4 in angiogenesis in vivo. To analyze the proangiogenic potential of BH4 in tumor stromal fibroblasts, BALB/c SCID mice were implanted with a construction where the gene nhibited . Interesnhibited ,116,117.3 in size inhibited AKT/eNOS pathway activation and decreased BH4 and NO concentration in hepatocellular carcinoma tissue. Hence, CD31 staining was significantly lower in DAHP-treated mice showing impairment of angiogenesis and tumor growth [On the other hand, Dai et al. observed that DAHP treatment (80 mg/kg/day) for two weeks once tumors reached 100 mmr growth . Some far growth . Finallyr growth .2•− amount or NOS expression; therefore, the relationship between BH4 and enzyme activity was not discussed. However, studies have indicated that NO can induce Ang1/Tie2 interaction [CAFs are found in the tumor microenvironmental surrounding the malignant lesion and, through dysregulated cell signaling and communication with tumor cells, contribute to malignant transformation, proliferation, metabolic program, and metastasis . Furthereraction ,123. Zaceraction ,125, BH4eraction . Therefolike phenotype were infiltrating tumor microenvironmental. These data are in accordance with the studies showing that macrophages polarized towards an anti-inflammatory M2 phenotype instead of M1-like phenotype are involved in high angiogenic activity [Showing that the anti-angiogenic and consequently anti-tumor role of DAHP is time-dependent, Pickert et al. showed that the administration of DAHP (100 mg/kg) twice daily orally immediately after tumor cells inoculation reduced the formation of blood vessels as indicated by decreased CD31 and αvβ3 integrin staining . Importaactivity ,128. Howactivity . TherefoImportant components of the TME in various types of cancer, TAMs exhibit a wide spectrum of phenotypic and functional profiles regulated by different signals of the surrounding environment ,131. TheGch1 specifically for these cells, there was a reduction in BH4 levels accompanied by a decrease in the proliferation of mature T cells induced via TCR (T cell receptors). A similar result was shown by inhibiting sepiapterin reductase through the SPRi3 inhibitor. On the other hand, the increased BH4 levels by GCH1 overexpression or L-sep/BH4 treatment enhanced the proliferation of stimulated CD4+ and CD8+ T cells. Interestingly, on the orthotopic breast cancer model (E0771 cells), the tumor growth was rejected in GCH1-overexpressing mice. Consistently, BH4 supplementation  decreased tumor growth and increased tumor-infiltrating CD4+ and CD8+ T cells. Furthermore, stimulated BH4-deficient T cells showed decreased iron levels and reduced mitochondrial respiration and oxygen consumption [c as a mechanism for the effects of BH4 deficiency in TCR-activated T cells. However, the antioxidant activity of BH4 is controversial [Gch1-ablated T cells do not show a difference in nitrite levels compared to control cells does not completely exclude the participation of NOS, since the enzyme in stimulated T cells may be in a state of partial uncoupling, that is, producing NO and O2•− [2•− levels were found in stimulated T cells, which were potentiated in BH4-deficient cells [Recently, the effects of BH4 on T cells and its role in breast cancer were also investigated. Although the normal development of T cells in mice knocked to sumption . Thus, toversial  and its oversial ,143. In and O2•− . Consistnt cells .The fact that hypoxia induces GTPCH1 expression and BH4 synthesis corroborate their critical role in angiogenesis . ReducedAngiogenic vasculature structure found in solid tumors is poorly organized, leading to heterogeneous blood flow, increased interstitial pressure, and intermittent hypoxia contributing to radioresistance and the low ratio of drug delivery . One of Metastasis is a complex process comprising a sequence of steps that begins with local invasion (migration and extracellular matrix degradation), then intravasation into lymphatic and blood vessels, survival in hematogenous and lymphatic systems, extravasation into the parenchyma of distant tissues, and finally the formation and growth of small niches of cells, the metastatic lesions ,150.2O2 levels and downregulated MMP-2 and MMP-9 transcripts, inhibiting the invasive potential of the cells, which was reversed in the presence of a catalase inhibitor [2O2 levels reduction and the anti-invasive effect of fluvastatin in these cells [Kanugula et al. showed that statin treatment reduced invasion and induced apoptosis of triple-negative breast cancer cells, by iNOS-mediated NO production . Statinsnhibitor . Since snhibitor ,154,155,se cells . Althougse cells ,157,158.2•− synthesis at the same time.In ovarian cancer cells, sepiapterin abrogated the increase in migration and proliferation triggered by vascular endothelial growth factor-A (VEGF-A) and p70S6K-dependent VEGRF2 expression in a NO-independent mechanism. Surprisingly, sepiapterin increased migration and proliferation of ovarian cancer cells in the absence of growth factors, which is mediated by a NO-dependent activation of ERK, AKT, and p70S6K signaling . Since Vanoikis. This type of apoptosis is induced by loss of attachment to extracellular matrix or inappropriate cell-matrix interactions, preventing cell adhesion to inadequate locations [anoikis-resistance of melanocytes submitted to anchorage impediment [2•− and reduced NO levels in these cells. Furthermore, sepiapterin and Mn(III)TBAP (a superoxide scavenger) were able to decrease melanocyte and metastatic melanoma cell survival in suspension, but not in adhesion [2•− production and decreased NO levels compared to parental melanocytes. In addition, in vivo analysis showed that the treatment with L-NAME before and during sequential cycles of anchorage blockade impairs melanocyte malignant transformation [Some important acquisitions of cancer cells during tumor progression and metastatic colonization are the ability of anchorage-independent growth and resistance to a programmed cell death known as ocations ,166. Campediment . Superoxadhesion . In factadhesion . The conadhesion . Cell liormation . Taken tAltered signaling pathways are associated with malignant transformation and the acquisition of an aggressiveness phenotype. The expression, activity, and cell localization of these transduction signals are regulated by innumerous factors, including ROS and NO-induced post-translational modifications. Different studies have shown that ROS and NO amount and the balance between these reactive species contribute to tumor growth, chemoresistance, and metastasis. Moreover, the contribution of ROS and RNS from tumor microenvironmental in cancer development has to be considered. Since NOS can be a source of both ROS and NO, understanding its activity is essential to improve cancer management. Therefore, the dual role of the BH4/NOS pathway in carcinogenesis can be explained by the status of transduction pathways found in distinct cancers .


Colon adenocarcinoma (COAD) is a malignancy with a high incidence and is associated with poor quality of life. Dysfunction of circadian clock genes and disruption of normal rhythms are associated with the occurrence and progression of many cancer types. However, studies that systematically describe the prognostic value and immune-related functions of circadian clock genes in COAD are lacking.  Genomic data obtained from The Cancer Genome Atlas (TCGA) database was analyzed for expression level, mutation status, potential biological functions, and prognostic performance of core circadian clock genes in COAD. Their correlations with immune infiltration and TMB/MSI score were analyzed by Spearman's correlation analysis. Pearson's correlation analysis was performed to analyze their associations with drug sensitivity. Lasso Cox regression analysis was performed to construct a prognosis signature. Moreover, an mRNA-miRNA-lncRNA regulatory axis was also detected by ceRNA network. CLOCK, CRY1, and NR1D1 were increased, while the mRNA levels of ARNTL, CRY2, PER1, PER3, and RORA were decreased. We also summarized the relative genetic mutation variation landscape. GO and KEGG pathway analyses demonstrated that these circadian clock genes were primarily correlated with the regulation of circadian rhythms and glucocorticoid receptor signaling pathways. COAD patients with high CRY2, NR1D1, and PER2 expression had worse prognosis. A prognostic model constructed based on the 9 core circadian clock genes predicted the COAD patients' overall survival with medium to high accuracy. A significant association between prognostic circadian clock genes and immune cell infiltration was found. Moreover, the lncRNA KCNQ1OT1/hsa-miRNA-32-5p/PER2/CRY2 regulatory axis in COAD was also detected through a mRNA-miRNA-lncRNA network.  In COAD tissues, the mRNA levels of CRY2, NR1D1, and PER2 as potential prognostic biomarkers for COAD patients and correlated their expression with immune cell infiltration. The lncRNA KCNQ1OT1/hsa-miRNA-32-5p/PER2/CRY2 regulatory axis was detected in COAD and might play a vital role in the occurrence and progression of COAD. Our results identified  Colon adenocarcinoma (COAD) has become a severe health burden that causes over half a million cancer-related deaths annually . COAD isCLOCK, ARNTL, PER1, PER2, PER3, CRY1, CRY2, NR1D1, and RORA [RORγ synthetic agonists and RORα synthetic agonists, could promote the activation of anticancer immunity [Physiological circadian rhythms are vital for various biological functions, such as metabolism, regeneration, immunology, and endocrinology . All humand RORA , 7. In rand RORA –11. For and RORA . Some stimmunity , 14. NevWith the rapid development of bioinformatics databases and the enrichment of genomic data, comprehensive analysis of the role of the circadian clock in COAD has become feasible. The present study explores the expression profiles, prognostic value, and related regulatory axis of the circadian clock in COAD, which may offer novel insights into prognostic biomarkers and biological clock targets for COAD.N = 455) was selected to perform the analyses. The corresponding clinical data, mainly including sex, age, tumor grade, and survival data, were also obtained from TCGA database on May 20, 2021. Furthermore, the copy number variation (CNV) and somatic mutation data in COAD were also extracted from TCGA database using UCSC Xena (https://xena.ucsc.edu/), an easy-to-use web portal for data files derived from TCGA. Setting the P value cutoff as 0.05, statistical analyses were conducted using R software V4.0.3. Before subsequent processing, the expression data were standardized to transcripts per kilobase million (TPM). To further analyze the expression of circadian clock genes, another gene expression profile of COAD was obtained from Oncomine (https://www.oncomine.org) [On May 20, 2021, COAD patient genomic data were downloaded from The Cancer Genome Atlas (TCGA) database. TCGA-COAD dataset (ine.org) , a canceCLOCK, ARNTL, PER1, PER2, PER3, CRY1, CRY2, NR1D1, and RORA [https://circadb.hogeneschlab.org) [x-axis represented the CYCLOPS-estimated sample phase in radians (from 0 to 2𝝿). The y-axis represented the expression level (TPM). The time 𝝿 represented E-box phase . The datasets concerning the expression levels of the 9 core circadian clock genes were also excavated from the Oncomine (https://www.oncomine.org) database [t-test was utilized to compare the transcriptional levels of the core circadian clock genes in COAD tissues with those in normal colon tissues using the “reshape2” and “limma” packages in R software.A total of 9 genes were selected to perform further analysis, which included and RORA , 7. The lab.org) . For eaclab.org) . In the database . To furthttps://string-db.org/), a database of predicted functional associations between genes and proteins [CLOCK, ARNTL, PER1, PER2, PER3, CRY1, CRY2, NR1D1, and RORA as well as their waterfall plot, were constructed using the “maftools” package. The locations of variants of the 9 genes on 23 chromosomes were shown using the “RCircos” package.To study the correlation between each member of the 9 core circadian clock genes, we constructed a PPI network with the Search Tool for the Retrieval of Interacting Genes  analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, was performed using the “ggplot2” package in R software. In addition, GO analysis included biological process (BP), cellular component (CC), and molecular function (MF) analyses.To assess the potential prognostic value of core circadian clock genes, Kaplan–Meier plots, including overall survival (OS) plots, disease-free survival (DFS) plots, disease-specific survival (DSS) plots, and progression-free survival (PFS) plots, were drawn. The hazard ratio (HR) and its 95% confidence interval (CI) are also listed in the plots. Based on the 9 core circadian clock genes, a LASSO Cox regression model was used to predict the influence of the expression of core circadian clock genes on the prognosis of COAD patients. According to the median risk score, patients were divided into the low- and high-risk groups, and the OS curves of the two groups were compared. The predictive accuracy of this prognostic model was assessed by time ROC analysis. Moreover, core circadian clock genes with potential prognostic value were selected for further analyses.https://cistrome.shinyapps.io/timer/) is a bioinformatics platform that is aimed at visualizing the association between immune cell infiltration and target gene expression in cancer cells [P value cutoff of 0.05. Moreover, to study the effect of prognostic circadian clock gene expression on drug resistance, drug sensitivity data in the Genomics of Drug Sensitivity in Cancer (GDSC) database were extracted.TIMER (er cells . The “Gehttp://mirtarbase.mbc.nctu.edu.tw/) [https://starbase.sysu.edu.cn/index.php) [https://starbase.sysu.edu.cn/index.php) and lncBase (https://diana.e-ce.uth.gr/lncbasev3) [An mRNA-miRNA-lncRNA network was constructed to determine the potential regulatory axis of prognostic circadian clock genes in COAD. The miRNA targets binding to prognostic circadian clock genes were identified by the miRTarBase (edu.tw/)  and stardex.php)  databasecbasev3) , and theWork flow of the present study is shown in CRY1 . At the transcriptional level, these core circadian clock genes were greatly altered in COAD tissues. Moreover, a protein–protein interaction (PPI) network was constructed to illustrate the associations among the 9 core circadian clock genes  and copy number variations (CNVs) of the 9 circadian clock genes in COAD. As shown in Figures To clarify the potential biological functions and molecular mechanisms of core circadian clock genes in COAD, GO and KEGG pathway analyses were performed . GO analP values and their HRs with 95% CIs are shown in CRY2 expression showed worse OS ), DFS ), DSS ), and PFS ). COAD patients with high NR1D1 expression showed worse DFS ) and PFS ). In addition, COAD patients with high PER2 expression showed worse OS ). The above results demonstrated that CRY2, NR1D1 and PER2 might be of great significance in the prognostic evaluation of COAD patients. Moreover, a prognostic model including the 9 core circadian clock genes was constructed by Lasso Cox regression analysis (CLOCK + (0.4144)∗CRY2 + (0.0934)∗NR1D1. According to the risk score, patients were divided into the high- and low-risk groups. The score distribution, survival status, and expression levels of PER2, NR1D1, and CLOCK are shown in P value = 0.015, Kaplan–Meier plots, including OS, DFS, PFS, and DSS plots, were used to assess the potential prognostic value of the 9 core circadian clock genes. The worse OS  ), DFS  ), DSS  , macrophages , neutrophils , and dendritic cells . The expression level of PER2 was positively associated with the infiltration levels of B cells , CD8+ T cells , CD4+ T cells , macrophages , neutrophils , and dendritic cells   and negatively associated with the infiltration levels of B cells  and CD8+ T cells   . In addi6E − 02) . We wereCRY2, NR1D1, and PER2 in COAD immunotherapy. In TMB analysis, negative correlations were detected between TMB and CRY2 expression  and NR1D1 expression , while no significant correlation was found between TMB and PER2 expression . In MSI analysis, unexpectedly, no significant associations between MSI and CRY2 expression , NR1D1 expression , or PER2 expression  were detected. Next, drug sensitivity analysis was performed to detect the potential functions of prognostic circadian clock genes in drug screening. Based on data from the GDSC database, high NR1D1 expression in COAD tissues was mainly correlated with drug resistance to 38 kinds of drugs and drug sensitivity to 6 kinds of drugs. High PER2 expression was primarily associated with drug sensitivity in 58 kinds of drugs and drug resistance in 13 kinds of drugs, while high CRY2 expression was mainly associated with drug sensitivity in 60 kinds of drugs and drug resistance in 18 kinds of drugs  and microsatellite instability (MSI) are of great significance in predicting the efficacy of cancer immunotherapy. The above results demonstrated that the prognostic circadian clock genes were associated with immune cell infiltration. Therefore, we conducted TMB and MSI analyses to assess the potential clinical applications of of drugs , indicatCRY2 , NR1D1 , and PER2  were associated with the clinical stage of COAD patients, which demonstrated that these 3 genes might be involved in the development of COAD. However, for potential applications in drug screening and prognostic performance, PER2 and CRY2 were selected to construct an mRNA-miRNA-lncRNA network. The miRNAs targeting PER2 and CRY2 identified by starBase and miRTarBase are shown in hsa-miRNA-32-5p, hsa-miRNA-340-5p, hsa-miRNA-20b-5p, and hsa-let-7b-5p were identified as the top highly connected miRNAs  mice [RORγ synthetic agonists could increase the expression levels of IL-17A, IL-17F, GM-CSF, and IL-22, thus boosting Th17 cells and Tregs to block immunosuppression [BMAL1, CLOCK, Rev-Erbα, and PER2 in intraperitoneal macrophages regulated the expression levels of F4/80 and CD11c in tumor tissues [Tumor-infiltrating immune cells play pivotal roles in eliminating cancer cells and hindering cancer progression. Exploring the association between core circadian clock genes and tumor-infiltrating immune cells is of significant importance. A previous study concluded the role of the circadian clock and its core genes in cancer immunity . Xu et ack genes . Cao et ut) mice . Howeverpression . The exp tissues . HoweverPER2 was an integral component of the glucocorticoid regulatory pathway and was directly regulated by the glucocorticoid receptor [CLOCK signaling contributed to mineralocorticoid receptor-mediated cardiac inflammation and fibrosis [GO and KEGG pathway analyses could predict the biological functions and molecular mechanisms of the target gene set. In our study, the main functions and mechanisms of the 9 core circadian clock genes were involved in the regulation of circadian rhythms and some signaling pathways. The corticosteroid receptor signaling pathway includes the glucocorticoid receptor signaling pathway and mineralocorticoid receptor signaling pathway. In a mouse model, So et al. identified glucocorticoid response elements in the molecular circadian clock and found that receptor . Dickmeireceptor . For thefibrosis . The aboKCNQ1OT1/hsa-miRNA-32-5p/PER2/CRY2 regulatory axis was identified. Previous studies have reported that miRNA-32-5p regulates the progression of gastric cancer and epithelial-mesenchymal transition and metastasis in lung adenocarcinoma [miRNA-32-5p in COAD are lacking. The upstream lncRNA KCNQ1OT1 was also detected and is considered a potential diagnostic biomarker for colon and rectal cancer and enhanced drug resistance in colon cancer cells [miRNA-32-5p and lncRNA KCNQ1OT1 and found that they were correlated with COAD patient prognosis. The above findings revealed that the lncRNA KCNQ1OT1/hsa-miRNA-32-5p/PER2/CRY2 regulatory axis might be of significant importance in the occurrence and progression of COAD and may have potential applications in the diagnosis and treatment of COAD. Nevertheless, in vivo and vitro studies are urgently needed to clarify the role of this regulatory axis in COAD.Through mRNA-miRNA-lncRNA network construction, the lncRNA arcinoma , 44. Hower cells , 46. In Some limitations still exist in the current study. First, our study and analysis were mainly performed at the transcriptional level, and the results may not be applicable in studies based on the protein level. Second, related fundamental and clinical studies that focus on the molecular mechanisms of circadian clock genes in colon or rectal cancer are rare. Third, the genetic background and etiology of COAD patients are influenced by many factors, such as patient race, sex, and age. Thus, more in-depth studies are necessary to validate the role of circadian clock genes in COAD.CRY2, NR1D1, and PER2. The lncRNA KCNQ1OT1/hsa-miRNA-32-5p/PER2/CRY2 regulatory axis was identified and might be of great significance in the occurrence and progression of COAD.In this study, a comprehensive bioinformatics analysis was used to identify the prognostic circadian clock gene signature in COAD, which included