Fragilariopsis cylindrus in producing Fx and Ddx+Dtx by means of the manipulation of the growth light climate  and temperature. We further compared its best capacity to the strongest xanthophyll production levels reported for temperate counterparts grown under comparable conditions. In our hands, the best growing conditions for F. cylindrus were a semi-continuous growth at 7 °C and under a 12 h light:12 h dark photoperiod of monochromatic blue light (445 nm) at a PUR of 11.7 μmol photons m−2 s−1. This allowed the highest Fx productivity of 43.80 µg L−1 day−1 and the highest Fx yield of 7.53 µg Wh−1, more than two times higher than under ‘white’ light. For Ddx+Dtx, the highest productivity (4.55 µg L−1 day−1) was reached under the same conditions of ‘white light’ and at 0 °C. Our results show that F. cylindrus, and potentially other polar diatom strains, are very well suited for Fx and Ddx+Dtx production under conditions of low temperature and light intensity, reaching similar productivity levels as model temperate counterparts such as Phaeodactylum tricornutum. The present work supports the possibility of using polar diatoms as an efficient cold and low light-adapted bioresource for xanthophyll pigments, especially usable in Nordic countries.Carotenoid xanthophyll pigments are receiving growing interest in various industrial fields due to their broad and diverse bioactive and health beneficial properties. Fucoxanthin (Fx) and the inter-convertible couple diadinoxanthin–diatoxanthin (Ddx+Dtx) are acknowledged as some of the most promising xanthophylls; they are mainly synthesized by diatoms (Bacillariophyta). While temperate strains of diatoms have been widely investigated, recent years showed a growing interest in using polar strains, which are better adapted to the natural growth conditions of Nordic countries. The aim of the present study was to explore the potential of the polar diatom  Carotenoid xanthophyll pigments are receiving growing interest in various industrial fields such as the functional foods industry, nutraceuticals and cosmetics, etc. ,2,3,4, wFucoxanthin Fx,  and the −1) .,78.F. cyF. cylindrus to that of P. tricornutum under similar conditions  suggest species ,42,78,94 species . The mos species ,46,91; a−2 s−1 with a 12 h light:12 h dark photoperiod appeared to be the sweet spot for F. cylindrus growth at 0 °C. Additionally, in the context of indoor growth and/or light supplemented cultures, the use of monochromatic lights solely or mixed proved to be efficient in increasing Fx production [F. cylindrus. Obviously, the sea ice and underneath ice light spectra, which show a very specific blue:red ratio depending on ice type [The light dose needs to be adjusted for the best balance between a fast growth rate (supported by a strong light dose up to a certain limit to avoid harmful excess) and a high Fx content (supported by a low light dose), which can be achieved by adjusting the photoperiod and intensity. Due to the high latitude and low-light natural habitat resulting from the snow and ice cover over the water column, most polar strains are adapted to a large range of photoperiods but to a limited range of intensities: this factor thus needs to be carefully adjusted. In our study, a PUR of 11.7 μmol photons moduction ,42,95,96oduction ,42,78,95ice type , will beF. cylindrus, independent of the temperature. Medium enrichment is a common way of increasing Fx production [2 enrichment of the cultures [The unselective enrichment of the culture medium f > f/2,  additionoduction  (by up toduction ), eitheroduction ,76,93,98oduction , silicatcultures ,99. Addicultures ,101,102)F. cylindrus could be further improved by means of genetic engineering [P. tricornutum [F. cylindrus based on the wealth of genomic, transcriptomic and physiological data already available [T. gravida and C. neogracilis produce and accumulate high amounts of Fx and Ddx+Dtx  and humidity was permanently controlled to avoid condensation (Dew point below −13 °C). Culturing and experiments were performed in axenic conditions with sterile and/or autoclaved equipment handled under a laminar flow hood inside the ‘cold’ laboratory to avoid contamination. Axenic ylindrus . Photosys growth . The ligF. cylindrus, by exploring the effects of varying light spectrum, intensity and temperature, and (2) a second series to compare F. cylindrus Fx productivity with the one of diatom temperate counterparts.All samplings and measurements were performed in the ‘cold’ laboratory under a green light of low intensity to limit the alteration of cell photosynthetic activity and pigments due to ambient light. We designed two distinct experiments: (1) a first series of experiments to define the best growing conditions for fucoxanthin (Fx) productivity in F. cylindrus cultures were grown in f/2 medium, at 0 °C and 7 °C under different photoperiods, PAR intensities and spectra  as in Wang et al. [F. cylindrus cultures were inoculated with 106 cells mL−1 and grown in batch mode in f/2 and f media, at 0 °C and 7 °C, and under a ‘white’ light spectrum of 30 µmol photons m−2 s−1 PAR. Here, the light intensity was set after the inoculation of the culture and left unchanged throughout the experiment . Sampling was performed every two days for cell counting, and the analysis of elemental composition and pigments.In order to directly compare the potential of g et al. . F. cyli−2 s−1), which is the part of PAR that is effectively absorbed by F. cylindrus cells and which varies depending upon their pigment composition. For determining F. cylindrus PUR, its absorption spectrum was measured using a double beam spectrophotometer  equipped with an integrative sphere of 15 cm in diameter , and following the procedure described by Babin and Stramski [F. cylindrus culture sample. From this measurement, the in vivo specific absorption spectrum of ph(λ)F. cylindrus a ((mg chla m−3)−1) was calculated as: l = 0.01 m. Finally, the determination of PUR was carried out as follows (Equation (1)) [A(λ) is ph(λ)a normalized to its maximum (A(λ) ≡ 1, λ = 440 nm) and 𝐸(λ) is the emission spectrum of the light source.Differing from PAR, PUR is the photosynthetically useable radiation ) :(1)PUR=∫F. cylindrus . The growth rate  was calculated according to Andersen [Cell concentration was measured using a particle sizing and counting analyser . Analyses were performed for cells between 2 and 15 μm in size. A dilution of the sample was performed before analysis in order to remain within the cell concentration measurement range of the analyser. Dilution was carried out with the electrolyte of the analyser, i.e., salted Milli-Q™ water . The CHN analyses were performed with a PerkinElmer 2400 Series II CHNS/O elemental analyser , and acetanilide was used as a standard.The total particulate carbon (TPC), total particulate nitrogen (TPN), and the dry algal biomass were determined by filtering culture samples on pre-burned (450 °C for 4 h) 25 mm GF/F glass-fibre filters . To remove the seawater salt from the filters without inducing an osmotic stress for F. cylindrus cultures were filtered on 25 mm GF/F glass-fibre filters  and immediately frozen in liquid nitrogen and stored at −80 °C until further analysis. Samples were extracted in 3 mL of extraction buffer  as internal standard) stored at −20 °C by grounding the filters by means of sonication (15 s followed by a 1 h storage at −20 °C). After centrifugation , the supernatants were filtered through 25 mm GA55 glass-fibre filters . Then, 700 μL of the filtered supernatants was transferred to chromatography vials complemented with 300 μL of a tetrabutylammonium acetate (TBAA) solution (28 mM) for polarization. A final volume of 50 μL was injected into the HPLC system. Separation was achieved with a gradient between a solution (A) of TBAA 28 mM:methanol 100%  and a solution (B) of 100% methanol according to Ras et al. [a (Chl a) at 640 nm. Pigment quantification was carried out using pigment standards provided by D.H.I. Water & Environment  and the internal standard (vitamin E acetate).Pigments were quantified by high-performance liquid chromatography  equipped with a C8 reverse-phase column , following the extraction and analytical procedure of Ras et al. . The ents et al. . FucoxanF. cylindrus cells on dark-acclimated (30 min) samples. Rapid light curves (RLCs) were performed with 12 steps of 30 s and of increasing light intensity from 0 to 611 µmol photons m−2 s−1. The dark-acclimated photochemical efficiency of photosystem II-PS II (FV/FM) was calculated as in Equation (2):0 and FM are, respectively, the minimum and maximum levels of dark-acclimated chlorophyll fluorescence. The relative electron transport rate (rETR) was calculated as in Equation (3):M′ are, respectively, the actual and maximum levels of light acclimated chlorophyll fluorescence. The determination of rETR for each of the 12 intensities of the RLCs allowed us to build rETR vs. E curves that were fitted according to Eilers and Peeters [max (the maximum relative electron transport rate), α (the maximum light efficiency use), Ek  and Eopt .A blue light source (λ = 460 ± 20 nm) pulse amplitude modulated (PAM) fluorometer  was used to measure the photosynthetic performance of  Peeters  in order Peeters ): rETRmaThe output of the RLCs also allowed us to calculate the non-photochemical quenching (NPQ) as in Equation (4):max) was the maximal value obtained for the highest RLC step (611 µmol photons m−2 s−1), and the non-photochemical quenching at growing PUR intensity (NPQgE) was the NPQ obtained for the RLC step with the closest PUR intensity to the growing PUR intensity available.The maximal NPQ (NPQPSII (NPQ (NO (M′ were measured during RLC at the light step with the closest PUR intensity to the growing PUR intensity.The partitioning of absorbed excitation energy in photosystem II (PSII) was determined by the complementary PSII quantum yields method ,111, wherescence ,111,112.F. cylindrus physiology and productivity (A 1-way ANOVA followed by Tukey’s HSD post hoc test was used to test differences in the measured parameters’ means between treatments see . A 3-wayuctivity .Fragilariopsis cylindrus that show even stronger potential in fucoxanthin production, such as Chaetoceros neogracile, could also be used. We have furthermore demonstrated that, beyond fucoxanthin, polar diatom biomass valorisation can include other bioactive xanthophyll pigments of interest, namely diadinoxanthin+diatoxanthin [The present work supports the possibility of using polar diatoms as an efficient cold and low light-adapted bioresource for xanthophyll pigments, especially usable in Nordic countries to develop new economics based on marine resources ,23. Beyooxanthin . Since doxanthin ,90,91,96oxanthin ,114, poloxanthin .


N = 74). We found that relationship length was associated with sexual consent and couples who accurately perceived each other’s consent communication cues reported elevated levels of internal consent feelings. Communicating willingness to engage in sexual activity remains important even within committed relationships. Preliminary findings suggest that further investigations of dyadic nuances of sexual consent are warranted.Sexual consent is a multidimensional construct that requires the participation of all involved in a sexual encounter; however, previous research has almost exclusively relied on one person’s perspective. To address this, we collected open- and closed-ended data on sexual consent from 37 dyads in committed sexual relationships ( Sexual consent refers to people’s willingness to engage in partnered sexual activity. Typically, one person initiates or requests a sexual behavior, and another person responds based on their willingness to take part in that sexual behavior  on people’s sexual consent communication  to participate in this study. Data from all 37 dyads were included in our assessment of participants’ typical consent communication. However, eight dyads were excluded from the event-level analytic sample for not referencing the same partnered sexual event when responding to items in the survey; another one dyad was excluded because one participant in the dyad did not respond to key constructs. Thus, the event-level analytic sample included 28 dyads (n = 56). We recruited 37 dyads .To assess internal feelings of sexual consent, we administered the Internal Consent Scale , safety/comfort , arousal , agreement/want , and readiness . The ICS demonstrated strong internal reliability across these factors in the present study (αs ≥ .86) as well as its validation study . We provided a list of 12 relationship-based contextual consent cues based on previous research  as a measure of effect size for each of the chi-squared tests. A φC-value of .10 indicates a small effect size, .30 medium, and .50 large , nonverbal cues , explicit cues , and implicit cues . Reflecting participants’ use of words to communicate their consent, responses coded as verbal cues included “We ask if the other one wants to have sex” and “One of us might say ‘you look hot’ or something to that effect.” Regarding the use of behaviors or actions as consent indicators, nonverbal cues were represented by responses like “We usually let each other know nonverbally that one of us wants to have sex” and “We may start by rubbing the other person first.” Responses also received codes if they were explicit , such as “Using direct expressions we will ask if the other person wants to or not” and “Sometimes we do explicitly ask verbally.” Conversely, responses coded as implicit  included statements like “She usually begins to gently scratch my back” and “If he’s interested, he will usually stay in bed in the morning.”The first set of codes represents certain types of consent cues people described using. Specifically, participants indicated that they and their partner typically consent to sexual activity using verbal cues , the sexual behavior just happening , and the presence or absence of refusals . Responses coded for behaviors building toward a sexual act included “Sometimes kissing just leads on to another thing” and “We touch each other excessively, and that leads to sex.” Regarding sexual behavior occurring apparently spontaneously, participants made statements like “It usually just happens” and “Sometimes we just end up having sex.” The presence or absence of refusals captured instances in which participants report they infer consent because neither partner has communicated that they were unwilling: “If the other is not in the mood, they will say so” or “We tell each other when we are too tired to.”In addition to the specific types of consent cues listed above, some participants described the process—or lack thereof—regarding how they and their partner typically consent to sexual activity. Relevant aspects of how people experience sexual consent included the escalation of behaviors  referred to how they communicate their own willingness with phrases like “I’ll usually ask if she would potentially be in the mood” and “I perform oral sex on her.” Other participants  indicated consent cues they perceive their partner uses by making statements such as “He will start gently touching to indicate that he is thinking about sex” and “My partner will typically give me cues by cleaning up her room, lighting candles, and dressing up.” But most participants  described how they and their partner communicate their willingness together; for example, “We just start touching each other” and “As a couple we are more upfront than most people.”Finally, we coded the perspective participants used when describing how sexual consent is typically communicated in their relationship. Some participants  = 5.01, p = .025, φC = .26. Specifically, women (20.0%) more frequently than men (2.9%) endorsed that they or their partners would refuse a sexual advance if they were not willing, were not interested, or did not want to.As seen in 2(1) = 8.32, p = .040, φC = .34. Further, the youngest relationships were associated with indicating that sexual activity just happens: less than 1 year (25.0%), 1–3 years (5.0%), 3–5 years (4.5%), and greater than 5 years (0.0%), χ2(1) = 8.17, p = .043, φC = .33.Two of these themes varied by relationship length . Only 50Descriptive statistics for the event-level sexual consent variables measured in this study are presented in rs > .52, ps < .001). Further, many types of consent communication cues—self-reported or perceived—were associated with other types of cues. For example, perceiving a partner had used explicit verbal cues was negatively associated with perceiving they had used implicit nonverbal  or explicit nonverbal  cues. There were no significant associations between internal consent feelings and external consent communication—self-reported or perceived.Bivariate correlations between each of the sexual consent variables are presented in 2(1) = 3.62, p = .057, φC = .25. Further, men more frequently endorsed using implicit verbal cues (66.7%) than did women (41.4%), χ2(1) = 3.60, p = .058, φC = .25; more men also reported using no response cues (29.6%) during their most recent vaginal intercourse event than did women (10.3%), χ2(1) = 3.29, p = .070, φC = .24.Neither internal consent feelings nor external consent communication significantly varied by gender. However, three of the five types of consent cues demonstrated effect sizes worth noting given our restricted sample size. Proportionally more women (79.3%) than men (55.6%) reported that they used explicit verbal cues during their most recent vaginal intercourse event, χr = −.34, p = .011, and agreement/want, r = −.28, p = .038, during their most recent vaginal intercourse event. Relationship length was not associated with the active types of consent communication cues participants reported having used or perceived their partner had used. However, participants who had been in their committed relationship relatively longer were less likely to report having used no response cues during their most recent vaginal intercourse event, r = −.28, p = .036.Two of the internal consent feelings factors were negatively associated with relationship length. Participants who had been in their committed relationship longer reported lower levels of arousal, r = .41, p = .002.Participants overwhelmingly reported that they perceived their partner’s sexual consent during their most recent vaginal intercourse event by relying on certain context cues rather than communication cues alone . Of the Participants were slightly better than chance at accurately perceiving the types of sexual consent communication cues their partners self-reported using . Almost r = .30, p = .026) and agreement/want  during their most recent vaginal intercourse event. Further, participants whose partners accurately perceived that they had used explicit nonverbal cues reported greater levels of arousal  during this event. Even though there were no gender differences in self-reported or perceived cues, a participant’s use of no response cues was more accurately perceived by their partner if they were a woman than if they were a man . Conversely, relationship length was not associated with whether dyads accurately perceived each other’s use of any type of consent communication cue.Accuracy regarding sexual consent communication at the dyad-level was associated with a participant’s internal consent feelings. Participants who accurately perceived that their partner had used explicit verbal cues reported greater levels of safety/comfort , which can occur in committed relationships when the acquiescent partner wants to please the other to show them love or to stop their nagging for sex  to reduce memory biases inherent to sexual behavior data (Willis & Jozkowski, Finally, although sexual consent is often discussed in the context of sexual violence, we do not recommend extrapolating any conclusions about sexual violence based on the present data. Instead, we focused on implications regarding sexual consent within the context of consensual sexual encounters. Our declination to speculate on the implications for sexual violence should not be interpreted to mean that nonconsensual sexual activity does not occur within the context of committed sexual relationships—or is even less likely to occur. In fact, evidence consistently demonstrates that people are at even greater risk of experiencing sexual violence from intimate partners than they are from strangers or acquaintances (Peterson et al., Sexual consent is a multidimensional construct that requires the participation of at least two people. By collecting data from dyads in committed sexual relationships, we provided preliminary insights regarding sexual consent feelings, communication, and perceptions that, to our knowledge, did not exist in the extant body of literature on sexual consent. Of note, we were able to measure accuracy of consent perceptions and how that is associated with consent feelings. These exploratory data suggest that further dyadic research on sexual consent is warranted. Future studies should include larger and more diverse samples and employ more sophisticated research designs to better understand dyadic-level nuances of sexual consent.


When the expression of NOS2 in M1-polarized macrophages is induced, huge amounts of nitric oxide (•NO) are produced from arginine and molecular oxygen as the substrates. While anti-microbial action is the primary function of M1 macrophages, excessive activation may result in inflammation being aggravated. The reaction of •NO with superoxide produces peroxynitrite, which is highly toxic to cells. Alternatively, however, this reaction eliminates radial electrons and may occasionally alleviate subsequent radical-mediated damage. Reactions of •NO with lipid radicals terminates the radical chain reaction in lipid peroxidation, which leads to the suppression of ferroptosis. •NO is involved in the metabolic remodeling of M1 macrophages. Enzymes in the tricarboxylic acid (TCA) cycle, notably aconitase 2, as well as respiratory chain enzymes, are preferential targets of •NO derivatives. Ornithine, an alternate compound produced from arginine instead of citrulline and •NO, is recruited to synthesize polyamines. Itaconate, which is produced from the remodeled TCA cycle, and polyamines function as defense systems against overresponses of M1 macrophages in a feedback manner. Herein, we overview the protective aspects of •NO against radical species and the autoregulatory systems that are enabled by metabolic remodeling in M9-polarized macrophages. Endothelial cells play an essential role in acetylcholine-induced relaxation of the vasculature. Endothelial cell-derived relaxing factor (EDRF) refers to compounds that cause the relaxation of vascular smooth muscle cells. After the identification of nitric oxide (•NO) as an EDRF, nitric oxide synthase (NOS) was isolated and extensively characterized . SeveralCell-mediated immunity is achieved by cytotoxic T cells, natural killer (NK) cells, and macrophages. Upon stimulation by lipopolysaccharide (LPS) and inflammatory cytokines such as interferon-γ (IFN-γ), macrophages are polarized to the M1 subtype and protect against pathogens and parasites . NOS2 is2•−) that is then converted to other reactive oxygen species (ROS) [–), which is formed from •NO and O2•−, is a powerful oxidant and may be responsible for oxidative damage to surrounding tissues [NADPH oxidase (NOX), notably NOX2, is also activated in M1 macrophages and produces superoxide (Oes (ROS) . •NO rea tissues . While • tissues . In this review article, we focus on the beneficial aspects of •NO produced by NOS against oxidative insult caused by oxygen radicals under conditions of inflammation. We also discuss metabolic remodeling that enhances M1 macrophage function and simultaneously activates its autoregulatory system to normalize overresponses.NOS was the first enzyme to be identified that produces •NO using arginine as the nitrogen source . It is nThree NOS isoforms, namely the neuronal form (NOS1), the inducible form (NOS2), and the endothelial form (NOS3), produce •NO in response to corresponding physiological stimuli and exert a variety of functions to maintain health . •NO is In addition to NOS, other enzymes including xanthine oxidase, sulfate oxidase, and aldehyde oxidase convert nitrite to •NO . In red Heme and nonheme iron are both preferential targets of •NO ,18,19. EWhile carbon monoxide and oxygen molecule bind ferrous iron that is mostly present in the form of heme, •NO exceptionally binds ferric iron too . The lab2•− and ONOO– releases one ferrous iron unit along with hydrogen peroxide, which results in the formation of 3Fe-4S [−1 s−1), while ONOO− inactivates it much faster (~1.4 × 105 M−1 s−1) [1/2 = 12 min), the protein structure is converted into the active form with aconitase activity [•NO also preferentially reacts with the iron–sulfur (Fe-S) cluster that is commonly involved in electron transfer reactions. The Fe-S cluster is not as rigid as heme and is more sensitive to being destroyed by ROS/RNOS ,23. AmonM−1 s−1) ,28. The M−1 s−1) . This ROactivity . Regardiactivity  as descractivity .2•−, and cytokines. O2•− and •NO along with other ROS/RNOS act primarily as host defense systems against microbial infection but may also cause detrimental effects on surrounding tissues [2•− is then produced to kill microbes. NOS2 expression is induced in macrophages, which results in the production of huge amounts of •NO [2•− and •NO proceeds in a diffusion-limited manner (k = 1.9 × 1010 M−1s−1) [– is a strong oxidant [2•− possess a negative charge, which prevents it from permeating through lipid bilayers. While O2•− diffuses only for a short distance within cells, •NO readily diffuses through lipid bilayers and can function in intercellular signaling. Accordingly, ONOO− tends to be formed at the site where O2•− is produced, dominantly in mitochondria [Activated macrophages produce a variety of inflammatory mediators, which include •NO, O tissues ,34,35. Ns of •NO . The co- M−1s−1) , and the oxidant ,37. O2•−chondria . − formation is estimated to be 0.1 to 0.5 µM s−1 and its steady-state concentration is ~1 nM [− in macrophages is as high as 50–100 µM min–1 [− is unstable with a half-life of ~10 ms at physiological pH because protonated form ONOOH (pKa = 6.8) is immediately isomerized to nitrate in aqueous conditions. Based on these properties, the distance of diffusion is assumed to be ~10–20 µm inside cells. During the decomposition of ONOOH, hydroxyl radicals (•OH) and nitrogen dioxide radicals (•NO2) may be produced under a hydrophobic environment such as inside a membrane bilayer [− interacts rapidly with carbon dioxide (CO2) (k = 4.6 × 104 M−1 s−1) and is consequently converted to carbonate radical (CO3•−) + •NO2, which also exerts bactericidal effects in phagosomes, or CO2 + NO3− [7 M−1 s−1 at 25 °C, pH 7.4) [7 M−1 s−1 at 25 °C, pH 7.8) [−. Nevertheless, ONOO− is considered to be a major oxidizing RNOS in vivo situation [In vivo ONOOis ~1 nM . HoweverµM min–1 . ONOO− i bilayer . ONOO− i2 + NO3− ,43. Seve2 + NO3− , PRDX2 ( pH 7.4) ,46, and  pH 7.8)  may detoituation . 2), which are relatively stable and hence can be useful as biomarkers [Organic compounds experience two types of reaction with •NO, i.e., nitration and nitrosylation. Nitration results in the formation of nitro compounds  is an amino acid modification that occurs frequently and has attracted the attention of researchers [− is a major reactant in the formation of 3-NT. For example, tyrosine nitration has been observed in ACO2 in several animal models with inflammation such as sepsis and diabetes [Streptococcus from a cell [Among the RNOearchers . 3-NT isearchers , neuroloearchers , and diaearchers . ONOO− idiabetes . Two spediabetes . Nitratidiabetes ,56 and 8diabetes , may exediabetes . 8-nitrom a cell .E. coli [1SNO with the other thiol compound (R2SH) produces either R1SH + R2SNO by transnitrosation or R1SSR2 + HNO. Because of its ability to transfer NO to other thiols, SNO can act as a signaling molecule, which is well-recognized in cardiovascular systems [While the RSNO formation by •NO is rather efficient process, enzymes designated as SNO synthases may accelerate the formation of SNO as reported in E. coli . RSNO unE. coli . Homolyt systems . S-nitro systems . Because systems . GSNO is systems ,66. Prot systems ,68. Thro systems .S-Nitroso-CoA may mediate protein S-nitrosylation [S-Nitroso-CoA by the genetic ablation of the responsible gene AKR1A impairs glycolysis in the reaction catalyzed by pyruvate kinase 2, which results in an elevation in NADPH production via the stimulation of glucose-6-phosphate flow to the pentose phosphate pathway [Either suppression of •NO production or denitrosylation may be potential therapeutics for the treatment of certain diseases caused by nitrosative stress that are associated with excessive •NO production ,69. GSNOsylation . As a re pathway .2•− increases the risk of production of more harmful radicals such as hydroxyl radicals [2•− by •NO at an early stage may prevent oxidative injury by outweighing the toxicity of ONOO− [2•− and hydrogen peroxide compared to the levels in wildtype mice [2•− by •NO in vivo. We recently showed that both intrinsically produced •NO and long-lasting •NO-donor compounds exert protection against oxidative damages caused by O2•− in LPS-treated SOD1-knockout macrophages [−, low stability at physiological pH and the presence of reducing enzymes may collectively overcome the cytotoxic effects of ONOO− in macrophages. Thus, from a different point of view, ONOO− is not always deleterious but protective in activated macrophages and some other cells expressing NOS2. Because the sustained presence of Oradicals , the remof ONOO− ,75. Alveype mice , which irophages . Primary9 M−1s−1 [•NO also exhibits a high reactivity for lipid radicals (L• and LOO•). The reaction of •NO with such radicals is in fact quite rapid, with rate constants of 1–3 × 109 M−1s−1 , which r9 M−1s−1   is a direct executer of ferroptosis, a type of iron-dependent necrotic cell death ,85. Ferrived •NO . Thus, •In response to inflammatory stimuli such as IFN-γ and LPS, macrophages are polarized to the M1 type with remodeled metabolic pathways and an altered gene expression . In spitLeishmania donovani and Mycobacterium tuberculosis [The NOS2 expressed in M1 macrophages produces large amounts of •NO that appear to be primary protectors against microbial infection ,99. It irculosis ,100. Howrculosis . The prorculosis ,102,103.κB and IRF1 are the main transcriptional regulatory factors for the induction of NOS2 and, as a result, their inhibition suppresses NOS2 induction [κB via blocking its phosphorylation in rat vascular smooth muscle cells [N-glycosylation as a suppressor [Calcium ions are the principle activators for NOS1 and NOS3 via binding to calmodulin, but NOS2, which contains bound calmodulin, is constitutively active. Accordingly, the production of •NO by NOS2 is mainly regulated through gene expression and the degradation of the NOS2 protein by proteasomes ,105. NF-nduction . NOS2 isnduction . •NO itsle cells . The NOSle cells ,110. Thele cells , but datle cells ,112. Thele cells  and N-glppressor . N-terminal oxygenase domain and a C-terminal reductase domain. Tightly bound calmodulin links these domains, which enables electron transfer from NADPH to the substrate arginine [2•− from NOS2, which aggravates organ damage, as is presumed for cardiovascular dysfunction under sepsis [In addition to antimicrobial effects, excessive ROS and RNOS produced in M1 macrophages may impair the surrounding host tissue. NOS2 consists of two functional domains: an arginine . The prer sepsis . Macrophr sepsis ,117. The2•− and •NO are stimulated , which, along with carbamoyl phosphate synthetase I, constitutes primary process for the detoxification of ammonia. However, the expression of OTC is limited to only a few organs and is nearly negligible in immune organs such as the thymus and the spleen . Hence, m values of arginases are ~10 mM, which is approximately three orders higher than that of NOS2 (Km = ~5 µM) [Arginase (ARG) is encoded by two genes, ARG1 and ARG2, and catalyzes the conversion of arginine to the urea and ornithine. Although NOS2 and ARG are assumed to be competing for the utilization of arginine, both ARG1 and ARG2 are actually co-induced with NOS2 in the macrophages upon inflammatory stimuli ,148. Giv= ~5 µM) . Neverth= ~5 µM) ,151. TheThe induction of ARG1/ARG2, along with the lack of OTC, leads to the accumulation of ornithine in M1 macrophages upon inflammatory stimuli . In addiH. pylori infections, which is not attributed to the direct inhibition of NOS2 activity but, rather, the inhibition of translation from the mRNA [In the immune system, polyamines negatively regulate inflammatory responses ,158, whithe mRNA . On the the mRNA . A subsethe mRNA . Consistthe mRNA . Thus, iRegarding the remodeling of these metabolic pathways, we hypothesize that a novel metabolic linkage between the fragmented TCA cycle and incomplete urea cycle exists . Fumaratm for arginine allows for the continuous production of •NO by NOS2, even under low concentrations of arginine. This implies that the stimulated uptake of arginine through the induction of CAT2 supports the robust production of ornithine and urea by ARG. The increase in ornithine, which is accomplished by the induced ARG and isocyanic acid synthetase, provides an advantage for the production of polyamines and, hence, cellular defense against excessive immune responses under conditions of inflammation [H. pylori degrades urea to ammonia, thus allowing it to survive in gastric fluids by neutralizing the acidic environment, e.g., [The low Kammation ,163. Cont, e.g., ,165. How− resulting from •NO and O2•− is highly toxic; however, the beneficial action of scavenging O2•− by •NO may outweigh such disadvantage in certain situations. Lipid peroxidation is enhanced by radical chain reactions and is associated with cellular dysfunction, such as ferroptosis, and a variety of diseases. The elimination of lipid radicals by interactions with •NO interferes with this chain reaction, and hence suppresses ferroptosis. Thus, the radical nature of •NO may contribute to defense against oxidative insults via scavenging toxic radical species, despite the fact that some RNOS are largely deleterious. Investigation of •NO function in terms of this issue is just beginning. When a methodology for in vivo evaluation of ferroptosis is established, the importance of antioxidation through radical scavenging by •NO will become unveiled in inflammatory diseases.ONOOThe TCA cycle and urea cycle are remodeled in M1 macrophages under conditions of inflammation. There is crosstalk between these metabolic pathways. Itaconate and polyamines that are produced from the remodeled TCA cycle and urea cycle, respectively, function to autoregulate M1 macrophage functions and host defenses against microbial infections. Further clarification of this type of metabolic remodeling would lead to a better understanding of the overall function of M1 macrophages and controlling them in cases of inflammatory diseases. Although induction of NOS2 occurs in some other cells, its relationship with metabolic systems, which has been revealed in M1 macrophages, remains largely unknown. Further elucidation of the role of NOS2-derived •NO in relation to other metabolic systems in a wide range of cells will lead to a comprehensive understanding of redox systems in vivo.


Moreover, PTH (1–34) exhibited repair of cartilage and SCB, analgesic, and anti-inflammatory effects. In vitro studies (n = 11) concluded that PTH (1–34) was important for chondrocytes via increasing the proliferation and matrix synthesis but preventing apoptosis or hypertrophy. All included studies were assessed with low or unclear risk of bias in methodological quality. The SR demonstrated that PTH (1–34) could alleviate the progression of OA. Moreover, PTH (1–34) had beneficial effects on osteoporotic OA (OPOA) models, which might be a therapeutic option for OA and OPOA treatment.Osteoarthritis (OA) is a common and prevalent degenerative joint disease characterized by degradation of the articular cartilage. However, none of disease-modifying OA drugs is approved currently. Teriparatide (PTH (1–34)) might stimulate chondrocyte proliferation and cartilage regeneration via some uncertain mechanisms. Relevant therapies of PTH (1–34) on OA with such effects have recently gained increasing interest, but have not become widespread practice. Thus, we launch this systematic review (SR) to update the latest evidence accordingly. A comprehensive literature search was conducted in PubMed, Web of Science, MEDLINE, the Cochrane Library, and Embase from their inception to February 2022. Studies investigating the effects of the PTH (1–34) on OA were obtained. The quality assessment and descriptive summary were made of all included studies. Overall, 307 records were identified, and 33 studies were included. In vivo studies (The online version contains supplementary material available at 10.1186/s13075-022-02981-w. Osteoarthritis (OA) is a common musculoskeletal disorder and prevalent degenerative disease worldwide , 2. BothRecommendation of OA treatment includes physiotherapy, pharmacological, and surgical interventions , 7. PhysTeriparatide (PTH (1–34)) contains 34 amino acids of parathyroid hormone, which was applied on the treatment of osteoporosis (OP) and bone fracture , 10 by mWe performed this SR in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) statements . We recoA comprehensive literature search was conducted in 5 databases  from their inception to February 2022. The Medical Subject Headings (MeSH) terms and keywords were combined with boolean operators, “OR” or “AND”. The MeSH terms and keywords were as follows: “Teriparatide,” “hPTH (1–34),” “Human Parathyroid Hormone (1–34),” “Parathar,” “Teriparatide Acetate,” “Forteo,” “Osteoarthritis,” “Osteoarthritides,” “Osteoarthrosis,” “Osteoarthroses,” “Arthritis,” “Degenerative,” “Arthritides,” “Degenerative,” “Degenerative Arthritides,” “Degenerative Arthritis,” “Arthrosis,” “Arthroses,” “Osteoarthrosis Deformans.” In addition, the reference lists of all retrieved papers were further obtained manually. The search strategy of these five databases is provided in Additional file 1.The eligible studies should meet the following criteria: (1) prospective and retrospective studies, randomized and controlled clinical trials; (2) patients or animal models with OA treated by PTH (1–34) directly or indirectly; and (3) studies published in the English language. Studies were excluded from this review if they were reviews, research protocols, abstracts only, commentaries, or editorials.All records of five databases were imported into the reference management software program Endnote X 9.3.3. After the removal of duplicates, two authors (GQL and SL) independently reviewed the titles and abstracts of the remaining records for relevance to the topic. Studies that potentially or completely met the inclusion criteria were kept and full texts were retrieved. The two authors (GQL and SL) independently assessed the full texts to decide whether to keep the records or not. A consensus meeting with a third reviewer (FY) was used to resolve discrepancies. The final included studies were reviewed by all authors for agreement.The information of in vivo and in vitro studies was extracted in the standardized information forms: (1) first author’s surname, year of publication, and country; (2) subjects; (3) intervention; (4) dose and duration of treatment; (5) route; and (6) findings. Two investigators (GQL and SL) independently reviewed and extracted information from included studies. Disagreements were discussed with a third author (JW) to reach a consensus.The methodological quality of the in vivo studies was assessed by SYRCLE’s risk of bias tool  while thn = 35), Web of Science (n = 26), MEDLINE (n = 90), the Cochrane Library (n = 101), and Embase (n = 44) ; (2) research protocol (n = 3); (3) abstract only (n = 2); (4) commentaries or editorials (n = 4); and (5) subjects treated without PTH (1–34) (n = 5). In addition, 11 additional records were added. Finally, 33 papers were considered and included.The initial literature search resulted in 296 articles from PubMed  exhibited protective effects on both cartilage and SCB. Shao et al. concluded similar findings among collagenase-induced OA (CIOA) mouse models in a dose-dependent manner via the JAK2/STAT3 and WNT5A/ROR2 signaling pathway , 40. At PTH 1–34) would reduce the predisposing factors for OA progression. At the dose of 40 μg/kg/day, Cui et al. believed that PTH (1–34) reduced the accumulation of senescent cells in SCB by inhibiting p16 for age-related OA . In addi would rePTH (1–34) prevents cartilage damage and retards the deterioration of SCB. Yan et al. concluded that 15 μg/kg/day of PTH (1–34) protected the cartilage among guinea pigs . Dai et Different routines would differ the effect of PTH (1–34). Eswaramoorthy et al. found that controlled-release property of PTH (1–34) via intra-articular (IA) injection suppressed early stages of OA in papain-induced OA (PIOA) rats . Chen etBased on the inclusion criteria, 11 in vitro investigations were included in the SR Table . These sAs for the effects on human articular chondrocytes, PTH (1–34) influenced its differentiation and regeneration. Tsukazaki et al. concluded that PTHrP was an important autocrine and paracrine factor for chondrocyte metabolism as for cell growth and differentiation . RutgersPTH (1–34) protected MSC with various effects. Shao et al. maintained that PTH (1–34) worked on MSC by increasing the migration, proliferation, ECM formation, and inhibiting proinflammatory cytokines . Mwale en = 16); detection bias ; performance bias , attrition bias (n = 17), reporting bias (n = 21), and other bias (n = 15) were found.Methodological quality was assessed for all 33 involved studies  on OA regarding in vivo and in vitro investigations. The chondro-protective and cartilage-regenerative effects were reviewed, indicating that PTH (1–34) might be a potential preventative and therapeutic treatment for OA.OA is the most prevalent degenerative joint disease with complicated pathogenesis characterized with damage to cartilage, narrow synovial cavity, invasion of the SCB, formation of osteophytes, and synovitis . OP is aQuantitative studies indicated that PTH 1–34) played a significant role in calcium metabolism with an anabolic effect in the treatment of OP, fracture healing, non-union and stress fracture, augmentation of implant fixation, and chondro-protection in OA , 60. In  played aThe anabolic effects of PTH 1–34) on both cartilage and SCB were explained by multiple mechanisms  alleviated OA progression by reducing terminal differentiation, reducing apoptosis, and increasing autophagy via the mechanistic target of rapamycin (mTOR) and p62 . ApoptosAn ideal DMOAD can not only repair and regenerate cartilage, but also alleviate inflammation of synovium and pain. Healthy synovial joints are capable of maintaining extraordinary lubrication, attributed to structures as well as the cellular constitutions. However, both synovitis and OP contributed to cartilage degradation  but all Clinically, resorption played a significant role while PTH (1–34) was a reasonable option for OP patients . SuccessThere are some limitations in our current review. Firstly, the present review cannot identify the mechanisms accounting for the precious mechanism of PTH (1–34) on OA. Further research evidence is needed to deepen our current review. Secondly, although a thorough search was performed from five English databases, some pertinent studies may still have been missed. Thirdly, limited information in the current reviewed investigations is an urgent call for subsequent studies to confirm the findings based on additional information. Finally, there are only included studies published in English; thereby, some studies in other languages would be missed out.In conclusion, the SR, which included both in vivo and in vitro studies, described the beneficial effects of PTH (1–34) on OA via alleviating cartilage damage progression, inhibiting the abnormal SCB remodeling, suppressing synovitis, reducing oxidative stress or apoptosis of chondrocytes, and elevating autophagy. Some of the OA or OAOP patients might benefit from PTH (1–34) as well. The present SR is a description of existing studies regarding the effectiveness of PTH (1–34) administration in OA together with mechanisms, which suggested the necessity for further clinical trials and animal investigations to achieve concise conclusions about the effects of PTH (1–34) on OA.Additional file 1. Search strategy


Background: Reports were recently published on hepatitis B virus reactivation (HBVr), tuberculosis (TB), and atypical mycobacterial infection (AMI) in patients with ustekinumab treatment. However, the literature is limited to case reports and series. The study was aimed to investigate their relationships by using an extensive population-based database.Methods: Using the United States Food and Drug Administration Adverse Event Reporting System (FAERS) database, we collected all cases of HBVr, TB, and AMI between 1 January 2009 and 30 September 2021, for ustekinumab and other drugs. Disproportionality was analyzed using the reporting odds ratio (ROR), which was considered significant when the lower limit of the 95% confidence interval (95% CI) was >1.Results: Of the 18,760,438 adverse cases reported to FAERS for all drugs, 56,581 cases had been exposed to ustekinumab. Adverse events of HBVr, TB, and AMI were reported in 21, 210, and 20 cases, respectively. The ROR for HBVr with ustekinumab was 2.33 , for TB was 5.09 , and for AMI was 2.09 . In the ustekinumab exposure group, no death occurred in patients with HBVr, but one patient experienced life-threatening liver failure. For those with TB, 24 cases experienced hospitalization and 2 deaths occurred. No death occurred in patients with AMI but eight experienced hospitalization.Conclusion: We identified positive signals between ustekinumab exposure and HBVr, TB, and AMI in FAERS. Although these complications are rare, clinicians using ustekinumab should be aware of the risks. It receMycobacterium Avium Complex Infection,” “Mycobacterium Abscessus Infection,” “Mycobacterium Avium Complex Infection,” “Mycobacterium Chelonae Infection,” “Mycobacterium Fortuitum Infection,” “Mycobacterium Hemophilum Infection,” “Mycobacterium Kansasii Infection,” “Mycobacterium Marinum Infection,” and “Mycobacterium Ulcerans Infection.”All AEs reported to FAERS were coded according to the Medical Dictionary for Regulatory Activities (MedDRA). For reports of HBV reactivation, we queried FAERS with the MedDRA term “Hepatitis B Reactivation.” For reports of TB, we queried using the following terms: “Adrenal Gland Tuberculosis,” “Bone Tuberculosis,” “Bovine Tuberculosis,” “Choroid Tubercles,” “Congenital Tuberculosis,” “Cutaneous Tuberculosis,” “Disseminated Tuberculosis,” “Ear Tuberculosis,” “Epididymitis Tuberculous,” “Extrapulmonary Tuberculosis,” “Female Genital Tract Tuberculosis,” “Intestinal Tuberculosis,” “Joint Tuberculosis,” “Lymph Node Tuberculosis,” “Male Genital Tract Tuberculosis,” “Meningitis Tuberculous,” “Esophageal Tuberculosis,” “Oral Tuberculosis,” “Pericarditis Tuberculous,” “Peritoneal Tuberculosis,” “Prostatitis Tuberculous,” “Pulmonary Tuberculoma,” “Pulmonary Tuberculosis,” “Renal Tuberculosis,” “Salpingitis Tuberculous,” “Spleen Tuberculosis,” “Thyroid Tuberculosis,” “Tuberculoma Of Central Nervous System,” “Tuberculosis,” “Tuberculosis Bladder,” “Tuberculosis Gastrointestinal,” “Tuberculosis Liver,” “Tuberculosis Of Central Nervous System,” “Tuberculosis Of Eye,” “Tuberculosis Of Genitourinary System,” “Tuberculosis Of Intrathoracic Lymph Nodes,” “Tuberculosis Of Peripheral Lymph Nodes,” “Tuberculosis Ureter,” “Tuberculous Abscess Central Nervous System,” “Tuberculous Laryngitis,” “Tuberculous Pleurisy,” and “Tuberculous Tenosynovitis.” For reports of AMI, we queried using the following terms: “Atypical Mycobacterial Infection,” “Atypical Mycobacterial Lower Respiratory Tract Infection,” “Atypical Mycobacterial Lymphadenitis,” “Atypical Mycobacterial Pneumonia,” “Atypical Mycobacterium Pericarditis,” “Disseminated HBVr, TB, and AMI cases among patients treated with ustekinumab were respectively compared to all HBVr, TB, and AMI events reported in the database due to other drugs. When available, the following clinical characteristics of reported cases were also collected and analyzed: sex, age, reporter type, report countries, indications, concomitant product names, and reaction outcomes. Duplicate reports were removed according to the unique case ID and the case characteristics. Cases were compiled into Microsoft Excel 2019.p < 0.05. Institutional review board approval was not required because the FAERS is open to the public and patient records are anonymized and deidentified. Our study adhered to the Guidelines for Accurate and Transparent Health Estimates Reporting (GATHER) statement  with its 95% confidence interval (CI)  Stricke. The RORtatement   individual cases were related to ustekinumab. Indication for use was reported in 43,474 cases (76.83%), with 26,509 (46.85%) for psoriasis/psoriatic arthritis, 15,005 (26.52%) for Crohn’s disease/ulcerative colitis, and 1,960 (3.46%) for other conditions. Country of AE origin was reported in 55,918 cases (98.83%), including 40,431 (71.46%) from the Americas, 12,493 (22.08%) from Europe, 1,720 (3.04%) from Asia, 1,234 (2.18%) from Oceania, and 40 (0.07%) from Africa. Most reported AEs related to ustekinumab were serious (61.31%). Deaths occurred in 1,269 (2.24%) patients with available follow-up .p < 0.001, 95% CI, 1.52–3.58). The evolution of ROR over time showed that the disproportionality remained significant for the association of ustekinumab and HBVr . The evolution of ROR over time showed that the disproportionality remained significant for the association of ustekinumab and TB . The evolution of ROR over time showed that the disproportionality remained significant for the association of ustekinumab and AMI  and Asia (40.00%). Males and females were eight cases each, whereas in four cases, sex was not specified. In 14 cases, ustekinumab was the only suspected drug. In the remaining six cases, concomitant drugs were also suspected for causing AEs. Infliximab, golimumab, and prednisolone were the most commonly used drugs concomitantly. In 13 of 20 patients who had AMI due to ustekinumab, additional AEs were reported including other infections (6 cases), musculoskeletal (6 cases), neurological (4 cases), dermatological (4 cases), infusion-related (3 cases), immunological (3 cases), pulmonary (2 cases), gastrointestinal (2 cases), ophthalmic (2 cases), cardiac (1 cases), haematological (1 cases), malignant (1 cases), vascular (1 cases), and other complications (8 cases). In the ustekinumab exposure group, no death occurred in patients with AMI, but eight patients experienced hospitalization, including two disabled.AMI was noted in 20 cases using ustekinumab compared with 3,164 cases using other medications . The ROR and AMI . Stratif and AMI . Eight pUstekinumab, as a first-in-class agent targeting the IL-12/23 pathway, was approved by the United States Food and Drug Administration in 2009 to treat moderate-to-severe plaque psoriasis in patients who are candidates for phototherapy or systemic therapy . CompareDue to the roles of the IL-12/IL-23 pathway in protecting the host from pathogens, however, ustekinumab may result in unintended downstream consequences such as the development of new infections or reactivation of underlying infections . Since uThrough data mining, we identified the signal between ustekinumab and possible increased risks of developing HBVr, TB, and AMI in the FAERS pharmacovigilance database. Data mining of pharmacovigilance databases might provide previously unknown or not well-established but clinically important associations . It is ePrevious evidence is only limited to case reports and series for the association of ustekinumab with HBVr. Eight studies investigating 40 patients with chronic HBV infection receiving ustekinumab uncovered 4 reactivations (10.0%) . RegardiExperience from other immunosuppressive agents showed that the mortality rate of HBVr is about 2% (0–7%) . The proAlthough cases of TB and AMI have been occasionally reported with ustekinumab, , increasIL-12 and IL-23 play central roles to regulate T cell-mediated immune responses. In patients with chronic hepatitis B, treatment with recombinant human IL-12 inhibits HBV replication in a dose-dependent manner . IL-12 cThe main strength of this study is our ability to detect serious AEs that were not observed during the clinical trial stage for ustekinumab. In clinical trials, participants with chronic hepatitis B or active TB were excluded, or conducted in populations with a low prevalence of exposure to HBV and TB. Sample size of clinical trials is far smaller than real-world data. To the best of our knowledge, this is the largest published series of ustekinumab-associated cases of HBVr, TB, and AMI to date. The data from these cases and significant signals detected in this study suggested caution should be taken with ustekinumab use in susceptible patients and will help prompt utilization of pretreatment screening and on-treatment monitoring and early recognition of these infections in such patients.Similar to many studies based on pharmacovigilance databases, our study had some limitations. First, due to the voluntary nature of reporting to FAERS, underreporting is expected and reporting bias exists . FAERS aIn summary, we identified positive signals between ustekinumab exposure with HBVr, TB, and AMI in FAERS. Although these complications are rare, clinicians using ustekinumab should be aware of the risks. Clinical trials, pharmacoepidemiological studies, and registries are warranted to confirm the relationship and provide evidence to develop strategies involving pre-treatment screening, monitoring, and utilization of prophylaxis in patients receiving ustekinumab.


The relationship between stable isotope ratios and dietary protein sources has been reported. However, few studies have examined the effect of stable isotope ratios on metabolic risk in humans. We investigated whether the stable isotope ratios of carbon and nitrogen in human hair are associated with blood pressure and hypertension.13C) and nitrogen (δ15N).We conducted a cross-sectional study of 392 subjects (228 men and 164 women). Hair samples of the subjects were used for the measurement of stable isotope ratios of carbon  for hypertension was 1.55 (1.04–2.30) per 1‰ increase in δ15N and 1.22 (0.86–1.73) per 1‰ increase in δ13C, respectively. However, in the subjects with antihypertensive medication, neither δ13C nor δ15N values showed a significant association with hypertension.The δ15N value might be used as a surrogate marker to screen a high-risk population for hypertension.The stable isotopic ratio of nitrogen in scalp hair is independently associated with hypertension in subjects without antihypertensive medication. The hair δ Hypertension is a major public health issue and is one of the most significant risk factors for cardiovascular disease, cerebrovascular accidents, and chronic kidney disease –4. It ha13C/12C, δ13C) and nitrogen  stable isotope ratios have lately been suggested as possible indicators for food intake and nutritional health  × 1000, where Rx is the 13C/12C or 15N/14N isotopic ratio of the sample, and Rs is the 13C/12C or 15N/14N isotopic ratio of the standard. The analytical precision was ±0.2‰ for δ13C and ± 0.3‰ for δ15N, respectively.Hair samples were taken by being cut from the subject’s crown and as close to the scalp as feasible. A standard process was used to prepare the hair samples , 25. Hai13C or δ15N values and other variables. We used multivariable logistic regression models to evaluate the independent association of δ13C or δ15N values with hypertension. Five logistic regression models were used, each with a different degree of confounder adjustment. In model 1, we adjusted for age and sex. In model 2, we additionally adjusted for total energy intake and dietary sodium intake. In model 3, we additionally adjusted for fasting blood glucose, HDL cholesterol, and triglyceride. In model 4, we additionally adjusted for smoking status, alcohol consumption, and regular exercise. In model 5, we additionally adjusted for waist circumference. Further, we assessed the association of δ13C or δ15N values with hypertension stratified by antihypertensive medication. SAS ver. 9.4  was used for all statistical analyses. All comparisons were considered statistically significant if the P-value was less than 0.05.We examined the distribution and normality of data and performed one-way analyses of variance, Kruskal-Wallis tests, or chi-square tests to compare differences in the profiles of study subjects. Pearson’s or Spearman’s correlation coefficients were calculated to test the correlation between δA total of 175 subjects had hypertension in 392 study subjects. Of the 175 hypertensive subjects, 45.1% were taking antihypertensive medications. Table 13C values showed positive correlations with diastolic blood pressure , waist circumference , and total energy intake , and showed negative correlation with age . The δ15N values showed positive correlations with diastolic blood pressure , body mass index , waist circumference , fasting glucose , and total energy intake , and showed negative correlations with age  and HDL cholesterol  in all subjects. In the subjects without antihypertensive medication, the δ13C values showed positive correlations with diastolic blood pressure , waist circumference , fasting glucose , and total energy intake , and showed negative correlation with age . The δ15N values showed positive correlations with systolic blood pressure , diastolic blood pressure , body mass index , waist circumference , fasting glucose , and total energy intake , and showed negative correlation with age  in the subjects without antihypertensive medication  and diastolic blood pressure . The δ13C values showed significant correlations with diastolic blood pressure . Furthermore, δ15N values progressively increased with the blood pressure classification of the Korean Society of Hypertension in the subjects without antihypertensive medication (P for trend =0.010)  and 2.19 , respectively, in multivariable models, adjusted for age, sex, total energy intake, dietary sodium intake, fasting blood glucose, triglyceride, HDL cholesterol, smoking, alcohol consumption, regular exercise, and waist circumference.Table 15N for hypertension was 1.17 (0.84–1.63) after adjusting for age, sex, total energy intake, dietary sodium intake, fasting blood glucose, triglyceride, HDL cholesterol, smoking, alcohol consumption, regular exercise, and waist circumference. When we divided subjects by antihypertensive medication, the positive association between a 1‰ increase in δ15N and hypertension was significant in subjects without antihypertensive medication , whereas the association between a 1‰ increase in δ15N and hypertension was not significant in subjects with antihypertensive medication . A 1‰ increase in δ13C was not associated with hypertension in all subjects and any subgroup of antihypertensive medication  per 1‰ increase in δ 0.84–1.6 after ad15N values of hair were associated with an increased risk of hypertension. However, in the subjects with antihypertensive medication, neither δ13C nor δ15N values showed a significant association with hypertension. This is the first research to explore the potential clinical implications of δ15N values in relation to hypertension.The nitrogen, but not carbon, stable isotopic ratio is independently associated with hypertension in the subjects without antihypertensive medication. Higher δ13C and δ15N values [15N values and hypertension. High amounts of meat intake have been associated with a higher risk of hypertension in previous studies [15N and hypertension. A UK research reported that those who consumed more animal protein showed higher δ15N values than vegans, suggesting that nitrogen stable isotope ratios might be utilized as a nutritional biomarker for dietary meat consumption [15N in the body [Stable isotopic analysis of hair has been known as a noninvasive technology that provides a long-term reflection of food consumption . PreviouN values . NitrogeN values , 13, 28, studies , 29, whisumption . Higher the body .15N values might be also an indicator of other metabolic risk factors, such as heavy metals or persistent organic pollutants, which are known to be associated with hypertension [15N values were related to fish consumption, the δ15N values associated with fish consumption might be linked to heavy metals or persistent organic pollutants, perhaps contributing to the positive correlation of δ15N values with hypertension [15N values and blood pressure.δrtension –32. Bioartension –35. The rtension , which crtension . Given trtension , 39. Mor15N values with hypertension was only found in the subjects not taking antihypertensive drugs, and no such association was observed in the subjects taking antihypertensive drugs. The use of antihypertensive drugs, which lower blood pressure, might alter the association between blood pressure levels and δ15N values. In addition, our findings might be attributed to the difference in lipid profiles between the subjects with and without antihypertensive medication. Dyslipidemia has been known to be associated with hypertension [In the present study, the association of δrtension , and prertension –43. Thos13C values of hair were not associated with hypertension. The proportion of C3 plants  and C4 plants  in a person’s diet determines the carbon stable isotope ratios in the human body [13C values might be employed as a biomarker for sugar-sweetened drinks containing C4 sugars [13C values have been reported to show no significant correlation with carbohydrate intake in Korean diets [13C values also have been reported to be inversely associated with dietary fish intake and diabetes [13C with various factors might explain the lack of association between δ13C and hypertension.Our findings showed that δman body , 44, δ134 sugars , 44. Die4 sugars , but δ13an diets . The δ13diabetes , 46. TheThere are several limitations to this study that should be considered. First, the direction of a causal relationship between nitrogen stable isotope ratios and hypertension could not be established because this study has a cross-sectional design. Longitudinal studies may be necessary to clarify the direction of causation. Second, because this study only included elderly Koreans, the findings may be limited in their generalizability. Third, measurement error may exist, particularly due to random within-person variation although lifestyle factors and dietary intake were measured according to standardized procedures.The stable isotopic ratio of nitrogen in scalp hair is independently associated with hypertension in subjects without antihypertensive medication. The hair δ15N value might be used as a surrogate marker to screen a high-risk population for hypertension. Further research is needed to fully comprehend the mechanism behind the association between stable isotope ratios and blood pressure.


Primary production showed significant seasonal variations, with higher values in the summer associated with lower euphotic depths, higher water temperatures, and higher nutrient concentrations. No spatial differences were found for primary production or photosynthetic parameters. Primary production values were lower than previous estimates, which reflects an improvement in water quality in the Ria Formosa, but values are higher than primary production estimates for other temperate coastal ecosystems, which reflects the highly productive nature of this coastal lagoon.Coastal lagoons are among the most productive ecosystems in the world, and they provide a wide range of ecosystem services and resources. In the Ria Formosa , phytoplankton production has rarely been addressed. The main goal of this study is thus to evaluate the variability of phytoplankton production and photosynthetic characteristics over the seasonal cycle and in different locations  of the Ria Formosa coastal lagoon, subjected to distinct natural and anthropogenic stressors. Primary production was evaluated using the  Coastal lagoons are shallow brackish or marine water bodies separated from the ocean by a barrier, such as islands, spits, sand banks, or reefs, and connected to the ocean by one or more inlets that remain open at least intermittently ,2. CoastCoastal lagoons are socio-ecological systems, important from both the ecological and the socio-economic perspectives, providing many valuable ecosystem goods and services, with societal, heritage, aesthetic, and scientific value . CoastalEutrophication in coastal lagoons is a major problem due to restricted flushing and long water residence times . EutrophPhytoplankton are usually the main source of organic matter for higher trophic levels in most aquatic ecosystems. In addition to their critical ecological function as primary producers, phytoplankton play pivotal roles in the ecological and biogeochemical dynamics of marine ecosystems influenced by connectivity to land . Phytopl14C are closer to net rather than gross primary production [Phytoplankton production can be easily determined using the carbon-14 incorporation method described by Steeman-Nielsen , which ioduction , and theoduction , light ioduction , and nutoduction , as welloduction  and endooduction . Therefooduction ,23,24, dThe Ria Formosa coastal lagoon  is one of the most important aquatic ecosystems in Portugal, both biologically and economically. It serves as a breeding and feeding ground for fish and birds, and supports a wide range of human activities, such as tourism, fishing, and shellfish farming . For insThe previous eutrophication studies in the Ria Formosa coastal lagoon were mostly based on the analysis of nutrient concentrations and ratios, and chlorophyll-a concentration. These indicators are rather static in relation to the dynamic nature of eutrophication, which includes enhancement of primary production in its definition. Measurements of primary production in the Ria Formosa, however, are scarce and somewhat conflicting. Regarding the relative contribution of each primary producer, different studies indicate either salt marsh plants, microphytobenthos, or phytoplankton as the main primary producers (see  and refe−2 s−1 (262.1 ± 183.3 µmol photons m−2 s−1). The depth of the euphotic zone varied between 2.0 and 20.3 m, with a mean value of 7.7 ± 4.4 m. Mean dissolved inorganic nitrogen (DIN), dissolved reactive phosphorus (DRP), and dissolved silicon (DSi) concentrations throughout the sampling period were 4.2 ± 3.8 µM, 0.2 ± 0.2 µM, and 3.8 ± 3.4 µM, whereas chlorophyll-a concentration ranged between undetectable values and 3.6 µg L−1, with a mean value of 0.85 ± 0.85 µg L−1. Spatial differences were found for euphotic depth (p < 0.001), with significantly higher values at the inlet (seaward boundary), and for DIN (p = 0.002), with significantly higher concentrations at the urban location in relation to the other three locations (p < 0.001), and for DRP (p < 0.001), with significantly higher concentrations in the summer and lower in the autumn and winter  (Bmax) and photosynthetic efficiency (α). PBmax varied between 0.5 and 178.3 mg C mg Chla−1 h−1 (20.6 ± 38.6 mg C mg Chla−1 h−1) and α presented values ranging between 0.00 and 1.19 mg C mg Chla−1 h−1 (µmol photons m−2 s−1)−1 (0.11 ± 0.24 mg C mg Chla−1 h−1 (µmol photons m−2 s−1)−1). Both PBmax (p = 0.008) and α (p = 0.009) were significantly higher in the summer in relation to the other seasons, but no spatial differences were observed for these parameters. The optimal light intensity (Eopt) ranged between 186.5 and 998.5 µmol photons m−2 s−1 (328.6 ± 165.3 µmol photons m−2 s−1), and no significant spatial or seasonal differences were detected (Primary production in the euphotic zone between May 2011 and April 2012 varied between 3.51 and 718.74 mg C m m−3 d−1 . Signifi< 0.001) . The samdetected . p < 0.001) and negatively correlated with the euphotic depth . Significant moderate and positive correlations were also found between primary production and water temperature  and chlorophyll-a concentration . Chla was, in turn, positively correlated with DIN . As for photosynthetic parameters, no significant correlations were found between those and environmental variables, but a strong and positive correlation was found between α and PBmax . Eopt was also correlated with Im , α , and PBmax .Significant moderate to strong correlations were found between primary production and environmental variables. Primary production was strongly and positively correlated with dissolved reactive phosphorus . Euphotic depth (score = −0.70) and phosphorus concentration (score = 0.83) were more strongly associated with the first axis, whereas nitrogen concentration (score = 0.53) and chlorophyll-a (score = 0.78) were associated with the second axis.The model obtained through redundancy analysis explained 38% of the constrained variance, with an adjusted RIn the Ria Formosa coastal lagoon, primary production showed significant seasonal variation, with higher values in the summer, associated with lower euphotic depths, higher water temperatures, and higher nutrient concentrations. No spatial differences were found for primary production or photosynthetic parameters. −3 d−1) are lower than values previously reported for this coastal lagoon. For instance, studies conducted in the early 1990s and the early 2000s reported mean (±SD) primary production values of 389 ± 273 mg C m−3 d−1 [−3 d−1 [−3 d−1 [−2 h−1 [−2 h−1). The estimates of phytoplankton primary production in the Ria Formosa  are higher than values reported for other temperate coastal ecosystems in Portugal. For instance, areal primary production in Portuguese estuaries ranges between 2 and 778 mg C m−2 d−1 in the Tagus estuary [−2 d−1 in the Guadiana estuary [−2 d−1 in the Douro estuary [−2 yr−1; mean = 223 mg C m−2 yr−1) are similar to values found in a compilation of 1148 values measured in 131 coastal ecosystems, with a median and mean annual primary production of 185 and 252 g C m−2 yr−1 [Overall, the primary production values estimated for the Ria Formosa coastal lagoon , but the differences in relation to the other locations were not significant . The inlet is the deepest location of the lagoon, with a deeper euphotic zone and lower mean light intensity in the mixed layer. Although the redundancy analysis resulted in a model that explained only 38% of the variability in primary production and photosynthetic parameters, euphotic depth was negatively and strongly associated with the response variables. Deeper lagoon locations, despite a well-mixed water column and a euphotic zone that extends to the bottom, will have a lower mean light intensity. Given that phytoplankton dynamics are strongly associated with light availability, it is expected that primary production will be lower at deeper lagoon locations, such as the inlets. Light is particularly important for phytoplankton in confined and/or turbid ecosystems [Significant spatial differences in primary production in the Ria Formosa coastal lagoon, with lower values at the inlet, were found in a previous study , whereasosystems ,43, reguosystems . In the osystems , particuosystems ), particosystems ).The nutritional environment is another major variable regulating phytoplankton growth and production. According to the constrained ordination, both nitrogen and phosphorus were related to the patterns observed in primary production and photosynthetic parameters, with higher nutrient concentrations associated with higher primary production. Co-limitation by N and P is frequently observed throughout the seasonal cycle in the Ria Formosa coastal lagoon , and higBmax) and photosynthetic efficiency (α) were strongly correlated with each other, which is not expected, as α is related to the concentration of photosynthetically active pigments [Bmax is related to electron transport capacities [Bmax and α indicates that phytoplankton with a lower photosynthetic efficiency tends to exhibit a lower maximum photosynthetic rate, which contradicts the typical photo-acclimation of phytoplankton, with lower photosynthetic efficiency but the higher maximum photosynthetic rate at surface waters, associated with higher light availability [opt) was positively correlated with light availability in the water column, as expected, given that the cells will typically adjust their light saturation coefficient in accordance with ambient irradiance [Bmax and α observed during summer. Higher summer values for photosynthetic parameters are typically observed in coastal ecosystems, associated with higher water temperature and light availability [Regarding photosynthetic parameters, no significant spatial variability was found, and no correlations with environmental variables. The maximal biomass-specific production rate , pacities . The poslability . The optradiance . Despitelability  and favolability . In the lability . The coalability , providiIn conclusion, planktonic primary production in the Ria Formosa coastal lagoon showed marked seasonal variability, with the summer months being more productive, associated with higher light intensity and higher temperature but no significant spatial differences were found. Estimates of primary production are within values reported for other coastal ecosystems, but are lower than previous estimates for the Ria Formosa, possibly due to an improvement in water quality in the last decade. Due to the shallowness of the Ria Formosa, a strong benthic-pelagic coupling is expected, and the relative contribution of benthic primary producers to the total productivity of the system should be as relevant as the planktonic component. Future studies should address not only the total productivity of the system but also the relative contribution of each component to overall primary production. Given that the primary productivity of an ecosystem constrains the availability of fish and other commercially relevant organisms, the assessment of primary production is of strategic economic importance in coastal ecosystems .The Ria Formosa coastal lagoon  is an eua concentration, and primary production  for each sampling date were estimated using the vertical profiles of PAR intensity, according to an exponential function (Equation (1)) as follows:z (µmol photons m−2 s−1) is the PAR intensity at depth level z (m) and I0 is the PAR intensity at the surface. Euphotic zone depth  was calculated as (Equation (2)) as follows:m, µmol photons m−2 s−1) for each sampling date was calculated as (Equation (3)) as follows:m (m) is the depth of the mixed layer. Given that our vertical profiles of PAR intensity were obtained at a specific time of the day that could not be representative of the whole day [0 was determined using daily solar irradiance data , considering that PAR constitutes 50% of the total solar radiation reaching the surface and a mean reflection of 6.6% at the surface [Light attenuation coefficients (kradiance . Mean PAhole day . I0 was s−1 W−1:  obtained surface . 4 Bq) of 14C-HCO3− were added to each flask. The flasks were incubated in a plant growth chamber under in situ temperature and different light intensities , and carbon uptake was stopped with formaldehyde after 2 h. Primary production for each light level was calculated as (Equation (4)) as follows:−1 h−1), Rs (dpm) is the activity in the sample, Rd (dpm) is the mean activity of the dark flasks, D (=1.05) is the isotopic discrimination (correction for the preference for 12C over 14C [−1) is the amount of dissolved inorganic carbon in the sample , R (dpm) is the total activity of the 14C added to each flask, and N (hours) is the incubation time. Phytoplankton primary production was determined using the . Fifty mover 14C ), W (mg −3 h−1) was converted to biomass-specific primary production, divided by the sample’s chlorophyll a concentration . Photosynthetic parameters were estimated using [B is biomass-specific primary production (mg C mg Chla−1 h−1) and E is the PAR irradiance during incubation (µmol photons m−2 s−1). Coefficients a, b, and c were used to estimate maximal biomass-specific production rate , optimal light intensity , and initial slope of the P-E curve, which represents photosynthetic efficiency −1), according to Equations (6)–(8), respectively, as follows:Primary production  was estimated by integration over depth in the euphotic zone, using non-normalized Pmax estimates. Daily volumetric primary production , phosphate (PO43−), and silicate (SiO44−) were determined immediately upon arrival to the laboratory, whilst samples for nitrate (NO3−) were frozen (−20 °C) until analysis. All nutrient analyses were made in triplicate, according to the spectrophotometric methods described by [a concentration was also determined spectrophotometrically, using glass fiber filters and acetone 90% to extract the pigment [Water samples for determination of dissolved inorganic nutrient concentration were immediately filtered after collection through cellulose acetate filters  to acid-cleaned vials. Ammonium  data using the function nls on R. Redundancy analysis (RDA) was used to examine relationships between environmental variables and primary production-related variables, to determine which predictor variables explain the most variation in multiple response variables [Descriptive statistics were used to explore the data in terms of averages and dispersion. Differences across locations  and seasons  were tested with analysis of variance (ANOVA) or Kruskall-Wallis ANOVA on ranks, depending on data normality, assessed with a Kolmogorov-Smirnov test. The strength of associations between variables was measured with Pearson’s correlation coefficient. These analyses were performed using IBM SPSS Statistics v. 28. A photosynthesis-irradiance model  was fittariables . Analyseariables  for R [6ariables  using thariables . All ana