This study explored the relationship between environmental polycyclic aromatic hydrocarbons (PAHs) and Chronic obstructive pulmonary disease (COPD), and identified systemic inflammation as a mediator of the increased risk of COPD from PAHs.Data were obtained from 60,936 middle-aged and older Americans recruited in the National Health and Nutrition Examination Survey 2005–2016. Environmental PAHs were measured in terms of urinary concentrations of PAHs metabolites . We used multifactor logical analysis to figure out the link between PAHs and COPD, and the non-linear relationship was examined using Restricted cubic spline. Spearman correlation analysis was utilized to analyze the connection between PAHs and systemic immune-inflammation index (SII).p < 0.001), FLU , PA , and PYR  levels than non-COPD population. In unadjusted logistics analysis, the risk of COPD with log NAP was higher . Upon taking into account, confounders like sex, age, race, and log NAP still increased a possible COPD risk . Similarly, FLU, PA, and PYR significantly increased the risk of COPD , both unadjusted and adjusted. Furthermore, Restricted cubic spline demonstrated a strong link between PAHs levels and COPD risk (p < 0.05). Additionally, a Spearman correlation analysis revealed a favorable association between log FLU and log SII , while NAP, PA, and PYR levels were not associated with log SII . Ultimately, the mediating effect analysis revealed a mediating effect capacity of 5.34% for the SII-mediated association between FLU and COPD.The results showed that the COPD population had higher NAP (3.550 vs. 3.282, The findings suggest that the risk of COPD is significantly increased when environmental PAHs exposure is at high levels, and that systemic inflammation may be involved in the process. As a hePolycyclic aromatic hydrocarbons (PAHs) are a collection of compounds that are produced through the incomplete combustion of oil, coal and natural waste, gas, and other organic materials. PAHs have been linked to a variety of health risks , 6. PAHsA recently developed inflammatory indicator is known as the systemic immune-inflammation index (SII) that simply combines the functions of neutrophil, platelet, and lymphocyte counts; due to its high stability, it can effectively reflect systemic inflammation , 11. SIIApparently, there is a paucity of direct robust evidence supporting the link between PAHs and the risk of COPD, and there is little known about whether PAHs progress by increasing systemic inflammation and thereby influence the development of COPD. Therefore, we desire to investigate the link between PAHs and COPD in middle-aged and older adults among participants recruited by the National Health and Nutrition Examination Survey (NHANES). In addition, we included SII as a mediator to assess whether SII was involved in the connection between COPD and PAHs in adults over 40 years old.22.1The NHANES is a survey that is carried out by the National Center for Health Statistics in conjunction with the Centers for Disease Control and Prevention. Since it stores data from the civilian population of the United States, it is nationally representative. The Ethics Review Committee gave their stamp of approval to the protocol for collecting the data, and before they were questioned or evaluated, each participant in the study gave their informed consent. Hence, the study did not require additional informed consent as well as ethical endorsement. The NHANES public data file from 2005 to 2016 was used to construct the dataset in this study, and this research was carried out with the assistance of a total of 60,936 people.2.2https://www.cdc.gov/nchs/nhanes/index.htm.The main exposure variable in this study was PAH with reference to the former studies , we used2.3Referring to previous studies , if the 2.49/L)  were included .p < 0.001] and the proportion of men  were significantly higher in the COPD group than in the non-COPD group  vs. (71.4%), p = 0.040], the two groups were not significantly different (p < 0.001] and “Now” smoking  behavior in the COPD population compared with the non-COPD population. Similarly, COPD patients consumed more alcohol than non-COPD patients  and diabetes  among people with COPD than among people without COPD  vs. (2.307), p < 0.001], PA , and PYR  levels compared with non-COPD patients.According to p < 0.001]. Taking into account sex, age, race, and BMI as covariates, with the increase of log NAP, the risk of developing COPD remained high [OR = 1.429, 95% CI (1.224–1.669), p < 0.001; p < 0.05; Also, in unadjusted logistics analysis , the risp > 0.05). Similarly, there was a similar intra-segmental variation around the inflection point for log PA , including a direct effect value was 0.044 . In addition, the indirect effect value for log FLU leading to COPD prevalence risk via log SII was 0.002 [(95% CI (0.001–0.003), p < 0.001); Finally, we analyzed the mediating effects of log FLU, log SII, and COPD risk after covariate adjustment. As shown in To conclude, the mediating effect of systemic inflammation mediating the association between FLU and COPD prevalence risk produced a mediating effect capacity of 5.34% .4In general, PAHs are mainly exposed to people through breathing indoor and outdoor polluted air and smoking . PAHs exp < 0.005) and FLU . Furthermore, we verified that COPD patients are generally older male, and have a higher prevalence of smoking as well as alcohol consumption in accordance with previous research (During large cross-sectional study of middle-aged and older adult people in the United States, higher concentrations of environmental PAHs were positively correlated with the risk of COPD. Previous studies have also shown similar results. For instance, Peng et al.  found thresearch . Previouresearch  have fouresearch  not beinresearch . PAHs caresearch . Our sturesearch , emphyseresearch , lung inresearch .Currently, research on the human hazards caused by PAHs primarily focuses on deformities, carcinogenicity, and neurotoxicity, while studies on the chronic lung inflammation caused by direct exposure to PAHs are relatively limited . ThroughPolycyclic aromatic hydrocarbons are primarily generated during the incomplete combustion of organic materials . PAHs arThe impact of PAHs on human health is significant, particularly in relation to lung health. Inhalation of PAHs can lead to both acute and chronic respiratory conditions . Prevent2-hydroxyfluorene, as a type of PAH, has been associated with various health effects largely related to its ability to induce oxidative stress and inflammation. In our study, after adjusting for covariates such as age, sex, race, and BMI, log FLU had a positive effect on the risk of COPD prevalence, and the indirect effect of log FLU leading to the risk of COPD prevalence through log SII accounted for 5.34% of the risk of COPD prevalence, suggesting that SII is an important pathway for FLU to increase the risk of COPD prevalence, and that the overall effect of 5.34% of the risk of COPD prevalence due to FLU may be realized through the systemic inflammatory response realized. Although this mediating effect may appear relatively small, it is statistically significant and represents an important contribution to the overall effect of the disease in a complex disease pathology. According to Ferguson et al.  urinary In light of these findings, we hypothesize that exposure to FLU may lead to systemic inflammation, as measured by SII, through the induction of oxidative stress and the production of pro-inflammatory cytokines. This increased systemic inflammation then may contribute to the development or exacerbation of COPD. However, the specific mechanisms through which FLU influences SII and COPD risk warrant further study.Nonetheless, there are still some limitations to our study. At first, the present study is a cross-sectional study and cannot analyze the association between PAHs and COPD over time, which needs to be complemented by a longitudinal study. Besides, PAHs or SII can only be used as a qualitative diagnostic tool for COPD and cannot assess the extent of COPD as well as lung function. Therefore, the correlation between the dose of PAHs exposure and the severity of COPD needs to be elucidated by further experiments. Also, the present study is an association study and cannot explain causes and effects of COPD caused by PAHs. Furthermore, how PAHs affect COPD is very complex, and more basic validation of the relationship is needed, and future studies need to fully elucidate the specific molecular biological mechanisms of NAP and FLU-induced COPD.5In the study, we found a strong association between exposure to PAHs and COPD risk among a representative sample across the middle-aged and older adult people in United States and that there is a mediation effect, a process mediated through systemic inflammation. In particular, FLU has the potential to elevate SII and thereby increase the risk of COPD.The original contributions presented in the study are included in the article/The studies involving humans were approved by Centers for Disease Control and Prevention in United States. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.YX carried out the acquisition and interpretation of data and was the major contributor to drafting the manuscript. YX and LZ carried out the clinical partial data collection and analysis. WH and YX participated in drawing tables and diagrams. HL was responsible for correcting the language and grammar. WH was responsible for reviewing and revising some drawings and tables. HL and WH contributed to the ideas of the article and reviewed the manuscript. All authors contributed to the article and approved the submitted version.


Zymomonas mobilis, a Gram-negative bacteria observed in some popular beverages, is considered safe and has been studied for its potential therapeutic benefits. In this study, we explored its effects on the inflammatory process, tissue integrity, differential gene expression, and microbiota composition in an experimental dextran sulfate sodium (DSS)-induced colitis model in mice. As a result, Z. mobilis alleviated the symptoms caused by DSS administration, as indicated by reduced weight loss, disease activity index, a significant reduction in the colon weight/length ratio, and histopathological improvement. Also, Z. mobilis could restore the mucosal barrier as well as increase the expression of Muc3 and Ocln genes. An analysis of 16S rRNA sequences showed that Z. mobilis alters gut microbiota, increasing Akkermansia muciniphila abundance and decreasing Escherichia coli. Furthermore, Z. mobilis seems to be involved in potentiating a regulatory phenotype by inducing immunomodulatory genes like Tgfb, Il5, Il10, and Foxp3 and reducing the relative mRNA expression of proinflammatory cytokines TNF, Il6, and Il17. Our data suggest that Z. mobilis could alleviate disease progression and be considered a possible probiotic adjuvant for pathologies of the bowel. Zymomonas mobilis was initially identified in the ritualistic fermented beverage pulque, popular in Mesoamerica. However, it attracted attention for its anaerobic metabolism, which uses the alternative Entner–Doudoroff (ED) pathway, and its efficient ethanol production. It is also widely studied for its metabolite production of ethanol, sorbitol, succinic acid, levan, and lactate .Three weeks before the beginning of the experiment, the mice went through a co-housing process: once a week, the animals were randomly mixed in the cages to homogenize the microbiota. Feces from days 5 and 10 of all animals were collected. To verify changes in the profile of microbial diversity in the group and not just individually, a pool of samples from each group was carried out on the established days. Total bacterial DNA from the feces pool was extracted with the GenElute™ Stool DNA Isolation Kit . The V3–V4 region of the 16S ribosomal RNA gene was sequenced to generate 250 bp paired-end raw reads using the HiSeq platform  by GenOne Biotechnologies .www.bioinformatics.com.cn/en (accessed on 1 July 2023)). The raw sequencing data have been uploaded to the NCBI Sequence Read Archive (SRA) database under BioProject number PRJNA997586.Paired-end reads were merged using FLASH (V1.2.7) . Quality® Protect Mini Kit , according to the manufacturer’s instructions. Quantifications of RNA samples were performed using a NanoDrop One spectrophotometer . To eliminate genomic DNA from RNA samples, TURBO™ DNase  was used, followed by reverse transcription with the High-Capacity cDNA Reverse Transcription Kit . Assays were performed in duplicate using the LightCycler® RNA Master SYBR Green I kit  in ABI Step One Plus Real-Time PCR System equipment . Relative mRNA expression was normalized from endogenous gene beta-2-microglobulin (B2m) expression, and the 2−ΔΔCt method was used to calculate transcript levels. The oligonucleotides are described in A fraction of the intestinal colon was macerated using TissueLyser LT  to disrupt the tissue. The extraction of total RNA was performed using the RNeasyp-values were considered significant <0.05.The results of body weight and DAI experiments are expressed as means ± SEM. Colon weight/length ratio results are expressed as medians ± SD and real-time PCR and histological score results are expressed as means ± SD. Statistical differences were determined by one-way ANOVA with a Tukey post hoc test for body weight and DAI curves and by the Mann–Whitney test for colon weight/length ratios, histological scores, and real-time PCR charts. All statistical analyses were performed with GraphPad Prism version 6.0  and Z. mobilis ingestion on DSS-induced colitis in mice were evaluated. The experiment lasted 10 days. From the first to the fifth day of the experiment, the animals in the DSS and DZM groups consumed DSS 3% in their water, while the NC and ZM groups only drank water. The animals were evaluated daily for weight, stool consistency, and degree of rectal bleeding. The animals were orally gavaged throughout the experiment: the ZM and DZM groups with 100 μL Z. mobilis (5 × 109 CFU/mL) and the NC and DSS groups with 100 μL of saline. On the 11th day, all animals were euthanized (The effects of thanized a.Z. mobilis (ZM) treatment did not impact body weight. The DZM group lost weight during DSS treatment, which stabilized after day 8.Body weights showed a gradual decrease in the experimental groups during the first five days of DSS treatment, with a markedly accelerated decline in the DSS-treated groups from the fifth day until the end of the experiment b. Z. mobZ. mobilis treatment on murine colitis was assessed using these indexes during the 10 days of the experiment. The DSS group had the highest DAI throughout the experiment and peaked on the sixth day. The peak was not observed for induced colitis mice treated with Z. mobilis (DZM group). After halting DSS administration, DAI scores declined until day 10, where most groups were comparable, except the DSS group (The disease activity index (DAI) quantifies the macroscopic parameters of DSS-induced colitis based on weight loss, stool consistency, and rectal bleeding. The higher this index, the more intense the macroscopic symptoms and the sicker the animals are. The effect of SS group c.Z. mobilis—DZM (p < 0.05)  d. The daZ. mobilis only in the last six days of the experiment ; crypt architectural disarray, crypt loss, irregular crypts, and crypts with hyperplasia (white arrowhead); loss of surface epithelium—erosion (black arrowhead); crypt abscess (yellow arrowhead); focal ulceration and decreased goblet cells a.Z. mobilis (ZM) only was morphologically similar to the healthy group (NC). In mice that received DSS and Z. mobilis (DZM), histological damage in the mucosa was ameliorated, and it was possible to observe few areas of erosion in the epithelium, a decrease in inflammatory cells, regular crypts with areas of hyperplasia, and an increase in goblet cells. In addition, the group that received only DSS showed stronger inflammatory signs in an overall damaged mucosa. The histological score is shown in The group that was orally gavaged with Z. mobilis on the gut microbiota. Fecal samples were collected from six animals from the NC, DSS, ZM, and DZM groups on days 5 and 10.The V3–V4 regions of the 16S rRNA gene were sequenced from pooled animal stool samples to evaluate the effects of Alpha diversity is an analytic method to assess community complexity in each sample through specific metrics . Part ofBacterial community diversity, assessed by the Shannon index, showed an increase in the ZM 10 and NC 10 groups, in contrast to groups DZM 10 and NC 5, which had a lower species diversity b. AnotheBacillota and Bacteroidota. Actinomycetota was more abundant in the DSS group on day 5. On day 10, there was an increase in Pseudomonadota in the DSS and DZM groups, and in the latter group, there was also an increase in Verrucomicrobiota. The phyla Cyanobacteriota, Thermodesulfobacteriota, and Chloroflexota remained stable during the analyzed days.The topmost relative abundant microbial communities were identified in the composition of each sample taxa at the phylum level a. In a gBifidobacterium pseudolongum on day 5 (DSS 5) and Escherichia coli on day 10 (DSS 10), as well as Romboutsia ilealis at both time points. In contrast, the group that received DSS plus treatment with Z. mobilis increased the relative abundance of the bacteria Akkermansia muciniphila at day 10 (DZM 10) after its increased abundance in the ZM group at day 5.At the species level, more intense changes occurred among the groups on days 5 and 10, as shown by ternary plots b. DSS trZ. mobilis (ZM and DZM), a heatmap analysis was used with the 20 most abundant common genera among all samples. The samples were clustered by their abundance distribution in different samples (by the Euclidean distance method)  and those that received  method) a. IntereAccording to the results of OTU clustering analysis, a Venn diagram was generated with common and unique information for the different samples on days 5  and 10  b,c. The Z. mobilis only, both clustering in the middle of the graph. However, neither DSS group clusters, and their day 5 and day 10 sample results are spread antagonistically . The control group measurements on days 5 and 10 appear close to each other, similarly to those for the group that received stically d.Z. mobilis treatment on modulating the immune response. The expression of these genes was evaluated by real-time PCR with colonic mRNA from animals in the NC, DSS, and DZM groups at the end of the experiment.Genes that play a key role in mediating the immune response in inflammatory diseases were analyzed to evaluate the impact of Muc3 expression, but treating with Z. mobilis activated it. Ocln expression was not affected after DSS treatment, but it was induced by Z. mobilis supplementation. Consistently with histopathological analyses, the expression of these markers increased in the DZM group compared to the DSS and NC group (Molecular markers such as Mucin 3 (Muc3) and Occludin (Ocln) are related to mucus production by goblet cells and the integrity of tight junctions in colonocytes, respectively. Their expression reflects tissue restoration. DSS treatment seems to have impaired NC group a,b.Il6, Il17 and Tnfa, and the two formers were reversed with Z. mobilis treatment. Moreover, the DZM group showed a significant increase in the gene expression of transcription factor Foxp3 and cytokines Tgfb, Il10, and Il5, associated with a regulatory profile, compared to NC and DSS groups  has been shown to induce remission of UC in patients and maintain it for a certain period [Bifidobacterium, Lactobacillus, and Streptococcus. This medication proved to be safe and effective in reducing the symptoms caused by UC, in addition to inducing and maintaining remission of this disease for a period in the patients tested [Z. mobilis appears as a possible therapeutic candidate as it presents probiotic characteristics associated with GRAS status and its cultural use [Chronic inflammation of the bowel is a modern-day disease that affects a large number of individuals, principally in industrialized countries. Even though no specific treatment is available, the use of probiotics could lead to benefits in terms of disease control. For example, n period ,31. Thisn period ,33,34. As tested ,36,37. Iural use , combineZ. mobilis consumption on DSS-induced colitis in mice were evaluated. This experimental design is accepted as a model for human IBD due to the clinical and histopathological damage it generates in the intestinal mucosa of mice, similar to the damage that occurs in humans affected with ulcerative colitis [The effects of  colitis ,39. ThisZ. mobilis-treated mice. The new protocol essentially resulted in the same observations. Animals in the DSS and DZM groups that drank water with DSS 3% during the first five days showed a gradual weight loss, which stabilized after the seventh day after DSS withdrawal. The NC and ZM groups remained stable and very similar throughout the experiment. Interestingly, the DZM group presented DAI results similar to the NC and ZM control groups, with a statistical difference between it and the DSS group, suggesting that this was possible due to Z. mobilis. Another evaluated parameter that suggested the use of Z. mobilis can alleviate the symptoms caused by DSS use is the significant reduction in colon weight/length ratio, whose increase is related to the intensity of the inflammatory process, observed in the DZM group in relation to the DSS group. These macroscopic evaluation features related to the severity of DSS-induced colitis suggest that disease progression and symptoms decreased in the presence of Z. mobilis in the gastrointestinal tract of mice.After a pilot test, we designed a 10-day protocol to test DSS administration in The effect of DSS administration on the murine colon is the loss of intestinal epithelial cell barrier integrity. Due to these injuries, the tissue is exposed to microorganisms in the intestinal lumen, causing an acute inflammatory response ,41. ThisAll these typical changes were found in the DSS group colonic sections, unlike those of the NC and ZM groups, which presented healthy colons with an integral histological architecture. In the DZM group, an improvement in the colonic mucosa was observed, such as decreased inflammatory cells in the lamina propria, crypts recovering their regular appearance, and an increase in goblet cells. However, as the DSS treatment was suspended on day 5, some of these microscopic changes was found to have been reversed at the end of the experiment, which was reflected in the low statistical relevance of the histological score. Part of this rapid recovery may have been affected by sex. DSS-induced colitis is known to be biased by sex. Hormonal issues seem to make female mice less prone to develop colitis-associated inflammatory symptoms ,43. In tMuc3 mRNA in DSS-treated mice, even after five days without DSS consumption, indicating a persistent degradation of the mucus layer, keeping inflammation levels high. Conversely, the DZM group showed a significant increase in the expression of Muc3 compared with the DSS group, suggesting a process of restoration of the mucosal barrier, possibly due to the ingestion of Z. mobilis.Mucins, the glycoproteins that make up mucus and are produced by goblet cells, play an important role in protecting the intestinal mucosal barrier. Mucin 3 is expressed in the colon and secreted in response to inflammatory cytokines. It is related to IBD pathophysiology, such as in ulcerative colitis ,45. HoweZ. mobilis (DZM) showed a significant increase in the Ocln gene in relation to the control group. These data corroborate the hypothesis that the presence of Z. mobilis could reestablish the integrity of the colonic epithelium, improving tissue recomposition and controlling the inflammation caused by DSS administration.The other structural protein studied, Occludin, is a component of intercellular tight junctions which promotes this barrier’s structural integrity ,47. Its Ulcerative colitis is also related to intestinal microbial dysbiosis in humans, with decreased diversity and abundance of microorganisms ,49. DSS Pseudomonadota (replacing Actinomycetota) occurred in the DSS group with an abundance of E. coli at the species level. The presence of this microorganism, often opportunistic, is present both in cases of ulcerative colitis in humans and in murine models and may worsen the severity of the disease [Actinomycetota phyla in the DSS group on day 5 and Pseudomonadota on day 10 was also found in murine models of colitis induced by DSS [Microbiota were observed to change during the experiment, and in the end, five days after DSS withdrawal (day 10), animals from the DSS and DZM groups showed a marked change in the distribution profile of the phyla. A marked increase in  disease ,53. Thisd by DSS ,41.Bacillota and an increase in the Verrucomicrobiota phylum, with an emphasis on the presence of the bacterium Akkermansia muciniphila. This bacterium plays a role in regulating intestinal barrier function and is involved in the host’s immune and metabolic responses [A. muciniphila can modulate the expression of genes related to mucus production, since it uses these glycoproteins as an energy source [A. muciniphila and increased Muc3 gene expression, also observed in our study in Z. mobilis-treated mice [A. muciniphila after Z. mobilis consumption needs further clarification due to the statistical limitation of this finding, but it could represent a mechanism of action for Z. mobilis on experimental colitis.The DZM group on day 10 showed a decrease in the esponses . Furthery source . Qu et ated mice . Moreoveted mice ,53. The In intestinal inflammation, the immune mechanism of action also occurs by activating genes that encode cytokines, chemokines, and other important factors for maintaining intestinal homeostasis . Given tIn ulcerative colitis, IL-6, together with TNF, mediates the intestinal inflammatory response ,59,60. TTnfa, Il6, and Il17 was induced by DSS, but Il6 and Il17 were significantly decreased in the DZM group, suggesting that Z. mobilis alleviated the inflammatory response.In our study, the relative mRNA expression of the proinflammatory cytokines Tgfb while Z. mobilis coadministration induces it, compared to non-treated mice. This, combined with a decrease in Il17, may result in the induction of regulatory cells. Another cytokine related to regulatory T cells is IL-10, which can reduce the expression of inflammatory cytokines such as TNF and IL-6. The inactivation of IL-10 causes the condition of chronic colitis, with an increase in several inflammatory cytokines [Z. mobilis induced Il10, but Z. mobilis administration seemed to potentiate the regulatory phenotype by efficiently inducing a broader range of immunomodulatory genes, including Tgfb, Il5 and Foxp3.Transforming growth factor β (TGF-β) is an important cytokine in T-cell-mediated tolerance, inducing regulatory T cell (Treg) differentiation concomitant to Foxp3 expression, in addition to being involved in cell growth control, production of extracellular matrix, and intestinal mucosa regeneration ,63. DSS ytokines . In our Z. mobilis to reduce disease progression in the intestinal colon in a murine model of DSS-induced colitis by reestablishing the integrity of the colonic epithelium, beneficially altering the microbiota, and significantly relieving inflammation caused by DSS administration. Despite the limitations of this work, this is a pioneering study using Z. mobilis in a murine model of DSS-induced colitis. It paves the way for new studies to be carried out using this innovative microorganism to ameliorate inflammatory intestinal diseases.The presented data suggest the ability of


Brain tumors account for 1% of all cancers diagnosed de novo. Due to the specificity of the anatomical area in which they grow, they can cause significant neurological disorders and lead to poor functional status and disability. Regardless of the results of biochemical markers of intracranial neoplasms, they are currently of no diagnostic significance. The aim of the study was to use LC-ESI-MS/MS in conjunction with multivariate statistical analyses to examine changes in amino acid metabolic profiles between patients with glioblastoma, meningioma, and a group of patients treated for osteoarthritis of the spine as a control group. Comparative analysis of amino acids between patients with glioblastoma, meningioma, and the control group allowed for the identification of statistically significant differences in the amino acid profile, including both exogenous and endogenous amino acids. The amino acids that showed statistically significant differences  were evaluated for diagnostic usefulness based on the ROC curve. The best results were obtained for phenylalanine. Classification trees were used to build a model allowing for the correct classification of patients into the study group (patients with glioblastoma multiforme) and the control group, in which cysteine turned out to be the most important amino acid in the decision-making algorithm. Our results indicate amino acids that may prove valuable, used alone or in combination, toward improving the diagnosis of patients with glioma and meningioma. To better assess the potential utility of these markers, their performance requires further validation in a larger cohort of samples. Intracranial neoplasms account for 2% of cancers, and deaths during these cancers account for 3% of all cancer deaths worldwide. Gliomas are among the most common CNS neoplasms and the most common primary malignant tumors. From a practical point of view, glial tumors are divided into low-grade gliomas (LGGs) and high-grade gliomas (HGGs) ,2,3,4,5.Meningiomas are the most common primary intracranial neoplasms. Most often, meningiomas are solitary intracranial tumors that arise without obvious risk factors. However, possible factors contributing to the development of meningiomas include radiation therapy, inflammation, intracranial trauma, and hormonal disorders ,11,12.Clinical symptoms of intracranial tumors have a twofold pathophysiological basis. Firstly, they result from increased intracranial pressure (ICP) and are non-specific, and secondly, they are related to the location of the lesion and depend on the neural centers and tracts occupied or compressed by the tumor. The most common symptom of all intracranial tumors is progressive neurological deficit ,14.The emergence and development of intracranial tumors are conditioned by genetic, molecular, cellular, and biochemical mechanisms. As in the case of any carcinogenesis, including CNS tumors, their growth is a multi-stage process underpinned by genetic mutations .Imaging tests  are the basic method of monitoring the course and treatment of brain tumors, while biochemical markers determined in urine and body fluids have limited use so far. Previous studies indicate the possibility of using various compounds as potential biomarkers in CNS tumors, such as interleukins ,17, the Metabolomics is a complementary approach to genomics and proteomics focused on the comprehensive profiling of all small molecular weight metabolites in biofluids, tissues, and cells. Metabolomics approaches can be targeted or non-targeted. Untargeted metabolomics focuses on the unbiased, hypothesis-free analysis of all detectable metabolites from a biological sample. Although this approach allows for the identification of a large pool of potentially relevant metabolites, the approach often represents a trade-off between specificity, selectivity, and analysis time. Targeted metabolomics refers to the quantitative measurement of a select group of metabolites  in order to investigate specific metabolic pathways or validate biomarkers identified using non-targeted metabolic profiling. Once a potentially relevant metabolic pathway has been identified using an untargeted approach, targeted metabolic profiling appears advisable to increase the number of metabolites associated with that particular pathway ,32.A wide range of analytical techniques are available for the qualitative and quantitative determination of metabolites; however, the most commonly used analytical techniques in cancer metabolomics are MS and NMR spectroscopy.NMR spectroscopy is a quantitative, high-throughput technology that provides both qualitative and quantitative information, making it a useful tool in metabolomics research. Despite the many advantages of NMR spectroscopy, due to its relatively low sensitivity and poor dynamic range, it is not suitable for the detection of metabolites present in trace amounts. Most metabolomics studies of brain tumors are based on NMR spectroscopy, which allows for the observation of changes in metabolism related to tumor development and differentiation of the degree of malignancy ,33,34,35Mass spectrometry (MS) provides a valuable analytical platform for metabolomics due to its high sensitivity and wide dynamic range. With the dissemination of this technology to routine medical laboratories, it has become possible to measure low molecular weight metabolites for diagnostic purposes. The use of LC-MS/MS methods for small molecules in biological fluids is becoming increasingly preferred because they have higher sensitivity and better reliability in complex biological fluids. Recent studies have shown that the use of metabolomic analysis with mass spectrometry detection allowed the identification of biomarkers differentiating patients with CNS tumors from healthy people or enabling the differentiation of low- and high-grade tumors ,39,40,41There are several ways to assess changes in the metabolome in patients with CNS tumors: through the metabolome of plasma collected from an antecubital vein or the metabolomic analysis of blood collected from resected tumor tissue or cerebrospinal fluid ,39,40,42Amino acids (AAs) are the building blocks of various biomacromolecules, as well as functional regulators of various metabolic processes. The altered metabolism of AAs promotes the proliferation of tumors and contribute to their immune escape and resistance to treatment. From a clinical point of view, it is estimated that changes in amino acid concentrations observed in specific body fluids provide the opportunity to develop new diagnostic and prognostic markers ,46. EarlThe aim of the study is to evaluate the potential of targeted dual-control serum amino acid metabolomics analysis in the diagnosis of patients with glioma and meningioma. In pilot studies, other groups have explored the use of a metabolomics approach to distinguish patients with glioblastoma and meningioma from healthy controls. However, to the best of our knowledge, no metabolomics study based on targeted amino acid analysis has attempted to remove the confounding effects of common pathophysiology, such as a generalized immune response.The present study used high-performance liquid chromatography–mass spectrometry (HPLC-MS) coupled with multivariate statistical analyses to accurately identify unique changes in amino acid metabolomic profiles that distinguish glioma patients from meningioma patients and other diseases that may be associated with overall inflammation, e.g., degenerative spine disease.The first stage of the study was to compare amino acid concentrations between patients diagnosed with glioblastoma and the control group. A comparative analysis of the analyzed AAs between patients with glioblastoma and the control group allowed the identification of statistically significant differences between the study groups. Statistically significant differences were observed both among endogenous  and exogp = 0.002245), ARG (p = 0.009292), CIT (p = 0.000000), ASN (p = 0.000939), GLY (p = 0.017846), GABA (p = 0.000000), SAR (p = 0.019362), ABA (p = 0.001831), ORN (p = 0.000012), and ASP (p = 0.000214).Among endogenous AAs, statistically significant differences concerned SER (p = 0.000032), MET (p = 0.000190), LYS (p = 0.000000), HIS (p = 0.000011), AAA (p = 0.000021), LEU (p = 0.036077), PHE (p = 0.000001), ILE (p = 0.000728), C-C (p = 0.000037), and TYR (p = 0.000007).In the case of exogenous amino acids, statistically significant differences were observed for THR , CIT (p = 0.000028), GABA (p = 0.000145), ORN (p = 0.005893), and ASP (p = 0.049001). A comparison of the group of patients with glioma and the control group also allowed for the confirmation of statistically significant differences for such exogenous amino acids such as THR (p = 0.005100), MET (p = 0.000003), LYS (p = 0.000019), AAA (p = 0.007040), PHE (p = 0.007552), C-C (p = 0.000000), and TYR (p = 0.000349).In the case of endogenous amino acids, significant differences were found for GLN . When comparing the group of patients with glioblastoma with the control group, statistically significant differences after post hoc analysis were observed for lysine, histidine, α-aminoadipic acid, and phenylalanine . In the In Only in the case of α-aminoadipic acid was the concentration of this amino acid higher in the group of men in patients with meningioma compared to the group with glioma and the control group .The next part of the analysis started to build a classification model that will classify patients as healthy or suffering from glioblastoma of the CNS with the highest efficiency. Receiver operating characteristic (ROC) analysis is a useful tool for evaluating the accuracy of a model’s prediction by plotting the sensitivity versus (1-specificity) a classification test. The model of the classification of patients into the group with glioblastoma and the control group included those AAs in which statistically significant differences were found between the study group and the control group, both in the group of women and men. Visualizations of the obtained results are presented in Then, the accuracy of the model was assessed in relation to the concentration of the analyzed amino acids. The obtained results are illustrated in The obtained cut-off, accuracy, sensitivity, and specificity values of the tested amino acids obtained using the ROC curve are summarized in The analysis was aimed at showing which amino acids would allow for a better classification of patients into a group with glioblastoma and a control group of healthy people. Classification trees are used to build predictive models (predicting subsequent data based on the model based on previously provided information) and descriptive models. The significance plot of the model variables based on standardized data primarily indicates the concentration of cysteine, citrulline, and glutamine .Importantly, the use of a model based on the above parameters gives a much better chance of correctly diagnosing a person with glioblastoma than diagnosing the same cancer in a healthy person .Many research projects focus on finding prognostic or diagnostic biomarkers for central nervous system tumors, including meningiomas and gliomas. Currently, classical metabolic pathways such as the citric acid cycle (TCA), glycolysis, and arachidonic acid/inflammatory pathway have been well-studied in glioma. However, in recent years, attention has also been paid to understanding new metabolic changes in glioma cells, such as metabolic changes in IDH mutant cells, amino acid metabolism, or nucleotide metabolism. Many characteristic metabolites have been discovered in glioma, such as 2-HG, fumaric acid, succinic acid, sarcosine, glycine, glutamine, aspartic acid, choline, serine, glucose, lactic acid, and polyamines, which suggest the dysregulation of multiple metabolic pathways  ,49,50,51This study was designed to evaluate changes in amino acid profiles between patients with glioblastoma, meningioma, and the control group. Available studies indicate that dysregulated AA metabolism is not characteristic of a given type of cancer. The selection of the study groups (glioblastoma vs. meninges vs. a group of patients treated for osteoarthritis of the spine as a control group) was aimed at capturing possible differences in the profiles of the tested amino acids, depending on the type of cancer.The conducted analysis allowed the observation of statistically significant differences between the study groups. When we compare the changes in the amino acid profiles of the glioblastoma and meningioma patients to the controls, we can see a group of amino acids that differ in both cases. This applies to endogenous amino acids  and exogenous . In the case of glioblastoma, we observe additional amino acids differentiating the study group and the control group, including SER, ARG, ASN, GLY, SAR, and ABA. This points to a more altered amino acid metabolism in the glioblastoma group. In the case of meningioma, attention is paid to glutamine, whose elevated concentration is observed in comparison with the control group. In patients with glioblastoma, these differences are not statistically significant. In most cases, we observe a higher concentration of amino acids in groups of cancer patients compared to the control group. Lower concentrations were observed with cysteine, the concentration of which is statistically significantly lower in patients with glioblastoma compared to the control group. In turn, the concentration of cysteine is reduced in both glioblastoma and meningioma patients compared to the control group.The biomarker should be resistant to inter- and intra-individual factors, such as gender. Therefore, in the proposed study, the study groups of both cancer patients and the control group were divided by gender. Among the compounds that showed statistically significant differences, only lysine, histidine, α-aminoadipic acid, and phenylalanine differentiated patients with glioblastoma from healthy ones both in the group of men and women. In patients with meningioma, differences in the group of men and women concerned only lysine.The AAs that showed statistically significant differences were evaluated for diagnostic usefulness. The diagnostic usefulness of AAs determined in the identification of glioblastoma was assessed based on the ROC curve with the proposed cut-off point and area under the AUC curve. Sensitivity, specificity, and accuracy are given for the designated cut-off points. The best results were obtained for phenylalanine. The ROC graph for phenylalanine showed a sensitivity of 66.7%, a specificity of 95.9%, and an accuracy of 79.5% for the proposed cut-off of 101.00 nmol/mL. The other analyzed amino acids were characterized by similar parameters of lysine , histidine , and α-aminoadipic acid .In the next stage of the research, classification trees were used, which allowed us to build a model allowing for the correct classification of patients into the study group (patients with glioblastoma) and the control group  . CysteinAnalyzing the results of our research, it seems that the most important amino acids in terms of diagnostic decisions are lysine, aminoadipic acid, histidine, and phenylalanine. The higher concentration of lysine observed in our study in patients with glioma and meningioma, considering the division by gender, was also observed in patients with head and neck cancers and sarcoma ,53,54. TWe found high levels of serum histidine in glioblastoma patients compared to the controls. This was parallel with the result of Toklu et al., where histidine levels were elevated in patients with high-grade glioblastoma . PreviouThe results of our study indicate an altered metabolism of phenylalanine in both glioblastoma and meningioma patients. In addition, we showed the statistical significance of phenylalanine differentiating patients with glioblastoma and the control group, considering a division by gender. Phenylalanine was also characterized by the best diagnostic values in the case of patients with glioblastoma. Phenylalanine is the precursor of tyrosine, an important AA for the biosynthesis of neurotransmitters like L-DOPA  and catecholamines dopamine, epinephrine, and norepinephrine. Available research results indicate an increased concentration of phenylalanine in cancer, which reflects increased muscle proteolysis. For example, elevated levels of phenylalanine have been observed in squamous cell carcinoma of the head and neck  and hepaThis study included 54 patients treated surgically for intracranial tumors in the Department of Neurosurgery, Neurotraumatology, and Paediatric Neurosurgery at the University Hospital No. 1, Collegium Medicum of the Nicolaus Copernicus University. The histological types of tumors in the examined patients were glial tumors and meningiomas. The control group consisted of 49 people  treated for osteoarthritis of the spine. The exclusion criteria for the control group were past or current neoplastic disease and (similarly to the study group) a surgical procedure performed less than 30 days ago. This study was conducted in accordance with all internationally approved human testing guidelines and was in accordance with the Declaration of Helsinki. The Ethics Committee of the Nicolaus Copernicus University in Toruń and Collegium Medicum in Bydgoszcz approved this study (consent number KB 499/2021).Antecubital whole-blood samples were drawn from a peripheral vein in the morning hours . Overnight fasting and 15 min of rest before the blood test were obligatory. Blood collected in the tube was kept for 30 min. Serum from the blood after clotting was separated, centrifuged, and frozen at −80 °C until analysis was performed. The EZ:faast amino acid analysis kit was utilized for serum sample preparation.The EZ:faast amino acid analysis procedure consists of a solid phase extraction step followed by derivatization and a liquid/liquid extraction. The solid phase extraction is performed via a sorbent-packed tip that binds amino acids while allowing interfering compounds to flow through. Amino acids on sorbent are then extruded into the sample vial and quickly derivatized with a reagent at room temperature in an aqueous solution. Derivatized amino acids concomitantly migrate to the organic layer for additional separation from interfering compounds. The organic layer is then removed, evaporated, and re-dissolved in an aqueous mobile phase and analyzed on an LC-MS system.The quantitative and qualitative analyses of samples were performed using High-Performance Liquid Chromatograph Nexera XR LC-20 AD pump  and a Nexera XR SIL-20AC autosampler  coupled with a mass spectrometer equipped with an electrospray ion source (ESI), an LCMS-8045 Mass Spectrometer . The instrument was controlled, and recorded data were processed using LabSolutions LCMS Ver.5.6 software.Chromatographic separation was carried on the EZ:faast amino acid analysis–mass spectrometry column  at a column temperature of 35 °C with the corresponding binary mobile phase. Solvent A was 10 mM ammonium formate in water and solvent B was 10 mM ammonium formate in methanol. The mobile phase flow was 0.25 mL/min and took place in a gradient system: 68% B–83% B in 13 min. The injection volume was 1 µL. The mass spectrometry (MS) data were acquired in positive ion mode. Multiple reacting monitoring was used for quantification by monitoring the ion transition of amino acids.Selected mass spectrometry parameters are summarized in TM amino acid analysis kit was obtained from Phenomenex, Inc. . HPLC grade methanol was obtained from Merck . Ammonium acetate and formic acid of analytical grade were purchased from Sigma-Aldrich Co.  with purity greater than 99%. Water was deionized and purified using a Milli-Q system  and used to prepare all aqueous solutions.The EZ:faastCalibration curves for each AA were constructed in a range from 0.01 to 75 nmol/mL. Calculation and calibration are based on an internal standard method. The kit contains as an internal standard an AA mixture of homoarginine (HARG), methionine-d3 (Met-d3), and homophenylalanine (HPHE).The statistical analysis was performed using Statistica 13.1 (StatSoft) and Excel 365 (Microsoft). The type of distribution was examined using the Shapiro–Wilk test. The Levene test was used to test the homogeneity of variance. The Kruskal–Wallis test and multiple comparisons of mean ranks for all trials were used to assess differences between more than two groups. Relationships between variables were described using the Spearman coefficient in the event of statistically significant differences. The data were visualized using a box plot. Principal component analysis (PCA) was used to classify correlation coefficients. The correlation matrix was used as the input. To isolate the number of principal components, Cattella’s and Kaiser’s criteria were used.The receiver operating characteristic (ROC) curves were prepared using the concentration values of amino acid compounds. Sensitivity (SEN), specificity (SPE), and accuracy (Acc) were used to evaluate the model. The general models of regression and classification trees were used to build the classification model. The collected data were divided by a 4:1 ratio into a learning and testing group. Cross-validation was used to verify the effectiveness of the models. All analyses were performed at a significance level of 5% (α = 0.05).A comparative analysis of AAs between patients with glioblastoma, meningioma, and the control group allowed for the identification of statistically significant differences between the study groups.A comparison of the amino acid profile between patients with glioma and the control group allowed for the identification of differences in both endogenous amino acids  and exogenous amino acids . Similarly, when comparing the amino acid profiles of patients with meningioma to the control group, the differences observed concerned endogenous amino acids  and exogenous amino acids . When comparing the amino acid profile by gender, only lysine showed statistically significantly higher concentrations in the group of patients with glioma and in the group of patients with meningioma compared to the control group. In the group of glioma patients, differences in amino acid profiles by gender also concerned histidine, α-aminoadipic acid, and phenylalanine. However, no statistically significant differences were observed when comparing amino acid profiles between the groups of patients with glioma and meningioma. The AAs that showed statistically significant differences were evaluated for diagnostic usefulness. The best results were obtained for phenylalanine.This study showed that amino acids may be involved in the mechanisms underlying the pathogenesis of glioblastoma and meningioma and may also be considered potential indicators of some pathways altered in CNS neoplastic diseases. However, given the multifactorial, heterogeneous, and complex nature of these diseases, further work with a larger study group is needed to correlate the results of previous studies with possible other malignancies to see if the amino acid profile of meningiomas and gliomas can be considered characteristic of these diseases.


Healthcare-associated infections have become a serious public health problem. Various types of information systems have begun to be applied in hospital infection prevention and control (IPC) practice. Clinicians are the key users of these systems, but few studies have assessed the use of infection prevention and control information systems (IPCISs) from their perspective.This study aimed to (1) apply the extended DeLone and McLean Information Systems Success model (D&M model) that incorporates IPC culture to examine how technical factors like information quality, system quality, and service quality, as well as organizational culture factors affect clinicians’ use intention, satisfaction, and perceived net benefits, and (2) identify which factors are the most important for clinicians’ use intention.A total of 12,317 clinicians from secondary and tertiary hospitals were surveyed online. Data were analyzed using partial least squares-structural equation modeling and the importance-performance matrix analysis.P<.001), information quality , and service quality  were significantly related to user satisfaction (R2=0.833), use intention (R2=0.821), and perceived net benefits . IPC culture had an effect on use intention , and it also indirectly affected perceived net benefits . In the importance-performance matrix analysis, the attributes of service quality (providing user training) and information quality (readability) were present in the fourth quadrant, indicating their high importance and low performance.Among the technical factors, system quality  and hospital IPC culture have an impact on the successful use of IPCISs after evaluating the application of IPCISs based on the extended D&M model. Furthermore, service quality and information quality showed higher importance and lower performance for use intention. These findings provide empirical evidence and specific practical directions for further improving the construction of IPCISs. Healthcare-associated infections (HAIs) have become a significant public health concern, posing a threat to the well-being of people and resulting in the spread of drug-resistant bacteria, an increase in unnecessary fatalities, and additional health care expenses . The WorThe information system is an integrated system of components to collect, store, and process data. The data are used to provide information and contribute knowledge to support operations, management, and decision-making . In thisThe DeLone and McLean Information Systems Success model (D&M model) is one of the widely used models for evaluating the successful application of information systems, providing classification metrics for information system success, as well as models of temporal and causal relationships between categories ,28. The This study focused on evaluating clinicians’ use of IPCISs for several reasons. First, policy requirements from the Association for Professionals in Infection Control and Epidemiology (APIC) highlight the need to strengthen the information ability of relevant personnel for prevention and control, including monitoring information technology and using electronic medical records and system data . Second,Motivated by the analyses above, this study proposed an extended D&M model as the basis of the theoretical framework that adds cultural factors to construct an IPCIS evaluation model of clinicians’ perceptions in the context of IPC. According to our developed model, this study assessed the success of IPCISs in a survey of 12,317 clinicians from secondary and tertiary hospitals.This study designed 8 constructs based on the D&M model: system quality ,44, infoDifferent types of information system qualities  positively affect users’ satisfaction and use intention, based on the D&M model and a review ,44,47. FTherefore, this study hypothesizes the following:H1: Technical factors  have a positive impact on users’ satisfaction.H2: Technical factors  have a positive impact on users’ use intention.Previous information system studies have found that cultural values influence the acceptance of information technology eg, ,51). In . In 51])H3: The culture of IPC has a positive impact on the use of IPCISs.User satisfaction is considered to be one of the necessary factors for evaluating the success of information systems. Studies confirmed a direct and significant relationship between user satisfaction and system use intention . Chow etH4: User satisfaction has a positive impact on the intention to use the system.The D&M model’s net benefits include individual and organizational benefits, representing the degree to which the user/organization believes that the use of the system will bring benefits, such as improving the user’s or organization’s job performance, productivity, etc . An incrH5: Use intention has a positive impact on individual benefits (decision-making [H5a] and communication [H5b]) and organizational benefits (H5c).H6: User satisfaction has a positive impact on individual benefits (decision-making [H6a] and communication [H6b]) and organizational benefits (H6c).Participants of the study were clinicians from 307 secondary and tertiary hospitals in Hubei Province, China. The inclusion criteria were as follows: clinicians with experience in using IPCISs, those who knew the purpose of the study, and those who were willing to cooperate. In addition, to ensure a balanced sample, we required a sample size of at least eight clinicians each  in the departments of respiratory medicine, urology, intensive care, neurology, endocrinology, and orthopedics, and at least five clinicians each in other departments.This study was approved by the ethics committee of Tongji Medical College, Huazhong University of Science and Technology (2021-S063). All participants provided written informed consent, which included survey purpose, survey period, privacy protection, and investigator information. The study data were anonymized and deidentified.This survey designed the constructs of information system quality , use intention, satisfaction, individual benefits, and organizational benefits based on the D&M model, and added the construct of IPC culture based on the organizational culture perspective. The measurement items we used were adapted from previous related studies with modifications to suit the context of the study. To ensure the validity of the content, we asked 6 experts  to check the clarity, redundancy, and understandability of the survey entries  was used to analyze the extended D&M model in this study by using the SmartPLS software (v3.39) . PLS-SEMA total of 12,493 clinicians from 246 secondary and tertiary medical institutions answered the questionnaire, and 12,317 questionnaires met the requirements after review, with a response rate of 98.6%. The demographic characteristics of the clinicians surveyed are shown in Composite reliability and Cronbach α values were used to assess the internal reliability of reflective constructs. The results showed that all composite reliability values were higher than the recommended value of 0.8 (range: 0.934-0.992), and Cronbach α values were above the recommended value of .7, indicating sufficient reliability for all constructs. The results for convergent validity showed that the average variance extracted of all constructs was greater than the recommended threshold of 0.5 (range: 0.806-0.967), suggesting that the variance of all constructs was greater than the variation induced by individual measurement error, which confirmed the convergence validity . The disR2 score. The R2 values of satisfaction (0.833), use intention (0.821), individual benefits in communication (0.624), individual benefits in decision-making (0.676), and organization benefits (0.656) were all higher than the level of 0.5. This indicates that the extended D&M model had a moderate to high level of explanatory power, accounting for 82.1% of the variance in use intention. Evaluating the effect size (f2), it was found that the impact size of system use on individual decision-making benefits was large (2.090).After confirming the reliability and validity of the measurement model, we tested the hypotheses by estimating the structural model. The explanatory power of the model was evaluated with the P<.001), individual communication benefits , and organizational benefits , which suggests that when individuals are willing to use IPCISs to support their IPC work, they are better able to communicate with each other and make decisions, as well as facilitate management at the organizational level. Therefore, the hypotheses H6a, H6b, and H6c are supported.Through the bootstrapping process, we provided normalized path coefficients for each path to compare the effects of different constructs. First, we found that users’ use intention was significantly related to individual decision-making benefits . Therefore, our data support hypotheses H1a, H1b, and H1c. With regard to use intention, there was significant influence by system quality , information quality , and service quality . Moreover, IPC culture  had a significant impact on use intention. Thus, hypotheses H2a, H2b, H2c, and H3 are supported. The structural model is shown in After understanding the effects of use intention and satisfaction, we further investigated the antecedents that may drive individuals to use IPCISs and their satisfaction. The results showed that all quality dimensions  had a significant effect on user satisfaction  and hospital IPC culture have an impact on the successful use of IPCISs. Among them, service quality and information quality showed higher importance and lower performance for use intention. Moreover, use intention had a greater impact on decision-making in net benefits. Furthermore, the results of the IPMA suggested that providing information system training and improving the legibility of information in the system could improve the use intention of clinicians. These findings provide empirical evidence and specific practical directions for further improving the construction of IPCISs. We suggest that hospitals conduct more training on the use of IPCISs, modify the information presentation form on the system, and adjust the amount of information to improve the efficient use of IPC information by clinicians.


INST-MFA entails fitting a (large) system of coupled ordinary differential equations, with metabolite pools and reaction fluxes as parameters. Here, we focus on the Calvin-Benson cycle (CBC) as a key pathway for carbon fixation in photosynthesizing organisms and ask if approaches other than classical INST-MFA can provide reliable estimation of fluxes for reactions comprising this pathway.Flux phenotypes from different organisms and growth conditions allow better understanding of differential metabolic networks functions. Fluxes of metabolic reactions represent the integrated outcome of transcription, translation, and post-translational modifications, and directly affect growth and fitness. However, fluxes of intracellular metabolic reactions cannot be directly measured, but are estimated via metabolic flux analysis (MFA) that integrates data on isotope labeling patterns of metabolites with metabolic models. While the application of metabolomics technologies in photosynthetic organisms have resulted in unprecedented data from First, we show that flux estimation with the labeling patterns of all CBC intermediates can be formulated as a single constrained regression problem, avoiding the need for repeated simulation of time-resolved labeling patterns.Chlamydomonas reinhardtii, Chlorella sorokiniana and Chlorella ohadii under different growth conditions.We then compare the flux estimates of the simulation-free constrained regression approach with those obtained from the classical INST-MFA based on labeling patterns of metabolites from the microalgae Our findings indicate that, in data-rich scenarios, simulation-free regression-based approaches provide a suitable alternative for flux estimation from classical INST-MFA since we observe a high qualitative agreement ( While metabolite concentrations and contents can be measured at cell- and (sub)compartment-specific level , reactioglobal MFA approaches is well established even for the isotopic nonstationary state and implemented in toolboxes like INCA  and related canonical processes, namely starch and sucrose syntheses. Estimating fluxes in the CBC and other downstream processes in metabolic networks of photosynthetic organism in photoautotrophic growth conditions necessitates the usage of isotopically nonstationary MFA (INST-MFA). This is the case since under photoautotrophic growth conditions, feeding 13CO13C-MFA, INCA  that can be regarded as a constraint-regression problem in which we minimize the chi-square goodness-of-fit measure for the change in MID concentration over time as well as external fluxes. The minimization is performed under the constraint that the entire system, represented by stoichiometric matrix with 2.2Chlamydomonas reinhardtii and Chlorella sorokiniana under low light , and in Chlorella ohadii under LL and extreme illumination levels . Like in Treves et al.  FBP, ADPG and UDPG were the only metabolites not at steady state  as well as flux values for starch and sucrose synthesis reactions . This approach was possible since all MIDs included in the ODEs were measured  and 520.6 (149 degrees of freedom), respectively, and were not considered statistically acceptable. Since the relative error in measurements under EIL is not significantly lower than in the other algae, there is no experimental basis for omitting the last time point, as there was for C. ohadii under LL. Our previous modeling efforts with INCA did not find acceptable fit for the data of C. ohadii under EIL  of bootstrap samples were within the acceptable range . For reactions PFP/FBA  the sum of flux through the entire CBC was the highest for . ohadii . For a dC. reinhardtii and C. sorokiniana. Interestingly, the Spearman correlation of the flux distribution for C. ohadii to that of either C. reinhardtii or C. sorokiniana was smaller , followed by C. reinhardtii (66s) and C. sorokiniana (6min). In line with the observed turnover of RuBP, we found that turnover of FBP was the fastest in C. ohadii (15s), followed by C. reinhardtii (22s) and C. sorokiniana (31s). However, while SBP exhibited the fastest turnover in C. ohadii (18s), C. sorokiniana (24s) showed similar turnover as C. ohadii, followed by C. reinhadtii (49s) with the slowest turnover for SBP. Together, these findings indicated that C. ohadii has more efficient photosynthesis in comparison to C. reinhardtii, indicated by higher carboxylation rates in comparison to C. reinhardtii, while metabolites pool sizes are in the same order of magnitude for both algae. In addition, it can be also concluded that C. ohadii has more efficient photosynthesis in comparison to C. sorokiniana, indicated by similar carboxylation rates for C. ohadii in comparison with C. sorokiniana, despite pool sizes in C. ohadii which are one order of magnitude smaller than those observed for C. sorokiniana . Hence, we investigated if the difference of flux estimates may be due to variance in metabolite pool sizes used in INCA and measured pool sizes integrated in the SFCR approach. While the estimated total pool size from INCA matched the measured content for ADPG, RuBP and UDPG, estimates of pool sizes for F6P, FBP, G1P and G6P were different from mean measurements by at least one order of magnitude . We found that the flux estimates obtained from the original time series and the time series with 3The availability of flux estimates for reactions in a metabolic network provides several possible applications. In scenarios in which fluxes are compared between conditions for the same organism, reactions with differential fluxes can be seen as main drivers for the differential behavior for a related trait at a higher level (e.g. growth). Further, in scenarios in which fluxes of the same pathway or network are compared across organisms , the reactions with differential fluxes between organisms, can indicate points of intervention to drive the high-level trait towards a desired biotechnological goal. To this end, the fluxes estimated from integration of labeling data can be integrated in genome-scale metabolic networks as constraints in various constraint-based approaches. However, obtaining flux estimates for photosynthetic organisms, particularly in plants, remains challenging due to the large experimental efforts as well as the related computational problems associated with the relevant mathematical formulations.C. reinhardtii and C. sorokiniana; however, it could be further refined by experimental knowledge in later studies. For C. ohadii, the compartmentation predicted in INCA indicates larger cytosolic than chloroplastic pools for T3P, FBP and G6P. Note that compartmentation values obtained from INCA correspond to those obtained from the pseudo-reaction added for compartmentation effects. Furthermore, we found differences between the compartmentation ratio from the ratio of estimated pool sizes used in INCA  and obtain flux estimates as well as consider multiple modeling scenarios which we discuss here. Interestingly, the SFCR formulation allowed us to investigate compartmentation via a simple sampling approach, significantly reducing the time for parameter estimation. The sampling approach assumed that the ratio between cytosolic and chloroplastic pool is between 0.1 and 0.9. This assumption is in line with predictions from INCA  total pool sizes, (2) sizes of pools actively involved in photosynthetic processes, (3) pool compartmentation, and (4) constraints on boundary fluxes to the modeled system.C. sorokiniana and no sugar production for C. reinhardtii and C. sorokiniana  differ by an order or magnitude, result in no acceptable fit for Another possibility is that only a fraction of the measured pool actively participates in photosynthesis, due to various reasons . RepeatLastly, it is known that boundary fluxes have a large effect on the flux estimates and often lead to numerical issues, which has prompted the usage of local approach that motivated our work. Rescaling the experimental exchange fluxes used by division with factor 100 results in starch and sucrose synthesis rate estimated from SFCR that match the observed pattern, underlining the issue of numerical problems. The rescaling of the exchange flux leads to a good qualitative agreement between flux estimated for individual algae from SFCR and INCA indicated by  studies .Our study showed that in data-rich scenarios, as it is the case for the CBC, as one of the major pathways in photosynthesis, SFCR provides a suitable alternative to INST-MFA approaches (e.g. as implemented in INCA). Recent developments in omics technologies result in high coverage in terms of data for other pathways, including: the TCA cycle and amino acid biosynthesis, and parts of lipid metabolism, for which SFCR can be applied in the future. In addition, the formulation of SFCR could be used to compare the extent to which flux distributions obtained from different constrained-based approaches fit experimentally measured labeling pattern. However, our study also pointed at several issues with flux estimation, even in data rich scenarios, and indicated that future efforts must focus on careful comparison of multiple alternative model structures and provide validations and plausible explanations for the discrepancy between measured and fitted (compartmented) pool sizes. To this end modifications to the approach may need to be introduced, such as rendering it applicable to estimation of fluxes akin to local approaches .44.1We used two variants of a metabolic model for the CBC differing in their complexity. The first model version, denoted as ‘simple CBC model’, considers only CBC with a branch towards starch synthesis and no compartmentation of metabolite pools. The second model version, denoted as ‘compartmented CBC model’, includes reactions for the CBC and related processes, i.e. photorespiration, starch and sucrose synthesis. The model considers metabolite compartmentation into chloroplast and cytosol. Under the assumptions indicated in the main text, ODEs for the MIDs of RuBP, F6P, FBP, G6P, G1P, and ADPG could be written for the ‘simple CBC model’ and ODEs for the MIDs of RuBP, F6P, FBP, G1P, G6P, UDPG, and ADPG could be written using the ‘compartmented CBC model’. A full list of model reactions and ordinary differential equations can be found in 4.2https://github.com/ankueken/MFA_Reg).To assess the quality of the fit we used: (i) the 4.3C. ohadii under LL we did not consider the last time point (see main text). Data on metabolite pool size, MID level and starch/sucrose synthesis rates that entered in the program above can be found in -6Having measurements of absolute pool size concentrations and MIDs at five time points 0, 5, 10, 20 and 40s, 4.4To obtain CI and The fit between model prediction and data can be accepted if the obtained rix N) .4.513C labeling of the total pool are the summation of the labeling dynamics observed in the plastid and those observed in the cytosol.For a metabolite present in both the chloroplast Hence, in the QP above matrix 4.6The SFCR approach also allows to treat the pool size parameter https://github.com/ankueken/MFA_Reg.Publicly available datasets were analyzed in this study. This data can be found here: Conceptualization, ZN; Experimental data, HT; Implementation, AK; Formal Analysis, AK, ZN; Writing – Original Draft, AK, ZN; Writing – Review and Editing, AK, HT, and ZN. All authors contributed to the article and approved the submitted version.


Central lymph node metastases (CLNM) in papillary thyroid microcarcinoma (PTMC) are common, but management through prophylactic central lymph node dissection (pCLND) remains controversial. In this study, the independent predictors of CLNM in PTMC were retrospectively studied based on ultrasound and pathological data, and we aim to establish the prediction model to predict CLNM in PTMC.This study included a total of 1,506 patients who underwent thyroid surgery for PTMC at the First Affiliated Hospital of Chongqing Medical University from 2015 to 2018. Ultrasound and clinicopathological features were summarized and analyzed. Univariate and multivariate analyses were performed to determine the risk factors associated with CLNM. The prediction model is established and verified according to the multivariate analysis results. The Kaplan–Meier curve was used to evaluate the effect of CLNM on survival.The CLNM rate was 44.5% . Multivariate analysis showed that men, younger age, smaller diameter, ETE, microcalcification, without Hashimoto’s thyroiditis, and multifocal were independent risk predictors of CLNM. Nomogram has a good discriminative ability , and the calibration effect is good. In the DCA curve, the CLNM prediction model performed better net benefit given any high-risk thresholds. The median follow-up time was 30 months (12–59 months), 116 cases were lost, and the follow-up rate was 92.8% . Of the 1,506 patients included, 12 (0.8%) experienced recurrence.The likelihood of CLNM can be objectively quantified before surgery by using this reliable and accurate nomogram that combines preoperative ultrasound with clinicopathological features. Clinicians can use this nomogram to assess central lymph node status in patients with PTMC and consider prophylactic CND in patients with high scores. Presently, thyroid carcinoma incidence happens more frequently, making it the most ordinary malignant neoplasm of the endocrine mechanism all around the world. Specifically, papillary thyroid microcarcinoma (PTMC) is defined as a subtype of papillary thyroid carcinoma (PTC) with a maximal size of less than 1 cm, accounting for 30% of PTC cases . The reaIt is important to balance the harms and benefits of pCLND in patients with PTMC for individualized and precise treatment. It is reasonable not to perform pCLND in low-risk PTMC patients. This was because it would reduce the extent of the procedure and the associated perplexities, particularly with regard to the protection of peripheral nerves, and facilitate the patient’s postoperative recovery. In high-risk PTMC patients, the implementation of pCLND can achieve effective radical treatment of the tumor. Therefore, this paper not only identified risk factors for CLNM through intraoperative and preoperative findings but also established a predictive model for CLNM in patients with PTMC. This accurate and intuitive nomogram offered the possibility to objectively quantify CLNM preoperatively. At the same time, it achieved good utility and convenience in developing individualized treatment plans for patients.The local institutional ethics committee backed up this retrospective research. Informed consent was waived after careful review by the ethics committee as there was no patient interest or privacy involved. The records of patients with PTMC who underwent surgery from January 2015 to December 2018 at the Department of Endocrinology and Breast of The First Affiliated Hospital of Chongqing Medical University were retrospectively reviewed. The total number of patients went through not less than a physical check, cervical ultrasound checking, and fine needle aspiration (FNA) before receiving surgery. Patients were the most vulnerable.Patients were excluded from this research on the condition that they owned any of the elements below:Non-PTMCs or mixed-type PTMC (14)Reoperation or other head and neck surgery history (55)Distant metastasis at diagnosis (7)The radiation exposure during childhood or family PTMC history (3)Incomplete clinicopathological data (144)Those not to be follow-up after receiving surgery (116)Adolescent (9)In total, 350 patients were excluded; additionally, 1,506 eligible patients were ultimately included in this research Figure 1After the patient finds a nodule of the thyroid through imaging inspection or palpation for the first time, we first perform the patient’s thyroid function test and the neck ultrasonography for diagnosis. Second, for nodules with suspected malignancy, the fine-needle aspiration (FNA) would be implemented. After all inspections are completed, we will communicate with the patient and their family members about the surgical method and surgical risks. The lobectomy plus ipsilateral central lymph node dissection (CLND) was the foundational therapy in a surgical manner. The total thyroidectomy plus bilateral multifocal CLND was deeply implemented for the cases suffering from bilateral multifocal tumors, tumors located in the thyroid isthmus with extrathyroidal extension (ETE), suspicious metastatic lymph nodes by preoperative examination or intraoperative frozen section biopsy showing CLNM. Lobectomy was stated as the removal of the implicated lobe, including the pyramidal lobe and the isthmus. Total thyroidectomy (TT) was stated as the removal of the two lobes: the pyramidal lobe and the isthmus lobe. Central lymph node dissection (CND) encompassed the removal of the pretracheal, prelaryngeal, and both the left and right paratracheal nodal basins . We routWe included the relevant data from ultrasonography and clinicopathological characteristics in the analysis. Data gathered included tumor size, age, multifocality, sex, ETE, preoperative cancer-related suspicion, central LN status, along with US features including shape, margin, location, echogenicity, length/width rate > 1, microcalcification emergence, the status of blood flow, and discontinuity of the capsule. Age has been dichotomized in view of the present stage criteria at 55 years. The biggest size of the neoplasm has been stated. The multifocalities have been characterized as > 1 overall neoplasm focus . In one single lobe, two or more PTMC foci were unilateral multifocality, whereas two or more PTMC foci in one lobe plus isthmus or both lobes were bilateral multifocality. Diagnosis of Hashimoto’s thyroiditis (HT) was made on the basis of the pathological data. ETE was based on pathological data and stated as the primary neoplasm spreading through the thyroid capsule to the perithyroidal soft tissue, including involving strap muscles or perithyroidal fat, or spreading to circumambient structures including trachea, larynx, recurrent laryngeal nerve, esophagus, skin, subcutaneous soft tissue, internal jugular vein, or carotid artery . The twoFor the total number of sick people, the initial follow-up was set at 1 month postoperatively; moreover, evaluation consisted of palpation and measurement of the levels of serum TSH. Serum thyroglobulin (Tg), along with Tg antibody levels, is merely for the patients who have undergone the overall thyroidectomy. Ultrasonography was performed 6 months after surgery. After that, the patient was re-examined every 6–12 months. On condition that the unstimulated Tg levels ≥ 2 ng/ml and stimulated Tg levels ≥ 20 ng/ml or serially elevated serum Tg (Tg-negative antibodies) are found, or if tumor recurrence is suspected on imaging studies , we willp-value was displayed in the univariate analysis. A significance level of 0.05 was considered statistically significant for all statistical analyses. Prior to multivariate analysis, abnormal influential observations were examined using Cook’s distance, and the abnormal line was set as greater than six times the mean of Cook’s distance. Observations beyond the abnormal line were excluded from the multivariate analysis. The logistic regression model was built for the prediction of CLNM with all candidate factors as an entry in the independent variables, and then the variables were selected stepwise. The estimated coefficient of each selected variable and its corresponding 95% confidence interval were calculated, along with the p-value of each estimated coefficient. The model’s prediction discrimination was evaluated by C-index and ROC curve. AUC was exhibited with the ROC curve. The model’s calibration ability was evaluated by a calibration plot. The Kaplan–Meier curve was plotted for the overall survival grouped by CLNM, and the log-rank test was conducted accordingly. At last, decision curve analysis (DCA) was carried out to evaluate the net benefit of the prediction model with different risk thresholds used. In addition, the clinical impact curve was plotted along with the DCA curve to evaluate the discrepancy between the number of predicted positive and true-positive observations with different risk thresholds used.Categorical variables were summarized as numbers (percentage), and continuous variables were summarized as means (± standard deviation). One-way ANOVA test was conducted to test the correlation relationship of each variable with CLNM, and the via one-way ANOVA test were sex, length, invasion, preoperative clinical suspicion of CLNM, Hashimoto’s thyroiditis, laterality, focal infection, ETE, age, and diameter. Women accounted for 83.3% in the no-CLNM group and 65.2% in the CLNM group. Patients without preoperative clinical suspicion of CLNM accounted for 99.2% in the no-CLNM group and 83.7% in the CLNM group. Unilateral accounted for 93.9% in the no-CLNM group and 82.7% in the CLNM group. Unifocal accounted for 86.7% of the no-CLNM group and 61.2% of the CLNM group. Patients without ETE accounted for 97.6% of the no-CLNM group and 89.0% of the CLNM group. The average age in the no-CLNM group is 45.02 (SD = 12.3), and in the CLNM group, it is 41.46 (SD = 10.68). The average diameter in the no-CLNM group is 6.64 (SD = 2.07), and it is 7.03 (SD = 2.08) in the CLNM group. Apart from these factors, the rest of the factors do not show a strong discrepancy in terms of the distribution between the no-CLNM and CLNM groups.The median follow-up time is 29 months. The baseline characteristics of risk factors for CLNM are shown in The influential observations were examined by Cook’s distance, as shown in p-value. Among the selected variables, the significant variables were sex, ETE, Hashimoto’s thyroiditis, focal infection, age, and diameter. Compared to men, women are less likely to experience CLNM, and their OR is 0.32 (95% CI: 0.22–0.45). Patients with ETE are much more likely to experience CLNM . Patients with Hashimoto’s thyroiditis are less likely to experience CLNM . Compared to unifocal infection, the odds of experiencing CLNM is 4.33 (95% CI: 3.04–6.17) times higher for multifocal infection. The age increases by 1 year, and the odds of CLNM would decrease by 0.03 . If the diameter increases by 1 mm, the odds of CLNM would increase by 0.08 . Patients with microcalcification are more likely to have CLNM , and coarse calcification is a protective factor for the occurrence of CLNM .A multivariate analysis of the risk factors for CLNM was carried out using a logistic regression model. Data were randomly split by a ratio of 3:2 as training dataset and a validation dataset, respectively. All candidate variables were included at the beginning of the model selection and were selected stepwise. The selected variables are shown in The final model achieved a C-index of 0.755 on the validation dataset. ROC curves for the training and validation sets are shown in p-value for the log-rank test is 0.3, which means that there is no significant difference between CLNM and no-CLNM group in terms of the recurrence rate. Both groups had a very low recurrence rate  after the initial surgery until December 2019. The interesting event is a recurrence of the PTMC. The nce rate Figure 6PTMC is a subtype of PTC that is less than 10 mm in diameter. In recent years, its incidence has gradually increased due to advances in ultrasonography and the importance individuals place on physical examination , 25. DesIn this research, the incidence of CLNM was 44.5%, which was in line with the results of earlier studies , 8. As oFor a long time, age was for a clinically important prognostic factor in PTMC. Research by Ito found that younger age was only an independent indicator of PTMC progression (included: (1) increase in volume; (2) new manifestations of LNM; (3) clinical disease progression with an increase in tumor volume of 12 mm or greater; and new manifestations of LNM). In other studies, age had the same effect on PTMC , 36, thap < 0.0001). It was traditionally assumed that the larger diameter of the tumor, the more aggressive it would be. ATA guidelines indicated that pCLND might be considered for patients with advanced primary neoplasms and clinically involved lateral cervical lymph nodes (cN1b). In contrast, in patients with marginal (T1 or T2), clinically negative PTC, and noninvasive patients  and the second significant variable in the nomogram.Bilateral or multifocal tumors might be caused by a single primary tumor spreading through the gland or by multiple concurrent primary tumors . It occuUltrasound was a convenient and quick way to evaluate PTMC. It might contain low central lymph nodes (grade VI) due to the presence of the thyroid or some anatomical areas  that were not clearly visible on ultrasound . RetrospETE was a tumor that breached the thyroid capsule. After the thyroid capsule ruptured, the tumor was more likely to invade lymph nodes through the lymphatic vessels on the capsule surface. It involved the pharynx, strap muscles , thyroid-bonding, larynx, prevertebral fascia, esophagus, mediastine, and even carotid artery, along with the PTMC artery. Various studies linked ETE to the invasiveness of PTMC. PTMC occurred when extrathyroidal structures were attacked and was shown to be a risk factor for CLNM , 51. AccThe results of the follow-up indicated that few patients suffered from central lymph node recurrence, which confirmed the reliability of examining the pathological status of the central lymph nodes. From the survival analysis, it was concluded that there was no significant difference in recurrence between patients with and without CLNM. However, some subclinical central lymph node metastases may lead to recurrence and distant metastasis. In addition, there is still controversy over whether PTMC patients need to undergo pCLND under domestic and foreign guidelines. Therefore, in order to achieve more accurate and individualized treatment, the model of this study was established to guide clinicians in selecting central lymph node dissection according to the scores, which were calculated based on disease status, so as to avoid unnecessary CLND and reduce postoperative complications. Moreover, in order to avoid recurrence, technical difficulties, hypoparathyroidism, and other complications caused by reoperation, CLND is still recommended when CLNM is found before or during surgery.This research still had some shortcomings: (1) It was only a single-center retrospective research. Additionally, selection bias was inevitable. (2) Deficiencies in external validation. Relevant prospective multicenter clinical research should be implemented in the coming years to assess the validity of the proposed model. (3) The relatively short follow-up period in this study did not allow for the analysis of potential confounding factors for future metastasis. Therefore, more data and longer follow-ups are needed in the subsequent work. On the other hand, improving existing proven models, such as by including new variables that have been shown to work (such as BRAF-V600E), can improve the accuracy of the “old” nomogram. In summary, a quantitative CLNM prediction model for PTMC was formulated in this paper. Physicians can use the nomogram in this model to assess patients with PTMC in clinical practice based on clinicopathological features. For PTMC with a high possibility of CLNM, unnecessary pCLND may be avoided.Above all, our study found that CLNM was independently associated with sex, age, diameter, ETE, calcification in ultrasound, Hashimoto’s thyroiditis, and focal infection. Using the above variables, we construct a nomogram. Clinicians can use the nomogram to assess central lymph node status in patients with PTMC. For patients with high scores, pCLND and meticulous postoperative evaluation can be considered.The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Ethics Committee of the First Affiliated Hospital of Chongqing Medical University. Written informed consent to participate was waivered by the ethics committee.DW and JH: designed this study, analyzed the data, and wrote this paper. DW, JZ, CD, and ZY: data collection. XS: designed this study, conducted the examination, and modified the manuscript. All authors contributed to the article and approved the submitted version.