CYP3A5 gene were associated with lower plasma midazolam levels. The concentration median in patients was 0.0 ng/mL, while in patients with the normal (C) allele, it was 438.17 ng/mL (Q25 135.75–Q75 580.24), p = 0.005. The midazolam plasmatic concentration in female patients with the minor (T) allele was 0.0 ng/mL (Q250.00–Q75204.3), while in patients with the normal (C) allele median it was 459.0 ng/mL (Q25296.9–Q75789.7), p = 0.002. Analysis of the dominant model for the rs2740574 variant in CYP3A4 revealed a median of 0.38 L/kg (Q250.02–Q751.5) for the volume of distribution parameter in female patients with the normal T allele, while female patients with the minor C allele showed a median of 18.1 L/kg (Q257.5–Q7528.7) p = 0.02. Our results suggest an altered midazolam metabolism due to the presence the allelic rs2740574 variants of CYP3A4 and rs776746 of CYP3A5, and also the strong influence of age and sex.Midazolam is a drug that is metabolized by cytochrome P450 (CYP450) enzymes, particularly CYP3A4 and CYP3A5. The present study aimed to determine the sex and age influence on association of CYP450 polymorphism with midazolam levels in critically ill children. Seventy-two DNA samples were genotyped by real-time PCR. Children ≤ five years of age who carry the rs776746 (T) allele in  CYP3A4*1.001 and CYP3A5*3.001 polymorphisms , one study reported no significant differences in the pharmacokinetic parameters of midazolam between Caucasian and African American healthy volunteers [CYP3A5 had increased plasma midazolam levels three hours after infusion discontinuation and greater drug clearance [CYP450 genes  have been analyzed, and an association of toxicity and response to treatment was reported in patients with solid tumors with the allelic CYP3A4 and CYP3A5 variants [CYP450 polymorphism with midazolam levels in critically ill children.Midazolam is a benzodiazepine used at the Pediatric Intensive Care Units, from premedication to maintenance of general anesthesia, mainly for effects such as sedation, hypnosis, decreased anxiety, and anterograde amnesia during diagnostic or therapeutic procedures ,2. Midazlearance . The timlearance . Physiollearance ,9. Most learance ,11. Excelearance ,13, a smlearance ,15, currlearance , or contlearance . In prevvariants ,19, so iThis study was approved with number INP-068/2014 by the Research, Biosafety, and Ethics Committee (IRB 00008064) of the National Institute of Pediatrics. This cohort is special because all patients requiring to be treated in both units during the study period were included. All informed consent forms were signed previously by parents or legal guardians.Pediatric patients 1–17 years old, of any sex, treated in the Pediatric Intensive Care Unit and Emergency Unit and sedation with midazolam through continuous intravenous (IV) infusion, starting at a 200 µg/kg/h (range 200 to 1200 µg/kg/h) rate and then adjusted according to the sedation level during hospitalization, were included. Patients with suspected hepatic or renal failure, who had undergone partial hepatectomy or partial or total nephrectomy, multiple organ failure, hypersensitivity to benzodiazepines, who had been treated with CYP3A4 inhibitors such as omeprazole, erythromycin, or clarithromycin, with blood transfusion during the last 6 months, who had suffered septic shock, or had any type of leukemia, were excluded. The elimination criteria were: incomplete sampling, serious adverse effects during treatment, and acute renal or hepatic dysfunction developed during the study.Concomitant medications were recorded during the hospital stay and classified as CYP3A4 substrates, inducers, or inhibitors.g , and plasma was separated and placed in microcentrifuge tubes, labeled, and stored at −80 °C  until analysis.Peripheral blood samples (3 mL) were collected in EDTA Vacutainer tubes  and centrifuged for lymphocyte isolation and further DNA extraction. For the pharmacological analysis, three blood samples (3 mL each) were obtained from every patient. The first sample was taken once the treatment with generic midazolam  was started at an initial IV infusion dose of 200 μg/kg/h, and was collected in a heparin Vacutainer tube . The next two samples were collected at three and twenty-four hours after the end of treatment. All samples were centrifuged for 10 min at 800× CYP3A4 and rs776746 in CYP3A5. Genotype analysis was performed by allelic discrimination assay using TaqMan probes. For each allelic discrimination assay, specific probes labeled with different fluorophores at the 5’ end, VIC for allele 1 and FAM for allele 2 were obtained from Applied Biosystems, with both probes characterized by having a “quencher” (TAMRA) at the 3’ end which, while the probe remains intact, inhibits fluorescence emission, and a passive signal (ROX) will be used. The PCR reaction was carried out as follows in an microtube, and the following was added for 48 samples: 240 μL of TaqMan PCR Master Mix 2X, 24 μL of 20X of each probe , and 120 μL of nuclease-free water (Ultra PureTM) DEPC treated water, Invitrogen, Carlsbad, CA, USA. Fluorescence for each sample was quantified in a StepOne using SDS 2.2.1 software for allelic discrimination .Genomic DNA was isolated from peripheral blood leukocytes (n = 72) using the QIAmp DNA Mini kit . For genotyping the allelic variants, rs2740574 in Finally, we analyzed the SNPs based on inheritance models: dominant  and recessive , by sex , and age (≤five and >five years old) stratification.The method is simple and reliable by high-performance liquid chromatography (HPLC) and requires only a small volume of plasma 200 µL), which has been previously validated and published by our group [0 µL, whiAnalytical validation met the following criteria: selectivity, linearity, reproducibility, repeatability, quantification limit, and stability to different conditions according to Official Mexican Standard NOM-177-SSA1, 2013 ,22.All reagents were analytical or HPLC grade. Midazolam hydrochloride, acetonitrile, and diethyl ether were obtained from Sigma , propranolol from ICN Biomedicals Inc. , potassium dihydrogen phosphate and sodium hydroxide from Merck , and deionized water was obtained from Elix 35/Milli Q water purification system .v/v) with an isocratic flow rate (1 mL/min); absorbance was monitored at 220 nm. Data were obtained and processed using Empower Pro™ version 2 software .A Waters  chromatographic system with a 515 pump, a 717 autosampler, a 2487 UV detector, and a degasser  were used. Analytical separation was realized in a reversed-phase column Pursuit C18  maintained at room temperature (25 °C), while the autosampler was kept at 4 °C. The mobile phase was 35 mM phosphate buffer solution pH 4.4 (adjusted with phosphoric acid) and acetonitrile . The solution was then vortexed for 1 min and centrifuged at 800× The data were adjusted to one-compartment pharmacokinetic model for calculating the pharmacokinetic parameters and the following formulae were considered:Elimination constant (ke) = lnCp2 − ln Cp1/t2 − t1; elimination half-life (t1/2) = ln2/ke; volume of distribution (Vd) = D/Cp0 and clearance (CL) = ke × Vd; where Cp is the plasma concentration, t denotes time, D is the dose administered, and Cp0 is the plasma concentration at zero time ,24.Once the pharmacokinetic parameters were obtained, stratification by sex  and age (≤five and >five years) was performed for statistical analysis.25–Q75) of the midazolam concentrations at three- and twenty-four hours post-infusion were compared between the allelic variants, stratified by sex and age. The Mann–Whitney U test between age, sex groups, and comparison between the presence of allelic variants and midazolam levels was performed. For the comparison of plasma levels of the drug at three- and twenty-four hours, the Wilcoxon test was used. A p-value < 0.05 indicated a statistically significant association. Variables were stratified by sex  and age (≤five and >five years old).The median and the interquartile range  was used.Eighty-one patients were invited to participate in the study, of whom 72 accepted and met the study criteria and were included by means of an informed consent that they signed. Forty-three of these patients were male and twenty-nine were female. The demographic and clinical data are shown in The genotyping analysis was performed on all 72 patients (data not shown), while, for the determination of pharmacokinetic parameters, nine patients were excluded because their clinical status was compromised due to organ failure, seven subjects were eliminated due to death, and one participant did not have a sufficient blood sample, leaving a total of 55 patients, of whom 21 were female and 34 were male.CYP3A4 and rs776746 in CYP3A5) stratified by age (≤five and >five years old).The genotype association’s magnitude with the midazolam plasmatic levels was determined through a comparison of the medians of the dominant and recessive inheritance models for both allelic variants (rs2740574 in 25 135.75–Q75 580.24), while in patients with the TT+TC (minor), the genotype was 0.0 ng/mL (Q250.00–Q750.00), p = 0.005  genotype was 438.17 ng/mL (Q = 0.005 .250.00–Q75196.64), and patients with the minor genotype had a midazolam concentration median of 0.00 ng/mL (Q250.00–Q750.00), p = 0.5. In children older than five years old, no statistically significant association was found.After twenty-four hours, patients with the major genotype had a concentration of 0.00 ng/mL  for both allelic variants (rs2740574 and rs776746) were compared to obtain the genotype association’s magnitude with the midazolam plasmatic levels.25296.9–Q75789.7) for the midazolam plasmatic concentration was found in female patients with the major genotype, while in patients with the minor genotype it was 0.0 ng/mL (Q250.00–Q75204.3), p = 0.002 , and patients with the minor genotype had a midazolam concentration median of 0.00 ng/mL (Q250.00–Q7579.0), p = 0.3. In male patients, no association was found with either allelic variant , stratifying by sex .250.02–Q751.5) for the Vd parameter in female patients with the TT (major) genotype. Meanwhile, female patients with the CC+CT (minor) genotype showed a median of 18.1 L/kg (Q257.5–Q7528.7) p = 0.02  .CYP3A5*3 and CYP3A4*1B polymorphisms, Miao et al. (2009) [CYP3A5 rs776746 variant comparing to girls with the normal allele, showing a decrease in plasma concentrations since three hours after the end of treatment and an increase in the volume of distribution females who carry the CYP3A4 rs2740574 variant comparing to girls with the normal allele.Sex-related differences in pharmacokinetics and pharmacodynamics include noticeable physiologically related differences, such as body fat content and hormonal influence, among others . The posCYP3A4 rs2740574 (CC+CT) variant had a larger volume of distribution compared to patients with the normal genotype (TT).In women, the volume of distribution of lipophilic drugs is increased ,37,38, iCYP3A5 rs776746 variant (TT+TC) did not show plasma levels of the drug compared to patients with the normal genotype (CC) three hours after the end of treatment.The same dose of a lipophilic drug will have a lower serum concentration in a female compared to a male with the same body weight because there is a relatively larger lipophilic compartment in which the drug resides. In our study, we observed that in the dominant model, female patients with the CYP3A5 variant in children were not found.Most studies have found no significant sex differences in midazolam metabolism ,11,39,40Studies have already been realized in adults evaluating the effect of age-related blood and plasma drug concentrations, with contradictory results, however. From seven studies conducted with intravenous midazolam, only five evaluated the age, and, just in one, with men participating, a significantly lower midazolam clearance was found as the age increased . HoweverCYP3A5 rs776746 variant; compared to children with the normal allele, they showed a decrease in plasma concentrations from three hours after the end of treatment, inferring an increase in clearance of the drug, and could require more doses of the drug to remain sedated, and also we could not find age-related differences in clearance because of the small sample size in this group. Some reports mentioned that the pharmacokinetics of xenobiotics could differ widely between children and adults due to physiological differences, metabolism, protein binding, immaturity of enzyme systems, the volume of distribution, and clearance mechanisms [This is the first study to show differences in midazolam plasma concentrations in critically ill patients ≤5 years old who carry the chanisms ,48,49,50Among the limitations of the study was the small sample size, and death and organ failure during follow-up, which made it difficult to determine the pharmacokinetic parameters. Possible adverse reactions with concomitant medications were not reported in the clinical records, and no information was found on the use of assessment scales in this population.This is a helpful strategy, since the results obtained could lay the foundations for the design of personalized therapeutic schemes, resulting in the best cost–benefit and monitoring of adverse events for different drugs metabolized by CYP3A in the Mexican pediatric and probably in the Latin American population.CYP3A4 and rs776746 of CYP3A5, and the strong influence of age and sex.Our results suggest an altered midazolam metabolism due to the presence of the allelic rs2740574 variants of CYP3A5, the plasma concentrations of midazolam decreased at three hours after the end of treatment compared to children with the normal allele. They may require more doses of midazolam to remain sedated.In critically ill patients ≤5 years old carrying the allelic rs776746 variant in CYP3A5 rs776746 variant had decreased plasma concentrations three hours after the end of treatment compared to girls with the normal allele, and also showed an increase in the volume of distribution in females who carry the CYP3A4 rs2740574 variant compared to those with the normal allele, and likely could need adjustment of doses of midazolam to achieve the desired effect.Females who carry the CYP3A4 and CYP3A5 genes.Further studies in larger samples are required to validate these findings and the relevance of these variants in


Anti-leucine-rich glioma inactivated 1(LGI1) encephalitis is one rare autoimmune encephalitis which is accompanied by inflammatory responses. (Anti-leucine-rich glioma inactivated 1 (anti-LGI1) encephalitis is an autoimmune disease mediated by inflammatory responses.)This study aimed to investigate the Chitinase 3-like 1(CHI3L1) in anti-LGI1encephalitis patients and evaluate its association with modified Rankin Scale (mRS) score in anti-LGI1 encephalitis at admission and 6 months follow-up.(This study looked into the relationship between Chitinase 3-like 1 (CHI3L1) and the modified Ranking Scale (mRS) score in anti-LGI1 encephalitis patients at admission and 6 months later.)Thirty-five patients with anti-LGI1 encephalitis and 22 patients with non-inflammatory neurological disease were enrolled in this study. Cerebrospinal fluid (CSF) and serum levels of CHI3L1 were measured by enzyme-linked immunosorbent assay.  by performing an enzyme-linked immunosorbent assay.)Patients were evaluated for mRS score at admission and at 6 months follow-up.(We recorded the mRS score of the patients at admission and 6 months later.)CHI3L1 levels in CSF and serum were highly elevated in patients with anti-LGI1 encephalitis at admission compared those with the controls. Additionally, patients presenting with cognitive impairment had significantly higher CSF CHI3L1 levels and mRS scores than those without cognitive impairment symptoms. Patients presenting with only faciobrachial dystonic seizures at admission had lower CSF CHI3L1 levels than those with other symptoms. Finally, CSF CHI3L1 levels were positively correlated with CSF lactate levels.CHI3L1 level in CSF is correlated with the severity and prognosis of anti-LGI1 encephalitis.  Anti-leucine-rich glioma inactivated 1 (anti-LGI1)encephalitis is a type of rare autoimmune encephalitis –4. ChitiWe retrospectively studied 35 patients with anti-LGI1 encephalitis. All included patients met the diagnostic criteria for anti-LGI1 encephalitis . The conThe local ethics committee at the Qilu Hospital of Shandong University approved this study, and all participants provided written informed consent.CSF and serum samples were centrifuged immediately after collection and then stored at −80°C for testing. Commercially available sandwich ELISA kits were used according to the manufacturer’s instructions to quantify CHI3L1 in the CSF and serum. (Elabscience Biotechnology Co. Ltd).p < 0.05 was considered statistically significant.All statistical analyses were performed using SPSS 26. Continuous variables of normally distributed data were presented as the mean ± standard deviation. Non-normally distributed datawere presented as the median and interquartile range (IQR). Groups were compared using Student’s t-test. Since CHI3L1 concentrations are non-normally distributed, we compared the anti-LGI1 encephalitis group and the control group using the Mann Whitney U. Spearman’s test was used to assess the correlation between CSF CHI3L1 levels and mRS scores. Correlations between the CHI3L1 levels and the clinical data were analyzed by multiple linear regression. Receiver operator characteristic (ROC) curves were used to assess the discriminating power of CHI3L1 levels in anti-LGI1 encephalitis patients. A value of p=0.005). The multiple linear regression analysis also revealed that the mRS score at admission was positively associated with hyponatremia and negatively correlated with FBDS . The mRS score at admission was not associated with serum/CSF LGI1 antibody titers, abnormal brain MRI, CSF protein concentrations, CSF white blood cell count, CSF immunoglobulin G, A, and M titers, and CSF lactate levels.A Simple linear regression analysis revealed that the mRS score at admission was positively associated with hyponatremia and negatively associated with FBDS  and serum samples (n =30) from patients with anti-LGI1 encephalitis (n =35), and controls (n = 22) using an ELISA assay. Patients with anti-LGI1 encephalitis had significantly higher CHI3L1 levels in the serum and CSF than controls.  of CSF and serum CHI3L1 levels were 0.7377 and 0.6742, respectively  had significantly higher CSF CHI3L1 levels than those without cognitive impairment symptoms  had significantly lower CSF CHI3L1 levels than those without FBDS symptoms , and 6-months later  and with the patients’ CSF lactate levels . The multiple linear regression analysis showed that CSF CHI3L1 levels were positively correlated with the patients’ mRS score at the 6-month follow-up examination  and the patients’ CSF lactate levels .The simple linear regression analysis revealed that CSF CHI3L1 levels were positively correlated with the patients’ mRS scores at admission (β=0.448, However, the simple linear regression analysis and multiple linear regression analysis revealed no correlation between serum CHI3L1 levels and the mRS score of the patients at onset and 6-months follow-up and other clinical features.Autoimmune encephalitis is often accompanied by inflammatory cell activation and cytokineproduction during pathogenesis , 16. AntSome studies have indicated that elevated CSF CHI3L1 are associated with Alzheimer’s disease, multiple sclerosis and Parkinson’s disease , 25–29. The present study, that patients with anti-LGI1 encephalitis had elevated serum and CSF CHI3L1 levels. CSF CHI3L1 levels were correlated with mRS scores at admission and 6 months later. Moreover, CSF CHI3L1 levels were higher in anti-LGI1 encephalitis patients presenting with cognitive impairment than those without cognitive impairment symptoms. Note-worthily, patients presenting with only FBDS at admission had significantly lower CSF CHI3L1 levels than patients with other symptoms.These results imply that CSF CHI3L1 levels were associated with severity and outcomes. Most patients presenting with only FBDS at admission did not suffer from serious anti-LGI1 encephalitis. A recent Mayo Clinic study have reported that more than half of patients with anti-LGI1 encephalitis presented with FBDS . Those pOur results revealed that the mRS score at admission was positively correlated with hyponatremia, suggesting that hyponatremia is also related to the severity of the disease. One study demonstrated that the prognosis of anti-LGI1 encephalitis patients with hyponatremia is poor . In thisThere are some limitations to our study. First, the patients population remains relatively small. Second, the serum and CSF samples obtained in the follow-up examination were not studied.CSF CHI3L1 levels are correlated with the severity and prognosis of anti-LGI1 encephalitis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by ethics committee of the Qilu Hospital of Shandong University. The patients/participants provided their written informed consent to participate in this study.JL and HL, drafting/revision of the manuscript, data acquisition, study concept and design, and data analysis and interpretation. YW, major role in sample collection. XZ, SW and LL, revision of the manuscript, study concept and design, and data analysis and interpretation. All authors contributed to the article and approved the submitted version.


Over recent years, traditional manufacturing factories have been accelerating their transformation and upgrade toward smart factories, which are an important concept within Industry 4.0. As a key communication technology in the industrial internet architecture, time-sensitive networks (TSNs) can break through communication barriers between subsystems within smart factories and form a common network for various network flows. Traditional routing algorithms are not applicable for this novel type of network, as they cause unnecessary congestion and latency. Therefore, this study examined the classification of TSN flows in smart factories, converted the routing problem into two graphical problems, and proposed two heuristic optimization algorithms, namely GATTRP and AACO, to find the optimal solution. The experiments showed that the algorithms proposed in this paper could provide a more reasonable routing arrangement for various TSN flows with different time sensitivities. The algorithms could effectively reduce the overall delay by up to 74% and 41%, respectively, with promising operating performances. With the rapid development of the industrial internet, real-time communication technologies with deterministic low latency have become a critical requirement in many industrial sectors. For example, most industrial automation networks require end-to-end latency to be strictly controlled at no more than 1 millisecond  that areHowever, incompatible network protocols lead to problems such as incompatible applications, a lack of interoperability, difficulty in portability, and expensive costs for development, deployment, and maintenance. With the relentless efforts of the AVnu Industry Alliance and the IEEE TSN Working Group (formerly the AVB Working Group), TSNs  have emeThe continuous improvement of TSN-related standardization has become a research trend in the building of a comprehensive industrial internet communication system that is based on a TSN with deep integration of IT and OT . Among mWe proposed an improved genetic algorithm to solve the TT flow routing problem (GATTRP). We modeled the TT flow routing problem in the TSN systems of smart factories and transformed the problem into a multiple traveling salesmen problem (MTSP) to solve. Based on the existing genetic algorithms, we optimized the design of the genetic evolution operators and algorithm processes and finally, formed an improved genetic algorithm with faster convergence speed and better results;We proposed an adversarial ant colony optimization (AACO) algorithm to solve the non-TT flow routing problem. We modeled the non-TT flow routing problem in the TSN systems of smart factories and transformed the problem into a load balancing pathfinding problem for multiple priority flows within a directed graph. Based on existing ACO algorithms, we designed a novel pheromone update rule to balance the impacts of higher priority tasks and path length on pathfinding. The algorithm could effectively equalize the non-TT network load and reduce the network latency;We established a simulation experiment platform for the smart factory TSN communication system and evaluated the performance of the proposed algorithm through experiments. The results showed that both algorithms produced a certain improvement in the corresponding evaluation indicators, which matched our expectations.Currently, research on TSNs is still in the development stage and existing data flow route planning mechanisms are relatively simple. In our previous studies ,17,18, wThe remainder of this paper is organized as follows. In Our study combined several fields, such as time-sensitive network routing, task/volume-balanced MTSPs, load balancing routing, etc. We reviewed many studies from the related fields, which are listed below.TSNs are composed of series of IEEE technical standards, including precise time synchronization, network traffic shaping, network configuration, and other aspects. The IEEE TSN Standardization Working Group issued a standard amendment  to addreHeuristic algorithms are intuitively or empirically constructed algorithms that search for a feasible solution to each instance of an optimization problem at a limited cost . Since heuristic algorithms can usually find promising solutions in a reasonable amount of time when dealing with many practical NP-hard problems, they have become a research hotspot in recent years. Inspired by various phenomena, such as animal behavior and natural laws, researchers have proposed many novel and effective optimization algorithms and have proven their value in practical applications. For example, based on the gravitational search algorithm (GSA), which was inspired by the law of gravity and interactions between mass entities, Precup et al.  proposedM travelers visiting a portion of cities separately and each city (except the starting city) is only visited by any traveler once and, eventually, finding the minimum cost to finish traversing all of the cities [The traveling salesman problem (TSP) is a typical NP-hard combinatorial optimization problem, which comprises finding the best traversal route at the lowest cost  through a given number of cities, in which all cities are visited only once by a single traveler, except for the starting city . The MTSe cities . When M= of MTSP .The genetic algorithm (GA) has definite advantages for solving the task-balanced MTSP problem. Carter et al.  proposedWhen routing in large-scale network systems, the load balancing problem needs to be fully considered to avoid partial network congestion. To solve the load balancing routing problem, some existing clustering protocols for wireless sensor networks (WSNs) have appreciable reference value. The LEACH (low-energy adaptive clustering hierarchy) algorithm  was the There are researchers continuously trying to apply ant colony optimization (ACO) algorithms to solving the load balancing routing assignment problem. ACO is an intelligent optimization algorithm that optimizes practical problems by imitating the foraging behavior of ants in nature, which was first proposed by Italian scholar Marco Dorigo in the 1990s . RamamooV and each SW had multiple incoming and outgoing ports, which were responsible for forwarding the data frames received from the incoming ports to the corresponding outgoing ports, according to the routing list. The ESs could be hard real-time control units, such as servo drivers, or soft real-time or non-real-time units, such as sensors, cameras, mobile operating terminals, etc. To study the TSN routing problem within a complex network topology, the network was abstracted as a directed graph Based on the common TSN traffic classification method ,46,47, wAVB and BE data cannot be transmitted in hard real-time, as with TT data, so we defined them as the same type of data, i.e., non-TT data, in our study. In the following sections, we define the two different routing problems, TT and non-TT, and propose two different optimization algorithms to solve these routing problems.When the controller conducts time-triggered communication with servo motors within TT subnetworks, the communication method is the master–slave method, in which the controller acts as the master station to send control-type frames to each slave station and slave stations return the processed frames to the master station. In the IT–OT converged industrial control network considered in this paper, the technical idea of aggregated forwarding frames was adopted, in which the whole subsystem has only one frame that runs in a loop. For the master, all devices with I/O information are considered as “logical” devices and the address of the field device corresponds to the physical location in the control frame, according to the protocol. When a message passes through a slave device, the slave only needs to read the command data from the corresponding mapped address and simultaneously resend the feedback data to the same place, so the effective utilization of the message can reach up to more than 90%. The frame format for the control data and communication mechanism design are shown in In industrial control networks, the number of slaves that need to be traversed in single time cycle increases as the network size continues to grow and the time cycle that is required to traverse the nodes increases linearly. In this case, we considered a TT control network based on the idea of distributed control, as depicted in m SW nodes and periodically sent m control-type data frames  ensured that every node T was less than the preset threshold In Equation , (c1)– but with a looser delay constraint than TT flows. Multiple types of data flows, including TT, AVB, and BE, are sent from multiple ESs, as well as presequential SWs, which are connected to ingress ports in a single SW. To solve the composite traffic scheduling problem, the IEEE 802.1 Qbv standard  defined Typically, the SWs keep a total of eight priority queues, so the sequence of gates is Gate 7 to Gate 0. In the examples in this paper, “o” means gate open, “C” means gate closed, and each action is based on the time window. Correspondingly, the GCL can tell the time sequence of each type of traffic that is allowed to be sent to the output ports in a scheduling cycle, as shown in In this scheduling mechanism, the number of time windows allocated to each priority queue within a common time period is limited. Therefore, as the number of queueing flows increases, it inevitably leads to relative congestion, which eventually causes an increase in the time needed to complete the transmission of each single task. For non-TT flows, ESs connected to each SW may send multiple types of non-TT data with different priorities, such as audio, video or sensor signals. The destination of non-TT flows is basically concentrated on one centralized data server. In this case, when the routing mechanism assigns too many time-consuming non-TT flows to the same data link, it is difficult to meet the soft deadlines of all flows due to severe time slot contention, resulting in a waste of bandwidth resources. When planning routes for low-priority non-TT flows, we considered changing their routing scheme from simply taking the shortest routing approach. When there were already higher priority flows causing queue congestion, we considered “bypassing” the congested SWs in exchange for a relative balance of SW loads at the cost of a partial loss of route length.n nodes, the planning of m routes for m non-TT flows with the sets of source nodes of these routes was defined as kth non-TT flow was kth non-TT flow was denoted as n nodes of Based on the above conditions, the non-TT routing problem in TSNs could be defined as follows: in a given kth non-TT flow between kth non-TT flow between kth flow, thereby making We retained the description of the time delay from T was:Since the traffic scheduling principle of a TSN requires planning higher priority non-TT flows first, the kth non-TT frame had visited the node In addition, we defined When non-TT flows with multiple priorities participate in queuing within the same SW, TAS schedules all priority queues in units of time windows. In a single SW, as the number of flows participating in the priority queue increases, the WCD of each data flow gradually increases, which is brought about by the deterioration of the queuing situation. The optimization goal of the AACO algorithm was to avoid unnecessary queuing as much as possible and, therefore, reduce the overall WCD for all flows, realize the relative balance of the TSN data link loads, and improve the response speed and bandwidth utilization of the non-TT networks, namely:kth non-TT flow no more than one time, so that there were no loops in the routes. Constraint (c3) ensured that the m non-TT flows all converged at the destination node T was less than the preset threshold In Equation , (c1)–(cm ant colonies, each of which represented a non-TT flow. Unlike classical ACO algorithms, there were strong and weak colonies among these ant colonies and the strength of the ith ant colony was equal to the priority ith non-TT flow. When the pheromone was updated, the pheromone left by the ant colonies with higher priorities greatly suppressed the pheromone increment of the weak colonies and reduced the probability of picking identical routes. Weak ant colonies would rather choose detouring than repeating the route of stronger colonies. Meanwhile, the distance to be detoured was also taken into account. When the extra cost of detouring was too large, weak ant colonies would choose to participate in the queuing process with stronger ant colonies, after weighing up the costs. Taking a small network consisting of six SWs with To solve the problem described above, we proposed a novel AACO algorithm to achieve load balancing routing among multi-priority non-TT flows. We set up m non-TT flows, we initialized m ant colonies with Z ants per colony. All ants from the ith colony were located at the corresponding source node m different pheromone concentration values for each edge in E, denoted as For the route assigning task of lth ant in the kth ant colony chooses to visit t, kth ant colony, and We then calculated the state transition probability of the ants and selected the next node to visit by roulette, based on the state transition probability. The state transition probability was calculated as follows:kth colony:kth population, which is accumulated by the pheromone increment of each ant Q is a preset pheromone increment constant. In each time window, the pheromone kth pheromone increment, the pheromone increments of the previous s and whose destination is p. The colonies with higher priorities than the kth colony were Compared to existing ACO algorithms, the main improvement of the ACO in this paper was the calculation of pheromone increment Algorithm 2: AACO algorithm for non-TT flow route planning.Based on the above theory, the pseudo-code of the AACO algorithm proposed in this paper is described in Algorithm 2.m new routes. Starting from the next time period, all non-TT flows were transmitted with m new routes to complete the registration of the new non-TT application.We added a non-TT routing program based on the AACO into the central control server of the smart factory TSN to optimize non-TT routing. When a new non-TT application sent a registration message to apply to set up a data flow with the data server, the transmission continued normally while the AACO process was created to perform offline route planning. The ES node where the application was running was treated as the source node AACO is also based on a heuristic algorithm and the average time complexity of its convergence is complex and can be influenced by various factors, including the number of non-TT flows, number of ants, number of iterations, and randomness. Thus, it was difficult to produce an accurate expression. For its convergence, we could only estimate its time complexity as For the non-TT routing problem in TSNs, one of the contributions of this paper is the selection of ACO as the basis of algorithm optimization. By reasonably adjusting the impact factor of the pheromones of stronger ant colonies on the update of the pheromones of other ant colonies, the improved ACO could effectively balance the relationship between the greedy principle and load balancing in order to better adapt to the TSN traffic shaping mechanisms, which is harder or even impossible for other algorithms to handle.In this section, we present simulation experiment environment that was used to evaluate the performances of the algorithms proposed in this paper when adopted in the routing scenarios of large-scale TSN networks in smart factories.Currently, there are two mainstream experimental frameworks in the field of TSN research, namely NeSTiNg  and COREOn the experimental network, we deployed the end-to-end latency optimization methods proposed in ,54. For m TT frames were represented by line segments, and different routes were labeled with different colors. Finally, on the longest route, all propagation and processing delays that were incurred while passing through each subnetwork were summed to calculate the final output T. The preset simulation parameters are shown in m and took the average value as the final result for each statistic in order to avoid misguidance by randomness. In order to verify the effectiveness of the GATTRP proposed in this paper, we built a simulation experiment environment for the TT routing problem on the MATLAB® R2021b software platform and developed a test program based on Algorithm 1. The simulation program is available online at . We simum changed. The results are shown in As the TT flows traversed through each SW, they consumed a certain amount of processing delay. We set a random processing delay for each SW with maximum values of 10 μs, 15 μs, and 20 μs to simulate the change of the optimization target m had to be properly set within a reasonable range. In addition, we observed that the higher the processing delay of the subnetworks, the better the optimization effect. Therefore, the larger the overall network size, the more obvious the optimization effect of the GATTRP.From the experimental results, we could confirm that it was feasible and effective to solve the routing problem of TT nodes based on the idea of solving the MTSP. With an increase in the number of salesmen, SW hops on the routes decreased and the propagation and processing delays on the longest route were correspondingly reduced. Considering that the number of parallel ingress and egress ports of the central control server is limited and that network wiring has comprehensive limitations from environment and financial costs, the value of To illustrate the operational efficiency of Algorithm 1, we conducted comparative experiments using several other algorithms. In order to verify the effectiveness of Algorithm 2, as proposed in this paper, we developed a non-TT routing test program, which is available online at . The simm non-TT flows from industrial sensors to a data server and compared the AACO to two other routing algorithms, as depicted in m value of 5 and set the starting and end nodes as the two farthest nodes within the network. The starting node was labeled with a diamond and the end node with a pentagram. The route of each non-TT flow was represented by a line segment, which were distinguished from each other by color.We simulated a case of sending m flows, the obtained multiple paths completely coincided. In addition, we made appropriate modifications for our problem model based on the LB-DRR method proposed by Ojewale et al. [We developed a routing program based on the SPB algorithm proposed by  and the e et al. . The resWhen a flow chose to detour, the more SWs the route passes through, the longer the processing and propagation delays. We defined the sum of processing and propagation delays as the detouring delay. In addition, from the simulation results presented in This paper highlighted the routing problems in TSNs. Different from traditional bus-based real-time communication networks, TSNs have two main routing problems: those for TT flows and those for non-TT flows. To address the specific communication needs of smart factories, we classified network data from smart factories and analyzed the communication requirements and transmission mechanisms of various types of traffic. By mathematically modeling the problem, we transformed the routing problem into an MTSP problem and a load-balanced multi-priority route assignment problem. For the MTSP problem, we proposed an improved algorithm named GATTRP. For the multi-priority route assignment problem, we proposed an improved algorithm named AACO. The simulation results showed that transforming the traversal problem of TT flows into an MTSP can effectively reduce the traversal cycle time and that the GATTRP proposed in this paper has a strong convergence performance. For non-TT flows, the AACO proposed in this paper is more comprehensive and exhibits an excellent performance and better results. However, there were also some limitations in this paper. For upper-layer industrial applications, the transmission mechanisms and requirements of AVB flows and BE flows, which belong to the same non-TT flow type, are not exactly the same. If they can be distinguished for refined routing, the the routing performance could be further improved. In the future, we will focus on improving the routing algorithms for AVB flows.


Aelurodon ferox and Aelurodon stirtoni) are largely complete, while the baculum from Carpocyon compressus is complete but still embedded in matrix that obscures some of its features. The bacula of Paratomarctus euthos and Desmocyon thomsoni are incomplete, but they provide useful information nonetheless. These borophagine bacula are similar to extant canines in being robust, having a urethral groove, and a simple distal end. These features suggest that the Borophaginae had long-lasting copulation and possibly spontaneous ovulation, similar to the extant canines. However, unlike the straight baculum of extant canines, borophagine bacula are ventrally curved (arched), which is also observed in the hesperocyonine baculum. The implication of this curvature for the reproductive biology of these animals remains unknown.The baculum of mammals offers the opportunity to study the reproductive biology of extinct species given that it is a fossilizable part of the male genitalia and that its size and shape correlate with several aspects of the reproductive biology of extant mammals. Fossil bacula, however, are rare. Currently, bacula have been described from only two extinct species of canids, one from the subfamily Caninae and the other from the extinct subfamily Hesperocyoninae. Here, I describe the bacula of five extinct species of Borophaginae, each of which was found with other skeletal elements that have enabled identification to the species level. Two specimens ( While neontologists can observe directly the morphology, behavior, endocrine responses, and life cycles of their study organisms, it is extremely rare for paleontologists to find direct evidence of reproductive biology [s et al.  showed tos penis or os priapi) provides an opportunity for drawing inferences regarding the reproductive biology of extinct species [e.g., ossa genitalia homologous to the baculum, the baubellum or os clitoridis, but it is normally smaller than the male counterpart [e.g., In this context, the penis bone of mammals  to the Blancan  with at least 66 recognized species . They weI use the bacula of the extant canids [all from subfamily Caninae, Museum Abbreviations: AMNH—American Museum of Natural History, New York, NY, US; F:AM—Frick Collection, Department of Vertebrate Paleontology, American Museum of Natural History, New York, NY, USA; USNM PAL—Smithsonian Institution National Museum of Natural History Paleontology collections, Washington, D.C., USA; UNSM—University of Nebraska State Museum, Lincoln, NE, USA.Paratomarctus euthos was a coyote-sized species [Paratomarctus euthos  was found in the same locality of F:AM 50213.thomsoni  are assoUrocyon cinereoargenteus [Specimen F:AM 50213 has a complete proximal end where marks of soft tissue attachment are present on the lateral surface. These marks form a 1.5 cm long diagonal line that extends from the dorsal and proximal extremity to the ventral face of the shaft. On the lateral surface, and dorsal to this diagonal line, the bone is rougher but transitions to smoother bone distally. The fragment is mostly straight in lateral profile, except for a slight ventral curvature that starts about one centimeter from the broken end of the specimen. In ventral view there is a deep urethral groove that is narrow and cylindrical. The proximal end of the baculum is laterally compressed, corresponding to where the urethral groove disappears. This differs from most modern canids, in which the urethral groove extends all the way to the proximal end, but is similar to the baculum of the gray fox, rgenteus . The dorSpecimen F:AM 49097 is slightly longer than F:AM 50213 , but it is more abraded and is missing both the proximal and the distal ends. This specimen preserves more of the distal end than F:AM 50213, revealing a ventral curvature (concave down). The groove is not visible on the either the proximal or distal broken surfaces and is thus restricted to the middle portion of the bone; it is filled with sediment, which obscures the ends of the groove. The dorsal crest is present in the middle portion of the fragment, but not at the proximal and distal ends. Using the proximal end of the crest and lack of the urethral groove on the proximal break surface as reference points to align the two specimens, it is possible to infer that only a small portion of the proximal end of F:AM 49097 is missing. It is not possible to see where the groove ends distally due to sediment and abrasion, but the groove is not present on the distal break surface  and consists of rough and porous bone.Carpocyon compressus was a medium sized species [about 16kg, The specimen appears to be complete but has one break close to the proximal end. This break slightly alters the lateral profile, but a ventral curvature is clear. The ventral side contains a deep urethral groove that extends for almost the entire length of the bone, ending about 1 cm before the distal end and about 1 cm from the proximal end. Although the dorsal side is still embedded in matrix, in lateral view lateral expansions associated with the groove can be seen, as can the corresponding dorsal crest. The matrix covering the proximal end makes it difficult to determine if there were any soft tissue attachment scars. The distal tip is laterally compressed but has a height similar to the height along most of the shaft. This is in contrast to most canid bacula, which tend to show compressed and cylindrical distal ends.H. gregarius, and borophagines are similar in terms of: i) relatively large size; and, ii) a ventral urethral groove along much of the length of the shaft but not at the distal tip. In two traits the five borophagine bacula are similar to H. gregarius but differ from the extant canines: i) their ventral curvature (concave down) in lateral profile; and ii) the closed urethral groove at the proximal end. Finally, the simple and mostly straight distal end of borophagine bacula is similar to extant canids but differs from the up curved “hook like” distal end of H. gregarius’ baculum [The bacula of the five borophagine species described here display similarities to each other and to the bacula of all other canids. Specifically, the bacula of extant canines, the extinct hesperocyonine  baculum . Fig 7 sSimilar to modern canids, the bacula of the Borophaginae were large and robust bones. This supports the observation from Wyss and Flynn  that a le.g., Several studies have explored the relationship between baculum length and the reproductive biology of the living carnivorans e.g., , 37–40. Among extant carnivorans, there is a weak correlation between longer bacula and larger testis mass, when both body mass and phylogeny are accounted for , 41.Both of these relationships—copulatory duration and likelihood of sperm competition—suggest that the baculum plays a role in postcopulatory sexual selection, especially in species in which longer copulations may function as a form of mate-guarding that prevents a female from mating with additional males  and/or gH. gregarius [The urethral groove is present in all bacula described from canids, including regarius , the borregarius –26. Thise.g.Aelurodon is shallower than in other canids.Interestingly, a groove is also present in other families within the Suborder Caniformia, including the extinct and basal Amphicyonidae , 14, somLontra, Lutra, and Pteronuria), weasels (Mustela), and bearded seals (Erignathus barbatus) . The deep distal groove is also observed in two fossil bacula described from the Amphicyonidae [Amphicyon ingens and Daphoenus, e.g., spectacled bear Tremarctos ornatus, and polar bear Ursus maritimus, personal observation) have a urethral groove in a similar position , but much shallower than that of canids.In contrast, in the members of other carnivoran families that have a deep groove, the groove continues to the distal most tip of the baculum, and may even be restricted to the distal tip, for example, extant otters , both of which are more similar to the borophagine condition of having a closed proximal end. Interestingly, the gray fox is the most basal species in phylogenetic reconstructions of canines that include fossils [e.g., Finally, while most canine species have an open urethral groove at the proximal end e.g., there als e.g., , but seels e.g., , for a dIn silico functional models of grooves more similar to those seen in canids [It has been suggested that the urethral groove protects the urethra during copulation, facilitating the delivery of sperm . Function canids  have demn canids , it seemet al. [The presence of a urethral groove may also have implications for borophagine mating systems. The urethral groove is a major contributor of bacula complexity when its morphology is quantified from 3D digital scans , 46. Braet al.  found thH. gregarius had a dorsally-curved distal tip  having a secondary dorsal curvature on the distal end. Only two specimens (both Aelurodon) have complete bacula without any indication of preservational distortion (such as bending or warping), while for the other three species the bacula are either incomplete or had the lateral profile slightly altered by a break (C. compressus). Nevertheless, a primarily ventral curvature is seen in all specimens. In this regard, the bacula of the Borophaginae differ from the mostly straight or even dorsally curved bacula of almost all extant canids  in lateral profile , with ont canids . For thein silico biomechanical analyses, Brassey et al. [Aelurodon ferox (et al. [Using y et al.  showed ty et al. . Based oon ferox  is a mas (et al.  should hThe baculum of borophagines shows an overall similarity with extant canines, in relative size, a simple distal end, and presence and configuration of a urethral groove. Drawing upon correlations between bacular morphology and reproductive biology of the extant carnivorans, it is possible to generate preliminary inferences regarding some aspects of borophagine reproductive biology. Specifically, based on comparative bacular morphology, I suggest the borophagines likely had long copulatory durations and spontaneous ovulation, based on the size and simple distal tip of their bacula, respectively. It is possible that with the description of more bacula and further quantitative analysis more robust inferences can be made. The study of reproductive biology in extinct species is challenging and depends on well established relationships between morphology and behaviour or physiology. Nevertheless, further inferences for extinct species could improve our understanding of the selective pressures and consequent evolutionary dynamics of reproductive strategies changing through time. In the long term, it might be possible to better distinguish the role of reproductive function from “phylogenetic inertia” in determining the morphology of the baculum.


This study aimed at evaluating the incidence of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in thyme and thyme-based products, related dietary exposure, and cancer risk for regular and high consumption. A total of 160 samples were collected, and 32 composite samples were analyzed. AFB1 and OTA were respectively found in 84% (27/32) and 38% (12/32) of the samples. AFB1 exceeded the limits in 41% (13/32) and 25% (8/32) of the samples according to the Lebanese and European standards, respectively. OTA was unacceptable in only 6% (2/32) and 3% (1/32) of the samples according to the Lebanese and European standards, respectively. AFB1 and OTA daily exposure was shown to be 4.270 and 1.345 ng/kg bw/day, respectively. AFB1 was shown to be associated with 0.41 and 0.35 additional cancer cases per 100,000 persons per year for regular consumption, respectively; while for high consumption, an increase of 0.911 and 0.639 cancer cases per 100,000 person per year was noted, respectively. The margin of exposure (MOE) for OTA was >10,000 for the non-neoplastic effect and >200 for the neoplastic effect, representing no toxicological concerns for consumers. Alternaria, Aspergillus, Fusarium, and Penicillium genera. They contaminate various agricultural commodities such as cereals and cereal products, nuts, dried fruits, spices, herbs, coffee, wine, and beer . In. In1 and.8 µg/kg . For certandards ,36. All tandards . The contandards . In controm AFB1 .1 concentration of 16.79 µg/kg , the contamination of the thyme-based products varied depending on the AFB1 concentration in the ingredients used in the final product  of the samples, with a mean of 1.4 µg/kg and a concentration ranging between 0.04 and 8.00 µg/kg. Only 6% (2/32) and 3% (1/32) of the samples were contaminated with levels of OTA above the Lebanese and European standards, respectively . All thyFor the dried herbs category, only thyme regular mix exceeded the acceptable limit according to the Lebanese standards, with a concentration of 4.83 µg/kg. Other studies conducted in Lebanon reported higher OTA concentrations of 9.13 µg/kg  and 7.0–OTA incidence in food items in the current study was not only due to thyme, but also could be due to any of the raw materials in the food items, and could appear in the final products, since OTA is thermally stable . For exa1 for regular and high consumption of thyme-based products and dried thyme herbs. For thyme-based products, the average daily exposures to AFB1 were 4.270 and 7.701 ng/kg bw/day for regular and high consumers, respectively. The dried thyme herbs category showed higher exposure values of 4.977 ng/kg bw/day and 10.980 ng/kg bw/day, respectively, for the same consumption levels.1 in this study were similarly higher than those reported in other countries. They were higher than the exposure from spices in Malaysia (0.59 ng/kg bw/day) [1 from various food products were 0.57 ng/kg bw/day and 0.433 ng/kg bw/day, much lower compared to the current study [1 in thyme and thyme-based products, as well as the high consumption of these products by the Lebanese population, contributed to the high dietary exposure levels found in the current study. The average dietary exposure values for AFB1 for regular consumption of thyme-based products and thyme dried herbs in the present study (4.270–4.977 ng/kg bw/day) were higher than those reported in a study on wheat products in Lebanon (0.92 ng/kg bw/day) . In addi bw/day) , from br bw/day) , and front study ,71. Diet1 in thyme-based products was pizza and pasta (30%); followed by cheese with thyme, thyme regular mix sandwich, and fresh thyme salad (17–12%); then cheese and thyme pie, thyme mix with nuts and seeds sandwich, and thyme pie (7–6%); with the remaining ones below 3% ; followed by cheese and thyme pie, fresh thyme salad, and thyme mix with nuts and seeds (12–10%). This increase in the contributions of thyme in thyme regular mix sandwich, cheese and thyme pie, and thyme mix with nuts and seeds reflected the observed increase in thyme consumption during thyme cultivation season, lent meals, school snacks, and other meals in Lebanon.The main contributor to the dietary exposure to AFBbelow 3% . For dribelow 3% . For hig1 was “bread and toast” (79.4%–82.2%) while “pizza and pies” contributed 4.9% to the dietary exposure to AFB1 [1, followed by nuts, cereal products, and snacks [In a study done in Lebanon, the main contributor to the average exposure to AFBt” 79.4%–2.2% whild snacks .1 determined using the dietary intake levels of each sample and the BMDL10 of 0.4 µg/kg bw/day [The MOE to AFBg bw/day  are show1.The MOEs for thyme-based products for regular and high consumption were 94 and 52, respectively. These very low MOEs  indicated a high risk due to consumption of thyme-based products, since the risk increased when the MOE decreased. On the individual product level, the lowest MOE was for pizza and pasta (317) followed by cheese with thyme (556), then thyme regular mix sandwich (675). For dried thyme herbs, the MOEs were 80 and 36 for regular and high consumption, respectively, highlighting the high daily intake and contamination of thyme herbs. As all the obtained MOE values were less than 10,000, the consumption of thyme and thyme-based products in Lebanon can put consumers at an exposure risk to AFB1 through spices used in Lebanese cooking was reported to range between 108 and 444 [1 contamination in spices was reported to be 520 [No study regarding the MOE in thyme or thyme-based products was reported previously. However, the MOE calculated for other spices in past studies was similarly high. For example, the MOE to AFB and 444 . In Malao be 520 .1 would induce 0.083 liver cancer cases per 100,000 persons per year, according to the Joint FAO/WHO Expert Committee on Food Additives [1 (ng/kg body weight/day) from thyme and thyme-based products can be calculated as: liver cancer risk= daily exposure to AFB1 (ng/kg bw/day) × 0.083 cancer cases per 100,000 persons. Therefore, according to the aforementioned, the Lebanese population would have 0.35 and 0.41 additional cancer cases per 100,000 persons per year for thyme-based products and dried thyme herbs, respectively. These corresponding values in the current study were higher than in studies conducted in Malaysia on spices (0.01) [It is estimated that for non-European countries, the ingestion of 1 ng per kg of body weigh per day of AFBdditives . Thus, ls (0.01)  and in Ls (0.01) . For higThe dietary exposure to OTA for regular consumption was calculated, and is shown in The estimated regular and high consumption weekly exposure levels to OTA from thyme-based products were 9.415 and 16.048 ng/kg bw/week, respectively . RegularAccording to EFSA (2020), the PTWI of 120 ng/kg bw/week for OTA is no longer valid, after a recent study that reported uncertainty regarding the mode of action for kidney carcinogenicity . Thus, tThe percentage contribution of each thyme-based product to the average dietary exposure to OTA was calculated . Pizza a1 and OTA, dietary exposure, and risk assessment related to the consumption of thyme herbs and thyme-based products was investigated. Consumption of thyme and thyme-based products could lead to an increase in health risks associated mainly with AFB1, while no potential risk was associated with OTA. This is critical, especially since this food group is highly consumed in Lebanon and the Mediterranean area. This study highlighted the importance of developing maximum mycotoxin limits specific to each product and country. These limits must be based on risk assessment and consumption patterns. Our study also highlighted the importance of monitoring herbs to prevent exceeding the maximum limits to ensure the safety of the thyme and its products, especially since mycotoxins have the ability to resist processing. In addition, future studies should focus on testing individual samples, analyzing contamination trends over time, and assessing the effects of supply chain control and law enforcement on the contamination levels in thyme products.In this study, the occurrence of AFB


The accurate diagnosis of neuroinflammatory (NIDs) and neurodegenerative (NDDs) diseases and the stratification of patients into disease subgroups with distinct disease-related characteristics that reflect the underlying pathology represents an unmet clinical need that is of particular interest in the era of emerging disease-modifying therapies (DMT). Proper patient selection for clinical trials and identifying those in the prodromal stages of the diseases or those at high risk will pave the way for precision medicine approaches and halt neuroinflammation and/or neurodegeneration in early stages where this is possible. Towards this direction, novel cerebrospinal fluid (CSF) biomarker candidates were developed to reflect the diseased organ’s pathology better. Μisfolded protein accumulation, microglial activation, synaptic dysfunction, and finally, neuronal death are some of the pathophysiological aspects captured by these biomarkers to support proper diagnosis and screening. We also describe advances in the field of molecular biomarkers, including miRNAs and extracellular nucleic acids known as cell-free DNA and mitochondrial DNA molecules. Here we review the most important of these novel CSF biomarkers of NIDs and NDDs, focusing on their involvement in disease development and emphasizing their ability to define homogeneous disease phenotypes and track potential treatment outcomes that can be mirrored in the CSF compartment. One hundred and thirty-one years since Heinrich Quincke carried out the first lumbar puncture and attempted cerebrospinal fluid (CSF) cytological examination, significant progress has been made in CSF analysis and diagnostics. Over the years, this “third circulation” or “the vital spirit” has become a readily available and important means of diagnosing and studying diseases affecting the nervous system with such accuracy that is considered—not unfairly—as the “liquid biopsy” of the brain. Nowadays, successful scientific and technical efforts have resulted in overcoming laboratory “barriers” in CSF analysis, such as the low concentrations of diagnostically important molecules and the mixture of blood vs. brain-derived proteins in the intrathecal compartment, rendering CSF analysis a powerful diagnostic tool at any time an immune-inflammatory or a neurodegenerative disease is suspected .With regard to autoimmune-demyelinating disorders, with multiple sclerosis (MS) the major representative, CSF’s contribution to diagnosis has long been appreciated. The recognition of “oligoclonal bands” (OBs)—known since the 1970s—along with the determination of intrathecal immunoglobulin synthesis is part of daily clinical practice . The fluIn the last 30 years, great advances have also been made in the field of biological markers of neurodegeneration, particularly in dementia-related disorders, mainly Alzheimer’s disease (AD). Biomarker discovery has been based on the identification of proteins proven to be closely associated with relevant biological processes, and cell or tissue pathologies. The pathophysiology of AD, the most common form of dementia, involves the extracellular aggregation of misfolded Aβ species, and the accumulation of tau proteins into the neurons, leading to synaptic loss, axonal damage, neurodegeneration, and finally cell death. Established CSF biomarkers already in use indicative of Aβ pathology are CSF Aβ42 and/or Aβ42/Aβ40 ratio, while tangle-related pathology can be captured by measurement of CSF phospho-tau protein (p-tau) levels. Biomarkers indicative of neurodegeneration in general, include CSF total tau (t-tau), neurofilaments, and 14-3-3 protein, among others . All theThe objective of the present study is not aspiring to be an exhaustive review of the ever-growing literature but to highlight the most important substances having the potential to emerge as novel CSF biomarkers tracking neurodegenerative and/or autoimmune-inflammatory aspects of CNS diseases.The most reliable biomarker for MS diagnosis is the presence of OBs that correspond to immunoglobulins produced by plasma cells and are primarily identified in CSF, compared to serum, indicative of their intrathecal synthesis. Antigens involved in the stimulation, activation, and differentiation of B cells in antibody-secreting plasma cells in MS are largely unknown . The higp > 0.99) and specificities 87.1% vs. 89.4%, respectively PBR28 PET imaging , suggesting that sTREM2 could be a novel marker for in vivo microglia activation [A recent study suggests a model in which Aβ and microglial activation as two partially independent processes that, when acting synergistically, lead to neocortical tau pathology. Pascoal et al. (2021), by using novel positron emission tomography (PET) brain imaging for capturing microglial, amyloid, and tau pathology, found that microglia activation correlates with tau pathology progression according to the stereotypical pattern of propagation of tau tangles from the transentorhinal/entorhinal to sensorimotor cortices in AD (known as Braak stages) . CSF sTRtivation .CHMP2B and GRN mutation carriers of FTD patients [Other studies evaluating CSF sTREM2 have revealed increased levels in Parkinson’s disease (PD) vs. controls that also correlated with CSF α-synuclein  as well patients  and in Cpatients .sCD163, a marker of activated microglia and macrophages ; and CXCA hot topic in the era of fluid-based biomarkers is the identification of those able to distinguish between NMO and MS patients. One potential candidate biomarker for such differentiation is CSF IL-6 levels. IL-6 is a proinflammatory cytokine, mainly produced by macrophages/monocytes and some activated B cells, and is considered a Th17 polarizing cytokine, thus fostering subsequent autoantibody production ,62. NMO Other potential CSF biomarkers discriminating MS from NMO are CSF complement components  as well as Th2 and Th17-related cytokines/chemokines  ,68. MostMonocyte chemoattractant protein-1 (MCP-1) is an important chemokine for the recruitment of monocytes and macrophages to the CNS through its interaction with its receptor CCR2. Tissue macrophages and microglia cells in active white matter lesions of MS patients have been found to overexpress CCR2. CCL2 expression was defined as activated astrocytes, pointing to their role as active players in orchestrating the inflammatory milieu in MS white matter lesions ,70. MCP-MCP-1 has been also reported significantly increased in prodromal AD compared to healthy controls and correlated with a short time interval to cognitive decline and development of dementia. In combination with CSF Aβ42 and t-tau and p-tau protein levels, it may be a potentially useful biomarker for monitoring disease progression .Glial fibrillary acidic protein (GFAP) is a cytoskeletal protein expressed by mature astrocytes and is widely used as a cell type marker in brain histopathology . IncreasGFAP, when measured in CSF, exhibited higher values in patients with NMO spectrum disorders (NMOSD) when compared to patients with MS or healthy controls ,83,84,85The above-mentioned novel CSF biomarkers capturing inflammatory aspects either in autoimmune or neurodegenerative neurological diseases affecting CNS are summarized in Neurofilaments are the major cytoskeletal proteins of neurons in both CNS and PNS, comprising light (NFL), medium, and heavy (NFH) neurofilament chains . The mosIntensive research over the past decades showed that in all clinical forms of MS, increased CSF NFL concentration reflects disease activity and progression . CSF NFLIncreased levels of NFL have been found in other neuroinflammatory conditions, such as MOGAD ,93. We rWidespread axonal injury is a prominent feature of NMO pathophysiology, compared to MS, with optic nerves and spinal cord being severely affected. Initially, high CSF levels of NF heavy subunit (NFH) in NMO patients but not in MS patients had been shown ,95. SubsSeveral studies have also found that CSF NFLs levels may be a marker of clinical severity of AD and MCI when compared to Aβ and tau proteins, reflecting future cognitive decline. Nevertheless, NFLs are not a specific marker for AD pathophysiology, as they are also found increased in other neurodegenerative diseases, while they normally also increase with age ,98.In movement disorders, such as PD-plus syndromes, NFLs show a good discriminatory power for the differentiation of PD, DLB, and PD dementia (PDD) but not multiple system atrophy (MSA). Scientific efforts for the inclusion of CSF NFL determination in the diagnostics of ALS are currently in progress ,101,102.2+ homeostasis in AD and thus serve as a marker of neuronal injury [VILIP-1, visinin-like protein 1, belongs to a family of proteins that are neuronal calcium sensors (NCS) and is highly expressed predominantly in neurons from pyramidal and non-pyramidal areas in AD brains . VILIP-1l injury . Regardil injury . A recenl injury . Furtherl injury ,110. TheNo solid evidence exists for the role of this biomarker in neuroinflammatory diseases.C9orf72 gene mutation showed that UCHL1 was among the most highly upregulated proteins in ALS patients [CSF ubiquitin C-terminal hydrolase L1 (UCH-L1), a protein involved in the maintenance of axonal integrity, is involved in the pathway leading to the degradation of highly ubiquitinylated aggregated and/or damaged proteins by the 26S ubiquitin–proteasome system (UPS) ,112. UCHpatients . No signpatients .Neurogranin (Ng) is a calmodulin-binding postsynaptic protein playing a role in synaptic plasticity. Reduced protein expression of Ng has been described in the brains of AD patients but was found conversely increased in the CSF. The most important finding with clinical applications is high CSF levels of Ng reported in MCI patients progressing to AD compared to cognitively stable MCI patients and control individuals, making CSF Ng a surrogate biomarker for identifying patients in early disease stages ,119,120.SNAP-25 is a component of the SNARE complex, which is central to synaptic vesicle exocytosis, and by directly interacting with different calcium channels subunits, it negatively modulates neuronal voltage-gated calcium channels, thus regulating intracellular calcium dynamics. ,123. A rGAP-43 is a presynaptic protein expressed in various brain regions  and is critically involved in synaptogenesis and neuronal plasticity in the adult brain . CSF GAPThe above-mentioned novel CSF biomarkers capturing neurodegenerative aspects as well as synaptic pathology either in autoimmune or neurodegenerative neurological diseases affecting CNS are summarized in Alpha-synuclein (α-Syn) is a neuronal protein that regulates synaptic vesicle trafficking and subsequent neurotransmitter release. It is the major constituent of Lewy bodies the pathogenic hallmark of PD and DLB, as well as in variable aggregated species in MSA, disorders collectively called synucleinopathies. A-Syn is readily secreted into extracellular space and can be found in different forms  in CSF. Quantification of total α-Syn levels in CSF in multiple studies has shown a general trend of decreased α-Syn levels in patients with PD compared to healthy individuals. Interestingly, a recent study of our group showed that in general synucleinopathies exhibit lower total-α-Syn and higher phosphoS129-α-syn/total-α-Syn ratios compared to tauopathies .A major limitation in measuring CSF α-Syn levels is the possible contamination by red blood cells, which is a big source of the protein and gives false-positive results . The useTARDBP gene, is the major aggregated protein involved in the formation of the characteristic inclusions, especially in its hyperphosphorylated and ubiquitin-bound form, in the brains of patients with ubiquitin-positive frontotemporal lobar degeneration (FTLD-U) and or ALS [TARDBP gene have been identified across the FTD/ALS spectrum [Frontotemporal lobar degenerations (FTLDs) comprise a spectrum of complex and heterogeneous neurodegenerative disorders characterized by degeneration of the frontal and anterior temporal lobes involving multiple clinical phenotypes, different protein aggregates  in tissue pathology and many genetic loci explaining up to 40% of familial FTLD . The mosd or ALS . More thspectrum ,144.About 50% of FTLDs have positive TDP-43 aggregates, thus rendering TDP-43 an emerging disease-specific biological marker for the FTD/ALS spectrum . RecentlPlasma levels of phosphorylated TDP-43 protein have been also associated with the presence of histopathological lesions of FTLD . It seemGRN gene, a cysteine-rich protein that is glycosylated and secreted as a glycoprotein [The protein progranulin (PGRN) is the product of the oprotein . The stroprotein . PGRN isoprotein . Progranoprotein . Moreoveoprotein ,158,159.GRN gene account for 20% of familial and 5% of sporadic cases of FTD, with variation in clinical expression, mainly expressed as primary progressive aphasia (PPA) and bvFTD , but rare phenotypes resembling AD and parkinsonism have also been reported [GRN mutations, in contrast to those of MAPT gene encoding tau, are associated with FTLD-TDP proteinopathy [Most attention has been given to PGRN due to the loss-of-function mutations in the progranulin gene (GRN) that have a pathogenic role in familial forms of FTD . Pathogereported ,162. At inopathy . CSF proinopathy ,164,165.inopathy .Previous studies had reported no differences in CSF progranulin levels among AD, MCI, and controls . RecentlAs regards MS patients, CSF levels of PGRN had shown contradictory results. A recent study has unraveled minor only perturbations of this biomarker in CSF of patients with different clinical forms of MS . InteresMicroRNAs (miRNAs), are single-stranded 19–23 nucleotides long, nonprotein-coding RNA molecules that act as post-transcriptional regulators to fine-tune protein expression levels either by promoting mRNA degradation or by diminishing protein translation. miRNAs are secreted from cells to extracellular spaces through vehicle mediated  and not-vehicle-mediated pathways (bound with high-density lipoproteins and Ago2 proteins) . A valuaIn MS, miRNAs could be a very useful and informative tool because it includes and transports material outside the cell originating from many cell types. This is the feature that renders miRNAs an important surrogate biomarker providing information on elusive cells and difficult-to-access tissues . VariousRegarding AD pathogenesis, miRNAs have been shown to target molecular pathways associated with pathologic processes that are implicated in disease evolution, such as synaptic and mitochondrial dysfunction, Aβ accumulation, and tau toxicity. In 2013 Sala Frigerio et al.  by applyApart from AD, the application of miRNA as a potential biomarker has been tested in many other neurodegenerative diseases. miR-9-3p and miR-106b-5p levels in CSF have been found capable of discriminating PD from MSA patients with good diagnostic accuracy by receiver operating characteristics curve evaluation (area under the curve = 0.73). In the same study, a single microRNA, miR-106b-5p, provided the best discrimination between PD and PSP (area under the curve = 0.85) in the CSF .De Felice et al. suggested that miR-338-3P, which is found in both blood and CSF, could be used as a biomarker for patients with ALS. They have shown that miR-338-3p is highly expressed in ALS tissues by in situ hybridization staining and that was specifically localized in the grey matter of spinal cord tissues from sALS autopsied patients .The limitations in the wide use of miRNAs mainly rely on the high cost of the techniques applied for their measurement, the need for bioinformatic tools for analyzing complex networks in which they are involved, the variability in levels , and the need for universal clear-cut off levels that require standardized protocols.Cell-free DNA (cfDNA) constitutes externalized, short, fragmented DNA in various lengths found in bodily fluids and is the product of either programmed cell death, necrosis, or cell activation . A breakThe role of cfDNA in AD has emerged in the last years. DNA methylation has been shown to be altered in various tissues and brain areas of AD patients. Other genes have been found hypermethylated  and other hypomethylated (PINI1) . Highly Methylation analysis patterns in cfDNA have gained much attention as they provide information on the tissue of origin. In a pioneer study by Lehmann-Werman and colleagues in 2016, oligodendrocyte-derived DNA was found to be enriched in the cell-free DNA of patients with relapsing MS . AdditioPerturbations in the methylation status of various genes extend beyond AD. A differentially methylated region located in the promoter–enhancer region of the rhomboid 5 homolog 2 (RHBDF2) gene was identified in ALS patients in cfDNA in the plasma . OverallThe release of DNA of mitochondrial origin (cf-mtDNA) in the CSF has been studied more extensively than cfDNA. mtDNA that have been misplaced into the cytosol or released into the bloodstream holds inflammatory properties as it could serve as damage-associated molecular patterns activating cytosolic receptors and TLRs . Again, Studies assessing the application of cf-mtDNA as a reliable biomarker in AD have revealed contradictory results. For example, reports of a significant decrease in CSF cf-mtDNA levels in AD patients have not been replicated or even resulted in opposite results . One groThe most critical step for future biomarkers discoveries is understanding disease-related pathogenetic mechanisms and unraveling the disturbed architecture of the tissue, cells, and the corresponding implicated biological pathways. An overall picture of the localization and distribution of all novel biomarkers in CNS, presented in this review, is illustrated in Established and novel biomarker for use in clinical practice for diagnosis, prognosis, and monitoring disease evolution, especially in the era of new drug discovery imposes the need for sensitive and accurate analytical methods producing homogeneous quantification results, such as those produced on fully automated laboratory instruments.Standardization among platforms, improvement of coefficients of variation, and the use of internal and external quality control programs are necessary both in clinical and research settings.Recent advances in methodologies have brought novel research tools, such as ultrasensitive immune-based technologies, such as SiMoA, capable of quantifying proteins at very low concentrations, and mass spectrometry-based proteomics both bearing promises for the discovery of new CSF biomarkers, tracking neuroinflammation and neurodegeneration aspects. In addition, novel “seeding” assays are under development, which could be used for amplification and capture of the abnormally aggregated misfolded proteins with prion-like properties, hallmarks of neurodegenerative diseases.Finally, novel molecular biomarkers are to be recognized with molecular techniques such as new-generation RNA sequencing, found at both cell-free and cell-rich compartments of CSF. Future delegate CSF DNA studies of methylation analysis or whole sequencing analysis will pave the way for new molecular and genetic/epigenetic markers to be identified thus, expanding the discovery of biomarkers to the nucleome.After more than a century following the introduction of lumbar puncture in medical practice, CSF laboratory analysis has never stopped being used as a powerful diagnostic tool mainly for infectious and autoimmune/demyelinating diseases of the CNS. The introduction of biomarkers for neurodegeneration in recent years marks another milestone in CSF diagnostics. Nowadays, there is an urgent need to open the field of biomarkers research beyond those already established. Under this perspective, we have tried to provide new evidence on novel biomarkers either for autoimmune inflammatory or neurodegenerative disorders.Studies have shown that single biomarker measurements are not able to capture disease pathobiology as a whole, while many biomarkers are not disease-specific but common in both neurodegenerative and neuroinflammatory diseases. Thus, future biomarker combination approach(es) (in the best possible scheme) integrating both old and new ones would permit proper identification of disease-specific mechanisms and contribute to a more precise diagnosis and subsequent treatment, while facilitating the enrollment of biologically homogeneous cohorts of patients in clinical trials.


Lachnospiraceae in both donors. LMW-HDE and HMW-LDE lemon pectins both increased total SCFAs  and increased acetic acid by 1.64-fold. Additionally, LMW-HDE lemon pectin led to an average 1.41-fold increase in butanoic acid. Together, these data provide valuable information linking chemical structure of pectin to its effect on the gut microbiota structure and function, which is important to understanding its prebiotic potential.Pectins are plant polysaccharides consumed as part of a diet containing fruits and vegetables. Inside the gastrointestinal tract, pectin cannot be metabolized by the mammalian cells but is fermented by the gut microbiota in the colon with the subsequent release of end products including short-chain fatty acids (SCFA). The prebiotic effects of pectin have been previously evaluated but reports are inconsistent, most likely due to differences in the pectin chemical structure which can vary by molecular weight (MW) and degree of esterification (DE). Here, the effects of two different MW lemon pectins with varying DEs on the gut microbiota of two donors were evaluated in vitro. The results demonstrated that low MW, high DE lemon pectin (LMW-HDE) altered community structure in a donor-dependent manner, whereas high MW, low DE lemon pectin (HMW-LDE) increased taxa within  Pectin is a heteropolysaccharide widely distributed throughout the plant cell wall and middle lamella ,2. It isBacteroides species are thought to be the primary pectin degraders within the community and in particular, Bacteroides thetaiotamicron has been previously found to carry the PULs capable of degrading the pectin structure .,23.22,23For HMW-LDE lemon pectin, both donor communities responded with an increase in richness B. HoweveNext, whether or not treatment with either LMW-HDE or HMW-LDE lemon pectin would affect beta diversity was tested using a principal coordinate analysis (PCoA) based on weighted and unweighted UniFrac distances . This tyIn both the weighted and unweighted analyses, the communities for the control, lemon pectin treatment, and post-treatment periods for each donor are clustered together and indicated in the figure with colored circles . The larAlthough the results of PCoA did not show a significant change in community structure, whether or not the addition of LMW-HDE or HMW-LDE lemon pectin would have an impact on specific taxonomic groups was questioned. Using 16S rRNA gene sequencing data, the relative abundance of taxa for each donor during the control, lemon pectin treatment, and post-treatment periods was determined and formatted as heat maps illustrating the community profiles at the class level . Donor 1Acidaminococcus and Paraprevotella, and a corresponding increase in Psuedoramibacter. In donor 2 communities, there is a decrease in Alistipes and an increase in Oscillospira and Fusobacterium.While these results were interesting, it was considered that changes might have occurred at a lower taxonomic level that were missed. Therefore, the statistically significant changes that occurred at the genus level were determined to provide granularity regarding the communities’ response to these lemon pectins . At the Lachnospiraceae, although the specific genus level taxa varied between donors. For donor 1, changes to family Lachnospiraceae came from the genera Blautia, Clostridium, and one unidentified genus, and for donor 2 they came from the genera Blautia, Coprococcus, and Ruminococcus. For HMW-LDE lemon pectin, there were also a few donor-dependent changes that occurred as well. Interestingly, donor 1 communities only responded with changes in taxa from class Clostridia, even though as a whole this class was not altered significantly, as shown in Alistipes, Sutterella, and Fusobacterium, and a decrease in Bacteroides.Comparatively, treatment with HMW-LDE lemon pectin produced a larger number of changes, a few of which were donor- independent, or were observed in both donor communities B. In parBifidobacterium, which is commonly associated with positive health outcomes and some reports have described pectin as bifidogenic [Bifidobacteriales 16S rRNA gene is not recovered well with the V1-V2 sequencing primers [Bifidobacterium to analyze changes to this taxon (Bifidobacterium levels for either donor communities when provided LMW-HDE or HMW-LDE pectin.The one taxon that was missing from this community profile was idogenic . This ab primers . The 16Sis taxon . The resThe gut microbiota is well known for its fermentation properties, and the end result of fermentation is the release of SCFAs, which are typically considered as beneficial to human health . The mosThe results showed that the levels of total SCFAs were significantly increased in response to both LMW-HDE and HMW-LDE pectin, and this occurred for both donor communities A. For LMWhen evaluated more in-depth, it was discovered that the increase in total SCFAs was largely due to significantly increased levels of acetic acid, which was the predominant SCFA measured in these communities B. This sThe prebiotic function of pectin has been described in previous studies that have demonstrated its ability to promote the growth of beneficial bacteria ,45,46,47The results of this study showed that both LMW-HDE and HMW-LDE lemon pectins were able to produce changes to the gut microbiota community structure, although the type of changes observed were variable and many were donor-dependent or were reliant on the composition of the individual donor’s community. In terms of alpha diversity, here, richness was enhanced by both lemon pectins, although this did not reach statistical significance for both donors and was more apparent for HMW-LDE lemon pectin . These rAn underlaying observation for community structure in this study was that the effect of both LMW-HDE and HMW-LDE lemon pectins were highly contingent on the composition of the individual donor communities. This was most apparent in the results of the weighted and unweighted UniFrac analyses, which showed that for both the LMW-HDE and the HMW-LDE lemon pectins the largest divergence was due to donor composition and not pectin treatment . Yet, thLachnospiraceae that occurred in both donor communities and was therefore not donor-dependent. This observation aligns with a previous report that found pectin treatment to increase levels of Lachnospiraceae, which contains some of the enzymes required to degrade the pectin structure [Alistipes, Sutterella, and Fusobacterium, and a decrease in Bacteroides. These changes were remarkable because when donor 2 communities were treated with LMW-HDE pectin, there was a decrease in Alistipes, whereas the HMW-LDE pectin enhanced levels of this taxa. The decrease in Bacteroides was unexpected, as many members of this genus carry enzymes capable of performing pectin fermentation [Alistipes, as a member of the Bacteroides, may be pectinolytic, increases in the non-pectinolytic Sutterella have also been observed with pectin additions, with the latter being attributed to cross-feeding. Thus, the responses observed here are likely inclusive of both direct and indirect metabolic benefit from the pectin [When the community structure was analyzed at higher resolution, looking at the genus level, LMW-HDE lemon pectin affected levels of multiple taxa, yet these were still considered as donor-dependent . Alternatructure . Howeverentation ,9. Whilee pectin ,50.Bifidobacterium was assessed using qPCR. Here, neither lemon pectin significantly altered Bifidobacterium levels for either donor (Bifidobacterium, or Bifidobacterium may be unable to properly utilize lemon pectin or cross-feed with taxa capable of metabolizing these pectin structures. Taken together, results generated from 16S sequencing data and qPCR quantification provided some interesting, yet conflicting results. The addition of lemon pectin elicited changes to the community structure, yet these were somewhat variable and depended on the donor-specific communities, and did not necessarily reproduce results as previously reported, i.e., no bifidogenic response [Several previous studies have reported the bifidogenic effects of pectin ,20. Baseer donor . Howeverresponse . HoweverNext, the ability for LMW-HDE and HMW-LDE lemon pectins to alter community function was analyzed based on SCFA quantification . Here, aInterestingly, the results showed that while both lemon pectins enhanced total levels of SCFAS, they did so through the promotion of different types of SCFAs. This supported previous findings on total levels SCFAs  and addsThe production of SCFAs through fermentation is an extremely important factor to consider in gut microbiota studies because they can be metabolized by the cells of the GIT or can be taken up and circulated throughout the body via the bloodstream. Butanoic acid is particularly important for the colonic epithelium as its oxidation supplies approximately 70% of the energy needed for the cells there . Acetic,Acidaminococcus, Paraprevotella, Psuedoramibacter, Alistipes, Oscillospira, and Fusobacterium. For HMW-HDE lemon pectin, there were donor-independent shift in taxa within Lachnospiraceae. When looking at function, the data showed a donor-independent enhancement of SCFA production for both LMW-HDE and HMW-LDE lemon pectin. This was predominantly from acetic acid which increased 1.64-fold during the treatment period for both lemon pectins used. However, treatment with LMW-HDE lemon pectin also increased butanoic acid by 1.41-fold, which was a novel finding. This data provides further evidence that pectin structure influences its ability to be utilized by the gut microbiota, which directly relates its potential prebiotic effect.In conclusion, the results of this study demonstrated that lemon pectins of varying MW and DE had differential effects on the gut microbiota structure and function. For LMW-HDE lemon pectin, community structure was altered in a donor-dependent manner, with changes noted in