Obesity is a public health issue worldwide. Conversational agents (CAs), also frequently called chatbots, are computer programs that simulate dialogue between people. Owing to better accessibility, cost-effectiveness, personalization, and compassionate patient-centered treatments, CAs are expected to have the potential to provide sustainable lifestyle counseling for weight management.This systematic review aimed to critically summarize and evaluate clinical studies on the effectiveness and feasibility of CAs with unconstrained natural language input for weight management.PubMed, Embase, the Cochrane Library (CENTRAL), PsycINFO, and ACM Digital Library were searched up to December 2022. Studies were included if CAs were used for weight management and had a capability for unconstrained natural language input. No restrictions were imposed on study design, language, or publication type. The quality of the included studies was assessed using the Cochrane risk-of-bias assessment tool or the Critical Appraisal Skills Programme checklist. The extracted data from the included studies were tabulated and narratively summarized as substantial heterogeneity was expected.In total, 8 studies met the eligibility criteria: 3 (38%) randomized controlled trials and 5 (62%) uncontrolled before-and-after studies. The CAs in the included studies were aimed at behavior changes through education, advice on food choices, or counseling via psychological approaches. Of the included studies, only 38% (3/8) reported a substantial weight loss outcome (1.3-2.4 kg decrease at 12-15 weeks of CA use). The overall quality of the included studies was judged as low.The findings of this systematic review suggest that CAs with unconstrained natural language input can be used as a feasible interpersonal weight management intervention by promoting engagement in psychiatric intervention-based conversations simulating treatments by health care professionals, but currently there is a paucity of evidence. Well-designed rigorous randomized controlled trials with larger sample sizes, longer treatment duration, and follow-up focusing on CAs’ acceptability, efficacy, and safety are warranted. Obesity, nearly tripled between 1975 and 2016 , cancersConversational agents (CAs), often called chatbots, are computer programs that replicate human dialogue  . The emeAlthough personalized, humanlike, unconstrained CAs provide long-term efficacies that enable users to better engage in conversations and adhere to customized intervention messages, previous reviews have included heterogeneous CAs or participants, the intervention’s goal was not solely weight management, and there were few unconstrained CAs ,28. ConsIn this context, this systematic review was conducted to critically evaluate the effectiveness of CAs using unconstrained natural language input on weight loss or obesity-related outcomes  and their feasibility in clinical practice.The protocol for this systematic review was registered with the Research Registry system (Review Registry Unique Identifying Number: reviewregistry960). This systematic review was compliant with the PRISMA  statement . The seaWizard of Oz methods, in which the dialogues were generated by an unseen human operator, only predetermined, and not generated in response to user input [Studies were included if the user communicated directly with a CA for the purpose of weight management; no restrictions on participants were imposed  if they interacted with a CA intended for weight management. As the definition of CAs differed and there was a lack of consensus, CAs in this systematic review were sought if digital tools mimicked humanlike behaviors and provided a task-oriented framework with participation in conversation . CAs thaer input ; focusedCA or chatbot was not used, it may be reasonable to assume that automated interactive digital tools with participation in conversation were regarded as CAs. If a reduction in body weight was reported, we assumed that the CA was used for weight management and participants had an intention of weight management. A variety of adherence measurements that could be evaluated were considered, for example, session attendance, retention or dropout rates, or the frequency and duration of CA use  [To determine the eligibility of the studies, they were first screened based on the titles and abstracts to determine whether the full texts should be further evaluated. Full texts were then obtained, read in full, and excluded with specific reasons. The areas in which CAs contributed to weight management and the strategy of CAs for weight management were identified and divided into two categories: (1) study characteristics and (2) CA characteristics. Regarding study characteristics, predefined extraction forms were used to collect relevant information, which included study design, year and country of study, first author, sample size, participants , intervention duration, control group description if relevant, CA contributing to conventional weight management program , and study outcomes. Regarding the characteristics of CAs, delivery device or platform, type of CA , input or output modality (written or spoken), whether the CA mimicked practice , theories integrated into CAs, commercialization, and personalization were extracted. If the CA was capable of personalization, personalization methods were also extracted  . The artecorded) ,31. For The included studies were assessed by 2 reviewers to evaluate their quality, and disagreements were resolved through discussion. For randomized controlled trials (RCTs), the revised Cochrane risk-of-bias tool for randomized trials was used . For theA narrative systematic review was conducted for all the included studies because of the expected heterogeneity of study characteristics, CAs, and reported outcomes. However, for the RCTs that provided analyzable outcomes, data were pooled and expressed as mean difference (MD) for continuous outcomes and risk ratio for dichotomous outcomes with 95% CIs using a random-effects model to incorporate expected heterogeneity with the ReviewManager program .P<.05) improvement was reported in a given outcome. If no significance or significant worsening was reported, the CA was regarded as having no significant evidence supporting it. User experience, such as usefulness, helpfulness, or satisfaction, was rated as positive, mixed, neutral, or negative [In addition, a structured analysis of the outcomes was conducted to derive conclusions regarding the effectiveness and feasibility of CAs for weight management . The CA negative .The initial electronic database search yielded 872 citations, and a further 15 studies were identified by hand searching the reference lists of relevant studies. After identifying and removing 17.9% (159/887) of duplicates, the titles and abstracts of the remaining 728 studies were screened, and 95.1% (692/728) of the studies were excluded. The remaining 36 studies were then read in full and excluded if applicable, leaving the final 8 (22%) studies from 9 publications. One of the studies that were finally excluded involved CAs without natural language input capability . The detThe 8 included studies were conducted in the United States , Australia , and South Korea . The authors of the included studies were mostly related to psychology, medicine, and computer science, that is, the fields of research in each study were as follows: psychology, neuroscience, and biological science ; public The number of study participants varied from 23  to 220 , with 712  of the studies included BMI in the inclusion criteria—BMI ≥25 kg/m/obesity ) or yout/obesity ,40. A to/obesity ,41-44, b/obesity , and the/obesity ,39-44. T/obesity ), and 12/obesity . The mai/obesity ,38,40,41The characteristics of the CAs, including name and type of CA, delivery channel, input or output modality, whether they mimicked practice, theories of CAs, and personalization, were summarized and tabulated .The included studies tested 3 types of CAs: an ECA named GistFit ; text-baThe CAs in the included studies were designed to mimic in-person treatment using AI techniques. In 12% (1/8) of the studies , 3 femalAll the CAs in the included studies  were developed based on psychological theories, such as social cognitive theory or fuzzy-trace theory ,40, theoPersonalization was available for 75% (6/8) of the CAs ,39,41-44The methodological quality of the included studies was evaluated. RCTs ,40,43 wesome concerns [high risk [some concerns in the domain of the randomization process as reporting was unclear both for the randomization and allocation concealment methods. In the domain of deviations from the intended interventions, 33% (1/3) of the studies [low risk considering that they used an intention-to-treat approach. However, another study [some concerns as blinding may not have been feasible because of the nature of the waitlist control group and as the intervention also included an electronic health record behavioral counseling tool used by the primary care clinician during well-child follow-up visits. The other study [high risk as it excluded eligible trial participants after randomization and did not use an intention-to-treat analysis. In addition, the trial by Piao et al [As shown in concerns ,40 and high risk , mainly igh risk  were jud studies  were rater study  was rateao et al  had someao et al ,40 had sYes answers was between 2 [No on question 1 (Did the study address a clearly focused issue?) [Can’t tell was given as they did not report power analyses and insufficient information was provided to determine whether the follow-up of participants was complete enough (question 6a). As maximal weight loss is generally considered to occur at 6 months [Can’t tell or No with regard to the applicability of the results to the local population, mainly because of small sample sizes, convenience samples, or unbalanced male to female participant ratios of the samples leading to less generalizability. It was difficult to determine whether the results were consistent with those of other studies or whether they had implications for practice as there were few studies on CAs’ weight loss effects (questions 11 and 12).As shown in etween 2  and 6 [2etween 2 ,41,44, m issue?) . Selecti issue?) , and ano issue?) , and the issue?) ,44. Most issue?) ,44 adjus issue?) ,39,42, C6 months , the fol6 months  were givThe effect estimates of the RCTs are listed in Weight change was reported in 33% (1/3) of the RCTs  and 40% A total of 33% (1/3) of the RCTs reported that there was no significant difference between the groups in diet change. The CA group’s total calorie intake  and total fat intake  were similar to those of a waitlist control . In totaP<.01) [In total, 100% (3/3) of the RCTs reported outcomes associated with physical activity. In 33% (1/3) of the RCTs, the CA group’s screen time (hours per day) considerably decreased  when compared with a waitlist control group . In anotP<.01) .P value not reported) [P value not reported).In the remaining 80% (4/5) of the uncontrolled studies, inconsistent results were reported. Total physical exercise time per week rose by 109.8 minutes (95% CI 1.9-217.7) , weekly eported) , daily seported) . Stepheneported)  reportedUser experiences such as satisfaction with or usefulness of the CAs were measured using a predefined survey ,44 or usokay (78.8%) or good (10.6%) [Positive user experiences were reported in 50% (4/8) of the studies. More than 75% of users agreed that the CA was useful and helped them eat healthy foods. When it came to watching less television, 78% of parents agreed that the CA was helpful, but only 35% of children did so . The res (10.6%) , whereas (10.6%) .Negative user experiences were also reported. Although 79% of users believed that the CA may help them modify their lifestyles, they had disappointing experiences, such as the CA answering questions incorrectly, having limited access, the CA not acting quite like a human, and taking a lot of time .A total of 88% (7/8) of the studies reported CA adherence using different types of measurements, such as retention or dropout rate ,40,41,43P value not reported) [P=.001) [P=.26 and P=.59, respectively) [The more users were engaged with the CA interventions, the better outcomes related to weight control were reported in 62% (5/8) of the studies ,40,41,44eported) . High usIn another study, the effect of engagement with the CA on outcomes was not tested, but it descriptively reported positive progress toward their goals ≥80% of the time based on interviews with active participants .Only 12% (1/8) of the studies reported adverse effects, showing no adverse events related to participation in CA research . There wThis systematic review aimed to critically evaluate the effectiveness of CAs on weight loss and obesity-related outcomes and their feasibility in clinical practice. In total, 33% (1/3) of the RCTs and 40% (2/5) of the uncontrolled studies reported weight loss of 1.3 to 2.4 kg at 12 to 15 weeks, and the results, including physical activity change, positive user experience , and adherence to CAs, largely supported the effectiveness and feasibility of CAs. On the basis of the main findings of this systematic review, the use of CAs is promising for behavior change and engagement in weight management programs even though it is difficult to be certain of their impact on weight reduction at present. CAs with unconstrained natural language input largely rely on psychological approaches and personalized content and support close to interpersonal interventions by human teachers or coaches. However, owing to the paucity of adequately designed and well-conducted controlled trials, further research is warranted to establish the role of CAs with unconstrained natural language input in weight management in clinical practice.P=.07) over 12 weeks [Although it was difficult to make a direct comparison, the weight loss of 1.3-2.4 kg  was lowe12 weeks . Compare12 weeks , better 12 weeks . SMS tex12 weeks ,49. AlthWith regard to the components of CAs contributing to weight loss, mimicking practices such as human health coaching or empathetic health counseling, theories applied to CAs such as psychological approaches , LM programs (such as the Diabetes Prevention Program), and personalization  were common.The CAs in the included studies can be divided into a logging and a health coaching part. Previous studies examining calorie-tracking or physical activity–logging apps without health coaching showed no additional weight loss when compared with a control group ,51. ThisThe engagement level of CAs in this review was related to better learning outcomes regarding healthy nutrition and exercise  and greaIn this review, only studies on CAs that used unconstrained natural language input through AI and machine learning were considered to reduce DHIs’ drawbacks such as dehumanization and low adherence. A protocol was developed and registered in the Research Registry database, and this systematic review was conducted as per protocol. However, because of a dearth of well-designed and rigorously conducted RCTs, a firm conclusion on whether CAs for weight management actually help maintain or lower weight in the overweight or obese population could not be drawn. Although a thorough literature search was conducted in a range of databases, including not just core medical databases but also psychology and computing or machinery databases, we cannot be completely certain that other potential studies were not overlooked.black box effect, which can generate unpredictable CA responses [The diverse characteristics of the CAs and outcome measures in each study also precluded drawing a definite conclusion. Self-reported weight reduction cannot be entirely free from reporting bias or inaccuracy, and this would have to be considered when interpreting the results. Another limitation is that few studies reported potential adverse events associated with CAs. Adverse event reporting in CA studies is important as machine learning–based CAs are not completely free from the concerns of the esponses . In addiGiven the breadth of the reported findings in this review, some may argue that a scoping review would better serve our study indication. Although a scoping review identifies knowledge gaps, scopes a body of literature, clarifies concepts, investigates research conduct, and informs a future systematic review, a systematic review summarizes and critically evaluates the current evidence to answer well-crafted specific questions addressing the feasibility, appropriateness, meaningfulness, or effectiveness of a certain treatment or practice . ConsideAlthough CAs have been widely used in various fields, research on CAs with unconstrained natural language input in weight management has only just begun. All included studies in this systematic review were published in the last decade. CAs are generally reported to have a positive effect on healthy behaviors, favorable experiences among users, and better adherence. Although the World Health Organization recommends via the global strategy on digital health 2020-2025 that all DHIs be evaluated and their effectiveness be verified in scientific research , it was Although it is impossible to draw firm conclusions on CAs’ effects on weight loss at the moment, their use seems promising for behavior change and active participation in weight management programs. Unconstrained CAs seem to have a potential for effective and patient-centered interventions providing education, advice on food selection, and psychological approaches to body weight management via complex and flexible conversation. These findings warrant future controlled studies examining the use of CAs with unconstrained natural language input as an option for weight management.


During embryonic and fetal development, the cerebellum undergoes several histological changes that require a specific microenvironment. Pleiotrophin (PTN) has been related to cerebral and cerebellar cortex ontogenesis in different species. PTN signaling includes PTPRZ1, ALK, and NRP-1 receptors, which are implicated in cell differentiation, migration, and proliferation. However, its involvement in human cerebellar development has not been described so far. Therefore, we investigated whether PTN and its receptors were expressed in the human cerebellar cortex during fetal and early neonatal development. The expression profile of PTN and its receptors was analyzed using an immunohistochemical method. PTN, PTPRZ1, and NRP-1 were expressed from week 17 to the postnatal stage, with variable expression among granule cell precursors, glial cells, and Purkinje cells. ALK was only expressed during week 31. These results suggest that, in the fetal and neonatal human cerebellum, PTN is involved in cell communication through granule cell precursors, Bergmann glia, and Purkinje cells via PTPRZ1, NRP-1, and ALK signaling. This communication could be involved in cell proliferation and cellular migration. Overall, the present study represents the first characterization of PTN, PTPRZ1, ALK, and NRP-1 expression in human tissues, suggesting their involvement in cerebellar cortex development. In humans, cerebellar development starts during the fourth gestational week (gw), and it continuously changes until it matures . This prPTN is a protein composed of 168 amino acids (a.a.) that is secreted into the extracellular matrix ,23, wherEven though these molecules are reported in nervous system development, most knowledge is based on animal models, and their cerebellar cortex localization is still debated. For this reason, this study aims to describe the expression of PTN and its receptors in the human fetal and neonatal cerebellar cortex.The present study was approved by the Ethics Committee of Centro Médico Nacional de Occidente (México) and Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Jalisco, México . Five fetal samples and one neonatal sample were collected from pathology autopsy studies between 2013 and 2015. For human fetal samples, there were three females at 31, 37.4, and 38 gw; two males at 17 and 36.6 gw. For human neonatal samples, there was one female at 38 gw. Any infectious process was discarded in the clinical history. Fetal ages were calculated from the first day of the mother’s last menstrual period and fetal somatometry study. The human fetal cerebellum was fixed in 4% paraformaldehyde with a pH of 7.4. They were dehydrated with ethanol and paraffin embedded. Then, 3 µm thick sections were made, and hematoxylin and eosin staining (H&E) was performed for histological examination.TM Polymer Detection System was used . Antigen retrieval was performed using citrate buffer solution  to analyze PTN, PTPRZ1, and ALK; EDTA buffer solution  was used for NRP-1 antigen retrieval. Endogenous peroxidase was blocked with 3% (v/v) H2O2. NovocastraTM protein block was used for 30 min to reduce the non-specific binding of primary antibodies and polymers. Anti-PTN  and anti-PTPRZ1  primary antibodies were incubated for 60 min; anti-ALK  was hatched for 90 min; anti-NRP-1  and anti-GFAP  were incubated for 30 min. The tissues were then treated with post-primary (rabbit anti-mouse IgG) and Novolink TM polymer solutions (anti-rabbit poly-HRP-IgG). The chromogen was 5% 3,3′-diaminobenzidine in a buffer solution containing 0.1% H2O2. The sections were counterstained with hematoxylin, dehydrated, and cover slipped. The specificity of antibody labeling was previously verified in several control tissues , 2+ (moderate), and 3+ (strong) . The analysis was conducted on a total of 4 different locations in each sample using Qupath  . EGL cel(strong) .The cerebellar cortex was analyzed for all human samples. The majority of them were female (67%), with a median age of 37 gw (range: 17–38 gw). The cause of death of the unique neonatal sample was respiratory failure. The clinical characteristics of the human cases are summarized in Cerebellar cortex cellularity was observed by using hematoxylin and eosin staining. The EGL was constantly detected in all gestational ages analyzed. The cell thickness and density showed variations at different gestational ages. The age with the highest EGL cell thickness and density of EGL was 36.6 gw, and the lowest density was at 17 gw A,B,F. AdInterestingly, the fetal and neonatal samples at 38 gw showed histological differences. The EGL density was higher in the fetal cerebellar cortex than in the neonatal cerebellar cortex. The Purkinje axonal projections were more prominent in the neonatal cerebellar cortex than in the fetal cerebellum . A summaThe anti-PTN antibody recognizes a 168 a.a. protein, the expression and localization of which were variable through the different stages of the human fetal cerebellar cortex development evaluated . As showAs PTN receptors, the expression of PTPRZ1, ALK, and NRP-1 proteins was evaluated using immunohistochemistry. All characterizations are shown in At this stage, the EGL was clearly detected in cerebellar cortex tissue A. The imAt this stage, cell density began to increase in the EGL, and PC bodies were easily seen B. The inAt this stage, PC body size was increased C. The exAt this stage, PTPRZ1 and GFAP staining patterns were consistently similar B,E. PTPRAt this stage, PTPRZ1 expression showed high-intensity staining along the axon projection of PCs, which was extended to the EGL. In the ML, PTPRZ1 continued showing diffuse distribution, as the GFAP pattern did, and it was positive at the neuropil zone of all cerebellar cortex layers. In the IGL, PTPRZ1 was observed in the soma of PCs . The ALKAt this stage, the thickness and cell density of the EGL were gradually reduced . The staThis study described, for the first time, the expression pattern of PTN and its receptors—PTPRZ1, NRP-1, and ALK—throughout the development of the human cerebellar cortex, from 17 gw to the early neonatal stage, using an immunohistochemical technique. The results indicate that, in the cases analyzed, the localization and intensity of PTN, PTPRZ1, and NRP-1 staining changed according to the gestational weeks, but the ALK protein was only expressed in the cerebellar cortex at 31 gw. We also contrasted the cell type that expresses them using the GFAP marker as an astroglial phenotype in human fetal stages.This work shows that PTN is predominantly expressed in the EGL at 17 gw and increases at 31 gw. This immunoreactivity retains a pattern similar to Bergmann glia only at 17 gw. This shows that, in humans, PTN could be produced by Bergmann glia at 17 gw, but granular cell precursors may be the major sources of PTN at 31 gw. Even though PTN protein expression has been described in Bergmann glia, it was only reported for the postnatal stage in rats . HoweverAlkKO [Additionally, the ALK protein was absent at 17 gw, where PTN and PTPRZ1 were weakly expressed, too, but they were highly expressed in the ML at 31 gw. This could suggest another cell type in which ALK and PTPRZ1 are co-expressed in the ML at 31 gw. It has been demonstrated that the PTN–PTPRZ1 complex promotes ALK activation in humans  and ALK AlkKO , but theAlkKO ,44,45. TAlkKO . These rAlkKO . Thus, aFrom 36.6 gw to the postnatal stage, the ALK protein was negative in all cerebellar cortex layers. Consistent with this result, ALK protein expression has been described in the central and peripheral nervous systems in mouse embryos ,47, but WT [It is also important to consider the change in the localization of PTN expression in the postnatal stage . We founWT .We are aware that this is an exploratory study based on a small number of cases, and we hypothesize rather than provide conclusive results about the role of each protein studied in human cerebellar maturation. Nevertheless, we believe that the information presented here is valuable because it provides insights into the biology of cerebellar development. In addition, since it is known that there are neoplasms originating from immature cerebella, such as medulloblastomas, these results raise the question of whether these molecules—the involvement of which has been demonstrated in other neoplasms of the nervous system—may also play a role in medulloblastomas. Further studies with larger samples are needed to deepen the investigation of signaling pathways that confirm the degree to which PTN and its receptors are involved in human cerebellar development, both prenatally and postnatally.The present study represents the first spatial–temporal characterization of PTN and the expression of its receptors in the human fetal and neonatal cerebellum. PTN, PTPRZ1, and NRP-1 change their expression and localization, indicating that PTN could activate different signaling pathways and may be involved in generating intercellular communication in the fetal cerebellum and neonatal cells during their development. Thus, PTN could be involved in the morphogenesis of PCs as well as granular cell proliferation and migration in the human cerebellar cortex. Furthermore, PTPRZ1 expression in all cerebellar cortex layers suggests that these proteoglycans play an important role in the maturation of the human cerebellum. Given the exploratory nature of this work, the extent to which these molecules are expressed in immature human cerebellar tissue needs to be confirmed by using other methodological approaches and larger samples.


Sporothrix spp. in clinical specimens. Therefore, the following five non-culture-based methods were evaluated for the detection of Sporothrix spp. in 30 CSF samples: (i) species-specific polymerase chain reaction (PCR); (ii) nested PCR; (iii) quantitative PCR; (iv) enzyme-linked immunosorbent assay (ELISA) for IgG detection; and (v) ELISA for IgM detection. The species-specific PCR was unsuccessful in the diagnosis of the meningeal sporotrichosis. The other four methods presented substantial levels of sensitivity (78.6% to 92.9%) and specificity (75% to 100%) for the indirect detection of Sporothrix spp. Both DNA-based methods presented similar accuracy (84.6%). Both ELISA methods were concomitantly positive only for patients with sporotrichosis and clinical signs of meningitis. We suggest that these methods should be implemented in clinical practice to detect Sporothrix spp. in CSF early, which may optimize treatment, augment the chances of a cure, and improve the prognosis of affected individuals.Sporotrichosis is the main subcutaneous mycosis worldwide. Several complications, including meningeal forms, can be observed in immunocompromised individuals. The sporotrichosis diagnosis is time-consuming due to the culture’s limitations. The low fungal burden in cerebrospinal fluid (CSF) samples is another important drawback in the diagnosis of meningeal sporotrichosis. Molecular and immunological tests can improve the detection of  Cryptococcus neoformans/gattii complex species are the main agents of fungal meningitis, occurring in both immunocompetent and immunocompromised individuals [Histoplasma capsulatum, may lead to CNS involvement in cases of immunosuppression [Paracoccidioides can cause neuroparacoccidioidomycosis, an uncommon but severe presentation of paracoccidioidomycosis [Candida spp., Trichosporon spp., Aspergillus spp., Fusarium spp., Mucor spp., Rhizopus spp., Blastomyces dermatitidis, Cladophialophora bantiana, and Exophiala dermatitidis, which may present fatal consequences [The central nervous system (CNS) involvement by fungal species is one of the most serious complications of the systemic mycoses. The ividuals . Histoplpression . The CNSpression . The patomycosis . Severalequences .Sporothrix brasiliensis, driving the increasing number of sporotrichosis cases in humans and cats [Sporotrichosis is the most common subcutaneous mycosis, representing a global distribution. It is highly predominant in regions with tropical/subtropical climate and reached hyperendemic levels in several parts of Brazil, especially in Rio de Janeiro state. In this area, few cases are associated with trauma involving organic matter, whereas the zoonotic transmission prevails, notably due to the species and cats .S. brasiliensis is usually high, and it is suggested that this species may present neurotropism in immunosuppressive settings, which occurs in PLHIV, due to failure in antiretroviral therapy, low adherence to treatment, or the unawareness of their HIV status [Complications in sporotrichosis have been mostly seen and described in people living with HIV/AIDS (PLHIV), in whom the disease presents a poor prognosis. In addition, they were also described in other immunosuppressive conditions, such as diabetes and alcohol abuse . MeningeV status ,8,9.Sporothrix spp. is the gold standard for the sporotrichosis diagnosis, regardless of its clinical manifestations. However, this method is time-consuming and lacks sensitivity in clinical materials with an associated microbiota or in cases of low fungal burden [Culture isolation of l burden , which fl burden . Immunoal burden . In addil burden .In fact, serological and molecular methods have presented good performances for the sporotrichosis diagnosis in some clinical samples, but few studies have been applied to CSF samples up to the present time . MethodsTwo cross-sectional studies, approved by the Institutional Review Board (#88551018.9.0000.5262 and #54249721.9.0000.5262), were carried out at the Instituto Nacional de Infectologia Evandro Chagas, Fundação Oswaldo Cruz (INI/FIOCRUZ), a reference center for infectious diseases, including sporotrichosis in the hyperendemic area of Rio de Janeiro, Brazil.Sporothrix spp. would be involved in asymptomatic meningitis (hereinafter referred to as the test group). All patients underwent lumbar puncture for a routine CNS infection investigation in the study place, using good medical practices.We included 14 CSF samples from six patients with disseminated sporotrichosis and meningitis, hereinafter referred to as the case group. One patient of this group had one sample, two patients had two different samples and three had three different samples, collected at distinct times. In addition, 12 CSF samples from 12 patients with other diseases, infectious or not, were used as controls (hereinafter referred to as the control group). Finally, four CSF samples from four patients with disseminated sporotrichosis, but without diagnostic criteria for meningitis, were additionally investigated to check whether Sporothrix sp. In culture from any clinical specimen.Sporotrichosis was defined as the development of clinical signs and/or symptoms in a patient with the isolation of Sporothrix sp. Isolation in culture from the CSF or as the detection of biochemical or cytological alterations in the CSF in patients with culture-proven sporotrichosis from any other site and with no other confirmed neurological cause.Meningeal sporotrichosis was defined as the Mycobacterium tuberculosis/resistance to rifampin (MTB/RIF), acid-fast bacilli detection, specific cultures for mycobacteria, regular bacteria, and fungi, multiplex polymerase chain reaction (PCR) for human herpesviruses , and PCR for the John Cunningham (JC) virus [The institutional routine for CSF analyses included biochemistry, cytology, nontreponemal syphilis evaluation (VDRL), lateral flow assay for cryptococcal antigen, GeneXpert for C) virus .g, according to the manufacturer’s instructions. Extracted DNA was quantified using the Biophotometer Plus  equipment. DNA samples were maintained at −20 °C until use. The 79 base pairs (bp) fragment of human β-globin gene was amplified, using 50 p-moles of primers, 5′-GCAAGAAAGTGCTCGGTGC-3′ (Forward) and 5′-TCACTCAGTGTGGCAAAGGTG-3′ (Reverse), and 2.5 p-moles of probe (5′-FAM-TAGTGATGGCCTGGCTCACCTGGAC-3′-TAMRA) aiming to evaluate potential PCR inhibitors, using the Agilent AriaMX qPCR thermal cycler , as previously described [Histoplasma capsulatum (ATCC 26032), Trichosporon asahii (isolate 53897), Candida albicans (ATCC 18804), Cryptococcus neoformans (ATCC 208821) and Cryptococcus gattii (ATCC 56990), as previously described [The DNA extraction was performed with the QIAamp DNA mini kit  using 200 μL of the CSF samples previously centrifuged at 13,000× escribed . Samplesescribed .Sporothrix detection in extracted DNA from CSF using the QIAamp DNA mini kit .Three molecular methods were performed for the 2, 0.4 µM of each primer, 1.5 U of Taq DNA polymerase , and 200 µM of each dNTP (Invitrogen). The template for the first reaction consisted of 4 µL of total DNA extracted from CSF and, for the second, 4 µL of the products amplified in the first reaction. Both cycles were performed in a T100 thermal cycler , as follows: 95 °C for 5 min, 35 cycles at 95 °C for 30 s, 60 °C for 45 s, 72 °C for 30 s, and a final extension of 72 °C for 10 min. The DNA of the reference strain S. brasiliensis (CBS 120339) was included as positive control, and the negative control was performed substituting the DNA with the same volume of ultrapure water .Firstly, a nested PCR targeting the 18S rRNA gene  was perfS. brasiliensis, using the primers Sbra-F→(5′-CCCCCGTTTGACGCTTGG-3′) and Sbra-R (5′-CCCGGATAACCGTGTGTCATAAT-3′), was performed according to a previously described test [The species-specific PCR for bed test , aiming S. brasiliensis detection was performed as previously described [A quantitative PCR (qPCR) aimed at escribed , with miS. brasiliensis (CBS 120339) DNA were performed in artificial CSF . Dilutions ranging from 100 ng/µL to 1 fg/µL of DNA were used to test the sensitivity of each molecular method. The detection limit was defined as the lowest concentration of target DNA detected by each method.To simulate the conditions of meningeal sporotrichosis, serial 10-fold dilutions of Sporothrix spp. Optimization of our ELISA applied to serum samples was performed for the use of CSF as a biological sample. CSF samples were diluted from 1:2 to 1:32,000 and the best dilution to discriminate between positive and negative was 1:100. Following optimization, the ELISAs were used to evaluate the CSF samples. This experiment was performed in triplicate, and the ELISA cutoff point was calculated as the mean optical densities (ODs) plus three standard deviations of the controls. ODs above the cutoff were considered positive.Indirect enzyme-linked immunosorbent assay (ELISA), in two different formats, was performed, as described previously , with slFor IgG detection, goat anti-human IgG peroxidase conjugate  was used as the secondary antibody diluted 1:32,000 in incubation buffer . The enzymatic reaction for IgG detection was developed with the addition of 100 μL per well of 0.4 mg of o-phenylenediamine dihydrochloride (OPD)/mL and 0.04% hydrogen peroxide in 10 mM sodium citrate buffer (pH 5.5). The reaction was stopped by the addition of 50 μL of 3 M HCl per well. Absorbances were measured using the SpectraMax Plus spectrophotometer  at 490 nm.2 and 1 mM ZnCl2 (pH 10.4), and the reaction was stopped by the addition of 25 μL of 3 M NaOH per well. The same Spectra Max Plus spectrophotometer , at 405 nm, was used to measure the absorbances in this reaction.The secondary antibody for IgM detection was goat anti-human IgM alkaline phosphatase conjugate  diluted 1:2000 in 10 mM TBS, 0.1% Tween 20, and 5% nonfat skimmed milk powder (pH 7.2) at a final volume of 100 μL per well. The enzymatic reaction was developed with the addition of 100 μL per well of 1.0 mg of p-nitrophenyl phosphate (PNPP)/mL in 0.1 M glycine buffer containing 1 mM MgClhttps://www.medcalc.org/calc/diagnostic_test.php (accessed on 08 March 2023).Clinical performance of these methods in the population of patients with meningeal sporotrichosis and other CNS diseases was assessed using 2 × 2 tables to calculate sensitivity, specificity, accuracy, and positive and negative likelihood ratios using the MedCalc Software Ltd., Ostend, Belgium. Diagnostic test evaluation calculator, version 20.211, freely available at The six patients with meningeal sporotrichosis (case group) were males from the metropolitan region of Rio de Janeiro, with a median age of 40 (range: 25–57) years. Among them, four mentioned previous contact with a sick cat suspected of sporotrichosis, and one reported scratches from a cat with sporotrichosis. The other patient was a gardener who was in daily contact with soil. All patients were PLHIV, with a median CD4+ T lymphocyte count of 104.5 (range: 29–302) cells/μL at the first lumbar puncture. By the end of this analysis, four patients had died due to sporotrichosis or other AIDS-related conditions, and two patients were cured of sporotrichosis.In the control group (patients without sporotrichosis), eleven were males and one was female, being nine PLHIV, with a median age of 41 (range: 30–66) years. Six patients had distinct CNS infections, as follows: meningeal tuberculosis, cryptococcal meningitis, neuroparacoccidioidomycosis, varicella-zoster virus, cytomegalovirus with herpes simplex 2 virus, and Epstein Barr virus, one patient each. Among the other six patients, four were PLHIV with neurological symptoms, one patient had a headache, and another presented a posterior uveitis, all with no specific neurological diagnosis.In the test group (four patients with sporotrichosis without criteria for meningitis after the routine investigation), three were males and one was female. Their median age was 42 (range 35–66) years and they also lived in the metropolitan region of Rio de Janeiro. Two reported contact with a sick cat and one of them was scratched. The other two patients could not remember any risk activities for sporotrichosis. Three patients were PLHIV, with a median CD4+ T lymphocyte count of 88 (range: 77–197) cells/μL and two of them are currently cured of sporotrichosis, while the woman was lost to follow-up. The fourth patient had uncontrolled diabetes and died due to sporotrichosis complications.−2 pg/µL, 102 pg/µL, and 105 pg/µL, respectively.Quality control tests revealed amplification of 79 bp fragments in all 30 CSF samples, using the qPCR (β-globin-FAM-TAMRA). Three other potential samples were excluded due to the absence of beta-globin amplification and were not submitted to the methods. The DNA samples extracted from fungal species related to CNS infections were negative in all three studied DNA-based methods. The LOD of the nested PCR, qPCR, and species-specific PCR methods were 10Sporothrix spp. species-specific PCR. Among the DNA-based methods herein studied, the nested PCR was the method with the highest number of positive samples. This method detected 19 positive samples, 13 in the case group, and three positive samples in each one of the other two groups. The qPCR detected 13 positive samples, 11 in the case group, and one in each of the other two groups. All samples were negative in the The ELISA for the detection of IgG antibodies was reactive in 13/14 CSF samples from the case group and in 3/4 samples from patients in the test group, while 12/12 samples from the control group were negative. When the ELISA was used for IgM detection, similar results were found, with 13 reactive samples from the case group, one reactive sample from a patient from the test group, and no reactivity in control samples. S. brasiliensis, from a cohort of 17 cases, did not present neurological symptoms at the first lumbar puncture, which were seen later. When we compared these patients with meningeal sporotrichosis (n = 17) vs. cutaneous-disseminated/disseminated sporotrichosis without meningitis (n = 36), lethality among patients with meningeal sporotrichosis was higher (64.7%), with a higher chance of death (HR = 3.87) [Sporothrix spp., possibly making the prognosis of these patients more favorable.Sporotrichosis is an infection of a benign course in the majority of the cases, restricted to the skin . In immu = 3.87) . Some meSeveral diagnostic methods can overcome the limitations of gold-standard diagnosis. The increase in sensitivity and specificity values are the main important advantages of the DNA-based methods and immunoassays. Other important advantages of these assays are the reduced turnaround time, which is associated with an improvement in the quality of life of affected individuals . The comSporothrix DNA detection in clinical samples, such as biopsy [S. brasiliensis isolation in culture, which can be attributed to the lower limit of detection of this method, associated to the low fungal burden expected to be found in CSF samples from patients with meningeal sporotrichosis [In sporotrichosis, different diagnostic DNA-based methods are reported. The species-specific PCR was developed for species identification , but cans biopsy , and in s biopsy . Howeverrichosis .S. brasiliensis. The false negative result for the nested PCR of sample 2a, from which there was the isolation of S. brasiliensis in culture, is something to be answered. Perhaps the analysis of more samples will help the understanding of what we currently believe to be a limitation of the method. The negative results for the qPCR of samples 3a and 3b may also be a limitation of the method, which, in this case, can be partially explained by the lower LOD when compared to the nested PCR. Currently, S. brasiliensis is the main agent of meningeal sporotrichosis in Brazil [Sporothrix species could benefit from the same technique, following the necessary modifications.Nested PCR and qPCR are reported as methods with higher sensitivity and specificity. For several fungal infections, these methods are used as diagnostic tests in the laboratorial routine ,29, but n Brazil , with a n Brazil . FurtherSporothrix spp., but demonstrated unspecific amplification, detecting several fungi of the Ophiostomataceae family after sequencing [Sporothrix spp., that can infect the CNS, which supports that, in the CSF, this nested PCR detects pathogenic species of the genus Sporothrix. Thus, the nested PCR can be used in the diagnosis of sporotrichosis, but carefully. As this test presented the lower specificity among the methodologies used, its use without other tests could lead to a misdiagnosis of meningeal sporotrichosis in patients without this condition, leading to erroneous medication administration and possible unfavorable outcomes. Moreover, an important universal precaution to be taken when performing nested PCR is to avoid cross-contamination, which has high odds of occurring between the first and second reactions [In a single-center study, a nested PCR for fungi was successfully used as a diagnostic test in 50 clinical samples, with a better performance than culture (21/50 vs. 0/50) . The nesquencing . Most ofeactions .Sporothrix spp., some studies demonstrate the usefulness of these tools in the diagnosis of sporotrichosis affecting the CNS [Sporothrix antibodies in a CSF sample. Additionally, the literature also reports the possibility of using the detection of anti-Sporothrix antibodies in CSF to monitor patients with meningeal sporotrichosis. Some studies describe an increase in the level of antibodies in the CSF in episodes of recurrence and a decrease with clinical improvement [The ELISA test and the antigen used in this study were previously analyzed in serum samples for IgG detection, with substantial sensitivity and specificity values  . The sam the CNS ,34. Usin the CNS  describerovement ,34.Our results also showed that it is important to evaluate both IgM and IgG responses, because patients with sporotrichosis, but without meningeal sporotrichosis, may present IgG antibodies in the CSF, possibly due to the antibody crossing the blood-brain barrier.Some samples had different results from the expected. In sample 5, the negativity in both ELISAs can be explained by a lack of immune response in a PLHIV with comorbidities and abandonment of treatment. Among the controls, sample 9 was from an undiagnosed patient in our institute. The most probable is that a cross-reactivity, which occurs in very sensitive techniques, may have happened, because the clinical picture was not compatible with sporotrichosis. Among the samples from the four patients with sporotrichosis but without clinical criteria for meningitis, none had concomitant positive IgG and IgM results in the CSF sample. Sample 19 had isolated positive IgG in the CSF. IgG may have crossed the blood-brain barrier. Samples 20 and 22 came from patients who did not have criteria for CNS involvement and were discharged from follow-up after sporotrichosis cure. Therefore, the isolated positive nested PCR for both seems nonspecific and, more likely, a false positive result. While in sample 21, the combination of positive nested PCR, qPCR, and IgG antibody in the CSF highlights the probability of the presence of the fungus in the CNS with no meningitis signs, in both clinical and laboratory settings. A hypothesis is that the fungus entered the CNS but did not manage to produce meningitis. It is interesting to note that this patient received treatment for disseminated sporotrichosis, with amphotericin B, until clinical improvement, followed by oral itraconazole . By the S. brasiliensis, two cases with culture confirmation and four presumed through the qPCR result, making it difficult to know whether meningitis caused by other Sporothrix species could be diagnosed with the same efficiency, and (iii) the real prevalence of meningeal sporotrichosis is unknown, which hinders the calculation of predictive values.Some limitations of this study include: (i) small number of CSF samples, due to the fact that meningeal sporotrichosis is a rare clinical manifestation, (ii) evaluation of meningeal sporotrichosis caused only by Meningeal sporotrichosis is associated with poor prognosis, and better diagnosis strategies are needed to improve the approach of these patients. The combination of molecular and immunological methods can improve the diagnosis of meningeal sporotrichosis, overcoming the limitations of conventional diagnostic methods. According to our results, the species-specific PCR is not useful for this purpose. Immunoassays, together with qPCR, should be used as the first diagnostic tool due to the high chance of detecting a positive sample. Finally, nested PCR can be used, considering the necessary precautions during the performance of this technique, to further screen negative samples in the previous techniques or in places that cannot afford qPCR.


Ascophyllum nodosum extract  and Camellia sinensis-leaf extract  have been reported to promote positive metabolic effects through different mechanisms. We investigated the effects of SE and TE on metabolic homeostasis in diet-induced obese mice and discussed their functional characteristics.Obesity is often associated with glucolipid and/or energy metabolism disorders. 1, EE2, and EE3), inflammatory factors, lipid anabolism enzymes, and gut flora of the obese mice.Male C57BL/6J mice fed with high-fat diets for 8 weeks were established as obese models and subsequently divided into different intervention groups, followed by SE, TE, and their joint interventions for 10 weeks. Body weight and food intake were monitored. Fasting glucose and oral glucose tolerance tests were interspersed during the experiment. After the intervention, the effects on obesity control were assessed based on body composition, liver pathology section, blood lipids and glucose, respiratory exchange ratio (RER), energy expenditure  and promoted the growth of Akkermansia and Bacteroides, thus affecting the chronic inflammatory state.After continuous gavage intervention, the mice in the intervention groups exhibited lower body weight , peri-testicular fat masses , and perirenal fat masses . All interventions prevented diet-induced increases in plasma levels of glucose, adiponectin, leptin, and the inflammatory factors IL-1β and TNF-α. The RER was modified by the interventions, while the rhythm of the RER was not. Blood lipids  decreased and were associated with lower lipid anabolism enzymes. In addition, the SE and TE interventions altered the structure and abundance of specific flora. Different interventions inhibited the growth of different genera positively associated with obesity  has a better effect on regulating energy metabolism and inhibiting chronic inflammation. Economic and social development has facilitated people's lives. However, uplifting their lifestyles has also exposed them to obesity. Obesity is a chronic metabolic disease wherein the body deviates from orderly energy homeostasis due to impaired fat accumulation . The WorA range of different pharmacotherapies have been developed for obesity in clinical practice; however, obesity's complex and diverse pathophysiological mechanisms render single-route pharmacological interventions often incomprehensible. Thus, an appropriate option to tackle multifactorial fat accumulation and obesity is to enable the simultaneous regulation of impaired metabolic pathways in multiple organs.Ascophyllum nodosum is a species of brown seaweed in the family Fucaceae rich in unique natural molecules. Ascophyllum nodosum extract  processed through water extraction, ultrafiltration, spray drying, etc. has at least 35% phlorotannin content. Phlorotannin inhibits pro-inflammatory cytokine expression and interferes with transcriptional regulation  could promote hepatic fatty acid oxidation and reduce lipogenic enzyme expression  of Ascophyllum nodosum extract, Camellia sinensis-leaf extract, and their joint interventions on weight loss and glycolipid metabolism in high-fat diet-induced (HFD-induced) obese mice.Both Yili Technology Development Co., Ltd. . Both were extracted using an aqueous solution. The fresh materials were sequentially subjected to aqueous extraction, screening, decantation, ultrafiltration, pre-sterilization, spray drying, and homogenization to obtain dry powders. The extraction ratio of each extract was 7:1. The active ingredients are iodine , α-amylase inhibition , lipase inhibition , minerals , and tannins  in SE, and polyphenols  in TE. The recipe groups are shown in SE and TE were provided by Beijing This study conforms to the Institutional Animal Care and Use Committee (IACUC) at Xiamen University and complies with guidelines described in the Guide for the Care and Use of Laboratory Animals . The protocols for animal use were reviewed and approved by the Animal Ethical and Welfare Committee of the Laboratory Animal Center of Xiamen University .n = 192) with an average body weight of 20.38 ± 0.87 g) were reared at 22°C, 10%−60% humidity, 12-h/12-h light–dark cycle, with free access to water. After adaptive feeding, 12 mice (ND group) were selected randomly and fed normal chow  for 8 weeks. The remaining mice were fed a high-fat diet  for 8 weeks to establish obesity models. Mice displaying a 40% increase in body weight after a high-fat diet were considered successful obesity models and were randomly divided into one model control group (HFD) and five intervention groups, with 12 mice in each group. The sample size calculation was performed according to Mera et al.  levels were measured at weeks 0, 4, 8, and 10. Ten mice from each group were randomly selected for oral glucose tolerance tests (OGTTs) at the end of the intervention: After 12-h fasting, the mice were immediately administered 20% glucose solution by gavage (10 μL/g body weight). Blood was drawn from the tail vein at 0, 30, 60, and 120 min after glucose administration to measure the blood glucose concentration using a blood glucose meter .ad libitum. ND mice were still fed a normal diet, whereas the other groups were fed a high-fat diet. After the adaptation period (the first 48 h), stable metabolic monitoring was initiated at 0:00 and ended at 0:00 the next day. One data point was recorded every 5 min for each mouse.At the end of the intervention, four mice were randomly selected from each group and monitored using the TSE PhenoMaster (12-channel) system, which measured the respiratory entropy metabolism and voluntary movement of mice for 5 days. Each mouse was caged independently, feeding 1) were calculated based on the O2 consumption and CO2 production measured in the exhaust gas of each cage . Body weight was used to correct EE1 and obtain EE2, and lean body mass was used to correct EE2 and obtain EE3.The respiratory exchange ratio (RER) and energy expenditure  and liver tissues were rapidly collected. The blood was centrifuged at 2,000 rpm (382 g) and 4°C for 15 min, and the supernatant was collected to measure biochemical indicators. All samples were frozen at −80°C for later use.Serum was collected using an automatic biochemical analyzer (Mindray BS-220) and supporting kits to detect total cholesterol (TC), triglycerides (TGs), low-density lipoproteins (LDLs), and high-density lipoproteins (HDL).Serum was taken to measure INS, LEP, ADP, IL-1β, and TNF-α levels in mice using a mouse enzyme-linked immunosorbent assay kit (ELISA). The insulin resistance index  and islet β cell function  were calculated according to the following formula , 20.Liver tissue was homogenized and centrifuged at 3,000 rpm for 10 min. The supernatant was used to measure the expression of lipoprotein lipase (LPL), fatty acid synthase (FAS), sterol regulatory element-binding transcription factor (SREBP-1c), and cholesterol-activated 7α-hydroxylase (CYP7A1) protein in the liver tissue by ELISA.Murine fecal samples were collected the day before the end of the experimental period and stored at −80°C. HiPure Stool DNA Kits  were used to extract total DNA from these samples, after which the conserved 16S rDNA region  was amplified by PCR  using appropriate primers and barcodes. The resultant amplicons were extracted using 2% agarose gel electrophoresis. They were purified with AMPure XP Beads  based on provided directions, then quantified with an ABI Step One Plus Real-Time PCR System. Equimolar amounts of these amplicons were pooled and subjected to paired-end sequencing (PE250) on an Illumina instrument  using standard protocols. Raw reads were filtered to remove low-quality reads, assembled, and filtered using FASTP (v. 0.18.0). Clean tags were clustered into operational taxonomic units (OTUs) at a ≥97% similarity threshold using the UPARSE pipeline (v. 9.2.64). The UCHIME algorithm was employed to remove chimeric tags, and the remaining tags were subjected to downstream analyses. The most abundant sequence within each OTU was selected as a representative sequence. Following OTU determination, gut microbiota analyses were conducted, including community composition, alpha diversity, and beta-diversity. Taxonomical abundance statistics were visualized using Krona (v 2.6), with R being used to analyze dominant bacteria at the phylum and genus levels. Species comparisons among groups were performed using Tukey's HSD test, and the Kruskal–Wallis H-test. QIIME  was used to assess changes in the overall microbiota community composition in the different groups. Principal coordinate analysis (PCoA) was conducted using the R Vegan package (v 2.5.3) and plotted using the ggplot2 package (v. 2.2.1).The data were analyzed using IBM SPSS Statistics . Descriptions are expressed as means, medians, standard deviations, and percentiles. Repeated-measures data were analyzed using the body weight, FBG, and OGTT values of the mice obtained for variance using a multivariate variance. If the data distribution was normal and the variance was homogeneous, a one-way analysis of variance (one-way ANOVA) was used to determine the variation between groups of one-way measurement indicators, and the LSD method was used for pairwise comparisons between groups. If the data distribution did not satisfy the normal distribution, the non-parametric Kruskal–Wallis H-test was used for comparison between groups, and the Nemenyi method was used for pairwise comparison of the overall mean between groups. The hypothesis test level was α = 0.05.P < 0.001). After random grouping, no significant difference in body weight was observed among the obese mice groups (P > 0.05). Except for the S1T3 group, all interventions lowered food intake in mice, with SE exhibiting the most obvious appetite suppression . After 1 week, the obese mice rebounded in weight in each group. The presumption is that the previous reduction in body weight reduced the volume of the gavage and brought it down to a volume that was more adaptable to the mice. Additionally, the SE itself has a natural seaweed odor, which is not a familiar odor to rodents. After a week, the mice gradually accepted the odor and regained their appetite. At the end of week 6, the differences in weight in all the plant extract groups were statistically significant (P < 0.01) compared with the HFD mice. At the end of the intervention, the average body weight of each intervention group was lower than that of the HFD group (P < 0.05).The average body weights of the HFD mice and ND mice were significantly different at week 0, being 32.68 ± 1.27 g and 24.72 ± 0.82 g . Notably, single SE had a better lipid-lowering effect than combined SE. No significant difference was found in the lipid-lowering effect between the three joint interventions with different proportions of SE to TE. Similar effects were observed for the lipid body ratios calculated for each group of mice.The peri-testicular and perirenal fat masses of mice in all intervention groups were lower than those in the HFD group .The composition of lean and fat tissues in mice was used to assess the degree of body composition balance. Different diets did not significantly alter the lean tissue composition of the mice, or at least did not increase it, either with the HFD or with the interventions. However, the HFD significantly increased the fat composition of the mice (P > 0.05), except for the TG levels in some intervention groups, which remained relatively higher (P < 0.05 vs. the ND group). SE and TE may regulate the level of HDL, thus promoting the carriage of TC, TG, and LDL into the liver for catabolism and efflux.HFD increased TG, TC, and LDL levels and decreased HDL in the serum of obese mice . SubsequP < 0.05; vs. ND: P > 0.05) at week 4, with the same situation appearing in the other groups at week 8. However, all plant extract groups except the SE group exhibited a rebound in FBG levels compared to the ND group at week 10. Meanwhile, the FBG levels of SE mice were significantly lower than those of TE mice at week 10 (P < 0.05), and the FBG levels of the joint intervention groups with a higher proportion of SE were significantly lower than those of the group with a higher proportion of TE (P < 0.05).As opposed to the HFD group, the SE mice showed a downward trend in FBG levels, while the other groups trended toward a transient increase followed by a decrease . This inP < 0.05). Among the three joint intervention groups, the relative AUC values decreased as the proportion of SE increased, implying that SE controlled blood glucose more efficiently than the single TE supplement, both for the SE alone and for the mixtures.The areas under the OGTT test curve (AUC) of each intervention group were smaller than those of the HFD group, indicating that all interventions enhanced the regulation of blood glucose in obese mice . Obese mP < 0.05, P > 0.05). In the extract intervention groups, SE mice had significantly higher INS levels than TE mice (P < 0.05). Notably, HOMA-β levels in the SE group were higher than those in the TE and S1T3 groups, which have higher proportions of Camellia sinensis-leaf extract.HFD impaired islet cell function and triggered insulin resistance in mice, as reflected by the increased INS and HOMA-IR levels, as well as the decreased HOMA-β levels in the HFD group. INS and HOMA-IR levels in the intervention groups were significantly downregulated compared with those in the HFD group (The respiratory exchange ratio (RER) is regularly used to evaluate the utilization of energy-supplying substances. RER is calculated as 0.7 when the body solely uses fat as its predominant energy source. This value increases, at which point the energy-producing substance transitions from fat to carbohydrate. An RER value of 1 indicates that the body relies only on carbohydrate consumption for energy supply .P < 0.001, intervention groups vs. HFD group, RER in the ND group showed a significant rhythmic fluctuation. As expected, the RER of normal organisms was relatively higher at night than during the daytime, reaching a level close to 1.0. HFD-induced obesity, however, alters the main energy substrates of the organism, making it predominantly fat metabolizing (~0.79) and losing rhythmicity . The int1, EE2, and EE3 line graphs over time. However, HFD-induced obesity was considered to increase resting energy consumption through weight gain, as evidenced by a higher energy expenditure in the HFD group mice, while exercise was significantly decreased (Body energy expenditure (EE) is influenced by resting energy consumption, food thermogenic effects, and activity energy consumption. Both normal-weight and obese mice had rhythmic energy expenditures and peaked at night, as shown by EEecreased . This re1, EE2, and EE3 than the other groups, despite having a lower body weight than the HFD group, as evidenced by an increase in exercise and a decrease in food intake. This implies that S3T1 might be able to increase energy expenditure by enhancing basal metabolic capacity.The results varied between different interventions. Energy expenditure capacity is frequently reduced by the loss of fat and lean tissue (SE group mice) or decrease reduction in physical activity (HFD and TE group mice). Nevertheless, the S3T1 group had consistently higher EEIL-1β  and TNF-P < 0.05, P < 0.05).Intervention groups showed a reduction in IL-1β and TNF-α levels in serum compared with the HFD group . The secretion of LEP may partly contribute to the appetite-regulating effect, which could potentially explain the greater decrease in food intake in the SE group.The serum levels of LEP and ADP in each intervention group were lower than those in the HFD group (P < 0.05). When compared to the results of the SE and TE groups only, TE tended to show a better ability to regulate fat metabolism than SE (P < 0.05), as assessed by the level of FAS, CYP7A1, and SREBP-1c activities.LPL, CYP7A1, FAS, and SREBP-1c are key enzymes and factors that regulate lipid homeostasis and metabolism in the body. As shown in Bacteroidetes (P < 0.05), and a higher abundance of Proteobacteria (P < 0.001) and Patescibacteria (P < 0.01). The relative abundance of Proteobacteria was significantly reduced after plant extract interventions compared to HFD mice (P < 0.001). SE, TE, and their joint interventions increased the relative abundance of Bacteroidetes and Verrucomicrobia and depressed the F:B ratio. Due to several individual instabilities, the dominant flora in the S3T1 group of obese mice failed to be significantly reversed and remained similar to the HFD group. (2) At the genus level, Enterococcus, Escherichia–Shigella, Helicobacter, Staphylococcus, Mucispirillum, Candidatus_Saccharimonas, Enterorhabdus, Rikenella etc., which were positively correlated with obesity, were significantly increased in the HFD mice, whereas the relative abundances of Roseburia, Intestinimonas, and Oscillibacter were lower than the ND group (P < 0.05). When compared with the HFD group, the SE treatment increased the percentage of not only Intestinimonas, Oscillibacter but also Dubosiella, Lactobacillus, and Bacteroides, and reduced Enterococcus, Escherichia–Shigella, and Helicobacter. The TE treatment increased the percentages of Akkermansia, Blautia, Alistipes, and Bacteroides while decreasing Prevotella and reducing Coriobacteriaceae_UCG-002.The relative abundance of the major phyla present in the gut of different groups after treatments is depicted in Proteobacteria was positively correlated with the concentration of those inflammatory factors, whereas the abundance of the Bacteroidetes and Firmicutes were negatively correlated with them. Furthermore, at the genus level, these inflammatory indicators had a positive correlation with Escherichia–Shigella and Enterococcus, with the highest abundance in HFD. The bacterial taxa of the HFD and ND groups separated in different directions at the phylum and genus levels, and the intestinal flora of obese mice treated with plant extracts tended to revert to the ND group. Notably, the SE and SE-containing intervention groups most closely matched the bacterial taxon distribution of ND mice. The distribution of these plant extract intervention groups all shifted away from the TNF-α and IL-1β arrows.In the RDA analysis , I, inflAscophyllum nodosum and Camellia sinensis-leaf extracts on metabolic homeostasis in the mouse model of HFD-induced obesity and explored the functional characteristics and synergies of the different ingredients. All mice in the intervention groups were effectively protected against the persistent body weight increase that afflicted HFD mice, even without any mandatory control of the dietary intake, suggesting that the plant extracts elicited some intrinsically beneficial alterations in the organism. Plant extracts' supplementation alleviated fat accumulation, dyslipidemia, and dysbiosis caused by HFD. Some improvements in voluntary exercise and energy metabolism were also observed. We predict that the interventions slowed down the digestion, absorption, and utilization of exogenous lipids and inhibited endogenous lipid synthesis in the liver. Meanwhile, interventions accelerated lipolysis and improved glucose metabolism by alleviating chronic inflammation and insulin resistance. Having achieved the regulation of glucolipid metabolism, enzyme level, and intestinal flora structure, the energy metabolism function of the organism was enhanced, thus achieving the control of obesity.This study mainly investigated the effects of Bacteroides is considered to be a microorganism closely associated with the lipid absorption capacity , and further cause disturbances in glucose, lipids, and energy metabolism , 30. MacThe interventions might inhibit liver lipid synthesis while accelerating oxidative lipolysis. Increased levels of SREBP-1c and FAS in HFD mice might be an overexpression driven by endoplasmic reticulum stress through the regulation of the PERK-SREBP-1c-FAS signaling pathway, triggered as a result of chronic low-grade inflammation , 36. SE The regulation of glucose and lipid metabolism is directly related to the status of energy metabolism. Interestingly, HFD-induced obesity significantly altered the RER of the mice, not only in terms of a decrease in the values but also in terms of a disruption of the rhythm in respiratory entropy. RER could characterize the variation in energy metabolism in the body. Moreover, the circadian rhythm of adipose tissue lipid metabolism plays a critical role in the development of hyperphagia and obesity . Our finEscherichia–Shigella, Enterococcus, Enterorhabdus, Rikenella, and Helicobacter, were significantly higher in the stools of HFD mice. The administration of SE effectively reduced the growth of some of these harmful bacteria (Escherichia–Shigella and Helicobacter) while increasing the proportion of genera such as Roseburia, Intestinimonas, and Oscillibacter. In contrast, the TE intervention boosted the growth of several other groups . Bacteroides is a microorganism closely associated with lipid absorption capacity , can maintain intestinal homeostasis by binding to the Toll-like receptor (TLR) of colonic epithelial cells and regulating the expression of various proteins, resulting in the improvement of diseases such as high-fat diet-induced obesity and inflammatory bowel disease  and hormones  associated with energy metabolism. The above indicates that SE is more aggressive in decreasing blood glucose in obese mice with the addition of the Camellia sinensis-leaf component, making this process milder. Joint interventions, especially those with a higher proportion of Ascophyllum nodosum extracts, often showed better anti-inflammatory effects. In addition, S3T1 more notably regulated various key enzymes involved in lipid synthesis and metabolism; certainly, TE alone showed the same benefits in some of those indicators. S3T1 also had a better effect on regulating metabolic energy balance than a single supplement.SE alone showed a more effective capacity for fat loss and weight maintenance. The joint intervention groups also showed a trend toward better weight control with higher proportions of 3 values, despite moderate exercise and stable weight control. The higher equilibrium indices of the S3T1 group might imply that it allowed the body to obtain more beneficial effects on glucolipid metabolism, inflammation suppression, and balanced energy expenditure under milder conditions.The SE and S3T1 groups were prominent among the groups, with the strength of SE reflected in the control of body weight and blood glucose and that of S3T1 in the alleviation of inflammation and the improvement of energy metabolism. We noticed that SE mice often consumed less food than other mice. Apart from the effect of SE on appetite-regulating hormones or enzymes such as LEP and FAS, we could not rule out the possibility of sensory discomfort caused by the bitter taste. With proper food intake, S3T1 mice performed well in terms of glucolipid metabolism and inflammation improvement. Furthermore, when examining energy metabolism, S3T1 mice showed higher EEAscophyllum nodosum extracts and Camellia sinensis-leaf extracts controlled body weight and repaired the fatty lesions of the liver cells. By measuring the levels of various biomarkers, the interventions improved obesity-related dysglycemia, alleviated chronic inflammation, and downregulated the expression of enzymes related to lipid metabolism. While balancing the lipid glucose energy supply ratio (RER), S3T1 can even increase the body's energy expenditure value. Both SE and TE promoted the growth of Bacteroidetes in the gut, while TE also promoted the growth of Akkermansia. Behind this might be the fact that the interventions slow down the digestion, absorption, and utilization of exogenous lipids by modulating the biometabolic function of the gut flora or by affecting the absorption characteristics of the intestinal epithelium. Meanwhile, the interventions maintained the balance of lipid synthesis and consumption by improving chronic inflammation and glucose metabolism and promoting lipid mobilization. Alterations in glucolipid metabolism, cytokines, enzymes, and gut flora might together affect metabolic function in obese mice. Joint supplementation with them has more internalized and systemic health benefits as assessed by energy metabolism and anti-inflammatory effects. Metabolomic analysis of SCFAs and various types of complex O-glycans in feces will be carried out in subsequent experiments. Algorithms such as DESeq2 or ALDEx2 will be used in subsequent analyses to improve the sensitivity and specificity of the examination. Further experiments are required to explore the impact of Ascophyllum nodosum and Camellia sinensis-leaf extracts on obesity, not only focusing on blood glucose, inflammatory factors, enzymes, etc. but also on the circadian rhythm of lipid metabolism and exercise joint interventions, all of which can contribute to their beneficial effects.In conclusion, The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by the Animal Ethical and Welfare Committee of the Laboratory Animal Center of Xiamen University . The study was conducted in accordance with the local legislation and institutional requirements.YX was responsible for formal analysis, investigation, extracting and analyzing data, supervision, validation, and writing the original draft. XJ contributed to writing the protocol and report, supervision, extracting and analyzing data, and visualization. WZ was responsible for data curation, validation, and writing-reviewing and editing. QX conducted the data analysis, formal analysis, and writing-original draft. MZ contributed resources and data curation. ZZ, JHa, HaL, JD, YL, and HF provided feedback on the report. JHe was responsible for validation, supervision, and writing-review and editing. HoL was responsible for conceptualization, funding acquisition, project administration, and writing-reviewing and editing. All authors contributed to the article and approved the submitted version.


Probenecid is an old uricosuric agent used in clinics to treat gout and reduce the renal excretion of antibiotics. In recent years, probenecid has gained attention due to its ability to interact with membrane proteins such as TRPV2 channels, organic anion transporters, and pannexin 1 hemichannels, which suggests new potential therapeutic utilities in medicine. Some current functions of probenecid include their use as an adjuvant to increase the bioavailability of several drugs in the Central Nervous System (CNS). Numerous studies also suggest that this drug has important neuroprotective, antiepileptic, and anti-inflammatory properties, as evidenced by their effect against neurological and neurodegenerative diseases. In these studies, the use of probenecid as a Panx1 hemichannel blocker to reduce neuroinflammation is highlighted since neuroinflammation is a major trigger for diverse CNS alterations. Although the clinical use of probenecid has declined over the years, advances in its use in preclinical research indicate that it may be useful to improve conventional therapies in the psychiatric field where the drugs used have a low bioavailability, either because of a deficient passage through the blood–brain barrier or a high efflux from the CNS or also a high urinary clearance. This review summarizes the history, pharmacological properties, and recent research uses of probenecid and discusses its future projections as a potential pharmacological strategy to intervene in neurodegeneration as an outcome of neuroinflammation. Probenecid (PBN) is a fat-soluble derivative of benzoic acid, synthesized by Miller, Ziegler, and Sprague in 1949 . InitialBetween the 1950s and 1960s, some studies indicated that PBN blocks the active transport across the blood–brain barrier (BBB). It decreases the excretion of the acidic metabolites of dopamine (homovanillic acid) and serotonin (5-hydroxy indoleacetic acid), which led to several investigations in the psychiatric field . SimilarIn the field of basic research, PBN has gained significant relevance due to its reported ability to modify the activity of some membrane channels and transporters, including the blockade of pannexin-1 (Panx1) hemichannels and organic anion transporters (OAT) and the activation of TRPV2 channels ,12,13. IPBN is a white crystalline powder that is poorly soluble in water but soluble in alcohol, chloroform, and acetone, and is also highly soluble in lipids. It has a pKa of 3.7, meaning it is a weak acid  4]. It . It 4]. PBN has several pharmacological interactions, including increasing the plasma concentration of some antibiotics, antivirals, antiretrovirals, nonsteroidal anti-inflammatory drugs, benzodiazepines, and loop diuretics ,4,14. ItThe organic anion transporter (OAT) subfamily is constituted of amphiphilic solute transporters that belong to the solute carrier 22 (SLC22) transporter family (Slc22a) . OATs arPioneering studies indicated that PBN blocks the transport of neurotransmitter metabolites, including homovanillic acid , vanillyl-mandelic acid , and 5-hydroxy indoleacetic acid  from the cerebrospinal fluid (CSF) to circulating blood, which limits their subsequent excretion in urine ,24,25,26A further study reported that PBN administration increased the rat brain content of kynurenate, a metabolic product of tryptophan capable of antagonizing excitatory amino acid activity . Other s2+ ions are transported into the cell  is an ion channel belonging to the TRP-type channel family that is present in yeast, invertebrates, and vertebrates . TRPV2 ithe cell . Their t2+, which could be detrimental to the cell. Indeed, PBN has been reported to induce mechanical hyperalgesia and allodynia by increasing the synaptic transmission in the rat dorsal horn [PBN specifically activates TRPV2 in mice over the other TRP-like receptors, as has been revealed by using calcium imaging techniques and electrophysiological recordings in HEK293T cells expressing the receptor . In thatsal horn . Petitjesal horn .Panxs were originally described as proteins homologous to innexins found in invertebrates . They arPANX1, PANX2, and PANX3. The first two genes are highly expressed in the CNS and are abundantly expressed in the cerebellum, thalamus, cerebral cortex, and hippocampus. It is specifically expressed in neurons, interneurons, astrocytes, and microglia [The genes composing this family are icroglia ,56,57,58icroglia ,60,61. Iicroglia .Panx1 hemichannels exhibit two modes of activity: constitutive small-pore ion channel activity characterized by the low conductance (50–80 pS) of slightly selective anion permeation (chloride ions) and small molecules such as DAPI and ATP ,64,65,6650 of PBN observed was 150 μM in oocytes. However, in the human cell line (HEK293), PBN inhibited Panx1 hemichannel currents with an IC50 350 μM without altering purinergic signaling (P2X7R) [Silverman et al., using electrophysiological techniques, showed that PBN abolished Panx1 hemichannel currents at a 1mM concentration in oocytes expressing Panx1 in a concentration-dependent fashion . The IC5 (P2X7R) . Interes (P2X7R) ,51. Neur (P2X7R) ,51. Acti (P2X7R) . It is i (P2X7R) .Purinergic receptors are a family of plasma membrane proteins classified into two categories, P1 or adenosine receptors (ARs) and P2 receptors . The P2 50 of 203 μM [+ monocytes and P2X7R calcium currents in HEK-293 cells at a 1 mM concentration, which shows that PBN is involved in inflammation and directly blocks the human P2X7Rs [Since Panx1 hemichannels can release ATP, which is inhibited by PBN , and conf 203 μM . Furthern P2X7Rs .Some evidence indicates that PBN can also decrease the renal clearance of organic cations ,86,87, w+ concentrations activate NLRP. The latter acts as membrane receptors that sense and bind these signals to recruit and promote inflammasome complex assembly [Neuroinflammation refers to the inflammatory response within the CNS caused by various pathological factors or insults, including infection, trauma, ischemia, misfolded proteins, and toxins . Inflammassembly ,99. Otheassembly , and pyrassembly . These rassembly . Once th+, whereas in Panx1-knockout cells, caspase activation was undetectable, which supports the idea that Panx1 hemichannels mediate inflammation [The outflow of ATP and the consequent activation of P2X7Rs also promotes inflammasome activation  , which lammation . Moreoveammation .3 production. High levels of extracellular ATP induce a rapid outflow of K+ from the innate immune cells [2+ influx through purinergic receptors  that interact with Panx1 hemichannels [Panx1 participates in ATP-induced ATP release, and the mechanism involves purinergic receptors (P2YRs) that are coupled with G-protein and activate phospholipase C and IPne cells ,103,104 channels . Therefochannels ,107. Notchannels ,109. In channels ,110, a mchannels . This inchannels .Regarding the inhibition of Panx1 hemichannels with PBN, it has been described that the activation of the inflammasome has been evaluated in cultured astrocytes after oxygen–glucose deprivation (OGD) using different PBN concentrations. In that study, the authors concluded that the protein expression levels of aquaporin-4 (AQP4), NLRP3, and caspase-1 increased concomitantly after 6 h of OGD. This increase was strongly inhibited by PBN treatment applied before OGD . AdditioAccording to the above findings, inhibition at the early stages of the inflammatory process with PBN could be crucial to stop neuroinflammation and neuronal death, which is a common hallmark of several pathologies; thus, this opens the possibility of new uses of PBN.Epilepsy is one of the most common neurological disorders that affects brain function. It is characterized by spontaneous seizures leading to cognitive, neurological, and psychosocial consequences . EpilepsParkinson´s disease (PD) is the second most common neurodegenerative disorder affecting 2–3% of the population over 65 years of age; it is characterized by the deterioration of motor activities due to the progressive loss of dopaminergic neurons in the substantia nigra . This coOn the other hand, the effects of the intraperitoneal administration of L-kynurenine (kynurenic acid precursor), a competitive antagonist of the glutamate receptor type (NMDAR), together with PBN was evaluated in a 6-hydroxydopamine (6-OHDA)-induced PD mice model. In that study, the coadministration of L-kynurenine and PBN at doses of 75 mg/kg and 50 mg/kg, respectively, for 7 days reduced the decay in the total dopamine levels and protected mice from striatal damage and neurodegeneration . Silva-AAs mentioned above, PBN has also been used to increase 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) levels in the brain, and this can be used to generate a mouse model for PD. In that model, two studies were conducted. The first study examined the benefits of methylene blue when administered in an MPTP/PBN model mouse , and theAmong the neurological conditions affecting older adults, AD is the most prevalent chronic neurodegenerative disease and the most common cause of dementia worldwide. AD is characterized by brain atrophy and the accumulation of amyloid plaques and neurofibrillary tangles that constitute the hallmarks of the disease . AD compIn that pathological context, the soluble Aβ oligomers impair synaptic plasticity by reducing the size and number of dendritic spines, which prevents long-term potentiation (LTP), promotes long-term depression (LTD), and leads to cognitive dysfunctions and an impaired spatial memory . InteresThe use of PBN as a therapeutic agent for AD has still not been entirely explored since, in most of the exposed cases, it was used to increase the bioavailability in the CNS of other molecules, and in the case where it was administered as a mono drug for AD treatment, it did not achieve sufficient benefits, and the evidence is still preliminary.PBN has also been used in several animal and cell models of CNS disorders. For instance, PBN was shown to have a protective effect in a Huntington’s disease (HD) model, a progressive and autosomal dominant neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin gene which encodes an abnormally long polyglutamine repeat in the huntingtin protein. HD is characterized by movement disorders (including chorea and a loss of coordination) and cognitive decline defects . In the PBN was also used in an animal model of multiple sclerosis (MS), an experimental autoimmune encephalomyelitis (EAE) model ,128. MS In another study, Wei et al. evaluated the protective effect of the intravenous, intraperitoneal, and gavage administration of PBN against cerebral ischemia/reperfusion (I/R) injury in rats. Independent of the route of administration, PBN decreased neuronal death in the CA1 area, and some inflammatory markers were induced by I/R . A similOn the other hand, PBN also has been used to alleviate migraine headaches . CorticaIn addition to its use in the mentioned diseases, PBN has also been used in other studies to discriminate between the effect of connexin and Panx1 hemichannels since PBN only blocks Panx1 hemichannels and not the gap junction channels formed by Panx1 . Thus, iThis review summarizes the recent research uses of PBN, a drug formerly used for gout treatment, as a pharmacological agent for some CNS disorders. PBN uses are related to their identified targets, including the blockade of OATs and Panx1 hemichannels and the activation of TRPV2 channels. Among the identified functions of PBN are its use as an adjuvant to increase the bioavailability of several drugs in the CNS. More recent preclinical evidence suggests the neuroprotective, antiepileptic, and anti-inflammatory properties of PBN in animal models of neurological and neurodegenerative conditions. Among these studies, the use of PBN as a Panx1 hemichannel blocker that reduce neuroinflammation is highlighted, and the neuroinflammation is a major cause of diverse CNS alterations. In this regard, it is important to note that neuroinflammation is a broad and complex response that involves several molecular cascades activated by specific molecular events that result in the release of inflammatory mediators and the activation of cellular targets such as receptors, channels, and enzymes. Therefore, the intervention of specific pathways does not discard the recruitment of other routes. Likewise, potential differences between rodent and human inflammasome types and species-dependent activation mechanisms should be considered. In this regard, it is noteworthy that in many cases, the evidence obtained from animal studies can not necessarily be translated into human studies. Regardless, the preclinical evidence of an anti-inflammatory-like effect of PBN reviewed here paves the way for future studies. The evidence summarized here also highlights the ability of PBN to reduce epileptic discharges in studies in rats and the severity of convulsions upon the induction of status epilepticus.Although its clinical use has declined over the years, advances in the use of PBN in the preclinical research suggest that it may be useful to improve conventional therapies in the psychiatric field where the drugs used have a low bioavailability, either because of their deficient passage through the BBB or a high efflux from the CNS, or also a high urinary elimination rate. Based on the information gathered, it is recommended to continue studying the drug PBN in neuroinflammatory diseases, and particularly its possible benefit at the hippocampal level in AD since positive results of its use were evidenced in these cases. In addition, their capability to cross the BBB and their ability to increase the bioavailability of several metabolites in the CNS support their use.