Giardia duodenalis is a common intestinal protozoan that can cause diarrhea and intestinal disease in animals and in humans. However, the prevalence and assemblages of G. duodenalis in pigs from Guangxi Zhuang Autonomous Region have not been reported. In this study, a total of 724 fecal samples  were obtained in four areas of the region . The gene of the small subunit ribosomal RNA (SSU rRNA) of G. duodenalis was amplified by nested PCR. The results show that the prevalence of G. duodenalis in pigs was 3.59% (26/724), of which 14 samples belonged to assemblage A (53.85%) and 12 samples belonged to assemblage E (46.15%). The infection rates of G. duodenalis in Hezhou, Yulin, Nanning, and Guigang were 0%, 0.7%, 10.8% and 1.1%, respectively ; whereas 5.1% of breeding pigs, 6.0% of piglets, 2.4% of fattening pigs, and 1.0% of nursery pigs were infected with G. duodenalis . The SSU rRNA-positive samples were amplified by PCR based on the β-giardin (bg), glutamate dehydrogenase (gdh), and triphosphate isomerase (tpi) genes. Ten, eight and seven positive samples were detected, respectively. Based on phylogenetic analysis of the three genetic loci sequences, a multilocus genotyping A1 was found. The findings of this study provide basic data for the development of prevention and control of G. duodenalis infections in pigs and humans in the Guangxi Zhuang Autonomous Region. Giardia spp. are common intestinal parasites, affecting both humans and a wide variety of other animals [Giardia species have been described [Giardia species  infect a wide range of animals including birds, amphibians, rodents, and mammals [Giardia duodenalis is an important zoonotic parasite that can cause giardiasis [G. duodenalis is simple. It consists of two key stages: rapidly multiplying trophozoites which attach to intestinal epithelial cells, and cysts with high resistance to environmental degradation, that are released in the feces and spread through the fecal-oral route [G. duodenalis shows a wide range of clinical symptoms, such as acute or chronic diarrhea, nausea, abdominal pain, vomiting, and weight loss [ animals . The parescribed . The six mammals . Giardiaardiasis , 35. Theal route , 19, 23.ght loss , 39. Furght loss , 34.G. duodenalis through molecular biological analysis has greatly contributed to the understanding of its taxonomy, population genetics, and epidemiology. The small subunit ribosomal RNA (SSU rRNA), β-giardin (bg), glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) genes are four commonly used gene loci in the genotyping of G. duodenalis, but a single gene may not correctly identify G. duodenalis or completely describe its genetic characterization [G. duodenalis, and is considered to be useful for detecting and identifying mixed infections by different assemblages of the parasite [G. duodenalis can be divided into eight assemblages (A–H) based on genetic analysis, with each assemblage exhibiting a distinct host range [G. duodenalis.The comprehension of rization , 43. Mulparasite , 37. To st range , 20, 27.st range , 31. Othst range , 13, 26.st range , 35, 44.G. duodenalis in pigs [G. duodenalis in pigs in Guangxi Zhuang Autonomous Region, China. Pigs infected with G. duodenalis may cause malabsorption and weight loss, resulting in a decline in pig production [G. duodenalis infection and identify the genotypes present in pigs from Guangxi Zhuang Autonomous Region, China. The study provides essential data concerning G. duodenalis infections in pigs in southern China, which can contribute to the development of targeted public health and effective strategies for prevention and control of giardiasis in this area.Although some studies have reported prevalence and distribution of  in pigs , 18, 41,oduction . CurrentFrom March 2021 to May 2022, a total of 724 fecal samples  were collected from four cities  in Guangxi Zhuang Autonomous Region, China . Fecal s® Stool DNA Kit , according to the manufacturer’s instructions. The extracted DNA was subsequently stored at −20 °C to maintain its integrity for subsequent analyses.Approximately 200 mg of each fecal sample was aseptically transferred to individual 1.5 mL centrifuge tubes. Genomic DNA extraction was performed using an E.Z.N.A.SSU rRNA gene, as previously described [et al. [SSU rRNA-positive samples were subjected to amplification using nested PCR targeting the bg, gdh, and tpi genes, as previously described [G. duodenalis stored in the laboratory at −80 °C) and a negative control (distilled water) were included in each PCR assay. Each sample was processed three times at the SSU rRNA gene and each SSU rRNA-positive sample was processed three times at the bg, gdh, and tpi genes. The PCR products were identified by 1.5% agarose gel electrophoresis .All samples were amplified by using nested PCR targeting the escribed . The PCR [et al. . All SSUescribed , 16, 36.https://www.ncbi.nlm.nih.gov/). The MLG method was only used to analyze the genetic diversity of positive samples which were successfully sequenced at all three gene loci . Neighbor-joining trees [PCR products of positive samples were processed for two-directional sequencing by Sangon Biotech . The obtained DNA sequences were aligned with homologous sequences available in the GenBank database of the National Center for Biotechnology Information  was used to assess differences in prevalence between regions, feeding stages and farming practices. Differences of OQ943958–OQ943959 for the SSU rRNA gene, OQ934094–OQ934095 for the bg gene, OQ934096–OQ934101 for the gdh gene, and OQ934102–OQ934104 for the tpi gene.All nucleotide sequences have been submitted to the GenBank database in NCBI and allocated accession numbers as follows: G. duodenalis based on the SSU rRNA gene. The highest infection rate of G. duodenalis was in Nanning , followed by Guigang , Yulin , and Hezhou . Statistical analysis demonstrated significant variations in the positive rate of G. duodenalis infection with pigs among different regions . Among the various feeding stages, piglets had the highest infection rate , followed by breeding pigs , fattening pigs , and nursery pigs  . The prevalence of G. duodenalis infection in intensive and free-range farms were 3.7% (15/410) and 3.5% (11/314), respectively. Statistical analysis showed no significant difference between the two groups (p > 0.05)  tested positive for  > 0.05) .SSU rRNA gene showed two assemblages (A and E) of G. duodenalis in pigs. Of the 26 SSU rRNA-positive samples, 14 isolates  belong to zoonotic assemblage A, while the other 12 isolates  were identified as assemblage E  in Canada, 3.4% (3/90) in Brazil, 14% (120/856) in Denmark, 14.8% (110/745) in Korea, 25.4% (53/209) in Nigeria, and 31.1% (90/289) in Western Australia [G. duodenalis in this investigation was similar to the prevalence in Sichuan Province  [e groups . In thiswas 3.2% . Therefo to pigs . The infch as 1% /633 in Czil, 14% 0/856 in ustralia , 28. In  11/357) ; it was  11/357) , 18, 40, 11/357) , 46–48. χ2 = 45.616, p < 0.01; χ2 = 8.874, p < 0.05). The highest prevalence  was observed in Nanning, and the lowest prevalence  in Hezhou. Among the different feeding stages, the infection rates of breeding pigs  and piglets  were higher than those of fattening pigs  and nursery pigs . Similarly, a study in Henan Province found that piglets had higher infection rates  compared to fattening pigs  [G. duodenalis and other pathogens.The positive rate in different regions and for different feeding stages was significantly different  . In cont, 1/439) . Previou, 1/439) . The hig5.8%, 14/3 comparep > 0.05). The positive rate of intensive feeding farms was 3.7% (15/410), while the positive rate of free-range farms was 3.5% (11/314). The low positive rates of both intensive feeding and free-range farms indicates that people have begun to pay attention to the prevention and control of giardiasis. However, the possibility that insufficient samples on free-range farms led to lower infection rates cannot be ruled out.No significant differences were found in the positive rates for different feeding methods in our study  and assemblage E  were identified among the 26 samples. Assemblage A was the predominant genotype in this study. However, in Australia, Armson et al. identified 56 G. duodenalis positive samples belonging to assemblage E  and assemblage A  [G. duodenalis positive samples belonged to assemblage E  and assemblage A  [SSU rRNA locus means the possibility of zoonotic transmission in this areas. Notably, we also identified assemblage E, which has been reported to infect humans [n = 14, 5.85% and , 15.4%) . The finG. duodenalis in pigs, the sequence characters of the bg, gdh, and tpi genes were analyzed for the 26 SSU rRNA positive samples and the MLGs were characterized in pigs based on data from these three loci. Amplification success rates at the bg, gdh, and tpi loci varied from 26.92% to 38.46%. The genetic loci of Giardia have different substitution rates, leading to different resolution for parasite typing at varied loci [e.g. SSU rRNA) have higher diagnostic sensitivities than those based on single-copy genes  [SSU rRNA-positive samples not amplified at the other three loci may be due to the limited sensitivity of PCR in detecting the single-copy genes. The present study confirmed that more positive samples of G. duodenalis can be amplified based on the SSU rRNA gene. The same problem has been reported in similar studies in pigs in Xinjiang [SSU rRNA-positive samples were simultaneously amplified at three loci, with four MLGs [SSU rRNA-positive samples were simultaneously amplified at three loci, with four MLGs [SSU rRNA-positive samples were simultaneously amplified at three loci, with one MLG [SSU rRNA-positive samples from humans were simultaneously amplified at three loci, with 23 MLGs [SSU rRNA-positive samples from humans were simultaneously amplified at three loci, with two MLGs [G. duodenalis is different in different regions.To further expand knowledge of the genetic diversity of ied loci . Nested and tpi) . TherefoXinjiang . The resour MLGs . In Shaaour MLGs . In Fuji 23 MLGs . In Souttwo MLGs . These rG. duodenalis in Guangxi Zhuang Autonomous Region may be a potential source of infectious cysts that affect humans. Workers on pig farms and in slaughterhouses are at risk of infection with G. duodenalis. Understanding the transmission dynamics between animals and humans is crucial for effective disease control and prevention strategies. Therefore, public health problems caused by G. duodenalis should be investigated.In short, pigs infected with G. duodenalis  was found in this study, the identification of assemblages A and E, and the predominance of assemblage A, suggest that pigs may play an important role in the transmission of G. duodenalis. The multilocus genotyping results of bg, tpi, and gdh loci showed only one MLG A1. The identification and understanding of the distribution of G. duodenalis in animals, such as pigs, are crucial for the development and implementation of effective prevention and control measures. These findings highlight the importance of continued surveillance and targeted interventions to mitigate the risk of G. duodenalis transmission between animals and humans. The data presented in this study serve as a foundation for future research on G. duodenalis.In conclusion, this study provides the first report of giardiasis in pigs from Guangxi Zhuang Autonomous Region, China. Although a low infection rate of All authors declare that they have no conflict of interest.


Among the human T-lymphotropic virus (HTLV) types, HTLV-1 is the most prevalent, and it has been linked to a spectrum of diseases, including HAM/TSP, ATLL, and hyperinfection syndrome or disseminated strongyloidiasis. There is currently no globally standard first-line treatment for HTLV-1 infection and its related diseases. To address this, a comprehensive review was conducted, analyzing 30 recent papers from databases PubMed, CAPES journals, and the Virtual Health Library (VHL). The studies encompassed a wide range of therapeutic approaches, including antiretrovirals, immunomodulators, antineoplastics, amino acids, antiparasitics, and even natural products and plant extracts. Notably, the category with the highest number of articles was related to drugs for the treatment of ATLL. Studies employing mogamulizumab as a new perspective for ATLL received greater attention in the last 5 years, demonstrating efficacy, safe use in the elderly, significant antitumor activity, and increased survival time for refractory patients. Concerning HAM/TSP, despite corticosteroid being recommended, a more randomized clinical trial is needed to support treatment other than corticoids. The study also included a comprehensive review of the drugs used to treat disseminated strongyloidiasis in co-infection with HTLV-1, including their administration form, in order to emphasize gaps and facilitate the development of other studies aiming at better-directed methodologies. Additionally, docking molecules and computer simulations show promise in identifying novel therapeutic targets and repurposing existing drugs. These advances are crucial in developing more effective and targeted treatments against HTLV-1 and its related diseases. Retroviridae family was discovered and isolated for the first time in 1980, and since then ..E)-3-pheA drug that has been studied is pomalidomide (pom). A group of researchers who had already tested this drug in vitro and proved it increased the susceptibility of HTLV-1-infected cells to NK and CTL killing used the rhesus macaque model to determine if pom treatment of infected individuals activates the host immune system and allows for the recognition and clearance of HTLV-1-infected cells. The results suggested pomalidomide can enhance the immune response to HTLV-1 infection, but this response is not maintained, and the authors suggested a combination with other well-tolerated drugs such as lenalidomide  24]..24].Bidens pilosa extracts have demonstrated growth suppressive effects on HTLV-1-infected T-cell lines and ATLL cells .Although clinical trials are extremely important to validate treatments, in vitro studies also play an essential role in the development of treatments. They help elucidate mechanisms of action and contribute to the search for new drugs or even the improvement of existing ones.Among the drugs authorized for use by the US Food and Drug Administration and the European Medicines Agency, dimethyl fumarate (DMF) has been highlighted as a promising treatment for ATLL due to its effects in cancer cells, including cell signaling, proliferation, and cell death . A studyThese results were observed by Maeta et al., and Sato et al., who evaluated the specific mechanism of DMF effects on the caspase recruitment domain family member 11 (CARD11)–BCL10 immune signaling adaptor (BCL10)–mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) (CBM) complex and upstream signaling molecules that are critical for NF-ΚB signaling in MT-2 cells by immunoblotting. In the cited study, DMF inhibited proliferation and influenced the apoptosis of HTLV-1-infected cells by suppressing the CBM complex in the NF-ΚB pathway  43,44].,44.43,44In the literature, apoptosis is highlighted as one of the main routes by which drugs act in the treatment of ATLL. In this context, we have dorsomorphin, an AMPK inhibitor that induced apoptosis in PBMCs from ATLL patients and in HTLV-1-infected T-cell lines in a dose- and time-dependent form. It increased the production of intracellular reactive oxygen species (ROS) that mediated DNA damage in HTLV-1-infected T-cell lines and suppressed the growth of human ATLL tumor xenografts in NOD/SCID mice . E)-3-phenyl-5-(phenylamino)-2-styryl-1,3,4-thiadiazol-3-ium chloride derivatives were promising against cells, with IC50 values lower than 10 μM. The compounds with electron donor substituents (R=CH3 or OCH3) were more active, showing cell death by necrosis, interacting with DNA, and exhibiting lower hydrophobicity. Also, the preliminary pharmacokinetic profile obtained through human serum albumin (HSA) binding affinity using multiple spectroscopic techniques, zeta potential, and molecular docking calculations revealed that all the mesoionic compounds presented good binding parameters toward serum albumin, indicating feasible biodistribution in the human bloodstream . These (odstream  23]..E)-3-pheAnother important strategy to treat ATLL would be the use of antibody drug conjugates (ADCs) that have been recently introduced as part of anticancer therapy . Yokota Hypericum perforatum L., in photodynamic therapy (PDT), was tested against ATL cell lines. It was highly effective in inducing the inhibition of cell proliferation in an ATL cell with minimal effect on peripheral blood CD4+ T lymphocytes and caused apoptosis and G2/M phase cell cycle arrest in leukemic cells. Western blot analyses also revealed the downregulation of Bcl-2 and enhanced expression of Bad, cytochrome C, and AIF. The luciferase assay showed an enhanced expression of Bax and p53 proteins. Finally, the treatment suppressed the expression of viral protein HBZ and Tax by blocking the promoter activity via HTLV-1 5′LTR and 3′LTR, which highlighted the promising use of hypericin-PDT as therapy for ATLL ..Nigella Recently, an innovative therapeutic perspective for the treatment of ATLL was discovered, paving the way for innovative research through viral biology. One study observed that the synergy between two transcription factors (IRF4/BATF3) makes ATLL viable, and when these factors are genetically knocked out, the cell cycle of these types of cancer is interrupted, as the MYC oncogene (target product of the activation of transcription factors’ transcription) is directly inhibited. Additionally, the study demonstrated that bromodomain-and-extra-terminal-domain (BET) chromatin protein inhibitors were able to act on the HBZ/BATF3 interface of cell lines from patients diagnosed with ATLL or xenotransplantation, performing cytotoxic action on all strains in in vitro preclinical tests .E)-3-phenyl-5-(phenylamino)-2-styryl-1,3,4-thiadiazol-3-i lead to necrosis of infected cell lineages. The Fv-Fc anti-CD70 antibody induces the selective death of peripheral blood mononuclear cells in ATLL patients. Various therapies, such as dimethyl fumarate, dorsomorphin, etretinate, arsenic/interferon-alpha (As/IFN-α) with thymoquinone (TQ) ..56].Schneidermann et al. (2022) sought to demonstrate in vitro the effects of antiretrovirals already used in therapy against the human immunodeficiency virus (HIV) as possible inhibitors for blocking HTLV-1 transmission. The study used a recombinant HTLV-1 integrase treated with increasing concentrations of cabotegravir, a recently developed drug that has been associated with efficacy in blocking HIV-1 transmission and replication, demonstrating similar results as raltegravir as an HTLV-1 integrase inhibitor. This study supports the possible use of antiretrovirals preventively in cases of exposure to HTLV-1 .Teriflunomide, a dihydrooratate dehydrogenase inhibitor, was tested on samples from 12 patients with HAM/TSP caused by HTLV-1. Patients were not using corticosteroids and had been diagnosed with HTLV-1 for over 10 years. The collected cells were cultured and subjected to tests to assess lymphocyte proliferation; expression of activation markers, Tax, and HBZ mRNA; and Tax protein expression. The results showed that teriflunomide exerted a dose-dependent regulatory action on the spontaneous proliferation of lymphocytes. The best response was observed at the 100 μM dose, resulting in a 90.7% inhibition, preventing the abnormal proliferation of CD4+ and CD8+ T lymphocytes, although markers of lymphocyte activation, such as CD25, were still expressed. There was no change in Tax and HBZ mRNA expression, as well as in Tax protein expression  58]..58].A phase 2, open-label, single-arm clinical trial was conducted with 20 patients diagnosed with HAM/TSP to evaluate the efficacy and safety of short-term treatment with L-arginine. During the study, patients received 20 g of L-arginine three times a day for one week; then, the amino acid was discontinued, and patients were followed for 3 weeks, evaluating them by the 10 m walk test, timed Up and Go test (TUGT), and neopterin levels produced after stimulation via interferon-gamma (IFN-γ). Patients demonstrated an increase in walking speed on the 10 m walk test on day 14 and improvement in gait functions on the TUGT on days 14 and 28, in addition to a reduction in neopterin levels in the cerebrospinal fluid of 12.7 pmol/mL to 10.6 pmol/mL. Adverse effects were minimal  61]..61].HAM/TSP also causes urinary disorders, such as an overactive bladder, and affects patients’ quality of life. In the search for alternatives to treat these symptoms, a prospective, single-center, open-label study evaluated the 12 week use of 300 mg of prosultiamine, a homologue of allithiamine, in 16 patients with HAM/TSP-related overactive bladder, evaluating nocturnal urination frequency, urinary urgency, and nerve growth factor/creatinine and adenosine. There was a decrease in biomarker concentrations and less nighttime urination and urinary urgency, with no reports of relevant adverse effects during the trial .Below is an illustrative figure depicting the pathological process of leukocyte infiltration and the consequent injury that leads to the development of clinical manifestations associated with HAM/TSP, as well as the possible sites of action of the studied molecules. The HAM/TSP is related to the production of IFN-γ (interferon-gamma) and NFG (nerve growth factor) by HTLV-1-infected cells and HTLV-1-specific cytotoxic cells infiltrating the cerebrospinal fluid, leading to damage to neural tissues, both directly through the exacerbation of inflammatory responses and indirectly by recruiting more IFN-γ-producing cells into the cerebrospinal fluid. Thus, the action of antiretrovirals raltegravir  and teriStrongyloides stercoralis, a nematode of the Strongyloididae family that causes chronic and asymptomatic infections ..S. stercS. stercoralis resistance due to repeated mass drug administrations as part of control programs for onchocerciasis or lymphatic filariasis elimination. Thus, the development of alternative treatment options to ivermectin that serve as additions to the depleted drug armamentarium are urgently needed .,66.65,66S. stercoralis larvae. In view of this, the patient was tested for HTLV-1, demonstrating the co-infection. Then, he was treated with subcutaneous ivermectin and albendazole due to the extensive involvement of the intestine, which prevents good drug absorption. Gradually, an improvement in the clinical condition was observed, proving that subcutaneous administration could be an option in similar cases [Hunter et al. (2019) reports the clinical case of a man residing in an Argentine province who reported diarrhea, abdominal pain, and weight loss in recent months. This case was treated as a severe inflammatory bowel disease; however, imaging tests and biopsy revealed the presence of ar cases .S. stercoralis, in which six were cases of co-infection with HTLV-1. The participants received treatment with ivermectin of 200 mcg/kg over 2 consecutive days, and 15 days later, the effectiveness of the treatment was assessed through the Bearmann technique in the stool, confirmed by PCR, and the response of cytokines was verified through flow cytometry. The study demonstrated Tregs are indeed influenced by S. stercoralis, especially in co-infected patients; therefore, an effective treatment for strongyloidiasis can confer benefits by inhibiting the parasite’s synergistic effect on the expansion of Tregs and promoting better immune responses in HTLV-1-positive patients  analyzed the hypothesis that the ivermectin could act on the expansion of regulatory T cells (Tregs) in contribution to the restoration of the cytokine response, improving the antigen-specific response. The study analyzed twenty asymptomatic people, but with active infection by patients . Furtherermectin .Finally, Dykie et al. (2020) argue that a relevant point to find new drugs to strongyloidiasis is the need to develop new animal models with a more appropriate size to allow for detailed studies of how the interaction between these two pathogens occurs and what drugs may interfere with co-infection and spread of the parasites .A method prominent in recent studies for drug discovery is molecular docking (MD). This technique is used to predict the preferred position of a binding molecule in a receptor macromolecule and evaluate physical-chemical properties related to their biological activity . MD can Studies have described the progression of HTLV-1 infection, inflammatory conditions, and the occurrence and severity of related diseases as consequences of HTLV-1 replication in CD4+ T and CD8+ T lymphocytes, as well as the imbalance between proinflammatory and anti-inflammatory cytokines ,71. TherIn addition, the cost of drug discovery and development can reach up to 2.6 billion dollars , but by The recent study by Jahantigh et al. (2022) used molecular docking to identify potential protease inhibitors among approved antiviral drugs as a potential treatment against HTLV-1. In general, protease is an essential enzyme for the viral replication cycle and therefore represents a promising target for the development of antiviral therapies. In the study, molecular docking techniques and molecular dynamics simulation were used to evaluate the interaction of different antiviral drugs with HTLV-1 protease . The resTo take advantage of knowledge about existing antivirals and reconstruct the binding pathway of an anti-HIV drug indinavir, in a complex with HTLV-1 protease, Sohraby and Hassan (2021) used molecular dynamics simulations. Indinavir is a protease inhibitor, and the simulation made it possible to follow the molecular pathway of indinavir binding involved in this process. The reconstruction of the binding pathway of indinavir with the HTLV-1 protease provided valuable information on the molecular mechanisms involved in this interaction for developing new treatment solutions against HTLV-1 .Barski et al. (2020) analyzed a structural basis for the inhibition of HTLV-1 integration through the analysis of deltaretroviral intasome structures obtained by electron cryomicroscopy (cryo-EM). HTLV-1 integration is an essential process for viral replication, and understanding the molecular details involved in this process may lead to the development of more effective therapeutic strategies against HTLV-1. Analysis of the structures revealed specific interactions between intasome components and viral DNA, providing insights into the mechanisms of DNA recognition and binding during the integration process. Furthermore, the researchers identified critical regions of the intasome that could be targets for the development of specific inhibitors .Sousa-Pereira et al. (2020) also used MD to obtain theoretical results to evaluate the activity of chloride derivatives, synthetic compounds that are promising chemotherapy agents in HTLV-1 infection. Their study showed some of the thiadiazole chloride derivatives demonstrated potent activity against HTLV-1, suggesting that the mechanism of action of the most promising compound could be related to its capacity to intercalate into DNA . An interesting alternative against viruses is vaccines. The study carried out by Alam et al. (2020) explored all the possible strains of HTLV-1 using different data-driven tools, including immunoinformatics, to predict an efficient vaccine. Using Vaxijen, which predicts antigens based on the biophysical and physicochemical properties of proteins, as well as other tools, the authors identified an envelope glycoprotein, GP62, of HTLV-1 as a probable antigen. It was targeted for designing the vaccine because glycoproteins are located on the outer layer of the cell membrane and can easily be recognized by the immune system. It also fits into parameters such as the Shannon entropy (H), which was selected as a variability metric, and the variability threshold was set at 0.5. Afterward, only the conserved epitopes were picked, and they coincided entirely through their whole length. After conducting a rigorous analysis with numerous databases and using tools of docking methods, nine epitopes for MHC (major histocompatibility complex) class I and five epitopes for MHC class II were selected. Among all the predicted peptides, the epitopes ALQTGITLV and VPSSSTPL interacted with three MHC alleles, and the author estimated that worldwide coverage for these alleles was about 70%. Docking revealed that ALQTGITLV and VPSSSTPL epitopes interact strongly with the epitope-binding groove of HLA-A02:03 and HLA-B35:01, respectively, and that molecular dynamics simulations of the bound complexes revealed that they form stable complexes. Therefore, this study revealed promising options for the development of a vaccine against HTLV-1 .In the same sense, Jahanting et al. (2021) used bioinformatics programs to investigate possible candidates for producing immunobiologicals against HTLV-1. Investigating five proteins , the researchers identified a strong candidate with a domain of eight epitopes highlighted both in B cells and in T cells. To verify the possible interactions between the epitopes of the projected protein and immune cell receptors, in silico docking studies were conducted, demonstrating strong interaction and the formation of complexes O2–HLA-A02:01 and D8–HLA-A02:01, which remained stable throughout the simulation. The results demonstrated an interaction between the projected protein and B- and T-cell receptors, producing humoral and cellular immune responses .Another interesting perspective was tested by Pandey et al. (2019), which used computer simulations to present a combinatorial screening algorithm to design a multiepitope vaccine targeting HTLV-1 infection. The authors combined different amino acid sequences to form a multiepitope vaccine and subjected it to analyses and computer simulations to assess its structural stability and its ability to interact with the immune system. They concluded this vaccine can induce an effective immune response against HTLV-1, targeting multiple antigenic regions of the virus and increasing the likelihood of protection against infection .The human T-lymphotropic virus type 1, HTLV-1, is a high-impact viral infectious agent whose mechanisms of pathogenesis are not completely defined. Furthermore, the existence of diseases related to the infection, such as HAM/TSP, strongyloidiasis, and ATLL, of varying courses and severities, make treatment against the infection a challenge for the scientific community. There is an international consensus regarding the treatment of ATLL and HAM/TSP to define prognostic factors, clinical subclassifications, treatment strategies, and response criteria. It uses GRADE criteria for quality of evidence and affirms that for ATLL, the level of evidence should be regarded as low or very low—the equivalent of a GRADE evidence score of C or D. For HAM/TSP, there is evidence to support the use of both methylprednisolone and prednisolone as for progressive disease, but there is no evidence to support the use of other therapies including antiretrovirals and interferon-α. Therefore, although the consensus exists, it does not determine the standardized use of drugs as the first line of treatment, because not all drugs are available and approved for use in countries with the highest prevalence of HTLV-1 infection.However, the observation of existing literature as well as the use of molecular techniques and in vitro and in vivo studies can help to keep the consensus updated and, in the future, help in the elaboration of guidelines with alternative treatments and/or more effective drugs. In our review, the category with the highest number of articles was “drugs for the treatment of ATLL”, perhaps due to the severity associated with this disease or the fact that the virus was first isolated from a person with a cutaneous T-cell malignancy. Another interesting thing was the fact that compounds from very different classes and origins were studied and showed promise for the treatment of ATLL. Among the selected articles, we observed compounds from various classes, ranging from mesoionic compounds to antineoplastics such as arsenic trioxide, as well as antibody drug conjugates, and moreover natural products such as thymoquinone and hypericin. This demonstrates that many classes of compounds can be used to treat ATLL.Articles regarding the treatment of HAM/TSP also demonstrated a wide variety of classes, including immunomodulators; antiretrovirals; and even arginine, which is an amino acid that seems promising in the palliative treatment of the inflammatory process, thus increasing the patient’s quality of life.Regarding strongyloidiasis, only a few articles on this topic were included, with most of them being case reports. In these reports, the authors highlighted that co-infection may be a determining factor in the difficulty of responding to treatment with ivermectin, which is considered the first choice for strongyloidiasis. Additionally, the negative prognosis has been attributed to late diagnosis or association with HTLV-1, suggesting that this factor could directly influence treatment failure.Further studies are needed to assess the effectiveness of these compounds, including the use of molecular docking and computer simulations to discover new therapeutic targets, design vaccines, or even repurpose existing antiviral drugs. The weaknesses of this study may reside in the few controlled and randomized clinical trials in the last 5 years, which may make it difficult to obtain conclusive evidence on the efficacy of the drugs under analysis. In addition, the heterogeneity of the included studies can make comparison and generalization difficult, due to differences in patient populations, diagnostic criteria, treatment protocols, and evaluated outcomes, as well as the intrinsic limitations of primary studies, such as small samples, lack of adequate control of variables, and insufficient duration of follow-up.This study provides a careful and comprehensive review of the most up-to-date evidence on new drugs and therapies under development, identifying opportunities for developing more effective and targeted treatments, as well as promoting the visualization of gaps in knowledge and providing guidance for future research and clinical trials because it systematized promising therapeutic targets and potential treatments for various conditions associated with HTLV-1. Through data synthesis, this document is expected to provide better insights for the scientific community, professionals, and health managers regarding the latest research on drugs against HTLV-1 infection and related diseases, supporting proposals for treatment.


The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) play a crucial role as pattern-recognition receptors within the innate immune system. These receptors, present in various cell and tissue types, serve as essential sensors for viral infections, enhancing the immune system’s capacity to combat infections through the induction of type I interferons (IFN-I) and inflammatory cytokines. RLRs are involved in a variety of physiological and pathological processes, including viral infections, autoimmune disorders, and cancer. An increasing body of research has examined the possibility of RLRs or microRNAs as therapeutic targets for antiviral infections and malignancies, despite the fact that few studies have focused on the regulatory function of microRNAs on RLR signaling. Consequently, our main emphasis in this review is on elucidating the role of microRNAs in modulating the signaling pathways of RLRs in the context of cancer and viral infections. The aim is to establish a robust knowledge base that can serve as a basis for future comprehensive investigations into the interplay between microRNAs and RIG-I, while also facilitating the advancement of therapeutic drug development. DDX58, IFIH1, and DHX58, respectively. These three proteins exhibit 30% similarity in their amino acid sequences, particularly in their key domains  [RLRs are classified within the DExD/H-box RNA helicase family and encompass three distinct members, namely RIG-I, melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), which are encoded by the genes RIG-I primarily detects two main types of RNA: single-stranded RNAs (ssRNAs) with a triphosphate group (PPP) at their 5′ end, and short double-stranded RNAs (dsRNAs) (<300 bp) ,5,6,7,8.Under normal circumstances, RIG-I/MDA5 remains in an inactive and inhibited state within the cytoplasm ,25. HoweIn parallel, linker proteins TRAF family-member-associated NF-κB activator (TANK), nucleosome assembly protein 1 (NAP1), and, similar to NAP1, TBK1 adaptor (SINTBAD), are recruited by the TRAF3 bound to MAVS. TANK binding to TANK-binding kinase 1 (TBK1), which is linked to upstream RLR signaling through TANK, leads to the phosphorylation of IRF3. Phosphorylated IRF3 (p-IRF3) dimerizes and translocates to the nucleus to initiate the transcription of the IFN-β gene ,29. ThisWhile LGP2 lacks the ability to directly recognize RNA and activate downstream pathways, it plays a crucial role in the regulation of RLR signaling. LGP2 exerts its inhibitory effects on the RIG-I-mediated signaling pathway through various mechanisms. Firstly, the C-terminal RD domain of LGP2 forms a heterodimeric complex with the RD domain of RIG-I, hindering RIG-I multimerization and thus impeding its recognition of viral dsRNAs. Also, LGP2 can hinder the interaction between RIG-I and MAVS, attenuating RIG-I signaling. Furthermore, LGP2 can also dampen RIG-I signaling by inhibiting RIG-I’s binding to MAVS . MoreoveEarlier studies have identified several E3 ubiquitin ligases, including Riplet, tripartite motif-containing protein 25 (TRIM25), and ring finger protein 123 (RNF123), which modulate RIG-I activity ,36. InitThe short-length, noncoding RNA molecules referred to as microRNAs (miRNAs) occur in a size range of 19–25 nucleotides and are reported to regulate the post-transcriptional silencing of target genes. A single miRNA can target hundreds of mRNAs simultaneously, thereby affecting the expression of numerous genes that are frequently involved in a functionally interconnected pathway . The majTwo miRNAs named lin-4 and let-7 have been verified in previous studies to be partially complementary to the 3′ region of several messenger RNA (mRNA) targets of interest. The noncoding regions (3′-UTRs) are responsible for the inhibition of protein translation, thereby inhibiting protein synthesis ultimately . MiR-125RIG-I and MDA5, which are pivotal upstream molecules of the RLR signaling pathway, play crucial roles in response to viral infections and relay signals downstream. Activation of the RLR signaling pathway triggers the production of IFN-I. The antiviral effects of IFNs mainly involve the following major pathways: I. Phosphorylation of IFN-induced proteins and RNA degradation facilitated by the protein kinase R (PKR) kinase and 2′-5′-oligoadenylate-dependent RNase L; II. RNA editing mediated by IFN-induced RNA-specific adenosine deaminase (ADAR1) and Mx protein GTPase; III. Induction of inducible nitric oxide synthase (iNOS2) and major histocompatibility complex class I and II proteins . FurtherWhile several proteins have been reported to modulate the RLR pathway and subsequent IFN signal transduction ,66, studFurthermore, miR-136 serves as a RIG-I immune agonist . In geneStudies have reported that miR-146a holds potential as a novel biomarker for distinguishing between the acute and post-acute phases of COVID-19 . HoweverNevertheless, a fascinating investigation revealed that the Epstein–Barr virus (EBV)-encoded miR-BART6-3p, which targets RIG-I, promoted viral infection and was linked to the immune surveillance evasion of EBV . AnotherOn the other hand, the overexpression of miR-34b-5p inhibited the MDA5 signaling pathway but not the RIG-I signaling pathway, thereby promoting the proliferation of the avian leukosis virus subgroup J (ALV-J)-infected DF-1 cells and facilitating the replication of ALV-J . Recent Apart from targeting RIG-I/MDA5 to modulate RLR signaling during viral infections, microRNAs also exhibit similar effects by targeting other molecules within the RLR signaling pathway. For example, extensive research has highlighted the functional involvement of miR-33 in cellular metabolism, particularly in the maintenance of cholesterol homeostasis ,82. HoweSimilar to miR-146a, let-7b, a microRNA, has been implicated in various conditions, including inflammation ,86, cancA separate investigation demonstrated that the upregulation of miRNA-146a in macrophages infected with VSV was dependent on both the RIG-I/NF-kB pathways. This miR-146a encourages VSV replication by targeting IRAK1, IRAK2, and TRAF6 and by preventing the release of IFN-I that is activated by the virus . On the Notably, miR-30a inhibits TRIM25 expression and TRIM25-mediated RIG-I ubiquitination to enhance the replication of coxsackievirus B3 (CVB3) . In addiThe miR-302/367 cluster is one of the most studied microRNAs and consists of five members: miR-367, miR-302a, miR-302b, miR-302c, and miR-302d. The miR-302/367 cluster is widely involved in cellular differentiation and development, tumor progression, immune regulation, and other biological processes ,101. MiRThe above-stated findings provide fresh insights into how microRNAs are involved in the regulation of RLR signaling. Therefore, this field of research is of great importance in the development of novel antiviral therapies in the future.The infection of COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), involves the activation of the host immune response through the recognition of the viral RNA by RIG-I/MDA5. Consequently, our research focuses on investigating the involvement of RLR signaling and microRNAs in the infection process. The two cytoplasmic viral RNA sensors, RIG-I and MDA5, are vital for the immune escape of SARS-CoV-2. Several coronavirus-encoded proteins have been confirmed to inhibit the IFN response, although the precise mechanism remains to be elucidated ,106. NonPrevious studies have extensively documented the importance of microRNAs in viral infections. Two key aspects have been elucidated: Firstly, host microRNAs enhance the immune response, thereby impacting viral replication. Secondly, viral microRNAs have been demonstrated to target host immune cells, impeding viral clearance ,112. TheIn a recent study, a correlation was observed between the upregulation of hsa_circ_0000479, RIG-I, and IL-6 and the downregulation of has-miR-149-5p, suggesting that the has-circ-0000479/hsa-miR-149-5p/RIG-I/IL-6 axis may be involved in regulating the immune response to SARS-CoV-2 . As a whFurthermore, several other microRNAs have been implicated in relation to COVID-19, including miR-1207-5p , miR-320MicroRNAs in RLR signaling are also involved in regulating cancer development. Once activated, the RIG-I pathway will induce target cell death through a variety of pathways, including endogenous apoptosis, exogenous apoptosis, pyroptosis, and autophagy ,128,129.Except for the above two aspects, studies have also reported that certain microRNAs stimulate the RIG-I signaling pathway to affect IFN-I production. For instance, miR-139 promotes IFN secretion in prostate cancer cells by acting as an immunological agonist of RIG-I . MoreoveSimilarly, studies have revealed that miR-145-5p, miR-122, and miR-489 can target MDA5, DAK, and TRAF6, respectively, in the miiuy croaker to modulate the RLR signaling pathway upon poly(I:C) stimulation ,145,146.The present review summarizes several roles that microRNAs play in the regulation of various physiological processes, such as cell division, apoptosis, etc. During recent years, numerous microRNAs have been discovered through advancements in high-throughput sequencing technology. Moreover, the regulatory functions of microRNAs have been receiving greater attention all over the world. Innate antiviral immune responses are mainly mediated by RLRs, particularly RIG-I/MDA5. Modulating RLR signaling through noncoding RNAs presents a promising approach for optimizing the immune response against viral infections and inhibiting tumor progression. The regulatory role of miRNAs in RLR signaling primarily involves binding to the 3′ UTR of the mRNAs associated with RLR signaling, thereby modulating the mRNA levels of target genes and subsequently influencing protein expression. This, in turn, enhances or suppresses RLR signaling, leading to the secretion of IFNs and inflammatory cytokines that impact viral replication and cancer progression. While previous studies have demonstrated the regulatory impact of microRNAs on RLR signaling, further investigation is required to elucidate the intricacies of their interactions.2+ uptake, and prevented apoptosis in cancer cells [The “energy factories” within the cell, mitochondria are essential organelles in the metabolic processes of the cell. Studies have demonstrated that the mitochondrial route may be used by microRNAs to regulate cellular processes. For instance, in adipose tissue, miR-494-3p directly reduces PGC1 expression and the ensuing mitochondrial biogenesis . MiR-125er cells . The conRIG-I/MDA5 induces antitumor immune responses through the IFN-dependent activation of effector T cells. On the other hand, RIG-I/MDA5 also induces cancer cell apoptosis in an IFN-independent manner. Therefore, targeting RIG-I/MDA5 has several advantages over conventional cancer therapies. First, RIG-I/MDA5 may be activated using the corresponding ligands and, therefore, significant tumor-cell-specific apoptosis may be induced. Second, the expression of RIG-I/MDA5 is also associated with the prognosis of certain cancers. For instance, liver cancer patients with low RIG-I expression have a poor prognosis , while oThe emergence of drug resistance to anticancer medications is a major obstacle to successful treatment. MicroRNAs have gained attention due to their ability to regulate genes involved in the cellular responses to drugs. Several miRNAs have been identified to sensitize cancer cells to therapy and serve as biomarkers of drug resistance. For example, a study investigating the co-delivery of miR-345 and gemcitabine in pancreatic cancer demonstrated superior outcomes compared with treatment with either miR-345 or gemcitabine alone . SimilarMoreover, microRNAs and RLRs also have distinct roles in SARS-CoV-2 infection. While microRNAs such as miR-146a, let-7b, and miR-200c have been implicated in mediating RLR signaling during SARS-CoV-2 infection, it remains to be empirically validated whether these alterations in RLR signaling impact the infection itself.MicroRNA-based therapies are promising. However, how to deliver microRNAs safely and effectively to target organs and target cells is the biggest challenge at present. Currently, the following major delivery modes have been developed, which may help miRNA therapeutics to move toward clinical application: (I) conjugation ; (II) viIn conclusion, the immune system utilizes various miRNAs to finely regulate its functional capacity, maintaining a delicate balance between activation and inhibition. However, the precise regulatory mechanisms of miRNAs in the RIG-I response are not fully understood, particularly how these miRNA networks collaboratively optimize the immune response. A deeper understanding of the miRNA-mediated regulation of RIG-I signaling will uncover novel therapeutic targets for inflammatory diseases and cancer.


Giardia duodenalis are still unclear. In recent years, evidence has pointed to the roles of host factors as well as parasite’s genetic heterogeneity as major contributing factors in the development of symptomatic human giardiasis. However, it remains contested as to how only a small fraction of individuals infected with G. duodenalis develop clinical gastrointestinal manifestations, whereas the majority of infected individuals remain asymptomatic. Here, we demonstrate that diversity in the fecal microbiome correlates with the clinical outcome of human giardiasis.The mechanisms underlying the clinical outcome disparity during human infection with G. duodenalis clinical isolates from human subjects with asymptomatic and symptomatic giardiasis was determined using a multilocus analysis approach. We also assessed the genetic proximity of G. duodenalis isolates by constructing phylogenetic trees using the maximum likelihood. Total genomic DNA (gDNA) from fecal specimens was utilized to construct DNA libraries, followed by performing paired-end sequencing using the HiSeq X platform. The Kraken2-generated, filtered FASTQ files were assigned to microbial metabolic pathways and functions using HUMAnN 3.04 and the UniRef90 diamond annotated full reference database (version 201901b). Results from HUMAnN for each sample were evaluated for differences among the biological groups using the Kruskal–Wallis non-parametric test with a post hoc Dunn test.The genetic heterogeneity of G. duodenalis, whereas 3/11 (27.27%) human subjects in the current study were infected with assemblage B of the parasite. We also found that the parasite’s genetic diversity was not associated with the clinical outcome of the infection. Further phylogenetic analysis based on the tpi and gdh loci indicated that those clinical isolates belonging to assemblage A of G. duodenalis subjects clustered compactly together in a monophyletic clade despite being isolated from human subjects with asymptomatic and symptomatic human giardiasis. Using a metagenomic shotgun sequencing approach, we observed that infected individuals with asymptomatic and symptomatic giardiasis represented distinctive microbial diversity profiles, and that both were distinguishable from the profiles of healthy volunteers.We found that a total of 8/11 (72.73%) human subjects were infected with assemblage A (sub-assemblage AII) of These findings identify a potential association between host microbiome disparity with the development of clinical disease during human giardiasis, and may provide insights into the mechanisms by which the parasite induces pathological changes in the gut. These observations may also lead to the development of novel selective therapeutic targets for preventing human enteric microbial infections.The online version contains supplementary material available at 10.1186/s13071-023-05821-1. Giardia duodenalis, is one of the most commonly identified intestinal protozoan infections, with 280 million new cases annually worldwide . The ability of a given G. duodenalis isolate to elicit T cell-dependent immune responses has been proposed as a mechanism by which immunopathological changes, including disaccharide deficiency, are induced [+ T cells (beta-2 microglobulin [β2m]−/−) failed to develop disaccharidase deficiency  following G. duodenalis infection, whereas the ability of these mice to clear the parasite was unaffected . The  induced –31. Miceaffected . Consistcontrols , 33. Furcontrols . These oG. duodenalis infection, and the resistance to infection was readily transferable to the susceptible mice upon being co-housed before G. duodenalis infection  HS Assay Kit  according to the manufacturer’s specifications.® dsDNA HS Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. A paired-end sequencing (2 × 150-bp) was performed on the HiSeq X platform.DNA libraries were constructed using the Illumina Nextera XT DNA Library Preparation Kit , followed by indexing utilizing the Integrated DNA Technologies (IDT) unique dual indexes with a total gDNA input of 1 ng. The gDNA enzymatic fragmentation was performed using an Illumina Nextera XT kit . Subsequently, the unique dual indexes were added to each sample, followed by 12 cycles of PCR to construct libraries. The DNA libraries were further enriched using AMPure magnetic beads (Beckman Coulter) and eluted in Buffer EB (Qiagen). The purified DNA libraries were quantified using Qubit 4 fluorometer and QubitThe HiSeq X platform utilizes a high-performance data-mining k-mer-based algorithm that efficiently groups millions of short sequence reads into distinct genome sequences. This platform consists of a pre-computation phase for reference databases and a per-sample computation phase. The inputs for the pre-computation phase are databases of reference genomes, virulence, and antimicrobial resistance markers, whereas the outputs for the pre-computational phase consist of phylogenetic relationships of microbes with a set of variable-length k-mer fingerprints. The per-sample computational phase, however, searches and compares hundreds of millions of short sequence reads or contigs from de novo assemblies against the fingerprint profiles. This query strategy enables the sensitive and accurate detection and the taxonomic classifications of microbial NGS reads, resulting in high-resolution taxonomic profiling and relative abundance estimates for the microbial NGS datasets. Relative abundance estimates were calculated by taking the abundance score for an individual taxon and dividing it by the total abundance score per group of samples. A filtering threshold based on the internal statistical scores derived from examining a large number of diverse metagenomes was further employed to exclude false positive identifications.https://cran.r-project.org/web/packages/microbial/index.html) R packages. Principal coordinate analysis (PCoA) plots using Bray–Curtis and Jaccard dissimilarity indices were generated based on genus-level bacterial read counts to identify differences between groups. Differential abundance analysis between groups was performed using the DESeq2 package (version 1.34.0) with a P < 0.05 and absolute log2fold change > 1 based on genus-level bacterial read counts.Classified reads from CosmosID  were used to create a phyloseq object which was assessed for diversity metrics based on bacterial counts at the species level using the phyloseq (version 1.38.0) and microbial (http://huttenhower.sph.harvard.edu/humann) and the UniRef90 diamond full reference database (version 201901b) [The raw paired-end sequencing data were obtained from CosmosID and assessed for quality using FastQC (version 0.11.9) and cleaned using Trimmomatic (version 0.39). Filtered reads were reassessed for quality using FastQC and classified taxonomically using Kraken2 (version 2.12) based on the Kraken2 PlusPF database (version 20210517). The Kraken2-generated, filtered FASTQ files were assigned to microbial metabolic pathways and functions using HUMAnN 3.04 (201901b) . ResultsBile acid (BA) internal standards, including taurocholic-d5, cholic-d4, glycocholic-d4 (1 ng/each), and 10 ng of chenodeoxycholic-d4 , were added to human fecal suspensions. Fecal suspensions were subsequently centrifuged at 12,000 g for 10 min and the soluble  and the insoluble  fractions were dried under vacuum. The fecal pellets were used to calculate fecal dry weights for normalization. Pellets and dried supernatants were re-suspended in 50 µl of 75% methanol, and a total of 10 µl of the resuspension injected into the ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) system for quantitative analysis.The BAs were resolved on an ACQUITY UPLC HSS T3 column  with an ACQUITY UPLC HSS T3 precolumn  at 55 °C . SolventG. duodenalis  and six healthy controls, were enrolled in this study. Infected individuals with asymptomatic human giardiasis did not show any overt clinical manifestations of human giardiasis, whereas those subjects with symptomatic giardiasis predominantly manifested gastrointestinal symptoms, including intermittent diarrhea, abdominal pain, and nausea, at the time of the diagnosis.Seventeen individuals, including eleven individuals infected with G. duodenalis clinical isolates from human subjects with asymptomatic and symptomatic giardiasis was determined using a multilocus analysis approach as described earlier [G. duodenalis-specific tpi, bg, and gdh loci were amplified using specific primers as described earlier [G. duodenalis infection by microscopy on wet-mount preparations.The genetic heterogeneity of  earlier –52. The  earlier , 60–64. G. duodenalis, whereas 3/11 (27.27%) human subjects in the current study were infected with assemblage B of the parasite  subtype. Analysis of the bg locus indicated that six clinical isolates belonged to the A3 subtype of G. duodenalis (AII-A3), whereas only two isolates were identified as the A2 subtype (AII-A2)  or intra-assemblage levels (A + B) were detected  human subjects were infected with assemblage A (sub-assemblage AII) of .73% humad Tables .Table 2Itpi loci of the eight clinical isolates belonging to assemblage A revealed two substitution patterns   Table . This nuY) Table . The mulrn Table . The nucG. duodenalis based on the multiple alignments of sequences obtained from the amplifications of the gdh locus  of us Table . Of thosly Table . Those iG. duodenalis, representing two distinctive unique multilocus genotypes  indicated six isolates belonged to the assemblage A of tpi and gdh loci indicated that those clinical isolates belonging to assemblage A of G. duodenalis subjects clustered compactly together in a monophyletic clade despite being isolated from human subjects with asymptomatic and symptomatic human giardiasis  were interspersed within two closely related adjacent clades  further demonstrated the closely related genetic structures of G. duodenalis parasites irrespective of whether or not they were isolated from individuals with asymptomatic or symptomatic human giardiasis , it did approach significance using the Bray–Curtis dissimilarity index (P = 0.051)  revealed a separation between healthy and G. duodenalis-infected patients  Fig. C. The Brnts Fig. C.Fig. 2MG. duodenalis-infected individuals. Our further analyses indicated an increase in the relative abundance of Actinobacteria and Proteobacteria, whereas the relative abundance of Bacteroidetes decreased following G. duodenalis infection as compared with healthy controls . We observed no significant differences in alpha diversity between groups Fig. A. This iG. duodenalis infection as compared with healthy controls, with those with symptomatic giardiasis having the highest relative abundance of Proteobacteria, followed by individuals with asymptomatic giardiasis . Many of these pathways are linked to metabolic degradation, biosynthesis, and recycling pathways. Pairwise comparisons of pathway results using Dunn’s test provided further insight into functional differences associated with giardiasis infection and symptomatic disease.Sequencing files were also subject to pathway enrichment analysis using HUMAnN 3.0 and the UniRef90 diamond full database (version 201901b). Overall, 95 pathways were significant across groups (P < 0.019) was differentially enriched between infected individuals (asymptomatic and symptomatic) as well as between individuals with asymptomatic giardiasis (P < 0.019) as compared with healthy controls. However, the pathway required for the biosynthesis of L-serine and glycine (L-serine and glycine biosynthesis I) was the main metabolic pathway enriched between individuals with symptomatic giardiasis and healthy controls (P < 0.03), followed by the 2-oxobutanoate degradation I pathway (P < 0.034). We also found that the C4 photosynthetic carbon assimilation cycle both nicotinamide adenine dinucleotide phosphate–malic enzyme (NADP-ME) type (P < 0.017) and phosphoenolpyruvate carboxykinase (PEPCK) type (P < 0.02) were significantly enriched between infected individuals with asymptomatic and symptomatic giardiasis  as compared with healthy controls  and secondary  BAs in the feces of healthy volunteers  by intestinal epithelial cells (IECs), thereby impeding the proliferation of the IECs as well as compromising the ability of parasitized IECs to directly kill Giardia trophozoites [G. duodenalis impairs the production of pro-inflammatory cytokines by macrophages  and DCs in the LP of the small intestine following colonization [ntestine –68. The nfection . This laironment . For inshozoites , 71. Deshozoites , G. duodnization , 74.G. duodenalis  as compared with healthy controls. It is well-established that multiple metabolites derived from bacterial metabolism, including secondary BAs, and short-chain fatty acids, have drastic immunomodulatory effects on the host and have been implicated in the pathogenesis of infectious and non-infectious diseases [Our analyses demonstrated distinct microbial metabolic pathways between individuals infected with diseases , 76.We found that the 2-oxobutanoate degradation I pathway was differentially enriched between infected individuals (asymptomatic and symptomatic) as compared with healthy controls  and infected individuals with asymptomatic (Asym) and symptomatic (Sym) giardiasis were determined by the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system for quantitative analysis.Additional file 2: Table S1. Target genes and primer sequences utilized for identification of G. duodenalis assemblages and multilocus sequence analysis. Table S2. Pathways enriched between healthy controls vs. G. duodenalis-Infected individuals.Table S3. Pathways enriched between healthy controls vs. infected individuals with symptomatic giardiasis. Table S4. Pathways enriched between infected individuals with asymptomatic vs. symptomatic giardiasis. Table S5. Pathways enriched between healthy controls vs. infected individuals with asymptomatic giardiasis.


Non-small cell lung cancer (NSCLC) patients, accounting for approximately 85% of lung cancer cases, are usually diagnosed in advanced stages. Traditional surgical resection and radiotherapy have very limited clinical benefits. The objective of this study was to develop and evaluate a targeted therapy, antibody-drug conjugate (ADC), for NSCLC treatment. Specifically, the CD276 receptor was evaluated and confirmed as an ideal surface target of NSCLC in the immunohistochemistry (IHC) staining of seventy-three patient tumor microarrays and western blotting analysis of eight cell lines. Our anti-CD276 monoclonal antibody (mAb) with cross-activity to both human and mouse receptors showed high surface binding, effective drug delivery and tumor-specific targeting in flow cytometry, confocal microscopy, and in vivo imaging system analysis. The ADC constructed with our CD276 mAb and payload monomethyl auristatin F (MMAF) showed high anti-NSCLC cytotoxicity to multiple lines and effective anti-tumor efficacy in both immunocompromised and immunocompetent NSCLC xenograft mouse models. The brief mechanism study revealed the integration of cell proliferation inhibition and immune cell reactivation in tumor microenvironments. The toxicity study did not detect off-target immune toxicity or peripheral toxicity. Altogether, this study suggested that anti-CD276 ADC could be a promising candidate for NSCLC treatment. Lung cancer, representing a significant global health challenge, is one of the most prevalent cancers and a leading cause of cancer-related deaths . Among tOver the years, significant progress has been made in the treatment of NSCLC, with the incorporation of traditional radiotherapies, chemotherapies inhibiting cell division and DNA replication with limited clinical benefits, and targeted therapies dealing with certain gene mutations ,5. As re+ T cells and enhance the anti-tumor immune response in the tumor microenvironment (TME) by blocking the PD-L1/PD-1 signaling pathway [Recently, the FDA approved immune checkpoint blockers (ICBs) ,6, such  pathway ,14,18,19 pathway . Moreove pathway ,22. As a pathway ,24,25, C pathway ,27,28.The objectives of this study were to develop and evaluate the CD276-targeting antibody-drug conjugate (ADC) for NSCLC treatment. The high surface expression of CD276 was confirmed in NSCLC cell lines and patient tissues. ADC was constructed using our murine anti-CD276 monoclonal antibody (mAb) and microtubule depolarizing payload monomethyl auristatin F (MMAF). The in vitro evaluations showed high surface binding, drug delivery, and cytotoxicity to NSCLC lines. Further, in vivo studies demonstrated effective anti-NSCLC efficacy in both immunocompetent and immunocompromised xenograft mouse models. In addition to cell proliferation inhibition and apoptosis, tumoral immunity was also detected in the NSCLC tissues post-ADC treatment. This study suggested that CD276 targeting therapy is a promising syngenetic therapy to treat NSCLC.The animal studies were conducted according to the Institutional Animal Care and Use Committee (IACUC) Protocol 2022A00000071, which was approved by the Institutional Biosafety Committee of Ohio State University (OSU).v/v; Gibco) and 1% penicillin streptomycin . The mouse Lewis Lung Cancer cell line LLC-Fluc (ATCC) was cultivated in a DMEM medium supplemented with 10% HI FBS and 1% P/S. The seed culture of hybridoma-producing murine anti-CD276 mAb was maintained in a serum-free medium and Hybridoma-SFM supplemented with 4 g/L of glucose and 4 mM of L-glutamine in 125 or 250 mL shaker flasks at 130 rpm or 250 mL spinner flasks at 175 rpm. All these cell lines were incubated in a humidified CellXpert C170 CO2 incubator  with a set point of 37 °C and 5% CO2. All the cell culture media, medium supplements, and bioreagents used in this project were purchased from Gibco or Fisher Scientific  unless otherwise specified.The human NSCLC cell lines, H-460 , H460-FLuc (Perkinelmer), and H358  were cultivated in an RPMI-1640 medium  supplemented with 10% heat-inactivated fetal bovine serum  with duplication (LC1461), was purchased from US Biomax . The TMA slides were processed following the procedure provided by the biomanufacturer and stained with recombinant rabbit anti-mouse/human CD276 antibody  and horseradish peroxidase (HRP)-conjugated secondary anti-rabbit antibody  to detect CD276 expression. The positive staining was visualized with DAB  and imaged with a Vectra 3 automated quantitative pathology imaging system . Following our previously established TMA image analysis ,30, we uv/v). The 30 or 60 mL cultures in SF125 or SF250 shaker flasks were seeded at a viable cell density (VCD) of 0.2 × 106 cells/mL and cultivated at an agitation speed of 130 rpm. Fed-batch production was performed by feeding 4 mM L-glutamine, 4 g/L glucose, and 3.5 g/L Cell Boost #6 on days 3 and 5. CD276 mAb was harvested from supernatant when the cell viability was lower than 50% and purified using NGC liquid chromatography equipped with protein A column [The murine anti-CD276 mAb-producing hybridoma cells were cultivated in the Hybridoma-SFM medium  supplemented with 4 mM L-glutamine and 4 g/L glucose and 1% P/S  were suspended in a 1 mL flow cytometry buffer ; then, 1 µg  or 5 µg (for LLC-FLuc) of CD276 mAb-AF647 was added, followed with incubation at room temperature for 30 min or 2 h, respectively, in the dark. Then, the reaction mixture was washed with PBS twice before applying it to a flow cytometer. A uniform cell suspension of 20,000 H460-FLuc or H358 cells and 10,000 LLC-FLuc cells were applied for surface binding analysis.The cell surface-binding rates of CD276 mAb to H460-FLuc, LLC-FLuc, and H358 cells were detected using a BD LSRII flow cytometer . Briefly, CD276 mAb was labeled with an Alexa Fluor™ 647  kit to generate CD276 mAb-AF647. About 1 × 10The expressions of CD276 in normal human lungs, human NSCLC cell lines, and human NSCLC cell lines were also analyzed by western blotting. Specifically, cells were washed three times with cold PBS and lysed using a RIPA buffer to extract whole cellular proteins. Protein concentration was quantified using the bicinchoninic acid (BCA) method with a Pierce protein assay kit . Next, a total of 30 µg of proteins were loaded per lane onto a gradient SDS-PAGE, NuPAGE 4–12% gradient gel (Invitrogen), along with a protein size marker, Bio-Rad Precision Plus , to separate proteins by molecular weight. The separated proteins were then transferred onto a PVDF membrane using a Bio-Rad power supply (Bio-Rad Laboratories) at a constant voltage of 100 V for 90 min. After transfer, the PVDF membrane was blocked with 5% non-fat milk in a TBST buffer and agitated at room temperature for 1 h. The primary antibodies of CD276  and β-actin  were purchased from Abcam  and Santa Cruz Antibodies , respectively. The membrane was then incubated at 4 °C with continuous agitation overnight. On the following day, the primary antibodies were discarded, and the membrane was washed three times with a TBST buffer on a shaker, with each wash lasting 5, 5, and 10 min, respectively. After the washing steps, horseradish peroxidase (HRP)-conjugated secondary antibodies (dilution 1:2000) specific to the mouse or rabbit from CST  in 3% non-fat milk were applied to the membrane at room temperature for 1 h. After discarding the secondary antibody, the membrane was washed three times with TBST for 5, 5, and 10 min, respectively. Finally, the protein bands were visualized and quantified using an Odyssey Fc imaging system . The expression of CD276 in TNBC cells was compared to the internal control, β-actin.4 cells per dish in 1.5 mL of medium. To visualize the cytoplasm and nucleus, BacMam GFP Transduction Control (Invitrogen) and NucBlue™ Live ReadyProbes™ Reagent (Invitrogen), respectively, were used for staining following manufacturing protocols. Next, the AF647-labeled CD276 mAb was added to the cells at a final concentration of 1 or 5 µg per mL. Live-cell images were captured at 0–24 h after the addition of CD276 mAb-AF647 using a Nikon A1R-HD25 confocal microscope . This experimental approach allowed us to dynamically monitor the binding and internalization of CD276 mAb into NSCLC over different time points.We investigated the surface binding and internalization of our anti-CD276 mAb using live-cell confocal imaging following established protocols in our previous publications ,30,33. H2 incubator for 24 h, followed by the addition of free MMAF or ADC at dosages of 0, 10, 20, 40, 80, 120, 160, and 200 nM. The 3--2,5-Diphenyltetrazolium Bromide Cell Proliferation Assay (MTT Assay) was performed following the biomanufacturer procedure after 5-day treatment. At the end of treatment, the spent medium was removed and replaced with 100 µL of the fresh culture medium supplemented with 10 µL of the MTT reagent in each well. After 2 h of incubation in the dark, 100 µL of the detergent reagent was added to each well until a purple color was developed. The OD value was read at 590 nm using a SpectraMax iD3 plate reader , and IC50 values were calculated.The cytotoxicity assay was performed using human NSCLC cell line H460-FLuc and mouse LLC-FLuc. With different cell division rates, 3000 H460-FLuc cells or 1000 LLC-FLuc cells with 200 uL of RPMI-1640 or a DMEM complete growth medium were seeded into each well in the 96-well plates. The cells were incubated at 37 °C in a COThe navigation of our anti-CD276 mAb to human NSCLC H460-FLuc xenograft tumor and mouse LLC-FLuc allograft tumor was monitored by detecting bioluminescent signal (tumor) and fluorescence signal (mAb) through IVIS Lumina Series III. The mice inoculated with FLuc-labeled NSCLC cells were intravenously (i.v.) injected with 50 µg of Cy5.5-labeled CD276 mAb. At 24 h post-mAb injection, 100 µL of luciferin (30 mg/mL stock) was intraperitoneally (i.p.) injected. After 10 min, the live mice were imaged under the IVIS machine for both bioluminescence at a wavelength of 550 nm and the targeted Cy5.5-labeled CD276 mAb at a wavelength of 750 nm. The major organs  and tumor were harvested to collect ex vivo images to confirm the tumor-specific targeting of CD276 mAb.At the end of the in vivo treatment, we collected mice blood for whole blood analysis after isoflurane was administrated to deeply anesthetize the mice. The cardiac puncture using a 39-gauge syringe was performed to obtain the largest volume of mice blood. We collected the blood, aliquoted it in two EDTA tubes, and immediately ran a whole blood analysis. For the remaining blood sample, we centrifuged and kept the serum samples at −80 °C for longer storage.6 of LLC-Fluc cells in each mouse when they were about 6 weeks old. Given the fast growth rate of LLC-Fluc, we started the i.v. administration of ADC at dosages of 12 mg/kg, 24 mg/kg, mAb only at a dosage of 12 mg/kg, or saline (control) two days after tumor cell implantation. The 9-week-old xenograft immunodeficient nude mice were s.c. injected with 3 × 106 cells H460-FLuc cells each mouse. The i.v. administration of ADC or the control was initiated when tumor volume reached ~25 mm3, and the same dosage was given to C57BL/6 black mice. The C57BL/6 black mice and nude mice were treated twice a week until the biggest tumor volume reached >1500 mm3 and 1000 mm3, respectively. Mice’s body weight and tumor volume were measured twice a week during treatment.The in vivo NSCLC treatments were tested in two mice models (immunocompetent and immunocompromised). The allograft C57BL/6 black mice were subcutaneously (s.c.) injected with 1 × 10For paraffin section preparation, lung cancer tissue samples were fixed in 4% formalin for >24 h. After fixation, the samples were dehydrated through graded ethanol solutions and embedded in paraffin blocks. Sections of 4–5 μm thickness were cut from the paraffin blocks using a microtome and mounted onto glass slides. Subsequently, the sections were deparaffinized and rehydrated for staining. H&E staining was performed to visualize the cellular and tissue structures. The deparaffinized sections were incubated in a hematoxylin solution to stain the nuclei, followed by a brief rinse in running water. Next, the sections were differentiated in acid alcohol to remove excess hematoxylin. After rinsing, the sections were counterstained with an eosin solution, highlighting the cytoplasm and extracellular components. The stained sections were dehydrated using ethanol, cleared with xylene, and mounted with a coverslip using a mounting medium.For H&E-stained sections, the assessment of cellular morphology and tissue architecture was carried out using an Eco microscope at 40× magnification. The characteristics of individual cells, including their size, shape, and staining patterns, as well as the overall arrangement and integrity of the tissue, were analyzed.t-test was used to analyze the significance of differences among groups, and the difference between the two groups was analyzed using one-way ANOVA. A statistical analysis was performed using GraphPad Prism software , and a p-value of <0.05 was used for all in vivo studies.All the in vitro experiments were performed with duplication, and the data were reported as mean ± standard error of the mean. A two-tailed This study evaluated the surface receptor CD276 as a therapeutic target of NSCLC and developed an anti-CD276 mAb-derived ADC, i.e., CD276 mAb-MMAF, which integrates the anti-cancer mechanism of blocking the immune checkpoint, reactivating immune cells in tumor microenvironments, and inhibiting microtubule polymerization, to treat NSCLC.n = 2), including stages of IB, IIA/B, and IIIA of subtypes ADC, SqCC, and other carcinoma, and normal lung tissue , were stained with anti-human CD276 mAb. The IHC images of NSCLC cores described in To assess the expression of CD276 receptor in NSCLC, the parafilm sectioned TMA slides with 73 cores  and 5% (LLC) post-treatment with MMAF at titers of 120–200 nM, 15% (H460), and 34% (LLC) treated with 80–200 nM of ADC C. IC50 vn = 6). The body weight of these mice had no obvious change at doses of 8–24 mg/kg ADC , CD276 mAb (8 mg/kg), and saline were injected into C57BL mice via tail vein , 12 mg/kg anti-CD276 mAb (control), and 12 and 24 mg/kg anti-CD276 mAb-MMAF (n = 6). As shown in To evaluate the synergetic anti-NSCLC efficacy of CD276 mAb-MMAF, about 3 × 10To understand the anti-NSCLC mechanisms, we first performed a Luminex assay using the tumor tissues harvested at the end of treatment to titrate the cytokine secretion in the tumor microenvironment. As presented in In addition to NSCLC treatment, we also tested the general toxicity in peripheral blood two weeks after ADC treatment by performing whole blood analysis with HemaVet 950FS. As summarized in All these data demonstrated that our anti-CD276 mAb-MMAF can effectively reduce NSCLC tumor burden via inhibiting microtubule polymerization by MMAF, reactivating immune cells , increasing immune cells infiltration, and/or enhancing the secretion of cytokines in the tumor. The anti-mouse NSCLC efficacy could be further improved by optimizing the ADC treatment schedule and dosage. Importantly, the off-target toxicity or side effect was minimal, as confirmed in both the H&E staining of major organs and whole blood analysis, suggesting that our anti-CD276 ADC is a promising targeted therapy for NSCLC treatment.6 H460-FLuc cells into nude mice. As described in Human NSCLC xenografted models were established by s.c. implanting 3 × 10An antibody-drug conjugate has been investigated and widely used as an effective targeted therapy to treat various cancers ,31,32,33+ NSCLC and exhibits strong cross-activity to both human and mouse CD276. Therefore, the cancer treatment evaluation using xenograft mouse models is translational. The constructed ADC  demonstrated high anti-tumor efficacy in both in vitro studies using multiple cell lines and in vivo studies using two animal models. The post-treatment analysis in immunocompetent models indicated strong synergetic anti-NSCLC mechanisms of our ADC, including direct tumor cell death by MMAF, which has been used to treat other cancers, immune responses, such as cytokine secretion and reactivation in the tumor microenvironment, and the blockage of the CD276 immune checkpoint, which needs further evaluation. The synergism of these cancer treatment mechanisms resulted in significant tumor growth inhibition (up to 92%) in xenograft models. Moreover, the toxicity studies did not detect any damage, inflammation, or other off-target side effects in major organs, immune toxicity in peripheral blood, adverse body weight changes, or behavior changes at a dose of 24 mg/kg. Taken together, the anti-CD276 ADC developed in this study could specifically target CD276-positive NSCLC cell lines and xenografts and deliver potent small molecules to effectively treat NSCLC with minimal side effects.To our knowledge, the CD276-targeting therapy (mAb or ADC) has not been developed for NSCLC treatment so far. As reported in this study, CD276 is a promising surface receptor to target NSCLC due to its high patient coverage rate (over 70%), despite the genome background or driving mutations. Our anti-human CD276 mAb has high specificity to CD27650 values  using new linkers ; investigate tumoral immunity in patient-derived xenograft humanized mouse models; and/or optimize ADC dosage by testing a high dosage of 60–80 mg/kg and treatment schedules. Combining the developed CD276-targeted ADC with other therapies could further improve NSCLC treatment efficacy.To translate the developed CD276-targeting ADC for NSCLC patient treatment in the future, we need further development and evaluation to provide guidance for future preclinical or clinical studies. For instance, we need to construct chimeric and humanized CD276 mAb to increase its half-life and plasma stability; evaluate and conjugate higher potent drugs with lower ICThis study reported a new antibody-drug conjugate to treat NSCLC by integrating multiple anti-cancer mechanisms. Both in vitro and in vivo evaluations indicated that our anti-CD276 ADC has great potential to target and effectively treat CD276 overexpressing NSCLC with undetectable side effects.


Background: Sarcopenia has emerged as a significant public health concern. Uric acid (UA), as a metabolite with excellent antioxidant capacity, has been found to be associated with sarcopenia. However, the casual effects of UA on incident sarcopenia still remain unclear. Our study aimed to explore the longitudinal association between UA and incident sarcopenia among middle-aged and older adults. Method: A total of 5086 participants aged ≥45 years old without sarcopenia at baseline were included from the China Health and Retirement Longitudinal Study (CHARLS). Due to the sex differences, the UA levels were analyzed by categorizing into sex-specific quartiles or by using UA levels as a continuous variable (per 1 mg/dL). The longitudinal association between UA and incident sarcopenia was evaluated using Cox proportional hazards regression models. Results: During the 4-year follow-up period, 552 (10.85%) participants with incident sarcopenia were identified, of which 370 cases were males and 182 cases were females. Compared to the first quartile (Q1) UA levels, the Q3 and Q4 UA levels were significantly associated with lower risk of incident sarcopenia in males (Q3: adjusted hazard ratio (HR), 0.72; 95% CI , 0.54–0.97; Q4: HR, 0.57; 95% CI, 0.41–0.80). When UA was as a continuous variable (per 1 mg/dL), the association in males remained significant . No significant association was observed in females. Conclusion: Our findings indicated that UA was negatively associated with incident sarcopenia in males but not in females among middle-aged and older Chinese. Sarcopenia is a generalized and progressive disease characterized by the accelerated loss of muscle mass and function, which could increase the risk of various adverse outcomes such as falls, frailty, and even mortality . SarcopeUric acid (UA) is a metabolite produced through the breakdown of purines in the body, which has excellent antioxidant capacity . UA leveThe study population was derived from the CHARLS, an ongoing nationally representative longitudinal survey of individuals aged 45 years and above in China. The CHARLS employed a multistage probability proportional to size sampling strategy to select households from 450 villages or residential communities in 28 provinces throughout China All recruited participants were interviewed through standardized face-to-face computer-assisted personal interview (CAPI) by well-trained interviewers. The baseline survey was conducted between June 2011 and March 2012, with a total of 17,708 participants being included. The follow-up survey was conducted every two years. A detailed description of the CHARLS design has been reported previously .n = 4635), aged <45 or missing age information (n = 258), missing sex information (n = 9) and lacking the data of UA (n = 3550) were excluded. A total of 9256 participants had complete information at wave1. We further excluded participants with sarcopenia at baseline  and participants missing information in the follow-ups (n = 3162). Finally, 5086 participants were included in the final analysis , 2013 (wave 2) and 2015 (wave 3). In wave 1, the sample size was 17,708. Participants lacking the information of sarcopenia , and written informed consents were obtained from all participants prior to enrollment in this survey . This st2). Based on previous studies, the criterion of low muscle mass was set as the sex-specific lowest 20% of SMI in the study population, which corresponded to a value of <6.99 kg/m2 in male and <5.27 kg/m2 in female [According to the 2019 consensus of the Asian Working Group for Sarcopenia (AWGS), sarcopenia diagnosis was assessed upon three parameters: muscle strength, appendicular skeletal muscle mass (ASM), and physical performance . Particin female . All participants were asked to fast overnight and the blood samples of participants were collected by medically-trained staff from Chinese center for disease control and prevention (Chinese CDC), based on a standard protocol. Venous blood was separated into plasma and buffy coat and stored in cryovials. These cryovials were promptly frozen at −20 °C and transported to the Chinese CDC in Beijing within two weeks. Upon arrival, the samples were transferred to a deep freezer and stored at −80 °C until they were assayed at the laboratory of Capital Medical University. UA levels (mg/dL) were quantified using the UA Plus method . The following baseline variables were selected as covariates in this study, including demographic, health behaviors, chronic diseases, and biochemical parameters. The baseline demographic variables included age, sex, residence , highest educational level , and marital status (married and living with spouse and others). Health behaviors included drinking status (yes and no), smoking status (yes and no) and self-reported nighttime sleep duration. Diabetes mellitus (DM) was diagnosed when participants self-reported having DM diagnosed by a physician or the fasting glucose levels ≥7 mmol/L or the random glucose levels ≥11.1 mmol/L or the glycosylated hemoglobin (HbA1c) ≥6.5%. Hypertension was defined as self-reported having hypertension diagnosed by a physician or the systolic blood pressure (BP) ≥140 mmHg or the diastolic BP ≥90 mmHg. Depression was assessed by the 10-item Center for Epidemiologic Studies Depression Scale (CES-D), and participants with scores ≥12 were considered to have depression. Dyslipidemia was defined as the ratio of cholesterol and high-density lipoprotein cholesterol (HDL-c) of ≥5.0 or self-reported having dyslipidemia diagnosed by a physician. Other chronic diseases such as pulmonary disease, kidney disease, cardiovascular disease, and arthritis were assessed by the physician-diagnosed history. The estimated glomerular filtration rate (eGFR) was calculated using creatinine values, age and sex . The sex differences in UA are well reported in several studies ,23. TherThe baseline characteristics of including participants were described and compared according to the sex-specific quartiles of UA. Continuous variables and categorical variables were presented as means with standard deviations (SDs) and numbers with percentages, respectively. The characteristic differences between quartiles groups were compared using one-way analysis of variance for continuous variables and chi-square test for categorical variables. To assess the association between UA and sarcopenia in this four-year follow-up study, we employed Cox proportional hazards models to estimate the hazard ratios (HRs) with corresponding 95% confidence intervals (CIs). The proportional hazard assumption in the Cox proportional hazards models was verified using Schoenfeld residuals . The ful2 were excluded. Several sensitivity analyses were conducted to exam the robustness of the findings. First, we compared the baseline characteristics of included and excluded participants to explore the potential selection bias. Second, participants with hyperuricemia were excluded. Hyperuricemia was defined as baseline plasma UA > 7.0 mg/dL in males and >6.0 mg/dL in females . Third, p < 0.05 (two-sided).All statistical analyses in this study were conducted by STATA/MP, version 17.0. The level of statistical significance was set at A total of 5086 participants were included in this study. Of these, 2359 (46.38%) participants were males and 2727 (53.62%) participants were females. The baseline characteristics of the total sample based on the sex were shown in During the 4 years follow-up, a total of 552 (10.85%) cases with incident sarcopenia were identified, of which 370 cases were males and 182 cases were females. Among males, the incidence rate of sarcopenia was 60.13 per 1000 person-years in Q1 UA group, 44.24 per 1000 person-years in Q2 UA group, 38.18 per 1000 person-years in Q3 UA group and 28.27 per 1000 person-years in Q4 UA group. In females, the incidence rate of sarcopenia in Q1–Q4 groups were 17.26, 18.46, 16.48 and 16.21 per 1000 person-years, respectively. There was no significant longitudinal association between UA and incident sarcopenia in females according to the quartiles of UA or as a continuous variable (per 1 mg/dL) B Table . SubgrouAccording to the subgroup analyses, males who were younger (≤58 years old), living in rural, smokers, non-drinkers, and without hypertension, DM, dyslipidemia, as well as with depression were more responsive to the protective effects of UA against the incident of sarcopenia . In fema2, we reanalyzed the data and found that the association between UA and sarcopenia did not change significantly (We compared the baseline characteristic between included and excluded participants and the difference was not significant . After eficantly .We conducted a longitudinal study using nationally representative data to assess the association between UA and incident sarcopenia, diagnosed using AWGS 2019 algorithm, in middle-aged and older Chinese population. When the UA was analyzed by sex-specific quartiles, the Q3 and Q4 groups in males were significantly associated with decreased risk of incident sarcopenia. This negative association remained significant when considering UA as a continuous variable in males. In our analysis of females, we did not find a significant association between UA and sarcopenia. Our findings indicated a significant negative association between UA and incident sarcopenia in males but not in females. The potential protective effect of UA against sarcopenia are in line with previous studies. A cross-sectional study demonstrated that UA levels were positively associated with muscle mass and muscle strength in kidney transplant patients . AnotherThe negative association between UA and incident sarcopenia may be related with the excellent antioxidant capacity of UA. It has been well established that oxidative stress was an essential pathogenetic process in age-related sarcopenia, which was accompanied by the overproduction of reactive oxygen species ,28. UA mFor the sex difference of the association between UA and incident sarcopenia in our study, it may be related with the hormonal influence. Most females included in this study were menopausal, which resulted in a decrease of estrogen. Estrogen has been reported to promote the excretion of UA, which may result in elevated UA levels in menopausal females . In addiThe strength of this study was based on a large and nationally representative sample, which allows for the extension of the findings to the middle-aged and older population in China. To our knowledge, this is the first study to explore the longitudinal association between UA and incident sarcopenia, assessed using the AWGS 2019 algorithm. However, there are also several limitations should be noted. First, although the formula has been previously validated in Chinese population and showed good consistency with DXA in various studies, the muscle mass was estimated by a formula, not by DXA or bioelectrical impedance analysis . Second,In conclusion, our findings indicated that UA was negatively associated with incident sarcopenia in males but not in females in middle-aged and older Chinese. The findings supported the association between UA and sarcopenia, and suggested the gender difference existed in the effects of UA on sarcopenia.