Helicobacter pylori (H. pylori) is the main cause of gastric adenocarcinoma. However, the traditional antibiotic treatment of H. pylori is limited due to increased antibiotic resistance and low efficacy; low drug delivery efficiency and difficulties in eradicating H. pylori that is present intracellularly or in biofilms cause further setbacks. Biomaterials that can protect drugs against stomach acid, target lesions, control drug release, destroy biofilms, and exhibit unique antibacterial mechanisms and excellent biocompatibility have emerged as attractive tools for H. pylori eradication, particularly for drug-resistant strains. Herein, we review the virulence mechanisms, current drug treatments, and antibiotic resistance of H. pylori strains. Furthermore, recent advances in the development of biomaterials, including nanoparticles , microspheres, and hydrogels, for effective and precise therapy of H. pylori and different types of therapeutic mechanisms, as well as future perspectives, have also been summarized. Helicobacter pylori (H. pylori), a gram-negative spiral-shaped bacterium that colonizes the gastric mucus and gastric epithelium, has been estimated to infect nearly 4.4 billion people worldwide.1 It was reported that H. pylori is usually acquired before the age of 10, and by inducing inflammation of the host in the gastric epithelium, H. pylori can cause a series of gastric diseases, including peptic ulcer disease, gastric adenocarcinoma, atrophic gastritis, and mucosa-associated lymphoid tissue lymphoma.2–6 Additionally, H. pylori is closely associated with iron deficiency anemia, idiopathic thrombocytopenic purpura, and vitamin B12 deficiency.7–9 In 2015, the Kyoto Global Consensus Report formally defined H. pylori as an infectious disease and recommended treatment for all patients with it because it plays an important role in the development of gastric adenocarcinoma.10 In addition, a nationwide multicenter study has also indicated H. pylori infection to be a high-risk factor for gastric cancer.6 An updated meta-analysis of randomized controlled trials indicated that its eradication could reduce the incidence and mortality of gastric cancer by 46% and 39%, respectively.11 Furthermore, recent guidelines recommend the treatment of individuals infected with H. pylori to prevent gastric cancer.12 Therefore, the eradication of H. pylori from the global population is of great significance.H. pylori eradication are based mainly on pharmacological treatments that include antibiotics.3 Early experiments have indicated a high susceptibility of H. pylori to antibiotics in vitro; however, it is difficult to achieve similar effects in vivo.13 This could be attributed to the acidic environment of the stomach  and the thick gastric mucosa (which makes it difficult for most antibiotics to reach the target effectively).13 Therefore, treatments to efficiently eradicate H. pylori, such as clarithromycin triple therapy and bismuth quadruple therapy, involve the simultaneous administration of several drugs for long periods .3 However, complex drug eradication programs are associated with several limitations, including large pill burdens, adverse effects, and high cost,14 which cause reduced adherence and low cure rates. Additionally, the progressive development of antimicrobial resistance due to the widespread misuse of antibiotics seriously undermines treatment efficacy.15 Moreover, persistent and slow-growing infections enable H. pylori to form biofilms or survive inside epithelial cells and macrophages by regulating autophagy, making their eradication by antibiotics extremely challenging.16 Therefore, new treatment strategies that can overcome the acidic gastric environment, increase drug delivery efficiency, and eradicate H. pylori that exists intracellularly or in biofilms are urgently needed for effective alternatives.Currently, regimens for 17–20 Biomaterials have also been used for the treatment of H. pylori infection due to their unique properties compared to conventional antibiotic therapy. First, biomaterials can conjugate with or encapsulate conventional antibiotics, which can reduce the administered dose and side effects and enhance bioavailability. In addition, the site-specific targeting of H. pylori and controlled drug release by biomaterials also improve antibacterial efficacy and slow down the emergence of drug-resistant bacteria.21 Additionally, biomaterials (such as inorganic nanoparticles) can enter bacterial cells directly and destroy biofilms, eliminating the H. pylori present there.23 Some biomaterials  exhibit unique antibacterial mechanisms ) and can eradicate H. pylori without any loaded drugs; they hinder the development of drug resistance in H. pylori.24–26 Currently, new H. pylori treatment strategies, including Chinese herbal medicines, probiotics, antimicrobial peptides, and urease inhibitors, are also being studied by researchers to eradicate H. pylori. However, compared with biomaterials, these strategies are either cytotoxic, intolerant to gastric acid and pepsin, or have a lower eradication rate.27–30 Therefore, biomaterials are effective alternatives for efficient H. pylori therapy; liposomes, nanostructured lipid carriers, nanoemulsions, polymeric nanoparticles, inorganic nanoparticles, microspheres, and hydrogels are commonly used.31–33Biomaterials formed by synthetic material engineering exhibit several important applications in cancer treatment, antibacterial, immunomodulatory, wound healing processes, and so on.H. pylori infection have been described, along with its current drug treatments and drug-resistance mechanisms. Furthermore, recent progress in biomaterials for H. pylori eradication has been summarized. Thus, biomaterials can be used for the effective and precise therapy of H. pylori infections in the future.Herein, the pathogenic mechanisms of H. pylori is a spiral microaerobic bacterium with five to seven flagella and demanding growth conditions.34H. pylori infection will first cause chronic gastritis and then gradually lead to gastric ulcers and gastric atrophy;35 in severe cases, it can lead to gastric cancer.36 It has been reported that there is a parallel relationship between H. pylori and gastric cancer mortality, and eradication of H. pylori is closely related to the prevention of gastric cancer.38Isolated and cultured by Warren and Marshall for the first time, H. pylori infection is shown in H. pylori produces urease upon entering the stomach, which promotes the hydrolysis of urea, forming ammonia and carbon dioxide. Ammonia neutralizes gastric acid, while carbon dioxide helps bacteria survive in the stomach by resisting gastric acidity. In this way, the bacteria can resist gastric acidity and survive in the stomach. H. pylori, with the help of its helical shape and flagellar movement, easily penetrates the mucus layer to reach the gastric epithelium,39 where it colonizes gastric epithelial cells under the synergistic action of adhesins and outer membrane proteins .40–42 Then, the proteins and toxins located in the host cells subsequently cause an inflammatory response.43The pathogenesis of H. pylori, cytotoxin-associated gene A (cagA) and vacuolating cytotoxin A (VacA) are the most extensively investigated virulence factors. Cytotoxin-associated protein A (CagA) is a protein produced by H. pylori, with masses in the range of 120–145 kDa. CagA, along with the type 4 secretion system (T4SS), is encoded by the cag pathogenicity island (cagPAI), which comprises approximately 40 kb of chromosomal DNA.44 According to the presence and absence of CagA, H. pylori can be classified into two subtypes, CagA-positive and CagA-negative. After attachment to gastric cells, the CagA-positive H. pylori injects CagA into the host cells through the T4SS, similar to a needle. Compared to those infected with CagA-negative H. pylori strains, patients with a CagA-positive H. pylori infection have a higher risk of gastric cancer or peptic ulcer disease.45 Xie et al. revealed autophagy was decreased in the duration of H. pylori infection in a CagA-dependent manner, resulted in ubiquitination degradation of RAD51 and genomic instability in gastric cells, which inducing progression of gastric epithelial cells to cancer.45 VacA is a cytotoxin encoded by the vagA gene; its main function is cell vacuolation through pores on the epithelial cell membrane. Additionally, it helps H. pylori resist immune clearance, improves its viability, and enables the host to continue infecting through membrane depolarization, apoptosis stimulation, intercellular adhesion, and T-cell obstruction.40 VacA also interferes with autophagy, enabling the survival of H. pylori in host cells.46Among the many pathogenic factors of H. pylori treatment integrate one PPI with a combination of antimicrobial agents (3 After failure of the first-line treatment (clarithromycin-containing triple therapy or bismuth quadruple therapy), fluoroquinolone-containing  triple or quadruple therapy is recommended as the second-line treatment. After failure, the patient is recommended to undergo susceptibility testing or molecular determination to guide further treatment.3 In addition to conventional regimens, researchers have modified drug-administration sequences and created sequential, hybrid, and concomitant therapies. However, due to resistance to clarithromycin and metronidazole, sequential therapy gradually fades out of the field of research.49 Although hybrid and concomitant therapies exhibit satisfactory H. pylori eradication rates,51 they involve complex drug administration, which may affect patient compliance, and the exposure of patients to at least one unnecessary antibiotic, which may promote H. pylori resistance. Thus, the promotion and application of these regimens should be investigated further.52 Graham et al. reported a rifabutin-based triple therapy randomized controlled study and indicated that the regimen is unaffected by clarithromycin or metronidazole resistance.53 However, this study excludes individuals of Asian descent; therefore, the applicability of rifabutin-based triple therapy to Asian people, who have a high risk of stomach cancer, remains unknown.53 High-dose PPI-amoxicillin dual therapy and vonoprazan (a new potent acid-inhibitory drug) triple or dual therapy are popular new treatment methods. Numerous studies have confirmed the high H. pylori eradication rates and low side effects of both methods.54–56 Interestingly, Horii et al. reported that vonoprazan dual therapy has a low impact on gut microbiota.57 However, vonoprazan is expensive; thus, economic aspects should be considered when using it for treatment. Additionally, probiotics have been poven to improve the eradication effect.58 A study recruiting 234 H. pylori-positive gastritis patients from seven local centers indicated that probiotic administration before or after standard triple therapy improves H. pylori eradication rates.58 In addition, to reduce the chance of reinfection after H. pylori eradication, the strategy of “family-based H. pylori management” could be vital.59The current regimens for l agents . AccordiH. pylori.60 At present, the drug resistance of H. pylori mainly refers to resistance against antibiotics used in eradication therapies . The mechanism of antibiotic resistance primarily involves structural changes in the gene sequence, the destruction of antibiotic activity by changing drug targets, and the inhibition of intracellular drug activation, affecting drug-efflux mechanisms and causing enzymatic deactivation of the drug  also affect the efficacy of antibiotic delivery, which reduces the cure rate.15 Furthermore, H. pylori can form biofilms composed of bacteria and a self-secreted extracellular matrix that adhere to inert or living surfaces.61 Biofilms play an imperative role in antibiotic resistance by reducing drug penetration, promoting gene mutation, and overexpressing efflux pumps involved in drug resistance.15 The matrix includes an effective and nonspecific barrier, which prevents drug penetration and promotes the overexpression of efflux pumps in the process of drug resistance; thus, encapsulating H. pylori can significantly enhance its viability. Compared to free-floating strains, H. pylori growth in biofilms may occur for a longer time, with a greater susceptibility to developing antibiotic resistance.63 Given the aforementioned reasons, investigating alternatives to increase drug-delivery efficiency and eradicate H. pylori present intracellularly or in biofilms may be the key to resolving several issues related to its rapidly increasing drug resistance.However, rapidly increasing antibiotic resistance has reduced the efficacy of traditional antibiotic therapy against the drug .15 In ad64 their application in disease treatment is an important aspect of modern medicine. Owing to their unique antibacterial mechanisms and efficiency in eradicating drug-resistant strains, biomaterials are gradually being used in H. pylori treatment. Currently, biomaterials are mainly used as delivery systems for drugs to eradicate H. pylori, which can increase the drug delivery efficiency. Apart from encapsulating antibacterial agents, biomaterials  have been directly applied in H. pylori treatments because of their inherent antibacterial activity. Herein, recent studies on the progress of biomaterials in the treatment of H. pylori infection have been described and summarized.Biomaterials possess many unique properties, such as a large specific surface area, targeted lesion sites, satisfactory biocompatibility, and excellent stability;21 Based on the matrix material, nanoparticles have been classified into several categories, such as lipid-based nanoparticles , polymeric nanoparticles, and inorganic nanoparticles.Nanoparticles, defined as nanosized delivery systems that encapsulate drugs, can increase drug solubility and modulate drug-release characteristics and are valuable tools in fighting antibiotic resistance.66 With compositional similarity to cell membranes, lipid-based nanoparticles exhibit high biocompatibility and low toxicity.68 Combined with other materials, they can present excellent features, such as target sites and controlled drug release, enabling a high drug-delivery efficiency and low occurrence of antibiotic resistance in H. pylori.69 Notably, lipid-based nanoparticles (such as liposomes) can enter cells and kill H. pylori inside epithelial cells, macrophages, and biofilms.70 In this section, diverse lipid-based nanoparticles (mainly including liposomes and nanostructured lipid carriers) used for the eradication of H. pylori have been outlined, focusing on their stability, cargo transportation, and antimicrobial activity.Lipid-based nanoparticles  are among the most widely used drug delivery systems for antimicrobial therapy.71 As delivery systems, they exhibit numerous advantageous properties. First, they are composed of phospholipid bilayers encapsulating water-phase vesicles, and liposomes have high elasticity and good biocompatibility. Additionally, they exhibit high drug-loading and entrapment capacity as the water-soluble and fat-soluble drugs are dissolved in the water phase and lipid membrane, respectively. Furthermore, phospholipids themselves are cell-membrane components; thus, liposomes are nontoxic with high bioavailability.73 As one of the most widely investigated antibiotic delivery systems, liposomes are often used to deliver antibiotics  in H. pylori treatments.74–77 In particular, liposomes are ideal vehicles for delivering antimicrobial lipids ), making it difficult for H. pylori to develop resistance. For example, Obonyo et al. designed a system named liposomal linolenic acid (LipoLLA) by incorporating lauric acid, myristoleic acid, linoleic acid, and LLA, which solves the problem of poor solubility of LLA in aqueous solutions  of H. pylori by damaging its membrane  to exhibit target specificity and antiadhesion.82 For example, Gottesmann et al. reported the fabrication of pectin-coated liposomes with encapsulated amoxicillin that utilize a specific interaction of pectin with mucins (BabA) and the surface structures of H. pylori.76 Their results showed that pectin-coated liposomes exhibit stronger bactericidal effects than nonpectin-coated liposomes.76Liposomes are defined as smooth, continuous, and bilayered structures that contain mainly phospholipid molecules.olutions .24 Aftermembrane .81 AdditH. pylori. First, liposomes alone exhibit low encapsulation efficiencies for antibiotics,84 which may be because liposomes are too unstable for gastric antibiotic delivery.32 Adding cholesterol to liposomal formulations can improve liposome stability and increase the interactions between liposomes and H. pylori.76 However, excessive cholesterol may disrupt the linear structure of liposomal membranes, decreasing the encapsulation efficiency and drug release rate.74 Thus, selecting an appropriate proportion of cholesterol is vital for generating stable liposomes. Alam et al. investigated the effects of different cholesterol amounts on the encapsulation efficiency and drug release rate of different liposome formulations. The formulation containing 5 mg of furazolidone, 53 mg of cholesterol, and 106 mg of lipid exhibits optimal encapsulation and drug release efficacy.74 Combining liposomes with polymers is another helpful way to improve their stability. For example, Cai et al. reported amoxicillin-loaded liposome-polymer hybrid nanoparticles with high stability in the acidic environment of the stomach.78 Interestingly, the addition of rhamnolipids imparted a strong biofilm-disrupting capacity to the nanoparticles , enabling their large-scale production and long-term preservation.66 In regard to antimicrobials, nanoemulsions exhibit antibacterial activity and can destroy bacteria without the addition of any antimicrobial substances.86 Furthermore, they improve the stability, solubility, and bioavailability of loaded  drugs.87 For instance, Tran et al. developed a nanoemulsion-based delivery system loaded with erythromycin, which exhibits low solubility in water and is easily destroyed in acidic environments, for the eradication of H. pylori. Their results showed that the stability of erythromycin was significantly enhanced by the nanoemulsion system.87 Additionally, nanoemulsions exhibit effective adjuvant activity for vaccines. Yang et al. designed an intranasal HpaA epitope peptide-loaded nanoemulsion delivery system to protect against H. pylori infection.79 They found that this system could extend antigen release and induce Th1 responses, which indicated that nanoemulsions are an ideal delivery system for intranasal vaccines against H. pylori  that are stabilized by surfactants and typically exhibit sizes in the range of 20–200 nm.. pylori .79 Howev68 They can be grouped into three classes  according to their mixture composition and fabrication method.88 Nanostructured lipid carriers exhibit good biocompatibility and stability and can lead to high storage capacities and encapsulation efficiencies.88 For example, Seabra et al. fabricated docosahexaenoic acid (DHA)-loaded nanostructured lipid carriers that inhibit H. pylori growth at a very low concentration (25 mM), indicating that the nanoparticles effectively protect the properties of DHA. In addition, they also found that the antibacterial mechanism of DHA against H. pylori primarily involved the destruction of the bacterial cell membrane.89 Interestingly, in a later study by Seabra et al., they found that nanostructured lipid carriers without any loaded drugs can eradicate H. pylori, even at very low concentrations.80 To explore the underlying mechanism, H. pylori was cultured with nanostructured lipid carriers (1.25%) and analyzed by transmission electron microscopy and atomic force microscopy, and they found that most bacterial membranes were disrupted after 12 h 90 and involve complicated preparation processes , making routine clinical applications challenging. Therefore, developing methods to improve their controlled drug-release capacity and simplifying their synthetic procedures could make lipid-based nanoparticles an effective alternative for H. pylori therapy in the future.In summary, lipid-based nanoparticles exhibit high biocompatibility and can enhance the solubility and bioavailability of anti-92 Compared with lipid-based nanoparticles, polymeric nanoparticles exhibit higher loading capacities and mechanical stabilities.93 Chitosan, the most widely studied natural cationic polysaccharide polymer, exhibits good adhesion and antibacterial properties.94 Interestingly, chitosan nanoparticles can bind to H. pylori through electrostatic interactions and inhibit its growth by changing the permeability of bacterial cell membranes, even without any drug loading. For example, Luo et al. have developed chitosan nanoparticles that exhibit the highest bacteriostatic activity at pH 4. Notably, they found that chitosan nanoparticles with a 95% degree of deacetylation exhibited a higher anti-H. pylori effect than those with an 88.5% degree of deacetylation, indicating that a stronger antibacterial effect is exhibited by chitosans with a higher degree of deacetylation.94 Furthermore, chitosan and its derivatives can be utilized as drug delivery systems because of their special mucoadhesive properties (due to the electrostatic interaction between the positively charged chitosan and negatively charged cells/mucus), which can significantly extend the residence time of drugs in the stomach.96 For example, Lin et al. developed pH-responsive chitosan nanoparticles by integrating chitosan and heparin.97 Their results showed that the nanoparticles enhanced the drug-membrane epithelium interaction through the electrostatic interaction between chitosan and mucosa and promoted gastric ulcer healing by the effect of heparin.97 Under acidic conditions (pH in the range of 1.2–6.5), heparin and chitosan form spherical polyelectrolyte complexes that disintegrate upon reaching the infection site .97 In their later study, they fabricated amoxicillin-loaded genipin-cross-linked fucose-conjugated chitosan/heparin nanoparticles (genipin-FCS/Hep nanoparticles).96 Fucose can directly adhere to H. pylori, binding to it, while genipin can slow down the release of amoxicillin. The results showed that the effect of amoxicillin-loaded genipin-FCS/Hep nanoparticles in clearing H. pylori infection was stronger than that of amoxicillin alone  to eradicate H. pylori biofilms under the mucus layer at minimal inhibitory concentrations of 32 and 132 µg/ml, respectively  have also been studied.101Polymeric nanoparticles are produced using polymeric materials ) and colloidal organic compounds in nanosizes.in alone .32,96 Taic field .95 Rhamnectively 98,100 Th102 For example, Chehelgerdi et al. prepared a cagW‑based DNA vaccine loaded with chitosan nanoparticles;103 upon injecting mice with this vaccine, the expression of cagW increased on the 7th day and then gradually decreased, reaching a minimum on the 45th day. Furthermore, they found that vaccination significantly increases cellular immunity. Interestingly, during the H. pylori challenge, the vaccinated mice can clear the H. pylori infection. This suggested that the vaccine exhibited a protective effect against H. pylori in mice.103 In addition to chitosan, polymers such as HP55 /D and poly(lactic-co-glycolic acid) (PLGA) have also been used as delivery systems for vaccines.104 For example, Tan et al. designed an oral dual-antigen epitope and dual-adjuvant vaccine based on the urease of H. pylori and cholera toxin B subunits and delivered it with HP55/PLGA nanoparticles.104 This vaccine exhibits pH-dependent antigen release, with a release rate of less than 5% in acidic environments (pH ≤ 4.5) and 75% in neutral environments (pH = 7.5). In vivo results indicate that vaccine-encapsulated HP55/PLGA nanoparticles elicit high levels of systemic and localized antigen-specific antibodies and Th1/Th17-biased immune responses. During H. pylori challenge, mice injected with this vaccine exhibited 43% complete protection.105 Oral vaccines are considered to be the best way to elicit an immune response in the human stomach. However, owing to the acidic environment in the stomach, oral vaccines are easily hydrolyzed, resulting in low antigen uptake efficiency. The use of polymer nanoparticles to deliver oral vaccines is undoubtedly a good way to solve this problem.Polymers are also used as delivery vehicles for mRNA, DNA, and/or adjuvants in vaccines in various forms and morphologies because of their excellent stability.H. pylori vaccine began in the early 1990s,106 effective vaccines are currently lacking. The main reason for this could be their inability to induce a completely protective immune response against H. pylori.107 Moreover, the necessity for developing an H. pylori vaccine remains controversial. Nevertheless, rapidly increasing drug resistance rates, the high prevalence of H. pylori infection, and frequent reinfections due to ineffective antibiotic treatment indicate that childhood vaccination to prevent H. pylori infection could be an effective and economical strategy. This is especially true for countries with a high incidence of gastric cancer (such as China and Japan).108 At present, identifying suitable antigens and adjuvants is vital to facilitate the development of H. pylori vaccines. The delivery of H. pylori vaccines using nanotechnology could improve vaccine delivery effectiveness and prevent its degradation, thereby improving vaccine efficacy. With the declining success rate of current H. pylori treatment strategies, designing an effective vaccine could be a cost-effective way to control H. pylori epidemics.Although efforts to develop an H. pylori eradication regimens, particularly for hydrophobic drugs. They cause high drug stability and controlled release, leading to a high drug delivery efficiency and low H. pylori drug resistance. Some polymer-based polymeric nanoparticles, such as chitosan, exhibit excellent adhesion and the ability to change the permeability of H. pylori membranes, which enables chitosan nanoparticles to inhibit the growth of H. pylori, even without any drug loading.To summarize, polymer-based polymeric nanoparticles are mainly investigated as drug delivery systems in 109 As an effective strategy to eradicate H. pylori, anti-H. pylori mechanisms of inorganic nanoparticles mainly involve metal-ion release, ROS production, and disruption of the H. pylori cell membrane and biofilm.110 Owing to inorganic nanoparticles’ unique antimicrobial mechanism, it is difficult for H. pylori to develop resistance.Inorganic nanoparticles are defined as inorganic salts  or oxides with nanoscale sizes (1–100 nm); they exhibit small particle sizes, large specific surface areas, numerous surface-active centers, and strong catalytic abilities.H. pylori and reduce biofilm formation, are the most representative.111 Gopalakrishnan et al. designed N-acylhomoserine lactonase-stabilized silver nanoparticles (AiiAAgnanoparticles) that inhibit biofilm formation by degrading quorum-sensing molecules and inhibit urea production in H. pylori.22 Interestingly, AiiAAgNPs exhibit no toxic side effects at effective concentrations (1–5 μM), but they exhibit cytotoxic effects against tumor cells at high concentrations (80–100 μM).22 Similarly, Camargo et al. also synthesized a silver ion-based complex (Ag(PhTSC∙HCl)2](NO3)∙H2O) and transported it using polymeric nanoparticles.111 Their results showed that these complexes exhibited high efficacy against floating H. pylori and H. pylori present in biofilms, with no in vivo toxicity for Galleria mellonella.111Among the inorganic nanoparticles, silver nanoparticles, which inhibit the urease activity of H. pylori eradication.112–114 Gold nanoparticles can deposit on the surface of H. pylori, which kills H. pylori by membrane disruption. In addition, gold nanoparticles can also enter the cell and produce ROS, which kill H. pylori by affecting its metabolism.25 For example, Zhi et al. synthesized pH-sensitive acid-sensitive cis-aconitic anhydride-modified anti-H. pylori-conjugated gold nanostars (GNS@Ab).25 Notably, they improved the bactericidal effect using near-infrared laser photothermal treatment, which reduced the emergence of H. pylori resistance and even eradicated H. pylori-resistant strains isolated from clinical patients @ZIF nanoparticles with ascorbate palmitate   nanoparticles can absorb and release hydrogen (which changes the permeability of the cell membrane and interferes with H. pylori metabolism), and the zinc-based zeolitic-imidazolate framework (ZIF-8) can generate Zn2+ .23 Notably, Pd(H)@ZIF-8@AP not only kills H. pylori but also inhibits the inflammatory response and repairs damaged mucosa by inhibiting the secretion of inflammatory factors in macrophages and removing ROS. In addition, the effect of Pd(H)@ZIF-8@AP on intestinal flora is minimal.23 The hydrogen-generating ability of Zn-based micromotors in an acidic environment has been used to design mobile antibacterial platforms. For example, Lin et al. have integrated Zn-based micromotors with gallium ions. The gallium ions present a good ability to eradicate H. pylori, and the Ga/Zn micromotors exhibit good locomotivity in a simulated gastric acid environment (pH = 1.5). Moreover, Zn and Ga can degrade completely after 15 min  .23 AP car 15 min .115H. pylori mechanisms of inorganic nanoparticles mainly involve oxidative stress, nonoxidative stress, and metal-ion release.112 Inorganic nanomaterials have gradually gained importance in the field of anti-H. pylori, particularly for eradicating drug-resistant bacteria, due to their anti-multidrug resistance and good antibacterial effects. They can effectively eradicate H. pylori by multiple antibacterial mechanisms, making it difficult for H. pylori to develop resistance, and they can be integrated with other bioactive materials to form multifunctional anti-H. pylori platforms. However, their refractory degradation and potential nanotoxicity require further investigation before use in clinical H. pylori therapy.In summary, anti-118 Compared with nanoparticles, microspheres exhibit better loading capacities and controlled-release capabilities, facilitating the reduction of drug doses and long-term maintenance of therapeutic effects.120 Microspheres are often combined with other materials to form multifunctional anti-H. pylori platforms. For example, Tripathi et al. designed amoxicillin-loaded pectin/gellan gum-blended sodium alginate microspheres. The results showed that these delivery systems exhibit targeted drug delivery to gastric sites for the treatment of H. pylori, along with a suitable drug-release pattern.121 Chitosan exhibits excellent adhesion and antibacterial properties,122 and chitosan microspheres impart a good loading capacity and controlled-release capability to delivery systems, along with specific adhesion to H. pylori.124 Gonçalves et al. reported that chitosan microspheres exhibit the highest adherence to H. pylori at pH 6, and the microspheres are significantly more effective against BabA/SabA+H. pylori (76% reduction) strain than the BabA+/SabA H. pylori strain (50% reduction).125 Moreover, their results showed that chitosan microspheres can eradicate H. pylori through adhesion and are subsequently eliminated from the gastrointestinal tract.126 Based on this idea, Adebisi et al. also designed a clarithromycin-loaded chitosan hybrid microsphere and found that chitosan causes a significantly high adhesion of the microsphere to H. pylori.124Microspheres are homogeneous monolithic particles ranging from 0.1–1000 µm in size that are widely used as controlled-release drug carriers.H. pylori drug-delivery efficiency while maintaining the bactericidal concentration. In addition, combining with other materials makes microspheres a strong multifunctional anti-H. pylori platforms.Collectively, microspheres exhibit high loading capacities and can sustain drug release over time, increasing the anti-127 As another efficient delivery system for H. pylori therapeutic agents, hydrogels exhibit good biocompatibility, degradability, and controlled drug-release capacity.128–130 Chitosan and its derivatives are often used as base materials to prepare hydrogels because of their antibacterial and adhesion properties with H. pylori.132 For example, El-Mahrouk et al. reported metronidazole-loaded pH-sensitive chitosan hydrogels for H. pylori eradication therapy.131 The results showed that the swelling and drug-release ability of the hydrogels were highly dependent on pH; they were significant at pH 1.2 but low in phosphate buffers at pH 7.4. In vivo experiments indicated that the system can exist in a dog’s stomach for at least 48 h, and its ability to kill H. pylori is stronger than that of oral metronidazole alone.131 Similarly, Mohamed et al. have developed a novel chitosan hydrogel. Notably, they linked the chitosan hydrogel with benzophenone tetracarboxylimide benzoyl thiourea, which selectively inhibited cyclooxygenase-2 activity.129 This means that hydrogels can also function as multifaceted platforms for H. pylori eradication. Additionally, hydrogels also exhibit high drug-loading capacity and the ability to load multiple anti-H. pylori agents.133 For example, Silva et al. fabricated cross-linked sodium alginate-carboxymethyl cellulose hydrogels loaded with furazolidone (encapsulation efficiency: 71–76%) and bismuth(III) (encapsulation efficiency: 88%).133 The hydrogels swelled to approximately 150% in the first 2 h, remaining for at least 6 days in simulated gastric tissue (pH = 1.2), indicating that the hydrogels can exhibit sustained drug release. Although this study did not show the role of hydrogels in eradicating H. pylori, it reported their drug-loading capacity and therapeutic potential toward H. pylori.Hydrogels are hydrophilic three-dimensional network-structured gels formed by water-soluble or hydrophilic polymers through chemical or physical cross-linking.H. pylori drug-delivery systems. They exhibit numerous advantageous properties; their three-dimensional network structure improves drug stability, and they exhibit good biocompatibility and sustained drug-release ability.134 Targeting the low drug utilization rate of anti-H. pylori drugs, hydrogels can effectively improve medication utilization by maintaining a sustained release of drugs in the stomach, effectively eradicating H. pylori and hindering the development of drug resistance.135Hydrogels, with high drug-carrying capacity, are efficient anti-H. pylori treatment strategies. Thus, extensive research has been carried out to find effective strategies to combat H. pylori infections and antibiotic resistance, including designing new drug combinations, extending the treatment periods, and performing susceptibility testing before treatment. However, the existence of gastric acid milieud and pepsin, which can destroy most of the antibiotics in the stomach, and gastric mucosa, which is approximately 200 μm thick with a pH gradient from pH 1–2 to 7,32 cripple effective antibiotic delivery efficiency. This makes drug eradication programs extremely complicated, which not only significantly affects patient compliance but also increases eradication costs and consequently limits the applicability of H. pylori therapy. Therefore, improving drug delivery efficiencies and developing effective antibiotic-independent antimicrobial moieties could be promising emerging approaches to resolve the challenges associated with anti-H. pylori processes. As a new strategy to eradicate H. pylori, biomaterials have the following advantages  delivered by microspheres or hydrogels in the future. Targeted lesions are another strategy for improving delivery efficiency, thereby increasing eradication rates and reducing the development of H. pylori resistance. Biomaterials  can adhere to H. pylori through electrostatic interactions, targeting lesions, and indirectly prolonging the duration of drug action. However, Gottesmann et al. indicated that electrostatic interactions alone are not sufficient for specific interactions with H. pylori, and direct interactions with the H. pylori surface (such as specific binding with the mucin of H. pylori) are critical.76 Hence, this should be considered when developing bacteria-targeting biomaterials. (2) Destroys the biofilm. Biofilms play an important role in the emergence of H. pylori antibiotic resistance, and the antimicrobial resistance of bacteria growing inside biofilms can be 1000 times higher than that of external bacteria.100 Biomaterials, such as rhamnolipids and silver, can bind to the extracellular substance of biofilms and disrupt them by mediating signals or by other means. Therefore, strategies involving these biomaterials could be powerful “weapons” against the antibiotic-resistance crisis. (3) Changes in the permeability of the H. pylori membrane. Biomaterials such as linolenic acid, docosahexaenoic acid, and nanostructured lipid carriers can change the permeability of the H. pylori membrane, disrupting its integrity. Additionally, owing to the presence of components similar to those of cell membranes, these biomaterials could also have sterilizing effects on intracellular H. pylori compared with other biomaterials. Hence, these biomaterials could be used for the development of antibiotic-free anti-H. pylori agents. In addition, chitosan can also change the permeability of the H. pylori membrane; unlike the aforementioned biomaterials, its mechanism could involve its adhesion to H. pylori, followed by membrane destruction by the physiochemical properties of the nanomaterial. (4) Produced reactive oxygen species (ROS) or affected the metabolic activities of H. pylori. Apart from mechanically disrupting its membrane through the direct contact of nanomaterials with H. pylori, some biomaterials  eradicate H. pylori by generating ROS or by disrupting its metabolic activities. A large amount of ROS causes changes in bacterial morphology and DNA damage. Owing to their unique anti-H. pylori mechanisms, these biomaterials could also be suitable for developing antibiotic-free anti-H. pylori agents  Proi agents .Table 2.H. pylori infections in the future. First, the designed biomaterials should exhibit high stability in the harsh acidic environment of the stomach to improve the bioavailability of drugs. Second, to ensure good anti-H. pylori activity by biomaterials, bacterial targeting should be applied in the formulation design. Third, rapidly increasing H. pylori resistance is largely attributable to the misuse of antibiotics. Delivering antibiotics with low resistance rates (such as amoxicillin), improving delivery efficiency, and developing antibiotic-independent antimicrobial moieties could be the focus of future studies. Fourth, the persistent survival of H. pylori inside cells and biofilms is also an important reason for therapeutic failure and the emergence of H. pylori resistance. Therefore, fabricating biomaterials that can eradicate H. pylori present intracellularly and inside biofilms is crucial for improving the cure rate. Additionally, it is also vital to maintain the ecological homeostasis of the gut ecosystem and develop biodegradable biomaterials for H. pylori treatment. Most importantly, clinical translation and widespread applications are the ultimate goals of biomaterial design, making cost effectiveness and large-scale production important considerations.From bench to bedside, numerous challenges need to be addressed before designing biomaterials for the efficient treatment of H. pylori. Their unique multiple antimicrobial mechanisms eradicate H. pylori that is present intracellularly and inside biofilms and could solve the current problem of high drug resistance. This review discusses recent progress in biomaterials for the eradication of H. pylori and summarizes the main anti-H. pylori mechanisms. In conclusion, biomaterials exhibit high potential to be integrated into standard treatments in the future because of their safety and H. pylori eradication efficiency. The application of biomaterials could help achieve the ultimate goal of complete elimination of H. pylori in the future.In summary, biomaterials can be used to encapsulate and deliver conventional antibiotics, antimicrobial lipids, vaccines, or phytomedicines with antibacterial effects to increase the drug delivery efficiency and hinder the development of drug resistance in


Glossina morsitans morsitans. We generated antennae-specific transcriptomes from adult male G. m. morsitans flies fed/unfed on bloodmeal and/or exposed to an attractant , a repellant  or paraffin diluent. Using bioinformatics approach, we mapped raw reads onto G. m. morsitans gene-set from VectorBase and collected un-mapped reads (constituting the gaps in annotation). We de novo assembled these reads (un-mapped) into transcript and identified corresponding genes of the transcripts in G. m. morsitans gene-set and protein homologs in UniProt protein database to further annotate the gaps. We predicted potential protein-coding gene regions associated with these transcripts in G. m. morsitans genome, annotated/curated these genes and identified their putative annotated orthologs/homologs in Drosophila melanogaster, Musca domestica or Anopheles gambiae genomes. We finally evaluated differential expression of the novel genes in relation to odor exposures relative to no-odor control (unfed flies). About 45.21% of the sequenced reads had no corresponding transcripts within G. m. morsitans gene-set, corresponding to the gap in existing annotation of the tsetse fly genome. The total reads assembled into 72,428 unique transcripts, most (74.43%) of which had no corresponding genes in the UniProt database. We annotated/curated 592 genes from these transcripts, among which 202 were novel while 390 were improvements of existing genes in the G. m. morsitans genome. Among the novel genes, 94 had orthologs in D. melanogaster, M. domestica or An. gambiae while 88 had homologs in UniProt. These orthologs were putatively associated with oxidative regulation, protein synthesis, transcriptional and/or translational regulation, detoxification and metal ion binding, thus providing insight into their specific roles in antennal physiological processes in male G. m. morsitans. A novel gene (GMOY014237.R1396) was differentially expressed in response to the attractant. We thus established significant gaps in G. m. morsitans genome annotation and identified novel male antennae-expressed genes in the genome, among which > 53% (108) are potentially G. m. morsitans specific.Tsetse flies use antennal expressed genes to navigate their environment. While most canonical genes associated with chemoreception are annotated, potential gaps with important antennal genes are uncharacterized in  Glossina fuscipes fuscipes, Glossina palpalis and Glossina tachinoides, prefer feeding on reptilian hosts, unlike the savannah species, such as Glossina morsitans morsitans and Glossina pallidipes, that feed largely on ungulates and other large mammals, a differential behavioural choice that is very likely associated with differences in their olfactory architectures. These olfactory attributes have been exploited in the characterization of tsetse fly species-specific attractant or repellent constituents and blends, which can be exploited for protection of humans and their livestock [Management of Human African Trypanosomiasis (HAT) and Animal African Trypanosomiasis (AAT) initiative is a longstanding development preoccupation in sub-Saharan Africa, with control of different species of tsetse fly vectors considered a potentially effective means of disease control and suppression. All tsetse fly species are susceptible to different trypanosome infections and can transmit HAT and/or AAT to humans and livestock respectively. The critical determinant of differential transmission of trypanosomiasis among tsetse fly species is hinged on their host preferences mediated mainly by antennae resident olfactory processes in the different species. Tsetse flies typically navigate their environment by detecting and responding to volatile and non-volatile cues and display polymorphic responses between species, sexes, and allopatric populations –7. Diffeivestock –10. Furtivestock  or repelivestock . The finG. m. morsitans, G. f. fuscipes, G. pallidipes, Glossina brevipalpis, Glossina austeni and Glossina palpalis gambiensis) [Search for novel tsetse fly responsive compounds and blends (repellent or attractant) can be facilitated by characterizing molecular processes mediating species and/or sex specific differential responses. These can then be incorporated into existing optimized blends for possible enhancement of repellence or attraction to target tsetse fly species. Impetus to explore this novel approach is supported by availability of genomes of a suite of tsetse fly species (biensis) . Initialbiensis) –17 and tbiensis) . Most ofbiensis) –20. The biensis) –25, all G. m. morsitans genome, were however based in part on transcript evidence that were not antennae specific [G. m. morsitans tsetse would be enriched with novel antennae expressed genes absent in the current annotations of G. m. morsitans genome in VectorBase [G. m. morsitans because males are available in larger proportions, compared to females that were limited by need for their deployment in the sustenance of our tsetse fly colony. Secondly, the males equally sufficed as females or mixed gender to establish proof-of-concept in the application RNA-Seq approach, and our associated data analyses pipeline in the identification and filling of gaps in genome annotations. Once established, the concept can be adopted to improve annotation of female specific antennae expressed genes.Currently, six genomes of tsetse fly species have been annotated and published by the VectorBase community , 14. Amospecific . This suspecific  and Apolspecific . We hypoctorBase . Thus, iG. m. morsitans, 2) annotate and curate genes associated with these gaps and 3) establish transcriptional responses of the novel genes associated with the gaps to feeding, an attractant  or a repellent  [Anopheles gambiae species complex and Anopheles sinensis mosquitoes to blood feeding [Drosophila melanogaster [G. m. morsitans [We thus initiated this study specifically to (1) establish gaps in annotation of antennae expressed genes in male lactone)  to the f feeding , 31, andnogaster  and G. morsitans . Herein G. m. morsitans tsetse fly colony maintained at Yale University, New Haven, CT, USA, insectary. The flies originated from a small population originally collected from Zimbabwe. The flies were maintained at 24°C and 50–60% relative humidity (RH) and received defibrinated bovine blood , via an artificial feeding system every 48h [s fed on defibrinated bovine blood- , to putatively prime their chemosensory apparatus. We sourced for δ-nonalactone repellent (98–99% pure) as racemic mixture from Sigma-Aldrich  and synthesized racemic blend of ε-nonalactone attractant  in the laboratory as previously described [13C NMR), Hydrogen Nuclear Magnetic Resonance (1H-NMR), and Fourier Transform Infrared (FTIR) spectrophotometry as detailed and reported in Wachira et al. [−3 dilutions in paraffin oil (1% vol/vol), as previously used in the assessment of laboratory responses of G. m. morsitans to odors [We obtained antennae from male very 48h , 35. Sama et al. . We testto odors .G. m. morsitans (1–3 days old) and starved some for 72hrs to ‘induce’ hunger and potentially prime them into ‘host seeking’ physiological state. We kept the other normally fed tsetse to assess the effect of feeding on antennal transcriptional changes. We hypothesized that the unfed flies would be conditioned to distinguish host (attractant) or non-host (repellent) odor compounds compared to paraffin oil as control. Briefly, we separately placed three independent replicates each consisting of 50 flies in 1L transparent glass jars, with 100 μl of the paraffin oil diluted attractant or repellent via Whatman filter paper (1 cm in diameter) [−3 dilution in paraffin oil within each jar. We did not assess empirical prevalence/concentration of the odors in headspace. We similarly handled the fed flies, but in two independent replicates due to limitations in biological samples/materials. It has previously been demonstrated that five hours exposure is optimal for desensitization of receptors and subsequent transcriptional change in response to odors in Dm [G. m. morsitans show marked diel changes in their biting activity in the field, with their peak activity in the mornings and afternoons [et al., [beta-tubulin gene primers as documented in Bateta et al. [Illumina TruSeq RNA Sample Preparation Kit  and the cDNA (101 bp paired end read) sequenced on Illumina HiSeq 2500 at Yale University Center of Genome Analysis (YCGA), New Haven, CT, USA. We selected paired rather than unpaired-end read sequencing platform to facilitate subsequent more accurate alignment of the reads on our reference transcripts [We collected the blood fed teneral male iameter) . We simirs in Dm . Since Gternoons , we perfternoons . Briefly[et al., . We thena et al. . We verinscripts . We thushttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/). We then used the FastQC results to clean (trim) and remove low quality reads from respective transcriptomes using Trimmomatic software version 3.8 [G. m. morsitans transcripts gene-set version 1.9 or genome version 1.0 from Vectorbase [de novo assembled them (unmapped reads) into transcripts and assessed quality of the assembled transcripts using the short reads Trinity de novo assembly software 2.10.0 [We established quality of the reads in each individual transcriptome library using FastQC version 11.0 (Babraham Bioinformatics) software package  that could putatively yield peptides with at least 100 amino acids long using TransDecoder software [G. m. morsitans transcripts gene-set version 1.9 from VectorBase [de novo transcript 1) a homolog of a Uniport database gene if it had an e-value < 0.001, a query coverage and length at least 95% and 100 amino acids respectively, and 2) corresponding transcript of G. m. morsitans transcript if it had an e-value < 0.001, query coverage and identity of at least 95% and length of 300 nucleotides. We considered transcripts with neither homologs nor corresponding transcripts in either database as novel transcripts. Those transcripts lent further insight into the gap in genome annotation. We separately used the longest transcripts with open reading frames to predict novel protein-coding genes in the G. m. morsitans genome version 1.0 from Vectorbase [ab initio gene predictions, transcript evidence and homologous protein evidence [https://www.uniprot.org/ (accessed on 10 June 2020) served as transcript and protein evidence respectively within the pipeline. We finally assessed for proportion of our longest transcripts with open reading frames that MAKER used in the prediction of protein-coding genes by searching for the corresponding genes for our transcripts using BLASTn [de novo transcript correspondent with the predicted genes if the e-value was < 0.001, and the query coverage and identity were at least 95% each.We mapped back our unmapped reads onto our on 2.3.5  to estabsoftware . We quersoftware  or correctorBase  using BactorBase  respectictorbase  using MActorbase . This pievidence . Our traevidence  publiclyg BLASTn . We consD. melanogaster within VectorBase [G. m. morsitans gene-set (Var 1.9) [de novo assembled transcripts utilized/accepted by MAKER computational pipeline in processing of the gene predictions by performing a nucleotide search using Basic Alignment Search Tool (BLASTn) [de novo assembled transcripts as query against the newly curated genes as subject. We isolated novel genes (predicted protein-coding genes) from among our newly annotated genes by performing a nucleotide search using BLASTn [G. m. morsitans gene-set (Var 1.9). We then identified putative functions of these novel genes (without corresponding gene in the G. m. morsitans gene-set (Var 1.9) by identifying their 1) homologs in Uniport/Swiss-prot protein database [M. domestica or An. gambiae mosquito genomes obtained from VectorBase release 53 [D. melanogaster genome from FlyBase [We manually curated the final gene models generated by the MAKER software by inspecting and refining the precise gene structure and putative function in graphical browser-based curation Apollo software  platformctorBase  and 3), ctorBase . We thenVar 1.9)  and the Var 1.9) . We asse(BLASTn)  of the dg BLASTn  of the adatabase  using BLdatabase  search adatabase  (accessedatabase  and 3) olease 53  or D. me FlyBase  using de FlyBase .G. m. morsitans transcripts gene-set version 1.9 from VectorBase [We assessed any differential expressions of the novel genes in response to the attractant  or the repellent  structural analogues, and compared these with the effect of feeding, using RSEM (RNA-Seq by Expectation Maximization) EBSeq pipeline . ResponsctorBase  to thesectorBase . In the ctorBase . We extrctorBase . This anctorBase . We haveG. m. morsitans. More than 99% of these libraries passed the quality control test as clean paired (94.94%) or unpaired (4.43%) reads  (G. m. morsitans gene set. This was indicative of their G. m. morsitans origin (not contaminants or of parasitic nature), while potentially revealing the gap in the annotations of active genes in the genome. The less than 4% of the clean paired reads that did not map onto the genome potentially represent gaps in assembly of the genome and/or presence of symbionts or pathogens in the fly population.We obtained 587 million reads from sequencing all the total RNA libraries from the antennae of adult male %) reads . More thnpaired) . Of the de novo assembly of the unmapped paired reads yielded to 311,970 transcript contigs, grouped into 213,184 putative genes. The assembly had a GC quality content of 34.18% and contig N50 quality statistic of 1,445 for all genes, and 903 (N50) for the longest isoforms. The median contig base length was 478 and 367 bases, with average base length of 857.16 and 626.51 bases for genes or longest isoforms respectively  (G. m. morsitans transcripts gene-set version 1.9 [de novo assembled transcripts associated with the unmapped reads (gaps in genome annotation) were potentially linked to functionally annotated genes, while others were novel without corresponding transcripts/genes in the databases. The functionally annotated genes homologous to assembled transcripts in both databases were putatively associated with chemoreception, regulation of gene expression and translation, detoxification of xenobiotics and responses to stress among other attributes (Our ectively . Mappingnpaired) . We isolnpaired) . Similarsion 1.9  comprisitributes  Tables.de novo assembled transcript in G. m. morsitans genome in Vectorbase [de novo assembled transcripts used in the prediction of protein-coding genes of the transcripts revealed that 68.86%  of these were deployed in the predictions, and about 30% were not, likely due to insufficient evidence. Our interrogation of these predicted genes in a VectorBase community platform [M. domestica, D. melanogaster or An. gambiae genome revealed that 46.53% (94) of the novel genes had orthologs in at least one of the target genomes  in VectorBase [We have summarized the variants/isoforms of the existing genes in ctorBase . These vctorBase .G. m. morsitans specific transcripts without any ortholog in any of the three  genomes or homologues among Uniprot database genes [Our assessment of differentially expressed transcripts revealed significant induction (3.5-fold change and FDR of <0.05) of GMOY014237.R1396 novel gene transcript in response to attractant exposure relative to the no-odor control (unfed). This (GMOY014237.R1396) is among the novel putative se genes  . These reads are typically discarded from further downstream analyses. We consequently identified 983 antennae-expressed genes in male G. m. morsitans as valid structural and putatively functional genes in the antennae. Among these, 592 gene models showed sufficient evidence for curation, validation and adoption for community use in VectorBase [de novo assembled transcripts were not integrated in the predicted gene models/coding genes by MAKER computational pipeline [G. m. morsitans genome using the same pipeline [de novo assembled transcripts might also have been components of noncoding RNA (ncRNA) genes in the genome. The Encyclopedia of DNA Elements (ENCODE) Program established significant transcription of DNA as RNA, vast majority of which are ncRNA [G. m. morsitans genome by at least 4.55%, potentially adding to the 13,018 number of genes in the G. m. morsitans transcripts gene-set version 1.9 [G. m. morsitans transcripts. The significant (> 96%) mapping of the antennae expressed reads onto the genome indicate sufficient  assembly of the reference genome. However, mapping statistics of the reads onto the transcripts points to 1) a major gap (incompleteness) in annotations of G. m. morsitans genome, 2) potential under representation of composition of genes present in G. m. morsitans genome, and 3) deficiency in annotation of some tissues specific/expressed genes in G. m. morsitans. Total number of annotated coding genes in G. m. morsitans is considerably smaller (by more than 50%) than for D. melanogaster [G. m. morsitans genome. Similar relationship has previously been observed between coding genes in avian and tetrapod genomes [G. m. morsitans genome annotation, the extent of this gap (genes misassembled or not annotated) and identification of the genes that miss in G. m. morsitans compared with D. melanogaster and related taxa/species remain to be established. However, this can be addressed by application of our approach in annotations of unmapped reads associated with tissue specific RNA-seq libraries as has recently been implemented in improving annotation of 9,206 genes and identification of 2,000 novel genes in the genome of Rhodnius prolixus Triatominae insect species [In this study, we annotated ctorBase , and of ctorBase  at the ppipeline , probablpipeline , which wpipeline . These dre ncRNA . We nevesion 1.9 , and est genomes –54 and a species .G. m. morsitans. None of the orthologs or homologs of the novel genes in selected genomes  and Uniprot database genes, respectively, were putative canonical chemosensory genes. The antennae are principal olfactory organs in insect with diverse sensilla [G. m. morsitans tissues and physiological states . Tsetse specific novel chemosensory genes might also be part of the 108 novel genes without orthologs in the selected genomes or homolog in the Uniprot database. This is supported in part by the apparent significant differential transcriptional responses in GMOY014237.R1396 novel gene in that category to attractant exposure relative to the no-odor control in the current study. However more insight into biological role of this gene (GMOY014237.R1396), can be better facilitated by validation of its expression using RT-qPCR [G. m. morsitans antennae. How these processes relate to phenotypic/behavioral and olfactory responses of the tsetse flies to odor is not clear but play a critical regulatory role in modulating such behavior. Further annotations of the missing genes in this tissue, including the novel putative G. m. morsitans specific genes, might provide further insight on the additional genes involved with mechanoreception and chemoreception typically associated with antennal functions in insects, including tsetse flies. However, such annotations would provide better resolution of the missing genes if RNA-Seq library originated from both gender and wild population of G. m. morsitans of various physiological states.At a functional level, this study provides us with new insights into the novel genes that potentially mediate molecular functions in the antennae of male sensilla , 57 exprsensilla –18, thatsensilla , and 2)  RT-qPCR  and func RT-qPCR  that wer RT-qPCR ,23–25 anG. m. morsitans genome. These genes can provide further insights on those that potentially mediate molecular functions in the antennae of male G. m. morsitans.In conclusion, we have shown missing genes with biologically significant information can be annotated, from reads that do not align to existing gene transcript (unmapped reads), but specific to the S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Cryptococcus species are a major cause of life-threatening infections in immunocompromised and immunocompetent hosts. While most disease is caused by Cryptococcus neoformans, Cryptococcus gattii, a genotypically and phenotypically distinct species, is responsible for 11–33% of global cases of cryptococcosis. Despite best treatment, C. gattii infections are associated with early mortality rates of 10–25%. The World Health Organization’s recently released Fungal Priority Pathogen List classified C. gattii as a medium-priority pathogen due to the lack of effective therapies and robust clinical and epidemiological data. This narrative review summarizes the latest research on the taxonomy, epidemiology, pathogenesis, laboratory testing, and management of C. gattii infections. Cryptococcus can cause life threatening infections in both immunocompromised and immunocompetent hosts. Although most disease is caused by Cryptococcus neoformans, C. gattii—a genotypically and phenotypically distinct species—causes 11–33% of invasive cryptococcosis globally [C. gattii is associated with 28 day case fatality rates in range 10–25% [C. gattii as a medium priority pathogen in light of the poor availability of safe and effective therapies and a lack of robust clinical and epidemiological data [globally ,3. Even e 10–25% ,5,6,7. TCryptococcus gattii infections—contrasting this, where relevant, with what is known about the more common pathogen, Cryptococcus neoformans. Data, as far as possible, are from the last 10 years. Only English-language publications were included.We conducted a narrative review of the literature, summarising what is new in epidemiology, pathogenesis, diagnosis, and management of Cryptococcus neoformans and Cryptococcus gattii are haploid encapsulated yeast-like fungi of the class Trellomycetes [Cryptococcus neoformans includes two varieties, Cryptococcus neoformans var. grubii and Cryptococcus neoformans var. neoformans, though further sub-divisions have been proposed; Cryptococcus gattii is a separate species [Cryptococcus neoformans and Cryptococcus gattii complexes [omycetes . First domycetes . Cryptoc species ,10,11. Tomplexes .Cryptococcus spp. were distinguished by serotype with serotypes A, B and C recognized two decades before serotype D [LAC1) and capsule gene (CAP64) [Cryptococcus neoformans: A is C. neoformans var grubii and D is var neoformans. Serotypes B and C are Cryptococcus gattii. Hybrids also exist. A minority of Cryptococcus spp. (ranging from 2–21%), are acapsular or pauci-capsular, and so untypeable [Historically, rotype D . Serotyp (CAP64) ,14,15. Stypeable ,17,18.C. gattii (VGI-VGIV) and C. neoformans  , VNIII (hybrid of serotypes A and D), VNIV–serotype D) [The advent of molecular taxonomy has revolutionized fungal nomenclature. Various techniques have been used including PCR-fingerprinting, Restriction Fragment Length Polymorphism (RFLP), Amplified Fragment Length Polymorphism (AFLP) and Multi-Locus Sequence Typing (MLST) to identify distinct lineages within the two species complexes: otype D)  (see TabC. neoformans and C. gattii be divided into 7 sibling species based on the phylogenetic (evolutionary) species concept. Namely, for neoformans: Cryptococcus neoformans (genotype VNI), Cryptococcus deneoformans (genotype VNIV), plus C. neoformans × C. deoneoformans hybrid (genotype VNIII). For gattii: Cryptococcus gattii (genotype VGI), Cryptococcus deuterogattii (VGII), Cryptococcus bacillispora (VGIII), Cryptococcus tetragattii (VGIV), and Cryptococcus decagattii [C. gattii discovered in Zambian woodland environments in association with nitrogen-rich middens of a small mammal known as Hyrax, has been designated VGV, and C. decagattii as VGVI [Adding a further genotype based on a single clinical isolate from Mexico, Hagen et al. in 2015 proposed that  as VGVI ,22.C. neoformans and C. gattii should be considered species complexes, supported by clinical, epidemiological and ecological data, or divided into separate clades (species) with corresponding changes in nomenclature, based on genotyping alone, without clearcut biological differences [C. gattii lineage in Zambian woodlands introduced above suggests that further lineages will be discovered [Debate continues within the scientific community as to whether ferences ,20,23. Iferences  and lateferences  that spescovered .URA5, CAP59, GPD1, LAC1, SOD1, PLB1, and IGS). A database of quality-controlled reference sequences is available at https://mlst.mycologylab.org/ (accessed on 30 June 2022). and in the CBS-KNAW database ). As of 30 June 2022, this data base contained 566 entries for C. gattii and 662 for C. neoformans.MLST-based DNA Barcoding, which uses short diverse genetic sequences that are stable and unique to a given species, is currently the gold standard for cryptococcal typing ,24,25. AC. neoformans and C. gattii can be distinguished readily [Accurate species identification from fungal colonies can also be achieved reliably and rapidly by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS)  with min readily ,26,27. HC. gattii, may have spread from Australia and Northern Brazil to North America and Canada [C. neoformans, ST5 is the predominant serotype in China and Taiwan, ST93 in Brazil, ST41 in Japan and the US, with greater diversity in African countries [MLST has revealed geographical associations with clonal populations with increased heterogeneity possibly related to the origins of the species; for example, d Canada ,29 whereountries ,31,32.C. neoformans was published in 2005 [C. neoformans (NCBI accessed 30 June 2022). This has enabled further elucidation of origins, evolution, and genetic variation amongst the species complexes [C. gattii isolates in Zambia, and sub-lineages VNBI and VNBII within VNB of sub-Saharan African isolates associated with phenotypic differences, as well as the new designation of VGVI (putative C. decagattii) [Despite the value and comparatively low cost of MLST, the highest resolution is achieved with whole genome (next-generation) sequencing (WGS or NGS). This allows greater discrimination between strains and offers a method for species identification, detection of drug resistance mutations and epidemiological information. WGS has broadly confirmed the relationships between the different genotypes identified by MLST. The first whole genome sequence for  in 2005 . There aomplexes ,34. Assoomplexes ,36. Becaomplexes ,37. Noneagattii) ,20,21.C. gattii and C. neoformans species complexes. For clinical purposes, the use of species complexes is the more useful terminology and will be used from this point. Meanwhile, important research continues into the implications of different genetic groupings.In summary, debate persists regarding the delineation of species within both C. gattii and C. neoformans are adapted to different ecological niches and exhibit differences in epidemiology, clinical features, and virulence mechanisms [chanisms ,39.C. gattii, often eucalypts but including a huge range from oaks to baobabs [C. neoformans, on the other hand, is primarily associated with weathered pigeon guano.Several arboreal species have been identified as the predominant environmental reservoir of  baobabs . C. neofC. gattii infections occur predominantly in Australia, British Columbia, Canada, the Pacific Northwest of the USA, parts of South America and Africa. Molecular type VGI, has a global distribution but is most prevalent in Africa, Australia, and Europe. Molecular type VGII occurs globally but is mostly reported from Australia and the Americas. Molecular type VGIII is concentrated in, but not limited to, the Americas. The relatively rare molecular type VGIV has been reported in southern Africa and India, while the very rare molecular type VGVI has been reported from Mexico [Despite potential sampling bias across the globe, evidence to date indicates that m Mexico ,21,40,41There is some evidence that subgroups (subclades) within the VG lineages are also geographically defined, for example VGIa is found in Australia and the Pacific Northwest of the US, whereas VGI b has been found in Zambia; VGII a and c predominate in the Vancouver area and the Pacific Northwest of the US whereas VGIIb has been reported in far Northern Australia. These associations may reflect microevolution within these lineages over time.C. gattii. Modelling performed by Cogliati et al. determined that in Europe, C. gattii was concentrated along the Mediterranean coast, with its distribution limited by low temperatures during the coldest season, and by heavy precipitations in the driest season; C. neoformans var. grubii colonized the same areas but it tolerated cold winter temperatures and summer precipitations better [C gattii from 1980 to 2009 in the same region followed by doubling of the potential environmental niche in 2010–2019 and a predicted further extension inland from the coastal Mediterranean basin as a result of climatic change and global warming [Interest has recently turned to environmental factors affecting the distribution and virulence of fungal species, including s better . More ex warming .C. gattii, the discovery of VGV coexisting with VGI and VGII in Zambia in an entirely new ecological niche, namely, middens of the Southern tree hyrax (Dendrohyrax arboreus) [Cryptococcus.In relation to evolution of rboreus)  is of grrboreus) . They haC. gattii infection occurred predominantly in immunocompetent people, evidence increasingly points to potential immune and genetic factors predisposing to infection. In Australia the incidence of cryptococcosis in aboriginal populations is 10.4/million/year compared with 0.7/million/year in non-indigenous populations, and the difference cannot be wholly explained by differences in places of domicile or work [C. gattii infection [C. gattii.Although it was traditionally believed that  or work . In the  or work ,47. Subtnfection ,49. OverC. gattii infections, clinicians must consider the possibility of an underlying immune defect. Although many major causes of immunocompromise may be obvious on history taking, such as transplant or haematological malignancy, others require specific questioning about history of prior infections, family history of unusual infections, early deaths and/or malignancies. Positive findings may indicate further testing is desirable, such as blood smear/film, CD4/CD8/natural killer (NK) cell subsets by flow cytometry, immunoglobulin levels, complement testing and others. Where inborn errors of immunity are suspected, genetic testing will be required, and a clinical immunologist should be involved in discussions.When dealing with C. gattii is less likely to present with CNS involvement (varying by genotype), when it does, there is a higher incidence of CNS imaging abnormalities, mass lesions and hydrocephalus requiring neurosurgical intervention. There appear to be significant differences in cerebrospinal fluid (CSF) examination between the two species, with C. gattii CNS infections demonstrating lower CSF glucose and protein, higher median white blood cell counts and more frequent India ink positivity. Immune reconstitution inflammatory syndrome (IRIS) is more frequent in C. gattii infections than C. neoformans. Despite all this, CNS C. gattii infection has a lower mortality rate than C. neoformans. It must be noted, however, that mortality in patients with cryptococcal infections is often related to the predisposing disease rather than direct cryptococcal-induced mortality and this may partially explain this observation [Although ervation ,39. See After inhaled Cryptococcus cells are phagocytosed by alveolar macrophages, they alter the macrophage transcriptome, preventing significant acidification, calcium efflux and protease activity, and thereby allowing intracellular pathogen proliferation .C. gattii can modulate macrophage polarisation to M2, thereby evading immune recognition and clearance [C. gattii impacts signalling, cell differentiation and regulation of immune system processes, while C. neoformans modulates the cellular response to stimulus, response to DNA damage and cell death [Classically activated M1 macrophages that result from the usual Th1 cytokine immune profiles are essential for clearing Cryptococcus, while alternatively activated M2 macrophages resulting from Th2 cytokine stimulation are associated with a higher burden of disease ,53. C. glearance ,52,53. Tlearance . C. gattll death .Deficiencies in alveolar macrophage functions, including phagocytosis and pattern recognition receptors, result in an intracellular proliferation of Cryptococcus. Subsequent abnormalities in cytokine and protein signalling pathways then initiate a suboptimal or potentially pathogenic immune response, depending on where the deficiency lies . AdvanceEndogenous interferon (IFN)-gamma production is associated with protection against Cryptococcus disease, leading to increased phagocytosis and fungicidal activity of phagocytes. Although exogneous IFN-gamma is sometimes useful as a therapy for refractory IRIS , as discC. gattii VGI and VGII genotypes cause most infections in immunocompetent hosts, while VGIII and VGIV are mainly isolated from immunocompromised hosts. Genotype VGII is the most heat-resistant of the known strains, especially at 37 °C. Different subtypes have different gene expressions identified in vitro, which correlate with their observed virulence clinically. Notably, the outbreak strain VGIIa, in a whole genome sequencing study, demonstrated genetic transformation and mitotic microevolution from 12 missense mutations and one shift mutation. Hypervirulent strains of C. gattii, but not C. neoformans, demonstrate enhanced mitochondrial tubularisation, which positively correlates with intracellular proliferative capacity within macrophages, a possible species-specific virulence mechanism [echanism .C. gattii compared with C. neoformans, perhaps reflecting their different environmental niches and contributing to different clinical manifestations [Both species elicit a robust host immune response. However, murine studies note a considerably different species-dependent host transcriptomic profile . There istations .C. gattii [C. gattii has an additional xylose residue compared to C. neoformans [Another difference is related to the cryptococcal capsule. The cryptococcal capsular polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan, simultaneously evoke an immune response, enhancing pathogen virulence and allowing evasion of immune recognition by coating antigens on . gattii . The GXMoformans .C. neoformans and C. gattii, hypothetically, are subjected to different herbicidal and environmental pressures, which may differentially alter capsule size and melanin production.Two structural factors associated with increased virulence, the larger size of the dynamic capsule and increased melanin production protecting against reactive oxygen species, are impacted by environmental conditions, with the former shown to be altered by some common herbicides . The difC. gattii seems to have a greater propensity to form titan cells [Capsule size correlates with virulence, although not case-fatalit . Notablyan cells . Titan can cells .C. gattii melanise more slowly than C. neoformans [oformans . Melaninoformans , and theC. gattii have a greater ability to form biofilms in vitro, which may also contribute to differences in disease phenotype between the species [Some strains of  species . Along w species ,65,66.C. gattii genotypes, phenotypes, virulence and regulatory mechanisms is recommended for those interested in further reading on this topic [A recent in-depth review summarising is topic .C. gattii infection, and recommendations are based on expert opinion as, to date, no prospective randomized controlled treatment trials have been conducted for C. gattii infection. The rarity of C. gattii infection presents a significant challenge to conducting such trials. For severe infection, many clinicians favour a prolonged induction therapy with liposomal amphotericin B and 5-flucytosine (4–6 weeks) followed by consolidation therapy for 12–18 months, and this is supported by an Australian case series [C. gattii infections in the last decade—more research is needed to optimise this aspect of care.Six international consensus guidelines have been released in the past decade covering cryptococcal infections ,70,71,72C. gattii and C. neoformans. Olorofim has no activity against either. Rezafungin, as consistent with data for the echinocandin class of antifungal, is not active against C. neoformans and is unlikely to be effective for C. gattii . There were no data for ibrexafungerp. Not included in that review was oteseconazole, a novel tetrazole, which shows activity against C. neoformans, though no data are available for C. gattii [C. neoformans but not C. gattii.A comprehensive review of the antifungal drug pipeline in 2021  reported. gattii ,75. A le. gattii ,77. SeveC. neoformans patients.Given the relative dearth of agents in the antifungal pipeline and the ongoing high mortality and morbidity rates with existing antifungal therapy, researchers have enthusiastically pursued adjunctive therapies. Despite their theoretical promise, trials have returned disappointing results. However, it must be highlighted that the following studies for “cryptococcal meningitis” were not species-specific and mainly recruited ClinicalTrials.gov as of 26 November 2022.Both pathogen and host play roles in tissue damage, so immunomodulation is a natural target for adjunctive therapies. Under this category, both IFN-gamma and dexamethasone have been subject to clinical trials in HIV co-infected patients. In 2004, a double-blind placebo-controlled trial (n = 70) of adjunctive IFN-gamma Ib (100 or 200 μg thrice weekly for ten weeks) showed a non-significant trend towards improved CSF clearance of cryptococci . HoweverC. gattii) and animal models. In 2016, results of a double-blind, randomised placebo-controlled trial  which recruited patients in Vietnam, Thailand, Indonesia, Uganda, and Malawi, were released [C. gattii infection.Dexamethasone was considered a promising therapy for HIV-associated CM based on mortality benefits in tuberculous meningitis and confirmed bacterial meningitis , and supreleased . The trireleased . Dexametreleased . NotablyC. gattii infections and patients with mass CNS lesions [Unfortunately, there is insufficient evidence to inform clinicians about the harms or benefits of steroids in HIV-uninfected cryptococcal meningitis patients. These patients have a very different baseline immune response to HIV-co-infected patients, so responses are likely to differ. Case series have described benefits, especially in  lesions ,83, but  lesions .Other agents have been identified as promising non-immunomodulating adjuncts, although again, results have been disappointing. Between 2018–2020 three trials were published reporting on the efficacy of the selective serotonin reuptake inhibitor sertraline in cryptococcosis ,86,87. AC. gattii and dendritic cell-based vaccines are under investigation as vaccine antigens [C. gattii and C. neoformans regardless of whether the cells are titan or not, but with a more pronounced affinity for the yeast form and reduced the size and number of pulmonary titan cells in cryptococcus infected mice [Work on cryptococcal vaccinations has been undertaken since the 1950s, with many different antigens and delivery systems protecting against infection or attenuation of disease in animal studies. More recently, acapsular strains of antigens ,89,90. Aantigens . Althougted mice ,92.Although there are multiple strategies to diagnose cryptococcosis, with and without fungal culture, diagnosis can be challenging. Delays in treatment because of missed early diagnoses or incorrect diagnoses, lead to worse clinical outcomes .C. gattii turns the culture medium blue, whereas C. neoformans does not change the colour of the medium [Traditionally, fungal culture has been used to diagnose cryptococcosis see . Whilst e medium .In terms of microscopy, Cryptococcus can be seen in body fluid with India ink see  examinatX-ray, computerised tomography (CT) scans and magnetic resonance images (MRI) of the lungs (pulmonary cryptococcosis), brain , and other parts of the body are valuable adjunctive tools for diagnosing cryptococcosis and especially the extent of disease, but radiography results alone are unlikely to be diagnostic ,97,98.C. gattii infections, including infections caused by C. gattii and C. neoformans hybrids [Serological diagnostic tests are used to detect cryptococcal capsular polysaccharide in serum and CSF by Latex agglutination test (LATs), enzyme-linked immunoassays (ELISA). These tests have an overall sensitivity and specificity of 93–100% and 93–98%, respectively [ hybrids .C. gattii infections which may be missed by other serological tests.The introduction of cryptococcal antigen lateral flow assays (CrAg-LFA) marked a major revolution in the diagnosis of cryptococcal infections . CrAG-LFC. neoformans and C. gattii. Recently, a real-time PCR assay targeting the multicopy mitochondrial cytochrome b (cyt b) gene to detect C. neoformans and C. gattii has been developed [C. neoformans and C. gattii. The overall reported assay sensitivity was 96.4%, and the specificity was 100%, and it can diagnose cryptococcosis in patients within four hours [Although molecular assays, such as polymerase chain reactions (PCRs), are potentially a more rapid diagnostic method than culture and antigen testing, such assays are not routienly required. When in use, commercial PCR-based assays that target Cryptococcus cannot distinguish between eveloped . The assur hours . The tarC. neoformans and C. gattii in clinical CSF specimens [C. neoformans per reaction within 10 min and be highly specific to Cryptococcus species. The assay sensitivity was 95.2%, and the specificity was 95.8% [C. neoformans or C. gattii to detect cryptococcosis in CSF [C. neoformans and C. gattii, and may not be able to detect cases where CSF is clear of infection.The novel lateral flow strips combined with recombinase polymerase amplification (LF-RPA) assays were developed to detect the specific DNA sequences of pecimens ,104. Theas 95.8% . MeanwhiC. gattii and C. neoformans [Other recent advances include identification systems such as MALDI-TOF MS. This is a rapid identification tool that can identify both oformans . It offeoformans .C. neoformans and C. gattii [C. neoformans and C. gattii in clinical samples directly. SERS is rapid and nondestructive and has relatively low equipment cost. Briefly, it was shown that AgNPs-self-assembled on the fungal cell wall surface via electrostatic aggregation, leading to enhanced SERS signals that were better than the standard substarte negatively charged AgNPs. The SERS spectra could then be used as a sample database in the multivariate analysis via orthogonal partial least-squares discriminant analysis. The SERS detection method can accurately (was shown to be 100%) distinguish between C. neoformans and C. gattii using principal component analysis. SERS seems to be a breakthrough, though further evaluation will be required before it can be introduced into routine clinical practice.Surface-enhanced Raman scattering (SERS) and spectral analysis offer an additional potential diagnostic tool for . gattii . This noC. gattii and C. neoformans at the individual patient level given their differences in patient predisposition, disease phenotype, and treatment approaches. Treatment trials should include patients infected with both strains and, where possible, run subgroup analyses to identify any differences.In conclusion, it is valuable to distinguish between C. gattii and C. neoformans. Ongoing basic science research is vital to identify potential new targets for both organisms.At the population/public health level, molecular characterization of the strains circulating in specific geographies can provide valuable information on virulence and impacts on at-risk groups. Although there is currently no evidence of differences in MICs impacting treatment outcome it is important that in vitro and in vivo studies of new antifungals include both Ensuring that diagnostic tools are effective against both strains is also crucial. Further research comparing IRIS manifestations and therapies between the two species would be valuable in reducing morbidity and mortality.C. neoformans as the causative pathogen. Consequently, our understanding of cryptococcosis in other settings remains poor, and we struggle to know how much can be directly translated. These gaps can only be filled by establishing large international collaborative networks designed to describe the protean manifestations of cryptococcosis in well-established and emerging niche host groups. These large networks would be fertile ground for investigating novel antifungal or adjunctive therapies, including proper assessment of pharmacokinetic-pharmacodynamic and drug-drug interactions in disparate groups.The vast majority of high-grade evidence on the management of cryptococcosis comes from HIV-associated cryptococcal meningitis trials, which are increasingly conducted in low- and middle-income countries with the highest disease burden and a predominance of


Background: Platelets (PLT) have a significant effect in promoting cancer progression and hematogenous metastasis. However, the effect of platelet activation-related lncRNAs (PLT-related lncRNAs) in gastric cancer (GC) is still poorly understood. In this study, we screened and validated PLT-related lncRNAs as potential biomarkers for prognosis and immunotherapy in GC patients.Methods: We obtained relevant datasets from the Cancer Genome Atlas (TCGA) and Gene Ontology (GO) Resource Database. Pearson correlation analysis was used to identify PLT-related lncRNAs. By using the univariate, least absolute shrinkage and selection operator (LASSO) Cox regression analyses, we constructed the PLT-related lncRNAs model. Kaplan-Meier survival analysis, univariate, multivariate Cox regression analysis, and nomogram were used to verify the model. The Gene Set Enrichment Analysis (GSEA), drug screening, tumor immune microenvironment analysis, epithelial-mesenchymal transition (EMT), and DNA methylation regulators correlation analysis were performed in the high- and low-risk groups. Patients were regrouped based on the risk model, and candidate compounds and immunotherapeutic responses aimed at GC subgroups were also identified. The expression of seven PLT-related lncRNAs was validated in clinical medical samples using quantitative reverse transcription-polymerase chain reaction (qRT-PCR).Results: In this study, a risk prediction model was established using seven PLT-related lncRNAs -, whose expression were validated in GC patients. Kaplan-Meier survival analysis, the receiver operating characteristic (ROC) curve analysis, univariate, multivariate Cox regression analysis verified the accuracy of the model. We screened multiple targeted drugs for the high-risk patients. Patients in the high-risk group had a poorer prognosis since low infiltration of immune killer cells, activation of immunosuppressive pathways, and poor response to immunotherapy. In addition, we revealed a close relationship between risk scores and EMT and DNA methylation regulators. The nomogram based on risk score suggested a good ability to predict prognosis and high clinical benefits.Conclusion: Our findings provide new insights into how PLT-related lncRNAs biomarkers affect prognosis and immunotherapy. Also, these lncRNAs may become potential biomarkers and therapeutic targets for GC patients. According to the most recent statistics from the American Cancer Society, the quantity of new cases and deaths cases of gastric cancer (GC) still remain a high level, and GC is the most common malignant tumor of the digestive system . Althougetc  regulates tumorigenesis and tumor progression, such as GC, prostate cancer, lung cancer, breast cancer, and colorectal cancer etc . For exaetc . In tumoetc . PLT-proetc . In addit tumors . All thiet al. suggested using circulating lncRNAs between tumor-educated platelets (TEPs) and serum can be used as a potential diagnostic and discriminative biomarkers for colorectal cancer (Long non-coding RNAs (lncRNAs) are about 200 nt or more non-coding protein RNAs, which significantly affect tumor immunity . Recentll cancer . Bioinfol cancer . Yet, thIn this study, we first extracted 14,087 lncRNAs expression matrix of GC patients from the Cancer Genome Atlas (TCGA) database, and ninety-four genes related to PLT activation were extracted from the Gene Ontology (GO) Resource Database. Then, bioinformatics analysis was performed to identify PLT-related lncRNAs using Pearson’s correlation analysis, after which prognostic risk models were established, and related signaling pathways were screened. Then, we screened for candidate drugs through the Connectivity Map (CMap) database. In addition, we explored the relationship between EMT markers, DNA methylation regulators, and immunotherapy responses and the risk model. Finally, we constructed a nomogram that can predict the overall survival (OS) of GC patients. The study workflow showed in A total of 417 cases  with clinical data and RNA sequencing dataset were downloaded from the Cancer Genome Atlas (TCGA). lncRNAs and mRNAs were classified by the Ensemble Human Genome browser GRCh38.p13. Ten gastric cancer and adjacent tissue specimens (specimen collection time: June 2021 to December 2021) were additionally collected from Jinan Central Hospital affiliated to Shandong First Medical University, which had been approved by the Ethics Committee of Jinan Central Hospital affiliated to Shandong First Medical University, and all patients had signed informed consent. http://geneontology.org/). For the purpose of evaluating the relationship between PLT activation genes and lncRNAs, Pearson correlation analysis was conducted with R software (R 4.2.1), and the intersection of lncRNA expression in GC patients with a correlation coefficient of 0.4 and p value <0.001 was obtained. A total of 848 lncRNAs associated with PLT activation and their co-expression networks were obtained using “limma” package  Resource database ( package  (Ritchie package .p < 0.05). Using “survival” package and pfilter = 0.05, univariate Cox analysis showed that 18 PLT-related lncRNAs were significantly correlated with OS  to obtain drugs that reduce risk in high-risk patients (p < 0.05 showed that drugs could be significantly enriched (https://pubchem.ncbi.nim.nih.gov/) was used to obtain the molecular structure of the effective drugs  . GC patihttp://tcia.at). Also, TIDE (http://tide.dfc-i.harvard.edu/) algorithm was used to assess the different responses to immune checkpoint inhibitors in high- and low-risk groups. When the TIDE score increased, tumors were more likely to have immune escape  in risk model we constructed. CIBERSORT was used to count the immune infiltration statuses of GC patients . Differee escape .A 5-methylcytosine (5mC) methylated regulator was used to assess the correlation between risk models and DNA methylation. Eleven methylated tuning nodes were obtained from the literature . EMT-assTotal RNA was extracted from 10 gastric cancer patients by TRIzon method. cDNA synthesis was then performed using reverse transcription reagents. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed using 2× SYBR Green HS Premix (AG) on Roche 480 instrument with β-actin as an internal reference. Gene expression levels were calculated using the 2-ΔΔCT method . Supplemp value <0.05 was considered to be statistically significant.R software 4.1.2 and GraphPad Prism 8 were used to analyze the data. The R software package “survival” and “survminer” were used for univariate Cox proportional risk regression analysis, multivariate Cox proportional risk regression analysis, and nomogram analysis. The Wilcoxon rank sum test or Kruskal–Wallis rank sum test was used to analyze the differences between the two groups; logarithmic rank testing was used to calculate the statistical difference of OS between high-and low-risk group. The R software package “glmnet” was used for lasso Cox proportional regression, and the R package “survival ROC” was used as the ROC curve . The p vp < 0.001). PLT-related genes and lncRNAs co-expression network is shown in p < 0.05). LASSO-penalized Cox analysis was then performed on the 18 lncRNAs, and vertical dashed lines were drawn at the optimal value when the order of Log(λ) was the least likely deviation for OS-related adjustment parameters, and seven lncRNAs related to the prognosis of PLT activation in GC were extracted  in GC patient tissues and corresponding adjacent tissues using qRT-PCR. We observed that the expression levels of AL355574.1, AC002401.4, LINC01094, and AL356417.2 were up-regulated in most GC tissues, while LINC01697 and AC129507.1 were down-regulated , which ip < 0.05). However, the survival analysis of the entire set showed the same results ; in the testing set, HR was 1.152,95% CI was 1.087–1.221 (p < 0.001) respectively; HR was 1.161, 95% CI was 1.092–1.234 (p < 0.001) in the testing set  and 95% confidence interval (CI) of the training set were 2.034 and 1.357–3.049 (87–1.221 . HR and ting set . For theting set . This rep = 0.0087) and immune score (p < 0.001), but not to age, gender, and TM stage, Stages (p < 0.05)  .https://portals.broadinstitute.org/cmap/). For enrichment scores, negative values indicate that the drugs can inhibit the expression of high-risk genes and improve the survival rate of patients. Positive values represent that it can promote the expression of high-risk genes (p < 0.05). All screened compounds could reduce the death risk in high-risk patients, and thus deserve further analysis in GC patients  and low-risk (C1) groups. Then, KEGG pathway enrichment analysis was performed with GSEA software. The pathways enriched by high- and low-risk genes were obtained, respectively. Pathways enriched in the C2 group mainly included complement and coagulation cascades, hematopoietic cell lineage, neuroactive ligand-receptor interaction, ECM receptor interaction, and other signaling pathways . The patet al. . Figure et al. .p < 0.05)  . Besides < 0.05) . Our stu < 0.05) . Further < 0.05) .p < 0.05), indicating that the risk model we constructed is correlated with DNA methylation. This result suggests that DNA methylation is one of the major biological characteristics of the high-risk group.DNA methylation and lncRNA regulation are generally considered to be important factors in cell differentiation and development . Some stPLT could produce TGFβ, a cytokine highly related to EMT, which has an extremely important role in EMT(13). Therefore, we tried to explore the correlation between the risk model and EMT markers. EMT-related genes came from EMTome. Ten genes  were selected for correlation analysis. We found that all the EMT-related genes we picked were significantly correlated with the risk model and the high-risk group patients had higher EMT gene expression . The resWe also used 1-year, 2-year, and 3-year calibration charts to prove that the nomogram was in good agreement with the prediction of 1-, 2-, and 3-year OS . NomograMore and more studies have been conducted on lncRNAs in recent years, which have an important role in cancer progression . Howeveret al. proposed a novel PLT-related gene signature as a practical tool for patients with triple-negative breast cancer (TNBC) with independent value in assessing clinical prognosis , providing guidance for the immunotherapy of patients with LUAD   were identified by LASSO regression analysis and used to construct the prognostic model for predicting OS in GC patients. AL355574.1 was identified as a protective lncRNA associated with autophagy in GC, which can be used as a promising therapeutic target for immunotherapy in GC patients . Zhang eGC cells . Moreovearcinoma . Zha andarcinoma . Sun et r (BRCA) . In addireatment . LINC010reatment . Li et apatients . In thiset al. showed that activation of the classical complement pathway promotes in situ tumor growth in mice , driven by DNA methylation, is a marker of PLT activation variability (DNA methylation modification is the most common covalent modification method. Many recent studies have confirmed the correlation between methylation modification and malignancy . 5mC is iability . Howeveriability . This stHowever, further experiments are needed to prove the effect of PLT-related lncRNAs on the prognosis of GC and related molecular mechanisms. The related signaling pathways screened out in this study and the effectiveness of immunotherapy drugs should be further investigated. In this study, we only analyzed and validated the data in the TCGA database. Although we have carried out some experimental verification using the collected specimens, there may still be deviations and deficiencies. Therefore, the risk model we constructed needs more external data for verification. We plan to collect more clinical samples to further validate the value of these lncRNAs in a future study.In this study, we constructed a model containing seven PLT-related lncRNAs. This study provides new clues for predicting the prognosis of GC patients and may help to elucidate the process and mechanism of PLT-related lncRNAs. In addition, small-molecule drugs were found to target PLT-related lncRNAs, and risk models showed sensitivity in distinguishing GC patients who benefited from immunotherapy. Our study further explored the role of PLT-related lncRNAs in TME, drug screening, and immunotherapy prediction in GC, providing new directions and therapeutic targets for further research and clinical practice.