Optimizing CAR-T cell Culture: Differential effects of IL-2, IL-12, and IL-21 on CAR-T cells.

BACKGROUND: Chimeric antigen receptor (CAR)-T therapy has demonstrated sustained clinical remission in numerous hematologic malignancies and has expanded to encompass solid tumors and autoimmune diseases. While progress is being made in establishing optimal culture conditions for CAR-T cells, the identification of the most effective cytokine for promoting their persistence in vitro remains elusive. METHODS: Here, we employed scRNA-seq (single-cell RNA sequencing) analysis to investigate the potential alterations in biological processes within CAR-T cells following exposure to cytokines (IL-2, IL-12, and IL-21) and antigens. Transcriptomic changes in diverse CAR-T groups were compared following various treatments, with a focus on epigenetic modifications, metabolic shifts, cellular senescence, and exhaustion. RESULTS: Our study reveals that CAR-T cells treated with antigen, IL-2, and IL-12 exhibit signs of exhaustion and senescence, whereas those treated with IL-21 do not display these characteristics. The activities of glycolysis and epigenetic changes were significantly increased by treatments with antigens, IL-2, and IL-12, while IL-21 treatment maintained the oxidative phosphorylation (OXPHOS) of CAR-T cells. CONCLUSIONS: Our findings suggest that IL-21 may play a role in preventing senescence and could be utilized in combination with other strategies, such as IL-2 and IL-12, for CAR-T culture.

Investigation of Interferences of Wearable Sensors with Plant Growth.

Plant wearable sensors have shown exceptional promise in continuously monitoring plant health. However, the potential adverse effects of these sensors on plant growth remain unclear. This study systematically quantifies wearable sensors' interference with plant growth using two ornamental species, Peperomia tetraphylla and Epipremnum aureum. We evaluated the impacts of four common disturbances-mechanical pressure, hindrance of gas exchange, hindrance of light acquisition, and mechanical constraint-on leaf growth. Our results indicated that the combination of light hindrance and mechanical constraint demonstrated the most significant interference. When the sensor weight was no greater than 0.6 g and the coverage was no greater than 5% of the leaf area, these four disturbances resulted in slight impacts on leaf growth. Additionally, we fabricated a minimally interfering wearable sensor capable of measuring the air temperature of the microclimate of the plant while maintaining plant growth. This research provides valuable insights into optimizing plant wearable sensors, balancing functionality with minimal plant interference.

Multi-omics analysis reveals a feedback loop amplifying immune responses in acute graft-versus-host disease due to imbalanced gut microbiota and bile acid metabolism.

Acute graft-versus-host disease (aGVHD) is primarily driven by allogeneic donor T cells associated with an altered composition of the host gut microbiome and its metabolites. The severity of aGVHD after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not solely determined by the host and donor characteristics; however, the underlying mechanisms remain unclear. Using single-cell RNA sequencing, we decoded the immune cell atlas of 12 patients who underwent allo-HSCT: six with aGVHD and six with non-aGVHD. We performed a fecal microbiota (16SrRNA sequencing) analysis to investigate the fecal bacterial composition of 82 patients: 30 with aGVHD and 52 with non-aGVHD. Fecal samples from these patients were analyzed for bile acid metabolism. Through multi-omic analysis, we identified a feedback loop involving "immune cell-gut microbes-bile acid metabolites" contributing to heightened immune responses in patients with aGVHD. The dysbiosis of the gut microbiota and disruption of bile acid metabolism contributed to an exaggerated interleukin-1 mediated immune response. Our findings suggest that resistin and defensins are crucial in mitigating against aGVHD. Therefore, a comprehensive multi-omic atlas incorporating immune cells, gut microbes, and bile acid metabolites was developed in this study and used to propose novel, non-immunosuppressive approaches to prevent aGVHD.

Dysregulated T-cell homeostasis and decreased CD30+ Treg proliferating in aplastic anemia.

Aplastic anemia (AA) is an autoimmune hematopoietic disease mediated by autoreactive T cells leading to bone marrow failure. However, the precise role of autoreactive T cells in the development of AA is not fully understood, hindering the advancement of therapeutic and diagnostic strategies. In this study, we conducted a single-cell transcriptome analysis of CD8+ T cells, conventional CD4+ T (CD4+ Tconv) cells, and Treg cells, to elucidate the potential disruption of T cell homeostasis in patients with AA. We identified changes in CD4+ Tconv cells, including loss of homeostasis in naïve and memory cells and increased differentiation potential in T helper type 1 (TH1), T helper type 2 (TH2), and T helper type 17 (TH17) cells. Additionally, we identified naïve and memory CD8+ T cells that were enforced into an effector state. CD127 is an ideal surface marker for assessing the immune state of CD8+ T cells，as identified by flow cytometry. Abnormal expression of TNFSF8 has been observed in AA and other autoimmune diseases. Flow cytometry analysis revealed that TNFRSF8 (CD30), a receptor for TNFSF8, was predominantly present in human Treg cells. Importantly, patients with AA have a decreased CD30+ Treg subset. RNA-sequencing analysis revealed, that the CD30+ Treg cells are characterized by high proliferation and a remarkable immunosuppressive phenotype. Taken, together, we propose that abnormal TNFSF8/TNFRSF8 signaling is involved in dysfunctional T cell immunity by increasing the destruction of CD30+ Treg cells.

Response of solar-induced chlorophyll fluorescence-based spatial and temporal evolution of vegetation in Xinjiang to multiscale drought.

Climate change and human activities have increased droughts, especially overgrazing and deforestation, which seriously threaten the balance of terrestrial ecosystems. The ecological carrying capacity and vegetation cover in the arid zone of Xinjiang, China, are generally low, necessitating research on vegetation response to drought in such arid regions. In this study, we analyzed the spatial and temporal characteristics of drought in Xinjiang from 2001 to 2020 and revealed the response mechanism of SIF to multi-timescale drought in different vegetation types using standardized precipitation evapotranspiration index (SPEI), solar-induced chlorophyll fluorescence (SIF), normalized difference vegetation index (NDVI), and enhanced vegetation index (EVI) data. We employed trend analysis, standardized anomaly index (SAI), Pearson correlation, and trend prediction techniques. Our investigation focused on the correlations between GOSIF (a new SIF product based on the Global Orbital Carbon Observatory-2), NDVI, and EVI with SPEI12 for different vegetation types over the past two decades. Additionally, we examined the sensitivities of vegetation GOSIF to various scales of SPEI in a typical drought year and predicted future drought trends in Xinjiang. The results revealed that the spatial distribution characteristics of GOSIF, normalized difference vegetation index (NDVI), and enhanced vegetation index (EVI) were consistent, with mean correlations with SPEI at 0.197, 0.156, and 0.128, respectively. GOSIF exhibited the strongest correlation with SPEI, reflecting the impact of drought stress on vegetation photosynthesis. Therefore, GOSIF proves advantageous for drought monitoring purposes. Most vegetation types showed a robust response of GOSIF to SPEI at a 9-month scale during a typical drought year, with grassland GOSIF being particularly sensitive to drought. Our trend predictions indicate a decreasing trend in GOSIF vegetation in Xinjiang, coupled with an increasing trend in drought. This study found that compared with that of the traditional greenness vegetation index, GOSIF has obvious advantages in monitoring drought in the arid zone of Xinjiang. Furthermore, it makes up for the lack of research on the mechanism of vegetation GOSIF response to drought on multiple timescales in the arid zone. These results provide strong theoretical support for investigating the monitoring, assessment, and prediction of vegetation response to drought in Xinjiang, which is vital for comprehending the mechanisms of carbon and water cycles in terrestrial ecosystems.

Origami-inspired highly stretchable and breathable 3D wearable sensors for in-situ and online monitoring of plant growth and microclimate.

The emerging wearable plant sensors demonstrate the capability of in-situ measurement of physiological and micro-environmental information of plants. However, the stretchability and breathability of current wearable plant sensors are restricted mainly due to their 2D planar structures, which interfere with plant growth and development. Here, origami-inspired 3D wearable sensors have been developed for plant growth and microclimate monitoring. Unlike 2D counterparts, the 3D sensors demonstrate theoretically infinitely high stretchability and breathability derived from the structure rather than the material. They are adjusted to 100% and 111.55 mg cm-2·h-1 in the optimized design. In addition to stretchability and breathability, the structural parameters are also used to control the strain distribution of the 3D sensors to enhance sensitivity and minimize interference. After integrating with corresponding sensing materials, electrodes, data acquisition and transmission circuits, and a mobile App, a miniaturized sensing system is produced with the capability of in-situ and online monitoring of plant elongation and microclimate. As a demonstration, the 3D sensors are worn on pumpkin leaves, which can accurately monitor the leaf elongation and microclimate with negligible hindrance to plant growth. Finally, the effects of the microclimate on the plant growth is resolved by analyzing the monitored data. This study would significantly promote the development of wearable plant sensors and their applications in the fields of plant phenomics, plant-environment interface, and smart agriculture.

Gut microbiota dynamics and fecal SCFAs after colonoscopy: accelerating microbiome stabilization by Clostridium butyricum.

BACKGROUND: Colonoscopy is a classic diagnostic method with possible complications including abdominal pain and diarrhoea. In this study, gut microbiota dynamics and related metabolic products during and after colonoscopy were explored to accelerate gut microbiome balance through probiotics. METHODS: The gut microbiota and fecal short-chain fatty acids (SCFAs) were analyzed in four healthy subjects before and after colonoscopy, along with seven individuals supplemented with Clostridium butyricum. We employed 16S rRNA sequencing and GC-MS to investigate these changes. We also conducted bioinformatic analysis to explore the buk gene, encoding butyrate kinase, across C. butyricum strains from the human gut. RESULTS: The gut microbiota and fecal short-chain fatty acids (SCFAs) of four healthy subjects were recovered on the 7th day after colonoscopy. We found that Clostridium and other bacteria might have efficient butyric acid production through bioinformatic analysis of the buk and assessment of the transcriptional level of the buk. Supplementation of seven healthy subjects with Clostridium butyricum after colonoscopy resulted in a quicker recovery and stabilization of gut microbiota and fecal SCFAs on the third day. CONCLUSION: We suggest that supplementation of Clostridium butyricum after colonoscopy should be considered in future routine clinical practice.

Effects of HMGB1/TLR4 on secretion IL-10 and VEGF in human jaw bone-marrow mesenchymal stem cells.

OBJECTIVE: We aimed to investigate the regulatory effects of HMGB1/TLR4 signaling pathway on the expression of IL-10 and VEGF in human bone marrow mesenchymal stem cells. METHODOLOGY: Human JBMSCs were isolated and cultured. Then, HMGB1 was added into the JBMSCs culture medium, and the protein and mRNA expression levels of IL-10 and VEGF were assessed. Moreover, cells were pretreated with a specific TLR4 inhibitor (TAK-242), and the expression changes of IL-10 and VEGF were compared. RESULTS: Compared with the control group, exposure to HMGB1 in human JBMSCs up-regulated TLR4, IL-10, and VEGF secretion at both protein and mRNA levels (P<0. 05). In addition, the increased expression of IL-10 and VEGF could be restrained in TAK-242 group compared with the HMGB1 group (P<0.05). CONCLUSIONS: The results indicated that HMGB1 activate TLR4 signaling pathway in Human JBMSCs, which plays a regulatory role in cytokines expression.

Unbiased Single-Cell Sequencing of Hematopoietic and Immune Cells from Aplastic Anemia Reveals the Contributors of Hematopoiesis Failure and Dysfunctional Immune Regulation.

Aplastic anemia (AA) is a bone marrow (BM) failure syndrome mediated by hyperactivated T-cells with heterogeneous pathogenic factors. The onset of BM failure cannot be accurately determined in humans; therefore, exact pathogenesis remains unclear. In this study, a cellular atlas and microenvironment interactions is established using unbiased single-cell RNA-seq, along with multi-omics analyses (mass cytometry, cytokine profiling, and oxidized fatty acid metabolomics). A new KIR+ CD8+ regulatory T cells (Treg) subset is identified in patients with AA that engages in immune homeostasis. Conventional CD4+ T-cells differentiate into highly differentiated T helper cells with type 2 cytokines (IL-4, IL-6, and IL-13), GM-SCF, and IL-1ß. Immunosuppressive homeostasis is impaired by enhanced apoptosis of activated Treg cells. Pathological Vδ1 cells dominated the main fraction of γδ T-cells. The B/plasma, erythroid, and myeloid lineages also exhibit substantial pathological features. Interactions between TNFSF12-TNFRSF12A, TNF-TNFRSF1A, and granzyme-gasdermin are associated with the cell death of hematopoietic stem/progenitor (HSPCs), Treg, and early erythroid cells. Ferroptosis, a major driver of HSPCs destruction, is identified in patients with AA. Furthermore, a case of twins with AA is reported to enhance the persuasiveness of the analysis. These results collectively constitute the cellular atlas and microenvironment interactions in patients with AA and provide novel insights into the development of new therapeutic opportunities.

Effects of HMGB1/TLR4 on secretion IL-10 and VEGF in human jaw bone-marrow mesenchymal stem cells

Abstract Objective: We aimed to investigate the regulatory effects of HMGB1/TLR4 signaling pathway on the expression of IL-10 and VEGF in human bone marrow mesenchymal stem cells. Methodology: Human JBMSCs were isolated and cultured. Then, HMGB1 was added into the JBMSCs culture medium, and the protein and mRNA expression levels of IL-10 and VEGF were assessed. Moreover, cells were pretreated with a specific TLR4 inhibitor (TAK-242), and the expression changes of IL-10 and VEGF were compared. Results: Compared with the control group, exposure to HMGB1 in human JBMSCs up-regulated TLR4, IL-10, and VEGF secretion at both protein and mRNA levels (P<0. 05). In addition, the increased expression of IL-10 and VEGF could be restrained in TAK-242 group compared with the HMGB1 group (P<0.05). Conclusions: The results indicated that HMGB1 activate TLR4 signaling pathway in Human JBMSCs, which plays a regulatory role in cytokines expression.

Cerebrospinal fluid metabolomic and proteomic characterization of neurologic post-acute sequelae of SARS-CoV-2 infection.

The mechanism by which SARS-CoV-2 causes neurological post-acute sequelae of SARS-CoV-2 (neuro-PASC) remains unclear. Herein, we conducted proteomic and metabolomic analyses of cerebrospinal fluid (CSF) samples from 21 neuro-PASC patients, 45 healthy volunteers, and 26 inflammatory neurological diseases patients. Our data showed 69 differentially expressed metabolites and six differentially expressed proteins between neuro-PASC patients and healthy individuals. Elevated sphinganine and ST1A1, sphingolipid metabolism disorder, and attenuated inflammatory responses may contribute to the occurrence of neuro-PASC, whereas decreased levels of 7,8-dihydropterin and activation of steroid hormone biosynthesis may play a role in the repair process. Additionally, a biomarker cohort consisting of sphinganine, 7,8-dihydroneopterin, and ST1A1 was preliminarily demonstrated to have high value in diagnosing neuro-PASC. In summary, our study represents the first attempt to integrate the diagnostic benefits of CSF with the methodological advantages of multi-omics, thereby offering valuable insights into the pathogenesis of neuro-PASC and facilitating the work of neuroscientists in disclosing different neurological dimensions associated with COVID-19.

Anti-Helicobacter pylori activity and gastroprotective effects of human stomach-derived Lactobacillus paragasseri strain LPG-9.

The eradication of Helicobacter pylori is an urgent global issue. However, the traditional regimens have several limitations. Thus, we propose the idea of treating bacterial gastric disease with the objective of eliminating gastric pathogenic bacteria and enhancing gastroprotective effects using gastric probiotics. In this study, a total of 12 Lactobacillus strains were isolated from the gastric mucosa of healthy donors. After evaluation using a weight scoring system, Lactobacillus paragasseri strain LPG-9 was identified as the most promising probiotic for gastric disease, with the highest acid-resistance and the best adhesion characteristics. Gastric colonisation, H. pylori inhibition, anti-inflammatory, and gastric homeostasis effects of LPG-9 were confirmed in C57BL/6 mice. Finally, a safety evaluation and whole-genome sequencing were performed. Based on the results of this study, LPG-9 originates from the gastric microbiota and is a promising probiotic for gastric disease, particularly H. pylori-induced gastritis, providing a solution to this global issue.

Targeted single-cell RNA sequencing analysis reveals metabolic reprogramming and the ferroptosis-resistant state in hematologic malignancies.

Hematologic malignancies are the most common hematopoietic diseases and a major public health concern. However, the mechanisms underlying myeloid tumors remain unknown owing to the intricate interplay between mutations and diverse clonal evolution patterns, as evidenced by the analysis of bulk cell-derived omics data. Several single-cell omics techniques have been used to characterize the hierarchies and altered immune microenvironments of hematologic malignancies. The comprehensive single-cell atlas of hematologic malignancies provides novel opportunities for personalized combinatorial targeted treatments, avoiding unwanted chemo-toxicity. In the present study, we performed transcriptome sequencing by combining single-cell RNA sequencing (scRNA-seq) with a targeted oncogenic gene panel for acute myeloid leukemia, overcoming the limitations of scRNA-seq in detecting oncogenic mutations. The distribution of oncogenic IDH1, IDH2, and KRAS mutations in each cell type was identified in the bone marrow (BM) samples of each patient. Our findings suggest that ferroptosis and metabolic reprogramming are involved in the tumorigenesis and chemotherapy resistance of oncogenic mutation-carrying cells. Biological progression via IDH1, IDH2, and KRAS mutations arrests hematopoietic maturation. Our study findings provide a rationale for using primary BM cells for personalized treatment in clinical settings.

Wearable Sensor: An Emerging Data Collection Tool for Plant Phenotyping.

The advancement of plant phenomics by using optical imaging-based phenotyping techniques has markedly improved breeding and crop management. However, there remains a challenge in increasing the spatial resolution and accuracy due to their noncontact measurement mode. Wearable sensors, an emerging data collection tool, present a promising solution to address these challenges. By using a contact measurement mode, wearable sensors enable in-situ monitoring of plant phenotypes and their surrounding environments. Although a few pioneering works have been reported in monitoring plant growth and microclimate, the utilization of wearable sensors in plant phenotyping has yet reach its full potential. This review aims to systematically examine the progress of wearable sensors in monitoring plant phenotypes and the environment from an interdisciplinary perspective, including materials science, signal communication, manufacturing technology, and plant physiology. Additionally, this review discusses the challenges and future directions of wearable sensors in the field of plant phenotyping.

[Design and Validation of Accuracy Assessment Tools and Algorithms for Optical Positioning System in Surgical Navigation].

The precision of optical positioning system is one of the most important factors which affects the precision of navigation guided surgery. In this study, an efficient and low-cost tool and its algorithm were proposed to evaluate the accuracy of optical positioning system based on the ablation scenario of liver cancer, and two validation experiments were designed. Experimental results show that the tool and its algorithm can evaluate the accuracy of the current positioning system accurately and efficiently.

Progress in Data Acquisition of Wearable Sensors.

Wearable sensors have demonstrated wide applications from medical treatment, health monitoring to real-time tracking, human-machine interface, smart home, and motion capture because of the capability of in situ and online monitoring. Data acquisition is extremely important for wearable sensors, including modules of probes, signal conditioning, and analog-to-digital conversion. However, signal conditioning, analog-to-digital conversion, and data transmission have received less attention than probes, especially flexible sensing materials, in research on wearable sensors. Here, as a supplement, this paper systematically reviews the recent progress of characteristics, applications, and optimizations of transistor amplifiers and typical filters in signal conditioning, and mainstream analog-to-digital conversion strategies. Moreover, possible research directions on the data acquisition of wearable sensors are discussed at the end of the paper.

Lactobacillus casei SYF-08 Protects Against Pb-Induced Injury in Young Mice by Regulating Bile Acid Metabolism and Increasing Pb Excretion.

Pb poisoning affects infant growth and development. However, dimercaptosuccinic acid (DMSA) as the current therapy for Pb poisoning exerts relatively significant toxic side effects in infants. Therefore, identifying a non-toxic treatment in this regard is particularly important. In this study, we aimed to investigate the therapeutic effect of an infant feces-derived probiotic strain, Lactobacillus casei SYF-08 (SYF-08), on Pb poisoning in young mice. The Pb levels in the organisms were detected via inductively coupled plasma mass spectrometry, while the therapeutic effect of SYF-08 on Pb-induced neural system damage was explored via the Morris water maze test, hematoxylin-eosin staining, and immunohistochemistry. Additionally, the molecular mechanisms underlying the protective effects of SYF-08 against Pb-induced intestinal damage were also explored via histological staining, 16S rRNA sequencing, untargeted metabolomics, qRT-PCR, and western blotting. In vivo experiments revealed that SYF-08 reduced blood and bone Pb levels and increased urinary Pb excretion. Additionally, SYF-08 alleviated Pb-induced pathological damage to the brain and ultimately improved the learning and cognitive abilities of the young mice. This treatment also restored intestinal microflora dysbiosis, regulated bile acid metabolism, and inhibited the FXR-NLRP3 signaling pathway. It also resulted in fewer adverse events than the DMSA treatment. In conclusion, our results provided valuable insights into the therapeutic role of SYF-08 in Pb poisoning and also suggested that its administration can significantly alleviate the Pb-induced damage.

The Effect of Acupuncture Combined with Aerobic Exercise for Coronary Heart Disease as Cardiac Rehabilitation.

Background: The mortality of coronary heart disease continues to rise. Cardiac rehabilitation intervenes the risk factors of cardiovascular disease, improves cardiopulmonary function, maintains healthy psychology, improves the quality of life of patients, and reduces cardiovascular mortality. Objective: To explore the effect of acupuncture combined with aerobic exercise on cardiopulmonary exercise ability, blood lipid, fatty acid oxidation, and psychology in patients with coronary heart disease. Methods: Sixty patients with coronary heart disease from February 2018 to October 2020 were randomly divided into two groups: the control group and experimental group. The control group was given an exercise prescription, and the experimental group was given acupuncture combined with an exercise prescription. Before and after the intervention, the cardiopulmonary exercise test, blood lipid, carnitine acyltransferase (CACT), the Self-Rating Somatic Symptom Scale (SSS), the Generalized Anxiety Disorder-7 (GAD-7), and the Patient Health Questionnaire-9 (PHQ-9) of the two groups were compared. Results: The PHQ-9 score was better in the experimental group than in the control group. In both groups, after the intervention, the peak oxygen uptake and anaerobic threshold were increased, and blood lipid and PHQ-9 scores were decreased. In the experimental group, the carbon dioxide metabolic equivalent was decreased, CACT was increased, and SSS and GAD-7 scores were decreased, with statistical difference (P < 0.05). Conclusion: Acupuncture combined with aerobic exercise can improve the cardiopulmonary exercise ability, increase fatty acid oxidation, decrease blood lipid, and ameliorate anxiety and depression symptoms of patients with coronary heart disease as cardiac rehabilitation.

Role of ultramicropores in the remarkable gas storage in hypercrosslinked polystyrene networks studied by positron annihilation.

In this paper, hypercrosslinked polystyrene (HCLPS) networks were synthesized by radical bulk polymerization and Friedel-Crafts alkylation reactions using vinylbenzyl-co-divinylbenzene chloride (VBC-DVB) as the precursors. A series of HCLPS was prepared with varying content of DVB from 0 to 10% in the precursor. Both N2 adsorption and positron annihilation measurements reveal micropores in the HCLPS. Especially, the existence of ultramicropores with a size in the range of 0.63-0.7 nm is confirmed by positron lifetime measurements. With increasing DVB content from 0 to 10%, the number of ultramicropores shows a gradual increase. Both the H2 and CO2 adsorption capacity increase monotonously with the increase of the DVB content. With 10% DVB in the HCLPS, the H2 storage increases to 10.3 mmol g-1 (2.05 wt%) at 77 K and 1 bar and the CO2 capture reaches 2.81 mmol g-1 (12.4 wt%) at 273 K and 1 bar. The remarkable gas storage ability is ascribed to the existence of the ultramicropores, which result in a stronger affinity to the gas molecules. By using positrons as a new probe for the pores, our results provide convincing evidence of the role of ultramicropores in the gas adsorption performance in microporous organic polymers.

Bioinformatic identification of key pathways, hub genes, and microbiota for therapeutic intervention in Helicobacter pylori infection.

The pathogenic mechanisms of Helicobacter pylori infection remain to be defined, and potential interventional microbiota are just beginning to be identified. In this study, gene-set enrichment analysis (GSEA) was used to integrate three H. pylori infection microarray data sets from the gene expression omnibus database and identified ten hallmark gene sets and 35 Kyoto encyclopedia of genes and genomes (KEGG) pathways that differed between healthy and Helicobacter pylori-infected individuals. Weighted gene co-expression network analysis (WGCNA) performed on two of the data sets identified three key gene coexpression modules. These modules contained 54 enriched KEGG pathways, 25 of which overlapped with the GSEA analysis, suggesting potentially important roles in H. pylori-infection. We selected 116 hub genes from the three key modules for in vitro validation at the transcriptional level using H. pylori Sydney Strain 1 and verified the upregulation of 80. WGCNA of the microbiomes based on 20 mucosal samples and a sequence read archive data set revealed four microbiota modules correlated with H. pylori infection. The negatively correlated modules contained 11 microbiome families. These findings provide new insight into the pathogenesis of H. pylori infection and systematically identify 25 key pathways, 80 upregulated hub genes, and 11 families of candidate interventional microbiota for further research.