Whole-transcriptome sequencing of phagocytes reveals a ceRNA network contributing to natural resistance to tuberculosis infection.

Tuberculosis (TB) is a major fatal infectious disease globally, exhibiting high morbidity rates and impacting public health and other socio-economic factors. However, some individuals are resistant to TB infection and are referred to as "Resisters". Resisters remain uninfected even after exposure to high load of Mycobacterium tuberculosis (Mtb). To delineate this further, this study aimed to investigate the factors and mechanisms influencing the Mtb resistance phenotype. We assayed the phagocytic capacity of peripheral blood mononuclear cells (PBMCs) collected from Resisters, patients with latent TB infection (LTBI), and patients with active TB (ATB), following infection with fluorescent Mycobacterium bovis Bacillus Calmette-Guérin (BCG). Phagocytosis was stronger in PBMCs from ATB patients, and comparable in LTBI patients and Resisters. Subsequently, phagocytes were isolated and subjected to whole transcriptome sequencing and small RNA sequencing to analyze transcriptional expression profiles and identify potential targets associated with the resistance phenotype. The results revealed that a total of 277 mRNAs, 589 long non-coding RNAs, 523 circular RNAs, and 35 microRNAs were differentially expressed in Resisters and LTBI patients. Further, the endogenous competitive RNA (ceRNA) network was constructed from differentially expressed genes after screening. Bioinformatics, statistical analysis, and quantitative real-time polymerase chain reaction were used for the identification and validation of potential crucial targets in the ceRNA network. As a result, we obtained a ceRNA network that contributes to the resistance phenotype. TCONS_00034796-F3, ENST00000629441-DDX43, hsa-ATAD3A_0003-CYP17A1, and XR_932996.2-CERS1 may be crucial association pairs for resistance to TB infection. Overall, this study demonstrated that the phagocytic capacity of PBMCs was not a determinant of the resistance phenotype and that some non-coding RNAs could be involved in the natural resistance to TB infection through a ceRNA mechanism.

Affinity maturation of an antibody for the UV-induced DNA lesions 6,4 pyrimidine-pyrimidones.

DNA lesions, associated mostly with minor changes in DNA structure, may induce permanent change in heritable coding information. Biochemically, these minor structural changes are difficult to be explored for generating high-affinity antibodies to detect specific DNA lesions in varying sequence contexts. Herein, we established a platform of bacterial display to facilitate antibodies to be matured with high affinity and high specificity against DNA lesions. To achieve this goal, we, for the first time, developed a two-round mutation/screening strategy: (1) using multiple lesion-containing DNA probes for primary maturation and (2) using single lesion-containing DNA probes for second maturation. Specifically, we capitalized on 64M-2 as a parental template to improve affinity for 6-4PP by 710-fold, compared with the model one. In addition, the matured antibody (9c3) is found to be much less dependent on the bases surrounding 6-4PPs than the model one. The mechanistic study from both computational simulation and reverse mutations revealed the critical roles of the two-round mutations in the enhanced binding affinity and independence of surrounding bases. This selection strategy opens a new way to improve affinity and specificity of antibodies for other DNA lesions.

In vitro evolution of diagnostic antibodies targeting native antigens in plasma by sandwich flow cytometry.

Monoclonal antibodies (mAbs) that recognize and bind to specific antigens (Ags) have a wide range of applications in research, therapy, and diagnostics. However, many of these antibodies cannot bind well to the native Ags. In this study, based on the Chinese hamster ovary (CHO) cell display platform developed previously in our lab, we reported a novel artificial evolution procedure to improve the affinity of mAb against the native Ag directly using the plasma samples without purification of the native Ag. In this procedure, a pair of antibodies able to bind the Ag in sandwich manner are first confirmed (Ab1/Ab2) and the antibody (Ab) to be affinity-improved (Ab1) is displayed on CHO cells for Ab mutation. Then the cells were detected and sorted with flow cytometry in the form of Ab1-Ag-fluorescence labeled Ab2, which we named sandwich flow cytometry. Here, we used soluble isoform of suppression of tumorigenicity 2 (sST2) protein as model Ag, carried out "sandwich" maturation directly using the plasma samples containing the native sST2 protein and optimized a pair of antibodies with significantly improved sensitivity in the detection of the native sST2 in plasma. This method could be very useful in optimization of the diagnostic Ab pairs working in a "sandwich" manner if more antibodies were also successfully affinity-matured with this method.

Identification of a Diagnostic Multiomics-Based Biomarker Cluster in a Mouse Model of Pulmonary Tuberculosis.

BACKGROUND: Tuberculosis (TB) stands as the second most prevalent infectious agent-related cause of death worldwide in 2022, trailing only COVID-19. With 1.13 million reported deaths, this figure is more than half of the mortality associated with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), which accounted for 0.63 million deaths. Diagnosing Mycobacterium tuberculosis (MTB) infection remains a formidable challenge due to the inability to isolate and detect MTB in sputum and within the human body. The absence of universally reliable diagnostic criteria for MTB infection globally poses a significant obstacle to preventing the progression of tuberculosis from the MTB infection stage. METHODS: In this study, our objective was to formulate a diagnostic biomarker cluster capable of discerning the progression of MTB infection and disease. This was achieved through a comprehensive joint multiomics analysis, encompassing transcriptome, proteome, and metabolome, conducted on lung tissue samples obtained from both normal control mice and those infected with MTB. RESULTS: A total of 1690 differentially expressed genes and 94 differentially expressed proteins were systematically screened. From this pool, 10 core genes were singled out. Additionally, eight long non-coding ribonucleic acids and eight metabolites linked to these core genes were identified to establish a cohesive cluster of biomarkers. This multiomics-based biomarker cluster demonstrated its capability to differentiate uninfected samples from MTB-infected samples effectively in both principle component analysis and the construction of a random forest model. CONCLUSION: The outcomes of our study strongly suggest that the multiomics-based biomarker cluster holds significant potential for enhancing the diagnosis of MTB infection.

The Humanization and Maturation of an Anti-PrPc Antibody.

The cellular prion protein (PrPc) is a cell surface glycoprotein that is highly expressed in a variety of cancer tissues in addition to the nervous system, and its elevated expression is correlated to poor prognosis in many cancer patients. Our team previously found that patients with colorectal cancer (CRC) with high-level PrPc expression had significantly poorer survival than those with no or low-level PrPc expression. Mouse antibodies for PrPc inhibited tumor initiation and liver metastasis of PrPc-positive human CRC cells in mouse model experiments. PrPc is a candidate target for CRC therapy. In this study, we newly cloned a mouse anti-PrPc antibody (Clone 6) and humanized it, then affinity-matured this antibody using a CHO cell display with a peptide antigen and full-length PrPc, respectively. We obtained two humanized antibody clones with affinities toward a full-length PrPc of about 10- and 100-fold of that of the original antibody. The two humanized antibodies bound to the PrPc displayed significantly better on the cell surface than Clone 6. Used for Western blotting and immunohistochemistry, the humanized antibody with the highest affinity is superior to the two most frequently used commercial antibodies (8H4 and 3F4). The two new antibodies have the potential to be developed as useful reagents for PrPc detection and even therapeutic antibodies targeting PrPc-positive cancers.

Genetic factors associated with acquired phenotypic drug resistance and its compensatory evolution during tuberculosis treatment.

OBJECTIVES: We elucidated the factors, evolution, and compensation of antimicrobial resistance (AMR) in Mycobacterium tuberculosis (MTB) isolates under dual pressure from the intra-host environment and anti-tuberculosis (anti-TB) drugs. METHODS: This retrospective case-control study included 337 patients with pulmonary tuberculosis from 15 clinics in Tianjin, China, with phenotypic drug susceptibility testing results available for at least two time points between January 1, 2009 and December 31, 2016. Patients in the case group exhibited acquired AMR to isoniazid (INH) or rifampicin (RIF), while those in the control group lacked acquired AMR. The whole-genome sequencing (WGS) was conducted on 149 serial longitudinal MTB isolates from 46 patients who acquired or reversed phenotypic INH/RIF-resistance during treatment. The genetic basis, associated factors, and intra-host evolution of acquired phenotypic INH/RIF-resistance were elucidated using a combined analysis. RESULTS: Anti-TB interruption duration of ≥30 days showed association with acquired phenotypic INH/RIF resistance (aOR = 2·2, 95% CI, 1·0-5·1) and new rpoB mutations (p = 0·024). The MTB evolution was 1·2 (95% CI, 1·02-1·38) single nucleotide polymorphisms per genome per year under dual pressure from the intra-host environment and anti-TB drugs. AMR-associated mutations occurred before phenotypic AMR appearance in cases with acquired phenotypic INH (10 of 16) and RIF (9 of 22) resistances. DISCUSSION: Compensatory evolution may promote the fixation of INH/RIF-resistance mutations and affect phenotypic AMR. The TB treatment should be adjusted based on gene sequencing results, especially in persistent culture positivity during treatment, which highlights the clinical importance of WGS in identifying reinfection and AMR acquisition before phenotypic drug susceptibility testing.

EspB and HtpG interact with the type III-A CRISPR/Cas system of Mycobacterium tuberculosis.

Introduction: Mycobacterium tuberculosis (MTB) has a type III-A clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system consisting of a Csm1-5 and CRISPR RNA (crRNA) complex involved in the defense against invading nucleic acids. However, CRISPR/Cas system in the MTB still is clearly unknown and needs to be further explored. Methods: In our work, two non-Cas system proteins EspB and HtpG protein were found and identified by LC-MS/MS. The effect of EspB and HtpG on Type III-A CRISPR/Cas System of M. tuberculosis was examined by using Plasmid interference assay and Co-immunoprecipitation analyses. We explored that EspB could interact with the crRNA RNP complex, but HtpG could inhibit the accumulation of the MTB Csm proteins and defense the mechanism of CRISPR/Cas system. Results: The proteins ESAT-6 secretion system-1(Esx-1) secreted protein B (EspB) and high-temperature protein G (HtpG), which were not previously associated with CRISPR/Cas systems, are involved in mycobacterial CRISPR/Cas systems with distinct functions. Conclusion: EspB is a novel crRNA-binding protein that interacts directly with the MTB crRNP complex. Meanwhile, HtpG influences the accumulation of MTB Csm proteins and EspB and interferes with the defense mechanism of the crRNP complex against foreign DNA in vivo. Thereby, our study not only leads to developing more precise clinical diagnostic tool to quickly detect for MTB infection, but also knows these proteins merits for TB biomarkers/vaccine candidates.

An Efficient Exogenous Gene Insertion Site in CHO Cells with High Transcription Level to Enhance AID-Induced Mutation.

Antibodies have been extensively used for the purpose of scientific research, clinical diagnosis, and therapy. Combination of in vitro somatic hypermutation and mammalian cell surface display has been an efficient technology for antibody or other proteins optimization, in which the efficiency of activation-induced cytidine deaminase (AID) mutations in genes is one of the most important key factors. Gene transcriptional level has been found to be positively proportional to AID-induced mutation frequency. Thus, construction of the cell clone bearing a gene of interest (GOI) with high transcription level can increase AID-induced mutations. In this study, a retargetable gene cassette is inserted onto predetermined chromosome site (ywhae gene site) which is among the genes with the highest as well as stable transcription, and is found that one subsite is suitable to be retargeted for efficient protein display in Chinese hamster ovary (CHO) cells. The resultant cell clone (T31) has higher and more stable transcription/expression than CHO-puro clone which was previously established through the strategy of random insertion followed by a high-throughput selection. It also possesses a significantly higher mutation frequency to GOI than CHO-puro cells; thus, it is a better clone for the in vitro improvement of antibody affinity, and probably other properties.

The Trends in Global Gene Expression in Mouse Embryonic Stem Cells During Spaceflight.

The environment in space differs greatly from the environment on the ground. Spaceflight causes a number of physiological changes in astronauts, such as bone loss and immune system dysregulation. These effects threaten astronauts' space missions, and understanding the underlying cellular and molecular mechanisms is important to manage the risks of space missions. The biological effects of spaceflight on mammalian cells, especially with regards to DNA damage, have attracted much attention. Rad9 -/- mouse embryonic stem cells (mESCs) are known to be extremely sensitive to DNA damage agents. In this study, a project of the SJ-10 satellite programme, we investigated the gene expression profiles of both Rad9 -/- mESCs and Rad9 +/+ (wild-type) mESCs in space with a focus on genes critical for inducing, preventing, or repairing genomic DNA lesions. We found that spaceflight downregulated more genes than it upregulated in both wild-type and Rad9 -/- mESCs, indicating a suppressive effect of spaceflight on global gene expression. In contrast, Rad9 deletion upregulated more genes than it downregulated. Of note, spaceflight mainly affected organ development and influenced a wide range of cellular functions in mESCs, while Rad9 deletion mainly affected the development and function of the hematological system, especially the development, differentiation and function of immune cells. The patterns of gene expression in mouse embryonic stem cells in space is distinct from those in other types of cells. In addition, both spaceflight and Rad9 deletion downregulated DNA repair genes, suggesting a possibility that spaceflight has negative effects on genome for embryonic stem cells and the effects are likely worsen when the genome maintenance mechanism is defective.

Simulated microgravity potentiates generation of reactive oxygen species in cells.

Microgravity (MG) and space radiation are two major environmental factors of space environment. Ionizing radiation generates reactive oxygen species (ROS) which plays a key role in radiation-induced DNA damage. Interestingly, simulated microgravity (SMG) also increases ROS production in various cell types. Thus, it is important to detect whether SMG could potentiate ROS production induced by genotoxins including radiation, especially at a minimal level not sufficient to induce detectable ROS. In this study, we treated mouse embryonic stem (MES) cells with H2O2 and SMG for 24 h. The concentration of H2O2 used was within 30 µmol/L at which intracellular ROS was the same as that in untreated cells. Exposure of cells to SMG for 24 h did not induce significantly higher levels of intracellular ROS than that of control cells either. Simultaneous exposure of cells to both SMG- and H2O2-induced ROS and apoptosis in MES cells. Although incubation in medium containing 5 or 30 µmol/L H2O2 induced a small enhancement of DNA double-strand breaks (DSBs), the addition of SMG treatment dramatically increased DSB levels. Taken together, SMG can significantly potentiate the effects of H2O2 at a low concentration that induce a small or negligible change in cells on ROS, apoptosis, and DNA damage. The results were discussed in relation to the combined effects of space radiation and MG on human body in this study.

The DmtA methyltransferase contributes to Aspergillus flavus conidiation, sclerotial production, aflatoxin biosynthesis and virulence.

DNA methylation is essential for epigenetic regulation of gene transcription and development in many animals, plants and fungi. We investigated whether DNA methylation plays a role in the development and secondary metabolism of Aspergillus flavus, identified the DmtA methyltransferase from A. flavus, and produced a dmtA knock-out mutant by replacing the dmtA coding sequence with the pyrG selectable marker. The A. flavus dmtA null mutant lines produced white fluffy mycelium in liquid medium, and displayed a slightly flavescent conidial pigmentation compared with the normal yellow of the wild-type strain when grown on agar. The ΔdmtA lines exhibited decreased conidiation and aflatoxin (AF) biosynthesis, compared with the wild-type line, suggesting that the DmtA knock-out affected the transcriptional level of genes in the AF cluster. In particular, sclerotia development and host colonization were altered in the dmtA null mutants. Green fluorescent protein tagging at the C-terminus of DmtA showed that DmtA localized to the nucleus and cytoplasm. DNA methylation content measurements in the dmtA mutants revealed no widespread DNA methylation in the mutants or wild-type lines. Thus, our findings suggest that DmtA, apart from being a C-5 cytosine methyltransferase in A. flavus, contributes to asexual development, aflatoxin biosynthesis, sclerotial production and virulence.

Inhibition of aflatoxin metabolism and growth of Aspergillus flavus in liquid culture by a DNA methylation inhibitor.

Aflatoxins (AFs) are a group of highly oxygenated polyketidese-derived toxins mainly produced by Aspergillus flavus and A. parasiticus, whose biosynthesis mechanisms are extremely sophisticated. Methylation is known as the major form of epigenetic regulation, which is correlated with gene expression. As the DNA methylation inhibitor 5-azacytidine (5-AC) blocks AF production, we studied AFB1 metabolism and morphological changes of A. flavus by treatment with 5-AC in liquid culture. The results show that 5-AC caused a decrease in AF production and concurrent changes in morphology. In addition, we isolated a non-aflatoxigenic mutant of A. flavus, showing a significant reduction in pigment production, after 5-AC treatment. This mutant showed significant reduction in the expression of genes in the AF biosynthesis pathway, and conidia formation. Furthermore, as AF biosynthesis and oxidative stress are intimately related events, we assessed the viability of A. flavus to oxidative stress after treatment with 5-AC, which showed that the mutant was more sensitive to the strong oxidant hydrogen peroxide. We found that the non-aflatoxigenic mutant showed a decrease in reactive oxygen species (ROS) and metabolites indicative of oxidative stress, which may be caused by the disruption of the defence system against excessive ROS formation after 5-AC treatment. These data indicate that 5-AC, as an inactivator of DNA methyltransferase, plays a very important role in AFB1 metabolism and the development of A. flavus, which might provide an effective strategy to pre- or post-harvest control of AFs.