DHX9 maintains epithelial homeostasis by restraining R-loop-mediated genomic instability in intestinal stem cells.

Epithelial barrier dysfunction and crypt destruction are hallmarks of inflammatory bowel disease (IBD). Intestinal stem cells (ISCs) residing in the crypts play a crucial role in the continuous self-renewal and rapid recovery of intestinal epithelial cells (IECs). However, how ISCs are dysregulated in IBD remains poorly understood. Here, we observe reduced DHX9 protein levels in IBD patients, and mice with conditional DHX9 depletion in the intestinal epithelium (Dhx9ΔIEC) exhibit an increased susceptibility to experimental colitis. Notably, Dhx9ΔIEC mice display a significant reduction in the numbers of ISCs and Paneth cells. Further investigation using ISC-specific or Paneth cell-specific Dhx9-deficient mice demonstrates the involvement of ISC-expressed DHX9 in maintaining epithelial homeostasis. Mechanistically, DHX9 deficiency leads to abnormal R-loop accumulation, resulting in genomic instability and the cGAS-STING-mediated inflammatory response, which together impair ISC function and contribute to the pathogenesis of IBD. Collectively, our findings highlight R-loop-mediated genomic instability in ISCs as a risk factor in IBD.

Research progress of Ustekinumab in the treatment of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic, recurrent gastrointestinal disorder with elusive etiology. Interleukin-12 (IL-12) and IL-23 have emerged as key proinflammatory mediators/cytokines in IBD pathogenesis. Ustekinumab (UST), targeting IL-12 and IL-23, has demonstrated promising efficacy and safety in the treatment of IBD. Recently, UST has become increasingly favored as a potential first-line treatment option. This review delineates UST's mechanism of action, its clinical applications in IBD, including the response rates, strategies for dose optimization for case of partial or lost response, and potential adverse events. This review aims to offer a comprehensive understanding of UST's role as a therapeutic option in IBD management.

Ligustilide enhances pregnancy outcomes via improvement of endometrial receptivity and promotion of endometrial angiogenesis in rats.

Ligustilide (LIG) is the main active ingredient of Angelica sinensis (Oliv.) Diels, which could promote focal angiogenesis to exert neuroprotection. However, there was no report that verified the exact effects of LIG on endometrial angiogenesis and the pregnancy outcomes. To explore the effects of LIG on low endometrial receptivity (LER) and angiogenesis, pregnancy rats were assigned into Control (saline treatment), LER (hydroxyurea-adrenaline treatment), LIG 20 mg/kg and LIG 40 mg/kg groups. Hematoxylin and eosin (H&E) staining was performed to evaluate endometrial morphology. Quantitative real-time PCR, immunofluorescence staining, western blot and immunohistochemistry staining were employed to assess the expression of endometrial receptivity factors and angiogenesis-related gene/protein, respectively. RNA sequencing was used to analyze the effects of LIG on LER caused by Kidney deficiency and blood stasis. We found that endometrial thickness and the implanted embryo number were substantially reduced in the hydroxyurea-adrenaline-treated pregnancy rats. At the same time, the gene and protein expressions of ERα, LIF, VEGFA and CD31 in the endometrium were markedly reduced, while the expressions of MUC1, E-cadherin were increased in the LER group. Administration of LIG raised the endometrial thickness and implanted embryos, as well as reversed the expressions of these factors. Collectively, our findings revealed that LIG could facilitate embryo implantation via recovery of the endometrium receptivity and promotion of endometrial angiogenesis.

ß-Galactosidase-Activatable Fluorescent and Photoacoustic Imaging of Tumor Senescence.

ß-Galactosidase (ß-gal) is the gold standard marker of cellular senescence, which is linked with various age-related diseases. Therefore, it is essential to develop more excellent probes that can real-time monitor ß-gal activity in cellular senescence in vivo. Fluorescent/photoacoustic (FL/PA) dual-modal imaging possesses excellent sensitivity and spatial resolution. To our knowledge, there has been no tumor-targeted FL/PA probe to image cellular senescence by monitoring the activity of ß-gal in vivo. Therefore, we developed a tumor-targeted FL/PA probe (Gal-HCy-Biotin) for ß-gal-activatable imaging of tumor senescence. Gal-HCy without tumor-targeted biotin is used as a control probe. Gal-HCy-Biotin is superior to Gal-HCy due to the higher kinetic parameter of Gal-HCy-Biotin than Gal-HCy in vitro. Moreover, biotin could help Gal-HCy-Biotin enter and accumulate in tumor cells with higher FL/PA signal. In detail, Gal-HCy-Biotin or Gal-HCy could image senescent tumor cells with 4.6-fold or 3.5-fold FL enhancement and 4.1-fold or 3.3-fold PA enhancement. Gal-HCy-Biotin or Gal-HCy could image tumor senescence with 2.9-fold or 1.7-fold FL enhancement and 3.8-fold or 1.3-fold PA enhancement. We envision that Gal-HCy-Biotin will be applied for FL/PA imaging of tumor senescence in clinic.

Nucleic DHX9 cooperates with STAT1 to transcribe interferon-stimulated genes.

RNA helicase DHX9 has been extensively characterized as a transcriptional regulator, which is consistent with its mostly nucleic localization. It is also involved in recognizing RNA viruses in the cytoplasm. However, there is no in vivo data to support the antiviral role of DHX9; meanwhile, as a nuclear protein, if and how nucleic DHX9 promotes antiviral immunity remains largely unknown. Here, we generated myeloid-specific and hepatocyte-specific DHX9 knockout mice and confirmed that DHX9 is crucial for host resistance to RNA virus infections in vivo. By additional knockout MAVS or STAT1 in DHX9-deficient mice, we demonstrated that nucleic DHX9 plays a positive role in regulating interferon-stimulated gene (ISG) expression downstream of type I interferon. Mechanistically, upon interferon stimulation, DHX9 is directly bound to STAT1 and recruits Pol II to the ISG promoter region to participate in STAT1-mediated transcription of ISGs. Collectively, these findings uncover an important role for nucleic DHX9 in antiviral immunity.

Caspase-1-responsive fluorescence biosensors for monitoring endogenous inflammasome activation.

The activation of inflammasome leads to secretion of inflammatory factors and cell pyroptosis that are critical in the pathogenesis of various chronic and acute inflammatory diseases. Recruitment and activation of caspase-1 is a marker of inflammasome activation. However, there is still lack of real-time and efficient methods to detect the activation of inflammasome, especially in vivo. Herein, we developed two activatable caspase-1-responsive fluorescence biosensors, WEHD-HCy and YVAD-HCy, to specifically monitor the activation of inflammasome in vivo. Our in vitro study demonstrated that WEHD-HCy and YVAD-HCy can sensitively and specifically respond to caspase-1 activation. Moreover, these biosensors can efficiency and specifically activated in the common inflammatory disease model, including inflammatory bowel disease, Salmonella infection, and acute arthritis. In particular, WEHD-HCy is more advantageous than YVAD-HCy to specifically image of caspase-1 activity both in vitro and in vivo. These caspase-1-responsive fluorescence biosensors provide an efficient, rapid, and in situ tool for monitoring inflammasome activation, and have the potential to be suitable for clinical diagnosis of various inflammatory diseases associated with inflammasome activation.

Intracellular Formation of Hemicyanine Nanoparticle Enhances Tumor-Targeting Photoacoustic Imaging and Photothermal Therapy.

Alkaline phosphatase (ALP) is a tumor marker for early diagnosis and treatment. Tumor targeting can recognize and fight tumor cells more accurately from healthy cells. Glycyrrhetinic acid (GA) is a targeting ligand of liver tumors. Photoacoustic imaging (PAI) and photothermal therapy (PTT) are promising techniques for tumor diagnosis and treatment. The outstanding characteristics of Hemicyanine (HCy) dye make it suitable for tumor diagnosis and treatment. However, using HCy nanoparticle (HCy NP) for liver tumor-targeting PAI and PTT has not been reported. Herein, Probe-1 is developed to enhance PAI and PTT of liver tumors due to GA targeting and intracellular ALP-instructed self-assembly of HCy NP. Compared to Probe-2 without self-assembly ability, Probe-1 displays a 4.6-fold higher PAI signal or 1.7-fold lower half inhibitory concentrations in HepG2 cells. Moreover, Probe-1 shows extended retention time (10 vs 6 h) and 2.1-fold higher PAI signal than Probe-2 in HepG2 tumors. The HepG2 tumors in Group Probe-1 obviously increase 18 °C (Tmax : 55 °C) with a 3.3-fold decreased volume while that in Group Probe-2 mildly increase 9.8 °C (Tmax : 46.8 °C) with a 4.3-fold increased volume. It is envisioned that this smart self-assembly strategy can be easily adjusted for PAI and PTT of more tumors.

High resolution intravital photoacoustic microscopy reveals VEGF-induced bone regeneration in mouse tibia.

Osteogenesis and angiogenesis are essential for bone homeostasis and repair. Newly formed vessels convey osteogenic progenitors during bone regeneration. However, the lack of continuous and label-free visualization of the bone microvasculature has resulted in little understanding of the neovascular dynamics. Here, we take advantage of optical-resolution photoacoustic microscopy (ORPAM) for label-free, intravital, long-term observation of the bone vascular dynamics, including angiogenesis, remodeling and quantified angiogenic effect of locally-applied vascular endothelial growth factor (VEGF) in the murine tibial defect model. We employed ex vivo confocal microscopy and micro-computed tomography (micro-CT) imaging to verify the positive role of VEGF treatment. VEGF treatment increased the concentration of total hemoglobin, vascular branching, and vascular density, which correlated with more osteoprogenitors and increased bone formation within the defect. These data demonstrated ORPAM as a useful imaging tool that detected functional capillaries to understand hemodynamics, and revealed the effectiveness of locally delivered therapeutic agents with sufficient sensitivity, contributing to the understanding of spatiotemporal regulatory mechanisms on blood vessels during bone regeneration.

Left atrial strain predicts risk and prognosis in patients with acute coronary syndrome: A retrospective study with external validation.

Objectives: To explore the association between left atrial (LA) strain and the GRACE score in patients with acute coronary syndrome (ACS) and to investigate the utility of LA function in predicting short-term adverse cardiovascular events post ACS. Methods: This retrospective study included ACS patients who underwent coronary angiography (CAG) in two independent cohorts from October 2020 to July 2022. The patients were classified into low-intermediate risk group and high-risk group based on the GRACE score. All participants underwent a transthoracic echocardiogram, with LA strain analysis before CAG. Correlation analysis was used to determine the relationship between LA strain and the GRACE score. The predictive value of LA strain was examined utilizing the area under the curve (AUC). Participants were followed for 10.5 ± 2.9 months for the primary endpoint of major adverse cardiovascular events (MACE). Results: A total of 229 patients were included in this study, including 196 in the primary group and 33 in the validation group. Spearman's correlation analysis showed there was a moderate negative correlation between the GRACE and left atrial reservoir strain (LASr) in both the primary (r = -0.63, P < 0.001) and validation (r = -0.73, P < 0.001) cohorts. Receiver operator characteristic (ROC) curve analysis showed that the AUC of LASr for prediction of the high-risk group was 0.86. Taking LASr 19.6% as the cut-off value, the sensitivity and specificity were 0.71 and 0.92, respectively. The cut-off value of 19.6% remains good at identifying high-risk group in the validation group (AUC = 0.87, sensitivity: 77.8%, specificity: 95.8%). Furthermore, 49 patients reached the endpoint in the primary cohort during the follow-up. On multivariable regression analysis, LASr (P = 0.03) was the independent echocardiographic predictor for the primary endpoint, rather than left atrial volume index (LAVI). Conclusions: LASr can identify high-risk patients with ACS as defined by the GRACE score and may be superior to Max LAVI in predicting incidents of MACE in the short-term following ACS.

Sequential self-assembly and disassembly of curcumin hydrogel effectively alleviates inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disorder of the gastrointestinal tract with unclear etiology and insufficient therapeutic efficacy. The development of specific, effective and safe IBD treatment drugs is of great clinical significance. Curcumin (Cur) is a good candidate to prevent and manage inflammatory diseases (such as IBD) due to its antioxidant and anti-inflammatory effects with safety profile. However, its poor aqueous solubility and instability under physiological conditions greatly limit its therapeutic efficacy. Herein, we exploited a Cur precursor Cur-FFEYp to locally deliver and slowly release Cur at inflamed regions for treatment of IBD by a sequential self-assembly and disassembly strategy. The much higher catalytic efficiency of alkaline phosphatase (ALP) than esterase towards Cur-FFEYp validated the sequential ALP-induced self-assembly with the formation of Cur hydrogel and esterase-guided disassembly with the slow release of Cur. In cell and animal experiments, Cur-FFEYp can effectively enhance the anti-inflammatory effect of Cur on inflammatory macrophages and significantly alleviate two types of IBD. We envision that by using other biomarkers to conduct the sequential self-assembly and disassembly processes and replacing other drugs, our smart strategy could be easily adjusted for the treatment of more diseases or cancers.

Establishment and Validation of a Predictive Model for Long-Term Severe Functional Tricuspid Regurgitation after Mitral Valve Replacement.

BACKGROUND: The objective was to develop and validate an individualized nomogram to predict severe functional tricuspid regurgitation (S-FTR) after mitral valve replacement (MVR) via retrospective analysis of rheumatic heart disease (RHD) patients' pre-clinical characteristics. METHODS: Between 2001-2015, 442 MVR patients of RHD were examined. Transthoracic echocardiography detected S-FTR, and logistic regression model analyzed its independent predictors. R software established a nomogram prediction model, and Bootstrap determined its theoretical probability, which subsequently was compared with the actual patient probability to calculate the area under the curve (AUC) and calibration plots. Decision curve analysis (DCA) identified its clinical utility. RESULTS: Ninety-six patients developed S-FTR during the follow-up period. Both uni- and multivariate analyses found significant correlations between S-FTR occurrence with gender, age, atrial fibrillation (AF), pulmonary arterial hypertension (PH), left atrial diameter (LAD), and tricuspid regurgitation area (TRA). The individualized nomogram model had the AUC of 0.99 in internal verification. Calibration test indicated high agreement of predicted and actual S-FTR onset. DCA also showed that utilization of those six aforementioned factors was clinically useful. CONCLUSION: The nomogram for the patient characteristics of age, gender, AF, PH, LAD, and TRA found that they were highly predictive for future S-FTR onset within 5 years. This predictive ability therefore allows clinicians to optimize postoperative patient care and avoid unnecessary tricuspid valve surgeries.

Combined insertion of basic and non-basic amino acids at hemagglutinin cleavage site of highly pathogenic H7N9 virus promotes replication and pathogenicity in chickens and mice.

Since mid-2016, the low pathogenic H7N9 influenza virus has evolved into a highly pathogenic (HP) phenotype in China, raising many concerns about public health and poultry industry. The insertion of a "KRTA" motif at hemagglutinin cleavage site (HACS) occurred in the early stage of HP H7N9 variants. During the co-circulation, the HACS of HP-H7N9 variants were more polymorphic in birds and humans. Although HP-H7N9 variants, unlike the H5 subtype virus, exhibited the insertions of basic and non-basic amino acids, the underlying function of those insertions and substitutions remains unclear. The results of bioinformatics analysis indicated that the PEVPKRKRTAR/G motif of HACS had become the dominant motif in China. Then, we generated six H7N9 viruses bearing the PEIPKGR/G, PEVPKGR/G, PEVPKRKRTAR/G, PEVPKGKRTAR/G, PEVPKGKRIAR/G, and PEVPKRKRR/G motifs. Interestingly, after the deletion of threonine and alanine (TA) at HACS, the H7N9 viruses manifested decreased thermostability and virulence in mice, and the PEVPKRKRTAR/G-motif virus is prevalent in birds and humans probably due to its increased transmissibility and moderate virulence. By contrast, the insertion of non-basic amino acid isoleucine and alanine (IA) decreased the transmissibility in chickens and virulence in mice. Remarkably, the I335V substitution of H7N9 virus enhanced infectivity and transmission in chickens, suggesting that the combination of mutations and insertions of amino acids at the HACS promoted replication and pathogenicity in chickens and mice. The ongoing evolution of H7N9 increasingly threatens public health and poultry industry, so, its comprehensive surveillance and prevention of H7N9 viruses should be pursued.

Tollip Orchestrates Macrophage Polarization to Alleviate Intestinal Mucosal Inflammation.

BACKGROUND AND AIMS: Regulation of macrophage polarization is a promising strategy for treating inflammatory bowel disease [IBD]. Tollip is an important negative regulator of Toll-like receptor [TLR]-mediated innate immunity with downregulated expression in the colon tissues of patients with IBD. This study aimed to regulate the expression of Tollip to affect macrophage polarization. METHODS: A molecular, targeted immunotherapy method was developed by linking mannose-modified trimethyl chitosan [MTC] with Tollip-expressing plasmids via ionic cross-linking, forming MTC-Tollip nanoparticles with a targeting function. MTC-Tollip selectively targeted mouse intestinal macrophages to regulate the polarization of macrophages for mucosal repair. RESULTS: Orally administered MTC-Tollip significantly elevated Tollip expression in intestinal tissue. Compared with MTC-negative control [NC]-treated mice in which colitis was induced with dextran sodium sulphate [DSS], the MTC-Tollip nanoparticle-treated mice exhibited decreased body weight loss and colon shortening, lower proinflammatory cytokine expression in colon tissues, and greater mucosal barrier integrity. MTC-Tollip treatment decreased TNF-α and iNOS expression but increased CD206 and Arg-1 expression in colon tissue. Tollip overexpression in mouse peritoneal macrophages inhibited lipopolysaccharide [LPS]-induced proinflammatory cytokine production and promoted IL-4-induced M2 expression. The progression of peritoneal macrophages extracted from Tollip-/- mice confirmed the effect of Tollip on macrophage polarization. Western blots showed that Tollip overexpression attenuated the upregulation of TLR pathway-associated targets in M1 macrophages. CONCLUSIONS: MTC nanoparticles can be 'intelligent' carriers in immunotherapy. The modulation of Tollip expression in macrophages may be a novel treatment approach for IBD.

Effects of multi-factors on the spatiotemporal variations of deep confined groundwater in coal mining regions, North China.

Natural processes and anthropogenic activities simultaneously control the long-term spatial and temporal variations of groundwater hydrogeochemistry in coalfields. In this study, the spatiotemporal variations and primary controlling factors of deep groundwater hydrogeochemistry in the Carboniferous limestone aquifer of the Huaibei coalfield, North China were investigated using cluster analysis combined with geological conditions, water-rock interactions and mining activities. The analysis data of 176 groundwater samples collected over five years from 20 monitoring wells were subdivided into six clusters through hierarchical cluster analysis. Moreover, principal component analysis, box plots and Piper and Stiff diagrams were employed to analyze the statistical and hydrogeochemical characteristics of each cluster, and to reveal the differences and connections between the clusters. The results show that there are significantly spatial variations in groundwater hydrogeochemistry, while the temporal variations are not evident with only a few notable exceptions. Geological conditions dominate the groundwater hydrogeochemistry by controlling the hydraulic connection between groundwater and meteoric water and the flow conditions of groundwater. Moreover, the types and degrees of diverse water-rock interactions in different regions are another important factor controlling the spatial variations of groundwater hydrogeochemistry. Anthropogenic activities are mainly pumping and drainage, which has led to the overall decline in groundwater levels and the temporal variations of hydrogeochemistry in some zones. The findings of this study not only have important implications for deep groundwater resources management in the Huaibei coalfield, but also provide a research template for other highly exploited coalfields in North China.

m6A modifications regulate intestinal immunity and rotavirus infection.

N6-methyladenosine (m6A) is an abundant mRNA modification and affects many biological processes. However, how m6A levels are regulated during physiological or pathological processes such as virus infections, and the in vivo function of m6A in the intestinal immune defense against virus infections are largely unknown. Here, we uncover a novel antiviral function of m6A modification during rotavirus (RV) infection in small bowel intestinal epithelial cells (IECs). We found that rotavirus infection induced global m6A modifications on mRNA transcripts by down-regulating the m6a eraser ALKBH5. Mice lacking the m6A writer enzymes METTL3 in IECs (Mettl3ΔIEC) were resistant to RV infection and showed increased expression of interferons (IFNs) and IFN-stimulated genes (ISGs). Using RNA-sequencing and m6A RNA immuno-precipitation (RIP)-sequencing, we identified IRF7, a master regulator of IFN responses, as one of the primary m6A targets during virus infection. In the absence of METTL3, IECs showed increased Irf7 mRNA stability and enhanced type I and III IFN expression. Deficiency in IRF7 attenuated the elevated expression of IFNs and ISGs and restored susceptibility to RV infection in Mettl3ΔIEC mice. Moreover, the global m6A modification on mRNA transcripts declined with age in mice, with a significant drop from 2 weeks to 3 weeks post birth, which likely has broad implications for the development of intestinal immune system against enteric viruses early in life. Collectively, we demonstrated a novel host m6A-IRF7-IFN antiviral signaling cascade that restricts rotavirus infection in vivo.

The PB2 co-adaptation of H10N8 avian influenza virus increases the pathogenicity to chickens and mice.

Avian influenza (AI) is an important zoonotic disease, which can be transmitted across species barriers to other hosts, especially humans, posing a serious threat to the poultry industry and public health. In recent years, human cases infected with the H10N8, H9N2, and H7N9 of avian influenza viruses (AIVs) have been identified frequently as have the internal genes of H7N9 and H10N8, which are derived from H9N2 viruses. The adaptive mutation of the PB2 gene is an important way for the H10N8, H9N2, and H7N9 AIVs to spread across species to adapt to new hosts. Several well-known adaptive mutations in the PB2 gene, such as E627K, D701N, and A588V, significantly enhanced the virulence of the AIVs in mammals. However, the co-adaptation of AIVs to avian and mammalian hosts is rarely studied. In this study, we found that the mutations of PB2-I292V, PB2-R389K, PB2-A588V, PB2-T598M/V, PB2-L648V, and PB2-T676M substitutions significantly increased after 2012. In addition, in our previous studies, we found that the human-origin and avian-origin of H10N8 AIVs with very high homology also have these six mutation differences in PB2 gene, and the avian-origin H10N8 strain known as JX102 with all the key amino acids on the PB2 protein in the pre-evolutionary stage, so JX102 was chosen as a model strain. Among them, PB2-A588V significantly enhanced the activity of polymerase in avian and mammalian cells. Notably, animal experiments showed that PB2-A588V substitution increased the pathogenicity and transmissibility in chickens and the virulence of mice. The combined mutations of PB2-F6 (including PB2-I292V, PB2-R389K, PB2-A588V, PB2-T598M, PB2-L648V, and PB2-T676M) obtained higher adaptability of AIVs in avians and mammals than that of the single mutation of PB2-A588V, which suggested that the PB2 588 site is a key co-adaptation site and that synergies with other mutation sites can further enhance this co-adaptability. The results of this study show that the emergence of co-adaptation not only increases the threat to avians and mammals but may also contribute to a pandemic among avians and cross the interspecies barrier to mammals.

Multi-isotopes and hydrochemistry combined to reveal the major factors affecting Carboniferous groundwater evolution in the Huaibei coalfield, North China.

Both natural processes and anthropogenic activities have significant effects on groundwater evolution in coal mining regions. In this study, the primary controlling mechanism of the groundwater chemistry evolution for the Carboniferous groundwater in the Huaibei coalfield, North China was proposed based on the hydrogeochemical indicators combining with multiple isotope tracers. The diversity of hydrochemical types indicates the complexity of the hydrogeochemical environment in the groundwater, which is recharged by precipitation infiltration with minimal evaporation according to the distributions of δD and δ18O. Additionally, ion correlation analysis suggests that minerals dissolution and cation exchange between Na+ and Ca2+ are the dominant processes within that groundwater. The hydrochemical and δ13CDIC characteristics of the groundwater demonstrate that HCO3- is mainly controlled by the dissolution of carbonate minerals and soil CO2, and the proportion of the latter is believed to be dominated by the hydrogeologic conditions. Similarly, the values of SO42- and δ34SSO4 indicate that a small portion of SO42- in the groundwater in the northern part originates from the meteoric precipitation, while it is mainly derived from the dissolution of gypsum in the southern part. Furthermore, mining activities also alter the groundwater level and flow conditions through pumping and drainage, which enhances the interaction between groundwater and aquifer lithologies, thereby affects the hydrogeochemical processes. The findings of this work are of great significance for promoting the safe exploitation of deep coal resources and the sustainable utilization of groundwater in the Huaibei coalfield, as well as the most of other coalfields in North China.

Cathepsin B-Activated Fluorescent and Photoacoustic Imaging of Tumor.

Early diagnosis is crucial to the treatment of cancer. Cathepsin B (CTB) plays an important role in numerous cancers, which is a promising biomarker for early diagnosis of cancer. It is necessary to exploit new probes for visualization of CTB in vivo. Fluorescent/photoacoustic (FL/PA) imaging is a powerful tool for in vivo study which possesses both excellent sensitivity and spatial resolution. To our knowledge, there has been no FL/PA probe to image CTB in vitro or in vivo. Therefore, we developed two CTB-activated FL/PA probes HCy-Cit-Val and HCy-Gly-Leu-Phe-Gly, which could successfully monitor CTB activity in vivo. Both two probes had excellent sensitivity and selectivity in vitro. Cell imaging showed that HCy-Cit-Val or HCy-Gly-Leu-Phe-Gly could image endogenous CTB in lysosome with 6.8-fold or 5.1-fold enhancement of the FL signal and 5.8-fold or 3.4-fold enhancement of the PA signal compared to their inhibitor contrast groups. Tumor imaging in vivo further confirmed the good applicability of these two probes to monitor CTB activity with high sensitivity and spatial resolution. Moreover, the property of HCy-Cit-Val is superior to HCy-Gly-Leu-Phe-Gly due to the higher catalytic efficiency of CTB toward HCy-Cit-Val than HCy-Gly-Leu-Phe-Gly. We envision that our FL/PA probe HCy-Cit-Val will be suitable for clinical early diagnosis of CTB-related cancer in the near future.

Pooled CRISPR screening identifies m6A as a positive regulator of macrophage activation.

m6A RNA modification is implicated in multiple cellular responses. However, its function in the innate immune cells is poorly understood. Here, we identified major m6A "writers" as the top candidate genes regulating macrophage activation by LPS in an RNA binding protein focused CRISPR screening. We have confirmed that Mettl3-deficient macrophages exhibited reduced TNF-α production upon LPS stimulation in vitro. Consistently, Mettl3 flox/flox;Lyzm-Cre mice displayed increased susceptibility to bacterial infection and showed faster tumor growth. Mechanistically, the transcripts of the Irakm gene encoding a negative regulator of TLR4 signaling were highly decorated by m6A modification. METTL3 deficiency led to the loss of m6A modification on Irakm mRNA and slowed down its degradation, resulting in a higher level of IRAKM, which ultimately suppressed TLR signaling-mediated macrophage activation. Our findings demonstrate a previously unknown role for METTL3-mediated m6A modification in innate immune responses and implicate the m6A machinery as a potential cancer immunotherapy target.

Generation of recombinant influenza virus bearing strep tagged PB2 and effective identification of interactional host factors.

The genome of influenza A virus is negative-sense and segmented RNA, which is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRp) during the virus life cycle. The viral RdRp is thought to be an important host range and virulence determinant factor, and the 627 site of PB2 subunit is a highly acceptable key site of RdRp function. Besides, the function of RdRp is modulated by several host factors. Identification of the host factors interacting with RdRp is of great interest. Here, we tried to explore an effective method to study virus-host interaction by rescuing replication-competent recombinant influenza viruses carrying Strep tagged PB2. Subsequently, we tested several biological characteristics of recombinant viruses in cells and pathogenicity in mice. Then, we purified of protein complex of Strep tagged PB2 and host factors of interest from 293 T cells infected with recombinant viruses. After purification, we performed mass spectrometry to identify these proteins that interacting with PB2. We identified 57 host factors in total. Through Gene Ontology (GO) and Protein-Protein interaction (PPI) network analysis, we revealed the function and network of these proteins. In summary, we generated replication-competent recombinant influenza viruses by inserting a Strep-Tag into PB2 and purified host factors interacting with viral RdRp bearing a 627 K or 627E PB2. These proteins might function as host range and virulence determinants of influenza virus.