GHITM regulates malignant phenotype and sensitivity to PD-1 blockade of renal cancer cells via Notch signalling.

Growth hormone inducible transmembrane protein (GHITM), one member of Bax inhibitory protein-like family, has been rarely studied, and the clinical importance and biological functions of GHITM in kidney renal clear cell carcinoma (KIRC) still remain unknown. In the present study, we found that GHITM was downregulated in KIRC. Aberrant GHITM downregulation related to clinicopathological feature and unfavourable prognosis of KIRC patients. GHITM overexpression inhibited KIRC cell proliferation, migration and invasion in vitro and in vivo. Mechanistically, GHITM overexpression could induce the downregulation of Notch1, which acts as an oncogene in KIRC. Overexpression of Notch1 effectively rescued the inhibitory effect induced by GHITM upregulation. More importantly, GHITM could regulate PD-L1 protein abundance and ectopic overexpression of GHITM enhanced the antitumour efficiency of PD-1 blockade in KIRC, which provided new insights into antitumour therapy. Furthermore, we also showed that YY1 could decrease GHITM level via binding to its promoter. Taken together, our study revealed that GHITM was a promising therapeutic target for KIRC, which could modulate malignant phenotype and sensitivity to PD-1 blockade of renal cancer cells via Notch signalling pathway.

The palmitoylation of gasdermin D directs its membrane translocation and pore formation during pyroptosis.

Plasma membrane perforation elicited by caspase cleavage of the gasdermin D (GSDMD) N-terminal domain (GSDMD-NT) triggers pyroptosis. The mechanisms underlying GSDMD membrane translocation and pore formation are not fully understood. Here, using a proteomic approach, we identified fatty acid synthase (FASN) as a GSDMD-binding partner. S-palmitoylation of GSDMD at Cys191/Cys192 (human/mouse), catalyzed by palmitoyl acyltransferases ZDHHC5 and ZDHHC9 and facilitated by reactive oxygen species (ROS), directly mediated membrane translocation of GSDMD-NT but not full-length GSDMD (GSDMD-FL). Palmitoylation of GSDMD-FL could be induced before inflammasome activation by stimuli such as lipopolysaccharide (LPS), consequently serving as an essential molecular event in macrophage priming. Inhibition of GSDMD palmitoylation suppressed macrophage pyroptosis and IL-1ß release, mitigated organ damage, and enhanced the survival of septic mice. Thus, GSDMD-NT palmitoylation is a key regulatory mechanism controlling GSDMD membrane localization and activation, which may offer an additional target for modulating immune activity in infectious and inflammatory diseases.

Gasdermin E dictates inflammatory responses by controlling the mode of neutrophil death.

Both lytic and apoptotic cell death remove senescent and damaged cells in living organisms. However, they elicit contrasting pro- and anti-inflammatory responses, respectively. The precise cellular mechanism that governs the choice between these two modes of death remains incompletely understood. Here we identify Gasdermin E (GSDME) as a master switch for neutrophil lytic pyroptotic death. The tightly regulated GSDME cleavage and activation in aging neutrophils are mediated by proteinase-3 and caspase-3, leading to pyroptosis. GSDME deficiency does not alter neutrophil overall survival rate; instead, it specifically precludes pyroptosis and skews neutrophil death towards apoptosis, thereby attenuating inflammatory responses due to augmented efferocytosis of apoptotic neutrophils by macrophages. In a clinically relevant acid-aspiration-induced lung injury model, neutrophil-specific deletion of GSDME reduces pulmonary inflammation, facilitates inflammation resolution, and alleviates lung injury. Thus, by controlling the mode of neutrophil death, GSDME dictates host inflammatory outcomes, providing a potential therapeutic target for infectious and inflammatory diseases.

Acoustic resolution photoacoustic Doppler flowmetry for assessment of patient rectal cancer blood perfusion.

Significance: Photoacoustic Doppler flowmetry offers quantitative blood perfusion information in addition to photoacoustic vascular contrast for rectal cancer assessment. Aim: We aim to develop and validate a correlational Doppler flowmetry utilizing an acoustic resolution photoacoustic microscopy (AR-PAM) system for blood perfusion analysis. Approach: To extract blood perfusion information, we implemented AR-PAM Doppler flowmetry consisting of signal filtering and conditioning, A-line correlation, and angle compensation. We developed flow phantoms and contrast agent to systemically investigate the flowmetry's efficacy in a series of phantom studies. The developed correlational Doppler flowmetry was applied to images collected during in vivo AR-PAM for post-treatment rectal cancer evaluation. Results: The linearity and accuracy of the Doppler flow measurement system were validated in phantom studies. Imaging rectal cancer patients treated with chemoradiation demonstrated the feasibility of using correlational Doppler flowmetry to assess treatment response and distinguish residual cancer from cancer-free tumor bed tissue and normal rectal tissue. Conclusions: A new correlational Doppler flowmetry was developed and validated through systematic phantom evaluations. The results of its application to in vivo patients suggest it could be a useful addition to photoacoustic endoscopy for post-treatment rectal cancer assessment.

Nitronyl Nitroxide Ameliorates Hypobaric Hypoxia-Induced Cognitive Impairment in Mice by Suppressing the Oxidative Stress, Inflammatory Response and Apoptosis.

Abundant investigations have shown that hypobaric hypoxia (HH) causes cognitive impairment, mostly attributed to oxidative stress, inflammation, and apoptosis. HPN (4'-hydroxyl-2-subsitiuted phenylnitronyl nitroxide) is an excellent free radical scavenger with anti-inflammatory and anti-apoptotic activities. Our previous study has found that HPN exhibited neuroprotective effect on HH induced brain injury. In the present study, we examined the protective effect and potential mechanism of HPN on HH-induced cognitive impairment. Male mice were exposed to HH at 8000 m for 3 days with and without HPN treatment. Cognitive performance was assessed by the eight-arm radical maze. The histological changes were assayed by Nissle staining. The hippocampus cell apoptosis was detected by Tunnel staining. The levels of inflammatory cytokines and oxidative stress markers were detected. The expression of oxidative stress, inflammation-related and apoptosis-related proteins was determined by western blot. HPN administration significantly and mitigated HH induced histological damages and spatial memory loss with the evidence of decreased working memory error (WME), reference memory error (RME), total errors (TE) and total time (TT). In addition, HPN treatment significantly decreased the content of H2O2 and MDA, increased the levels of SOD, CAT, GSH-Px and GSH, and inhibited the synthesis of TNF-α, IL-1ß and IL-6. Moreover, HPN administration could down-regulate the expression of NF-κB, TNF-α, Bax, and cleaved caspase-3 and up-regulate the expression of Nrf2, HO-1 and Bcl-2. The number of apoptotic cells was also significantly decreased in the hippocampus of mice in the HPN group. There results indicate that HPN improve HH-induced cognitive impairment by alleviating oxidative stress damage, suppressing inflammatory response and apoptosis and may be a powerful candidate compound for alleviating memory loss induced by HH.

HDAC6 deacetylates IDH1 to promote the homeostasis of hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are cells mainly present in the bone marrow and capable of forming mature blood cells. However, the epigenetic mechanisms governing the homeostasis of HSPCs remain elusive. Here, we demonstrate an important role for histone deacetylase 6 (HDAC6) in regulating this process. Our data show that the percentage of HSPCs in Hdac6 knockout mice is lower than in wild-type mice due to decreased HSPC proliferation. HDAC6 interacts with isocitrate dehydrogenase 1 (IDH1) and deacetylates IDH1 at lysine 233. The deacetylation of IDH1 inhibits its catalytic activity and thereby decreases the 5-hydroxymethylcytosine level of ten-eleven translocation 2 (TET2) target genes, changing gene expression patterns to promote the proliferation of HSPCs. These findings uncover a role for HDAC6 and IDH1 in regulating the homeostasis of HSPCs and may have implications for the treatment of hematological diseases.

Enhanced 3D visualization of human fallopian tube morphology using a miniature optical coherence tomography catheter.

We demonstrate the use of our miniature optical coherence tomography catheter to acquire three-dimensional human fallopian tube images. Images of the fallopian tube's tissue morphology, vasculature, and tissue heterogeneity distribution are enhanced by adaptive thresholding, masking, and intensity inverting, making it easier to differentiate malignant tissue from normal tissue. The results show that normal fallopian tubes tend to have rich vasculature accompanied by a patterned tissue scattering background, features that do not appear in malignant cases. This finding suggests that miniature OCT catheters may have great potential for fast optical biopsy of the fallopian tube.

Improved anti-tumor activity of fluorinated camptothecin derivatives 9-fluorocamptothecin and 7-ethyl-9-fluorocamptothecin on hepatocellular carcinoma by targeting topoisomerase I.

Primary liver cancer is one of the most common malignant cancers of the digestive system that lacks effective chemotherapeutic drugs in clinical settings. Camptothecin (CPT) and its derivatives have been approved for cancer treatment; however, their application is limited by their systemic toxicity. For lead optimization in new drug discovery stages, fluorination is an effective and robust approach to increase the bioavailability and optimize the pharmacokinetics of candidate compounds, thereby improving their efficacy. To obtain new and highly active CPT derivatives, we designed, synthesized, and evaluated two new fluorinated CPT derivatives, 9-fluorocamptothecin (A1) and 7-ethyl-9-fluorocamptothecin (A2), in this study. In vitro, A1 and A2 exhibited more robust anti-tumor activity than topotecan (TPT) in various cancer cells, particularly hepatocellular carcinoma (HCC) cells. In vivo, A1 and A2 exhibited greater anti-tumor activity than TPT in both AKT/Met induced primary HCC mouse models and implanted HepG2 cell xenografts. Acute toxicity tests revealed that A1 and A2 were not lethal and did not cause significant body weight loss at high doses. Moreover, A1 and A2 exhibited no significant toxicity in the mouse liver, heart, lung, spleen, kidney, and hematopoietic systems at therapeutic doses. Mechanistically, A1 and A2 blocked HCC cell proliferation by inhibiting the enzymatic activity of Topo I, subsequently inducing DNA damage, cell cycle arrest, and apoptosis. In summary, our results indicate that fluorination improves the anti-tumor activity of CPT while decreasing its toxicity and highlight the application potential of fluorination products A1 and A2 in clinical settings.

Screening of immunotherapy-related genes in bladder cancer based on GEO datasets.

Background: As one of the most prevalent genitourinary cancers, bladder cancer (BLCA) is associated with high morbidity and mortality. Currently, limited indicators are available for early detection and diagnosis of bladder cancer, and there is a lack of specific biomarkers for evaluating the prognosis of BLCA patients. This study aims to identify critical genes that affect bladder cancer immunity to improve the diagnosis and prognosis of bladder cancer and to identify new biomarkers and targets for immunotherapy. Methods: Two GEO datasets were used to screen differentially expressed genes (DEGs). The STRING database was used to construct a protein-protein interaction network of DEGs, and plug-in APP CytoHubba in Cytoscape was used to identify critical genes in the network. GO and KEGG analyses explored the functions and pathways of differential gene enrichment. We used GEPIA to validate the expression of differential genes, their impact on patient survival, and their relationship to clinicopathological parameters. Additionally, hub genes were verified using qRT-PCR and Western blotting. Immune infiltration analysis and multiple immunohistochemistry reveal the impact of Hub genes on the tumor microenvironment. Result: We screened out 259 differential genes, and identified 10 key hub genes by the degree algorithm. Four genes (ACTA2, FLNA, TAGLN, and TPM1) were associated with overall or disease-free survival in BLCA patients and were significantly associated with clinical parameters. We experimentally confirmed that the mRNA and protein levels of these four genes were significantly decreased in bladder cancer cells. Immunoassays revealed that these four genes affect immune cell infiltration in the tumor microenvironment; they increased the polarization of M2 macrophages. Conclusion: These four genes affect the tumor microenvironment of bladder cancer, provide a new direction for tumor immunotherapy, and have significant potential in the diagnosis and prognosis of bladder cancer.

microRNA-203 functions as a natural Ras inhibitor in hepatocellular carcinoma.

microRNA-203 (miR203) plays an important role in the formation and development of multiple types of cancers. However, its role in hepatic carcinogenesis has not been well studied. Mitogen-activated protein kinase signaling is known to be activated in hepatocellular carcinoma (HCC), but there is a lack of effective drugs targeting this pathway for HCC treatment. In this study, we investigated the role of miR203 in HCC and the underlying mechanism. We found that miR203 was significantly downregulated in HCC cell lines and patient tissues compared with a hepatocyte cell line (L02) or normal liver tissues. Restoration of miR203 inhibited HCC cell growth and induced cell cycle arrest and apoptosis. In primary and xenograft HCC mouse models, miR203 also significantly blocked HCC growth. Bioinformatic analysis indicated that miR203 directly binds to the 3'UTR of NRas mRNA, resulting in decreased expression of NRas and inactivation of mitogen-activated protein kinase (MAPK) signaling. Activation of MAPK signaling by ectopic NRas expression rescued the cell proliferation blocked by miR203. Together, our findings illustrate the fundamental role of miR203 as a natural inhibitor of RAS/MAPK signaling in hepatic carcinogenesis in vitro and in vivo. In light of the critical and universal activation of the MAPK pathway in HCC, miR203 has the potential to serve as a nucleotide drug for the treatment of HCC with activated MAPK signaling.

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay.

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous research indicated that multiple pathways could mediate the spontaneous death of neutrophils. A cocktail was developed by simultaneously targeting these pathways, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which prolonged the neutrophil lifespan to greater than 5 days without significantly compromising the neutrophil function. Concurrently, a reliable and stable protocol for assessing and evaluating neutrophil death was also developed. In this work, we show that CLON-G can prolong the neutrophil lifespan in vitro to more than 5 days, and we exhibit the lengthening of the neutrophil lifespan with FACS and confocal fluorescence microscopy. This report introduces procedures for the preparation of CLON-G and showcases an in vitro spontaneous death assay of neutrophils, which can be used for the study of neutrophils and for subsequently interrogating neutrophil death, thus providing a reliable resource for the neutrophil community.

Deep learning based on co-registered ultrasound and photoacoustic imaging improves the assessment of rectal cancer treatment response.

Identifying complete response (CR) after rectal cancer preoperative treatment is critical to deciding subsequent management. Imaging techniques, including endorectal ultrasound and MRI, have been investigated but have low negative predictive values. By imaging post-treatment vascular normalization using photoacoustic microscopy, we hypothesize that co-registered ultrasound and photoacoustic imaging will better identify complete responders. In this study, we used in vivo data from 21 patients to develop a robust deep learning model (US-PAM DenseNet) based on co-registered dual-modality ultrasound (US) and photoacoustic microscopy (PAM) images and individualized normal reference images. We tested the model's accuracy in differentiating malignant from non-cancer tissue. Compared to models based on US alone (classification accuracy 82.9 ± 1.3%, AUC 0.917(95%CI: 0.897-0.937)), the addition of PAM and normal reference images improved the model performance significantly (accuracy 92.4 ± 0.6%, AUC 0.968(95%CI: 0.960-0.976)) without increasing model complexity. Additionally, while US models could not reliably differentiate images of cancer from those of normalized tissue with complete treatment response, US-PAM DenseNet made accurate predictions from these images. For use in the clinical settings, US-PAM DenseNet was extended to classify entire US-PAM B-scans through sequential ROI classification. Finally, to help focus surgical evaluation in real time, we computed attention heat maps from the model predictions to highlight suspicious cancer regions. We conclude that US-PAM DenseNet could improve the clinical care of rectal cancer patients by identifying complete responders with higher accuracy than current imaging techniques.

GOLM1 Promotes Epithelial-Mesenchymal Transition by Activating TGFß1/Smad2 Signaling in Prostate Cancer.

Background: Prostate cancer (PC) is one of the most commonly diagnosed cancer in men worldwide. Epithelial-mesenchymal transition (EMT) is considered to play a crucial role in the development of the metastatic castration-resistant prostate cancer, which causes the majority of the death cases in PC. Golgi membrane protein 1 (GOLM1) is highly expressed in PC and has been identified as a driver factor for EMT in various cancers. However, its biological functions and underlying mechanisms remain ambiguous in PC. Method: GOLM1 expression level of PC was detected by Western blot and immunohistochemistry analyses. To investigate GOLM1 functions in cancer cells, we overexpressed and knocked down GOLM1 in different prostate cancer cell lines. Transwell assay and wound healing assay were used to determine the role of GOLM1 in cell EMT, such as migration and invasion abilities. TGF-ß1/Smad2 signaling pathway downstream of GOLM1 was detected by Western blot and Transwell assay. Result: GOLM1 expression is up-regulated in PC and correlated with a worse prognosis. GOLM1 promotes the abilities of migration and invasion in PC cell lines (DU145 and LNCaP). Furthermore, TGF-ß1/Smad2 signaling is positively regulated by GOLM1 to facilitate EMT in PC, whereas this role can be restored by TGF-ß1 after GOLM1 knockdown or be abrogated by p-Smad inhibitor SB431542. Conclusion: GOLM1 is significantly upregulated in PC and acts as a critical oncogene by promoting PC cell EMT process by activating TGF-ß1/Smad2 signaling pathway. Therefore, GOLM1 has the potential to be a biomarker for PC diagnosis and to predict the prognosis of PC patients. It is of great significance to seek effective and specific inhibitor of GOLM1 for PC treatment as well.

Depth-resolved attenuation mapping of the human ovary and fallopian tube using optical coherence tomography.

Due to the lack of reliable early-diagnostic tools, most ovarian cancers are diagnosed at late stages. Although optical coherence tomography (OCT) has shown promise for identifying diseased ovaries and fallopian tubes at an earlier stage, previous studies either did not provide quantitative scattering mapping or simply used Beer's law to fit the scattering coefficients of each A-line. In this paper, we calculated the pixel-wise attenuation coefficients of ovaries and fallopian tubes in OCT images. Data from 73 freshly excised human ovaries and fallopian tubes from 36 patients have shown that statistical features are statistically different between cancerous ovaries, infundibula, and fimbriae and normal ones.

A Coregistered Ultrasound and Photoacoustic Imaging Protocol for the Transvaginal Imaging of Ovarian Lesions.

Ovarian cancer remains the deadliest of all the gynecological malignancies due to the lack of reliable screening tools for early detection and diagnosis. Photoacoustic imaging or tomography (PAT) is an emerging imaging modality that can provide the total hemoglobin concentration (relative scale, rHbT) and blood oxygen saturation (%sO2) of ovarian/adnexal lesions, which are important parameters for cancer diagnosis. Combined with coregistered ultrasound (US), PAT has demonstrated great potential for detecting ovarian cancers and for accurately diagnosing ovarian lesions for effective risk assessment and the reduction of unnecessary surgeries of benign lesions. However, PAT imaging protocols in clinical applications, to our knowledge, largely vary among different studies. Here, we report a transvaginal ovarian cancer imaging protocol that can be beneficial to other clinical studies, especially those using commercial ultrasound arrays for the detection of photoacoustic signals and standard delay-and-sum beamforming algorithms for imaging.

CD11c regulates neutrophil maturation.

Sepsis continues to be associated with high morbidity and mortality. Currently, sepsis is managed only conservatively. In sepsis, a substantial number of neutrophils is required, leading to accelerated neutrophil production. Immature neutrophils are released into the circulation to meet a demand, despite their less effective functioning in microbial eradication. Although an intervention to provide more mature neutrophils may serve as a potential sepsis treatment, the mechanism of neutrophil differentiation and maturation remains poorly understood. We discovered that CD11c, traditionally known as a dendritic cell marker, was expressed in neutrophils and regulated neutrophil maturation and effector functions. In the absence of CD11c, neutrophil maturation was impaired in the bone marrow, concomitant with a significant increase in the proliferation and apoptosis of preneutrophils, associated with less effector functions. Under lipopolysaccharide challenge, inducing an emergent neutrophil production in the bone marrow, CD11c deficiency exaggerated the release of immature neutrophils into the circulation, associated with a significant proliferation and apoptosis of preneutrophils. In contrast, constitutively active CD11c knock-in mice showed accelerated neutrophil maturation associated with enhanced effector functions, which further supports the notion that CD11c regulates neutrophil maturation. Furthermore, the constitutively active CD11c knock-in mice offered enhanced bacterial eradication. Taken together, we discovered that CD11c was critical for the regulation of neutrophil maturation, and CD11c activation could serve as a potential target for sepsis treatment.

Heterogeneity of neutrophils in cancer: one size does not fit all.

Neutrophils play an essential role in the defense against bacterial infections and orchestrate both the innate and adaptive immune responses. With their abundant numbers, diverse function and short life span, these cells are at the forefront of immune responses, and have gained attention in recent years because of their presence in tumor sites. Neutrophil involvement pertains to tumor cells' ability to construct a suitable tumor microenvironment (TME) that accelerates their own growth and malignancy, by facilitating their interaction with surrounding cells through the circulatory and lymphatic systems, thereby influencing tumor development and progression. Studies have indicated both pro- and anti-tumor properties of infiltrating neutrophils. The TME can exploit neutrophil function, recruitment, and even production, thus resulting in pro-tumor properties of neutrophils, including promotion of genetic instability, tumor cell proliferation, angiogenesis and suppression of anti-tumor or inflammatory response. In contrast, neutrophils can mediate anti-tumor resistance by direct cytotoxicity to the tumor cells or by facilitating anti-tumor functions via crosstalk with T cells. Here, we summarize current knowledge regarding the effects of neutrophil heterogeneity under homeostatic and tumor conditions, including neutrophil phenotype and function, in cancer biology.

Temporal molecular program of human hematopoietic stem and progenitor cells after birth.

Hematopoietic stem and progenitor cells (HSPCs) give rise to the blood system and maintain hematopoiesis throughout the human lifespan. Here, we report a transcriptional census of human bone-marrow-derived HSPCs from the neonate, infant, child, adult, and aging stages, showing two subpopulations of multipotent progenitors separated by CD52 expression. From birth to the adult stage, stem and multipotent progenitors shared similar transcriptional alterations, and erythroid potential was enhanced after the infant stage. By integrating transcriptome, chromatin accessibility, and functional data, we further showed that aging hematopoietic stem cells (HSCs) exhibited a bias toward megakaryocytic differentiation. Finally, in comparison with the HSCs from the cord blood, neonate bone-marrow-derived HSCs were more quiescent and had higher long-term regeneration capability and durable self-renewal. Taken together, this work provides an integral transcriptome landscape of HSPCs and identifies their dynamics in post-natal steady-state hemopoiesis, thereby helping explore hematopoiesis in development and diseases.

αDß2 as a novel target of experimental polymicrobial sepsis.

Since sepsis was defined three decades ago, it has been a target of intensive study. However, there is no specific sepsis treatment available, with its high mortality and morbidity. αDß2 (CD11d/CD18) is one of the four ß2 integrin members. Its role in sepsis has been limitedly studied. Using an experimental polymicrobial sepsis model, we found that the deficiency of αDß2 was associated with less lung injury and better outcome, which was in sharp contrast to other ß2 integrin member αLß2 (CD11a/CD18), and αMß2 (CD11b/CD18). This phenotype was supported by a reduction of bacterial loads in αDß2 knockout mice. Further analysis showed that the deficiency of αDß2 led to a reduction of neutrophil cell death as well as an increase in neutrophil phagocytosis in both murine and human systems. Our data showed a unique role of αDß2 among the ß2 integrin members, which would serve as a potential target to improve the outcome of sepsis.