Background: Nanobodies, named as VHHs (variable domain of heavy chain of HCAb [heavy-chain antibodies]), are derived from heavy-chain-only antibodies that circulate in sera of camelids. Their exceptional physicochemical properties, possibility of humanization, and unique antigen recognition properties make them excellent candidates for targeted delivery of biologically active components, including immunotoxins. In our previous efforts, we have successfully generated the monovalent and bivalent CD7 nanobody-based immunotoxins, which can effectively trigger the apoptosis of CD7-positive malignant cells. To pursue the possibility of translating those immunotoxins into clinics, we humanized the nanobody sequences (designated as dhuVHH6) as well as further truncated the Pseudomonas exotoxin A (PE)-derived PE38 toxin to produce a more protease-resistant form, which is named as PE-LR, by deleting majority of PE domain II. Methods and results: Three new types of immunotoxins, dhuVHH6-PE38, dVHH6-PE-LR, and dhuVHH6-PE-LR, were successfully constructed. These recombinant immunotoxins were expressed in Escherichia coli and showed that nanobody immunotoxins have the benefits of easy soluble expression in a prokaryotic expression system. Flow cytometry results revealed that all immunotoxins still maintained the ability to bind specifically to CD7-positive T lymphocyte strains without binding to CD7-negative control cells. Laser scanning confocal microscopy revealed that these proteins can be endocytosed into the cytoplasm after binding with CD7-positive cells and that this phenomenon was not observed in CD7-negative cells. WST-8 experiments showed that all immunotoxins retained the highly effective and specific growth inhibition activity in CD7-positive cell lines and primary T-cell acute lymphoblastic leukemia (T-ALL) cells. Further in vivo animal model experiments showed that humanized dhuVHH6-PE38 immunotoxin can tolerate higher doses and extend the survival of NOD-Prkdc^em26Il2rg^em26Nju (NCG) mice transplanted with CEM cells without any obvious decrease in body weight. Further studies on NCG mice model with patient-derived T-ALL cells, dhuVHH6-PE38 treatment, significantly prolonged mice survival with ~40% survival improvement. However, it was also noticed that although dhuVHH6-PE-LR showed strong antitumor effect in vitro, its in vivo antitumor efficacy was disappointing. Conclusion: We have successfully constructed a targeted CD7 molecule-modified nanobody (CD7 molecule-improved nanobody) immunotoxin dhuVHH6-PE38 and demonstrated its potential for treating CD7-positive malignant tumors, especially T-cell acute lymphoblastic leukemia.

Objectives: MicroRNAs are powerful regulators in hepatocellular carcinoma (HCC) tumorigenesis. MicoRNA‐191 (miR‐191) has been reported to play an important role in HCC, However, the regulatory mechanism is still unclear. In this study, we investigated the role of miR‐191 in HCC and studied its underlying mechanisms of action. Materials and methods: The expression of miR‐191 in HCC tissues was determined by quantitative real‐time PCR (qRT‐PCR). The role of miR‐191 in HCC cells was examined by using both in vitro and in vivo assays. Downstream targets of miR‐191 were determined by qRT‐PCR and Western blot analysis. Dual‐luciferase assays were performed to validate the interaction between miR‐191 and its targets. Results: The expression of miR‐191 was significantly higher in HCC patients and a higher miR‐191 expression predicted poorer prognosis. Analysis of The Cancer Genome Atlas data sets suggested that miR‐191 positively correlated with cell cycle progression. Gain and loss of function assays showed that miR‐191 promoted cell cycle progression and proliferation. Luciferase reporter assay showed that miR‐191 directly targeted the 3'‐untranslated region of KLF6 mRNA. Furthermore, circular RNA has_circ_0000204 could sponge with miR‐191, resulting in inactivation of miR‐191. Conclusions: Our study sheds light on the novel underlying mechanism of miR‐191 in HCC, which may accelerate the development of cancer therapy.

Objectives: Circular RNA, a type of RNA formed by a covalently closed loop, possesses sophisticated abilities of gene regulation in tumorigenesis and metastasis. However, the role of circRNAs on lung adenocarcinoma (LUAD) remains largely unknown.Materials and methods: The role of cMras was examined both in vitro and in vivo. cMras expression in LUAD tissues was determined by quantitative real‐time PCR (qRT‐PCR). Downstream targets of cMras were predicted by bioinformatics tools and confirmed by RNA immunoprecipitation assay and luciferase assay. qRT‐PCR and western blot assay were used to detect the expression of specific targets.Results: Thirty‐six paired LUAD and healthy tissues were collected and cMras resulted significantly downregulated in cancerous tissues. Its expression was negatively associated with tumour stages. cMras overexpression suppressed LUAD growth and metastasis, while endogenous cMras silencing resulted in the opposite effects. Bioinformatics analysis and experimental evidence confirmed that cMras was a sponge of miRNA‐567 and released its direct target, PTPRG. cMras overexpression decreased miR‐567 expression and subsequently increased PTPRG expression, while increased miRNA‐567 expression blocked the effects induced by cMras. Moreover, PTPRG was downregulated in LUAD and patients with low PTPRG expression exhibited significantly poor prognosis. These results suggested that cMras/miR‐567/PTPRG regulatory pathway might be associated to LUAD tumorigenesis and development.Conclusions: A novel circular RNA cMras and its functions were identified, discovering a cMras/miR‐567/PTPRG regulatory pathway in LUAD tumorigenesis and development.

Purpose: Modified Xi-Jiao-Di-Huang decoction (MXJDH) has significant clinical efficacy for the treatment of sepsis; however, its mechanism of action remains unclear. The purpose of this study was to investigate the protective effects of MXJDH in septic mice and explore its mechanism of action. Methods: Utilizing UPLC-Q-TOF-MS, we identified the primary constituents of the compound MXJDH. Subsequently, we created a mouse model for sepsis, observing their overall condition, including specific symptoms and behavior. We also monitored key inflammatory markers and pathological changes in their organs. Flow cytometry was then employed to assess the polarization of macrophages. Transcriptome sequencing was used to identify genes with altered expression patterns. We investigated the connection between MXJDH and the Pim2/NF-κB signaling pathway, a crucial regulatory mechanism in inflammation. Finally, we examined the expression and tissue distribution of macrophages in the sepsis-induced mice. Results: MXJDH effectively reduces inflammation in sepsis mice, leading to a progressive recovery of organ functions. Moreover, MXJDH facilitates the conversion of macrophages from pro-inflammatory M1 phenotype to anti-inflammatory M2 phenotype. This transformation is potentially mediated through the Pim2/NF-κB signaling pathway. By suppressing Pim2 expression, MXJDH mitigates the nuclear translocation of NF-κB, thereby modulating the expression of downstream inflammatory mediators. The role of MXJDH in regulating macrophage polarization has also been confirmed in sepsis mouse tissues. Conclusion: MXJDH regulates macrophage polarization, inhibits CRS, and alleviates sepsis by inhibiting the Pim2/NF-κB signaling pathway.

Systemic sclerosis (SSc) is a rare chronic autoimmune disorder, mainly characterized by skin sclerosis. In this study, Bufei Qingyu Granules (BQG), a Chinese herbal formula, was used to treat SSc. To better understand the effects and molecular mechanisms of BQG, we successfully established a Bleomycin- (BLM-) induced SSc mouse model, and the mice were treated by BQG. Meanwhile, transcriptomic and bioinformatics analyses were conducted on those samples. As a result, we visually showed that BQG ameliorated the overall health of mice, including body weight, spleen, and thymus index. Thus, it also significantly alleviated inflammation presented by Chemokine (C-X-C motif) ligand 2 (Cxcl2), vasculopathy characterized by α-smooth muscle actin (α-SMA), and fibrotic changes elaborated by not only pathological images, but also the hydroxyproline (HYP) content. After testing by transcriptomic analysis, Cxcl2, Synaptosomal-associated protein 25 (Snap25), and Eukaryotic translation initiation factor 3, and subunit J2 (Eif3j2) which were differentially expressed genes, were verified, so that the data were credible. We further found that BQG could regulate Notch signaling pathway by significantly decreasing both mRNA and protein expression levels of Notch-1 and Jagged-2. Hence, this study demonstrated that BQG could ameliorate the sclerotic skin in mice model involved in inflammation, vascular changes, and fibrosis effects, which was partly mediated by Notch signaling pathway.