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The CRISPR/Cas9 system has recently been adapted for generating knockout mice to investigate physiological functions and pathological mechanisms. Here, we report a highly efficient procedure for brain-specific disruption of genes of interest in vivo . We constructed pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor and is responsible for callosal axon projections in the developing mouse brain. We first confirmed that these constructs efficiently induced double-strand breaks (DSBs) in target sites of exogenous plasmids both in vitro and in vivo . We then found that the introduction of pX330-Satb2 into the developing mouse brain using in utero electroporation led to a dramatic reduction of Satb2 expression in the transfected cerebral cortex, suggesting DSBs had occurred in the Satb2 gene with high efficiency. Furthermore, we found that Cas9-mediated targeting of the Satb2 gene induced abnormalities in axonal projection patterns, which is consistent with the phenotypes previously observed in Satb2 mutant mice. Introduction of pX330-NeuN using our procedure also resulted in the efficient disruption of the NeuN gene. Thus, our procedure combining the CRISPR/Cas9 system and in utero electroporation is an effective and rapid approach to achieve brain-specific gene knockout in vivo .

Acquired point mutations in the ABL1 gene are widely recognized as a cause of Philadelphia chromosome-positive B cell precursor acute lymphoblastic leukemia (Ph + B-ALL) that is resistant to tyrosine kinase inhibitors, whereas there are few reports about other types of the ABL1 mutation. Here, we report 2 cases of Ph + B-ALL gaining a partial deletion type mutation of the ABL1 gene (Δ184-274 mutation), which resulted in truncation of the ABL1 molecule and loss of kinase activity. In both cases, the disease was refractory to multiple agents in the recurrent phase after allogeneic hematopoietic cell transplantation. This is a case report of a truncated ABL1 mutation in 2 patients with Ph + B-ALL.

Tumor‐derived endothelial‐like cells (tEC) were prepared by culturing human umbilical vein endothelial cells (HUVEC) in the presence of HT1080 human fibrosarcoma‐conditioned medium. tEC showed higher permeability and less cell‐adhesion activity than normal HUVEC (nEC). Tumor necrosis factor‐α (TNF) is known to have tumor‐vasculature disrupting activity. tEC showed higher cytotoxicity to recombinant human TNF (rhTNF) than nEC, and was not observed using HUVEC cultured with WI38 human diploid cell‐conditioned medium as a medium‐control. These results demonstrate that tEC acquire physiological properties of tumor‐associated vasculature, and may be a useful model system for the study of the mechanisms of TNF antitumor action. The TNF‐mutant RGD‐V29 (code No. F4614), which has an inserted 4Arg‐Gly‐Asp sequence and an 29Arg→Val replacement, was found to induce greater preferential destruction of tEC compared to rhTNF. When the preferential activities were evaluated in terms of 30% cytotoxicity (IC30) ratio (nEC/tEC), the ratio was 460 for RGD‐V29 compared to 4.2 for rhTNF. RGD‐V29 also exhibited cell‐adhesive function and bound preferentially to the p55 TNF‐receptor. Both these properties of RGD‐V29 contributed to the tEC selective cytotoxicity, indicating that the RGD ligands and selective p55 receptor binding on the cells, although uncharacterized, are involved in tEC targeting. Therefore, the TNF mutant RGD‐V29 may show greater selectivity toward tumor vasculature than wild‐type TNF.

We previously reported that the optimally PEGylated tumor necrosis factor‐α (MPEG‐TNF‐α), in which 56% of the TNF‐α‐lysine amino groups were coupled with polyethylene glycol (PEG), had about 100‐fold greater anti‐tumor effect than native TNF‐α. Here, we assessed the usefulness of MPEG‐TNF‐α as a systemic anti‐tumor therapeutic drug, using B16‐BL6 melanoma and colon‐26 adenocarcinoma, which have been reported to be resistant to TNF‐αin vivo, as compared with Meth‐A fibrosarcoma. MPEG‐TNF‐α markedly inhibited the growth of both tumors without causing any TNF‐α‐mediated side‐effects, whereas native TNF‐α had no anti‐tumor effects and caused adverse side‐effects. In addition, MPEG‐TNF‐α drastically inhibited the metastatic colony formation of B16‐BL6 melanoma. MPEG‐TNF‐α may, thus, be a potential systemic anti‐tumor therapeutic agent.

Currently, first-line immune checkpoint inhibitors (ICIs), including programmed cell death protein-1 (PD-1) inhibitors, are utilized as monotherapy in advanced non-small cell lung cancer (NSCLC) patients with high programmed death ligand-1 (PD-L1) expression (≧50%). Pre-treatment or post-treatment serum soluble PD-L1 (sPD-L1) has been identified as a potential biomarker for assessing ICI efficacy through fixed-point observations. However, existing studies on sPD-L1 changes have produced inconsistent results or have had sample sizes too small to detect clinically meaningful effect sizes. To elucidate the role of sPD-L1, we conducted a collaborative individual patient data meta-analysis of PD-1 inhibitor treatments. We conducted a thorough search of articles in PubMed via Medline, Embase, Scopus, and Cochrane databases from inception to October 20, 2023. Trials were deemed eligible if they contained individual datasets for advanced NSCLC patients, including data on overall survival (OS)/progression-free survival (PFS), as well as pre- and post-treatment sPD-L1 levels after 3-4 cycles of PD-1 inhibitor treatments. Our analysis focused on patients who completed 3-4 cycles of PD-1 inhibitor treatments. The primary outcome measure was OS/PFS, and we assessed changes in sPD-L1 concentration pre- and post-treatment through ELISA analyses. From our search, we identified a potential seven trials, encompassing 256 patients. Among these, two trials with 26 patients met the criteria for inclusion in our primary analyses. Over a median follow-up period of 10 months, pooled univariate analysis revealed that increases in sPD-L1 levels during PD-1 inhibitor treatment were not associated with OS (HR = 1.25; CI: 0.52-3.02)/PFS (HR = 1.42; CI: 0.61-3.30) when compared to cases with sPD-L1 decreases. Subgroup analyses indicated that the impact of sPD-L1 changes on overall mortality/progression-related mortality remained consistent regardless of gender, age, or the type of treatment (nivolumab or pembrolizumab). Our findings suggest that changes in sPD-L1 levels during PD-1 inhibitor treatment do not significantly influence the prognosis of advanced NSCLC patients, regardless of gender, age, or treatment type. Continuous monitoring of sPD-L1 may not offer significant advantages compared to fixed-point observations.

The low response rate of immune checkpoint inhibitors (ICIs) is a challenge. The efficacy of ICIs is influenced by the tumour microenvironment, which is controlled by the gut microbiota. In particular, intestinal bacteria and their metabolites, such as short chain fatty acids (SCFAs), are important regulators of cancer immunity; however, our knowledge on the effects of individual SCFAs remains limited. Here, we show that isobutyric acid has the strongest effect among SCFAs on both immune activity and tumour growth. In vitro, cancer cell numbers were suppressed by approximately 75% in humans and mice compared with those in controls. Oral administration of isobutyric acid to carcinoma-bearing mice enhanced the effect of anti-PD-1 immunotherapy, reducing tumour volume by approximately 80% and 60% compared with those in the control group and anti-PD-1 antibody alone group, respectively. Taken together, these findings may support the development of novel cancer therapies that can improve the response rate to ICIs.

The Poisson’s ratio (μ) of natural rubber (NR) undergoing strain-induced crystallization (SIC) during uniaxial stretching was investigated as a function of the applied stretch ( λ ∣ ∣ ) over a broad λ ∣ ∣ range, encompassing the SIC onset stretch ( λ ∣ ∣ * ≈ 4.1). Below λ ∣ ∣ * , μ remains near 1/2, indicating the incompressible behavior of NR in its fully rubbery amorphous state. However, once λ ∣ ∣ exceeds λ ∣ ∣ * , μ decreases as the degree of crystallinity ( χ c ) increases. As λ ∣ ∣ increases from λ ∣ ∣ * to fracture stretch ( λ ∣ ∣ ≈ 7.1), χ c increases to 18%, and μ gradually decreases to 0.33. This reduction in μ reflects the transformation of the NR matrix from a rubbery amorphous state to a semicrystalline state. In fact, the true stress (force per cross-sectional area in the deformed state) at the fracture point, obtained via actual lateral contraction, is approximately 85% of that estimated under the assumption μ = 1/2. These findings provide a critical foundation for accurately modeling the mechanical behavior of strain-crystallizing elastomers. The Poisson's ratio ( μ ) of natural rubber undergoing strain-induced crystallization was studied across a broad stretch range. Below the crystallization onset stretch ( λ ∣ ∣ * ≈ 4.1), μ remains near 0.5, indicating incompressibility. Beyond λ ∣ ∣ *, μ decreases with increasing crystallinity, reaching 0.33 at fracture ( λ ∣ ∣ ≈ 7.1), as the matrix transforms from amorphous to semicrystalline. The true stress at fracture, accounting for lateral contraction, is approximately 85% of the estimated value assuming μ = 0.5. These insights are vital for modeling the mechanical behavior of strain-crystallizing elastomers.

Gemcitabine is a key drug for the treatment of pancreatic cancer; however, with its limitation in clinical benefits, the development of another potent therapeutic is necessary. Vascular endothelial growth factor receptor 2 is an essential target for tumor angiogenesis, and we have conducted a phase I clinical trial using gemcitabine and vascular endothelial growth factor receptor 2 peptide (elpamotide). Based on the promising results of this phase I trial, a multicenter, randomized, placebo‐controlled, double‐blind phase II/III clinical trial has been carried out for pancreatic cancer. The eligibility criteria included locally advanced or metastatic pancreatic cancer. Patients were assigned to either the Active group (elpamotide + gemcitabine) or Placebo group (placebo + gemcitabine) in a 2:1 ratio by the dynamic allocation method. The primary endpoint was overall survival. The Harrington–Fleming test was applied to the statistical analysis in this study to evaluate the time‐lagged effect of immunotherapy appropriately. A total of 153 patients (Active group, n = 100; Placebo group, n = 53) were included in the analysis. No statistically significant differences were found between the two groups in the prolongation of overall survival (Harrington–Fleming P‐value, 0.918; log–rank P‐value, 0.897; hazard ratio, 0.87, 95% confidence interval [CI], 0.486–1.557). Median survival time was 8.36 months (95% CI, 7.46–10.18) for the Active group and 8.54 months (95% CI, 7.33–10.84) for the Placebo group. The toxicity observed in both groups was manageable. Combination therapy of elpamotide with gemcitabine was well tolerated. Despite the lack of benefit in overall survival, subgroup analysis suggested that the patients who experienced severe injection site reaction, such as ulceration and erosion, might have better survival. Phase II/III trial of elpamotide was performed to evaluate the clinical effect for advanced pancreatic cancer. Despite the lack of benefit in OS, sub‐group analysis suggested that the patients with severe ISR might have better survival.

[This corrects the article DOI: 10.3389/fimmu.2023.1308381.].

Strain‐induced crystallization (SIC) in natural rubber (NR) near crack tips significantly enhances crack growth resistance, but understanding the interplay between local strain field and crystallization remains challenging due to confined and heterogeneous characteristics. Using micro‐scale digital image correlation (DIC) and scanning wide‐angle X‐ray diffraction (WAXD, with a narrow 10 µm square beam), this study maps local strain tensor properties and SIC in the vicinity of the crack tip and its peripheral zone (≈3 mm × 1 mm area). The analysis reveals a significant correlation between these properties. In the peripheral zone, there is a noticeable deviation of both the principal strain axis and the crystal orientation from the crack opening direction. These deviations are linearly correlated, which indicates that shear strain plays a significant role in determining the crystal orientation. Crucially, the maximum tensile component in the tensor of local principal strains predominantly dictates local crystallinity. This simplicity is attributed to the limited variation in types of deformation within the SIC region, with corresponding to deformations falling between planar and uniaxial stretching. These findings pave the way for predicting crystallinity distribution using solely strain field data, offering valuable insights into the role of SIC in enhancing the crack growth resistance of NR. This study explores the interplay between non‐uniform strain and crystallization near crack‐tips in natural rubber, employing micro‐scale DIC and micro‐beam scanning X‐ray scattering. Local maximum tensile strain predominantly dictates local crystallinity, while shear strain governs crystal orientation. These findings shed light on how crystallization contributes to the crack growth resistance of natural rubber.