An anti-CTLA-4 heavy chain-only antibody with enhanced Treg depletion shows excellent preclinical efficacy and safety profile.

The value of anti-CTLA-4 antibodies in cancer therapy is well established. However, the broad application of currently available anti-CTLA-4 therapeutic antibodies is hampered by their narrow therapeutic index. It is therefore challenging and attractive to develop the next generation of anti-CTLA-4 therapeutics with improved safety and efficacy. To this end, we generated fully human heavy chain-only antibodies (HCAbs) against CTLA-4. The hIgG1 Fc domain of the top candidate, HCAb 4003-1, was further engineered to enhance its regulatory T (Treg) cell depletion effect and to decrease its half-life, resulting in HCAb 4003-2. We tested these HCAbs in in vitro and in vivo experiments in comparison with ipilimumab and other anti-CTLA4 antibodies. The results show that human HCAb 4003-2 binds human CTLA-4 with high affinity and potently blocks the binding of B7-1 (CD80) and B7-2 (CD86) to CTLA-4. The results also show efficient tumor penetration. HCAb 4003-2 exhibits enhanced antibody-dependent cellular cytotoxicity function, lower serum exposure, and more potent anti-tumor activity than ipilimumab in murine tumor models, which is partly driven by a substantial depletion of intratumoral Tregs. Importantly, the enhanced efficacy combined with the shorter serum half-life and less systemic drug exposure in vivo potentially provides an improved therapeutic window in cynomolgus monkeys and preliminary clinical applications. With its augmented efficacy via Treg depletion and improved safety profile, HCAb 4003-2 is a promising candidate for the development of next generation anti-CTLA-4 therapy.

Singleton effect decreases under time pressure: An fNIRS study.

Time pressure affects multiple cognitive processes but how it affects attention capture remains unclear. Two experiments were carried out in the present study to assess whether time pressure prevents attention from capturing by salient distractors and explore the underlying neural mechanisms using functional near-infrared spectroscopy. The results of behavioral tests showed that the singleton effect decreased (Experiment 2) or even disappeared (Experiment 1) when the subject was under time pressure. Neuroimaging data showed that under time pressure, a salient distractor elicited greater activation in the left middle frontal gyrus/inferior frontal gyrus and bilateral superior parietal lobule, brain areas that are thought to be involved in cognitive inhibition and control of spatial attentional shifts. These findings suggest that the reduction or disappearance of the singleton effect under time pressure results from enhanced inhibition of and/or accelerated disengagement from salient distractors.

Hprt Serves as an Ideal Reference Gene for qRT-PCR Normalization in Rat DRG Neurons.

OBJECTIVE: To identify suitable reference genes for gene expression studies in rat dorsal root ganglia (DRG) neurons. METHODS: The raw cycle threshold (Ct) values of 12 selected reference genes were obtained via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in neurons at different developmental stages or under different treatments. Two strategies were employed to screen the most stable reference genes: the genes were ranked according to the coefficient of biological variation and further validated using geNorm and NormFinder programs. The stable and unstable reference genes were subsequently used as internal controls to assess their effects on target gene expression. RESULTS: All reference genes showed varying degrees of fluctuation in Ct values during the growth process of neurons or after different treatments. 18S ribosomal RNA (Rn18s) and ß-actin (Actb) exhibited the most significant changes, while ubiquitin C (Ubc), hypoxanthine phosphoribosyl transferase (Hprt), and mitochondrial ribosomal protein L10 (Mrpl10) showed relatively minor changes. The most stable and unstable genes obtained by different evaluation methods varied slightly. Overall, Actb was found to be the most unstable reference gene, while Hprt was the relatively most stable reference gene. The use of unstable reference genes Actb and ankyrin repeat domain 27 (Ankrd27) as internal controls led to high variability within the control group, ultimately affecting the determination of target gene expression. In contrast, the stable reference gene Hprt had small inter-assay variation and high stability. CONCLUSIONS: Our observations indicate that Hprt is a proper endogenous reference gene for qRT-PCR analysis in rat DRG neurons and thus provides a critical molecular basis for the genetic characterization in neurological disorders.

miR-140-3p suppresses the proliferation and migration of macrophages.

Macrophages benefit myelin debris removal, blood vessel formation, and Schwann cell activation following peripheral nerve injury. Identifying factors that modulate macrophage phenotype may advantage the repair and regeneration of injured peripheral nerves. microRNAs (miRNAs) are important regulators of many physiological and pathological processes, including peripheral nerve regeneration. Herein, we investigated the regulatory roles of miR-140-3p, a miRNA that was differentially expressed in injured rat sciatic nerves, in macrophage RAW264.7 cells. Observations from EdU proliferation assay demonstrated that elevated miR-140-3p decreased the proliferation rates of RAW264.7 cells while suppressed miR-140-3p increased the proliferation rates of RAW264.7 cells. Transwell-based migration assay showed that up-regulated and down-regulated miR-140-3p led to elevated and reduced migration abilities, respectively. However, the abundances of numerous phenotypic markers of M1 and M2 macrophages were not significantly altered by miR-140-3p mimic or inhibitor transfection. Bioinformatic analysis and miR-140-3p-induced gene suppression examination suggested that Smad3 might be the target gene of miR-140-3p. These findings illuminate the inhibitory effects of miR-140-3p on the proliferation and migration of macrophages and contribute to the cognition of the essential roles of miRNAs during peripheral nerve regeneration.

MALT1 is a potential therapeutic target in glioblastoma and plays a crucial role in EGFR-induced NF-κB activation.

Glioblastoma multiforme (GBM) is the most common malignant tumour in the adult brain and hard to treat. Nuclear factor κB (NF-κB) signalling has a crucial role in the tumorigenesis of GBM. EGFR signalling is an important driver of NF-κB activation in GBM; however, the correlation between EGFR and the NF-κB pathway remains unclear. In this study, we investigated the role of mucosa-associated lymphoma antigen 1 (MALT1) in glioma progression and evaluated the anti-tumour activity and effectiveness of MI-2, a MALT1 inhibitor in a pre-clinical GBM model. We identified a paracaspase MALT1 that is involved in EGFR-induced NF-kB activation in GBM. MALT1 deficiency or inhibition significantly affected the proliferation, survival, migration and invasion of GBM cells both in vitro and in vivo. Moreover, MALT1 inhibition caused G1 cell cycle arrest by regulating multiple cell cycle-associated proteins. Mechanistically, MALTI inhibition blocks the degradation of IκBα and prevents the nuclear accumulation of the NF-κB p65 subunit in GBM cells. This study found that MALT1, a key signal transduction cascade, can mediate EGFR-induced NF-kB activation in GBM and may be potentially used as a novel therapeutic target for GBM.

Inhibition of chromosomal region maintenance 1 suppresses the migration and invasion of glioma cells via inactivation of the STAT3/MMP2 signaling pathway.

Chromosomal region maintenance 1 (CRM1) is associated with an adverse prognosis in glioma. We previously reported that CRM1 inhibition suppressed glioma cell proliferation both in vitro and in vivo. In this study, we investigated the role of CRM1 in the migration and invasion of glioma cells. S109, a novel reversible selective inhibitor of CRM1, was used to treat Human glioma U87 and U251 cells. Cell migration and invasion were evaluated by wound-healing and transwell invasion assays. The results showed that S109 significantly inhibited the migration and invasion of U87 and U251 cells. However, mutation of Cys528 in CRM1 abolished the inhibitory activity of S109 in glioma cells. Furthermore, we found that S109 treatment decreased the expression level and activity of MMP2 and reduced the level of phosphorylated STAT3 but not total STAT3. Therefore, the inhibition of migration and invasion induced by S109 may be associated with the downregulation of MMP2 activity and expression, and inactivation of the STAT3 signaling pathway. These results support our previous conclusion that inhibition of CRM1 is an attractive strategy for the treatment of glioma.

Mrpl10 and Tbp Are Suitable Reference Genes for Peripheral Nerve Crush Injury.

Peripheral nerve injury triggers the dysregulation of a large number of genes at multiple sites, including neurons, peripheral nerve stump, and the target organ. Housekeeping genes were frequently used as reference genes to normalize the expression values of target genes. Suitable selection of housekeeping genes that are stably expressed after nerve injury minimizes bias elicited by reference genes and thus helps to better and more sensitively reflect gene expression changes. However, many housekeeping genes have been used as reference genes without testing the expression patterns of themselves. In the current study, we calculated the expression stability of nine commonly used housekeeping genes, such as 18S (18S ribosomal RNA), Actb (ß-actin), CypA (cyclophilin A), Gapdh (glyceraldehydes-3-phosphate dehydrogenase), Hprt (hypoxanthine guanine phosphoribosyl transferase), Pgk1 (phosphoglycerate kinase 1), Tbp (TATA box binding protein), Ubc (ubiquitin C), YwhaZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation), and four newly identified housekeeping genes, including Ankrd27 (Ankyrin repeat domain 27), Mrpl10 (mitochondrial ribosomal protein L10), Rictor (rapamycin-insensitive companion of mTOR, Complex 2), and Ubxn 11 (UBX domain protein 11), in both distal sciatic nerve samples and dorsal root ganglion (DRG) samples after sciatic nerve injury. Our results suggested that following peripheral nerve injury, Mrpl10 and Tbp might be used as suitable reference genes for sciatic nerve stump and DRGs, respectively.

Reflectance confocal microscopy versus dermoscopy for the diagnosis of cutaneous melanoma: a head-to-head comparative meta-analysis.

This meta-analysis aimed to evaluate the comparative diagnostic performance of reflectance confocal microscopy (RCM) and dermoscopy in detecting cutaneous melanoma patients. An extensive search was conducted in the PubMed and Embase databases to identify available publications up to December 2023. Studies were included if they evaluated the diagnostic performance of RCM and dermoscopy in patients with cutaneous melanoma. The quality of the included studies was assessed using the Quality Assessment of Diagnostic Performance Studies (QUADAS-2) tool. A total of 14 articles involving 2013 patients were included in the meta-analysis. The overall sensitivity of RCM was 0.94 [95% confidence interval (CI), 0.87-0.98], while the overall sensitivity of dermoscopy was 0.84 (95% CI, 0.71-0.95). These results suggested that RCM has a similar level of sensitivity compared with dermoscopy ( P  = 0.15). In contrast, the overall specificity of RCM was 0.76 (95% CI, 0.67-0.85), while the overall specificity of dermoscopy was 0.47 (95% CI, 0.31-0.63). The results indicated that RCM appears to have a higher specificity in comparison to dermoscopy ( P  < 0.01). Our meta-analysis indicates that RCM demonstrates superior specificity and similar sensitivity to dermoscopy in detecting cutaneous melanoma patients. The high heterogeneity, however, may impact the evidence of the current study, further larger sample prospective research is required to confirm these findings.

Selection of suitable internal controls for gene expression normalization in rats with spinal cord injury.

There is a lack of systematic research on the expression of internal control genes used for gene expression normalization in real-time reverse transcription polymerase chain reaction in spinal cord injury research. In this study, we used rat models of spinal cord hemisection to analyze the expression stability of 13 commonly applied reference genes: Actb, Ankrd27, CypA, Gapdh, Hprt1, Mrpl10, Pgk1, Rictor, Rn18s, Tbp, Ubc, Ubxn11, and Ywhaz. Our results show that the expression of Ankrd27, Ubc, and Tbp were stable after spinal cord injury, while Actb was the most unstable internal control gene. Ankrd27, Ubc, Tbp, and Actb were consequently used to investigate the effects of internal control genes with differing stabilities on the normalization of target gene expression. Target gene expression levels and changes over time were similar when Ankrd27, Ubc, and Tbp were used as internal controls but different when Actb was used as an internal control. We recommend that Ankrd27, Ubc, and Tbp are used as internal control genes for real-time reverse transcription polymerase chain reaction in spinal cord injury research. This study was approved by the Administration Committee of Experimental Animals, Jiangsu Province, China (approval No. 20180304-008) on March 4, 2018.

Paeonol-loaded PLGA nanoparticles as an oral drug delivery system: Design, optimization and evaluation.

Herein, we report a novel type of NPs by loading paeonol (Pae) into PLGA NPs, to enhance drug stability and oral bioavailability. The paeonol (Pae)-loaded polylactic-co-Gly-colic acid (PLGA) nanoparticles (Pae-PLGA-NPs) were prepared by nanoprecipitation method. The resultant NPs were in spherical shape with an average particle size around 237.7 ± 4.92 nm, and the PDI and zeta potential were 0.110 ± 0.01 and -25.33 ± 1.37 mV, respectively. The encapsulation efficiency (EE) and drug loading (DL) of the Pae-PLGA-NPs were 86.26 ± 1.12 and 12.74 ± 0.37% respectively. The in vitro drug release, in vivo pharmacokinetics and in situ single-pass intestinal perfusion (SPIPs) of Pae-PLGA-NPs was investigated. In vivo, the AUC(0-t), C max, MRT(0-t), and T1/2z of the Pae-PLGA-NPs group were 3.79-, 1.89-, 1.40- and 1.49-fold greater than those of the Pae suspension group, respectively. The in situ single-pass intestinal perfusion of NPs results showed the Ka values in the duodenum, jejunum, ileum and colon were 1.12-, 1.40-, 1.52- and 2.21-fold higher than those of Pae solution, respectively. Moreover, the Papp values of the ileum and colon were 1.27- and 1.31-fold higher than those of the solution group. Such findings suggested the Pae-PLGA-NPs can significantly improve the intestinal absorption characteristics, and have a beneficial effect on oral administration as a nanometer-sized carrier.

Risk factors for oral mucositis in patients with malignant tumors: a prospective cohort study.

BACKGROUND: To investigate the incidence and risk factors of oral mucositis in patients with malignant tumors. METHODS: A total of 74 patients with malignant tumors who were hospitalized in the Affiliated Hospital of Nantong University from January 2020 to December 2020 were selected and divided into two groups according to whether oral mucositis occurred (n=45) or not (n=29). Chi-square test was used to compare the general data between the two groups, and multivariate logistic regression analysis was used to investigate the risk factors of oral mucositis in patients with malignant tumors. RESULTS: Oral mucositis occurred in 45 of 74 malignant tumor patients (60.8%), and the incidence in patients with head and neck tumors was significantly higher than in those with chest and abdomen tumors (P<0.05). A significantly higher incidence was also seen in patients with poor oral cleanliness in comparison to those with high oral cleanliness; in radiotherapy patients in comparison to non-radiotherapy patients; in patients who received Nituzumab during radiotherapy in comparison to those who did not, and in patients receiving eight cycles of chemotherapy in comparison to those not receiving chemotherapy. Multivariate logistic regression analysis showed that oral cleanliness, radiotherapy, and duration of radiotherapy were independent risk factors for oral mucositis in patients with malignant tumors (P<0.05). CONCLUSIONS: Poor oral cleanliness, radiotherapy, and longer duration of radiotherapy lead to the occurrence of oral mucositis in patients with malignant tumors, and these risk factors should be targeted for intervention to reduce the occurrence of oral mucositis.

Seasonal and spatial variabilities of dissolved iron in southern Yellow Sea.

The southern Yellow Sea (SYS) is considered to be of the most prolific fishing grounds in the China Sea. In this study, the seasonal and spatial distributions of total dissolved iron (DFe) are investigated across four seasons in the SYS, from July 2013 to January 2016. This investigation showed that the DFe values of all samples exhibited seasonal variations: summer (1.7-15.8 nM), autumn (0.9-38.5 nM), winter (3.0-69.8 nM), and spring (3.0-100.2 nM). The DFe values in both surface and bottom waters also exhibited distinct temporal changes. The influence of water masses on the distribution of DFe as well as other factors, such as major nutrient concentrations of total dissolved nitrogen, total dissolved phosphate, and hydrologic factors, was investigated in this study. Based on the investigation of DFe and major nutrients in the SYS, the Yellow Sea Cold Water Mass was identified as the most conservable water mass in the study area. The results of this study further indicate that in winter, DFe0.4 µm (using a 0.4 µm filter) is considerably higher than DFe0.2 µm (using a 0.2 µm filter) in the surface water of the SYS coastal area. Therefore, the dissolution of colloidal Fe in this area had a significant effect on DFe.

Cubic and hexagonal liquid crystals as drug carriers for the transdermal delivery of triptolide.

The purpose of this study was to develop and evaluate triptolide-loaded cubic and hexagonal liquid crystals for transdermal drug delivery systems (TDDSs). We prepared and characterized triptolide-loaded lyotropic liquid crystals and evaluated for their percutaneous permeation properties in vitro and in vivo. We then used the adjuvant arthritic rat model and HaCaT cells to analyze the pharmacodynamics and conduct cell-stimulating studies of these liquid crystals. The optimized preparations were identified as cubic and hexagonal phase structures, respectively. Moreover, the in vitro percutaneous penetration studies demonstrated that compared to the homemade triptolide gel, cubic and hexagonal liquid crystals could significantly increase the percutaneous cumulative penetration of drugs within 48 h. Besides, the results of skin-blood synchronous microdialysis showed that the triptolide concentration in skin was higher than that in blood, and the cubic and hexagonal liquid crystals significantly increased the bioavailability of triptolide. Triptolide-loaded cubic and hexagonal liquid crystals presented excellent anti-arthritic effects, alleviating paw swelling and inhibiting inflammation by downregulating the levels of TNF-α and IL-1ß. In vitro cell-stimulating studies displayed that triptolide-loaded cubic and hexagonal liquid crystals exhibited no obvious toxicity, which exhibited that triptolide-loaded cubic and hexagonal liquid crystals were remarkable biocompatibility. Collectively, triptolide-loaded cubic and hexagonal liquid crystals represented a promising candidate for rheumatoid arthritis therapy.

The third-generation EGFR inhibitor AZD9291 overcomes primary resistance by continuously blocking ERK signaling in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is a fatal brain tumor, lacking effective treatment. Epidermal growth factor receptor (EGFR) is recognized as an attractive target for GBM treatment. However, GBMs have very poor responses to the first- and second-generation EGFR inhibitors. The third-generation EGFR-targeted drug, AZD9291, is a novel and irreversible inhibitor. It is noteworthy that AZD9291 shows excellent blood-brain barrier penetration and has potential for the treatment of brain tumors. METHODS: In this study, we evaluated the anti-tumor activity and effectiveness of AZD9291 in a preclinical GBM model. RESULTS: AZD9291 showed dose-responsive growth inhibitory activity against six GBM cell lines. Importantly, AZD9291 inhibited GBM cell proliferation > 10 times more efficiently than the first-generation EGFR inhibitors. AZD9291 induced GBM cell cycle arrest and significantly inhibited colony formation, migration, and invasion of GBM cells. In an orthotopic GBM model, AZD9291 treatment significantly inhibited tumor survival and prolonged animal survival. The underlying anti-GBM mechanism of AZD9291 was shown to be different from that of the first-generation EGFR inhibitors. In contrast to erlotinib, AZD9291 continuously and efficiently inhibited the EGFR/ERK signaling in GBM cells. CONCLUSION: AZD9291 demonstrated an efficient preclinical activity in GBM in vitro and in vivo models. AZD9291 has been approved for the treatment of lung cancer with good safety and tolerability. Our results support the possibility of conducting clinical trials of anti-GBM therapy using AZD9291.

Actin Cytoskeleton Affects Schwann Cell Migration and Peripheral Nerve Regeneration.

Actin cytoskeleton regulates many essential biological functions, including cellular development, shape, polarity, and motility. The organization of actin cytoskeleton has also been associated with numerous physiological and pathological conditions, for instance, the elongation of axonal growth cone during peripheral nerve regeneration. However, the spatio-temporal expression patterns of actin cytoskeleton-related genes and the specific roles of actin cytoskeleton following peripheral nerve injury have not been fully revealed. To address this question, we made rat sciatic nerve crush surgery, collected injured sciatic nerve stumps, analyzed RNA deep sequencing outcomes, and specifically studied two significantly involved canonical pathways that were related with actin, actin cytoskeleton signaling and regulation of actin-based motility by Rho. By using bioinformatic tools and qRT-PCR, We identified and validated differentially expressed genes in these two signaling pathways. Moreover, by applying actin polymerization inhibitor cytochalasin D to sciatic nerve crushed rats, we studied the in vivo effect of cytochalasin D and demonstrated that inhibiting actin polymerization would delay the migration of Schwann cells and hinder the repair and regeneration of injured peripheral nerves. Overall, our data revealed the changes of actin cytoskeleton-related genes following peripheral nerve injury and stated the importance of actin cytoskeleton during peripheral nerve regeneration.

Phytantriol-based lyotropic liquid crystal as a transdermal delivery system.

The purpose of this study was examined the feasibility of using phytantriol-based cubic and hexagonal liquid crystal preparation for the percutaneous administration of trans­cinnamaldehyde (TCA). TCA-loaded lyotropic liquid crystal formulations were prepared and characterized, their skin permeability in vitro and in vivo was evaluated. Preliminary pharmacodynamics were also investigated in adjuvant arthritics (AA) rats. The formulations were identified respectively as cubic and hexagonal structure. The in vitro permeability study exhibited that both cubic and hexagonal liquid crystal improved the cumulative permeation quantity and permeation rates of TCA compared with home-made gel. The results of an in vivo transdermal permeability experiment showed that the area under the curve [AUC(0-∞)] of the hexagonal and cubic liquid crystal was 1.62 and 1.53 times higher than that of the gel group, respectively. Preliminary pharmacodynamics studies indicated that the group of high-dose TCA-loaded (200 mg·kg-1) hexagonal liquid crystal was shown to inhibit the paw swelling of AA rats, improve synovial hyperplasia and inflammatory cell infiltration, and down-regulate the levels of serum interleukin (IL)­1ß and tumor necrosis factor (TNF)­α. Furthermore, there was no significant difference in the anti-inflammatory effects of TCA-loaded hexagonal liquid crystal and the commercially available product Voltaren® emulgel®. Thus, hexagonal liquid crystal was considered as an effective delivery system for TCA.

Dissolving Microneedles Integrated With Liquid Crystals Facilitate Transdermal Delivery of Sinomenine Hydrochloride.

The purpose of this study was to investigate the feasibility of delivering sinomenine hydrochloride (SH) transdermally using composite dissolving microneedles (DM) which integrated with liquid crystals (H2). The fabricated SH-loaded composite DM was evaluated for their appearance, mechanical strength, irritation, and efficiency of transdermal delivery of SH. Results depicted that optimized SH-loaded composite DM had sufficient mechanical strength to pierce into rat skin. The in vitro permeability study in rat skin was successfully performed using Franz diffusion cell which showed that the cumulative permeation of SH in the composite DM were significantly increased compared with H2 group (p <0.05), and the penetration rate of SH in the composite DM kept constant during 48 h compared with DM group. The study of in vivo pharmacokinetics effects showed that the transdermal absorption of SH was enhanced and sustained by the SH-loaded composite DM compared with SH-loaded H2 and SH-loaded DM. The skin irritation study in rats indicated that there were no irritation for normal skin, and the slight irritation for impaired skin recovered 24 h later. In conclusion, the novel composite DM may be a promising transdermal drug delivery system owing to its physical enhancing permeation function, drug reservoir effect, and biocompatibility.