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CRISPR/Cas systems have found widespread applications in gene editing due to their high accuracy, high programmability, ease of use, and affordability. Benefiting from the cleavage properties (trans- or cis-) of Cas enzymes, the scope of CRISPR/Cas systems has expanded beyond gene editing and they have been utilized in various fields, particularly in live-cell imaging and bioanalysis. In this review, we summarize some fundamental working mechanisms and concepts of the CRISPR/Cas systems, describe the recent advances and design principles of CRISPR/Cas mediated techniques employed in live-cell imaging and bioanalysis, highlight the main applications in the imaging and biosensing of a wide range of molecular targets, and discuss the challenges and prospects of CRISPR/Cas systems in live-cell imaging and biosensing. By illustrating the imaging and bio-sensing processes, we hope this review will guide the best use of the CRISPR/Cas in imaging and quantifying biological and clinical elements and inspire new ideas for better tool design in live-cell imaging and bioanalysis.

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Introduction: Oral Banzhilian formula (BZLF) is effective in the clinical treatment of psoriasis. However, the effectiveness and mechanism of different drug delivery routes deserve further study. Methods: First, we established the mouse model of psoriasis using imiquimod (IMQ), and high-performance liquid chromatography (HPLC) was used for the quality control of BZLF. Secondly, Total RNA Sequencing and bioinformatics analysis were used to explore the regulatory mechanism of BZLF in improving psoriatic lesions. Finally, further verification was based on animal experiments. Results: we externally applied BZLF for skin lesions in an imiquimod-induced psoriasis mouse model and found that BZLF alleviated psoriasis-like skin lesions while inhibiting the expression of Ki67 and inflammatory factors (Il17a, Tnf-α, S100a7 and Cxcl1) in skin lesions. Transcriptome sequencing results suggested that BZLF inhibited signalling pathways closely related to psoriatic inflammation, such as the IL-17 signalling pathway, chemokine signalling pathway, TNF signalling pathway, and NF-kappa B signalling pathway, and the protein-protein interaction (PPI) network identified LCN2 as one of the core target genes and screened out its regulated downstream gene MMP9. Discussion: Our findings suggest that the anti-psoriatic mechanism of BZLF involved in downregulating the LCN2/MMP-9 axis.

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High drug resistance, which is usually mediated by drug resistance­associated genes, is a characteristic of human tumours. CD44s, an ATP­binding cassette multidrug resistance transporter, is expressed in a variety of human cancers. In the present study, the effect of CD44s expression was investigated on BCC resistance against vismodegib. Lentiviral vectors were constructed to allow efficient CD44s expression. Cell clones expressing the CD44s construct were selected and expanded and then identified using qRT­PCR and western blotting. A lentiviral vector containing a blank sequence was used as a control. Cellular growth capacity and cell sensitivity to vismodegib were detected by MTT and Transwell assays, respectively. BCC cell growth was evaluated in vivo with a transplanted BCC nude mouse model. The cell clones expressing CD44s at high levels were identified by qRT­PCR and western blotting, and the difference in the cell proliferation rate between these cells and LV­CON BCC cells was assessed by growth curve analysis. The in vitro study revealed that treatment with vismodegib decreased BCC cell growth and migration; however, these effects were reversed by LV­CD44s overexpression. The in vivo study revealed that BCC tumour growth was significantly increased in nude mice transplanted with cells stably infected with CD44s compared with nude mice transplanted with cells infected with a control vector. Our investigation demonstrated that lentivirus­mediated CD44s expression may reverse the effects of vismodegib treatment on BCC.

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Retinoic acid (RA), the bioactive metabolite of vitamin A, has demonstrated efficacy in the treatment of photoaged skin; however, the mechanism of action of RA remains unclear. The aim of the present study was to examine whether the therapeutic effects of RA on photoaged skin are mediated by retinoic acid receptor (RAR) and/or retinoid X receptor (RXR) in mice, and to investigate the underlying mechanism. Photoaged skin in Imprinting Control Region mice was induced by repeated exposure to ultraviolet (UV) irradiation. Mice were randomly divided into nine groups: Normal; UV control; all­trans retinoic acid (ATRA); ATRA + RAR antagonist; ATRA + RXR antagonist; RAR agonist; RAR agonist + RAR antagonist; RXR agonist; and RXR agonist + RXR antagonist. Masson's trichrome staining was used to examine skin collagen fibers. Hydroxyproline assays were used to determine collagen content. The protein expression of matrix metalloproteinase (MMP)­3, MMP­13, type I procollagen, c­Jun and c­Fos was detected using western blot analysis. The results demonstrated that ATRA and RAR agonist ameliorated the UV­induced damage to skin collagen fibers, and increased the collagen content in photoaged skin through RAR. Furthermore, ATRA and RAR agonist stimulated type I procollagen protein expression, and inhibited MMP­3, MMP­13 and c­Jun protein expression through RAR in photoaged skin. However, ATRA and RAR agonist exhibited no significant effect on the protein expression of c­Fos in photoaged skin. These findings suggest that RA ameliorates photoaged skin through a RAR­mediated signaling pathway in mice.

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Alterations in the levels and functions of microRNAs (miRs) have been associated with carcinogenesis. In this study, we investigated the role and underlying mechanism of miR-4262 in the proliferation of human cutaneous malignant melanoma (CMM) cells. The expression levels of miR-4262 were significantly upregulated in cancerous tissues compared with those in matched adjacent normal tissues from 110 CMM patients. miR-4262 was also regulated in five types of CMM cell lines, displaying an opposite expression pattern to that of Kruppel-like 6 (KLF6), a proven tumor suppressor in several cancers other than CMM. KLF6 overexpression sharply reduced A375 cell proliferation, suppressed the activation of epidermal growth factor receptor (EGFR) and increased p21 expression levels, while knockdown of KLF6 by siRNA transfection had an opposite effect. Furthermore, KLF6 was proven to be a direct target gene of miR-4262 by bioinformatic analysis and KLF6­3'UTR luciferase reporter assay. Finally, our data on miR-4262 mimic and inhibitor transfection indicated that miR-4262 could markedly reduce the expression of KLF6 protein and had a stimulatory effect on A375 cell proliferation. Our findings indicate that KLF6 acts as a tumor suppressor in CMM cells and miR-4262 promotes the proliferation of CMM cells through KLF6-mediated EGFR inactivation and p21 upregulation.

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Objective. The objective of this study was to systematically evaluate the association between vitiligo and human leukocyte antigen- (HLA-) A. Methods. PubMed, Embase, Web of Science, Chinese National Knowledge Infrastructure, and reference lists were searched for relevant original articles. Results. Nineteen case-control studies comprising 3042 patients and 5614 controls were included, in which 33 HLA-A alleles were reported. Overall, three alleles (HLA-A⁎02, A⁎33, and Aw⁎31) were significantly associated with increased risk of vitiligo, two (HLA-A⁎09 and Aw⁎19) were associated with decreased risk, and the remaining 28 were unassociated. Twelve alleles, seven alleles, and 19 alleles were common to three ethnicities, both types of vitiligo, and both typing methods, respectively. In the subgroup analysis by ethnicity and typing methods, the association of six alleles and five alleles was inconsistent in three populations and both typing methods, respectively. In the subgroup analysis by clinical type, the association of all seven alleles was consistent in both types of vitiligo. Conclusion. The meta-analysis suggests that HLA-A⁎02, A⁎33, and Aw⁎31 are associated with increased risk of vitiligo, while HLA-A⁎09 and Aw⁎19 are associated with decreased risk of vitiligo. The association of some alleles varies in terms of ethnicity and typing methods.

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Malignant melanoma is the deadliest form of all skin cancers. Recently, microRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by targeted repression of transcription and translation and play essential roles during cancer development. Our study showed that miR-135a is upregulated in malignant melanoma tissues and cell lines by using Real-time PCR assay. Enforced expression of miR-135a in malignant melanoma cells promotes cell proliferation, tumorigenicity, and cell cycle progression, whereas inhibition of miR-135a reverses the function. Additionally, we demonstrated FOXO1 is a direct target of miR-135a and transcriptionally down-regulated by miR-135a. Ectopic expression of miR-135a led to downregulation of the FOXO1 protein, resulting in upregulation of Cyclin D1, and downregulation of P21(Cip1) and P27(Kip1) through AKT pathway. Our findings suggested that miR-135a represents a potential onco-miRNA and plays an important role in malignant melanoma progression by suppressing FOXO1 expression.

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The present study was designed to investigate the effects of ketamine on lipopolysaccharide (LPS)-induced depressive-like behavior and the expression of inflammatory cytokines in the rat prefrontal cortex. Thirty male Wistar rats were randomly divided into 3 groups (n=10): saline group (S group), LPS only group (L group) and LPS plus ketamine group (LK group). A forced swimming test (FST) was performed. On the first day, rats were placed into water for 15 min. Twenty-four hours later, rats were treated again as in the first test for a 5 min session, and the immobility time was recorded. The prefrontal cortex was harvested for the determination of the interleukin (IL)­1ß, IL-6 and IL-10 levels. Compared with the S group, rats in the L group had significantly increased immobility time in the FST and expression of IL-1ß and IL-6, and significantly decreased expression of IL-10 in the prefrontal cortex (P<0.05). However, rats in the LK group had significantly decreased immobility times in the FST and expression of IL-1ß and IL-6, and significantly increased expression of IL-10 in the prefrontal cortex compared with the L group (P<0.05). Ketamine can alleviate LPS-induced depressive-like behavior, and its effect is likely associated with changes in the expression of inflammatory cytokines in the rat prefrontal cortex.

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Several lines of evidence have indicated that rapamycin acts as an inhibitor of mammalian target of rapamycin (mTOR) and this produces therapeutic benefits as a treatment for Alzheimer's disease (AD) by activating an autophagic pathway. Similarly, postoperative cognitive dysfunction (POCD) is a decline in cognitive function for weeks or months after surgery. POCD and AD are both characterized by cognitive dysfunction, and more importantly, are both related to aging. We therefore hypothesized that rapamycin may have a therapeutic effect to relieve POCD. Inhibition of mTOR induces autophagic effect, thereby leading to a slower aging process, so this would be a novel target for the prevention and treatment of POCD.

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AIM: The present study aimed to elucidate the role of T-subtype calcium channels (Cav3.1, Cav3.2, and Cav3.3) in the pathogenesis of neuropathic pain at spinal level. METHODS: The chronic compression of the dorsal root ganglion (CCD) rat model was adopted. The antisense oligonucleotide of Cav3.1, Cav3.2, and Cav3.3 or normal saline (NS) were intrathecally administered twice per day from the first day to the fourth day after operation. Paw mechanical withdrawal threshold and paw thermal withdrawal latency were measured to evaluate the tactile allodynia and thermal hyperalgesia, respectively. RESULTS: CCD rats developed reliable tactile allodynia and thermal hyperalgesia after operation. Intrathecal administration of antisense oligonucleotide of Cav3.2 and Cav3.3 significantly relieved tactile allodynia and thermal hyperalgesia in CCD rats, but not Cav3.1. CONCLUSION: Cav3.2 and Cav3.3 subtype calcium channels in the spinal cord may play an important role in the pathogenesis of neuropathic pain, which may contribute to the management of the neuropathic pain.

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