Herein, we proposed novel three-in-one DNA nanowheels with simultaneous chemo and gene therapy to treat tumor, especially to prevent simultaneous drug resistance, which could be disassembled via a cascaded hybridization reactions triggered by the highly expressed microRNA in cancer cells for smart and efficient cancer therapy. Typically, with breast cancer as a model, microRNA 21 could trigger the self-disassembly of DNA nanowheel 1 via hybridization with a specially designed oligonucleotide (anti-microRNA 21) in DNA nanowheel 1, releasing another special oligonucleotide (Contact sequence) to trigger the self-disassembly of DNA nanowheel 2 with releasing of a special oligonucleotide (anti-Contact sequence) to trigger the self-disassembly of DNA nanowheel 1 cyclically, and thus the cascaded hybridization reactions with three-in-one anti-cancer functions could be generated based on three main therapeutic effects via releasing doxorubicin to inhibit macromolecular biosynthesis, antisense oligonucleotide of microRNA 21 to activate the apoptotic cell pathway and antisense oligonucleotide of MDR1 to prevent the drug resistance respectively. As expected, the proposed method showed improved therapeutic efficacy on the cancer cells with about 80 % apoptosis ratio, especially on the drug resistant cancer cells with about 75 % apoptosis ratio, compared with that in the conventional anti-cancer systems of about 70% on cancer cells and below 40 % on drug resistant cancer cells, respectively. Most importantly, this strategy opened the door for generation of complex functional DNA-based structures for target triggering drugs releasing system combining with chemo- and gene-therapy to generate tumor regression and prevent drug resistance with an optimized therapeutic efficacy, providing a new avenue for efficient cancer treatment, especially drug resistant cancers.

Purpose: Immune checkpoint inhibitors (ICIs) have become a standard therapy for recurrent or metastatic head and neck squamous cell carcinoma (R/MHNSCC), however, there are still unanswered questions about immunotherapy. Furthermore, immunotherapy for R/MHNSCC of the mainland Chinese population are lacking. The aim of this study is to evaluate the efficacy and safety of ICIs in real-world settings in China.Materials and methods: We retrospectively reviewed 59 patients with R/MHNSCC who received immunotherapy between May 2019 and December 2021. We assessed demographics, efficacy, survival and safety.Results: Fifty-nine patients were included in the study, all of whom had R/MHNSCC affecting the oral cavity, oropharynx, hypopharynx, larynx, nasal cavity, paranasal sinuses and metastatic cancer in the neck with an unknown primary. The objective response rate (ORR) for all patients was found to be 40.6 %. Out of these patients, 11 patients achieved a complete response and 13 achieved a partial response. The median progressionfree survival (PFS) was calculated to be 10.64 months (range: 1.15-29.24 months), while the median overall survival (OS) was 21.75 months (range 2.0-37.55 months). The addition of local radiotherapy resulted in higher ORR and PFS compared to previous reports. Notably, patients with R/MHNSCC in the paranasal sinuses and nasal cavity also showed benefits from immunotherapy. Additionally, patients who achieved stable disease (SD) had similar survival rates to those who achieved partial response (PR), indicating that SD is also an indicator of clinical benefit from immunotherapy. The overall incidence of immune-related adverse reactions in this study was low, with fatigue and rash being the most common side effects.Conclusion: These findings highlight the effectiveness and safety of immunotherapy for R/MHNSCC in a realworld setting in China. Further investigation is warranted to explore the potential benefits of incorporating local radiotherapy into the treatment of R/MHNSCC.

IntroductionMicroRNAs (miRNAs) have been recognized as promising diagnostic biomarkers for Diabetic Retinopathy (DR) due to their notable upregulation in individuals with the condition. However, the development of highly sensitive miRNAs assays for the rapid diagnosis of DR in clinical settings remains a challenging task.MethodsIn this study, we introduce an enhanced CRISPR/Cas12a assay, leveraging suboptimal PAM (sPAM)-mediated Cas12a trans-cleavage in conjunction with rolling circle amplification (RCA). sPAM was found to perform better than canonical PAM (cPAM) in the detection of Cas12a-mediated ssDNA detection at low concentrations and was used instead of canonical PAM (cPAM) to mediate the detection. The parameters of reactions have also been optimized.Results and discussionIn comparison with cPAM, sPAM has higher sensitivity in the detection of ssDNA at concentrations lower than 10 pM by Cas12a. By replacing cPAM with sPAM in the padlock template of RCA, ultra-high sensitivity for miR-183 detection is achieved, with a detection limit of 0.40 aM. within 25 min and a linear range spanning from 1 aM. to 1 pM. Our assay also exhibits exceptional specificity in detecting miR-183 from other miRNAs. Furthermore, the applicability of our assay for the sensitive detection of miR-183 in clinical serum samples is also validated. This study introduces a groundbreaking assay with excellent performance through a simple modification, which not only addresses existing diagnostic challenges, but also opens exciting new avenues for clinical diagnosis in the realm of DR.

The utilization of immune checkpoint inhibitors (ICIs) has received authorization for the treatment of head and neck squamous cell carcinoma (HNSC) and various other tumor types. However, a large proportion of HNSC patients exhibit unresponsiveness to ICIs. Dickkopf-1, a secreted protein with pivotal functions in Wnt signaling pathways, is increasingly recognized for its role in modulating anti-tumor immunity. In this study, the authors employed TCGA and GEO datasets to investigate the correlation between tumoral DKK1 expression and survival outcomes, as well as the suppressive nature of the tumor immunosuppressive microenvironment (TIME) in HNSC patients. The findings reveal a strong association between elevated DKK1 expression in HNSC tumors and poorer survival rates, along with an immunosuppressive TIME. In vivo studies utilizing subcutaneous tumor graft mouse models demonstrate that knockdown of DKK1 inhibit the growth of HNSC tumor cells in a CD8(+) T cell-dependent manner. Additionally, an inverse relationship between DKK1 expression and CD8(+) T cell infiltration and activation is observed, while a positive correlation is observed with infiltration of myeloid-derived suppressor cells (MDSCs) and CD8(+) T cell exhaustion. Moreover, combining DKK1 knockdown with neutralizing antibodies against PD-1 significantly suppress the growth of HNSC cells. Consequently, The study proposes DKK1 as a promising target for immunotherapy to enhance PD-1 blockade therapy in HNSC. These findings show that DKK1 can thus be regarded as a potential target for immunotherapy and need further study.

PurposeIn this study, the aim was to comprehensively analyze the current status, hotspots, and trends of trans-oral endoscopic thyroidectomy (TOET) through bibliometric analysis and by presenting the field atlas.MethodsWeb of Science Core Collection database was adopted to screen studies regarding TOET published between January 1, 2008 and August 1, 2022. The evaluation covered the criteria total number of studies, keywords, and contributions from countries/regions, institutions, journals, and authors.ResultsA total of 229 studies were covered. SURGICAL ENDOSCOPY AND OTHER INTERVENTIONAL TECHNIQUES is the largest publication in the field of TOET. The three countries that contributed the most studies were Korea, China, and the USA. The most frequently occurring core keywords in the field of TOET are vestibular approach, outcomes, experience, safety, robotic thyroidectomy, scar, video-assisted thyroidectomy and quality-of-life. The seven clusters were generated in this study: intraoperative monitoring of the laryngeal return nerve (# 0), learning curve (# 1), postoperative quality of life (# 2), central lymph node dissection and safety (# 3), complications (# 4), minimally invasive surgery (# 5), and robotic surgery (# 6).ConclusionThe main research topics in the field of TOET place focuses on learning curves, laryngeal nerve monitoring, carbon dioxide gas bolus, chin nerve injury, surgical complications, and surgical safety. In the future, more academics will focus on the safety of the procedure and reducing complications..

The identification of a lower boundary for the central lymph node (CLN) of the neck in total endoscopic thyroidectomy via the areola approach (ETA) is important for its radical dissection. We found that resection of the suprasternal fossa fat (SFF) was beneficial for exposing the lower boundary and preventing suprasternal swelling after the operation. This retrospective analysis included 470 papillary thyroid carcinoma (PTC) cases, with some treated by unilateral lobectomy, some by central lymph node dissection (CLND) through ETA (n = 193), and the others by conventional open thyroidectomy (COT, n = 277). The main observation indicators included the total number of CLNs, CLND operative time, visualization of the upper pole of the thymus before removing the CLN, and postoperative suprasternal swelling. The SFF retention group and COT group had a similar percentage of women (78.65% vs. 79.42%, P = 0.876) lower than that in the SFF resection group (95.19%, P < 0.001). The percentage of the visualized upper pole of the thymus before CLN removal was notably higher in the SFF resection group than that in the SFF retention group (63.46 vs. 29.21%, P < 0.001) but notably lower than that in the COT group (63.46% vs. 100%, P < 0.001). A total of 43.82% and 23.1% of patients in the SFF retention and COT groups showed suprasternal swelling, respectively. No patient exhibited such swelling in the SFF resection group (23.1% vs. 0, P < 0.001). Resection of SFF in ETA easily identified the lower boundary for CLND and prevented suprasternal fossa swelling.

Today, a lot of attention is being paid to the pre-miRNAs/miRNAs or activity of Dicer due to their important functions in various physiological processes. Especially, the intrinsic relationship among these associated targets is of significant importance for more in-depth research on the mechanism of disease formation and early diagnosis. Herein, a strategy for simultaneous bioanalysis of miRNAs/pre-miRNAs and Dicer enzyme based on the self-designed multi-path nucleic acid amplification technology was proposed. Typically, in the presence of pre-miRNA-155, it can hybridize with Helper to generate a structure with two new toeholds, one of which could react with H1, H2, and H3, performing a modified CHA reaction with obvious fluorescence responses of FAM, and another of which could hybridize with H4, H5, and H6 to construct the [H4-H5-H6]n DNA nanosphere with obvious fluorescence responses of Cy5. Similarly, miRNA-155 could just hybridize with H1, H2, and H3 to generate the same modified CHA reaction with obvious fluorescence responses of FAM. Due to the successful multi-path nucleic acid amplification, the proposed bioanalysis strategy could be successfully employed for miRNA-155 and pre-miRNA-155 analysis in the range from 500 pM to 100 nM and 1 to 300 nM, respectively. The proposed strategy could be applied to explore another inter-related nucleic acid relationship also, providing great potential in bioanalysis of various nucleic acids.

Aims/Introduction To investigate the association of subtle alterations in thyroid function with presarcopenia among patients with type 2 diabetes mellitus. Materials and Methods A total of 1,865 adult patients with type 2 diabetes mellitus were enrolled in this cross-sectional study, excluding patients with overt thyroid dysfunction. Skeletal muscle mass measured by dual energy X-ray absorptiometry was used to assess presarcopenia. Logistic regression models were used to estimate the effects of thyroid hormones on presarcopenia, and subgroup analyses were carried out in different strata of age, sex and body mass index, respectively. Results Compared with the euthyroid group (Euthy), the subclinical hyperthyroidism group had an increased odds of presarcopenia (multivariate-adjusted odds ratio 1.99, 95% confidence interval 1.09-3.63), but the subclinical hypothyroidism group did not (P > 0.05). In the subclinical hyperthyroidism group, age and body mass index <24 kg/m(2) were independent risk factors for presarcopenia. In the overall Euthy group, an increased odds of presarcopenia was correlated with the elevated free thyroxine : free triiodothyronine ratio (all P for trend <0.05), whereas not with increment in free triiodothyronine level (P for trend >0.05). Additionally, in Euthy subgroup analyses stratified by middle-age, sex and body mass index, a similar association was noted (all P for trend <0.05), but not in the older-aged patients (P for trend >0.05). Conclusions Subclinical hyperthyroidism was an independent risk factor for presarcopenia in patients with type 2 diabetes mellitus, but subclinical hypothyroidism was not. In the Euthy group with type 2 diabetes mellitus, a high free thyroxine : free triiodothyronine ratio was a good index of presarcopenia in addition to older age.

Biomechanical factors play an important role in tumor distribution, epithelial-mesenchymal transition (EMT), invasion and other important processes. Despite fewer reports investigating biomechanical function in papillary thyroid carcinoma (PTC), a large number of PTC cases are located close to the trachea and the majority of advanced cases of PTC have been associated with invasion of the trachea. However, the effect of trachea stiffness on PTC distribution and growth remains unknown. To clarify this issue, two types of PTC cells (TPC-1 and KTC-1) were seeded on a substrate with different stiffness to observe cell proliferation and movement. To identify the effect of trachea stiffness on the thyroid, two thyroid lobes (left and right) were evenly divided into interior (close to the trachea) and lateral (away from the trachea) parts, based on the vertical line between the trachea and thyroid lateral margin with different von Mises stress values. As PTC originates from papillary thyroid microcarcinoma (PTMC) with a maximum diameter of <1 cm, the present study selected PTMC as the study subject to reflect initial PTC distribution in the thyroid. The association between the percentage of PTMC distribution in different parts of the thyroid and von Mises stress values was analyzed. Both PTC cells exhibited stronger proliferation and mobility on the stiff substrate compared with that on the soft substrate. Furthermore, the results of finite element analysis revealed that the von Mises stress values of the interior parts of the trachea were notably higher compared with that in the lateral parts. PTMC distribution in the interior trachea was notably greater compared with that in the lateral section. There was also an observed association between von Mises stress values and PTMC distribution. In addition, the results of RNA-sequencing and reverse transcription-quantitative PCR demonstrated that three biomechanical genes were overexpressed in PTMC located in the interior section compared with that in adjacent normal tissue, and the related signaling pathways were also activated in these tissues. On the whole, these results indicated that trachea stiffness may supply a suitable biomechanical environment for PTMC growth, and the related biomechanical genes may serve as novel targets for PTMC diagnosis and prognostic estimation.

Bone resorption is mainly mediated by osteoclasts (OCs), whose formation and function are regulated by intracellular Ca2+ oscillation. Our previous studies demonstrated that fluid shear stress (FSS) lead to Ca2+ oscillation through mechanosensitive cation-selective channels. However, the specific channels responsible for this FSS-induced Ca2+ oscillation remain unknown. In the present study, we examined the expression of several Ca2+ channels in OCs, including STIM1, ORAI1, TRPV1, TRPV4, TRPV5, and TRPV6, by western blotting and reverse transcription-polymerase chain reaction. The results showed that STIM1 was highly expressed in early stage OCs, while TRPV4 was highly expressed in late stage OCs. We observed intracellular Ca2+ responses in OCs that were mechanically stimulated by FSS. When we blocked STIM1-dependent store-operated Ca2+ entry or inhibited TRPV4 using siRNA or drug inhibition, FSS-induced Ca2+ oscillations were almost undetectable in early and late stage OCs, respectively. These results indicate that STIM1 and TRPV4 act as mechanical transduction channels for OCs during the early and late differentiation stages, respectively, suggesting that these calcium channel could serve as markers of osteoclastogenesis or bone resorption.

A sensitive and highly efficient approach to monitor the expression of proteins on live cells was urgently needed to demonstrate its factor and mechanism and most important for clinical diagnostics and molecular biology. Herein, we developed a simple and highly efficient strategy, nonlinear hybridization chain reaction (nonlinear HCR), for the sensitive determination of proteins on live cells with transient receptor potential vanilloid 4 (TRPV4) and RAW264.7 cells as a model. Unlike the normal hybridization chain reaction (HCR) with multiplicative amplification, an exponential amplified fluorescent response could be obtained in theory based on the proposed nonlinear HCR. As a result, the nonlinear HCR generated a significant enhancement about 3 times compared with the normal HCR and 10 times compared with the directly immunofluorescence assay. Based on the proposed nonlinear HCR, the fluorescent signals increased with the concentration of TRPV4 in the range from 10 pg/mL to 100 ng/mL with a detection limit of 2.8 pg/mL, which would be useful for the sensitive detection of proteins in cell lysis or on cell surface. At the same time, the significant improvements via nonlinear HCR were achieved in the fluorescent imaging system compared with traditional immunofluorescence staining and normal HCR, proving the significant value of nonlinear HCR-based amplification strategy. Success in the establishment of the highly efficient nonlinear HCR strategy offered a simple and sensitive approach to demonstrate the concentration of special proteins on cell and other proteins and nucleotide potentially, revealing a simple and efficient technology for research fields of clinical diagnostics and molecular biology.

Metastasis is a major cause of poor prognosis in patients suffered with salivary adenoid cystic carcinoma (SACC), in which many factors are implicated. In this study, we identified that IGFBP2, overexpressed in SACC, correlated positively with perineural invasion or metastasis and indicated worse outcome. Moreover, IGFBP2 overexpression could dramatically improve motility and invasion capacity of SACC cells in vitro. Mechanically, IGFBP2 enhanced expression of ZEB1 in a NF-κB (p65)-dependent manner and then promoted epithelial-mesenchymal transition (EMT) in SACC. In addition, IGFBP2 mutation in the nuclear localization signal could impede nuclear translocation of p65, lower ZEB1 expression, and abrogate the EMT process. In xenograft models, IGFBP2 overexpression promoted lung and liver metastases of SACC cells; while if nuclear IGFBP2 was reduced, the formation of metastases in lung and liver was weakened. Together, these results for the first time demonstrate that IGFBP2 plays an important role in invasion and metastasis of SACC through the NF-κB/ZEB1 signaling pathway and IGFBP2 may be a novel biomarker and target for SACC.

Homeobox (HOX) transcript antisense RNA (HOTAIR), a long nuclear-retained noncoding RNA (lncRNA), is overexpressed in a variety of human cancers. Increasing evidence shows that HOTAIR plays a vital role in cancer initiation and progression by affecting cell cycle progress, apoptosis and invasion. However, whether HOTAIR serves as a target of therapeutic potential and the underlying mechanism in head and neck squamous cell carcinoma (HNSCC) is still unclear. Thus, we employed a HOTAIR specific siRNA to deplete its expression in two human HNSCC cell lines, Tca8113 and Tscca. The flow cytometry (FCM) analysis showed that HOTAIR depletion induced tumor cell apoptosis in vitro. JC-1 probe examination showed that the mitochondrial membrane potential was changed significantly by HOTAIR blockage. Mitochondrial calcium uptake 1(MICU1) dependent cell death was induced by HOTAIR depletion. Protein expression analysis indicated that mitochondrial related cell death pathway (Bcl-2, BAX, Caspase-3, Cleaved Caspase-3, Cytochrome c) involved in HOTAIR dependent apoptosis process. Moreover, a Tscca derived xenograft tumor model was employed to further validate that injection of HOTAIR siRNA inhibited tumor growth. In summary, we suggested that HOTAIR inhibition could be developed as a new therapeutic in HNSCC treatments.

Abnormalities in signal transducer and activator of transcription 3 (STAT3) are involved in the oncogenesis of oral squamous cell carcinoma (OSCC). MicroRNA-21 (miR-21) is an important gene expression regulator to OSCC. miR-21 induction by STAT3 has been reported in multiple human cancers. In the present study, we found that STAT3 (-/p) expression was positively correlated with miR-21 in 60 OSCC samples. A reporter gene assay showed that miR-21 overexpression was dependent on STAT3 activation. WP1066, a small molecular inhibitor of STAT3, was used to suppress STAT3 expression in OSCC cells. TSCCA and TCA8113 showed reduction in tumor cell proliferation, invasion ability and miR-21 expression by WP1066 treatment. In addition, the expression of miR-21 target proteins [programmed cell death 4 (PDCD4), tissue inhibitor of metalloproteinase 3 (TIMP-3) and phosphatase and tensin homolog (PTEN)] was upregulated. Restored STAT3 expression by IL-6 induced miR-21 overexpression, which further confirmed the correlation between STAT3 and miR-21. WP1066 inhibited tumor growth and induced tumor cell apoptosis in the TSCCA xenograft tumor model. Western blotting and immunohistochemistry staining indicated that STAT3 (-/p), Ki67, Bcl-2 and MMP-2 expressions decreased in the WP1066-treated group; PDCD4, TIMP-3 and PTEN expression increased simulta-neously. The present study provides evidence that targeting STAT3 could regulate OSCC cell growth in a miR-21-dependent manner and WP1066 could be a novel candidate drug to treat OSCC by inhibiting STAT3/miR-21 axis.

Connexin 43 (Cx43), a gap junctional protein, regulates osteocyte viability, and modulates mechanical stimulation-induced bone remodeling. However, the underlying mechanisms of its action remain unclear. In the current study, osteocyte-like MLO-Y4 cells were exposed to fluid shear stress (FSS) of 16 (physiological) or 30 (high) dyne/cm(2) for the indicated time points. Cx43 gene (Gja1) was silenced using siRNA or the protein was blocked chemically. The signaling molecules related to osteocyte apoptosis, osteogenesis, or osteoclastogenesis were detected at mRNA or protein levels. The results showed that physiological FSS significantly upregulated Cx43, which further inhibited apoptosis pathways (e.g., caspase-3) and osteoclastogenesis signaling (e.g., RANKL), but activated osteogenesis signaling (Sost/sclerostin). Suppressing Cx43 gene (Gja1) by siRNA or chemically blocking gap junction communication enhanced caspase-3, RANKL, and Sost/sclerostin, which could be restored with physiological FSS over 8 h. In addition, high FSS decreased Cx43 expression and adversely affected signaling molecules compared with physiological FSS. The findings indicate the involvement of Cx43 in mechanotransduction of FSS and in the modulation of mechanical loading-related apoptosis, osteogenesis, and osteoclastogenesis of osteocytes. This may provide a cellular and molecular basis for interpreting the biomechanical mechanism of bone absorption and remodeling.