Aligning tumor mutational burden (TMB) quantification across diagnostic platforms: phase II of the Friends of Cancer Research TMB Harmonization Project.

BACKGROUND: Tumor mutational burden (TMB) measurements aid in identifying patients who are likely to benefit from immunotherapy; however, there is empirical variability across panel assays and factors contributing to this variability have not been comprehensively investigated. Identifying sources of variability can help facilitate comparability across different panel assays, which may aid in broader adoption of panel assays and development of clinical applications. MATERIALS AND METHODS: Twenty-nine tumor samples and 10 human-derived cell lines were processed and distributed to 16 laboratories; each used their own bioinformatics pipelines to calculate TMB and compare to whole exome results. Additionally, theoretical positive percent agreement (PPA) and negative percent agreement (NPA) of TMB were estimated. The impact of filtering pathogenic and germline variants on TMB estimates was assessed. Calibration curves specific to each panel assay were developed to facilitate translation of panel TMB values to whole exome sequencing (WES) TMB values. RESULTS: Panel sizes >667 Kb are necessary to maintain adequate PPA and NPA for calling TMB high versus TMB low across the range of cut-offs used in practice. Failure to filter out pathogenic variants when estimating panel TMB resulted in overestimating TMB relative to WES for all assays. Filtering out potential germline variants at >0% population minor allele frequency resulted in the strongest correlation to WES TMB. Application of a calibration approach derived from The Cancer Genome Atlas data, tailored to each panel assay, reduced the spread of panel TMB values around the WES TMB as reflected in lower root mean squared error (RMSE) for 26/29 (90%) of the clinical samples. CONCLUSIONS: Estimation of TMB varies across different panels, with panel size, gene content, and bioinformatics pipelines contributing to empirical variability. Statistical calibration can achieve more consistent results across panels and allows for comparison of TMB values across various panel assays. To promote reproducibility and comparability across assays, a software tool was developed and made publicly available.

CCL20-CCR6 axis directs sperm-oocyte interaction and its dysregulation correlates/associates with male infertility‡.

The interaction of sperm with the oocyte is pivotal during the process of mammalian fertilization. The limited numbers of sperm that reach the fallopian tube as well as anatomic restrictions indicate that human sperm-oocyte encounter is not a matter of chance but a directed process. Chemotaxis is the proposed mechanism for re-orientating sperm toward the source of a chemoattractant and hence to the oocyte. Chemokines represent a superfamily of small (8-11 kDa), cytokine-like proteins that have been shown to mediate chemotaxis and tissue-specific homing of leukocytes through binding to specific chemokine receptors such as CCRs. Here we show that CCR6 is abundantly expressed on human sperms and in human testes. Furthermore, radioligand-binding experiments showed that CCL20 bound human sperm in a specific manner. Conversely, granulosa cells of the oocyte-surrounding cumulus complex as well as human oocytes represent an abundant source of the CCR6-specific ligand CCL20. In human ovaries, CCL20 shows a cycle-dependent expression pattern with peak expression in the preovulatory phase and CCL20 protein induces chemotactic responses of human sperm. Neutralization of CCL20 in ovarian follicular fluid significantly impairs sperm migratory responses. Conversely, analyses in infertile men with inflammatory conditions of the reproductive organs demonstrate a significant increase of CCL20/CCR6 expression in testis and ejaculate. Taken together, findings of the present study suggest that CCR6-CCL20 interaction may represent an important factor in directing sperm-oocyte interaction.

In vivo reflectance confocal microscopy in daily practice: Image features correlated to histopathology.

BACKGROUND: In vivo reflectance confocal microscopy (RCM) represents a promising technique for noninvasive visualization of skin lesions. In the clinical daily practice, doctors want to know the relationship between the RCM images and the skin pathological changes. OBJECTIVE: The aim of this study was to identify the basic skin pathological changes under RCM, and use RCM terminology to describe these pathological changes. METHODS: A total of 100 patients were recruited and were evaluated both by RCM and histopathologic examination. Ten healthy volunteers were also recruited as control. RCM examinations were done and biopsies of the lesions at the same site of RCM examination were performed for histopathology analysis. RESULTS: The pathological changes including hyperkeratosis, parakeratosis, acanthosis, papilloma, spongiosis, pustule, vacuolar degeneration, hyperpigmentation, changes of collagen fibers, and vascular changes can be imaged by RCM and corresponded well to their histopathology. RCM failed to find the atypical keratinocytes in two squamous cell carcinoma cases because of the hyperkeratosis and failed to find the vascular changes in one port wine stain cases because of the limitation of detecting depth. CONCLUSION: Features correlating well to histopathology are observed on RCM. RCM can be used as an auxiliary diagnosis tool for the clinical diagnosis.

Time-Bin-Encoded Boson Sampling with a Single-Photon Device.

Boson sampling is a problem strongly believed to be intractable for classical computers, but can be naturally solved on a specialized photonic quantum simulator. Here, we implement the first time-bin-encoded boson sampling using a highly indistinguishable (∼94%) single-photon source based on a single quantum-dot-micropillar device. The protocol requires only one single-photon source, two detectors, and a loop-based interferometer for an arbitrary number of photons. The single-photon pulse train is time-bin encoded and deterministically injected into an electrically programmable multimode network. The observed three- and four-photon boson sampling rates are 18.8 and 0.2 Hz, respectively, which are more than 100 times faster than previous experiments based on parametric down-conversion.

Galectins expressed differently in genetically susceptible C57BL/6 and resistant BALB/c mice during acute ocular Toxoplasma gondii infection.

Ocular toxoplasmosis (OT) caused by Toxoplasma gondii is a major cause of infectious uveitis, however little is known about its immunopathological mechanism. Susceptible C57BL/6 (B6) and resistant BALB/c mice were intravitreally infected with 500 tachyzoites of the RH strain of T. gondii. B6 mice showed more severe ocular pathology and higher parasite loads in the eyes. The levels of galectin (Gal)-9 and its receptors (Tim-3 and CD137), interferon (IFN)-γ, IL-6 and IL-10 were significantly higher in the eyes of B6 mice than those of BALB/c mice; however, the levels of IFN-α and -ß were significantly decreased in the eyes and CLNs of B6 mice but significantly increased in BALB/c mice after infection. After blockage of galectin-receptor interactions by α-lactose, neither ocular immunopathology nor parasite loads were different from those of infected BALB/c mice without α-lactose treatment. Although the expressions of Gal-9/receptor were significantly increased in B6 mice and Gal-1 and -3 were upregulated in both strains of mice upon ocular T. gondii infection, blockage of galectins did not change the ocular pathogenesis of genetic resistant BALB/c mice. However, IFN-α and -ß were differently expressed in B6 and BALB/c mice, suggesting that type I IFNs may play a protective role in experimental OT.

Predomination of IL-17-producing tryptase-positive/chymase-positive mast cells in azoospermic chronic testicular inflammation.

Chronic testicular inflammation and infection have been regarded as important factors in the pathogenesis of azoospermia. As key effector cells in innate and adaptive immune system, mast cells (MCs) were observed in inflammation and autoimmune disease. Furthermore, increased expression of tryptase-positive MCs has been reported in testicular disorders associated with male infertility/subfertility. However, little is known about the potential relationship between MCs and chronic testicular inflammation in azoospermic patients. Moreover, the preferential expression of MCs' subtypes in testis of these patients is still far from being understood. Thus, this study aimed to investigate characteristics of testicular MCs as well as their subtypes in azoospermic men with chronic testicular inflammation (AZI, n = 5) by immunohistochemical techniques. Our results showed significant increase of MCs in AZI, and more importantly, considerable numbers of tryptase-positive/chymase-positive MCs could also be demonstrated in AZI, when compared to control groups representing azoospermia without chronic testicular inflammation (AZW, n = 5) and normal spermatogenesis (NT, n = 5) respectively. Most interestingly, immunofluorescence staining revealed autoimmune-associated interleukin (IL)-17-producing MCs in AZI, whereas co-expression of MC markers with tumour necrosis factor (TNF)-α, IL-10 and IL-1ß could not be detected. In conclusion, AZI is associated with significant increase of tryptase-positive/chymase-positive MCs expressing IL-17, and these MCs might contribute to the pathogenesis of AZI.

Synergistic targeted therapy for acute promyelocytic leukaemia: a model of translational research in human cancer.

Acute promyelocytic leukaemia (APL), the M3 subtype of acute myeloid leukaemia, was once a lethal disease, yet nowadays the majority of patients with APL can be successfully cured by molecularly targeted therapy. This dramatic improvement in the survival rate is an example of the advantage of modern medicine. APL is characterized by a balanced reciprocal chromosomal translocation fusing the promyelocytic leukaemia (PML) gene on chromosome 15 with the retinoic acid receptor α (RARα) gene on chromosome 17. It has been found that all-trans-retinoic acid (ATRA) or arsenic trioxide (ATO) alone exerts therapeutic effect on APL patients with the PML-RARα fusion gene, and the combination of both drugs can act synergistically to further enhance the cure rate of the patients. Here, we provide an insight into the pathogenesis of APL and the mechanisms underlying the respective roles of ATRA and ATO. In addition, treatments that lead to more effective differentiation and apoptosis of APL cells, including leukaemia-initiating cells, and more thorough eradication of the disease will be discussed. Moreover, as a model of translational research, the development of a cure for APL has followed a bidirectional approach of 'bench to bedside' and 'bedside to bench', which can serve as a valuable example for the diagnosis and treatment of other malignancies.

Targeting adipose tissue via systemic gene therapy.

Adipose tissue has a critical role in energy and metabolic homeostasis, but it is challenging to adapt techniques to modulate adipose function in vivo. Here we develop an in vivo, systemic method of gene transfer specifically targeting adipose tissue using adeno-associated virus (AAV) vectors. We constructed AAV vectors containing cytomegalovirus promoter-regulated reporter genes, intravenously injected adult mice with vectors using multiple AAV serotypes, and determined that AAV2/8 best targeted adipose tissue. Altering vectors to contain adiponectin promoter/enhancer elements and liver-specific microRNA-122 target sites restricted reporter gene expression to adipose tissue. As proof of efficacy, the leptin gene was incorporated into the adipose-targeted expression vector, package into AAV2/8 and administered intravenously to 9- to 10-week-old ob/ob mice. Phenotypic changes were measured over an 8-week period. Leptin mRNA and protein were expressed in adipose and leptin protein was secreted into plasma. Mice responded with reversal of weight gain, decreased hyperinsulinemia and improved glucose tolerance. AAV2/8-mediated systemic delivery of an adipose-targeted expression vector can replace a gene lacking in adipose tissue and correct a mouse model of human disease, demonstrating experimental application and therapeutic potential in disorders of adipose.

Recent trend of pylephlebitis in Taiwan: Klebsiella pneumoniae liver abscess as an emerging etiology.

BACKGROUND AND AIMS: Pylephlebitis (septic thrombophlebitis of the portal venous system) is a rare complication of intra-abdominal infection. We aimed to investigate the recent trend of its etiology, clinical manifestation, and prognosis. METHODS: We retrospectively studied the etiology, clinical manifestation, and outcome by reviewing the medical records of all imaging-confirmed pylephlebitis cases diagnosed during the period 2002-2011 in a university hospital in Taiwan. To identify the risk factors for pylephlebitis, we randomly selected 160 patients with intra-abdominal infections but without pylephlebitis as the comparison group. RESULTS: We identified 35 cases of pylephlebitis. Most patients were men [29/35 (83 %)]. The median age of the patients was 57 years (range 35-90 years). Unspecified abdominal pain (18/35) and fever (10/35) were the most common clinical manifestations. Klebsiella pneumoniae liver abscess (7/35) and cholangitis (7/35) were the most common etiologies. Liver abscess was a risk factor for pylephlebitis (13/35 vs. 27/160, P = 0.01). With antibiotic therapy, there was no in-hospital mortality, but pylephlebitis was still associated with an excess hospital stay (22.2 ± 17.6 vs. 9.8 ± 7.1 days, P < 0.001). CONCLUSIONS: Our study results suggested a different pattern of pylephlebitis from previous Western literature. K. pneumoniae liver abscess (7/35) is an emerging etiology of pylephlebitis in Taiwan.

Smallest critical region for microcephaly in a patient with mosaic ring chromosome 13.

A ring chromosome 13 or r(13) exhibits breakage and reunion at breakage points on the long and short arms of chromosome 13, with deletions of the chromosomal segments distal to the breakage points. The r(13) chromosome accounts for approximately 20% of ring chromosomes compatible with life. We describe a female patient with mental retardation, growth retardation, microcephaly, craniofacial dysmorphy, hearing impairment, and prolonged prothrombin time. Chromosomal analysis via GTG banding of peripheral blood lymphocytes revealed a karyotype of 46,XX,r(13)(p13q34)[71]/45,XX,-13[12]/ 46,XX,dic r(13;13)(p13q34;p13q34)[9]/46,XX,-13,+mar[5]/47, XX,+r(13) (p13q34)x2[2]/46,XX[1] at the age of 6 years and 46,XX,r(13)(p13q34)[82]/45,XX,-13[14]/46,XX,dic r(13;13)(p13q34; p13q34)[2]/46,XX, -13,+mar[2]. Array comparative genomic hybridization analysis of the blood demonstrated a 4.37-Mb deletion on chromosome 13q [arr cgh 13q34q34(109,743,729-144,110,721)]. A cytogenetic study of peripheral blood revealed a rare chromosomal abnormality associated with different cell lines that included structural and numerical abnormalities of chromosome 13. This case, along with 14 previously reported cases, indicate that the smallest critical region for chromosome 13 microcephaly is 109,743,729-144,110,721.

Research trends of Janus Kinase inhibitors: a bibliometric and visualized study from 2012 to 2023.

OBJECTIVE: Janus Kinase (JAK) inhibitors have been extensively evaluated for their potential in the management of various diseases. Despite previous research on this topic, there is a lack of bibliometric analysis that summarizes research trends on JAK inhibitors. This study aims to provide a comprehensive overview of the top 100 most frequently cited studies on JAK inhibitors over the last ten years. MATERIALS AND METHODS: The Web of Science database was used to screen and extract relevant studies on JAK inhibitors. The top 100 studies most cited within the JAK inhibitor-related research were identified and evaluated, and various data such as the year of publication, study focus and keywords, author information, and number of citations were extracted and analyzed for further examination. RESULTS: In the top 100 most cited studies of JAK inhibitors, more than 70% of studies focused on the role of JAK inhibitors in disease treatments, with 42% of these studies focused on using JAK inhibitors as treatment for autoimmune diseases and 19 of them focused on the treatment of neoplasms. Time trend analysis revealed that the keywords "tofacitinib", "atopic dermatitis", and "rheumatoid arthritis" were widely mentioned in 2016, while new trends emerged in 2018, with "ruxolitinib" and "baricitinib" being more commonly mentioned. CONCLUSIONS: The top 100 most frequently cited studies on JAK inhibitors focused primarily on the safety and efficacy of these inhibitors in the management of various diseases, particularly inflammatory diseases and neoplasms. The results can serve as a valuable reference for rheumatologists and immunologists interested in the development of JAK inhibitors and expanding future research fields.

Facial asymmetry and clinical manifestations in patients with novel insertion of the TCOF1 gene.

This study explored the role of TCOF1 insertion mutations in Taiwanese patients with craniofacial anomalies. Twelve patients with single or multiple, asymmetrical congenital craniofacial anomalies were enrolled. Genomic DNA was prepared from leukocytes; the coding regions of TCOF1 were analyzed by polymerase chain reaction and direct sequencing. Clinical manifestations were correlated to the TCOF1 mutation. Six of 12 patients diagnosed with hemifacial microsomia exhibited a novel insertion mutation 4127 ins G (frameshift) in exon 24 in the TCOF1 gene. All six patients were diagnosed with anomalies on the left side. In addition, four of these six patients had hearing impairment; three had other major anomalies; and two had developmental delay. The insertion caused a frameshift, an early truncation, the loss of two putative nuclear localization signals (residues 1404-1420 and 1424-1440), and the loss of coiled coil domain (1406-1426) in treacle protein. These findings support the existence of two regulators of growth of the mandibular condyles.

Clinical characteristics and outcomes of bacteremia due to different genomic species of Acinetobacter baumannii complex in patients with solid tumors.

PURPOSE: Acinetobacter baumannii, Acinetobacter genomic species 3 (AGS 3), and Acinetobacter genomic species sensu Tjernberg and Ursing (AGS 13TU) are phenotypically indistinguishable and are often reported together as the A. baumannii complex (ABC). Few studies have investigated the difference in outcome caused by these different species, and all involved heterogeneous groups of patients. This study aimed to delineate whether there are differences in the clinical characteristics and outcome among patients with solid tumors and bacteremia caused by A. baumannii or two other non-baumannii ABC species (AGS 3 plus AGS 13TU). METHODS: Patients with solid tumors and ABC bacteremia over a period of 5 years in a medical center were identified. The patient data were retrospectively reviewed and analyzed. RESULTS: We identified 103 patients with ABC bacteremia during the study period. Bacteremia was due to A. baumannii in 30 patients, AGS 3 in 24 patients, and AGS 13TU in 49 patients. Among the 103 patients with ABC bacteremia, recent stay in the intensive care unit (ICU) (p = 0.008) was independently associated with the acquisition of A. baumannii bacteremia. Multivariate analysis revealed that bacteremia caused by A. baumannii (hazard ratio [HR] 2.990, 95% confidence interval [CI], 1.021-8.752, p = 0.046) and Acute Physiology and Chronic Health Evaluation (APACHE) II score ≥21 (HR 4.623, 95% CI 1.348-15.859, p = 0.015) were independent factors associated with 14-day mortality. CONCLUSIONS: Infection with A. baumannii and a high APACHE II score (≥21) might be associated with poor outcome in patients with solid tumors and ABC bacteremia.

Application of allele-specific RNAi in hepatitis B virus lamivudine resistance.

Understanding the consequences of mutation in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of hepatitis B virus (HBV) genome on replication is critical for treating chronic hepatitis B with lamivudine. Allele-specific gene silencing by RNAi (allele-specific RNAi: ASP-RNAi) is an advanced application of RNAi techniques. Use of this strategy as a means for specifically inhibiting an allele expression of interest suggested that it can specifically suppress the expression of alleles causing disease without inhibiting the expression of corresponding wild-type alleles. However, no studies have used ASP-RNAi to address the issue of HBV lamivudine resistance. In this study, we applied ASP-RNAi into two long-term eukaryotic cell lines of full-length HBV containing either lamivudine-resistant mutants (HBV-YIDD) or wild type (HBV-WT) which we generated in previously. The designed siRNAs were also used in this eukaryotic expression system together with lamivudine. ELISA and real-time PCR were performed to monitor virus-specific protein synthesis and viral DNA replication. The results showed that the base substitutions conferring marked ASP-RNAi appeared to be largely present in positions 1, 3, 6, 11, 12, 15 and 19 of the sense strand of siRNAs which were different from the most sensitive positions of this application in eukaryotes. In addition, siRNA-lamivudine combinations did not possess the prominent anti-HBV activity we expected because of some unknown mechanisms. These findings recapitulated many of the features of ASP-RNAi in hepadnaviruses which provided a new insight into the development of a potent strategy against HBV drug resistance.

Comparative molecular analysis of meticillin-resistant Staphylococcus aureus isolates from children with atopic dermatitis and healthy subjects in Taiwan.

BACKGROUND: Children with atopic dermatitis (AD) are more frequently colonized by Staphylococcus aureus than healthy children. OBJECTIVES: To assess whether any relationship exists between nasal meticillin-resistant S. aureus (MRSA) colonization and subsequent skin and soft-tissue infections (SSTI). PATIENTS AND METHODS: From 2005 through 2006, comparative molecular analyses of 23 MRSA-colonizing isolates from 133 children with AD, 44 MRSA-colonizing isolates from 490 healthy controls, and 12 MRSA-infecting isolates from 20 children with AD and concurrent SSTI were conducted. RESULTS: Nasal MRSA colonization in children with AD was significantly higher compared with normal individuals (17.3% vs. 9.0%; P = 0.01). The molecular characteristics differed significantly between the MRSA isolates from children with AD and the MRSA-colonizing isolates from healthy controls. The clone characterized as sequence type (ST)59 (338)/pulsotype B/staphylococcal cassette chromosome mec (SCCmec) V(T)/Panton-Valentine leucocidin (PVL)-positive/staphylococcal enterotoxin B (SEB)-positive accounted for half of the MRSA isolates from children with AD, and another clone, characterized as ST59/pulsotype A/SCCmec IV/PVL-negative/SEB-positive accounted for 61% of the MRSA-colonizing isolates from healthy controls. CONCLUSIONS: We found MRSA colonizing the anterior nares of a large number of Taiwanese children, especially among those with AD. Analysis of our data provides evidence that links MRSA-colonizing isolates to MRSA-infecting isolates from concurrent SSTI in children with AD.

Combined central retinal artery and vein occlusion in a patient with systemic lupus erythematosus and anti-phospholipid syndrome.

Combined central retinal artery occlusion and central retinal venous occlusion have been rarely reported in patients with systemic lupus erythematosus and anti-phospholipid syndrome. The impact of this severe vaso-occlusive disease on vision is usually devastating and permanent in spite of vigorous treatment. We report herein a 35-year-old female patient displaying a transient and reversible process. Her best-corrected visual acuity improved from 6/60 to 6/8.6 1 day later, before the initiation of systemic corticosteroid and anticoagulant treatment. The retina regained a normal appearance with her vision recovering to 6/6 2 weeks after the episode of temporary vision loss. Her rapid recovery suggests that continued anti-coagulation therapy and close follow-up to prevent severe complications and recurrent thrombosis is warranted.

MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma.

MicroRNAs (miRNAs) are a family of small non-coding RNA molecules of about 20-23 nucleotides in length, which negatively regulate protein-coding genes at post-transcriptional level. Using a stem-loop real-time-PCR method, we quantified the expression levels of 270 human miRNAs in 13 nasopharyngeal carcinoma (NPC) samples and 9 adjacent normal tissues, and identified 35 miRNAs whose expression levels were significantly altered in NPC samples. Several known oncogenic miRNAs, including miR-17-92 cluster and miR-155, are among the miRNAs upregulated in NPC. Tumour suppressive miRNAs, including miR-34 family, miR-143, and miR-145, are significantly downregulated in NPC. To explore the roles of these dysregulated miRNAs in the pathogenesis of NPC, a computational analysis was performed to predict the pathways collectively targeted by the 22 significantly downregulated miRNAs. Several biological pathways that are well characterised in cancer are significantly targeted by the downregulated miRNAs. These pathways include TGF-Wnt pathways, G1-S cell cycle progression, VEGF signalling pathway, apoptosis and survival pathways, and IP3 signalling pathways. Expression levels of several predicted target genes in G1-S progression and VEGF signalling pathways were elevated in NPC tissues and showed inverse correlation with the down-modulated miRNAs. These results indicate that these downregulated miRNAs coordinately regulate several oncogenic pathways in NPC.

Activation of the mammalian target of rapamycin signalling pathway in epidermal tumours and its correlation with cyclin-dependent kinase 2.

BACKGROUND: The enzyme mammalian target of rapamycin (mTOR) integrates many different cellular signals to control cell growth and proliferation, protein synthesis and breakdown, and other processes. Dysregulation of mTOR is implicated in a range of human diseases, including cancers and cardiovascular disorders. To date, there has been no report on the expression of protein kinase B (AKT)/mTOR cell signalling in epidermal tumours. OBJECTIVES: This study was designed to investigate the activation of the mTOR signalling pathway in epidermal tumours and to correlate this with cyclin-dependent kinase 2 (CDK2) expression. METHODS: Immunohistological staining was performed with phosphorylated (p-) AKT, p-mTOR, p-4E-binding protein 1 (p-4EBP1), p-ribosomal protein S6 (p-S6), p-p70 ribosomal protein S6 kinase 1 (p-p70S6K1) and CDK2 in 15 cases each of seborrhoeic keratosis, actinic keratosis, keratoacanthoma and Bowen's disease (BD), and 25 cases of squamous cell carcinoma (SCC). Fifteen normal skin (NS) samples served as control. RESULTS: Among 85 tumours, 40 (47%) were positive for p-AKT, 31 (36%) for p-mTOR, 44 (52%) for p-4EBP1, 38 (45%) for p-S6, and 39 (46%) for p-p70S6K1. CDK2 immunostaining was positive in all cases of SCC and BD, and in 67% of benign tumours. All of these markers were stained much more frequently in malignant tumours than in benign tumours or NS. p-AKT, p-mTOR, p-4EBP1, p-p70S6K1 and p-S6 each showed high correlation with CDK2. CONCLUSIONS: Constitutive activation of the AKT/mTOR pathway was frequent in epidermal tumours, especially in malignant tumours. Activation was highly correlated with CDK2 expression, suggesting that the AKT/mTOR pathway may induce the malignant transition through CDK2 in epidermal tumours.

MiR-140-3p overexpression activates the Wnt signaling pathway to promote fracture healing.

OBJECTIVE: To study the mechanism of micro ribonucleic acid (miR)-140-3p participating in the regulation of fracture healing in rats. MATERIALS AND METHODS: A total of 50 male Sprague-Dawley (SD) rats were randomly divided into five groups, namely, group A [phosphate-buffered saline (PBS)] (n=10), group B (miR-140-3p mimics) (n=10), group C [ mimics negative control (NC)] (n=10), group D [antisense oligonucleotide (ASO)-miR-140-3p] (n=10), and group E (ASO NC) (n=10). A rat model of fracture was established on all the rats through the operation. From the successful establishment of the model, the rats in group A were intraperitoneally injected with 50 µL PBS (2 nmol) once a week for 6 weeks, and those in group B, C, D, and E were injected with equivalent volume of miR-140-3p mimics, mimics NC, ASO-miR-140-3p, and ASO NC, respectively, once a week since the successful establishment of model for 6 weeks. The fracture healing in the rats was evaluated via imaging. Meanwhile, Real Time-Polymerase Chain Reaction (RT-PCR) was applied to detect the expression of miR-140-3p in the five groups. Wnt and ß-catenin expressions in the five groups were detected by means of Western blotting (WB). Alkaline phosphatase (ALP) and its quantized statistical value in the five groups were detected through immunohistochemical staining. RESULTS: The expression of miR-140-3p was stimulated in miR-140-3p mimics group and inhibited in ASO-miR-140-3p group. The detection of the miR-140-3p expression level in the five groups via RT-PCR showed that miR-140-3p mimics group had a remarkably higher miR-140-3p expression than the other four groups. The differences were statistically significant (p<0.05). The WB assay verified that the Wnt and ß-catenin expressions in miR-140-3p mimics group were notably higher than those in control groups, and there were statistically significant differences (p<0.05). Compared with those in the groups injected with PBS, ASO miR-140-3p, mimics NC, and ASO NC, there were evidently more callus tissues, better healed and more blurred fracture lines, as well as no translocation and looseness of internal fixation, in the group injected with miR-140-3p mimics, suggesting that the stimulation of the miR-140-3p expression promotes the fracture healing in the rats. The results of immunohistochemical staining indicated that the number of ALP-positive osteoblasts in the rats in miR-140-3p mimics group was increased markedly in comparison with that in the remaining groups (p<0.05), implying that the differentiation of osteoblasts in the rats was affected in miR-140-3p mimics group. CONCLUSIONS: The overexpressed miR-140-3p in the rats with fracture can promote fracture healing by activating the Wnt signaling pathway.