Desflurane improves electrical activity of neurons and alleviates oxygen-glucose deprivation-induced neuronal injury by activating the Kcna1-dependent Kv1.1 channel.

Several volatile anesthetics have presented neuroprotective functions in ischemic injury. This study investigates the effect of desflurane (Des) on neurons following oxygen-glucose deprivation (OGD) challenge and explores the underpinning mechanism. Mouse neurons HT22 were subjected to OGD, which significantly reduced cell viability, increased lactate dehydrogenase release, and promoted cell apoptosis. In addition, the OGD condition increased oxidative stress in HT22 cells, as manifested by increased ROS and MDA contents, decreased SOD activity and GSH/GSSG ratio, and reduced nuclear protein level of Nrf2. Notably, the oxidative stress and neuronal apoptosis were substantially blocked by Des treatment. Bioinformatics suggested potassium voltage-gated channel subfamily A member 1 (Kcna1) as a target of Des. Indeed, the Kcna1 expression in HT22 cells was decreased by OGD but restored by Des treatment. Artificial knockdown of Kcna1 negated the neuroprotective effects of Des. By upregulating Kcna1, Des activated the Kv1.1 channel, therefore enhancing K+ currents and inducing neuronal repolarization. Pharmacological inhibition of the Kv1.1 channel reversed the protective effects of Des against OGD-induced injury. Collectively, this study demonstrates that Des improves electrical activity of neurons and alleviates OGD-induced neuronal injury by activating the Kcna1-dependent Kv1.1 channel.

Inferior displacement of the lateral rectus muscle insertion in exotropia with mild V-pattern or vertical deviation.

BACKGROUND: To describe clinical features and intraoperative findings of the patients with exotropia who presented mild V-pattern or vertical deviation, and to investigate the surgical outcomes of anatomical relocation of inferiorly displaced lateral rectus (LR) muscle insertion. METHODS: Detailed ophthalmological evaluations were obtained in 42 consecutive patients, and the horizontal rectus muscle insertions were detected intraoperatively. The displaced insertion of LR muscle was corrected accompanied with classic recession-resection procedure. RESULTS: The inferiorly displaced LR muscle insertions were detected in 19 patients (Group A), while the remaining 23 patients (Group B) had normal insertions. The mean distance of displaced insertion from the normal position was 2.92 ± 1.05 mm (range: 1.0-4.0). Mild V-pattern was more common in Group A (78.9%, 15/19) than Group B (47.8%, 11/23), and the magnitude of V-pattern in Group A (6.16 ± 3.91 PD) was also greater than Group B (3.43 ± 3.92 PD). The fundus extorsions of the affected eyes (9.68 ± 4.77 °) were greater than the contralateral eyes (5.91 ± 5.82 °) in Group A. At the 2 months follow-up, mild V-pattern and mild vertical deviation were corrected by upward transposition. The significant correlations were identified between the pre-operative misalignments and the amounts of misalignments correction. CONCLUSIONS: Nearly half of the cases with mild V-pattern or vertical deviation resulted from the inferiorly displaced LR muscle insertion, so the intraoperative exploration of the LR muscle insertion is strongly suggested. Upward transposition may effectively correct both the mild V-pattern and vertical deviation.

Laser Direct Writing of Flexible Thermal Flow Sensors.

Thin film-based thermal flow sensors afford applications in healthcare and industries owing to their merits in preserving initial flow distributions. However, traditional thermal flow sensors are primarily applied to track flow intensities based on hot-wire or hot-film sensing mechanisms due to their relatively facile device configurations and fabrication strategies. Herein, a calorimetric thermal flow sensor is proposed based on laser direct writing to form laser-induced graphene as heaters and temperature sensors, resulting in monitoring both flow intensities and orientations. Via homogeneously surrounding spiral heaters with multiple temperature sensors, the device exhibits high sensitivity (∼162 K·s/m) at small flows with an extended flow detection range (∼25 m/s). Integrating the device with a data-acquisition board and a dual-mode graphical user interface enables wirelessly and dynamically monitoring respiration and the motion of robotic arms. This versatile flow sensor with facile manufacturing affords potentials in health inspection, remote monitoring, and studying hydrodynamics.

Identifying circRNA-miRNA-mRNA Regulatory Networks in Chemotherapy-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a frequent and severe side effect of first-line chemotherapeutic agents. The association between circular RNAs (circRNAs) and CIPN remains unclear. In this study, CIPN models were constructed with Taxol, while 134 differentially expressed circRNAs, 353 differentially expressed long non-coding RNAs, and 86 differentially expressed messenger RNAs (mRNAs) were identified utilizing RNA sequencing. CircRNA-targeted microRNAs (miRNAs) were predicted using miRanda, and miRNA-targeted mRNAs were predicted using TargetScan and miRDB. The intersection of sequencing and mRNA prediction results was selected to establish the circRNA-miRNA-mRNA networks, which include 15 circRNAs, 18 miRNAs, and 11 mRNAs. Functional enrichment pathway analyses and immune infiltration analyses revealed that differentially expressed mRNAs were enriched in the immune system, especially in T cells, monocytes, and macrophages. Cdh1, Satb2, Fas, P2ry2, and Zfhx2 were further identified as hub genes and validated by RT-qPCR, correlating with macrophages, plasmacytoid dendritic cells, and central memory CD4 T cells in CIPN. Additionally, we predicted the associated diseases, 36 potential transcription factors (TFs), and 30 putative drugs for hub genes using the DisGeNET, TRRUST, and DGIdb databases, respectively. Our results indicated the crucial role of circRNAs, and the immune microenvironment played in CIPN, providing novel insights for further research.

Antagonizing pathological α-synuclein-mediated neurodegeneration by J24335 via the activation of immunoproteasome.

The aggregation of misfolded proteins, such as α-synuclein in Parkinson's disease (PD), occurs intracellularly or extracellularly in the majority of neurodegenerative diseases. The immunoproteasome has more potent chymotrypsin-like activity than normal proteasome. Thus, degradation of α-synuclein aggregation via immunoproteasome is an attractive approach for PD drug development. Herein, we aimed to determine if novel compound, 11-Hydroxy-1-(8-methoxy-5-(trifluoromethyl)quinolin-2-yl)undecan-1-one oxime (named as J24335), is a promising candidate for disease-modifying therapy to prevent the pathological progression of neurodegenerative diseases, such as PD. The effects of J24335 on inducible PC12/A53T-α-syn cell viability and cytotoxicity were evaluated by MTT assay and LDH assay, respectively. Evaluation of various proteasome activities was done by measuring the luminescence of enzymatic activity after the addition of different amounts of aminoluciferin. Immunoblotting and real-time PCR were employed to detect the expression of various proteins and genes, respectively. We also used a transgenic mouse model for behavioral testing and immunochemical analysis, to assess the neuroprotective effects of J24335. J24335 inhibited wild-type and mutant α-synuclein aggregation without affecting the growth or death of neuronal cells. The inhibition of α-synuclein aggregation by J24335 was caused by activation of immunoproteasome, as mediated by upregulation of LMP7, and increased cellular chymotrypsin-like activity in 20S proteasome. J24335-enhanced immunoproteasome activity was mediated by PKA/Akt/mTOR pathway activation. Moreover, animal studies revealed that J24335 treatment markedly mitigated both the loss of tyrosine hydroxylase-positive (TH-) neurons and impaired motor skill development. This is the first report to use J24335 as an immunoproteasome enhancing agent to antagonize pathological α-synuclein-mediated neurodegeneration.

Design, synthesis, and evaluation of pyrido.[3,4-b]pyrazin-2(1H)-one derivatives as potent FLT3 inhibitors.

Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as driver mutations in a subgroup of AML patients. Herein, we describe the design, synthesis, and biological evaluation of a novel series of potent pyrido.[3,4-b]pyrazin-2(1H)-one derivatives as FLT3 inhibitors. The compounds exhibited moderate to potent FLT3 kinase inhibitory potency and excellent antiproliferative activities against MV4-11 cells. Among them, compound 13 demonstrated the most potent kinase activity against FLT3-D835Y (IC50 = 29.54 ± 4.76 nM) and cellular potency against MV4-11 cells (IC50 = 15.77 ± 0.15 nM). Compound 13 also efficiently inhibited the growth of multiple mutant BaF3 cells expressing FLT3-D835V/F, FLT3-F691L, and FLT3-ITD/D835Y. Furthermore, compound 13 was metabolically stable in mouse liver microsomes. Moreover, the treatment with compound 13 led to robust inhibition of FLT3 autophosphorylation on Tyr589/591 in MV4-11 cells. In summary, our data demonstrated that 13 was worthy of further study for the treatment of AML.

Discovery of pyrido[4,3-d]pyrimidinone derivatives as novel Wee1 inhibitors.

Wee1 has emerged as a potential target in cancer therapy due to its critical role in the regulation of the cell cycle. Here, we describe a series of Wee1 inhibitors with a novel scaffold that are potent inhibitors of this kinase (IC50 = 19-1485 nM). These inhibitors demonstrated robust cytotoxicity in MV-4-11 and T47D cell lines (MV-4-11 IC50 = 660-2690 nM, T47D IC50 = 2670-20,000 nM) and displayed good stability in mouse liver microsomes in vitro. Additionally, compound 34 showed remarkable selectivity (more than 500-fold) over the other 9 kinases. Further mechanistic studies demonstrated that compound 34 displayed measurable effects on downstream biomarkers and induced cancer cell apoptosis and cell cycle arrest in the G0/G1 phase. Taken together, these results show that compound 34, potentially a leading Wee1 inhibitor, warrants further investigation.

Safety and efficacy of anlotinib hydrochloride capsules in advanced non-small-cell lung cancer: a multicenter, real-world study.

Objective: To investigate the safety and efficacy of anlotinib hydrochloride capsules in stage III-IV non-small-cell lung cancer (NSCLC). Methods: NSCLC patients received anlotinib monotherapy or combination therapy. The primary end point was adverse reactions during anlotinib treatment and the secondary end point was progression-free survival. Results: During anlotinib treatement, 41.85% (167/399) of patients experienced adverse reactions, and the monotherapy group had a lower incidence than the combination group (36.89 vs 49.68%; p = 0.012). The median progression-free survival of patients in the monotherapy group was significantly lower than that in the combination group (5 vs 6 months; p = 0.0119). Conclusion: Compared with anlotinib monotherapy, combination therapy resulted in longer PFS and a higher incidence of adverse reactions in patients with NSCLC.

Substituent-Controllable Cascade Regioselective Annulation of ß-Enaminones with N-Sulfonyl Triazoles for Modular Access to Imidazoles and Pyrroles.

A controllable synthesis of trisubstituted imidazoles and pyrroles has been developed through rhodium(II)-catalyzed regioselective annulation of N-sulfonyl-1,2,3-trizaoles with ß-enaminones. The imidazole ring was formed through a 1,1-insertion of the N-H bond to α-imino rhodium carbene, followed by a subsequent intramolecular 1,4-conjugate addition. This occurred when the α-carbon atom of the amino group was bearing a methyl group. Additionally, the pyrrole ring was constructed by utilizing a phenyl substituent and undergoing intramolecular nucleophilic addition. The mild conditions, good tolerance towards functional groups, gram-scale synthesis capability, and ability to undergo valuable transformations of the products qualify this unique protocol as an efficient tool for the synthesis of N-heterocycles.

Fabrication of Superhydrophobic Porous Brass by Chemical Dealloying for Efficient Emulsion Separation.

By taking advantage of typical dealloying and subsequent aging methods, a novel homogeneous porous brass with a micro/nano hierarchical structure was prepared without any chemical modification. The treatment of commercial brass with hot concentrated HCl solution caused preferential etching of Zn from Cu62Zn38 alloy foil, leaving a microporous skeleton with an average tortuous channel size of 1.6 µm for liquid transfer. After storage in the atmosphere for 7 days, the wettability of the dealloyed brass changed from superhydrophilic to superhydrophobic with a contact angle > 156° and sliding angle < 7°. The aging treatment enhanced the hydrophobicity of the brass by the formation of Cu2O on the surface. By virtue of the opposite wettability to water and oil, the aged brass separated surfactant-stabilized water-in-oil emulsions with separation efficiency of over 99.4% and permeate flux of about 851 L·m-2·h-1 even after recycling for 60 times. After 10 times of tape peeling or sandpaper abrasion, the aged brass maintained its superhydrophobicity, indicating its excellent mechanical stability. Moreover, the aged brass still retained its superhydrophobicity after exposure to high temperatures or corrosive solutions, displaying high resistance to extreme environments. The reason may be that the bicontinuous porous structure throughout the whole foil endows stable mechanical properties to tolerate extreme environments. This method should have a promising future in expanding the applications of alloys.

IMP3 promotes re-endothelialization after arterial injury via increasing stability of VEGF mRNAhv.

IMP3, an RNA-binding protein (RBP) that participates in the process of post-transcriptional modifications of mRNA transcripts, is capable of altering cellular functions, and in some cases, be involved in specific disease progression. We aimed to investigate whether IMP3 has the ability to regulate the functional properties of endothelial cells and re-endothelialization in response to arterial injury. Wire injury was introduced to the right carotid arteries of wildtype C57/BL6 mice. As a result, IMPs' expressions were up-regulated in the induced arterial lesions, and IMP3 was the most up-regulated RNA among other IMPs. We overexpressed IMP3 before the wire-injured surgery using adeno-associated virus AAV2-IMP3. In vivo studies confirmed that IMP3 overexpression accelerated the progress of re-endothelialization after arterial injury. In vitro, endothelial cells were transfected with either ad-IMP3 or Si-IMP3, cell functional studies showed that IMP3 could promote endothelial cell proliferation and migration, while reducing apoptosis. Mechanistic studies also revealed that IMP3 could enhance VEGF mRNA stability and therefore up-regulate activities of VEGF/PI3K/Akt signalling pathway. Our data indicated that IMP3 promotes re-endothelialization after arterial injury and regulates endothelial cell proliferation, migration and apoptosis via increasing stability of VEGF mRNA and activation of VEGF/PI3K/Akt signalling pathway.

Insights into the Dermal Absorption, Deposition, and Elimination of Poly- and Perfluoroalkyl Substances in Rats: The Importance of Skin Exposure.

Humans are frequently exposed to poly- and perfluoroalkyl substances (PFASs) via direct skin contact with personal care and consumer products containing them. Here, we used a rat model to estimate the dermal penetration efficiency of 15 representative PFASs. After 144 h post-dosing, 4.1-18.0 and 5.3-15.1% of the applied PFASs in the low (L) and high (H) groups, respectively, were absorbed into the rats. PFAS absorption and permeation were parabolically associated with the perfluorinated carbon chain length (CF), peaking for perfluoroheptanoic acid (PFHpA). The lipid-rich stratum corneum of the skin barrier substantially suppressed the penetration of less hydrophobic short-chain PFASs, whereas the water-rich viable epidermis and dermis served as obstacles to hydrophobic long-chain PFAS permeation. However, the renal clearance (CLrenal) of the target PFAS decreased with increasing CF, suggesting that urinary excretion is crucial to eliminate less hydrophobic short-chain PFASs. Notably, the peak times of PFASs in the systemic circulation of rats (8-72 h) were remarkably longer than those after oral administration (1-24 h). These results suggest that dermal penetration can be long-lasting and contribute considerably to the body burden of PFASs, especially for those with moderate hydrophobicity due to their favorable skin permeation and unfavorable urinary excretion.

Reversal of hepatorenal syndrome and kidney recovery: When renal has more to offer.

Hepatorenal syndrome (HRS) is one of the most severe complications in advanced cirrhosis. Type-1 HRS is relatively uncommon, yet carries considerably higher mortality rate. Effective treatment for HRS, especially therapy towards survival benefits, is still limited. However, the role for dialysis in HRS has been questioned over the years. The initiation of dialysis remains controversial for those who aren't transplant candidates. Meanwhile, there's a growing attention towards the successful use of peritoneal dialysis (PD) in cirrhotic patients. Herein, we report a case of HRS-1 in a 76-year-old male patient with decompensated cirrhosis. Through a series of adjustments of hemodialysis regimens and pharmacological prescriptions, patient stabilized and the opportunity for transjugular intrahepatic portosystemic shunt (TIPS) insertion was gained. PD was initiated after TIPS placement. With a gradual decrease of dialysis dose, patient successfully weaned off PD and achieved both reversal of HRS and kidney recovery. Markedly improved nutritional status and quality of life were reported. The potential role of dialysis and TIPS in HRS may be worth revisiting. Further studies regarding the optimal timing of dialysis initiation, choices of dialysis modality, and efficacy of dialysis therapy in combination with TIPS in HRS patients are warranted.

Rh-Catalyzed Cascade C-H Activation/Annulation of N-Hydroxybenzamides and Propargylic Acetates for Modular Access to Isoquinolones.

A sequential Rh(III)-catalyzed C-H activation/annulation of N-hydroxybenzamides with propargylic acetates leading to the formation of NH-free isoquinolones is described. This reaction proceeds through a sequential C-H activation/alkyne insertion/intramolecular annulation/N-O bond cleavage procedure, affording a broad spectrum of products with diverse substituents in moderate-to-excellent yields. Notably, this protocol features the simultaneous formation of two new C-C/C-N bonds and one heterocycle in one pot with the release of water as the sole byproduct.

High concentration of nitrogen recovery from anaerobic digested slurry (ADS) using biochars: adsorption and improvement.

Biochar produced from biomass has been increasingly used as an environmentally friendly and low-cost adsorbent. This study systemically evaluated the effects of raw materials including corn straw (CS), cattle manure (CM), and cherry woods (CW) as well as pyrolysis temperature (400, 500, and 600 °C) on the physicochemical properties, such as morphological structure, element content, and surface functionality of biochars. The batch experiments of NH4+-N adsorption using anaerobic digested slurry (ADS) confirmed that CM600 (biochar derived from CM at 600 °C) had the highest adsorption capacity of 18.16 mg·g-1. The effects of coexisting ions in ADS, biochar dosage, adsorption time and initial concentration on NH4+-N adsorption from ADS by the biochars were evaluated. The results of the batch equilibrium and kinetics experiments showed that Langmuir isotherm model and pseudo-second-order kinetic model well described NH4+-N adsorption by the biochars, indicating that physical and chemical adsorption occurred simultaneously. Furthermore, compared to the biochar-modified method, the raw material-modified biochar (CM600-modified biochar) showed excellent adsorption capacity with a maximum of 69.82 mg·g-1 (284% increase) for the high NH4+-N concentration (4,000 mg·L-1) from ADS. Therefore, it was concluded that high-concentration nitrogen recovery from ADS using modified biochar was an effective method.

Extracellular Vesicle-Mediated Delivery of Circular RNA SCMH1 Promotes Functional Recovery in Rodent and Nonhuman Primate Ischemic Stroke Models.

BACKGROUND: Stroke is a leading cause of adult disability that can severely compromise the quality of life of patients, yet no effective medication currently exists to accelerate rehabilitation. A variety of circular RNA (circRNA) molecules are known to function in ischemic brain injury. Lentivirus-based expression systems have been widely used in basic studies of circRNAs, but safety issues with such delivery systems have limited exploration of the potential therapeutic roles for circRNAs. METHODS: Circular RNA SCMH1 (circSCMH1) was screened from the plasma of patients with acute ischemic stroke by using circRNA microarrays. Engineered rabies virus glycoprotein-circSCMH1-extracellular vesicles were generated to selectively deliver circSCMH1 to the brain. Nissl staining was used to examine infarct size. Behavioral tasks were performed to evaluate motor functions in both rodent and nonhuman primate ischemic stroke models. Golgi staining and immunostaining were used to examine neuroplasticity and glial activation. Proteomic assays and RNA-sequencing data combined with transcriptional profiling were used to identify downstream targets of circSCMH1. RESULTS: CircSCMH1 levels were significantly decreased in the plasma of patients with acute ischemic stroke, offering significant power in predicting stroke outcomes. The decreased levels of circSCMH1 were further confirmed in the plasma and peri-infarct cortex of photothrombotic stroke mice. Beyond demonstrating proof-of-concept for an RNA drug delivery technology, we observed that circSCMH1 treatment improved functional recovery after stroke in both mice and monkeys, and we discovered that circSCMH1 enhanced the neuronal plasticity and inhibited glial activation and peripheral immune cell infiltration. CircSCMH1 binds mechanistically to the transcription factor MeCP2 (methyl-CpG binding protein 2), thereby releasing repression of MeCP2 target gene transcription. CONCLUSIONS: Rabies virus glycoprotein-circSCMH1-extracellular vesicles afford protection by promoting functional recovery in the rodent and the nonhuman primate ischemic stroke models. Our study presents a potentially widely applicable nucleotide drug delivery technology and demonstrates the basic mechanism of how circRNAs can be therapeutically exploited to improve poststroke outcomes.

Infection Control Precautions and Care Delivery in Hemodialysis Unit during Coronavirus Disease 2019 Outbreak: A Case Series.

BACKGROUND: With an estimated basic reproductive number of 3.77, the Coronavirus Disease 2019 (COVID-19) continues to spread. It is urgent to exert adequate efforts for the management of dialysis patients, caregivers, and healthcare personnel (HCP). This study aimed at reporting practical workflow, identification of high-risk or suspected cases of CO-VID-19, and subsequent response measures. METHODS: At the time of the COVID-19 outbreak, precautions and practice protocols were applied in our dialysis units (DUs). This single-center study retrospectively reviewed all high-risk/suspected cases from January 23, 2020, to February 10, 2020. Epidemiological, clinical feature, and detailed data on all cases were recorded. RESULTS: Practical workflow for the clinical management of dialysis patients, caregivers, and HCP was initiated. A total of 6 high-risk/suspected cases were identified. Female gender, older age, presence of cardiovascular disease, diabetes, anuresis, immunocompromised status, hypoalbuminemia, and underweight were noticeable features in these cases. Direct evidence of infection or epidemiological risk was detected in five cases. Close monitoring for temperature and oxygen saturation during hemodialysis sessions may be reasonable. No confirmed COVID-19 cases were reported in our DU, but certain cases showed rapid deterioration due to other critically severe condition needing hospitalization. Portable dialysis machines are of great need to ensure dialysis care provision. CONCLUSIONS: Our study described a practical workflow for patient-centered management during COVID-19 outbreak. Potential risk factors and underlying clinical patterns were reported. Further studies regarding the efficacy of infection control precautions and practice protocols tailored for dialysis settings are warranted.

Circular RNA TLK1 Aggravates Neuronal Injury and Neurological Deficits after Ischemic Stroke via miR-335-3p/TIPARP.

Circular RNAs (circRNAs) are expressed at high levels in the brain and are involved in various CNS diseases. However, the potential role of circRNAs in ischemic stroke-associated neuronal injury remains largely unknown. Here, we investigated the important functions of circRNA TLK1 (circTLK1) in this process. The levels of circTLK1 were significantly increased in brain tissues in a mouse model of focal cerebral ischemia and reperfusion. Knockdown of circTLK1 significantly decreased infarct volumes, attenuated neuronal injury, and improved neurological deficits. Furthermore, circTLK1 functioned as an endogenous miR-335-3p sponge to inhibit miR-335-3p activity, resulting in the increase of 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible poly (ADP-ribose) polymerase expression and a subsequent exacerbation of neuronal injury. Clinical studies confirmed increased levels of circTLK1 in the plasma of patients with acute ischemic stroke (59 males and 12 females). Our findings reveal a detrimental role of circTLK1 in ischemic brain injury.SIGNIFICANCE STATEMENT The extent of neuronal injury after brain ischemia is a primary factor determining stroke outcomes. However, the molecular switches that control the death of ischemic neurons are poorly understood. While our previous studies indicated the involvement of circRNAs in ischemic stroke, the potential role of circRNAs in neuronal injury remains largely unknown. The levels of circTLK1 were significantly increased in the brain tissue and plasma isolated from animal models of ischemic stroke and patients. Knockdown of circTLK1 significantly decreased infarct volumes, attenuated neuronal injury, and improved subsequent long-term neurological deficits. To our knowledge, these results provide the first definitive evidence that circTLK1 is detrimental in ischemic stroke.

Effect of RSK4 on biological characteristics of colorectal cancer.

BACKGROUND: This study aimed to investigate the expression of P90 Ribosomal Protein S6 kinase 4 (RSK4) in colorectal cancer cells and its biological function. METHODS: We selected early SW480 and HCT116 colorectal cancer cell lines, using Lipofectamine™ 2000 transfection reagent carrying RSK4 gene transfected into cells to establish the colorectal cancer cell lines with high expression of RSK4. RT-PCR and western blot (WB) analysis confirmed RSK4 expression in SW480 and HCT116 cancer cell lines. We used methylthiazoltetrazolium (MTT) assay and flow cytometry to detect the proliferation of colorectal cancer cells. After transfection of RSK4, the effect of RSK4 on the RNA levels associated with epithelial-mesenchymal transition (EMT) of colorectal cancer cells was analyzed by real-time fluorescence quantitative PCR and the expression of EMT-related protein was detected by WB analysis. RESULTS: After transfection of RSK4 overexpression, the MTT assay detected that RSK4 could significantly inhibit the growth of colorectal cancer cells in vitro; flow cytometry detected that S-phase cells decreased significantly, and G0/1 cells increased significantly (P < 0.05). The invasion ability of SW480 and HCT116 cells transfected with RSK4 was markedly lower than that in the control group, and the difference was statistically significant (P < 0.05). Fluorescent quantitative PCR and WB analysis showed that the expression of EMT-associated molecular E-cadherin was remarkably increased and the expression of Snail was significantly decreased (P < 0.01). CONCLUSION: RSK4 gene in colorectal cancer cell lines with low expression of RSK4 after transfection can inhibit the growth and invasion of tumor cells. RSK4 gene may inhibit EMT and inhibit metastasis of colorectal cancer cells, may be a potential tumor suppressor gene and inhibit tumor distant metastasis, and may provide the biological basis for new therapeutic targets.

An evaluation and recommendation of the optimal methodologies to detect RET gene rearrangements in papillary thyroid carcinoma.

To recommend a reliable and clinically realistic RET/PTC rearrangement detection assay for papillary thyroid carcinoma (PTC), we compared multiplex quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). RET/PTC rearrangement was detected using either RET break-apart FISH followed by multicolor FISH to confirm CCDC6/RET or NCOA4/RET fusions, or by multiplex qPCR to detect 14 RET/PTC subtypes with simultaneous RET mRNA expression. RET protein expression was detected by IHC. The specificity and sensitivity of multiplex qPCR and IHC were calculated using break-apart FISH as a reference. Among 73 PTC patients with sufficient tissue available for FISH and multiplex qPCR, 10 cases were defined as RET/PTC positive by both assays, including eight CCDC6/RET and two NCOA4/RET fusions with relatively high RET mRNA. In addition, multiplex qPCR identified another two CCDC6/RET fusion positive cases, but with low RET mRNA expression. IHC staining identified 11 RET positive cases among 39 patients with available samples. In comparison to FISH, multiplex qPCR displayed 100% sensitivity and 97% specificity to detect RET/PTC fusions, while IHC was neither sensitive nor specific. Our data reveal that both multiplex qPCR and FISH assays are equally applicable for detection of RET/PTC rearrangements. Break-apart FISH methodology is highly recommended for the wider screening of RET rearrangements (regardless of partner genes), while multiplex qPCR is preferred to identify all known fusion types using one assay, provided mRNA expression is also measured. IHC analysis could potentially provide an additional method of fusion detection dependent on further optimization of assay conditions and scoring cutoffs.