Regulation of epidermal growth factor receptor tyrosine kinase inhibitor resistance via Ambra1-mediated autophagy in non-small cell lung cancer.

To explore the molecular mechanisms related to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) resistance, along with potential therapeutic targets and strategies. The autophagy and Beclin 1 regulator 1 (Ambra1) short hairpin ribonucleic acid (shRNA) lentivirus vector and Ambra1 overexpression plasmid, constructed with a plasmid cloning deoxyribonucleic acid (pcDNA) 3.1 vector, were used to down-regulate and up-regulate Ambra1 expression in the human lung adenocarcinoma erlotinib-resistant cell line (PC9/ER), respectively, as well as to screen stable transgenic cell lines. The IC50 of Erlotinib in these cell lines were measured to determine their resistance status. The real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure messenger ribonucleic acid (mRNA) expression of resistance-related genes like multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1 (MRP1), and lung drug-resistant-related protein (LRP). Western blot was performed to analyze the protein expressions of the autophagy-related genes Beclin 1, LC3II/I, and p62. Each stable transgenic line formed a tumor under the skin in nude mice; the mice with subcutaneous tumorigenesis of PC9/ER cells and shAmbra1-PC9/ER cells were subsequently treated with rapamycin (RAPA) and chloroquine (CQ), respectively. The mRNA expressions of MDR1, MRP1, and LRP in each tumor tissue sample were detected by qRT-PCR. The protein expressions of adenosine monophosphate-activated protein kinase (AMPK), phosphorylated-AMPK (p-AMPK), forkhead box O3 (FoxO3a), and phosphorylated forkhead box O3 (p-FoxO3a) in the AMPK/FoxO3a signaling pathway were analyzed via Western blot. The qRT-PCR result revealed that the level of Ambra1 in EGFR-TKI-resistant cells had increased. This was further exacerbated by the overexpression of Ambra1 and was reduced after its inhibition. Additionally, Ambra1 upregulated the mRNA expression of drug-resistant genes and the expression of autophagy-related proteins. Subcutaneous tumorigenesis of RAPA-treated shAmbra1-PC9/ER cells resulted in increased expression of drug resistance-related genes and a concomitant decrease in p-AMPK and increase in p-FoxO3a. The results revealed that Beclin-1/ß-actin, p62/ß-actin, and LC3II/I in the model group were all significantly increased compared to the control group, with P<0.05. Compared to the model group, Beclin-1/ß-actin, p62/ß-actin, and LC3II/I were all significantly higher in the pcDNA-Ambra1 group, with P<0.05. Compared to the model group, Beclin-1/ß-actin, p62/ß-actin, and LC3II/I were all significantly decreased in the shAmbra1 group, with P<0.05. Thus, these data suggest that Ambra1 promotes cellular autophagy. In addition, subcutaneous tumorigenesis of CQ-treated shAmbra1-PC9/ER cells resulted in reduced expression of drug resistance-related genes, and a concomitant increase in p-AMPK and decrease in p-FoxO3a. The results of this study revealed that Ambra1-mediated autophagy regulated EGFR-TKI resistance in NSCLC, most probably through the AMPK/FoxO3a signaling pathway.

Social association predicts immunological similarity in rewilded mice.

Environmental influences on immune phenotypes are well-documented, but our understanding of which elements of the environment affect immune systems, and how, remains vague. Behaviors, including socializing with others, are central to an individual's interaction with its environment. We tracked behavior of rewilded laboratory mice of three inbred strains in outdoor enclosures and examined contributions of behavior, including social associations, to immune phenotypes. We found that the more associated two individuals were, the more similar their immune phenotypes were. Social association was particularly predictive of similar memory T and B cell profiles and was more influential than sibling relationships or worm infection status. These results highlight the importance of social networks for immune phenotype and reveal important immunological correlates of social life.

Predicting adverse drug effects: A heterogeneous graph convolution network with a multi-layer perceptron approach.

We apply a heterogeneous graph convolution network (GCN) combined with a multi-layer perceptron (MLP) denoted by GCNMLP to explore the potential side effects of drugs. Here the SIDER, OFFSIDERS, and FAERS are used as the datasets. We integrate the drug information with similar characteristics from the datasets of known drugs and side effect networks. The heterogeneous graph networks explore the potential side effects of drugs by inferring the relationship between similar drugs and related side effects. This novel in silico method will shorten the time spent in uncovering the unseen side effects within routine drug prescriptions while highlighting the relevance of exploring drug mechanisms from well-documented drugs. In our experiments, we inquire about the drugs Vancomycin, Amlodipine, Cisplatin, and Glimepiride from a trained model, where the parameters are acquired from the dataset SIDER after training. Our results show that the performance of the GCNMLP on these three datasets is superior to the non-negative matrix factorization method (NMF) and some well-known machine learning methods with respect to various evaluation scales. Moreover, new side effects of drugs can be obtained using the GCNMLP.

Supplementation with heat-inactivated Lacticaseibacillus paracasei K47 ameliorates allergic asthma in mice by regulating the Th1/Th2 balance.

Asthma is a chronic inflammatory disease related to the immune response of type 2 T helper cells (Th2), which affects all age groups. The incidence of asthma is increasing worldwide, and it has become a significant public health problem. This study aimed to investigate the immunomodulatory effects of Lacticaseibacillus (formerly Lactobacillus) paracasei K47 on mice with ovalbumin (OVA)-induced allergy. The consequences of orally administered heat-inactivated K47 in OVA-sensitised/challenged BALB/c mice were evaluated by assessing the serum levels of immunoglobulins (Igs), airway hyperresponsiveness (AHR), and bronchoalveolar lavage fluid (BALF) cytokine. In addition, the effect of K47 on type 1 T helper cells (Th1)/Th2 cytokine production in splenocytes from OVA-sensitised mice was evaluated. The results revealed that supplementation with K47 remarkably reduced serum levels of total IgE, OVA-specific IgE, and OVA-specific IgG1 in OVA-sensitised/challenged mice. In addition, K47 intervention ameliorated AHR and suppressed the accumulation of inflammatory cells in the BALF of OVA-sensitised/challenged mice. Furthermore, the immunomodulatory ability of K47 was mediated by regulation of the cytokine profile toward the Th1 response in the BALF, and splenocytes of OVA-sensitised mice. Taken together, these results suggested that K47 can modulate the host immune response to ameliorate AHR and inflammation in allergic asthma.

Transcriptional profiling of primary prostate tumor in metastatic hormone-sensitive prostate cancer and association with clinical outcomes: correlative analysis of the E3805 CHAARTED trial.

BACKGROUND: The phase III CHAARTED trial established upfront androgen-deprivation therapy (ADT) plus docetaxel (D) as a standard for metastatic hormone-sensitive prostate cancer (mHSPC) based on meaningful improvement in overall survival (OS). Biological prognostic markers of outcomes and predictors of chemotherapy benefit are undefined. PATIENTS AND METHODS: Whole transcriptomic profiling was performed on primary PC tissue obtained from patients enrolled in CHAARTED prior to systemic therapy. We adopted an a priori analytical plan to test defined RNA signatures and their associations with HSPC clinical phenotypes and outcomes. Multivariable analyses (MVAs) were adjusted for age, Eastern Cooperative Oncology Group status, de novo metastasis presentation, volume of disease, and treatment arm. The primary endpoint was OS; the secondary endpoint was time to castration-resistant PC. RESULTS: The analytic cohort of 160 patients demonstrated marked differences in transcriptional profile compared with localized PC, with a predominance of luminal B (50%) and basal (48%) subtypes, lower androgen receptor activity (AR-A), and high Decipher risk disease. Luminal B subtype was associated with poorer prognosis on ADT alone but benefited significantly from ADT + D [OS: hazard ratio (HR) 0.45; P = 0.007], in contrast to basal subtype which showed no OS benefit (HR 0.85; P = 0.58), even in those with high-volume disease. Higher Decipher risk and lower AR-A were significantly associated with poorer OS in MVA. In addition, higher Decipher risk showed greater improvements in OS with ADT + D (HR 0.41; P = 0.015). CONCLUSION: This study demonstrates the utility of transcriptomic subtyping to guide prognostication in mHSPC and potential selection of patients for chemohormonal therapy, and provides proof of concept for the possibility of biomarker-guided selection of established combination therapies in mHSPC.

Prothrombin complex concentrates and andexanet for management of direct factor Xa inhibitor related bleeding: a meta-analysis.

There are potential concerns related to bleeding caused by oral anticoagulants, especially in the elderly. Andexanet alfa has been authorized for use to reverse the effects of oral anticoagulants. Off-label use of four factor prothrombin complex concentrate (4F-PCC) for the reversal of oral factor Xa inhibitors is common. However, not much is known about their efficacy and safety profile. The intent of this meta-analysis was to evaluate the efficacy and safety of 4F-PCC and andexanet alfa for management of major bleeding due to oral factor Xa inhibitors. Comprehensive searches were done systematically through PubMed, Scopus and Google scholar databases. Studies that were retrospective record based or adopted prospective cohort approach and reported either of the three main outcomes, i.e., achieved hemostasis rate or rate of thrombotic events or mortality rate were included in the meta-analysis. Statistical analyses were done using STATA version 13.0. A total of 22 studies were included in the meta-analysis. All the studies had a single arm with no control/comparator group. The pooled rate of good to excellent hemostatic control upon use of andexanet was 80% (95% CI; 72% to 88%) and for 4F-PCC, it was 76% (95% CI; 70% to 83%). A comparatively higher pooled rate of thrombotic complications upon use of andexanet [13% (95% CI; 5% to 20%) was noted, compared to use of aPCC/4F-PCC [4% (95% CI; 3% to 5%). The pooled all-cause mortality rate within 30 days of administration was 24% (95% CI; 12% to 35%) with andexanet use and 19% (95% CI; 14% to 25%) for aPCC/4F-PCC. The findings suggest that use of both andexanet and aPCC/4F-PCC achieves a good hemostasis but there is an associated risk of thrombotic events and mortality. Future studies should have a control group to better establish evidence on efficacy and safety of these agents.

To safely reopen after a lockdown, masks are crucial: lessons from Taiwan.

Taiwan had been using many important public health management strategies to beat Coronavirus disease 2019 (COVID-19) without a lockdown. Mask wearing by the general public was thought to be the major factor for the success of Taiwan to stop the spread of COVID-19. We share our experience in Taiwan as an example for other countries to safely reopen from a lockdown.

Iron based nanotherapeutics for ferroptosis-induced cancer therapy.

Traditional anti-cancer treatments are far from satisfactory. There is an urgent to combine new therapeutics with traditional treatments to improve anti-cancer effectiveness. Ferroptosis is a new type of iron dependent non-apoptotic cell death could still offer benefits to patients who failed in apoptosis and necroptosis induction treatment. Iron plays a vital role during ferroptosis induction. While iron is a double-edged sword in cancer treatment, tumor specific distribution of iron is especially important. Nanotechnology is an efficient way to help drugs targeting distribution. We intended to review the latest progress in ferroptosis and iron based nanotherapeutics. First, the relationship between ferroptosis and iron metabolism was reviewed briefly to demonstrate the central role of iron in ferroptosis induction. Second, the latest progress of iron-based nanotechnology was presented and discussed according to the different designs. Finally, the future expectations of iron based nanotherapeutics for ferroptosis were spotlighted.

EZH2-mediated H3K27me3 enrichment on the lncRNA MEG3 promoter regulates the growth and metastasis of glioma cells by regulating miR-21-3p.

OBJECTIVE: Glioma is one of the most common and invasive brain tumors worldwide. Long non-coding RNAs (LncRNAs) play an important role in the development of glioma. However, the regulatory mechanism of LncRNAs in glioma has not been fully elucidated. This study aimed to explore the interaction of lncRNA maternally expressed gene 3 (MEG3) and aberrant histone modification in glioma. MATERIALS AND METHODS: The expression levels of MEG3 and miR-21-3p in glioma cells were measured by quantitative polymerase chain reaction (qPCR). EZH2 (enhancer of zeste homolog 2) and H3K27me3 expression in glioma cells were detected by Western Blot (WB). The binding site of the promoter of MEG3 by H3K27me3 was confirmed by ChIP-Real-time PCR. The direct target of MEG3 and miR-21-3p in glioma cells was measured by a luciferase reporter assay. Cell proliferation was detected by Cell Counting Kit-8 (CCK8), and cell invasion and migration were measured by Transwell assays. RESULTS: EZH2 and miR-21-3p were upregulated and MEG3 was downregulated in glioma cells. Silencing of EZH2 inhibited cell proliferation, migration, and invasion in U87 and U251 cells. Meanwhile, the expression of H3K27me3 could be significantly inhibited by EZH2 interference. H3K27me3 protein can bind to MEG3 promoter directly. EZH2 inhibition and MEG3 down-expression in U87 cells reversed the effects of silencing of EZH2 on glioma cell growth and metastasis. However, EZH2 inhibition and MEG3 overexpression in U251 cells restricted cell proliferation, migration, and invasion. Furthermore, miR-21-3p was verified to interact with MEG3 by direct binding. Inhibition of MEG3 promoted U87 cell growth and metastasis, which was further strengthened following the co-transfection of si-MEG3 and miR-21-3p. Overexpressed MEG3 inhibited U251 cell growth and metastasis and a complete reversal of the results observed in the co-transfection of LV-MEG3 and miR-21-3p. CONCLUSIONS: EZH2 was highly expressed in glioma cells and EZH2-mediated H3K27me3 enrichment on the MEG3 promoter regulated the growth and metastasis of glioma cells by targeting miR-21-3p. It potentially provided a new therapeutic marker targeting glioma.

Extending Phenomenological Crystal-Field Methods to C_{1} Point-Group Symmetry: Characterization of the Optically Excited Hyperfine Structure of

We show that crystal-field calculations for C_{1} point-group symmetry are possible, and that such calculations can be performed with sufficient accuracy to have substantial utility for rare-earth based quantum information applications. In particular, we perform crystal-field fitting for a C_{1}-symmetry site in ^{167}Er^{3+}:Y_{2}SiO_{5}. The calculation simultaneously includes site-selective spectroscopic data up to 20 000 cm^{-1}, rotational Zeeman data, and ground- and excited-state hyperfine structure determined from high-resolution Raman-heterodyne spectroscopy on the 1.5 µm telecom transition. We achieve an agreement of better than 50 MHz for assigned hyperfine transitions. The success of this analysis opens the possibility of systematically evaluating the coherence properties, as well as transition energies and intensities, of any rare-earth ion doped into Y_{2}SiO_{5}.

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.

Ten-year fracture risk by FRAX and osteoporotic fractures in patients with systemic autoimmune diseases.

The Fracture Risk Assessment Tool (FRAX) has been used universally for the purpose of fracture risk assessment. However, the predictive capacity of FRAX for autoimmune diseases remains inconclusive. This study aimed to compare the applicability of FRAX for autoimmune disease patients. This retrospective study recruited rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and primary Sjögren syndrome (pSS) patients with bone mineral density (BMD) tests. Patients with any osteoporotic fractures were identified. Taiwan-specific FRAX with and without BMD were then calculated. In total, 802 patients (451 RA, 233 SLE and 118 pSS) were enrolled in this study. The cumulative incidences of osteoporotic fractures in the RA, SLE and pSS patients were 43.0%, 29.2% and 33.1%, respectively. For those with a previous osteoporotic fracture, T-scores were classified as low bone mass. Overall, the patients' 10-year probability of major fracture risk by FRAX without BMD was 15.8%, which then increased to 20.3% after incorporation of BMD measurement. When analyzed by disease group, the fracture risk in RA patients was accurately predicted by FRAX. In contrast, current FRAX, either with or without BMD measurement, underestimated the fracture risk both in SLE and pSS patients, even after stratification by age and glucocorticoid treatment. For pSS patients with major osteoporotic fractures, FRAX risks imputed by RA were comparable to major osteoporotic fracture risks of RA patients. Current FRAX accurately predicted fracture probability in RA patients, but not in SLE and pSS patients. RA-imputed FRAX risk scores could be used as a temporary substitute for SLE and pSS patients.

Do dysplastic proximal resection margins predict the risk of anastomotic recurrence and overall survival in patients with esophageal squamous cell carcinoma?

Positive proximal resection margins are strongly associated with anastomotic recurrence in esophageal cancer. However, the prognostic significance of dysplastic proximal resection margins remains unclear. The aim of this study is to investigate whether the dysplastic proximal resection margin can predict anastomotic recurrence and overall survival in patients with esophageal squamous cell carcinoma. Between 2000 and 2014, patients with esophageal squamous cell carcinoma who received a nonpalliative resection and survived the perioperative period were included. Two expert pathologists independently reviewed the proximal resection margin status, which was classified as negative, dysplastic, or positive. The kappa statistic was used to test interobserver reliability. Anastomotic recurrence and overall survival served as the main outcome measures. The study cohort consisted of 469 patients (445 males and 27 females). There was an excellent interobserver agreement for negative (kappa = 0.88), dysplastic (kappa = 0.88), and positive (kappa = 1) proximal resection margins-which were identified in 418 (89.1%), 37 (7.9%), and 14 (3.0%) patients, respectively. After a median follow-up of 21.6 months, 30 (6.4%) patients developed an anastomotic recurrence. Compared with patients with negative proximal resection margins (24/418, 5.7%), the occurrence of anastomotic recurrence was more commonly observed in those with positive proximal resection margins (3/14, 21.4%, P = 0.017) but not in those with dysplastic proximal resection margins (3/37, 8.1%, P = 0.56). Multivariable Cox regression analysis identified positive proximal resection margins (hazard ratio: 5.93, P = 0.010) and advanced clinical stage (hazard ratio: 12.04, P = 0.023) as independent risk factors for anastomotic recurrence. Dysplastic proximal resection margins were not retained in the model as an independent predictor (hazard ratio: 1.38, P = 0.602). The 5-year overall survival rates of patients with negative (38.2%) and dysplastic margins (27.0%) were similar (P = 0.814), and significantly higher than that observed in those with positive proximal resection margins (9.5%, P = 0.015). In conclusion, dysplastic proximal resection margins can be identified in at least 7.9% of patients with esophageal squamous cell carcinoma, but neither they are associated with an increased risk of anastomotic recurrence nor they portend a poor overall survival.

Long-term outcomes and quality of life following ligation of the intersphincteric fistula tract for high transsphincteric fistulas.

AIM: Published outcomes following ligation of the intersphincteric fistula tract (LIFT) for high transsphincteric fistulas (HTFs) are equivocal probably because most trials are small and comprise mixed patient populations. The aim of this study was to highlight the long-term efficacy of LIFT for HTFs in a large homogeneous sample and to determine the risk factors that contribute to non-healing resulting in failure and recurrence. METHOD: A retrospective study was performed which assessed patients with HTFs treated by LIFT without prior loose setons from September 2012 to December 2017. Continence function was evaluated by the Wexner incontinence scale and anal manometry. Quality of life was assessed by using the faecal incontinence quality of life (FIQL) scale with four domains: lifestyle, coping, depression and embarrassment. RESULTS: Seventy patients with HTFs underwent 71 LIFT procedures. The primary healing rate was 81.7% with a median follow-up duration of 16.5 (range 4.5-68) months. The healing rates of mature and immature fistulas were 83.7% and 77.3%, respectively. Two patients suffered failure with an unhealed intersphincteric wound. Recurrence occurred in 11 patients. Incontinence of flatus, present in four patients before surgery, improved postoperatively. Two patients undergoing LIFT combined with fistulotomy complained of flatus incontinence after surgery. No significant differences between preoperative and postoperative Wexner score, maximum resting pressure and maximum squeeze pressure were detected. The FIQL was improved in lifestyle, coping and depression. No risk factor for non-healing was found. CONCLUSION: LIFT has a promising long-term outcome for HTFs, with negligible impairment on continence and improved quality of life.

Cost-effectiveness analysis of oral probiotics for the prevention of Clostridium difficile-associated diarrhoea in children and adolescents.

BACKGROUND: The incidence of Clostridium difficile-associated diarrhoea (CDAD) in hospitalized children and adolescents has been increasing year-on-year. Paediatric CDAD places a significant economic burden on healthcare systems. Probiotics are live organisms thought to improve the microbial balance of the host, counteract disturbances in intestinal flora, and reduce the risk of colonization by pathogenic bacteria. AIM: A cost-effectiveness analysis was conducted to assess the economy of probiotics for the prevention of CDAD in children and adolescents receiving antibiotics. METHODS: A decision tree model combining clinical effectiveness, utility and cost data was used. Sensitivity analyses were conducted to determine the robustness of the model outcomes. FINDING: The 'oral probiotics' strategy and 'no probiotics' strategy offered patients 0.05876 and 0.056 quality-adjusted life years (QALYs) at a cost of $16,668.70 and $20,355.28, respectively. The oral probiotics strategy exhibited higher QALY and lower cost, and represents the cost-saving strategy. The results were robust for sensitivity analyses. CONCLUSION: From the perspective of the medical system, oral probiotics as a preventive strategy for CDAD in hospitalized children and adolescents receiving a therapeutic course of antibiotics reduced the risk of CDAD and represents a cost-saving strategy.