In Vitro Functional Profiling of Fentanyl and Nitazene Analogs at the µ-Opioid Receptor Reveals High Efficacy for Gi Protein Signaling.

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as µ-opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G-protein-biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains the reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at the MOR using adenylate cyclase inhibition and ß-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach. By fitting the concentration-response data to the operational model of agonism, we deduced the intrinsic efficacy and affinity for each opioid in the Gi protein signaling and ß-arrestin2 recruitment pathways. Compared to the reference agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin, we found that several fentanyl analogs were more efficacious at inhibiting cAMP production, whereas all fentanyl analogs were less efficacious at recruiting ß-arrestin2. In contrast, the non-fentanyl 2-benzylbenzimidazole (i.e., nitazene) analogs were highly efficacious and potent in both the cAMP and ß-arrestin2 assays. Our findings suggest that the high intrinsic efficacy of the NSOs in Gi protein signaling is a common property that may underlie their high risk of intoxication and overdose, highlighting the limitation of using in vitro functional bias to predict the adverse effects of opioids. In addition, the extremely high potency of many NSOs now infiltrating illicit drug markets further contributes to the danger posed to public health.

The in vitro functional profiles of fentanyl and nitazene analogs at the µ-opioid receptor - high efficacy is dangerous regardless of signaling bias.

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as the µ-opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G protein biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at MOR using adenylate cyclase inhibition and ß-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach. By fitting the concentration-response data to the operational model of agonism, we deduced the intrinsic efficacy and affinity for each opioid in the Gi protein signaling and ß-arrestin2 recruitment pathways. Compared to the reference agonist DAMGO, we found that several fentanyl analogs were more efficacious at inhibiting cAMP production, whereas all fentanyl analogs were less efficacious at recruiting ß-arrestin2. In contrast, the non-fentanyl 2-benzylbenzimidazole (i.e., nitazene) analogs were highly efficacious and potent in both the cAMP and ß-arrestin2 assays. Our findings suggest that the high intrinsic efficacy of the NSOs in Gi protein signaling is a common property that may underlie their high risk of intoxication and overdose, highlighting the limitation of using in vitro functional bias to predict the adverse effects of opioids. Instead, our results show that, regardless of bias, opioids with sufficiently high intrinsic efficacy can be lethal, especially given the extremely high potency of many of these compounds that are now pervading the illicit drug market.

Alkoxy chain length governs the potency of 2-benzylbenzimidazole 'nitazene' opioids associated with human overdose.

RATIONALE: Novel synthetic opioids (NSOs) are emerging in recreational drug markets worldwide. In particular, 2-benzylbenzimidazole 'nitazene' compounds are problematic NSOs associated with serious clinical consequences, including fatal respiratory depression. Evidence from in vitro studies shows that alkoxy chain length can influence the potency of nitazenes at the mu-opioid receptor (MOR). However, structure-activity relationships (SARs) of nitazenes for inducing opioid-like effects in animal models are not well understood compared to relevant opioids contributing to the ongoing opioid crisis (e.g., fentanyl). OBJECTIVES: Here, we examined the in vitro and in vivo effects of nitazene analogues with varying alkoxy chain lengths (i.e., metonitazene, etonitazene, isotonitazene, protonitazene, and butonitazene) as compared to reference opioids (i.e., morphine and fentanyl). METHODS AND RESULTS: Nitazene analogues displayed nanomolar affinities for MOR in rat brain membranes and picomolar potencies to activate MOR in transfected cells. All compounds induced opioid-like effects on locomotor activity, hot plate latency, and body temperature in male mice, and alkoxy chain length markedly influenced potency. Etonitazene, with an ethoxy chain, was the most potent analogue in MOR functional assays (EC50 = 30 pM, Emax = 103%) and across all in vivo endpoints (ED50 = 3-12 µg/kg). In vivo SARs revealed that ethoxy, isopropoxy, and propoxy chains engendered higher potencies than fentanyl, whereas methoxy and butoxy analogues were less potent. MOR functional potencies, but not MOR affinities, were positively correlated with in vivo potencies to induce opioid effects. CONCLUSIONS: Overall, our data show that certain nitazene NSOs are more potent than fentanyl as MOR agonists in mice, highlighting concerns regarding the high potential for overdose in humans who are exposed to these compounds.

Comparative neuropharmacology of structurally distinct non-fentanyl opioids that are appearing on recreational drug markets worldwide.

BACKGROUND: The emergence of novel synthetic opioids (NSOs) is contributing to the opioid overdose crisis. While fentanyl analogs have historically dominated the NSO market, a shift towards non-fentanyl compounds is now occurring. METHODS: Here, we examined the neuropharmacology of structurally distinct non-fentanyl NSOs, including U-47700, isotonitazene, brorphine, and N-desethyl isotonitazene, as compared to morphine and fentanyl. Compounds were tested in vitro using opioid receptor binding assays in rat brain tissue and by monitoring forskolin-stimulated cAMP accumulation in cells expressing the human mu-opioid receptor (MOR). Compounds were administered subcutaneously to male Sprague-Dawley rats, and hot plate antinociception, catalepsy score, and body temperature changes were measured. RESULTS: Receptor binding results revealed high MOR selectivity for all compounds, with MOR affinities comparable to those of morphine and fentanyl (i.e., nM). All drugs acted as full-efficacy MOR agonists in the cyclic AMP assay, but nitazene analogs had greater functional potencies (i.e., pM) compared to the other drugs (i.e., nM). When administered to rats, all compounds induced opioid-like antinociception, catalepsy, and body temperature changes, but nitazenes were the most potent. Similar to fentanyl, the nitazenes had faster onset and decline of in vivo effects when compared to morphine. In vivo potencies to induce antinociception and catalepsy (i.e., ED50s) correlated with in vitro functional potencies (i.e., EC50s) but not binding affinities (i.e., Kis) at MOR. CONCLUSIONS: Collectively, our findings indicate that non-fentanyl NSOs pose grave danger to those individuals who use opioids. Continued vigilance is needed to identify and characterize synthetic opioids as they emerge in clandestine drug markets.

Binding preference at the µ-opioid receptor underlies distinct pharmacology of cyclopropyl versus valeryl analogs of fentanyl.

Illicitly manufactured fentanyl is driving the current opioid crisis, and various fentanyl analogs are appearing in recreational drug markets worldwide. To assess the potential health risks posed by fentanyl analogs, it is necessary to understand structure-activity relationships for these compounds. Here we compared the pharmacology of two structurally related fentanyl analogs implicated in opioid overdose: cyclopropylfentanyl and valerylfentanyl. Cyclopropylfentanyl has a three-carbon ring attached to the carbonyl group on the fentanyl scaffold, whereas valerylfentanyl has a four-carbon chain at the same position. In vitro assays examining µ-opioid receptor (MOR) coupling to G proteins in CHO cells showed that cyclopropylfentanyl is a full agonist (EC50 = 8.6 nM, %Emax = 113%), with potency and efficacy similar to fentanyl (EC50 = 10.3 nM, %Emax = 113%). By contrast, valerylfentanyl is a partial agonist at MOR (EC50 = 179.8 nM, %Emax = 60%). Similar results were found in assays assessing MOR-mediated ß-arrestin recruitment in HEK cells. In vivo studies in male CD-1 mice demonstrated that both fentanyl analogs induce naloxone-reversible antinociception and respiratory suppression, but cyclopropylfentanyl is 100-times more potent as an antinociceptive agent (ED50 = 0.04 mg/kg, s. c.) than valerylfentanyl (ED50 = 4.0 mg/kg, s. c.). Molecular simulation results revealed that the alkyl chain of valerylfentanyl cannot be well accommodated by the active state of MOR and may transition the receptor toward an inactive state, converting the fentanyl scaffold to a partial agonist. Taken together, our results suggest that cyclopropylfentanyl presents much greater risk of adverse effects when compared to valerylfentanyl. Moreover, the summed findings may provide clues to the design of therapeutic opioids with reduced adverse side effects.

Bronchial asthma and risk of 4 specific cardiovascular diseases and cardiovascular mortality: a meta-analysis of cohort studies.

OBJECTIVE: It has been shown that asthma is significantly associated with the risk of cardiovascular disease (CVD). Under this background, this study aimed to systematically classify and summarize the epidemiological evidence of asthma and the risk of 4 specific cardiovascular diseases (CVDs) and cardiovascular mortality (CVM). MATERIALS AND METHODS: PubMed and Embase databases were searched from inception to December 1st, 2021 in order to identify relevant studies. The random-model was used to assess the pooled results. All pooled results were expressed as risk ratios (RRs) and corresponding 95% confidence intervals (CIs). RESULTS: Finally, a total of 18 studies were included in the present meta-analysis. Compared with non-asthmatic group, patients with asthma had significantly increased risks of subsequent cardiovascular heart disease (CHD, RR 1.33; 1.19-1.50, I2=80.3%; p<0.001), and CVM (RR 1.35; 1.15-1.59, I2=0%; p<0.001). Similarly, the risks of heart failure (HF, RR 2.10; 1.98-2.22, I2=17.4%; p<0.001) and myocardial infraction (MI, RR 1.39; 1.16-1.66, I2=59.3%; p<0.001) were higher in the asthmatic population. However, the higher risk of atrial fibrillation (RR 1.70; 1.45-2.00, I2=0%; p<0.001) was observed only in the active asthmatic population. CONCLUSIONS: In general, asthma is associated with subsequent increased risks of CHD, MI, AF, HF, and CVM. In addition, among patients with asthma, females have a higher risk of CHD than males, while active asthmatic patients have a higher risk of CVM than non-active asthmatic patients.

Active Precipitation of Radiation Belt Electrons Using Rocket Exhaust Driven Amplification (REDA) of Man-Made Whistlers.

Ground-based very low frequency (VLF) transmitters located around the world generate signals that leak through the bottom side of the ionosphere in the form of whistler mode waves. Wave and particle measurements on satellites have observed that these man-made VLF waves can be strong enough to scatter trapped energetic electrons into low pitch angle orbits, causing loss by absorption in the lower atmosphere. This precipitation loss process is greatly enhanced by intentional amplification of the whistler waves using a newly discovered process called rocket exhaust driven amplification (REDA). Satellite measurements of REDA have shown between 30 and 50 dB intensification of VLF waves in space using a 60 s burn of the 150 g/s thruster on the Cygnus satellite that services the International Space Station. This controlled amplification process is adequate to deplete the energetic particle population on the affected field lines in a few minutes rather than the multi-day period it would take naturally. Numerical simulations of the pitch angle diffusion for radiation belt particles use the UCLA quasi-linear Fokker Planck model to assess the impact of REDA on radiation belt remediation of newly injected energetic electrons. The simulated precipitation fluxes of energetic electrons are applied to models of D-region electron density and bremsstrahlung X-rays for predictions of the modified environment that can be observed with satellite and ground-based sensors.

[Protective effect of vitamin D in mice with acute liver failure].

Objective: To explore the protective effect of vitamin D in acute liver failure through a mouse model. Methods: Acute liver failure was induced by combining D-galactosamine (D-GalN) lipopolysaccharide (LPS) to observe the effect of long-term vitamin D deficiency on liver injury and inflammatory signals in a mouse model. Acute liver failure was induced by thioacetamide (TAA) to observe the effect of vitamin D deficiency on the survival rate, and further high-dose of vitamin D supplementation protective effect was determined in a mouse model. Liver function was evaluated by measuring serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and liver inflammation by hematoxylin-eosin staining. The expressions of tumor necrosis factor (TNF-α), interleukin (IL) -1ß, NOD-like receptor family, pyrin domain containing 3 (NLRP-3), chemokines (CCL2, CXCL1 and CXCL2), etc. in liver tissues were detected by RT-qPCR. The quantitation of macrophages in liver tissue was detected by immunohistochemistry. The comparison between groups were performed by t-test. The survival curve was analyzed by log-rank (Mantel-Cox) test. Results: Long-term vitamin D deficiency had increased acute liver failure sensitivity in mice, which was manifested by increased blood cell extravasation, massive necrosis of parenchymal cells, up-regulation of TNF-α, IL-1ß, and NLRP-3 mRNA expression (P < 0.05), and increased macrophages quantitation (P < 0.05) in liver tissues. At the same time, vitamin D deficiency had increased the mice mortality rate because of liver injury (P < 0.01). On the contrary, pre-administration of high dose of vitamin D (100 IU/g) had significantly reduced liver injury, inhibited ALT and AST rise (P < 0.01), alleviated liver necrosis, and down-regulated the mRNA expression of inflammatory factors in liver tissues (P < 0.05). Conclusion: Mouse model shows that long-term vitamin D deficiency can aggravate drug-induced acute liver failure and reduce survival rates. Furthermore, high-dose of vitamin D has a certain hepatoprotective effect, which can significantly improve liver necrosis condition and inhibit inflammation. Therefore, adequate vitamin D can retain liver physiological balance to resist liver injury.

[The influence of peripheral blood sample storage and delivery on the quantitative detection result of BCR-ABL (P210) transcript levels].

Objective: To explore the influence of storage and delivery conditions of the peripheral blood samples from patients with chronic myeloid leukemia (CML) on the real-time quantitative PCR (RQ-PCR) detection of the BCR-ABL (P210) transcript levels. Methods: The peripheral blood samples of 84 CML patients were collected. The same sample was divided into different groups according to storage time (0, 6, 12, 24, 48, and 72 h) , temperature (room temperature, 18-24 ℃; low temperature, 2-8 ℃) , and vibration conditions (3, 6, and 12 h) . RQ-PCR was used to detect BCR-ABL (P210) transcript levels of the different groups. This study logarithmically transformed (log(10N)) the original data [BCR-ABL copy number, ABL copy number, and BCR-ABL (P210) transcript levels]. Results: ①Agarose gel electrophoresis showed significant RNA degradation of samples after storage for 48 and 72 h at room temperature. ②Among the overall samples, the BCR-ABL copy number of the samples stored at room temperature for 48 and 72 h was significantly lower than that of the samples stored at low temperature (P<0.05) . However, the BCR-ABL (P210) transcript levels had no significant difference between samples stored at low temperature and room temperature. ③No significant changes were noted in the BCR-ABL (P210) transcript levels at different storage times (6, 12, 24, 48, and 72 h) regardless of storage temperature (P>0.05) compared with that at baseline (0 h, -0.56±1.51) . ④ The BCR-ABL copy number of the overall sample only decreased significantly (P<0.05) at 48 h (2.93±1.59) and 72 h (2.79±1.42) compared with that at baseline (0 h, 3.35±1.60) when stored at room temperature. The ABL copy number in the overall sample decreased significantly at 48 and 72 h (whether low and room temperature; P<0.05) . However, no significant changes were noted in the BCR-ABL (P210) transcript levels after vibration for 3 h (-1.29±1.81) , 6 h (-1.24±1.72) , and 12 h (-1.18±1.68; P>0.05) compared with that at baseline (0 h, -0.60±1.37) . Conclusion: Sample storage time, storage temperature, and vibration can interfere with the results of BCR-ABL and ABL copy number but have no significant effect on the quantitative determination of BCR-ABL (P210) transcript levels. This study provides strong support for the feasibility of transregional transportation of peripheral blood samples from patients with CML.

Author Correction: The dual role of anti-viral therapy in the treatment of Coronavirus disease 2019.

Correction to: European Review for Medical and Pharmacological Sciences 2020; 24 (22): 11939-11944-DOI: 10.26355/eurrev_202011_23854-PMID: 33275267, published online 30 November, 2020. The authors state that "Figures 3 and 4 were used twice due to a careless mistake during the preparation of Figures". There are amendments to this paper.  The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/23854.

FOXN2 suppresses the proliferation and invasion of human hepatocellular carcinoma cells.

OBJECTIVE: The aim of this study was to explore the roles of FOXN2 (Fork head Box N2) in mediating the proliferation and invasion of hepatocellular carcinoma (HCC) cells. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to determine expression of FOXN2 in HCC tissues and cells. Transfection of plasmid containing FOXN2 was used to exogenously overexpress FOXN2 in vitro. Cell Counting Kit-8 (CCK-8) assay and transwell assay were applied to detect the proliferation and invasion of HCC cells, respectively. RESULTS: FOXN2 expression decreased significantly in both HCC tissues and cells (p<0.05). Upregulation of FOXN2 significantly inhibited the proliferation and invasion of HCC cells (p<0.05). CONCLUSIONS: FOXN2 acts as a regulator in the progression of HCC. Our findings suggest that FOXN2 may be a novel therapeutic monitoring and prognosis biomarker in HCC.

The dual role of anti-viral therapy in the treatment of Coronavirus disease 2019.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) has become a worldwide public health emergency; unfortunately, there is currently no treatment for improving outcomes or reducing viral-clearance times in infected patients. The aim of the present study was to evaluate the efficacy of interferon (IFN) with or without lopinavir and ritonavir as antiviral therapeutic option for treating COVID-19 infection. PATIENTS AND METHODS: The present study enrolled 148 patients that received either standard care, treatment with IFN alfa-2b, or IFN alfa-2b combined with lopinavir plus ritonavir. Viral testing was performed using Reverse-Transcription Polymerase Chain Reaction (RT-PCR). RESULTS: There was no significant difference in the viral-clearance time at 28 days after treatment between patients receiving standard care and those receiving anti-viral treatments. However, the average viral-clearance time of patients receiving standard care (14 days) was shorter than that for patients receiving IFN alfa-2b or IFN alfa-2b combined with lopinavir plus ritonavir (15.5 or 17.5 days) (p<0.05). Patients treated with IFN alfa-2b within five days or IFN alfa-2b combined with lopinavir plus ritonavir after three days of symptoms exhibited shorter viral-clearance times than the other groups (p<0.05). Moreover, viral-clearance times were significantly longer in patients receiving standard care or anti-viral treatment 5 days after symptoms appeared than those of patients who received these treatments within five days of symptom onset (p<0.05). CONCLUSIONS: Early symptomatic treatment is most critical for maximizing amelioration of COVID-19 infection. Anti-viral treatment might have complicated effect on viral-clearance.

[Proteomics analysis of erythrocyte membrane in rats with high altitude polycythemia before and after intervention with salidroside].

To investigate the effect of salidroside on the proteomics of erythrocyte membrane in high altitude erythrocytosis(HAPC) rats, in order to explore the mechanism of salidroside in improving HAPC based on the proteomics analysis. First, HPAC rat models were established, and 16 rats were randomly divided into HAPC model group and salidroside(100 mg·kg~(-1)) treatment group(8 rats per group). Saline was administered to the HAPC model group, while salidroside treatment group was given 100 mg·kg~(-1) salidroside once a day. After continuous oral administration with salidroside for 40 days(once a day), blood was collected from the femoral artery to obtain total red blood cell membrane proteins. Two-dimensional electrophoresis was used to separate total proteins. The two-dimensional electrophoresis of erythrocyte membrane proteins was analyzed before and after salidroside intervention, and the proteins with significant differences were identified by mass spectrometry. Finally, biological functions were analyzed using bioinformatics. A two-dimensional electrophoresis method was used to establish a protein expression profile with a high resolution and reproducibility of erythrocyte membranes in HAPC rats. Salidroside treatment significantly changed 18 protein spots in the 2-DE map of erythrocyte membranes, of which 13 proteins were up-regulated and 5 proteins were down-regulated. Eight differential proteins were successfully identified by mass spectrometry. Moreover, bioinformatics analysis found that these differential proteins were involved in such biological processes as oxidative stress, redox, and peroxisome pathway, which are mainly associated with peroxisome and MAPK signaling pathways. Therefore, salidroside could significantly change the expressions of erythrocyte membrane proteins in HAPC rats. Eight differential proteins were identified by a proteomic-based approach. The differential proteins were involved in such biological processes as oxidative stress, redox, peroxisome pathway.

Strain-Dependent Modifier Genes Determine Survival in Zfp423 Mice.

Zfp423 encodes a transcriptional regulatory protein that interacts with canonical signaling and lineage pathways. Mutations in mouse Zfp423 or its human ortholog ZNF423 are associated with a range of developmental abnormalities reminiscent of ciliopathies, including cerebellar vermis hypoplasia and other midline brain defects. Null mice have reduced viability in most strain backgrounds. Here we show complete lethality on a C57BL/6J background, dominant rescue in backcrosses to any of 13 partner strains, with strain-dependent survival frequencies, and evidence for a BALB/c-derived survival modifier locus on chromosome 5. Survival data indicate both perinatal and postnatal periods of lethality. Anatomical data from a hypomorphic gene trap allele observed on both C57BL/6J and BALB/c congenic backgrounds shows an aggregate effect of background on sensitivity to Zfp423 loss rather than a binary effect on viability.

Immunorelated gene polymorphisms associated with acute myeloid leukemia.

Although the pathogenesis of acute myeloid leukemia (AML) is still unknown, accumulating evidence has revealed that immune response plays a vital part in the pathogenesis. Here, we investigated the involvement of 21 single nucleotide polymorphisms (SNPs) of immunorelated genes, including cytokines [interleukin (IL)-2, IL-4, IL-9, IL-12A, IL-22, interferon (IFN-α) and transforming growth factor (TGF)-ß1], transcriptional regulatory genes (TBX21, STAT1, STAT3, STAT5B, STAT6, GATA3, FOXP3 and IRF4) and others (IL2RA, IL6R, NFKBIA) in 269 AML in-patients and 200 healthy controls. Furthermore, we analyzed the relationship between the SNPs and clinical characteristics. Immunorelated SNP genotyping was performed on the Sequenom MassARRAY iPLEX platform. All the SNPs in healthy controls were consistent with Hardy-Weinberg equilibrium. All final P-values were adjusted by Bonferroni multiple testing. Our results showed that IL-22 (rs2227491) was significantly associated with the white blood cell (WBC) counts. Signal transducer and activator of transcription 5B (STAT-5B) (rs6503691) showed a close relationship with the recurrent genetic abnormalities in patients with AML. We verified the negatively independent effect of age and risk of cytogenetics on overall survival (OS). More importantly, the GG genotype of IL-12A (rs6887695) showed a negative impact on AML prognosis independently. Furthermore, the relative expression of IL-12 was decreased in GG genotype, no matter under a co-dominant or recessive model. However, no correlation was observed between the SNPs mentioned above and disease susceptibility, risk stratification and survival. Our findings suggest that immunorelated gene polymorphisms are associated with prognosis in AML, which may perform as novel inspection targets for AML patients.

A Comparative Analysis of Medical Education Models and Curriculums of A Medical University and A Medical Education Center.

INTRODUCTION: In order to solve the shortage of competent healthcare manpower at the village level of Yunnan Province, We compared the training mode of Kunming Medical University and The Medical Educational Center, Hat Yai of PSU. The aim of this study is to compare the difference of the two institutions and learn from each other's advantages. METHODS: The review covered relevant policy areas and stipulations governing general practitioner training for both countries. Qualitative research was done by using a questionnaire developed in house by the project team, students from the inaugural cohort at KMU and students from the MECH. In Qualitative research, in-depth interviews were carried out with the teaching administration and students from both schools. RESULTS: In Kunming Medical University, besides the conventional lectures, teaching methods such as case based learning and problem based learning have been worked into the basic science, laboratory, and clerkship/internship sessions. The desired end product is a general practitioner. The curriculum emphasizes general practice and clinical exposure during the course being guided and informed by the "Undergraduate Medical Education Standard-Clinical Medicine" and the "General Practitioner Training Guidelines" about teaching methods. In Prince of Songkla University, the first and second phases consist of basic science and preclinical integrated topics taught at PSU. For the third and final phase, the students have core clinical modules and selective at MECH where the methods are learner centered, problem based, integrated and set in the context of community primary healthcare practice. CONCLUSIONS: We should start with the integration of the medical disciplines and the humanities, so as to restore the lost "art of doctoring". We need to integrate the various foundational and clinical disciplines into an organ system based curriculum, not just in form, but also in function and purpose.

[Promoting migration and apoptosis of glioma cells by macrophage migration inhibitory factor].

Objective: To investigate the effect of macrophage migration inhibitory factor (MIF) on the biology of glioma U87MG and U251 cells. Methods: Silencing MIF gene expression in U87MG cells by RNA interference was monitored by Western blot. MIF low expressing U251 cells were treated at different concentrations of recombinant human MIF (rhMIF) and scratching test and flow cytometry were used to detect cell migration and apoptosis. The protein expression of bcl-2, bax, AKT, p-AKT was detected by Western blot. Results: The ability of migration and anti-apoptosis of U87MG cells silenced by siRNA decreased significantly, and the expression levels of p-AKT and anti-apoptotic protein bcl-2 also decreased; in contrast, the expression level of apoptosis protein bax increased. With increase of rhMIF treatment concentration, the expression levels of MIF protein, p-AKT and bcl-2 in U251 cells were gradually enhanced, whereas the level of apoptosis protein bax was inhibited. Conclusion: MIF promotes cell migration and inhibits apoptosis of both U87MG and U251 cells, likely through the regulation of PI3K/AKT signaling pathway.

A composite of exhaled LTB4 , LXA4 , FeNO, and FEV1 as an "asthma classification ratio" characterizes childhood asthma.

BACKGROUND: Aberrant generation of eicosanoids is associated with asthma, but the evidence remains incomplete and its potential utility as biomarkers is unclear. Major eicosanoids in exhaled breath condensates (EBCs) were assessed as candidate markers for childhood asthma. METHODS: Ten exhaled eicosanoid species was evaluated using ELISA in the discovery phase, followed by prediction model-building and validation phases. RESULTS: Exhaled LTB4 , LTE4 , PGE2, and LXA4 showed significant difference between asthmatics (N = 60) and controls (N = 20). For validation, an expanded study population consisting of 626 subjects with asthma and 161 healthy controls was partitioned into a training subset to establish a prediction model and a test sample subset for validation. Receiver operating characteristic (ROC) analyses of the training subset revealed the level of exhaled LTB4 to be the most discriminative among all parameters, including FeNO, and a composite of exhaled LTB4 , LXA4 , together with FeNO and FEV1 , distinguishing asthma with high sensitivity and specificity. Further, the Youden index (J) indicated the cut point value of 0.598 for this composite of markers as having the strongest discriminatory ability (sensitivity = 85.2% and specificity = 83.6%). The predictive algorithm as "asthma classification ratio" was further validated in an independent test sample with sensitivity and specificity being 84.4% and 84.8%, respectively. CONCLUSIONS: In a pediatric study population in Taiwan, the levels of exhaled LTB4 , LTE4 , LXA4, and PGE2 in asthmatic children were significantly different from those of healthy controls, and the combination of exhaled LTB4 and LXA4 , together with FeNO and FEV1 , best characterized childhood asthma.

Posterior amorphous corneal dystrophy caused by a de novo deletion.

We present a newborn diagnosed with posterior amorphous corneal dystrophy (PACD). PACD is a rare disorder with partial or complete posterior lamellar corneal opacification. Genetic screening showed a deletion of chromosome 12q21.33-q22 containing the identified four small leucine-rich proteoglycans (SLRP's) associated with this particular dystrophy. Neither parents were carrier of the deletion. To our knowledge, this is the first report of a de novo mutation causing PACD.

Longitudinal investigation of nasopharyngeal methicillin-resistant Staphylococcus aureus colonization in early infancy: The PATCH birth cohort study.

OBJECTIVES: The study aimed to determine the long-term Staphylococcus aureus colonization patterns and strain relatedness, and the association between maternal and infant colonization in infancy. METHODS: A birth cohort study was conducted from January 2012 to November 2014. Nasopharyngeal swabs for S. aureus detection were collected from infants at the age of 1, 2, 4, 6 and 12 months and from mothers when their children were 1-month-old. RESULTS: In total, 254 samples were collected at each planned visit during the first 12-month study. The prevalence of S. aureus colonization decreased in the first year of life, ranging from 61.0% (155/254) at the age of 1 month to 12.2% (31/254) at 12 months. Persistent colonization, defined as a positive culture on four or five occasions, was detected in only 13.8% (35/254) of carriers. Most of the persistent carriers were colonized with methicillin-resistant S. aureus (MRSA) only, and among persistent MRSA carriers, 61.1% (11/18) had indistinguishable genotypes. Of the mothers with MRSA colonization, 77.1% (27/35) had infants who were concomitantly colonized at the age of 1 month; 70.4% (19/27) of the infant-mother paired isolates belonged to indistinguishable or related subtypes, which suggests that surrounding carriers, probably their mothers, may be the possible source for MRSA acquisition in early infancy. CONCLUSIONS: Staphylococcus aureus colonization including MRSA was commonly observed in our cohort. Strains of persistent MRSA among infant-mother pairs were usually of indistinguishable genotypes. Therefore, horizontal spread within households is possibly an important factor related to infant MRSA colonization.