Palmitoylation in apoptosis.

Palmitoylation, a critical lipid modification of proteins, is involved in various physiological processes such as altering protein localization, transport, and stability, which perform essential roles in protein function. Palmitoyltransferases are specific enzymes involved in the palmitoylation modification of substrates. S-palmitoylation, as the only reversible palmitoylation modification, is able to be deacylated by deacyltransferases. As an important mode of programmed cell death, apoptosis functions in the maintenance of organismal homeostasis as well as being associated with inflammatory and immune diseases. Recently, studies have found that palmitoylation and apoptosis have been demonstrated to be related in many human diseases. In this review, we will focus on the role of palmitoylation modifications in apoptosis.

Discovery and mechanism of action of Thonzonium bromide from an FDA-approved drug library with potent and broad-spectrum inhibitory activity against main proteases of human coronaviruses.

Although the effective drugs or vaccines have been developed to prevent the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), their efficacy may be limited for the viral evolution and immune escape. Thus, it is urgently needed to develop the novel broad-spectrum antiviral agents to control the coronavirus disease 2019 (COVID-19) global pandemic. The 3C-like protease (3CLpro) is a highly conserved cysteine proteinase that plays a pivotal role in processing the viral polyprotein to create non-structural proteins (nsps) for replication and transcription of SARS-CoV-2, making it an attractive antiviral target for developing broad-spectrum antiviral agents against SARS-CoV-2. In this study, we identified Thonzonium bromide as an inhibitor of SARS-CoV-2 3CLpro with an IC50 value of 2.04 ± 0.25 µM by fluorescence resonance energy transfer (FRET)-based enzymatic inhibition assay from the FDA-approved drug library. Next, we determined the inhibitory activity of Thonzonium bromide analogues against SARS-CoV-2 3CLpro and analyzed their structure-activity relationship (SAR). Interestingly, Thonzonium bromide showed better inhibitory activity than other analogues. Further fluorescence quenching assay, enzyme kinetics analysis, circular dichroism (CD) analysis and molecular docking studies showed that Thonzonium bromide inhibited SARS-CoV-2 3CLpro activity by firmly occupying the catalytic site and inducing conformational changes of the protease. In addition, Thonzonium bromide didn't exhibit inhibitory activity on human chymotrypsin C (CTRC) and Dipeptidyl peptidase IV (DPP-IV), indicating that it had a certain selectivity. Finally, we measured the inhibitory activities of Thonzonium bromide against 3CLpro of SARS-CoV, MERS-CoV and HCoV-229E and found that it had the broad-spectrum inhibitory activity against the proteases of human coronaviruses. These results provide the possible mechanism of action of Thonzonium bromide, highlighting its potential efficacy against multiple human coronaviruses.

Comparison of Multiple Quantitative Evaluation Indices of Theoretical Knowledge and Clinical Practice Skills and Training of Medical Interns in Cardiovascular Imaging Using Blended Teaching and the Case Resource Network Platform (CRNP).

BACKGROUND This study aimed to compare multiple quantitative evaluation indices of levels of theoretical knowledge and clinical practice skills in training medical interns in cardiovascular imaging based on the use of the blended teaching (BT) online artificial intelligence (AI) case resource network platform (CRNP), including time and frequency indices and effectiveness of the CRNP. MATERIAL AND METHODS The study included 110 medical interns who were divided into the routine teaching (RT) group (n=55) and the blended teaching (BT) group (n=55). The two were assessed using the mini-clinical evaluation exercise (mini-CEX) that assessed clinical skills, attitudes, and behaviors and using an objective written questionnaire. The following four indices were compared between the RT and BT groups: the X-ray score (XS), the computed tomography angiography (CTA) score (CS), the cardiac magnetic resonance imaging (CMRI) score (MS), and the average score (AS). Seven assessment indicators included: the imaging description (ID), the qualitative diagnosis (QD), the differential diagnosis (DD), examination preparation (EP), interview skill (IS), position display (PD), and human care (HC). Indicators of CRNP use included: number of times (TN), average duration (AD), single maximum duration (SMD), and total duration (TD). RESULTS AS significantly correlated with AD (rAD=0.761) and TD (rTD=0.754), and showed moderate correlation with TN (rTN=0.595), but weak correlation with SMD (rSMD=0.404). CONCLUSIONS Levels of theoretical knowledge and clinical practice skills during medical intern training in cardiovascular imaging based on BT using the CRNP teaching technology improved theoretical knowledge and practical skills.

Analysis of thermolabile residual solvents in a spray dried dispersion using static headspace gas chromatography.

In this study, we discuss the development of a static headspace gas chromatography method for the analysis of residual acetone as well as its enriched impurities including mesityl oxide and diacetone alcohol, in a spray dried dispersion. The major challenges include the instability of mesityl oxide and diacetone alcohol at high temperature and peak tailing of diacetone alcohol. It was found that the headspace oven temperature has to be controlled to 150°C or below to prevent degradation beyond an acceptable level (< 1%). The peak tailing of diacetone alcohol was attributed to the "Phase Soaking" effect due to excessive diluent, which may condense and temporarily modify the stationary phase. The peak shape of diacetone alcohol is dependent on the column loading capacity and the peak area of N-methyl pyrrolidone, the solvent that elutes after diacetone alcohol. The headspace oven temperature was set at 140°C, where the highest response ratio of diacetone alcohol/N-methyl pyrrolidone at 1.46 and thus the best sensitivity was obtained. The calculated quantitation limits were 1 ppm for acetone, 3 ppm for mesityl oxide and 31 ppm for diacetone alcohol. The method successfully passed validation criteria for specificity, linearity, accuracy, and precision.

Assessment of coulometric array electrochemical detection coupled with HPLC-UV for the absolute quantitation of pharmaceuticals.

The use of a coulometric array detector in tandem with HPLC-UV was evaluated for the absolute quantitation of pharmaceutical compounds without standards, an important capability gap in contemporary pharmaceutical research and development. The high-efficiency LC flow-through electrochemical detector system allows for the rapid evaluation of up to 16 different potentials, aiding in the identification and quantitation of electrochemically reactive species. By quantifying the number of electrons added or removed from an analyte during its passage through the detector, the number of moles of the analyte can be established. Herein we demonstrate that molecules containing common electroactive functional groups (e.g. anilines, phenols, parabens and tertiary alkyl amines) can in some cases be reliably quantified in HPLC-EC-UV without the need for authentic standards. Furthermore, the multichannel nature of the CoulArray detector makes it well suited for optimizing the conditions for electrochemical reaction, allowing the impact of changes in potential, flow rate, temperature and pH to be conveniently studied. The electrochemical oxidation of albacivir, zomepirac, diclofenac, rosiglitazone and several other marketed drugs resulted in large linear ranges, predictable recoveries and excellent quantitation using the total moles of electrons and back-calculating using Faraday's law. Importantly, we observed several instances where subtle structural changes within a given class of molecules (e.g. aromatic ring isomers) led to unanticipated changes in electrochemical behavior. Consequently, some care should be taken when applying the technique to the routine quantitation of compound libraries where standards are not available.

[Retracted] Long non­coding RNA receptor activator of nuclear factor­κB ligand promotes cisplatin resistance in non­small cell lung cancer cells.

[This retracts the article DOI: 10.3892/etm.2021.9949.].

Resveratrol alleviates heat-stress-induced impairment of the jejunal mucosa through TLR4/MAPK signaling pathway in black-boned chicken.

Heat stress in chickens caused by high temperatures in summer is a serious issue faced by the poultry industry globally, which reduces product quality. The aim of this study is to investigate the role of resveratrol in alleviating heat stress injury and inflammatory response of jejunal mucosa in black-boned chickens through TLR4/MAPK signaling pathway. In total, 240 black-boned chickens (28-day old) were randomly divided into 4 treatment groups as follows. The normal temperature (NT) and normal temperature with resveratrol (NT+Res) groups received a basal diet without and with 400 mg/kg resveratrol, respectively, and treated at 24℃ ± 2℃, 24 h/d. The high temperature (HT) and high temperature with resveratrol (HT+Res) groups received basal diet without and with 400 mg/kg resveratrol, respectively, and treated at 37℃ ± 2℃ for 8 h/d and 24°C ± 2°C for the rest of the time for 12 d. The results revealed the heat-stress responses impaired the villous structure of the jejunum, causing a rough and uneven surface of the jejunal villus, and local intestinal villi were even more prone to rupture. However, resveratrol significantly improved the morphology and structure of jejunal mucosa under heat stress. Heat stress increased the mRNA levels of toll-like receptor 4 (TLR4), c-Jun, c-fos, caspase-3, and p38 (P < 0.05), reduced mRNA level of Bcl-2, and reduced the expression of tight junction proteins Occludin, ZO-1, and Claudin1 (P < 0.05) in the jejunal mucosa. However, resveratrol inhibited the TLR4/ mitogen-activated protein kinase (MAPK) signaling pathway via downregulating TLR4, c-Jun, p38, and caspase-3 (P < 0.05); upregulating Bcl-2 (P < 0.05); decreasing the protein levels of MKK3, p53, and myeloid differentiation factor 88 (MYD88); and increasing the protein levels of Occludin, ZO-1, and Claudin1. In addition, it reduced the levels of JNK and p38 proteins (P < 0.05) and inflammatory factors like tumor necrosis factor-α (TNF-α) in the jejunal mucosa of black-boned chickens under heat stress. In conclusion, resveratrol may play a regulatory role in heat-stress-induced damage and inflammatory response in the intestinal mucosa of black-boned chickens under heat stress.

Improved chiral SFC screening for analytical method development.

In this study we describe the evaluation of a recently developed supercritical fluid chromatography (SFC) instrument for automated chiral SFC method development. The greatly improved gradient dwell volume and liquid flow control of the new instrument in combination with the use of shorter columns containing smaller stationary phase particles affords chiral SFC method development that is faster and more universal than previous systems.

Role of transcription factors in apoptotic cells clearance.

The human body generates 10-100 billion cells every day, and the same number of cells die to maintain homeostasis. The genetically controlled, autonomously ordered cell death mainly proceeds by apoptosis. Apoptosis is an important way of programmed cell death in multicellular organisms, timely and effective elimination of apoptotic cells plays a key role in the growth and development of organisms and the maintenance of homeostasis. During the clearance of apoptotic cells, transcription factors bind to specific target promoters and act as activators or repressors to regulate multiple genes expression, how transcription factors regulate apoptosis is an important and poorly understood aspect of normal development. This paper summarizes the regulatory mechanisms of transcription factors in the clearance of apoptotic cells to date.

A novel chiral oxazoline copper(II)-based complex inhibits ovarian cancer growth in vitro and in vivo by regulating VEGF/VEGFR2 downstream signaling pathways and apoptosis factors.

A novel chiral oxazoline copper(II)-based complex {[Cu(C13H14NO3S)2]}2 (Cu-A) was synthesized by an in situ reaction using L-methioninol, 4-hydroxyisophthalaldehyde, sodium hydroxide and copper(II) nitrate trihydrate as reactants. Its crystal structure was characterized. In vitro, Cu-A was superior to cis-dichlorodiammineplatinum (DDP) in cytotoxicity and angiogenesis inhibition. Cu-A significantly induced apoptosis of ovarian cancer cells (SKOV3) and human umbilical vein endothelial cells (HUVECs), showing significant anti-ovarian cancer and anti-angiogenesis effects. Notably, Cu-A significantly inhibits the growth of ovarian cancer in nude mice xenografted with SKOV3 cells, and it is less renal toxic than DDP. The molecular mechanism of anti-ovarian cancer and anti-angiogenesis is possibly that it down-regulates the expression of the proteins ERK1/2, AKT, FAK, and VEGFR2 and their phosphorylated proteins p-ERK1/2, p-AKT, p-FAK, and p-VEGFR2 in the VEGF/VEGFR2 signal transduction pathway to inhibit SKOV3 cell and HUVEC proliferation, induce apoptosis, suppress migration and metastasis, and inhibit angiogenesis. What's more, Cu-A significantly inhibits ovarian tumor growth in vivo by inhibiting tumor cells from inducing vascular endothelial cells to form their own vasculature and by inhibiting the expression of the anti-apoptotic protein Bcl-2 and up-regulating the expression of the pro-apoptotic proteins Caspase-9 and Bax to induce apoptosis of tumor cells.

Clinical efficacy of low-dose glucocorticoid therapy for critically ill patients with severe fever with thrombocytopenia syndrome: A retrospective cohort study.

OBJECTIVES: To determine whether glucocorticoids can improve clinical outcomes of severe fever with thrombocytopenia syndrome (SFTS) patients, and how to identify patients who may benefit from the treatment. METHODS: A retrospective study was performed to include patients with confirmed SFTS from designated hospitals. The effect of glucocorticoids in reducing case fatality rate (CFR) and improving clinical recovery was evaluated by multivariate logistic regression models. RESULTS: A total of 2478 eligible patients were analyzed, of whom 331 received glucocorticoids. An integrated parameter (L-index) based on Log10(lactate dehydrogenase*blood urea nitrogen/lymphocyte count) was constructed to discriminate disease severity. In patients with L-index >3.823 indicating severe SFTS, significantly reduced CFR was observed in patients receiving low-moderate glucocorticoid doses with ≤60 mg daily methylprednisolone or equivalent (odds ratio [OR] 0.46, 95% confidence interval [CI], 0.23-0.88), but not in patients receiving high doses. In patients with L-index ≤3.823 indicating mild SFTS, glucocorticoid treatment was significantly associated with increased CFR (OR 3.34, 95% CI, 1.35-9.51), and mainly attributable to high-dose glucocorticoids (OR 2.83, 95% CI, 1.72-4.96). Disaggregated data analysis revealed a significant effect only in patients ≤65 years old, male, and early admission within 7 days after onset, but not in their counterparts. CONCLUSION: Glucocorticoids are not recommended for mild patients defined by L-index <3.823; however, patients with severe SFTS may benefit from low-moderate doses of glucocorticoids.

The design and synthesis of potent, selective benzodiazepine sulfonamide bombesin receptor subtype 3 (BRS-3) agonists with an increased barrier of atropisomerization.

Bombesin receptor subtype 3 (BRS-3) is an orphan G-protein coupled receptor expressed primarily in the hypothalamus which plays a role in the onset of both diabetes and obesity. We report herein our progress made towards identifying a potent, selective bombesin receptor subtype-3 (BRS-3) agonist related to the previously described MK-7725(1) Chobanian et al. (2012) that would prevent atropisomerization through the increase of steric bulk at the C-2 position. This would thereby make clinical development of this class of compounds more cost effective by inhibiting racemization which can occur over long periods of time at room/elevated temperature.

LINC00511 Knockdown Suppresses Resistance to Cisplatin in Lung Adenocarcinoma by Interacting with miR-182-3p and BIRC5.

We studied the role of long intergenic non-protein coding RNA 00,511 (LINC00511) in lung adenocarcinoma (LUAD), with a specific focus on acquired chemoresistance. LINC00511 expression was higher in responders to cisplatin (DDP, another name for cisplantin) than non-responders, in A549/DDP cells than in parental A549 cells and normal human bronchial epithelial cells (16HBE). LINC00511 knockdown decreased the half maximal inhibitory concentration (IC50) value, suppressed A549/DDP cell viability, but induced apoptosis. LINC00511 bound with miR-182 and increased the expression of baculoviral inhibitor of apoptosis protein (IAP) repeat containing 5 (BIRC5). BIRC5 knockdown mimicked the effects of LINC00511 knockdown on the IC50 value, A549/DDP cell viability, and apoptosis. BIRC5 overexpression negated the effects of LINC00511 knockdown on A549/DDP cells. In vivo, LINC00511 knockdown attenuated the tumorigenesis of A549/DDP cells after DDP injection. These results provide a novel LINC00511/miR-182/BIRC5 paradigm to explain the mechanism of acquired DDP resistance.

Impact of glycemia and insulin treatment in fatal outcome of severe fever with thrombocytopenia syndrome.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease with a high fatality rate. How the glucose level might affect the clinical outcome remains obscure. METHODS: A multicenter study was performed in 2 hospitals from 2011 to 2021. Patients with SFTS and acute hyperglycemia (admission fasting plasma glucose [FPG] ≥7 mmol/L), postadmission hyperglycemia (admission FPG <7 mmol/L but FPG ≥7 mmol/L after admission), and euglycemia (FPG <7 mmol/L throughout hospitalization) were compared for their clinical progress and outcomes. RESULTS: A total of 3225 patients were included in this study, 37.9% of whom developed acute hyperglycemia and 7.6% postadmission hyperglycemia. The presence of acute hyperglycemia, with or without known diabetes, was associated with increased risk of death (odds ratio [OR]: 1.63; 95% confidence interval [CI]: 1.29-2.05) compared with euglycemia. This effect, however, was only determined in female patients (OR: 2.15; 95% CI: 1.54-2.93). Insulin treatment of patients with SFTS and acute hyperglycemia without previous diabetes was associated with significantly increased mortality (OR: 1.58; 95% CI: 1.16-2.16). CONCLUSION: Acute hyperglycemia can act as a strong predictor of SFTS-related death in female patients. Insulin treatment of hyperglycemia in patients with SFTS without pre-existing diabetes has adverse effects.

Estimating chromatographic enantioselectivity (α) from gradient enantioselective chromatography data.

Calculation of chromatographic enantioselectivity (α) is critically important in enantioselective chromatographic method development studies. The fact that α can only be calculated from isocratic elution conditions, whereas gradient elution conditions are predominantly used in method development screening, presents some problems for the use of α as a scoring indicator for automated, intelligent enantioselective chromatography method development systems. In this study, an empirical algorithm was developed to estimate α at isocratic conditions based upon information collected from a gradient elution. The algorithm was validated for SFC applications and has been shown to accurately predict enantioselectivity for a wide variety of racemic test analytes eluted on different chiral column and mobile phase conditions.

Long non-coding RNA receptor activator of nuclear factor-κ B ligand promotes cisplatin resistance in non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is a common malignancy associated with poor clinical outcomes and high mortality rate. The association between NSCLC development and long non-coding RNA (lncRNA) expression remains to be elucidated. The current study investigated the role of a novel lncRNA, receptor activator of nuclear factor-κ B ligand (RANKL), in the resistance of NSCLC to chemotherapy. RANKL expression was assessed via reverse transcription-quantitative PCR, cell death rate was evaluated using flow cytometry and sensitivity of cisplatin (DDP)-resistant A549/DDP cells to chemotherapy was determined using the Cell Counting Kit-8 assay. Western blotting was performed to quantify p53 protein levels. Compared with matched A549 cells, A549/DDP cells exhibited significant upregulation of RANKL expression. Sensitivity of A549/DDP cells to DDP was restored following RANKL knockdown. A549 cells overexpressing RANKL exhibited notably impaired DDP sensitivity compared with controls. Conversely, downregulated RANKL expression triggered cell death and inhibited cell migration via p53 stimulation and phosphatidylinositol 3-kinase/protein kinase B pathway suppression. The current findings indicate that RANKL contributes to DDP resistance in NSCLC and may represent a novel therapeutic target in this malignancy.

Development of a simple liquid chromatography method for dissolution testing of low dose desogestrel and ethinylestradiol tablets.

Accurate quantitation of low dose, multi-active dissolution samples poses unique challenges in the pharmaceutical industry, often resulting in separate HPLC methods for each active or the use of multiple detectors for increased sensitivity. In this study, we report a fast, isocratic HPLC method utilizing only UV detection for dissolution testing of low dose desogestrel and ethinylestradiol tablets. Rapid separation is completed in 5 min using isocratic elution at a flow rate of 0.45 mL/min, with a column temperature at 30 °C, an injection volume of 50 µL and the detection wavelength at 200 nm. After extensive method development and optimization, the cyano stationary phase was used to overcome the large difference in hydrophobicity for desogestrel and ethinylestradiol, providing balanced retention for both analytes under isocratic elution. Chromatography modeling software was used to provide a rapid analysis of multiple columns and chromatography conditions. The optimized method boasts fast and efficient separation through use of a short, small I.D. column and a large injection volume of dissolution solution to achieve high sensitivity. The stable baseline from an isocratic separation allows low detection wavelengths to be used, resulting in accurate and precise quantitation of both desogestrel and ethinylestradiol. The method has been successfully validated for specificity, linearity, accuracy and precision in the range of 75 - 600 ng/mL for desogestrel and 10 - 80 ng/mL for ethinylestradiol using both HPLC and UHPLC systems. The method robustness was characterized using a design of experiment approach, and the operational design region of the method was established.

Factors influencing the interconversion of a new class of dibenzodiazepine sulfonamide atropisomers.

A novel family of atropisomers based on a conformationally constrained seven membered ring system is investigated using a combination of preparative chiral chromatography, circular dichroism, and other analytical techniques. The influence of structure on the rate of atropisomer interconversion was explored with a series of analogs showing a range of interconversion rates ranging from very fast (undetectable on the HPLC timescale) to very slow (half life of many days).

Microscale HPLC enables a new paradigm for commercialization of complex chiral stationary phases.

The small column size (0.3 mm i.d. x 15 cm) used in microscale HPLC contains only a small fraction (<1%) of the chromatographic packing material of a typical analytical HPLC column. Consequently, chromatographic stationary phases that are prohibitively expensive in conventional HPLC, owing either to synthetic complexity or costly starting materials, may become commercially viable in the microscale format. To illustrate this point, a previously described, synthetically complex, crown ether chiral stationary phase was prepared and evaluated in the microscale format, showing excellent separation of the enantiomers of underivatized amine analytes.

Multiparallel microfluidic high-performance liquid chromatography for high-throughput normal-phase chiral analysis.

The suitability of the Eksigent Express 800 microfluidic eight-channel HPLC instrument for multiparallel normal-phase chiral analysis in support of high-throughput pharmaceutical process research was investigated. Analysis of test mixtures containing the two enantiomers of benzoin and the closely related (R,S)-dihydrobenzoin, was carried out in a 96-well microplate, affording rapid (<2 h) and accurate assessment of enantiopurity. In a second example, use of the instrument to support high-throughput catalyst screening of the asymmetric hydrogenation of a prochiral unsaturated ester is presented, in which method development (gradient screening of four columns and two eluents, followed by optimization to afford a fast analytical method) and analysis of a 96-well microplate was carried out within a single working day. This represents a considerable improvement over conventional analysis techniques that usually take several days to complete.