ErbB3 is required for hyperaminoacidemia-induced pancreatic α cell hyperplasia.

Blood amino acid levels are maintained in a narrow physiological range. The pancreatic α cells have emerged as the primary aminoacidemia regulator through glucagon secretion to promote hepatic amino acid catabolism. Interruption of glucagon signaling disrupts the liver-α cells axis leading to hyperaminoacidemia, which triggers a compensatory rise in glucagon secretion and α cell hyperplasia. The mechanisms of hyperaminoacidemia-induced α cell hyperplasia remain incompletely understood. Using a mouse α cell line and in vivo studies in zebrafish and mice, we found that hyperaminoacidemia-induced α cell hyperplasia requires ErbB3 signaling. In addition to mechanistic target of rapamycin complex 1, another ErbB3 downstream effector signal transducer and activator of transcription 3 also plays a role in α cell hyperplasia. Mechanistically, ErbB3 may partner with ErbB2 to stimulate cyclin D2 and suppress p27 via mechanistic target of rapamycin complex 1 and signal transducer and activator of transcription 3. Our study identifies ErbB3 as a new regulator for hyperaminoacidemia-induced α cell proliferation and a critical component of the liver-α cells axis that regulates aminoacidemia.

Celastrol-Induced Nur77 Interaction with TRAF2 Alleviates Inflammation by Promoting Mitochondrial Ubiquitination and Autophagy.

Mitochondria play an integral role in cell death, autophagy, immunity, and inflammation. We previously showed that Nur77, an orphan nuclear receptor, induces apoptosis by targeting mitochondria. Here, we report that celastrol, a potent anti-inflammatory pentacyclic triterpene, binds Nur77 to inhibit inflammation and induce autophagy in a Nur77-dependent manner. Celastrol promotes Nur77 translocation from the nucleus to mitochondria, where it interacts with tumor necrosis factor receptor-associated factor 2 (TRAF2), a scaffold protein and E3 ubiquitin ligase important for inflammatory signaling. The interaction is mediated by an LxxLL motif in TRAF2 and results not only in the inhibition of TRAF2 ubiquitination but also in Lys63-linked Nur77 ubiquitination. Under inflammatory conditions, ubiquitinated Nur77 resides at mitochondria, rendering them sensitive to autophagy, an event involving Nur77 interaction with p62/SQSTM1. Together, our results identify Nur77 as a critical intracellular target for celastrol and unravel a mechanism of Nur77-dependent clearance of inflamed mitochondria to alleviate inflammation.

Nuclear receptor RXRα binds the precursor of miR-103 to inhibit its maturation.

BACKGROUND: The maturation of microRNAs (miRNAs) successively undergoes Drosha, Dicer, and Argonaute -mediated processing, however, the intricate regulations of the individual miRNA maturation are largely unknown. Retinoid x receptor alpha (RXRα) belongs to nuclear receptors that regulate gene transcription by binding to DNA elements, however, whether RXRα binds to miRNAs to exert physiological functions is not known. RESULTS: In this work, we found that RXRα directly binds to the precursor of miR-103 (pre-miR-103a-2) via its DNA-binding domain with a preferred binding sequence of AGGUCA. The binding of RXRα inhibits the processing of miR-103 maturation from pre-miR-103a-2. Mechanistically, RXRα prevents the nuclear export of pre-miR-103a-2 for further processing by inhibiting the association of exportin-5 with pre-miR-103a-2. Pathophysiologically, the negative effect of RXRα on miR-103 maturation correlates to the positive effects of RXRα on the expression of Dicer, a target of miR-103, and on the inhibition of breast cancer. CONCLUSIONS: Our findings unravel an unexpected role of transcription factor RXRα in specific miRNA maturation at post-transcriptional level through pre-miRNA binding, and present a mechanistic insight regarding RXRα role in breast cancer progression.

Dexmedetomidine adjunct to ropivacaine for ultrasound-guided transversus abdominis plane block for open inguinal hernia repair in the older adults: A randomised clinical trial.

OBJECTIVE: The aim of this study was to evaluate the effect of adding dexmedetomidine to ropivacaine on pain relief and quality of recovery in older patients undergoing open inguinal hernia repair surgeries. METHODS: This was a prospective and randomised clinical trial of 102 patients aged over 65 years who received an ultrasound-guided transversus open mesh herniorrhaphy abdominis plane (TAP) block with either 0.375% ropivacaine 20 ml (Group R, n = 47) or 0.375% ropivacaine combined with 1 µg/kg dexmedetomidine 20 ml (Group RD, n = 45) in the pre-anaesthesia care unit before elective open inguinal hernia surgeries. The primary outcome measure was Visual Analogue Scale (VAS) pain scores at rest and on movement at 2, 4, 8, 12 and 24 h and at 1 and 3 months' postoperatively. The secondary outcome measures were the incidence of post-operative delirium (POD), nausea and vomiting and the occurrence of side effects or complications on post-operative day 1. RESULTS: Group RD had lower VAS scores at rest and on movement at 8 and 12 h postoperatively and a lower incidence of POD on the post-operative day 1 than Group R. Transient bradycardia was more frequent in Group RD than in Group R, and side effects or post-operative complications were reported in either group. CONCLUSION: The addition of dexmedetomidine to ropivacaine in a TAP block enhances postoperative analgesia during hospitalisation and improves the quality of recovery without affecting chronic pain in older patients undergoing open inguinal hernia repair surgery.

Disruption of the glucagon receptor increases glucagon expression beyond α-cell hyperplasia in zebrafish.

The glucagon receptor (GCGR) is a potential target for diabetes therapy. Several emerging GCGR antagonism-based therapies are under preclinical and clinical development. However, GCGR antagonism, as well as genetically engineered GCGR deficiency in animal models, are accompanied by α-cell hyperplasia and hyperglucagonemia, which may limit the application of GCGR antagonism. To better understand the physiological changes in α cells following GCGR disruption, we performed single cell sequencing of α cells isolated from control and gcgr-/- (glucagon receptor deficient) zebrafish. Interestingly, beyond the α-cell hyperplasia, we also found that the expression of gcga, gcgb, pnoca, and several glucagon-regulatory transcription factors were dramatically increased in one cluster of gcgr-/- α cells. We further confirmed that glucagon mRNA was upregulated in gcgr-/- animals by in situ hybridization and that glucagon promoter activity was increased in gcgr-/-;Tg(gcga:GFP) reporter zebrafish. We also demonstrated that gcgr-/- α cells had increased glucagon protein levels and increased granules after GCGR disruption. Intriguingly, the increased mRNA and protein levels could be suppressed by treatment with high-level glucose or knockdown of the pnoca gene. In conclusion, these data demonstrated that GCGR deficiency not only induced α-cell hyperplasia but also increased glucagon expression in α cells, findings which provide more information about physiological changes in α-cells when the GCGR is disrupted.

Visible-Light-Driven [2 + 2] Photocycloaddition for Constructing Dimers of N,N'-Diacyl-1,4-dihydropyrazines: Experimental and Theoretical Investigation.

In this study, the visible-light-driven [2 + 2] photocycloaddition of 1,4-dihydropyrazines in solution was reported. The N,N'-diacyl-1,4-dihydropyrazines with different substituents showed completely different reactivity under the irradiation of a 430 nm blue light-emitting diode (LED) lamp. N,N'-Diacetyl-1,4-dihydropyrazine and N,N'-dipropionyl-1,4-dihydropyrazine were the only compounds capable of undergoing a [2 + 2] photocycloaddition reaction, yielding syn-dimers and cage-dimers (known as 3,6,9,12-tetraazatetraasteranes) with overall yields of 76 and 83%, correspondingly. The substituent-reactivity effect on [2 + 2] photocycloaddition of N,N'-diacyl-1,4-dihydropyrazines was investigated by density functional theory calculations. The results show that the substituents have little influence on Gibbs free energy for the [2 + 2] photocycloaddition and mainly affect the excited energy, reaction sites, and the triplet excited-state structures of 1,4-dihydropyrazines, which are closely related to whether the reaction occurs. The results offer insights into the photochemical reactivity of 1,4-dihydropyrazines and an approach for constructing dimers of N,N'-diacyl-1,4-dihydropyrazines through a solution-based visible-light-driven [2 + 2] photocycloaddition, especially for the construction of 3,6,9,12-tetraazatetraasteranes. Compared with the solid-state [2 + 2] photocycloaddition of 1,4-dihydropyrazine, this photocycloaddition will be an efficient and environmentally friendly method for synthesizing tetraazatetraasteranes with the advantages of milder reaction conditions, simple operation, adjustable reaction amounts by omitting the cocrystal growth step, etc.

Chemoselective [2 + 2]/[3 + 2] Photocycloaddition of 1,4-Dihydropyridines: Experimental and Theoretical Investigation.

The photocycloaddition of 1,4-dihydropyridines (1,4-DHPs) is a main approach to synthesize structurally complex compounds, which are important intermediates for the preparation of cage compounds, such as 3,9-diazatetraasterane, 3,6-diazatetraasterane, 3,9-diazatetracyclododecane, and 6,12-diazaterakishomocubanes. The acquisition of different cage compounds depended on the chemoselectivity, which is mainly caused by the reaction conditions and structural characteristics of 1,4-DHPs. This study aimed to investigate the effect of the structural characteristics on chemoselectivity in [2 + 2]/[3 + 2] photocycloaddition of 1,4-DHPs. The photocycloadditions were conducted on the 1,4-diaryl-1,4-dihydropyridine-3-carboxylic ester with steric hindrance groups at the C3 position or chirality at the C4 position irradiated by a 430 nm blue LED lamp. When the 1,4-DHPs contained high steric hindrance groups at the C3 position, [2 + 2] photocycloaddition was the main reaction, resulting in 3,9-diazatetraasteranes with a yield of 57%. Conversely, when the 1,4-DHPs were resolved to a chiral isomer, the main reaction was [3 + 2] photocycloaddition, producing 6,12-diazaterakishomocubanes with a yield of 87%. To investigate the chemoselectivity and understand the photocycloaddition of 1,4-DHPs, density functional theory (DFT) and time-dependent DFT (TDDFT) calculations were performed at the B3LYP-D3/def-SVP//M06-2X-D3/def2-TZVP level. The steric hindrance and excitation energy modulated by substituents at the C3 position and chiral carbon at the C4 position were crucial for the chemoselectivity in [2 + 2]/[3 + 2] photocycloaddition of 1,4-DHPs.

A Single Preoperative Dose of S-Ketamine Has No Beneficial Effect on Chronic Postsurgical Pain in Patients Undergoing Video-Assisted Thoracoscopic Surgical Lung Lesion Resection: A Prospective Randomized Controlled Study.

OBJECTIVES: To evaluate the efficacy of a single preoperative dose of S-ketamine for chronic postsurgical pain (CPSP) in patients undergoing video-assisted thoracoscopic surgical lung lesion resection (VATS). DESIGN: A prospective randomized, double-blind controlled study. SETTING: Patients were enrolled from March 17, 2021, to November 18, 2021, at a single tertiary academic hospital. PARTICIPANTS: Patients were 18-to-65 years of age and undergoing VATS. INTERVENTIONS: The experiment was divided into an S-ketamine group (0.5 mg/kg intravenous injection before anesthesia induction) or a placebo group (the same volume of normal saline). MEASUREMENTS AND MAIN RESULTS: The primary endpoint was the incidence of CPSP and its neuropathic component. The secondary endpoints included acute postoperative pain, the use of postoperative analgesics, anxiety and sleep quality scores, and the occurrence of adverse effects. There were no significant differences between the 2 groups in the incidences of CPSP, neuropathic pain, acute postoperative pain, and postoperative use of analgesics. The sleep quality scores on the first postoperative day differed significantly between the groups (47.45 ± 27.58 v . 52.97 ± 27.57, p = 0.049), but not the anxiety scores. In addition, adverse effects were similar between the 2 groups. CONCLUSIONS: A single preoperative dose of S-ketamine in patients who underwent VATS had no significant effect on acute and chronic postoperative pain or the consumption of analgesics after surgery. A single preoperative dose of S-ketamine could improve sleep on the first day after surgery, whereas it had no significant effect on anxiety levels.

Nur77 Serves as a Potential Prognostic Biomarker That Correlates with Immune Infiltration and May Act as a Good Target for Prostate adenocarcinoma.

Prostate adenocarcinoma (PRAD) is the most frequent malignancy, and is the second leading cause of death due to cancer in men. Thus, new prognostic biomarkers and drug targets for PRAD are urgently needed. As we know, nuclear receptor Nur77 is important in cancer development and changes in the tumor microenvironment; whereas, the function of Nur77 in PRAD remains to be elucidated. The TCGA database was used to explore the Nur77 expression and its role in the prognosis of PRAD. It was shown that Nur77 was down regulated in PRAD, and low Nur77 expression was correlated with advanced clinical pathologic characteristics (high grade, histological type, age) and poor prognosis. Furthermore, key genes screening was examined by univariate Cox analysis and Kaplan-Meier survival. Additionally, Nur77 was closely related to immune infiltration and some anti-tumor immune functions. The differentially expressed genes (DEGs) were presented by protein-protein interaction (PPI) network analysis. Therefore, the expression level of Nur77 might help predict the survival of PRAD cases, which presents a new insight and a new target for the treatment of PRAD. In vitro experiments verified that natural product malayoside targeting Nur77 exhibited significant therapeutic effects on PRAD and largely induced cell apoptosis by up-regulating the expression of Nur77 and its mitochondrial localization. Taken together, Nur77 is a prognostic biomarker for patients with PRAD, which may refresh the profound understanding of PRAD individualized treatment.

[Stapled anoplin peptide combined with photothermal therapy enhances oncolytic immunotherapy of triple-negative breast cancer].

This study constructed a nano-drug delivery system, A3@GMH, by co-delivering the stapled anoplin peptide(Ano-3, A3) with the light-harvesting material graphene oxide(GO), and evaluated its oncolytic immunotherapy effect on triple-negative breast cancer(TNBC). A3@GMH was prepared using an emulsion template method and its physicochemical properties were characterized. The in vivo and in vitro photothermal conversion abilities of A3@GMH were investigated using an infrared thermal imager. The oncoly-tic activity of A3@GMH against TNBC 4T1 cells was evaluated through cell counting kit-8(CCK-8), lactate dehydrogenase(LDH) release, live/dead cell staining, and super-resolution microscopy. The targeting properties of A3@GMH on 4T1 cells were assessed using a high-content imaging system and flow cytometry. In vitro and in vivo studies were conducted to investigate the antitumor mechanism of A3@GMH in combination with photothermal therapy(PTT) through inducing immunogenic cell death(ICD) in 4T1 cells. The results showed that the prepared A3@GMH exhibited distinct mesoporous and coated structures with an average particle size of(308.9±7.5) nm and a surface potential of(-6.79±0.58) mV. The encapsulation efficiency and drug loading of A3 were 23.9%±0.6% and 20.5%±0.5%, respectively. A3@GMH demonstrated excellent photothermal conversion ability and biological safety. A3@GMH actively mediated oncolytic features such as 4T1 cell lysis and LDH release, as well as ICD effects, and showed enhanced in vitro antitumor activity when combined with PTT. In vivo, A3@GMH efficiently induced ICD effects with two rounds of PTT, activated the host's antitumor immune response, and effectively suppressed tumor growth in 4T1 tumor-bearing mice, achieving an 88.9% tumor inhibition rate with no apparent toxic side effects. This study suggests that the combination of stapled anoplin peptide and PTT significantly enhances the oncolytic immunotherapy for TNBC and provides a basis for the innovative application of anti-tumor peptides derived from TCM in TNBC treatment.

The transcription factor CCAAT/enhancer-binding protein ß in spinal microglia contributes to pre-operative stress-induced prolongation of postsurgical pain.

Prolongation of postsurgical pain caused by pre-operative stress is a clinically significant problem, although the mechanisms are not fully understood. Stress can promote the pro-inflammatory activation of microglia, and the transcription factor CCAAT/enhancer-binding protein (C/EBP) ß regulates pro-inflammatory gene expression in microglia. Therefore, we speculated that C/EBPß in spinal microglia may have critical roles in the development of chronic postsurgical pain. Accordingly, in this study, we used a single prolonged stress (SPS) procedure and plantar incisions to evaluate the roles of C/EBPß in postsurgical pain. Our experiments showed that SPS exposure prolonged mechanical allodynia, increased the expression of C/EBPß and pro-inflammatory cytokines, and potentiated the activation of spinal microglia. Subsequently, microinjection of C/EBPß siRNA attenuated the duration of SPS-prolonged postoperative mechanical allodynia and inhibited microglial activation in the spinal cord. Conversely, mimicking this increase in C/EBPß promoted microglial activation via pretreatment with a pre-injection of AAV5-C/EBPß, leading to prolongation of postsurgical pain. Overall, these results suggested that spinal microglia may play key roles in prolongation of postsurgical pain induced by pre-operative stress and that C/EBPß may be a potential target for disease treatment.

Merbromin is a mixed-type inhibitor of 3-chyomotrypsin like protease of SARS-CoV-2.

3-chyomotrypsin like protease (3CLpro) has been considered as a promising target for developing anti-SARS-CoV-2 drugs. Herein, about 6000 compounds were analyzed by high-throughput screening using enzyme activity model, and Merbromin, an antibacterial agent, was identified as a potent inhibitor of 3CLpro. Merbromin strongly inhibited the proteolytic activity of 3CLpro but not the other three proteases Proteinase K, Trypsin and Papain. Michaelis-Menten kinetic analysis showed that Merbromin was a mixed-type inhibitor of 3CLpro, due to its ability of increasing the KM and decreasing the Kcat of 3CLpro. The binding assays and molecular docking suggested that 3CLpro possessed two binding sites for Merbromin. Consistently, Merbromin showed a weak binding to the other three proteases. Together, these findings demonstrated that Merbromin is a selective inhibitor of 3CLpro and provided a scaffold to design effective inhibitors of SARS-CoV-2.

Stable Lithium Plating and Stripping Enabled by a LiPON Nanolayer on PP Separator.

The practical application of the Li metal anode (LMA) is hindered by its low coulombic efficiency and dendrite formation. Although solid-state electrolytes hold promise as ideal partners for LMA, their effectiveness is limited by the poor workability and ionic conductivity. Herein, a modified separator combining the rapid Li+ transport of a liquid electrolyte and the interfacial stability of a solid-state electrolyte is explored to realize stable cycling of the LMA. A conformal nanolayer of LiPON is coated on a polypropylene separator by a scalable magnetron sputtering method, which is compatible with current Li-ion battery production lines and promising for the practical applications. The resulting LMA-electrolyte/separator interface is Li+ -conductive, electron-insulating, mechanically and chemically stable. Consequently, Li|Li cells maintain stable dendrite-free cycling with overpotentials of 10 and 40 mV over 2000 h at 1 and 5 mA cm-2 , respectively. Additionally, the Li|LiFePO4 full cells achieve a capacity retention of 92% after 550 cycles, confirming its application potential.

Design, synthesis and biological evaluation of acyl hydrazones-based derivatives as RXRα-targeted anti-mitotic agents.

RXRα, a unique and important nuclear receptor, plays a vital role in various biological and pathological pathways, including growth, differentiation, and apoptosis. We recently reported a transcription-independent function of RXRα in cancer cells in which RXRα is phosphorylated by Cdk1 at the onset of mitosis, resulting in its translocation to the centrosome, where the phosphorylated RXRα (p-RXRα) interacts with polo-like kinase 1 (PLK1) to promote centrosome maturation and mitotic progression. Significantly, we also identified that a small molecule XS-060 binds to RXRα and selectively inhibits the p-RXRα/PLK1 interaction to induce mitotic arrest and catastrophe in cancer cells. Here, we report our design, synthesis, and biological evaluation of a series of XS-060 analogs as RXRα-targeted anti-mitotic agents. Our results identified B10 as an improved anti-mitotic agent. B10 bound to RXRα (Kd = 3.04 ± 0.58 µM) and inhibited the growth of cervical cancer cells (HeLa, IC50 = 1.46 ± 0.10 µM) and hepatoma cells (HepG2, IC50 = 3.89 ± 0.45 µM and SK-hep-1, IC50 = 5.74 ± 0.50 µM) with low cytotoxicity to nonmalignant cells(LO2, IC50 > 50 µM). Furthermore, our mechanistic studies confirmed that B10 acted as an anticancer agent by inhibiting the p-RXRα/PLK1 pathway. These results provide a basis for further investigation and optimization of RXRα-targeted anti-mitotic molecules for cancer therapy.

SAR study of oxidative DIMs analogs targeting the Nur77-mediated apoptotic pathway of cancer cells.

Nur77, an orphan nuclear receptor, is implicated in regulating diverse cellular biological processes including apoptosis and inflammation. We previously identified BI1071 (DIM-C-pPhCF3+MeSO3-), an oxidized methanesulfonate salt of (4-CF3-Ph-C-DIM), was a direct ligand of Nur77, which could activate the Nur77-Bcl-2 apoptotic pathway. To obtain more effective compounds targeting the Nur77-mediated apoptotic pathway, we designed and synthesized a series of BI1071 analogs by introducing various substituent groups in the indolyl-rings of BI1071. Structure-activity relationship study identified A11, B5 and B15 as improved analogs with stronger binding affinity to Nur77 and enhanced apoptotic activity compared to BI1071. Nur77-binding studies demonstrated that A11, B5 and B15 bind to Nur77 with a Kd of 34 nM, 19 nM and 16 nM, respectively. Furthermore, mechanism studies showed that A11, B5 and B15 induced apoptosis through utilizing the Nur77-Bcl-2 pathway.

Chemical Constituents of the Deep-Sea-Derived Penicillium citreonigrum MCCC 3A00169 and Their Antiproliferative Effects.

Six new citreoviridins (citreoviridins J-O, 1-6) and twenty-two known compounds (7-28) were isolated from the deep-sea-derived Penicillium citreonigrum MCCC 3A00169. The structures of the new compounds were determined by spectroscopic methods, including the HRESIMS, NMR, ECD calculations, and dimolybdenum tetraacetate-induced CD (ICD) experiments. Citreoviridins J-O (1-6) are diastereomers of 6,7-epoxycitreoviridin with different chiral centers at C-2-C-7. Pyrenocine A (7), terrein (14), and citreoviridin (20) significantly induced apoptosis for HeLa cells with IC50 values of 5.4 µM, 11.3 µM, and 0.7 µM, respectively. To be specific, pyrenocine A could induce S phase arrest, while terrein and citreoviridin could obviously induce G0-G1 phase arrest. Citreoviridin could inhibit mTOR activity in HeLa cells.

Self-Healing Nucleation Seeds Induced Long-Term Dendrite-Free Lithium Metal Anode.

Seeded lithium (Li) nucleation has been considered as a promising strategy to achieve uniform Li deposition. However, problems of agglomeration and pulverization quickly invalidate the nucleation seeds, resulting in Li dendrite growth during repeated charge/discharge processes. Herein, liquid gallium-indium (GaIn) nanoparticles with structural self-healing properties are utilized to guide uniform metallic Li nucleation and deposition. Ultrafine GaIn nanoparticles (∼25 nm) uniformly decorated on the surface of carbon layers effectively homogenize the lithium-ion flux. After fully Li stripping, lithiophilic GaIn nanoparticles return to the liquid binary eutectic phase, thereby healing the deformed structure and enabling them to continuously guide dendrite-free Li deposition. Li metal anodes with such nucleation seeds exhibit nearly zero nucleation overpotential even after hundreds of cycles and a high average Coulombic efficiency of 99.03% for more than 400 cycles. The design of self-healing nucleation seeds provides important insights for obtaining high-performance lithium metal anodes.

The impact of age on anxiety in Covid-19 patients in quarantine wardwards.

This study aimed to explore which age group out of the patients in quarantine wards with novel coronavirus pneumonia is the most susceptible to anxiety. The data of 32 Covid-19 patients isolated in the quarantine wards of the second Infectious Diseases Department of Baoding Hospital and 71 Covid-19 patients in Tangshan City Infectious Disease Hospital from January 24th to March 5th, 2020, a total of 103 patients, were analyzed. Among these patients, 97 isolated patients were scored with a self-rating anxiety scale (SAS) score seven days after quarantine, and the correlation between age and score was analyzed. These 97 isolated patients were then divided into three groups according to age: group A (up to 35 years old), group B (36-60 years), and group C (over 60 years). One-way analysis of variance was used to compare the scores among groups. The Q-test was used for pairwise comparison.P < 0.05 was considered statistically significant.There was a negative correlation between age and SAS score in isolated Covid-19 patients, and the differences in the score among groups were statistically significant. Patients under 35 years old were more prone to anxiety when they were isolated for seven days. Isolated patients aged up to 35 years old need more attention from quarantine medical staff, communication should be strengthened, and psychological intervention from psychotherapists should be given if necessary.

Free energy of micellization of dodecyl phosphocholine (DPC) from molecular simulation: Hybrid PEACH-BAR method.

A new method to extract the free energy of aggregation versus aggregate size from molecular simulation data is proposed and applied to a united atom model of the zwitterionic surfactant dodecyl phosphocholine in water. This system's slow dissociation rate and low critical micelle concentration (CMC of approximately 1-2 mM) make extraction of cluster free energies directly from simulation results using the "partition-enabled analysis of cluster histogram" (PEACH) method impractical. The new approach applies PEACH to a model with weakened attractions between aggregants, which allows sampling of a continuous range of cluster sizes, then recovers the free energy of aggregation under the original fully-attractive force field using the BAR free energy difference method. PEACH-BAR results are compared with free energy differences calculated via umbrella sampling, and are used to make predictions of CMC, average cluster size, and SAXS scattering profiles that are in fair agreement with experiment.

Fc-specific and covalent conjugation of a fluorescent protein to a native antibody through a photoconjugation strategy for fabrication of a novel photostable fluorescent antibody.

Fluorophore-antibody conjugates with high photobleaching resistance, high chemical stability, and Fc-specific attachment is a great advantage for immunofluorescence imaging. Here, an Fc-binding protein (Z-domain) carrying a photo-cross-linker (p-benzoylphenylalanine, Bpa) fused with enhanced green fluorescent protein (EGFP), namely photoactivatable ZBpa-EGFP recombinant, was directly generated using the aminoacyl-tRNA synthetase/suppressor tRNA technique without any further modification. By employing the photoactivatable ZBpa-EGFP, an optimal approach was successfully developed which enabled EGFP to site-selectively and covalently attach to native antibody (IgG) with approximately 90% conjugation efficiency. After characterizing the Fc-specific and covalent manner of the EGFP-photoconjugated antibody, its excellent photobleaching resistance for immunofluorescence imaging was demonstrated in a model study by monitoring the toll-like receptor 4 (TLR4) expression in HepG2 cells. The proposed approach here for the preparation of a novel fluorescent antibody is available and reliable, which would play an important role in fluorescence immunoassay, and is expected to be extended to the generation of other biomolecule-photoconjugated antibodies, such as other fluorescent proteins for multiplex immunofluorescence imaging or reporter enzymes for highly sensitive enzyme immunoassays.Graphical abstract.