EGFR activation limits the response of liver cancer to lenvatinib.

Hepatocellular carcinoma (HCC)-the most common form of liver cancer-is an aggressive malignancy with few effective treatment options1. Lenvatinib is a small-molecule inhibitor of multiple receptor tyrosine kinases that is used for the treatment of patients with advanced HCC, but this drug has only limited clinical benefit2. Here, using a kinome-centred CRISPR-Cas9 genetic screen, we show that inhibition of epidermal growth factor receptor (EGFR) is synthetic lethal with lenvatinib in liver cancer. The combination of the EGFR inhibitor gefitinib and lenvatinib displays potent anti-proliferative effects in vitro in liver cancer cell lines that express EGFR and in vivo in xenografted liver cancer cell lines, immunocompetent mouse models and patient-derived HCC tumours in mice. Mechanistically, inhibition of fibroblast growth factor receptor (FGFR)  by lenvatinib treatment leads to feedback activation of the EGFR-PAK2-ERK5 signalling axis, which is blocked by EGFR inhibition. Treatment of 12 patients with advanced HCC who were unresponsive to lenvatinib treatment with the combination of lenvatinib plus gefitinib (trial identifier NCT04642547) resulted in meaningful clinical responses. The combination therapy identified here may represent a promising strategy for the approximately 50% of patients with advanced HCC who have high levels of EGFR.

Orange/far-red hybrid voltage indicators with reduced phototoxicity enable reliable long-term imaging in neurons and cardiomyocytes.

Hybrid voltage indicators (HVIs) are chemogenetic sensors that combines the superior photophysical properties of organic dyes and the genetic targetability of protein sensors to report transient membrane voltage changes. They exhibit boosted sensitivity in excitable cells such as neurons and cardiomyocytes. However, the voltage signals recorded during long-term imaging are severely diminished or distorted due to phototoxicity and photobleaching issues. To capture stable electrophysiological activities over a long time, we employ cyanine dyes conjugated with a cyclooctatetraene (COT) molecule as the fluorescence reporter of HVI. The resulting orange-emitting HVI-COT-Cy3 enables high-fidelity voltage imaging for up to 30 min in cultured primary neurons with a sensitivity of ~ -30% ΔF/F0 per action potential (AP). It also maximally preserves the signal of individual APs in cardiomyocytes. The far-red-emitting HVI-COT-Cy5 allows two-color voltage/calcium imaging with GCaMP6s in neurons and cardiomyocytes for 15 min. We leverage the HVI-COT series with reduced phototoxicity and photobleaching to evaluate the impact of drug candidates on the electrophysiology of excitable cells.

Molecular Mechanism of P53 Peptide Permeation through Lipid Membranes from Solid-State NMR Spectroscopy and Molecular Dynamics Simulations.

Macrocyclic peptides show promise in targeting high-value therapeutically relevant binding sites due to their high affinity and specificity. However, their clinical application is often hindered by low membrane permeability, which limits their effectiveness against intracellular targets. Previous studies focused on peptide conformations in various solvents, leaving a gap in understanding their interactions with and translocation through lipid bilayers. Addressing this, our study explores the membrane interactions of stapled peptides, a subclass of macrocyclic peptides, using solid-state nuclear magnetic resonance (ssNMR) spectroscopy and molecular dynamics (MD) simulations. We conducted ssNMR measurements on ATSP-7041M, a prototypical stapled peptide, to understand its interaction with lipid membranes, leading to an MD-informed model for peptide membrane permeation. Our findings reveal that ATSP-7041M adopts a stable α-helical structure upon membrane binding, facilitated by a cation-π interaction between its phenylalanine side chain and the lipid headgroup. This interaction makes the membrane-bound state energetically favorable, facilitating membrane affinity and insertion. The bound peptide displayed asymmetric insertion depths, with the C-terminus penetrating deeper (approximately 9 Å) than the N-terminus (approximately 4.3 Å) relative to the lipid headgroups. Contrary to expectations, peptide dynamics was not hindered by membrane binding and exhibited rapid motions similar to cell-penetrating peptides. These dynamic interactions and peptide-lipid affinity appear to be crucial for membrane permeation. MD simulations indicated a thermodynamically stable transmembrane conformation of ATSP-7041M, reducing the energy barrier for translocation. Our study offers an in silico view of ATSP-7041M's translocation from the extracellular to the intracellular region, highlighting the significance of peptide-lipid interactions and dynamics in enabling peptide transit through membranes.

Targeted Quantification of Glutathione/Arginine Redox Metabolism Based on a Novel Paired Mass Spectrometry Probe Approach for the Functional Assessment of Redox Status.

Glutathione (GSH) redox control and arginine metabolism are critical in regulating the physiological response to injury and oxidative stress. Quantification assessment of the GSH/arginine redox metabolism supports monitoring metabolic pathway shifts during pathological processes and their linkages to redox regulation. However, assessing the redox status of organisms with complex matrices is challenging, and single redox molecule analysis may not be accurate for interrogating the redox status in cells and in vivo. Herein, guided by a paired derivatization strategy, we present a new ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)-based approach for the functional assessment of biological redox status. Two structurally analogous probes, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and newly synthesized 2-methyl-6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (MeAQC), were set for paired derivatization. The developed approach was successfully applied to LPS-stimulated RAW 264.7 cells and HDM-induced asthma mice to obtain quantitative information on GSH/arginine redox metabolism. The results suggest that the redox status was remarkably altered upon LPS and HDM stimulation. We expect that this approach will be of good use in a clinical biomarker assay and potential drug screening associated with redox metabolism, oxidative damage, and redox signaling.

Iterative outlier detection and refinement rule of compensation for phase aberrations in digital holographic microscopy.

We propose a robust and accurate compensation method for phase aberrations based on the iterative outlier detection and refinement (ODR) rule. This method does not require additional steps to select the known flat region manually or by image segmentation. Based on the proposed method, the phase aberration in regions of a specimen can be detected and refined iteratively. Then, the least squares fitting can be carried out to estimate the coefficients of Zernike polynomials and obtain the accurate phase aberration finally. Computer simulations and real experiments validate the feasibility and effectiveness, and the results show that the proposed method is robust to noise and has superior accuracy even when the specimen occupies half of the field of view.

Reduced doses of emicizumab achieve good efficacy: Results from a national-wide multicentre real-world study in China.

INTRODUCTION: Reduced doses of emicizumab improve the affordability among patients in developing countries. However, the relationship between variant dose selection and efficacy in the real world of China is still unclear. AIM: This study aimed to investigate the efficacy and safety of emicizumab especially in those on reduced dose regimens in a real-world setting. METHODS: We carried out a multicentre study from 28 hospitals between June 2019 and June 2023 in China and retrospectively analysed the characteristics including demographics, diagnosis, treatment, bleeding episodes, and surgical procedures. RESULTS: In total, 127 patients with haemophilia A, including 42 with inhibitors, were followed for a median duration of 16.0 (IQR: 9.0-30.0) months. Median age at emicizumab initiation was 2.0 (IQR: 1.0-4.0) years. Median (IQR) consumption for loading and maintenance was 12.0 (8.0-12.0) and 4.2 (3.0-6.0) mg/kg/4 weeks, respectively. While on emicizumab, 67 (52.8%) patients had no bleeds, whereas 60 (47.2%) patients had any bleeds, including 26 with treated bleeds. Compared to previous treatments, patients on emicizumab had significantly decreased annualized bleeding rate, annualized joint bleeding rate, target joints and intracerebral haemorrhage. Different dosages had similar efficacy except the proportion of patients with treated spontaneous bleeds and target joints. Adverse events were reported in 12 (9.4%) patients. Postoperative excessive bleeding occurred following two of nine procedures. CONCLUSION: This is the largest study describing patients with HA receiving emicizumab prophylaxis on variant dose regimens in China. We confirmed that nonstandard dose is efficacious and can be considered where full-dose emicizumab is ill affordable.

Head-to-Head Comparison of Caco-2 Transwell and Gut-on-a-Chip Models for Assessing Oral Peptide Formulations.

Oral delivery of potent peptide drugs provides key formulation challenges in the pharmaceutical industry: stability, solubility, and permeability. Intestinal permeation enhancers (PEs) can overcome the low oral bioavailability by improving the drug permeability. Conventional in vitro and ex vivo models for assessing PEs fail to predict efficacy in vivo. Here, we compared Caco-2 cells cultured in the conventional static Transwell model to a commercially available continuous flow microfluidic Gut-on-a-Chip model. We determined baseline permeability of FITC-Dextan 3 kDa (FD3) in Transwell (5.3 ± 0.8 × 10-8 cm/s) vs Chip (3.2 ± 1.8 × 10-7 cm/s). We screened the concentration impact of two established PEs sodium caprate and sucrose monolaurate and indicated a requirement for higher enhancer concentration in the Chip model to elicit equivalent efficacy e.g., 10 mM sodium caprate in Transwells vs 25 mM in Chips. Fasted and fed state simulated intestinal fluids (FaSSIF/FeSSIF) were introduced into the Chip and increased basal FD3 permeability by 3-fold and 20-fold, respectively, compared to 4-fold and 4000-fold in Transwells. We assessed the utility of this model to peptides (Insulin and Octreotide) with PEs and observed much more modest permeability enhancement in the Chip model in line with observations in ex vivo and in vivo preclinical models. These data indicate that microfluidic Chip models are well suited to bridge the gap between conventional in vitro and in vivo models.

Conversion of Propargylic Amines with CO2 Catalyzed by a Highly Stable Copper(I) Iodide Thorium-Based Heterometal-Organic Framework.

The conversion of CO2 to generate high-value-added chemicals has become one of the hot research topics in green synthesis. Thereinto, the cyclization reaction of propargylic amines with CO2 is highly attractive because the resultant oxazolidinones are widely found in pharmaceutical chemistry. Cu(I)-based metal-organic frameworks (MOFs) as catalysts exhibit promising application prospects for CO2 conversion. However, their practical application was greatly limited due to Cu(I) being liable to disproportionation or oxidization. Herein, the solid copper(I) iodide thorium-based porous framework {[Cu5I6Th6(µ3-O)4(µ3-OH)4(H2O)10(L)10]·OH·4DMF·H2O}n (1) (HL = 2-methylpyridine-4-carboxylic acid) constructed by [Th6] clusters and [CuxIy] subunits was successfully prepared and structurally characterized. To our knowledge, this is the first copper(I) iodide-based actinide organic framework. Catalytic investigations indicate that 1 can effectively catalyze the cyclization of propargylic amines with CO2 under ambient conditions, which can be reused at least five times without a remarkable decline of catalytic activity. Importantly, 1 exhibits excellent chemical stability and the oxidation state of Cu(I) in it can remain stable under various conditions. This work can provide a valuable strategy for the synthesis of stable Cu(I)-MOF materials.

Utility of cystatin C and serum creatinine-based glomerular filtration rate equations in predicting vancomycin clearance: A population pharmacokinetics analysis in elderly Chinese patients.

Renal function is an important factor affecting the pharmacokinetics of vancomycin. The renal function in elderly patients gradually decreases with age. An accurate estimated glomerular filtration rate (GFR) is essential in drug dosing. The study aimed to determine the most appropriate renal function estimation equations to describe vancomycin pharmacokinetics in elderly patients using population pharmacokinetic analysis. Data were obtained retrospectively from elderly patients aged ≥65 years who received vancomycin for infection from September 2016 to January 2022. Renal function was estimated using the Cockcroft-Gault equation (CG), Modification of Diet in Renal Disease equation (MDRD), three Chronic Kidney Disease Epidemiology Collaboration equations (CKD-EPIcys-scr , CKD-EPIscr , and CKD-EPIcys ) and two Berlin Initiative Study equations (BIS-1 and BIS-2). The CKD-EPIcys-scr and BIS-2 equations were based on cystatin C (Cys C) and serum creatinine (Scr). The others were based on Cys C or Scr. A nonlinear mixed effects model (NONMEM) was used to develop the population pharmacokinetic model. A total of 471 serum concentrations from 313 elderly patients were used to develop the population pharmacokinetic model. Weight and GFR were identified as significant covariates affecting the pharmacokinetics of vancomycin. Cys C and Scr-based GFR (CKD-EPIcys-scr and BIS-2) yielded significant improvement performance compared with the other equations in model building. The interindividual variability of CL was reduced from 49.4% to 23.6% and 49.4% to 23.7% in CKD-EPIcys-scr and BIS-2 based models, respectively. However, greater interindividual variabilities of CL (from 26.6% to 29.0%) were represented in the other five models which were based on either Cys C or Scr. The GFR estimated by EPIcys-scr and BIS-2 equations and vancomycin CL exhibited a good correlation (r = 0.834 and 0.833). In the external validation with 124 serum concentrations, the predictive performances of the CKD-EPIcys-scr and BIS-2 based models (the mean relative prediction errors were less than 1%, the mean relative absolute prediction errors were about 23%) were also superior to the other five models (the mean relative prediction errors were about 2%, the mean relative absolute prediction errors were greater than 25%) which are based on either Cys C or Scr. In this study, we determined that the equation used to estimate GFR can affect the population pharmacokinetic model fitting result. Population pharmacokinetics model with CKD-EPIcys-scr or BIS-2 can be used to optimize vancomycin dosage in elderly Chinese patients.

The expression mechanism of programmed cell death 1 ligand 1 and its role in immunomodulatory ability of mesenchymal stem cells.

Programmed cell death 1 ligand 1 (PD-L1) is an important immunosuppressive molecule, which inhibits the function of T cells and other immune cells by binding to the receptor programmed cell death-1. The PD-L1 expression disorder plays an important role in the occurrence, development, and treatment of sepsis or other inflammatory diseases, and has become an important target for the treatment of these diseases. Mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells with multiple differentiation potential. In recent years, MSCs have been found to have a strong immunosuppressive ability and are used to treat various inflammatory insults caused by hyperimmune diseases. Moreover, PD-L1 is deeply involved in the immunosuppressive events of MSCs and plays an important role in the treatment of various diseases. In this review, we will summarize the main regulatory mechanism of PD-L1 expression, and discuss various biological functions of PD-L1 in the immune regulation of MSCs.

[Mechanism of mitochondrial dysfunction in polycystic ovary syndrome and traditional Chinese medicine intervention: a review].

Polycystic ovary syndrome(PCOS) is a highly prevalent endocrine and reproductive disorder characterized by ovulatory dysfunction, hyperandrogenism(HA), and polycystic ovarian morphology(PCOM). It is often accompanied by insulin resistance(IR), obesity, and metabolic disorders and can lead to cardiovascular diseases, endometrial carcinoma and many other late complications, seriously affecting the physical and mental health and quality of life in premenopausal women. The etiology of PCOS is still unknown and many scholars assume that mitochondrial dysfunction may represent a major pathogenic factor in PCOS in recent years. With a holistic view, treatment based on syndrome differentiation, and multi-system and multi-target treatment manner, traditional Chinese medicine(TCM) can mitigate the symptoms and signs of PCOS from multiple aspects. Although there have been reviews on the mechanism of mitochondrial dysfunction in PCOS, there is still a lack of reviews on the intervention of mitochondrial function by TCM to treat PCOS. Therefore, this paper focuses on the role of mitochondrial dysfunction in PCOS and summarizes the studies about the TCM intervention of PCOS by regulating the mitochondrial function, inflammation, oxidative stress(OS), autophagy, and apoptosis in the last five years, aiming to shed new light on the prevention and treatment of PCOS with TCM.

[Effect and mechanism of free total rhubarb anthraquinones on pyroptosis of J774A.1 macrophages].

In this study, J774A.1 macrophages stimulated by lipopolysaccharide(LPS) and adenosine triphosphate(ATP) were used to establish an in vitro model of pyroptosis, and the intervention mechanism of free total rhubarb anthraquinones(FTRAs) on pyroptosis was investigated. J774A.1 macrophages were cultured in vitro, and the experiment was assigned to the control group and groups with different concentrations of LPS(0.25, 0.5, and 1 µg·mL~(-1)) and ATP(1.25, 2.5, and 5 mmol·L~(-1)). An in vitro model of macrophage pyroptosis was established by detecting cell viability through CCK-8, propidium iodide(PI) apoptotic cell staining, lactate dehydrogenase(LDH), interleukin(IL)-18, and tumor necrosis factor(TNF)-α release. Then, J774A.1 macrophages were randomly divided into six groups: blank control group, LPS+ATP group, high-dose FTRA group, and low, medium, and high-dose FTRA pre-protection group. The phenotypic characteristics and key indicators of pyroptosis were detected as the basis for evaluating the effect of FTRAs on pyroptosis induced by LPS and ATP. Western blot and RT-PCR were used to detect the expression levels of protein and mRNA related to the pyroptosis pathway in caspase-1/11 and elucidate the molecular mechanism of the anti-pyroptosis effect. The results showed that the stimulation condition of 0.50 µg·mL~(-1) LPS+5.00 mmol·L~(-1) ATP was the most effective in the in vitro model of macrophage pyroptosis. FTRAs pre-protected cells for 24 h and then can increase cell viability under pyroptosis conditions, alleviate cell damage, lower the positive rate of PI staining, and reduce the release of LDH, IL-18, and TNF-α. FTRAs were able to significantly inhibit the activation of GSDMD proteins and significantly down-regulate the protein expression of the pyroptosis pathway signature molecules, TLR4, NLRP3, cleaved-caspase-1, and cleaved-caspase-11, but they had no significant effect on ASC proteins. FTRAs were also able to significantly inhibit the mRNA expression of caspase-1, caspase-11, and GSDMD. These results indicate that FTRAs have an inhibitory effect on the pyroptosis model induced by LPS and ATP and play an anti-pyroptosis effect by regulating classical and non-classical pyroptosis signaling pathways and reducing the production of inflammatory cytokines.

General Strategy To Improve the Photon Budget of Thiol-Conjugated Cyanine Dyes.

Maleimide-cysteine chemistry has been a routine practice for the site-specific labeling of fluorophores to proteins since the 1950s. This approach, however, cannot bring out the best photon budget of fluorophores. Here, we systematically measured the Cyanine3/5 dye conjugates via maleimide-thiol and amide linkages by counting the total emitted photons at the single-molecule level. While brightness and signal-to-noise ratios do not change significantly, dyes with thioether linkages exhibit more severe photobleaching than amide linkers. We then screened modern arylation-type bioconjugation strategies to alleviate this damage. Labeling thiols with phenyloxadiazole (POD) methyl sulfone, p-chloronitrobenzene, and fluorobenzene probes gave rise to electron-deficient aryl thioethers, effectively increasing the total emitted photons by 1.5-3 fold. Among the linkers, POD maintains labeling efficiency and specificity that are comparable to maleimide. Such an increase has proved to be universal among bulk and single-molecule assays, with or without triplet-state quenchers and oxygen scavengers, and on conformationally unrestricted or restricted cyanines. We demonstrated that cyanine-POD conjugates are general and superior fluorophores for thiol labeling in single-molecule FRET measurements of biomolecular conformational dynamics and in two-color STED nanoscopy using site-selectively labeled nanobodies. This work sheds light on the photobleaching mechanism of cyanines under single-molecule imaging while highlighting the interplay between the protein microenvironment, bioconjugation chemistry, and fluorophore photochemistry.

Overexpression of POU3F2 promotes radioresistance in triple-negative breast cancer via Akt pathway activation.

PURPOSE: POU3F2 is associated with malignant behaviors and poor prognosis in cancer. However, the function and mechanism of POU3F2 in breast cancer remain to be elucidated. Our study aimed to explore the role of POU3F2 in triple-negative breast cancer and radiotherapy. METHODS: POU3F2 expression was examined by RT-PCR and Western blot. The proliferation of cancer cells was measured by MTT assay. Migration of cancer cells was determined by Transwell assay and wound healing assay. To determine which protein interacts with POU3F2, Co-IP was performed. Survival analysis was performed based on the online database GEPIA. DNA damage after radiation was examined by Comet Assay. Radiosensitivity was evaluated with clonogenic survival assays. A tumor xenograft model was established with MDA-MB-231 breast cancer cells in BALB/c nude mice to explore the effect of POU3F2 in vivo. RESULTS: We found that the expression of POU3F2 was significantly elevated in breast cancer cells, especially in TNBC, and higher POU3F2 expression was related to poor prognosis of patients with breast cancer. Functional assays revealed that POU3F2 promoted proliferation, migration, and invasion of triple-negative breast cancer (TNBC) cells in vitro and in vivo. In addition, the knockdown of POU3F2 decreased the radioresistance of TNBC cells in vitro. Furthermore, POU3F2 could enhance the activation of the Akt pathway by interacting with ARNT2, thereby promoting proliferation and radioresistance in TNBC cells. CONCLUSIONS: Our results provide evidence that high expression of POU3F2 promotes radioresistance in triple-negative breast cancer via Akt pathway activation by interacting with ARNT2.

Genetic spectrum and clinical features in a cohort of Chinese patients with isolated dystonia.

Dystonia is a genetically and phenotypically heterogeneous disorder that occurs in isolation (isolated dystonia) or in combination with other movement disorders. To determine the genetic spectrum in isolated dystonia, we enrolled 88 patients with isolated dystonia for whole-exome sequencing (WES). Seventeen mutations, including nine novel ones, were identified in 19 of the 88 patients, providing a 21.59% positive molecular diagnostic rate. Eleven distinct genes were involved, of which TOR1A and THAP1 accounted for 47.37% (9/19) of the positive cases. A novel missense variant, p.S225R in TOR1A, was found in a patient with adolescence-onset generalized dystonia. Cellular experiments revealed that p.S255R results in the abnormal aggregation of Torsin-1A encoding by TOR1A. In addition, we reviewed the clinical and genetic features of the isolated dystonia patients carrying TOR1A, THAP1, ANO3, and GNAL mutations in the Chinese population. Our results expand the genetic spectrum and clinical profiles of patients with isolated dystonia and demonstrate WES as an effective strategy for the molecular diagnosis of isolated dystonia.

Functional evaluation of CYP2C19 and CYP3A4 gene polymorphism on ibuprofen metabolism.

AIM: Ibuprofen is the most commonly used analgesic. CYP polymorphisms are mainly responsible for the differences in drug metabolism among individuals. Variations in the ability of populations to metabolize ibuprofen can lead to drug exposure events. The aim of this study was to evaluate the effects of CYP2C19 and CYP3A4 polymorphisms on ibuprofen metabolism in a Chinese population. METHODS: First, 31 CYP2C19 and 12 CYP3A4 microsomal enzymes were identified using an insect expression system. Then, variants were evaluated using a mature incubation system. Moreover, ibuprofen metabolite content was determined via ultra-performance liquid chromatography-tandem mass spectrometry analysis. Finally, kinetic parameters of CYP2C19 and CYP3A4 genotypes were determined via Michaelis-Menten curve fitting. RESULTS: Most variants exhibited significantly altered intrinsic clearance compared to the wild type. In the CYP2C19 metabolic pathway, seven variants exhibited no significant alterations in intrinsic clearance (CLint), six variants exhibited significantly high CLint (121-291%), and the remaining 15 variants exhibited substantially reduced CLint (1-71%). In the CYP3A4 metabolic pathway, CYP3A4*30 was not detected in the metabolite content due to the absence of activity, and 10 variants exhibited significantly reduced CLint. CONCLUSION: To the best of our knowledge, this is the first study to assess the kinetic characteristics of 31 CYP2C19 and 12 CYP3A4 genotypes on ibuprofen metabolism. However, further studies are needed on poor metabolizers as they are more susceptible to drug exposure. Our findings suggest that the kinetic characteristics in combination with artificial intelligence to predict the toxicity of ibuprofen and reduce any adverse drug reactions.

Effect of Buffer Salts on Physical Stability of Lyophilized and Spray-Dried Protein Formulations Containing Bovine Serum Albumin and Trehalose.

This study examined the effect of buffer salts on the physical stability of spray-dried and lyophilized formulations of a model protein, bovine serum albumin (BSA). BSA formulations with various buffers were dried by either lyophilization or spray drying. The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), powder X-ray diffraction (PXRD), size exclusion chromatography (SEC), solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS), and solid-state nuclear magnetic resonance spectroscopy (ssNMR). Particle characterizations such as Brunauer-Emmett-Teller (BET) surface area, particle size distribution, and particle morphology were also performed. Results from conventional techniques such as ssFTIR did not exhibit correlations with the physical stability of studied formulations. Deconvoluted peak areas of deuterated samples from the ssHDX-MS study showed a satisfactory correlation with the loss of the monomeric peak area measured by SEC (R2 of 0.8722 for spray-dried formulations and 0.8428 for lyophilized formulations) in the 90-day accelerated stability study conducted at 40°C. mDSC and PXRD was unable to measure phase separation in the samples right after drying. In contrast, ssNMR successfully detected the occurrence of phase separation between the succinic buffer component and protein in the lyophilized formulation, which results in a distribution of microenvironmental acidity and the subsequent loss of long-term stability. Moreover, our results suggested that buffer salts have less impact on physical stability for the spray-dried formulations than the lyophilized solids.

Determination of valproic acid and its six metabolites in human serum using LC-MS/MS and application to interaction with carbapenems in epileptic patients.

Valproic acid (VPA) is a classic medication for several types of epilepsy and mood disorders, and some of its effectiveness and toxicity is associated with metabolites. Although many reports have reported the drug-drug interactions of VPA, no study has focused on the influence of carbapenems (CBPMs) on VPA's active metabolites. An LC-MS/MS method for determining VPA and its six metabolites (3-hydroxy valproic acid, 4-hydroxy valproic acid, 2-propyl-2-pentenoic acid, 2-propyl-4-pentenoic acid, 3-keto valproic acid, and 2-propylglutaric acid) in human serum was established and applied to evaluate the drug-drug interaction with CBPMs in epileptic patients. The stable isotope valproic acid-d6 was used as an internal standard. Analytes in serum samples (50 µl) were isolated using a Kinetex C18 column (3 × 100 mm, 2.6 µm) and detected via negative electrospray ionization after protein precipitation. It was linear (r > 0.99) over the calibration range for different analytes. The accuracy was 91.44-110.92%, and the precision was less than 9.98%. The matrix effect, recovery, and stability met the acceptance criteria. According to the data collected from 150 epileptic patients, the concentration-dose ratio for VPA and its metabolites decreased with CBPM polytherapy. This method is simple and rapid with great accuracy and precision. It is suitable for routine clinical analysis of VPA and its metabolites in human serum.

Transauricular Vagal Nerve Stimulation at 40 Hz Inhibits Hippocampal P2X7R/NLRP3/Caspase-1 Signaling and Improves Spatial Learning and Memory in 6-Month-Old APP/PS1 Mice.

OBJECTIVES: Transauricular vagal nerve stimulation (taVNS) at 40 Hz attenuates hippocampal amyloid load in 6-month-old amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice, but it is unclear whether 40-Hz taVNS can improve cognition in these mice. Moreover, the underlying mechanisms are still unclear. MATERIALS AND METHODS: 6-month-old C57BL/6 (wild type [WT]) and APP/PS1 mice were subjected to 40-Hz taVNS. Novel Object Recognition and the Morris Water Maze were used to evaluate cognition. Hippocampal amyloid-ß (Aß)1-40, Aß1-42, pro-interleukin (IL)-1ß, and pro-IL-18 were measured using enzyme-linked immunosorbent assays. Hippocampal Aß42, purinergic 2X7 receptor (P2X7R), nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3), Caspase-1, IL-1ß, and IL-18 expression were evaluated by western blotting. Histologic assessments including immunofluorescence, immunohistochemistry, Nissl staining, and Congo red staining were used to assess microglial phagocytosis, neuroprotective effects, and Aß plaque load. RESULTS: 40-Hz taVNS improved spatial memory and learning in 6-month-old APP/PS1 mice but did not affect recognition memory. There were no effects on the cognitive behaviors of 6-month-old WT mice. taVNS at 40 Hz modulated microglia; significantly decreased levels of Aß1-40, Aß1-42, pro-IL-1ß, and pro-IL-18; inhibited Aß42, P2X7R, NLRP3, Caspase-1, IL-1ß, and IL-18 expression; reduced Aß deposits; and had neuroprotective effects in the hippocampus of 6-month-old APP/PS1 mice. These changes were not observed in 6-month-old WT mice. CONCLUSION: Our results show that 40-Hz taVNS inhibits the hippocampal P2X7R/NLRP3/Caspase-1 signaling and improves spatial learning and memory in 6-month-old APP/PS1 mice.

Research Progresses and Applications of Fluorescent Protein Antibodies: A Review Focusing on Nanobodies.

Since the discovery of fluorescent proteins (FPs), their rich fluorescence spectra and photochemical properties have promoted widespread biological research applications. FPs can be classified into green fluorescent protein (GFP) and its derivates, red fluorescent protein (RFP) and its derivates, and near-infrared FPs. With the continuous development of FPs, antibodies targeting FPs have emerged. The antibody, a class of immunoglobulin, is the main component of humoral immunity that explicitly recognizes and binds antigens. Monoclonal antibody, originating from a single B cell, has been widely applied in immunoassay, in vitro diagnostics, and drug development. The nanobody is a new type of antibody entirely composed of the variable domain of a heavy-chain antibody. Compared with conventional antibodies, these small and stable nanobodies can be expressed and functional in living cells. In addition, they can easily access grooves, seams, or hidden antigenic epitopes on the surface of the target. This review provides an overview of various FPs, the research progress of their antibodies, particularly nanobodies, and advanced applications of nanobodies targeting FPs. This review will be helpful for further research on nanobodies targeting FPs, making FPs more valuable in biological research.