Simultaneous Screening of 172 Veterinary Drugs by Modified QuEChERS-LC-MS/MS in TCM Galli Gigerii Endothelium Corneum.

An analytical method was developed for the screening of 172 veterinary drugs in traditional Chinese medicine Galli Gigerii Endothelium Corneum by high-performance liquid chromatography tandem mass spectrometry. The samples were pretreated by a modified QuEChERS method. A Zorbax Eclipse plus C18 column (1.8 µm, 3.0 × 150 mm2, Agilent) was used for the separation of analytes by gradient elution. All analytes were detected by electrospray ionization mass spectrometry with multiple reaction monitoring mode. Good linearity with R ≥ 0.99 was exhibited for all analytes within the respective range. The recoveries of all monitored analytes ranged from 55.4 to 127.6% at three spiked levels (limit of quantitation-LOQ, 2-fold LOQ, 10-fold LOQ), with relative standard deviations <17.8%. The estimated LOQ levels were 0.2-20 µg/kg. The application of this method provides a reference for the safety control of traditional Chinese medicines.

An Enzyme-Cleavable Solubilizing-Tag Facilitates the Chemical Synthesis of Mirror-Image Proteins.

Mirror-image proteins (D-proteins) are useful in biomedical research for purposes such as mirror-image screening for D-peptide drug discovery, but the chemical synthesis of many D-proteins is often low yielding due to the poor solubility or aggregation of their constituent peptide segments. Here, we report a Lys-C protease-cleavable solubilizing tag and its use to synthesize difficult-to-obtain D-proteins. Our tag is easily installed onto multiple amino acids such as DLys, DSer, DThr, and/or the N-terminal amino acid of hydrophobic D-peptides, is impervious to various reaction conditions, such as peptide synthesis, ligation, desulfurization, and transition metal-mediated deprotection, and yet can be completely removed by Lys-C protease under denaturing conditions to give the desired D-protein. The efficacy and practicality of the new method were exemplified in the synthesis of two challenging D-proteins: D-enantiomers of programmed cell death protein 1 IgV domain and SARS-CoV-2 envelope protein, in high yield. This work demonstrates that the enzymatic cleavage of solubilizing tags under denaturing conditions is feasible, thus paving the way for the production of more D-proteins.

L-Glycosidase-Cleavable Natural Glycans Facilitate the Chemical Synthesis of Correctly Folded Disulfide-Bonded D-Proteins.

D-peptide ligands can be screened for therapeutic potency and enzymatic stability using synthetic mirror-image proteins (D-proteins), but efficient acquisition of these D-proteins can be hampered by the need to accomplish their in vitro folding, which often requires the formation of correctly linked disulfide bonds. Here, we report the finding that temporary installation of natural O-linked-ß-N-acetyl-D-glucosamine (O-GlcNAc) groups onto selected D-serine or D-threonine residues of the synthetic disulfide-bonded D-proteins can facilitate their folding in vitro, and that the natural glycosyl groups can be completely removed from the folded D-proteins to afford the desired chirally inverted D-protein targets using naturally occurring O-GlcNAcase. This approach enabled the efficient chemical syntheses of several important but difficult-to-fold D-proteins incorporating disulfide bonds including the mirror-image tumor necrosis factor alpha (D-TNFα) homotrimer and the mirror-image receptor-binding domain of the Omicron spike protein (D-RBD). Our work establishes the use of O-GlcNAc to facilitate D-protein synthesis and folding and proves that D-proteins bearing O-GlcNAc can be good substrates for naturally occurring O-GlcNAcase.

Hydrophobic Cross-Linked Chains Regulate High Wet Tissue Adhesion Hydrogel with Toughness, Anti-hydration for Dynamic Tissue Repair.

Hydrogel adhesion materials are widely reported for tissue engineering repair applications, however, wet tissue surface moisture can reduce the wet-adhesion properties and mechanical strength of hydrogels limiting their application. Here, anti-hydration gelatin-acrylic acid-ethylene dimethacrylate (GAE) hydrogels with hydrophobic cross-linked chains are constructed. The prepared GAE hydrogel is soaked in PBS (3 days) with a volume change of 0.6 times of the original and the adhesive strength, Young's modulus, toughness, and burst pressure are maintained by ≈70% of the original. A simple and universal method is used to introduce hydrophobic chains as cross-linking points to prepare hydrogels with anti-hydration, toughness, and high wet state adhesion. The hydrophobic cross-linked chains not only restrict the movement of molecular chains but also hinder the intrusion of water molecules. Antihydration GAE hydrogels exhibit good biocompatibility, slow drug release, and dynamic oral wet-state tissue repair properties. Therefore, the anti-hydration hydrogel has excellent toughness, wet tissue adhesion properties, and good prospects for biological applications.

Species discrimination of multiple botanical origins of Fritillaria species based on infrared spectroscopy, thin layer chromatography-image analysis and untargeted metabolomics.

BACKGROUND: Fritillaria Bulbus (FB), a precious medicinal herb renowned for its heat-clearing, lung-moistening, cough-relieving and phlegm-eliminating effects. In pursuit of profits, unscrupulous merchants have engaged in the substitution or adulteration of valuable varieties with cheaper alternatives. It is, therefore, urgent to develop effective technical approaches to identify FBs from adulterants. METHODS: This paper employed infrared spectroscopy (IR), thin layer chromatography-image analysis (TLC-IA), and untargeted metabolomics techniques to discriminate ten species of FBs. RESULTS: Five species of FBs were successfully differentiated using mid-infrared spectroscopy. Furthermore, the power of TLC-IA technology allowed the differentiation of five species of FBs and two origins of FCBs (Fritillariae Cirrhosae Bulbus). Remarkably, through the application of untargeted metabolomics technique, the precise discrimination of five species of FBs, as well as three origins of FCBs were accomplished. Moreover, a comprehensive identification of 101 markers that reliably distinguished diverse FBs was achieved through the employment of untargeted metabolomics technique. CONCLUSION: The investigation presented powerful means of detection for assuring the quality control of Fritillaria herbs.

Chemical synthesis of a 28 kDa full-length PET degrading enzyme ICCG by the removable backbone modification strategy.

Chemical protein synthesis offers a powerful way to access otherwise-difficult-to-obtain proteins such as mirror-image proteins. Although a large number of proteins have been chemically synthesized to date, the acquisition to proteins containing hydrophobic peptide fragments has proven challenging. Here, we describe an approach that combines the removable backbone modification strategy and the peptide hydrazide-based native chemical ligation for the chemical synthesis of a 28 kDa full-length PET degrading enzyme IGGC (a higher depolymerization efficiency of variant leaf-branch compost cutinase (LCC)) containing hydrophobic peptide segments. The synthetic ICCG exhibits the enzymatic activity and will be useful in establishing the corresponding mirror-image version of ICCG.

Characterization of natural peptides in Pheretima by integrating proteogenomics and label-free peptidomics.

Pheretima, also called "earthworms", is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines (CPMs) in Chinese Pharmacopoeia (2020 edition). However, its zoological origin is unclear, both in the herbal market and CPMs. In this study, a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing algorithms (restricted search, open search, and de novo) was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima, including Pheretima aspergillum (PA), Pheretima vulgaris (PV), and Metaphire magna (MM). We identified 10,477 natural peptides in the PA, 7,451 in PV, and 5,896 in MM samples. Five specific signature peptides were screened and then validated using synthetic peptides; these demonstrated robust specificity for the authentication of PA, PV, and MM. Finally, all marker peptides were successfully applied to identify the zoological origins of Brain Heart capsules and Xiaohuoluo pills, revealing the inconsistent Pheretima species used in these CPMs. In conclusion, our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines, especially non-model species with poorly annotated protein databases.

Dealloying-Induced Zeolite-like Metal Framework of AB2 Laves Phase Intermetallic Electrocatalysts.

Exploring an efficient and robust electrocatalyst for hydrogen evolution reaction (HER) at high pH and temperature holds the key to the industrial application of alkaline water electrolysis (AWE). Herein, we design an open tunnel structure by dealloying a series of Laves phase intermetallics, i.e., MCo2 and MRu0.25Co1.75 (M = Sc and Zr). The dealloying process can induce a zeolite-like metal framework for ScCo2 and ScRu0.25Co1.75 by stripping Sc metal from the center of a tunnel structure. This structural engineering significantly lowers their overpotentials at a current density of 500 mA/cm2 (η500) ca. 80 mV in 1.0 M KOH. Through a simple process, ScRu0.25Co1.75 can be easily decorated on a carbon cloth substrate and only requires 132 mV to reach 500 mA/cm2. More importantly it can maintain activity over 1000 h in industrial conditions (6.0 M KOH at 333 K), showing its potential for practical industrial applications.

A MS-feature-based medicinal plant database-driven strategy for ingredient identification of Chinese medicine prescriptions.

Identification of the individual herbs that constitute the Chinese medicine prescription (CMP) is a key step to control the quality and ensure the efficacy of traditional Chinese medicine (TCM), but also a challenging task for analysts from all over the world. In this study, a MS-feature-based medicinal plant database-driven strategy was proposed for quick and automatic interpretation of CMP ingredients. The single herb database consisting of stable ions of sixty-one common TCM medicinal herbs was first constructed. And then, the data of CMP was imported into a self-built searching program to achieve quick and automatic identification with four steps including level 1 candidate herb screening based on stable ions (step 1), level 2 candidate herb screening based on unique ions (step 2), difficult-to-distinguish herb differentiation (step 3) and results integration (step 4). The identification model was optimized and validated with homemade Shaoyaogancao Decoction, Mahuang Decoction, Banxiaxiexin Decoction, and their related negative prescriptions and homemade fakes. Another nine batches of homemade and commercial CMPs were applied to this new approach and most of composed herbs in the corresponding CMPs were correctly identified. This work provided a promising and universal strategy for the clarification of CMP ingredients.

Isolation and identification of a novel vardenafil analogue, propoxy-vardenafil, found as an adulterant in a health supplement.

Background: A novel vardenafil analogue was detected from a health wine claimed to be anti-impotence during a special inspection in an online store. Methods: The unknown compound was found by using ultra-high performance liquid chromatography coupled to quadrupole time of flight mass spectrometry (UHPLC/Q-TOF MS). The characteristic product ions were similar to those of vardenafil. The UV spectrum of the compound closely mirrored that of vardenafil. The analogue underwent purification by semi-preparative HPLC and structurally identified by FT-IR and NMR analysis. Results: Based on the data, The structure of the analogue was characterized as 2-[2-propyloxy-5-(4-ethylpiperazin-1-yl)sulfonylphenyl]-5-methyl-7-propyl-3H-imidazo [5,15,1-f] [1,2,4]triazin-4-one, simplified as propoxy-vardenafil. Conclusion: To the best of our knowledge, the analogue has not been reported and is even only ninth vardenafil analogue, which was confirmed that a n-propyloxy group had replaced the ethoxy group on the aromatic ring of vardenafil. Therefore, It is essential to pay more attention to vardenafil analogues in the routine inspection of health supplements.

Backbone-Installed Split Intein-Assisted Ligation for the Chemical Synthesis of Mirror-Image Proteins.

Membrane-associated D-proteins are an important class of synthetic molecules needed for D-peptide drug discovery, but their chemical synthesis using canonical ligation methods such as native chemical ligation is often hampered by the poor solubility of their constituent peptide segments. Here, we describe a Backbone-Installed Split Intein-Assisted Ligation (BISIAL) method for the synthesis of these proteins, wherein the native L-forms of the N- and C-intein fragments of the unique consensus-fast (Cfa) (i.e. L-CfaN and L-CfaC ) are separately installed onto the two D-peptide segments to be ligated via a removable backbone modification. The ligation proceeds smoothly at micromolar (µM) concentrations under strongly chaotropic conditions (8.0 M urea), and the subsequent removal of the backbone modification groups affords the desired D-proteins without leaving any "ligation scar" on the products. The effectiveness and practicality of the BISIAL method are exemplified by the synthesis of the D-enantiomers of the extracellular domains of T cell immunoglobulin and ITIM domain (TIGIT) and tropomyosin receptor kinase C (TrkC). The BISIAL method further expands the chemical protein synthesis ligation toolkit and provides practical access to challenging D-protein targets.

Chemically Synthetic d-Sortase Enables Enzymatic Ligation of d-Peptides.

We have described the chemical synthesis of d-Sortase A in large quantity and high purity by a hydrazide ligation strategy. The d-Sortase was fully active toward d-peptides and D/L hybrid proteins, and the ligation efficiency was unaffected by the chirality of the C-terminus substrate. This study points toward using d-sortase ligation as a modern ligation method for d-proteins and D/L hybrid proteins and expands the chemical protein synthesis toolbox in biotechnology.

Low-Temperature Cleanup Followed by Dispersive Solid-Phase Extraction for Determination of Nine Polar Plant Growth Regulators in Herbal Matrices Using Liquid Chromatography-Tandem Mass Spectrometry.

Polar plant growth regulators, used alone or doped in fertilizers, are most effective and widely utilized plant growth regulators (PGRs) in agriculture, which play important roles in mediating the yield and quality of crops and foodstuffs. The application scope has been extended to herbal medicines in the past 2 decades and relevant study is inadequate. The aim of this study is to establish a QuPPe-based extraction method containing low-temperature and d-SPE cleanup procedure followed by the detection on a selective multiresidue ultrahigh-performance liquid chromatography - triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) in three herbal matrices. This simple, accurate, versatile and robust method was verified according to the validation criteria of the SANTE/12682/2019 guideline document. The analytical range was from 2.5 to 200 µg/L, and the average recoveries were in the range of 64.6-117.8% (n = 6). The optimized method was applied to 135 herbal medicines thereof. Result showed that the detection frequency of chlormequat was the highest in the investigated PGRs, with the positive rate of 15.6%. Improvement of the detection method for polar PGRs will enrich the coverage of PGRs, which is conducive to safeguard public health and ensure drug safety. Supplementary Information: The online version contains supplementary material available at 10.1007/s10337-023-04254-3.

Evolving longer for a mate: A new scorpionfly (Mecoptera: Panorpoidea: Panorpidae) with exaggeratedly elongated male abdominal segments.

Male scorpionflies often evolve exaggeratedly elongated abdominal segments (EEAS) that are used as a sexual display to the females, and a combat weapon in intra-sexual disputes. EEAS have independently evolved several times since the Jurassic till the present day in at least eight genera and four families. In this paper, Neopanorpa exaggerata sp. n. with EEAS is described from Yunnan Province, China. Through comparisons among extinct and extant species, our current knowledge is summarized for the evolution, copulatory mechanism, and biogeography of the scorpionflies with EEAS. Also discussed are the trade-offs between the benefits of "looking sexy" and the negative impacts associated with a long abdomen. Additionally, further clarification is provided on the multiple origins of EEAS in Panorpoidea.

Isolation and characterization of a new oxyphenisatin analogue, oxyphenisatin propionate, from a processed plum intended as a weight loss product.

A new oxyphenisatin analogue was detected from a processed plum claiming to be a weight loss product without any side effects during the daily inspecting and monitoring of illegal adulterants in health supplements. An abundant peak caused our interest firstly owing to its identical fragments of m/z 224 and 196 in the MS/MS experiments with those of oxyphenisatin acetate. The chemical structure of the unknown compound was characterized by ultra-high performance liquid chromatography equipped with diode array detector and quadrupole time-of-flight tandem mass spectrometry (UHPLC-DAD-Q-TOF/MS), followed by nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy experiments. Based on the data, it was defined that the two symmetrical acetyl groups in oxyphenisatin acetate were replaced by two propionyl groups for the unknown structure. Finally, the new oxyphenisatin analogue was identified as 3,3-bis[4'-(propionyloxy)phenyl]-1,3-dihydroindole-2-one and designated as oxyphenisatin propionate. Thereafter, the content of the new analogue was quantitatively determined to be 681 mg/kg, which would inevitably cause adverse health effect because there was not specification for daily consumption of this product. To the best of our knowledge, this is the first report for identification of oxyphenisatin propionate.

Dual Function of Hypo-d-electronic Transition Metals in the Brewer Intermetallic Phase for the Highly Efficient Electrocatalytic Hydrogen Evolution Reaction in Alkaline Electrolytes.

Reported are the synthesis, material characterization, and electrocatalytic hydrogen evolution reaction (HER) in acid and alkaline electrolytes for the Brewer intermetallic phase, Nb6Co7 and Mo6Co7. It was realized that the overpotential at a current density of 10 mA/cm2 (η10) for Nb6Co7 (η10 = 62 mV) and Mo6Co7 (η10 = 143 mV) are both much lower than that of using a single Co metal (η10 = 253 mV) in alkaline electrolytes. The enhancement of electrocatalytic HER activity of Nb6Co7 and Mo6Co7 can be attributed to the hypo-hyper-d-electronic interaction between Nb/Mo and Co elements. Based on the result of density functional theory calculation, alloying between Nb/Mo and Co elements will increase the antibonding state population of the Co-Co bond near the Fermi level (EF), which induces the synergistic effect to influence the adsorption energy of the H atom (ΔGH) on the surface of Nb6Co7 and Mo6Co7. Moreover, the role of the Nb element is not only a simple electron donor but is also an anchor position for the OH molecule (i.e., dual function) due to the bonding character of the Nb-Co bond near EF. It can reduce the OH position effect as well as the activation energy for water dissociation, which rationalizes the high and robust HER performance of Nb6Co7 to that of commercial Pt/C (η10 = 67 mV) in alkaline electrolytes.

MRTCM: A comprehensive dataset for probabilistic risk assessment of metals and metalloids in traditional Chinese medicine.

Traditional Chinese medicine (TCM) is still considered a global complementary or alternative medical system, but exogenous hazardous contaminants remain in TCM even after decocting. Besides, it is time-consuming to conduct a risk assessment of trace elements in TCMs with a non-automatic approach due to the wide variety of TCMs. Here, we present MRTCM, a cloud-computing infrastructure for automating the probabilistic risk assessment of metals and metalloids in TCM. MRTCM includes a consumption database and a pollutant database involving forty million rows of consumption data and fourteen types of TCM potentially toxic elements concentrations. The algorithm of probabilistic risk assessment was also packaged in MRTCM to assess the risks of eight elements with Monte Carlo simulation. The results demonstrated that 96.64% and 99.46% had no non-carcinogenic risk (hazard indices (HI) were < 1.0) for animal and herbal medicines consumers, respectively. After twenty years of exposure, less than 1% of the total carcinogenic risk (CRt) was > 10-4 for TCM consumers, indicating that they are at potential risk for carcinogenicity. Sensitivity analysis revealed that annual consumption and concentration were the main variables affecting the assessment results. Ultimately, a priority management list of TCMs was also generated, indicating that more attention should be paid to the non-carcinogenic risks of As, Mn, and Hg and the carcinogenic risks of As and Cr in Pheretima and Cr in Arcae Conch. In general, MRTCM could significantly enhance the efficiency of risk assessment in TCM and provide reasonable guidance for policymakers to optimize risk management.

Combined effects of low calf circumference and vitamin D deficiency on all-cause mortality among community-dwelling older adults / 中华老年医学杂志

Objective:To investigate the individual and combined effects of calf circumference and vitamin D levels on all-cause mortality risk in community-dwelling older adults based on the Chinese Longitudinal Healthy Longevity Survey.Methods:The calf circumference was measured in the baseline survey in 2012 and 2014.Low calf circumference was defined as <34 cm for men or 33 cm for women at screening.Vitamin D deficiency was defined as plasma 25-hydroxyvitamin D[25(OH)D]levels <50 nmol/L.All participants were followed up until 2018, when death outcomes and survival time were collected.Cox proportional hazard regression models were used to analyze the effects of calf circumference and 25(OH)D levels on the risk of all-cause mortality.Results:A total of 3 052 older adults were included in the analysis, of which 1 960(64.22%)had low calf circumference and 2 245(73.56%)had vitamin D deficiency.After 10 559.9 person-years of follow-up period, 1 312 death events were recorded.After adjusting for sociodemographic characteristics, physical activities, cognitive function, and multiple chronic diseases, calf circumference and 25(OH)D levels were negatively associated with the risk of all-cause mortality(both P<0.05). In the combined analysis, compared with the normal group, the risk of death was highest in the participants with both low calf circumference and vitamin D deficiency, which was higher than those with low calf circumference or vitamin D deficiency alone, with a hazard ratios( HR)(95% CI)of 2.51(1.81-3.45), 1.71(1.22-2.42)and 1.53(1.09-2.15), respectively.There was a significant additive interaction between low calf circumference and vitamin D deficiency on mortality(RERI>0). Conclusions:Low calf circumference and vitamin D deficiency are associated with higher mortality.Older adults with combined conditions had a even higher risk of death.Attention should be paid to joint screening and comprehensive intervention for older adults with both low calf circumference and vitamin D deficiency.

Dose Titration of Solid Dosage Forms via FDM 3D-Printed Mini-Tablets.

The robustness of 3D-printed mini-tablets as a platform to administer milligram dosages, intended for age-specific therapy, without the need of tablet splitting while maintaining similar release profiles, was investigated. Griseofulvin, as a model poorly water-soluble drug, and hydroxypropyl cellulose along with Kollicoat Protect as polymers were used to prepare filaments at 1-20% drug concentrations via hot-melt extrusion (HME). Higher drug concentrations served for testing the feasibility of a reduced number of mini-tablets to be administered. A reliable dose titration in the range 0.19-3.91 mg at a high accuracy (R2 of 0.999) was achieved through composite unit (multi-unit) mini-tablets. All mini-tablets produced had excellent content uniformity and their label claim values were within the acceptable range, proving that HME processing followed by 3D printing promotes content uniformity even for mini-tablets containing low drug doses (0.19 mg). Remarkably, the proposed approach allowed achieving similar drug release profiles via composite unit mini-tablets as well as single mini-tablets at high drug concentrations. In contrast, split tablets demonstrated different release behaviors, attributed to their size and shape differences. Overall, the distinct advantages of mini-tablets to provide dose flexibility while maintaining similar release profiles was demonstrated.