Targeting spike protein-induced TLR/NET axis by COVID-19 therapeutic NRICM102 ameliorates pulmonary embolism and fibrosis.

The global COVID-19 pandemic remains a critical public health threat, as existing vaccines and drugs appear insufficient to halt the rapid transmission. During an outbreak from May to August 2021 in Taiwan, patients with severe COVID-19 were administered NRICM102, which was a traditional Chinese medicine (TCM) formula developed based on its predecessor NRICM101 approved for treating mild cases. This study aimed to explore the mechanism of NRICM102 in ameliorating severe COVID-19-related embolic and fibrotic pulmonary injury. NRICM102 was found to disrupt spike protein/ACE2 interaction, 3CL protease activity, reduce activation of neutrophils, monocytes and expression of cytokines (TNF-α, IL-1ß, IL-6, IL-8), chemokines (MCP-1, MIP-1, RANTES) and proinflammatory receptor (TLR4). NRICM102 also inhibited the spread of virus and progression to embolic and fibrotic pulmonary injury through reducing prothrombotic (vWF, PAI-1, NET) and fibrotic (c-Kit, SCF) factors, and reducing alveolar type I (AT1) and type II (AT2) cell apoptosis. NRICM102 may exhibit its protective capability via regulation of TLRs, JAK/STAT, PI3K/AKT, and NET signaling pathways. The study demonstrates the ability of NRICM102 to ameliorate severe COVID-19-related embolic and fibrotic pulmonary injury in vitro and in vivo and elucidates the underlying mechanisms.

Curbing COVID-19 progression and mortality with traditional Chinese medicine among hospitalized patients with COVID-19: A propensity score-matched analysis.

BACKGROUND: Viral- and host-targeted traditional Chinese medicine (TCM) formulae NRICM101 and NRICM102 were administered to hospitalized patients with COVID-19 during the mid-2021 outbreak in Taiwan. We report the outcomes by measuring the risks of intubation or admission to intensive care unit (ICU) for patients requiring no oxygen support, and death for those requiring oxygen therapy. METHODS: This multicenter retrospective study retrieved data of 840 patients admitted to 9 hospitals between May 1 and July 26, 2021. After propensity score matching, 302 patients (151 received NRICM101 and 151 did not) and 246 patients (123 received NRICM102 and 123 did not) were included in the analysis to assess relative risks. RESULTS: During the 30-day observation period, no endpoint occurred in the patients receiving NRICM101 plus usual care while 14 (9.27%) in the group receiving only usual care were intubated or admitted to ICU. The numbers of deceased patients were 7 (5.69%) in the group receiving NRICM102 plus usual care and 27 (21.95%) in the usual care group. No patients receiving NRICM101 transitioned to a more severe status; NRICM102 users were 74.07% less likely to die than non-users (relative risk= 25.93%, 95% confidence interval 11.73%-57.29%). CONCLUSION: NRICM101 and NRICM102 were significantly associated with a lower risk of intubation/ICU admission or death among patients with mild-to-severe COVID-19. This study provides real-world evidence of adopting broad-spectrum oral therapeutics and shortening the gap between outbreak and effective response. It offers a new vision in our preparation for future pandemics.

Innovative Hybrid-Alignment Annotation Method for Bioinformatics Identification and Functional Verification of a Novel Nitric Oxide Synthase in Trichomonas vaginalis.

Both the annotation and identification of genes in pathogenic parasites are still challenging. Although, as a survival factor, nitric oxide (NO) has been proven to be synthesized in Trichomonas vaginalis (TV), nitric oxide synthase (NOS) has not yet been annotated in the TV genome. We developed a witness-to-suspect strategy to identify incorrectly annotated genes in TV via the Smith-Waterman and Needleman-Wunsch algorithms through in-depth and repeated alignment of whole coding sequences of TV against thousands of sequences of known proteins from other organisms. A novel NOS of TV (TV NOS), which was annotated as hydrogenase in the NCBI database, was successfully identified; this TV NOS had a high witness-to-suspect ratio and contained all the NOS cofactor-binding motifs (NADPH, tetrahydrobiopterin (BH4), heme and flavin adenine dinucleotide (FAD) motifs). To confirm this identification, we performed in silico modeling of the protein structure and cofactor docking, cloned the gene, expressed and purified the protein, performed mass spectrometry analysis, and ultimately performed an assay to measure enzymatic activity. Our data showed that although the predicted structure of the TV NOS protein was not similar to the structure of NOSs of other species, all cofactor-binding motifs could interact with their ligands with high affinities. We clearly showed that the purified protein had high enzymatic activity for generating NO in vitro. This study provides an innovative approach to identify incorrectly annotated genes in TV and highlights a novel NOS that might serve as a virulence factor of TV.

12-Plex UHPLC-MS/MS analysis of sarcosine in human urine using integrated principle of multiplex tags chemical isotope labeling and selective imprint enriching.

Urinary sarcosine was considered to be a potential biomarker for prostate cancer (Pca). In this work, an integrated strategy of multiplex tags chemical isotope labeling (MTCIL) combined with magnetic dispersive solid phase extraction (MDSPE), was proposed for specific extraction and high-throughput determination of sarcosine by ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). In the past three months, we have developed 8-plex MTCIL reagents with excellent qualitative and quantitative performance. In this work, the multiplexing capacity of MTCIL reagents (MTCIL360/361/362/363/364/365/366/375/376/378/379/381) was increased 1.5-fold from 8-plex to 12-plex. MTCIL359 was prepared and used to label sarcosine standard as internal standard (IS). The structural analogue derivative (MTCIL373-sarcosine) of all targeted MTCIL-sarcosine derivatives was synthesized and used as a novel dummy template to prepare dummy magnetic molecularly imprinted polymers (DMMIPs). The integration of MTCIL and DMMIPs procedures were extremely favorable to excellent chromatographic separation and efficient mass spectrometric detection. The labeling efficiency, chromatographic retention and mass spectrometry responses of MTCIL reagents were consistent for sarcosine. In a single UHPLC-MS/MS run (2.0 min), this method can simultaneously quantify sarcosine in 12-plex urine samples and achieve unbiased concentrations comparison between different urine samples. Analytical parameters including linearity (R2 0.989-0.997), detection limits (0.02 nM), precision (2.6-11.5%), accuracy (96.1-107.4%), matrix effect, labeling and extraction efficiency were carefully validated. The proposed method was successfully applied for urinary sarcosine determination of healthy male individuals and Pca patients. It was found that the sarcosine concentrations in these two groups were statistically extremely significantly different (P < 0.001). The developed method was a powerful analytical tool to substantially promote the analysis throughput and large-scale experiments about the potential biomarker research.

Risk of Acquired Cholesteatoma and External Auditory Canal Stenosis in Traumatic Brain Injury: A Nationwide Population-Based Cohort Study.

The aim of study is to investigate the risk of developing acquired cholesteatoma and external auditory canal (EAC) stenosis after traumatic brain injury (TBI) from the Taiwan National Health Insurance Research Database (NHIRD). Each subject was individually traced from their index date to identify those who received a diagnosis of acquired cholesteatoma and EAC stenosis. Cox regression analyses were applied to determine the risk of TBI-related acquired cholesteatoma and EAC stenosis. The follow-up data collected over 10 years were obtained from the TBI and comparison cohorts, of 455,834 and 911,668 patients, respectively. Multivariate analysis demonstrated that TBI significantly increased the risk of cholesteatoma (adjusted hazard ratio (HR), 1.777; 95% confidence interval (CI), 1.494-2.114, p < 0.001) and EAC stenosis (adjusted (HR), 3.549; 95% (CI), 2.713-4.644, p < 0.001). In our subgroup injury analysis, falls had the highest associated risk (4.308 times), followed by traffic injuries (66.73%; 3.718 times that of the control group). Otolaryngologists should not neglect the clinical importance and carefully investigate the possibility of subsequent cholesteatoma and EAC stenosis, which leads to hearing impairment in patients with TBI. Our research also shows the important role in preventing TBI, especially as a result of traffic injuries and falls.

13-Plex UHPLC-MS/MS Analysis of Hexanal and Heptanal Using Multiplex Tags Chemical Isotope Labeling Technology.

In this work, a new series of chemical isotope labeling reagents, levofloxacin-hydrazide-based mass tags (LHMTs) named as LHMT359/360/361/362/363/364/365/366/373/375/376/378/379/381 were first designed and synthesized for the high-throughput analysis of potential biomarkers containing hexanal and heptanal of lung cancer. We exploited a new core structure of levofloxacin-d3, which significantly enhanced the multiplexing capability. Among them, LHMT359 was used for labeling standard compounds as internal standards for quantification. Using LHMT373-heptanal as dummy template, dummy magnetic molecularly imprinted polymers (DMMIPs) were prepared for magnetic dispersive solid-phase extraction after derivatization procedure. Other 12 LHMTs were established for high-throughput labeling hexanal and heptanal in human serum samples. The presynthesized DMMIPs can selectively extract LHMTs-derivatives of hexanal and heptanal from equally mixed derivatization solutions. The enriched derivatives of hexanal and heptanal were quantified by ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). A single UHPLC-MS/MS run enabled simultaneously quantifying hexanal and heptanal from 12 serum samples only within 2 min. The limits of detection were all 0.5 pM for hexanal and heptanal. The accuracies from human serum samples ranged from -10.2% to +11.0% with the intra- and interday precisions less than 11.3%. Meanwhile, this method was successfully applied for the analysis of hexanal and heptanal in serum samples from healthy people and lung cancer patients. The results show that this method has the significant advantages of high sensitivity, accuracy, selectivity, and analysis-throughput. The method application indicates that the developed method is promising in the screening of suspected lung cancer patients.

8-Plex stable isotope labeling absolute quantitation strategy combined with dual-targeted recognizing function material for simultaneous separation and determination of glucosylsphingosine and galactosylsphingosine in human plasma.

Glucosylsphingosine (GlcS) in plasma is considered to be a reliable biomarker of Gaucher disease. The detection difficulty of GlcS is that it is difficult to achieve simultaneous separation and quantification with its isomer galactosylsphingosine (GalS), a biomarker of Krabbe disease. In this work, a multiplexed stable isotope labeling absolute quantization strategy coupled with magnetic dispersive solid phase extraction using new prepared dummy magnetic molecularly imprinted polymers (DMMIPs) has been developed for this purpose by ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). 8-Plex Amine-reactive Mass Difference Tags (M360/361/362/363/373/375/376/378-AMDTs), were designed, synthesized and used to label GalS and GlcS in different 8 plasma samples, respectively. Synchronously, M359-AMDTs was prepared and used to label mixed standards of GalS and GlcS, which served as internal standards in UHPLC-MS/MS quantitation. Then DMMIPs possessing dual recognition function were applied for specific enrichment and purification of all GlcS and GalS derivatives from a combined solution of labeled 8-plex plasma samples and mixed standards before UHPLC-MS/MS injection. The labeling efficiency, chromatographic retention and mass spectrometry responses of all the 9 AMDTs reagents were consistent for GlcS and GalS. The established and validated method enabled 8-plex plasma samples quantification in a single UHPLC-MS/MS run (<2.0 min). Good linearity of AMDTs-GlcS/GalS derivatives was obtained in the range of 0.02-800 nM. LODs of GlcS and GalS were both 0.005 nM. The recoveries were in the range of 96.1-107.2%. The method was successfully applied for multiplex quantitative analysis of GlcS and GalS in human plasma samples. The results indicated that this method was capable of better realizing the simultaneous separation and quantification of GalS and GlcS compared to reported methods.

Improvement of chicken plasma protein hydrolysate angiotensin I-converting enzyme inhibitory activity by optimizing plastein reaction.

Chicken plasma protein hydrolysate (CPPH) was prepared by trypsin with angiotensin I-converting enzyme (ACE) inhibitory activity of 53.5% ± 0.14% and the degree of hydrolysis (DH) of 16.22% ± 0.21% at 1 mg·ml-1; then, five proteases, including pepsin, trypsin, papain, alcalase, and neutrase, were employed to improve ACE inhibitory ability by catalyzing plastein reaction. The results indicated that trypsin-catalyzed plastein reaction showed the highest ACE inhibitory activity. The exogenous amino acids of leucine, histidine, tyrosine, valine, and cysteine were selected to modify the CPPH. The leucine-modified plastein reaction released the highest ACE inhibitory activity. The effects of four reaction parameters on plastein reaction were studied, and the optimal conditions with the purpose of obtaining the most powerful ACE inhibitory peptides from modified products were obtained by response surface methodology (RSM). The maximum ACE inhibition rate of the modified hydrolysate reached 82.07% ± 0.03% prepared at concentration of hydrolysates of 30%, reaction time of 4.9 hr, pH value of 8.0, temperature of 40°C, and E/S ratio of 5,681.62 U·g-1. The results indicated that trypsin-catalyzed plastein reaction increased ACE inhibitory activity of chicken plasma protein hydrolysates by 28.57%.

Multiplexed derivatization strategy-based dummy molecularly imprinted polymers as sorbents for magnetic dispersive solid phase extraction of globotriaosylsphingosine prior to UHPLC-MS/MS quantitation.

A new series of 9-plex chemical isotope-labeling reagents, levofloxacin-based mass tags (LMTs) named as LMT359, 360, 361, 362, 363, 373, 375, 376, and 378, was firstly designed and synthesized for the high-throughput labeling of globotriaosylsphingosine (lyso-Gb3), a disease biomarker of Fabry disease. Creatively based on derivatization strategy-dummy template technique, dummy magnetic molecularly imprinted polymers (DMMIPs) were designed and prepared using LMT387-labeled lyso-Gb3 as a dummy template. The novel DMMIP material was used as sorbents for magnetic dispersive solid-phase extraction of 9-plexed LMT derivatives of lyso-Gb3 from equally mixed derivatization solutions. The enriched 8-plexed lyso-Gb3 derivatives from 8 real samples were quantified by ultra-high-performance liquid chromatography tandem mass spectrometry in a single run using simultaneously extracted LMT359-labeled standard lyso-Gb3 as internal standards. DMMIPs were characterized by using the transmission electron microscope (TEM), Fourier transform infrared, X-ray photoelectron spectroscopy, and some other characterization techniques. TEM micrograph showed that the prepared DMMIPs had an apparent imprinting layer. Triple-recognition abilities of DMMIPs towards LMT-lyso-Gb3 mainly rely on the hydrogen bonding, electrostatic attraction, hydrophobic interaction, and boronate affinity. The imprinting factor of DMMIPs towards LMT-lyso-Gb3 was 5.1. This method shows the advantages of high selectivity (triple recognition), high sensitivity, high accuracy (recovery 93.5-108.8%), and high throughput (8 samples in a single run). The proposed method was successfully applied to the determination of lyso-Gb3 in plasma samples with spiked recoveries in the range of 95.0-102.4%. This indicates that the method is promising in bioanalysis and medical testing of lyso-Gb3 in the future. Graphical abstract Synthesis of multiplexed derivatization reagents and its correlative molecularly imprinted polymers for magnetic extraction of globotriaosylsphingosine.

Immunogenicity of adenovirus and DNA vaccines co-expressing P39 and lumazine synthase proteins of Brucella abortus in BALB/c mice.

Brucella poses a great threat to animal and human health. Vaccination is the most promising strategy in the effort to control Brucella abortus (B. abortus) infection, but the currently used live vaccines interfere with diagnostic tests and could potentially result in disease outbreak. Therefore, new subunit vaccines and combined immunization strategies are currently under investigation. In this study, immunogenicity and protection ability of a recombinant adenovirus and plasmid DNA vaccine co-expressing P39 and lumazine synthase proteins of B. abortus were evaluated based on the construction of the two molecular vaccines. Four immunization strategies (single adenovirus, single DNA, adenovirus/DNA, DNA/adenovirus) were investigated. The results showed that the immunization strategy of DNA priming followed by adenovirus boosting induced robust humoral and cellular immune responses, and it significantly reduced the numbers of B. abortus in a mouse model. These results suggest that it could be a potential antigen candidate for development of a new subunit vaccine against B. abortus infection.

Alarelin active immunization influences expression levels of GnRHR, FSHR and LHR proteins in the ovary and enhances follicular development in ewes.

We investigated the effects of gonadotropin releasing hormone (GnRH) agonist on expressions of GnRH receptor (GnRHR), follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) proteins in the ovaries and follicular development in the ewes. Forty-two pre-pubertal ewes were assigned to experimental groups 1 to 5 (EG-I to EG-V) and control group (CG). Ewes in EG-I, EG-II and EG-III were subcutaneously injected with 200, 300 or 400 µg alarelin antigens twice (on days 0 and 14), respectively. Ewes in EG-IV and EG-V were subcutaneously injected with 200 µg and 300 µg alarelin antigen four times (on days 0, 7, 14 and 21). Ewes in CG were subcutaneously injected with a solvent twice (on days 0 and 14). Serum concentrations of GnRH antibody in the EGs increased and were higher than (P<0.05) that of CG from day 14 to day 60. GnRH antibody concentrations in EG-IV and EG-V were higher than that in EG-I, EG-II and EG-III from days 35 to 45. Expressions of GnRHR protein in EG-IV and EG-V were lower than that in CG (P<0. 01). Expressions of FSHR and LHR proteins in EGs increased. Levels of FSHR and LHR proteins in EG-IV and EG-V (P<0.05) were higher than CG. Ovarian weights in EGs increased. Values of follicle vertical diameter, follicle transverse diameter, follicle wall thickness, follicle externatheca thickness and follicle internatheca thickness in EG-III and EG-V were greater than other groups. Primordial follicles and primary follicles developed quickly in alarelin-immunized animals. Secondary follicles and mature follicles became more abundant. Mitochondria, mitochondrial cristaes and cortical granules increased. Serum FSH concentrations of EGs remained higher than that in CG from days 28 to 70 (P<0.05). Alarelin immunization stimulated GnRH antibody production, suppressed expression of GnRHR protein, enhanced expressions of FSHR and LHR proteins in ovaries, promoted FSH secretion and thereby accelerated the development of ovaries and follicles in ewes.

Two new species of Schizorchis (Cestoda: Anoplocephalidae) from leporids (Lagomorpha: Leporidae) in China.

Four specimens of wild leporids, Lepus capensis Linnaeus, 1758, in Longde County, Ningxia Hui Autonomous Region (NXHAR), and 40 domestic rabbits, Oryctolagus cuniculus (Linnaeus, 1758) (20 from Xiji County, NXHAR, and 20 from Jingning County, Gansu Province) were examined for the presence of helminth parasites. Two new cestode species of Schizorchis Hansen, 1948 (Anoplocephalidae), were found: Schizorchis sinensis sp. n. from L. capensis and Schizorchis oryctolagi sp. n. from domestic rabbits. Schizorchis sinensis sp. n. is distinguished from all the species in the genus from pikas, Ochotona spp. (Lagomorpha: Ochotonidae) by having much longer strobila, a larger cirrus sac, a greater number of segments, and more numerous testes. The morphology of S. oryctolagi n. sp. is most similar to that of S. sinensis. It is distinguished from the latter species by its relatively small strobila, larger number of segments, smaller size of eggs, and much larger cirrus sac, which is substantially extending in a medial direction. In addition, a genital papilla is present in S. sinensis , but absent in S. oryctolagi. These findings indicate that species of Schizorchis are widespread in leporid mammals, contrary to the widespread opinion that the latter genus is specific to ochotonid lagomorph mammals. They are congruent, however, with the hypothesis of a close phylogenetic relationship between species of Schizorchis and Mosgovoyia. The occurrence of Schizorchis spp. in leporids is considered relict, vestigial from wider geographical and host ranges of this parasite genus during the Pleistocene.