The mechanism of action of Ophiocordyceps sinensis mycelia for prevention of acute lung injury based on non-targeted serum metabolomics.

Ophiocordyceps sinensis is a fungus with medicinal value in treating lung diseases, but no study has reported how to prevent acute lung injury using this fungus. The mice were divided into normal, model, positive control, and O. sinensis groups to observe lung histopathological sections and transmission electron microscopy, along with liquid chromatography-mass spectrometry and hematoxylin and eosin (H&E) staining to closely identify structural differences resulting from destruction between the groups. The results of the H&E staining showed that, compared with the normal group, the model group showed alveolar collapse. Compared with the model group, the infiltration of inflammatory cells in the alveolar cavity of the O. sinensis group was significantly reduced. Mitochondrial plate-like cristae were observed in type II alveolar cells of the normal group, with normal coloration of the mitochondrial matrix. Type II alveolar cells in the model group showed obvious edema. The statuses of type II alveolar cells in the O. sinensis and positive groups were similar to that in the normal group. Twenty-nine biomarkers and 10 related metabolic pathways were identified by serum metabolomics screening. The results showed that O. sinensis mycelia had a significant effect on the prevention of lipopolysaccharide-induced inflammation.

Preclinical safety evaluation of intradermal SARS-CoV-2 inactivated vaccine (Vero cells) administration in macaques.

Coronavirus disease 2019 (COVID-19) is an acute and highly pathogenic infectious disease in humans caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Six months after immunization with the SARS-CoV-2 vaccine, however, antibodies are almost depleted. Intradermal immunization could be a new way to solve the problem of nondurable antibody responses against SARS-CoV-2 or the poor immune protection against variant strains. We evaluated the preclinical safety of a SARS-CoV-2 vaccine for intradermal immunization in rhesus monkeys. The results showed that there were no obvious abnormalities in the general clinical condition, food intake, body weight or ophthalmologic examination except for a reaction at the local vaccination site. In the hematology examination, bone marrow imaging, serum biochemistry, and routine urine testing, the related indexes of each group fluctuated to different degrees after administration, but there was no dose-response or time-response correlation. The neutralization antibody and ELISpot results also showed that strong humoral and cellular immunity could be induced after vaccination, and the levels of neutralizing antibodies increased with certain dose- and time-response trends. The results of a repeated-administration toxicity test in rhesus monkeys intradermally inoculated with a SARS-CoV-2 inactivated vaccine showed good safety and immunogenicity.

Human exposure to trace elements in central Cambodia: Influence of seasonal hydrology and food-chain bioaccumulation behaviour.

Exposure to mercury and other trace elements remains an important public health concern, worldwide. The present study involved a comprehensive field study to determine concentrations of fourteen trace elements (Al, As, Cr, Co, Cd, Cu, Fe, Hg, Mn, Ni, Pb, Se, V and Zn) in surface water and different fish species from Tonlé Sap Lake in central Cambodia, during both the dry and wet seasons. Total arsenic (tAs) and Mn in surface water during the dry season exceeded WHO drinking water guidelines. Total mercury (tHg) concentrations (µg/g wet wt.) in fish during the wet season (GM = 0.055; CI95 = 0.01-0.26) were approximately 15 times higher (P < 0.05) compared to those during the dry season (GM = 0.0035; CI95 = 0.0004-0.033). Mean target hazard quotients (THQs) for inorganic arsenic (iAs), methyl mercury (MeHg), Mn and Pb were > 1, with estimated maximum values greatly exceeding 1. Mean THQs of Zn, Cd, Ni and Se were very near 1, with estimated maximum values exceeding 1. The MeHg THQ (min-max range: 0.16-9.09) during the wet season was 7 times higher than in the dry season (min-max range: 0.05-1.35). Concentrations of Hg and other trace elements varied widely between fish species. The findings suggest that exposure of some trace elements via water and food is of concern in this region. High consumption rates of fish and rice key factors related to trace element exposure. Seasonal hydrology and species-specific bioaccumulation behaviour in the Tonlé Sap Lake watershed also play an important role. The generated information will be useful to better mitigate trace element exposure in this region.

The uptake of mixed PAHs and PBDEs in wastewater by mangrove plants under different tidal flushing regimes.

Wastewater often contains mixed toxic pollutants, and the contribution of plant uptake in constructed wetland treatment systems is affected by environmental conditions, particularly tidal flushing. In this study, the uptake of wastewater-borne pollutants, including a mixture of polycyclic aromatic hydrocarbons (PAHs) and polybrominated diphenyl ethers (PBDEs) congeners, by two mangrove plant species, namely Excoecaria agallocha L. and Kandelia obovata Sheue, Liu & Yong, under different tidal flushing regimes was investigated. Results showed that Fe plaque formed on root surfaces could immobilize wastewater-borne PAHs and PBDEs. At the end of 8-month wastewater treatment, most of the pollutants removed by plants ended up in Fe plaque, with 0.12-20.83% of total PAHs and 0.78-24.76% of total PBDEs added to the microcosm retained in Fe plaque. On the contrary, the percentages of PAHs and PBDEs taken up by plant tissues were relatively small, ranging from not detected to 0.09% and from 0.01 to 2.00%, respectively. More uptake of Fe plaque-immobilized PAHs and PBDEs was found in K. obovata than in E. agallocha, leading to more plant damages in the former species due to its weaker root outer layers. While E. agallocha with stronger root protective outer layer was able to uptake more PAHs and PBDEs from wastewater but immobilize in Fe plaque than that of K. obovata. In both plant species, tidal flushing regimes significantly affected the immobilization of PAHs and PBDEs in Fe plaque, and more frequent tidal flushing led to higher percentages of immobilization. This is the first study demonstrating that E. agallocha was a more suitable mangrove plant species to remove wastewater-borne PAHs and PBDEs than K. obovata, and the significance of tidal flushing on performance of constructed mangrove wetlands.

USPIO assisting degradation of MXC by host/guest-type immobilized laccase in AOT reverse micelle system.

The laccase and ultrasmall superparamagnetic iron oxide nanoparticles (USPIO) have been assembled inside the tubular mesoporous silica via co-adsorption technology to prepare host/guest-type immobilized laccase, which is applied to degrade methoxychlor (MXC) in aqueous and reverse micelle environments. The effects of various parameters on degradation of MXC were studied. Under the optimum conditions, the degradation rate could reach maximum value of 45.6 % and remain at 20.8 % after seven cycles. Moreover, the addition of small molecular compound 2, 2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) to the system could greatly improve the degradation efficiency. The MXC degradation process is a first-order reaction, and the activation energy of MXC degradation catalyzed by immobilized laccase (41.46 kJ mol(-1)) is relatively lower than that catalyzed by free laccase (44.91 kJ mol(-1)). Based on the degradation products measured by gas chromatograph-mass spectrometer (GC-MS) and nuclear magnetic resonance (NMR), the degradation mechanism of MXC has also been proposed.

Associations of serum organohalogen levels and prostate cancer risk: Results from a case-control study in Singapore.

There is increasing evidence that elevated exposure to organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) may lead to an increased risk of prostate cancer. As part of a hospital-based case-control study of the Singaporean male population, we investigated associations between organohalogen exposure and risk of prostate cancer. Trace residue concentrations of 74 organohalogen contaminants, including several PCBs, OCPs and halogenated flame retardants (HFRs), were determined in serum samples (n = 120) using gas chromatography tandem mass spectrometry (GC-MS/MS). A variety of OCPs, PCBs and HFRs were detected in samples of both patients and controls. Mean concentrations of p,p' DDT, p,p' DDE, PCB 118, PCB 138, PCB 153 and PCB 187 were significantly higher (p < 0.05) in serum of patients. p,p' DDE measured in serum of patients was relatively high (mean:13,700 ng/g lipid, 95% CI:7000-26,800). Odds ratios (ORs) of OCPs and PCBs in many cases exceeded 1. ORs for p,p' DDE and PCB-153 at the highest tertile (>67th) were 5.67 (95% CI, 2.37-13.54) and 2.14 (95% CI, 0.99 to 4.66), respectively. The results suggest that exposure to DDTs and PCBs may be associated with prostate cancer risk in Singaporean males. No such association was observed for the organohalogen flame retardants studied, including polybrominated diphenyl ethers (PBDEs). The study provides novel information regarding the occurrence, levels and potential associations with prostate cancer risk for several organohalogen contaminants in the Singapore population. However, further investigation and analyses should be conducted to confirm these findings.

Differential GFP expression patterns induced by different heavy metals in Tg(hsp70:gfp) transgenic medaka (Oryzias latipes).

Heat shock protein 70 (Hsp70) is one of the most widely used biomarker for monitoring environment perturbations in biological systems. To facilitate the analysis of hsp70 expression as a biomarker, we generated a Tg(hsp70:gfp) transgenic medaka line in which green fluorescence protein (GFP) reporter gene was driven by the medaka hsp70 promoter. Here, we characterized Tg(hsp70:gfp) medaka for inducible GFP expression by seven environment-relevant heavy metals, including mercury, arsenic, lead, cadmium, copper, chromium, and zinc. We found that four of them (mercury, arsenic, lead, and cadmium) induced GFP expression in multiple and different organs. In general, the liver, kidney, gut, and skin are among the most frequent organs to show induced GFP expression. In contrast, no detectable GFP induction was observed to copper, chromium, or zinc, indicating that the transgenic line was not responsive to all heavy metals. RT-qPCR determination of hsp70 mRNA showed similar induction and non-induction by these metals, which also correlated with the levels of metal uptake in medaka exposed to these metals. Our observations suggested that these heavy metals have different mechanisms of toxicity and/or differential bioaccumulation in various organs; different patterns of GFP expression induced by different metals may be used to determine or exclude metals in water samples tested. Furthermore, we also tested several non-metal toxicants such as bisphenol A, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 4-introphenol, and lindane; none of them induced significant GFP expression in Tg(hsp70:gfp) medaka, further suggesting that the inducibility of Tg(hsp70:gfp) for GFP expression is specific to a subset of heavy metals.

Investigation of microbial community structure in constructed mangrove microcosms receiving wastewater-borne polycyclic aromatic hydrocarbons (PAHs) and polybrominated diphenyl ethers (PBDEs).

The study aims to examine relationships between microbial community structure and mixed pollutants of polycyclic aromatic hydrocarbons (PAHs) and polybrominated diphenyl ethers (PBDEs) in constructed wetland microcosms, planted with Excoecaria agallocha or Kandelia obovata, two common mangrove plant species, and under two tidal regimes, everyday tidal (Te) and no tidal flooding (Tn). Results showed both microbial community structure and the retained amounts of pollutants were significantly determined by tidal regime, while the effect of plant species was small. Higher amounts of PAHs but lower amounts of PBDEs were always retained in sediments under Te than Tn regimes. Accordingly, temporal and vertical distributions of microbial community structure differed greatly between the two tidal regimes. Redundancy analysis further revealed significant correlation between a subgroup of the mixed PAHs and PBDEs with variation in microbial community structure. The findings will help to propose specific strategies to improve the bioremediation efficiency of constructed wetland.

Kinetic measurements and mechanism determination of Stf0 sulfotransferase using mass spectrometry.

Mycobacterial carbohydrate sulfotransferase Stf0 catalyzes the sulfuryl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to trehalose. The sulfation of trehalose is required for the biosynthesis of sulfolipid-1, the most abundant sulfated metabolite found in Mycobacterium tuberculosis. In this paper, an efficient enzyme kinetics assay for Stf0 using electrospray ionization (ESI) mass spectrometry is presented. The kinetic constants of Stf0 were measured, and the catalytic mechanism of the sulfuryl group transfer reaction was investigated in initial rate kinetics and product inhibition experiments. In addition, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry was employed to detect the noncovalent complexes, the Stf0-PAPS and Stf0-trehalose binary complexes, and a Stf0-3'-phosphoadenosine 5'-phosphate-trehalose ternary complex. The results from our study strongly suggest a rapid equilibrium random sequential Bi-Bi mechanism for Stf0 with formation of a ternary complex intermediate. In this mechanism, PAPS and trehalose bind and their products are released in random fashion. To our knowledge, this is the first detailed mechanistic data reported for Stf0, which further demonstrates the power of mass spectrometry in elucidating the reaction pathway and catalytic mechanism of promising enzymatic systems.

Characterization of noncovalent protein-ligand complexes and associated enzyme intermediates of GlcNAc-6-O-sulfotransferase by electrospray ionization FT-ICR mass spectrometry.

In this study, a GlcNAc-6-O-Sulfotransferase, NodST and its complexation with the substrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and the inhibitor 3'-phosphoadenosine 5'-phosphate (PAP) were studied using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. In addition, using isotopically labeled substrate, we have successfully confirmed a sulfated enzyme intermediate, which was predicted by the MS kinetic measurement. It is also shown that information regarding solution binding affinities can be obtained using electrospray ionization (ESI)-FTICR mass spectrometry. The relative binding constants, Kd(PAPS)/Kd(PAP), derived from the solution and gas phase were very similar, which suggests that the binding domain of this particular enzyme system, given known structures of other sulfotransferases, may be preserved during the transmission of the complex from solution to the gas phase.

Mass spectrometric characterization of a three-enzyme tandem reaction for assembly and modification of the novobiocin skeleton.

The tripartite scaffold of the natural product antibiotic novobiocin is assembled by the tandem action of novobiocin ligase (NovL) and novobiocic acid noviosyl transferase (NovM). The noviosyl ring of the tripartite scaffold is further decorated by a methyltransferase (NovP) and a carbamoyltransferase (NovN), resulting in the formation of novobiocin. To facilitate kinetic evaluation of alternate substrate usage by NovL and NovM toward the creation of variant antibiotic scaffolds, an electrospray ionization/MS assay for obtaining kinetic measurements is presented for NovL and NovM separately, in each case with natural substrate and the 3-methyl-4-hydroxybenzoic acid analog. Additionally, assays of tandem two-enzyme (NovL/NovM) and three-enzyme (NovL/NovM/NovP) incubations were developed. The development of these assays allows for the direct detection of each intermediate followed by its utilization as substrate for the next enzyme, as well as the subsequent formation of final product as a function of time. This MS tandem assay is useful for optimization of conditions for chemoenzymatic generation of novobiocin and is also suitable for evaluation of competitive usage of variant substrate analogs by multiple enzymes. The studies presented here serve as a platform for the subsequent expansion of the repertoire of coumarin-based antibiotics.

Observation of a hybrid random ping-pong mechanism of catalysis for NodST: a mass spectrometry approach.

An efficient enzyme kinetics assay using electrospray ionization mass spectrometry (ESI-MS) was initially applied to the catalytic mechanism investigation of a carbohydrate sulfotransferase, NodST. Herein, the recombinant NodST was overexpressed with a His(6)-tag and purified via Ni-NTA metal-affinity chromatography. In this bisubstrate enzymatic system, an internal standard similar in structure and ionization efficiency to the product was chosen in the ESI-MS assay, and a single point normalization factor was determined and used to quantify the product concentration. The catalytic mechanism of NodST was rapidly determined by fitting the MS kinetic data into a nonlinear regression analysis program. The initial rate kinetics analysis and product inhibition study described support a hybrid double-displacement, two-site ping-pong mechanism of NodST with formation of a sulfated NodST intermediate. This covalent intermediate was further isolated and detected via trypsin digestion and Fourier transform ion cyclotron resonance mass spectrometry. To our knowledge, these are the first mechanistic data reported for the bacterial sulfotransferase, NodST, which demonstrated the power of mass spectrometry in elucidating the reaction pathway and catalytic mechanism of promising enzymatic systems.

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay.

The traditional method used to investigate the reaction specificity of an enzyme with different substrates is to perform individual kinetic measurements. In this case, a series of varied concentrations are required to study each substrate and a non-regression analysis program is used several times to obtain all the specificity constants for comparison. To avoid the large amount of experimental materials, long analysis time, and redundant data processing procedures involved in the traditional method, we have developed a novel strategy for rapid determination of enzyme substrate specificity using one reaction system containing multiple competing substrates. In this multiplex assay method, the electrospray ionization mass spectrometry (ESI-MS) technique was used for simultaneous quantification of multiple products and a steady-state kinetics model was established for efficient specificity constant calculation. The system investigated was the bacterial sulfotransferase NodH (NodST), which is a host specific nod gene product that catalyzes the sulfate group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to natural Nod factors or synthetic chitooligosaccharides. Herein, the reaction specificity of NodST for four chitooligosaccharide acceptor substrates of different chain length (chitobiose, chitotriose, chitotetraose, and chitopentaose) was determined by both individual kinetic measurements and the new multiplex ESI-MS assay. The results obtained from the two methods were compared and found to be consistent. The multiplex ESI-MS assay is an accurate and valid method for substrate specificity evaluation, in which multiple substrates can be evaluated in one assay.

Kinetic analysis of NodST sulfotransferase using an electrospray ionization mass spectrometry assay.

A novel and efficient enzyme kinetics assay using electrospray ionization mass spectrometry was developed and applied to the bacterial carbohydrate sulfotransferase (NodST). NodST catalyzes the sulfuryl group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to chitobiose, generating 3'-phosphoadenosine 5'-phosphate (PAP) and chitobiose-6-OSO(3)(-) as products. Traditional spectrophotometric assays are not applicable to the NodST system since no shift in absorption accompanies sulfuryl group transfer. Alternative assays have employed thin-layer chromatography, but this procedure is time-consuming and requires radioactive materials. The ESI-MS assay presented herein requires no chromophoric substrate or product, and the analysis time is very short. The ESI-MS assay is used to determine NodST kinetic parameters, including K(M), V(max), and K(i) (for PAP). In addition, the mode of inhibition for PAP was rapidly determined. The results were in excellent agreement with those obtained from previous assays, verifying the accuracy and reliability of the ESI-MS assay. This unique technique is currently being used to investigate the enzymatic mechanism of NodST and to identify sulfotransferase inhibitors.