Co-transplantation of autologous Treg cells in a cell therapy for Parkinson's disease.

The specific loss of midbrain dopamine neurons (mDANs) causes major motor dysfunction in Parkinson's disease, which makes cell replacement a promising therapeutic approach1-4. However, poor survival of grafted mDANs remains an obstacle to successful clinical outcomes5-8. Here we show that the surgical procedure itself (referred to here as 'needle trauma') triggers a profound host response that is characterized by acute neuroinflammation, robust infiltration of peripheral immune cells and brain cell death. When midbrain dopamine (mDA) cells derived from human induced pluripotent stem (iPS) cells were transplanted into the rodent striatum, less than 10% of implanted tyrosine hydroxylase (TH)+ mDANs survived at two weeks after transplantation. By contrast, TH- grafted cells mostly survived. Notably, transplantation of autologous regulatory T (Treg) cells greatly modified the response to needle trauma, suppressing acute neuroinflammation and immune cell infiltration. Furthermore, intra-striatal co-transplantation of Treg cells and human-iPS-cell-derived mDA cells significantly protected grafted mDANs from needle-trauma-associated death and improved therapeutic outcomes in rodent models of Parkinson's disease with 6-hydroxydopamine lesions. Co-transplantation with Treg cells also suppressed the undesirable proliferation of TH- grafted cells, resulting in more compact grafts with a higher proportion and higher absolute numbers of TH+ neurons. Together, these data emphasize the importance of the initial inflammatory response to surgical injury in the differential survival of cellular components of the graft, and suggest that co-transplanting autologous Treg cells effectively reduces the needle-trauma-induced death of mDANs, providing a potential strategy to achieve better clinical outcomes for cell therapy in Parkinson's disease.

Ionization and electron excitation of C60 in a carbon nanotube: A variable temperature/voltage transmission electron microscopic study.

There is increasing attention to chemical applications of transmission electron microscopy, which is often plagued by radiation damage. The damage in organic matter predominantly occurs via radiolysis. Although radiolysis is highly important, previous studies on radiolysis have largely been descriptive and qualitative, lacking in such fundamental information as the product structure, the influence of the energy of the electrons, and the reaction kinetics. We need a chemically well-defined system to obtain such data and have chosen as a model a variable-temperature and variable-voltage (VT/VV) study of the [2 + 2] dimerization of a van der Waals dimer [60]fullerene (C60) to C120 in a carbon nanotube (CNT), as studied for several hundred individual reaction events at atomic resolution. We report here the identification of five reaction pathways that serve as mechanistic models of radiolysis damage. Two of them occur via a radical cation of the specimen generated by specimen ionization, and three involve singlet or triplet excited states of the specimen, as initiated by electron excitation of the CNT, followed by energy transfer to the specimen. The [2 + 2] product was identified by measuring the distance between the two C60 moieties, and the mechanisms were distinguished by the pre-exponential factor and the Arrhenius activation energy­the standard protocol of chemical kinetic studies. The results illustrate the importance of VT/VV kinetic analysis in the studies of radiation damage and show that chemical ionization and electron excitation are inseparable, but different, mechanisms of radiation damage, which has so far been classified loosely under the single term "ionization."

Impact of schizophrenia GWAS loci converge onto distinct pathways in cortical interneurons vs glutamatergic neurons during development.

Remarkable advances have been made in schizophrenia (SCZ) GWAS, but gleaning biological insight from these loci is challenging. Genetic influences on gene expression (e.g., eQTLs) are cell type-specific, but most studies that attempt to clarify GWAS loci's influence on gene expression have employed tissues with mixed cell compositions that can obscure cell-specific effects. Furthermore, enriched SCZ heritability in the fetal brain underscores the need to study the impact of SCZ risk loci in specific developing neurons. MGE-derived cortical interneurons (cINs) are consistently affected in SCZ brains and show enriched SCZ heritability in human fetal brains. We identified SCZ GWAS risk genes that are dysregulated in iPSC-derived homogeneous populations of developing SCZ cINs. These SCZ GWAS loci differential expression (DE) genes converge on the PKC pathway. Their disruption results in PKC hyperactivity in developing cINs, leading to arborization deficits. We show that the fine-mapped GWAS locus in the ATP2A2 gene of the PKC pathway harbors enhancer marks by ATACseq and ChIPseq, and regulates ATP2A2 expression. We also generated developing glutamatergic neurons (GNs), another population with enriched SCZ heritability, and confirmed their functionality after transplantation into the mouse brain. Then, we identified SCZ GWAS risk genes that are dysregulated in developing SCZ GNs. GN-specific SCZ GWAS loci DE genes converge on the ion transporter pathway, distinct from those for cINs. Disruption of the pathway gene CACNA1D resulted in deficits of Ca2+ currents in developing GNs, suggesting compromised neuronal function by GWAS loci pathway deficits during development. This study allows us to identify cell type-specific and developmental stage-specific mechanisms of SCZ risk gene function, and may aid in identifying mechanism-based novel therapeutic targets.

Diagnosis of pulmonary tuberculosis via identification of core genes and pathways utilizing blood transcriptional signatures: a multicohort analysis.

BACKGROUND: Blood transcriptomics can be used for confirmation of tuberculosis diagnosis or sputumless triage, and a comparison of their practical diagnostic accuracy is needed to assess their usefulness. In this study, we investigated potential biomarkers to improve our understanding of the pathogenesis of active pulmonary tuberculosis (PTB) using bioinformatics methods. METHODS: Differentially expressed genes (DEGs) were analyzed between PTB and healthy controls (HCs) based on two microarray datasets. Pathways and functional annotation of DEGs were identified and ten hub genes were selected. They were further analyzed and selected, then verified with an independent sample set. Finally, their diagnostic power was further evaluated between PTB and HCs or other diseases. RESULTS: 62 DEGs mostly related to type I IFN pathway, IFN-γ-mediated pathway, etc. in GO term and immune process, and especially RIG-I-like receptor pathway were acquired. Among them, OAS1, IFIT1 and IFIT3 were upregulated and were the main risk factors for predicting PTB, with adjusted risk ratios of 1.36, 3.10, and 1.32, respectively. These results further verified that peripheral blood mRNA expression levels of OAS1, IFIT1 and IFIT3 were significantly higher in PTB patients than HCs (all P < 0.01). The performance of a combination of these three genes (three-gene set) had exceeded that of all pairwise combinations of them in discriminating TB from HCs, with mean AUC reaching as high as 0.975 with a sensitivity of 94.4% and a specificity of 100%. The good discernibility capacity was evaluated d via 7 independent datasets with an AUC of 0.902, as well as mean sensitivity of 87.9% and mean specificity of 90.2%. In regards to discriminating PTB from other diseases (i.e., initially considered to be possible TB, but rejected in differential diagnosis), the three-gene set equally exhibited an overall strong ability to separate PTB from other diseases with an AUC of 0.999 (sensitivity: 99.0%; specificity: 100%) in the training set, and 0.974 with a sensitivity of 96.4% and a specificity of 98.6% in the test set. CONCLUSION: The described commonalities and unique signatures in the blood profiles of PTB and the other control samples have considerable implications for PTB biosignature design and future diagnosis, and provide insights into the biological processes underlying PTB.

A CASPR1-ATP1B3 protein interaction modulates plasma membrane localization of Na+/K+-ATPase in brain microvascular endothelial cells.

Contactin-associated protein 1 (CASPR1 or CNTNAP1) was recently reported to be expressed in brain microvascular endothelial cells (BMECs), the major component of the blood-brain barrier. To investigate CASPR1's physiological role in BMECs, here we used CASPR1 as a bait in a yeast two-hybrid screen to identify CASPR1-interacting proteins and identified the ß3 subunit of Na+/K+-ATPase (ATP1B3) as a CASPR1-binding protein. Using recombinant and purified CASPR1, RNAi, GST-pulldown, immunofluorescence, immunoprecipitation, and Na+/K+-ATPase activity assays, we found that ATP1B3's core proteins, but not its glycosylated forms, interact with CASPR1, which was primarily located in the endoplasmic reticulum of BMECs. CASPR1 knockdown reduced ATP1B3 glycosylation and prevented its plasma membrane localization, phenotypes that were reversed by expression of full-length CASPR1. We also found that the CASPR1 knockdown reduces the plasma membrane distribution of the α1 subunit of Na+/K+-ATPase, which is the major component assembled with ATP1B3 in the complete Na+/K+-ATPase complex. The binding of CASPR1 with ATP1B3, but not the α1 subunit, indicated that CASPR1 binds with ATP1B3 to facilitate the assembly of Na+/K+-ATPase. Furthermore, the activity of Na+/K+-ATPase was reduced in CASPR1-silenced BMECs. Interestingly, shRNA-mediated CASPR1 silencing reduced glutamate efflux through the BMECs. These results demonstrate that CASPR1 binds with ATP1B3 and thereby contributes to the regulation of Na+/K+-ATPase maturation and trafficking to the plasma membrane in BMECs. We conclude that CASPR1-mediated regulation of Na+/K+-ATPase activity is important for glutamate transport across the blood-brain barrier.

δ-C-H Mono- and Dihalogenation of Alcohols.

Alkoxy radicals have long been known to enable remote C-H functionalization via 1,5-hydrogen atom abstraction. However, methods for their generation traditionally have relied upon highly oxidizing metals, ultraviolet radiation, or preformed peroxide intermediates, which has prevented the development of many desirable transformations. Herein we report a new bench-stable precursor that decomposes to free alkoxy radicals via a previously unreported single-electron oxidation pathway. This new precursor enables the fluorination and chlorination of remote C-H bonds under exceptionally mild conditions with exceedingly high monoselectivity. Iterative use of this precursor enables the introduction of a second halogen atom, granting access to remote dihalide motifs, including CF2 and CFCl.

Inhibitor discovery for the E. coli meningitis virulence factor IbeA from homology modeling and virtual screening.

Escherichia coli (E. coli) K1 is the most common Gram-negative bacteria cause of neonatal meningitis. The penetration of E. coli through the blood-brain barrier is a key step of the meningitis pathogenesis. A host receptor protein, Caspr1, interacts with the E. coli virulence factor IbeA and thus facilitates bacterial penetration through the blood-brain barrier. Based on this result, we have now predicted the binding pattern between Caspr1 and IbeA by an integrated computational protocol. Based on the predicted model, we have identified a putative molecular binding pocket in IbeA, that directly bind with Caspr1. This evidence indicates that the IbeA (229-343aa) region might play a key role in mediating the bacteria invasion. Virtual screening with the molecular model was conducted to search for potential inhibitors from 213,279 commercially available chemical compounds. From the top 50 identified compounds, 9 demonstrated a direct binding ability to the residues within the Caspr1 binding site on IbeA. By using human brain microvascular endothelial cells (hBMEC) with E. coli strain RS218, four molecules were characterized that significantly attenuated the bacteria invasions at concentrations devoid of cell toxicity. Our study provides useful structural information for understanding the pathogenesis of neonatal meningitis, and have identified drug-like compounds that could be used to develop effective anti-meningitis agents.

Learning a Second Language in Adulthood Changes Subcortical Neural Encoding.

Second language learning has been shown to impact and reshape the central nervous system, anatomically and functionally. Most of the studies on second language learning and neuroplasticity have been focused on cortical areas, whereas the subcortical neural encoding mechanism and its relationship with L2 learning have not been examined extensively. The purpose of this study was to utilize frequency-following response (FFR) to examine if and how learning a tonal language in adulthood changes the subcortical neural encoding in hearing adults. Three groups of subjects were recruited: native speakers of Mandarin Chinese (native speakers (NS)), learners of the language (L2 learners), and those with no experience (native speakers of foreign languages (NSFL)). It is hypothesized that differences would exist in FFRs obtained from the three language experience groups. Results revealed that FFRs obtained from L2 learners were found to be more robust than the NSFL group, yet not on a par with the NS group. Such results may suggest that in human adulthood, subcortical neural encoding ability may be trainable with the acquisition of a new language and that neuroplasticity at the brainstem level can indeed be influenced by L2 learning.

Genetic Correlation of Antibiotic Susceptibility and Resistance Genotyping for the Mycobacterium abscessus Group.

Treatment efficacy of Mycobacterium abscessus infections depends on bacterial genotype. Here, the relationship between genotype, as determined by sequence analysis, and antibiotic resistance phenotype was analyzed. The results demonstrate that M. abscessus genotype characteristics, including erm(41) sequevar and mutations of rrl and rrs, are predictive of clarithromycin and amikacin resistance.

CXCL1 promotes the proliferation of neural stem cells by stimulating the generation of reactive oxygen species in APP/PS1 mice.

PURPOSE: Elevated levels of CXCL1 were observed in the cerebrospinal fluid of patients with early Alzheimer's disease, which may affect neural stem cells in the subventricular zone. We used APP/PS1 mice and neural stem cells to elucidate the role of CXCL1 in Alzheimer's disease. METHODS & RESULTS: We detected CXCL1 in cerebrospinal fluid (CSF), activated macrophages, and microglia suggesting that macrophages may contribute to elevated CXCL1 in the CSF of middle-aged APP/PS1 mice. Proliferation and differentiation of neural stem cells were further analyzed and the results suggested that CXCL1 promotes the proliferation of neural stem cells and inhibits their differentiation into astrocytes. In order to determine how CXCL1 exerts these effects, we analyzed intracellular reactive oxygen species, cell signaling, and performed in vivo recovery experiments. Our results suggest that CXCL1 promotes neural stem cell proliferation through a mechanism involving the production of reactive oxygen species and the PI3K/Akt pathway. CONCLUSION: In APP/PS1 mice, macrophage-derived CXCL1 can promote the proliferation of neural stem cells in the subventricular zone via the NOX2-ROS-PI3K/Akt pathway.

Atg7 Regulates Brain Angiogenesis via NF-κB-Dependent IL-6 Production.

The formation of brain vasculature is an essential step during central nervous system development. The molecular mechanism underlying brain angiogenesis remains incompletely understood. The role of Atg7, an autophagy-related protein, in brain angiogenesis was investigated in this study. We found that the microvessel density in mice brains with endothelial-specific knockout of Atg7 (Atg7 EKO) was significantly decreased compared to wild-type control. Consistently, in vitro angiogenesis assays showed that Atg7 knockdown impaired angiogenesis in brain microvascular endothelial cells. Further results indicated that knockdown of Atg7 reduced interleukin-6 (IL-6) expression in brain microvascular endothelial cells, which is mediated by NF-κB-dependent transcriptional control. Interestingly, exogenous IL-6 restored the impaired angiogenesis and reduced cell motility caused by Atg7 knockdown. These results demonstrated that Atg7 has proangiogenic activity in brain angiogenesis which is mediated by IL-6 production in a NF-κB-dependent manner.

The Relative Contributions of Temporal Envelope and Fine Structure to Mandarin Lexical Tone Perception in Auditory Neuropathy Spectrum Disorder.

Previous studies have demonstrated that temporal envelope (E) is sufficient for speech perception, while fine structure (FS) is important for pitch perception for normal-hearing (NH) listeners. Listeners with sensorineural hearing loss (SNHL) have an impaired ability to use FS in lexical tone perception due to the reduced frequency resolution. Listeners with auditory neuropathy spectrum disorder (ANSD) may have deficits in temporal resolution. Little is known about how such deficits may impact their ability to use E and FS to perceive lexical tone, and whether it is the deficit in temporal resolution or frequency resolution that may lead to more detrimental effects on FS processing in pitch perception. Three experiments were conducted in the present study. Experiment I used the "auditory chimera" technique to investigate how SNHL and ANSD listeners would achieve lexical tone recognition using either the E or the FS cues. Experiment II tested their frequency resolution as measured with their psychophysical tuning curves (PTCs). Experiment III tested their temporal resolution as measured with the temporal gap detection (TGD) threshold. The results showed that the SNHL listeners had reduced frequency selectivity, but intact temporal resolution ability, while the ANSD listeners had degraded temporal resolution ability, but intact frequency selectivity. In comparison with the SNHL listeners, the ANSD listeners had severely degraded ability to process the FS cues and thus their ability to perceive lexical tone mainly depended on the ability to use the E cues. These results suggested that, in comparison with the detrimental impact of the reduced frequency selectivity, the impaired temporal resolution may lead to more degraded FS processing in pitch perception.

The Novel Multiple Inner Primers-Loop-Mediated Isothermal Amplification (MIP-LAMP) for Rapid Detection and Differentiation of Listeria monocytogenes.

Here, a novel model of loop-mediated isothermal amplification (LAMP), termed multiple inner primers-LAMP (MIP-LAMP), was devised and successfully applied to detect Listeria monocytogenes. A set of 10 specific MIP-LAMP primers, which recognized 14 different regions of target gene, was designed to target a sequence in the hlyA gene. The MIP-LAMP assay efficiently amplified the target element within 35 min at 63 °C and was evaluated for sensitivity and specificity. The templates were specially amplified in the presence of the genomic DNA from L. monocytogenes. The limit of detection (LoD) of MIP-LAMP assay was 62.5 fg/reaction using purified L. monocytogenes DNA. The LoD for DNA isolated from serial dilutions of L. monocytogenes cells in buffer and in milk corresponded to 2.4 CFU and 24 CFU, respectively. The amplified products were analyzed by real-time monitoring of changes in turbidity, and visualized by adding Loop Fluorescent Detection Reagent (FD), or as a ladder-like banding pattern on gel electrophoresis. A total of 48 pork samples were investigated for L. monocytogenes by the novel MIP-LAMP method, and the diagnostic accuracy was shown to be 100% when compared to the culture-biotechnical method. In conclusion, the MIP-LAMP methodology was demonstrated to be a reliable, sensitive and specific tool for rapid detection of L. monocytogenes strains.

Melting entropy of crystals determined by electron-beam-induced configurational disordering.

Upon melting, the molecules in a crystal explore numerous configurations, reflecting an increase in disorder. The molar entropy of disorder can be defined by Boltzmann's formula ΔSd = Rln(Wd), where Wd is the increase in the number of microscopic states, so far inaccessible experimentally. We found that the Arrhenius frequency factor A of the electron diffraction signal decay provides Wd through an experimental equation A = AINTWd, where AINT is an inelastic scattering cross section. The method connects Clausius and Boltzmann experimentally and supplements the Clausius approach, being applicable to a femtogram quantity of thermally unstable and biomolecular crystals. The data also showed that crystal disordering and crystallization of melt are reciprocal, both governed by the entropy change but manifesting in opposite directions.

γ-Cyclodextrin metal-organic frameworks as the promising carrier for pulmonary delivery of cyclosporine A.

γ-Cyclodextrin metal-organic frameworks (CD-MOFs) are considered as a green and biocompatible material with great potential in drug delivery systems. Original CD-MOFs show the poor aerosol properties, which limit the application in pulmonary drug delivery. To improve the in vitro deposition properties, herein, we synthesized CD-MOFs by the vapor diffusion method using a series of modulators to achieve better pulmonary delivery of cyclosporine A (CsA). The results showed that blank CD-MOFs and drug loaded CD-MOFs prepared with different modulators all preserved the cubical shape, and exhibited the similar crystal form, structural characteristics, thermal behaviors and release properties. In addition, drug loaded CD-MOFs prepared with polyethylene glycol 10000 (PEG 10000) as a modulator exhibited better in vitro aerosol performance than those of synthesized using other modulators, and the in vivo pharmacokinetics data demonstrated that the bioavailability of CsA could be significantly enhanced by inhalation administration of drug loaded CD-MOFs compared with oral administration of Neoral®. The repeated dose inhalation toxicity also confirmed the fine biocompatibility of CD-MOFs as the carrier for pulmonary drug delivery. Therefore, the results demonstrated CD-MOFs as the promising carrier could be used for pulmonary drug delivery.

fosA11, a novel chromosomal-encoded fosfomycin resistance gene identified in Providencia rettgeri.

This study investigated resistance genes corresponding to the fosfomycin resistance phenotype in clinical isolate Providencia rettgeri W986, as well as characterizing the enzymatic activity of FosA11 and the genetic environment. Antimicrobial susceptibility testing was performed using the agar microdilution method based on the Clinical and Laboratory Standards Institute guidelines. The whole genomic sequence of Providencia rettgeri W986 was obtained using Illumina sequencing and the PacBio platform. The fosA-11 gene was amplified by PCR and cloned into the pUCP20 vector. The recombinant strain pCold1-fosA11-BL21 was expressed to extract the target protein, and absorbance photometry was applied for enzymatic parameter determination. Minimal inhibitory concentration (MIC) tests showed that W986 conferred fosfomycin resistance and was inhibited by phosphonoformate, thereby indicating the presence of a FosA protein. A novel resistance gene designated as fosA11 was identified by whole-genome sequencing and bioinformatics analysis, and it shared 54.41%-64.23% amino acid identity with known FosA proteins. Cloning fosA11 into Escherichia coli obtained a significant increase (32-fold) in the MIC with fosfomycin. Determination of the enzyme kinetics showed that FosA11 had a high catalytic effect on fosfomycin, with Km = 18 ± 4 and Kcat = 56.1 ± 3.2. We also found that fosA11 was located on the chromosome, but the difference in the GC content between the chromosome and fosA11 was dubious, and thus further investigation is required. In this study, we identified and characterized a novel fosfomycin inactivation enzyme called FosA11. The origin and prevalence of the fosA11 gene in other bacteria require further investigation.IMPORTANCEFosfomycin is an effective antimicrobial agent against Enterobacterales strains. However, the resistance rate of fosfomycin is increasing year by year. Therefore, it is necessary to study the deep molecular mechanism of bacterial resistance to fosfomycin. We identified a novel chromosomal fosfomycin glutathione S-transferase, FosA11 from Providencia rettgeri, which shares a very low identity (54.41%-64.23%) with the previously known FosA and exhibits highly efficient catalytic ability against fosfomycin. Analysis of the genetic context and origin of fosA11 displays that the gene and its surrounding environments are widely conserved in Providencia and no mobile elements are discovered, implying that FosA11 may be broadly important in the natural resistance to fosfomycin of Providencia species.

Revealing nitrogenous VX metabolites and the whole-molecule VX metabolism in the urine of guinea pigs.

VX, a well-known organophosphorus nerve agent (OPNA), poses a significant threat to public safety if employed by terrorists. Obtaining complete metabolites is critical to unequivocally confirm its alleged use/exposure and elucidate its whole-molecular metabolism. However, the nitrogenous VX metabolites containing 2-diisopropylaminoethyl moiety from urinary excretion remain unknown. Therefore, this study applied a newly developed untargeted workflow platform to discover and identify them using VX-exposed guinea pigs as animal models. 2-(N,N-diisopropylamino)ethanesulfonic acid (DiPSA) was revealed as a novel nitrogenous VX metabolite in urine, and 2-(Diisopropylaminoethyl) methyl sulfide (DAEMS) was confirmed as another in plasma, indicating that VX metabolism differed between urine and plasma. It is the first report of a nitrogenous VX metabolite in urine and a complete elucidation of the VX metabolic pathway. DiPSA was evaluated as an excellent VX exposure biomarker. The whole-molecule VX metabolism in urine was characterized entirely for the first time via the simultaneous quantification of DiPSA and two known P-based biomarkers. About 52.1% and 32.4% of VX were excreted in urine as P-based and nitrogenous biomarkers within 24 h. These findings provide valuable insights into the unambiguous detection of OPNA exposure/intoxication and human and environmental exposure risk assessment.

Impact of MSMEG5257 Deletion on Mycolicibacterium smegmatis Growth.

Mycobacterial membrane proteins play a pivotal role in the bacterial invasion of host cells; however, the precise mechanisms underlying certain membrane proteins remain elusive. Mycolicibacterium smegmatis (Ms) msmeg5257 is a hemolysin III family protein that is homologous to Mycobacterium tuberculosis (Mtb) Rv1085c, but it has an unclear function in growth. To address this issue, we utilized the CRISPR/Cas9 gene editor to construct Δmsmeg5257 strains and combined RNA transcription and LC-MS/MS protein profiling to determine the functional role of msmeg5257 in Ms growth. The correlative analysis showed that the deletion of msmeg5257 inhibits ABC transporters in the cytomembrane and inhibits the biosynthesis of amino acids in the cell wall. Corresponding to these results, we confirmed that MSMEG5257 localizes in the cytomembrane via subcellular fractionation and also plays a role in facilitating the transport of iron ions in environments with low iron levels. Our data provide insights that msmeg5257 plays a role in maintaining Ms metabolic homeostasis, and the deletion of msmeg5257 significantly impacts the growth rate of Ms. Furthermore, msmeg5257, a promising drug target, offers a direction for the development of novel therapeutic strategies against mycobacterial diseases.

Transmission characteristics in Tuberculosis by WGS: nationwide cross-sectional surveillance in China.

China, with the third largest share of global tuberculosis cases, faces a substantial challenge in its healthcare system as a result of the high burden of multidrug-resistant and rifampicin-resistant tuberculosis (MDR/RR-TB). This study employs a genomic epidemiological approach to assess recent tuberculosis transmissions between individuals, identifying potential risk factors and discerning the role of transmitted resistant isolates in the emergence of drug-resistant tuberculosis in China. We conducted a population-based retrospective study on 5052 Mycobacterium tuberculosis (MTB) isolates from 70 surveillance sites using whole genome sequencing (WGS). Minimum spanning tree analysis identified resistance mutations, while epidemiological data analysis pinpointed transmission risk factors. Of the 5052 isolates, 23% (1160) formed 452 genomic clusters, with 85.6% (387) of the transmissions occurring within the same counties. Individuals with younger age, larger family size, new cases, smear positive, and MDR/RR were at higher odds for recent transmission, while higher education (university and above) and occupation as a non-physical workers emerged as protective factors. At least 61.4% (251/409) of MDR/RR-TB were likely a result of recent transmission of MDR/RR isolates, with previous treatment (crude OR = 2.77), smear-positive (cOR = 2.07) and larger family population (cOR = 1.13) established as risk factors. Our findings highlight that local transmission remains the predominant form of TB transmission in China. Correspondingly, drug-resistant tuberculosis is primarily driven by the transmission of resistant tuberculosis isolates. Targeted interventions for high-risk populations to interrupt transmission within the country will likely provide an opportunity to reduce the prevalence of both tuberculosis and drug-resistant tuberculosis.

A retrospective screening method for carbamate toxicant exposure based on butyrylcholinesterase adducts in human plasma with ultra-high performance liquid chromatography-tandem mass spectrometry.

Carbamate pesticides are extensively used in agriculture for their inhibition to acetylcholinesterase and damages to the insects' neural systems. Because of their toxicity, human poisoning incidents caused by carbamate pesticide exposure have occurred from time to time. What's more, some lethally toxic carbamate toxicants known as carbamate nerve agents (CMNAs) have been supplemented in Schedule 1 of the Annex on Chemicals in the Chemical Weapons Convention (CWC) by Organisation of the Prohibition of Chemical Weapons (OPCW) from 2020. And some other carbamates, like physostigmine, have been used in clinical treatment as anticholinergic drugs and their misuse may also cause damages to the body. Similar to organophosphorus toxicants, carbamate toxicants would react with butyrylcholinesterase (BChE) in plasma when entering the human body, resulting in the BChE adducts, based on which the exposure of carbamate toxicants could be detected retrospectively. In this study, methylcarbamyl nonapeptide and dimethylcarbamyl nonapeptide from pepsin digestion of BChE adducts were identified with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in product ion scan mode. Carbofuran was chosen as the target to establish the detection method of carbamate toxicant exposure based on methylcarbamyl nonapeptide digested from methylcarbamyl BChE. Procainamide-gel affinity purification, pepsin digestion and UHPLC-MS/MS analysis in multiple reaction monitoring (MRM) mode were applied. Under the optimized conditions of sample preparation and UHPLC-MS/MS MRM analysis, the limits of detection (LODs) reached 10.0 ng/mL of plasma exposed to carbofuran with satisfactory specificity. The quantitation approach was established with d3-carbofuran-exposed plasma as the internal standard (IS) and the linearity range was 30.0-1.00 × 103 nmol/L (R2 >0.998) with the accuracy of 95.6%-107% and precision of ≤9% relative standard deviation (RSD). The applicability was also evaluated by N,N-dimethyl-carbamates with the LODs of 30.0 nmol/L for pirimicarb-exposed plasma based on dimethylcarbamyl nonapeptide. Because most of carbamate toxicants has methylcarbamyl or dimethylcarbamyl groups, this approach could be applied on the retrospective screening of carbamate toxicant exposure including CMNAs, carbamate pesticides or carbamate drugs. This study could provide an effective means in the fields of CWC verification, toxicological mechanism investigation and down-selection of potential treatment options.