In vitro Antibacterial Effect Study of Plasma-Activated Saline on Mycobacterium Tuberculosis.

Purpose: This study aimed to investigate the antibacterial effects of plasma-activated saline (PAS) on My-cobacterium tuberculosis (Mtb). Methods: We conducted a growth assay on 3 strains of Mtb and an antibiotic sensitivity test on 4 strains of Mtb. Both tests included groups treated with normal saline (NS), PAS, and hydrochloric acid (HCl). The test of antibiotic sensitivity consisted of parallel tests with two concentrations of bacteria suspension: 10-2 and 10-4. The selected antibiotics were rifampicin (RIF), isoniazid (INH), ethambutol (EMB), and streptomycin (SM). The number of bacteria was determined after one month of culture under different conditions. The Kruskal-Wallis test was used to analyze the differences in grouping factors at representative time points. Results: The growth assay indicated that PAS significantly inhibited the growth of 3 strains of Mtb compared with NS and HCl treatment groups. Furthermore, except for the initial observation time point, the remaining three observation time points consistently demonstrate no significant differences between the NS group and the HCl group. The antibiotic sensitivity test of INH, SM, and RIF indicated that PAS could inhibit the growth of antibiotic-resistant Mtb, and the antibiotic sensitivity test of INH and SM with bacterial suspension concentration of 10-2 and SM with bacterial suspension concentration of 10-4 showed statistically different results. The antibiotic sensitivity test of EMB indicated that the growth of Mtb in PAS was slower than that in NS and HCl in both antibiotic-resistant and sensitive Mtb, but there was no statistical difference. Conclusion: The study indicates that PAS contains a significant amount of active substances and exhibits high oxidizability and an acidic pH state. The unique physicochemical properties of PAS significantly delayed the growth of Mtb, compared to the NS and the HCl. PAS not only inhibited the growth of drug-sensitive strains but also significantly enhanced the sensitivity of drug-resistant strains to anti-tuberculosis drugs, which may provide a new therapeutic strategy for the treatment of tuberculosis.

Advancements in LAM-based diagnostic kit for tuberculosis detection: enhancing TB diagnosis in HIV-negative individuals.

Objective: The purpose of this study was to investigate the diagnostic value of urine lipoarabinomannan (LAM) detection based on chemiluminescence assay for pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) in HIV-negative individuals. Methods: A total of 215 patients and 37 healthy individuals were included according to inclusion and exclusion criteria, including 173 cases of PTB and 42 cases of EPTB. Sputum smears, sputum culture, TB-RNA, GeneXpert, and urine LAM results were obtained from all patients before treatment. Using the composite reference standard as the reference, the diagnostic performance of these methods for PTB and EPTB was evaluated, and the diagnostic performance and cost-effectiveness of different combinations were analyzed. Results: In PTB, LAM exhibited the highest sensitivity (55.49%), followed by GeneXpert (44.51%). In EPTB, LAM also had the highest sensitivity (40.48%), followed by GeneXpert (33.33%). When combined with one method, LAM combined with GeneXpert showed the highest sensitivity for both PTB (68.79%) and EPTB (61.9%). When combined with two methods, culture, GeneXpert, and LAM showed the highest sensitivity for both PTB (73.99%) and EPTB (69.05%). In terms of cost-effectiveness analysis, the price of LAM was significantly lower than that of GeneXpert ($129.82 vs. $275.79 in PTB and 275.79 vs. 502.33 in EPTB). Among all combinations, the combination of LAM and sputum smear had the lowest cost, with prices of $124.94 for PTB and $263.72 for EPTB. Conclusion: Urine LAM detection based on chemiluminescence assay can be used as an adjunct diagnostic tool for PTB and EPTB in HIV-negative individuals. This facilitates expanding the current application of urine LAM from solely HIV-positive populations to the general population. LAM detection can overcome the limitations of obtaining clinical samples, and its ease of sample acquisition will be beneficial for its broader application in a larger scope. For economically better-off areas, we recommend using a combination of LAM + GeneXpert+culture for higher sensitivity; for economically disadvantaged areas, LAM + smear microscopy combination can provide a quick and accurate diagnosis of tuberculosis at a lower cost.

The emerging threat of fluroquinolone-, bedaquiline-, and linezolid-resistant Mycobacterium tuberculosis in China: Observations on surveillance data.

BACKGROUND: Drug-resistant tuberculosis (TB), especially multidrug-resistant tuberculosis (MDR-TB), constitutes a major obstacle to fulfill end TB strategy globally. Although fluoroquinolones (FQs), linezolid (LZD) and bedaquiline (BDQ) were classified as Group A drugs for MDR-TB treatment, our knowledge of the prevalence of TB which were resistant to Group A drugs in China is quite limited. METHODS: In this study, we conducted a prospective multicenter surveillance study in China to determine the proportion of TB patients that were resistant to Group A drugs. A total of 1877 TB patients were enrolled from 2022 at four TB specialized hospitals. The drug susceptibility of isolated strains was conducted using the MGIT 960 system and the molecular mechanisms conferring drug resistance were investigated by Sanger sequencing. RESULTS: 12.9% of isolates were resistant to levofloxacin (LFX), 13.2% were resistant to moxifloxacin (MOX), 0.2% were resistant to bedaquiline (BDQ), and 0.8% were resistant to linezolid (LZD). Totally, 14.0% and 0.4% were classified as multidrug resistant- (MDR-) and extensively drug resistant- (XDR-) TB. The drug resistance was more common in retreated TB cases compared to new cases. In addition, 70.0% of fluoroquinolone (FQ)-resistant isolates harbored mutations in the gyrA and gyrB gene. By contrast, the common drug-resistant mutations were only found in 50% BDQ-resistant and 20% LZD-resistant isolates. CONCLUSIONS: Our data demonstrate that approximate half of MDR -TB patients are resistant to fluoroquinolones, with extremely low prevalence of initial BDQ and LZD resistance. Findings from this study provide important implications for the current management of MDR-TB patients.

Accuracy of the InnowaveDX MTB/RIF test for detection of Mycobacterium tuberculosis and rifampicin resistance: a prospective multicentre study.

Early and accurate diagnosis of tuberculosis (TB) is necessary to initiate proper therapy for the benefit of the patients and to prevent disease transmission in the community. In this study, we developed the InnowaveDX MTB/RIF (InnowaveDX) to detect Mycobacterium tuberculosis (MTB) and rifampicin resistance simultaneously. A prospective multicentre study was conducted to evaluate the diagnostic performance of InnowaveDX for the detection MTB in sputum samples as compared with Xpert and culture. The calculated limit of detection (LOD) for InnowaveDX was 9.6 CFU/ml for TB detection and 374.9 CFU/ml for RIF susceptibility. None of the other bacteria tested produced signals that fulfilled the positive TB criteria, demonstrating a species-specificity of InnowaveDX. Then 951 individuals were enrolled at 7 hospitals, of which 607 were definite TB cases with positive culture and/or Xpert results, including 354 smear-positive and 253 smear-negative cases. InnowaveDX sensitivity was 92.7% versus bacteriologically TB standard. Further follow-up revealed that 61 (91.0%) out of 67 false-positive patients with no bacteriological evidence met the criteria of clinically diagnosed TB. Among 125 RIF-resistant TB patients diagnosed by Xpert, 108 cases were correctly identified by InnowaveDX, yielding a sensitivity of 86.4%. Additionally, the proportion of very low bacterial load in the discordant susceptibility group was significantly higher than in the concordant susceptibility group (P = 0.029). To conclude, we have developed a novel molecular diagnostic with promising detection capabilities of TB and RIF susceptibility. In addition, the discordant RIF susceptibility results between InnowaveDX and Xpert are more frequently observed in samples with very low bacterial load.

Genetic basis of kernel starch content decoded in a maize multi-parent population.

Starch is the most abundant storage carbohydrate in maize kernels and provides calories for humans and other animals as well as raw materials for various industrial applications. Decoding the genetic basis of natural variation in kernel starch content is needed to manipulate starch quantity and quality via molecular breeding to meet future needs. Here, we identified 50 unique single quantitative trait loci (QTLs) for starch content with 18 novel QTLs via single linkage mapping, joint linkage mapping and a genome-wide association study in a multi-parent population containing six recombinant inbred line populations. Only five QTLs explained over 10% of phenotypic variation in single populations. In addition to a few large-effect and many small-effect additive QTLs, limited pairs of epistatic QTLs also contributed to the genetic basis of the variation in kernel starch content. A regional association study identified five non-starch-pathway genes that were the causal candidate genes underlying the identified QTLs for starch content. The pathway-driven analysis identified ZmTPS9, which encodes a trehalose-6-phosphate synthase in the trehalose pathway, as the causal gene for the QTL qSTA4-2, which was detected by all three statistical analyses. Knockout of ZmTPS9 increased kernel starch content and, in turn, kernel weight in maize, suggesting potential applications for ZmTPS9 in maize starch and yield improvement. These findings extend our knowledge about the genetic basis of starch content in maize kernels and provide valuable information for maize genetic improvement of starch quantity and quality.

Low Rate of Acquired Linezolid Resistance in Multidrug-Resistant Tuberculosis Treated With Bedaquiline-Linezolid Combination.

In this retrospective study in China, we aimed to: (1) determine the prevalence of linezolid (LZD) resistance among multidrug-resistant tuberculosis (MDR-TB)-infected patients; (2) monitor for dynamic LZD susceptibility changes during anti-TB treatment; and (3) explore molecular mechanisms conferring LZD resistance. A total of 277 MDR-TB patients receiving bedaquiline (BDQ)-containing regimens in 13 TB specialized hospitals across China were enrolled in the study. LZD and BDQ susceptibility rates were determined using the minimum inhibitory concentration (MIC) method, then DNA sequences of patient isolates were analyzed using Sanger sequencing to detect mutations conferring LZD resistance. Of 277 patients in our cohort, 115 (115/277, 41.5%) with prior LZD exposure yielded 19 (19/277, 6.9%) isolates exhibiting LZD resistance. The LZD resistance rate of LZD-exposed group isolates significantly exceeded the corresponding rate for non-exposed group isolates (P = 0.047). Genetic mutations were observed in 10 (52.6%, 10/19) LZD-resistant isolates, of which a Cys154Arg (36.8%, 7/19) substitution within ribosomal protein L3 was most prevalent. Analysis of sequential positive cultures obtained from 81 LZD-treated patients indicated that cultured organisms obtained from most patients (85.2%, 69/81) retained original LZD MIC values; however, organisms cultured later from two patients exhibited significantly increased MIC values that were attributed to the rplC substitution T460C. Overall, LZD resistance was detected in 6.9% of patients of an MDR-TB cohort in China. Low rate of acquired LZD resistance was noted in MDR-TB treated with BDQ-LZD combination.

Heterologous Expression of GbTCP4, a Class II TCP Transcription Factor, Regulates Trichome Formation and Root Hair Development in Arabidopsis.

Two class I family teosinte branched1/cycloidea/proliferating cell factor1 (TCP) proteins from allotetraploid cotton are involved in cotton fiber cell differentiation and elongation and root hair development. However, the biological function of most class II TCP proteins is unclear. This study sought to reveal the characteristics and functions of the sea-island cotton class II TCP gene GbTCP4 by biochemical, genetic, and molecular biology methods. GbTCP4 protein localizes to nuclei, binding two types of TCP-binding cis-acting elements, including the one in its promoter. Expression pattern analysis revealed that GbTCP4 is widely expressed in tissues, with the highest level in flowers. GbTCP4 is expressed at different fiber development stages and has high transcription in fibers beginning at 5 days post anthesis (DPA). GbTCP4 overexpression increases primary root hair length and density and leaf and stem trichomes in transgenic Arabidopsis relative to wild-type plants (WT). GbTCP4 binds directly to the CAPRICE (CPC) promoter, increasing CPC transcript levels in roots and reducing them in leaves. Compared with WT plants, lignin content in the stems of transgenic Arabidopsis overexpressing GbTCP4 increased, and AtCAD5 gene transcript levels increased. These results suggest that GbTCP4 regulates trichome formation and root hair development in Arabidopsis and may be a candidate gene for regulating cotton fiber elongation.

SEED CAROTENOID DEFICIENT Functions in Isoprenoid Biosynthesis via the Plastid MEP Pathway.

Plastid isoprenoids, a diverse group of compounds that includes carotenoids, chlorophylls, tocopherols, and multiple hormones, are essential for plant growth and development. Here, we identified and characterized SEED CAROTENOID DEFICIENT (SCD), which encodes an enzyme that functions in the biosynthesis of plastid isoprenoids in maize (Zea mays). SCD converts 2C-methyl-d-erytrithol 2,4-cyclodiphosphate to 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate in the penultimate step of the methylerythritol phosphate (MEP) pathway. In scd mutants, plant growth and development are impaired and the levels of MEP-derived isoprenoids, such as carotenoids, chlorophylls, and tocopherols, as well as abscisic and gibberellic acids, are reduced in leaves and seeds. This scd metabolic alteration varies among plant tissues and under different light conditions. RNA-sequencing of the scd mutant and wild type identified a limited number of differentially expressed genes in the MEP pathway, although isoprenoid levels were significantly reduced in scd seeds and dark-grown leaves. Furthermore, SCD-overexpressing transgenic lines showed little or no differences in isoprenoid levels, indicating that SCD may be subject to posttranslational regulation or not represent a rate-limiting step in the MEP pathway. These results enhance our understanding of the transcriptomic and metabolic regulatory roles of enzymes in the MEP pathway and of their effects on downstream isoprenoid pathways in various plant tissues and under different light conditions.

Relationship, evolutionary fate and function of two maize co-orthologs of rice GW2 associated with kernel size and weight.

BACKGROUND: In rice, the GW2 gene, found on chromosome 2, controls grain width and weight. Two homologs of this gene, ZmGW2-CHR4 and ZmGW2-CHR5, have been found in maize. In this study, we investigated the relationship, evolutionary fate and putative function of these two maize genes. RESULTS: The two genes are located on duplicated maize chromosomal regions that show co-orthologous relationships with the rice region containing GW2. ZmGW2-CHR5 is more closely related to the sorghum counterpart than to ZmGW2-CHR4. Sequence comparisons between the two genes in eight diverse maize inbred lines revealed that the functional protein domain of both genes is completely conserved, with no non-synonymous polymorphisms identified. This suggests that both genes may have conserved functions, a hypothesis that was further confirmed through linkage, association, and expression analyses. Linkage analysis showed that ZmGW2-CHR4 is located within a consistent quantitative trait locus (QTL) for one-hundred kernel weight (HKW). Association analysis with a diverse panel of 121 maize inbred lines identified one single nucleotide polymorphism (SNP) in the promoter region of ZmGW2-CHR4 that was significantly associated with kernel width (KW) and HKW across all three field experiments examined in this study. SNPs or insertion/deletion polymorphisms (InDels) in other regions of ZmGW2-CHR4 and ZmGW2-CHR5 were also found to be significantly associated with at least one of the four yield-related traits (kernel length (KL), kernel thickness (KT), KW and HKW). None of the polymorphisms in either maize gene are similar to each other or to the 1 bp InDel causing phenotypic variation in rice. Expression levels of both maize genes vary over ear and kernel developmental stages, and the expression level of ZmGW2-CHR4 is significantly negatively correlated with KW. CONCLUSIONS: The sequence, linkage, association and expression analyses collectively showed that the two maize genes represent chromosomal duplicates, both of which function to control some of the phenotypic variation for kernel size and weight in maize, as does their counterpart in rice. However, the different polymorphisms identified in the two maize genes and in the rice gene indicate that they may cause phenotypic variation through different mechanisms.

Genetic analysis and characterization of a new maize association mapping panel for quantitative trait loci dissection.

Association mapping based on the linkage disequilibrium provides a promising tool to identify genes responsible for quantitative variations underlying complex traits. Presented here is a maize association mapping panel consisting of 155 inbred lines with mainly temperate germplasm, which was phenotyped for 34 traits and genotyped using 82 SSRs and 1,536 SNPs. Abundant phenotypic and genetic diversities were observed within the panel based on the phenotypic and genotypic analysis. A model-based analysis using 82 SSRs assigned all inbred lines to two groups with eight subgroups. The relative kinship matrix was calculated using 884 SNPs with minor allele frequency > or = 20% indicating that no or weak relationships were identified for most individual pairs. Three traits (total tocopherol content in maize kernel, plant height and kernel length) and 1,414 SNPs with missing data < 20% were used to evaluate the performance of four models for association mapping analysis. For all traits, the model controlling relative kinship (K) performed better than the model controlling population structure (Q), and similarly to the model controlling both population structure and relative kinship (Q + K) in this panel. Our results suggest this maize panel can be used for association mapping analysis targeting multiple agronomic and quality traits with optimal association model.

Cloning and characterization of a putative GS3 ortholog involved in maize kernel development.

The GS3 gene was the first identified gene controlling the grain size in rice. It has been proven to be involved in the evolution of grain size during domestication. We isolated the maize ortholog, ZmGS3 and investigated its role in the evolution of maize grain size. ZmGS3 has five exons encoding a protein with 198 amino acids, and has domains in common with the rice GS3 protein. Compared with teosinte, maize has reduced nucleotide diversity at ZmGS3, and the reduction is comparable to that found in neutrally evolving maize genes. No positive selection was detected along the length of the gene using either the Hudson-Kreitman-Aguadé or Tajima's D tests. Phylogenetic analysis reveals a distribution of maize sequences among two different clades, with one clade including related teosinte sequences. The nucleotide polymorphism analysis, selection test and phylogenetic analysis reveal that ZmGS3 has not been subjected to selection, and appears to be a neutrally evolving gene. In maize, ZmGS3 is primarily expressed in immature ears and kernels, implying a role in maize kernel development. Association mapping analysis revealed one polymorphism in the fifth exon that is significantly associated with kernel length in two environments. Also one polymorphism in the promoter region was found to affect hundred kernel weight in both environments. Collectively, these results imply that ZmGS3 is involved in maize kernel development but with different functional polymorphisms and thus, possibly different mechanisms from that of the rice GS3 gene.