Mycobacterium tuberculosis ( Mtb ), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS) generated within host macrophages during infection. The melH gene in Mtb and Mycobacterium marinum ( Mm ) plays a crucial role in defense mechanisms against ROS generated during infection. We demonstrate that melH encodes an epoxide hydrolase and contributes to ROS detoxification. Deletion of melH in Mm resulted in a mutant with increased sensitivity to oxidative stress, increased accumulation of aldehyde species, and decreased production of mycothiol and ergothioneine. This heightened vulnerability is attributed to the increased expression of whiB3 , a universal stress sensor. The absence of melH also resulted in reduced intracellular levels of NAD + , NADH, and ATP. Bacterial growth was impaired, even in the absence of external stressors, and the impairment was carbon-source-dependent. Initial MelH substrate specificity studies demonstrate a preference for epoxides with a single aromatic substituent. Taken together, these results highlight the role of melH in mycobacterial bioenergetic metabolism and provide new insights into the complex interplay between redox homeostasis and generation of reactive aldehyde species in mycobacteria. Importance:This study unveils the pivotal role played by the melH gene in Mycobacterium tuberculosis and Mycobacterium marinum in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, para -hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of melH . Moreover, changes in crucial cofactors responsible for electron transfer highlighted melH 's crucial function in maintaining a delicate equilibrium of redox and bioenergetic processes. MelH prefers epoxide small substrates with a phenyl substituted substrate. These findings collectively emphasize the potential of melH as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis.

By illuminating key 6-azasteroid–protein interactions in both Mycobacterium tuberculosis (Mtb) and the closely related model organism Mycobacterium marinum (Mm), we sought to improve the antimycobacterial potency of 6-azasteroids and further our understanding of the mechanisms responsible for their potentiation of the antituberculosis drug bedaquiline. We selected a newly developed 6-azasteroid analog and an analog reported previously (ACS Infect. Dis. 2019, 5 (7), 1239–1251) to study their phenotypic effects on Mtb and Mm, both alone and in combination with bedaquiline. The 6-azasteroid analog, 17β-[N-(4-trifluoromethoxy-diphenylmethyl)carbamoyl]-6-propyl-azaandrostan-3-one, robustly potentiated bedaquiline-mediated antimycobacterial activity, with a nearly 8-fold reduction in Mm bedaquiline minimal inhibitory concentration (85 nM alone versus 11 nM with 20 μM 6-azasteroid). This analog displayed minimal inhibitory activity against recombinant mycobacterial 3β-hydroxysteroid dehydrogenase, a previously identified target of several 6-azasteroids. Dose-dependent potentiation of bedaquiline by this analog reduced mycobacterial intracellular ATP levels and impeded the ability of Mtb to neutralize exogenous oxidative stress in culture. We developed two 6-azasteroid photoaffinity probes to investigate azasteroid–protein interactions in Mm whole cells. Using bottom-up mass spectrometric profiling of the cross-linked proteins, we identified eight potential Mm/Mtb protein targets for 6-azasteroids. The nature of these potential targets indicates that proteins related to oxidative stress resistance play a key role in the BDQ-potentiating activity of azasteroids and highlights the potential impact of inhibition of these targets on the generation of drug sensitivity.

This study aims to extract an active heteropolysaccharide Chia seed polysaccharide (CSP-A) and further purified by DEAE Sepharose Fast Flow and Sepharose CL-6B chromatographic column, characterize its structure, and evaluate its antioxidant and immunomodulatory activities. Structural analysis revealed that CSP-A was composed of d-mannose, d-glucuronic acid and d-xylose in a molar ratio of 1:3:4 with molecular weight of 1.688 × 105 Da, owning 4 sugar residues of β-d-Manp-(1→, →4)-α-d-GlcpA-(1→, →2,4)-β-d-Xylp-(1→, and → 4)-β-d-Manp-(1 →. Congo red assay and microscopic characteristics showed that CSP-A in its solution may possess a helical conformation. In vitro experiments showed that CSP-A had moderate DPPH· and OH· scavenging activities. CSP-A also enhanced the phagocytosis ability of RAW 264.7 cells and prompted the release of NO, TNF-α, IL-6 and IL-1β from RAW 264.7 cells, which indicated CSP-A had immune regulation effect. This experiment provides scientific basis for further utilization and development of chia seeds, a kind of functional food.

目的 利用超高效液相色谱-线性离子阱-静电场轨道阱联用质谱(UPLC-LTQ-Orbitrap-MS)技术快速鉴别瓜拉那Paullinia cupana叶中的化学成分,建立高效液相色谱(HPLC)法测定瓜拉那叶中咖啡因的含量.方法 定性分析采用Waters Acquity UPLC HSS T3色谱柱(100 mm×2.1 mm,1.8 μm),流动相为0.1％甲酸水-乙腈,梯度洗脱,体积流量0.3 mL·min-1,进样量3μL,在电喷雾正负离子模式下采集数据.定量分析采用Prevail色谱柱(250 mm×4.6mm,5μm),柱温30 ℃,流动相为纯水-乙腈,梯度洗脱,体积流量为1 mL·min-1,进样量10 μL,检测波长275 nm.结果 利用UPLC-LTQ-Orbitrap-MS技术,根据质谱一级精确相对分子质量和二级特征碎片,从瓜拉那叶中共鉴定出46个化学成分,包括22个酚酸类,7个黄酮类,7个生物碱类,5个长链脂肪酸类和5个其他类化合物.建立HPLC-UV测定瓜拉那叶咖啡因含量的方法中,咖啡因在0.2018～2.0180 μg线性关系良好(R2=0.9998),平均加样回收率(n=9)为99.52％,RSD=1.16％,瓜拉那叶中咖啡因质量分数在2.8％左右.结论 首次采用UPLC-LTQ-Orbitrap-MS技术鉴定瓜拉那叶中的化学成分,提供了一种快速、高效的分析方法,所建立的HPLC-UV测定咖啡因含量的方法准确、可靠,为瓜拉那叶后续的深入研究奠定了基础.

Background Current TB diagnostic methods available have been developed for adults and development efforts have neglected the differences in disease and sampling that occur between adults and children. Diagnostic challenges are even greater in HIV co-infected children and infants. Methods and results We established a sandwich ELISA assay to detect Mycobacterium tuberculosis modified lipoprotein (TLP) ex vivo in plasma. The study population contains plasma samples from 21 patients with active TB and 24 control samples with no TB, collected in the International Maternal Pediatric Adolescent AIDS Clinical Trails (IMPAACT) P1041 study. Retrospective analysis was performed and the result demonstrate that TLP level is associated with TB disease. Conclusions Plasma levels of TLP associate with active TB disease in HIV positive subjects and can be used as an indicator for TB detection in children.

目的:建立高效阴离子交换色谱-脉冲安培检测法(HPAEC-PAD)测定枸杞粗多糖及其不同组分单糖和糖醛酸的种类及含量.方法:枸杞子经水提醇沉,Hiprep DEAE FF16/10柱分离,再由HPAEC-PAD法测定枸杞粗多糖及其不同组分中单糖及糖醛酸的组成.结果:测得各单糖及糖醛酸在2.5～50mg/L线性关系良好(r≥0.9990),最低检出限为0.010～0.028mg/L,日内精密度RSD为0.98％～2.20％,日间精密度RSD为1.38％～2.46％,加标回收率为94.60％～112.62％.枸杞粗多糖及其不同组分均由阿拉伯糖、氨基葡萄糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖、半乳糖醛酸和葡萄糖醛酸组成.结论:本研究所建立的方法简便、准确、分离效果好、灵敏度高,可用于枸杞粗多糖及其不同组分单糖及糖醛酸组成和测定的分析研究.

目的 利用UPLC-LTQ-Orbitrap-MS技术对刺果番荔枝叶的化学成分进行定性分析.方法 采用Waters Acquity UPLC HSS T3色谱柱(2.1 mm×100 mm,1.8μm),流动相为0.1％甲酸水(A)和乙腈(B)梯度洗脱,流速为0.3 mL/min,进样量2μL,在电喷雾正负离子模式下采集数据.经Reaxys数据库检索番荔枝属类化合物信息,通过质谱信息比对各化合物的m/z值、保留时间、质谱特征碎片等,并结合文献数据对鉴定的化合物进行验证.结果 根据各化合物的特征裂解规律,从刺果番荔枝叶中共鉴定出45个化合物,包括16个生物碱类,14个番荔枝内酯类,7个黄酮类和8个其他类化合物,其中以番荔枝内酯类和生物碱类成分居多,与文献报道番荔枝内酯与生物碱类化合物是发挥抗癌的主要活性成分一致.结论 利用UPLC-LTQ-Orbitrap-MS技术对刺果番荔枝叶中的化学成分进行了快速、准确的定性分析,为刺果番荔枝叶的提取分离与药效物质基础的研究提供依据.

目的 利用UPLC-LTQ-Orbitrap-MS技术对4种秦艽药材的化学成分进行定性分析.方法 采用Waters Acquity UPLC HSS T3色谱柱(2.1 mm×50 mm,1.7 μm),在电喷雾正负离子模式下采集数据.将母离子列表(PIL)-MS2和高分辨质谱结合,用于秦艽药材中化学成分的鉴别.结果 结合质谱数据和文献数据共鉴定、推断出66个成分,其中包括环烯醚萜类36个、木脂素类5个、黄酮类5个、三萜类12个、其他类8个.其中19个成分为首次报道.结论 4种秦艽成分大致相同,利用UPLC-LTQ-Orbitrap-MS技术结合母离子列表的方法可以快速筛选《中国药典》规定的4种秦艽中的化学成分,为秦艽化学成分的提取分离及其药效物质基础研究提供参考.

Steroid-degrading bacteria, including Mycobacterium tuberculosis (Mtb), utilize an architecturally distinct subfamily of acyl coenzyme A dehydrogenases (ACADs) for steroid catabolism. These ACADs are α2β2 heterotetramers that are usually encoded by adjacent fadE-like genes. In mycobacteria, ipdE1 and ipdE2 (formerly fadE30 and fadE33) occur in divergently transcribed operons associated with the catabolism of 3aα-H-4α(3′-propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP), a steroid metabolite. In Mycobacterium smegmatis, ΔipdE1 and ΔipdE2 mutants had similar phenotypes, showing impaired growth on cholesterol and accumulating 5-OH HIP in the culture supernatant. Bioinformatic analyses revealed that IpdE1 and IpdE2 share many of the features of the α- and β-subunits, respectively, of heterotetrameric ACADs that are encoded by adjacent genes in many steroid-degrading proteobacteria. When coproduced in a rhodococcal strain, IpdE1 and IpdE2 of Mtb formed a complex that catalyzed the dehydrogenation of 5OH-HIP coenzyme A (5OH-HIP-CoA) to 5OH-3aα-H-4α(3′-prop-1-enoate)-7aβ-methylhexa-hydro-1,5-indanedione coenzyme A ((E)-5OH-HIPE-CoA). This corresponds to the initial step in the pathway that leads to degradation of steroid C and D rings via β-oxidation. Small-angle X-ray scattering revealed that the IpdE1-IpdE2 complex was an α2β2 heterotetramer typical of other ACADs involved in steroid catabolism. These results provide insight into an important class of steroid catabolic enzymes and a potential virulence determinant in Mtb.

目的采用超高效液相色谱-四级杆-飞行时间质谱（UPLC-Q-TOF/HRMS E ）技术对活血解毒方中的化学成分进行研究。方法采用ACQUITY UPLC BEH C 18 色谱柱（100 mm×2.1 mm,1.8μm）,以0.1%甲酸溶液（A）和0.1%甲酸乙腈（B）作为流动相进行梯度洗脱,体积流量为0.3 mL/min,采用ESI源在正、负离子模式下采集数据。根据化合物精确相对分子质量及二级特征碎片离子信息,结合参考文献数据与UNIFI数据库,鉴定活血解毒方的主要化学成分。结果从活血解毒方中鉴定出66个化学成分,其中黄酮类化合物8个、生物碱类化合物7个、酚类化合物6个、皂苷类化合物30个、木质素类化合物6个、萜类化合物4个和其他类化合物5个。结论建立的UPLC-Q-TOF/HRMS E 联用技术与UNIFI结合法能够系统、快速、准确地鉴定多种化学成分,为其质量评价指标选择及药效物质基础深入研究提供了参考。

This study is to establish a qualitative method for rapid identification of bile acids in Suis Fellis Pulvis based on UHPLC-LTQ-Orbitrap-MS technology,and an HPLC-ELSD internal standard method for the quantitative determination of two glycine-conjugated BAs in Suis Fellis Pulvis.The chromatographic separation of the UHPLC-LTQ-Orbitrap-MS qualitative analysis was achieved on a Waters Acquity UPLC HSS T_3column(2.1 mm×100 mm,1.8μm),with 0.2%formic acid aqueous solution(A)-acetonitrile(B)as mobile phase ingradient elution.Electrospray ionization(ESI)source was applied and operated in negative ion mode.Quantitative analysis was performed at 30℃on a Diamonsil-C_(18)column(4.6 mm×250 mm,5μm).The mobile phase consisted of 0.2%formic acid solution and acetonitrile with gradient elution and the flow rate was 1.0 m L·min~(-1).An ELSD was used with a nitrogen flow-rate of1.4 L·min~(-1)at a drift tube temperature of 60℃and the gain was 1.A total of 14 bile acids in Suis Fellis Pulvis were characterized based on the accurate mass measurements,fragmentation patterns,chromatographic retention times,and reference materials.For the quantitative analysis method,the glycohyodeoxycholic acid and glycochenodeoxycholic acid had good linear relationship in the range of26.52-265.20 mg·L~(-1)(r=0.999 8)and 19.84-198.40 mg·L~(-1)(r=0.999 1),respectively.The average recoveries(n=6)were104.1%and 103.1%,and the RSD were 2.0%and 2.4%.The UHPLC-LTQ-Orbitrap-MS technology provides a fast and efficient qualitative analysis method for identification of bile acids in Suis Fellis Pulvis.The HPLC-ELSD internal standard method is accurate and reliable,which has reference value for the quality control of Suis Fellis Pulvis.

雷公藤是一味传统中药,所含化学成分丰富,具有多方面的药理活性.在刘昌孝院士提出中药质量标志物(Q-marker)新概念的基础上,系统综述了雷公藤化学成分及药理活性,并根据质量标志物的定义结合雷公藤的传统药效、传统药性、临床用药新用途、可测成分、入血成分和配伍研究进行分析,对雷公藤质量标志物进行预测,以期为雷公藤药材质量的深入研究提供参考.

盆腔炎性疾病(PID)为妇科常见病和多发病,属炎性反应性感染性疾病,以腹痛或腰痛为临床特征,有些患者热象明显,可伴有发热、带下黄臭,尿赤便秘、甚则咽痛身痛、舌红苔黄腻等湿热内蕴征象.清利三焦治法是针对湿热邪气困遏三焦证候的治法,具有清热、利湿,宣畅气机的功效,广泛应用于外感热病及各科内伤杂病的治疗.借鉴清利三焦湿热治疗思路,应用温病学名方黄芩滑石汤治疗PID,取得良好效果,故本文对清利三焦湿热治疗PID的理论依据及治疗机制进行探讨,以供临床同道参考.

One-third of the world’s population carries Mycobacterium tuberculosis (Mtb), the infectious agent that causes tuberculosis (TB), and every 17 seconds someone dies of TB. After infection, Mtbcan live dormant within macrophages for decades in a granuloma structure arising from the host immune response; and cholesterol is important for this persistence of Mtb. Current treatments require long-duration drug regimens with many associated toxicities, which are compounded by the high doses required. We phenotypically screened 35 6-azasteroid analogues against Mtband found that at low micromolar concentrations, a subset of the analogues sensitized Mtbto multiple TB drugs. Two analogues were selected for further study to characterize the bactericidal activity of bedaquiline and isoniazid under normoxic and low-oxygen conditions. These two 6-azasteroids showed strong synergy with bedaquiline(fractional inhibitory concentration index = 0.21, bedaquiline minimal inhibitory concentration = 16 nM at 1 μM 6-azasteroid). The rate at which spontaneous resistance to one of the 6-azasteroids arose in the presence of bedaquiline was approximately 10−9, and the 6-azasteroid-resistant mutants retained their isoniazid and bedaquiline sensitivity. Genes in the cholesterol-regulated Mce3R regulon were required for 6-azasteroid activity, whereas genes in the cholesterol catabolism pathway were not. Expression of a subset of Mce3R genes was down-regulated upon 6-azasteroid treatment. The Mce3R regulon is implicated in stress resistance and is absent in saprophytic mycobacteria. This regulon encodes a cholesterol-regulated stress-resistance pathway that we conclude is important for pathogenesis and contributes to drug tolerance, and that this pathway is vulnerable to small-molecule targeting in live mycobacteria.

Objective:To sort out the prescription medication rules of lumbar pain in Unprinted Medical Cases of Ye Tian-shi based on the platform of TCM inheritance assistant platform.Methods:Through 19 cases of lumbar pain in Unprinted Medical Cases of Ye Tian-shi, the prescription medication rules of lumbar pain and its accompanying symptoms were analyzed.Results:Among the 19 prescriptions included in the study, 59 Chinese materia medica were involved and 8 were used more than three times, they were as follows successively:Shudihuang (Radix Rehmanniae Praeparata), Fuling (Poria), Duzhong (Cortex Eucommiae), Tusizi (Semen Cuscutae), Fushen (Sclerotium Poriae Pararadicis), Bajitian (Radix Morindae Officinalis), Shayuanzi (Semen Astragali Complanati), Gouqizi (Fructus Lycii).Through the analysis of the prescription medication rules, we could get the drug pairs as "Tusizi-Duzhong", "Bajitian-Duzhong", "Shayuanzi-Duzhong".Fuling was the most frequently used medicine for the treatment of lumbar pain and its accompanying symptoms.Among the drugs used twice, kidney-tonifying and yang-warming drugs were the most frequently used.Conclusion:In treating lumbar pain, Unprinted Medical Cases of Ye Tian-shi uses nourishing liver and kidney as its general principle, and pays attention to the role of spleen and stomach.Its drug use not only considers the main therapeutic effect of drugs, but also pays attention to the meridian tropism and peculiar meridian function of drugs.Drugs for using in govern vessel are especially emphasized.

Cholesterol is important for Mycobacterium tuberculosis (Mtb) survival and persistence in the host. We describe a series of 6-azasteroids tested as cholesterol analogs, that are synergistic inhibitors of Mtb viability. The analogs improve Mtb killing efficacy by isoniaizid and bedaquiline, two approved drugs for the treatment of drug-sensitive and drug-resistant Mtb, respectively. In addition, the 6-azasteroids work with existing TB drugs under anaerobic conditions and lower their MICs by greater than 10-fold, suggesting they will work in hypoxic conditions found in infected lungs. Our work provides strong evidence for an expanded repertoire of cholesterol utilization in Mtb infection that extends beyond the commonly accepted role of cholesterol in nutrition and primary energy generation. We discovered that a stress-resistance regulon (Mce3R) is required for the synergistic activity of 6-azasteroids. Moreover, we demonstrate that cholesterol catabolism is not required for the activity of these inhibitors. Earlier Cirillo, Jacobs and coworkers, established that the mel2 operon residing in the Mce3R regulon is important for persistence and dissemination of Mtb infection in mice (DOI: 10.1128/IAI.01481-08). Thus, an important role of cholesterol utilization for survival of Mtb infection is stress resistance.Importantly, we demonstrate that this stress-resistance pathway is vulnerable to small molecule targeting.

目的 对药典规定的不同产地、不同生长环境的4种秦艽的有效成分进行含量测定和特征图谱研究,以评价不同产地、不同生长环境药典规定秦艽的质量.方法 采用HPLC外标法对30批秦艽样品中4种有效成分进行测定,利用《中药色谱指纹图谱相似度评价系统》(2012版)对30批药材进行特征图谱建立及特征图谱的质谱成分指认.结果 30批药材中龙胆苦苷和马钱苷酸含量均符合2015年版《中国药典》的规定,其中栽培品平均含量高于野生品,云南栽培秦艽平均有效成分含量较高;30批秦艽药材相似度在0.957～0.998,有良好的相似度,形成的特种图谱具有10个共有峰.而后由质谱分析指认14个成分,并对部分成分进行了归属.结论 本方法能确切地评价秦艽的质量.

目的:通过计算机医案数据分析,探索孔光一教授治疗2型糖尿病用药特点,整理总结名老中医诊疗经验.方法:收集整理孔光一教授治疗2型糖尿病共41份门诊病历,录入中医传承辅助平台(V2.5)分析其用药特点.结果:孔光一教授临床治疗2型糖尿病共涉及94味中药,频次≥21次的药物有16味,关键药物前6位分别为赤芍、丹参、麦冬、黄柏、黄芩、炒白术;常用药对15对,其中置信度最高的是“麦冬,赤芍”,常用赤芍、丹参、麦冬、黄柏、黄芩、炒白术作为基本处方加减.结论:孔光一教授在临床治疗2型糖尿病时,体现以三焦膜系学说为指导,清热通络,气血并调的治疗特色.

This paper discusses the pathogenesis and treatment of childhood psoriasis,based on syndrome differentiation of weifen,qifen,yingfen and xuefen of warm disease,syndrome differentiation of macula,and hidden pathogen theory.Connected with aetiological agent,pathogenesis and paediatric physiology,the pathogenic factors,classification,clinical features and attack of childhood psoriasis are studied and analyzed.A case report about childhood psoriasis is listed,to investigate its characteristics of warm disease theory.