Alkaloids of Nelumbo nucifera

The alkaloids of leaves of Nelumbo nucifera Gaertn. were examined using combined GLC-MS. The occurrence of four new alkaloids, dehydroroemerine (XII), dehydronuciferine (XI), dehydroanonaine (XIII) and N-methylisococlaurine (III) were revealed, besides the known roemerine (V), nuciferine (VI), anonaine (VII), pronuciferine (IV), N-nornuciferine (VIII), nornuciferine (IX), amepavine (I) and N-methylcoclaurine (II).     

MRI Plaque Imaging Detects Carotid Plaques with a High Risk for Future Cerebrovascular Events in Asymptomatic Patients

Purpose

The aim of this study was to investigate prospectively whether MRI plaque imaging can identify patients with asymptomatic carotid artery stenosis who have an increased risk for future cerebral events. MRI plaque imaging allows categorization of carotid stenosis into different lesion types (I–VIII). Within these lesion types, lesion types IV–V and VI are regarded as rupture-prone plaques, whereas the other lesion types represent stable ones.

Methods

Eighty-three consecutive patients (45 male (54.2%); age 54–88 years (mean 73.2 years)) presenting with an asymptomatic carotid stenosis of 50–99% according to ECST-criteria were recruited. Patients were imaged with a 1.5-T scanner. T1-, T2-, time-of-flight-, and proton-density weighted studies were performed. The carotid plaques were classified as lesion type I–VIII. Clinical endpoints were ischemic stroke, TIA or amaurosis fugax. Survival analysis and log rank test were used to ascertain statistical significance.

Results

Six out of 83 patients (7.2%) were excluded: 4 patients had insufficient MR image quality; 1 patient was lost-to-follow-up; 1 patient died shortly after the baseline MRI plaque imaging. The following results were obtained by analyzing the remaining 77 patients. The mean time of follow-up was 41.1 months.During follow-up, n = 9 (11.7%) ipsilateral ischemic cerebrovascular events occurred. Only patients presenting with the high-risk lesion types IV–V and VI developed an ipsilateral cerebrovascular event versus none of the patients presenting with the stable lesion types III, VII, and VIII (n = 9 (11.7%) vs. n = 0 (0%) during follow-up). Event-free survival was higher among patients with the MRI-defined stable lesion types (III, VII, and VIII) than in patients with the high-risk lesion types (IV–V and VI) (log rank test P<0.0001).

Conclusions

MRI plaque imaging has the potential to identify patients with asymptomatic carotid stenosis who are particularly at risk of developing future cerebral ischemia. MRI could improve selection criteria for invasive therapy in the future.     

A Chemical Genetics Analysis of the Roles of Bypass Polymerase DinB and DNA Repair Protein AlkB in Processing N 2-Alkylguanine Lesions In Vivo

DinB, the E. coli translesion synthesis polymerase, has been shown to bypass several N ²-alkylguanine adducts in vitro, including N ²-furfurylguanine, the structural analog of the DNA adduct formed by the antibacterial agent nitrofurazone. Recently, it was demonstrated that the Fe(II)- and α-ketoglutarate-dependent dioxygenase AlkB, a DNA repair enzyme, can dealkylate in vitro a series of N ²-alkyguanines, including N ²-furfurylguanine. The present study explored, head to head, the in vivo relative contributions of these two DNA maintenance pathways (replicative bypass vs. repair) as they processed a series of structurally varied, biologically relevant N ²-alkylguanine lesions: N ²-furfurylguanine (FF), 2-tetrahydrofuran-2-yl-methylguanine (HF), 2-methylguanine, and 2-ethylguanine. Each lesion was chemically synthesized and incorporated site-specifically into an M13 bacteriophage genome, which was then replicated in E. coli cells deficient or proficient for DinB and AlkB (4 strains in total). Biochemical tools were employed to analyze the relative replication efficiencies of the phage (a measure of the bypass efficiency of each lesion) and the base composition at the lesion site after replication (a measure of the mutagenesis profile of each lesion). The main findings were: 1) Among the lesions studied, the bulky FF and HF lesions proved to be strong replication blocks when introduced site-specifically on a single-stranded vector in DinB deficient cells. This toxic effect disappeared in the strains expressing physiological levels of DinB. 2) AlkB is known to repair N ²-alkylguanine lesions in vitro; however, the presence of AlkB showed no relief from the replication blocks induced by FF and HF in vivo. 3) The mutagenic properties of the entire series of N ²-alkyguanines adducts were investigated in vivo for the first time. None of the adducts were mutagenic under the conditions evaluated, regardless of the DinB or AlkB cellular status. Taken together, the data indicated that the cellular pathway to combat bulky N ²-alkylguanine DNA adducts was DinB-dependent lesion bypass.     

Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine,guanine lesions induced by nitric oxide and nitrous acid

Nitrosation of guanine in DNA by nitrogen oxides such as nitric oxide (NO) and nitrous acid leads to formation of xanthine (Xan) and oxanine (Oxa), potentially cytotoxic and mutagenic lesions. In the present study, we have examined the repair capacity of DNA N-glycosylases from Escherichia coli for Xan and Oxa. The nicking assay with the defined substrates containing Xan and Oxa revealed that AlkA [in combination with endonuclease (Endo) IV] and Endo VIII recognized Xan in the tested enzymes. The activity (V_max/K_m) of AlkA for Xan was 5-fold lower than that for 7-methylguanine, and that of Endo VIII was 50-fold lower than that for thymine glycol. The activity of AlkA and Endo VIII for Xan was further substantiated by the release of [³H]Xan from the substrate. The treatment of E.coli with N-methyl-N′-nitro-N-nitrosoguanidine increased the Xan-excising activity in the cell extract from alkA⁺ but not alkA^– strains. The alkA and nei (the Endo VIII gene) double mutant, but not the single mutants, exhibited increased sensitivity to nitrous acid relative to the wild type strain. AlkA and Endo VIII also exhibited excision activity for Oxa, but the activity was much lower than that for Xan.     

Permanent translocation heterozygosity in Viscum hildebrandtii Engl. and V. engleri Tiegh. (viscaceae) in East Africa

Translocation heterozygosity is characteristic of male plants of Viscum hildebrandtii Engl. (n=14) and V. engleri Tiegh. (n=14). Both species are polymorphic with regard to the known chromosome associations which include: 12II, 1IV; 11II, 1VI; 10II, 2IV; 10II, 1VIII; 9II, 1IV, 1VI and 6II, 1IV, 2VI. All multivalents are rings. The translocation heterozygosity is not associated with a distortion of the sex ratio in favour of female plants, as occurs in V. fischeri Engl., but probably with the differentiation and adaptation of biotypes.     

Phylogenomic Analysis Reveals Deep Divergence and Recombination in an Economically Important Grapevine Virus

The evolutionary history of the exclusively grapevine (Vitis spp.) infecting, grapevine leafroll-associated virus 3 (GLRaV-3) has not been studied extensively, partly due to limited available sequence data. In this study we trace the evolutionary history of GLRaV-3, focussing on isolate GH24, a newly discovered variant. GH24 was discovered through the use of next-generation sequencing (NGS) and the whole genome sequence determined and validated with Sanger sequencing. We assembled an alignment of all 13 available whole genomes of GLRaV-3 isolates and all other publicly available GLRaV-3 sequence data. Using multiple recombination detection methods we identified a clear signal for recombination in one whole genome sequence and further evidence for recombination in two more, including GH24. We inferred phylogenetic trees and networks and estimated the ages of common ancestors of GLRaV-3 clades by means of relaxed clock models calibrated with asynchronous sampling dates. Our results generally confirm previously identified variant groups as well as two new groups (VII and VIII). Higher order groups were defined as supergroups designated A to D. Supergroup A includes variant groups I-V and supergroup B group VI and its related unclassified isolates. Supergroups C and D are less well known, including the newly identified groups VII (including isolate GH24) and VIII respectively. The inferred node ages suggest that the origins of the major groups of GLRaV-3, including isolate GH24, may have occurred prior to worldwide cultivation of grapevines, whilst the current diversity represents closely related isolates that diverged from common ancestors within the last century.     

Conformational dynamics of the interaction of Escherichia coli endonuclease VIII with DNA substrates

Endonuclease VIII (Nei) from Escherichia coli is a DNA repair enzyme that removes a wide range of oxidized pyrimidine bases from DNA. As inferred from the crystal structures and biochemical studies, recognition of DNA lesions by Nei involves several conformational changes in both protein and DNA, such as DNA kinking, damaged base eversion into the enzyme's active site, and insertion of a loop of the enzyme into the void formed by the eversion. Excision of the damaged base by Nei also proceeds through several chemical steps: N-glycosidic bond breakage, β-elimination and δ-elimination of the phosphates flanking the lesion. We have used stopped-flow kinetics with fluorescence detection to follow conformational changes in the Nei molecule when the enzyme binds normal DNA, damaged but uncleavable DNA, or several cleavable damaged DNA substrates. Binding normal or damaged uncleavable DNA proceeded in two fluorescently discernible reversible stages, while processing of cleavable substrates involved three reversible stages followed by and irreversible stage and equilibrium with the reaction product. Individual rate constants were calculated for each reaction step. Based on the stopped-flow data, crystal structure, and a comparison with the stopped-flow kinetics of E. coli formamidopyrimidine-DNA glycosylase, a homolog of Nei, we propose the nature of some of the steps that may be involved into the recognition and excision of damaged bases by Nei.     

Pig carcass tail lesions: the influence of record keeping through an advisory service and the relationship with farm performance parameters

Tail lesions are important pig welfare indicators that could be recorded during meat inspection as they are more visible on the carcass than on the live animal. Tail biting is associated with reduced performance in the bitten pig, but it is not clear whether problems with tail biting are reflected in general farm performance figures. Farm advisory services aim to improve farm productivity which could be associated with improvements in pig welfare. Record keeping forms an integral part of such advisory services. The aim of this study was to examine the influence of record keeping in the Teagasc eProfit Monitor (ePM herds) on the prevalence of tail lesion severity scores in Irish slaughter pigs. In addition, we investigated associations between the prevalence of tail lesion scores and production parameters at farm level in ePM herds. Pigs were observed after scalding/dehairing and tail lesion score (0 to 4), sex and farm identification were recorded. Tail lesion scores were collapsed into none/mild lesions (score ⩽1), moderate lesions (score 2) and severe lesions (score ⩾3). The effect of record keeping (ePM herd) on the different tail lesion outcomes was analysed at batch level using the events/trials structure in generalized linear mixed models (PROC GLIMMIX). Spearman’s rank correlations were calculated between average tail lesion score of a batch and production parameters. A total of 13 133 pigs were assessed from 73 batches coming from 61 farms. In all, 23 farms were identified as ePM herds. The average prevalence of moderate tail lesions was 26.8% and of severe tail lesions was 3.4% in a batch. Batches coming from ePM herds had a lower prevalence of moderate tail lesions than non-ePM herds (P<0.001). Average tail lesion score was negatively associated with age (P<0.05) and weight (P<0.05) at sale/transfer of weaners, and tended to be positively associated with the number of finishing days (P=0.06). In addition, the prevalence of severe tail lesions was negatively associated with average daily gain in weaners (P<0.05) and tended to do so with average daily gain in finishers (P=0.08). This study provides the first indication that record keeping through an advisory service may help to lower the risk of tail biting, which is associated with improved farm performance.     

Determination of the Pressure Drop Across an Arterial Stenosis Utilizing Angiocinedensitometry

A method is presented for an evaluation of the hemodynamic significance of a stenotic lesion in the arterial tree.Twenty-three patients were examined with arteriosclerosis obliterans and intermittent claudication of the same severity. Flow velocity data obtained by angiodensitometry and viscosity values calculated from the hematocrit were inserted into the Poiseuille''s flow formula to obtain the pressure drop across a stenotic lesion in the left external iliac artery. By the same way, the pressure gradient was calculated in 33 “normal” subjects.The normal pressure gradient along the external iliac artery varied between 23 to 110 dynes/cm² (52 ± 24 dynes/cm² for mean and S.d), and the normal resistance to flow was 6.08 ± 4.1 dyne sec/cm⁵).Stenotic lesions of similar dimensions gave widely varying pressure drops (114-4,736 dynes/cm²) (mean and S.d 1,309 ± 1,224 dynes/cm²) indicating a difference in the hemodynamic significance of the various lesions. These values were significantly different (p(t) < 0.001) from the normal values. The resistance across these stenotic lesions ranged between 21 to 768 dyne sec/cm⁵ (196 ± 192 dyne sec/cm⁵) for the mean and S.d and this was significantly different from the normal group; p(t) < 0.001.Direct measurement of blood viscosity coupled with angiocinedensitometry at rest and after forced vasodilatation should provide an accurate means of determining the relative significance of a stenotic lesion and distal vessel disease in a given patient on blood flow to the leg.     

Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia

Epigenetic modifications, such as aberrant DNA promoter methylation, are frequently observed in cervical cancer. Identification of hypermethylated regions allowing discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3), or worse, may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions was studied using genome-wide DNA methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methylated DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium. Hypermethylated differentially methylated regions (DMRs) were identified. Validation of nine selected DMRs using BSP and MSP in cervical tissue revealed methylation in 63.2–94.7% high-grade CIN and in 59.3–100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was conducted exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples. Clinical validation of both markers in cervical scrapings from patients with an abnormal cervical smear confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion and that ROC analysis was discriminative. These markers represent the COL25A1 and KATNAL2 and their observed increased methylation upon progression could intimate the regulatory role in carcinogenesis. In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and are candidate biomarkers for early detection.     

Effects of scalding and dehairing of pig carcasses at abattoirs on the visibility of welfare-related lesions

There is increasing interest in developing abattoir-based measures to assist in determining the welfare status of pigs. The primary aim of this study was to determine the most appropriate place on the slaughter line to conduct assessments of welfare-related lesions, namely apparent aggression-related skin lesions (hereafter referred to as ‘skin lesions’), loin bruising and apparent tail biting damage. The study also lent itself to an assessment of the prevalence of these lesions, and the extent to which they were linked with production variables. Finishing pigs processed at two abattoirs on the Island of Ireland (n=1950 in abattoir A, and n=1939 in abattoir B) were used. Data were collected over 6 days in each abattoir in July 2014. Lesion scoring took place at two points on the slaughter line: (1) at exsanguination (slaughter stage 1 (SS1)), and (2) following scalding and dehairing of carcasses (slaughter stage 2 (SS2)). At both points, each carcass was assigned a skin and tail lesion score ranging from 0 (lesion absent) to 3 or 4 (severe lesions), respectively. Loin bruising was recorded as present or absent. Differences in the percentage of pigs with observable lesions of each type were compared between SS1 and SS2 using McNemar/McNemar-Bowker tests. The associations between each lesion type, and both cold carcass weight and condemnations, were examined at batch level using Pearson’s correlations. Batch was defined as the group of animals with a particular farm identification code on a given day. The overall percentage of pigs with a visible skin lesion (i.e. score>0) decreased between SS1 and SS2 (P<0.001). However, the percentage of pigs with a severe skin lesion increased numerically from SS1 to SS2. The percentage of pigs with a visible tail lesion and with loin bruising also increased between SS1 and SS2 (P<0.001). There was a positive correlation between the percentage of carcasses that were partially condemned, and the percentage of pigs with skin lesions, tail lesions and loin bruising (P<0.05). In addition, as the batch-level frequency of each lesion type increased, average cold carcass weight decreased (P<0.001). These findings suggest that severe skin lesions, tail lesions and loin bruising are more visible on pig carcasses after they have been scalded and dehaired, and that this is when abattoir-based lesion scoring should take place. The high prevalence of all three lesion types, and the links with economically important production parameters, suggests that more research into identifying key risk factors is warranted.     

Early lesions induced in rat colon epithelium by N-methyl-N′-nitro-N-nitrosoguanidine

The development of ACF (aberrant crypt foci), adenoma and cancer following intrarectal administration of the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) has been described. However, microscopic lesions not previously reported were observed as soon as two weeks following carcinogen treatment. These lesions protrude slightly over the epithelial lining of the colon, with a micropolyp-like appearance. Oriented sections show that the centre of these lesions present pseudo-“cystic” appearance, with disorganized crypts made of normal cells. The chorion of the lesion is invaded by numerous inflammatory cells and some ACF may be present nearby. The epithelium lining the cysts and the distorted crypts shows expression of gastric mucin M1/MUC5AC, an early marker of colonic carcinogenesis which is not present in normal colon. This mucin is retained within the “cysts” together with some inflammatory cells.The micropolyps observed contain in a minute form some histological elements described in ulcerative colitis or short-term radiotherapy (distortion of crypts, crypt abscesses, increase of chorion cellularity, infiltration by immune cells). In addition, the presence of bifid crypts nearby suggests mucosal regeneration.Our hypothesis is that these modifications are steps in a normal healing pathway that may in some cases degenerate into precancerous lesions and cancer.     

吞咽困难对食管早期肿瘤及癌前病变患者食管动力、心理状态和临床相关症状的影响

摘要 目的：探讨吞咽困难对食管早期肿瘤及癌前病变患者食管动力、心理状态和临床相关症状的影响。方法：纳入2020年3月-2021年9月在南京医科大学第一附属医院消化内科住院确诊为食管早期肿瘤及癌前病变拟行内镜治疗的60例患者，根据有无合并吞咽困难将患者分为吞咽困难组和无吞咽困难组，分别比较两组患者在临床特征、高分辨率食管测压（HRM）参数以及焦虑自评量表（SAS）、抑郁自评量表（SDS）、胃食管反流病自评量表（GERDQ）评分的差异，分析HRM异常参数与原发病灶参数的相关性。结果：吞咽困难组患者的食管下括约肌（LES）中心点位置低于无吞咽困难组患者，LES静息压、LES残余压、无效吞咽百分比显著高于无吞咽困难组患者（P<0.05），HRM异常参数与原发病灶参数之间未见明显相关性（P>0.05），吞咽困难组患者的SAS、SDS评分高于无吞咽困难组（P<0.05），而两组患者GERDQ评分比较差异无统计学意义（P>0.05）。结论：食管早期肿瘤及癌前病变患者出现的吞咽困难症状与合并的异常食管动力有关，同时在一定程度上影响了患者的心理状态，对患者的临床相关症状无明显影响。     

Detection and first characterization of an uncommon haptoglobin in porcine saliva of pigs with rectal prolapse by using boronic acid sample enrichment

Salivary glycoprotein profiles, obtained after boronic acid enrichment, were studied for the first time in pigs in order to search for specific overall alterations related to acute inflammatory condition. Five healthy pigs and five pigs suffering from rectal prolapse were used, and the levels of acute phase proteins were measured to determine the degree of inflammation of the animals. The enriched glycoprotein profiles, achieved by two-dimensional gel electrophoresis (2DE) were statistically evaluated and spots that appeared differentially regulated between states were subjected to MS analysis for protein identification. Spots from three unique proteins were identified: carbonic anhydrase VI (CA VI), α-1-antichymotrypsin and haptoglobin (Hp). CA VI appeared as two adjacent horizontal spot trains in the glycoprotein profile of healthy animals in its regular isoelectric points (pI). One spot of α-1-antichymotrypsin was found in saliva from pigs with rectal prolapse in an unusual basic pI, and was considered as a breakdown product. Hp was identified as several spot trains in saliva from pigs with rectal prolapse in an unusual alkaline pI and was consequently further investigated. SDS-PAGE and 2DE of paired serum and saliva samples combined with Western blot analysis showed that the unusual Hp position observed in saliva samples was absent in serum. Furthermore, N-glycans from serum and saliva Hp glycopatterns were evaluated from SDS-PAGE Hp bands and showed that the serum N-glycan distribution in Hp β-chain was comparable in quantity and quality in both groups of animals. In saliva, no Hp β-chain derived N-glycans could unambiguously be identified from this sample set, thus needing further detailed investigations in the future.     

A comparison of the carcinogen-DNA adducts formed in rat liver in vivo after administration of single or multiple doses of N-methyl-4-aminoazobenzene

N-Methyl-4-aminoazobenzene (MAB) is believed to be metabolized in the liver to an electrophilic N-sulfonyloxy ester which binds covalently to cellular macromolecules, resulting in the induction of hepatic neoplasia. Previous in vivo studies in the rat detected only two hepatic MAB-DNA adducts, 3-(deoxyguanosin-N²-yl)-MAB(N²-dG) and N-(deoxyguanosin-8-yl)-MAB(C8-dG), which respectively accounted for 25% and 70% of the total MAB bound to DNA at 8 h after a single dose of the carcinogen. Subsequently, the C8-dG adduct was shown to be rapidly lost from the DNA while the N²-dG adduct was a persistent lesion. Since a single dose of MAB is not sufficient for complete carcinogenic activity, we sought to identify the MAB-DNA adducts present in rat liver after multiple oral doses of [³H]MAB. The MAB was administered by intubation at a level of 0.2 mmol/kg for 1, 3 or 4 doses and animals were sacrificed at 8 h after the last dose. Hepatic DNA was isolated by extraction and hydroxylapatite chromatography and was enzymatically hydrolyzed to MAB-mononucleoside adducts, which were quantitated by high pressure liquid chromatography (HPLC). After 3 doses, N²-dG, C8-dG, and an unknown adduct were detected. By 4 doses, these accounted for 51%, 25% and 23% of the total adducts. This data is consistent with rapid removal of the C8-dG derivative and the relative persistence of the N²-dG and the unknown adduct. The latter was shown to exhibit chromatographic and pH-dependent solvent partitioning properties that were identical to a product also present in DNA treated with the synthetic ultimate carcinogen, N-benzoyloxy-MAB. Analysis of this adduct by field desorption mass spectrometry (M⁺ = 460) and, after perdeuteromethylation, by electron impact mass spectrometry (M⁺ = 528; M-N(CH₃)(CD₃) = 481) indicated the structure to be a deoxyadenosin-N⁶-yl derivative substituted through an aromatic ring of MAB. Further analysis by 270 MHz ¹H-NMR spectroscopy allowed complete assignment of the MAB and adenyl resonances and was uniquely consistent with a 3-(deoxyadenosin-N⁶-yl)-MAB structure. Since this persistent adduct is potentially mutagenic due to possible tautomeric equilibria between the N⁶-amino and N⁶-imino structures, it may represent an initiating lesion in MAB hepatocarcinogenesis.     

Isolation and Characterization of a Virus Inhibitor from Spinach (Spinacia oleracea L.)

A virus inhibiting protein (VI) was isolated from spinach (Spinacia oleracea L.). The VI inhibited infections of test plants with plus- and minus-strand RNA viruses. Inoculation of both local lesion and systemic hosts with TMV in the presence of varying amounts of the VI resulted in typical dose response curves for the number of local lesions or the amount of virus respectively. The lowest concentration of VI leading to a significant reduction in the number of local lesions was 0.06 μg/ml. The VI was found to inhibit local lesion formation only when applied within 2–3 h p.i. but still reduced the number of local lesions when applied up to 9 h prior to virus inoculation. The antiviral activity could be attributed to a protein of molecular weight 29,000 dalton with an isoelectric point of 10.3. Its activity was destroyed by heating for 30 min to 70°C. These characteristics resemble those of other virus inhibiting proteins described for members of the order Caryophyllales such as the Phytolacca inhibitor against which a serological relationship was obtained.     

Differential Expression Patterns of N-Acetylglucosaminyl Transferases and Polylactosamines in Uterine Lesions

Polylactosamine (polyLacNAc) is a fundamental structure in glycoconjugates and it is expressed in specific cells/tissues associated with the development and carcinogenesis. β1,3-N-acetylglucosaminyl transferases ((β3GnTs) play an important role in polyLacNAc synthesis, however the roles of these glycosyltransferases and their products in cancer progression are still unclear. In this sense, this work aimed to evaluate differential expression pattern of the N-acetylglucosaminyl transferases and polylactosamines in invasive and premalignant lesions of the uterus cervix. The expression of β3GnT2 and β3GnT3 were evaluated in normal (n=10) and uterine cervix lesions (n=120), both malignant [squamous carcinoma (SC)] and premalignant [cervical intraepithelial neoplasia (CIN), grades 1, 2 and 3] using immunohistochemistry. Besides, lectin histochemistry with Phytolacca americana lectin (PWM) and Wheat germ agglutinin (WGA) was also carried out to observe the presence of polyLacNAc chains and N-acetylglucosamine (GlcNAc), respectively. The β3GnT3 was expressed in almost all samples (99%) and β3GnT2 was higher expressed in disease samples mainly in CIN 3, when compared with normal (P=0.002), CIN 1 (P=0.009) and CIN 2 (P=0.03). The expression of polyLacNAc was higher is SC samples, when compared with normal (P=0.03), CIN 1 (P=0.02) and CIN 3 (P=0.004), and was observed only nuclear expression in nearly 50% of the SC samples, showing a statistically significant when compared with normal (P=0.01), CIN 1 (P=0.002), CIN 2 (P=0.007) and CIN 3 (P=0.04). Deferring from transferases and polyLacNAc chains, GlcNAc (WGA ligand) reveals a gradual staining pattern decrease with the increase of the lesion degree, being more expressed in CIN 1 lesions when compared with normal (P<0.0001), CIN 2 (P<0.0001), SC (P<0.0001) and CIN 3 (P=0.0003). Our data reveal that β3GnT2 and polyLacNAc may be involved in the progression of the pre-malignant lesions of the human uterine cervix. In addition, polyLacNAc expression only in the nucleus can be associated a poor prognostic in uterine lesions.     

Evaluation of aid to diagnosis of pigmented skin lesions in general practice: controlled trial randomised by practice

Objectives To determine whether an aid to the diagnosis of pigmented skin lesions reduces the ratio of benign lesions to melanomas excised in general practice.Design Controlled trial randomised by practice.Setting General practices in Perth, Western Australia.Participants 468 general practitioners in 223 practices.Interventions Intervention practices were given an algorithm and instant camera to assist with the diagnosis of pigmented skin lesions. All practices were given national guidelines on managing melanoma.Main outcome measures Ratio of benign pigmented lesions to melanomas excised. Analyses conducted with and without inclusion of seborrhoeic keratoses.Results At baseline the ratios of benign to malignant lesions were lower in the intervention group than in the control group. During the trial period the ratios were higher in the intervention group (19:1 v 17:1 without seborrhoeic keratoses and 29:1 v 26:1 with seborrhoeic keratoses). After adjustment for patients'' age, sex, and socioeconomic status, the ratio was 1.02 times higher (95% confidence interval 0.68 to 1.51, P = 0.94) in the intervention group when seborrhoeic keratoses were not included and 1.03 times higher (0.71 to 1.50, P = 0.88) when seborrhoeic keratoses were included. General practitioners in the intervention group were less likely than those in the control group to excise the most recent pigmented skin lesion they managed (22% v 48%, P < 0.001) and to refer the patient to a specialist (16% v 27%, P = 0.06).Conclusions Provision of the algorithm and camera did not decrease the ratio of benign pigmented skin lesions to melanomas excised by general practitioners.     

Characteristics of the Cervicovaginal Microenvironment in Childbearing-Age Women with Different Degrees of Cervical Lesions and HR-HPV Positivity

Persistent infection with high-risk human papillomavirus (HR-HPV) is the most important determinate in the development of cervical cancer, and cervical microecology can modulate cervical viral infection. However, few studies have been conducted on the microecological analysis of cervical diseases using strict physiological factors. This study investigated the characteristics and dynamics of cervical microecology in childbearing-age Chinese women with different degrees of HR-HPV-positive cervical lesions. A total of 168 subjects were selected according to the selection criteria, including healthy HPV-negative individuals (n = 29), HR-HPV-infected individuals (n = 29), low-grade squamous intraepithelial lesion individuals (LSIL, n = 32), high-grade squamous intraepithelial lesion individuals (HSIL, n = 40), and cervical cancer individuals (n = 38). We sampled cervical secretions from each subject and performed comparative analysis using the 16S rRNA sequencing method. Comparison analysis showed that Lactobacillus and Ignatzschineria were the dominant genera in the healthy group, while Gardnerella and Prevotella were more enriched in the disease groups. Based on the taxa composition, we roughly divided the development of cervical cancer into two phases: phase I was from healthy status to HR-HPV infection and LSIL; phase II was from LSIL to HSIL and cervical cancer. Different interactions among different genera were observed in different groups. Prevotella inhibited the abundance of Lactobacillus in the healthy group, while Prevotella inhabited the abundance of Gardnerella in the other groups. In the HR-HPV infection group, Ignatzschineria and Enterococcus showed a positive interaction but dissociated with the increase in cervical lesions, which might eventually lead to a continuous decrease in the abundances of Lactobacillus and Ignatzschineria.     

Design and synthesis of novel imidazo[4,5-b]pyridine based compounds as potent anticancer agents with CDK9 inhibitory activity

New imidazo[4,5-b]pyridine derivatives were designed, synthesized and screened for their anticancer activity against breast (MCF-7) and colon (HCT116) cancer cell lines. Nine compounds (I, II, IIIa, IIIb, IV, VI, VIIa, VIII, IX) showed significant activity against MCF-7, while six compounds (I, VIIc, VIIe, VIIf, VIII, IX) elicited a remarkable activity against HCT116. Compounds showing significant anticancer activity revealed remarkable CDK9 inhibitory potential (IC₅₀ = 0.63–1.32 μM) relative to sorafenib (IC₅₀ = 0.76 μM). Moreover, a molecular docking study was performed to illustrate the binding mode of the most active compounds in the active site of CDK9 where it revealed superior binding affinity relative to the natural ligand (T3C).