Deep Learning-Based Classification and Targeted Gene Alteration Prediction from Pleural Effusion Cell Block Whole-Slide Images.

Cytopathological examination is one of the main examinations for pleural effusion, and especially for many patients with advanced cancer, pleural effusion is the only accessible specimen for establishing a pathological diagnosis. The lack of cytopathologists and the high cost of gene detection present opportunities for the application of deep learning. In this retrospective analysis, data representing 1321 consecutive cases of pleural effusion were collected. We trained and evaluated our deep learning model based on several tasks, including the diagnosis of benign and malignant pleural effusion, the identification of the primary location of common metastatic cancer from pleural effusion, and the prediction of genetic alterations associated with targeted therapy. We achieved good results in identifying benign and malignant pleural effusions (0.932 AUC (area under the ROC curve)) and the primary location of common metastatic cancer (0.910 AUC). In addition, we analyzed ten genes related to targeted therapy in specimens and used them to train the model regarding four alteration statuses, which also yielded reasonable results (0.869 AUC for ALK fusion, 0.804 AUC for KRAS mutation, 0.644 AUC for EGFR mutation and 0.774 AUC for NONE alteration). Our research shows the feasibility and benefits of deep learning to assist in cytopathological diagnosis in clinical settings.

A retrospective analysis of serous effusions based on the newly proposed international system for reporting serous fluid cytopathology: a report of 3633 cases in an oncological center.

BACKGROUND: The International System for Reporting Serous Fluid Cytopathology (TIS) was recently proposed. We retrospectively applied TIS recommendations for reporting the cytological diagnosis of serous effusions and reported our experience. METHODS: All the serous effusions from January 2018 to September 2021 were retrieved from the database. Recategorization was performed using the TIS classification, the risk of malignancy (ROM) was calculated for each TIS category. In addition, on the basis of the original TIS classification, we further subdivided the TIS category IV (suspicious for malignancy, SFM) into 2 groups (IVa and IVb) according to cytological characteristics (quality and quantity) to explore the necessity of SFM subclassification. The performance evaluation was carried out using different samples (pleural, peritoneal and pericardial effusions) and preparation methods (conventional smears, liquid-based preparations and cell blocks). RESULTS: A total of 3633 cases were studied: 17 (0.5%) were diagnosed as 'nondiagnostic' (I, ND), 1100 (30.3%) as 'negative for malignancy' (II, NFM), 101 (2.8%) as 'atypia of undetermined significance' (III, AUS), 677 (18.6%) as 'suspicious for malignancy' (IV, SFM), and 1738 (47.8%) as 'malignant' (V, MAL). The ROMs for the categories were 38.5%, 28.6%, 52.1%, 99.4% and 100%, respectively. The ROM for SFM was significantly higher than that for AUS (P < 0.001), while the difference between the ROMs for IVa and IVb was insignificant. The sensitivity, negative predictive value (NPV) and diagnostic accuracy of liquid-based preparations were all superior to those of conventional smears and cell blocks in detecting abnormalities. Using the three preparation methods simultaneously had the highest sensitivity, NPV and diagnostic accuracy. CONCLUSION: Serous effusion cytology has a high specificity and positive predictive value (PPV), and TIS is a user-friendly reporting system. Liquid-based preparations could improve the sensitivity of diagnosis, and it is best to use three different preparation methods simultaneously for serous effusion cytologic examination.

Cytomorphologic features as predictors of aggressiveness in patients with pT1 papillary thyroid carcinoma: a retrospective study of associations with clinicopathological parameters in 226 fine-needle aspirates.

BACKGROUND: Distinguishing aggressive pT1 papillary thyroid carcinomas (PTCs) from indolent PTCs before or during surgery is important. To the best of our knowledge, few reports in the literature have examined the value of the cytomorphologic features of PTC as predictors of aggressiveness. METHODS: This retrospective study included 226 pT1 PTC patients who underwent preoperative fine-needle aspiration cytology (FNAC) and surgery at Peking University Cancer Hospital between January 2018 and December 2019. Data on the clinical characteristics and pathological results were obtained from the electronic medical record database. All FNAC smears were blindly reviewed by two independent cytopathologists, and the associations between nine cytomorphologic features (lymphocytes, multinucleated giant cells, cellularity, cellular adhesiveness, nuclear size, nuclear pleomorphism, nuclear membrane regularity, intranuclear pseudoinclusions and the amount of cytoplasm) and clinicopathological parameters were statistically analyzed. RESULTS: Univariate analysis showed that cellularity, intranuclear pseudoinclusions, cellular adhesiveness, nuclear size, and nuclear pleomorphism were strong predictors of some clinicopathological parameters such as extracapsular invasion (ECI) and lymph node metastasis (LNM). Multivariate analysis confirmed that cellular adhesiveness was a strong independent predictor of ECI (P=0.001) and LNM (P<0.001), and the amount of cytoplasm can also predict LNM (P=0.024). CONCLUSIONS: Cytomorphologic features including cellular adhesiveness and the amount of cytoplasm in preoperative FNAC smears could be a valuable tool for predicting ECI or LNM and may be predictors of aggressiveness in patients with pT1 PTC.

Acute and repeated dose 26-week oral toxicity study of 20(S)-ginsenoside Rg3 in Kunming mice and Sprague-Dawley rats.

BACKGROUND: 20(S)-ginsenoside-Rg3 (C42H72O13), a natural triterpenoid saponin, is extracted from red ginseng. The increasing use of 20(S)-ginsenoside Rg3 has raised product safety concerns. METHODS: In acute toxicity, 20(S)-ginsenoside Rg3 was singly and orally administrated to Kunming mice and Sprague-Dawley (SD) rats at the maximum doses of 1600 mg/kg and 800 mg/kg, respectively. In the 26-week toxicity study, we used repeated oral administration of 20(S)-ginsenoside Rg3 in SD rats over 26 weeks at doses of 0, 20, 60, or 180 mg/kg. Moreover, a 4-week recovery period was scheduled to observe the persistence, delayed occurrence, and reversibility of toxic effects. RESULTS: The result of acute toxicity shows that oral administration of 20(S)-ginsenoside Rg3 to mice and rats did not induce mortality or toxicity up to 1600 and 800 mg/kg, respectively. During a 26-week administration period and a 4-week withdrawal period (recovery period), there were no significant differences in clinical signs, body weight, food consumption, urinalysis parameters, biochemical and hematological values, or histopathological findings. CONCLUSION: The mean oral lethal dose (LD50) of 20(S)-ginsenoside Rg3, in acute toxicity, is above 1600 mg/kg and 800 mg/kg in mice and rats, respectively. In a repeated-dose 26-week oral toxicity study, the no-observed-adverse-effect level for female and male SD rats was 180 mg/kg.

Chemical constituents and their bioactivities from the fruits of Vitex negundo var. cannabifolia.

Phytochemical investigation of the fruits of Vitex negundo var. cannabifolia led to the isolation of 22 compounds (1-22). Their structures were elucidated mainly by spectroscopic analysis and comparison with the literature data. Among them, compounds 1 and 2 were two new artificial lignans. Primary bioassay showed that the polymethoxyflavones 9-12 displayed moderate-to-weak cytotoxicity against human HepG2 and rat C6 cell lines, while the triterpenoids 13-17 exhibited significant brine shrimp lethality with LC50values of 7.5-29.4 µM.

Effect of structure variation of the aptamer-DNA duplex probe on the performance of displacement-based electrochemical aptamer sensors.

Electrochemical aptamer-based (E-AB) sensors employing electrode-immobilized, redox-tagged aptamer probes have emerged as a promising platform for the sensitive and quick detection of target analytes ranging from small molecules to proteins. Signal generation in this class of sensor is linked to change in electron transfer efficiency upon binding-induced change in flexibility/conformation of the aptamer probe. Because of this signaling mechanism, signal gains of these sensors can be improved by employing a displacement-based recognition system, which links target binding with a large-scale flexibility/conformation shift from the aptamer-DNA duplex to the single-stranded DNA or the native aptamer. Despite the relatively large number of displacement-based E-AB sensor samples, little attention has been paid to the structure variation of the aptamer-DNA duplex probe. Here we detail the effects of complementary length and position of the aptamer-DNA duplex probe on the performance of a model displacement-based E-AB sensor for ATP. We find that, greater background suppression and signal gain are observed with longer complementary length of the aptamer-DNA duplex probe. However, sensor equilibration time slows monotonically with increasing complementary length; and with too many target binding sites in aptamer sequence being occupied by the complementary DNA, the aptamer-target binding does not occur and no signal gain observed. We also demonstrate that signal gain of the displacement-based E-AB sensor is strongly dependent on the complementary position of the aptamer-DNA duplex probe, with complementary position located at the electrode-attached or redox-tagged end of the duplex probe, larger background suppression and signal increase than that of the middle position are observed. These results highlight the importance of rational structure design of the aptamer-DNA duplex probe and provide new insights into the optimization of displacement-based E-AB sensors.

Separation of vitexin-4″-O-glucoside and vitexin-2″-O-rhamnoside from hawthorn leaves extracts using macroporous resins.

Vitexin-4″-O-glucoside and vitexin-2″-O-rhamnoside are the major flavonoids of hawthorn leaves. In this work, the adsorption and desorption characteristics of vitexin-4″-O-glucoside and vitexin-2″-O-rhamnoside on seven macroporous resins were evaluated. Among the tested resins, the HPD-400 resin showed the best adsorption and desorption capacities. Adsorption isotherms were constructed for the HPD-400 resin and well fitted to Langmuir and Freundlich models. Dynamic adsorption and desorption tests were performed on column packed with the HPD-400 resin to optimize the chromatographic parameters. After one run treatment with the HPD-400 resin, the contents of vitexin-4″-O-glucoside and vitexin-2″-O-rhamnoside in the product were increased 8.44-fold and 8.43-fold from 0.720% and 2.63% to 6.08% and 22.2% with recovery yields of 79.1% and 81.2%, respectively. These results show that the developed method is a promising basis for the large-scale purification of vitexin-4″-O-glucoside and vitexin-2″-O-rhamnoside from hawthorn leaves and other plant materials.

A subchronic toxicity study, preceded by an in utero exposure phase, with refined arachidonic acid-rich oil (RAO) derived from Mortierella alpina XM027 in rats.

To evaluate the potential toxicity of refined arachidonic acid-rich oil (RAO) derived from Mortierella alpina (M. alpina) XM027, we performed a 90-day subchronic study in F1 Sprague Dawley (SD) rats. This study was preceded by a 4-week pretreatment period of parental (F0) rats and exposure of the F0 dams throughout mating, gestation, and lactation. The results indicated that RAO, at dose levels of 0.5%, 1.5%, and 5%, did not affect either reproductive performance of the parental rats, or any characteristics of the pups. In the subchronic study with the offspring (F1) rats, no treatment related abnormalities were observed. In summary, no observable adverse effect level (NOAEL) in this study was placed at 5% RAO, the highest level tested. This level corresponds to approximately 3750mg/kg in F0 females, 2850mg/kg in F0 males, 4850mg/kg in F1 females, and 4480mg/kg in F1 males.

Fucoidan, a sulfated polysaccharide from brown algae, improves cognitive impairment induced by infusion of Aß peptide in rats.

Fucoidan is a complex sulfated polysaccharide, derived from marine brown seaweed. In the present study, we investigated the effects of fucoidan on improving learning and memory impairment in rats induced by infusion of Aß (1-40), and its possible mechanisms. The results indicated that fucoidan could ameliorate Aß-induced learning and memory impairment in animal behavioral tests. Furthermore, fucoidan reversed the decreased activity of choline acetyl transferase (ChAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and content of acetylcholine (Ach), as well as the increased activity of acetylcholine esterase (AchE) and content of malondialdehyde (MDA) in hippocampal tissue of Aß-injected rats. Moreover, these were accompanied by an increase of Bcl-2/Bax ratio and a decrease of caspase-3 activity. These results suggested that fucoidan could ameliorate the learning and memory abilities in Aß-induced AD rats, and the mechanisms appeared to be due to regulating the cholinergic system, reducing oxidative stress and inhibiting the cell apoptosis.

Anti-inflammatory effects of sophocarpine in LPS-induced RAW 264.7 cells via NF-κB and MAPKs signaling pathways.

Sophocarpine, a tetracyclic quinolizidine alkaloid, is one of the most abundant active ingredients in Sophora alopecuroides L. Our previous studies have showed that sophocarpine exerts anti-inflammatory activity in animal models. In the present study, anti-inflammatory mechanisms of sophocarpine were investigated in lipopolysaccharide (LPS)-induced responses in RAW 264.7 cells. Furthermore, the cytotoxicity of sophocarpine was tested. The results indicated that sophocarpine could increase the LDH level and inhibit cell viability up to 800µg/ml, and which was far higher than that of the plasma concentration of sophocarpine in clinical effective dosage. The results also demonstrated that sophocarpine (50 and 100µg/ml) suppressed LPS-stimulated NO production and pro-inflammatory cytokines secretion, including tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). These were associated with the decrease of the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, sophocarpine inhibited LPS-mediated nuclear factor-κB (NF-κB) activation via the prevention of inhibitor κB (IκB) phosphorylation. Sophocarpine had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2), whereas it attenuated the phosphorylation of p38 mitogen-activated protein (MAP) kinase and c-Jun NH(2)-terminal kinase (JNK). Our data suggested that sophocarpine exerted anti-inflammatory activity in vitro, and it might attribute to the inhibition of iNOS and COX-2 expressions via down-regulation of the JNK and p38 MAP kinase signal pathways and inhibition of NF-κB activation.

Molecular authentication of the Oriental medicines Pericarpium citri reticulatae and Citri unshius pericarpium using SNP markers.

Pericarpium citri reticulatae and Citri unshius pericarpium are important Oriental medicinal materials used in many prescriptions. Among the Citrus species, the dried peels of C. japonica, C. maxima, and C. trifoliata are found to be adulterants and substitutes of Pericarpium citri reticulatae and Citri unshius pericarpium. In order to develop a simple and reliable DNA method for authentication of these two medicinal materials, nuclear ribosomal internal transcribed spacer (ITS) region was targeted for molecular analysis. A host of single nucleotide polymorphism (SNP) sites were detected among ITS sequences of five Citrus species. From two SNP sites, two modified specific primers were designed for authentication of Pericarpium citri reticulatae and Citri unshius pericarpium using multiplex PCR. The established multiplex allele-specific PCR system was proven to be effective for simultaneous authentication of Pericarpium citri reticulatae and Citri unshius pericarpium. The scheme used in this study could be adapted for determination of the botanical identity and origin of other medicinal materials.

Development of a multiplex amplification refractory mutation system for simultaneous authentication of Korean ginseng cultivars "Gumpoong" and "Chungsun".

BACKGROUND AND AIMS. Molecular authentication of Korean ginseng cultivars was investigated using the mitochondrial nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 7 (nad7) intron 3 region. MATERIALS AND METHODS. A mutation site specific to Panax ginseng "Gumpoong" and "Chungsun" cultivars was detected within the sequence data. Based on this mutation site and the "Gumpoong"-specific single nucleotide polymorphism site reported in 26S rDNA, two modified allele-specific primer pairs were designed and a multiplex amplification refractory mutation system (MARMS) was applied to identify "Gumpoong" and "Chungsun." RESULTS. The results showed that "Gumpoong" and "Chungsun" can be clearly discriminated from the other Korean ginseng cultivars by simultaneously identifying the haplotype of "Gumpoong" and the specific allele of "Chungsun" by applying the MARMS. CONCLUSION. This study, therefore, provides a simple and reliable method for simultaneous authentication of "Gumpoong" and "Chungsun" cultivars.

Molecular authentication of Panax ginseng and ginseng products using robust SNP markers in ribosomal external transcribed spacer region.

Panax ginseng and Panax quinquefolius are the most widely used Panax species, but they are known to have different properties and medicinal values. The aim of this study is to develop a robust and accurate DNA marker for identifying P. ginseng and the origins of ginseng products. Two single nucleotide polymorphism (SNP) sites specific to P. ginseng were exploited from nuclear ribosomal external transcribed spacer (ETS) region. Based on the SNP sites, two specific primers were designed for P. ginseng and P. quinquefolius respectively. P. ginseng can be easily discriminated from P. quinquefolius by amplifying the two specific alleles using multiplex allele-specific PCR. Favorable results can also be obtained from commercial ginseng products. The established method is highly sensitive and can detect 1% of intentional adulteration of P. quinquefolius into P. ginseng down to the 0.1ng level of total DNA. Therefore this study provides a reliable and simple DNA method for authentication of the origins and purities of ginseng products.

Effect of food azo dye tartrazine on learning and memory functions in mice and rats, and the possible mechanisms involved.

UNLABELLED: Tartrazine is an artificial azo dye commonly used in human food and pharmaceutical products. The present study was conducted to evaluate the toxic effect of tartrazine on the learning and memory functions in mice and rats. Animals were administered different doses of tartrazine for a period of 30 d and were evaluated by open-field test, step-through test, and Morris water maze test, respectively. Furthermore, the biomarkers of the oxidative stress and pathohistology were also measured to explore the possible mechanisms involved. The results indicated that tartrazine extract significantly enhanced active behavioral response to the open field, increased the escape latency in Morris water maze test and decreased the retention latency in step-through tests. The decline in the activities of catalase, glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) as well as a rise in the level of malonaldehyde (MDA) were observed in the brain of tartrazine-treated rats, and these changes were associated with the brain from oxidative damage. The dose levels of tartrazine in the present study produced a few adverse effects in learning and memory functions in animals. The mechanisms might be attributed to promoting lipid peroxidation products and reactive oxygen species, inhibiting endogenous antioxidant defense enzymes and the brain tissue damage. PRACTICAL APPLICATION: Tartrazine is an artificial azo dye commonly used in human food and pharmaceutical products. Since the last assessment carried out by the Joint FAO/WHO Expert Committee on Food Additives in 1964, many new studies have been conducted. However, there is a little information about the effects on learning and memory performance. The present study was conducted to evaluate the toxic effect of tartrazine on the learning and memory functions in animals and its possible mechanism involved. Based on our results, we believe that more extensive assessment of food additives in current use is warranted.

A simplified method for identifying the Panax ginseng cultivar Gumpoong based on 26S rDNA.

Molecular identification of PANAX GINSENG (Korean ginseng) cultivars is very important for their management. We have developed a simple and reliable method for specific identification of the superior cultivar "Gumpoong." The 26S rDNA was targeted for our molecular analysis, and a single-nucleotide polymorphism (SNP) was identified between Gumpoong and the other cultivars within the sequence data. From this SNP site, a modified allele-specific primer and a novel primer set have been developed to identify the Gumpoong cultivar via multiplex PCR. The established multiplex PCR method was determined to be effective, and the SNP marker showed high specificity to Gumpoong. Therefore, the described method may serve as a useful tool for rapid selection of Gumpoong cultivar.

A PCR-based SNP marker for specific authentication of Korean ginseng (panax ginseng) cultivar "Chunpoong".

Korean ginseng (Panax ginseng) has been developed as a horticultural crop due to the increasing demand in the world market. "Chunpoong" is an economically important cultivar with superior quality and high yield among nine cultivars of Korean ginseng. The aim of this work was to develop a simple technique for specific authentication of Chunpoong using DNA method. Molecular authentication of Chunpoong was investigated using DNA sequences of mitochondrial cytochrome oxidase subunit 2 (cox2) intron I and intron II regions. A single nucleotide polymorphism (SNP) specific to Chunpoong was detected and amplification refractory mutation system (ARMS)-PCR method was applied to specific identification of Chunpoong based on the SNP site. Ginseng samples collected from other locations were used to validate the SNP marker and the established method was determined to be effective. Thus, this work provides a rapid and reliable method for the specific identification of Chunpoong cultivar.

Molecular identification of the Korean ginseng cultivar "Chunpoong" using the mitochondrial nad7 intron 4 region.

BACKGROUND AND AIMS: Korean ginseng (Panax ginseng) is one of the most important medicinal plants in the Orient. Among the nine cultivars of Korean ginseng, "Chunpoong" commands a much greater market value and has been planted widely. MATERIALS AND METHODS: A rapid and reliable method for discriminating the Chunpoong cultivar was developed by exploiting a single nucleotide polymorphism (SNP) in the mitochondrial nad7 intron 4 region of nine Korean ginseng cultivars using universal primers. RESULTS: A SNP was detected between Chunpoong and other cultivars, and modified allele-specific primers were designed from this SNP site to specifically identify Chunpoong cultivar via multiplex PCR. CONCLUSION: We therefore present an effective method for the genetic identification of the Chunpoong cultivar of ginseng.

Burkholderia ginsengisoli sp. nov., a beta-glucosidase-producing bacterium isolated from soil of a ginseng field.

A bacterial strain (designated KMY03T) that possesses beta-glucosidase activity was isolated from soil from a ginseng field in South Korea and was characterized in order to determine its taxonomic position. The bacterium was found to comprise Gram-negative, rod-shaped, motile cells with unipolar polytrichous flagella. On the basis of 16S rRNA gene sequence similarity, strain KMY03T was shown to belong to the family Burkholderiaceae of the Betaproteobacteria, being most closely related to Burkholderia caledonica LMG 19076T (97.8%), Burkholderia terricola LMG 20594T (97.5%), Burkholderia xenovorans LMG 21463T (97.4%) and Burkholderia phytofirmans LMG 22146T (97.3%). Chemotaxonomic data (major ubiquinone, Q-8; major fatty acids, C17:0 cyclo, C16:0, C19:0 cyclo omega8c and summed feature 2) supported the affiliation of the novel strain with the genus Burkholderia. The results of DNA-DNA hybridizations and physiological and biochemical tests allowed the strain to be differentiated genotypically and phenotypically from Burkholderia species with validly published names. On the basis of these data, strain KMY03T represents a novel species of the genus Burkholderia, for which the name Burkholderia ginsengisoli sp. nov. is proposed. The type strain is KMY03T (=KCTC 12389T=NBRC 100965T).