Evaluation of clinical relevance of examining K-ras, p16 and p53 mutations along with allelic losses at 9p and 18q in EUS-guided fine needle aspiration samples of patients with chronic pancreatitis and pancreatic cancer

AIM： To establish an optimum combination of molecular markers resulting in best overall diagnostic sensitivity and specificity for evaluation of suspicious pancreatic mass. METHODS： Endoscopic ultrasound （EUS）-guided fine needle aspiration cytology （FNA） was performed on 101 consecutive patients （63 males, 38 females, 60 ± 12 years; 81 with subsequently diagnosed pancreatic cancer, 20 with chronic pancreatitis） with focal pancreatic mass. Samples were evaluated on-site by an experienced cytopathologist. DNA was extracted from Giemsa stained cells selected by laser microdissection and the presence of K-ras, p53 and p16 somatic mutations was tested by cycling-gradient capillary electrophoresis （CGCE） and single-strand conformation polymorphism （SSCP） techniques. In addition, allelic losses of tumor suppressor genes p16 （INK4, CDKN2A） and DPC4 （MADH4, SMAD4） were detected by monitoring the loss of heterozygosity （LOH） at 9p and 18q, respectively. RESULTS： Sensitivity and specificity of EUS-guided FNA were 75% and 85%, positive and negative predictive value reached 100%. The remaining 26% samples were assigned as inconclusive. Testing of molecular markers revealed sensitivity and specificity of 70% and 100% for K-ras mutations （P 〈 0.001）, 24% and 90% for p53 mutations （NS）, 13% and 100% for p16 mutations （NS）, 85% and 64% for aUelic losses at 9p （P 〈 0.001） and 78% and 57% for allelic losses at 18q （P 〈 0.05）. When tests for different molecular markers were combined, the best results were obtained with K-ras ＋ LOH at 9p （92% and 64%, P 〈 0.001）, K-ras ＋ LOH at 18q （92% and 57%, P 〈 0.001）, and K-ras ＋ LOH 9q ＋ LOH 18q （96% and 43%, P 〈 0.001）. When the molecular markers were used as complements to FNA cytology to evaluate inconclusive samples only, the overall sensitivity of cancer detection was 100% in all patients enrolled in the study. CONCLUSION： EUS-guided FNA cytology combined with screening of K-ras mutations and allelic losses of tumor suppressors p16 and DPC4 represents a very sensitive approach in screening for pancreatic malignancy. Molecular markers may find its use particularly in cases where FNA cytology has been inconclusive.

Impact of humic acids on the colonic microbiome in healthy volunteers

AIMTo test the effects of humic acids on innate microbial communities of the colon.METHODSWe followed the effects of oral supplementation with humic acids (Activomin®) on concentrations and composition of colonic microbiome in 14 healthy volunteers for 45 d. 3 × 800 mg Activomin® were taken orally for 10 d followed by 3 × 400 mg for 35 d. Colonic microbiota were investigated using multicolor fluorescence in situ hybridization (FISH) of Carnoy fixated and paraffin embedded stool cylinders. Two stool samples were collected a week prior to therapy and one stool sample on days 10, 31 and 45. Forty-one FISH probes representing different bacterial groups were used.RESULTSThe sum concentration of colonic microbiota increased from 20% at day 10 to 30% by day 31 and remained stable until day 45 (32%) of humic acid supplementation (P < 0.001). The increase in the concentrations in each person was due to growth of preexisting groups. The individual microbial profile of the patients remained unchanged. Similarly, the bacterial diversity remained stable. Concentrations of 24 of the 35 substantial groups increased from 20% to 96%. Two bacterial groups detected with Bac303 (Bacteroides) and Myc657 (mycolic acid-containing Actinomycetes) FISH probes decreased (P > 0.05). The others remained unaffected. Bacterial groups with initially marginal concentrations (< 0.1 × 109/mL) demonstrated no response to humic acids. The concentrations of pioneer groups of Bifidobacteriaceae, Enterobacteriaceae and Clostridium difficile increased but the observed differences were statistically not significant.CONCLUSIONHumic acids have a profound effect on healthy colonic microbiome and may be potentially interesting substances for the development of drugs that control the innate colonic microbiome.

The Use of Restriction Fragment Length Polymorphism and Fluorescence in Situ Hybridization to Investigate Microbiota of Piglets after Feeding Oregano

A total of 80 piglets (7.9 ± 1.0 kg) were used in a feeding experiment with dried oregano. The diets differed in their oregano content: 0 g, 2 g, 4 g and 8 g oregano/kg feed, corresponding to 0, 23.5, 46.9 and 93.9 mg carvacrol/kg DM. After the experimental period of 5 weeks, 20 piglets of both extreme feeding groups were slaughtered: 10 animals of the control group and 10 animals of the group that received 8 g oregano/kg. Ingesta samples of jejunum, caecum and colon were collected and analyzed by FISH and PCR RFLP to compare the diversity of microbiota. The results showed no significant changes in microbiota in response to oregano. The patterns of the PCR-RFLP showed a similarity of 61.8% - 91.8% in both feeding groups. In conclusion, an effect of oregano on the in- testinal microbiota could not be shown under the methods used.

Dihydromyricetin Alleviates H9C2 Cell Apoptosis and Autophagy by Regulating CircHIPK3 Expression and PI3K/AKT/mTOR Pathway

Objective: To investigate the effect and potential mechanism of dihydromyricetin(Dmy) on H9C2 cell proliferation, apoptosis, and autophagy. Methods: H9C2 cells were randomly divided into 7 groups, namely control, model, EV(empty p CDH-CMV-MCS-EF1-Cop GFP-T2A-Puro vector), IV(circHIPK3 interference), Dmy(50 μmol/L), Dmy+IV, and Dmy+EV groups. Cell proliferation and apoptosis were detected by cell counting kit-8 assay and flow cytometry, respectivley. Western blot was used to evaluate the levels of light chain 3 Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ), phospho-phosphoinositide 3-kinase(p-PI3K), protein kinase B(p-AKT), and phospho-mammalian target of rapamycin(p-mTOR). The level of circHIPK3 was determined using reverse transcriptase polymerase chain reaction. Electron microscopy was used to observe autophagosomes in H9C2 cells. Results: Compared to H9C2 cells, the expression of circHIPK in H9C2 hypoxia model cells increased significantly(P<0.05). Compared to the control group, the cell apoptosis and autophagosomes increased, cell proliferation rate decreased significantly, and the expression of LC3Ⅱ/Ⅰ significantly increased(all P<0.05). Compared to the model group, the rate of apoptosis and autophagosomes in IV, Dmy, and Dmy+IV group decreased, the cell proliferation rate increased, and the expression of LC3Ⅱ/Ⅰ decreased significantly(all P<0.05). Compared to the control group, the expressions of p-PI3K, p-AKT, and p-mTOR in the model group significantly reduced(P<0.05), whereas after treatment with Dmy and sh-circHIPK3, the above situation was reversed(P<0.05). Conclusion: Dmy plays a protective role in H9C2 cells by inhibiting circHIPK expression and cell apoptosis and autophagy, and the mechanism may be related to PI3K/AKT/mTOR pathway.