Association between intestinal microflora and renal function in patients with chronic renal failure: A case-control analysis.

Objective: To identify the association between the changes in intestinal microflora and renal function in patients with chronic renal failure (CRF). Methods: This retrospective case-control study included 50 patients with CRF (study group), admitted to the Clinical Laboratory Department of Shenzhen People's Hospital from March 2021 to May 2022, and 50 healthy individuals (control group). The association between the distribution of intestinal microflora and the glomerular filtration rate (GFR), levels of serum creatinine (SCr), blood urea nitrogen (BUN), and serum cystatin C (CysC) were analyzed. Results: Intestinal microflora of CRF patients had significantly higher levels of Enterococci compared to the control group (p-Value <0.05), while the levels of Bifidobacterium spp. and Escherichia coli were lower in the study group (p-Value <0.05). GFR was lower, and the levels of BUN, SCr, and CysC were higher in the study group compared to the control group (all p-Value <0.05). GFR, BUN, SCr and CysC levels in the study group negatively correlated with the levels of Bifidobacterium spp. and Lactobacillus spp. (r<0, P<0.05), and positively correlated with the abundance of Enterococcus spp. and Escherichia coli (r>0, P<0.05) in the intestinal microflora. Conclusions: Changes in intestinal microbiota are associated with a significant decrease in GFR and a marked increase in serum levels of renal function indicators, and alterations in the balance of intestinal microbiota may lead to further aggravation of the renal function damage in patients with CRF.

Pseudomonas aeruginosa detection based on droplets incubation using an integrated microfluidic chip, laser spectroscopy, and machine learning.

Pseudomonas aeruginosa is an opportunist pathogen responsible for causing several infections in the human body, especially in patients with weak immune systems. The proposed approach reports a novel pathogens detection system based on cultivating microdroplets and acquiring the scattered light signals from the incubated droplets using a microfluidic device. Initially, the microdroplets were generated and incubated to cultivate bacteria inside the microdroplets. The second part of the microfluidic chip is the detection module, embedded with three optical fibers to connect laser light and photosensors. The incubated droplets were reinjected in the detection module and passed through the laser light. The surrounding photosensors were arranged symmetrically at 45° to the flowing channel for acquiring the scattered light signal. The noise was removed from the acquired data, and time-domain waveform features were evaluated. The acquired features were trained using machine learning classifiers to classify P. aeruginosa. The k-nearest neighbors (KNN) showed superior classification performance with 95.6 % accuracy among other classifiers, including logistic regression (LR), support vector machines (SVM), and naïve Bayes (NB). The proposed research was performed to validate the method for pathogens detection with a concentration of 105 CFU/mL. The total duration of 6 h is required to test the sample, including five hours for droplets incubation and one hour for sample preparation and detection using light scattering module. The results indicate that acquiring the light scattering patterns from incubated droplets can detect P. aeruginosa using machine learning classification. The proposed system is anticipated to be helpful as a rapid device for diagnosing pathogenic infections.

Isoginkgetin, a potential CDK6 inhibitor, suppresses SLC2A1/GLUT1 enhancer activity to induce AMPK-ULK1-mediated cytotoxic autophagy in hepatocellular carcinoma.

Isoginkgetin (ISO), a natural biflavonoid, exhibited cytotoxic activity against several types of cancer cells. However, its effects on hepatocellular carcinoma (HCC) cells and mechanism remain unclear. Here, we revealed that ISO effectively inhibited HCC cell proliferation and migration in vitro. LC3-II expression and autophagosomes were increased under ISO treatment. In addition, ISO-induced cell death was attenuated by treatment with chloroquine or knockdown of autophagy-related genes (ATG5 or ULK1). ISO significantly suppressed SLC2A1/GLUT1 (solute carrier family 2 member 1) expression and glucose uptake, leading to activation of the AMPK-ULK1 axis in HepG2 cells. Overexpression of SLC2A1/GLUT1 abrogated ISO-induced autophagy. Combining molecular docking with thermal shift analysis, we confirmed that ISO directly bound to the N terminus of CDK6 (cyclin-dependent kinase 6) and promoted its degradation. Overexpression of CDK6 abrogated ISO-induced inhibition of SLC2A1/GLUT1 transcription and induction of autophagy. Furthermore, ISO treatment significantly decreased the H3K27ac, H4K8ac and H3K4me1 levels on the SLC2A1/GLUT1 enhancer in HepG2 cells. Finally, ISO suppressed the hepatocarcinogenesis in the HepG2 xenograft mice and the diethylnitrosamine+carbon tetrachloride (DEN+CCl4)-induced primary HCC mice and we confirmed SLC2A1/GLUT1 and CDK6 as promising oncogenes in HCC by analysis of TCGA data and human HCC tissues. Our results provide a new molecular mechanism by which ISO treatment or CDK6 deletion promotes autophagy; that is, ISO targeting the N terminus of CDK6 for degradation inhibits the expression of SLC2A1/GLUT1 by decreasing the enhancer activity of SLC2A1/GLUT1, resulting in decreased glucose levels and inducing the AMPK-ULK1 pathway.

Rapid detection of Pseudomonas aeruginosa based on lab-on-a-chip platform using immunomagnetic separation, light scattering, and machine learning.

The rapid detection of the pathogenic bacteria in patient samples is crucial to expedient patient care. The proposed approach reports the development of a novel lab-on-a-chip device for the rapid detection of P. aeruginosa based on immunomagnetic separation, optical scattering, and machine learning. The immunomagnetic particles with a diameter of 5 µm were synthesized for isolating P. aeruginosa from the test sample. A microfluidic chip was fabricated, and three optical fibers were embedded for connecting a laser light and two photodetectors. The laser light was pointed towards the channel to pass light through the sample. A pair of photodetectors via optical fibers were arranged symmetrically at 45° to the channel. The photodetectors acquired scattered light from the flowing sample and converted the light to an electrical signal. The sample containing immunomagnetic beads linked with bacteria was injected into the microfluidic chip. The optimized conditions for performing the experiments were characterized for real-time detection of P. aeruginosa. The data acquisition system recorded the real-time light scattering from the test sample. After removing noise from the output waveform, five different time-domain statistical features were extracted from each waveform: standard mean, standard variance, skewness, kurtosis, and coefficient of variation. The pathogens classification was performed by training the discrimination model using extracted features based on machine learning algorithms. The support vector machines (SVM) with a sigmoid function kernel showed superior classification performance with 97.9% accuracy among other classifiers, including k-nearest neighbors (KNN), logistic regression (LR), and naïve Bayes (NB). The method can detect P. aeruginosa specifically and quantitatively with a limit of detection of 102 CFU/mL. The device can classify P. aeruginosa within 10 min with a total assay time of 25 min. The device was used to test its ability to detect pathogen from the serum and sputum specimens spiked with 105 CFU/mL concentration of P. aeruginosa. The results indicate that light scattering combined with machine learning can be used to detect P. aeruginosa. The proposed technique is anticipated to be helpful as a rapid device for diagnosing P. aeruginosa related infections.

Altered fecal microbial and metabolic profile reveals potential mechanisms underlying iron deficiency anemia in pregnant women in China.

The gut microbiome and its metabolism may provide crucial insight into the cause of iron deficiency anemia (IDA) in pregnant women. This study aimed to investigate the effect of the gut microbiome and its related metabolites on pregnant women with iron deficiency (ID) and IDA. Maternal cubital venous blood and stool samples were collected from healthy control pregnant women (HC, non-anemic, n=10), pregnant women with ID non-anemia (ID, n=10), and IDA (n=10). All groups were subjected to fecal metagenomics and metabolomics. The composition and function of the gut microbiome were then compared in pregnant women with ID and IDA with HC after excluding the possibility of inflammation and insufficient iron absorption capacity. Whole-genome shotgun libraries were prepared by quantifying metagenomic DNA samples with Quant-iT PicoGreen dsDNA Assay. The levels of 41 microbial species, including 21 Streptococci and ten metabolites (catechol), which could serve as siderophores, were increased. In contrast, 3 Bacteroides and six metabolites were decreased in pregnant women with IDA (p<0.05). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the bio-pathways, including biosynthesis of siderophore group non-ribosomal peptides (p<0.01), ABC transporters (p<0.05) and membrane transport of the gut microbiota (p<0.01) in IDA patients were expressed differently compared with HC. Correlation analysis also indicates that these increased bacteria formed strong co-occurring relationships with metabolites in the occurrence and development of IDA in pregnant women. The current study identified that streptococci and catechol (fecal metabolite) were significantly increased in pregnant women with IDA. Therefore, adjusting the intestinal homeostasis using long-term living and eating habits on oral Streptococcus in pregnant women with IDA before iron supplementation may be more conducive to iron supplementation, thus providing novel therapies for IDA.

The Anti-inflammatory Effects of Lignan Glycosides from Cistanche tubulosa stems on LPS/IFN-γ-induced RAW264.7 Macrophage Cells via PI3K/ AKT Pathway.

BACKGROUND: Cistanche tubulosa is a tonic in traditional Chinese medicines and has a broad spectrum of biological activity, including anti-inflammatory. However, the anti-inflammatory major constituents of C. tubulosa and their underlying mechanisms are still unknown. OBJECTIVE: The aim of the current study was to explore the separation and structural characterization of lignan glycosides from C. tubulosa (Schenk) Wight., their anti-inflammatory activity and the underlying mechanism. MATERIALS AND METHODS: Fractionation and isolation of the 85% EtOH extract of C. tubulosa (Schenk) Wight. were carried out and the primary ingredients lignan glycosides (1-6) were structurally characterized. CCK8 methods were used to evaluate the cytotoxic effect of lignan glycosides (1-6). Effects of lignan glycosides (1-6) on NO production in LPS/IFN-γ-induced RAW264.7 macrophages cells were measured using Griess reagent by reaction with nitrite. The mRNA expression levels of iNOS, COX-2, IL-1ß, IL-6, TNF-a, and TGF-ß treated RAW264.7 cells with various concentrations (0, 25 and 50 µg/ml) of lignan glycosides (1, 4) in the presence of LPS (10 ng/ml) and IFN-γ (20 ng/ml) for 24 h were analyzed by quantitative RT-PCR. Also, the protein expressions of iNOS, COX-2, PI3K, AKT, p-AKT and ß -actin were determined using Western blot analysis. A molecular docking study was performed to investigate the interactions between the lignan glycosides and the PI3K using Autodock vina 1.1.2 package. RESULTS: Six lignan glycosides (1-6) were isolated from stems of C. tubulosa. Among them, (+)- pinoresinol-4-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranoside (5) and eleutheroside E (6) were firstly isolated from C. tubulosa. Of these lignans, 1 and 4 exhibited pronounced inhibitions on NO production with the values of 33.63 ± 4.78 and 39.28 ± 5.52 % at 50 µg/ml, respectively. Additionally, LPS/IFN-γ-induced expression of inducible Nitric Oxide Synthase (iNOS), Cyclooxygenase-2 (COX-2), Interleukin-1ß (IL-1ß), IL-6, and Tumor Necrosis Factor-a (TNF-a) was significantly suppressed by pre-treatment of 1 and 4 in a dose-dependent manner. While 1 and 4 increased the mRNA levels of anti-inflammatory cytokines (TGF-ß). Furthermore, 1 and 4 significantly inhibited the protein levels of PI3K and p-AKT in a dose-dependent manner. CONCLUSION: Taken together, these results suggest that 1 and 4 play an important role in the attenuation of LPS/IFN-γ-induced inflammatory responses in RAW264.7 cells and that the mechanisms involve down-regulation of the PI3K/AKT pathway.

Knockdown of NHP2 inhibits hepatitis B virus X protein-induced hepatocarcinogenesis via repressing TERT expression and disrupting the stability of telomerase complex.

Hepatitis B virus X protein (HBx) is highly expressed in HBV-infected hepatocellular carcinoma (HCC) and upregulates transcriptional activation of telomerase reverse transcriptase (TERT). NHP2 is a component of the telomerase complex and also increased in HCC. However, whether NHP2 could accelerate HCC caused by HBx overexpression remains unknown. This study intended to investigate the effects of NHP2 knockdown on HBx-overexpressed HCC and uncover the potential mechanism. Results showed that after HBx overexpression, the expression of TERT and NHP2 was increased. NHP2 knockdown inhibited cell proliferation, colony formation and telomerase activity, while promoting cell apoptosis in PLC/PRF5 cells with or without HBx overexpression. Moreover, the protein expression of TERT and HBx was inhibited, pro-apoptotic proteins Bax and cleaved-caspase3 expression was enhanced, whereas anti-apoptotic protein Bcl-2 level was reduced upon NHP2 silencing in PLC/PRF5 cells with or without HBx upregulation. The interaction between NHP2 and TERT was also confirmed. Treatment with shRNA-NHP2-1 inhibited tumor growth in xenograft model, and the alterations of related proteins were consisted with in vitro results. In conclusion, knockdown of NHP2 could inhibit the proliferation of hepatoma cells overexpressing HBx via inhibiting TERT expression.

Novel caffeoylquinic acid derivatives from Lonicera japonica Thunb. flower buds exert pronounced anti-HBV activities.

Lonicera japonica Thunb., possesses antiviral and hepatoprotective activities, and is widely used as a health food and in cosmetics. However, its major constituents, caffeoylquinic acid derivatives, and their anti-HBV activity were lacking systematic research. In this study, four novel caffeoylquinic acids, five simple caffeic acids and fourteen known caffeoylquinic acids are isolated and identified from L. japonica. Most caffeoylquinic acids inhibited HBsAg and HBeAg secretion, and HBV DNA replication. In particular, 100 µg ml-1 monocaffeoylquinic acid 9 inhibits HBsAg and HBeAg secretion, and HBV DNA replication by 83.82, 70.76 and 39.36% compared to the control. Unfortunately, 50 µg ml-1 tricaffeoylquinic acid 23 promotes HBsAg and HBeAg secretion, and HBV DNA replication by 172.39, 9.92 and 55.40%. Finally, structure-activity relationships reveal that caffeoylquinic acids containing a caffeoyl group have better inhibitory activities. The results indicate that caffeoylquinic acids from L. japonica could serve as anti-HBV agents for functional food or medicinal use.

[Correlation of serum cystatin C with blood pressure: a cross-sectional study of 912 subjects].

OBJECTIVE: To study the association between serum cystatin C level and blood pressure. METHODS: We conducted a cross-sectional study of 912 subjects randomly sampled from a cohort visiting for routine physical examinations. The epidemiological data were obtained using questionnaires and from the database of physical examination results. Pearson analysis and multiple stepwise regression analysis were used to analyze the relationship between blood pressure and cystatin C. RESULTS: The levels of serum cystatin C differed significantly among the normotensive, prehypertensive, and hypertensive subjects (P<0.05). Pearson analysis revealed that regardless of gender, serum cystatin C was positively correlated with SBP, DBP, MAP, BMI, TC, TG, LDL-C, UA and BUN (P<0.05). With MAP, SBP and DBP as dependent variables, multiple stepwise regression analysis of the factors affecting blood pressure indicated that cystatin C had the strongest effect on SBP and MAP (P<0.05) but did not significantly affect DBP (P>0.05). CONCLUSION: Serum cystatin C level is significantly correlated with SBP and MAP and can be used as a biomarker for alert of hypertension.