Impact of particulate matter 2.5 on the liver function of mice.

OBJECTIVE: The aim of this study was to evaluate the impact of particulate matter 2.5 (PM2.5) on liver function at the animal level and to study its impact targets. MATERIALS AND METHODS: 60 male and female BALB/c mice of SPF grade, aged 6-8 weeks, were randomly divided into four groups, with 15 mice in each, including the normal saline control group, the PM2.5 low dose group [2 µg/(100 g/d)], the PM2.5 medium dose group [8 µg/(100 g/d)] and the PM2.5 high dose group [16 µg/(100 g/d)]. Each day, 0.9% saline or PM2.5 particles were administered through the nasal route, and samples were taken after 3 weeks of continuous exposure. Hematoxylin-eosin staining (HE) was used to observe the liver damage caused by PM2.5. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected by using an automatic biochemical analyzer to detect the content of liver glycogen and blood glucose. Multiple indicators were observed, including plasma tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) levels, oxidative stress response indicators reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) detection, RT-PCR and Western blot detection of glycogen synthase (GS), glucokinase (GK), nuclear factor erythroid 2-related factor 2 (Nrf2) expression and phosphorylation level of phospho-c-Jun N-terminal kinases (p-JNK). RESULTS: PM2.5 can cause damage to the liver by increasing PM2.5 concentrations, raising the metabolic rate of liver cells, resulting in a substantial amount of inflammatory infiltration and vacuolar degeneration of cells, and increasing the liver/body weight. TNF-α and IL-6 inflammatory factor expression increased (p<0.05). An increase in the serum ALT and AST levels were also observed. The blood glucose of mice increased, whereas the content of liver glycogen declined (p<0.05). ROS, MDA, and SOD levels all increased considerably. PM2.5 can drastically lower the expression of GS and GK, increase the expression of Nrf2, and raise the phosphorylation level of p-JNK (p<0.05). CONCLUSIONS: PM2.5 can induce oxidative stress in mouse liver through the Nrf2/JNK pathway, induce liver inflammation in mice, and inhibit glycogen synthesis.

Viral kinetics and factors associated with rapid viral clearance during lopinavir/ritonavir-based combination therapy in non-severe COVID-19 patients.

OBJECTIVE: Lopinavir/ritonavir has modest antiviral activity against severe acute respiratory syndrome coronavirus 2. The aim was to investigate the viral kinetics and factors associated with viral clearance during lopinavir/ritonavir-based combination treatment in non-severe patients. PATIENTS AND METHODS: Sixty-four patients were retrospectively enrolled. Viral RNA was detected by real-time RT-PCR assay from sputum or throat swab samples at different time points. The patterns of viral kinetics were characterized, and factors associated with rapid viral clearance, which was defined as viral RNA undetectable within two weeks, were analyzed using multivariate logistic regression analyses. RESULTS: All patients achieved viral RNA negativity and were discharged from the hospital. Furthermore, 48 (75%) and 16 (25%) patients achieved rapid and delayed viral clearance, respectively. The lymphocyte counts of rapid viral clearance patients (1.40 [1.20-1.80] × 109/L) were higher, when compared to delayed viral clearance patients (1.00 [0.70-1.47] × 109/L) (p=0.024). The multivariate logistic analysis revealed that high lymphocyte count (≥1.3×109/L) is an independent factor associated with rapid viral clearance (OR=7.62, 95% CI=1.15-50.34, p=0.035). CONCLUSIONS: The viral shedding exhibited different patterns during treatment. Immune insufficiency is responsible for the delayed viral clearance, suggesting that an immunomodulator should be considered to promote viral clearance in patients with low lymphocyte counts.

Ocnus is essential for male germ cell development in Drosophila melanogaster.

The ocnus (ocn) gene encodes a protein abundant in the testes, implying its role in testis development. When Drosophila melanogaster is infected with the endosymbiont wMel Wolbachia, which affects the spermatogenesis of its hosts, ocn is downregulated in the third-instar larval testes, suggesting a role of ocn in spermatogenesis. In this study, we knocked down ocn in the testes and found that the hatch rates of embryos derived from ocn-knockdown males were significantly decreased, and 84.38% of the testes were much smaller in comparison to controls. Analysis of the smaller testes showed no germ cells but they had an extended hub. Using RNA-sequencing (RNA-Seq), we identified 69 genes with at least a twofold change (q-value < 5%) in their expression after ocn knockdown; of these, eight testes-specific and three reproduction-related genes were verified to be significantly downregulated using quantitative reverse transcription-PCR. Three genes (orientation disruptor, p24-2 and CG13541) were also significantly downregulated in the presence of Wolbachia. Furthermore, 98 genes were not expressed when ocn was knocked down in testes. These results suggest that ocn plays a crucial role in male germ cell development in Drosophila, possibly by regulating the expression of multiple spermatogenesis-related genes. Our data provide important information to help understand the molecular regulatory mechanisms underlying spermatogenesis.

Molecular cloning and characterization of a short peptidoglycan recognition protein from silkworm Bombyx mori.

Peptidoglycan is the major bacterial component recognized by the insect immune system. Peptidoglycan recognition proteins (PGRPs) are a family of pattern-recognition receptors that recognize peptidoglycans and modulate innate immune responses. Some PGRPs retain N-acetylmuramoyl-L-alanine amidase (Enzyme Commission number: 3.5.1.28) activity to hydrolyse bacterial peptidoglycans. Others have lost the enzymatic activity and work only as immune receptors. They are all important modulators for innate immunity. Here, we report the cloning and functional analysis of PGRP-S4, a short-form PGRP from the domesticated silkworm, Bombyx mori. The PGRP-S4 gene encodes a protein of 199 amino acids with a signal peptide and a PGRP domain. PGRP-S4 was expressed in the fat body, haemocytes and midgut. Its expression level was significantly induced by bacterial challenges in the midgut. The recombinant PGRP-S4 bound bacteria and different peptidoglycans. In addition, it inhibited bacterial growth and hydrolysed an Escherichia coli peptidoglycan in the presence of Zn2+ . Scanning electron microscopy showed that PGRP-S4 disrupted the bacterial cell surface. PGRP-S4 further increased prophenoloxidase activation caused by peptidoglycans. Taken together, our data suggest that B. mori PGRP-S4 has multiple functions in immunity.

Cloning and functional identification of moricins from the diamondback moth, Plutella xylostella (L.).

Antimicrobial peptides (AMPs) are small-molecule peptides that play crucial roles in insect innate immune responses. To better understand the function of AMPs in Plutella xylostella, one of the main pests of cruciferous vegetables, three full-length cDNAs encoding moricins were cloned from Pl. xylostella. Two variants of the moricin named PxMor2 and PxMor3 were heterologously expressed and purified. A secondary structure analysis using circular dichroism demonstrated that the two peptides adopted an α-helical structure in the membrane-like environment, but in aqueous solution, they were present in random coiled conformation. Antimicrobial activity assays demonstrated that PxMor2 exhibited high activity against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli; however, PxMor3 only demonstrated high activity against E. coli. Scanning electron microscopy and confocal laser-scanning microscopy analyses suggest that PxMors can lead to the disruption of bacterial membrane, which might be the mechanism by which PxMors inhibit bacterial growth. This study contributes to the understanding of Pl. xylostella AMPs and immune responses, and also enriches the knowledge of insect moricin.

Interleukin-27 augments the inhibitory effects of sorafenib on bladder cancer cells

Both sorafenib and interleukin-27 (IL-27) are antineoplastic drugs. This study aimed to investigate the synergistic effect of these two drugs on bladder cancer cells. HTB-9 and T24 cells were stimulated with IL-27 (50 ng/mL), sorafenib (2 μM) or the synergistic action of these two drugs. The cells without treatment acted as control. Cell proliferation, apoptosis and invasion were measured by bromodeoxyuridine assay, flow cytometry and modified Boyden chamber, respectively. Simultaneously, both modified Boyden chamber and scratch assay were used to assess cell migration. Finally, the phosphorylation levels of key kinases in the Akt/mechanistic target of rapamycin (mTOR)/mitogen-activated protein kinase (MAPK) pathway, and expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 were detected by western blot analysis. Stimulation with IL-27 or sorafenib repressed proliferation, migration and invasion but promoted apoptosis, and the effects were all enhanced by the combination of these two drugs in HTB-9 cells. The effect of the combined treatment on bladder cancer cells was verified in T24 cells. Additionally, the phosphorylation levels of AKT, mTOR and MAPK as well as the expression levels of MMP-2 and MMP-9 were all decreased by a single treatment of IL-27 or sorafenib, and further decreased by the combined treatment of these two drugs. The combination of IL-27 and sorafenib inhibited proliferation, migration and invasion and promoted apoptosis of bladder cancer cells compared with mono-drug treatment. Additionally, the AKT/mTOR/MAPK pathway might be implicated in the functional effects by down-regulations of MMP-2 and MMP-9.

Association between daily cigarette consumption and hypertension moderated by CYP2A6 genotypes in Chinese male current smokers.

The purpose of this study was to assess whether cytochrome P450 enzyme 2A6 (CYP2A6) genotypes moderate the association between smoking and hypertension. In this study, 954 Chinese male current smokers from a community-based chronic disease screening project in Guangzhou were interviewed with a structured questionnaire about socio-demographic status, smoking and other health-related behaviors. Blood was collected for DNA extraction and CYP2A6 genotyping. Hypertension was defined according to 2007 ESH-ESC Practice Guidelines. A multivariate logistic regression was performed to examine the interaction between smoking quantity and CYP2A6 genotypes on hypertension after adjusting for age, education level and other potential confounders. Multivariate analyses indicated that smoking more than 15 cigarettes per day significantly increased the risk of hypertension (odds ratio (OR)=1.59, 95% confidence interval (CI)=1.21-2.10) compared with smoking 1-15 cigarettes per day, and further suggested that smoking interacted with normal CYP2A6 metabolizer genotype to increase the risk of hypertension. Smokers consuming more than 15 cigarettes per day with normal CYP2A6 metabolizer genotypes had the highest risk of hypertension (OR=2.04, 95% CI=1.11-3.75) compared with those consuming 1-15 cigarettes per day with slower CYP2A6 metabolizer genotypes. These findings demonstrated that smoking quantity was positively associated with hypertension and that CYP2A6 genotypes may moderate this relationship.

Prevalence and determinants of diabetes and impaired fasting glucose among urban community-dwelling adults in Guangzhou, China.

AIM: This study aimed to estimate the prevalence of diabetes and impaired fasting glucose (IFG) in the adult population aged >or= 20 years in Guangzhou and to evaluate the associated risk factors. METHOD: A total of 6197 randomly selected adults, aged >or= 20 years and living for at least 5 years in Guangzhou, participated in questionnaire-based interviews between 2006 and 2007, and had their clinical characteristics and standard blood chemistries measured. A 75 g OGTT was conducted for those subjects with fasting glucose levels >or= 5.6 mmol/L. Diabetes and IFG were defined according to WHO 1999 criteria. RESULTS: Based on Chinese census data, the age- and gender-standardized prevalences of diabetes and IFG were 5.5% and 3.3%, respectively. Among the identified diabetic individuals in the present investigation, 42.3% were newly diagnosed. The prevalence of diabetes and IFG increased with age. The results of multivariate logistic-regression analyses showed that diabetes and IFG were significantly associated with age, a family history of diabetes, obesity, hypertension and hyperlipidaemia. CONCLUSION: The prevalences of diabetes and IFG have increased dramatically over the past decade. Yet, a large proportion of cases go undiagnosed. These results suggest an urgent need to establish regular population-based diabetes screening in Guangzhou.

Improved GFR estimation by combined creatinine and cystatin C measurements.

Plasma creatinine may not reflect glomerular filtration rate (GFR) especially in the early stages of chronic kidney disease (CKD). Plasma cystatin C (cysC), however, has the potential to more accurately determine early GFR reduction. We sought to improve the creatinine-based GFR estimation by including cysC measurements. We derived a reference GFR from standard dual plasma sampling (99m)Tc-DTPA clearance in a training cohort of 376 randomly selected adult Chinese patients with CKD. We compared reference values to estimated GFR and applied multiple regression models to one equation based solely on cysC, and to another combining plasma creatinine (Pcr) and cysC measurements of the training cohort. The results were validated by testing an additional 191 patients. The difference, precision, and accuracy of the two estimates were compared with the modified Modification of Diet in Renal Disease (MDRD) equation for Chinese patients, and another estimate combining cysC and modified MDRD calculations. The estimated GFR combining Pcr and cysC measurements more accurately matched the reference GFR at all stages of CKD than the other equations, particularly in patients with near-normal kidney function.

Molecular diversity of tobacco vein banding mosaic virus.

Tobacco vein banding mosaic virus (TVBMV) is of increasing importance in tobacco production on the Chinese mainland. The 3'-terminal genomic sequences (1624 nucleotides) of 12 TVBMV isolates from China were determined and compared to the sequences of only four TVBMV isolates available in databanks. The results revealed that TVBMV consists of several phylogenetically distinguishable strains that show a degree of correlation with the geographical origin. Two isolates from Yunnan had a unique putative NIb/CP proteolytic cleavage site of Q/N that is uncommon for potyviruses, whereas other TVBMV isolates had the more typical Q/G amino acids at that site. One isolate (ZB6) from Zibo, Shandong Province, was predicted to have experienced recombination within the characterized genomic region.

Role of ATP-binding cassette drug transporters in the intestinal absorption of tanshinone IIB, one of the major active diterpenoids from the root of Salvia miltiorrhiza.

There is an increasing use of herbal medicines worldwide, and the extracts from the root of Salvia miltiorrhiza are widely used in the treatment of angina and stroke. In this study, we investigated the mechanism for the intestinal absorption of tanshinone IIB (TSB), a major constituent of S. miltiorrhiza. The oral bioavailability of TSB was about 3% in rats with less proportional increase in its maximum plasma concentration (C(max)) and area under the plasma concentration-time curve (AUC) with increasing dosage. The time to C(max) (T(max)) was prolonged at higher oral dosage. In a single pass rat intestinal perfusion model, the permeability coefficients (P(app)) based on TSB disappearance from the lumen (P(lumen)) were 6.2- to 7.2-fold higher (p < 0.01) than those based on drug appearance in mesenteric venous blood (P(blood)). The uptake and efflux of TSB in Caco-2 cells were also significantly altered in the presence of an inhibitor for P-glycoprotein (PgP) or for multi-drug resistance associated protein (MRP1/2). TSB transport from the apical (AP) to basolateral (BL) side in Caco-2 monolayers was 3.3- to 5.7-fold lower than that from BL to AP side, but this polarized transport was attenuated by co-incubation of PgP or MRP1/2 inhibitors. The P(app) values of TSB in the BL-AP direction were significantly higher in MDCKII cells over-expressing MDR1 or MRP1, but not in cells over-expressing MRP2-5, as compared with the wild-type cells. The plasma AUC(0-24hr) in mdr1a and mrp1 gene-deficient mice was 10.2- to 1.7-fold higher than that in the wild-type mice. Furthermore, TSB significantly inhibited the uptake of digoxin and vinblastine in membrane vesicles containing PgP or MRP1. TSB also moderately stimulated PgP ATPase activity. Taken collectively, our findings indicate that TSB is a substrate for PgP and MRP1 and that drug resistance to TSB therapy and drug interactions may occur through PgP and MRP1 modulation.

Immulectin-4 from the tobacco hornworm Manduca sexta binds to lipopolysaccharide and lipoteichoic acid.

Insect C-type lectins function as pattern recognition receptors in innate immunity. In the tobacco hornworm Manduca sexta, we have previously isolated three C-type lectins named immulectins, which are involved in innate immune responses. Here, we report a new member of the immulectin family, immulectin-4 (IML-4). IML-4 mRNA was detected in the fat body of control larvae and was induced in the fat body when larvae were injected with bacteria. Recombinant IML-4 bound to bacterial lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and the binding activity was not affected by addition of calcium or EGTA. IML-4 agglutinated bacteria and yeast, and agglutination of Escherichia coli by IML-4 was concentration- and calcium-dependent. IML-4 also enhanced haemocyte encapsulation and melanization.

Pattern recognition proteins in Manduca sexta plasma.

Recognition of nonself is the first step in mounting immune responses. In the innate immune systems of both vertebrates and arthropods, such recognition, termed pattern recognition, is mediated by a group of proteins, known as pattern recognition proteins or receptors. Different pattern recognition proteins recognize and bind to molecules (molecular patterns) present on the surface of microorganisms but absent from animals. These molecular patterns include microbial cell wall components such as bacterial lipopolysaccharide, lipoteichoic acid and peptidoglycan, and fungal beta-1,3-glucans. Binding of pattern recognition proteins to these molecular patterns triggers responses such as phagocytosis, nodule formation, encapsulation, activation of proteinase cascades, and synthesis of antimicrobial peptides. In this article, we describe four classes of pattern recognition proteins, hemolin, peptidoglycan recognition protein, beta-1,3-glucan recognition proteins, and immulectins (C-type lectins) involved in immune responses of the tobacco hornworm, Manduca sexta.