Characteristics of bacterial community in eyelashes of patients with Demodex blepharitis.

BACKGROUND: Demodex blepharitis (DB) is a common disease of the ocular surface. The characteristics of the bacterial community in eyelash roots after Demodex infestation are still unknown. Knowledge of the characteristics of the bacterial community of eyelash follicles in patients with DB can provide valuable insights for guiding the diagnosis and treatment of DB. METHODS: Twenty-five patients with DB (DB group) and 21 non-DB volunteers (control group) were enrolled in the study. Eyelashes from the upper eyelid of the right eye were sampled, and 16S ribosomal DNA (rDNA) sequencing was performed to determine the V3-V4 regions of the microbial 16S rDNA gene within 1 month of infestation. The sequencing data of the two groups were analyzed and compared. The effect of the bacterium Burkholderia on the survival of Demodex mites was evaluated using Demodex obtained from 12 patients with DB other that the patients in the DB group. RESULTS: A total of 31 phyla and 862 genera were identified in the DB and control groups. The five most abundant phyla in the two groups were Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Cyanobacteria. The abundance of Actinomycetes was significantly higher in the DB group than in the control group. At the genus level, the five most abundant genera in the two groups were Pseudomonas, Burkholderia-Caballeronia-Paraburkholderia, Rolstonia and Acinetobacter; Clostridium sensu stricto 1 was abundant in the control group and Corynebacterium_1 was abundant in the DB group. Compared with the control group, the abundance of Burkholderia-Caballeronia-Paraburkholderia was 2.36-fold lower in the DB group. Linear discriminant analysis Effect Size (LEfSe) analysis revealed Burkholderia-Caballeronia-Paraburkholderia, SC_I_84_unclassified, Nonmyxobacteria and Succinvibrio to be the major biomarkers in the control group and Catenibacterium and Lachnospiraceae NK4A136 group to be the major biomarkers in the DB group. To explore the performance of these optimal marker models, receiver operational characteristic curve analysis was performed, and the average area under the curve value of Burkholderia-Caballeronia-Paraburkholderia was 0.7448. Burkholderia cepacia isolated from normal human eyelashes was fermented, and the Demodex mites isolated from patient eyelashes were cultured together with its fermented supernatant. The results showed that the fermentation supernatant could significantly reduce the survival time of the Demodex mites, suggesting the potential therapeutic value of this bacterium against Demodex. CONCLUSIONS: The composition of the bacterial community in the eyelashes of DB patients differed from that in eyelashes of healthy volunteers, revealing a decrease in bacterial diversity in infested eyelashes. This decrease may be related to the occurrence and development of DB. The supernatant of Burkholderia cepacia culture medium was found to inhibit the growth of Demodex in eyelash hair follicles, providing a new insight with potential applications for the clinical treatment of Demodex infestation.

PDCD5 as a Potential Biomarker for Improved Prediction of the Incidence and Remission for Patients with Rheumatoid Arthritis.

INTRODUCTION: Rheumatoid arthritis (RA) often involves an altered T-cell subpopulation, higher levels of inflammatory cytokines, and auto-antibodies. This study investigated whether PDCD5 could be a biomarker to predict the incidence and remission of RA so as to guide the therapeutic management of clinical RA. METHODS: One hundred fifty-two patients (41 being in both active status and stable remission status) who were newly diagnosed with RA and 38 healthy controls were enrolled. Basic clinical data were collected before using blood samples remaining in the clinic after routine complete blood count. The ability of PDCD5 and important indicators to predict the remission of RA was estimated based on receiver operating characteristic curve (ROC) analysis. RESULTS: PDCD5 expression was found to be significantly increased in RA patients in active status in comparison with healthy controls or those in stable remission status. Compared with anti-CCP, ESR and DAS28 score, PDCD5 was of better predictive value with an AUC of 0.846 (95% CI 0.780-0.912) for RA remission. The incidence risk of RA increased with higher levels of PDCD5 (OR = 1.73, 95% CI = 1.45-1.98, P = 0.005) in multiple logistic regression analysis, with the risk increasing by 2.94-times for high-risk group in comparison with low-risk group (OR = 2.94, 95% CI = 2.35-4.62, P < 0.001). The association between PDCD5 and RA remission showed a similar result. For correlation analysis, significant associations were eventually found between PDCD5 and indicated genes (FOXP3, TNF-α, IL-17A, IFN-γ and IL-6) as well as several important clinical parameters including IgG, RF, CRP, ESR, anti-CCP and DAS28 score. CONCLUSIONS: This study suggested that increased PDCD5 expression was significantly linked to the incidence and remission of RA. PDCD5 may be used as a novel biomarker for the prediction of RA incidence and remission, especially due to its potential involvement in the development of the condition.

Gibson assembly interposition improves amplification efficiency of long DNA and multifragment overlap extension PCR.

For difficult overlap extension PCR, a Gibson assembly process was inserted between the two PCR rounds to facilitate the formation of complete gene templates at a moderate temperature. That is, after amplifying each DNA fragment, they were preluded by a Gibson assembly process in equal proportion. Then, the assembled mixture was used as a template for the second PCR round. This idea was tested and verified by taking the cloning example of a single and a double site mutation of the retinoblastoma gene. This scheme associates overlap extension PCR with Gibson assembly exquisitely, significantly improving gene amplification efficiency, particularly in the fusion of long genes and multifragments using overlap extension PCR.

Kirenol inhibits inflammation challenged by lipopolysaccharide through the AMPK-mTOR-ULK1 autophagy pathway.

Kirenol is a bioactive substance isolated from Herba Siegesbeckiae. Although the anti-inflammatory activity of kirenol has been well documented, its role in autophagy remains unknown. The present study aimed to investigate the protective role of kirenol on inflammation challenged by lipopolysaccharide (LPS) in acute lung injury (ALI) cell and mouse models and unravel the underlying mechanisms, with a particular focus on autophagy. For this purpose, an ALI cell and mouse models were established, and the effects of kirenol on the expression of molecules related to inflammation and autophagy were examined. The present results revealed that kirenol could significantly inhibit inflammatory cytokines secretion in cells and in the mice injured by LPS; this effect may be attributed to enhanced autophagy as evidenced by the up-regulation of LC3-II and the down-regulation of p62 both in vitro and in vivo. Phosphorylated AMPK and ULK1 increased, while phosphorylated mTOR decreased in the kirenol-treated ALI cell model. Moreover, inhibition of autophagy using AMPK inhibitor or 3-MA or chloroquine (CQ) reversed the anti-inflammatory and autophagy-enhancement effects of kirenol exposure in vitro, indicating that kirenol could enhance autophagy by activating the AMPK-mTOR-ULK1 pathway. The results of RNA sequencing suggested that kirenol was strongly related to the biological functions of acute inflammatory response and the AMPK signaling pathway. Further in vivo ALI mouse model studies demonstrated the protective role of kirenol against lung inflammation, such as improved histopathology, decreased lung edema, and leukocyte infiltration were abolished by 3-MA. These findings implicate that kirenol can inhibit LPS-induced inflammation via the AMPK-mTOR-ULK1 autophagy pathway.

Delineation of the bacterial composition in exogenous endophthalmitis using 16S rDNA sequencing.

PURPOSE: To determine the bacterial spectrum of exogenous endophthalmitis of different origins, namely, posttraumatic, postcataract surgery, filtering bleb-associated, and intravitreal treatment-related endophthalmitis, using the 16S rDNA sequencing method. METHODS: Aqueous humor or vitreous humor samples were collected from 24 endophthalmitis patients. Traditional cultivation and 16S rDNA sequencing were conducted with these samples. Three senile cataract controls and one intraocular irrigating solution were used as sequencing control. RESULTS: Eleven of the 24 samples (45.8%) obtained positive bacterial cultivation, and each sample positive for only one species. The 11 culture-positive species could all be identified in their corresponding sequencing results, but only four strains being the top one pathogen in the sequencing. A total of 567 species were isolated using 16S rDNA sequencing, with the top five species being Pseudomonas sp., Staphylococcus epidermidis, Staphylococcus sp., Streptococcus sp., and Enterococcus faecalis. The dominant bacterial strains varied among the different endophthalmitis categories but with no significant difference in the overall bacterial spectrum. Bacterial atlas containing Pseudomonas, Streptococcus, Staphylococcus, Actinomycetales_unclassified, Thermus, and Janibacter was shared by the four categories. Aqueous humor bacterial profile showed a higher overlap with contaminating bacteria from the environment. CONCLUSIONS: 16S rDNA sequencing is more efficient for endophthalmitis pathogen screening than the traditional cultivation method in terms of positive detection rate and the number of bacteria identified. But the risk of environmental contamination exists when using 16S rDNA sequencing method for endophthalmitis diagnosis. Different categories of endophthalmitis displayed diversified bacterial composition.

TM9SF1 knockdown decreases inflammation by enhancing autophagy in a mouse model of acute lung injury.

TM9SF1 is a member of the TM9SF (Transmembrane 9 Superfamily Member) family, which usually has a long N-terminal extracellular region and nine transmembrane domains. TM9SF1's biological function and mechanisms in inflammation are yet unknown. Tm9sf1 was shown to be upregulated in the lung tissues of mice suffering from LPS-induced acute lung injury (ALI). Tm9sf1 knockout mice were studied, and it was shown that Tm9sf1 knockout significantly alleviated LPS-induced ALI, as evidenced by higher survival rate, improved pulmonary vascular permeability, decreased inflammatory cell infiltration, and downregulated inflammatory cytokines. TM9SF1 was also demonstrated to be a negative regulator of autophagy in the LPS-induced ALI model in vitro and in vivo. The autophagy inhibitor 3-MA could counteract the beneficial effects of Tm9sf1 knockout on ALI. Therefore, we discover for the first time the role and mechanism of TM9SF1 in LPS-induced ALI and establish a relationship between TM9SF1 regulated autophagy and ALI progression, which may provide novel targets for the treatment of ALI.

[Using multiple-fragment amplification combined with Gibson assembly to clone genes with site-directed mutations].

In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.

Preparation and in vitro/in vivo evaluations of novel ocular micelle formulations of hesperetin with glycyrrhizin as a nanocarrier.

The purpose of this study was to explore the potential of formulating hesperetin into an ophthalmic solution with dipotassium glycyrrhizinate (DG) as a micelle nanocarrier. A DG-based micelle ophthalmic solution encapsulating hesperetin (DG-Hes) was developed and its in vitro/in vivo characterizations were evaluated. The optimal formulation featured a DG/hesperetin (Hes) weight ratio of 12:1 and an encapsulation efficiency of 90.4 ± 1.7%; The optimized DG-Hes was characterized as small uniform spheres with an average micelle size of 70.93 ± 3.41 nm, a polydispersity index of 0.11 ± 0.02, and an electrically negative surface (-36.12 ± 2.79 mV). The DG-Hes ophthalmic solution had good tolerance in rabbit eyes. DG-Hes significantly improved the in vitro passive permeation, ex vivo corneal permeation, and in vivo ocular bioavailability of Hes. DG-Hes showed markedly increases in in vitro antioxidant activity. In vitro antibacterial activity tests revealed a lower minimum inhibitory concentration and lower minimum bactericidal concentration for DG-Hes ophthalmic solution were lower than for free Hes. DG-Hes ophthalmic solution also significantly reduced symptoms of eye infection in the rabbit bacterial keratitis model when compared to a Hes suspension. These results suggest that DG-Hes eye drops may be useful as a new ophthalmic preparation for the treatment of ocular diseases, especially bacterial ophthalmopathy.

RNA-Seq analysis of differentially expressed genes of Staphylococcus epidermidis isolated from postoperative endophthalmitis and the healthy conjunctiva.

Staphylococcus epidermidis (S. epidermidis) is one of the primary pathogens in postoperative endophthalmitis, which is a devastating complication of cataract surgery and often results in irreversible visual loss and even blindness. Meanwhile, it is the most frequently isolated commensal bacterium in the healthy conjunctiva. In this study, we investigated the differentially expressed genes (DEGs) of S. epidermidis isolated from the patients with postoperative endophthalmitis and the healthy conjunctiva to predict their functions and pathways by Illumina high-throughput RNA sequencing. Using genome-wide transcriptional analysis, 281 genes (142 upregulated and 139 downregulated genes) were found to be differentially expressed (fold change ≥ 2, p ≤ 0.05) in the strains from endophthalmitis. Ten randomly selected DEGs were further validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). GO enrichment analysis suggested that more DEGs were associated with the thioredoxin system and iron ion metabolism. KEGG pathway analysis revealed that more DEGs were associated with the pathways of the two-component system and pyruvate metabolism. Moreover, the gene SE1634 code for staphylococcal toxin was significantly upregulated in S. epidermidis strains of the endophthalmitis, which might be directly responsible for the pathogenesis of endophthalmitis. In conclusion, this research is helpful for further investigations on genes or pathways related with the pathogenesis and therapeutic targets of S. epidermidis endophthalmitis.

[Site-directed mutagenesis of long gene by partial amplification combining with double fragments ligation].

Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.

Ocular bacterial infections at a tertiary eye center in China: a 5-year review of pathogen distribution and antibiotic sensitivity.

AIM: To provide statistical evidence for the use of antibiotics in ophthalmology by assessing the distribution and antibiotic sensitivity of bacterial isolates from ocular specimens with suspected microbial infections. METHODS: This study applied a retrospective analysis of 3690 bacterial isolates from ocular specimens, which were obtained from the conjunctiva, cornea, aqueous humor, vitreous body, and other ocular sites of the patients at Shandong Eye Institute in northern China from January 2013 to December 2017. The parameters assessed mainly included the distribution of isolated bacteria and the results of susceptibility tests for antibiotics. In the analysis of antibiotic sensitivities, the bacteria were divided into four groups according to gram staining, and statistical methods were used to compare their antibiotic sensitivities. RESULTS: Among the 3690 isolated bacterial strains, Staphylococcus epidermidis (2007, 54.39%) accounted for the highest proportion. As for the total isolates, their sensitivity rate to gatifloxacin was up to 90.01%, with four types of gram-stained bacteria being all highly sensitive to it, but their sensitivity rate to levofloxacin was only 51.91%. The sensitivity rate of gram-negative bacilli (G-B) to levofloxacin was 83.66%, significantly higher than the other three types of gram-stained bacteria (P<0.05). Gram-positive cocci (G+C, 97.95%) and gram-positive bacilli (G+B, 97.54%) were more sensitive to vancomycin than gram-negative cocci (G-C, 70.59%) and G-B (68.57%; P<0.05). For fusidic acid, the sensitivity rates of G+C (89.83%) and G+B (73.37%) were significantly higher than that of G-B (29.83%; P<0.05). The gram-negative bacteria's sensitivity rate to cefuroxime was as low as 59.25%, but only G-B was less sensitive to cefuroxime (57.28%), while G-C was still highly sensitive (89.29%). The sensitivity rate of gram-positive bacteria to moxifloxacin was as high as 80.28%, but only G+C was highly sensitive to moxifloxacin (81.21%), while G+B was still less sensitive (32.00%). CONCLUSION: Staphylococcus epidermidis is the predominant isolate in all ocular specimens with bacteria. Gatifloxacin is more suitable for topical prophylactic use than levofloxacin in ophthalmology when necessary. Vancomycin and fusidic acid both have better effects on gram-positive bacteria than gram-negative bacteria. More accurate antibiotic sensitivity analysis results can be obtained when a more detailed bacterial classification and more appropriate statistical methods are performed.

Synthesis of uniform Ag nanosponges and its SERS application.

With the aid of amino acid, various Ag nanostructures were successfully synthesized via the reaction between silver nitrate and hydrazine hydrate at room temperature. The as-prepared products were characterized by X-ray diffraction and scanning electron microscopy. It was found that the morphology of the as-prepared Ag products depended on the sorts of amino acid and solvents. The uniform Ag nanosponges could be obtained in glycol with aid of glycine. Using rhodamine 6G (R6G) as probe, the surface-enhanced Raman scattering (SERS) performance was also investigated, which showed that the uniform Ag nanosponges exhibited an intensive and enhanced Raman scattering. Pazufloxacin mesilate (PM) were detected conveniently using these uniform nanosponges as SERS substrates. The present work might afford some guidance for the rationally controllable synthesis of other metal nanomaterials.

Construction of (3,6)-connected polyoxometalate-based metal-organic frameworks (POMOFs) from triangular carboxylate and dimerized Zn4-ε-Keggin.

Using the methodology of extension of reduced transition metal-grafted ε-Keggin polyoxoanions with two types of terphenyl-based tricarboxylates of H3L1 (3,5',3''-position substitution) and H3L2 (4,5',4''-position substitution) we isolated two (3,6)-connected 3D polyoxometalate-based metal-organic frameworks, [TBA]3[H3PMo12O40][Zn4L2] (1, YZU-105), and [TPA]3[H3PMo12O40][Zn4L1]·0.5H2O (2, YZU-106) (H3L1 = [1,1';3',1''-terphenyl]-3,5',3''-tricarboxylic acid; H3L2 = [1,1';3',1''-terphenyl]-4,5',4''-tricarboxylic acid; TBA = tetrabutylammonium; TPA = tetrapropylammonium). In both compounds, the building block was the dimerized form of Zn4-{ε-H3PMo12O40}. Such dimerization left six anchoring points for each dimer and, as a result, a 6-connected node was formed. Compounds 1 and 2 exhibited topologies of (4·85)3(4·82)6 and (65·10)3(63)6, respectively. This work illustrates that use of tri-carboxylate substitutions in different positions (3,5',3''-position/4,5',4''-position) in tripodal terphenyl-based ligands allows different extents of twisting of the peripheral aromatic ring with respect to the central ring, thereby giving rise to different extending directions and symmetries.

CDK4 protein is degraded by anaphase-promoting complex/cyclosome in mitosis and reaccumulates in early G1 phase to initiate a new cell cycle in HeLa cells.

CDK4 regulates G1/S phase transition in the mammalian cell cycle by phosphorylating retinoblastoma family proteins. However, the mechanism underlying the regulation of CDK4 activity is not fully understood. Here, we show that CDK4 protein is degraded by anaphase-promoting complex/cyclosome (APC/C) during metaphase-anaphase transition in HeLa cells, whereas its main regulator, cyclin D1, remains intact but is sequestered in cytoplasm. CDK4 protein reaccumulates in the following G1 phase and shuttles between the nucleus and the cytoplasm to facilitate the nuclear import of cyclin D1. Without CDK4, cyclin D1 cannot enter the nucleus. Point mutations that disrupt CDK4 and cyclin D1 interaction impair the nuclear import of cyclin D1 and the activity of CDK4. RNAi knockdown of CDK4 also induces cytoplasmic retention of cyclin D1 and G0/G1 phase arrest of the cells. Collectively, our data demonstrate that CDK4 protein is degraded in late mitosis and reaccumulates in the following G1 phase to facilitate the nuclear import of cyclin D1 for activation of CKD4 to initiate a new cell cycle in HeLa cells.

[HBV genotypes distribution and YMDD spontaneous mutation in Qingdao population].

OBJECTIVE: To investigate the distribution of hepatitis B virus (HBV) genotypes in Qingdao, and the relationship of HBV genotypes with the serum HBV-DNA levels and HBV YMDD spontaneous mutation of patients, then to discuss the clinical significance. METHODS: Hepatitis B virus genotypes and YMDD spontaneous mutation of 144 patients were detected by real time PCR (Taqman probe), then the results were analyzed by statistical method. RESULTS: Of the 144 patients, 130 (90.3%) were genotype C, 12 (8.3%) were genotype B, and 2 (1.4%) were neither genotype B nor genotype C; 33 (22.9%) were detected to have YMDD mutation, and 25 (75.5%) were YVDD positive, 3 (9.1%) were YIDD positive, 5 (15.2%) were YVDD and YIDD positive. There were no significant differences between clinical diagnosis, serum HBV-DNA levels, YMDD spontaneous mutation and HBV genotypes (P > 0.05). CONCLUSION: Genotype C is the dominant position for HBV genotype in Qingdao. Untreated patients with chronic hepatitis B have YMDD spontaneous mutation. HBV genotypes have no association with YMDD spontaneous mutation and the development of diseases.

[Relationship between polymorphism sites of IRF5, TLR-9 and SLE patients in Shandong Han population].

OBJECTIVE: To investigate two single nucleotide polymorphism sites of IRF5 and TLR-9 and to detect their relationship with SLE (systemic lupus erythematosus) in a Han population from Shandong province. METHODS: The polymorphisms of rs2004640 G/T, rs10954213 G/A in IRF-5 and rs187084C/T, rs352139A/G in TLR-9 were detected with PCR-RFLP in 92 cases of SLE and 88 healthy controls. The genotype and allele frequencies were calculated and analyzed. RESULTS: (1) The genotype frequencies of GG, GT and TT in rs2004640 site in SLE were 0.198, 0.521 and 0.281 respectively. The difference was significant between SLE and controls (chi(2) = 8.73, P < 0.05); the genotype frequencies of GG, GA and AA at rs109542130 site in SLE were 0.318, 0.409 and 0.273 respectively. The difference was significant between SLE and controls (chi(2) = 6.36, P < 0.05). (2) The genotype frequencies of CC, CT and TT at rs187084 site in SLE were 0.185, 0.413 and 0.402 respectively. There was no difference between SLE and controls (chi(2) = 2.99, P > 0.05); the genotype frequencies of AA, AG and GG at rs352139 site in SLE were 0.228, 0.533 and 0.239 respectively. There was no difference between SLE and controls (chi(2) = 4.54, P > 0.05). CONCLUSION: The polymorphism of rs2004640 and rs10954213 in IRF5 may be associated with SLE in the population of Han nationality from Shandong province. However, the polymorphism of rs187084 and rs352139 in TLR-9 is not associated with SLE.