Performance enhancement, bacterial communities optimization and emerging pollutants elimination by microalgal-bacterial consortium for treating aquaculture pond sediments.

Aquaculture pond sediments have a notable influence on the ecosystem balance and farmed animal health. In this study, microalgal-bacterial immobilization (MBI) was designed to improve aquaculture pond sediments via synergistic interactions. The physicochemical characteristics, bacterial communities, and the removal efficiencies of emerging pollutants were systematically investigated. The consortium containing diatom Navicula seminulum and Alcaligenes faecalis was cultivated and established in the free and immobilized forms for evaluating the treatment performance. The results indicated that the immobilized group exhibited superior performance in controlling nutrient pollutants, shaping and optimizing the bacterial community compositions with the enrichment of functional bacteria. Additionally, it showed a stronger positive correlation between the bacterial community shifts and nutrient pollutants removal compared to free cells. Furthermore, the immobilized system maintained the higher removal performance of emerging pollutants (heavy metals, antibiotics, and pathogenic Vibrios) than free group. These findings confirmed that the employment of immobilized N. seminulum and A. faecalis produced more synergistic benefits and exerted more improvements than free cells in ameliorating aquaculture pond sediments, suggesting the potential for engineering application of functional microalgal-bacterial consortium in aquaculture.

Dimethyl Bisphenolate Ameliorates Carbon Tetrachloride-Induced Liver Injury by Regulating Oxidative Stress-Related Genes.

Liver disease accounts for millions of deaths per year all over the world due to complications from cirrhosis and liver injury. In this study, a novel compound, dimethyl bisphenolate (DMB), was synthesized to investigate its role in ameliorating carbon tetrachloride (CCl4)-induced liver injury through the regulation of oxidative stress-related genes. The structure of DMB was confirmed based on its hydrogen spectrum and mass spectrometry. DMB significantly reduced the high levels of ALT, AST, DBIL, TBIL, ALP, and LDH in a dose-dependent manner in the sera of CCl4-treated rats. The protective effects of DMB on biochemical indicators were similar to those of silymarin. The ROS fluorescence intensity increased in CCl4-treated cells but significantly weakened in DMB-treated cells compared with the controls. DMB significantly increased the content of oxidative stress-related GSH, Nrf2, and GCLC dose-dependently but reduced MDA levels in CCl4-treated cells or the liver tissues of CCl4-treated rats. Moreover, DMB treatment decreased the expression levels of P53 and Bax but increased those of Bcl2. In summary, DMB demonstrated protective effects on CCl4-induced liver injury by regulating oxidative stress-related genes.

Characterization of a novel sucrose phosphorylase from Paenibacillus elgii and its use in biosynthesis of α-arbutin.

α-Arbutin, a naturally occurring glycosylated derivative of hydroquinone (HQ), effectively inhibits melanin biosynthesis in epidermal cells. It is widely recognized as a fourth-generation whitening agent within the cosmetic industry. Currently, enzymatic catalysis is universally deemed the safest and most efficient method for α-arbutin synthesis. Sucrose phosphorylase (SPase), one of the most frequently employed glycosyltransferases, has been extensively reported for α-arbutin synthesis. In this study, a previously reported SPase known for its effectiveness in synthesizing α-arbutin, was used as a probe sequence to identify a novel SPase from Paenibacillus elgii (PeSP) in the protein database. The sequence similarity between PeSP and the probe was 39.71%, indicating a degree of novelty. Subsequently, the gene encoding PeSP was coexpressed with the molecular chaperone pG-Tf2 in Escherichia coli, significantly improving PeSP's solubility. Following this, PeSP was characterized and employed for α-arbutin biosynthesis. The specific activity of co-expressed PeSP reached 169.72 U/mg, exhibited optimal activity at 35℃ and pH 7.0, with a half-life of 3.6 h under the condition of 35℃. PeSP demonstrated excellent stability at pH 6.5-8.5 and sensitivity to high concentrations of metal ions. The kinetic parameters Km and kcat/Km were determined to be 14.50 mM and 9.79 min- 1·mM- 1, respectively.The reaction conditions for α-arbutin biosynthesis using recombinant PeSP were optimized, resulting in a maximum α-arbutin concentration of 52.60 g/L and a HQ conversion rate of 60.9%. The optimal conditions were achieved at 30℃ and pH 7.0 with 200 U/mL of PeSP, and by combining sucrose and hydroquinone at a molar ratio of 5:1 for a duration of 25 h.

Cardiac performance and heat shock response variation related to shell colour morphs in the mudflat snail Batillaria attramentaria.

Gastropods exhibit remarkable variation in shell colour within and among populations, but the function of shell colour is often not clear. In the present study, body temperature in the field and physiological and transcriptomic responses to thermal stress were investigated in different shell colour morphs of the mudflat snail Batillaria attramentaria. Using biomimetic models, we found that the body temperature of snails with a dark unbanded shell (D-type morph) was slightly higher than that of snails with a white line on the upper side of each whorl (UL-type morph) when exposed to sunlight. Despite no differences in upper lethal temperature among shell colour morphs, their Arrhenius breakpoint temperature (ABT) for cardiac thermal performance differed significantly, and the ABT of snails with the D-type morph was higher than that of snails with the UL-type morph. Transcriptomic analysis showed that D-type snails exhibit higher levels of four heat shock proteins (HSPs) than UL-type snails at control temperature. The unfolded protein response was activated in UL-type snails but not in D-type snails under moderate thermal stress. And 11 HSPs showed an increase in UL-type snails in contrast to 1 HSP in D-type snails, suggesting a 'preparative defence' strategy of the heat shock response in D-type snails under moderate thermal stress. When exposed to sublethal temperature, eight molecular chaperones were uniquely upregulated in D-type snails, suggesting these genes may allow D-type snails to improve their cardiac thermal tolerance. Our results suggest that the preparative defence strategies and higher ABT for cardiac thermal performance may allow the dark shell snails to adapt to rapid and stronger thermal stress in the field.

Identification of castration-dependent and -independent driver genes and pathways in castration-resistant prostate cancer (CRPC).

BACKGROUND: Prostate cancer (PCa) is one of the most diagnosed cancers in the world. PCa inevitably progresses to castration-resistant prostate cancer (CRPC) after androgen deprivation therapy treatment, and castration-resistant state means a shorter survival time than other causes. Here we aimed to define castration-dependent and -independent diver genes and molecular pathways in CRPC which are responsible for such lethal metastatic events. METHODS: By employing digital gene expression (DGE) profiling, the alterations of the epididymal gene expression profile in the mature and bilateral castrated rat were explored. Then we detect and characterize the castration-dependent and -independent genes and pathways with two data set of CPRC-associated gene expression profiles publicly available on the NCBI. RESULTS: We identified 1,632 up-regulated and 816 down-regulated genes in rat's epididymis after bilateral castration. Differential expression analysis of CRPC samples compared with the primary PCa samples was also done. In contrast to castration, we identified 97 up-regulated genes and 128 down-regulated genes that changed in both GEO dataset and DGE profile, and 120 up-regulated genes and 136 down-regulated genes changed only in CRPC, considered as CRPC-specific genes independent of castration. CRPC-specific DEGs were mainly enriched in cell proliferation, while CRPC-castration genes were associated with prostate gland development. NUSAP1 and NCAPG were identified as key genes, which might be promising biomarkers of the diagnosis and prognosis of CRPC. CONCLUSION: Our study will provide insights into gene regulation of CRPC dependent or independent of castration and will improve understandings of CRPC development and progression.

Functional analyses of inhibitor of apoptosis protein 1 (IAP1) of Antheraea pernyi multinucleocapsid nucleopolyhedrovirus (AnpeNPV) in viral replication and occlusion body production.

Inhibitor of apoptosis protein 1 (IAP1) of Antheraea pernyi multinucleocapsid nucleopolyhedrovirus (AnpeNPV) belongs to the baculovirus IAP1 type. The function of AnpeNPV-IAP1 in viral replication and occlusion body (OB) production remains unknown. In this study, we demonstrated that AnpeNPV-iap1 is a late gene. AnpeNPV-IAP1 mainly localizes to the nuclear ring zone and exhibits dynamic distribution in the cytoplasm and the virogenic stroma during AnpeNPV infection. AnpeNPV-IAP1 impacted the expression of a variety of viral genes at the very late phase of infection in Tn-Hi5 cells. The deletion of AnpeNPV-iap1 caused decreased expression levels of polyhedrin, morphological changes to OBs and reduced OB production in A. pernyi pupae, along with a lengthening of the lethal time of A. pernyi larvae. These results suggest that AnpeNPV-iap1 is involved in regulating viral gene expression, OB production and morphogenesis in A. pernyi.

Two Foreign Antimicrobial Peptides Expressed in the Chloroplast of Porphyridium purpureum Possessed Antibacterial Properties.

To solve the problem of antibiotic abuse in aquaculture and to utilize the application potential of antimicrobial peptides (AMPs), a chloroplast transformation system of Porphyridium purpureum was successfully constructed for effectively expressing two exogenous AMPs. The endogenous fragments of 16S rDNA/trnA-23S rDNA were used as flanking fragments for the homologous recombination in the chloroplast genome. Two AMPs encoded by the transformation vector were controlled by the native promoter psbB in a polycistron. The plasmids were transferred into P. purpureum via particle bombardment and the transformation vectors were screened using phosphinothricin (bar), a dominant selection marker under the control of the psbA promoter. Subsequently, in the positive transformed colonies, the exogenous fragments were found to be inserted in the flanking fragments directionally as expected and two foreign AMPs were successfully obtained. Finally, two exogenous peptides with antibacterial properties were obtained from the transformed strain. The two AMPs expressed by the transformed strain were shown to have similar inhibitory effects to antibiotics by inhibition tests. This suggested that AMPs can be introduced into aquaculture using baited microalgae, providing new ideas and ways to solve a series of aquaculture diseases caused by bacteria.

Correction to: YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma.

An amendment to this paper has been published and can be accessed via the original article.

Construction of the Antheraea pernyi (Lepidoptera: Saturniidae) Multicapsid Nucleopolyhedrovirus Bacmid System.

In this study, we established the Antheraea pernyi multicapsid nucleopolyhedrovirus (AnpeNPV) bacmid system for the construction of a Bac-to-Bac expression system and the generation of virus mutants. The CopyRight pSMART BAC cloning vector harboring the chloramphenicol resistance gene was introduced into the AnpeNPV genome to produce the AnpeNPV bacmid that could be propagated in Escherichia coli with stable replication. The enhanced green fluorescent protein (EGFP) was successfully expressed in both Tn-Hi5 cells and A. pernyi pupae using the AnpeNPV Bac-to-Bac expression system. To generate the AnpeNPV mutants, we developed the AnpeNPV bacmid/λ Red recombination system that facilitated the deletion of viral genes from the AnpeNPV genome. The genes cathepsin and chitinase were deleted and a derivative AnpeNPV Bac-to-Bac expression system was constructed. Furthermore, we demonstrated that the novel expression system could be used to express human epidermal growth factor in A. pernyi pupae. Taken together, the AnpeNPV bacmid system provides a powerful tool to create the AnpeNPV Bac-to-Bac expression system for protein expression in A. pernyi pupae. Further, it helps to knock-out genes from the AnpeNPV genome with λ Red recombination system for identification of the role of viral genes involved in regulating gene expression, DNA replication, virion structure, and infectivity during the AnpeNPV infection process.

Construction of a Baculovirus Derivative to Produce Linearized Antheraea pernyi (Lepidoptera: Saturniidae) Multicapsid Nucleopolyhedrovirus Genomic DNA.

In the Antheraea pernyi multicapsid nucleopolyhedrovirus (AnpeNPV)-based expression vector system, the frequency of homologous recombination events between wild-type AnpeNPV DNA and the transfer vector is low, resulting in a small amount of recombinant virus. Previous reports have indicated that linearized baculovirus DNA can increase the proportion of recombinant virus relative to the total progeny. To improve the recombination efficiency, we constructed a linearized derivative of AnpeNPV, referred to as AnpeNPVPhEGFP-AvrII, in which egfp flanked by AvrII restriction sites was located at the polyhedrin locus and driven by the polyhedrin promoter. Linear AnpeNPV DNA was obtained by the treatment of AnpeNPVPhEGFP-AvrII genomic DNA with AvrII endonuclease. The infectivity and recombinogenic activity between the linearized and circular viral DNA were evaluated by quantitative real-time polymerase chain reactions. We demonstrated that the linearized AnpeNPV DNA produced only small numbers of infectious budded viruses, accounting for approximately 4.5% of the budded virus production of wild-type AnpeNPV DNA in A. pernyi pupae. However, the linearized AnpeNPV DNA substantially increased recombinant virus production after cotransfection with an appropriate transfer vector; relative abundance of the recombinant virus was approximately 5.5-fold higher than that of the wild-type AnpeNPV DNA in A. pernyi pupae. The linearization of AnpeNPV DNA will facilitate the purification of recombinant viruses using the AnpeNPV-based expression vector system and the construction of an AnpeNPV-based bacmid system.

YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma.

BACKGROUND: Dynamic N6-methyladenosine (m6A) modification was previously identified as a ubiquitous post-transcriptional regulation that affected mRNA homeostasis. However, the m6A-related epitranscriptomic alterations and functions remain elusive in human cancer. Here we aim to identify the profile and outcome of m6A-methylation in hepatocellular carcinoma (HCC). RESULTS: Using liquid chromatography-tandem mass spectrometry and m6A-immunoprecipitation in combination with high-throughput sequencing, we determined the m6A-mRNA levels in human HCC. Human HCC exhibited a characteristic gain of m6A modification in tandem with an increase of mRNA expression, owing to YTH domain family 2 (YTHDF2) reduction. The latter predicted poor classification and prognosis of HCC patients, and highly correlated with HCC m6A landscape. YTHDF2 silenced in human HCC cells or ablated in mouse hepatocytes provoked inflammation, vascular reconstruction and metastatic progression. Mechanistically, YTHDF2 processed the decay of m6A-containing interleukin 11 (IL11) and serpin family E member 2 (SERPINE2) mRNAs, which were responsible for the inflammation-mediated malignancy and disruption of vascular normalization. Reciprocally, YTHDF2 transcription succumbed to hypoxia-inducible factor-2α (HIF-2α). Administration of a HIF-2α antagonist (PT2385) restored YTHDF2-programed epigenetic machinery and repressed liver cancer. CONCLUSION: Our results have characterized the m6A-mRNA landscape in human HCC and revealed YTHDF2 as a molecular 'rheostat' in epitranscriptome and cancer progression.

Quantitative real-time PCR with high-throughput automatable DNA preparation for molecular screening of Nosema spp. in Antheraea pernyi.

Accurate diagnosis of pathogenic Nosema spp. in Antheraea pernyi samples is considered especially useful for reducing economic losses in sericulture and improving food safety by maintaining pathogen-free pupae. However, microscopy and immunologic methods have poor diagnostic sensitivity, while the more sensitive PCR methods remain costly and time-consuming for template preparation. To address this issue, we introduce a sensitive ALMS-qPCR method that combines fast, simple DNA extraction using Alkali Lysis followed by Magnetic bead Separation (ALMS) and quantitative real-time PCR (qPCR). This approach is especially fit for large-scale pathogen molecular screening, because the DNA preparation procedure is fast (<0.94 min per sample) and is high-throughput (performs on a 96-well plate). It is cost-effective, since the most expensive materials can be made in the lab and can be recycled, while the automated procedure can help to minimize labor cost. Though the DNA preparation procedure was substantially simplified, common PCR inhibitory factors were not observed. The sensitivity of ALMS-qPCR is high and the limit of detection is 0.045 parasites/µL. Large-scale screening of Nosema spp. in 3000 Antheraea pernyi samples confirmed the efficacy of the ALMS-qPCR method. Sensitivity is much higher than clinical microscopy, especially for host groups with low infection prevalence and levels. High-throughput ALMS-qPCR, combining automated DNA preparation and sensitive qPCR, provides an enhanced approach for pébrine screening and epidemiological studies. The application of ALMS-qPCR in the sericulture industry will help to strengthen pébrine control and breed pathogen-free species, which means much safer food provision and better genetic resource conservation.

Evaluation of Reference Genes for Normalization of RT-qPCR Gene Expression Data for Trichoplusia ni Cells During Antheraea pernyi (Lepidoptera: Saturniidae) Multicapsid Nucleopolyhedrovirus (AnpeNPV) Infection.

Baculovirus infection impacts global gene expression in the host cell, including the expression of housekeeping genes. Evaluation of candidate reference genes during a viral infection will inform the selection of appropriate reference gene(s) for the normalization of expression data generated by Reverse Transcription Quantitative Real-timePolymerase Chain Reaction (RT-qPCR). Antheraea pernyi multicapsid nucleopolyhedrovirus (AnpeNPV) is able to infect the High Five cells (Tn-Hi5). In the present study, 10 candidate reference genes were evaluated in AnpeNPV-infected Tn-Hi5 cells. Gene expression data were analyzed using geNorm, NormFinder, BestKeeper, and RefFinder. The candidate genes were further validated as reliable reference genes for RT-qPCR by analyzing the expression of three target genes. The results of data analysis using four statistical methods showed that RPS18 was the least stable among the candidate reference genes tested. 18S rRNA and 28S rRNA were not suitable as reference genes for RT-qPCR analysis in AnpeNPV-infected Tn-Hi5 cells. Comprehensive ranking of the 10 candidate reference genes by RefFinder analysis indicated that Ann B, c45128_g1, and ACT were the top three genes. Normalization of the expression of three target genes using the candidate reference genes indicated the same expression pattern when Ann B and c45128_g1 were used as reference genes, with slight differences in the relative expression at each infection time point. Overall, Ann B and c45128_g1 were recommended to be more suitable than the most commonly used reference genes, such as ACT, GAPDH, and TUB, for RT-qPCR data normalization in AnpeNPV-infected Tn-Hi5 cells up to 48 hpi.

Radiation-Stimulated Translocation of CD166 and CRYAB to the Endothelial Surface Provides Potential Vascular Targets on Irradiated Brain Arteriovenous Malformations.

Vascular targeting with pro-thrombotic antibody-conjugates is a promising biological treatment for brain arteriovenous malformations (bAVMs). However, targeted drug delivery relies on the identification of unique or overexpressed markers on the surface of a target cell. In the absence of inherent biological markers, stereotactic radiosurgery may be used to prime induction of site-specific and targetable molecular changes on the endothelial surface. To investigate lumen-accessible, endothelial targets induced by radiation, we combined Gamma knife surgery in an AVM animal model with in vivo biotin-labeling and comparative proteomics. Two proteins, αB-crystallin (CRYAB)-a small heat shock protein that normally acts as an intracellular chaperone to misfolded proteins-and activated leukocyte cell adhesion molecule CD166, were further validated for endothelial surface expression after irradiation. Immunostaining of endothelial cells in vitro and rat AVM tissue ex vivo confirmed de novo induction of CRYAB following irradiation (20 Gy). Western analysis demonstrated that CRYAB accumulated intracellularly as a 20 kDa monomer, but, at the cell surface, a novel 65 kDa protein was observed, suggesting radiation stimulates translocation of an atypical CRYAB isoform. In contrast, CD166 had relatively high expression in non-irradiated cells, localized predominantly to the lateral surfaces. Radiation increased CD166 surface exposure by inducing translocation from intercellular junctions to the apical surface without significantly altering total protein levels. These findings reinforce the dynamic molecular changes induced by radiation exposure, particularly at the cell surface, and support further investigation of radiation as a priming mechanism and these molecules as putative targets for focused drug delivery in irradiated tissue.

Comparison of methods for detecting asymptomatic malaria infections in the China-Myanmar border area.

BACKGROUND: Sensitive methods for detecting asymptomatic malaria infections are essential for identifying potential transmission reservoirs and obtaining an accurate assessment of malaria epidemiology in low-endemicity areas aiming to eliminate malaria. PCR techniques to detect parasite nucleic acids (DNA or RNA) are among the most commonly used molecular methods. However, most of these methods are of low throughput and cannot be used for large-scale molecular epidemiological studies. A recently developed capture and ligation probe-PCR (CLIP-PCR) is claimed to have the sensitivity of molecular techniques and the high throughput capacity needed for screening purposes. This study aimed to compare several molecular methods for detecting asymptomatic and submicroscopic Plasmodium infections in healthy residents of a malaria-hypoendemic region in Southeast Asia, where malaria elimination is in sight. METHOD: This study compared three molecular detection methods side-by-side, namely nested PCR targeting the rRNA genes, nested RT-PCR to detect parasite rRNA, and CLIP-PCR to detect parasite rRNA in 1005 healthy individuals in northeastern Myanmar. For nested PCR and RT-PCR, parasite DNA and total RNA were extracted from ~100 µL of blood, whereas RNA used for CLIP-PCR was from a 3 mm disk of dried blood filter paper. The sensitivity and specificity of these methods were compared with those of conventional light microscopy. In addition, RT-PCR and quantitative RT-PCR (qRT-PCR) targeting the Pvs25 gene in Plasmodium vivax were used to assess gametocyte prevalence in the samples. RESULTS: Light microscopy detected Plasmodium infections in only 1.19% of the residents harbouring the parasites. CLIP-PCR had slightly better performance and detected Plasmodium infections in 1.89% of the population. Further improvement was achieved by nested PCR to detect parasite DNA, which detected P. vivax and Plasmodium falciparum infections in 2.39% of the residents. The nested RT-PCR targeting rRNA, however, detected as many as 187 (18.61%) individuals having Plasmodium infections with P. vivax being the predominant species (176 P. vivax, 5 P. falciparum and 6 P. falciparum/P. vivax mixed infections). Of the 210 Plasmodium-positive samples detected by all molecular methods, 115 were Pvs25-positive by qRT-PCR, indicating that a large proportion of asymptomatic individuals were gametocyte carriers. CONCLUSION: Nested RT-PCR based on the detection of asexual-stage parasite rRNA was the most sensitive, with a more than sixfold higher sensitivity than the other two molecular methods of parasite detection. CLIP-PCR has an increased throughput, but its sensitivity in this study was much lower than those of other molecular methods, which may be partially due to the smaller amount of RNA input used.

Circular RNA participates in the carcinogenesis and the malignant behavior of cancer.

Circular RNAs (circRNAs) are long, non-coding RNAs that result from the non-canonical splicing of linear pre-mRNAs. However, the characteristics and the critical role of circRNA in co-/post-transcriptional regulation were not well recognized until the "microRNA sponge" function of circRNA is discovered. Recent studies have mainly been devoted to the function of the circular RNA sponge for miR-7 (ciRS-7) and sex-determining region Y (SRY) by targeting microRNA-7 (miR-7) and microRNA-138 (miR-138), respectively. In this review, we illustrate the specific role of circRNAs in a wide variety of cancers and in regulating the biological behavior of cancers via miR-7 or miR-138 regulation. Furthermore, circRNA, together with its gene silencing ability, also shows its potential in RNA interference (RNAi) therapy by binding to target RNAs, which provides a novel perspective in cancer treatment. Thus, this review concerns the biogenesis, biological function, oncogenesis, progression and possible therapies for cancer involving circRNAs.

Contact Lens-Induced Discomfort and Inflammatory Mediator Changes in Tears.

PURPOSE: Studies indicate that contact lens (CL) discontinuation mostly occurs because of dryness and discomfort symptoms. This study aimed to investigate relationships between changes in the concentration of tear inflammatory mediators with subjective comfort ratings with CL wear and no contact lens wear between morning and evening. METHOD: Forty-five subjects collected tears twice daily in the morning and in the evening with or without lenses. Comfort was rated subjectively on a scale from 1 to 100 (where 100 was extremely comfortable) just before each tear collection. Tear samples were assayed for complement components (C3 and C3a), leukotriene B4 (LTB4), secretory phospholipase A2 (sPLA2), secretory immunoglobulin A (sIgA), and bradykinin using commercially available immuno-based assay kits. RESULTS: Comfort ratings showed a statistically significant decline from morning to evening both with CL (89.0±10.1 AM vs. 76.7±15.2 PM; P<0.001) and without CL (89.1±10.2 AM vs. 84.2±12.6 PM; P<0.005) wear. The decline was steeper with lens wear (P<0.001). Bradykinin and sPLA2 levels did not change between morning and evening or with CL wear (P>0.05). Leukotriene B4 levels were slightly higher in CL (CL 43.4±12.6 pg/ml vs. No CL 39.4±13.4 pg/mL; P=0.034), whereas the concentration of LTB4, C3, C3a, and sIgA dropped by the end of the day in the presence or absence of lens wear (P<0.001). For most mediators, tear levels were not correlated with comfort ratings in any of the conditions. Leukotriene B4 had a higher concentration in the evening, and when measured as a ratio to sIgA, there was a trend for increased concentration of this mediator during CL wear. CONCLUSION: Although specific mediators showed changes from morning to evening with and without lens wear, most of these were not correlated with subjective comfort ratings in lens wear. The only mediator that showed an increase in concentration during the day and during lens wear was LTB4, and further studies on this mediator are warranted.

Glycan involvement in the adhesion of Pseudomonas aeruginosa to tears.

The human eye is constantly bathed by tears, which protect the ocular surface via a variety of mechanisms. The O-linked glycans of tear mucins have long been considered to play a role in binding to pathogens and facilitating their removal in the tear flow. Other conjugated glycans in tears could similarly contribute to pathogen binding and removal but have received less attention. In the work presented here we assessed the contribution of glycan moieties, in particular the protein attached N-glycans, presented by the broad complement of tear proteins to the adhesion of the opportunistic pathogen Pseudomonas aeruginosa, a leading cause of microbial keratitis and ulceration of the cornea. Our adhesion assay involved immobilising the macromolecular components of tears into the wells of a polyvinyl difluoride (PVDF) microtitre filter plate and probing the binding of fluorescently labelled bacteria. Three P. aeruginosa strains were studied: a cytotoxic strain (6206) and an invasive strain (6294) from eye infections, and an invasive strain (320) from a urinary tract infection (UTI). The ocular isolates adhered two to three times more to human tears than to human saliva or porcine gastric mucin, suggesting ocular niche-specific adaptation. Support for the role of the N-glycans carried by human tear proteins in the binding and removal of P. aeruginosa from the eye was shown by: 1) pre-incubation of the bacteria with free component sugars, galactose, mannose, fucose and sialyl lactose (or combination thereof) inhibiting adhesion of all the P. aeruginosa strains to the immobilised tear proteins, with the greatest inhibition of binding of the ocular cytotoxic 6206 and least for the invasive 6294 strain; 2) pre-incubation of the bacteria with N-glycans released from the commercially available human milk lactoferrin, an abundant protein that carries N-linked glycans in tears, inhibiting the adhesion to tears of the ocular bacteria by up to 70%, which was significantly more binding inhibition than by the same amount of intact human lactoferrin or by the plant-derived N-glycans released from the rice recombinant lactoferrin; 3) pre-incubation of the bacteria with N-linked glycans released from human tear proteins inhibiting the adhesion of the ocular P. aeruginosa strains to immobilised tear proteins; 4) inhibition by the N-glycans from lactoferrin of the ability of an ocular strain of P. aeruginosa to invade corneal epithelial cells; 5) removal of terminal sialic acid and fucose moieties from the tear glycoproteins with α2-3,6,8 neuraminidase (sialidase) and α1-2,3,4 fucosidase resulting in a reduction in binding of the UTI P. aeruginosa isolate, but not the adhesion of the ocular cytotoxic (6206) or invasive (6294) isolates. Glycosidase activity was validated by mass spectrometry. In all cases, the magnitude of inhibition of bacterial adhesion by the N-glycans was consistently greater for the cytotoxic ocular strain than for the invasive ocular strain. Ocular P. aeruginosa isolates seems to exhibit different adhesion mechanism than previously known PAI and PAII lectin adhesion. The work may contribute towards the development of glycan-focused therapies to prevent P. aeruginosa infection of the eye.

Plasmodium malariae and Plasmodium ovale infections in the China-Myanmar border area.

BACKGROUND: The Greater Mekong Subregion is aiming to achieve regional malaria elimination by 2030. Though a shift in malaria parasite species predominance by Plasmodium vivax has been recently documented, the transmission of the two minor Plasmodium species, Plasmodium malariae and Plasmodium ovale spp., is poorly characterized in the region. This study aims to determine the prevalence of these minor species in the China-Myanmar border area and their genetic diversity. METHODS: Epidemiology study was conducted during passive case detection in hospitals and clinics in Myanmar and four counties in China along the China-Myanmar border. Cross-sectional surveys were conducted in villages and camps for internally displaced persons to determine the prevalence of malaria infections. Malaria infections were diagnosed initially by microscopy and later in the laboratory using nested PCR for the SSU rRNA genes. Plasmodium malariae and P. ovale infections were confirmed by sequencing the PCR products. The P. ovale subtypes were determined by sequencing the Pocytb, Pocox1 and Pog3p genes. Parasite populations were evaluated by PCR amplification and sequencing of the MSP-1 genes. Antifolate sensitivity was assessed by sequencing the dhfr-ts and dhps genes from the P. malariae and P. ovale isolates. RESULTS: Analysis of 2701 blood samples collected from the China-Myanmar border by nested PCR targeting the parasite SSU rRNA genes identified 561 malaria cases, including 161 Plasmodium falciparum, 327 P. vivax, 66 P. falciparum/P. vivax mixed infections, 4 P. malariae and 3 P. ovale spp. P. vivax and P. falciparum accounted for >60 and ~30% of all malaria cases, respectively. In comparison, the prevalence of P. malariae and P. ovale spp. was very low and only made up ~1% of all PCR-positive cases. Nevertheless, these two species were often misidentified as P. vivax infections or completely missed by microscopy even among symptomatic patients. Phylogenetic analysis of the SSU rRNA, Pocytb, Pocox1 and Pog3p genes confirmed that the three P. ovale spp. isolates belonged to the subtype P. ovale curtisi. Low-level genetic diversity was detected in the MSP-1, dhfr and dhps genes of these minor parasite species, potentially stemming from the low prevalence of these parasites preventing their mixing. Whereas most of the dhfr and dhps positions equivalent to those conferring antifolate resistance in P. falciparum and P. vivax were wild type, a new mutation S113C corresponding to the S108 position in pfdhfr was identified in two P. ovale curtisi isolates. CONCLUSIONS: The four human malaria parasite species all occurred sympatrically at the China-Myanmar border. While P. vivax has become the predominant species, the two minor parasite species also occurred at very low prevalence but were often misidentified or missed by conventional microscopy. These minor parasite species displayed low levels of polymorphisms in the msp-1, dhfr and dhps genes.

Glycomic characterization of basal tears and changes with diabetes and diabetic retinopathy.

As a secreted fluid, the state of tear glycosylation is particularly important in the role of immunity of the ocular surface. Tears are a valuable source of non-invasive biomarkers for disease and there are continued efforts to characterize their components thoroughly. In this study, a small volume of basal tears (5 µL) was collected from healthy controls, patients with diabetes without retinopathy and patients with diabetes and retinopathy. The detailed N- and O-linked tear protein glycome was characterized and the relative abundance of each structure determined. Of the 50 N-linked glycans found, 89% were complex with 50% containing a bisecting N-acetylglucosamine, 65% containing a core fucose whilst 33% were sialylated. Of the 8 O-linked glycans detected, 3 were of cores 1 and 5 of core 2 type, with a majority of them being sialylated (90%). Additionally, these glycan structures were profiled across the three diabetic disease groups. Whilst the higher abundant structures did not alter across the three groups, only five low abundance N-linked glycans and 1 O-linked glycan did alter with the onset of diabetes mellitus and diabetic retinopathy (DR). These results suggest the conservation of glycan types on basal tear proteins between individuals and point to only small changes in glycan expression on the proteins in tears with the development of diabetes and DR.