Therapeutic application of decellularized porcine small intestinal submucosa scaffold in conjunctiva reconstruction.

The objective of this study was to investigate the biological feasibility and surgical applicability of decellularized porcine small intestinal submucosa (DSIS) in conjunctiva reconstruction. A total of 52 Balb/c mice were included in the study. We obtained the DSIS by decellularization, evaluated the physical and biological properties of DSIS in vitro, and further evaluated the effect of surgical transplantation of DSIS scaffold in vivo. The histopathology and ultrastructural analysis results showed that the scaffold retained the integrity of the fibrous morphology while removing cells. Biomechanical analysis showed that the elongation at break of the DSIS (239.00 ± 12.51%) were better than that of natural mouse conjunctiva (170.70 ± 9.41%, P < 0.05). Moreover, in vivo experiments confirmed the excellent biocompatibility of the decellularized scaffolds. In the DSIS group, partial epithelialization occurred at day-3 after operation, and the conjunctival injury healed at day-7, which was significantly faster than that in human amniotic membrane (AM) and sham surgery (SHAM) group (P < 0.05). The number and distribution of goblet cells of transplanted DSIS were significantly better than those of the AM and SHAM groups. Consequently, the DSIS scaffold shows excellent biological characteristics and surgical applicability in the mouse conjunctival defect model, and DSIS is expected to be an alternative scaffold for conjunctival reconstruction.

Exudates of Microcystis aeruginosa on oxidative stress and inflammatory responses in gills of Sinocyclocheilus grahami.

Early cyanobacterial blooms studies observed that exposure to blue-green algae led to fish gills impairment. The objective of this work was to evaluate the toxic mechanisms of exudates of Microcystis aeruginosa (MaE) on fish gills. In this study, the toxic mechanism of MaE (2×106 cells/mL) and one of its main components phytosphingosine (PHS) with two concentrations 2.9 ng/mL and 145 ng/mL were conducted by integrating histopathology, biochemical biomarkers, and transcriptomics techniques in Sinocyclocheilus grahami (S. grahami) for 96 h exposure. Damaged gill tissue with epithelial hyperplasia and hypertrophy, remarkable Na+/K+-ATPase (NKA) enzyme activity, disrupted the redox homeostats including lipid peroxidation and inflammatory responses were observed in the fish of MaE exposure group. Compare to MaE exposure, two concentrations of PHS exposure appeared to be a trend of lower degree of tissue damage, NKA activity and oxidative stress, but induced obviously lipid metabolism disorder with higher triglycerides, total cholesterol and total bile acid, which might be responsible for inflammation responses in fish gill. By transcriptome analysis, MaE exposure were primarily enriched in pathways related to gill function and immune response. PHS exposure, with higher number of differentially expressed genes (DEGs), were enriched in Toll-like receptor (TLR), Mitogen-Activated Protein Kinase (MAPK) and NOD-like receptor protein 3 (NLRP3) pathways. We concluded that MaE and PHS were induced the inflammatory responses, with oxidative stress-induced inflammation for MaE exposure but lipid metabolism disorder-induced inflammation for PHS exposure. The present study provided two toxin-induced gill inflammation response pathways under cyanobacterial blooms, which could be a scientific basis for the ecological and health risk assessment in the aquatic environment.

A ratiometric fluorescent dark box and smartphone integrated portable sensing platform based on hydrogen bonding induction for on-site determination of enrofloxacin.

Enrofloxacin (ENR) residues in animal-derived food and water threaten human health. Simple, low-cost and on-site detection methods are urgently needed. Blue emitting carbon quantum dots (CQDs) and orange rhodamine B (RhB) were used as recognition and reference signals, respectively, to construct a ratiometric fluorescence sensor. After the addition of ENR, the color of the sensor changed from orange to blue because hydrogen bonding induced a considerable increase in CQDs fluorescence. Based on this mechanism, a simple and low cost on-site portable sensing platform was constructed, which integrated a stable UV light strip and a smartphone with voice-controlled phototaking function and an RGB app. The t-test results of spiked ENR recoveries for diluted milk, honey and drinking water revealed no significant differences between the ratiometric fluorescent sensor and portable sensing platform. Thus, this portable sensing platform provides a novel strategy for on-site quantification of quinolone antibiotics in foodstuffs and environmental water.

Proteomics coupled transcriptomics reveals Slc34a1 and Slc34a3 downregulation as potential features of nephrotoxin-induced acute kidney injury.

Acute kidney injury (AKI) stands as a prevalent and economically burdensome condition worldwide, yet its complex molecular mechanisms remain incompletely understood. To address this gap, our study employs a multifaceted approach, combining mass spectrometry and RNA sequencing technologies, to elucidate the intricate molecular landscape underlying nephrotoxin-induced AKI in mice by cisplatin- and LPS-induced. By examining the protein and RNA expression profiles, we aimed to uncover novel insights into the pathogenesis of AKI and identify potential diagnostic and therapeutic targets. Our results demonstrate significant down-regulation of Slc34a1 and Slc34a3, shedding light on their crucial roles in AKI pathology and highlighting their promise as actionable targets for diagnosis and treatment. This comprehensive analysis not only enhances our understanding of AKI pathophysiology but also offers valuable avenues for the development of targeted interventions to mitigate its clinical impact. SIGNIFICANCE: Nephrotoxicity acute kidney injury (AKI) is a common clinical condition whose pathogenesis is the process by which some drugs, chemicals or other factors cause damage to the kidneys, resulting in impaired kidney function. Although it has been proved that different nephrotoxic substances can affect the kidney through different pathways, whether they have a commonality has not been registered. Here, we combined transcriptomics and proteomics to study the molecular mechanism of LPS and cisplatin-induced nephrotoxic acute kidney injury finding that the down-regulation of Slc34a1 and Slc34a3 may be a critical link in nephrotoxic acute kidney injury, which can be used as a marker for its early diagnosis.

A compact and high-performance setup of capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D).

Capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) has the advantages of high throughput (simultaneous detection of multiple ions), high separation efficiency (higher than 105 theoretical plates) and rapid analysis capability (less than 5 min for common inorganic ions). A compact CE-C4D system is ideal for water quality control and on-site analysis. It is suitable not only for common cations (e.g. Na+, K+, Li+, NH4+, Ca2+, etc.) and anions (e.g. Cl-, SO42-, BrO3-, etc.) but also for some ions (e.g. lanthanide ions, Pb2+, Cd2+, etc.) that require complex derivatization procedures to be detected by ion chromatography (IC). However, an obvious limitation of the CE-C4D method is that its sensitivity (e.g. 0.3-1 µM for common inorganic ions) is often insufficient for trace analysis (e.g. 1 ppb or 20 nM level for common inorganic ions) without preconcentration. For this technology to become a powerful and routine analytical technique, the system should be made compact while maintaining trace analysis sensitivity. In this study, we developed an all-in-one version of the CE-C4D instrument with custom-made modular components to make it a convenient, compact and high-performance system. The system was designed using direct digital synthesis (DDS) technology to generate programmable sinusoidal waveforms with any frequency for excitation, a kilovolt high-voltage power supply for capillary electrophoresis separation, and an "effective" differential C4D cell with a low-noise circuitry for high-sensitivity detection. We characterized the system with different concentrations of Cs+, and even a low concentration of 20 nM was detectable without preconcentration. Moreover, the optimized CE-C4D setup was applied to analyse mixed ions at a trace concentration of 200 nM with excellent signal-to-noise ratios. In typical applications, the limits of detection based on the 3σ criterion (without baseline filtering) were 9, 10, 24, 5, and 12 nM for K+, Cs+, Li+, Ca2+, and Mg2+, respectively, and about 7, 6, 6 and 6 nM for Br-, ClO4-, BrO3- and SO42-, respectively. Finally, the setup was also applied for the analysis of all 14 lanthanide ions and rare-earth minerals, and it showed an improvement in sensitivity by more than 25 times.

Proteomics coupled transcriptomics reveals lipopolysaccharide inhibiting peroxisome proliferator-activated receptors signalling pathway to reduce lipid droplets accumulation in mouse liver.

Lipid droplets (LDs) are multifunctional organelles consisting of a central compartment of non-polar lipids shielded from the cytoplasm by a phospholipid monolayer. The excessive accumulation of LDs in cells is closely related to the development and progression of many diseases in humans and animals, such as liver-related and cardiovascular diseases. Thus, regulating the LDs size and abundance is necessary to maintain metabolic homeostasis. This study found that lipopolysaccharide (LPS) stimulation reduced the LDs content in the mouse liver. We tried to explain the possible molecular mechanisms at the broad protein and mRNA levels, finding that inhibition of the peroxisome proliferator-activated receptors (PPAR) signalling pathway by LPS may be a critical factor in reducing LDs content.

The role of regulated necrosis in inflammation and ocular surface diseases.

In recent decades, numerous types of regulated cell death have been identified, including pyroptosis, ferroptosis and necroptosis. Regulated necrosis is characterized by a series of amplified inflammatory responses that result in cell death. Therefore, it has been suggested to play an essential role in the pathogenesis of ocular surface diseases. The cell morphological features and molecular mechanisms of regulated necrosis are discussed in this review. Furthermore, it summarizes the role of ocular surface diseases, such as dry eye, keratitis, and cornea alkali burn, as potential disease prevention and treatment targets.

Association between poor sleep quality and an increased risk of dry eye disease in patients with obstructive sleep apnea syndrome.

Purpose: Obstructive sleep apnea (OSA) is related to an increased incidence of dry eye disease (DED). However, their exact relationship is unknown and requires further well-designed studies with advanced mechanisms detection. Patients and methods: This case-control study included 125 OSA cases and 125 age-gender-matched controls enrolled in the hospital between 1 January and 1 October 2021. OSA diagnosis and classification were performed using a polysomnography (PSG) assay. Detailed ophthalmological examinations, including the Schirmer I test, corneal staining, and ocular surface disease index (OSDI), were used to detect DED-related parameters. A comprehensive ocular surface assay was performed to measure a series of parameters, including first non-invasive first tear film break-up time (f-NIBUT), average non-invasive first tear film break-up time (av-NIBUT), tear meniscus height (TMH), and loss of meibomian gland. In addition, the Pittsburgh Sleep Quality Index (PSQI) scale was used to assess sleep quality. Results: Compared to the control, the OSA group showed an increased DED risk (P = 0.016) along with an increased PSQI score and a higher rate of poor quality sleep (P < 0.001 and P = 0.007, respectively). Stratification of OSA cases indicated that DED-related parameters were impaired in patients with severe OSA (P < 0.05). The analysis of DED-parameters-related factors showed significant correlations between OSA-related indexes and PSQI (P < 0.05). Moreover, the poor sleep quality group in the OSA cases showed worse DED-related parameters (P < 0.05), which was not observed in the control group. Conclusion: OSA, especially the severe stage OSA, was related to an increased risk of DED. Also, sleep quality was correlated with the onset of both OSA and DED, where poor sleep quality revealed a relationship between OSA and the risk of DED. Overall, our findings provided evidence for advanced management of DED and OSA in future.

Expression Plasticity of Transposable Elements Is Highly Associated with Organismal Re-adaptation to Ancestral Environments.

Understanding the roles of phenotypic plasticity in adaptive evolution has gained recognition for decades. Studies involving multiple taxa have shown that gene expression plasticity serves as "long-term memory" to facilitate re-adaptations to ancestral environments. Nevertheless, the general pattern and the underlying genetic basis of expression plasticity remain unclear. The transposable elements (TEs) play crucial roles in gene expression regulation and are widely distributed within the genome. Given this, we re-analyzed the transcriptomic data of chicken (Gallus gallus) generated from a reciprocal transplant experiment to examine whether expression shifts of TEs are involved in the re-adaptation process. Similar to the protein-coding genes, the plastic changes of TEs overwhelmingly exceed the genetic changes in the re-adaptation process. Further, the associated TEs co-expressed with diverse genes to perform a regulatory activity. Thus, our study supports the general function of phenotypic plasticity in adaptive evolution, and suggests a regulatory functions of TEs in this process.

Nicotinamide mononucleotide increases cell viability and restores tight junctions in high-glucose-treated human corneal epithelial cells via the SIRT1/Nrf2/HO-1 pathway.

BACKGROUND: Diabetes mellitus (DM)-related corneal epithelial dysfunction is a severe ocular disorder; however, the effects of nicotinamide mononucleotide (NMN) on high-glucose (HG)-treated human corneal epithelial cells (HCECs) remain unclear. METHODS: We conducted an in-vitro study to examine the effects of NMN treatment on HG-treated HCECs. Cell viability was measured using trypan blue stain, mitochondrial membrane potential was measured using JC-1 stain, and intracellular reactive oxygen species and apoptosis assays were conducted using flow cytometry. Transepithelial electrical resistance (TEER) and zonula occludens-1 (ZO-1) immunofluorescence for tight junction examinations were conducted. Immunoblot analyses were conducted to analyze the expression of silent information regulator-1 (SIRT1), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) of the SIRT1/Nrf2/HO-1 pathway. RESULTS: NMN increased cell viability by reducing cell damage, reducing apoptosis, increasing cell migration, and restoring tight junctions in HG-treated HCECs. By analyzing the expressions of SIRT1, Nrf2, HO-1, NMN demonstrated protective effects via the SIRT1/Nrf2/HO-1 pathway. CONCLUSIONS: NMN increases cell viability by reversing cell damage, reducing apoptosis, increasing cell migration, and restoring tight junctions in HG-treated HCECs, and these effects may be mediated by the SIRT1/Nrf2/HO-1 pathway.

Soft substrate stiffness modifies corneal epithelial stem cell phenotype through hippo-YAP/notch pathway crosstalk.

Corneal disease remains to be one of the leading causes of blindness in the world and limbal stem cell (LSC) therapy is a promising therapy for LSC deficiency, which is associated with the diseased corneal epithelium repair. Soft substrate could effectively promote the stemness maintenance of LSC and thus modification of cell culture substrate would help in the potential LSC deficiency therapy. Both Hippo-Yes-associated protein (YAP) and Notch pathway have been reported to affect the LSC function, however, the detailed mechanisms remain unclear. Instead of some soft but biologically toxic substrates, we present a hypothesis on the application of soft substrate generated by HA/PTX3, an FDA approved nontoxic drug, on the LSC culture in this current study. Soft substrate could help in the stemness maintenance and thus promote the LSC deficiency treatment. In more detailed mechanism detection, we hypothesize that soft substrate would block the activation of Hippo-YAP pathway and thus decrease the activity of Notch pathway. This proposed hypothesis should be evaluated by both a series of in-vitro experiments based on soft and stiff substrates and in-vivo treatment with LSC cultured in different conditions. Advanced experiments on related cellular behaviors and detailed molecular mechanisms would provide us more knowledge on the molecular mechanism detection as well as cell transplantation therapy.

Neutrophil/Lymphocyte Ratio as an Inflammatory Predictor of Dry Eye Disease: A Case-Control Study.

BACKGROUND: The neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been used as indicators of inflammation, however, their roles in dry eye disease (DED) patients require advanced study. MATERIALS AND METHODS: A total of 104 DED cases and 97 healthy controls from January 2020 to May 2020 were enrolled in this study. The dry eye related clinical variables, including Schirmer I test, tear break-up time (TBUT), corneal fluorescein staining (CFS) and Ocular Surface Disease Index (OSDI), were detected in all the participants. Besides, the NLR and PLR pattern in DED cases were detected and their potential value as inflammatory predictors of DED were evaluated. In advanced analyses, the correlation between NLR and DED severity was examined. RESULTS: The NLR and PLR were 2.59 ± 1.25 and 117.48 ± 54.68 in the DED group, respectively, while they were 2.20 ± 1.24 and 115.48 ± 54.33 in the control group, respectively. The NLR was higher in the DED group (p = 0.027), however, PLR was not significantly different compared with the control group (p = 0.951). In advanced analyses, it was found that more severe TBUT, CFS, and OSDI scores were detected in the high NLR group (NLR ≥2.145, p = 0.003, 0.013, and 0.017, respectively) compared with the low NLR group (NLR <2.145). CONCLUSION: The NLR value, but not PLR, of DED patients was higher than that of healthy controls. The NLR could be used as an inflammatory predictor to estimate the severity of DED.

Nicotinamide Mononucleotide Alleviates Hyperosmolarity-Induced IL-17a Secretion and Macrophage Activation in Corneal Epithelial Cells/Macrophage Co-Culture System.

BACKGROUND: Hyperosmosis stress (HS) was a key pathological factor in the development of dry eye disease (DED). Nicotinamide mononucleotide (NMN) demonstrated protective effects in the corneal damage, however, its role in the HS-induced DED remained unclear. METHODS: A NaCl based HS in-vitro model (500 mOsm) was generated and used in a co-culture system including corneal epithelial cells (CEC) and macrophage cell line RAW264.7. The effect of NMN on NAD+ metabolism and the expression of HS biomarker, tonicity-responsive element binding protein (TonEBP), was studied in the CEC. The cellular activity, including cell viability, apoptosis status and lactate dehydrogenase (LDH) release through trypan blue staining, flow cytometry and LDH assay, respectively. The mitochondrial membrane potential (MMP) assay would be conducted using the JC1 kit. The expression of IL-17a were detected using RT-PCR, ELISA and Western blot. After co-culture with the CEC in different group for 24 h, the phagocytosis ability and macrophage polarization were assessed in RAW264.7 cells co-cultured with CEC with or without HS or NMN treatment. Besides, the involvement of Notch pathway in the RAW264.7 would be analyzed. The potential involvement of Sirtuin 1 (SIRT1) and IL-17a in the crosstalk between CEC and macrophage was studied with SIRT1 inhibitor EX 527 and anti-IL-17a monoclonal antibody, respectively. RESULTS: NMN treatment increased NAD+ concentration and thus improved cell viability, reduced apoptotic rate and decreased the LDH release in HS-treated CEC. Besides, NMN alleviated HS-induced MMP, intracellular ROS and LDH release. Besides, it was confirmed NMN improve SIRT1 function and decreased the HS related IL-17a expression in CEC and then alleviated macrophage phagocytosis ability and M1 polarization based on a CEC-macrophage co-culture system. Moreover, NMN treatment of CEC in the CEC could moderate the subsequent macrophage activation through Notch pathway. SIRT1 activation and IL-17a inhibition was regarded as key progress in the function of NMN based on the application of EX 527 and anti-IL-17a antibody in the CEC-macrophage co-culture system. CONCLUSION: The findings demonstrated that NMN could alleviated HS-induced DED status through regulating the CEC/macrophage interaction. Our data pointed to the role of SIRT1, IL-17a and Notch pathway in the function of NMN and then provided updated knowledge of potential NMN application in the management of DED.

Selection and Validation of Reference Genes for Quantitative Real-Time PCR in White Clover (Trifolium repens L.) Involved in Five Abiotic Stresses.

White clover (Trifolium repens L.) is a widely cultivated cool-season perennial forage legume in temperate grassland systems. Many studies have analyzed the gene expression in this grass species using quantitative real-time reverse transcription PCR (qRT-PCR). The selection of stable reference genes for qRT-PCR is crucial. However, there was no detailed study on reference genes in different tissues of white clover under various abiotic stress conditions. Herein, 14 candidate reference genes (ACT7, ACT101, TUA1109, TUB, CYP, 60SrRNA, UBQ, E3, GAPDH1, GAPDH2, PP2A, BAM3, SAMDC, and ABC) were selected and analyzed by four programs (GeNorm, NormFinder, BestKeeper, and RefFinder). Samples were taken from two tissues (leaves and roots) under five different abiotic stresses (drought, salt, heat, cold, and heavy metal stress). Our results showed that 60SrRNA and ACT101 were the two top-ranked genes for all samples. Under various experimental conditions, the most stable gene was different; however, SAMDC, UBQ, 60SrRNA, and ACT101 were always top ranked. The most suitable reference genes should be selected according to different plant tissues and growth conditions. Validation of these reference genes by expression analysis of Cyt-Cu/Zn SOD and CAT confirmed their reliability. Our study will benefit the subsequent research of gene function in this species.

Spermine Regulates Water Balance Associated with Ca2+-Dependent Aquaporin (TrTIP2-1, TrTIP2-2 and TrPIP2-7) Expression in Plants under Water Stress.

Spermine (Spm) regulates water balance involved in water channel proteins, aquaporins (AQPs), in plants. An increase in endogenous Spm content via exogenous Spm application significantly improved cell membrane stability, photosynthesis, osmotic adjustment (OA) and water use efficiency (WUE) contributing to enhanced tolerance to water stress in white clover. Spm upregulated TrTIP2-1, TrTIP2-2 and TrPIP2-7 expressions and also increased the abundance of TIP2 and PIP2-7 proteins in white clover under water stress. Spm quickly activated intracellular Ca2+ signaling and Spm-induced TrTIP2-2 and TrPIP2-7 expressions could be blocked by Ca2+ channel blockers and the inhibitor of Ca2+-dependent protein kinase in leaves of white clover. TrSAMS in relation to Spm biosynthesis was first cloned from white clover and the TrSAMS was located in the nucleus. Transgenic Arabidopsis overexpressing the TrSAMS had significantly higher endogenous Spm content and improved cell membrane stability, photosynthesis, OA, WUE and transcript levels of AtSIP1-1, AtSIP1-2, AtTIP2-1, AtTIP2-2, AtPIP1-2, AtPIP2-1 and AtNIP2-1 than wild type in response to water stress. Current findings indicate that Spm regulates water balance via an enhancement in OA, WUE and water transport related to Ca2+-dependent AQP expression in plants under water stress.

Novel Stapling by Lysine Tethering Provides Stable and Low Hemolytic Cationic Antimicrobial Peptides.

Cationic antimicrobial peptides (CAMPs) are potent therapeutics for drug-resistant bacterial infections. However, the clinical application of CAMPs is hampered by its poor proteolytic stability and hemolytic activity toward eukaryotic cells. Great efforts have been made to design and generate derivatives of CAMPs with improved pharmacological properties. Here, we report a novel stapling protocol, which tethers two ε-amino groups of the lysine residue by the N-alkylation reaction on the hydrophilic face of amphiphilic antimicrobial peptides. A series of lysine-tethered stapled CAMPs were synthesized, employing the antimicrobial peptide OH-CM6 as a model. Biological screening of the stapled CAMPs provided an analogue with strong antimicrobial activity, high proteolytic stability, and low hemolytic activity. This novel stapling approach offers an important chemical tool for developing CAMP-based antibiotics.

[Sterilization Effect of an Atmospheric Low Temperature Plasma Jet on Candida albicans Biofilm].

OBJECTIVE: To evaluate the sterilization effect of new designed atmospheric low temperature plasma jet on Candida albicans ( C. albicans) biofilm. METHODS: C. albicans was grown into the logarithmic phase, and then was added to polystyrene 24-well microtitre plate. The amount of germs were calculated by viable plate counting to determine the reproducibility of each biofilm well. The germs in biofilm were treated by plasma for different exposure time and then the survived germs were quantified by plate counting, the dead cells were determined by staining the biofilm with propidium iodide (PI), and the ultrastructural changes of the germs in biofilm were observed by transmission electron microscopy (TEM). RESULTS: When incubated for 72 h, germs tightly polymerized and classical mature biofilm were formed. This atmospheric low temperature plasma jet could inactivate C. albicans biofilm within a short exposure time. C. albicans were 90% inactivated when treated 20 s and 55 s of plasma treatment reduced bacteria populations to undetectable levels. With the increase of treatment time, enlarged fluorescent positive area appeared, and more bacteria died with the extending of exposure. The TEM scanning results showed that the new plasma jet inactivated C. albicans biofilm mainly via disrupting cell envelopes and then leading the release of cellular components, thus resulting in loss of cell viability. CONCLUSION: Plasma generated from atmospheric low temperature plasma jet could damage the cell structure of C. albicans and efficiently sterilize C. albicans biofilm.

Expansion and maintenance of primary corneal epithelial stem/progenitor cells by inhibition of TGFß receptor I-mediated signaling.

Transforming growth factor ß (TGFß) signaling is one of the most important signaling pathways regulating cell behavior in ocular tissues. Its functions are mainly linked to tissue fibrosis and inflammatory responses in ophthalmology. In epithelial cells, however, the growth inhibitory activity of TGFß was reported in both non-ocular and ocular tissues. Since TGFß is a bifunctional regulator that either inhibits or stimulates cell proliferation according to the specific context, we examined the effect of inhibition of TGFß receptor (TßR) I-mediated signaling on primary corneal epithelial cells (CECs) in serum- and feeder-free conditions. The mouse CECs were isolated from the eyeballs of 6-8 weeks old female C57BL/6 mice using dispase and trypsin separately, cultivated in defined Keratinocyte serum-free medium (KSFM) with supplements (the complete medium) without feeder layer. Cells were divided into three groups, those cultured in complete medium additionally supplemented with 10 µM SB-431542, a specific inhibitor of TßR-I, were SB-CECs; those cultured in complete medium additionally supplemented with 10 ng/ml SRI-011381, a TGF-beta signaling agonist, were SRI-CECs; those cultured in complete medium without SB-431542 or SRI-011381 were control CECs. The growth rate and morphology were analyzed by light microscopy. The identity and stemness of cells was investigated through marker staining of p63, inhibitor of differentiation 1 (ID1), cytokeratin 12 (K12), cytokeratin 14 (K14), PAX6, pSmad3, alpha smooth muscle Actin (αSMA) and E-cadherin (E-cad); Real-time quantitative (RT-PCR) analysis of p63; Western blot analysis of ID1; as well as colony forming assay, sphere forming assay, healing wound in vitro assay and air-lifting interface assay. The results showed SB-CECs subcultured steadily, achieved sustained expansion, and expanded almost thrice faster than control CECs. Expanded SB-CECs exhibited smaller and more compact morphology, up-regulated p63 and ID1, as well as better performed colony-forming capacity, sphere-forming capacity, in vitro wound healing capacity, and the capacity to stratify and differentiate on air-lifting interface. Preliminary tests on human limbal epithelial cells (HLECs) showed the same results as mouse CECs. Interestingly, the ID1 expression pattern was almost identical to p63, the typical marker for corneal epithelial stem/progenitor cell (CESC/CEPC), in cultured CECs and normal corneal sections. Since ID1 has been proven to be regulated negatively by TGFß signaling in epithelial cells and plays a role in blocking cell differentiation, its derepression by TßR-I inhibitor could be, at least in part, the underlying cause of CESC/CEPC expansion and the synchronously up-regulated expression of p63 in SB-CECs. In conclusion, inhibition of TßR-I-mediated signaling, CESCs/CEPCs achieved efficient long-term expansion in a feeder- and serum-free condition in vitro. And derepression of ID1 could be the underlying cause. Meanwhile, ID1 could serve as a marker for CESC/CEPC. These results may advance the basic and clinical CESC/CEPC research.

[Prediction and Analysis of Epitopes in clpP2 of Mycobacterium tuberculosis].

OBJECTIVES: To predict and analyze the antigenic epitopes in Mycobacterium tuberculosis protein caseinolytic protease P2 (clpP2), and explore its possibility to be applied as a new tuberculosis (TB) vaccine and drug development target. METHODS: Secondary structure of clpP2 based on nucleic sequence was predicted by DNA Star software. The homologous sequence conformation were analyzed by Swiss-Model online software. T cells antigenic epitopes were predicted through VaxiPred, and B cell epitopes were predicted by combining use of several different prediction programs, such as ABCpred, COBEPro and BepiPredPred. The immune characteristics of clpP2 were analyzed by DNA Star, SignalP, TMHMM online software and were searched through NCBI database. RESULTS: clpP2protein was diverse in structure, composing with a great deal of CTL and Th cell epitopes. clpP2 was also predicted to comprise rich potential liner and discontinuous B-cell epitopes. These epitopes were accessible on the protein surface, located in flexible and hydrophilic regions. CONCLUSION: clpP2 is prompted to induce immune responses and developes a novel target in surveillance, treatment and vaccine.

[Genetic Recombiniation and Protein Expression Detection of Listeria-based tuberculosis Vaccine Candidates.]

OBJECTIVES: Genetic construction of tuberculosis vaccine candidates based on Listeria(L.) monocytogenes,L.ivanovii,and evaluation their protein expression,in order to provide a novel method for research on tuberculosis controlling. METHODS: Two kinds of gene cassettes carrying tuberculosis antigen encoding gene Rv3875 or Rv0129c were inserted into targeting vector harboring L.monocytogenes,L.ivanovii homologous sequences via genetic connection methods and plasmid transformation technology in vitro.Targeting plasmids were electroporated into L.monocytogenes,L.ivanovii,and the recombinant strains were experienced serial passage at 42 °C and 30 °C.Subsequently,the tuberculosis antigen gene cassettes in targeting plasmids were integrated into L.monocytogenes and L.ivanovii attenuated strain (knocking out of virulence gene actA and plcB) and L.ivanovii wild type strain by homologous recombination and gene targeting technology.The recombinant strains were screened by blue-white spot and antibiotic resistance test;the intracellular and extracellular proteins of the recombinant strains were tested by Western blot. RESULTS: Five recombination strains carried antigen gene cassette were constructed,and the recombinant genome were confirmed by PCR and sequencing.No erythromycin resistance gene was found in 5 strains,which was coincident to expection.Recombination strains Li-Rv0129c,Li-ΔactAplcB-Rv0129c and Li-ΔactAplcB-Rv3875 expressed Mycobacterium tuberculosis antigenic protein,Ag85C or ESAT-6,as expected.But L.monocytogenes strains did not express proper antigenic protein. CONCLUSIONS: Three novel L.ivanovii-based tuberculosis vaccine candicates,carrying Mycobacterium tuberculosis Rv0129c antigen gene cassette (coding for Ag85C) or Rv3875 gene cassette (coding for ESAT-6),and expressing relevant antigenic proteins have been successfully selected.