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Nitrite is widespread in the environment, and is frequently used as an additive to extend the shelf life of meat products. However, the excess intake of nitrite can be harmful to human health. Hence, it is very important to know and control the content of nitrite in foodstuffs. In this work, by the means of self-assembly induced by solvent evaporation, we used the amphiphilic PEO-b-PS diblock copolymers resol and cobalt nitrate as a template to synthesize ordered mesoporous CoOx/C nanocomposites. Then, the CoOx/C nanocomposites were modified on a glassy carbon electrode (GCE), which showed excellent sensitivity, good selectivity, and a wide detection range for nitrite. Through cyclic voltammetry and current–time techniques, the electrochemical performance of the GCE modified with CoOx/C nanocomposites was analyzed. Under the optimized conditions, we found that anodic currents were linearly related to nitrite concentrations with a regression equation of lp (µA) = 0.36388 + 0.01616C (R2 = 0.9987) from 0.2 µM to 2500 µM, and the detection limit was 0.05 µM. Furthermore, the electrochemical sensor behaved with high reproducibility and anti-interference ability towards various organic and inorganic ions, such as NO3−, SO42−, Cl−, COOH− (Ac−), Na+, K+, Mg2+, and NH4+. Our results indicated that these CoOx/C nanocomposites could be applied in electrochemical sensors for the rapid and sensitive detection of the food preservative nitrite. [ABSTRACT FROM AUTHOR]

of bis(o-haloaryl)carbodiimides with primary amines [ABSTRACT FROM AUTHOR]

In the course of a large-scale program of ENU mutagenesis, we isolated a dominant mutation, called Velvet. The mutation was found to be uniformly lethal to homozygotes, which do not survive E13.5. Mice heterozygous for the Velvet mutation are born with eyelids open and demonstrate a wavy coat and curly vibrissae. The mutation was mapped to the proximal end of chromosome 11 by genome-wide linkage analysis. On 249 meioses, the locus was confined to a 2.7-Mb region, which included the epidermal growth factor receptor gene (Egfr). An A → G transition in the Egfr coding region of Velvet mice was identified, causing the amino acid substitution D833G. This substitution alters an essential triad of amino acids (DFG → GFG) that is normally required for coordination of the ATP substrate. As such, kinase activity is at least mostly abolished, but quaternary structure of the receptor is presumably maintained, accounting for the dominant effect. Velvet is the first known dominant representative of the Egfr allelic series that is fully viable, a fact that makes it particularly useful for developmental studies. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) is an important zoonotic foodborne pathogen that can induce acute gastrointestinal symptoms related to human infection. In this study, we characterized 49 STEC strains isolated from 816 retail raw meats from 2010 to 2016 in Southeast China for their serotype, virulence-associated genes, phylogenetic group, antibiotic resistance, and biofilm-forming ability. These isolates belonged to 33 divergent O: H serotypes, among which O157: H7 (12.24%, 6/49) was the most common serotype. Moreover, 22.45% (11/49) isolates belonged to the "top six" serotypes most frequently implicated in human infections. Among the 11 virulence-associated genes studied, stx2 (71.43%, 35/49) was the most prevalent virulence factor, with 87.76% (43/49) of isolates carrying two or more types of virulence factors. The B1 (55.10%, 27/49) and D (28.57%, 14/49) phylogenetic groups were predominant among isolates, whereas no strain belonged to group B2. A total of 59.18% (29/49) of isolates were resistant to 13 out of 16 tested antimicrobials, with resistance rate ranging from 4.08% (2/49) for both ceftazidime and chloramphenicol to 46.94% (23/49) for streptomycin. We detected eight out of 30 antibiotic-resistance genes (tetA , tetB , strA , strB , gyrA , floR , sulI , and sulII), with obvious mismatch between the phenotypes and genotypes of these isolates. Most strains (87.76%, 43/49) produced biofilms and 23.26% (10/43) were categorized as high producers. In conclusion, raw meats retailed in Southeast China contain STEC with multifarious serotypes, virulence-associated genes, antimicrobial resistance and biofilm-forming ability, some of which pose potential health risks to consumers. • Retail raw meats in Southeast China were contaminated with STEC. • STEC serogroups O157 and "top six" were isolated from the meat samples and analyzed. • 59% of isolates showed resistant to one or more types of antibiotics tested. • Biofilms were produced by 87% of strains, among which 23% were strong producers. [ABSTRACT FROM AUTHOR]

Background: Intervertebral disc (IVD) degeneration (IVDD) is characterized by structural destruction accompanied by accelerated signs of aging. This study aimed to investigate the mechanism of lysine methyltransferase 2D (KMT2D) in the proliferation, apoptosis, and inflammation of nucleus pulposus cells (NPCs) in IVDD., Methods: Mouse-derived NPCs were cultured and induced with interleukin-1 beta (IL-1β) to establish cell models. KMT2D expression was detected by western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). KMT2D expression was interfered with, and the contents of IL-18, IL-6, and tumor necrosis factor (TNF) were detected by enzyme-linked immunosorbent assay. Cell proliferation, apoptosis, and the expression of miR-133a-5p and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2) were measured. The enrichment of KMT2D and Histone 3 Lysine 4 monomethylation/dimethylation (H3K4me1/2) on the miR-133a-5p promoter was analyzed by chromatin immunoprecipitation and qPCR. The binding of miR-133a-5p and PFKFB2 was analyzed by a dual-luciferase assay., Results: IL-1β treatment promoted KMT2D expression in NPCs. KMT2D knockdown reduced inflammation and apoptosis and promoted the proliferation of IL-1β-induced NPCs. Mechanistically, KMT2D upregulated miR-133a-5p expression by increasing the level of H3K4me2 at the miR-133a-5p promoter, thereby promoting the binding between miR-133a-5p and PFKFB2 and downregulating the transcription of PFKFB2. miR-133a-5p overexpression or PFKFB2 knockdown alleviated the protective effect of KMT2D knockdown on IL-1β-induced NPCs., Conclusion: KMT2D promoted miR-133a-5p expression through H3K4me2 methylation, thereby promoting the binding of miR-133a-5p to PFKFB2, reducing the mRNA level of PFKFB2, promoting inflammation and apoptosis of IL-1β-induced NPCs, and inhibiting NPC proliferation., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

Listeria monocytogenes , which is abundant in environment, can lead to many kinds of serious illnesses and even death. Nowadays, indirectly detecting the metabolite biomarker of L. monocytogenes , 3-hydroxy-2-butanone, has been verified to be an effective way to evaluate the contamination of L. monocytogenes . However, this detection approach is still limited by sensitivity, selectivity, and ppb-level detection limit. Herein, low-cost and highly sensitive and selective 3-hydroxy-2-butanone sensors have been proposed based on the bimetallic AuPd decorated hierarchical flower-like WO 3 nanospheres. Notably, the 1.0 wt % AuPd-WO 3 based sensors displayed the highest sensitivity ( R a / R g = 84 @ 1 ppm) at 250 °C. In addition, the sensors showed outstanding selectivity, rapid response/recovery (8/4 s @ 10 ppm), and low detection limit (100 ppb). Furthermore, the evaluation of L. monocytogenes with high sensitivity and specificity has been achieved using 1.0 wt % AuPd-WO 3 based sensors. Such a marvelous sensing performance benefits from the synergistic effect of bimetallic AuPd nanoparticles, which lead to thicker electron depletion layer and increased adsorbed oxygen species. Meanwhile, the unique hierarchical nanostructure of the flower-like WO 3 nanospheres benefits the gas-sensing performance. The AuPd-WO 3 nanosphere-based sensors exhibit a particular and highly selective method to detect 3-hydroxy-2-butanone, foreseeing a feasible route for the rapid and nondestructive evaluation of foodborne pathogens.

Based on network pharmacology tools and public bioinformatics databases, the pharmacodynamic target and key mechanism of icariin (ICA) in the treatment of ovarian cancer (OC) were identified and experimentally verified. Our previous research showed that TNF, MMP9, STAT3, PIK3CA, ERBB2, MTOR, IL2, PTGS2, KDR and F2 are important targets of ICA in the treatment of OC. TNF, as a hub gene in tumor tissues, was associated with poor prognosis. ICA acted on OC mainly through the biological functions of various kinases, and the pathway with the highest accuracy ([Formula: see text]-value) was PI3K. Meanwhile, we observed a close upstream and downstream relationship between NF-[Formula: see text]B and the Pl3K-AKT pathway. This study further verified the mechanism of ICA in promoting apoptosis of SKOV3 cells through the NF-[Formula: see text]B signaling pathway and the tandem relationship between NF-[Formula: see text]B and the Pl3K-AKT pathway. The assay results demonstrated that ICA can promote the apoptosis of SKOV3 cells as indicated by the proapoptotic markers Bax, Bcl-xl and Caspase-3 and the key factors of the NF-[Formula: see text]B signaling pathway (NF-[Formula: see text]Bp65, p-NF-[Formula: see text]Bp65, p-I[Formula: see text]B[Formula: see text] and I[Formula: see text]B[Formula: see text]. ICA can block the classical NF-[Formula: see text]B pathway by inhibiting I[Formula: see text]B[Formula: see text] phosphorylation and consequently blocking the activation of the NF-[Formula: see text]B pathway in SKOV3 cells. ICA can also promote apoptosis by blocking the activation of the NF-[Formula: see text]B pathway in SKOV3 cells via inhibition of NF-[Formula: see text]Bp65 nuclear translocation. After using a PI3K pathway inhibitor, we further discovered that ICA may reduce AKT signal transduction by inhibiting the level of Akt phosphorylation, resulting in a loss of PI3K/Akt-dependent activation of the NF-[Formula: see text]B pathway.

In this work, we present a new metal oxide semiconductor gas sensor for detecting trimethylamine (TMA) by bimetal Au@Pt-modified α-Fe 2 O 3 hollow nanocubes (NCs) as sensing materials. The structure and morphological characteristics of Au@Pt/α-Fe 2 O 3 were evaluated through multiple analyses, and their gas-sensitive performance was investigated. Compared with the pristine α-Fe 2 O 3 NC sensor, the sensor based on Au@Pt/α-Fe 2 O 3 NCs exhibited faster response time (5 s) and higher response ( R a / R g = 32) toward 100 ppm TMA gas at a lower temperature (150 °C). Furthermore, we also assessed the Au@Pt/α-Fe 2 O 3 NC sensor for detecting the freshness of Larimichthys crocea which have been observed by headspace solid-phase microextraction and gas chromatography-mass spectrometry. The high performance of the Au@Pt/α-Fe 2 O 3 NCs is attributed to the special hollow morphology with a high specific surface area (212.9 m 2 /g) and the synergistic effect of the Au@Pt bimetal. The Au@Pt/α-Fe 2 O 3 sensor shows promising application prospects in estimating seafood freshness on the spot.

Food-borne pathogens pose great risks to human health and public safety, and the formation of biofilm exacerbates their pathogenicity and antimicrobial resistance. Enzymes can target special substances in the biofilm to disintegrate the biofilm of food-borne pathogens, which has great potential for applications. This review summarized the progress of using enzymes to disintegrate the biofilms of food-borne pathogens, highlighting quorum-quenching enzymes, C-di-GMP metabolic enzymes, as well as extracellular matrix hydrolases. Finally, challenges and perspectives on developing enzymes into effective products for disintegrating the biofilms of food-borne pathogens were discussed.

Listeria monocytogenes (L. monocytogenes) has been recognized as one of the extremely hazardous and potentially life-threatening food-borne pathogens, its real-time monitoring is of great importance to human health. Herein, a simple and effective method based on platinum sensitized tin dioxide semiconductor gas sensors has been proposed for selective and rapid detection of L. monocytogenes . Pt doped SnO 2 nanospheres with particular mesoporous hollow structure have been synthesized successfully through a robust and template-free approach and used for the detection of 3-hydroxy-2-butanone biomarker of L. monocytogenes . The steady crystal structure, unique micromorphology, good monodispersit, and large specific surface area of the obtained materials have been confirmed by X-ray diffraction (XRD), Raman spectroscopy, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-ray Photoelectron Spectroscopy (XPS), Brunauer-Emmett-Teller (BET), and Photoluminescence spectra (PL). Pt doped SnO 2 mesoporous hollow nanosphere sensors reach the maximum response of 3-hydroxy-2-butanone at 250°C. Remarkably, sensors based on SnO 2 mesoporous hollow nanospheres with 0.16 wt% Pt dopant exhibit excellent sensitivity (R air /R gas = 48.69) and short response/recovery time (11/20 s, respectively) to 10 ppm 3-hydroxy-2-butanone at the optimum working temperature. Moreover, 0.16 wt% Pt doped SnO 2 gas sensors also present particularly low limit of detection (LOD = 0.5 ppm), superb long-term stability and prominent selectivity to 3-hydroxy-2-butanone. Such a gas sensor with high sensing performance foresees its tremendous application prospects for accurate and efficient detection of foodborne pathogens for the food security and public health., (Copyright © 2019 Cai, Liu, Ni, Pan, Zhao and Zhu.)

This paper reports the successful design of an UC fluorescence nanosystem for distinguishable and sequential detection of MnO4- and Cr2O72- based on the inner filter effect (IFE) between Gd2O3:Yb,Er upconversion nanospheres (UCNS) and 3,3',5,5'-tetramethylbenzidine (TMB). The detection of MnO4- was realized by a stoichiometric oxidation reaction between TMB and Mn(vii), which resulted in quenching of the upconversion luminescence (UCL) by the blue product through IFE. The assay of Cr2O72-, in contrast, was based on the formation of complexes and the consumption of the oxidation product of the previous reaction. The sensing of MnO4- did not interfere with that of Cr2O72- when Pb2+ was added as a masking agent. This provides a new strategy for sensitive and selective detection of MnO4- and Cr2O72-. The detection of MnO4- showed not only a low LOD of 0.243 μM but also high selectivity over other possible interfering ions. The sensitivity assay of MnO4- can be performed in the linear range of 1.85 to 69 μM. An excellent linear relationship was also found in the sensing of Cr2O72-, with a linear range of 18.3 to 250 μM and a LOD of 2.95 μM. The selectivity towards Cr2O72- was also proved to be good. Based on these properties, the UCNS-TMB nanoplatform is a potential chemosensor for both MnO4- and Cr2O72- ions in practical assays for waste water.

Pathogenic and non-pathogenic Vibrio parahaemolyticus strains were simultaneously detected and quantified using a novel viable multiplex real-time PCR (novel qPCR). We used a new PCR primer and probe, ure R, as a surrogate for detection of the toxin trh gene as the primer was better at identifying variant V. parahaemolyticus trh strains. The specificity of all primers and probes used in this study were validated on three standard strains of V. parahaemolyticus , 42 clinical strains, 12 wild strains, 4 strains of Vibrio spp., and 4 strains of other bacteria. Then, propidium monoazide (PMA) was applied to inhibit DNA of dead cell, and the results of PMA optimized treatments were 15 μM concentration, 5 min incubation periods, 15 min light exposure periods and 30 RPM rotational speed, which resulted in time and cost savings. Pathogenic and non-pathogenic strains were quantified using a two-reaction tube method where the tlh , tdh , and ure R genes were amplified. Additionally, standard curves with a 7-log dynamic range were generated for quantifying viable V. parahaemolyticus and the amplification efficiencies were 108.68, 105.17, and 115.61% for tlh + , tdh + , and ure R + . This novel qPCR accurately monitored V. parahaemolyticus contamination rates in shrimps ( Penaeus vannamei ) and clams ( Ruditapes philippinarum ) sampled from retail stores located in a major district in Shanghai. In conclusion, our assay can prioritize the detection and quantification of viable pathogenic V. parahaemolyticus and can prove to be a more effective tool for reducing infection risks from consumption of seafood in Shanghai.

We evaluated the tolerance and pathogenesis of foodborne pathogens with a simulated gastro-intestinal tract model that simulates the chemical, physical and biological effects of human digestion process under laboratory conditions. This could be used to study the tolerance, pathogenesis, gut microbiota interaction and vaccine development of foodborne pathogens, so as to contribute to control and treatment of foodborne pathogens. This review introduces the applications of simulated gastro-intestinal tract model tp evaluate foodborne pathogens, which includes in-vitro static gastro-intestinal model, in-vitro dynamic gastro-intestinal model, conventional animal model and humanized animal model. And the concepts and characteristics of all models are described in detail. Also, the shortcomings of existing models are analyzed, and improvements of artificial gastro-intestinal tract model are prospected. In conclusion, this review could provide comprehensive data for promoting the progress of studying tolerance and pathogenesis of foodborne pathogens.

A novel TaqMan-based multiplex real-time PCR method combined with propidium monoazide (PMA) treatment was firstly developed for the simultaneous quantification of viable Vibrio parahaemolyticus and Listeria monocytogenes in raw shrimp. The optimization of PMA concentration showed that 100 μM was considered optimal to effectively inhibit 10(8) CFU/mL dead cells of both bacteria. The high specificity of this method was confirmed on tests using 96 target and non-target strains. The optimized assay could detect as low as 10(1)-10(2) CFU/g of each strain on the artificially contaminated shrimp, and its amplification efficiencies were up to 100 and 106 % for V. parahaemolyticus and L. monocytogenes, respectively. Furthermore, this assay has been successfully applied to describe the behavior of these two pathogens in raw shrimps stored at 4 °C. In conclusion, this PMA TaqMan-based multiplex real-time PCR technique, where the whole procedure takes less than 5 h, provides an effective and rapid tool for monitoring contamination of viable V. parahaemolyticus and L. monocytogenes in seafood, improving seafood safety and protecting public health.

A new N-ethyl-N-nitrosourea (ENU)-induced mouse recessive mutation, identified by fundus examination of the eye, develops depigmented patches, indicating retinal disorder. Histology data show aberrant retinal pigment epithelium (RPE) and late-onset photoreceptor cell loss in the mutant retina. Chromosomal mapping and DNA sequencing reveal a point mutation (T to A) of the Slc9a8 gene, resulting in mutant sodium/proton exchanger 8 (NHE8)-M120K protein. The lysine substitution decreases the probability of forming the 3(rd) transmembrane helix, which impairs the pore structure of the Na(+)/H(+) exchanger. Various RPE defects, including mislocalization of the apical marker ezrin, and disrupted apical microvilli and basal infoldings are observed in mutant mice. We have further generated NHE8 knockout mice and confirmed similar phenotypes, including abnormal RPE cells and late-onset photoreceptor cell loss. Both in vivo and in vitro data indicate that NHE8 co-localizes with ER, Golgi and intracellular vesicles in RPE cells. Thus, NHE8 function is necessary for the survival of photoreceptor cells and NHE8 is important for RPE cell polarity and function. Dysfunctional RPE may ultimately lead to photoreceptor cell death in the NHE8 mutants. Further studies will be needed to elucidate whether or not NHE8 regulates pH homeostasis in the protein secretory pathways of RPE.

Interneuronal subtype diversity lies at the heart of the distinct molecular properties and synaptic connections that shape the formation of the neuronal circuits that are necessary for the complex spatial and temporal processing of sensory information. Here, we investigate the role of Irx6, a member of the Iroquois homeodomain transcription factor family, in regulating the development of retinal bipolar interneurons. Using a knock-in reporter approach, we show that, in the mouse retina, Irx6 is expressed in type 2 and 3a OFF bipolar interneurons and is required for the expression of cell type-specific markers in these cells, likely through direct transcriptional regulation. In Irx6 mutant mice, presumptive type 3a bipolar cells exhibit an expansion of their axonal projection domain to the entire OFF region of the inner plexiform layer, and adopt molecular features of both type 2 and 3a bipolar cells, highlighted by the ectopic upregulation of neurokinin 3 receptor (Nk3r) and Vsx1. These findings reveal Irx6 as a key regulator of type 3a bipolar cell identity that prevents these cells from adopting characteristic features of type 2 bipolar cells. Analysis of the Irx6;Vsx1 double null retina suggests that the terminal differentiation of type 2 bipolar cells is dependent on the combined expression of the transcription factors Irx6 and Vsx1, but also points to the existence of Irx6;Vsx1-independent mechanisms in regulating OFF bipolar subtype-specific gene expression. This work provides insight into the generation of neuronal subtypes by revealing a mechanism in which opposing, yet interdependent, transcription factors regulate subtype identity.

Objective: To investigate morphological appearance of ductal carcinoma in situ of breast on MRI and to correlate the appearances with some factors., Methods and Materials: MRI feature of 41 DCISs were analyzed retrospectively according to ACR BI-RADS, twenty-three of the 41 were pure DCIS and 18 were DCIS with microinvasion (DCIS-MI). The shape was categorized as mass lesion and non-mass-like lesion. The shape was correlated with histological grade, ER status and expression of e-erbB2 as well as pure DCIS or DCIS-MI., Results: Percentage of high grade in non-mass-like lesion was higher than that in mass type group, Average size of tumor in group of high grade, negative ER status and positive c-erbB2 expression were larger than that in non-high grade, positive ER status and negative c-erbB2 expression. Comparing pure DCIS and DCIS-MI, high grade in DCIS-MI was significantly higher than that in pure DCIS, Average size of DCIS-MI was larger than that in pure DCIS. Percentage of non-mass-like lesion was higher in DCIS-IM than that in pure DCIS, the difference was marginally significant., Conclusion: DCIS can be classified as mass type and non-mass-like type morphologically on MRI. The two types reflect different biological behavior., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Purpose: To study the radiological appearance and pathological features of breast phyllodes tumors (PTs), and to enhance the recognition of the tumor., Materials and Methods: Clinical and imaging findings were retrospectively reviewed in 24 women with PTs confirmed by surgical pathology. All of the 24 patients had preoperative MRI and sonography, and 10 had preoperative mammography., Results: The histologic findings were benign, borderline and malignant PTs in 16.7% (4/24), 45.8% (11/24) and 37.5% (9/24) of cases, respectively. The tumor size (p=0.001), irregular shape on sonographic imaging (p=0.039), internal non-enhanced septations (p=0.009), silt-like changes in enhanced images (p=0.006) and signal changes from T2-weighted to enhanced images on MRI (p=0.001) correlated significantly with the histologic grade; the BI-RADS category of the MRI could reflect the PT's histologic grade with a correlation coefficient of 0.440 (p=0.031). If the category BI-RADS ≥4a was considered to be a suspicious malignant lesion, the diagnostic accuracy of mammography, US and MRI would be 70% (7/10), 62.5% (15/24) and 95.8% (23/24), respectively., Conclusion: The tumor size and several US and MRI findings can be used to help preoperatively determine the histologic grade of breast PTs. When a patient presents with a progressively enlarging, painless breast mass, MRI should be recommended first., (Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.)

Objective: To investigate the factors influencing mammographic and sonographic features of mucinous breast carcinoma., Materials and Methods: Forty-six patients diagnosed as mucinous breast carcinoma between March 2005 and April 2009 were studied. Mammographies of all patients and sonographies of 41 patients were analyzed retrospectively. Microscopic slides were re-evaluated by a pathologist in order to estimate the volume of extracellular mucus which was classified as: (+), less than 50% of mucus; (++), 50-80% of mucus; and (+++), more than 80% of mucus., Results: Thirty-one (61.3%) cases of pure mucinous type and 2 (33.3%) of mixed mucinous carcinoma presented as masses had well-defined margins on mammography. No correlation was found between margin characteristics and histological type as well as extracellular mucus rates. Denser breasts and young age group had significantly higher ratios of tumors presented with ill-defined masses than its counterpart. Sonographically, 100% lesions showed a hypoechoic lesion, 76.5% lesions in pure type and 71.4% lesions in mixed type showed a heterogeneous internal echo. No correlation was found between sonographic findings and histological type as well as extracellular mucus rates. Lesions with >40 positivity PCNA had higher ratio presented as ill-defined margins than those of ≤40% positivity PCNA., Conclusions: The most common appearance of mucinous breast carcinoma is a mass on mammography and a hypoechoic lesion with heterogeneous internal echo on sonography. Mammographic margins and sonographic findings have no relation with histological type and extracellular mucus rate. Mammographic margin features are correlated with breast density and age and PCNA., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

PURPOSE. To identify and characterize the r26 mouse line, which displays depigmented patches in the retina, and to determine the causative gene mutation and study the underlying mechanism. METHODS. Fundus examination, fluorescein angiography, histology, and immunostaining were used to determine the retinal phenotypes. Genome-wide linkage analysis, DNA sequencing, and an allelic test were used to identify the causative gene mutation. Wild-type and mutant gene products were examined by Western blot and transient transfection. RESULTS. Homozygous r26/r26 mice displayed depigmented patches in the fundus that overlapped the hyperfluorescent spots in the angiogram. Histology showed overgrown retinal vessels in the subretinal space. Immunostaining verified the presence of endothelial cells in the photoreceptor layer. Chromosome mapping and DNA sequencing revealed a point mutation, c.2239C>T, in the very-low-density lipoprotein receptor (Vldlr) gene. An allelic test in Vldlr knockout (-/-) mice confirmed that r26/(-) mice display a phenotype similar to that of r26/r26 mice. The Vldlr protein was predominantly localized at the plasma membrane of transfected cells, whereas the truncated Vldlr was diffusely expressed in the cell cytosol. The r26 truncated Vldlr was undetectable in mutant retinas by Western blot. CONCLUSIONS. The r26 is a recessive mutant caused by a missense mutation in the Vldlr gene. This results in a truncated Vldlr protein that lacks the C-terminal 127 amino acid residues including the single transmembrane domain and fails to localize at the plasma membrane. Thus, the r26 is a loss-of-function Vldlr mutation. Vldlr on the cell surface probably mediates an antiangiogenic signal to prevent retinal endothelial cells from migrating into the photoreceptor cell layer.

Transcriptional programs guide the specification of neural cell types in the developing nervous system. However, it is unclear whether such programs also control specific aspects of neural circuit function at maturity. In the mammalian retina, Vsx1 and Irx5 transcription factors are present in a subset of bipolar interneurons that convey signals from photoreceptors to ganglion cells. The biased expression of Vsx1 and Irx5 in hyperpolarizing OFF compared with depolarizing ON bipolar cells suggests that these transcription factors may selectively regulate signal processing in OFF circuits. To test this hypothesis, we generated mice lacking both Vsx1 and Irx5. Bipolar cells in these mice were morphologically normal, but the expression of cell-specific markers in some OFF but not ON bipolar cells was reduced or absent. To assess visual function in Vsx1(-/-)Irx5(-/-) retinas, we recorded light responses from ensembles of retinal ganglion cells (RGCs). We first identified functional RGC types in control mice and describe their response properties and adaptation to temporal contrast using a simple linear-nonlinear model. We found that space-time receptive fields of RGCs are unchanged in Vsx1(-/-)Irx5(-/-) mice compared with control retinas. In contrast, response threshold, gain, and range were lowered in a cell-type-specific manner in OFF but not ON RGCs in Vsx1(-/-)Irx5(-/-) retinas. Finally, we discovered that the ability to adapt to temporal contrast is greatly reduced in OFF RGCs in the double mutant, suggesting that Vsx1 and Irx5 control specific aspects of visual function in circuits of the mammalian retina.

Purpose: To understand the underlying molecular mechanism for a dominant cataract caused by a point mutation in the gammaD-crystallin gene., Methods: A dominant cataractous mouse line was identified from chemically induced mouse mutations by phenotypic screening with slit lamp examination. Genomewide linkage analysis and DNA sequencing were used to determine the causative gene mutation. Histology, immunohistochemistry, Western blotting, and in vitro transfection studies were used to characterize mutant lenses., Results: Cataracts in mutant mice were caused by a point mutation in the gammaD-crystallin gene (gammaD-V76D). Intranuclear gamma-crystallin aggregates, incomplete denucleation, and decreased connexins were observed in mutant lens fiber cells. Mutant gammaD-V76D proteins became less soluble in the lens, and structural modeling suggested that the substituted aspartic acid residue (D) altered hydrogen bond formation and surface electrostatic potential of the protein. Unexpectedly, the formation of cold cataracts, which occurred in wild-type lenses at low temperature, was abolished in gammaD-V76D mutant lenses. In vitro transfection studies revealed that wild-type gammaD proteins were uniformly distributed in the cytosol and nucleus of transfected cells, whereas gammaD-V76D proteins formed cytosolic and nuclear aggregates., Conclusions: Mutant gammaD-V76D reduces protein solubility in the lens and forms substantial intranuclear aggregates that disrupt the denucleation process of inner lens fiber cells. Sustained fiber cell nuclei and nuclear remnants scatter light, whereas other downstream events, such as decreased connexins, presumably disrupt gap junction communication and lens homeostasis, further contributing to the cataract phenotype in mutant lenses. This work also suggests that gammaD-crystallin is one of the crucial components for the formation of cold cataracts in vivo.

Purpose: To identify new mouse models for studying roles of alphaAlpha-crystallin in vivo and to investigate why and how different mutations of the alphaAlpha-crystallin gene lead to dominant or recessive cataracts., Methods: Using mouse genetic approaches and slit lamp screening, we identified two mouse cataractous mutant lines. Causative genes were mapped by a genome-wide linkage analysis. DNA sequencing verified missense mutations of alphaA-crystallin gene in both mutant lines. Histology, imaging of green fluorescent protein (GFP)-positive lenses, and protein 2-DE gel were used to determine the morphologic and biochemical properties of mutant lenses., Results: Two new alphaA-crystallin gene mutations were identified, alphaA-R54C (alphaA-Cys) and alphaA-Y118D, which cause recessive whole cataracts and dominant nuclear cataracts, respectively. In homozygous alphaA-Cys mutant mice, lens epithelial and fiber cells lost their characteristic cellular features and developed disrupted subcellular structures, such as actin filaments and mitochondria. The nuclear cataract caused by alphaA-Y118D mutation was associated with increased water-insoluble crystallins (alpha, beta, and gamma classes). These results suggest that the Arg54 residue in the N-terminal region is crucial for alphaA-crystallin to perform its roles in lens epithelial and fiber cells during development, whereas the Y118D mutation in the central alpha-crystallin domain impairs alphaA-crystallin's ability to maintain the solubility of crystallin proteins in the lens., Conclusions: This work demonstrates that different regions of alphaA-crystallin mediate distinct functions in vivo. These two mutant mouse lines provide useful animal models for further investigating the multiple roles of alphaA-crystallin in the lens.

A new mouse mutant line, Clapper, identified from N-ethyl-N-nitrosurea (ENU)-mutagenized mice, develops a dominant lamellar cataract. The cataract blocks the image of retinal fundus and transmits a fuzzy fluorescein image of retinal vasculature during angiography. The cataractous lens opacity decreases as the mice age. The Clapper mutation has been identified to be a missense mutation of the gammaB-crystallin gene that replaces the 4th isoleucine residue with a phenylalanine (gammaB-I4F). Unlike wild type gammaB, the gammaB-I4F mutant protein binds to alpha-crystallin to form high molecular weight complexes in vivo and in vitro. Circular dichroism measurements indicate that gammaB-I4F protein is less stable than wild type gammaB at high temperature. Darkly stained aggregates, enlarged interfiber spaces, and disorganized and smaller inner mature fibers were found in the regions of the cataract in homozygous Clapper mutant lenses. Thus, the lamellar cataract is likely due to the light-scattering effects of the enlarged interfiber spaces and protein aggregates caused by gammaB-I4F mutant proteins interacting with alpha-crystallin in the lens.

Purpose: To investigate the expression of p53, MDM2 and Ref1 gene in cultured retina neurons of SD rats treated with Vitamin B1 and (or) elevated pressure., Methods: The retinal neuron of postnatal SD rats were cultured in vivo, the elevated pressure was produced after 7 days, and the total RNA was extracted after another 2 days, expression of p53, MDM2 and Ref1 gene were analyzed with RT-PCR., Results: The expression level of p53 and MDM2 gene were increased in elevated pressure group, normal with Ref1 gene expression. But the expression of p53 and MDM2 gene were decreased significantly in elevated pressure group treated with vitamine B1 compare to the elevated group., Conclusion: Apoptosis seem to be a mechanism of cell death in retinal neurons of SD rats with elevated pressure.Vitamine B1 have protect effects against elevated pressure.

Objective: To investigate the effects of N-methyl-D-aspartate (NMDA), MK801 (non-competitive blocking agent of NMDA receptor) and Ca(2+) on cultured retinal neurons and the interaction of these reagents., Methods: NMDA, MK801 and Ca(2+) were singly and jointly added to the retinal neurons after primary culture for 7 days which obtained from 1 - 3 day-old postnatal Sprague-Dawley rat. The cells were identified with anti-neurofilament protein (NF), Thy1.1 and anti-glial fibrillary acidic protein (GFAP) monoclonal anti-body. The effects were assessed quantitatively using the trypan blue exclusion methods., Results: Neuronal death which induced by 200 micromol/L NMDA and 10 mmol/L Ca(2+) was significantly different from that of the control, 20 micromol/L MK801 had no significant effect on cultured retinal neurons, and did not protect the cell from neurotoxicity of Ca(2+), but could block the neurotoxicty of NMDA., Conclusions: NMDA and certain concentration of Ca(2+) are neurotoxic to retinal neurons in the primary cultured. MK801 can block the neurotoxicity of NMDA, but not the neurotoxicity of Ca(2+). Neuronal death induced by Ca(2+) may get through another mechanism.