Search

A recent challenge in bioimaging is the observation and imaging of vital, thick, and complex tissues in real time and in non-invasive mode. In the last decade, non-linear excitation microscopy showed several advantages for in-vivo imaging compared to conventional confocal techniques. Nevertheless, deep tissue imaging remains challenging, especially for thick media, due to spherical aberrations induced on focused beams by the tissue. A low numerical aperture objective lens coupled to high dioptric power microlenses, implanted in the tissue, can be beneficial for the reduction of optical aberrations. In this context, we fabricated a system of plano-convex microlenses and microscaffolds on a single chip by means of two-photon polymerization), to be used for non-linear imaging of biological specimens.

‘Braeburn’ apples from three harvests after 6-month storage in controlled atmosphere were measured at 670 nm by time-resolved reflectance spectroscopy (TRS), ranked on decreasing μa670 (increasing maturity), classified as less (LeM), medium and more mature (MoM), randomised into three batches per harvest and analysed after 1, 8 and 14 days of shelf life. LeM and MoM apples were measured in the 630-900 nm range by TRS, and analysed for sensory profile (firm, crispy, juicy, mealy) and pulp mechanical characteristics (firmness, stiffness, energy-to-rupture). All data were processed by Principal Component Analysis (PCA). According to sensory intensity scores, fruits were either divided into five classes (very low – VL; low – L; medium – M; high – H; very high – VH) separately for every attribute, or clustered into four groups, each one representing a specific sensory profile. The absorption spectra showed a maximum at 670 nm (chlorophyll-a) and μa670 was higher in the VH class for firm, crispy and juicy and in the VL and L classes for mealy. The scattering spectra had a decreasing trend with the wavelength increase, and μs’ values were lower in the VH class for firm and crispy, and higher in the VH class for mealy and in the VL ones for juicy. PCA underlined that μs’ values were negatively related to firmness and μa670, and that μa690, μa730, μa830, μa850 and μa900 were opposed to mealiness. PC scores differed among the four sensory profiles and increased from VL to VH classes for firmness, crispiness and juiciness and from VH to VL classes for mealiness.

We investigate the presence and behavior of Cu, Pb, and Cd in runoff waters from a residential area located in the city of Curitiba, Brazil. Samples were collected in twenty-one stormwater events in an intermediate traffic way located in the Bacacheri watershed. Metal concentrations were similar to range values compiled worldwide and followed the order: Pb>Cu>>Cd. Unit loads were estimated to the watershed, which cover an area of 30 km², revealing values of 1520, 950, 25 kg for Pb, Cu, and Cd, respectively. Among the three metals, lead showed the greatest affinity for the suspended solids (82%) followed by Cd (66%) and Cu (48%). Meanwhile, an association of the metals with the organic matter in the dissolved phase, following the order Cu>Pb>Cd. Metal loads and behavior in urban runoff also depends on factors such as the first flush and the dry period preceding a storm event.

The sorption of copper ions from aqueous solutions onto modified aluminum oxide was investigated as a function of pH, solids concentration and both particulate and dissolved organic matter. The aluminum oxide studied was modified by adsorption of humic acid. The pH value, organic matter content, and solids concentration dominated copper adsorption onto modified aluminum oxide. The adsorption of copper increased with increasing pH. An increase in particulate organic carbon resulted in an enhanced copper uptake. The partition coefficient (Kd) of copper decreased as the solids concentration increased. Copper partitioning was maximal in the neutral pH range and decreased at either low or high pH values. There was also a decrease in copper adsorption with increasing dissolved organic matter concentration.

Abstract Background Synthetic biology allows the development of new biochemical pathways for the production of chemicals from renewable sources. One major challenge is the identification of suitable microorganisms to hold these pathways with sufficient robustness and high yield. In this work we analyzed the genome of the propionic acid producer Actinobacteria Propionibacterium acidipropionici (ATCC 4875). Results The assembled P. acidipropionici genome has 3,656,170 base pairs (bp) with 68.8% G + C content and a low-copy plasmid of 6,868 bp. We identified 3,336 protein coding genes, approximately 1000 more than P. freudenreichii and P. acnes, with an increase in the number of genes putatively involved in maintenance of genome integrity, as well as the presence of an invertase and genes putatively involved in carbon catabolite repression. In addition, we made an experimental confirmation of the ability of P. acidipropionici to fix CO2, but no phosphoenolpyruvate carboxylase coding gene was found in the genome. Instead, we identified the pyruvate carboxylase gene and confirmed the presence of the corresponding enzyme in proteome analysis as a potential candidate for this activity. Similarly, the phosphate acetyltransferase and acetate kinase genes, which are considered responsible for acetate formation, were not present in the genome. In P. acidipropionici, a similar function seems to be performed by an ADP forming acetate-CoA ligase gene and its corresponding enzyme was confirmed in the proteome analysis. Conclusions Our data shows that P. acidipropionici has several of the desired features that are required to become a platform for the production of chemical commodities: multiple pathways for efficient feedstock utilization, ability to fix CO2, robustness, and efficient production of propionic acid, a potential precursor for valuable 3-carbon compounds.

Abstract Background Frontotemporal Lobar Degeneration (FTLD) thus recently renamed, refers to a spectrum of heterogeneous conditions. This same heterogeneity of presentation represents the major methodological limit for the correct evaluation of clinical designation and brain functional correlates. At present, no study has investigated clinical clusters due to specific cognitive and behavioural disturbances beyond current clinical criteria. The aim of this study was to identify clinical FTLD presentation, based on cognitive and behavioural profile, and to define their SPECT functional correlations. Methods Ninety-seven FTLD patients entered the study. A clinical evaluation and standardised assessment were preformed, as well as a brain SPECT perfusion imaging study. Latent Profile Analysis on clinical, neuropsychological, and behavioural data was performed. Voxel-basis analysis of SPECT data was computed. Results Three specific clusters were identified and named "pseudomanic behaviour" (LC1), "cognitive" (LC2), and "pseudodepressed behaviour" (LC3) endophenotypes. These endophenotypes showed a comparable hypoperfusion in left temporal lobe, but a specific pattern involving: medial and orbitobasal frontal cortex in LC1, subcortical brain region in LC2, and right dorsolateral frontal cortex and insula in LC3. Conclusion These findings provide evidence that specific functional-cluster symptom relationship can be delineated in FTLD patients by a standardised assessment. The understanding of the different functional correlates of clinical presentations will hopefully lead to the possibility of individuating diagnostic and treatment algorithms.

Processed cheese is a commercial product characterized by high microbiological stability and extended shelf life obtained through the application of severe heat treatment. However, spore-forming bacteria can survive through thermal processes. Among them, microorganisms belonging to Bacillus genus have been reported. In this study, we examined the microbiological population of the first hours' production of processed cheeses in an Italian dairy plant during two seasons, between June and October 2020. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify bacteria colonies, allowing the isolation of Bacillus cereus and Bacillus subtilis strains. These results were further confirmed by amplification and sequencing of 16 rRNA bacterial region. A multi-locus sequence type (MLST) analysis was performed to assess the genetic similarity among a selection of isolates. The fourteen B. cereus strains showed two sequence types: ST-32 was observed in only one strain and the ST-371 in the remaining thirteen isolates. On the contrary, all twenty-one B. subtlis strains, included in the study, showed a new allelic profile for the pycA gene, resulting in a new sequence type: ST-249. For B. cereus strains, analysis of toxin genes was performed. All isolates were positive for nheABC , entFM , and cytK , while hblABCD , bceT , and ces were not detected. Moreover, the biofilm-forming ability of B. cereus and B. subtilis strains was assessed, and all selected isolates proved to be biofilm formers (most of them were stronger producers). Considering the genetical similarity between isolates, jointly with the capacity to produce biofilm, the presence of a recurring Bacillus population could be hypothesized.

A pathognomonic macular ripple sign has been reported with scanning laser ophthalmoscopy images in patients with foveal hypoplasia, though the optical basis of this sign is presently unknown. Here we present a case series of seven individuals with foveal hypoplasia (based on spectral domain optical coherence tomography). Each patient underwent infrared scanning laser ophthalmoscopy retinal imaging in both eyes, acquired with and without a polarization filter and assessment for a ripple-like effect in the fovea. On imaging, macular ripples were present in all eyes with foveal hypoplasia when using a polarization filter, but not when imaged without the filter. We conclude that the macular ripple sign is an imaging artifact attributable to the unique pattern of phase retardation of the Henle fiber layer in the setting of foveal hypoplasia. By utilizing a polarization filter with retinal photography, this feature can be exploited to promptly identify foveal hypoplasia in settings where OCT is not possible due to nystagmus.

Introduction: We present two patients, the proband and the affected sibling, with biallelic CRB1 mutations leading to a macular dystrophy., Case Presentation: We present two patients, the proband and the affected sibling, with biallelic CRB1 mutations leading to a macular dystrophy. With 15 years of follow-up for the proband, we illustrate the natural history of CRB1 maculopathy based on clinical examination, multimodal imaging, and electrophysiology. In addition, we demonstrate the wide phenotypic spectrum of the condition with the affected sister harboring the same variants but with much milder phenotypic manifestations., Conclusion: In addition to a previously described pathogenic variant, Ile167_Gly169del, one pathogenic missense variant in CRB1, Lys801Ter, not previously associated with macular dystrophy, is reported here. While CRB1 mutations have been more commonly described in retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), we demonstrate that mutations in CRB1 can cause a maculopathy with initial features similar to fenestrated sheen macular dystrophy (FSMD) that later evolves into severe macular atrophy.

We determined differential gene expression in response to high glucose in lymphoblastoid cell lines derived from matched individuals with type 1 diabetes with and without retinopathy. Those genes exhibiting the largest difference in glucose response were assessed for association with diabetic retinopathy in a genome-wide association study meta-analysis. Expression quantitative trait loci (eQTLs) of the glucose response genes were tested for association with diabetic retinopathy. We detected an enrichment of the eQTLs from the glucose response genes among small association p-values and identified folliculin ( FLCN ) as a susceptibility gene for diabetic retinopathy. Expression of FLCN in response to glucose was greater in individuals with diabetic retinopathy. Independent cohorts of individuals with diabetes revealed an association of FLCN eQTLs with diabetic retinopathy. Mendelian randomization confirmed a direct positive effect of increased FLCN expression on retinopathy. Integrating genetic association with gene expression implicated FLCN as a disease gene for diabetic retinopathy., Competing Interests: AS, SJ, AS, SC, SF, OS, PB, AL, MS, DC, AS, IB, BS, MG No competing interests declared, (© 2020, Skol et al.)

We report two unrelated male patients presenting at a young age with decreased vision from a macular dystrophy due to biallelic CRB1 mutations. In addition to a previously-described pathogenic variant, Ile167_Gly169del, two new pathogenic missense variants in CRB1, Thr745Met, and Cys948Tyr are reported here. While CRB1 mutations have been more commonly described in retinitis pigmentosa (RP) and Leber's congenital amaurosis (LCA), we demonstrate that mutations in CRB1 can cause a maculopathy whose initial features can resemble juvenile X-linked retinoschisis (JXLRS). We show that the accompanying macular edema is responsive to carbonic anhydrase inhibitors. With long-term follow-up for each case, we illustrate the natural history of CRB1 maculopathy based on clinical examination and diagnostic imaging., Competing Interests: Financial disclosure and competing interest statement: Each author warrants that he or she has no commercial associations that might pose a conflict of interest in connection with the submitted article. We hereby declare that none of the authors have a proprietary interest.

Background: Multifocal choroiditis (MFC) is a relatively uncommon bilateral inflammatory chorioretinopathy affecting Caucasian young women with myopia. We present images from a case of completely unilateral multifocal choroiditis following EBV-positive mononucleosis that demonstrated a dramatic clinical response to immunosuppression., Case Presentation: A 20-year-old woman with bilateral high myopia (-6D) and a documented normal prior retinal examination presented with visual loss in the right eye 2 months following confirmed Epstein-Barr virus (EBV) positive mononucleosis. Ophthalmoscopic examination showed completely unilateral placoid lesions of variable age. The left eye was unaffected. Fluorescein angiography revealed active leakage, especially in the parafovea. Spectral domain optical coherence tomography (SD-OCT) demonstrated sub-retinal pigment epithelial nodular deposits, some of which were confluent with overlying intra-retinal fluid and indistinct margins. Upon treatment with the immunosuppressant azathioprine there was significant resolution of the lesions in her right eye along with improvement in vision., Conclusion: This is a rare case of completely unilateral MFC following an episode of EBV positive mononucleosis that showed a dramatic response to immunosuppression.

Literature data regarding the survival of microorganisms on materials used for food package purposes are scarce. The aim of the current study is to assess the survival of Listeria monocytogenes on different packaging materials for dairy products during extended storage at different temperatures. Three packaging materials (5 × 5 cm) were contaminated with a cocktail of five strains of Listeria monocytogenes suspended in a cheese homogenate, including the cheese's native microbial population. Contaminated samples were incubated at 37°, 12°, and 4°C and periodically analyzed up to 56 days. The evolution of the total viable count and pathogen population was evaluated. At 37°C, the results showed that Listeria monocytogenes was no longer detected on polyethylene-coated nylon (B) by day 4 and on polyethylene-coated parchment (A) and greaseproof paper (C) by day 7. Interestingly, the initial cell population (ranging between 2.5 and 2.7 log CFU/cm 2 ) of Listeria monocytogenes increased to 3 log CFU/cm 2 within 4 days of storage at 12°C on A and C. During storage, the number remained fairly constant at 12°C and 4°C on two materials (A-C) and decreased slowly on the third one (B). This study shows that survival of Listeria monocytogenes on packaging materials for dairy products will be higher when stored at 4 or 12°C compared to 37°C. The survival of Listeria monocytogenes on the packaging materials raises concerns of cross-contamination during food handling and preparation at catering and retail premises and within the home, highlighting the importance of treating the packaging materials as a potential source of cross-contamination. These initial findings may aid in quantifying risks associated with contamination of food packaging materials., (Copyright © 2020 Di Ciccio, Rubiola, Grassi, Civera, Abbate and Chiesa.)

Purpose: To report a case of linezolid-induced reversible retinopathy., Methods: Case report with literature review., Results: Clinical examination and imaging are presented over a 7-month interval, from initial presentation to subsequent follow-up (6 months after discontinuation of linezolid). The subject was found to have not only an optic neuropathy but also severe reversible photoreceptor dysfunction as demonstrated by electrophysiologic testing. Upon discontinuation of linezolid, not only did the patient's visual acuity, visual fields, and visual evoked potential significantly improve, but the electroretinogram did as well., Conclusions: Linezolid has previously been reported to cause a toxic optic neuropathy. Reversible photoreceptor dysfunction on full-field electroretinography has never been reported in conjunction with linezolid toxicity. This novel case suggests that linezolid toxicity should be considered in cases of photoreceptor dysfunction.

To identify genetic variants associated with diabetic retinopathy (DR), we performed a large multiethnic genome-wide association study. Discovery included eight European cohorts ( n = 3,246) and seven African American cohorts ( n = 2,611). We meta-analyzed across cohorts using inverse-variance weighting, with and without liability threshold modeling of glycemic control and duration of diabetes. Variants with a P value <1 × 10 -5 were investigated in replication cohorts that included 18,545 European, 16,453 Asian, and 2,710 Hispanic subjects. After correction for multiple testing, the C allele of rs142293996 in an intron of nuclear VCP-like ( NVL ) was associated with DR in European discovery cohorts ( P = 2.1 × 10 -9 ), but did not reach genome-wide significance after meta-analysis with replication cohorts. We applied the Disease Association Protein-Protein Link Evaluator (DAPPLE) to our discovery results to test for evidence of risk being spread across underlying molecular pathways. One protein-protein interaction network built from genes in regions associated with proliferative DR was found to have significant connectivity ( P = 0.0009) and corroborated with gene set enrichment analyses. These findings suggest that genetic variation in NVL, as well as variation within a protein-protein interaction network that includes genes implicated in inflammation, may influence risk for DR., (© 2018 by the American Diabetes Association.)

Purpose: To assess whether mutations in Mer tyrosine kinase (MERTK) are associated with age-related macular degeneration (AMD)., Methods: An association study using whole-genome sequencing was performed to determine whether rare variants in MERTK are associated with AMD. The data set included 4787 propensity score-matched case-control samples: 2394 AMD cases and 2393 controls. Whole-genome sequencing was performed and variants in MERTK were identified. Combined annotation-dependent depletion (CADD) scores and allele frequencies were calculated for each variant identified in MERTK. Student's t-test was used to assess the mean number of MERTK variants per subject between case and control cohorts (Bonferroni adjusted α = 0.0125). The number of subjects carrying at least one high CADD score loss-of-function or nonsynonymous mutation in each cohort was compared using Fisher's exact test (p < 0.05)., Results: No significant difference was found in the mean number of MERTK variants in AMD versus control subjects (p = 0.0502). Additionally, there was no significant difference between cohorts in the number of subjects with at least one high CADD score loss-of-function or nonsynonymous variant (p = 0.15 at CADD > 10 and p = 0.91 at CADD > 20)., Conclusions: The present study provides a meaningfully negative result demonstrating that rare variants in MERTK are not associated with AMD. The study also demonstrates the role of large sample size genetic studies utilizing whole-genome sequencing as a powerful tool that can resolve clinically relevant questions regarding the genetic basis of ophthalmic disease.

Purpose: Diabetic retinopathy is the most common eye complication in patients with diabetes. The purpose of this study is to identify genetic factors contributing to severe diabetic retinopathy., Methods: A genome-wide association approach was applied. In the Genetics of Diabetes Audit and Research in Tayside Scotland (GoDARTS) datasets, cases of severe diabetic retinopathy were defined as type 2 diabetic patients who were ever graded as having severe background retinopathy (Level R3) or proliferative retinopathy (Level R4) in at least one eye according to the Scottish Diabetic Retinopathy Grading Scheme or who were once treated by laser photocoagulation. Controls were diabetic individuals whose longitudinal retinopathy screening records were either normal (Level R0) or only with mild background retinopathy (Level R1) in both eyes. Significant Single Nucleotide Polymorphisms (SNPs) were taken forward for meta-analysis using multiple Caucasian cohorts., Results: Five hundred and sixty cases of type 2 diabetes with severe diabetic retinopathy and 4,106 controls were identified in the GoDARTS cohort. We revealed that rs3913535 in the NADPH Oxidase 4 (NOX4) gene reached a p value of 4.05 × 10 -9 . Two nearby SNPs, rs10765219 and rs11018670 also showed promising p values (p values = 7.41 × 10 -8 and 1.23 × 10 -8 , respectively). In the meta-analysis using multiple Caucasian cohorts (excluding GoDARTS), rs10765219 and rs11018670 showed associations for diabetic retinopathy (p = 0.003 and 0.007, respectively), while the p value of rs3913535 was not significant (p = 0.429)., Conclusion: This genome-wide association study of severe diabetic retinopathy suggests new evidence for the involvement of the NOX4 gene., (© 2018 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.)

Purpose: To present a case of a patient with XXXXY syndrome, anomalous optic nerves, and dural ectasia in conjunction with macular detachment., Methods: Case report., Results: A 3-year-old boy with XXXXY chromosomal abnormality presented with bilateral maculopathy. On evaluation, he was found to have anomalous optic disks with serous detachment of the left eye. Magnetic resonance imaging of the brain revealed bilateral optic nerve dural ectasia without evidence of elevated intracranial pressure., Conclusion: XXXXY syndrome, like the related condition of Klinefelter syndrome, can manifest with ocular abnormalities. In the present case, the dural ectasia may have facilitated access of cerebrospinal fluid through anomalous optic nerves, resulting in neurosensory detachment.

Purpose: Retinitis pigmentosa (RP) is a genetically heterogeneous inherited retinal dystrophy. To date, over 80 genes have been implicated in RP. However, the disease demonstrates significant locus and allelic heterogeneity not entirely captured by current testing platforms. The purpose of the present study was to characterize the underlying mutation in a patient with RP without a molecular diagnosis after initial genetic testing., Methods: Whole-exome sequencing of the affected proband was performed. Candidate gene mutations were selected based on adherence to expected genetic inheritance pattern and predicted pathogenicity. Sanger sequencing of MERTK was completed on the patient's unaffected mother, affected brother, and unaffected sister to determine genetic phase., Results: Eight sequence variants were identified in the proband in known RP-associated genes. Sequence analysis revealed that the proband was a compound heterozygote with two independent mutations in MERTK, a novel nonsense mutation (c.2179C > T) and a previously reported missense variant (c.2530C > T). The proband's affected brother also had both mutations. Predicted phase was confirmed in unaffected family members., Conclusion: Our study identifies a novel nonsense mutation in MERTK in a family with RP and no prior molecular diagnosis. The present study also demonstrates the clinical value of exome sequencing in determining the genetic basis of Mendelian diseases when standard genetic testing is unsuccessful.

Purpose: To report a case of severe, bilateral, rapidly progressing peripheral retinal nonperfusion associated with underlying aplastic anemia., Methods: An interventional case report., Results: A 4-year-old girl presented with decreased visual acuity. On clinical examination, she was found to have a RAPD, elevated intraocular pressure, 360° rubeosis, vitreous hemorrhage, severe exudative retinal detachment, and telangiectasia with severe peripheral retinal nonperfusion. Laboratory workup was significant for pancytopenia, and a bone marrow biopsy showed extreme hypocellularity with no malignant cells. The patient was diagnosed with primary aplastic anemia. She developed dramatic progression of retinal nonperfusion in the left eye, as well as in the fellow right eye. This bilateral retinopathy was poorly responsive to aggressive management, which included laser photocoagulation and intravitreal injections of anti-vascular endothelial growth factor medications., Conclusion: Asymmetric, bilateral quickly progressing peripheral retinal ischemia, in conjunction with pancytopenia and otherwise negative workup may be related to underlying bone marrow failure and aplastic anemia.

Competing Interests: Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported.

Background: White blood cells have been shown in animal studies to play a central role in the pathogenesis of diabetic retinopathy. Lymphoblastoid cells are immortalized EBV-transformed primary B-cell leukocytes that have been extensively used as a model for conditions in which white blood cells play a primary role. The purpose of this study was to investigate whether lymphoblastoid cell lines, by retaining many of the key features of primary leukocytes, can be induced with glucose to demonstrate relevant biological responses to those found in diabetic retinopathy., Methods: Lymphoblastoid cell lines were obtained from twenty-three human subjects. Differences between high and standard glucose conditions were assessed for expression, endothelial adhesion, and reactive oxygen species., Results: Collectively, stimulation of the lymphoblastoid cell lines with high glucose demonstrated corresponding changes on molecular, cellular and functional levels. Lymphoblastoid cell lines up-regulated expression of a panel of genes associated with the leukocyte-mediated inflammation found in diabetic retinopathy that include: a cytokine (IL-1B fold change = 2.11, p-value = 0.02), an enzyme (PKCB fold change = 2.30, p-value = 0.01), transcription factors (NFKB-p50 fold change = 2.05, p-value = 0.01), (NFKB-p65 fold change = 2.82, p-value = 0.003), and an adhesion molecule (CD18 fold change = 2.59, 0.02). Protein expression of CD18 was also increased (p-value = 2.14x10-5). The lymphoblastoid cell lines demonstrated increased adhesiveness to endothelial cells (p = 1.28x10-5). Reactive oxygen species were increased (p = 2.56x10-6). Significant inter-individual variation among the lymphoblastoid cell lines in these responses was evident (F = 18.70, p < 0.0001)., Conclusions: Exposure of lymphoblastoid cell lines derived from different human subjects to high glucose demonstrated differential and heterogeneous gene expression, adhesion, and cellular effects that recapitulated features found in the diabetic state. Lymphoblastoid cells may represent a useful tool to guide an individualized understanding of the development and potential treatment of diabetic complications like retinopathy.

Anaerobiosis, critical for successful ensilage, constitutes a challenge in baled silages. The loss of complete anaerobiosis causes aerobic deterioration and silages undergo dry matter and nutrient losses, pathogen growth, and mycotoxin production. Silage may represent an ideal substrate for Listeria monocytogenes, a pathogen of primary concern in several cheeses. The aim of this research was to investigate the occurrence of Listeria in baled silage fed to cows producing milk for a protected designation of origin cheese, and to characterize isolates by repetitive sequence-based PCR. Listeria spp. were detected in 21 silages and L. monocytogenes in 6 out of 80 of the analyzed silages; 67% of positives were found in molded zones. Results of the PCR typing showed genotypic homogeneity: 72.9 and 78.8% similarity between strains of Listeria spp. (n=56) and L. monocytogenes (n=24), respectively. Identical profiles were recovered in molded and nonmolded areas, indicating that contamination may have occurred during production. The application of PCR allowed the unambiguous identification of Listeria isolated from baled silages, and repetitive sequence-based PCR allowed a rapid and effective typing of isolates. Results disclose the potential of the systematic typing of Listeria in primary production, which is needed for the understanding of its transmission pathways., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Purpose: To report a case of Batten disease due to a previously unreported mutation in PPT1., Methods: A 9-year-old girl presented with classic clinical findings of Batten Disease., Results: Genetic testing for the mutations in the most common Batten disease gene, CLN3, was negative. Evaluation of a panel of genes known to be implicated in neuronal ceroid lipofuscinoses revealed disease causing mutations in PPT1, one of which was novel., Conclusion: Mutations in PPT1 typically cause the infantile form of neuronal ceroid lipofuscinosis. Clinical diagnosis of the juvenile form of neuronal ceroid lipofuscinosis, Batten disease, should still be considered in cases with negative CLN3 genetic testing. Batten disease can occur due to genetic heterogeneity. Testing of other members of the neuronal ceroid lipofuscinosis gene family can lead to confirmation of the correct diagnosis.

This study was aimed at collecting data on presence, dissemination and persistence of Pseudomonas in small-scale dairy farms. Six farms (located in Piedmont) were visited three times over 2014: 116 waters (wells and different faucets/pipes) and 117 environmental samples (milking equipments and drains) were collected. Enumeration of Pseudomonadaceae was performed, 3-5 colonies/samples were selected for identification via 16SrDNA/ oprI polymerase chain reaction (PCR), and typed by enterobacterial-repetitive-intergenic-consensus (ERIC)-PCR. Pseudomonadaceae were detected in 77% of samples. No statistical differences were found among proportions of positives across farms, sample typologies and seasons. Most isolates were Pseudomonas fluorescens (45%), and ERIC-PCR showed 32 persistent types diffused across farms. All in all, Pseudomonas spp. represents a challenge, considering its presence over time in water as well as in teat cups, indicating a continuous source of contamination. Moreover, persistency of strains may indicate biofilm-formation and/or sanitisers resistance, therefore emphasising the role of primary production for preventing milk contamination by Pseudomonas spp., Competing Interests: the authors declare no potential conflict of interest.

Competing Interests: Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported.

Introduction: Immunotherapy has become a promising approach for the treatment of NSCLC. In order to stimulate the host immune system against tumour antigens, several cancer vaccines have been generated and evaluated. Belagenpumatucel-L is a whole tumour cell vaccine expressing the antisense strand of the TGF-β2 gene., Areas Covered: The purpose of this article is to review the most relevant findings of clinical trials testing belagenpumatucel-L in advanced NSCLC patients., Expert Opinion: Although the Phase III trial investigating belagenpumatucel-L in stage III/IV patients did not meet its primary end point, a survival benefit was observed in several subgroups of patients. Further studies are needed in order to select patients who may benefit from this vaccine.

Purpose: Genome-wide association studies have suggested an association between a previously uncharacterized gene, FAM18B, and diabetic retinopathy. This study explores the role of FAM18B in diabetic retinopathy. An improved understanding of FAM18B could yield important insights into the pathogenesis of this sight-threatening complication of diabetes mellitus., Methods: Postmortem human eyes were examined with immunohistochemistry and immunofluorescence for the presence of FAM18B. Expression of FAM18B in primary human retinal microvascular endothelial cells (HRMECs) exposed to hyperglycemia, vascular endothelial growth factor (VEGF), or advanced glycation end products (AGEs) was determined with quantitative reverse-transcription PCR (qRT-PCR) and/or western blot. The role of FAM18B in regulating human retinal microvascular endothelial cell viability, migration, and endothelial tube formation was determined following RNAi-mediated knockdown of FAM18B. The presence of FAM18B was determined with qRT-PCR in CD34+/VEGFR2+ mononuclear cells isolated from a cohort of 17 diabetic subjects with and without diabetic retinopathy., Results: Immunohistochemistry and immunofluorescence demonstrated the presence of FAM18B in the human retina with prominent vascular staining. Hyperglycemia, VEGF, and AGEs downregulated the expression of FAM18B in HRMECs. RNAi-mediated knockdown of FAM18B in HRMECs contributed to enhanced migration and tube formation as well as exacerbating the hyperglycemia-induced decrease in HRMEC viability. The enhanced migration, tube formation, and decrease in the viability of HRMECs as a result of FAM18B downregulation was reversed with pyrrolidine dithiocarbamate (PDTC), a specific nuclear factor-kappa B (NF-κB) inhibitor. CD34+/VEGFR2+ mononuclear cells from subjects with proliferative diabetic retinopathy demonstrated significantly reduced mRNA expression of FAM18B compared to diabetic subjects without retinopathy., Conclusions: FAM18B is expressed in the retina. Diabetic culture conditions decrease the expression of FAM18B in HRMECs. The downregulation of FAM18B by siRNA in HRMECs results in enhanced migration and tube formation, but also exacerbates the hyperglycemia-induced decrease in HRMEC viability. The pathogenic changes observed in HRMECs as a result of FAM18B downregulation were reversed with PDTC, a specific NF-κB inhibitor. This study is the first to demonstrate a potential role for FAM18B in the pathogenesis of diabetic retinopathy.

Introduction: Originally formulated as an intravenous (i.v.) agent, vinorelbine is also currently available as an oral chemotherapeutic agent. Oral vinorelbine has demonstrated significant activity in different settings for NSCLC, including adjuvant treatment for resected disease, concurrent chemoradiation for locally advanced NSCLC and palliative chemotherapy for recurrent/metastatic NSCLC, as part of combination schedules or as a single-agent treatment., Areas Covered: The authors explored the available data describing the use of oral vinorelbine in NSCLC. PubMed articles and abstracts presented at international conferences were analysed, and relevant trials were reported and discussed. Specific settings, including the treatment of elderly and unfit patients and metronomic schedules including oral vinorelbine, were evaluated. Available pharmacoeconomic data were also assessed., Expert Opinion: Oral vinorelbine is an appealing agent, particularly as part of combination regimens containing platinum derivatives, although it can have a role as a single-agent treatment as well. Its safety profile is generally favourable and its route of administration is generally preferred by patients receiving chemotherapy. Compared to i.v. vinorelbine and other antineoplastic agents, oral vinorelbine has been reported to be advantageous in terms of cost savings.

Lymphangioma circumscriptum (LC) results from the development of abnormal lymphatic vasculature and is characterized by the presence of grouped vesicles filled with clear or colored fluid. Vulvar localization is uncommon. Abnormalities of the lymphatic system, such as lymphedema and cystic hygroma, are well-known features of genetic disorders such as Noonan syndrome (NS) and Turner syndrome. We report the case of a patient with NS who presented with LC of the vulva. We also discuss the expanding spectrum of clinical anomalies associated with the presentation of NS.

Degenerative loss of photoreceptors occurs in inherited and age-related retinal degenerative diseases. A chemical screen facilitates development of new testing routes for neuroprotection and mechanistic investigation. Herein, we conducted a mouse-derived photoreceptor (661W cell)-based high throughput screen of the Food and Drug Administration-approved Prestwick drug library to identify putative cytoprotective compounds against light-induced, synthetic visual chromophore-precipitated cell death. Different classes of hit compounds were identified, some of which target known genes or pathways pathologically associated with retinitis pigmentosa. Sulfaphenazole (SFZ), a selective inhibitor of human cytochrome P450 (CYP) 2C9 isozyme, was identified as a novel and leading cytoprotective compound. Expression of CYP2C proteins was induced by light. Gene-targeted knockdown of CYP2C55, the homologous gene of CYP2C9, demonstrated viability rescue to light-induced cell death, whereas stable expression of functional CYP2C9-GFP fusion protein further exacerbated light-induced cell death. Mechanistically, SFZ inhibited light-induced necrosis and mitochondrial stress-initiated apoptosis. Light elicited calcium influx, which was mitigated by SFZ. Light provoked the release of arachidonic acid from membrane phospholipids and production of non-epoxyeicosatrienoic acid metabolites. Administration of SFZ further stimulated the production of non-epoxyeicosatrienoic acid metabolites, suggesting a metabolic shift of arachidonic acid under inhibition of the CYP2C pathway. Together, our findings indicate that CYP2C genes play a direct causative role in photochemical stress-induced death of photoreceptors and suggest that the CYP monooxygenase system is a risk factor for retinal photodamage, especially in individuals with Stargardt disease and age-related macular degeneration that deposit condensation products of retinoids.

Objective: To determine the genetic contribution to leukocyte endothelial adhesion., Methods: Leukocyte endothelial adhesion was assessed through a novel cell-based assay using human lymphoblastoid cell lines. A high-throughput screening method was developed to evaluate the inter-individual variability in leukocyte endothelial adhesion using lymphoblastoid cell lines derived from different donors. To assess heritability, ninety-two lymphoblastoid cell lines derived from twenty-three monozygotic twin pairs and twenty-three sibling pairs were compared. These lymphoblastoid cell lines were plated with the endothelial cell line EA.hy926 and labeled with Calcein AM dye. Fluorescence was assessed to determine endothelial cell adhesion to each lymphoblastoid cell line. Intra-pair similarity was determined for monozygotic twins and siblings using Pearson pairwise correlation coefficients., Results: A leukocyte endothelial adhesion assay for lymphoblastoid cell lines was developed and optimized (CV = 8.68, Z'-factor = 0.67, SNR = 18.41). A higher adhesion correlation was found between the twins than that between the siblings. Intra-pair similarity for leukocyte endothelial adhesion in monozygotic twins was 0.60 compared to 0.25 in the siblings. The extent to which these differences are attributable to underlying genetic factors was quantified and the heritability of leukocyte endothelial adhesion was calculated to be 69.66% (p-value<0.0001)., Conclusions: There is a heritable component to leukocyte endothelial adhesion. Underlying genetic predisposition plays a significant role in inter-individual variability of leukocyte endothelial adhesion.

Purpose: This study sought to determine the validity of self-report of prior panretinal photocoagulation (PRP) and focal photocoagulation (FP) compared with fundus photography., Design: Prospective cohort study., Participants: One thousand three hundred sixty-three type 1 diabetic subjects from the Epidemiology of Diabetes Interventions and Complications (EDIC) study, a subset of the 1441 subjects originally enrolled in the multicenter Diabetes Control and Complications Trial., Methods: At each annual visit, subjects were asked by EDIC staff whether they had undergone PRP, FP, or both since the last completed annual clinic visit. Fundus photographs were collected from one quarter of the cohort each year and from the entire cohort at EDIC years 4 and 10. Photographs were graded for the presence and extent of PRP and FP. Seventeen years of subject reporting and photograph grading of PRP and FP were compared in EDIC subjects., Main Outcome Measures: The κ, sensitivity, specificity, and positive and negative predictive values were calculated for subject-reported PRP and FP. Factors influencing subject misreporting were investigated., Results: For subject reporting, 1244 (96%) of 1296 subjects with gradable photographs accurately reported whether they had a history of PRP in one or both eyes, and 1259 (97.5%) of 1291 with valid photographs correctly reported their history of FP. For PRP and FP, sensitivities were 90.4% and 74.0%, respectively; specificities were 96.0% and 98.8%, respectively; positive predictive values were 75.9% and 80.3%, respectively; negative predictive values were 98.9% and 98.4%, respectively; and κ values were 0.80 and 0.76, respectively. Risk factors associated with misreporting included prior laser for diabetic retinopathy and prior ocular surgery (each P<0.04)., Conclusions: For subjects with type 1 diabetes, in the absence of a clinical examination or fundus photographs, subject self-report could be a reliable tool in a well-monitored study for assessing laser treatment type in diabetic retinopathy., (Copyright © 2013 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

This study investigates the microbiological conditions of large game animal carcasses following evisceration. Carcasses of animals (N=291) hunted in the Upper Susa Valley (Italian Alps) were analysed for pH, Aerobic Viable Count (AVC), Enterobacteriaceae, Yersinia spp., Listeria monocytogenes and Salmonella spp. After shooting, evisceration occurred within 60 min in 90.7% of animals and sampling within 90 min in 88.3% of animals. Mean pH values (5.97: ruminants; 5.77: wild boar) were similar to those of regularly slaughtered domestic species. AVC values were highest in animals shot in the abdomen. Within species, AVC and Enterobacteriaceae values did not differ across different shooting-evisceration/sampling times. However, these counts exceeded 5 and 2.5 log, respectively, in 18% of wild boar and 39% of ruminants; the highest values were detected in wild boar. No pathogens were detected in any species. These results reveal inadequate hygiene in game meat handling/harvesting, implicating the need for improved practices., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose: We investigated the function of Fas in photoreceptors., Methods: Postmortem human eyes and mouse-derived photoreceptor cells (661W) were examined for Fas expression by in situ hybridization and immunofluorescence. 661W cells were treated with FasL or Fas agonistic antibody, or exposed to light with/without pharmacological manipulation of Fas signaling, followed by apoptosis detection by TUNEL, immunofluorescence and fluorescence activated cell scanning (FACS). Fractionated cellular extracts were used to detect protein expression or protein phosphorylation after immunoprecipitation by Western blot., Results: Fas was expressed in the photoreceptor layer of human retina. Fas and a cleaved form of FasL were found on the cell surface of 661W cells. Treatment with FasL or Fas agonistic antibody induced apoptosis in 661W cells. Blocking the activity of FasL or administration of caspase-8 inhibitor z-IETD inhibited light-induced apoptosis. However, it simultaneously caused induction of necroptosis, which could be blocked by the receptor-interacting protein 1 (RIP1) inhibitor, necrostatin-1. Light exposure in the presence of z-IETD caused hyper-phosphorylation of RIP1. Light exposure did not elevate the expression of Fas, FasL, or the Fas-associated death domain adaptor protein (FADD). Cells or conditioned medium after light exposure induced apoptosis in dark-adapted cells, which could be attenuated by blockade of Fas., Conclusions: Fas has a pro-apoptotic role in photoreceptors. Under light stress, soluble and membrane-bound FasL can bind to Fas, inducing apoptosis via a paracrine mechanism. Although blocking Fas signaling inhibits apoptosis, it does not improve the overall photoreceptor survival due to a compensatory activation of necroptosis. Hence, prevention of photoreceptor loss from retinal photo-oxidative stress should target Fas and RIP1.

Purpose: The purpose of this study is to attempt to replicate the top single nucleotide polymorphism (SNP) associations from a previous genome-wide association study (GWAS) for the sight-threatening complications of diabetic retinopathy in an independent cohort of diabetic subjects from the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR)., Methods: This study included 469 type 1 diabetic, Caucasian subjects from WESDR. Cases (n = 208) were defined by prior laser treatment for either proliferative diabetic retinopathy or diabetic macular edema. Controls (n = 261) were all other subjects in the cohort. Three hundred eighty-nine SNPs were tested for association using the Illumina GoldenGate custom array. A retinopathy-only subanalysis was conducted in 437 subjects by removing those with end-stage renal disease. Evaluation for association between cases and controls was conducted by using chi-square tests. A combined analysis incorporated the results from WESDR with the prior GWAS., Results: No associations were significant at a genome-wide level. The analysis did identify SNPs that can be pursued in future replication studies. The top association was at rs4865047, an intronic SNP, in the gene CEP135 (P value 2.06 × 10(-5)). The top association from the subanalysis was at rs1902491 (P value 2.81 × 10(-5)), a SNP that sits upstream of the gene NPY2R., Conclusions: This study nominates several novel genetic loci that may be associated with severe diabetic retinopathy. In order to confirm these findings, replication and extension in additional cohorts will be necessary as susceptibility alleles for diabetic retinopathy appear to be of modest effect.

The purpose of this study was to evaluate the feasibility of measuring rod and cone electroretinograms (ERGs) at a single mesopic adaptation level. To accomplish this, a four-primary photostimulator was implemented using a commercially available ERG system (Diagnosys ColorDome) to generate three types of stimuli that temporally modulated rods alone, cones alone, and rods and cones simultaneously. For each stimulus type, ERGs were recorded as a function of temporal frequency (2, 4, 8, or 16 Hz) and mesopic light levels (0.02, 0.16, or 1.26 cd/m(2)) in normal observers and patients with retinitis pigmentosa (RP) or cone-rod degeneration. The normal observers ERG waveforms showed a clear periodic pattern, mirroring the sinusoidal stimuli. At all light levels, rod responses were always higher than cone responses for temporal frequencies between 2 and 8 Hz, suggesting that rods dominated the responses. Cone responses were minimal at the lowest light level and increased with increases in light level. The amplitude of the response to the combined stimuli was intermediate between that of the isolated cone and the isolated rod stimuli for all light levels. Good receptoral isolation was confirmed by the results showing (1) minimal or no rod ERGs but recordable cone ERGs in the patients and (2) high correlation between the ERG amplitudes obtained from the four-primary method and those from the ISCEV standard clinical protocol in normal observers.

Purpose: To describe clinical characteristics, including visual acuity (VA), genetic analysis, and management of complications, over a 30-year period in an African American family with macular dystrophy of the retina, locus 1 (MCDR1), commonly referred to as "North Carolina macular dystrophy.", Design: Observational, cohort study., Participants: Twelve family members from a 4-generation pedigree., Methods: A total of 12 African American patients in an affected family were examined. Clinical examination was documented during 2 different follow-up periods from 1979 to 1982 in 10 patients and from 2005 to 2009 in 11 patients. Genetic analysis was performed in 4 affected members during this time. Foveal microperimetry, fundus autofluorescence, and spectral domain optical coherence tomography (OCT) data were also obtained., Main Outcome Measures: Change in VA of 8 members followed over 3 decades and clinical data and management of complications for all patients., Results: Nine of 11 living family members had classic findings ranging from disease grade 2 (confluent foveal drusen, 8 eyes) to grade 3 (central coloboma-like lesion, 10 eyes). Two members developed choroidal neovascularization (CNV) requiring laser ablation, and 1 member developed non-clearing vitreous hemorrhage and underwent 25-gauge pars plana vitrectomy. Another family member developed exotropia and amblyopia in 1 eye by age 7 years. Those without CNV had no significant change in VA over 30 years. Linkage studies of 4 affected family members showed the same short tandem repeats on markers spanning D6S249 and D6S283 within the MCDR1 region of chromosome 6q16. Microperimetry analysis of an affected member with grade 3 MCDR1 revealed absent function in the region of the central coloboma-like lesions, corresponding to photoreceptor absence on OCT, although there were preserved foveal function and intact photoreceptors adjacent to the lesion., Conclusions: This African American family shares similar clinical findings as other MCDR1 pedigrees and the same haplotype as the originally described family from North Carolina. Clinical characteristics, including retinal features and stable VA in the absence of amblyopia and CNV, are similar to those in other reports. Eccentric viewing around impaired photoreceptors may explain good VA in patients with clinically severe-appearing macular lesions. Sequencing of the MCDR1 interval may help identify a protein responsible for early macular development., Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references., (Copyright © 2011 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)