Neuro-Ophthalmologic Variability in Presentation of Genetically Confirmed Wolfram Syndrome: A Case Series and Review.

Wolfram syndrome is a neurodegenerative disorder caused by pathogenic variants in the genes WFS1 or CISD2. Clinically, the classic phenotype is composed of optic atrophy, diabetes mellitus type 1, diabetes insipidus, and deafness. Wolfram syndrome, however, is phenotypically heterogenous with variable clinical manifestations and age of onset. We describe four cases of genetically confirmed Wolfram syndrome with variable presentations, including acute-on-chronic vision loss, dyschromatopsia, and tonic pupils. All patients had optic atrophy, only three had diabetes, and none exhibited the classic Wolfram phenotype. MRI revealed a varying degree of the classical features associated with the syndrome, including optic nerve, cerebellar, and brainstem atrophy. The cohort's genotype and presentation supported the reported phenotype-genotype correlations for Wolfram, where missense variants lead to milder, later-onset presentation of the Wolfram syndrome spectrum. When early onset optic atrophy and/or diabetes mellitus are present in a patient, a diagnosis of Wolfram syndrome should be considered, as early diagnosis is crucial for the appropriate referrals and management of the associated conditions. Nevertheless, the condition should also be considered in otherwise unexplained, later-onset optic atrophy, given the phenotypic spectrum.

Pathobionts in the tumour microbiota predict survival following resection for colorectal cancer.

BACKGROUND AND AIMS: The gut microbiota is implicated in the pathogenesis of colorectal cancer (CRC). We aimed to map the CRC mucosal microbiota and metabolome and define the influence of the tumoral microbiota on oncological outcomes. METHODS: A multicentre, prospective observational study was conducted of CRC patients undergoing primary surgical resection in the UK (n = 74) and Czech Republic (n = 61). Analysis was performed using metataxonomics, ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), targeted bacterial qPCR and tumour exome sequencing. Hierarchical clustering accounting for clinical and oncological covariates was performed to identify clusters of bacteria and metabolites linked to CRC. Cox proportional hazards regression was used to ascertain clusters associated with disease-free survival over median follow-up of 50 months. RESULTS: Thirteen mucosal microbiota clusters were identified, of which five were significantly different between tumour and paired normal mucosa. Cluster 7, containing the pathobionts Fusobacterium nucleatum and Granulicatella adiacens, was strongly associated with CRC (PFDR = 0.0002). Additionally, tumoral dominance of cluster 7 independently predicted favourable disease-free survival (adjusted p = 0.031). Cluster 1, containing Faecalibacterium prausnitzii and Ruminococcus gnavus, was negatively associated with cancer (PFDR = 0.0009), and abundance was independently predictive of worse disease-free survival (adjusted p = 0.0009). UPLC-MS analysis revealed two major metabolic (Met) clusters. Met 1, composed of medium chain (MCFA), long-chain (LCFA) and very long-chain (VLCFA) fatty acid species, ceramides and lysophospholipids, was negatively associated with CRC (PFDR = 2.61 × 10-11); Met 2, composed of phosphatidylcholine species, nucleosides and amino acids, was strongly associated with CRC (PFDR = 1.30 × 10-12), but metabolite clusters were not associated with disease-free survival (p = 0.358). An association was identified between Met 1 and DNA mismatch-repair deficiency (p = 0.005). FBXW7 mutations were only found in cancers predominant in microbiota cluster 7. CONCLUSIONS: Networks of pathobionts in the tumour mucosal niche are associated with tumour mutation and metabolic subtypes and predict favourable outcome following CRC resection. Video Abstract.

DESI-MSI-based technique to unravel spatial distribution of COMT inhibitor Tolcapone.

Ascertaining compound exposure and its spatial distribution are essential steps in the drug development process. Desorption electrospray ionization mass spectrometry (DESI-MSI) is a label-free imaging technique capable of simultaneously identify and visualize the distribution of a diverse range of biomolecules. In this study, DESI-MSI was employed to investigate spatial distribution of tolcapone in rat liver and brain coronal - frontal and striatal -sections after a single oral administration of 100 mg/Kg of tolcapone, brain-penetrant compound. Tolcapone was evenly distributed in liver tissue sections whereas in the brain it showed differential distribution across brain regions analyzed, being mainly located in the olfactory bulb, basal forebrain region, striatum, and pre-frontal cortex (PFC; cingulate, prelimbic and infralimbic area). Tolcapone concentration in tissues was compared using DESI-MSI and liquid-chromatography mass spectrometry (LC-MS/MS). DESI-MSI technique showed a higher specificity on detecting tolcapone in liver sections while in the brain samples DESI-MSI did not allow a feasible quantification. Indeed, DESI-MSI is a qualitative technique that allows to observe heterogeneity on distribution but more challenging regarding accurate measurements. Overall, tolcapone was successfully localized in liver and brain tissue sections using DESI-MSI, highlighting the added value that this technique could provide in assisting tissue-specific drug distribution studies.

Central Retinal Artery Visualization with Cone-Beam CT Angiography.

Background There are multiple tools available to visualize the retinal and choroidal vasculature of the posterior globe. However, there are currently no reliable in vivo imaging techniques that can visualize the entire retrobulbar course of the retinal and ciliary vessels. Purpose To identify and characterize the central retinal artery (CRA) using cone-beam CT (CBCT) images obtained as part of diagnostic cerebral angiography. Materials and Methods In this retrospective study, patients with catheter DSA performed between October 2019 and October 2020 were included if CBCT angiography included the orbit in the field of view. The CBCT angiography data sets were postprocessed with a small field-of-view volume centered in the posterior globe to a maximum resolution of 0.2 mm. The following were evaluated: CRA origin, CRA course, CRA point of penetration into the optic nerve sheath, bifurcation of the CRA at the papilla, visualization of anatomic variants, and visualization of the central retinal vein. Descriptive statistical analysis was performed. Results Twenty-one patients with 24 visualized orbits were included in the analysis (mean age, 55 years ± 15; 14 women). Indications for angiography were as follows: diagnostic angiography (n = 8), aneurysm treatment (n = 6), or other (n = 7). The CRA was identified in all orbits; the origin, course, point of penetration of the CRA into the optic nerve sheath, and termination in the papilla were visualized in all orbits. The average length of the intraneural segment was 10.6 mm (range, 7-18 mm). The central retinal vein was identified in six of 24 orbits. Conclusion Cone-beam CT, performed during diagnostic angiography, consistently demonstrated the in vivo central retinal artery, demonstrating excellent potential for multiple diagnostic and therapeutic applications. © RSNA, 2021 Online supplemental material is available for this article.

King-Devick Test Performance and Cognitive Dysfunction after Concussion: A Pilot Eye Movement Study.

(1) Background: The King-Devick (KD) rapid number naming test is sensitive for concussion diagnosis, with increased test time from baseline as the outcome measure. Eye tracking during KD performance in concussed individuals shows an association between inter-saccadic interval (ISI) (the time between saccades) prolongation and prolonged testing time. This pilot study retrospectively assesses the relation between ISI prolongation during KD testing and cognitive performance in persistently-symptomatic individuals post-concussion. (2) Results: Fourteen participants (median age 34 years; 6 women) with prior neuropsychological assessment and KD testing with eye tracking were included. KD test times (72.6 ± 20.7 s) and median ISI (379.1 ± 199.1 msec) were prolonged compared to published normative values. Greater ISI prolongation was associated with lower scores for processing speed (WAIS-IV Coding, r = 0.72, p = 0.0017), attention/working memory (Trails Making A, r = -0.65, p = 0.006) (Digit Span Forward, r = 0.57, p = -0.017) (Digit Span Backward, r= -0.55, p = 0.021) (Digit Span Total, r = -0.74, p = 0.001), and executive function (Stroop Color Word Interference, r = -0.8, p = 0.0003). (3) Conclusions: This pilot study provides preliminary evidence suggesting that cognitive dysfunction may be associated with prolonged ISI and KD test times in concussion.

OM-85 in the prevention of respiratory infections: State-of-the-art and future perspectives in clinical practice.

Respiratory infections (RI) significantly burden patients, their families, and society. Respiratory infection recurrence (RRI) usually depends on a defect of the immune response, which can be more or less transient and/or selective. In particular, children, older people, heavy smokers, and patients with chronic diseases, characterized by an inadequate immune response, may be at risk of developing RRI. In this context, OM- 85 could represent a valuable option in the management of RRI. OM-85 is a bacterial lysate containing the extracts of some common pathogens, including Branhamella catarrhalis, Klebsiella pneumoniae, Klebsiella ozaenae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, and Staphylococcus aureus. Methodologically rigorous studies have documented the mechanism of action, efficacy, and safety of OM-85. OM-85 enhances the natural and acquired immune response through multifaceted mechanisms. Substantial evidence has shown that OM-85 can prevent respiratory infections, reduce the number of COPD exacerbations, and shorten the disease duration at home or in hospital. OM-85 can enhance the effectiveness of the 'flu vaccination without affecting the vaccine tolerability. The preventive use of OM-85 can reduce the use of antibiotics, contributing to contrast antibiotic resistance and saving the high cost of chronic respiratory diseases. Further studies should define the ideal candidate to OM-85 treatment.

Neuro-ophthalmologic complications of coronavirus disease 2019 (COVID-19).

Multiple neuro-ophthalmological manifestations have been described in association with COVID-19. These symptoms and signs may be the result of a range of pathophysiological mechanisms throughout the course from acute illness to recovery phase. Optic nerve dysfunction, eye movement abnormalities and visual field defects have been described.

Binocular Diplopia Caused by an Epiretinal Membrane With Foveal Displacement.

A 73-year-old woman presented with 3 years of monocular visual distortion and progressive binocular diplopia. She was found to have a comitant left hypertropia due to an epiretinal membrane causing inferior foveal drag. Displacement of the fovea from an epiretinal membrane is a likely under-recognized cause ocular cause of a comitant binocular diplopia.

Efficacy of safety catheter devices in the prevention of occupational needlestick injuries: applied research in the Liguria Region (Italy).

Healthcare workers who use or may be exposed to needles are at risk of needlestick injuries, which can lead to serious infections by bloodborne pathogens. These injuries can be avoided by eliminating the unnecessary use of needles and using safety devices. The present study was aimed at evaluating the impact of a safety-engineered device, with passive fully automatic needlestick protection, on the rate of needlestick injuries among healthcare workers. The setting of the study was a network of five public healthcare institutions situated in a Northern Italian Region. Data on the type of device, the number of employees and the number of catheter devices used per year were collected through regular meetings with healthcare workers over a period of five years. The most notable result of this study was the huge risk reduction associated with safety devices. Indeed, the risk of needlestick injuries due to conventional devices was found to be 25-fold higher than that observed for safety devices. However, it is noteworthy that a considerable part of this excess can be explained by the different background number of devices used. Moreover, descriptive analysis suggested that individuals with a poor/moderate training level had a lower risk than those with good/high training, though the difference was not statistically significant. In conclusion, there is convincing evidence of a causal connection between the introduction of safety devices and the reduction in needlestick injuries. This consideration should prompt the introduction of safety devices into daily clinical practice.

Shotgun Lipidomic Profiling of the NCI60 Cell Line Panel Using Rapid Evaporative Ionization Mass Spectrometry.

Rapid evaporative ionization mass spectrometry (REIMS) was used for the rapid mass spectrometric profiling of cancer cell lines. Spectral reproducibility was assessed for three different cell lines, and the extent of interclass differences and intraclass variance was found to allow the identification of these cell lines based on the REIMS data. Subsequently, the NCI60 cell line panel was subjected to REIMS analysis, and the resulting data set was investigated for its distinction of individual cell lines and different tissue types of origin. Information content of REIMS spectral profiles of cell lines were found to be similar to those obtained from mammalian tissues although pronounced differences in relative lipid intensity were observed. Ultimately, REIMS was shown to detect changes in lipid content of cell lines due to mycoplasma infection. The data show that REIMS is an attractive means to study cell lines involving minimal sample preparation and analysis times in the range of seconds.

Improved frozen section examination of the retroareolar margin for prediction of nipple involvement in breast cancer.

INTRODUCTION: In this prospective ex vivo study, we propose a new technique for the intraoperative examination of retroareolar tissue and describe both surgical excision and pathological methods. We performed a nipple-sparing mastectomy simulation in patients selected to total mastectomy, in order to evaluate the accuracy of these new technique. MATERIALS AND METHODS: A total of 158 total mastectomy specimens from patients affected by ductal carcinoma in situ (n = 15) or invasive ductal carcinoma (stages I, II, or IIIA) (n = 143) were examined. To obtain the entire sample area, the terminal retroareolar milk duct bunch was isolated. Fragments approximately 1.5 cm in length were excised and sectioned in parallel at the base of the nipple using a cold bistoury. Three transverse histological sections (4 µm each) at 200 µm intervals that included the entire isolated fragments were subjected to frozen section examination. The sections were stained with hematoxylin-eosin and were evaluated. The remainder of each fragment was embedded in paraffin and 4 µm sections were subsequently stained with hematoxylin-eosin and examined. RESULTS: There were two false-negative (1.3%) and five false-positive (3.1%) findings among the frozen and paraffin sections analyzed. A statistical analysis of the frozen section examinations showed a sensitivity of 92.0%, a specificity of 96.2%, a positive predictive value of 82.1%, a negative predictive value of 98.4%, and an accuracy of 95.4%. CONCLUSION: The frozen section examination technique described here detected nipple involvement in breast cancer with greater accuracy than the frozen section usually performed by most surgeons.

Lipidomic approach towards deciphering anandamide effects in rat decidual cell.

Altered phospholipid (PL) metabolism has been associated with pregnancy disorders. Moreover, lipid molecules such as endocannabinoids (eCBs) and prostaglandins (PGs) are important mediators of reproductive events. In humans, abnormal decidualization has been linked with unexplained infertility, miscarriage and endometrial pathologies. Anandamide (AEA), the major eCB, induces apoptosis in rat decidual cells. In this study, the PL profile of rat decidual cells was characterized by a Mass spectrometry (MS) based lipidomic approach. Furthermore, we analyzed a possible correlation between changes in PL of rat decidual cells' membrane and AEA-induced apoptosis. We found an increase in phosphatidylserine and a reduction of cardiolipin and phophatidylinositol relative contents. In addition, we observed an increase in the content of alkyl(alkenyl) acylPL, plasmalogens, and of long chain fatty acids especially with high degrees of unsaturation, as well as an increase in lipid hydroperoxides in treated cells. These findings provide novel insights on deregulation of lipid metabolism by anandamide, which may display further implications in decidualization process.

The reliability and validity of outcome measures for atopic dermatitis in patients with pigmented skin: A grey area.

BACKGROUND: Outcome measures for atopic dermatitis (AD) patients with pigmented skin have neither been developed nor validated. OBJECTIVE: To compare the reliability and validity of four common AD outcome measures in patients with various levels of skin darkness. METHOD: The inter- and intra-rater reliability and construct validity of the EASI (Eczema Area and Severity Index), objective-SCORing Atopic Dermatitis (oSCORAD), Three Items Severity index (TIS) and Six Areas, Six Sites Atopic Dermatitis (SASSAD) were evaluated in 18 patients of various levels of skin darkness, using their full body photographs, by five trained clinicians. RESULTS: The inter-rater reliability intraclass coefficient (ICCs) and 95% confidence intervals were poor for highly pigmented patients: EASI -.054(-.200 to .657), oSCORAD -.089(-.206 to .598), TIS -.21(-.24 to .147), SASSAD -.071(-.200 to .631); fair for mildly pigmented patients: EASI .464(.140-.839), oSCORAD .588(.265-.89), TIS.524(.200-.865), SASSAD .41(.045-.775); and fair to good for non-pigmented patients: EASI .64(.330-.908), oSCORAD .586(.263-.889), TIS .403(.09-.809), SASSAD .667(.358-.916). Erythema likely contributed to the inter-rater variability. Construct validity had significant correlations across all measures in non-pigmented patients, but no correlations in highly pigmented patients. CONCLUSION: AD outcome measures have poor reliability and validity in highly pigmented patients, with variations in erythema perception being a contributor.

Fatty acid and phospholipid biosynthetic pathways are regulated throughout mammary epithelial cell differentiation and correlate to breast cancer survival.

This work combined gene and protein expression, gas chromatography-flame ionization detector, and hydrophilic interaction liquid chromatography-tandem mass spectrometry to compare lipid metabolism changes in undifferentiated/proliferating vs. functionally differentiated mammary epithelial cells (MECs) and to study their correlation to breast cancer survival. Sixty-eight genes involved in lipid metabolism were changed in MEC differentiation. Differentiated cells showed induction of Elovl6 (2-fold), Scd1 (4-fold), and Fads2 (2-fold), which correlated with increased levels of C16:1 n-7 and C18:1 n-9 (1.5-fold), C20:3 n-6 (2.5-fold), and C20:4 n-6 (6-fold) fatty acids (FAs) and more phospholipids (PLs) containing these species. Further, increased expression (2- to 3-fold) of genes in phosphatidylethanolamine (PE) de novo biosynthesis resulted in a 20% PE increase. Proliferating/undifferentiated cells showed higher C16:0 (1.7-fold) and C18:2 n-6 (4.2-fold) levels and more PLs containing C16:0 FAs [PC(16:0/16:1), PG(16:0/18:2), PG(16:0/18:1), and SM(16:0/18:0)]. Kaplan-Meier analysis of data from 3455 patients with breast cancer disclosed a positive correlation for 59% of genes expressed in differentiated MECs with better survival. PE biosynthesis and FA oxidation correlated with better prognosis in patients with breast cancer, including the basal-like subtype. Therefore, genes involved in mammary gland FA and PL metabolism and their resulting molecular species reflect the cellular proliferative ability and differentiation state and deserve further studies as potential markers of breast cancer progression

Subtypes of type I IFN differentially enhance cytokine expression by suboptimally stimulated CD4(+) T cells.

Human type I interferons (IFNs) include IFN-ß and 12 subtypes of IFN-α. During viral infection, infiltrating memory CD4(+) T cells are exposed to IFNs, but their impact on memory T-cell function is poorly understood. To address this, we pretreated PBMCs with different IFNs for 16 h before stimulation with Staphylococcus aureus enterotoxin B and measured cytokine expression by flow cytometry. IFN-α8 and -α10 most potently enhanced expression of IFN-γ, IL-2, and IL-4. Potency among the subtypes differed most at doses between 10 and 100 U/mL. While enhancement of IL-2 and IL-4 correlated with the time of preincubation with type I IFN, IFN-γ production was enhanced best when IFN-α was added immediately preceding or simultaneously with T-cell stimulation. Comparison of T-cell responses to multiple doses of Staphylococcus aureus enterotoxin B and to peptide libraries from RSV or CMV demonstrated that IFN-α best enhanced cytokine expression when CD4(+) T cells were suboptimally stimulated. We conclude that type I IFNs enhance Th1 and Th2 function with dose dependency and subtype specificity, and best when T-cell stimulation is suboptimal. While type I IFNs may beneficially enhance CD4(+) T-cell memory responses to vaccines or viral pathogens, they may also enhance the function of resident Th2 cells and exacerbate allergic inflammation.

Prospective phospholipid markers for skin sensitization prediction in keratinocytes: a phospholipidomic approach.

Prevalence of skin inflammatory disorders has increased in recent years being estimated that 15-20% of the general population suffers from allergic contact dermatitis (ACD). Currently, the sensitizing potential of chemicals is assessed through animal tests; however growing ethical concerns and actual legislative framework impose the development of new alternative tests. Several genomic and proteomic approaches have already indicated some potential biomarkers, but lipidomic analysis was not so far explored with this purpose. A growing body of data suggests that phospholipids (PLs) play important roles in the modulation of immune responses. Therefore, this work focused in identifying changes in the PLs profile of human keratinocytes (KCs). For that, HaCaT cell line was exposed to two immune stimulators: the strong skin allergen 2,4-dinitrofluorobenzene (DNFB) and the non-allergenic stimulus LPS, and to the irritant benzalkonium chloride (BC), using off line TLC-ESI-MS, HPLC-MS and MS/MS. LPS and DNFB reduced PS class relative content, corroborating with consistent changes observed in its molecular profile. PC profile was also altered by immune stimulators. These findings suggest that PC and PS molecular species may discriminate immunogenic compounds from irritants. Analysis of such alterations may be therefore valuable in a future in vitro test platform for skin sensitization prediction.

Photodynamic oxidation of Escherichia coli membrane phospholipids: new insights based on lipidomics.

RATIONALE: The irreversible oxidation of biological molecules, such as lipids, can be achieved with a photosensitizing agent and subsequent exposure to light, in the presence of molecular oxygen. Although lipid peroxidation is an important toxicity mechanism in bacteria, the alterations caused by the photodynamic therapy on bacterial phospholipids are still unknown. In this work, we studied the photodynamic oxidation of Escherichia coli membrane phospholipids using a lipidomic approach. METHODS: E. coli ATCC 25922 were irradiated for 90 min with white light (4 mW cm(-2), 21.6 J cm(-2)) in the presence of a tricationic porphyrin [(5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin triiodide, Tri-Py(+)-Me-PF]. Lipids were extracted and separated by thin-layer chromatography. Phospholipid classes were quantified by phosphorus assay and analyzed by electrospray ionization tandem mass spectrometry. Fatty acids were analyzed by gas chromatography. Quantification of lipid hydroperoxides was performed by FOX2 assay. Analysis of the photodynamic oxidation of a phospholipid standard was also performed. RESULTS: Our approach allowed us to see that the photodynamic treatment induced the formation of a high amount of lipid hydroperoxides in the E. coli lipid extract. Quantification of fatty acids revealed a decrease in the unsaturated C16:1 and C18:1 species suggesting that oxidative modifications were responsible for their variation. It was also observed that photosensitization induced the oxidation of phosphatidylethanolamines with C16:1, C18:1 and C18:2 fatty acyl chains, with formation of hydroxy and hydroperoxy derivatives. CONCLUSIONS: Membrane phospholipids of E. coli are molecular targets of the photodynamic effect induced by Tri-Py(+) -Me-PF. The overall change in the relative amount of unsaturated fatty acids and the formation of PE hydroxides and hydroperoxides evidence the damages in bacterial phospholipids caused by this lethal treatment.

Lipidomic analysis of phospholipids from human mammary epithelial and breast cancer cell lines.

Alterations of phospholipid (PL) profiles have been associated to disease and specific lipids may be involved in the onset and evolution of cancer; yet, analysis of PL profiles using mass spectrometry (MS) in breast cancer cells is a novel approach. Previously, we reported a lipidomic analysis of PLs from mouse mammary epithelial and breast cancer cells using off-line thin layer chromatography (TLC)-MS, where several changes in PL profile were found to be associated with the degree of malignancy of cells. In the present study, lipidomic analysis has been extended to human mammary epithelial cells and breast cancer cell lines (MCF10A, T47-D, and MDA-MB-231), using TLC-MS, validated by hydrophilic interaction liquid chromatography-MS. Differences in phosphatidylethanolamine (PE) content relative to total amount of PLs was highest in non-malignant cells while phosphatidic acid was present with highest relative abundance in metastatic cells. In addition, the following differences in PL molecular species associated to cancer phenotype, metastatic potential, and cell morphology were found: higher levels of alkylacyl PCs and phosphatidylinositol (PI; 22:5/18:0) were detected in migratory cells, epithelial cells had less unsaturated fatty acyl chains and shorter aliphatic tails in PE and sphingomyelin classes, while PI (18:0/18:1) was lowest in non-malignant cells compared to cancer cells. To date, information about PL changes in cancer progression is scarce, therefore results presented in this work will be useful as a starting point to define possible PLs with prospective as biomarkers and disclose metabolic pathways with potential for cancer therapy.