Salmonella species is among the most commonly reported bacterial causes of laboratory-acquired infections. We report on three cases of laboratory-acquired Salmonella enterica serotype Typhi  infection which occurred over the period 2012 to 2016 in South Africa.Workers in clinical microbiology laboratories are exposed to a variety of pathogenic microorganisms. Laboratory investigation included phenotypic and genotypic characterization of isolates. Phenotypic analysis included standard microbiological identification techniques, serotyping and antimicrobial susceptibility testing. Genotypic analysis included the molecular subtyping methodologies of pulsed-field gel electrophoresis analysis, multilocus sequence typing and whole-genome sequencing (WGS); with WGS data analysis including phylogenetic analysis based upon comparison of single nucleotide polymorphism profiles of isolates.Salmonella Typhi as nontyphoidal Salmonella by a diagnostic laboratory, with associated public health implications. We highlight issues concerning the importance of accurate fluoroquinolone susceptibility testing and interpretation of results according to updated guidelines. We describe potential shortcomings of a single disk susceptibility screening test for fluoroquinolone susceptibility and suggest that confirmatory minimum inhibitory concentration testing should always be performed in cases of invasive Salmonella infections. These antimicrobial susceptibility testing issues resulted in inappropriate ciprofloxacin therapy which may have been responsible for failure in clearance of pathogen from patients. Salmonella Typhi capsular polysaccharide vaccine was not protective in one case, possibly secondarily to a faulty vaccine.All cases of laboratory-acquired infection were most likely the result of lapses in good laboratory practice and laboratory safety. The following critical issues were highlighted. There was misdiagnosis and misreporting of Molecular subtyping of isolates proved effective to investigate the genetic relatedness of isolates. Molecular subtyping data interpreted together with epidemiological data allowed us to pinpoint the most likely sources for our cases of laboratory-acquired infection. Shigella species, Brucella species, Salmonella species, Mycobacterium tuberculosis and Neisseria meningitidis were the most commonly reported bacterial causes of laboratory-acquired infections  and susceptibility to azithromycin . The patient, though recovered, was still excreting Salmonella Typhi in her stool and was then treated with a combination of azithromycin and fluoroquinolone for 14 days. Subsequent stools tested negative for Salmonella Typhi. The patient, being a laboratory technologist with frequent exposure to Salmonella Typhi, had received Salmonella Typhi vaccination nine months earlier as part of an occupational health program. On investigation, it was found that this vaccine was part of a batch recalled by the manufacturer, because of potentially low (below specification) antigen content  and sub-cultured onto 5% Blood Agar (DMP), to check for viability and purity. Cultures were identified using standard phenotypic microbiological identification and serotyping techniques, briefly described as follows. As required, bacterial colonies were identified using the VITEK-2 COMPACT 15 automated microbial identification system . Serotyping was performed according to the White-Kauffmann-Le Minor Scheme. Antimicrobial susceptibility testing was performed using the VITEK-2 COMPACT 15 system (bioMérieux) and the Etest method (bioMérieux). Interpretation of antimicrobial susceptibility data was done in accordance with the Clinical and Laboratory Standards Institute (CLSI) .Salmonella Typhi isolates belonged to the H58 haplotype were performed according to the methodology described by Murgia and coworkers [PCR to determine whether oworkers .XbaI digested genomic DNA was performed using a Bio-Rad CHEF-DR III electrophoresis system , following a PulseNet protocol [PFGE analysis of protocol . PFGE paGenomic DNA was isolated from bacteria using the Qiagen QIAamp DNA Mini Kit . DNA libraries were prepared using a Nextera XT DNA Library Preparation Kit , followed by a 2 × 300 paired-end sequencing runs with 100× coverage using Illumina MiSeq equipment. Raw data generated on the MiSeq was further analyzed using tools available in the CLC Genomics Workbench Software, version 8.5 (Qiagen). Using the ‘Trim Sequences Tool’, sequence reads were trimmed to include quality trimming and ambiguity trimming, and length trimming to discard reads below a length of 50 bases. Trimmed reads were assembled using the ‘De novo Assembly Tool’; the assembly algorithm works by using de Bruijn graphs to produce contiguous (contig) sequences (minimum contig length was set at 200 bases).aroC, dnaN, hemD, hisD, purE, sucA and thrA), as described at the Salmonella MLST database [Assembled genome data was analyzed using the ‘multilocus sequence typing (MLST)’ on-line analysis pipeline available at the Center for Genomic Epidemiology (CGE) of the Technical University of Denmark . MLST prdatabase .Assembled genome data was analyzed using the ‘CSIPhylogeny 1.4’ on-line analysis pipeline available at the CGE . The CSIThe results for molecular subtyping of isolates are as follows.Salmonella Typhi isolates that the patient (laboratory technologist) had been working on in Laboratory-A  to that of a PFGE pattern shown by a cluster of and Asia , 28.Fig.Salmonella Typhi isolates that the patient (resident) could have been exposed to in Laboratory-B. Compared to three possible isolates, PFGE analysis showed a PFGE pattern match  could have been exposed to in Laboratory-C. Compared to two possible isolates (isolates B and C) that the patient could have been exposed to in Laboratory-C; PFGE analysis showed a PFGE pattern match  had not been vaccinated; so lack of vaccination is a definite risk factor [All cases highlight issues concerning reported , 34. ForSalmonella laboratory-acquired infections probably reflect the current increased awareness and practice of GLP and laboratory safety. Risky procedures such as mouth-pipetting are now no longer practiced. Today, clinical microbiology laboratories in most countries enforce uniform safety policies nationwide, which would include: safety training; restricted laboratory access; use of biological safety cabinets; personal protective equipment (PPE) ; hand-washing facilities with automatic-operating taps or elbow operating taps and automatic-operating paper towel dispensers; prohibiting eating, drinking and smoking in the laboratory; prohibiting cell phones in the laboratory; proper decontamination of laboratory benches before and after working with clinical specimens and bacterial cultures; etc. Laboratory accreditation to International Organization for Standardization (ISO) requirements ensures compliance and audit safety policies; diagnostic laboratories in South Africa are all accredited against the ISO 15189 standard [Salmonella Typhi. However, for some laboratories in Europe and the USA, it is mandatory to work with Salmonella Typhi within a biological safety cabinet (cabinets with a minimum of level-2 safety) or within a biosafety level-2 laboratory.All our cases of laboratory-acquired infection were most likely the result of lapses in good laboratory practice (GLP) and laboratory safety. Case one had six years of working experience in a clinical microbiology laboratory, yet laboratory-infection still occurred. Lapses in laboratory safety do occur, even among laboratory workers with many years of experience working in a clinical microbiology laboratory. A recent report in 2015 described an NTS infection involving a laboratory technician with 20 years of experience in a clinical microbiology laboratory . Over restandard . With reSalmonella species. In particular, Alexander and coworkers [Molecular subtyping of bacterial isolates is an effective methodology to investigate the relatedness of bacterial isolates and to investigate the source of the infections. Molecular subtyping, including PFGE analysis , 15–18 aFor our current cases, all isolates were investigated with PFGE analysis. Although PFGE analysis is an older molecular subtyping methodology, it still provides valuable information and still remains the primary bacterial subtyping methodology employed by PulseNet International, a molecular subtyping network for foodborne and waterborne disease surveillance. PFGE analysis is limited in that it may not always provide sufficient resolution; for example, if you have a case isolate and you query a database of isolates to find a closest match by genetic relatedness, you may find that the case isolate will equally match (by PFGE pattern) a number of isolates. This was the situation for our case one, where the PFGE pattern for the case isolate was found to match a PFGE pattern represented by a cluster of isolates Fig. , suggestFor any finer degree of resolution, alternative molecular subtyping methodologies must be employed. The ultimate methodology is WGS data analysis; this WGS methodology was used to investigate isolates for cases two and three, thereby providing a secondary analysis to the primary PFGE analysis. Our analysis of WGS data included a phylogenetic analysis based upon a comparison of SNP profiles. When the number of SNP differences is compared among isolates, the lowest number of SNP differences implies the closest genetic relationship. This allowed us to determine the exact genetic similarity between a case isolate and its probable source. For case two, the case isolate and the probable source isolate (isolate A) differed by only 41 SNPs and were located within close proximity on a phylogenetic tree Fig. . For casLastly, a third molecular subtyping methodology, MLST, was employed to investigate our case isolates. MLST is generally more suitable for a long-term epidemiological comparison of isolates or a global comparison of isolates. However, in some cases of short-term epidemiological investigations (outbreak investigations), it can sometimes provide valuable supporting data. This was the situation for case three, whereby the laboratory worker had acquired his infection by exposure to two possible isolates; isolate B with MLST ST 1 or isolate C with MLST ST 2. The case three isolate presented MLST ST 1, providing evidence supporting isolate B as the cause of the infection.Salmonella Typhi is uncommon, as the risk of infection is low if appropriate laboratory safety policies are in place and adhered to. Nonetheless, accidental infections still do occur and would most likely be the result of lapses in aseptic techniques. In these situations, molecular subtyping of bacterial isolates is very useful to investigate the source of infections.Laboratory-acquired infection of


Molecular plant breeding with the aid of molecular markers has played an important role in modern plant breeding over the last two decades. Many marker-based predictions for quantitative traits have been made to enhance parental selection, but the trait prediction accuracy remains generally low, even with the aid of dense, genome-wide SNP markers. To search for more accurate trait-specific prediction with informative SNP markers, we conducted a literature review on the prediction issues in molecular plant breeding and on the applicability of an RNA-Seq technique for developing function-associated specific trait (FAST) SNP markers. To understand whether and how FAST SNP markers could enhance trait prediction, we also performed a theoretical reasoning on the effectiveness of these markers in a trait-specific prediction, and verified the reasoning through computer simulation. To the end, the search yielded an alternative to regular genomic selection with FAST SNP markers that could be explored to achieve more accurate trait-specific prediction. Continuous search for better alternatives is encouraged to enhance marker-based predictions for an individual quantitative trait in molecular plant breeding. Molecular plant breeding with the aid of molecular markers has played an important role in modern plant breeding over the last two decades . Many usRNA-Sequencing (or RNA-Seq) is a recently developed genomic approach for transcriptome profiling, can be applied to study each transcript of genes affecting a trait at a developmental stage, and has opened many avenues to develop informative markers associated with genes controlling genetically complex traits of agronomical importance . Here weMolecular plant breeding is generally termed as the application of molecular markers to improve the characters of interest in plants , and is Phaseolus vulgaris L.) and the promotion made by The idea for the use of markers to assist plant selection could date back to the association analysis done by Triticum spp.) cultivars released for commercial cultivation in Canada . For example, a range of prediction accuracies from 0.31 to 0.63 for several traits were found in rice (Oryza sativa L.) (Hordeum vulgare L.) and 0.40 to 0.80 for winter barley in malting quality traits (Zea mays L.) root traits  ; 0.14 toy traits ; 0.10 tot traits ; and 0.3t traits . Second,t traits . These ft traits , and sugGossypium hirsutum; Our analysis of the GS applications with respect to prediction accuracy concurs well with the renewed argument that more research efforts are needed to develop functional markers for MAS, taking advantage of the recent advances in NGS application . This rede novo assemble without the genomic sequence, and analyze the alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression . Merr.] and wheat  selected for improved growth versus unselected genetic cohorts and developed many FAST SNP markers for growth traits for fish breeding. Similarly, Danio rerio) and developed 164 SNPs, five of which were associated with genes affecting fish growth. Sorghum bicolor) genotypes with contrasting responses to cold stress. Yr15, the yellow rust (Puccinia striiformis f.sp. tritici) resistance in wheat germplasm. Clearly, the most successful applications in plants were those using RNA-Seq in combination with bulked segregant analysis (Raphanus sativus L.) and onion (Allium cepa L.)  . These sOur literature review indicates the importance of using functional markers to increase the trait prediction accuracy for GS. This should not be surprised, as functional markers should be more informative to acquire genetic effects of causal genes for trait prediction than genome-wide neutral markers . Using mBos taurus). In one simulation on an ideal pattern of LD with marker loci either in complete LD or linkage equilibrium with QTL, they found only the prediction method of Bayes-B  displayed the highest prediction accuracies, ranging from 0.72 to 0.95 and increasing with fewer QTL. When there were more than 100 QTL, the highly linked markers (mp4) showed higher prediction accuracies than all genome-wide SNPs including QTL (mp3). Thus, these simulation results are consistent with the two expectations mentioned above for FAST SNPs, as FAST SNPs should approach the behavior of those highly linked markers to QTL (mp4) for a trait predication.To understand these two expectations, Fernando and his colleagues conducted extensive computer simulations e.g.,  to illus Bayes-B  could ac Bayes-B . Conside Bayes-B . These f Bayes-B . SpecifirrBLUP mixed.solve function . First, the patterns of prediction accuracy by Bayes-B in soybean are the same as in cattle for the QTL scenarios of QTL250 and QTL500. In any QTL scenario, soybean functional markers (mp1) always showed the highest accuracies of trait prediction, followed by highly linked markers (mp4) and all genome-wide SNP markers (mp3) (Table 1). Also, the patterns of decreased prediction accuracies by functional markers (mp1) with more QTL were also observed in soybean data (Table 1). Second, soybean markers with a little relaxed LD to QTL like mp6 or mp7 still displayed higher prediction accuracies than those genome-wide SNP markers (mp3) in any QTL scenarios assayed. For example, for a trait of heritability 0.5 with 100 QTL, 200 HLDr2 markers (mp6) displayed a correlation of 0.67 while all 36,543 SNP markers (mp3) had only a correlation of 0.61 (Table 1). Third, several extra patterns of prediction accuracy were also observed in soybean (Table 1 and Supplementary Table S2). Three different prediction methods did not show much difference in prediction accuracy. The prediction accuracies became lower for a trait of lower heritability. The prediction accuracies using 36,543 SNPs for a trait of heritability 0.2 ranged from 0.48 to 0.53, while those using highly linked markers (mp6 or mp7) ranged from 0.49 to 0.63. All together, these simulation results demonstrated the potential gain in prediction accuracy from the application of FAST SNP markers in molecular breeding. More evenly distributed markers unlinked to causal genes do not enhance, but rather reduce, trait prediction accuracy.Our simulation not only confirmed those observed in the Angus cattle, but also revealed some interesting patterns of trait prediction  for ease of interpretation and comparison to marker-specific selection and GS. It is our hope that this alternative or its modifications later can provide a useful breeding tool to improve the accuracy of marker-based prediction on individual trait performance.Figure 2). It requires the development and validation of FAST SNP markers for a given trait in other populations, before the application to genotype a breeding population of interest; estimates the marker “effects” in a training set of the breeding population; and applies the estimated marker “effects” to predict trait performance in the same breeding population. Such an approach differs from traditional MAS with QTL-specific markers in genotyping and prediction, but follows the same idea of GS to predict trait performance with RNA-Seq FAST SNP markers, rather than the genome-wide selectively neutral SNP markers. To make the strategy more understandable, we outline the two major components of TSMS in Figures 4 for developing FAST SNP markers through RNA-Seq technology and for performing SNP marker prediction of breeding values, respectively. The proposed RNA-Seq method  considers multiple pairs of individual plants with two extreme trait values and collects their sample tissues at given developmental stages for gene expressions associated with the trait of interest. The collected samples will be subjected to RNA-Seq analysis through RNA extraction, cDNA library preparation, multiplexing with barcoding and cDNA sequencing. The collected RNA-Seq data will be analyzed through de novo assembly using various bioinformatics tools to identify differential transcripts for each pair and to generate consensus differential transcripts from all the assayed pairs. Identification of a differential transcript in a pair is made based on the presence or absence of a transcript or the difference in abundance of the transcript detected in both plants. Multiple pairs are used to enhance the reliability of identifying differential transcripts for the trait. Based on the consensus differential transcripts, SNP call will be made from all the samples and the detected SNPs will be filtered to generate putative SNPs for the trait based on the differences in allelic frequency between two trait-extreme sets of assayed samples. An empirical validation of putative SNPs in separate population(s) is required to confirm if the acquired SNP markers are truly associated with the trait performance. The validated SNP markers can be applied to genotype all the breeding materials of interest, and some of these genotyped plants will also be assessed with their trait performance as a training set . These marker and trait data in the training set can be analyzed using existing marker prediction models for GS such as RR-BLUP or Bayes-B implemented in various R packages , we proposed a new procedure, following the principle of BSR-Seq developed by Figure 3). We suggest 10 or more pairs for the effort, but the optimum pairs to be used remain to be empirically determined and they may vary with respect to trait and plant mating system.The advantage of this alternative over regular GS mainly lies in the potential gains in marker prediction of individual trait performance through the application of FAST SNP markers. Realizing the gain in trait prediction highly depends on the development of the FAST SNP markers for individual traits, and requires further empirical investigations in breeding programs. To facilitate the development of FAST SNP markers through RNA-Seq  did not consider the multiple developmental stages of RNA sampling to capture all expressed genes associated with the trait performance and may miss some trans-regulatory genes  or using existing methods such as eQTL analysis, GWAS, or those methods used in fish breeding (e.g., Yr15, a major disease resistance gene for wheat yellow rust (In spite of these issues, FAST SNP markers for specific traits can be developed for plant breeding, either following our proposed procedure (ng e.g., . The goong e.g.,  and 175 low rust . Built uOur search for a better marker-based prediction of trait performance through literature review and theoretical reasoning yielded an alternative to regular genome selection for individual trait prediction. More accurate trait predictions can be theoretically achieved through the development of FAST SNP markers with RNA-Seq technique and the application of these markers to genotype plants and to predict breeding values following existing genomic prediction methods in breeding populations. Further empirical investigation is needed to realize how much gain in trait prediction with respect to breeding efficiency could be achieved from the derived alternative in a plant breeding program. The derived alternative may be questioned for its breeding efficiency in multiple-traits breeding, as function-associated SNP markers unspecified for specific traits could be more efficiently developed from exome capture technology than the proposed FAST SNP markers. However, our synthesis is encouraging, as continuous search for better alternatives based on the other genetic characteristics of a quantitative trait is possible and may yield more accurate trait prediction for molecular plant breeding.The writing process of this manuscript complies with the current laws of Canada.Y-BF conceived of the research, conducted the literature review, performed the computer simulation and wrote the paper. M-HY conducted the literature review, performed the computer simulation and revised the paper. FZ conducted the literature review and wrote the paper. BB conducted the literature review and revised the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arachnoid cysts can be associated with neurologic symptoms due to compression of underlying brain structures though indications for surgical intervention are unclear. We present the case of a 70-year-old man who presented with a two-year history of speech changes along with decreased initiation and talkativeness, shorter utterances, and dysnomia. [ Primary progressive apraxia of speech is a recently described neurodegenerative disorder in which patients present with an isolated apraxia of speech and show focal degeneration of superior premotor cortex . LongituA 70-year-old right-handed man was brought to the Emergency Department for an episode of confusion at work characterized by “saying things I didn't mean to say.” His family reported a 2-year nonprogressive history of word finding difficulty and shuffling gait. He had once worked as a computer database developer but in the last decade had shifted to working as a greeter in a department store.On examination, the patient displayed limited facial expression with mild right oral commissure depression and slowed voluntary face, lip, and tongue movements. Perceptual speech analysis revealed a harsh voice with episodes of decreased volume, imprecise speech sounds, occasional repetition of sounds, difficulty sequencing multisyllabic words, slowing, and diminished stress patterns. He did not initiate conversations. The patient generally answered questions with 1 to 4 words but occasionally said up to a 9-word, grammatically correct sentence. Testing revealed impaired word finding and impaired verbal fluency. Handwriting demonstrated decreased smoothness and extra loops. Overall, the patient's speech and language skills appeared more impaired than his global cognitive status. Together, these findings suggested a mixed disorder of both motor speech sequencing (praxis) and language (nonfluent aphasia). There was no evidence of gait apraxia.An EEG showed findings of left temporal slowing. Brain MRI demonstrated mild diffuse cerebral atrophy and mild convex contour deformity of the left frontal skull. FDG PET-CT of the brain  revealedExtra-axial arachnoid cysts may be associated with a variety of neurologic symptoms that sometimes may require surgical intervention . HoweverWe conclude that timely diagnosis and workup of focal cortical syndromes, such as motor speech apraxia, may uncover treatable or relatively benign mimics of neurodegenerative diseases. Understanding the natural history of such mimics, however, is still a work in progress.


Second, FXS patients showed increased spatial spreading of phase-synchronized high frequency neural activity in the gamma band. Third, we observed increased negative theta-to-gamma but decreased alpha-to-gamma band amplitude coupling, and the level of increased theta power was inversely related to the level of resting gamma power in FXS.Increased theta band power and coupling from frontal sources may represent a mechanism providing compensatory inhibition of high-frequency gamma band activity, potentially contributing to the widely varying level of neurophysiological and behavioral abnormalities and treatment response seen in full-mutation FXS patients. These findings extend preclinical observations and provide new mechanistic insights into brain alterations and their variability across FXS patients. Electrophysiological measures may provide useful translational biomarkers for advancing drug development and individualizing treatments for neurodevelopmental disorders with associated neuronal hyperexcitability.The online version of this article (doi:10.1186/s11689-017-9191-z) contains supplementary material, which is available to authorized users. FMR1) on the X chromosome, leading to reduced production of Fragile X Mental Retardation Protein (FMRP) [fmr1 knockout (KO) mice have shown enhanced activity of metabotropic glutamate receptors [Fragile X syndrome (FXS) is a neurodevelopmental disorder resulting from silencing of the fragile X mental retardation gene FMR on the Xeceptors  and redueceptors . These aeceptors –5.fmr1 KO mice have abnormally high synchrony of neocortical network activity and a threefold higher neuronal firing rate during Up states [fmr1 KO mice have also shown increased EEG responses to auditory stimuli via in vivo recordings [Neurophysiological studies can clarify the functional brain consequences of neurochemical and neuroanatomic changes in FXS. p states , 7. fmr1cordings –10. Simicordings –14.fmr1 KO mice, and with due consideration of the challenges integrating knowledge from intracranial recordings and clinical data, it is noteworthy that few EEG studies of resting brain function have yet been conducted with FXS patients. To our knowledge there has only been one quantitative study of resting state EEG in FXS presented in two reports [fmr1 KO mouse studies that have identified abnormalities in fast-spiking inhibitory GABAergic interneurons [Given the model of a neurophysiological imbalance leading to heightened neural excitability and the increased prevalence of seizures in FXS patients and  reports , 16. Exc reports –19. Thisrneurons , 20 whicrneurons . Third, fmr1 KO mice [Alpha rhythms are the most dominant oscillation during the resting state and play an inhibitory role in information processing systems . Theta r KO mice . As incrThe aim of the present study was to investigate resting state EEG activity in FXS patients focusing on resting EEG power—specifically gamma band power, functional connectivity, and gamma coupling. We hypothesized that resting state EEG power in FXS would be enhanced in both low- and high-frequency bands (theta and gamma) but reduced in middle range frequencies  relative to healthy controls. Second, we predicted that functional connectivity in FXS would be reduced in long-range connections but increased in short-range connections dominated by gamma band oscillations relative to controls. Third, we predicted that FXS patients would show reduced alpha-to-gamma coupling consistent with reduced top-down alpha-related inhibition on local cortical excitability in sensory systems. Fourth, we hypothesized that altered resting EEG power spectra in FXS individuals would be correlated with social and sensory processing difficulties.Twenty-one FXS participants with a full mutation (greater than 200 CGG repeats)  and 21 healthy age-matched controls  participated in this study , done to facilitate cooperation in the FXS patients as in previous studies . EEG datcontrol = 138, SD = 5.8; EpochsFXS = 139, SD = 4.7, p > 0.05) or number of interpolated channels . For each channel, every 2-s epoch was detrended, tapered with a Hanning window, and transformed in Matlab using a Fourier (FFT) algorithm, yielding Fourier coefficients in 0.5 Hz frequency steps. The Fourier coefficients were then squared to yield power values (uV2). To be comparable with previous resting-state EEG reports on FXS [There was no significant group difference in the number of artifact-free epochs .Functional connectivity among all electrode pairs was examined separately in each of the six frequency bands of interest using the debiased weighted phase lag index (dbWPLI) . This mehttp://www.ru.nl/neuroimaging/fieldtrip/) for calculating dbWPLI among all electrode pairs (8128 pairs) at each of the six frequency bands of interest. Then, at each frequency band, the obtained dbWPLI difference between FXS and control groups at each electrode pair was tested using a permutation approach [EEG data were imported to FieldTrip . Upper alpha connectivity in controls decreased with increased electrode distance ; this effect was not seen in FXS. In the beta band, both between-electrode distance  and its interaction with group  were significant. Although both groups showed decreased beta connectivity with increased between-electrode distance, the effect was significantly reduced in FXS patients. In the gamma band, both between-electrode distance  and its interaction with group  were highly significant. Although both groups showed decreased gamma connectivity with greater between-electrode distance, the FXS group showed stronger effects. Because connectivity at a scale on the order of even short electrode distances is well beyond the scale maintained by individual PV positive inhibitory neurons, the observation of increased functional connectivity of gamma band activity in FXS across a variety of inter-electrode distances indicates a greater spread of coherent high frequency neural activity in FXS than in controls.Paired sts Fig.  showed tsts Fig. . ResultsWhen evaluating amplitude coupling across electrodes, cluster-permutation testing showed that the inverse correlation between upper alpha and gamma power was significantly reduced in FXS patients compared to controls in the occipital, parietal, and frontal regions Fig. . There wThese results suggest that while upper alpha power negatively coupled with gamma power both within and across electrodes in controls, FXS patients displayed excessive gamma activity and reduced upper alpha power without showing a coupling of gamma and upper alpha band activity. In other words, reduced upper alpha-related inhibition of gamma power was observed in FXS. Instead, FXS showed stronger negative theta to gamma power coupling both within and across electrodes. Follow-up phase amplitude coupling analyses were not significant . Lower alpha power was significantly correlated with greater social impairment in SCQ scores  and with hypersensitivity to sensory stimuli on the Sensory Profile scale . For functional connectivity and cross-frequency coupling measurements, no correlations with clinical variables were significant. Within each group, no significant correlations for EEG parameters were observed with age or gender. The consistent and marked reductions in IQ, especially in male FXS patients , made correlations with electrophysiological parameters difficult to meaningfully evaluate.Exploratory correlational analyses were performed between EEG measurements showing significant alteration in FXS and SCQ and Adolescent/Adult Sensory Profile scores in FXS participants. Increased resting gamma power was significantly correlated with social-communication abnormalities as assessed with the SCQ  and high-frequency gamma band activity both within and across electrodes. We did this to determine whether the pattern of findings seen in gamma power and connectivity was related to a disruption in top-down modulation. Based on EEG data, it is not possible to determine whether a local circuit dysfunction is causing or resulting from the altered pattern of reduced long-distance functional connectivity in FXS. However, previous studies have shown that low frequency oscillations  provide top-down inhibitory and modulatory influences in large, distributed neural networks, whereas fast oscillations  at rest are more related to neurophysiological tone in local networks . Thus, tIn contrast to the reduced upper alpha-to-gamma coupling, FXS showed strong theta-to-gamma coupling with atypical theta connectivity being related to lower levels of gamma power Fig. . Thus, wFMRP is highly expressed in the hippocampus [fmr1 KO mice [The functional role of theta oscillation is related to its neural sources in the prefrontal and anterior cingulate cortex (ACC) , which ppocampus , and lon KO mice .Interest in systems biology alterations to complement understanding of local circuit pathology may be important not only for comprehensive models of pathology in FXS, but because individual variability in system-level modulatory factors may contribute to the wide range of clinical phenotypes seen even in patients with full mutation. This variability might explain the inconsistent treatment response to drugs targeting mGluR and other mechanisms aiming to reduce neuronal hyperexcitability, which have had more consistently positive effects in animal models. In this context, our findings not only provide new mechanistic understanding of FXS but also suggest that EEG studies of FXS may provide biomarkers for delineating disease heterogeneity and predicting and tracking response in human and mouse models to drugs targeting neuronal hyperactivity. Such approaches are urgently needed to advance drug development and personalized medicine for FXS patients. Our observation that quantitative EEG alterations were related to the severity of social communication and sensory reactivity problems supports the potential clinical utility of this approach.fmr1 KO mice suggest a relevance to FXS, more research is needed to determine whether similar findings might be seen in the subset of ASD patients who demonstrate sensory hypersensitivities or other signs of cortical excitability, as well as in multiple other neurodevelopmental disorders.This study has certain limitations, including the wide age range (12–57 years old) and the fact that younger children were not assessed. Although FXS and control groups were age-matched and no significant age effects were observed in the data, studies with younger populations remain an important target for future research. Other effects such as sex differences and medication effects were not statistically significant, but further research is needed to address those issues. Third, our study did not compare resting state abnormalities in FXS directly with other developmental disabilities to establish specificity of deficits to FXS relative to general effects of intellectual or developmental disability. While the parallel findings from our study and preclinical work in In summary, we found an abnormal U-shaped alteration of spectral power with reduced long-range inhibitory and enhanced excitatory shorter-range connectivity in FXS. Furthermore, we found stronger theta-to-gamma amplitude coupling in FXS, possibly serving as a compensatory response to reduced top-down alpha-band inhibitory modulation and intrinsic pathology of local neural circuit excitability in sensory systems. Taken in combination, our findings provide direct in vivo evidence in FXS patients of heightened cortical arousal, reduced top-down regulatory input in the alpha band, and increased modulation in the theta band that together may determine the level of circuit hyper-excitability in sensory cortex in vivo.


In presenting a case in which proton SBRT was performed safely and effectively for a medically complex patient with inoperable synchronous bilateral RCC, we suggest that proton therapy is a promising therapeutic approach for even unilateral primary RCC where preservation of renal function is of importance and should be considered for medically inoperable patients. Further experience is needed to determine the optimal dose and fractionation regimen for renal SBRT with proton therapy.Limited therapeutic options exist for inoperable bilateral kidney tumors. We report the first ever use of proton therapy to treat primary renal cell carcinoma (RCC) and the first report of proton stereotactic body radiation therapy (SBRT) for RCC in an inoperable patient with synchronous RCCs treated with proton SBRT. The patient is a 47-year-old 450-pound female with multiple medical comorbidities, including Stage 3 chronic kidney disease (CKD), who was found to have bilateral renal masses during work-up for cellulitis and sepsis. Following resolution of her sepsis, subsequent cross-sectional imaging demonstrated interval growth of the left renal mass to 4.4 x 4.8 cm and the right renal mass to 2.0 x 2.6 cm. Bilateral biopsies were performed, with pathology revealing Furhman Grade 1-2 clear cell RCC on both sides. A customized SBRT plan delivered a total dose of 3,000 cGy in five fractions to the bilateral kidneys every other day using proton beam therapy. The patient experienced no grade > 1 acute adverse toxicities from proton therapy, and now at one year after treatment, she has had no clinical symptoms of late radiation-induced toxicities. Although a marginal decline in post-treatment glomerular filtration rate (GFR) was observed (baseline 34 mL/min/1.73m Bilateral renal tumors are rare, accounting for only 4% of patients with renal cell carcinoma (RCC). Surgical resection is the standard curative treatment for bilateral RCC and delivers outcomes comparable to that of unilateral RCC . Many paConventional radiotherapy has been limited as a modality, given the relative radio-resistance of RCC and the risks of radiation-induced toxicities present with irradiating large volumes of the kidney. Stereotactic body radiation therapy (SBRT), also known as stereotactic ablative radiotherapy (SABR), however, may overcome these limitations by taking advantage of highly conformal dose delivery, image guidance, and rapid dose fall-off. This allows delivery of ablative doses to the tumor while sparing normal tissues at risk . Large fIn patients with pre-existing kidney disease, however, incidental irradiation of the uninvolved kidney with photon-based SBRT presents a high risk of kidney failure requiring chronic hemodialysis with resulting impairment of quality of life and decreased overall survival. This risk is magnified in patients with bilateral renal malignancies. Herein exists an opportunity where proton beam radiotherapy could offer a dosimetric advantage due to its characteristic Bragg peak and lack of exit dose. Minimization of dose to the uninvolved kidney and surrounding tissues could substantially improve clinical outcomes and reduce life-threatening toxicities when compared to plans delivered with photon therapy. For the first time, we herein report the safety and efficacy of using proton SBRT in a patient with inoperable bilateral synchronous RCC.History and physicalA 47-year-old 450-pound female with multiple comorbidities presented to our department for outpatient consultation with bilateral synchronous Stage I RCCs for consideration of external beam radiation therapy. Her bilateral renal masses were incidentally found during work-up for methicillin-resistant Staphylococcus aureus cellulitis and sepsis. Following recovery of her sepsis, she underwent repeat abdominal magnetic resonance imaging (MRI) that demonstrated interval growth of the left-sided mass to 4.4 x 4.8 cm and of the right-sided mass to 2.0 x 2.6 cm. The patient underwent image-guided biopsies of the bilateral masses, both of which revealed Furhman Grade 1-2 clear cell RCC. She was determined to be a poor surgical candidate due to her extensive comorbidities, including morbid obesity with a body mass index (BMI) > 90, pulmonary hypertension, chronic obstructive pulmonary disease, congestive heart failure, peripheral vascular disease, Stage 3 chronic kidney disease (CKD), adrenal insufficiency, obstructive sleep apnea, and recurrent wound infections. She was referred by her urologist to an outside radiation oncology practice but was not offered therapy due to concerns of excessive dose and resultant risk of toxicity to her remaining normal kidney tissue. She was referred to our institution for consideration of proton therapy, given its potential dosimetric advantages and the ability of our proton treatment couch to accommodate the patient’s weight.At the time of consultation, the patient was asymptomatic from her RCCs; however, her performance status was limited due to her underlying comorbidities. The patient required 3 liters of oxygen via nasal cannula, even while at rest in her wheelchair. Her mobility was limited to transfer from wheelchair to bed, and she was unable to walk more than a few steps, even with a walker. Her immobilization resulted in the development of recurrent pressure ulcers, the most significant of which required multiple incisions, drainages, debridements, and admissions for bacteremia and sepsis.2.Her previous surgical history included a Caesarean section, laparoscopic cholecystectomy, and multiple exploratory laparotomies. Her family history was notable for uterine and breast cancer in her mother and maternal aunt, respectively. She was a previous smoker of > 50 pack years, and she had no history of alcohol or intravenous (IV) drug use. Her exam revealed morbid obesity, distant heart sounds, 1+ bilateral pitting edema, damp desquamation in her skin folds at the flanks without ulceration, oxygen use, and an Eastern Cooperative Oncology Group (ECOG)  performance status of 3+. Her creatinine levels were stable at 1.62 mg/dL and her estimated glomerular filtration rate (GFR) was 34 mL/mg/1.73mPatient setup and treatment planThe patient underwent simulation in the supine position with her arms above her head; a full body vac-lock bag was used for immobilization. To assess and account for tumor motion, a four-dimensional (4D) computed tomography (CT) scan was performed at the time of simulation to monitor the location of the tumor during all phases of the respiratory cycle Figure .Given that the patient was not a candidate for IV contrast at the time of simulation due to her preexisting CKD, she underwent a 4D positron emission tomography (PET)/CT simulation to aid in target volume delineation. The bilateral renal masses and organs at risk were contoured using the simulation CT. Gross tumor volume (GTV) was defined as the radiographically visible tumor based on the CT/PET simulation images and fused MRI abdominal imaging. Bilateral internal gross tumor volumes (iGTVs) were created that represented an expansion of the GTV to account for tumor motion, as assessed by the 4D simulation imaging. Beam-specific planning treatment volumes (PTVs) were created for each tumor that were approximately a 5 mm expansion on the iGTVs. Customized SBRT plans using separate isocenters were designed to deliver a total dose of 3,000 cGy in five fractions to the bilateral kidney target volumes delivered every other day Figure . Plans wThe patient underwent daily pre-treatment kV-KV and cone-beam CT (CBCT) scans in the treatment position prior to each fraction to ensure optimal target localization, although CBCT image quality was suboptimal given the patient’s body habitus.Acute toxicitiesAcute toxicities were minimal and included Grade 1 urinary urgency, Grade 1 urinary incontinence, and Grade 1 fatigue; she had no hematuria, flank pain, nausea, changes in bowel habits, loss of appetite, or other toxicities during or in the weeks and first months following proton SBRT. Her performance status remained stable at ECOG 3 by the completion of treatment and at one month following treatment.Post-treatment follow-up2, previously 34 mL/min/1.73m2), the patient remains asymptomatic with no significant clinical toxicity.Three months following radiation treatment, the patient reported resolution of both her fatigue and urinary symptoms to pre-treatment levels, and she had no additional complaints. An MRI at this time demonstrated stable appearing bilateral renal masses, now 4.0 x 4.3 cm on the right and 3.0 x 2.7 cm on the left, consistent with early findings of a delayed response to treatment Figure . She denTo the best of our knowledge, this is the first reported case of primary RCC treated with proton therapy. This also is the first report of delivering SBRT with protons for RCC and the first report of proton therapy to treat bilateral synchronous renal malignancies. The novelty of this treatment regimen was commanded by the particular set of clinical dilemmas with which our patient presented: a body habitus posing technical limitations for photon-based delivery, multiple comorbidities precluding standard-of-care surgical intervention, and bilateral tumors in the setting of underlying CKD, placing paramount importance on the preservation of her renal function. With this case, we demonstrate that proton SBRT is feasible, safe, and well-tolerated, providing a rationale to consider extending the use of this modality for medically inoperable unilateral RCC, particularly for complex patients and/or where renal function preservation is critical.A limited number of retrospective and phase I/II studies have demonstrated excellent local control for primary RCC using photon-based SBRT. In 2012, Siva, et al. conducted a systematic review of 10 studies (three prospective and seven retrospective) with 126 patients treated with SBRT for inoperable RCC . These sSince that review, several additional studies have been published and continue to support the safety and efficacy of kidney SBRT with photon therapy. The University Hospitals Seidman Cancer Center in Cleveland reported on their Phase I dose-escalation study with 19 patients receiving 24 to 48 Gy in four fractions. They described a limited rate of early radiation-related side effects: one Grade 4 acute cardiac event (not thought to be treatment-related), one Grade 4 acute duodenal ulcer (possibly treatment-related), and no Grade 3 adverse events. Of 15 patients with evaluable responses, three had a partial response and 12 had stable disease . A groupBalancing the need for maximal oncologic control and the chance for a cure against the goal of maximal functional preservation is of particular importance for bilateral renal tumors, and this is a current clinical challenge for photon-based radiotherapy. The need to reduce incidental irradiation to the uninvolved kidney tissue is of critical importance in patients with bilateral RCCs, especially in the setting of pre-existing CKD as was the case for our patient. Such challenging cases are where the dosimetric advantages of proton radiotherapy compared to photon-based modalities are magnified. Photons deposit their peak dose shortly after entering tissue and experience an exponential decrease of deposited dose with increasing depth. In comparison, protons travel through tissue with limited dose deposition until reaching the end of their path, where they deposit a majority of their energy, termed the “Bragg peak,” and thereafter have essentially zero energy beyond that depth, resulting in greater sparing of normal tissues . AlthougTaken together, given the dosimetric benefits of proton therapy and the potentially curable nature of the patient’s synchronous tumors, we offered definitive proton SBRT to her bilateral tumors to maximize the biologically effective dose and the likelihood of long-term control while preserving her normal kidney parenchyma and minimizing skin dose. This treatment, however, was not without risks, particularly given her pre-existing CKD. The patient experienced no severe acute adverse events and now, at one year following proton SBRT, she has at least stable disease without the development of any late symptoms from treatment. Although she experienced a marginal decline in post-treatment GFR, the patient remains asymptomatic and without a requirement for intervention.Of note, we elected not to apply a dose constraint on the adrenal glands in our treatment plan since there is no well-established dose constraint for these organs at present and our priority in treatment was to minimize dose to her bilateral kidneys. The patient was referred to an endocrinology specialist prior to treatment, and she will continue to undergo surveillance lab work and potential replacement therapy should she develop a late endocrine deficiency from treatment. Fortunately, however, given the specific location of her lesions, her adrenal glands were significantly spared of collateral irradiation with proton beam radiotherapy.In this case report, we are limited by relatively short follow-up, especially as the patient will remain at risk for late renal toxicity for years following treatment. Additionally, despite early encouraging radiographic stability over serial post-treatment imaging, which is the most likely radiographic finding in patients with durable treatment responses following SBRT, we cannot definitely confirm a lack of viable residual tumor since a histologic confirmation of treatment response was not sought. Although radiological response assessment after ablative doses of radiation therapy is, at times, difficult to interpret, given the lack of radiographic enlargement and her medical comorbidities, biopsy at this time is not felt to be medically indicated. We were also limited in field design by her specific tumor locations and bilateral lesions, as well as her body habitus. It is likely that conformity can be optimized further for future patients who are treated with this potentially beneficial modality through different field designs and using more than two beams for each lesion. Despite these various limitations, this case report presents the feasibility of proton SBRT in inoperable primary RCC and should be used as support by other practitioners.We present the first application of proton SBRT in the treatment of synchronous bilateral RCC and provide early evidence that proton SBRT is feasible, efficacious, and associated with minimal toxicities. Proton SBRT should be considered as an emerging treatment modality for patients with inoperable primary RCC where preservation of renal function is of paramount importance, including those with pre-existing CKD, bilateral lesions, only one kidney, or particularly large tumors. Further experience is needed to determine a dose and fractionation regimen that optimizes both local control and safety.


Astroviruses are one of the leading causes of pediatric gastroenteritis worldwide and are clinically importantly pathogens in the elderly and immunocompromised populations. Although the use of cell culture systems and small animal models have enhanced our understanding of astrovirus infection and pathogenesis, little is known about the immune response to astrovirus infection. Studies from humans and animals suggest that adaptive immunity is important in restricting classic and novel astrovirus infections, while studies from animal models and cell culture systems suggest that an innate immune system plays a role in limiting astrovirus replication. The relative contribution of each arm of the immune system in restricting astrovirus infection remains unknown. This review summarizes our current understanding of the immune response to astrovirus infection and highlights some of the key questions that stem from these studies. A full understanding of the immune response to astrovirus infection is required to be able to treat and control astrovirus-induced gastroenteritis. Astroviruses (AstV) are small, nonenveloped, RNA viruses that are a major cause of gastroenteritis in infants, immunocompromised people, and the elderly, and they also cause disease in mammals and birds ,6,7,8,9.Astrovirus infection begins by binding to an unidentified receptor(s) on epithelial cells in the intestine after fecal-oral transmission ,11 and eAlthough astroviruses are considered gastrointestinal pathogens, viral RNA and infectious viral particles have been recovered from extraintestinal organs in both animals and humans . In the The innate immune response provides signals to recruit the adaptive immune response, which controls viral infection at later times during an infection. This response is pathogen specific and has a memory component that induces a more rapid and robust response following a second infection with the same pathogen. The adaptive immune response contains two arms: the humoral, or antibody-mediated/B cell response, and the cell-mediated response, which involves antigen-specific cytotoxic T cells. Although the findings between the current turkey model and the newly emerging mouse model yielded conflicting results, the reports showing the biphasic age distribution as well as immunocompromised individuals have formed a general conclusion that the adaptive immune system is a major component in controlling astrovirus disease (reviewed in ). Early studies in the turkey poult model found little adaptive immune responses after TAstV-2 infection . Koci etrag1) gene knockout mice (Rag1−/−), which lack mature B cells or T cells [−/− mice also contained 3–4 logs higher astrovirus RNA levels in the intestines compared to wild type controls [−/−) mice by Marvin et al., the Rag1−/− mice were positive for astrovirus prior to experimental inoculation [−/− mice are generated. Studies using the newly emerging mouse model and clinical studies in humans demonstrate that the adaptive immune response is key in controlling astrovirus infection and disease. Experimental infection of recombination activating gene 1  and saw a signification reduction in new capsid protein synthesis and mRNA levels at all HAstV-4 multiplicity of infection (MOIs) used . They alIn addition to limiting viral replication, both studies asked if type I IFN influences astrovirus-induced barrier permeability. Astrovirus infection has been shown to increase epithelial permeability, which correlated with the disruption of tight junction proteins and sodium malabsorption ,4,68. Gu-/- knockout mice. Oral gavage of wild-type C57BL/6 mice with MuAstV-positive fecal filtrates results in productive infection, viral shedding, and increased intestinal permeability [−/− mice used in the study were found to already be MuAstV-positive in their colony. The viral titers shed in the stool of wild-type mice peaked at 6 to 11 dpi, shedding similar levels as the IFNaR−/− mice. However, wild-type mice titers rapidly decreased after 11 dpi and cleared the infection by 53 dpi, compared to IFNaR−/− mice that shed virus at high levels (>106 copies/μg RNA) at 53 dpi. These data suggest that type I IFN signaling is important for viral clearance in vivo [−/− mice also had increased intestinal permeability compared to MuAstV-infected wild-type mice, which correlated with their in vitro observations, suggesting a role for type I IFN in protecting the epithelial barrier during infection, although MuAstV-free IFNaR−/− mice would need to be generated to definitively determine the role of type I IFN signaling in intestinal permeability [The effects of type I IFN signaling in astrovirus replication, clearance, and barrier permeability have been examined in vivo using IFNaReability . However in vivo . IFNaR−/eability .Overall, the studies of Guix et al. and Marvin et al. demonstrate that the type I IFN system can limit astrovirus replication in vitro and in vivo and protects against astrovirus-induced barrier permeability. How astrovirus replication induces type I IFN production, and why induction is only seen late in infection and at low levels remains unknown. Additionally, how type I IFN protects against astrovirus-induced barrier permeability remains to be resolved. Several observations in animals have described other innate immune responses during astrovirus infection. Astroviruses have been found in macrophages of lambs and described in calves with cells around Peyer’s patches ,69. Howe−/−) shed 10-fold more viral copies in the feces 14 dpi compared to wild-type mice and had viral RNA in the spleen and the mesenteric lymph nodes, while viral RNA was not detected in those organs of wild-type mice [Finally, a study in mice found a role for the signal transducer and activator of transcription 1 (STAT1) transcription factor, a factor known for controlling viral infection , in limiype mice . Like tyMultiple studies have concluded that astrovirus may evade the immune system by preventing complement activation ,73,74,75Escherichia coli (E. coli) challenge compared to uninfected macrophages [E. coli per macrophage compared to macrophages isolated from uninfected poults [E. coli in the spleens of TAstV-infected poults [Studies in turkey poults suggest that astrovirus infection suppresses the immune system, making the host more susceptible to subsequent infections ,76. Turkrophages . In vivorophages . These md poults . TAstV-id poults . These rd poults , suggestIn summary, although our understanding of the immune responses to astrovirus infection has progressed over the years, further studies are required to fully characterize the cellular and molecular factors that mediate the immune response to astrovirus infection, including identifying the correlates of protection that inhibit viral replication and pathogenesis. Future areas of inquiry include the absence of a potent inflammatory response following the rupture of endosomal membranes , as seen


The 11-cis chromophore is returned to photoreceptors, forming light-sensitive visual pigments with opsin GPCRs. Dysfunction of this process perturbs phototransduction because functional visual pigment cannot be generated. Mutations in visual cycle genes can result in monogenic inherited forms of blindness. Though key enzymatic processes are well characterized, questions remain as to the physiological role of visual cycle proteins in different retinal cell types, functional domains of these proteins in retinoid biochemistry and in vivo pathogenesis of disease mutations. Significant progress is needed to develop effective and accessible treatments for inherited blindness arising from mutations in visual cycle genes. Here, we review opportunities to apply gene editing technology to two crucial visual cycle components, RPE65 and CRALBP. Expressed exclusively in the human RPE, RPE65 enzymatically converts retinyl esters into 11-cis retinal. CRALBP is an 11-cis-retinal binding protein expressed in human RPE and Muller glia. Loss-of-function mutations in either protein results in autosomal recessive forms of blindness. Modeling these human conditions using RPE65 or CRALBP murine knockout models have enhanced our understanding of their biochemical function, associated disease pathogenesis and development of therapeutics. However, rod-dominated murine retinae provide a challenge to assess cone function. The cone-rich zebrafish model is amenable to cost-effective maintenance of a variety of strains. Interestingly, gene duplication in zebrafish resulted in three Rpe65 and two Cralbp isoforms with differential temporal and spatial expression patterns. Functional investigations of zebrafish Rpe65 and Cralbp were restricted to gene knockdown with morpholino oligonucleotides. However, transient silencing, off-target effects and discrepancies between knockdown and knockout models, highlight a need for more comprehensive alternatives for functional genomics. CRISPR/Cas9 in zebrafish has emerged as a formidable technology enabling targeted gene knockout, knock-in, activation, or silencing to single base-pair resolution. Effective, targeted gene editing by CRISPR/Cas9 in zebrafish enables unprecedented opportunities to create genetic research models. This review will discuss existing knowledge gaps regarding RPE65 and CRALBP. We explore the benefits of CRISPR/Cas9 to establish innovative zebrafish models to enhance knowledge of the visual cycle.During the vertebrate visual cycle, all- Cones also use the canonical pathway, though to a lesser extent  by all-trans-ROL dehydrogenase, and shuttled to the RPE by interphotoreceptor retinoid-binding protein (IRBP) where it encounters cellular retinol binding protein (CRBP)  to produce retinyl esters (REs). Subsequently, the REs are isomerized into 11-cis-ROL by RPE65 and then oxidized into 11-cis-RAL by 11-cis-ROL dehydrogenase, prior to transportation back to the photoreceptor outer segments  and a putative Isomerase II, all-trans-ROL is directly converted to 11-cis-ROL, which is subsequently transported to the cone inner segment by IRBP  is thought to have evolved through competition with rods for 11-This review focuses on the shortcomings in our understanding of the visual cycle, using two key proteins, RPE65 and CRALBP, as examples. Their pivotal role in human diseases as well as the emerging opportunities to develop zebrafish models harboring patient specific mutations using CRISPR/Cas9 genome editing technology is discussed.cis ROL from all-trans-ROL  resulted in morphologically altered rod outer segments which suggested impaired visual function in rpe65a deficient larvae. Although 11-cis-RAL levels were reduced following dark adaptation, full regeneration occurred following dark re-adaptation. This study concluded that an Rpe65-independent isomerization event is necessary to sustain cone-mediated vision. However more recently, a third isoform, rpe65c was identified in zebrafish which is believed to be the result of a tandem duplication event. Similarly to rpe65a, it generates 11-cis-ROL from all-trans retinyl esters  are present, with divergent expression patterns, allowing individual evaluation within the RPE and Müller glia, respectively  was reported  supporting an independent requirement of both isoforms for cone vision  of early onset retinal degenerations  , fundus albipunctatus (FA) and Newfoundland rod-cone dystrophy ; therefore, patients with RDH5 mutations display a mildly progressive form of FA  knockouts that lack rod photoreceptors  are a powerful vertebrate model to study physiology and disease. Key advantages of zebrafish for vision research include rapid eye development and functional vision by 5 days post fertilization . Additionally, complementary biochemical assays for CRALBP-retinoid binding or RPE65 isomerohydrolase activity must be performed in vitro.Owing to ~70% sequence identity between human and zebrafish orthologs, and ease of genome manipulation, zebrafish , mutant alleles for all rpe65 and rlbp1 genes are recently available  is introduced three nucleotides upstream of the PAM. Upon DSB, the native host DNA repair mechanism known as non-homologous end joining (NHEJ) promotes insertions or deletions (indels). These indels result, in most cases, frameshifts and incorporation of early stop codons, creating gene KOs, enabling functional genomic studies  to improve our understanding of the biochemical, molecular and cellular progression of diseases associated with the visual cycle and (2) to develop novel gene, cell and drug based therapies for human pathologies linked to defective RPE65 or CRALBP.We are still in the initial phase of exploring the possibilities that CRISPR technology offers for gene regulation and the control of cell identity and behavior. At present, gene activation, gene silencing, KI and KO are all feasible in zebrafish and their use in retinal regeneration studies has recently been reviewed (Campbell and Hyde, RPE65−/− and RLBP1−/− phenotype specifically in relation to cone photoreceptors. Unanswered questions regarding the visual cycle mainly relate to the cone specific visual cycle. In line with previous studies supporting the existence of a putative cone specific visual cycle (Das et al., trans-cis isomerization in zebrafish is an Rpe65 independent process (Schonthaler et al., In humans, high-acuity vision is predominantly mediated by cones. It is therefore imperative to understand the pathophysiology of the in vivo and extends its potential use to personalized zebrafish knockouts for specific patient mutations. For example, patients harboring a R91W RPE65 mutation retain low levels of 11-cis chromophore generation and some cone function during early life (Samardzija et al., Rpe65 hypomorphic murine model has been generated for this specific mutation and crossed with Nrl−/− to study cone function (Samardzija et al., in vivo (Bowne et al., The rapid advances in CRISPR/Cas9 technology may mean that virtually any mutation can be created In vitro studies show this mutation abolishes 11-cis retinoid binding and reduces CRALBP solubility (Maw et al., in vivo and the processes leading to CRALBP mediated RP degeneration.Interestingly, some vision loss patients retain residual RPE65 activity with reduced and partial vision, as a result. An alternative approach to study human variations in RPE65 is to constitutively activate the gene of interest through KI of the human specific mutation. This will be extremely valuable as the effect of retaining some RPE65 isomerization activity on photoreceptor integrity and function can be studied. Similarly, an arginine to glutamine substitution in CRALBP at position 150 results in autosomal dominant RP. cis-RAL analog is being evaluated in LCA patients following its beneficial use in restoring light sensitivity in Rpe65−/− albeit concerns remain over the adverse drug responses reported (Maeda et al., RPE65 and RLBP1 typically lead to slow progressing rod-cone dystrophies and therefore, the application of effective therapies during early disease presentation could preserve remaining photoreceptor structural integrity and retain functional vision. Zebrafish are useful in identifying lead compounds before application in a mammalian model and developing rlbp1−/− and rpe65−/− lines would provide an inexpensive platform to conduct high throughput pharmacological screens.Gene replacement therapies for RPE65 have recently received FDA approval and ones for recessive CRALBP mutations are in development (MacLachlan et al., in vivo models. Targeted gene editing using CRISPR/Cas9 KO and KI strategies are routinely applied in zebrafish with very high success rates. Development of a permanent KO or KI cone dominant models will provide inexpensive and reliable platform to study the resultant phenotypes in the long term and advance our understanding of the molecular processes which lead to cone degeneration in patients.Although our knowledge of visual cycle processes and related human pathologies has progressed significantly in the last decade, our understanding of the cone-specific visual cycle and the biochemical mechanisms leading to photoreceptor degeneration is lacking. To advance therapeutics for degeneration, caused by defective visual cycle genes, it is necessary to develop robust and reliable RW was the primary author of this review. HS and VD contributed in drafting sections for this review. HS prepared the figure. BK contributed significant intellectual input and approved the submission of the review. All authors edited the final draft.VD is employed by ZeClinics SL. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.