The zebrafish has become a valuable model for examining ocular lens development, physiology and disease. The zebrafish cloche mutant, first described for its loss of hematopoiesis, also shows reduced ...eye and lens size, interruption in lens cell differentiation and a cataract likely caused by abnormal protein aggregation. To facilitate the use of the cloche mutant for studies on cataract development and prevention we characterized variation in the lens phenotype, quantified changes in gene expression by qRT-PCR and RNA-Seq and compared the ability of two promoters to drive expression of introduced proteins into the cloche lens. We found that the severity of cloche embryo lens cataract varied, while the decrease in lens diameter and retention of nuclei in differentiating lens fiber cells was constant. We found very low expression of both αB-crystallin genes (cryaba and cryabb) at 4 days post fertilization (dpf) by both qRT-PCR and RNA-Seq in cloche, cloche sibling and wildtype embryos and no significant difference in αA-crystallin (cryaa) expression. RNA-Seq analysis of 4 dpf embryos identified transcripts from 25,281 genes, with 1,329 showing statistically significantly different expression between cloche and wildtype samples. Downregulation of eight lens β- and γM-crystallin genes and 22 retinal related genes may reflect a general reduction in eye development and growth. Six stress response genes were upregulated. We did not find misregulation of any known components of lens development gene regulatory networks. These results suggest that the cloche lens cataract is not caused by loss of αA-crystallin or changes to lens gene regulatory networks. Instead, we propose that the cataract results from general physiological stress related to loss of hematopoiesis. Our finding that the zebrafish αA-crystallin promoter drove strong GFP expression in the cloche lens demonstrates its use as a tool for examining the effects of introduced proteins on lens crystallin aggregation and cataract prevention.
The α-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens ...cataracts, the leading cause of human blindness. There are conflicting data in the literature as to what role the α-crystallins may play in early lens development. In this study, we used CRISPR gene editing to produce zebrafish lines with mutations in each of the three α-crystallin genes (cryaa, cryaba and cryabb) to prevent protein production. The absence of each α-crystallin protein was analyzed by mass spectrometry, and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of αA-crystallin produced a variety of lens defects with varying severity in larvae at 3 and 4 dpf but little substantial change in normal fiber cell denucleation. Loss of αBa-crystallin produced no substantial lens defects. Our cryabb mutant produced a truncated αBb-crystallin protein and showed no substantial change in lens development. Mutation of each α-crystallin gene did not alter the mRNA levels of the remaining two, suggesting a lack of genetic compensation. These data suggest that αA-crystallin plays some role in lens development, but the range of phenotype severity in null mutants indicates its loss simply increases the chance for defects and that the protein is not essential. Our finding that cryaba and cryabb mutants lack noticeable lens defects is congruent with insubstantial transcript levels for these genes in lens epithelial and fiber cells through five days of development. Future experiments can explore the molecular mechanisms leading to lens defects in cryaa null mutants and the impact of αA-crystallin loss during zebrafish lens aging.
•Loss of zebrafish αA-crystallin produced a variety of developmental lens defects with varying severity.•Mutation of neither zebrafish αB-crystallin gene substantially impacted zebrafish lens development.•Loss of alpha crystallin proteins did not delay fiber cell denucleation or alter expression of other α-crystallin genes.•Alpha A-crystallin is not required for lens development, but its loss makes the lens prone to various defects.•Understanding developmental role of α-crystallins can inform future cataract treatment.
Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. However, little is known about zebrafish α-crystallin ...promoter function, how it compares to that of mammals, or whether mammalian α-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of α-crystallin promoter fragments from each species combined with the coding sequence for green fluorescent protein (GFP) into zebrafish zygotes to determine the resulting spatiotemporal expression patterns in the developing embryo. We also measured mRNA levels and protein abundance for all three zebrafish α-crystallins. Our data showed that mouse and zebrafish αA-crystallin promoters generated similar GFP expression in the lens, but with earlier onset when using mouse promoters. Expression was also found in notochord and skeletal muscle in a smaller percentage of embryos. Mouse αB-crystallin promoter fragments drove GFP expression primarily in zebrafish skeletal muscle, with less common expression in notochord, lens, heart and in extraocular regions of the eye. A short fragment containing only a lens-specific enhancer region increased lens and notochord GFP expression while decreasing muscle expression, suggesting that the influence of mouse promoter control regions carries over into zebrafish embryos. The two paralogous zebrafish αB-crystallin promoters produced subtly different expression profiles, with the aBa promoter driving expression equally in notochord and skeletal muscle while the αBb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish αA increased between 1 and 2 days post fertilization (dpf), αBa increased between 4 and 5 dpf, but
Bb remained at baseline levels through 5 dpf. Parallel reaction monitoring (PRM) mass spectrometry was used to detect αA, aBa, and αBb peptides in digests of zebrafish embryos. In whole embryos, αA-crystallin was first detected by 2 dpf, peaked in abundance by 4-5 dpf, and was localized to the eye. αBa was detected in whole embryo at nearly constant levels from 1-6 dpf, was also localized primarily to the eye
and its abundance in extraocular tissues decreased from 4-7 dpf. In contrast, due to its low abundance, no αBb protein could be detected in whole embryo, or dissected eye and extraocular tissues. Our results show that mammalian α-crystallin promoters can be efficiently screened in zebrafish embryos and that their controlling regions are well conserved. An ontogenetic shift in zebrafish aBa-crystallin promoter activity provides an interesting system for examining the evolution and control of tissue specificity. Future studies that combine these promoter based approaches with the expanding ability to engineer the zebrafish genome via techniques such as CRISPR/Cas9 will allow the manipulation of protein expression to test hypotheses about lens crystallin function and its relation to lens biology and disease.
Mixed amphetamine salts extended release (MAS XR; Adderall XR®) and atomoxetine (Strattera®) were compared in children 6 to 12 years old with attention deficit/hyperactivity disorder (ADHD) combined ...or hyperactive/impulsive type in a randomized, double-blind, multicenter, parallel-group, forced-dose-escalation laboratory school study. Primary efficacy measure was the SKAMP (Swanson, Kotkin, Agler, M-Flynn, and Pelham) behavioral rating scale. Changes in mean SKAMP deportment scores from baseline were significantly greater for MAS XR (n = 102) than for atomoxetine (n = 101) overall (-0.56 and -0.13, respectively; p < .0001) and at each week (p < .001). Adverse events were similar for both treatment groups. The extended time course of action and greater therapeutic efficacy of MAS XR suggests that it is more effective than atomoxetine in children with ADHD.
Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. However, little is known about zebrafish alpha-crystallin ...promoter function, how it compares to that of mammals, or whether mammalian alpha-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of alpha-crystallin promoter fragments from each species combined with the coding sequence for green fluorescent protein (GFP) into zebrafish zygotes to determine the resulting spatiotemporal expression patterns in the developing embryo. We also measured mRNA levels and protein abundance for all three zebrafish alpha-crystallins. Our data showed that mouse and zebrafish alphaA-crystallin promoters generated similar GFP expression in the lens, but with earlier onset when using mouse promoters. Expression was also found in notochord and skeletal muscle in a smaller percentage of embryos. Mouse alphaB-crystallin promoter fragments drove GFP expression primarily in zebrafish skeletal muscle, with less common expression in notochord, lens, heart and in extraocular regions of the eye. A short fragment containing only a lens-specific enhancer region increased lens and notochord GFP expression while decreasing muscle expression, suggesting that the influence of mouse promoter control regions carries over into zebrafish embryos. The two paralogous zebrafish alphaB-crystallin promoters produced subtly different expression profiles, with the aBa promoter driving expression equally in notochord and skeletal muscle while the alphaBb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish alphaA increased between 1 and 2 days post fertilization (dpf), alphaBa increased between 4 and 5 dpf, but alphaBb remained at baseline levels through 5 dpf. Parallel reaction monitoring (PRM) mass spectrometry was used to detect alphaA, aBa, and alphaBb peptides in digests of zebrafish embryos. In whole embryos, alphaA-crystallin was first detected by 2 dpf, peaked in abundance by 4-5 dpf, and was localized to the eye. alphaBa was detected in whole embryo at nearly constant levels from 1-6 dpf, was also localized primarily to the eye, and its abundance in extraocular tissues decreased from 4-7 dpf. In contrast, due to its low abundance, no alphaBb protein could be detected in whole embryo, or dissected eye and extraocular tissues. Our results show that mammalian alpha-crystallin promoters can be efficiently screened in zebrafish embryos and that their controlling regions are well conserved. An ontogenetic shift in zebrafish aBa-crystallin promoter activity provides an interesting system for examining the evolution and control of tissue specificity. Future studies that combine these promoter based approaches with the expanding ability to engineer the zebrafish genome via techniques such as CRISPR/Cas9 will allow the manipulation of protein expression to test hypotheses about lens crystallin function and its relation to lens biology and disease.
Strategies to stage and treat cancer rely on a presumption of either localized or widespread metastatic disease. An intermediate state of metastasis termed oligometastasis(es) characterized by ...limited progression has been proposed. Oligometastases are amenable to treatment by surgical resection or radiotherapy.
We analyzed microRNA expression patterns from lung metastasis samples of patients with ≤ 5 initial metastases resected with curative intent.
Patients were stratified into subgroups based on their rate of metastatic progression. We prioritized microRNAs between patients with the highest and lowest rates of recurrence. We designated these as high rate of progression (HRP) and low rate of progression (LRP); the latter group included patients with no recurrences. The prioritized microRNAs distinguished HRP from LRP and were associated with rate of metastatic progression and survival in an independent validation dataset.
Oligo- and poly- metastasis are distinct entities at the clinical and molecular level.
Research on suicide prevention and interventions requires a standard method for assessing both suicidal ideation and behavior to identify those at risk and to track treatment response. The ...Columbia-Suicide Severity Rating Scale (C-SSRS) was designed to quantify the severity of suicidal ideation and behavior. The authors examined the psychometric properties of the scale.
The C-SSRS's validity relative to other measures of suicidal ideation and behavior and the internal consistency of its intensity of ideation subscale were analyzed in three multisite studies: a treatment study of adolescent suicide attempters (N=124); a medication efficacy trial with depressed adolescents (N=312); and a study of adults presenting to an emergency department for psychiatric reasons (N=237).
The C-SSRS demonstrated good convergent and divergent validity with other multi-informant suicidal ideation and behavior scales and had high sensitivity and specificity for suicidal behavior classifications compared with another behavior scale and an independent suicide evaluation board. Both the ideation and behavior subscales were sensitive to change over time. The intensity of ideation subscale demonstrated moderate to strong internal consistency. In the adolescent suicide attempters study, worst-point lifetime suicidal ideation on the C-SSRS predicted suicide attempts during the study, whereas the Scale for Suicide Ideation did not. Participants with the two highest levels of ideation severity (intent or intent with plan) at baseline had higher odds for attempting suicide during the study.
These findings suggest that the C-SSRS is suitable for assessment of suicidal ideation and behavior in clinical and research settings.
Genetic studies may help identify causal pathways; therefore, we sought to identify genetic determinants of ideal CVH and their association with CVD outcomes in the multi-population Veteran ...Administration Million Veteran Program.
An ideal health score (IHS) was calculated from 3 clinical factors (blood pressure, total cholesterol, and blood glucose levels) and 3 behavioral factors (smoking status, physical activity, and BMI), ascertained at baseline. Multi-population genome-wide association study (GWAS) was performed on IHS and binary ideal health using linear and logistic regression, respectively. Using the genome-wide significant SNPs from the IHS GWAS, we created a weighted IHS polygenic risk score (PRSIHS) which was used (i) to conduct a phenome-wide association study (PheWAS) of associations between PRSIHS and ICD-9 phenotypes and (ii) to further test for associations with mortality and selected CVD outcomes using logistic and Cox regression and, as an instrumental variable, in Mendelian Randomization.
The discovery and replication cohorts consisted of 142,404 (119,129 European American (EUR); 16,495 African American (AFR)), and 45,766 (37,646 EUR; 5,366 AFR) participants, respectively. The mean age was 65.8 years (SD = 11.2) and 92.7% were male. Overall, 4.2% exhibited ideal CVH based on the clinical and behavioral factors. In the multi-population meta-analysis, variants at 17 loci were associated with IHS and each had known GWAS associations with multiple components of the IHS. PheWAS analysis in 456,026 participants showed that increased PRSIHS was associated with a lower odds ratio for many CVD outcomes and risk factors. Both IHS and PRSIHS measures of ideal CVH were associated with significantly less CVD outcomes and CVD mortality.
A set of high interest genetic variants contribute to the presence of ideal CVH in a multi-ethnic cohort of US Veterans. Genetically influenced ideal CVH is associated with lower odds of CVD outcomes and mortality.
The COVID-19 pandemic remains a significant global threat. However, despite urgent need, there remains uncertainty surrounding best practices for pharmaceutical interventions to treat COVID-19. In ...particular, conflicting evidence has emerged surrounding the use of hydroxychloroquine and azithromycin, alone or in combination, for COVID-19. The COVID-19 Evidence Accelerator convened by the Reagan-Udall Foundation for the FDA, in collaboration with Friends of Cancer Research, assembled experts from the health systems research, regulatory science, data science, and epidemiology to participate in a large parallel analysis of different data sets to further explore the effectiveness of these treatments.
Electronic health record (EHR) and claims data were extracted from seven separate databases. Parallel analyses were undertaken on data extracted from each source. Each analysis examined time to mortality in hospitalized patients treated with hydroxychloroquine, azithromycin, and the two in combination as compared to patients not treated with either drug. Cox proportional hazards models were used, and propensity score methods were undertaken to adjust for confounding. Frequencies of adverse events in each treatment group were also examined.
Neither hydroxychloroquine nor azithromycin, alone or in combination, were significantly associated with time to mortality among hospitalized COVID-19 patients. No treatment groups appeared to have an elevated risk of adverse events.
Administration of hydroxychloroquine, azithromycin, and their combination appeared to have no effect on time to mortality in hospitalized COVID-19 patients. Continued research is needed to clarify best practices surrounding treatment of COVID-19.
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