Tissue-specific compensatory mechanisms maintain tissue architecture and body size independent of cell size in polyploid zebrafish.

Genome duplications and ploidy transitions have occurred in nearly every major taxon of eukaryotes, but they are far more common in plants than in animals. Due to the conservation of the nuclear:cytoplasmic volume ratio increased DNA content results in larger cells. In plants, polyploid organisms are larger than diploids as cell number remains relatively constant. Conversely, vertebrate body size does not correlate with cell size and ploidy as vertebrates compensate for increased cell size to maintain tissue architecture and body size. This has historically been explained by a simple reduction in cell number that matches the increase in cell size maintaining body size as ploidy increases, but here we show that the compensatory mechanisms that maintain body size in triploid zebrafish are tissue-specific: A) erythrocytes respond in the classical pattern with a reduced number of larger erythrocytes in circulation, B) muscle, a tissue comprised of polynucleated muscle fibers, compensates by reducing the number of larger nuclei such that myofiber and myotome size in unaffected by ploidy, and C) vascular tissue compensates by thickening blood vessel walls, possibly at the expense of luminal diameter. Understanding the physiological implications of ploidy on tissue function requires a detailed description of the specific mechanisms of morphological compensation occurring in each tissue to understand how ploidy changes affect development and physiology.

Investigating home-based opioid use among youth with sickle cell disease using ecological momentary assessment.

BACKGROUND: Opioids are a common and essential treatment for acute sickle cell disease (SCD) pain. However, opioids carry well-known adverse side effects, including potential development of hyperalgesia and nociplastic pain. We characterized opioid use in youth with SCD using ecological momentary assessment (EMA) data, and investigated the relationships between home-based opioid use, pain, and a range of biopsychosocial factors. METHOD: Eighty-eight youth with SCD (aged 8-17 years) completed EMAs assessing home-based opioid use, pain, and related factors. Analyses consisted of descriptive and multilevel logistic regression to predict daily home opioid use. RESULTS: Youth averaged 3.64 weeks of EMAs. Approximately 35% of the sample (n = 31) took an opioid during the EMA period, and used them on only 24% of reported pain days. Youth who took opioids reported a higher percentage of pain days (t = -2.67, p < .05) and mean pain severity scores (t = -2.30, p < .05) than youth who did not take opioids. Multilevel logistic regression analyses indicated that high daily pain severity (odds ratio [OR] = 1.02, p < .01), older age (OR = 1.324, p < .01), and low positive affect (OR = 0.91, p < .01) were each related to an increased likelihood of opioid use. CONCLUSION: Youth with SCD take opioids appropriately in response to their pain, based on daily self-report. Beyond daily pain severity, age, and daily variation in positive affect were related to home-based opioid use. This suggests that behavioral interventions that enhance positive affect may promote reduced opioid use among youth with SCD.

MeCP2-regulated miRNAs control early human neurogenesis through differential effects on ERK and AKT signaling.

Rett syndrome (RTT) is an X-linked, neurodevelopmental disorder caused primarily by mutations in the methyl-CpG-binding protein 2 (MECP2) gene, which encodes a multifunctional epigenetic regulator with known links to a wide spectrum of neuropsychiatric disorders. Although postnatal functions of MeCP2 have been thoroughly investigated, its role in prenatal brain development remains poorly understood. Given the well-established importance of microRNAs (miRNAs) in neurogenesis, we employed isogenic human RTT patient-derived induced pluripotent stem cell (iPSC) and MeCP2 short hairpin RNA knockdown approaches to identify novel MeCP2-regulated miRNAs enriched during early human neuronal development. Focusing on the most dysregulated miRNAs, we found miR-199 and miR-214 to be increased during early brain development and to differentially regulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase and protein kinase B (PKB/AKT) signaling. In parallel, we characterized the effects on human neurogenesis and neuronal differentiation brought about by MeCP2 deficiency using both monolayer and three-dimensional (cerebral organoid) patient-derived and MeCP2-deficient neuronal culture models. Inhibiting miR-199 or miR-214 expression in iPSC-derived neural progenitors deficient in MeCP2 restored AKT and ERK activation, respectively, and ameliorated the observed alterations in neuronal differentiation. Moreover, overexpression of miR-199 or miR-214 in the wild-type mouse embryonic brains was sufficient to disturb neurogenesis and neuronal migration in a similar manner to Mecp2 knockdown. Taken together, our data support a novel miRNA-mediated pathway downstream of MeCP2 that influences neurogenesis via interactions with central molecular hubs linked to autism spectrum disorders.

Carnitine therapy for the treatment of metabolic syndrome and cardiovascular disease: evidence and controversies.

As the incidence of metabolic syndrome increases, there is also a growing interest in finding safe and inexpensive treatments to help lower associated risk factors. L-carntine, a natural dietary supplement with the potential to ameliorate atherosclerosis, has been the subject of recent investigation and controversy. A majority of studies have shown benefit of L-C supplementation in the metabolic syndrome or cardiovascular risk factors. However, recent work has suggested that dietary L-C may accelerate atherosclerosis via gut microbiota metabolites, complicating the role of L-C supplementation in health.

Disease correction by combined neonatal intracranial AAV and systemic lentiviral gene therapy in Sanfilippo Syndrome type B mice.

Mucopolysaccharidosis type IIIB (MPS IIIB) or Sanfilippo Syndrome type B is a lysosomal storage disease resulting from the deficiency of N-acetyl glucosaminidase (NAGLU) activity. We previously showed that intracranial adeno-associated virus (AAV)-based gene therapy results in partial improvements of several aspects of the disease. In an attempt to further correct the disease, MPS IIIB mice were treated at 2-4 days of age with intracranial AAV2/5-NAGLU (IC-AAV), intravenous lentiviral-NAGLU (IV-LENTI) or the combination of both (BOTH). The BOTH group had the most complete biochemical and histological improvements of any treatment group. Compared with untreated MPS IIIB animals, all treatments resulted in significant improvements in motor function (rotarod) and hearing (auditory-evoked brainstem response). In addition, each treatment group had a significantly increased median life span compared with the untreated group (322 days). The combination arm had the greatest increase (612 days), followed by IC-AAV (463 days) and IV-LENTI (358 days). Finally, the BOTH group had nearly normal circadian rhythm measures with improvement in time to activity onset. In summary, targeting both the systemic and central nervous system disease of MPS IIIB early in life appears to be the most efficacious approach for this inherited metabolic disorder.

Assessing fibromyalgia-related fatigue: content validity and psychometric performance of the Fatigue Visual Analog Scale in adult patients with fibromyalgia.

OBJECTIVES: To document 1) the content validity and 2) measure improvements in fatigue, using the Fatigue Visual Analogue Scale (VAS) assessment tool in patients with fibromyalgia. METHODS: The relevance and comprehensiveness of the Fatigue VAS were tested through a qualitative analysis of 20 subjects' verbatim transcripts from semi-structured qualitative interviews. Data from two randomised, controller trials in fibromyalgia (n=1121) were used to conduct correlation analyses with the Fatigue and Tiredness items from the Fibromyalgia Impact Questionnaire (FIQ) and the Short Form-36 Vitality scale. Known-groups and cross classification analyses were conducted to demonstrate the ability to measure improvement in fatigue using the Fatigue VAS. RESULTS: All subjects spontaneously reported that fatigue was an important symptom to capture in fibromyalgia. The Fatigue VAS was well understood by most subjects (n=18/20). High correlations (Pearson r>0.75) and good agreement (k>0.66) were found between the Fatigue VAS and the FIQ tiredness items no. 16 and 17 and SF-36™ Vitality scale. In both clinical trials there was a substantial separation of approximately 20 points on the mean change in the Fatigue VAS score between responders (>30% improvement in pain VAS) and non-responders. CONCLUSIONS: Previous studies have confirmed that fatigue is a major component of the fibromyalgia experience. This current study reports that fibromyalgia patients spontaneously rated fatigue as a highly significant feature of their illness, and supports the use of the Fatigue VAS as a valid questionnaire in fibromyalgia clinical trials.

Assessing sleep in fibromyalgia: investigation of an alternative scoring method for the Jenkins Sleep Scale based on data from randomized controlled studies.

OBJECTIVES: To investigate the validity of a rescored version of the Jenkins Sleep Scale (JSS) to assess the extent of possible bias of a 4-week recall period in assessing sleep in patients with fibromyalgia. METHODS: A rescoring algorithm of the JSS was developed. The psychometric properties of the rescored JSS were examined using blinded, observed data from a Phase 2 trial (n=195) in subjects with fibromyalgia. In addition, data from two Phase 3, randomised, controlled trials (n=1,121) in subjects with fibromyalgia were used to further validate the rescored JSS by conducting correlation analyses with other assessments expected to correlate with sleep. These included fatigue and tiredness items from the Fibromyalgia Impact Questionnaire (FIQ), the Functional Outcomes of Sleep Questionnaire (FOSQ), and the Short Form-36 (SF-36™) Vitality scale. RESULTS: Construct validity of the rescored JSS was found to be acceptable, with an internal consistency reliability of α=0.70. Test-retest reliability on stable subjects, defined using the FIQ total score, was also acceptable (ICC=0.70). Moderate to high correlations (Pearson r>0.66) were found with two FIQ items, addressing fatigue and non-restorative sleep, and the SF-36™ Vitality scale; correlations with the original JSS were similar. Both JSS versions were found to be responsive (p<0.0001), and the rescored version accounted for 90% of the variance captured in the original version. CONCLUSIONS: These results showed the rescored JSS performed similarly to the original scale, suggesting the original scale's 4-week recall period did not introduce substantial bias in capturing the experience of fibromyalgia-related sleep disturbances.

Use of "spydergrams" to present and interpret SF-36 health-related quality of life data across rheumatic diseases.

The Medical Outcomes Study Short Form-36 (SF-36) is a generic measure of health-related quality of life (HRQOL), validated and cross-culturally translated, which has been extensively utilised in rheumatology. In randomised controlled trials and observational studies, SF-36 provides rich data regarding HRQOL; but as typically portrayed, patterns of disease and treatment-associated effects can be difficult to discern. "Spydergrams" offer a simplified means to visualise complex results across all domains of SF-36 in a single figure: depicting disease and population-specific patterns of decrements in HRQOL compared with age and gender-matched normative data, as well as providing a tool for interpreting complex treatment-associated or longitudinal changes. Utilising spydergrams as a standard format to illustrate and report changes in SF-36 across different rheumatic diseases can greatly facilitate analyses and interpretations of clinical trial results, as well as providing patients an accessible means to compare baseline scores and treatment-associated improvements with normative data from individuals without arthritis. Furthermore, SF-6D utility scores based on mean changes across all eight domains of SF-36 are suggested as a quantitative means of summarising changes illustrated by spydergrams, offering a universal metric for cost-effectiveness analyses of therapeutic interventions.

Cloned pigmented retinal epiehtlium. The role of microfilaments in the differentiation of cell shape.

3-wk-old clones of pigmented epithelial cells from chick retina can be divided into four zones on the basis of cellular morphology and pigmentation. These zones appear to represent different stages in the re-expression of differentiation: those cells with essentially no differentiated characteristics are at the outer edge and those with the greatest number are at the center. Cells of the colony exhibit three different types of movement when analyzed by time-lapse cinephotomicrography: focal contractions, extension and retraction of apical protrusions, and undulations of the lateral membranes. All the cells of the colony contain microfilaments, 4--7 nm in Diam, which are primarily arranged as apical and basal webs. In addition, less well defined filamentous networks are found in the apical protrusions and lateral interdigitations. When colonies are treated with 10 micrograms/ml of the drug cytochalasin B (CCB), the apical microfilament arrays are disrupted and movement stops. Both phenomena are reversible upon removal of the drug. During the process of redifferentiation, the cells change their shape from squamous to cuboidal, and the greatest change is found where the colony exhibits the greatest number of focal contractions. The evidence suggests that the apical microfilament arrays are directly responsible for the observed movements, particularly the focal contractions, and that focal contractions contribute to the development of the differentiated cellular shape. Possible roles for the other movements are discussed.

Experimental upper bound and theoretical expectations for parity-violating neutron spin rotation in 4He.

Neutron spin rotation is expected from quark-quark weak interactions in the standard model, which induce weak interactions among nucleons that violate parity. We present the results from an experiment searching for the effect of parity violation via the spin rotation of polarized neutrons in a liquid 4He medium. The value for the neutron spin rotation angle per unit length in 4He, d ϕ / d z = [ + 2.1 ± 8.3 (stat.) - 0.2 + 2.9 (sys.) ] × 10 - 7 rad/m, is consistent with zero. The result agrees with the best current theoretical estimates of the size of nucleon-nucleon weak amplitudes from other experiments and with the expectations from recent theoretical approaches to weak nucleon-nucleon interactions. In this paper we review the theoretical status of parity violation in the n → + 4He system and discuss details of the data analysis leading to the quoted result. Analysis tools are presented that quantify systematic uncertainties in this measurement and that are expected to be essential for future measurements.

Neoplastic conversion of preneoplastic Syrian hamster cells: rate estimation by fluctuation analysis.

Analysis of the role of gene mutations in the multistep process of neoplastic transformation requires that the discrete steps in carcinogenesis first be dissected. Toward this end, we have isolated and characterized preneoplastic Syrian hamster cells which exhibit in vitro a trait highly correlated with neoplastic conversion in vivo. Previous findings (J. C. Barrett, Cancer Res. 40:91-94, 1980) indicate that spontaneous neoplastic transformation of Syrian hamster cells occurs in at least two steps. An intermediate stage, characterized by an aneuploid established cell line which has a propensity to become neoplastic spontaneously upon further growth in vitro, has been described. These preneoplastic cells differ from diploid early-passage Syrian hamster cells in becoming capable of anchorage-independent growth in semisolid agar, as well as becoming neoplastic in vivo when attached to a solid substrate. Evidence presented here demonstrates that anchorage-independent conversion in vitro is a reliable marker for neoplastic conversion in this cell system. Fluctuation analyses, patterned after those described by Luria and Delbruck for microbial genetics, demonstrate that anchorage-independent variants are generated randomly from clonally derived preneoplastic cells at the rate of 10(-8) to 10(-7) variants per cell per generation. These results establish a multistep stochastic process for transformation in vitro and indicate that conversion to anchorage independence may be necessary for Syrian hamster cells to become tumorigenic. The possible role of gene mutation in this step during neoplastic progression is discussed.

Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosome 3: evidence for the role of chromosome rearrangement in gene amplification.

Cadmium resistant (Cdr) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cdr variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with a Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster X mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. We speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cdr hamster cell lines.

Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulating metallothionein gene expression.

We describe here the derivation, characterization, and use of clonal cadmium-resistant (Cdr) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylamide gel electrophoresis, we showed that the stable Cdr phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 microM, coordinate amplification of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which we have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cdr variants expressing abundant MT and their corresponding Cds parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expression of the isometallothioneins.

Methylomic profiling of cortex samples from completed suicide cases implicates a role for PSORS1C3 in major depression and suicide.

Major depressive disorder (MDD) represents a major social and economic health issue and constitutes a major risk factor for suicide. The molecular pathology of suicidal depression remains poorly understood, although it has been hypothesised that regulatory genomic processes are involved in the pathology of both MDD and suicidality. In this study, genome-wide patterns of DNA methylation were assessed in depressed suicide completers (n=20) and compared with non-psychiatric, sudden-death controls (n=20) using tissue from two cortical brain regions (Brodmann Area 11 (BA11) and Brodmann Area 25 (BA25)). Analyses focused on identifying differentially methylated regions (DMRs) associated with suicidal depression and epigenetic variation were explored in the context of polygenic risk scores for major depression and suicide. Weighted gene co-methylation network analysis was used to identify modules of co-methylated loci associated with depressed suicide completers and polygenic burden for MDD and suicide attempt. We identified a DMR upstream of the PSORS1C3 gene, subsequently validated using bisulfite pyrosequencing and replicated in a second set of suicide samples, which is characterised by significant hypomethylation in both cortical brain regions in MDD suicide cases. We also identified discrete modules of co-methylated loci associated with polygenic risk burden for suicide attempt, but not major depression. Suicide-associated co-methylation modules were enriched among gene networks implicating biological processes relevant to depression and suicidality, including nervous system development and mitochondria function. Our data suggest that there are coordinated changes in DNA methylation associated with suicide that may offer novel insights into the molecular pathology associated with depressed suicide completers.

Testing the gonadal regression-cytoprotection hypothesis.

Germinal damage is an almost universal accompaniment of cancer treatment as the result of bystander damage to the testis from cytotoxic drugs and/or irradiation. Cancer treatment for the most common cancers of the reproductive age group in men has improved such that most are now treated with curative intent, and many others are treated with likelihood of prolonged survival, so that the preservation of fertility is an important component of posttreatment quality of life. This has led to the consideration of developing adjuvant treatments that may reduce the gonadal toxicity of cancer therapy. One dominant hypothesis has been based on the supposition that the immature testis was resistant to cytotoxin damage. Hence, if hormonal treatment were able to cause spermatogenic regression to an immature state via an effective withdrawal of gonadotrophin secretion, the testis might be maintained temporarily in a protected state during cytotoxin exposure. However, clinical studies have been disappointing but have also been unable to test the hypothesis definitively thus far, due to the inability to completely suppress gonadotrophin secretion. Similarly, experimental models have also given conflicting results and, at best, a modest cytoprotection. To definitively test this hypothesis experimentally, we used the fact that the functionally hpg mouse has complete gonadotrophin deficiency but can undergo the induction of full spermatogenesis by testosterone. Thus, if complete gonadotrophin deficiency were an advantage during cytotoxin exposure, then the hpg mouse should exhibit some degree of germinal protection against cytotoxin-induced damage. We therefore administered three different cytotoxins (200 mg/kg procarbazine, 9 mg/kg doxorubicin, 8 Gy of X irradiation) to produce a range of severity in testicular damage and mechanism of action to either phenotypically normal or hpg mice. Testis weight and homogenization-resistant spermatid numbers were measured to evaluate the potential protective effects on spermatogenesis. Although the three cytotoxins produced a range of severity of spermatogenic damage, there was no evidence of cytoprotection in the hpg mice that were completely gonadotrophin deficient at the time of treatment. These findings cast doubt on the validity of the hypothesis that spermatogenic regression via gonadotrophin withdrawal can protect the mouse testis against cytotoxin-mediated spermatogenic damage.

Quantitation of fibrinolytic activity of Syrian hamster fibroblasts using 3H-labeled fibrinogen prepared by reductive alkylation.

Tritium-labeled fibrinogen with a specific activity of 2.0 X 10(7) cpm/mg was prepared by the method of reductive alkylation. The use of the 3H-fibrinogen as a substrate for detection of both intracellular and extracellular fibrinolytic activity derived from cultures of benzo(a)pyrene-transformed Syrian hamster cell lines was examined in cell-free assays, 3H-fibrinogen enabled reliable quantitation of the fibrinolytic activity associated with neoplastic cells. The elevated extracellular fibrinolytic activity in the transformed cell lines as compared to normal hamster embryo cultures was demonstrated with this substrate. The ease with which large quantities of 3H-fibrinogen of high specific activity and prolonged half-life can be prepared makes the use of this substrate an attractive alternative to 125I-labeled fibrinogen.