The metabolic consequences of 'yo-yo' dieting are markedly influenced by genetic diversity.

BACKGROUND: Weight loss can improve the metabolic complications of obesity. However, it is unclear whether insulin resistance persists despite weight loss and whether any protective benefits are preserved following weight regain (weight cycling). The impact of genetic background on weight cycling is undocumented. We aimed to investigate the effects of weight loss and weight cycling on metabolic outcomes and sought to clarify the role of genetics in this relationship. METHOD: Both C57BL/6 J and genetically heterogeneous Diversity Outbred Australia (DOz) mice were alternately fed high fat Western-style diet (WD) and a chow diet at 8-week intervals. Metabolic measures including body composition, glucose tolerance, pancreatic beta cell activity, liver lipid levels and adipose tissue insulin sensitivity were determined. RESULTS: After diet switch from WD (8-week) to chow (8-week), C57BL/6 J mice displayed a rapid normalisation of body weight, adiposity, hyperinsulinemia, liver lipid levels and glucose uptake into adipose tissue comparable to chow-fed controls. In response to the same dietary intervention, genetically diverse DOz mice conversely maintained significantly higher fat mass and insulin levels compared to chow-fed controls and exhibited much more profound interindividual variability than C57BL/6 J mice. Weight cycled (WC) animals were re-exposed to WD (8-week) and compared to age-matched controls fed 8-week WD for the first time (LOb). In C57BL/6 J but not DOz mice, WC animals had significantly higher blood insulin levels than LOb controls. All WC animals exhibited significantly greater beta cell activity than LOb controls despite similar fat mass, glucose tolerance, liver lipid levels and insulin-stimulated glucose uptake in adipose tissue. CONCLUSION: Following weight loss, metabolic outcomes return to baseline in C57BL/6 J mice with obesity. However, genetic diversity significantly impacts this response. A period of weight loss does not provide lasting benefits after weight regain, and weight cycling is detrimental and associated with hyperinsulinemia and elevated basal insulin secretion.

L-type voltage-gated calcium channel agonists mitigate hearing loss and modify ribbon synapse morphology in the zebrafish model of Usher syndrome type 1.

The mariner (myo7aa-/- ) mutant is a zebrafish model for Usher syndrome type 1 (USH1). To further characterize hair cell synaptic elements in myo7aa-/- mutants, we focused on the ribbon synapse and evaluated ultrastructure, number and distribution of immunolabeled ribbons, and postsynaptic densities. By transmission electron microscopy, we determined that myo7aa-/- zebrafish have fewer glutamatergic vesicles tethered to ribbon synapses, yet maintain a comparable ribbon area. In myo7aa-/- hair cells, immunolocalization of Ctbp2 showed fewer ribbon-containing cells in total and an altered distribution of Ctbp2 puncta compared to wild-type hair cells. myo7aa-/- mutants have fewer postsynaptic densities - as assessed by MAGUK immunolabeling - compared to wild-type zebrafish. We quantified the circular swimming behavior of myo7aa-/- mutant fish and measured a greater turning angle (absolute smooth orientation). It has previously been shown that L-type voltage-gated calcium channels are necessary for ribbon localization and occurrence of postsynaptic density; thus, we hypothesized and observed that L-type voltage-gated calcium channel agonists change behavioral and synaptic phenotypes in myo7aa-/- mutants in a drug-specific manner. Our results indicate that treatment with L-type voltage-gated calcium channel agonists alter hair cell synaptic elements and improve behavioral phenotypes of myo7aa-/- mutants. Our data support that L-type voltage-gated calcium channel agonists induce morphological changes at the ribbon synapse - in both the number of tethered vesicles and regarding the distribution of Ctbp2 puncta - shift swimming behavior and improve acoustic startle response.

Optimization of a microfluidics-based next generation sequencing assay for clinical oncology diagnostics.

BACKGROUND: Massively parallel, or next-generation, sequencing is a powerful technique for the assessment of somatic genomic alterations in cancer samples. Numerous gene targets can be interrogated simultaneously with a high degree of sensitivity. The clinical standard of care for many advanced solid and hematologic malignancies currently requires mutation analysis of several genes in the front-line setting, making focused next generation sequencing (NGS) assays an effective tool for clinical molecular diagnostic laboratories. METHODS: We have utilized an integrated microfluidics circuit (IFC) technology for multiplex PCR-based library preparation coupled with a bioinformatic method designed to enhance indel detection. A parallel low input PCR-based library preparation method was developed for challenging specimens with low DNA yield. Computational data filters were written to optimize analytic sensitivity and specificity for clinically relevant variants. RESULTS: Minimum sequencing coverage and precision of variant calls were the two primary criteria used to establish minimum DNA mass input onto the IFC. Wet-bench and bioinformatics protocols were modified based on data from the optimization and familiarization process to improve assay performance. The NGS platform was then clinically validated for single nucleotide and indel (up to 93 base pair) variant detection with overall analytic accuracy of 98% (97% sensitivity; 100% specificity) using as little as 3 ng of formalin-fixed, paraffin-embedded DNA or 0.3 ng of unfixed DNA. CONCLUSIONS: We created a targeted clinical NGS assay for common solid and hematologic cancers with high sensitivity, high specificity, and the flexibility to test very limited tissue samples often encountered in routine clinical practice.

Optimal detection of clinically relevant mutations in colorectal carcinoma: sample pooling overcomes intra-tumoral heterogeneity.

Intra-tumoral genomic heterogeneity is a well-established biologic property of human malignancies with emerging roles in cancer progression and therapy resistance. However, its impact on the clinical utility of genomic testing in patient management remains unclear. Furthermore, best practices to account for heterogeneity in the provision of highly accurate, clinically valid molecular testing have yet to be firmly established. Genomic biomarkers for the management of colorectal carcinoma are both well-established (ie, KRAS, NRAS) and emerging (BRAF, PIK3CA, and others) in respect to therapy selection and clinical trial eligibility. Critically, standard colorectal carcinoma management requires the exclusion of KRAS and NRAS mutations; thus optimal procedures to control for potential intra-tumoral heterogeneity are clinically important. Here, we assessed heterogeneity among three intra-tumoral sites within 99 colorectal carcinomas cases on a CLIA-validated oncology next generation sequencing assay and examined whether a pooling strategy overcame any discordant results. Overall, 11% of cases demonstrated discordant mutation results between sites; 2% of cases were discrepant for mutations within RAS genes while the remainder was discrepant in PIK3CA. Half of the discrepant cases were associated with borderline tumor cellularity assessment. Further, a sample pooling strategy across all three sites successfully detected the relevant mutation in all but one case. Our results indicate that intra-tumoral genomic heterogeneity of clinically relevant genes within colorectal carcinoma is a relatively infrequent occurrence and that a simple strategy to pool DNA from several tumor sites with adequate cellularity minimizes the risk of false negative results even further to ensure optimal patient management.

Abnormal differentiation of dopaminergic neurons in zebrafish trpm7 mutant larvae impairs development of the motor pattern.

Transient receptor potential, melastatin-like 7 (Trpm7) is a combined ion channel and kinase implicated in the differentiation or function of many cell types. Early lethality in mice and frogs depleted of the corresponding gene impedes investigation of the functions of this protein particularly during later stages of development. By contrast, zebrafish trpm7 mutant larvae undergo early morphogenesis normally and thus do not have this limitation. The mutant larvae are characterized by multiple defects including melanocyte cell death, transient paralysis, and an ion imbalance that leads to the development of kidney stones. Here we report a requirement for Trpm7 in differentiation or function of dopaminergic neurons in vivo. First, trpm7 mutant larvae are hypomotile and fail to make a dopamine-dependent developmental transition in swim-bout length. Both of these deficits are partially rescued by the application of levodopa or dopamine. Second, histological analysis reveals that in trpm7 mutants a significant fraction of dopaminergic neurons lack expression of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Third, trpm7 mutants are unusually sensitive to the neurotoxin 1-methyl-4-phenylpyridinium, an oxidative stressor, and their motility is partially rescued by application of the iron chelator deferoxamine, an anti-oxidant. Finally, in SH-SY5Y cells, which model aspects of human dopaminergic neurons, forced expression of a channel-dead variant of TRPM7 causes cell death. In summary, a forward genetic screen in zebrafish has revealed that both melanocytes and dopaminergic neurons depend on the ion channel Trpm7. The mechanistic underpinning of this dependence requires further investigation.

The conserved dopaminergic diencephalospinal tract mediates vertebrate locomotor development in zebrafish larvae.

The most conserved part of the vertebrate dopaminergic system is the orthopedia (otp)-expressing diencephalic neuronal population that constitutes the dopaminergic diencephalospinal tract (DDT). Although studies in the neonatal murine spinal cord in vitro suggest an early locomotor role of the DDT, the function of the DDT in developing vertebrates in vivo remains unknown. Here, we investigated the role of the DDT in the locomotor development of zebrafish larvae. To assess the development of the behavioral and neural locomotor pattern, we used high-throughput video tracking in combination with peripheral nerve recordings. We found a behavioral and neural correspondence in the developmental switch from an immature to mature locomotor pattern. Blocking endogenous dopamine receptor 4 (D(4)R) signaling in vivo either before or after the developmental switch prevented or reversed the switch, respectively. Spinal transections of post-switch larvae reestablished the immature locomotor pattern, which was rescued to a mature-like pattern via spinal D(4)R agonism. Selective chemogenetic ablation of otp b (otpb) neurons that contribute to the DDT perpetuated the immature locomotor pattern in vivo. This phenotype was recapitulated by diencephalic transections that removed the dopaminergic otpb population and was rescued to a mature-like locomotor pattern by D(4)R agonism. We conclude that the dopaminergic otpb population, via the DDT, is responsible for spinal D(4)R signaling to mediate the developmental switch to the mature locomotor pattern of zebrafish. These results, integrated with the mammalian literature, suggest that the DDT represents an evolutionarily conserved neuromodulatory system that is necessary for normal vertebrate locomotor development.

Mutation of zebrafish dihydrolipoamide branched-chain transacylase E2 results in motor dysfunction and models maple syrup urine disease.

Analysis of zebrafish mutants that demonstrate abnormal locomotive behavior can elucidate the molecular requirements for neural network function and provide new models of human disease. Here, we show that zebrafish quetschkommode (que) mutant larvae exhibit a progressive locomotor defect that culminates in unusual nose-to-tail compressions and an inability to swim. Correspondingly, extracellular peripheral nerve recordings show that que mutants demonstrate abnormal locomotor output to the axial muscles used for swimming. Using positional cloning and candidate gene analysis, we reveal that a point mutation disrupts the gene encoding dihydrolipoamide branched-chain transacylase E2 (Dbt), a component of a mitochondrial enzyme complex, to generate the que phenotype. In humans, mutation of the DBT gene causes maple syrup urine disease (MSUD), a disorder of branched-chain amino acid metabolism that can result in mental retardation, severe dystonia, profound neurological damage and death. que mutants harbor abnormal amino acid levels, similar to MSUD patients and consistent with an error in branched-chain amino acid metabolism. que mutants also contain markedly reduced levels of the neurotransmitter glutamate within the brain and spinal cord, which probably contributes to their abnormal spinal cord locomotor output and aberrant motility behavior, a trait that probably represents severe dystonia in larval zebrafish. Taken together, these data illustrate how defects in branched-chain amino acid metabolism can disrupt nervous system development and/or function, and establish zebrafish que mutants as a model to better understand MSUD.

Very high rates of vitamin D insufficiency in women of child-bearing age living in Beijing and Hong Kong.

We aimed to describe the vitamin D status of young women living in two Chinese cities in the spring--Beijing in the north (latitude 39 degrees north) and Hong Kong (latitude 22 degrees north) in the south. We also examined the relationship between serum 25-hydroxyvitamin D and parathyroid hormone (PTH) concentrations to determine a threshold for serum 25-hydroxyvitamin D above which there is no further suppression of PTH. Finally, we examined whether dietary Ca intake influences this relationship. Non-pregnant women aged 18-40 years (n 441) were recruited between February and June. Fasting blood was collected and dietary intakes were assessed using 5 d food records. Mean serum 25-hydroxyvitamin D concentration was lower in Beijing than Hong Kong women (29 v. 34 nmol/l; P < 0.001). Vitamin D deficiency (< or = 25 nmol/l) was indicated in 40% of Beijing and 18% of Hong Kong women, and over 90% of women in both cities were insufficient (< or = 50 nmol/l). Mean Ca and vitamin D intakes were 478 mg/d and 2.0 microg/d, respectively. The relationship between 25-hydroxyvitamin D concentration and PTH was linear throughout the range with a slope of -0.36 (different from 0; P < 0.001; R 0.26), with no apparent threshold. There was no influence of Ca intake on the relationship between 25-hydroxyvitamin D and PTH concentration. Vitamin D deficiency is common and insufficiency is very common in non-pregnant women in Hong Kong and Beijing during spring. Serum 25-hydroxyvitamin D was inversely associated with PTH with no apparent threshold. Strategies such as vitamin D fortification or supplementation may be required.

Raphe magnus neurons help protect reactions to visceral pain from interruption by cutaneous pain.

Suppression of reactions to one noxious stimulus by a spatially distant noxious stimulus is termed heterotopic antinociception. In lightly anesthetized rats, a noxious visceral stimulus, colorectal distension (CRD), suppressed motor withdrawals but not blood pressure or heart rate changes evoked by noxious hindpaw heat. Microinjection of muscimol, a GABA(A) receptor agonist, into raphe magnus (RM) reduced CRD-evoked suppression of withdrawals, evidence that RM neurons contribute to this heterotopic antinociception. To understand how brain stem neurons contribute to heterotopic antinociception, RM neurons were recorded during CRD-elicited suppression of hindpaw withdrawals. Although subsets of RM neurons that were excited (on cells) or inhibited (off cells) by noxious cutaneous stimulation were either excited or inhibited by CRD, on cells were inhibited and off cells excited by an intracerebroventricularly administered opioid, evidence that the nociception-facilitating and -inhibiting functions of on and off cells, respectively, are predicted by the cellular response to noxious cutaneous stimulation alone and not by the response to CRD. When recorded during CRD-elicited antinociception, RM cell discharge resembled the pattern observed in response to CRD stimulation alone. However, when hindpaw withdrawal suppression was incomplete, RM cell discharge resembled the pattern observed in response to heat alone. We propose that on cells excited by CRD facilitate responses to CRD itself, which in turn augments excitation of off cells that then act to suppress cutaneous nociception. RM cells may thereby contribute to the dominance of quiet recuperative reactions evoked by potentially life-threatening visceral stimuli over transient somatomotor activity elicited by less-injurious noxious cutaneous stimuli.