Inducible and Reversible Clock Gene Expression in Brain Using the tTA System for the Study of Circadian Behavior

The mechanism of circadian oscillations in mammals is cell autonomous and is generated by a set of genes that form a transcriptional autoregulatory feedback loop. While these “clock genes” are well conserved among animals, their specific functions remain to be fully understood and their roles in central versus peripheral circadian oscillators remain to be defined. We utilized the in vivo inducible tetracycline-controlled transactivator (tTA) system to regulate Clock gene expression conditionally in a tissue-specific and temporally controlled manner. Through the use of Secretogranin II to drive tTA expression, suprachiasmatic nucleus– and brain-directed expression of a tetO::Clock(Δ19) dominant-negative transgene lengthened the period of circadian locomotor rhythms in mice, whereas overexpression of a tetO::Clock(wt) wild-type transgene shortened the period. Low doses (10 μg/ml) of doxycycline (Dox) in the drinking water efficiently inactivated the tTA protein to silence the tetO transgenes and caused the circadian periodicity to return to a wild-type state. Importantly, low, but not high, doses of Dox were completely reversible and led to a rapid reactivation of the tetO transgenes. The rapid time course of tTA-regulated transgene expression demonstrates that the CLOCK protein is an excellent indicator for the kinetics of Dox-dependent induction/repression in the brain. Interestingly, the daily readout of circadian period in this system provides a real-time readout of the tTA transactivation state in vivo. In summary, the tTA system can manipulate circadian clock gene expression in a tissue-specific, conditional, and reversible manner in the central nervous system. The specific methods developed here should have general applicability for the study of brain and behavior in the mouse

A call to action: A need for initiatives that increase equitable access to COVID-19 therapeutics

Structural racism is endemic in the United States and causes inequitable health outcomes that have been amplified throughout the COVID-19 pandemic. Non-Hispanic Black, Hispanic/Latino, and Native American individuals have been disproportionately affected, and are twice as likely to be hospitalized or die from COVID-19 or related morbidities when compared to White Americans. Social determinants of health inequities contribute to these disparate outcomes, given that minoritized individuals are more likely to occupy essential worker roles and to live in high-density settings. Despite their higher risk of severe COVID-19 illness, racially and ethnically minoritized individuals are less likely to receive potentially lifesaving COVID-19 therapeutics.3 While several state health departments attempted to implement race-conscious interventions and narrow the disparities, these efforts have been met with fallacious claims of ‘reverse racism’ and the reversal of the proposed implementations

A multi-omics study of circulating phospholipid markers of blood pressure.

High-throughput techniques allow us to measure a wide-range of phospholipids which can provide insight into the mechanisms of hypertension. We aimed to conduct an in-depth multi-omics study of various phospholipids with systolic blood pressure (SBP) and diastolic blood pressure (DBP). The associations of blood pressure and 151 plasma phospholipids measured by electrospray ionization tandem mass spectrometry were performed by linear regression in five European cohorts (n = 2786 in discovery and n = 1185 in replication). We further explored the blood pressure-related phospholipids in Erasmus Rucphen Family (ERF) study by associating them with multiple cardiometabolic traits (linear regression) and predicting incident hypertension (Cox regression). Mendelian Randomization (MR) and phenome-wide association study (Phewas) were also explored to further investigate these association results. We identified six phosphatidylethanolamines (PE 38:3, PE 38:4, PE 38:6, PE 40:4, PE 40:5 and PE 40:6) and two phosphatidylcholines (PC 32:1 and PC 40:5) which together predicted incident hypertension with an area under the ROC curve (AUC) of 0.61. The identified eight phospholipids are strongly associated with triglycerides, obesity related traits (e.g. waist, waist-hip ratio, total fat percentage, body mass index, lipid-lowering medication, and leptin), diabetes related traits (e.g. glucose, insulin resistance and insulin) and prevalent type 2 diabetes. The genetic determinants of these phospholipids also associated with many lipoproteins, heart rate, pulse rate and blood cell counts. No significant association was identified by bi-directional MR approach. We identified eight blood pressure-related circulating phospholipids that have a predictive value for incident hypertension. Our cross-omics analyses show that phospholipid metabolites in the circulation may yield insight into blood pressure regulation and raise a number of testable hypothesis for future research

Identification of TBX15 as an adipose master trans regulator of abdominal obesity genes

Background: Obesity predisposes individuals to multiple cardiometabolic disorders, including type 2 diabetes (T2D). As body mass index (BMI) cannot reliably differentiate fat from lean mass, the metabolically detrimental abdominal obesity has been estimated using waist-hip ratio (WHR). Waist-hip ratio adjusted for body mass index (WHRadjBMI) in turn is a well-established sex-specific marker for abdominal fat and adiposity, and a predictor of adverse metabolic outcomes, such as T2D. However, the underlying genes and regulatory mechanisms orchestrating the sex differences in obesity and body fat distribution in humans are not well understood. Methods: We searched for genetic master regulators of WHRadjBMI by employing integrative genomics approaches on human subcutaneous adipose RNA sequencing (RNA-seq) data (n similar to 1400) and WHRadjBMI GWAS data (n similar to 700,000) from the WHRadjBMI GWAS cohorts and the UK Biobank (UKB), using co-expression network, transcriptome-wide association study (TWAS), and polygenic risk score (PRS) approaches. Finally, we functionally verified our genomic results using gene knockdown experiments in a human primary cell type that is critical for adipose tissue function. Results: Here, we identified an adipose gene co-expression network that contains 35 obesity GWAS genes and explains a significant amount of polygenic risk for abdominal obesity and T2D in the UKB (n = 392,551) in a sex-dependent way. We showed that this network is preserved in the adipose tissue data from the Finnish Kuopio Obesity Study and Mexican Obesity Study. The network is controlled by a novel adipose master transcription factor (TF), TBX15, a WHRadjBMI GWAS gene that regulates the network in trans. Knockdown of TBX15 in human primary preadipocytes resulted in changes in expression of 130 network genes, including the key adipose TFs, PPARG and KLF15, which were significantly impacted (FDR < 0.05), thus functionally verifying the trans regulatory effect of TBX15 on the WHRadjBMI co-expression network. Conclusions: Our study discovers a novel key function for the TBX15 TF in trans regulating an adipose co-expression network of 347 adipose, mitochondrial, and metabolically important genes, including PPARG, KLF15, PPARA, ADIPOQ, and 35 obesity GWAS genes. Thus, based on our converging genomic, transcriptional, and functional evidence, we interpret the role of TBX15 to be a main transcriptional regulator in the adipose tissue and discover its importance in human abdominal obesity.Peer reviewe

Kynurenic acid may underlie sex-specific immune responses to COVID-19

Coronavirus disease 2019 (COVID-19) has poorer clinical outcomes in males than in females, and immune responses underlie these sex-related differences. Because immune responses are, in part, regulated by metabolites, we examined the serum metabolomes of COVID-19 patients. In male patients, kynurenic acid (KA) and a high KA–to–kynurenine (K) ratio (KA:K) positively correlated with age and with inflammatory cytokines and chemokines and negatively correlated with T cell responses. Males that clinically deteriorated had a higher KA:K than those that stabilized. KA inhibits glutamate release, and glutamate abundance was lower in patients that clinically deteriorated and correlated with immune responses. Analysis of data from the Genotype-Tissue Expression (GTEx) project revealed that the expression of the gene encoding the enzyme that produces KA, kynurenine aminotransferase, correlated with cytokine abundance and activation of immune responses in older males. This study reveals that KA has a sex-specific link to immune responses and clinical outcomes in COVID-19, suggesting a positive feedback between metabolites and immune responses in males

Beyond the call of duty: Why customers contribute to firm-hosted commercial online communities

Firm-hosted commercial online communities, in which customers interact to solve each other's service problems, represent a fascinating context to study the motivations of collective action in the form of knowledge contribution to the community. We extend a model of social capital based on Wasko and Faraj (2005) to incorporate and contrast the direct impact of commitment to both the online community and the host firm, as well as reciprocity, on quality and quantity of knowledge contribution. In addition, we examine the moderating influence of three individual attributes that are particularly relevant to the firm-hosted community context: perceived informational value, sportsmanship, and online interaction propensity. We empirically test our framework using self-reported and objective data from 203 members of a firm-hosted technical support community. In addition to several interesting moderating effects, we find that a customer's online interaction propensity, commitment to the community, and the informational value s/he perceives in the community are the strongest drivers of knowledge contribution

New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk.

Levels of circulating glucose are tightly regulated. To identify new loci influencing glycemic traits, we performed meta-analyses of 21 genome-wide association studies informative for fasting glucose, fasting insulin and indices of beta-cell function (HOMA-B) and insulin resistance (HOMA-IR) in up to 46,186 nondiabetic participants. Follow-up of 25 loci in up to 76,558 additional subjects identified 16 loci associated with fasting glucose and HOMA-B and two loci associated with fasting insulin and HOMA-IR. These include nine loci newly associated with fasting glucose (in or near ADCY5, MADD, ADRA2A, CRY2, FADS1, GLIS3, SLC2A2, PROX1 and C2CD4B) and one influencing fasting insulin and HOMA-IR (near IGF1). We also demonstrated association of ADCY5, PROX1, GCK, GCKR and DGKB-TMEM195 with type 2 diabetes. Within these loci, likely biological candidate genes influence signal transduction, cell proliferation, development, glucose-sensing and circadian regulation. Our results demonstrate that genetic studies of glycemic traits can identify type 2 diabetes risk loci, as well as loci containing gene variants that are associated with a modest elevation in glucose levels but are not associated with overt diabetes

A novel piggybac transposon inducible expression system identifies a role for akt signalling in primordial germ cell migration

In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC) lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development