Understanding the limits to generalizability of experimental evolutionary models.

Post print version of article deposited in accordance with SHERPA RoMEO guidelines. The final definitive version is available online at: http://www.nature.com/nature/journal/v455/n7210/abs/nature07152.htmlGiven the difficulty of testing evolutionary and ecological theory in situ, in vitro model systems are attractive alternatives; however, can we appraise whether an experimental result is particular to the in vitro model, and, if so, characterize the systems likely to behave differently and understand why? Here we examine these issues using the relationship between phenotypic diversity and resource input in the T7-Escherichia coli co-evolving system as a case history. We establish a mathematical model of this interaction, framed as one instance of a super-class of host-parasite co-evolutionary models, and show that it captures experimental results. By tuning this model, we then ask how diversity as a function of resource input could behave for alternative co-evolving partners (for example, E. coli with lambda bacteriophages). In contrast to populations lacking bacteriophages, variation in diversity with differences in resources is always found for co-evolving populations, supporting the geographic mosaic theory of co-evolution. The form of this variation is not, however, universal. Details of infectivity are pivotal: in T7-E. coli with a modified gene-for-gene interaction, diversity is low at high resource input, whereas, for matching-allele interactions, maximal diversity is found at high resource input. A combination of in vitro systems and appropriately configured mathematical models is an effective means to isolate results particular to the in vitro system, to characterize systems likely to behave differently and to understand the biology underpinning those alternatives

Sleep health among people with severe mental ill health during the COVID-19 pandemic: Results from a linked UK population cohort study

Objectives: Sleep problems are a transdiagnostic feature of nearly all psychiatric conditions, and a strong risk factor for initial and recurrent episodes. However, people with severe mental ill health (SMI) are often excluded from general population surveys, and as such the extent and associates of poor sleep in this population are less well understood. This study explores sleep health in an SMI sample during the COVID-19 pandemic, using multiple regression to identify risk factors, including daily routine, wellbeing and demographics. Methods: An existing cohort of people with an SMI diagnosis were sampled. Participants were invited to complete a self-report survey about their health and the impacts of COVID-19 and associated public health measures. Sleep duration, efficiency, and quality were measured using items from the Pittsburgh Sleep Quality Index (PSQI). Results: Two hundred forty-nine adults (aged 21–84 years) completed the survey. Mean sleep duration and efficiency were similar to general population estimates, at 7 h 19 min and 78%, respectively. However, 43% reported “bad” sleep quality that was associated with being younger in age as well as disturbed routine and declined wellbeing. Indeed, 37% reported a disturbed routine during the pandemic. Conclusions: High estimates of perceived poor sleep quality in the SMI population align with previous findings. Supporting people with SMI to maintain routine regularity may work to protect sleep quality and wellbeing. Future research should more closely examine sleep health in people with SMI, using accessible and scalable measures of objective and subjective sleep, examining longitudinal trends

Continuous Glucose Monitors and Automated Insulin Dosing Systems in the Hospital Consensus Guideline.

This article is the work product of the Continuous Glucose Monitor and Automated Insulin Dosing Systems in the Hospital Consensus Guideline Panel, which was organized by Diabetes Technology Society and met virtually on April 23, 2020. The guideline panel consisted of 24 international experts in the use of continuous glucose monitors (CGMs) and automated insulin dosing (AID) systems representing adult endocrinology, pediatric endocrinology, obstetrics and gynecology, advanced practice nursing, diabetes care and education, clinical chemistry, bioengineering, and product liability law. The panelists reviewed the medical literature pertaining to five topics: (1) continuation of home CGMs after hospitalization, (2) initiation of CGMs in the hospital, (3) continuation of AID systems in the hospital, (4) logistics and hands-on care of hospitalized patients using CGMs and AID systems, and (5) data management of CGMs and AID systems in the hospital. The panelists then developed three types of recommendations for each topic, including clinical practice (to use the technology optimally), research (to improve the safety and effectiveness of the technology), and hospital policies (to build an environment for facilitating use of these devices) for each of the five topics. The panelists voted on 78 proposed recommendations. Based on the panel vote, 77 recommendations were classified as either strong or mild. One recommendation failed to reach consensus. Additional research is needed on CGMs and AID systems in the hospital setting regarding device accuracy, practices for deployment, data management, and achievable outcomes. This guideline is intended to support these technologies for the management of hospitalized patients with diabetes

Evidence Based Selection of Housekeeping Genes

For accurate and reliable gene expression analysis, normalization of gene expression data against housekeeping genes (reference or internal control genes) is required. It is known that commonly used housekeeping genes (e.g. ACTB, GAPDH, HPRT1, and B2M) vary considerably under different experimental conditions and therefore their use for normalization is limited. We performed a meta-analysis of 13,629 human gene array samples in order to identify the most stable expressed genes. Here we show novel candidate housekeeping genes (e.g. RPS13, RPL27, RPS20 and OAZ1) with enhanced stability among a multitude of different cell types and varying experimental conditions. None of the commonly used housekeeping genes were present in the top 50 of the most stable expressed genes. In addition, using 2,543 diverse mouse gene array samples we were able to confirm the enhanced stability of the candidate novel housekeeping genes in another mammalian species. Therefore, the identified novel candidate housekeeping genes seem to be the most appropriate choice for normalizing gene expression data

Validation of Endogenous Control Genes for Gene Expression Studies on Human Ocular Surface Epithelium

PURPOSE: To evaluate a panel of ten known endogenous control genes (ECG) with quantitative reverse transcription PCR (qPCR), for identification of stably expressed endogenous control genes in the ocular surface (OS) epithelial regions including cornea, limbus, limbal epithelial crypt and conjunctiva to normalise the quantitative reverse transcription PCR data of genes of interest expressed in above-mentioned regions. METHOD: The lasermicrodissected (LMD) OS epithelial regions of cryosectioned corneoscleral buttons from the cadaver eyes were processed for RNA extraction and cDNA synthesis to detect genes of interest with qPCR. Gene expression of 10 known ECG--glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin (ACTB), peptidylprolyl isomerase (PPIA), TATA-box binding protein (TBP1), hypoxanthine guanine phosphoribosyl transferase (HPRT1), beta glucuronidase (GUSB), Eucaryotic 18S ribosomal RNA (18S), phosphoglycerate kinase (PGK1), beta-2-microglobulin (B2M), ribosomal protein, large, P0 (RPLP0)--was measured in the OS epithelial regions by qPCR method and the data collected was further analysed using geNorm software. RESULTS: The expression stability of ecgs in the os epithelial regions in increasing order as determined with genorm software is as follows: ACTB<18S<TBP<B2M<PGK1<HPRT1<GUSB<GAPDH<PPIA-RPLP0. In this study, geNorm analysis has shown the following ECGs pairs to be most stably expressed in individual OS epithelial regions: HPRT1-TBP in cornea, GUSB-PPIA in limbus, B2M-PPIA and RPLP0-TBP in LEC and conjunctiva respectively. However, across the entire ocular surface including all the regions mentioned above, PPIA-RPLP0 pair was shown to be most stable. CONCLUSION: This study has identified stably expressed ECGs on the OS epithelial regions for effective qPCR results in genes of interest. The results from this study are broadly applicable to quantitative reverse transcription PCR studies on human OS epithelium and provide evidence for the use of PPIA-RPLP0 ECGs pair in quantitative reverse transcription PCR across the OS epithelium

Pseudorabies Virus Infection Alters Neuronal Activity and Connectivity In Vitro

Alpha-herpesviruses, including human herpes simplex virus 1 & 2, varicella zoster virus and the swine pseudorabies virus (PRV), infect the peripheral nervous system of their hosts. Symptoms of infection often include itching, numbness, or pain indicative of altered neurological function. To determine if there is an in vitro electrophysiological correlate to these characteristic in vivo symptoms, we infected cultured rat sympathetic neurons with well-characterized strains of PRV known to produce virulent or attenuated symptoms in animals. Whole-cell patch clamp recordings were made at various times after infection. By 8 hours of infection with virulent PRV, action potential (AP) firing rates increased substantially and were accompanied by hyperpolarized resting membrane potentials and spikelet-like events. Coincident with the increase in AP firing rate, adjacent neurons exhibited coupled firing events, first with AP-spikelets and later with near identical resting membrane potentials and AP firing. Small fusion pores between adjacent cell bodies formed early after infection as demonstrated by transfer of the low molecular weight dye, Lucifer Yellow. Later, larger pores formed as demonstrated by transfer of high molecular weight Texas red-dextran conjugates between infected cells. Further evidence for viral-induced fusion pores was obtained by infecting neurons with a viral mutant defective for glycoprotein B, a component of the viral membrane fusion complex. These infected neurons were essentially identical to mock infected neurons: no increased AP firing, no spikelet-like events, and no electrical or dye transfer. Infection with PRV Bartha, an attenuated circuit-tracing strain delayed, but did not eliminate the increased neuronal activity and coupling events. We suggest that formation of fusion pores between infected neurons results in electrical coupling and elevated firing rates, and that these processes may contribute to the altered neural function seen in PRV-infected animals

Somatic p16INK4a loss accelerates melanomagenesis

Loss of p16INK4a–RB and ARF–p53 tumor suppressor pathways, as well as activation of RAS–RAF signaling, is seen in a majority of human melanomas. Although heterozygous germline mutations of p16INK4a are associated with familial melanoma, most melanomas result from somatic genetic events: often p16INK4a loss and N-RAS or B-RAF mutational activation, with a minority possessing alternative genetic alterations such as activating mutations in K-RAS and/or p53 inactivation. To generate a murine model of melanoma featuring some of these somatic genetic events, we engineered a novel conditional p16INK4a-null allele and combined this allele with a melanocyte-specific, inducible CRE recombinase strain, a conditional p53-null allele and a loxP-stop-loxP activatable oncogenic K-Ras allele. We found potent synergy between melanocyte-specific activation of K-Ras and loss of p16INK4a and/or p53 in melanomagenesis. Mice harboring melanocyte-specific activated K-Ras and loss of p16INK4a and/or p53 developed invasive, unpigmented and nonmetastatic melanomas with short latency and high penetrance. In addition, the capacity of these somatic genetic events to rapidly induce melanomas in adult mice suggests that melanocytes remain susceptible to transformation throughout adulthood