Identification, characterization, and natural selection of mutations driving airborne transmission of A/H5N1 virus.

Recently, A/H5N1 influenza viruses were shown to acquire airborne transmissibility between ferrets upon targeted mutagenesis and virus passage. The critical genetic changes in airborne A/Indonesia/5/05 were not yet identified. Here, five substitutions proved to be sufficient to determine this airborne transmission phenotype. Substitutions in PB1 and PB2 collectively caused enhanced transcription and virus replication. One substitution increased HA thermostability and lowered the pH of membrane fusion. Two substitutions independently changed HA binding preference from α2,3-linked to α2,6-linked sialic acid receptors. The loss of a glycosylation site in HA enhanced overall binding to receptors. The acquired substitutions emerged early during ferret passage as minor variants and became dominant rapidly. Identification of substitutions that are essential for airborne transmission of avian influenza viruses between ferrets and their associated phenotypes advances our fundamental understanding of virus transmission and will increase the value of future surveillance programs and public health risk assessments.

Characterization of changes in the hemagglutinin that accompanied the emergence of H3N2/1968 pandemic influenza viruses.

The hemagglutinin (HA) of A/H3N2 pandemic influenza viruses (IAVs) of 1968 differed from its inferred avian precursor by eight amino acid substitutions. To determine their phenotypic effects, we studied recombinant variants of A/Hong Kong/1/1968 virus containing either human-type or avian-type amino acids in the corresponding positions of HA. The precursor HA displayed receptor binding profile and high conformational stability typical for duck IAVs. Substitutions Q226L and G228S, in addition to their known effects on receptor specificity and replication, marginally decreased HA stability. Substitutions R62I, D63N, D81N and N193S reduced HA binding avidity. Substitutions R62I, D81N and A144G promoted viral replication in human airway epithelial cultures. Analysis of HA sequences revealed that substitutions D63N and D81N accompanied by the addition of N-glycans represent common markers of avian H3 HA adaptation to mammals. Our results advance understanding of genotypic and phenotypic changes in IAV HA required for avian-to-human adaptation and pandemic emergence.

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa.

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.

Lassa and Crimean-Congo Hemorrhagic Fever Viruses, Mali.

We report detection of Lassa virus and Crimean-Congo hemorrhagic fever virus infections in the area of Bamako, the capital of Mali. Our investigation found 2 cases of infection with each of these viruses. These results show the potential for both of these viruses to be endemic to Mali.

TRAPPC13 modulates autophagy and the response to Golgi stress.

Tether complexes play important roles in endocytic and exocytic trafficking of lipids and proteins. In yeast, the multisubunit transport protein particle (TRAPP) tether regulates endoplasmic reticulum (ER)-to-Golgi and intra-Golgi transport and is also implicated in autophagy. In addition, the TRAPP complex acts as a guanine nucleotide exchange factor (GEF) for Ypt1, which is homologous to human Rab1a and Rab1b. Here, we show that human TRAPPC13 and other TRAPP subunits are critically involved in the survival response to several Golgi-disrupting agents. Loss of TRAPPC13 partially preserves the secretory pathway and viability in response to brefeldin A, in a manner that is dependent on ARF1 and the large GEF GBF1, and concomitant with reduced caspase activation and ER stress marker induction. TRAPPC13 depletion reduces Rab1a and Rab1b activity, impairs autophagy and leads to increased infectivity to the pathogenic bacterium Shigella flexneri in response to brefeldin A. Thus, our results lend support for the existence of a mammalian TRAPPIII complex containing TRAPPC13, which is important for autophagic flux under certain stress conditions.

Blood markers of cardiac stress after generalized convulsive seizures.

OBJECTIVE: Generalized convulsive seizures (GCS) are associated with high demands on the cardiovascular system, thereby facilitating cardiac complications. To investigate occurrence, influencing factors, and extent of cardiac stress or injury, the alterations and time course of the latest generation of cardiac blood markers were investigated after documented GCS. METHODS: Adult patients with refractory epilepsy who underwent video-electroencephalography (EEG) monitoring along with simultaneous one-lead electrocardiography (ECG) recordings were included. Cardiac biomarkers (cardiac troponin I [cTNI]; high-sensitive troponin T [hsTNT]; N-terminal prohormone of brain natriuretic peptide [NT-proBNP]; copeptin; suppression of tumorigenicity-2 [SST-2]; growth differentiation factor 15, [GDF-15]; soluble urokinase plasminogen activator receptor [suPAR]; and heart-type fatty acid binding protein [HFABP]) and catecholamines were measured at inclusion and at different time points after GCS. Periictal cardiac properties were assessed by analyzing heart rate (HR), HR variability (HRV), and corrected QT intervals(QTc). RESULTS: Thirty-six GCS (6 generalized-onset tonic-clonic seizures and 30 focal to bilateral tonic-clonic seizures) were recorded in 30 patients without a history of cardiac or renal disease. Postictal catecholamine levels were elevated more than twofold. A concomitant increase in HR and QTc, as well as a decrease in HRV, was observed. Elevations of cTNI and hsTNT were found in 3 of 30 patients (10%) and 6 of 23 patients (26%), respectively, which were associated with higher dopamine levels. Copeptin was increased considerably after most GCS, whereas SST-2, HFABP, and GDF-15 displayed only subtle variations, and suPAR was unaltered in the postictal period. Cardiac symptoms did not occur in any patient. SIGNIFICANCE: The use of more sensitive biomarkers such as hsTNT suggests that signs of cardiac stress occur in about 25% of the patients with GCS without apparent clinical symptoms. SuPAR may indicate clinically relevant troponin elevations. Copeptin could help to diagnose GCS, but specificity needs to be tested.

H1N1 Swine Influenza Viruses Differ from Avian Precursors by a Higher pH Optimum of Membrane Fusion.

UNLABELLED: The H1N1 Eurasian avian-like swine (EAsw) influenza viruses originated from an avian H1N1 virus. To characterize potential changes in the membrane fusion activity of the hemagglutinin (HA) during avian-to-swine adaptation of the virus, we studied EAsw viruses isolated in the first years of their circulation in pigs and closely related contemporary H1N1 viruses of wild aquatic birds. Compared to the avian viruses, the swine viruses were less sensitive to neutralization by lysosomotropic agent NH4Cl in MDCK cells, had a higher pH optimum of hemolytic activity, and were less stable at acidic pH. Eight amino acid substitutions in the HA were found to separate the EAsw viruses from their putative avian precursor; four substitutions-T492S, N722D, R752K, and S1132F-were located in the structural regions of the HA2 subunit known to play a role in acid-induced conformational transition of the HA. We also studied low-pH-induced syncytium formation by cell-expressed HA proteins and found that the HAs of the 1918, 1957, 1968, and 2009 pandemic viruses required a lower pH for fusion induction than did the HA of a representative EAsw virus. Our data show that transmission of an avian H1N1 virus to pigs was accompanied by changes in conformational stability and fusion promotion activity of the HA. We conclude that distinctive host-determined fusion characteristics of the HA may represent a barrier for avian-to-swine and swine-to-human transmission of influenza viruses. IMPORTANCE: Continuing cases of human infections with zoonotic influenza viruses highlight the necessity to understand which viral properties contribute to interspecies transmission. Efficient binding of the HA to cellular receptors in a new host species is known to be essential for the transmission. Less is known about required adaptive changes in the membrane fusion activity of the HA. Here we show that adaptation of an avian influenza virus to pigs in Europe in 1980s was accompanied by mutations in the HA, which decreased its conformational stability and increased pH optimum of membrane fusion activity. This finding represents the first formal evidence of alteration of the HA fusion activity/stability during interspecies transmission of influenza viruses under natural settings.

Role of Substitutions in the Hemagglutinin in the Emergence of the 1968 Pandemic Influenza Virus.

Hemagglutinin (HA) of H3N2/1968 pandemic influenza viruses differs from the putative avian precursor by seven amino acid substitutions. Substitutions Q226L and G228S are known to be essential for adaptation of avian HA to mammals. We found that introduction of avian-virus-like amino acids at five other HA positions (positions 62, 81, 92, 144, and 193) of A/Hong Kong/1/1968 virus decreased viral replication in human cells and transmission in pigs. Thus, substitutions at some of these positions facilitated emergence of the pandemic virus.

Palmitate-induced ER stress increases trastuzumab sensitivity in HER2/neu-positive breast cancer cells.

BACKGROUND: HER2/neu-positive breast cancer cells have recently been shown to use a unique Warburg-like metabolism for survival and aggressive behavior. These cells exhibit increased fatty acid synthesis and storage compared to normal breast cells or other tumor cells. Disruption of this synthetic process results in apoptosis. Since the addition of physiological doses of exogenous palmitate induces cell death in HER2/neu-positive breast cancer cells, the pathway is likely operating at its limits in these cells. We have studied the response of HER2/neu-positive breast cancer cells to physiological concentrations of exogenous palmitate to identify lipotoxicity-associated consequences of this physiology. Since epidemiological data show that a diet rich in saturated fatty acids is negatively associated with the development of HER2/neu-positive cancer, this cellular physiology may be relevant to the etiology and treatment of the disease. We sought to identify signaling pathways that are regulated by physiological concentrations of exogenous palmitate specifically in HER2/neu-positive breast cancer cells and gain insights into the molecular mechanism and its relevance to disease prevention and treatment. METHODS: Transcriptional profiling was performed to assess programs that are regulated in HER2-normal MCF7 and HER2/neu-positive SKBR3 breast cancer cells in response to exogenous palmitate. Computational analyses were used to define and predict functional relationships and identify networks that are differentially regulated in the two cell lines. These predictions were tested using reporter assays, fluorescence-based high content microscopy, flow cytometry and immunoblotting. Physiological effects were confirmed in HER2/neu-positive BT474 and HCC1569 breast cancer cell lines. RESULTS: Exogenous palmitate induces functionally distinct transcriptional programs in HER2/neu-positive breast cancer cells. In the lipogenic HER2/neu-positive SKBR3 cell line, palmitate induces a G2 phase cell cycle delay and CHOP-dependent apoptosis as well as a partial activation of the ER stress response network via XBP1 and ATF6. This response appears to be a general feature of HER2/neu-positive breast cancer cells but not cells that overexpress only HER2/neu. Exogenous palmitate reduces HER2 and HER3 protein levels without changes in phosphorylation and sensitizes HER2/neu-positive breast cancer cells to treatment with the HER2-targeted therapy trastuzumab. CONCLUSIONS: Several studies have shown that HER2, FASN and fatty acid synthesis are functionally linked. Exogenous palmitate exerts its toxic effects in part through inducing ER stress, reducing HER2 expression and thereby sensitizing cells to trastuzumab. These data provide further evidence that HER2 signaling and fatty acid metabolism are highly integrated processes that may be important for disease development and progression.

Lipid biology of breast cancer.

Alterations in lipid metabolism have been reported in many types of cancer. Lipids have been implicated in the regulation of proliferation, differentiation, apoptosis, inflammation, autophagy, motility and membrane homeostasis. It is required that their biosynthesis is tightly regulated to ensure homeostasis and to prevent unnecessary energy expenditure. This review focuses on the emerging understanding of the role of lipids and lipogenic pathway regulation in breast cancer, including parallels drawn from the study of metabolic disease models, and suggestions on how these findings can potentially be exploited to promote gains in HER2/neu-positive breast cancer research. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.

Short-term effects of transcutaneous auricular vagus nerve stimulation on T-wave alternans in people with focal epilepsy - An exploratory pilot study.

High levels of T-wave alternans (TWA) are linked to an increased risk of sudden cardiac death. People with epilepsy display elevated TWA levels that are decreased by chronic vagus nerve stimulation via implanted devices after 2-4 weeks or later. Our objective was to explore short-term effects of transcutaneous auricular vagus nerve stimulation (tVNS) on TWA. Five patients (3 female) with focal epilepsy undergoing video-EEG monitoring were included. TWA levels were determined using a one-channel modified lead I ECG via an open-source TWA-algorithm on two consecutive days, 1 h before, during and after tVNS via the left auricle. Data are given as mean ± SE. Mean TWA at baseline was 3.8 ± 0.4 µV and 3.0 ± 0.6 µV during stimulation on day 2. Stimulations on the second day were associated with TWA reductions by 22 ± 13 % that exceeded stimulation effects on the first day relative to baseline (p < 0.05). Linear mixed-models revealed effects of both stimulation (p < 0.05) and stimulation number (p < 0.005). Normalized TWA showed reproducible peak reductions at both days within 35 min after the initiation of tVNS (p < 0.05). Our observations suggest that tVNS has short-term effects on TWA, supporting the notion that vagus nerve stimulation has a beneficial impact on electrical cardiac properties.

A novel, lactase-based selection and strain improvement strategy for recombinant protein expression in Kluyveromyces lactis.

BACKGROUND: The Crabtree-negative yeast species Kluyveromyces lactis has been established as an attractive microbial expression system for recombinant proteins at industrial scale. Its LAC genes allow for utilization of the inexpensive sugar lactose as a sole source of carbon and energy. Lactose efficiently induces the LAC4 promoter, which can be used to drive regulated expression of heterologous genes. So far, strain manipulation of K. lactis by homologous recombination was hampered by the high rate of non-homologous end-joining. RESULTS: Selection for growth on lactose was applied to target the insertion of heterologous genes downstream of the LAC4 promoter into the K. lactis genome and found to yield high numbers of positive transformants. Concurrent reconstitution of the ß-galactosidase gene indicated the desired integration event of the expression cassette, and ß-galactosidase activity measurements were used to monitor gene expression for strain improvement and fermentation optimization. The system was particularly improved by usage of a cell lysis resistant strain, VAK367-D4, which allowed for protein accumulation in long-term fermentation. Further optimization was achieved by increased gene dosage of KlGAL4 encoding the activator of lactose and galactose metabolic genes that led to elevated transcription rates. Pilot experiments were performed with strains expressing a single-chain antibody fragment (scFvox) and a viral envelope protein (BVDV-E2), respectively. scFvox was shown to be secreted into the culture medium in an active, epitope-binding form indicating correct processing and protein folding; the E2 protein could be expressed intracellularly. Further data on the influence of protein toxicity on batch fermentation and potential post-transcriptional bottlenecks in protein accumulation were obtained. CONCLUSIONS: A novel Kluyveromyces lactis host-vector system was developed that places heterologous genes under the control of the chromosomal LAC4 promoter and that allows monitoring of its transcription rates by ß-galactosidase measurement. The procedure is rapid and efficient, and the resulting recombinant strains contain no foreign genes other than the gene of interest. The recombinant strains can be grown non-selectively in rich medium and stably maintained even when the gene product exerts protein toxicity.

Performance of ECG-based seizure detection algorithms strongly depends on training and test conditions.

OBJECTIVE: To identify non-EEG-based signals and algorithms for detection of motor and non-motor seizures in people lying in bed during video-EEG (VEEG) monitoring and to test whether these algorithms work in freely moving people during mobile EEG recordings. METHODS: Data of three groups of adult people with epilepsy (PwE) were analyzed. Group 1 underwent VEEG with additional devices (accelerometry, ECG, electrodermal activity); group 2 underwent VEEG; and group 3 underwent mobile EEG recordings both including one-lead ECG. All seizure types were analyzed. Feature extraction and machine-learning techniques were applied to develop seizure detection algorithms. Performance was expressed as sensitivity, precision, F1 score, and false positives per 24 hours. RESULTS: The algorithms were developed in group 1 (35 PwE, 33 seizures) and achieved best results (F1 score 56%, sensitivity 67%, precision 45%, false positives 0.7/24 hours) when ECG features alone were used, with no improvement by including accelerometry and electrodermal activity. In group 2 (97 PwE, 255 seizures), this ECG-based algorithm largely achieved the same performance (F1 score 51%, sensitivity 39%, precision 73%, false positives 0.4/24 hours). In group 3 (30 PwE, 51 seizures), the same ECG-based algorithm failed to meet up with the performance in groups 1 and 2 (F1 score 27%, sensitivity 31%, precision 23%, false positives 1.2/24 hours). ECG-based algorithms were also separately trained on data of groups 2 and 3 and tested on the data of the other groups, yielding maximal F1 scores between 8% and 26%. SIGNIFICANCE: Our results suggest that algorithms based on ECG features alone can provide clinically meaningful performance for automatic detection of all seizure types. Our study also underscores that the circumstances under which such algorithms were developed, and the selection of the training and test data sets need to be considered and limit the application of such systems to unseen patient groups behaving in different conditions.

German and international studies on SARS-CoV-2 seroprevalence.

Since the beginning of the year 2020, the SARS-CoV-2 coronavirus has spread globally at a tremendous pace. Studies on the prevalence of SARS-CoV-2 antibodies in the population help estimate the number of people that have already been infected. They also allow an estimate of the number of undetected infections i.e. infections that do not appear in data on officially reported cases. The interpretation of study results needs to consider bias from selective sampling and the diagnostic test properties. To promote networking and co-operation between scientists, the Robert Koch Institute has compiled an overview of the seroepidemiological studies conducted in Germany on its website, which is regularly updated. The RKI conducts searches, for example of press releases, study registry entries or preprint server publications, and contacts the lead investigators of these studies. Of the 40 studies contacted so far, 24 have already provided information (as of 25.06.2020). We can differentiate between studies of the general population, of selected population groups such as healthcare workers, or of ongoing cohorts. This article provides an overview of such studies from Germany, but also of selected international studies. A special focus is set on studies of children and adolescents, which are now of particular interest due to the planned reopening of childcare facilities and schools.

ICD-11 Adjustment Disorder among Organ Transplant Patients and Their Relatives.

Adjustment disorder (AD) is one of the most frequent mental health conditions after stressful life experiences in the medical setting. The diagnosis has been conceptually redefined in International Classification of Diseases (ICD-11) and now includes specific symptoms of preoccupations and failure to adapt. The current study assesses the prevalence of self-reported ICD-11 AD among organ transplantation patients and their relatives, explores the association of patients' demographic-, transplant-, and health-related characteristics and ICD-11 AD symptoms, and evaluates the role of social support in the post- transplant context. A total of N = 140 patient-relative dyads were examined cross-sectionally. Hierarchical linear regression analyses were conducted to explore potential predictive factors of AD. The results revealed an AD prevalence of 10.7% among patients and 16.4% among relatives at an average of 13.5 years after the transplantation. The time that had passed since the transplantation was unrelated to AD symptom severity. Women tended to be at a higher risk in both groups. Somatic issues were predictive for AD only among patients and social support was predictive mainly among relatives. The results suggest that ICD-11 AD is a relevant diagnosis after organ transplantations for patients and relatives and its specific symptom clusters may provide important information for developing intervention strategies.

Global metabolite profiling analysis of lipotoxicity in HER2/neu-positive breast cancer cells.

Recent work has shown that HER2/neu-positive breast cancer cells rely on a unique Warburg-like metabolism for survival and aggressive behavior. These cells are dependent on fatty acid (FA) synthesis, show markedly increased levels of stored fats and disruption of the synthetic process results in apoptosis. In this study, we used global metabolite profiling and a multi-omics network analysis approach to model the metabolic changes in this physiology under palmitate-supplemented growth conditions to gain insights into the molecular mechanism and its relevance to disease prevention and treatment. Computational analyses were used to define pathway enrichment based on the dataset of significantly altered metabolites and to integrate metabolomics and transcriptomics data in a multi-omics network analysis. Network-predicted changes and functional relationships were tested with cell assays in vitro. Palmitate-supplemented growth conditions induce distinct metabolic alterations. Growth of HER2-normal MCF7 cells is unaffected under these conditions whereas HER2/neu-positive cells display unchanged neutral lipid content, AMPK activation, inhibition of fatty acid synthesis and significantly altered glutamine, glucose and serine/glycine metabolism. The predominant upregulated lipid species is the novel bioactive lipid N-palmitoylglycine, which is non-toxic to these cells. Limiting the availability of glutamine significantly ameliorates the lipotoxic effects of palmitate, reduces CHOP and XBP1(s) induction and restores the expression levels of HER2 and HER3. The study shows that HER2/neu-positive breast cancer cells change their metabolic phenotype in the presence of palmitate. Palmitate induces AMPK activation and inhibition of fatty acid synthesis that feeds back into glycolysis as well as anaplerotic glutamine metabolism.

Golgi stress-induced transcriptional changes mediated by MAPK signaling and three ETS transcription factors regulate MCL1 splicing.

The secretory pathway is a major determinant of cellular homoeostasis. While research into secretory stress signaling has so far mostly focused on the endoplasmic reticulum (ER), emerging data suggest that the Golgi itself serves as an important signaling hub capable of initiating stress responses. To systematically identify novel Golgi stress mediators, we performed a transcriptomic analysis of cells exposed to three different pharmacological compounds known to elicit Golgi fragmentation: brefeldin A, golgicide A, and monensin. Subsequent gene-set enrichment analysis revealed a significant contribution of the ETS family transcription factors ELK1, GABPA/B, and ETS1 to the control of gene expression following compound treatment. Induction of Golgi stress leads to a late activation of the ETS upstream kinases MEK1/2 and ERK1/2, resulting in enhanced ETS factor activity and the transcription of ETS family target genes related to spliceosome function and cell death induction via alternate MCL1 splicing. Further genetic analyses using loss-of-function and gain-of-function experiments suggest that these transcription factors operate in parallel.

Image-based drug screen identifies HDAC inhibitors as novel Golgi disruptors synergizing with JQ1.

The Golgi apparatus is increasingly recognized as a major hub for cellular signaling and is involved in numerous pathologies, including neurodegenerative diseases and cancer. The study of Golgi stress-induced signaling pathways relies on the selectivity of the available tool compounds of which currently only a few are known. To discover novel Golgi-fragmenting agents, transcriptomic profiles of cells treated with brefeldin A, golgicide A, or monensin were generated and compared with a database of gene expression profiles from cells treated with other bioactive small molecules. In parallel, a phenotypic screen was performed for compounds that alter normal Golgi structure. Histone deacetylase (HDAC) inhibitors and DNA-damaging agents were identified as novel Golgi disruptors. Further analysis identified HDAC1/HDAC9 as well as BRD8 and DNA-PK as important regulators of Golgi breakdown mediated by HDAC inhibition. We provide evidence that combinatorial HDACi/(+)-JQ1 treatment spurs synergistic Golgi dispersal in several cancer cell lines, pinpointing a possible link between drug-induced toxicity and Golgi morphology alterations.

Rapid outbreak sequencing of Ebola virus in Sierra Leone identifies transmission chains linked to sporadic cases.

To end the largest known outbreak of Ebola virus disease (EVD) in West Africa and to prevent new transmissions, rapid epidemiological tracing of cases and contacts was required. The ability to quickly identify unknown sources and chains of transmission is key to ending the EVD epidemic and of even greater importance in the context of recent reports of Ebola virus (EBOV) persistence in survivors. Phylogenetic analysis of complete EBOV genomes can provide important information on the source of any new infection. A local deep sequencing facility was established at the Mateneh Ebola Treatment Centre in central Sierra Leone. The facility included all wetlab and computational resources to rapidly process EBOV diagnostic samples into full genome sequences. We produced 554 EBOV genomes from EVD cases across Sierra Leone. These genomes provided a detailed description of EBOV evolution and facilitated phylogenetic tracking of new EVD cases. Importantly, we show that linked genomic and epidemiological data can not only support contact tracing but also identify unconventional transmission chains involving body fluids, including semen. Rapid EBOV genome sequencing, when linked to epidemiological information and a comprehensive database of virus sequences across the outbreak, provided a powerful tool for public health epidemic control efforts.

Metabolic Assays for Detection of Neutral Fat Stores.

Lipid droplets (LDs) are ubiquitous intracellular structures whose formation, growth, and maintenance are highly regulated (Wang et al., 2013; Ranall et al., 2011; Goodman, 2009). Lipid metabolism and droplet dynamics are of considerable interest to agriculture, biofuel production, viral pathology, nutrition, and cancer biology (Walther and Farese, 2009; Liu et al., 2010). Accumulation of fatty acids and neutral lipids in nonadipose tissues is cytotoxic (Kourtidis et al., 2009). BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) is the standard dye to study LDs within adipocytes. BODIPY 493/503 contains a nonpolar structure that, upon binding to neutral lipid, emits a green fluorescence signal with a narrow wavelength range, making it an ideal fluorophore for multi-labeling experiments. The hydrophobic nature of the dye molecules promotes rapid entry into the nonpolar environment of LDs (Listenberge and Brown, 2007). Gocze and Freeman showed that the lipid fluorescent variability is significantly lower when using BODIPY493/503 compared to Nile Red, suggesting that it may be more specific for the LD (Gocze and Freeman, 1994). Here, we describe a BODIPY 493/503 assay for the detection of neural fat stores in cultured cells (Figure 1) (Wang et al., 2013).