Virus genomes reveal factors that spread and sustained the Ebola epidemic.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics.

Loss of Baiap2l2 destabilizes the transducing stereocilia of cochlear hair cells and leads to deafness.

KEY POINTS: Mechanoelectrical transduction at auditory hair cells requires highly specialized stereociliary bundles that project from their apical surface, forming a characteristic graded 'staircase' structure. The morphogenesis and maintenance of these stereociliary bundles is a tightly regulated process requiring the involvement of several actin-binding proteins, many of which are still unidentified. We identify a new stereociliary protein, the I-BAR protein BAIAP2L2, which localizes to the tips of the shorter transducing stereocilia in both inner and outer hair cells (IHCs and OHCs). We find that Baiap2l2 deficient mice lose their second and third rows of stereocilia, their mechanoelectrical transducer current, and develop progressive hearing loss, becoming deaf by 8 months of age. We demonstrate that BAIAP2L2 localization to stereocilia tips is dependent on the motor protein MYO15A and its cargo EPS8. We propose that BAIAP2L2 is a new key protein required for the maintenance of the transducing stereocilia in mature cochlear hair cells. ABSTRACT: The transduction of sound waves into electrical signals depends upon mechanosensitive stereociliary bundles that project from the apical surface of hair cells within the cochlea. The height and width of these actin-based stereocilia is tightly regulated throughout life to establish and maintain their characteristic staircase-like structure, which is essential for normal mechanoelectrical transduction. Here, we show that BAIAP2L2, a member of the I-BAR protein family, is a newly identified hair bundle protein that is localized to the tips of the shorter rows of transducing stereocilia in mouse cochlear hair cells. BAIAP2L2 was detected by immunohistochemistry from postnatal day 2.5 (P2.5) throughout adulthood. In Baiap2l2 deficient mice, outer hair cells (OHCs), but not inner hair cells (IHCs), began to lose their third row of stereocilia and showed a reduction in the size of the mechanoelectrical transducer current from just after P9. Over the following post-hearing weeks, the ordered staircase structure of the bundle progressively deteriorates, such that, by 8 months of age, both OHCs and IHCs of Baiap2l2 deficient mice have lost most of the second and third rows of stereocilia and become deaf. We also found that BAIAP2L2 interacts with other key stereociliary proteins involved in normal hair bundle morphogenesis, such as CDC42, RAC1, EPS8 and ESPNL. Furthermore, we show that BAIAP2L2 localization to the stereocilia tips depends on the motor protein MYO15A and its cargo EPS8. We propose that BAIAP2L2 is key to maintenance of the normal actin structure of the transducing stereocilia in mature mouse cochlear hair cells.

Selective expression of a dominant-negative type Iα PKA regulatory subunit in striatal medium spiny neurons impairs gene expression and leads to reduced feeding and locomotor activity.

Striatal medium spiny neurons (MSNs) mediate many of the physiological effects of dopamine, including the regulation of feeding and motor behaviors. Dopaminergic inputs from the midbrain modulate MSN excitability through pathways that involve cAMP and protein kinase A (PKA), but the physiological role of specific PKA isoforms in MSN neurons remains poorly understood. One of the major PKA regulatory (R) subunit isoforms expressed in MSNs is RIIß, which localizes the PKA holoenzyme primarily to dendrites by interaction with AKAP5 and other scaffolding proteins. However, RI (RIα and RIß) subunits are also expressed in MSNs and the RI holoenzyme has a weaker affinity for most scaffolding proteins and tends to localize in the cell body. We generated mice with selective expression of a dominant-negative RI subunit (RIαB) in striatal MSNs and show that this dominant-negative RIαB localizes to the cytoplasm and specifically inhibits type I PKA activity in the striatum. These mice are normal at birth; however, soon after weaning they exhibit growth retardation and the adult mice are hypophagic, lean, and resistant to high-fat diet-induced hyperphagia and obesity. The RIαB-expressing mice also exhibit decreased locomotor activity and decreased dopamine-regulated CREB phosphorylation and c-fos gene expression in the striatum. Our results demonstrate a critical role for cytoplasmic RI-PKA holoenzyme in gene regulation and the overall physiological function of MSNs.

Expression of a dominant negative PKA mutation in the kidney elicits a diabetes insipidus phenotype.

PKA plays a critical role in water excretion through regulation of the production and action of the antidiuretic hormone arginine vasopressin (AVP). The AVP prohormone is produced in the hypothalamus, where its transcription is regulated by cAMP. Once released into the circulation, AVP stimulates antidiuresis through activation of vasopressin 2 receptors in renal principal cells. Vasopressin 2 receptor activation increases cAMP and activates PKA, which, in turn, phosphorylates aquaporin (AQP)2, triggering apical membrane accumulation, increased collecting duct permeability, and water reabsorption. We used single-minded homolog 1 (Sim1)-Cre recombinase-mediated expression of a dominant negative PKA regulatory subunit (RIαB) to disrupt kinase activity in vivo and assess the role of PKA in fluid homeostasis. RIαB expression gave rise to marked polydipsia and polyuria; however, neither hypothalamic Avp mRNA expression nor urinary AVP levels were attenuated, indicating a primary physiological effect on the kidney. RIαB mice displayed a marked deficit in urinary concentrating ability and greatly reduced levels of AQP2 and phospho-AQP2. Dehydration induced Aqp2 mRNA in the kidney of both control and RIαB-expressing mice, but AQP2 protein levels were still reduced in RIαB-expressing mutants, and mice were unable to fully concentrate their urine and conserve water. We conclude that partial PKA inhibition in the kidney leads to posttranslational effects that reduce AQP2 protein levels and interfere with apical membrane localization. These findings demonstrate a distinct physiological role for PKA signaling in both short- and long-term regulation of AQP2 and characterize a novel mouse model of diabetes insipidus.

Cardiomyocytes from AKAP7 knockout mice respond normally to adrenergic stimulation.

Protein kinase A (PKA) is activated during sympathetic stimulation of the heart and phosphorylates key proteins involved in cardiac Ca(2+) handling, including the L-type Ca(2+) channel (Ca(V)1.2) and phospholamban (PLN). This results in acceleration and amplification of the beat-to-beat changes in cytosolic Ca(2+) in cardiomyocytes and, in turn, an increased rate and force of contraction. PKA is held in proximity to its substrates by protein scaffolds called A kinase anchoring proteins (AKAPs). It has been suggested that the short and long isoforms of AKAP7 (also called AKAP15/18) localize PKA in complexes with Ca(V)1.2 and PLN, respectively. We generated an AKAP7 KO mouse in which all isoforms were deleted and tested whether Ca(2+) current, intracellular Ca(2+) concentration, or Ca(2+) reuptake were impaired in isolated adult ventricular cardiomyocytes following stimulation with the ß-adrenergic agonist isoproterenol. KO cardiomyocytes responded normally to adrenergic stimulation, as measured by whole-cell patch clamp or a fluorescent intracellular Ca(2+) indicator. Phosphorylation of Ca(V)1.2 and PLN were also unaffected by genetic deletion of AKAP7. Immunoblot and RT-PCR revealed that only the long isoforms of AKAP7 were detectable in ventricular cardiomyocytes. The results indicate that AKAP7 is not required for regulation of Ca(2+) handling in mouse cardiomyocytes.

Thermoregulatory and metabolic defects in Huntington's disease transgenic mice implicate PGC-1alpha in Huntington's disease neurodegeneration.

Huntington's disease (HD) is a fatal, dominantly inherited disorder caused by polyglutamine repeat expansion in the huntingtin (htt) gene. Here, we observe that HD mice develop hypothermia associated with impaired activation of brown adipose tissue (BAT). Although sympathetic stimulation of PPARgamma coactivator 1alpha (PGC-1alpha) was intact in BAT of HD mice, uncoupling protein 1 (UCP-1) induction was blunted. In cultured cells, expression of mutant htt suppressed UCP-1 promoter activity; this was reversed by PGC-1alpha expression. HD mice showed reduced food intake and increased energy expenditure, with dysfunctional BAT mitochondria. PGC-1alpha is a known regulator of mitochondrial function; here, we document reduced expression of PGC-1alpha target genes in HD patient and mouse striatum. Mitochondria of HD mouse brain show reduced oxygen consumption rates. Finally, HD striatal neurons expressing exogenous PGC-1alpha were resistant to 3-nitropropionic acid treatment. Altered PGC-1alpha function may thus link transcription dysregulation and mitochondrial dysfunction in HD.

Mitigating the Impact of Antibacterial Drug Resistance through Host-Directed Therapies: Current Progress, Outlook, and Challenges.

Increasing incidences of multidrug resistance in pathogenic bacteria threaten our ability to treat and manage bacterial infection. The development and FDA approval of novel antibiotics have slowed over the past decade; therefore, the adoption and improvement of alternative therapeutic strategies are critical for addressing the threat posed by multidrug-resistant bacteria. Host-directed therapies utilize small-molecule drugs and proteins to alter the host response to pathogen infection. Here, we highlight strategies for modulating the host inflammatory response to enhance bacterial clearance, small-molecule potentiation of innate immunity, and targeting of host factors that are exploited by pathogen virulence factors. Application of state-of-the-art "omic" technologies, including proteomics, transcriptomics, and image-omics (image-based high-throughput phenotypic screening), combined with powerful bioinformatics tools will enable the modeling of key signaling pathways in the host-pathogen interplay and aid in the identification of host proteins for therapeutic targeting and the discovery of host-directed small molecules that will regulate bacterial infection. We conclude with an outlook on research needed to overcome the challenges associated with transitioning host-directed therapies into a clinical setting.

Contribution of calcium influx in mediating glucose-stimulated oxygen consumption in pancreatic islets.

In brain, muscle, and pancreatic islets, depolarization induces an increase in respiration, which is dependent on calcium influx. The goal of this study was to assess the quantitative significance of this effect in islets relative to glucose-stimulated ATP turnover, to examine the molecular mechanism mediating the changes, and to investigate the functional implications with respect to insulin secretion. Glucose (3-20 mmol/l) increased steady-state levels of cytochrome c reduction (32-66%) in isolated rat islets, reflecting an increased production of NADH, and oxygen consumption rate (OCR) by 0.32 nmol/min/100 islets. Glucose-stimulated OCR was inhibited 30% by inhibitors of calcium influx (diazoxide or nimodipine), whereas a protein synthesis inhibitor (emetine) decreased it by only 24%. None of the inhibitors affected cytochrome c reduction, suggesting that calcium's effect on steady-state OCR is mediated by changes in ATP usage rather than the rate of NADH generation. 3-isobutyl-1-methylxanthine increased insulin secretion but had little effect on OCR, indicating that the processes of movement and exocytosis of secretory granules do not significantly contribute to ATP turnover. At 20 mmol/l glucose, a blocker of sarcoendoplasmic reticulum calcium ATPase (SERCA) had little effect on OCR despite a large increase in cytosolic calcium, further supporting the notion that influx of calcium, not bulk cytosolic calcium, is associated with the increase in ATP turnover. The glucose dose response of calcium influx-dependent OCR showed a remarkable correlation with insulin secretion, suggesting that the process mediating the effect of calcium on ATP turnover has a role in the amplification pathway of insulin secretion.

Loss of AKAP150 promotes pathological remodelling and heart failure propensity by disrupting calcium cycling and contractile reserve.

AIMS: Impaired Ca2 + cycling and myocyte contractility are a hallmark of heart failure triggered by pathological stress such as hemodynamic overload. The A-Kinase anchoring protein AKAP150 has been shown to coordinate key aspects of adrenergic regulation of Ca2+ cycling and excitation-contraction in cardiomyocytes. However, the role of the AKAP150 signalling complexes in the pathogenesis of heart failure has not been investigated. METHODS AND RESULTS: Here we examined how AKAP150 signalling complexes impact Ca2+ cycling, myocyte contractility, and heart failure susceptibility following pathological stress. We detected a significant reduction of AKAP150 expression in the failing mouse heart induced by pressure overload. Importantly, cardiac-specific AKAP150 knockout mice were predisposed to develop dilated cardiomyopathy with severe cardiac dysfunction and fibrosis after pressure overload. Loss of AKAP150 also promoted pathological remodelling and heart failure progression following myocardial infarction. However, ablation of AKAP150 did not affect calcineurin-nuclear factor of activated T cells signalling in cardiomyocytes or pressure overload- or agonist-induced cardiac hypertrophy. Immunoprecipitation studies showed that AKAP150 was associated with SERCA2, phospholamban, and ryanodine receptor-2, providing a targeted control of sarcoplasmic reticulum Ca2+ regulatory proteins. Mechanistically, loss of AKAP150 led to impaired Ca2+ cycling and reduced myocyte contractility reserve following adrenergic stimulation or pressure overload. CONCLUSIONS: These findings define a critical role for AKAP150 in regulating Ca2+ cycling and myocardial ionotropy following pathological stress, suggesting the AKAP150 signalling pathway may serve as a novel therapeutic target for heart failure.

Reduced evolutionary rate in reemerged Ebola virus transmission chains.

On 29 June 2015, Liberia's respite from Ebola virus disease (EVD) was interrupted for the second time by a renewed outbreak ("flare-up") of seven confirmed cases. We demonstrate that, similar to the March 2015 flare-up associated with sexual transmission, this new flare-up was a reemergence of a Liberian transmission chain originating from a persistently infected source rather than a reintroduction from a reservoir or a neighboring country with active transmission. Although distinct, Ebola virus (EBOV) genomes from both flare-ups exhibit significantly low genetic divergence, indicating a reduced rate of EBOV evolution during persistent infection. Using this rate of change as a signature, we identified two additional EVD clusters that possibly arose from persistently infected sources. These findings highlight the risk of EVD flare-ups even after an outbreak is declared over.

Glucose stimulation of cytochrome C reduction and oxygen consumption as assessment of human islet quality.

BACKGROUND: An in vitro method to assess human islets could prevent transplantation of nonviable islets and facilitate the optimization of islet preparation. We hypothesize that glucose-stimulated cytochrome c reduction and oxygen consumption by human islets can be used as predictors of transplant success. METHODS: Isolated human islets were obtained from research-grade pancreata. Using a previously developed islet flow culture system, the response of cytochrome c reduction and oxygen consumption to glucose was compared to the ability of islets transplanted into nondiabetic NOD-SCID mice to secrete C-peptide in response to a glucose tolerance test conducted 7 days following transplant (n=10). RESULTS: In vitro responses by human islets were qualitatively similar to those seen in rat islets: glucose increased both oxygen consumption and cytochrome c reduction. However, the responses were smaller in magnitude and quite variable. Scatter plots of C-peptide and quantiles for ln(C-peptide) indicated that 12 ng/ml could be used as threshold of transplant success with which to evaluate the diagnostic potential of cytochrome c and oxygen consumption. Data was analyzed by generating receiver operating curves and the area under the curve was 0.889 (95% CI: 0.645-1.000) and 0.738 (95% CI: 0.413-1.000) for cytochrome c reduction and oxygen consumption respectively (1 indicates absolute predictive capability and 0.5 indicates no predictive capability). CONCLUSIONS: The detection of glucose-stimulated cytochrome c reduction and oxygen consumption may have utility as criteria for the assessment of human islet quality.

Evolution and Spread of Ebola Virus in Liberia, 2014-2015.

The 2013-present Western African Ebola virus disease (EVD) outbreak is the largest ever recorded with >28,000 reported cases. Ebola virus (EBOV) genome sequencing has played an important role throughout this outbreak; however, relatively few sequences have been determined from patients in Liberia, the second worst-affected country. Here, we report 140 EBOV genome sequences from the second wave of the Liberian outbreak and analyze them in combination with 782 previously published sequences from throughout the Western African outbreak. While multiple early introductions of EBOV to Liberia are evident, the majority of Liberian EVD cases are consistent with a single introduction, followed by spread and diversification within the country. Movement of the virus within Liberia was widespread, and reintroductions from Liberia served as an important source for the continuation of the already ongoing EVD outbreak in Guinea. Overall, little evidence was found for incremental adaptation of EBOV to the human host.

Islet oxygen consumption and insulin secretion tightly coupled to calcium derived from L-type calcium channels but not from the endoplasmic reticulum.

The aim of the study was to test whether the source of intracellular calcium (Ca2+) is a determinant of beta cell function. We hypothesized that elevations in cytosolic Ca2+ caused by the release of Ca2+ from the endoplasmic reticulum (ER) have little physiologic impact on oxygen consumption and insulin secretion. Ca2+ release from the ER was induced in isolated rat islets by acetylcholine and response of oxygen consumption rate (OCR), NAD(P)H, cytosolic Ca2+, and insulin secretory rate (ISR) were measured. Glucose increased all four parameters, and thereafter acetylcholine further increased cytosolic Ca2+, OCR, and ISR. To assess the contribution of Ca2+ release from the ER in mediating the effects of acetylcholine, ER Ca2+ stores were first emptied by inhibiting the sarcoendoplasmic reticulum Ca2+-ATPase, which subsequently reduced the effect of acetylcholine on cytosolic Ca2+ but not its effects on OCR or ISR. As predicted, OCR and ISR were acutely sensitive to changes in L-type Ca2+ channel activity; nimodipine completely inhibited glucose-stimulated ISR and suppressed OCR by 36%, despite only inhibiting cytosolic Ca2+ by 46%. Moreover, in the presence of nimodipine and high glucose, acetylcholine still elevated cytosolic Ca2+ levels above those observed in the presence of high glucose alone but did not significantly stimulate ISR. In conclusion, Ca2+ flux through L-type Ca2+ channels was tightly coupled to changes in OCR and ISR. In contrast, the results obtained support the notion that Ca2+ release from the ER has little or no access to the intracellular machinery that regulates OCR and ISR.

Treatment of diabetic rats with encapsulated islets.

Immunoprotection of islets using bioisolator systems permits introduction of allogeneic cells to diabetic patients without the need for immunosuppression. Using TheraCyte immunoisolation devices, we investigated two rat models of type 1 diabetes mellitus (T1DM), BB rats and rats made diabetic by streptozotocin (STZ) treatment. We chose to implant islets after the onset of diabetes to mimic the probable treatment of children with T1DM as they are usually diagnosed after disease onset. We encapsulated 1000 rat islets and implanted them subcutaneously (SQ) into diabetic biobreeding (BB) rats and STZ-induced diabetic rats, defined as two or more consecutive days of blood glucose>350 mg/dl. Rats were monitored for weight and blood glucose. Untreated BB rats rapidly lost weight and were euthanized at >20% weight loss that occurred between 4 and 10 days from implantation. For period of 30-40 days following islet implantation weights of treated rats remained steady or increased. Rapid weight loss occurred after surgical removal of devices that contained insulin positive islets. STZ-treated rats that received encapsulated islets showed steady weight gain for up to 130 days, whereas untreated control rats showed steady weight loss that achieved >20% at around 55 days. Although islet implants did not normalize blood glucose, treated rats were apparently healthy and groomed normally. Autologous or allogeneic islets were equally effective in providing treatment. TheraCyte devices can sustain islets, protect allogeneic cells from immune attack and provide treatment for diabetic-mediated weight loss in both BB rats and STZ-induced diabetic rats.