Rise of plasma ghrelin with weight loss is not sustained during weight maintenance

Objective: Ghrelin is postulated to be an orexigenic signal that promotes weight regain after weight loss (WL). However, it is not known whether this putative effect of ghrelin is sustained after weight stabilization. The objective of this study was to investigate the relationship of plasma ghrelin concentrations to active WL and weight maintenance in obese subjects. Research Methods and Procedures: This study was a randomized clinical trial, with a 12‐month follow‐up period. Obese Mexican‐American women matched for age and BMI were randomized to a 12‐month WL program (n = 25) or no intervention (controls, n = 23). Interventions included diet, exercise, and orlistat. Body weight and fasting ghrelin, leptin, insulin, and glucose concentrations were measured at baseline and 6 and 12 months. Results: The WL group lost 8.5% of body weight after 6 months and maintained the new weight for the next 6 months. Ghrelin concentrations increased significantly at 6 months but returned to baseline at 12 months. Baseline ghrelin concentrations were directly related to the degree of WL achieved after 12 months. Controls experienced no change in BMI or ghrelin levels. There were no associations between plasma ghrelin and leptin or insulin concentrations. Discussion: Consistent with previous results, ghrelin rises in response to WL, perhaps as a counterregulatory mechanism. However, the present results indicate that ghrelin concentrations return to baseline with sustained weight maintenance, suggesting that its effects are unlikely to regulate long‐term energy balance. Baseline ghrelin concentrations are related to the degree of WL that can be achieved by active weight reduction.     

Should symptomatic menopausal women be offered hormone therapy?

Many physicians remain uncertain about prescribing hormone therapy for symptomatic women at the onset of menopause. The American Society for Reproductive Medicine (ASRM) convened a multidisciplinary group of healthcare providers to discuss the efficacy and risks of hormone therapy for symptomatic women, and to determine whether it would be appropriate to treat women at the onset of menopause who were complaining of menopausal symptoms. MAJOR FINDINGS: Numerous controlled clinical trials consistently demonstrate that hormone therapy, administered via oral, transdermal, or vaginal routes, is the most effective treatment for vasomotor symptoms. Topical vaginal formulations of hormone therapy should be preferred when prescribing solely for the treatment of symptoms of vulvar and vaginal atrophy. Data from the Women's Health Initiative indicate that the overall attributable risk of invasive breast cancer in women receiving estrogen plus progestin was 8 more cases per 10,000 women-years. No increased risk for invasive breast cancer was detected for women who never used hormone therapy in the past or for those receiving estrogen only. Hormone therapy is not effective for the treatment of cardiovascular disease and that the risk of cardiovascular disease with hormone therapy is principally in older women who are considerably postmenopause. CONCLUSIONS: Healthy symptomatic women should be offered the option of hormone therapy for menopausal symptoms. Symptom relief with hormone therapy for many younger women (at the onset of menopause) with menopausal symptoms outweighs the risks and may provide an overall improvement in quality of life. Hormone therapy should be individualized for symptomatic women. This involves tailoring the regimen and dose to individual needs.     

Exploiting collider bias to apply two-sample summary data Mendelian randomization methods to one-sample individual level data

Over the last decade the availability of SNP-trait associations from genome-wide association studies has led to an array of methods for performing Mendelian randomization studies using only summary statistics. A common feature of these methods, besides their intuitive simplicity, is the ability to combine data from several sources, incorporate multiple variants and account for biases due to weak instruments and pleiotropy. With the advent of large and accessible fully-genotyped cohorts such as UK Biobank, there is now increasing interest in understanding how best to apply these well developed summary data methods to individual level data, and to explore the use of more sophisticated causal methods allowing for non-linearity and effect modification.In this paper we describe a general procedure for optimally applying any two sample summary data method using one sample data. Our procedure first performs a meta-analysis of summary data estimates that are intentionally contaminated by collider bias between the genetic instruments and unmeasured confounders, due to conditioning on the observed exposure. These estimates are then used to correct the standard observational association between an exposure and outcome. Simulations are conducted to demonstrate the method’s performance against naive applications of two sample summary data MR. We apply the approach to the UK Biobank cohort to investigate the causal role of sleep disturbance on HbA1c levels, an important determinant of diabetes.Our approach can be viewed as a generalization of Dudbridge et al. (Nat. Comm. 10: 1561), who developed a technique to adjust for index event bias when uncovering genetic predictors of disease progression based on case-only data. Our work serves to clarify that in any one sample MR analysis, it can be advantageous to estimate causal relationships by artificially inducing and then correcting for collider bias.     

Characterisation of Curli Production, Cell Surface Hydrophobicity, Autoaggregation and Attachment Behaviour of Escherichia coli O157

Escherichia coli O157 are an important group of foodborne pathogens with the ability to attach to materials commonly used in food processing environments such as slightly hydrophilic stainless steel. The aim of this study was to characterise six E. coli isolates, including five E. coli O157, for curli production, autoaggregation, hydrophobicity and attachment to highly hydrophilic glass and hydrophobic Teflon^®. Curli production and autoaggregation were determined using absorbance assays; hydrophobicity by bacterial adherence to hydrocarbons, hydrophobic interaction chromatography and contact angle measurements; and attachment using epifluorescence microscopy. Curli production varied between strains and for some strains correlated with autoaggregation. Curli production correlated with decreased hydrophobicity for two strains. Four of the six isolates increased attachment to glass, but decreased attachment to Teflon^® with increased curli production. In contrast, one of the six isolates decreased attachment to glass, but increased attachment to Teflon^® with increasing curli production. Curli production by the remaining isolate did not correlate with hydrophobicity or attachment. Attachment of some E. coli, including E. coli O157, to abiotic surfaces may be influenced by curli production, autoaggregation and hydrophobicity. However, for other strains, a variety of factors may be of greater influence on these properties and ability to attach to abiotic surfaces. This study highlights the complexity of bacterial surface properties and their relationship with bacterial attachment.     

The case of the disappearing bone: Dogs’ understanding of the physical properties of objects

To assess dogs’ memory for an occluded object, a gaze duration procedure was used similar to one often used with nonverbal infants. A bone shaped dog biscuit was placed behind a solid screen that then rotated in the depth plane through an arc front to back. Dogs were shown either of the two test events. In one event (the possible event), the screen rotated until it reached the point at which it would have reached the bone and then stopped (about 120°); in the other event (the impossible event), the screen rotated through a full 180° arc, as though it had passed through the bone. The dogs looked significantly longer at the impossible event. To control for the differential time it took for the screen to move, for a control group, a bone was placed behind the screen and the screen was rotated either 60° or 120° (both possible events). No difference in looking time was found. To control for the movement of the screen through 120° or 180° when both were possible, for a second control group, the bone was placed to the side of the screen rather than behind the screen and the screen was moved 120° or 180°. Again, no significant difference in looking time was found. Results suggest that much like young children, dogs understand the physical properties of an occluded object. That is they appear to understand that an object (such as a screen) should not be able to pass through another object (such as dog bone).     

Induced biosynthesis of resveratrol and the prenylated stilbenoids arachidin-1 and arachidin-3 in hairy root cultures of peanut: Effects of culture medium and growth stage

Previously, we have shown that hairy root cultures of peanut provide a controlled, sustainable and scalable production system that can be induced to produce stilbenoids. However to leverage peanut hairy roots to study the biosynthesis of this polyphenolic biosynthetic pathway, growing conditions and elicitation kinetics of these tissue cultures must be defined and understood. To this end, a new peanut cv. Hull hairy root (line 3) that produces resveratrol and its prenylated analogues arachidin-1 and arachidin-3 upon sodium acetate-mediated elicitation was established. Two culture media were compared for impact on root growth and stilbenoid biosynthesis/secretion. The levels of ammonium, nitrate, phosphate and residual sugars were monitored along growth and elicitation period. A modified MS (MSV) medium resulted in higher root biomass when compared to B5 medium. The stilbenoid profile after elicitation varied depending on the age of the culture (6, 9, 12, and 15-day old). After elicitation at day 9 (exponential growth in MSV medium), over 90% of the total resveratrol, arachidin-1 and arachidin-3 accumulated in the medium. Our studies demonstrate the benefits of the hairy root culture system to study the biosynthesis of stilbenoids including valuable prenylated polyphenolic compounds.     

Stochastic simulations of a multi-group compartmental model for Johne's disease on US dairy herds with test-based culling intervention

Infection elimination may be an important goal of control programs. Only in stochastic infection models can true infection elimination be observed as a fadeout. The phenomena of fadeout and variable prevalence are important in understanding the transmission dynamics of infectious diseases and these phenomena are essential to evaluate the effectiveness of control measures. To investigate the stochastic dynamics of Mycobacterium avium subsp. paratuberculosis (MAP) infection on US dairy herds with test-based culling intervention, we developed a multi-group stochastic compartmental model (a continuous time Markov chain model) with both horizontal and vertical transmission. The stochastic model predicted fadeout and within-herd prevalence to have a large variance. Although test-based culling intervention generally decreased prevalence over time, it took longer than desired by producers to eliminate the endemic MAP infection from a herd. Uncertainty analysis showed that, using annual culture test and culling of only high shedders or culling of both low and high shedders with a 12-month delay in culling of low shedders, MAP infection persisted in many herds beyond 20 years. While using semi-annual culture test and culling of low and high shedders with a 6-month delay in culling of low shedders, MAP infection in many herds would be extinct within 20 years. Sensitivity analysis of the cumulative density function of fadeout suggested that combining test-based culling intervention and reduction of transmission rates through improved management between susceptible calves and shedding animals may be more effective than either alone in eliminating endemic MAP infection. We also discussed the effects of other factors such as herd size, heifer replacement, and adult cow infection on the probability of fadeout.     

Host Insect Inhibitor-of-Apoptosis SfIAP Functionally Replaces Baculovirus IAP but Is Differentially Regulated by Its N-Terminal Leader

The inhibitor-of-apoptosis (IAP) proteins encoded by baculoviruses bear a striking resemblance to the cellular IAP homologs of their invertebrate hosts. By virtue of the acquired selective advantage of blocking virus-induced apoptosis, baculoviruses may have captured cellular IAP genes that subsequently evolved for virus-specific objectives. To compare viral and host IAPs, we defined antiapoptotic properties of SfIAP, the principal cellular IAP of the lepidopteran host Spodoptera frugiperda. We report here that SfIAP prevented virus-induced apoptosis as well as viral Op-IAP3 (which is encoded by the Orgyia pseudotsugata nucleopolyhedrovirus) when overexpressed from the baculovirus genome. Like Op-IAP3, SfIAP blocked apoptosis at a step prior to caspase activation. Both of the baculovirus IAP repeats (BIRs) were required for SfIAP function. Moreover, deletion of the C-terminal RING motif generated a loss-of-function SfIAP that interacted and dominantly interfered with wild-type SfIAP. Like Op-IAP3, wild-type SfIAP formed intracellular homodimers, suggesting that oligomerization is a functional requirement for both cellular and viral IAPs. SfIAP possesses a ∼100-residue N-terminal leader domain, which is absent among all viral IAPs. Remarkably, deletion of the leader yielded a fully functional SfIAP with dramatically increased protein stability. Thus, the SfIAP leader contains an instability motif that may confer regulatory options for cellular IAPs that baculovirus IAPs have evolved to bypass for maximal stability and antiapoptotic potency. Our findings that SfIAP and viral IAPs have common motifs, share multiple biochemical properties including oligomerization, and act at the same step to block apoptosis support the hypothesis that baculoviral IAPs were derived by acquisition of host insect IAPs.Apoptosis is a prevalent host cell response to virus infection. Representing an important antivirus defense, apoptotic cell death can limit multiplication and virus dissemination in the host. Thus, the mechanisms by which a host organism detects a viral intruder and initiates the apoptotic response are critical to the outcome of the infection for both the host and virus. The cellular inhibitor-of-apoptosis (IAP) proteins are important candidates for sensing virus infection and determining cell fate by virtue of their central position in the apoptosis pathway (reviewed in references 35, 36, and 44). Affirming their importance in regulation of apoptosis, IAPs are encoded by multiple DNA viruses, including baculoviruses, entomopoxviruses, iridoviruses, and African swine fever virus (reviewed in 3). Nonetheless, the molecular mechanisms by which viral IAPs regulate virus-induced apoptosis and how they biochemically differ from cellular IAPs are poorly understood.The IAPs were first discovered in baculoviruses because of their capacity to prevent virus-induced apoptosis and thereby facilitate virus multiplication (4, 8). The baculovirus IAPs bear a striking resemblance to the cellular IAPs carried by the host insects that they infect. Cellular IAPs are a highly conserved family of survival factors that regulate developmental and stress-induced apoptosis, as well as inflammation, the cell cycle, and other signaling processes (35, 38, 44). Importantly, misregulation or overexpression of IAPs is associated with neoplasia and tumor chemoresistance (24, 49). The IAPs are defined by the presence of one or more ∼80-residue baculovirus IAP repeat (BIR) domains. The BIRs consist of a conserved Zn²⁺-coordinating arrangement of Cys and His residues (CCHC) that interact with diverse proteins, including the cysteinyl aspartate-specific proteases called caspases that execute apoptosis (reviewed in 16 and 37). The antiapoptotic activity of some, but not all, IAPs is derived from their ability to bind and neutralize caspases (reviewed in 35 and 44). The BIRs also interact with proapoptotic factors that contain IAP binding motifs (IBMs). IBM-containing factors have the capacity to bind and dissociate the IAP-caspase complex, thereby liberating active caspases to execute apoptosis (16, 35, 36, 48). Many IAPs, including viral IAPs, also possess a C-terminal RING domain, which is a Zn²⁺-coordinating motif with E3-ubiquitin ligase activity, which can contribute to antiapoptotic activity (48).The best-studied baculovirus IAP is Op-IAP3, which is encoded by Orgyia pseudotsugata nucleopolyhedrovirus. This small IAP (268 residues) contains two BIRs and a C-terminal RING (Fig. ​(Fig.1A).1A). Both BIRs are required for Op-IAP3 antiapoptotic activity (19, 50, 53). Truncation of the Op-IAP3 RING creates a loss-of-function dominant inhibitor (19). Op-IAP3''s capacity to form a complex with this RING-lacking (RINGless) dominant inhibitor and with itself suggests that oligomerization is necessary for IAP function. Upon overexpression, Op-IAP3 blocks apoptosis triggered by diverse signals in cells from certain insects and mammals, suggesting that it acts through a conserved mechanism (7, 11, 15, 33, 51, 54, 56). In the baculovirus host moth Spodoptera frugiperda (Lepidoptera: Noctuidae), Op-IAP3 prevents apoptosis by blocking the activation of effector caspases (25, 32, 40). However, in contrast to host insect IAPs, Op-IAP3 fails to inhibit active caspases (45, 51, 54). Thus, the host cell target(s) and the mechanism by which they are neutralized by this viral IAP remain unclear.Open in a separate windowFIG. 1.SfIAP structure and mutagenesis. (A) Viral and cellular IAPs. Viral Op-IAP3 (268 residues) and SfIAP (377 residues) each contain two BIR motifs (black boxes) and an E3 ligase RING domain (cross-hatched box). Each representing a potential start site, four methionines (M1 to M4) exist in the N-terminal leader of SfIAP. (B) SfIAP^M4 mutations. SfIAP^M4 (281 residues) begins with the M4 methionine. SfIAP^M4ΔR (227 residues) lacks the C-terminal RING. Amino acid substitutions of Zn-coordinating residues are indicated. An epitope tag (HA) was inserted at the N terminus. (C) Marker rescue assay. The antiapoptotic activity of wild-type or mutated forms of SfIAP^M4 was assayed by virus marker rescue in which replication of p35-deficient vΔp35/lacZ was restored in proportion to the antiapoptotic activity of the mutated Sfiap gene acquired by integration of the SfIAP-encoding plasmid (2). Virus yields were determined by plaque assay using apoptosis-sensitive SF21 cells. Antiapoptotic activity is reported as the ratio of nonapoptotic, lacZ-expressing plaques produced by transfection of the indicated Sfiap to those produced by wild-type Sfiap. Values shown are the averages ± standard deviations obtained from triplicate transfections.Among the cellular IAPs, SfIAP from Spodoptera frugiperda is most closely related to viral Op-IAP3. SfIAP (Fig. ​(Fig.1A)1A) is 42% identical to Op-IAP3, with a higher degree of amino acid identity localized to its two BIRs and C-terminal RING (20). As the principal IAP in Spodoptera, SfIAP suppresses a constitutive push toward apoptosis (34); ablation of SfIAP leads to immediate apoptosis of cultured Spodoptera cells. Upon overexpression, SfIAP also rescues the multiplication of apoptosis-inducing baculoviruses and can prevent apoptosis in certain mammalian cell lines (20, 26). In contrast to viral Op-IAP3, SfIAP can bind and inhibit caspases, including Spodoptera frugiperda caspase-1 (Sf-caspase-1) and human caspase-9 (20, 45). Thus, despite their structural similarities, there exist fundamental differences in the biochemical activities of these two IAPs. Importantly, SfIAP fails to prevent baculovirus-induced apoptosis when produced at endogenous levels in permissive Spodoptera cells. Thus, it is expected that SfIAP also possesses regulatory motifs that respond to cellular signals triggered upon virus infection.SfIAP provides an unprecedented opportunity to investigate the functional and evolutionary relationships between host and viral IAPs and to test the intriguing hypothesis that viral IAPs were acquired by host gene capture (21). We have investigated the biochemical properties of SfIAP as a means to define its molecular mechanisms and to test its relatedness to viral IAPs. We report here that SfIAP shares many biochemical and functional features with viral IAPs. Like Op-IAP3, overexpressed SfIAP prevented virus-induced apoptosis at a step upstream of caspase activation by a mechanism that required BIR1, BIR2, and the RING. SfIAP formed a complex with itself and with a RINGless dominant inhibitor, suggesting that oligomerization is also required for function of cellular IAPs. Unlike viral IAPs, SfIAP possesses an N-terminal leader, which modulates intracellular SfIAP levels and may respond to apoptotic signals to regulate cell survival. Our data are consistent with a model in which baculoviruses acquired a host cell IAP and modified it for virus-specific needs, thereby increasing virus fitness by preventing virus-induced apoptosis.     

Attenuation of Vesicular Stomatitis Virus Encephalitis through MicroRNA Targeting

Vesicular stomatitis virus (VSV) has long been regarded as a promising recombinant vaccine platform and oncolytic agent but has not yet been tested in humans because it causes encephalomyelitis in rodents and primates. Recent studies have shown that specific tropisms of several viruses could be eliminated by engineering microRNA target sequences into their genomes, thereby inhibiting spread in tissues expressing cognate microRNAs. We therefore sought to determine whether microRNA targets could be engineered into VSV to ameliorate its neuropathogenicity. Using a panel of recombinant VSVs incorporating microRNA target sequences corresponding to neuron-specific or control microRNAs (in forward and reverse orientations), we tested viral replication kinetics in cell lines treated with microRNA mimics, neurotoxicity after direct intracerebral inoculation in mice, and antitumor efficacy. Compared to picornaviruses and adenoviruses, the engineered VSVs were relatively resistant to microRNA-mediated inhibition, but neurotoxicity could nevertheless be ameliorated significantly using this approach, without compromise to antitumor efficacy. Neurotoxicity was most profoundly reduced in a virus carrying four tandem copies of a neuronal mir125 target sequence inserted in the 3′-untranslated region of the viral polymerase (L) gene.Vesicular stomatitis virus (VSV) is a nonsegmented, negative-strand rhabdovirus widely used as a vaccine platform as well as an anticancer therapeutic. While VSV is predominantly a pathogen of livestock (34), it has a very broad species tropism. The cellular tropism of VSV is determined predominantly at postentry steps, since the G glycoprotein of the virus mediates entry into most tissues in nearly all animal species (10).Though viral entry can take place in nearly all cell types, in vivo models of VSV infection have revealed that the virus is highly sensitive to the innate immune response, limiting its pathogenesis (4). VSV is intensively responsive to type I interferon (IFN), as the double-stranded RNA (dsRNA)-dependent PKR (2), the downstream effector of pattern recognition receptors MyD88 (32), and other molecules mediate shutdown of viral translation and allow the adaptive immune response to clear the virus. The vulnerability of the virus to the type I IFN response, typically defective in many cancers, has been exploited to generate tumor-selective replication (49), such that the virus is now poised to enter phase I trials. However, the virus remains potently neurotoxic, causing lethal encephalitis not only in rodent models (7, 22, 53) but also in nonhuman primates (25).VSV very often infiltrates the central nervous system (CNS) through infection of the olfactory nerves (41). When administered intranasally, the virus replicates rapidly in the nasal epithelium and is transmitted to olfactory neurons, from which it then moves retrograde axonally to the brain and replicates robustly, causing neuropathogenesis. While intranasal inoculation does cause neuropathy in mice, neurotoxicity following viral administration also occurs when the virus is delivered intravascularly (47), intraperitoneally (42), and (not surprisingly) intracranially (13). Previously, other groups have modified the VSV genome to be more sensitive to cellular IFNs (49) and have actually encoded IFN in the virus (36). However, the former can result in attenuation of the virus, such that it has reduced anticancer potential, while the latter still results in lethal encephalitis (unpublished results). In order to mitigate the effects of VSV infection on the brain without perturbing the potent oncolytic activity of the virus, we utilized a microRNA (miRNA) targeting paradigm, whereby viral replication is restricted in the brain without altering the tropism of the virus for other tissues.To redirect the tissue tropism of anticancer therapeutics, we (26) and others (11, 14, 55) have previously exploited the tissue-specific expression of cellular miRNAs. miRNAs are ∼22-nucleotide (nt) regulatory RNAs that regulate a diverse and expansive array of cellular activities. Through recognition of sequence-complementary target elements, miRNAs can either translationally suppress or catalytically degrade both cellular (6) and viral (50) RNAs. We have determined that cellular miRNAs can potentially regulate numerous steps of a virus life cycle and that this regulation of the virus by endogenous miRNAs can then abrogate toxicities of replication-competent viruses (27; E. J. Kelly et al., unpublished data).miRNAs are known to be highly upregulated in many different tissues, including (but not limited to) muscle (40), lung (44), liver (15, 44), spleen (44, 46), and kidney (51). In addition, the brain has a number of upregulated miRNAs, with each different subtype of cell having a unique miRNA profile. miR-125 is highly upregulated in all cells in the brain (neurons, astrocytes, and glia cells), while miR-124 is found predominantly in neuronal cells (48). Glial cells and glioblastomas are thought to have decreased expression of miR-128 compared to neurons (17), while miR-134 is particularly abundant in dendrites of neurons in the hippocampus (43). In addition to these miRNAs, the tumor suppressor miRNA let-7 and miRs 9, 26, and 29 (51) are also found to be enriched in the brain, with expression varying not only between different cell types and regions of the brain but also temporally (48).MicroRNAs have previously been exploited to modulate the tissue tropism of nonreplicating lentiviral vectors (8, 9), as well as curbing known toxicities of replication-competent picornaviruses (5, 26), adenoviruses (11), herpes simplex virus 1 (33), and influenza A virus (39). In addition, a recombinant VSV encoding a tumor suppressor target was found to be responsive to sequence-complementary miRNAs in vitro, possibly by affecting expression of the matrix (M) protein (14), and evidence from Dicer-deficient mice suggests that endogenously expressed microRNA targets within the P and L genes of VSV could restrict enhanced pathogenicity of the virus (37). However, in vivo protection from neuropathogenesis by this means has not been demonstrated for VSV.Here we evaluate the efficiencies of different brain-specific miRNAs for shutting down gene expression and extensively characterize the ability of miRNA targeting to attenuate the neurotoxicity of vesicular stomatitis virus in vivo. We constructed and evaluated recombinant VSVs with miRNA target (miRT) insertions at different regions of the viral genome, with special focus upon those affecting viral L expression. In addition, we looked at the regulatory efficiency of different brain-specific miRNAs and the impact of miRT orientation on VSV replication and determined the impact of the virus on oncolytic activity in vivo.