Mutation in a "tesB-like" hydroxyacyl-coenzyme A-specific thioesterase gene causes hyperproduction of extracellular polyhydroxyalkanoates by Alcanivorax borkumensis SK2

A novel mutant of the marine oil-degrading bacterium Alcanivorax borkumensis SK2, containing a mini-Tn5 transposon disrupting a "tesB-like" acyl-coenzyme A (CoA) thioesterase gene, was found to hyperproduce polyhydroxyalkanoates (PHA), resulting in the extracellular deposition of this biotechnologically important polymer when grown on alkanes. The tesB-like gene encodes a distinct novel enzyme activity, which acts exclusively on hydroxylated acyl-CoAs and thus represents a hydroxyacyl-CoA-specific thioesterase. Inactivation of this enzyme results in the rechanneling of CoA-activated hydroxylated fatty acids, the cellular intermediates of alkane degradation, towards PHA production. These findings may open up new avenues for the development of simplified biotechnological processes for the production of PHA as a raw material for the production of bioplastics.     

Application of electron transfer dissociation (ETD) for the analysis of posttranslational modifications

Despite major advantages in the field of proteomics, the analysis of PTMs still poses a major challenge; thus far, preventing insights into the role and regulation of protein networks. Additionally, top-down sequencing of proteins is another powerful approach to reveal comprehensive information for biological function. A commonly used fragmentation technique in MS-based peptide sequencing is CID. As CID often fails in PTM-analysis and performs best on doubly-charged, short and middle-sized peptides, confident peptide identification may be hampered. A newly developed fragmentation technique, namely electron transfer dissociation (ETD), supports both, PTM- and top-down analysis, and generally results in more confident identification of long, highly charged or modified peptides. The following review presents the theoretical background of ETD and its technical implementation in mass analyzers. Furthermore, current improvements of ETD and approaches for the PTM-analysis and top-down sequencing are introduced. Alternating both fragmentation techniques, ETD and CID, increases the amount of information derived from peptide fragmentation, thereby enhancing both, peptide sequence coverage and the confidence of peptide and protein identification.     

Reproductive output of invasive versus native plants

Aim Propagule size and output are critical for the ability of a plant species to colonize new environments. If invasive species have a greater reproductive output than native species (via more and/or larger seeds), then they will have a greater dispersal and establishment ability. Previous comparisons within plant genera, families or environments have conflicted over the differences in reproductive traits between native and invasive species. We went beyond a genus‐, family‐ or habitat‐specific approach and analysed data for plant reproductive traits from the global literature, to investigate whether: (1) seed mass and production differ between the original and introduced ranges of invasive species; (2) seed mass and production differ between invasives and natives; and (3) invasives produce more seeds per unit seed mass than natives. Location Global. Methods We combined an existing data set of native plant reproductive data with a new data compilation for invasive species. We used t‐tests to compare original and introduced range populations, two‐way ANOVAs to compare natives and invasives, and an ANCOVA to examine the relationship between seed mass and production for natives and invasives. The ANCOVA was performed again incorporating phylogenetically independent contrasts to overcome any phylogenetic bias in the data sets. Results Neither seed mass nor seed production of invasive species differed between their introduced and original ranges. We found no significant difference in seed mass between invasives and natives after growth form had been accounted for. Seed production was greater for invasive species overall and within herb and woody growth forms. For a given seed mass, invasive species produced 6.7‐fold (all species), 6.9‐fold (herbs only) and 26.1‐fold (woody species only) more seeds per individual per year than native species. The phylogenetic ANCOVA verified that this trend did not appear to be influenced by phylogenetic bias within either data set. Main conclusions This study provides the first global examination of both seed mass and production traits in native and invasive species. Invasive species express a strategy of greater seed production both overall and per unit seed mass compared with natives. The consequent increased likelihood of establishment from long‐distance seed dispersal may significantly contribute to the invasiveness of many exotic species.     

2-Decarboxy-2-hydroxymethyl prostaglandin E1 (TR4161), a prostaglandin bronchodilator of low tracheobronchial irritancy

2-Decarboxy 2-hydroxymethyl prostaglandin E₁ (TR4161) relaxed isolated guinea-pig trachea with about double and relaxed human isolated bronchial muscle with about one half the potency of PGE₁. In conscious restrained cats an aerosol of TR4161 was about 100–1000 times less active than PGE₁ in inducing tracheobronchial irritation. When given intravenously or by aerosol to the anaesthetised spontaneously breathing guinea-pig, TR4161 was approximately equipotent with PGE₁ in inhibiting histamine-induced bronchoconstriction and in reducing basal inherent tone. The onset and duration of the bronchodilator effects of TR4161 administered intravenously, however, were significantly longer than those of PGE₁. In conscious guinea-pigs, TR4161 by aerosol was approximately three times more potent than PGE₁ in preventing histamine-induced convulsions, whereas only TR4161 was active in this test system when the test drugs were administered orally. These observations indicate that TR4161 might be therapeutically useful as a non-irritant prostaglandin bronchodilator in conditions of airway obstruction.     

Multiscale Investigation of Sodium-Ion Battery Anodes: Analytical Techniques and Applications

The anode/electrolyte interface behavior, and by extension, the overall cell performance of sodium-ion batteries is determined by a complex interaction of processes that occur at all components of the electrochemical cell across a wide range of size- and timescales. Single-scale studies may provide incomplete insights, as they cannot capture the full picture of this complex and intertwined behavior. Broad, multiscale studies are essential to elucidate these processes. Within this perspectives article, several analytical and theoretical techniques are introduced, and described how they can be combined to provide a more complete and comprehensive understanding of sodium-ion battery (SIB) performance throughout its lifetime, with a special focus on the interfaces of hard carbon anodes. These methods target various length- and time scales, ranging from micro to nano, from cell level to atomistic structures, and account for a broad spectrum of physical and (electro)chemical characteristics. Specifically, how mass spectrometric, microscopic, spectroscopic, electrochemical, thermodynamic, and physical methods can be employed to obtain the various types of information required to understand battery behavior will be explored. Ways are then discussed how these methods can be coupled together in order to elucidate the multiscale phenomena at the anode interface and develop a holistic understanding of their relationship to overall sodium-ion battery function.     

Opposing functions for retromer and Rab11 in extracellular vesicle traffic at presynaptic terminals

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer’s disease, and suggest that misregulated EV traffic may be an underlying defect.     

Marek’s disease virus prolongs survival of primary chicken B-cells by inducing a senescence-like phenotype

Marek’s disease virus (MDV) is an alphaherpesvirus that causes immunosuppression and deadly lymphoma in chickens. Lymphoid organs play a central role in MDV infection in animals. B-cells in the bursa of Fabricius facilitate high levels of MDV replication and contribute to dissemination at early stages of infection. Several studies investigated host responses in bursal tissue of MDV-infected chickens; however, the cellular responses specifically in bursal B-cells has never been investigated. We took advantage of our recently established in vitro infection system to decipher the cellular responses of bursal B-cells to infection with a very virulent MDV strain. Here, we demonstrate that MDV infection extends the survival of bursal B-cells in culture. Microarray analyses revealed that most cytokine/cytokine-receptor-, cell cycle- and apoptosis-associated genes are significantly down-regulated in these cells. Further functional assays validated these strong effects of MDV infections on cell cycle progression and thus, B-cell proliferation. In addition, we confirmed that MDV infections protect B-cells from apoptosis and trigger an accumulation of the autophagy marker Lc3-II. Taken together, our data indicate that MDV-infected bursal B-cells show hallmarks of a senescence-like phenotype, leading to a prolonged B-cell survival. This study provides an in-depth analysis of bursal B-cell responses to MDV infection and important insights into how the virus extends the survival of these cells.     

Predicting novel candidate human obesity genes and their site of action by systematic functional screening in Drosophila

The discovery of human obesity-associated genes can reveal new mechanisms to target for weight loss therapy. Genetic studies of obese individuals and the analysis of rare genetic variants can identify novel obesity-associated genes. However, establishing a functional relationship between these candidate genes and adiposity remains a significant challenge. We uncovered a large number of rare homozygous gene variants by exome sequencing of severely obese children, including those from consanguineous families. By assessing the function of these genes in vivo in Drosophila, we identified 4 genes, not previously linked to human obesity, that regulate adiposity (itpr, dachsous, calpA, and sdk). Dachsous is a transmembrane protein upstream of the Hippo signalling pathway. We found that 3 further members of the Hippo pathway, fat, four-jointed, and hippo, also regulate adiposity and that they act in neurons, rather than in adipose tissue (fat body). Screening Hippo pathway genes in larger human cohorts revealed rare variants in TAOK2 associated with human obesity. Knockdown of Drosophila tao increased adiposity in vivo demonstrating the strength of our approach in predicting novel human obesity genes and signalling pathways and their site of action.

This study set out to identify novel gene variants that may contribute to human obesity, by combining human exosome sequencing analyses with systematic functional screening in Drosophila. This identifies a number of novel obesity-associated genes which control adiposity in flies, and uncovers a potential role for the Hippo signaling pathway in obesity.

Obesity is a major risk factor for type 2 diabetes, cardiovascular disease, cancers, and, most recently, COVID-19 [1]. Despite the obvious environmental drivers to weight gain, multiple genetic studies have demonstrated that 40% to 70% of the variation in body weight is attributable to genetic variation [2]. The discovery of genes that contribute to the regulation of human body weight can provide insights into the mechanisms involved in energy homeostasis and identify potential targets for weight loss therapy. Moreover, drug targets supported by human genetic evidence are more likely to transit successfully through the drug discovery pipeline [3].A classical approach to the discovery of pathogenic variants is to investigate consanguineous populations with high degrees of parental relatedness (parents who are first or second cousins) where large portions of the genome are identical by descent as a result of family structure in preceding generations (long regions of homozygosity). Indeed, studies in consanguineous families led to the discovery of the first homozygous loss-of-function mutations in the genes encoding leptin (LEP; [4]) and the leptin receptor (LEPR; [5]) associated with severe obesity. However, at the time, the function of leptin and its receptor had already been established in ob/ob and db/db mice, respectively [6], so the pathogenicity of homozygous mutations that resulted in loss of function in cells was readily established.The situation is more complex when studying homozygous mutations in new candidate genes. Some of these genes may play a direct causal role in the development of obesity, others may increase susceptibility to obesity only in certain contexts, and some genes will play no role at all. Recent large-scale studies in healthy people in outbred populations have revealed that a significant proportion of rare homozygous variants that are predicted to cause a loss of function do not result in a clinically discernible phenotype [7,8]. As such, identifying the subset of genes that may be involved in the regulation of adiposity in large human genetic studies presents a major hurdle.For some diseases, functional screens in cultured cells permit rapid testing of candidate genes, as exemplified by studies of insulin secretion in islet cells for genes associated with type 2 diabetes [9]. However, obesity is a systems-level disorder that cannot be replicated in cells. As such, a functional screen in vivo is needed. Here, we use Drosophila to screen the functional consequences of knocking down expression of candidate human obesity genes and to explore the complex interactions between multiple organ systems that are regulated by environmental and genetic factors.Drosophila has been a useful tool in the functional characterisation of human disease-associated genes [10–12]. Many organ systems and metabolic enzymes are highly conserved in Drosophila, as are the major regulatory mechanisms involved in metabolic homeostasis [13,14]. As in humans, Drosophila accumulate lipids and become obese when raised on a high-fat or high-sugar diet, developing cardiomyopathy and diabetic phenotypes [15,16]. Furthermore, more than 60% of the genes identified in an unbiased genome-wide RNAi screen for increased fat levels in Drosophila have human orthologues [17]. Most studies in Drosophila have performed forward genetic screens resulting in obesity [18] before assessing whether misregulation of the corresponding mammalian orthologue affects adiposity [17]. Another report knocked down Drosophila orthologs of human genes near body mass index (BMI) loci from GWAS studies to identify genes regulating adiposity [19].Here, instead, we chose to take advantage of new data from a cohort of patients carrying rare genetic variants that might cause severe early-onset obesity. We set out to identify, in Drosophila, whether any of these genes are likely to be responsible for the obese phenotype. An additional advantage of working with Drosophila is the potential to identify interacting genes and signalling pathways. We proposed that it would then be possible to search for variants in human orthologues of these genes in larger cohorts of patients, to discover further as yet unidentified genes regulating human obesity.To increase our chances of finding pathogenic variants, we focused on rare homozygous variants identified in probands with severe obesity, many from consanguineous families. After knocking down expression of Drosophila orthologues of candidate human obesity genes, we discovered 4 genes that significantly increased triacylglyceride (TAG) levels. Importantly, none of these genes had been associated previously with human obesity, but the pathways in which they act are known and could be further analysed in Drosophila. Knockdown of further members of one of these signalling pathways, the Hippo pathway, also gave an obesity phenotype, highlighting the success of our approach. We then searched for variants in the novel obesity genes we identified in Drosophila, and their associated signalling pathways, in larger cohorts of unrelated obese people and healthy controls. This uncovered yet another gene, which, when knocked down in Drosophila, increased adiposity. We demonstrate that the cross-fertilisation of human and Drosophila genetics is a powerful system to provide novel insights into the genetic and cellular processes regulating adiposity and may ultimately contribute to strategies for the prevention and treatment of obesity.