Organochlorine contamination enriches virus-encoded metabolism and pesticide degradation associated auxiliary genes in soil microbiomes

Viruses significantly influence local and global biogeochemical cycles and help bacteria to survive in different environments by encoding various auxiliary metabolic genes (AMGs) associated with energy acquisition, stress tolerance and degradation of xenobiotics. Here we studied whether bacterial (dsDNA) virus encoded AMGs are enriched in organochlorine pesticide (OCP) contaminated soil in China and if viral AMGs include genes linked to OCP biodegradation. Using metagenomics, we found that OCP-contaminated soils displayed a lower bacterial, but higher diversity of viruses that harbored a higher relative abundance of AMGs linked to pesticide degradation and metabolism. Furthermore, the diversity and relative abundance of AMGs significantly increased along with the severity of pesticide contamination, and several biodegradation genes were identified bioinformatically in viral metagenomes. Functional assays were conducted to experimentally demonstrate that virus-encoded L-2-haloacid dehalogenase gene (L-DEX) is responsible for the degradation of L-2-haloacid pesticide precursors, improving bacterial growth at sub-inhibitory pesticide concentrations. Taken together, these results demonstrate that virus-encoded AMGs are linked to bacterial metabolism and biodegradation, being more abundant and diverse in soils contaminated with pesticides. Moreover, our findings highlight the importance of virus-encoded accessory genes for bacterial ecology in stressful environments, providing a novel avenue for using viruses in the bioremediation of contaminated soils.Subject terms: Metagenomics, Soil microbiology, Microbial ecology

As the most abundant biological entities on earth, viruses of bacteria (bacteriophages referred as viruses from here on) play a critical role in modulating the ecology of microbial communities through lytic infection and lysogenic conversion of their bacterial hosts [1, 2]. Viruses significantly influence the biogeochemical cycles via the release of organic carbon and nutrients through host cell lysis, and in addition to core viral genes (i.e., genes encoding viral structural proteins [3]), they also encode various auxiliary metabolic genes (AMGs [4, 5]), which contribute the metabolic capacity and survival of their bacterial hosts. The role of AMGs has been especially well demonstrated with marine viruses that encode a diversity of AMGs involved in photosynthesis [6], translation machinery [7], carbon metabolism [8], phosphate metabolism [9] and sulfur cycle [10, 11]. Furthermore, sequencing of whole marine viral communities has revealed a clear involvement of viral AMGs in central carbon metabolism of host bacteria [10, 12]. Compared with the study of viral communities in marine ecosystem, the diversity and functional role of viral AMGs in soils are less well understood.In soils, viruses reach abundances of up to ~10⁹ per gram of soil leading to frequent encounters with their host bacteria [13]. Similar to aquatic environments, viruses can regulate host bacterial densities, leading to indirect changes in the relative abundance of non-target bacterial taxa likely via release of niche space [14, 15]. Moreover, over longer time periods, viruses can coevolve with their host, following fluctuating selection dynamics [16] or patterns of local adaptation [17]. Viruses are also important mediators of horizontal gene transfer, promoting the transfer of antibiotic resistance genes, virulence factors and AMGs [18, 19]. However, these effects are less well understood at viral community level. Recent advances in viral purification have enabled a glimpse into soil viral communities of permafrost peatland [20, 21] and agricultural ecosystems [22, 23] based on metagenomics. These studies have demonstrated that viruses may alter the biogeochemical nutrient cycling [1, 2] and bacterial adaptation and evolution by carrying genes linked to carbon and nitrogen metabolism [20, 21]. Moreover, recent identification of atrazine chlorohydrolase trzN [24] and arsenic methyltransferase arsM [25] genes in soil-associated lysogenic viruses suggest that virus-encoded AMGs could shape bacterial metabolism under pollutant exposure. Therefore, we hypothesize, that contaminated soil microbiomes could contain a relatively higher abundance of viruses carrying AMGs linked to the degradation of pesticides and xenobiotics due to their potential benefit for the host bacteria.Pesticide contamination imposes a serious threat to natural ecosystems and public health globally. China is the leading producer of organochlorine pesticides (OCPs), which are synthetic pesticides with vast applications in chemical and agricultural industries. OCPs are especially notorious due to their high toxicity, slow degradation and bioaccumulation [26]. Following the implementation of the Stockholm Convention, hundreds of pesticide plants in China were closed or re-located, and contaminated soils around the plants left untreated. As microbial communities are often capable of degrading OCPs, there is growing biotechnological interest to identify important genes and microbial taxa behind pesticide biodegradation. Heavy OCP contaminations have previously been shown to adversely impact soil bacterial diversity, composition, and activity [27, 28]. Prolonged exposure to contaminants has resulted in selection for bacteria that have evolved their own degradation enzymes, such as dehalogenases, which protect from the toxic effects of pesticides [29]. Interestingly, if also viruses can carry and encode such genes, pesticide exposure could create a strong positive selection for virus-encoded AMGs associated with pesticide degradation, potentially shifting soil microbiome community composition [30] by favoring bacterial and virus taxa that carry these genes.To address this, we used a combination of metagenomics and direct experimentation to explore how pesticide exposure affects the abundance and type of bacterial and virus-encoded AMGs in the soil of former OCP production factory in Yangtze River Delta (China). We found that contaminated and clean control soils harbored very distinct bacterial and viral communities, and crucially, pesticide exposure was linked to higher diversity and abundance of virus-encoded metabolism and pesticide degradation AMGs. The functional activity of one candidate viral AMG, L-2-haloacid dehalogenase (L-DEX), was experimentally shown to improve bacterial growth at sub-inhibitory concentrations of haloacid, which is an important precursor of herbicides and insecticides. Together, our findings suggest that virus-encoded auxiliary genes could help bacteria to counteract pesticide stress, potentially explaining the benefits of virus carriage in stressful soil microbiomes.     

The NOTCH signaling pathway in normal and malignant blood cell production

The NOTCH pathway is an evolutionarily conserved signalling network, which is fundamental in regulating developmental processes in invertebrates and vertebrates (Gazave et al. in BMC Evol Biol 9:249, 2009). It regulates self-renewal (Butler et al. in Cell Stem Cell 6:251–264, 2010), differentiation (Auderset et al. in Curr Top Microbiol Immunol 360:115–134, 2012), proliferation (VanDussen et al. in Development 139:488–497, 2012) and apoptosis (Cao et al. in APMIS 120:441–450, 2012) of diverse cell types at various stages of their development. NOTCH signalling governs cell-cell interactions and the outcome of such responses is highly context specific. This makes it impossible to generalize about NOTCH functions as it stimulates survival and differentiation of certain cell types, whereas inhibiting these processes in others (Meier-Stiegen et al. in PLoS One 5:e11481, 2010). NOTCH was first identified in 1914 in Drosophila and was named after the indentations (notches) present in the wings of the mutant flies (Bigas et al. in Int J Dev Biol 54:1175–1188, 2010). Homologs of NOTCH in vertebrates were initially identified in Xenopus (Coffman et al. in Science 249:1438–1441, 1990) and in humans NOTCH was first identified in T-Acute Lymphoblastic Leukaemia (T-ALL) (Ellisen et al. in Cell 66:649–61, 1991). NOTCH signalling is integral in neurogenesis (Mead and Yutzey in Dev Dyn 241:376–389, 2012), myogenesis (Schuster-Gossler et al. in Proc Natl Acad Sci U S A 104:537–542, 2007), haematopoiesis (Bigas et al. in Int J Dev Biol 54:1175–1188, 2010), oogenesis (Xu and Gridley in Genet Res Int 2012:648207, 2012), differentiation of intestinal cells (Okamoto et al. in Am J Physiol Gastrointest Liver Physiol 296:G23–35, 2009) and pancreatic cells (Apelqvist et al. in Nature 400:877–881, 1999). The current review will focus on NOTCH signalling in normal and malignant blood cell production or haematopoiesis.     

Propagation of viral genomes by replicating ammonia-oxidising archaea during soil nitrification

Ammonia-oxidising archaea (AOA) are a ubiquitous component of microbial communities and dominate the first stage of nitrification in some soils. While we are beginning to understand soil virus dynamics, we have no knowledge of the composition or activity of those infecting nitrifiers or their potential to influence processes. This study aimed to characterise viruses having infected autotrophic AOA in two nitrifying soils of contrasting pH by following transfer of assimilated CO₂-derived ¹³C from host to virus via DNA stable-isotope probing and metagenomic analysis. Incorporation of ¹³C into low GC mol% AOA and virus genomes increased DNA buoyant density in CsCl gradients but resulted in co-migration with dominant non-enriched high GC mol% genomes, reducing sequencing depth and contig assembly. We therefore developed a hybrid approach where AOA and virus genomes were assembled from low buoyant density DNA with subsequent mapping of ¹³C isotopically enriched high buoyant density DNA reads to identify activity of AOA. Metagenome-assembled genomes were different between the two soils and represented a broad diversity of active populations. Sixty-four AOA-infecting viral operational taxonomic units (vOTUs) were identified with no clear relatedness to previously characterised prokaryote viruses. These vOTUs were also distinct between soils, with 42% enriched in ¹³C derived from hosts. The majority were predicted as capable of lysogeny and auxiliary metabolic genes included an AOA-specific multicopper oxidase suggesting infection may augment copper uptake essential for central metabolic functioning. These findings indicate virus infection of AOA may be a frequent process during nitrification with potential to influence host physiology and activity.Subject terms: Microbial ecology, Stable isotope analysis

Microbially mediated oxidation of ammonia to nitrate during nitrification is a central component of the global nitrogen (N) cycle. It is also responsible for major losses of applied fertiliser N in soil, generating atmospheric pollution via direct and indirect production of nitrous oxide (N₂O) as well as nitrate (NO₃^-) pollution of groundwater [1]. Autotrophic ammonia-oxidising archaea (AOA) of the class Nitrososphaeria are a ubiquitous component of soil microbial communities and often dominate ammonia oxidation and nitrification-associated N₂O emissions when ammonia is supplied at low rates via organic matter mineralisation [2], slow-release fertilisers [3] or in acidic soils [4]. Integrated temperate viruses (proviruses) and other virus-associated protein-encoding genes are found in most AOA genomes suggesting frequent interaction (see Supplementary Text). While viruses infecting marine AOA have been characterised through metagenomic approaches [5] and cultivation [6], those infecting soil AOA or other nitrifier groups are currently uncharacterised.Virus infection can influence biogeochemical cycling by augmenting host activity or causing cell mortality and subsequent release of nutrients [7]. Recent advances have demonstrated that soil virus communities are dynamic in a wide range of soils [e.g. 8, 9] and augmenting virus loads modulate C and N fluxes [10, 11]. Nevertheless, identifying active interactions with specific populations or functional groups in soil remains challenging due to structural complexity and the vast diversity of hosts and viruses. Recent use of stable-isotope approaches has investigated whole community host-virus dynamics [12, 13] or interactions between individual host-virus populations specific to a functional process and substrate [14]. The aim of this study was to utilise the latter approach with ¹³CO₂-based DNA-SIP to focus on nitrification-associated interactions and to test the hypothesis that viruses are a dynamic component of soil AOA activity.     

Investigating dynamic interdomain allostery in Pin1

Signaling proteins often sequester complementary functional sites in separate domains. How do the different domains communicate with one another? An attractive system to address this question is the mitotic regulator, human Pin1 (Lu et al., Nature 380:544–547, 1996). Pin-1 consists of two mutually tethered domains: a WW domain for substrate binding and a catalytic domain for peptidyl-prolyl isomerase (PPIase) activity. Pin1 accelerates the cis–trans isomerization of phospho-Ser/Thr-Pro (pS/T-P) motifs within proteins regulating the cell cycle and neuronal development. The early X-ray (Ranganathan et al., Cell 89:875–886, 1997; Verdecia et al., Nat Struct Biol 7:639–643, 2000) and solution NMR studies (Bayer et al., J Biol Chem 278:26183–26193, 2003; Jacobs et al., J Biol Chem 278:26174–26182, 2003) of Pin1 indicated inter- and intradomain motions. We have explored how such motions might affect interdomain communication, using NMR. Our accumulated results indicate substrate binding to Pin1 WW domain changes the intra/interdomain mobility, thereby altering substrate activity in the distal PPIase domain catalytic site. Thus, Pin1 shows evidence of dynamic allostery, in the sense of Cooper and Dryden (Eur J Biochem 11:103–109, 1984). We highlight our results supporting this conclusion and summarize them via a simple speculative model of conformational selection.     

Targeting subchondral bone in osteoporotic osteoarthritis

The identification of well-defined phenotypes along the course of the disease may open new avenues for personalized management in osteoarthritis (OA). In vivo research carried out in various animal models as well as epidemiological and clinical data support the existence of a particular phenotype – osteoporotic OA. In fact, subchondral bone has become a potential therapeutic target in OA. Depending on the ratio between formation and resorption, subchondral bone remodeling can culminate in either a sclerotic or an osteoporotic phenotype. Patients with osteoporotic OA may thus achieve clinical and structural benefit from treatment with bone-targeted interventions.Subchondral bone has become a potential therapeutic target in osteoarthritis (OA). In a previous issue of Arthritis Research & Therapy, Wang and colleagues demonstrate that osteoporosis aggravates cartilage damage in an experimental model of knee OA in rats [1]. Interestingly, the authors also describe that extracorporeal shockwave therapy (ESWT), a mechanical therapeutic intervention probably acting at subchondral bone, may reduce OA progression [1]. The significance of these findings in experimental osteoporotic OA relates to the search for well-defined phenotypes in human OA that will lead to personalized therapy.The controversy regarding the relationship between subchondral bone quality and cartilage integrity originates from the complex biological and mechanical nature of the osteochondral junction [2]. OA progression is often accompanied by increased subchondral bone remodeling that enables mechanical forces to dynamically modify its structure. Depending on the ratio between formation and resorption, subchondral bone can exhibit either a sclerotic or an osteoporotic phenotype [3]. These phenotypes may represent up to 70% and 30% of patients in daily practice, respectively [4]. Furthermore, OA in females can display a different pathogenic profile from OA in males. In this sense, it is reasonable to underline the consequences of estrogen deficiency during menopause [5]. A low estrogen state could induce a deleterious effect on all articular tissues of the knee joint, the subchondral bone being particularly affected due to its capacity for high bone turnover. Thus, during early post menopause, estrogen deficiency may be a risk factor for the development of knee OA. Taking all these facts into consideration, the characterization of patients with either sclerotic or osteoporotic OA phenotypes may enable individualized targeted therapy [3].The effects of estrogen deficiency on the knee joint have been reported in various experimental animal models of OA. The findings obtained by Wang and colleagues on subchondral bone quality and articular cartilage damage support previous research carried out in rabbits, in which osteoporosis aggravated instability-induced OA [6]. In this combined model, the induction of systemic and subchondral osteoporosis associated with increased bone remodeling resulted in worse cartilage damage compared with control animals. Greater fragility of the subchondral bone was suggested to account for the aggravation of cartilage damage when early OA and osteoporosis coexist [7]. In a further study carried out in the same model, the intermittent administration of parathyroid hormone 1-34, a bone-forming agent, was used to increase subchondral bone density and quality [8]. As a consequence, the improvement of subchondral bone integrity was associated with reduced progression of cartilage damage in OA preceded by osteoporosis. In a similar approach, the inhibition of bone resorption by pamidronate in osteoporotic mice alleviated the instability-induced OA histological score with a reduction in the expression of aggrecanases [9]. Several experimental models therefore indicate that osteopenia/osteoporosis induces an accelerated progression of knee OA that can be reversed not only by bone-forming agents but also by antiresorptive drugs.These findings in animal models could be translated to humans, and together with epidemiological and clinical data they support the existence of a particular phenotype – osteoporotic OA [10]. Indeed, this phenotype characterized by decreased density and high remodeling at subchondral bone defines a subgroup of patients treatable with specific agents. In fact, beneficial effects of bone-acting drugs in OA are increasingly reported, but reliable conclusions regarding their efficacy are hindered by methodological drawbacks in study design [10]. Identifying patients with osteoporotic OA may improve the success of bone-directed agents.The original approach of using ESWT in OA by Wang and colleagues remains intriguing. These authors have reported previously that the application of ESWT to subchondral bone of the proximal tibia showed a chondroprotective effect in the initiation of knee OA and regression of established OA of the knee in rats. These effects were attributed to the ESWT multifunctional actions on cartilage and bone. Yet achieving such beneficial effects in this osteoporotic OA model suggests that the main mechanism of action of ESWT may be improving subchondral bone structure [1]. However, some limitations on the study design and the lack of adequate standardization of dosages and optimal frequency, as well as little information regarding the molecular mechanisms underlying the effects of ESWT, hold back the achievement of solid results. In any case, this study points out the potential benefit of nonpharmacological interventions aiming to improve mechanical properties of articular tissues in OA.In summary, the study by Wang and colleagues further supports the existence of the osteoporotic OA subtype and the potential benefit of bone-acting therapeutic interventions. Consequently, the identification of patient phenotypes along with the discovery of specific therapeutic interventions targeting relevant pathogenic mechanisms during the course of the disease could lead to a personalized approach to the management of OA.     

Solutions in microbiome engineering: prioritizing barriers to organism establishment

Microbiome engineering is increasingly being employed as a solution to challenges in health, agriculture, and climate. Often manipulation involves inoculation of new microbes designed to improve function into a preexisting microbial community. Despite, increased efforts in microbiome engineering inoculants frequently fail to establish and/or confer long-lasting modifications on ecosystem function. We posit that one underlying cause of these shortfalls is the failure to consider barriers to organism establishment. This is a key challenge and focus of macroecology research, specifically invasion biology and restoration ecology. We adopt a framework from invasion biology that summarizes establishment barriers in three categories: (1) propagule pressure, (2) environmental filtering, and (3) biotic interactions factors. We suggest that biotic interactions is the most neglected factor in microbiome engineering research, and we recommend a number of actions to accelerate engineering solutions.Subject terms: Community ecology, Microbial ecology

Microbiome engineering is a rapidly evolving frontier for solutions to improve human health, agricultural productivity, and climate management. Microbiome engineering seeks to improve the function of an ecosystem by manipulating the composition of microbes. Two major challenges for successful microbiome engineering are (1) the design of a microbiome with improved function and (2) the establishment of an improved microbiome in a recipient system of interest. While multiple articles and reviews have addressed functional design [1–3], microbiome establishment has received less attention. Here, we propose a strategy to improve microbiome engineering by focusing on microbial establishment and leveraging insights from macrobial ecology.Two general engineering strategies are to manipulate indigenous microbes [4] or to introduce new members [5]. The latter involves the design and delivery of inoculants (a.k.a., probiotics in medical and agricultural arenas) and is a rapidly growing biotechnology sector. In their most general form, both strategies have been practiced crudely for thousands of years in human health [6] and agriculture [7]. However, despite current technical advances, inoculants frequently still fail to establish or confer long-lasting (months to years) modifications to ecosystem function [8]. We argue that this repeated failure is in part driven by lack of emphasis on establishment of inoculants.The problem of organism establishment in recipient ecosystems is not unique to microbiome engineering; it has roots in macrobiology, particularly invasion biology and restoration ecology. We propose that adopting a cross-disciplinary conceptual framework to identify barriers to organism establishment, and then prioritizing these barriers through targeted research will accelerate successful microbiome engineering. In addition, recognizing differences in terminology and experimental design within and across disciplines will facilitate research integration across diverse ecosystems and scales. The components of a more holistic strategy are discussed below.     

Borderline pulmonary pressures in scleroderma - a ‘pre-pulmonary arterial hypertension’ condition?

Patients with systemic sclerosis may develop borderline pulmonary arterial pressure. The clinical relevance of this condition is not always clear. Reported data support the evidence that this subgroup may represent an intermediate stage between normal pulmonary arterial pressure and manifest pulmonary arterial hypertension, a serious complication in scleroderma. Recognizing the clinical relevance of borderline pulmonary arterial pressure increase in scleroderma patients, future studies should aim for clear evidence for diagnostic and therapeutic algorithms for this population.In their recent study, Visovatti and colleagues [1] present a detailed analysis of patients with borderline pulmonary arterial pressure (PAP) as a subgroup analysis of the DETECT study, providing important clinical data for understanding early pulmonary vasculopathy in patients with systemic sclerosis.In fact, every physician who has observed the dramatic deterioration of patients with pulmonary arterial hypertension (PAH) and successive right ventricular failure would urge for the earlier recognition and therapy of this devastating condition. About 10% of all scleroderma patients may develop PAH [2], which - besides lung fibrosis - represents the most frequent cause of death in this patient population [3]. But can PAH be recognized at an early stage and maybe even prevented?If we assume that the increase of PAP is a process lasting for a longer period of time, there must be a phase of transition from normal (mean PAP ≤20 mmHg) pulmonary hemodynamic conditions to PAH (mean PAP ≥25 mmHg). Patients in this so-called ''borderline'' range may represent the early stage of PAH. Earlier studies found that such patients were more likely to develop pulmonary hypertension than patients with mean PAP ≤20 mmHg, with a hazard ratio of 3.7 [4]. The rate of borderline patients developing PAH was 19% after 3 years and 27% after 5 years. Accordingly, we may argue that borderline PAP is a ''pre-PAH'' condition in scleroderma. Of course, borderline elevation of PAP may be caused not only by pulmonary vasculopathy but also by cardiac or pulmonary co-morbidities [5]. In these cases borderline elevation of PAP may be considered as a general prognostic marker [5,6].The analysis of Visovatti and colleagues [1] includes several clinical (for example, current/past telangiectasis, presence of peripheral edema), laboratory (for example, ACA antibody, NT-proBNP), lung functional (for example, forced vital capacity (percentage predicted)/diffusion capacity for carbon monoxide ratio) and cardiac (for example, tricuspid annular plane systolic excursion) markers that may distinguish scleroderma patients with borderline PAP elevation from those with normal PAP or with manifest PAH. According to this analysis, borderline elevation of PAP in scleroderma patients may represent an intermediate stage in the continuum between normal PAP and manifest PAH.Among the DETECT population, 15% of all patients presented with borderline PAP hemodynamics. Although this number may be different in the general scleroderma population, due to the strict inclusion and exclusion criteria of the DETECT study [7], the borderline population seems to be a substantial subgroup. Unfortunately, follow-up data of the described patients in comparison with normal PAP and manifest PAH patients have not been provided. Such data might impact the development of clinical algorithms regarding further follow-up and treatment of these patients.In addition to the borderline elevation of resting PAP, another specific hemodynamic situation in scleroderma patients needs careful interpretation: exercise-induced PAP increase. Earlier studies showed that this may be a frequent condition among scleroderma patients and clinical deterioration and the development of PAH are frequent in this population [2]. In a recent analysis, a strong correlation between resting and exercise PAP values was evident [5], suggesting that patients with borderline hemodynamics and those with a strong PAP increase during exercise may strongly overlap, closing the gap between these two hemodynamic conditions.The most important question remains open: should targeted PAH therapy be offered to scleroderma patients with borderline PAP or exercise-induced PAP increase? Unfortunately there has been no clinical study investigating patients with borderline PAP so far and only two small studies have selected patients with exercise-induced PAP increase [8,9]. The results of these studies are promising, but need to be confirmed in adequately powered, randomized, prospective trials.Based on a series of studies indicating borderline hemodynamics has an important role in scleroderma patients with regard to the development of PAH and potentially for early treatment, future studies should aim for clear evidence for diagnostic and therapeutic algorithms for this patient population. This may contribute to a substantial prognostic improvement for patients with scleroderma who develop pulmonary vasculopathy     

Phospholamban phosphorylation,mutation, and structural dynamics: a biophysical approach to understanding and treating cardiomyopathy

We review the recent development of novel biochemical and spectroscopic methods to determine the site-specific phosphorylation, expression, mutation, and structural dynamics of phospholamban (PLB), in relation to its function (inhibition of the cardiac calcium pump, SERCA2a), with specific focus on cardiac physiology, pathology, and therapy. In the cardiomyocyte, SERCA2a actively transports Ca²⁺ into the sarcoplasmic reticulum (SR) during relaxation (diastole) to create the concentration gradient that drives the passive efflux of Ca²⁺ required for cardiac contraction (systole). Unphosphorylated PLB (U-PLB) inhibits SERCA2a, but phosphorylation at S16 and/or T17 (producing P-PLB) changes the structure of PLB to relieve SERCA2a inhibition. Because insufficient SERCA2a activity is a hallmark of heart failure, SERCA2a activation, by gene therapy (Andino et al. 2008; Fish et al. 2013; Hoshijima et al. 2002; Jessup et al. 2011) or drug therapy (Ferrandi et al. 2013; Huang 2013; Khan et al. 2009; Rocchetti et al. 2008; Zhang et al. 2012), is a widely sought goal for treatment of heart failure. This review describes rational approaches to this goal. Novel biophysical assays, using site-directed labeling and high-resolution spectroscopy, have been developed to resolve the structural states of SERCA2a-PLB complexes in vitro and in living cells. Novel biochemical assays, using synthetic standards and multidimensional immunofluorescence, have been developed to quantitate PLB expression and phosphorylation states in cells and human tissues. The biochemical and biophysical properties of U-PLB, P-PLB, and mutant PLB will ultimately resolve the mechanisms of loss of inhibition and gain of inhibition to guide therapeutic development. These assays will be powerful tools for investigating human tissue samples from the Sydney Heart Bank, for the purpose of analyzing and diagnosing specific disorders.     

High-resolution genomic analysis: the tumor-immune interface comes into focus

A genomic analysis of heterogeneous colorectal tumor samples has uncovered interactions between immunophenotype and various aspects of tumor biology, with implications for informing the choice of immunotherapies for specific patients and guiding the design of personalized neoantigen-based vaccines.Please see related article: http://dx.doi.org/10.1186/s13059-015-0620-6Immunotherapy is a promising new approach for treating human malignancies. Approximately 20% of melanoma and lung cancer patients receiving immune checkpoint inhibitors show responses [1,2]. Current major challenges include identification of patients most likely to respond to specific therapies and elucidation of novel targets to treat those who do not. To address these problems, a detailed understanding of the dynamic interactions between tumors and the immune system is required. In a new study, Zlatko Trajanoski and colleagues [3] describe a powerful approach to dissecting these issues through high-resolution analysis of patient genomic data. This study represents a significant advance over previous work from this group, which defined 28 immune-cell-type gene expression signatures and identified specific cell types as prognostic indicators in colorectal cancer (CRC) patients [4]. Here, the authors [3] integrate genomic analyses of CRC tumor molecular phenotypes, predicted antigenicity (called the ‘antigenome’), and immune-cell infiltration derived from multiple independent cohorts to gain refined insights into tumor-immune system interactions.     

Human-associated microbiota suppress invading bacteria even under disruption by antibiotics

In light of their adverse impacts on resident microbial communities, it is widely predicted that broad-spectrum antibiotics can promote the spread of resistance by releasing resistant strains from competition with other strains and species. We investigated the competitive suppression of a resistant strain of Escherichia coli inoculated into human-associated communities in the presence and absence of the broad and narrow spectrum antibiotics rifampicin and polymyxin B, respectively. We found strong evidence of community-level suppression of the resistant strain in the absence of antibiotics and, despite large changes in community composition and abundance following rifampicin exposure, suppression of the invading resistant strain was maintained in both antibiotic treatments. Instead, the strength of competitive suppression was more strongly associated with the source community (stool sample from individual human donor). This suggests microbiome composition strongly influences the competitive suppression of antibiotic-resistant strains, but at least some antibiotic-associated disruption can be tolerated before competitive release is observed. A deeper understanding of this association will aid the development of ecologically-aware strategies for managing antibiotic resistance.Subject terms: Microbial ecology, Community ecology, Antibiotics

The overuse of broad-spectrum antibiotics in clinical and agricultural settings is a key driver of the current antibiotic resistance crisis [1]. Research into antibiotic resistance has traditionally focused on the evolution of resistance in individual pathogens [2]. In the last decade, researchers have turned their attention to the collateral damage inflicted on commensal members of the microbiome, such as those belonging to the dense communities of the human gastrointestinal tract [3, 4]. Several studies have shown that antibiotics can leave gut communities vulnerable to colonisation by other pathogens [5–7], and, most recently, resistance evolution in invading strains can be facilitated by the absence of community suppression [8, 9]. Taken together, these two lines of enquiry appear to bear out conventional wisdom that relative to narrow-spectrum antibiotics or antibiotic-free conditions, broad spectrum antibiotics should increase the likelihood of communities being invaded by resistant strains [10, 11]. On the other hand, given evidence that community-level properties can sometimes be robust to changes in taxonomic composition [12], it is possible that some antibiotic-associated disruption can be tolerated before colonization resistance is affected. Despite the importance of these contrasting predictions, there have been few, if any, direct tests in human-associated microbiota.We investigated the effect of broad and narrow spectrum antibiotics on the strength of competitive suppression on a resistant variant (generated by in vitro selection for resistance mutations) of a focal strain (Escherichia coli K-12 MG1655) inoculated into gut microbiome communities collected from human faecal samples. The focal strain was jointly resistant to the broad-spectrum antibiotic rifampicin (targets Gram-positives and Gram-negatives via inhibition of the highly conserved bacterial RNA polymerase) and the narrow spectrum antibiotic polymyxin B (only targets Gram-negatives). The focal strain was inoculated alongside live or sterile slurry produced using a sample from one of three healthy human donors (described in [9]) into customized gut media without antibiotics or supplemented with 128 μg/ml rifampicin or 4 μg/ml polymyxin B (see Fig S1). Following 24 h incubation under anaerobic conditions, focal strain density and total biomass were measured via colony counting and flow cytometry, and community composition and diversity were analysed via 16S rRNA sequencing.In the absence of either antibiotic, focal strain density after 24 h was significantly lower in the presence of the three donor communities, indicative of strong competitive suppression (Fig. 1a). Surprisingly, we detected similarly strong competitive suppression in both the antibiotic treatments as we did in the antibiotic-free treatment. Instead, we found that focal strain performance was a stronger function of the specific donor community, irrespective of antibiotic treatment (Figs. 1b, and S2).Open in a separate windowFig. 1Effect of community, donor and antibiotic on focal strain abundance.a Violin plots showing the distribution of observed abundances of the focal strain in each antibiotic treatment. Blue denotes community free treatments; yellow denotes community treatment. Point shape denotes the individual human donor of live community or sterilized slurry: donor 1 = circles, donor 2 = squares, donor 3 = diamonds. b Treatment contrasts (posterior distributions of parameter estimates for a linear model with negative binomial errors) for focal strain abundance as a function of community (live vs sterile slurry), antibiotic (none, polymixin B or rifampicin), and donor (slurry prepared with samples from human donor 1, 2 or 3), and the interactions between community and antibiotic, and community and donor. Posterior parameter estimates in green have 95% credible intervals that do not overlap with 0 (i.e., there is less than 5% probability there is no effect of the variables/interactions captured by these coefficients). The reference level (vertical black line) = donor 1 in the no antibiotic treatment in the absence of the community (i.e., sterilized slurry).What makes these results particularly striking is that, consistent with previous studies [7, 10, 13], treatment with a broad-spectrum antibiotic was still associated with a marked shift in community composition (analysis of 16S amplicon data) (Fig. 2a). Based on OTU composition, all three donors in the rifampicin treatment cluster separately from the polymyxin B and antibiotic-free treatments, which cluster together (Fig. 2b). This divergence in composition appears to be largely driven by enrichment of both Enterobacteriaceae and Erysipelotrichaceae in the rifampicin treatment (Fig. 2a). In addition to strong shifts in composition, total bacterial abundance was significantly reduced in the rifampicin treatment (Figs. 2c and S3). Despite this, total richness and diversity (Shannon Index) after 24 h did not differ between the treatments (Fig. 2c). In contrast, diversity loss over time was more strongly associated with donor identity, with the donor community associated with the weakest competitive suppression (donor 3) also exhibiting the largest decline in richness and diversity across all treatments. This observation is consistent with previous work demonstrating that colonization resistance in the mouse gut is highly contingent on the complexity and composition of the resident microbiota [14].Open in a separate windowFig. 2Community response to antibiotic treatments.a Heatmap of relative abundance of the ten most abundant families of bacteria across treatments (derived from amplicon data). I = inoculum; AB free = Antibiotic free; Poly = polymyxin B; Rif = rifampicin. b NMDS ordination of family level composition in each treatment-donor combination. c Violin plots showing the abundance (top), species richness (middle) and diversity (Shannon Index) (bottom) distributions in each treatment. In b and c: circles = donor 1; squares = donor 2, diamonds = donor 3.A limitation of this study is that we only considered the effects of two antibiotics. Nevertheless, given the scale of community perturbation observed (Fig. 2), we can at least be sure our findings are not explained by a lack of antibiotic effects in our system. There must be some limit dictated by antibiotic concentration, combination, or duration of exposure, beyond which we would expect to observe stronger competitive release. Indeed, prior research has shown that antibiotics can greatly inhibit colonisation resistance [15, 16]. As such, characterizing where this limit lies (e.g., by investigating community-mediated suppression as a function of antibiotic concentration/duration) will be an important challenge for future work. Similarly, although we only considered a single focal strain, and other strains/species may have been more invasive (for example, those with fewer, different or less costly resistance mutations), key for our experiment was that the focal strain had a positive growth rate over the timescale of the experiment, despite exhibiting significant resistance costs in antibiotic-free assays (Fig. S1). This allowed us to test for sensitivity of competitive suppression to antibiotic treatment. We also note that in spite of a small boost in the focal strain’s performance in the presence of rifampicin independent of the community (a possible hormetic response [17] absent under aerobic growth in LB, Fig S1), we did not observe an increase in the magnitude of competitive release in the rifampicin treatment. Finally, the drop in diversity indicates, unsurprisingly, microcosms are a novel environment relative to the source environment. Despite this, key taxa in each community were stable over the course of the experiment, and previously over a longer timescale in the same set-up [9], demonstrating these conditions sustain diverse human-associated communities over relevant timescales.In conclusion, these results are consistent with prevailing wisdom that healthy gut communities can suppress invading strains and thereby reduce the likelihood of resistance emerging [8, 9, 18]. Nevertheless, the absence of a significant effect of broad, or even narrow, spectrum antibiotics on the degree of competitive suppression of our focal strain is much more surprising. Despite the limitations of scope discussed above, this shows that the functional diversity of gut communities may be more robust to disturbance by broad spectrum antibiotics than previously recognised. This is not to suggest that the use of broad-spectrum antibiotics does not drive marked changes in composition but rather that there is some degree of functional redundancy in diverse communities that facilitates the maintenance of competitive suppression [12, 19]. Notwithstanding the need to test how these findings translate to in vivo settings, this finding is relevant for optimizing personalised treatments that either account for disruption by antibiotics or that make microbiomes harder for pathogens to invade.     

Down on one knee: soft tissue knee injuries across the lifespan

Joint injury is a potent risk factor for osteoarthritis, the most important musculoskeletal disease affecting humankind. Yet the population incidence of soft tissue knee injury is not well documented. Using health-care register data from Sweden, Peat and colleagues report that soft tissue knee injuries are common, peak in adolescence and early adulthood, have a second spike in women who are 35 to 49 years old, and continue throughout the lifespan. The study highlights the need for more knowledge on the natural history of knee injuries, their impact on knee osteoarthritis development and progression, and the potential for prevention programs to reduce the incidence of these injuries.Joint injury is a potent risk factor for osteoarthritis (OA), the most important musculoskeletal disease affecting humankind. Although evidence is mounting that knee joint injury rates are high and increasing, it is also perhaps the lowest hanging fruit for primary OA prevention; several randomized clinical trials have shown that knee injuries can be dramatically reduced with relatively straightforward interventions. Yet outside of anterior cruciate ligament (ACL) injury and despite its potential public health impact, the population incidence of soft tissue knee injury requiring medical attention is not well documented: we have not known the extent or the nature of the problem, until now.In a recent issue of Arthritis Research & Therapy, Peat and colleagues [1] provided population-wide estimates of clinically diagnosed soft tissue knee injuries across all ages on the basis of an entire region of Sweden (approximately 1.3 million people). The opportunity to report and classify all clinically diagnosed knee injuries across the lifespan arises from unique and detailed health-care registries typical to Scandinavian countries. This overcomes weaknesses of previous epidemiological evaluations of knee injuries, which are limited to specific health-care settings, subgroups of people, and specific injury types. Of note, the findings of Peat and colleagues [1] have convergent validity - largely agreeing with previous reports of incidence for specific injury types and subgroups where data overlap.What emerges is that population exposure to soft tissue knee injury is a common problem; the annual incidences for males and females are 766 and 676 per 100,000 persons per year, respectively. This is approximately 10 times higher than ACL injuries alone. If these ‘less catastrophic’ but more common injuries are a risk for OA development (as risk factor studies measuring self-reported injury suggest [2]), then this study may be uncovering and detailing critical new exposure data. They are clearly more numerous though more difficult to accurately diagnose. This study begins to shed light on this challenge.Also revealed is new information on age and gender differences. The incidence of soft tissue knee injuries peaks in adolescence and early adulthood and is likely sports-related, matching seasonal fluctuations in popular sports in Sweden. The rates after this period decline over the lifespan with a notable exception: females from 35 to 49 experience a second peak. This is intriguing and the reasons are not clear, although the authors propose that the previously reported link between parity/child-bearing and knee OA may be mediated by injury. Although the reasons remain obscure, the finding is compelling and may help elucidate the consistently reported, but unexplained, higher prevalence of knee OA in females.Peat and colleagues [1] show that, although incidence rates are highest in the second and third decades of life, considerable rates of contusion, collateral ligament sprain, and other soft tissue strains continue into middle and old age. These injuries coincide with the age of onset of knee OA symptoms and illustrate the challenge of differentiating what is truly an injury from what is part of a previously latent or degenerative process or both. This also applies to meniscal injuries. Surgeries for meniscal tears peak in the mid to late 40s [3–5]. In contrast, Peat and colleagues [1] report a high incidence of meniscal tears in adolescents and young adults. As acknowledged by the authors, less severe injuries such as meniscal tears likely suffer from some misclassification. However, the relationship between diagnosis and surgery for meniscal tears requires further investigation.The high injury incidence among adolescents and young adults, together with the known risk of OA incidence from ACL and meniscal injuries, provides further impetus for implementing knee injury prevention programs, for which there is a strong body of level 1 evidence [6–11]. Efficacy has been demonstrated primarily in the sports team setting, implemented as novel 10- to 15-minute team warm-ups consisting of neuromuscular exercises to train athletes to land, decelerate, and push off with better lower limb alignment and improved trunk control, balance, and proprioception. The reported risk reductions range from 41% to 88% [7,8,11]. Given the age and frequency at which these injuries most often occur and their potential sequelae, perhaps targeting injury prevention programs to physical education classes in public schools could address a growing public health problem.The study by Peat and colleagues highlights several areas for further study. Knowledge is needed on the natural history of knee injuries in the development of knee OA as well as the potential for prevention programs to reduce the incidence. The spike of injuries in females between 35 and 49 requires confirmation and further investigation as to its causes, prevention, and potential role in OA development or progression. The same is true for injuries that occur in middle and older age, often coinciding with a time when knee OA has been diagnosed. Further clarity is needed around meniscal injury: what is traumatic injury and what is degenerative knee disease? There is still much to discover about the different knee injury types throughout the lifespan and the initiation and progression of knee OA. The study by Peat and colleagues [1] provides a good platform for this to be pursued.     

Gluten-free diet in the management of patients with irritable bowel syndrome,fibromyalgia and lymphocytic enteritis

An evaluation of the effect of 1 year of a gluten-free diet was performed in patients with irritable bowel syndrome and fibromyalgia syndrome displaying lymphocytic enteritis. Gluten withdrawal produced a slight but significant improvement of the functional symptoms, suggesting that gluten might be partly responsible for this clinical picture. This hypothesis should be confirmed by a double-blind placebo-controlled trial since it cannot be ruled out that the studied patients displayed a subjective sensation of improvement due to the placebo effect of gluten withdrawal. Further investigations are needed before recommending gluten withdrawal in patients with fibromyalgia and lymphocytic enteritis.In their paper published in a recent issue of Arthritis Research and Therapy, Rodrigo and colleagues evaluated the effect of 1 year of a gluten-free diet on the clinical evolution of irritable bowel syndrome (IBS) plus fibromyalgia syndrome (FMS) in patients with lymphocytic enteritis (LE) [1]. The study sample included 97 adult females with IBS and FMS, of whom 58 had LE and the remaining 39 had a normal intraepithelial lymphocytic (IEL) count. All subjects fulfilled the Rome III criteria for IBS and the American College of Rheumatology 1990 criteria for FMS and none of them satisfied the diagnostic criteria for celiac disease diagnosis (absence of villous atrophy and negativity for tissue transglutaminase antibodies).IBS and FMS are two chronic functional disorders that are found in a high number of people in the general population and are frequently detected in the same subject [2]. A subset of patients complaining of IBS and FMS displays LE, a morphological finding that by itself is not specific for celiac disease, also being found in many other pathological conditions such as food allergy, autoimmune disorders, Helicobacter pylori infection, nonsteroidal anti-inflammatory drug treatment and common variable immunodeficiency [3].The spectrum of gluten-related disorders has recently acquired a new syndrome, defined as nonceliac gluten sensitivity according to the criteria established in the two Consensus Conferences held in London and Munich [4]. This new clinical entity is characterized by IBS-like symptoms and several extraintestinal manifestations occurring after gluten ingestion in patients without celiac disease and wheat allergy. In a recent prospective multicenter survey of 486 patients with nonceliac gluten sensitivity, IBS and FMS were respectively detected in 47% and 31% of cases and about one-third of these patients had LE [5].Along with IBS-related and FMS-related symptoms, the patients studied by Rodrigo and colleagues also showed other manifestations resembling the clinical picture of nonceliac gluten sensitivity such as skin rash, cognitive dysfunction, headache, numbness, anxiety and depression [1].In Rodrigo and colleagues’ paper, the gluten-free diet produced a slight but significant improvement of both IBS-related (chronic abdominal pain, changes in intestinal habit, bloating) and FMS-related symptoms (chronic widespread pain, generalized tender points, fatigue and restless sleep) in the LE subgroup versus the non-LE subgroup. These results stress the potential role of gluten as a trigger of the clinical manifestations of IBS and FMS and indicate that LE might be useful to identify those patients who potentially benefit from gluten withdrawal. One relevant limitation of this study is the lack of a double-blind placebo-controlled challenge, which is the only procedure to confirm the role of gluten proteins in the development of these clinical manifestations. Indeed, it cannot be ruled out that some patients displayed a subjective sensation of improvement due to the placebo effect of a gluten-free diet [6]. The search for antigliadin antibodies could be of help to elucidate whether gluten can be partly responsible for the clinical picture observed in Rodrigo and colleagues’ patients. Indeed, antigliadin antibodies (particularly those belonging to the IgG class) are the only marker observed in patients with symptoms elicited by gluten ingestion, being positive in more than 50% of cases [7]. These antibodies are not specific for gluten-related symptoms, but their finding in patients with symptoms potentially evoked by gluten ingestion should be regarded as an indication for a gluten-free diet trial in patients with LE [8]. Antigliadin antibodies of the IgG class are closely related to the gluten-induced symptoms and tend to disappear very quickly (within a few weeks) together with the remission of symptoms after a gluten-free diet [9].An interesting finding emerging from the Spanish study is that about 20% of IBS/FMS patients with LE had relatives with celiac disease, whereas no familial case of celiac disease was observed among patients without LE [1]. In the same guise, familial cases of FMS were found, although to a lesser extent, only in the group with LE (7%). These data suggest that first-degree relatives of IBS/FMS patients with LE should be carefully investigated for the possible presence of undetected cases of celiac disease and FMS. For LE, the mean IEL number reported in Rodrigo and colleagues’ paper was 35/100. This result confirms that LE found in gluten-sensitive patients is mild, with a lower mean IEL number than that usually observed in celiac disease patients (usually >40/100) [10].The caution in the conclusions of Rodrigo and colleagues’ study is appreciable and shareable. A gluten-free diet is not appropriate in patients with IBS/FMS with normal intestinal mucosa (normal IEL count). Moreover, although the reported results suggest a significant improvement of symptomatology after a gluten-free diet in the LE subgroup, further studies including double-blind placebo-controlled trials are needed before proposing gluten withdrawal in IBS/FMS patients with LE.     

New Method for Monitoring the Process of Freeze Drying of Biological Materials

A capacitive sensor was proposed and tested for the monitoring and control of a freeze drying process of a vaccine against the Newcastle disease of birds. The residual moisture of the vaccine was measured by the thermogravimetric method. The vaccine activity was determined by titration in chicken embryos. It was shown that, at the stages of freezing and primary drying, a capacitive sensor measured the fraction of unfrozen liquid phase in a material and allowed one to control the sublimation stage of drying in an optimal way. This prevented the foaming of the material and shortened the total drying time approximately twice. The control range at the sublimation stage of drying expanded up to −70°C. It was found at the final stage of drying that the signal of a capacitive sensor passed through a maximum value. We supposed that this maximum corresponds to the minimum of intramolecular mobility of biological macromolecules and hence to the optimal residual moisture of the material, which ensures long-term preservation of its activity. We also suppose that using the capacitive sensor at the final stage of drying allows one to more precisely detect the time when the residual moisture of dried material reaches the optimal value.KEY WORDS: biological materials, capacitive sensor, freeze drying, optimal residual moistureAt present, most biological materials containing live viruses or bacteria are exposed to lyophilization (i.e., drying from the frozen state); this ensures long-term preservation of their activity. Typically, this process consists of preliminary freezing and subsequent freeze drying. The latter process, in turn, consists of two stages: primary drying and secondary drying. During primary drying or sublimation, frozen water is removed from a biological product under vacuum and at temperatures below 0°C. At this stage, the drying rate is limited because of the foaming of a product that occurs due to its high temperature and the excess amount of liquid phase in it. The secondary drying, or final stage, begins after the end of the sublimation stage and occurs at temperatures above 0°C. The goal of the secondary drying is to bring the residual moisture of a biological product to an optimum level, which provides long-term preservation of its activity. Note that the moisture content both above and below the optimum value reduces the effective life of biological materials (1,2)To increase the shelf life of biological products, the following should be investigated: (1) the influence of the composition of the dried biological product and the residual moisture on the change in its activity over the time (3); (2) it is needed to optimize the sublimation drying process for different types of biological products (4). For the investigation of the of the state of water in the dried biologic drugs and the influence of the humidity of the biological on the change in their activity during shelf life, different physical methods are used such as neutron scattering (5), nuclear magnetic resonance (NMR) (6,7), Raman spectroscopy (8), infrared spectroscopy, differential scanning calorimetry, thermal activity monitor (9), and gravimetric sorption analysis (10). The investigations using these methods allow to find an optimum composition of a protective medium for biologics and to determine its optimal residual moisture.At all stages of the freeze drying, the parameters of the material and the parameters of the drying process (temperature of a material, the shelf temperature, the condenser temperature, the pressure in the sublimation chamber, etc.) are also monitored. According to these data, the mode of the process is selected to conduct him for the minimum time and get the best product quality (11). Usually during the drying process, the temperature is measured in several vials with biologic located on different shelves. The sharp increase of the temperature indicates the end of primary drying and the beginning of the secondary drying. The finish of the sublimation stage is revealed by a sharp decrease of the partial pressure of water vapor in the sublimation chamber (12,13). Note that the partial pressure of water vapor in the sublimation chamber does not characterize the state of the biological product to be dried and it is an indirect parameter. For monitoring and controlling the process of freeze drying, it is important to use the own properties of biological materials. In (14), a resistivity sensor placed in a frozen biological material was proposed to control the primary stage of freeze drying. A disadvantage of this method is that one cannot establish an unambiguous relationship between the amount of liquid phase in the frozen material and the value of resistivity: the resistance of the sensor depends not only on the amount of liquid phase but also on the concentration of dissolved salts. Another disadvantage of the resistivity sensor is that, when the temperature decreases, the resistivity of the material sharply increases to values that are difficult to measure, which makes impossible the control of the sublimation stage with this sensor.In (15,16), the interesting methods for determining the moisture of biological materials during secondary drying were proposed. These methods are based on the measurement of the partial pressure of water vapors in the sublimation chamber by NIR spectroscopy or Raman spectroscopy. Note that this method is indirect and requires laborious calibration to establish a correspondence between the current moisture of the biological material in vials and the pressure of water vapor in the sublimation chamber.It should be noted that one has to carry out a series of long-term experiments to find the optimal residual moisture of a biological product. These experiments result in the lifetimes of biological samples with various residual moistures. As the optimal residual moisture of a biological product, one takes the value that provides the longest term preservation of its activity.However, finding the optimal conditions of freeze drying has traditionally been a process of trial and error and required several experimental runs (17). Note also that the freeze drying process is time-consuming and labor intensive.A promising method for the investigation of the properties of biological materials is dielcometry (18,19). This method is relatively simple and very informative since it gives information about the structure of biological macromolecules and the state and role of water in the biological material, etc. This method was used in (20–22) for monitoring biological materials at the primary stage of freeze drying. In (20), authors had found an anomalous low-frequency dispersion of the dielectric permittivity in the biological under study and explain this phenomenon by the proton transfer among water molecules, connected by hydrogen bonds The dielectric relaxation time turned out to be sensitive to the loss of moisture content in the product, and the authors suggested to use of this phenomenon to determine the end point of the freeze drying process. The authors mounted the electrodes of the capacitive sensor on the outer surface of vials with the material to be dried. This approach allows monitoring the sublimation rate and determining the end of the primary stage of freeze drying. Unfortunately, the sensitivity of the capacitive sensor of this design is not enough for the reliable monitoring of the stage of secondary drying.In this paper, a new design of a capacitive sensor and measurement technique are proposed that enable monitoring all stages of the drying process: the freezing stage, the sublimation stage, and the final stage. During freezing and the sublimation stages, the sensor monitors the amount of liquid phase in the frozen material. This allows an optimal control during the whole sublimation stage which prevents the foaming of the material and significantly reduces the total drying time. The sensor also fixes the end of the sublimation stage and the beginning of the final stage of drying. At this stage, the high sensitivity of the measuring system enables one to discover that there is a certain time interval when the signal of the capacitive sensor passes through a maximum. We believe that this maximum corresponds to the minimum of the molecular mobility of biological macromolecules and the optimal residual moisture of the material to be dried.     

Suberin-Associated Fatty Alcohols in Arabidopsis: Distributions in Roots and Contributions to Seed Coat Barrier Properties

Suberin is found in a variety of tissues, such as root endoderms and periderms, storage tuber periderms, tree cork layer, and seed coats. It acts as a hydrophobic barrier to control the movement of water, gases, and solutes as well as an antimicrobial barrier. Suberin consists of polymerized phenolics, glycerol, and a variety of fatty acid derivatives, including primary fatty alcohols. We have conducted an in-depth analysis of the distribution of the C18:0 to C22:0 fatty alcohols in Arabidopsis (Arabidopsis thaliana) roots and found that only 20% are part of the root suberin polymer, together representing about 5% of its aliphatic monomer composition, while the remaining 80% are found in the nonpolymeric (soluble) fraction. Down-regulation of Arabidopsis FATTY ACYL REDUCTASE1 (FAR1), FAR4, and FAR5, which collectively produce the fatty alcohols found in suberin, reduced their levels by 70% to 80% in (1) the polymeric and nonpolymeric fractions from roots of tissue culture-grown plants, (2) the suberin-associated root waxes from 7-week-old soil-grown plants, and (3) the seed coat suberin polymer. By contrast, the other main monomers of suberin were not altered, indicating that reduced levels of fatty alcohols did not influence the suberin polymerization process. Nevertheless, the 75% reduction in total fatty alcohol and diol loads in the seed coat resulted in increased permeability to tetrazolium salts and a higher sensitivity to abscisic acid. These results suggest that fatty alcohols and diols play an important role in determining the functional properties of the seed coat suberin barrier.Suberin is a cell wall-linked polymeric barrier that plays a critical role in the survival of plants by protecting them against various biotic and abiotic stresses. It primarily acts as a hydrophobic barrier to control the movement of water, gases, and solutes, but also contributes to the strength of the cell wall (Ranathunge et al., 2011). Suberin is deposited at the inner face of primary cell walls next to the plasma membrane (Kolattukudy, 1980; Franke and Schreiber, 2007). It is typically found as lamellae (alternating dark and light bands when viewed by transmission electron microscopy) in the endodermis, exodermis, and peridermis of roots, as well as in the peridermis of underground storage tubers (Bernards, 2002). Suberin is also found in shoot periderms of trees (i.e. cork layer) and in seed coats (Molina et al., 2006, 2008) and is deposited in response to wounding (Kolattukudy, 2001).Suberin is a polymer consisting of aliphatics (fatty acid derivatives), phenolics, and glycerol. The predominant aliphatic components of suberin are ω-hydroxy fatty acids, α,ω-dicarboxylic acids, very-long-chain fatty acids, and primary fatty alcohols, while the major phenolic components are p-hydroxycinnamic acids, especially ferulic acid (Kolatukudy, 1980; Bernards et al., 1995; Pollard et al., 2008; Ranathunge et al., 2011). In the periderm of underground storage organs, suberin is found in association with waxes, which are isolated either by extensive extraction in solvent (Soliday et al., 1979; Serra et al., 2009) or by brief immersion of tubers in chloroform (Espelie et al., 1980). These suberin-associated waxes consist of linear aliphatics (e.g. alkanes, fatty acids, and fatty alcohols), which are similar to cuticular wax components of aerial tissues but generally of shorter chain lengths (Espelie et al., 1980). In waxes extracted from 3-week-old wounded potato (Solanum tuberosum) periderms, alkyl ferulates (i.e. ferulic acid linked by an ester bond to a C16:0–C32:0 fatty alcohol) represent up to 60% of the total wax load (Schreiber et al., 2005). Root waxes are also found in 6- to 7-week-old mature taproots of Arabidopsis (Arabidopsis thaliana) with a fully developed periderm (Li et al., 2007; Kosma et al., 2012). They are enriched in alkyl hydroxycinnamates (AHCs) made of C18:0 to C22:0 fatty alcohols esterified with coumaric, caffeic, or ferulic acids (Kosma et al., 2012). The monomer composition (in terms of major chemical species and chain length) of both suberin and suberin-associated waxes varies considerably between plant species, tissues, and developmental stages. Aliphatic suberin and suberin-associated waxes are considered the major contributors to the diffusion resistance of suberized cell walls to radial transport of water and solutes (Soliday et al., 1979; Espelie et al., 1980; Zimmermann et al., 2000; Ranathunge and Schreiber, 2011). The organization of suberin components in the lamellated structure as well as how waxes may be associated with the polymer is a matter of debate (Graça and Santos, 2007).Primary fatty alcohols are long-chain hydrocarbons containing a single hydroxyl group at the terminal position. They are ubiquitously detected as components of the suberin polymer, representing 1% to 10% of the total monomer mass recovered after transesterification (Holloway, 1983; Bernards, 2002; Pollard et al., 2008). Primary fatty alcohols are also typical components of suberin-associated waxes, where they can be found either in free form or linked by an ester bond with a hydroxycinnamic acid (i.e. as AHCs; Soliday et al., 1979; Espelie et al., 1980; Bernards and Lewis 1992; Li et al., 2007; Kosma et al., 2012). In mechanically isolated endodermis of soybean (Glycine max) roots, fatty alcohols represent about 1.5% and 0.2% of the total aliphatics found in suberin-associated waxes and suberin polymer, respectively (Thomas et al., 2007). In onion (Allium cepa) root exodermis, fatty alcohols (C14:0–C28:0) account for 7% to 12% of the soluble fraction, while the suberin fraction contains only C22:0 fatty alcohol, which makes up 3% of the suberin fraction across all exodermal maturation zones (Meyer et al., 2011). In suberizing potato periderms 7 d post wounding, C16:0 to C28:0 fatty alcohols represent about 10% and 18% of the total aliphatics in the insoluble poly(aliphatic) domain (suberin polymer) and in the soluble (nonpolymeric) fraction, respectively (Yang and Bernards, 2006). A similar study on native periderms from 21-d-stored potato (Serra et al., 2009) reported that fatty alcohols represent about 20% of the total aliphatic components found in the suberin polyester, while unlinked fatty alcohols and alkyl ferulates accounted for about 23% and 44% of the total aliphatics in the soluble waxes.In Arabidopsis, C18:0, C20:0, and C22:0 fatty alcohols account for slightly less than 3% of the polymerized aliphatics in roots of soil-grown plants (Domergue et al., 2010), but as much as 36% [w/w] of the soluble wax load (Li et al., 2007). Arabidopsis fatty acyl reductases FAR1 (At5g22500), FAR4 (At3g44540), and FAR5 (At3g44550) generate, respectively, the C22:0, C20:0, and C18:0 fatty alcohol present in the suberin of root, seed coat, and wounded leaf tissues (Domergue et al., 2010). These three enzymes also generate the C18:0 to C22:0 fatty alcohol components that make up AHCs of root waxes (Kosma et al., 2012). Although one particular chain length of primary alcohol was reduced in each far single mutant line (C18:0-OH, C20:0-OH, and C22:0-OH in far5, far4, and far1, respectively), the total fatty alcohol load of the suberin polymer and its composition were only slightly affected and mutant plants had no obvious developmental or physiological defects (Domergue et al., 2010). In this study, we report on the distribution of primary fatty alcohols in the soluble (nonpolymeric) and insoluble (suberin polymer) fractions from mature roots of Arabidopsis. We report that far double and triple mutants have highly reduced fatty alcohol levels, in a chain length-specific manner, in both fractions as well as in the seed coat suberin polymer. The significant reductions in total fatty alcohol and diol levels in the seed coat of these mutants lead to increased permeability and higher sensitivity to abscisic acid (ABA), bringing to light insights on the roles of fatty alcohols and diols in determining functional properties of suberin.