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.     

Peroxisome-driven ether-linked phospholipids biosynthesis is essential for ferroptosis

It is well established that ferroptosis is primarily induced by peroxidation of long-chain poly-unsaturated fatty acid (PUFA) through nonenzymatic oxidation by free radicals or enzymatic stimulation of lipoxygenase. Although there is emerging evidence that long-chain saturated fatty acid (SFA) might be implicated in ferroptosis, it remains unclear whether and how SFA participates in the process of ferroptosis. Using endogenous metabolites and genome-wide CRISPR screening, we have identified FAR1 as a critical factor for SFA-mediated ferroptosis. FAR1 catalyzes the reduction of C16 or C18 saturated fatty acid to fatty alcohol, which is required for the synthesis of alkyl-ether lipids and plasmalogens. Inactivation of FAR1 diminishes SFA-dependent ferroptosis. Furthermore, FAR1-mediated ferroptosis is dependent on peroxisome-driven ether phospholipid biosynthesis. Strikingly, TMEM189, a newly identified gene which introduces vinyl-ether double bond into alkyl-ether lipids to generate plasmalogens abrogates FAR1-alkyl-ether lipids axis induced ferroptosis. Our study reveals a new FAR1-ether lipids-TMEM189 axis dependent ferroptosis pathway and suggests TMEM189 as a promising druggable target for anticancer therapy.Subject terms: Phospholipids, Cancer metabolism

Ether phospholipids represent an important group of phospholipids containing a glycerol backbone with an alkyl or a vinyl bond connecting a fatty alcohol at sn-1 position, usually polyunsaturated fatty acid (PUFA) including docosahexaenoic acid and arachidonic acid at sn-2. Ether phospholipids are initially synthesized in peroxisomes and processed in the endoplasmic reticulum (ER) [1–3]. Plasmalogens are the most abundant form of ether phospholipids which have a vinyl ether bond, enriched in the brain and heart tissues [1–3]. The plasmalogens have been found as endogenous antioxidants with vinyl ether bond susceptible to cleavage by reactive oxygen species (ROS). The deficiency of plasmalogens correlates with various human disorders, including Alzheimer’s disease and cancer [1, 2, 4].Ferroptosis is an iron-dependent form of non-apoptotic cell death induced by excess accumulation of peroxidized phopholipids, generated through oxidation of the PUFA moieties at sn-2 position of membrane phospholipids [5–9]. Ferroptosis is morphologically, biochemically and genetically distinct from other forms of cells death [5], which is tightly regulated by glutathione peroxidase 4 (GPX4) via converting lipid hydroperoxides (PUFA-OOH) into non-toxic lipid alcohols (PUFA-OH) [10, 11]. Emerging evidence indicates that ferroptosis is implicated in ischemia–reperfusion injury (IRI), neurodegeneration, antiviral immunity, cancer immunotherapy and tumor suppression [11–19].Accumulating evidence reveals a robust link between lipid metabolism and ferroptosis [14, 20–24]. However, little is known about the role of ether phospholipids in ferroptosis. In the present study, we revealed the FAR1-TMEM189 axis as a central pathway to drive the susceptibility of ferroptosis. FAR1-TMEM189 axis specifically synthesizes alkyl and vinyl ether phospholipid, where the two isoforms of ether phospholipid play distinct role in ferroptosis. Our findings provide an insight into the mechanism of ether phospholipid-mediated ferroptosis, with implications for novel treatment options for cancer therapy.     

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.     

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.     

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.     

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.     

A New Extracellular β-Galactosidase Producing Kluyveromyces sp. PCH397 from Yak Milk and Its Applications for Lactose Hydrolysis and Prebiotics Synthesis

β-Galactosidase is a crucial glycoside hydrolase enzyme with potential applications in the dairy, food, and pharmaceutical industries. The enzyme is produced in the intracellular environment by bacteria and yeast. The present study reports yeast Kluyveromyces sp. PCH397 isolated from yak milk, which has displayed extracellular β-galactosidase activity in cell-free supernatant through the growth phase. To investigate further, cell counting and methylene blue staining of culture collected at different growth stages were performed and suggested for possible autolysis or cell lysis, thereby releasing enzymes into the extracellular medium. The maximum enzyme production (9.94 ± 2.53U/ml) was achieved at 37 °C in a modified deMan, Rogosa, and Sharpe (MRS) medium supplemented with lactose (1.5%) as a carbon source. The enzyme showed activity at a wide temperature range (4–50 °C), maximum at 50 °C in neutral pH (7.0). In addition to the hydrolysis of lactose (5.0%), crude β-galactosidase also synthesized vital prebiotics (i.e., lactulose and galacto-oligosaccharides (GOS)). Additionally, β-fructofuranosidase (FFase) activity in the culture supernatant ensued the synthesis of a significant prebiotic, fructo-oligosaccharides (FOS). Hence, the unique features such as extracellular enzymes production, efficient lactose hydrolysis, and broad temperature functionality by yeast isolate PCH397 are of industrial relevance. In conclusion, the present study unrevealed for the first time, extracellular production of β-galactosidase from a new yeast source and its applications in milk lactose hydrolysis and synthesis of valuable prebiotics of industrial importance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-021-00955-1.Keyword: β-Galactosidase, Lactulose, Galacto-oligosaccharides, Fructo-oligosaccharides, Milk-microbes

β-Galactosidase (EC 3.2.1.23) hydrolyzes the glycosidic bond in β-galactosides and finds applications in the food industry [1, 2]. The trans-glycosylation property of β-galactosidase (β-gal) is widely used to produce various galactosylated products and prebiotics such as GOS and lactulose [3–7]. The β-gal enzyme is produced intracellularly by many bacteria and yeast, a major constraint for industrial production [1, 8]. Therefore, extracellular β-gal producing bacteria/yeast are of huge relevance. Hence, the present work revealed an efficient extracellular β-gal producing microbe from dairy products of the Indian Himalaya and evaluated its applications in lactose hydrolysis and prebiotics’ synthesis.In this study, twenty milk and four curd samples were collected from the Lahaul and Pangi valleys of Himachal Pradesh, India. The samples were plated on MRS and Elliker agar medium (Himedia, India) for 2–7 days at 28 °C and 37 °C until visible microbial growth. Morphologically distinct isolates were screened for β-gal activity using X-Gal and IPTG plate assay [6, 9]. The positive isolates were screened for β-gal production in liquid MRS medium. The β-gal activity was expressed as U/mg dcw (dry cell weight) for whole cells and U/ml for cell-free supernatant [10, 11]. Yeast isolate PCH397 showing the highest and extracellular enzymatic activity was selected. The culture and reaction conditions for maximum β-gal activity were optimized. FFase activity of whole cells and cell-free supernatant was estimated as described by Lincoln and More [12].The cell-free supernatant (β-gal) was employed for applications in lactose hydrolysis and prebiotic synthesis. The enzyme was incubated with lactose solution (5%, w/v) at 37 °C for lactose hydrolysis followed by thin layer chromatography (TLC) [13] analysis and quantification using the ImageJ program (http://rsbweb.nih.gov/ij/). Further, the cell-free supernatant was incubated with milk at 4 °C for milk lactose hydrolysis. Samples were withdrawn at different time intervals and analyzed for residual lactose concentration using ultra-high performance liquid chromatography-quadrupole-time of flight-ion mobility mass spectrometry (UHPLC-Q-TOF-IMS) [14]. Prebiotic production was carried out by mixing an equal volume of the enzyme with a sugar solution i.e., lactose (40%, w/v) for GOS, and lactose (20%, w/v) + fructose (20%, w/v) for lactulose and FOS production, respectively at 50 °C for 24 h [6]. Samples were analyzed by TLC for GOS, UHPLC-Q-TOF-IMS for FOS and lactulose synthesis.The study resulted in the isolation of 203 morphologically distinct microbes, 62 of which were tested positive for β-gal. Based on quantitative screening, eight isolates showing maximum β-gal activity were selected and examined for the intracellular and extracellular enzymatic activities (Table S1). Yeast isolate PCH397 exhibited maximum extracellular β-gal activity (9.94 ± 2.53 U/ml) along with FFase activity (0.59 ± 0.155) after 48 h of incubation. Isolate PCH397 was identified as Kluyveromyces marxianus by its morphological and molecular characterization (Fig. S1). Phylogenetic tree based on ITS DNA sequence showed similarity (99.63%) with Kluyveromyces marxianus CBS712. To the best of our knowledge, the genus Kluyveromyces has not been reported earlier for extracellular β-gal production. In the past, efforts were made to produce β-gal extracellularly through permeabilization or incorporation of signal peptide to β-gal gene in a fusion construct [15, 16]. The isolate PCH397 was selected due to its generally regarded as safe (GRAS) status and the novel feature of extracellular enzyme production.Highest β-gal activity in the extracellular environment was observed when PCH397 was grown in MRS medium supplemented with 1.5% (w/v) lactose as a substrate and incubated at 37 °C for 48 h (Fig. S2). PCH397 produced extracellular β-gal at lower lactose concentration (1.5%) as compared to various Kluyveromyces spp. [15] where 3% lactose has been used in the growth medium for intracellular β-gal production. Further, whether the extracellular enzyme activity is due to the secretion or cell lysis, the CFU count and cell viability were checked by the methylene blue test. The decreased cell count in the late stationary phase for live cells (Fig. S3) and increased number of methylene blue stained cells indicated cell death (Fig S4). These results suggested that cell lysis in the late stationary phase leads to the secretion of enzymes in extracellular medium. The extracellular production of enzyme would lead to a lower production costs of the enzyme.Cell-free supernatant showed the highest β-gal activity at pH 7.0 in 10 mM sodium phosphate buffer at 50 °C in 5 min (Fig S2). The β-gal enzyme from the current finding holds promise in the sweet whey and milk lactose hydrolysis [1] due to its neutral pH optima. Also, β-gal, which is functional at high temperatures, is used in the synthesis of oligosaccharides [1, 3]. High temperature increases the reaction rate as well as lactose solubility, thus, facilitating transgalactosylation reactions [17]. The β-gal activity (9 U/ml) in cell-free supernatant of PCH397 completely hydrolyzed 5.0% of lactose within 8 h at 37 °C (Fig. 1a, S5a). In a recent study, 5.0% lactose was also hydrolyzed by purified β-gal (5 U/ml) of Paenibacillus barengoltzii CAU904 within 8 h at 40 °C [13]. Under refrigerated conditions (4 °C), the cell free supernatant hydrolyzed ~ 50% milk lactose within 36 h and ~ 80% in 72 h (Fig. 1b, S5b). Since β-gal of PCH397 is active at 4 °C, the enzyme could be utilized to hydrolyze lactose in dairy products under refrigerated conditions. Lactose-free milk products or low-lactose milk products are important dietary constituents for lactose intolerant individuals and deliver essential nutrients to combat nutritional deficiencies [18]. Even with commercially purified enzymes, 100% milk-lactose hydrolysis could not be achieved at a low temperature [19]. However, the crude enzyme from the present investigation can efficiently hydrolyze milk lactose at ambient and refrigerated conditions, reducing the cost associated with enzyme purification. Additionally, the source of enzyme is Kluyveromyces sp. which has GRAS status, therefore, can be used in food applications.Open in a separate windowFig. 1Lactose hydrolysis by crude β-gal of PCH397. a Relative quantification of the hydrolysed products from lactose (5%, w/v) at 37 °C for 24 h. b Relative decrease in lactose concentration (%) at refrigerated conditions obtained by UHPLC-QTOF-IMSFurther, the enzyme was evaluated for its ability to catalyze transgalactosylation reactions at 50 °C. The crude enzyme was incubated with different substrate mixture viz. lactose and fructose. After 8 h of incubation, 50% of lactose was hydrolyzed into glucose, galactose, and GOS (Fig. S6a). Maximum GOS production was achieved after 12 h (Fig. 2a). The purified β-gal from Paenibacillus barengoltzii synthesized GOS from 350 g/L of lactose within 4 h [13]. Though GOS synthesis was faster in comparison to the current study, it is to be noted that we used a crude enzyme mixture instead of a purified enzyme. The crude enzyme has also shown FFase activity (Table S1), and was used for the synthesis of FOS from lactose and fructose mixture. UHPLC-Q-TOF-IMS analysis confirmed the formation of FOS (Fig. 2b). Multiple peaks were observed in the sample containing lactulose, one of which was identical with the peak of lactulose standard (Fig. 2c) as confirmed by HPAEC-PAD (Fig. S6b). The lactulose formation was maximum at 20 h of incubation (Fig. S6c).Open in a separate windowFig. 2Hydrolysis and transgalactosylation of lactose by crude enzyme from PCH397 having β-gal and FFase activity. a Relative quantification of the hydrolyzed and transgalactosylated products. UHPLC-QTOF-IMS detection of prebiotics b FOS and c lactulose with their respective standardIt is the first report of simultaneous co-synthesis of multiple prebiotics i.e., GOS, FOS, and lactulose using a yeast strain. Similar reports for GOS and FOS synthesis have been attempted by enzymatic means from fungal sources in the past [6]. The synthesis of multiple prebiotics is very advantageous. Numerous studies have shown that blended consumption of multiple prebiotics including GOS and FOS has many health benefits [20–24]. The combination of GOS, FOS, and lactulose can be of considerable importance for their prebiotic applications. In conclusion, our findings revealed a yeast source for the cost-effective production of β-galactosidase and a strategy for co-synthesis of valuable prebiotics, which is not reported in the past. The utilization of a yeast source with GRAS status for lactose hydrolysis and co-synthesis of prebiotics promises various health benefits and commercial relevance.     

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     

PDK1 Regulates Vascular Remodeling and Promotes Epithelial-Mesenchymal Transition in Cardiac Development

One essential downstream signaling pathway of receptor tyrosine kinases (RTKs), such as vascular endothelial growth factor receptor (VEGFR) and the Tie2 receptor, is the phosphoinositide-3 kinase (PI3K)-phosphoinositide-dependent protein kinase 1 (PDK1)-Akt/protein kinase B (PKB) cascade that plays a critical role in development and tumorigenesis. However, the role of PDK1 in cardiovascular development remains unknown. Here, we deleted PDK1 specifically in endothelial cells in mice. These mice displayed hemorrhage and hydropericardium and died at approximately embryonic day 11.5 (E11.5). Histological analysis revealed defective vascular remodeling and development and disrupted integrity between the endothelium and trabeculae/myocardium in the heart. The atrioventricular canal (AVC) cushion and valves failed to form, indicating a defect in epithelial-mesenchymal transition (EMT), together with increased endothelial apoptosis. Consistently, ex vivo AVC explant culture showed impeded mesenchymal outgrowth. Snail protein was reduced and was absent from the nucleus in AVC cells. Delivery of the Snail S6A mutant to the AVC explant effectively rescued EMT defects. Furthermore, adenoviral Akt delivery rescued EMT defects in AVC explant culture, and deletion of PTEN delayed embryonic lethality of PDK1 endothelial deletion mice by 1 day and rendered normal development of the AVC cushion in the PDK1-deficient heart. Taken together, these results have revealed an essential role of PDK1 in cardiovascular development through activation of Akt and Snail.Polypeptide growth factors, such as insulin, insulin-like growth factor 1 (IGF-I), vascular endothelial growth factor (VEGF), and angiopoietin 1 (Ang1), exert biological functions through binding to their transmembrane receptors that belong to a large family of receptor tyrosine kinases (RTKs) (4). Consequently, the receptor molecules form homo- or heterodimers, and the intracellular tyrosines at the carboxyl termini of the receptors become phosphorylated (37). Numerous distinct adaptor/regulatory proteins, through their Src homologous 2 (SH2) domains, bind to the phosphotyrosines and transduce the signal to downstream pathways, among which are two essential and well-characterized signaling cascades—the mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3K)-phosphoinositide-dependent protein kinase 1 (PDK1)-Akt signaling pathways (4, 13, 37).The regulatory subunit of PI3K, p85, possesses the SH2 domain and can, therefore, bind to phosphotyrosines on the RTKs and subsequently render activation of the catalytic subunit of PI3K, p110 (7, 8). Active p110 phosphorylates phosphoinositide biphosphate (PIP2), turning it into PIP3 that recruits PDK1 and Akt to the cellular membrane, where Akt is phosphorylated at threonine 308 (T308 for Akt1) by PDK (5, 23, 30). The serine 473 (S473) of Akt (Akt1) is phosphorylated by mTOR complex 2 (mTORC2) and other kinases (17, 36). Phosphorylation of Akt at these two amino acids brings it to full activation. In PDK1-deficient embryonic stem (ES) cells, T308 phosphorylation was abolished and most of the Akt activity was lost, although the S473 phosphorylation was intact (40).Akt plays an important role in multiple biological processes, such as cell survival, growth, glucose metabolism, and angiogenesis (2, 12, 14-16, 22, 23, 39, 41-43). In mammals, there are three Akt isoforms, termed Akt 1, -2, and -3. Previously, we generated Akt1- and Akt3-deficient mice and studied their roles in mouse development (2, 15, 39, 42, 43). We found that the Akt1 and -3 double knockout (KO) (DKO) mice were embryonically lethal at around embryonic day 12 (E12) and manifested developmental defects in multiple tissues, including the cardiovascular system (14, 15, 43). These studies suggest that the Akt signaling pathway is involved in cardiovascular development.Other than Akt isoforms, PDK1 also activates another group of AGC family kinases, such as p70 ribosomal S6 kinase (S6K) (32), serum, and glucocorticoid-induced protein kinase (SGK) (26), p90 ribosomal S6 kinase (RSK) (21), and atypical isoforms of protein kinase C (PKC) (31). Comprehensive and intensive mouse genetic studies performed mainly by Alessi and coworkers have confirmed the regulation of these AGC kinases by PDK1 (3, 9, 10, 27-29, 40).PDK1 knockout mice were severely growth retarded and died at around E9.0, indicating an essential role of PDK1 in development (27). However, its function and downstream targets in cardiovascular development are still elusive. To study this, we deleted PDK1 specifically in endothelial cells through Cre recombinase-mediated excision (25). The results have revealed an essential role of PDK1 in vascular remodeling and integrity and in cardiac development through activation of Akt and its downstream target of Snail.     

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.     

Activation of the PI3K/Akt Pathway Early during Vaccinia and Cowpox Virus Infections Is Required for both Host Survival and Viral Replication

Viral manipulation of the transduction pathways associated with key cellular functions such as actin remodeling, microtubule stabilization, and survival may favor a productive viral infection. Here we show that consistent with the vaccinia virus (VACV) and cowpox virus (CPXV) requirement for cytoskeleton alterations early during the infection cycle, PBK/Akt was phosphorylated at S473 [Akt(S473-P)], a modification associated with the mammalian target of rapamycin complex 2 (mTORC2), which was paralleled by phosphorylation at T308 [Akt(T308-P)] by PI3K/PDK1, which is required for host survival. Notably, while VACV stimulated Akt(S473-P/T308-P) at early (1 h postinfection [p.i.]) and late (24 h p.i.) times during the infective cycle, CPXV stimulated Akt at early times only. Pharmacological and genetic inhibition of PI3K ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) or Akt (Akt-X and a dominant-negative form of Akt-K179M) resulted in a significant decline in virus yield (from 80% to ≥90%). This decline was secondary to the inhibition of late viral gene expression, which in turn led to an arrest of virion morphogenesis at the immature-virion stage of the viral growth cycle. Furthermore, the cleavage of both caspase-3 and poly(ADP-ribose) polymerase and terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling assays confirmed that permissive, spontaneously immortalized cells such as A31 cells and mouse embryonic fibroblasts (MEFs) underwent apoptosis upon orthopoxvirus infection plus {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 treatment. Thus, in A31 cells and MEFs, early viral receptor-mediated signals transmitted via the PI3K/Akt pathway are required and precede the expression of viral antiapoptotic genes. Additionally, the inhibition of these signals resulted in the apoptosis of the infected cells and a significant decline in viral titers.The family Poxviridae is a family of large, linear, double-stranded DNA viruses that carry out their entire life cycle within the cytoplasmic compartment of infected cells. Vaccinia virus (VACV) is a prototypical member of the genus Orthopoxvirus, which also includes the closely related cowpox virus (CPXV) (12, 52). The genomes of these viruses are approximately 200 kbp in length, with a coding capacity of approximately 200 genes. The genes involved in virus-host interactions are situated at both ends of the genome and are associated with the evasion of host immune defenses (1). These evasion mechanisms operate mainly extracellularly. For example, the secretion of soluble cytokine and chemokine receptor homologues blocks the receptor recognition by intercepting the cognate cytokine/chemokine in the extracellular environment. This mechanism facilitates viral attachment and entry into cells (1, 70). Therefore, decoy receptors for alpha interferon (IFN-α), IFN-β, IFN-γ, and tumor necrosis factor alpha play an important immunomodulatory role by affecting both the host antiviral and apoptotic responses.To counteract the host proapoptotic response, poxviruses have developed a number of antiapoptotic strategies, including the inhibition of apoptotic signals triggered by the extrinsic pathway (those mediated by death receptors such as tumor necrosis factor and Fas ligand) or the intrinsic pathway (mediated by the mitochondria and triggered upon viral infection) (1, 25, 70, 74). Many studies previously identified viral inhibitors that block specific steps of the intrinsic pathway. These include the VACV-encoded E3L, F1L, and N1L genes and the myxoma virus (MYXV)-encoded M11L gene, which block cytochrome c release (14, 20, 34, 39, 45, 75, 90), and the CPXV-encoded cytokine response modifier gene (CrmA) as well as the VACV-encoded SPI-2 gene, which inhibits both caspase-1 and caspase-8 (25, 58, 61, 74).An emerging body of evidence has also highlighted the pivotal role played by intracellular signaling pathways in Orthopoxvirus biology (18, 48, 92). We and others have shown that poxvirus manipulation of signaling pathways can be virus specific. For example, while both VACV and CPXV stimulate the MEK/extracellular signal-regulated kinase (ERK)/EGR-1 pathway during a substantial length of time of their infective cycle, the pathway is required only for VACV replication, whereas its role in CPXV biology has yet to be identified (71). MYXV, a rabbit-specific poxvirus, also activates the MEK/ERK pathway in a mouse model of poxvirus-host interactions. However, this stimulation led to the expression of IFN-β, which consequently blocked virus replication and possibly explains why MYXV has such a restricted host range (87).Another signaling molecule associated with viral replication is Akt kinase (also known as protein kinase B). The MYXV host range factor M-T5 is able to reprogram the intracellular environment, thereby increasing human tumor cell permissiveness to viral replication, which is directly associated with levels of phosphorylated Akt (88). In addition, M-T5 is functionally replaced by the host phosphatidylinositol 3-kinase (PI3K) enhancer A protein (92).The transmission of intracellular signals mediated by the serine/threonine kinase Akt to downstream molecules in response to diverse stimuli such as growth factors, insulin, and hormones is dependent upon the phosphorylation of serine 473 (S473-P) and threonine 308 (T308-P). This phosphorylation is mediated by mammalian target of rapamycin complex 2 (mTORC2) and phosphoinositide-dependent protein kinase 1 (PDK1), which act as downstream effectors of the PI3K/Akt/mTORC1 pathway (2, 66). PI3Ks are a family of enzymes (classes I to III) that generate lipid second messengers by the phosphorylation of plasma membrane phosphoinositides. Class IA PI3Ks consist of a catalytic subunit (p110, comprising the three isoforms α, β, and δ) and an adaptor/regulatory subunit (p85, comprising the two isoforms α and β) (for a detailed review, see reference 80).The Akt family of proteins is comprised of the three isoforms α, β, and γ, which are composed of an N-terminal pleckstrin homology domain, a central catalytic domain, and a C-terminal hydrophobic domain. Akt is recruited to the plasma membrane through the binding of its pleckstrin homology domain to the phosphatidylinositol 3,4,5-triphosphate (PIP3), which is a product of PI3K that is anchored to the plasma membrane. PDK1 is also recruited to the plasma membrane through interactions with PIP3. As both PDK1 and Akt interact with PIP3, PDK1 colocalizes with Akt and activates it by phosphorylating threonine 308 (T308-P) (2, 66). Following its activation, Akt phosphorylates a number of downstream substrates such as caspase-9, BAD, glycogen synthase kinase 3β (GSK-3β), and FKHR. This leads to the suppression of apoptosis, cell growth, survival, and proliferation (11, 16, 56).Another downstream target of PI3K/Akt is mTOR, a serine/threonine kinase that plays a central role in the regulation of cell growth, proliferation, survival, and protein synthesis (26). mTOR kinase has recently been found to be associated with two functionally distinct complexes in mammalian cells, known as mTORC1 and mTORC2 (63, 66). Although these multiprotein complexes share molecules in common, distinct adaptor proteins are recruited into each complex: regulatory-associated protein of TOR (raptor) is recruited into mTORC1, while rapamycin-insensitive companion of TOR (rictor) is recruited into mTORC2 (33, 64). While mTORC1 controls cell growth and protein translation and has proven to be rapamycin sensitive, mTORC2 regulates the actin cytoskeleton and is assumed to be rapamycin insensitive, even though under conditions of prolonged exposure to the drug, it appears to inhibit mTORC2 assembly (29, 64, 65). Additionally, it has been demonstrated that mTORC2 regulates the activity of Akt through the phosphorylation of S473 (S473-P). S473-P appears to be required for the full activation of Akt, since S473-P has been shown to enhance the subsequent phosphorylation of T308 by PDK1 (66, 67, 94). Moreover, the phosphorylation of both S473 and T308 results in a four- to fivefold increase in Akt activity compared to T308-P by PDK1 alone (66).The PI3K/PDK1/Akt(T308)/mTORC1 pathway regulates vital cellular processes that are important for viral replication and propagation, including cell growth, proliferation, and protein translation. This pathway is particularly important for the replication of DNA viruses, as their replication is cap dependent. However, the Akt signaling pathway can also negatively affect viral replication. The stress response downstream of Akt signaling, including hypoxia and energy and amino acid depletion, inhibits mTORC1 (5, 9, 69). Therefore, DNA viruses must overcome these constraints to translate their mRNAs.Pharmacological disruption of the PI3K/Akt pathway with the specific PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (2-morpholino-8-phenyl-4H-1-benzopyran-4-one) (82) has been reported to not only increase the cleavage of downstream molecules associated with proapoptotic activity [e.g., poly(ADP-ribose) polymerase (PARP) and the executioner caspase-3] (38, 41) but also promote microtubule stabilization, actin filament remodeling/cell migration, and bleb formation/viral infectivity (10, 35, 49, 54, 59).Because the PI3K/Akt and PI3K/Akt/mTOR pathways influence diverse cellular functions and possibly a healthy antiviral response, usurping these pathways could support an increase in viral replication. In support of this, a number of reports have demonstrated that either the PI3K/Akt or the PI3K/Akt/mTOR pathway plays a role in the replication of many viruses including flavivirus (38), hepatitis C virus (27), human immunodeficiency virus type 1 (93), human papillomavirus (44, 96), respiratory syncytial virus (77), coxsackievirus B3 (19), Epstein-Barr virus (17, 50, 73), human cytomegalovirus (36, 37, 72), herpes simplex virus type 1 (7, 83), varicella-zoster virus (60), Kaposi''s sarcoma-associated herpesvirus (89), adenovirus (55), and simian virus 40 (SV40) (95). With this in mind, we also investigated whether the PI3K/Akt pathway played a pivotal role in orthopoxvirus biology. In this study, we show that the VACV- and CPXV-stimulated PI3K/Akt pathway not only contributes to the prevention of host-cell death but also plays a beneficial role in the viral replication cycle.     

Irreversible Electroporation for Microbial Control of Drugs in Solution

The purpose of this study was to examine the feasibility of using irreversible electroporation (IRE) as a non-chemical method for eliminating microorganisms of liquid drugs. The studied drug was a topical ophthalmic medication, a pharmaceutical field in which the problem of microbial contamination has not yet been adequately solved, especially in the case of eye drops prescribed for chronic use. Commercially available Hylo-Comod® preservative-free eye drop solution was subjected to contamination with Escherichia coli bacteria (10⁶ colony forming units/mL). Electroporation parameters for bacterial control were investigated by comparing the effects of electrical fields of 5.4, 7.2, and 10 kV/cm, delivered as 100-µs square pulses at 1 Hz in sequences of 10 pulses, 20 pulses, or 20 pulses delivered as four sets of five pulses with 1-min intervals between each set. Microorganism survival after treatment was determined by pour plate counting. Effects of the treatment parameters on temperature and pH were recorded. Bacterial survival was lowest (0.14% ± 0.03%) after application of 20 pulses delivered as four separate sets. With that application mode, the solution remained at pH 7.5 and the temperature rose to 35.6° ± 0.2°C. Because IRE can be efficiently delivered under conditions that avoid the potentially deleterious effects of electrical pulses on temperature and pH, it appears to be a feasible method for bacterial control of drugs in solution. The principles established in this study can be applied to any drug in solution and optimized individually according to the solution''s composition.Key words: contamination, eye drops, irreversible electroporation, microorganisms, preservatives, sterilization of drugsContamination of liquid drugs can have substantial detrimental effects on the health of patients using drugs (1,2), necessitating the addition of preservatives in many pharmaceutical preparations. A particularly significant problem is the presence of preservatives in pediatric vaccination and the possible association with neurodevelopmental disorders such as autism (3,4). While this association is highly controversial, eliminating the need for preservatives in the vaccination will serve to allay the apprehension among parents and may increase the use of vaccinations. We focus here on the contamination of topical ophthalmic medications. The problem of infections engendered by microorganisms in eye drops has not yet been adequately solved and is especially troublesome when eye drops are used chronically for many years, as in glaucoma patients. In addition, patients who suffer from dry eyes and do not use the more expensive single-unit dose preparations are exposed to similar risks of infection, as are contact lens users. The problem of contamination can also arise in cases of acute eye drop treatment spanning days or weeks.The prevalence of bacteria in anti-hypertensive glaucoma drops in the community setting has been documented in a number of studies. Geyer et al. found bacteria in more than 28% of in-use topical medications (bottle tips and drops) of 109 treated glaucoma patients (5). The contamination rate was significantly related to the time since the container was first opened; bacteria were detected in 40% of eye drops from bottles that had been opened more than 8 weeks earlier compared to 19% in bottles in use for less time. Similar findings were reported by Schein et al. (6) in drops used by patients suffering from ocular surface diseases. Lower rates (12.8% and 12.9%) have also been reported (7,8). The high contamination rate is not surprising given the way in which eye drop containers are handled by patients. More than half of all elderly patients in one study were found to touch the eyelid or conjunctiva with the container, undoubtedly causing the solution to become infected by flora of the skin and conjunctiva (9). In contrast, a much lower contamination rate (2.3%) was measured in drops used by medical personnel in a clinic (10).Growth of microorganisms in ophthalmic medications can be reduced to some extent by adding preservatives to the solution, typically benzalkonium chloride (BAK). However, since the contamination rates cited above were found in eye drops that contain preservatives, their presence obviously does not solve the problem. Moreover, all preservatives have considerable side effects, particularly when the medications are used on a chronic basis. BAK, which is used in most topical ophthalmic preparations, harms the surface of the eye and probably accounts for the finding that well over half of treated glaucoma patients suffer from symptoms and signs of dry eyes (11). This compound can actually be used to induce inflammation when producing a dry eye model in rabbits (12). Not only does BAK damage the superficial eye tissues but its chronic administration apparently also harms the trabecular meshwork and thus may counteract the anti-hypertensive ocular drugs in which it helps control the bacterial load (13). Newer preservatives might be less injurious to the eyes than BAK, but they too are not free of complications (14), and they have not been in use long enough for their possible effects to be precisely determined. As expected, preservative-free medications seem to produce the least complications (15). One possible way to overcome the problem might be through the use of eye drops packaged in single-unit dose containers. These, however, are expensive and are not generally used for the glaucoma drugs financed by health maintenance organizations. Furthermore, many elderly patients find the containers difficult or impossible to manipulate properly (9).Clearly, then, it is important to find a non-chemical, practical method of bacterial control in liquid fluids in their delivery containers. Accordingly, the aim of this study was to examine the feasibility of using irreversible electroporation (IRE) as a method for controlling bacterial contamination in liquid drugs. Electroporation is a physical phenomenon in which a cell membrane becomes permeabilized by application of short (microsecond-scale) electrical pulses across the living cell. The mechanism presumably operates by forming nanoscale defects in the cell membrane. The overall effect of the electrical pulses is a function of various pulse parameters such as pulse length, pulse amplitude, and number of pulse repeats. These parameters determine whether the cell membrane will remain intact or will become permeabilized, either reversibly (reversible electroporation) or irreversibly (IRE). The nature of both reversible and irreversible electroporation, and their uses, most widely in the food industry, are well documented and have been comprehensively reviewed in the scientific literature (16–26).We postulated that IRE can be used as a means of bacterial control in fluid drug containers, either for the whole volume or during the passage of fluid into and out of the container. The issues to be addressed when treating drugs by IRE are different from those documented in the case of foods. With drugs, the volume of the solvent is significantly smaller, its ionic content is proportionately much larger (25), and the solution conditions after IRE [in particular temperature and pH (25,26)] should remain unchanged to avoid their potentially undesirable effects on the drug. These issues are addressed in this preliminary study on the use of IRE in bacterial control in liquid drugs. We first studied the effects of IRE in a liquid ophthalmic preparation and at a volume typical of eye drop containers. We next investigated what are the IRE pulse modes capable of maximal reduction in bacterial contents of the solution in these small containers without substantially affecting its temperature. Finally, we examined the effects of the IRE pulses on the pH of a small volume of solution.     

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.     

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.     

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.