Hsf1 and the molecular chaperone Hsp90 support a 'rewiring stress response' leading to an adaptive cell size increase in chronic stress.

Cells are exposed to a wide variety of internal and external stresses. Although many studies have focused on cellular responses to acute and severe stresses, little is known about how cellular systems adapt to sublethal chronic stresses. Using mammalian cells in culture, we discovered that they adapt to chronic mild stresses of up to two weeks, notably proteotoxic stresses such as heat, by increasing their size and translation, thereby scaling the amount of total protein. These adaptations render them more resilient to persistent and subsequent stresses. We demonstrate that Hsf1, well known for its role in acute stress responses, is required for the cell size increase, and that the molecular chaperone Hsp90 is essential for coupling the cell size increase to augmented translation. We term this translational reprogramming the 'rewiring stress response', and propose that this protective process of chronic stress adaptation contributes to the increase in size as cells get older, and that its failure promotes aging.

SARS-CoV-2 convalescence and hybrid immunity elicits mucosal immune responses.

BACKGROUND: Mucosal antibodies play a key role in the protection against SARS-CoV-2 infection in the upper respiratory tract, and potentially in limiting virus replication and therefore onward transmission. While systemic immunity to SARS-CoV-2 is well understood, we have a limited understanding about the antibodies present on the nasal mucosal surfaces. METHODS: In this study, we evaluated SARS-CoV-2 mucosal antibodies following previous infection, vaccination, or a combination of both. Paired nasal fluid and serum samples were collected from 143 individuals, which include convalescent, vaccinated, or breakthrough infections. FINDINGS: We detected a high correlation between IgG responses in serum and nasal fluids, which were higher in both compartments in vaccinated compared to convalescent participants. Contrary, nasal and systemic SARS-CoV-2 IgA responses were weakly correlated, indicating a compartmentalization between the local and systemic IgA responses. SARS-CoV-2 secretory component IgA (s-IgA) antibodies, present exclusively on mucosal surfaces, were detected in the nasal fluid only in a minority of vaccinated subjects and were significantly higher in previously infected individuals. Depletion of IgA antibodies in nasal fluids resulted in a tremendous reduction of neutralization activity against SARS-CoV-2, indicating that IgA is the crucial contributor to neutralization in the nasal mucosa. Neutralization against SARS-CoV-2 was higher in the mucosa of subjects with previous SARS-CoV-2 infections compared to vaccinated participants. INTERPRETATION: In summary, we demonstrate that currently available vaccines elicit strong systemic antibody responses, but SARS-CoV-2 infection generates higher titers of binding and neutralizing mucosal antibodies. Our results support the importance to develop SARS-CoV-2 vaccines that elicit mucosal antibodies. FUNDING: The work was funded by the COVID-19 National Research Program 78 (grant number 198412) of the Swiss National Science Foundation.

Altered cell function and increased replication of rhinoviruses and EV-D68 in airway epithelia of asthma patients.

Introduction: Rhinovirus (RV) infections constitute one of the main triggers of asthma exacerbations and an important burden in pediatric yard. However, the mechanisms underlying this association remain poorly understood. Methods: In the present study, we compared infections of in vitro reconstituted airway epithelia originating from asthmatic versus healthy donors with representative strains of RV-A major group and minor groups, RV-C, RV-B, and the respiratory enterovirus EV-D68. Results: We found that viral replication was higher in tissues derived from asthmatic donors for all tested viruses. Viral receptor expression was comparable in non-infected tissues from both groups. After infection, ICAM1 and LDLR were upregulated, while CDHR3 was downregulated. Overall, these variations were related to viral replication levels. The presence of the CDHR3 asthma susceptibility allele (rs6967330) was not associated with increased RV-C replication. Regarding the tissue response, a significantly higher interferon (IFN) induction was demonstrated in infected tissues derived from asthmatic donors, which excludes a defect in IFN-response. Unbiased transcriptomic comparison of asthmatic versus control tissues revealed significant modifications, such as alterations of cilia structure and motility, in both infected and non-infected tissues. These observations were supported by a reduced mucociliary clearance and increased mucus secretion in non-infected tissues from asthmatic donors. Discussion: Altogether, we demonstrated an increased permissiveness and susceptibility to RV and respiratory EV infections in HAE derived from asthmatic patients, which was associated with a global alteration in epithelial cell functions. These results unveil the mechanisms underlying the pathogenesis of asthma exacerbation and suggest interesting therapeutic targets.

Nicotinamide Mononucleotide Administration Triggers Macrophages Reprogramming and Alleviates Inflammation During Sepsis Induced by Experimental Peritonitis.

Peritonitis and subsequent sepsis lead to high morbidity and mortality in response to uncontrolled systemic inflammation primarily mediated by macrophages. Nicotinamide adenine dinucleotide (NAD+) is an important regulator of oxidative stress and immunoinflammatory responses. However, the effects of NAD+ replenishment during inflammatory activation are still poorly defined. Hence, we investigated whether the administration of ß-nicotinamide mononucleotide (ß-NMN), a natural biosynthetic precursor of NAD+, could modulate the macrophage phenotype and thereby ameliorate the dysregulated inflammatory response during sepsis. For this purpose, C57BL6 mice were subjected to the cecal ligation and puncture (CLP) model to provoke sepsis or were injected with thioglycolate to induce sterile peritonitis with recruitment and differentiation of macrophages into the inflamed peritoneal cavity. ß-NMN was administered for 4 days after CLP and for 3 days post thioglycolate treatment where peritoneal macrophages were subsequently analyzed. In the CLP model, administration of ß-NMN decreased bacterial load in blood and reduced clinical signs of distress and mortality during sepsis. These results were supported by transcriptomic analysis of hearts and lungs 24 h post CLP-induction, which revealed that ß-NMN downregulated genes controlling the immuno-inflammatory response and upregulated genes involved in bioenergetic metabolism, mitochondria, and autophagy. In the thioglycolate model, a significant increase in the proportion of CD206 macrophages, marker of anti-inflammatory M2 phenotype, was detected on peritoneal exudate macrophages from ß-NMN-administered mice. Transcriptomic signature of these macrophages after bacterial stimulation confirmed that ß-NMN administration limited the pro-inflammatory M1 phenotype and induced the expression of specific markers of M2 type macrophages. Furthermore, our data show that ß-NMN treatment significantly impacts NAD + metabolism. This shift in the macrophage phenotype and metabolism was accompanied by a reduction in phagolysosome acidification and secretion of inflammatory mediators in macrophages from ß-NMN-treated mice suggesting a reduced pro-inflammatory activation. In conclusion, administration of ß-NMN prevented clinical deterioration and improved survival during sepsis. These effects relied on shifts in the metabolism of organs that face up an increased energy requirement caused by bacterial infection and in innate immunity response, including reprogramming of macrophages from a highly inflammatory phenotype to an anti-inflammatory/pro-resolving profile.

Infectious viral load in unvaccinated and vaccinated individuals infected with ancestral, Delta or Omicron SARS-CoV-2.

Infectious viral load (VL) expelled as droplets and aerosols by infected individuals partly determines transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RNA VL measured by qRT-PCR is only a weak proxy for infectiousness. Studies on the kinetics of infectious VL are important to understand the mechanisms behind the different transmissibility of SARS-CoV-2 variants and the effect of vaccination on transmission, which allows guidance of public health measures. In this study, we quantified infectious VL in individuals infected with SARS-CoV-2 during the first five symptomatic days by in vitro culturability assay in unvaccinated or vaccinated individuals infected with pre-variant of concern (pre-VOC) SARS-CoV-2, Delta or Omicron BA.1. Unvaccinated individuals infected with pre-VOC SARS-CoV-2 had lower infectious VL than Delta-infected unvaccinated individuals. Full vaccination (defined as >2 weeks after receipt of the second dose during the primary vaccination series) significantly reduced infectious VL for Delta breakthrough cases compared to unvaccinated individuals. For Omicron BA.1 breakthrough cases, reduced infectious VL was observed only in boosted but not in fully vaccinated individuals compared to unvaccinated individuals. In addition, infectious VL was lower in fully vaccinated Omicron BA.1-infected individuals compared to fully vaccinated Delta-infected individuals, suggesting that mechanisms other than increased infectious VL contribute to the high infectiousness of SARS-CoV-2 Omicron BA.1. Our findings indicate that vaccines may lower transmission risk and, therefore, have a public health benefit beyond the individual protection from severe disease.

Cooperative interaction between ERα and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis.

The epithelial to mesenchymal transition (EMT) has been proposed to contribute to the metastatic spread of breast cancer cells. EMT-promoting transcription factors determine a continuum of different EMT states. In contrast, estrogen receptor α (ERα) helps to maintain the epithelial phenotype of breast cancer cells and its expression is crucial for effective endocrine therapies. Determining whether and how EMT-associated transcription factors such as ZEB1 modulate ERα signaling during early stages of EMT could promote the discovery of therapeutic approaches to suppress metastasis. Here we show that, shortly after induction of EMT and while cells are still epithelial, ZEB1 modulates ERα-mediated transcription induced by estrogen or cAMP signaling in breast cancer cells. Based on these findings and our ex vivo and xenograft results, we suggest that the functional interaction between ZEB1 and ERα may alter the tissue tropism of metastatic breast cancer cells towards bone.

Essential role of a conserved aspartate for the enzymatic activity of plasmanylethanolamine desaturase.

Plasmalogens are an abundant class of glycerophospholipids in the mammalian body, with special occurrence in the brain and in immune cell membranes. Plasmanylethanolamine desaturase (PEDS1) is the final enzyme of plasmalogen biosynthesis, which introduces the characteristic 1-O-alk-1'-enyl double bond. The recent sequence identification of PEDS1 as transmembrane protein 189 showed that its protein sequence is related to a special class of plant desaturases (FAD4), with whom it shares a motif of 8 conserved histidines, which are essential for the enzymatic activity. In the present work, we wanted to gain more insight into the sequence-function relationship of this enzyme and mutated to alanine additional 28 amino acid residues of murine plasmanylethanolamine desaturase including those 20 residues, which are also totally conserved-in addition to the eight-histidine-motif-among the animal PEDS1 and plant FAD4 plant desaturases. We measured the enzymatic activity by transient transfection of tagged murine PEDS1 expression clones to a PEDS1-deficient human HAP1 cell line by monitoring of labeled plasmalogens formed from supplemented 1-O-pyrenedecyl-sn-glycerol in relation to recombinant protein expression. Surprisingly, only a single mutation, namely aspartate 100, led to a total loss of PEDS1 activity. The second strongest impact on enzymatic activity had mutation of phenylalanine 118, leaving only 6% residual activity. A structural model obtained by homology modelling to available structures of stearoyl-CoA reductase predicted that this aspartate 100 residue interacts with histidine 96, and phenylalanine 118 interacts with histidine 187, both being essential histidines assumed to be involved in the coordination of the di-metal center of the enzyme.

Nicotinamide Mononucleotide Administration Prevents Doxorubicin-Induced Cardiotoxicity and Loss in Physical Activity in Mice.

Doxorubicin (Doxo) is a widely used antineoplastic drug with limited clinical application due to its deleterious dose-related side effects. We investigated whether nicotinamide mononucleotide (NMN) could protect against Doxo-induced cardiotoxicity and physical dysfunction in vivo. To assess the short- and long-term toxicity, two Doxo regimens were tested, acute and chronic. In the acute study, C57BL6/J (B6) mice were injected intraperitoneally (i.p.) once with Doxo (20 mg/kg) and NMN (180 mg/kg/day, i.p.) was administered daily for five days before and after the Doxo injection. In the chronic study, B6 mice received a cumulative dose of 20 mg/kg Doxo administered in fractionated doses for five days. NMN (500 mg/kg/day) was supplied in the mice's drinking water beginning five days before the first injection of Doxo and continuing for 60 days after. We found that NMN significantly increased tissue levels of NAD+ and its metabolites and improved survival and bodyweight loss in both experimental models. In addition, NMN protected against Doxo-induced cardiotoxicity and loss of physical function in acute and chronic studies, respectively. In the heart, NMN prevented Doxo-induced transcriptomic changes related to mitochondrial function, apoptosis, oxidative stress, inflammation and p53, and promyelocytic leukemia nuclear body pathways. Overall, our results suggest that NMN could prevent Doxo-induced toxicity in heart and skeletal muscle.

Dynamic dichotomy of accumbal population activity underlies cocaine sensitization.

Locomotor sensitization (LS) is an early behavioral adaptation to addictive drugs, driven by the increase of dopamine in the Nucleus Accumbens (NAc). However, the effect on accumbal population activity remains elusive. Here, we used single-cell calcium imaging in mice to record the activity of dopamine-1-receptor (D1R) and dopamine-2-receptor (D2R) expressing spiny projection neurons (SPNs) during cocaine LS. Acute exposure to cocaine elevated D1R SPN activity and reduced D2R SPN activity, albeit with high variability between neurons. During LS, the number of D1R and D2R neurons responding in opposite directions increased. Moreover, preventing LS by inhibition of the ERK signaling pathway decreased the number of cocaine responsive D1R SPNs, but had little effect on D2R SPNs. These results indicate that accumbal population dichotomy is dynamic and contains a subgroup of D1R SPNs that eventually drives LS. Insights into the drug-related activity dynamics provides a foundation for understanding the circuit-level addiction pathogenesis.

The TMEM189 gene encodes plasmanylethanolamine desaturase which introduces the characteristic vinyl ether double bond into plasmalogens.

A significant fraction of the glycerophospholipids in the human body is composed of plasmalogens, particularly in the brain, cardiac, and immune cell membranes. A decline in these lipids has been observed in such diseases as Alzheimer's and chronic obstructive pulmonary disease. Plasmalogens contain a characteristic 1-O-alk-1'-enyl ether (vinyl ether) double bond that confers special biophysical, biochemical, and chemical properties to these lipids. However, the genetics of their biosynthesis is not fully understood, since no gene has been identified that encodes plasmanylethanolamine desaturase (E.C. 1.14.99.19), the enzyme introducing the crucial alk-1'-enyl ether double bond. The present work identifies this gene as transmembrane protein 189 (TMEM189). Inactivation of the TMEM189 gene in human HAP1 cells led to a total loss of plasmanylethanolamine desaturase activity, strongly decreased plasmalogen levels, and accumulation of plasmanylethanolamine substrates and resulted in an inability of these cells to form labeled plasmalogens from labeled alkylglycerols. Transient expression of TMEM189 protein, but not of other selected desaturases, recovered this deficit. TMEM189 proteins contain a conserved protein motif (pfam10520) with eight conserved histidines that is shared by an alternative type of plant desaturase but not by other mammalian proteins. Each of these histidines is essential for plasmanylethanolamine desaturase activity. Mice homozygous for an inactivated Tmem189 gene lacked plasmanylethanolamine desaturase activity and had dramatically lowered plasmalogen levels in their tissues. These results assign the TMEM189 gene to plasmanylethanolamine desaturase and suggest that the previously characterized phenotype of Tmem189-deficient mice may be caused by a lack of plasmalogens.

Transcriptomic analyses reveal rhythmic and CLOCK-driven pathways in human skeletal muscle.

Circadian regulation of transcriptional processes has a broad impact on cell metabolism. Here, we compared the diurnal transcriptome of human skeletal muscle conducted on serial muscle biopsies in vivo with profiles of human skeletal myotubes synchronized in vitro. More extensive rhythmic transcription was observed in human skeletal muscle compared to in vitro cell culture as a large part of the in vivo mRNA rhythmicity was lost in vitro. siRNA-mediated clock disruption in primary myotubes significantly affected the expression of ~8% of all genes, with impact on glucose homeostasis and lipid metabolism. Genes involved in GLUT4 expression, translocation and recycling were negatively affected, whereas lipid metabolic genes were altered to promote activation of lipid utilization. Moreover, basal and insulin-stimulated glucose uptake were significantly reduced upon CLOCK depletion. Our findings suggest an essential role for the circadian coordination of skeletal muscle glucose homeostasis and lipid metabolism in humans.

Peptidomic and transcriptomic profiling of four distinct spider venoms.

Venom based research is exploited to find novel candidates for the development of innovative pharmacological tools, drug candidates and new ingredients for cosmetic and agrochemical industries. Moreover, venomics, as a well-established approach in systems biology, helps to elucidate the genetic mechanisms of the production of such a great molecular biodiversity. Today the advances made in the proteomics, transcriptomics and bioinformatics fields, favor venomics, allowing the in depth study of complex matrices and the elucidation even of minor compounds present in minute biological samples. The present study illustrates a rapid and efficient method developed for the elucidation of venom composition based on NextGen mRNA sequencing of venom glands and LC-MS/MS venom proteome profiling. The analysis of the comprehensive data obtained was focused on cysteine rich peptide toxins from four spider species originating from phylogenetically distant families for comparison purposes. The studied species were Heteropoda davidbowie (Sparassidae), Poecilotheria formosa (Theraphosidae), Viridasius fasciatus (Viridasiidae) and Latrodectus mactans (Theridiidae). This led to a high resolution profiling of 284 characterized cysteine rich peptides, 111 of which belong to the Inhibitor Cysteine Knot (ICK) structural motif. The analysis of H. davidbowie venom revealed a high richness in term of venom diversity: 95 peptide sequences were identified; out of these, 32 peptides presented the ICK structural motif and could be classified in six distinct families. The profiling of P. formosa venom highlighted the presence of 126 peptide sequences, with 52 ICK toxins belonging to three structural distinct families. V. fasciatus venom was shown to contain 49 peptide sequences, out of which 22 presented the ICK structural motif and were attributed to five families. The venom of L. mactans, until now studied for its large neurotoxins (Latrotoxins), revealed the presence of 14 cysteine rich peptides, out of which five were ICK toxins belonging to the CSTX superfamily. This in depth profiling of distinct ICK peptide families identified across the four spider species highlighted the high conservation of these neurotoxins among spider families.

ER-mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem-like cells.

Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem-like cells (GSC) being more sensitive to cytotoxic lymphocyte-mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER-mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER-mitochondria contacts compared to GDCs. Forced ER-mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER-mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma.

The Pseudouridine Synthase RPUSD4 Is an Essential Component of Mitochondrial RNA Granules.

Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking. Here, we have applied a microscopy-based approach that has allowed us to identify novel components of the MRG proteome. Among these, we have focused our attention on RPUSD4, an uncharacterized mitochondrial putative pseudouridine synthase. We show that RPUSD4 depletion leads to a severe reduction of the steady-state level of the 16S mitochondrial (mt) rRNA with defects in the biogenesis of the mitoribosome large subunit and consequently in mitochondrial translation. We report that RPUSD4 binds 16S mt-rRNA, mt-tRNAMet, and mt-tRNAPhe, and we demonstrate that it is responsible for pseudouridylation of the latter. These data provide new insights into the relevance of RNA pseudouridylation in mitochondrial gene expression.

Monoubiquitination of Histone H2B Blocks Eviction of Histone Variant H2A.Z from Inducible Enhancers.

Covalent modifications of histones play a crucial role in the regulation of gene expression. Histone H2B monoubiquitination has mainly been described as a regulator of transcription elongation, but its role in transcription initiation is poorly documented. We investigated the role of this histone mark (H2Bub1) on different inducible enhancers, in particular those regulated by estrogen receptor α, by loss- and gain-of-function experiments with the specific E3-ubiquitin ligase complex of H2B: RNF20/RNF40. RNF20/RNF40 overexpression causes repression of the induced activity of these enhancers. Genome-wide profiles show that H2Bub1 levels are negatively correlated with the accessibility of enhancers to transcriptional activators. We found that the chromatin association of histone variant H2A.Z, which is evicted from enhancers for transcriptional activation, is stabilized by H2Bub1 by impairing access of the chromatin remodeler INO80. We propose that H2Bub1 acts as a gatekeeper of H2A.Z eviction and activation of inducible enhancers.

Uncovering intense protein diversification in a cone snail venom gland using an integrative venomics approach.

Marine cone snail venoms are highly complex mixtures of peptides and proteins. They have been studied in-depth over the past 3 decades, but the modus operandi of the venomous apparatus still remains unclear. Using the fish-hunting Conus consors as a model, we present an integrative venomics approach, based on new proteomic results from the venom gland and data previously obtained from the transcriptome and the injectable venom. We describe here the complete peptide content of the dissected venom by the identification of numerous new peptides using nanospray tandem mass spectrometry in combination with transcriptomic data. Results reveal extensive mature peptide diversification mechanisms at work in the venom gland. In addition, by integrating data from three different venom stages, transcriptome, dissected, and injectable venoms, from a single species, we obtain a global overview of the venom processing that occurs from the venom gland tissue to the venom delivery step. In the light of the successive steps in this venom production system, we demonstrate that each venom compartment is highly specific in terms of peptide and protein content. Moreover, the integrated investigative approach discussed here could become an essential part of pharmaceutical development, as it provides new potential drug candidates and opens the door to numerous analogues generated by the very mechanisms used by nature to diversify its peptide and protein arsenal.

New and continuing developments at PROSITE.

PROSITE (http://prosite.expasy.org/) consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule a collection of rules, which increases the discriminatory power of these profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. PROSITE signatures, together with ProRule, are used for the annotation of domains and features of UniProtKB/Swiss-Prot entries. Here, we describe recent developments that allow users to perform whole-proteome annotation as well as a number of filtering options that can be combined to perform powerful targeted searches for biological discovery. The latest version of PROSITE (release 20.85, of 30 August 2012) contains 1308 patterns, 1039 profiles and 1041 ProRules.

Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase.

Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure-function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis-Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase.

InterPro in 2011: new developments in the family and domain prediction database.

InterPro (http://www.ebi.ac.uk/interpro/) is a database that integrates diverse information about protein families, domains and functional sites, and makes it freely available to the public via Web-based interfaces and services. Central to the database are diagnostic models, known as signatures, against which protein sequences can be searched to determine their potential function. InterPro has utility in the large-scale analysis of whole genomes and meta-genomes, as well as in characterizing individual protein sequences. Herein we give an overview of new developments in the database and its associated software since 2009, including updates to database content, curation processes and Web and programmatic interfaces.