Using Machine Learning Imputed Outcomes to Assess Drug-Dependent Risk of Self-Harm in Patients with Bipolar Disorder: A Comparative Effectiveness Study.

BACKGROUND: Incomplete suicidality coding in administrative claims data is a known obstacle for observational studies. With most of the negative outcomes missing from the data, it is challenging to assess the evidence on treatment strategies for the prevention of self-harm in bipolar disorder (BD), including pharmacotherapy and psychotherapy. There are conflicting data from studies on the drug-dependent risk of self-harm, and there is major uncertainty regarding the preventive effect of monotherapy and drug combinations. OBJECTIVE: The aim of this study was to compare all commonly used BD pharmacotherapies, as well as psychotherapy for the risk of self-harm, in a large population of commercially insured individuals, using self-harm imputation to overcome the known limitations of this outcome being underrecorded within US electronic health care records. METHODS: The IBM MarketScan administrative claims database was used to compare self-harm risk in patients with BD following 65 drug regimens and drug-free periods. Probable but uncoded self-harm events were imputed via machine learning, with different probability thresholds examined in a sensitivity analysis. Comparators included lithium, mood-stabilizing anticonvulsants (MSAs), second-generation antipsychotics (SGAs), first-generation antipsychotics (FGAs), and five classes of antidepressants. Cox regression models with time-varying covariates were built for individual treatment regimens and for any pharmacotherapy with or without psychosocial interventions ("psychotherapy"). RESULTS: Among 529,359 patients, 1.66% (n=8813 events) had imputed and/or coded self-harm following the exposure of interest. A higher self-harm risk was observed during adolescence. After multiple testing adjustment (P≤.012), the following six regimens had higher risk of self-harm than lithium: tri/tetracyclic antidepressants + SGA, FGA + MSA, FGA, serotonin-norepinephrine reuptake inhibitor (SNRI) + SGA, lithium + MSA, and lithium + SGA (hazard ratios [HRs] 1.44-2.29), and the following nine had lower risk: lamotrigine, valproate, risperidone, aripiprazole, SNRI, selective serotonin reuptake inhibitor (SSRI), "no drug," bupropion, and bupropion + SSRI (HRs 0.28-0.74). Psychotherapy alone (without medication) had a lower self-harm risk than no treatment (HR 0.56, 95% CI 0.52-0.60; P=8.76×10-58). The sensitivity analysis showed that the direction of drug-outcome associations did not change as a function of the self-harm probability threshold. CONCLUSIONS: Our data support evidence on the effectiveness of antidepressants, MSAs, and psychotherapy for self-harm prevention in BD. TRIAL REGISTRATION: ClinicalTrials.gov NCT02893371; https://clinicaltrials.gov/ct2/show/NCT02893371.

Diabetes mellitus risk for 102 drugs and drug combinations used in patients with bipolar disorder.

OBJECTIVE: To compare the largest set of bipolar disorder pharmacotherapies to date (102 drugs and drug combinations) for risk of diabetes mellitus (DM). METHODS: The IBM MarketScan® database was used to retrospectively analyze data on 565,253 adults with bipolar disorder without prior glucose metabolism-related diagnoses. The pharmacotherapies compared were lithium, mood-stabilizing anticonvulsants, antipsychotics, and antidepressants (monotherapy and multi-class polypharmacy). Cox regression modeling included fixed pre-treatment covariates and time-varying drug exposure covariates to estimate the hazard ratio (HR) of each treatment versus "No drug". RESULTS: The annual incidence of new-onset diabetes during the exposure period was 3.09 % (22,951 patients). The HR of drug-dependent DM ranged from 0.79 to 2.37. One-third of the studied pharmacotherapies, including most of the antipsychotic-containing regimens, had a significantly higher risk of DM compared to "No drug". A significantly lower DM risk was associated with lithium, lamotrigine, oxcarbazepine and bupropion monotherapies, selective serotonin reuptake inhibitors (SSRI) mono-class therapy and several drug combinations containing bupropion and an SSRI. As additional drugs were combined in more complex polypharmacy, higher HRs were consistently observed. CONCLUSIONS: There is an increased risk of diabetes mellitus associated with antipsychotic and psychotropic polypharmacy use in bipolar disorder. The evidence of a lower-than-baseline risk of DM with lamotrigine, oxcarbazepine, lithium, and bupropion monotherapy should be further investigated.

Preferences of Information Dissemination on Treatment for Bipolar Disorder: Patient-Centered Focus Group Study.

BACKGROUND: Patient education has taken center stage in successfully shared decision making between patients and health care providers. However, little is known about how patients with bipolar disorder typically obtain information on their illness and the treatment options available to them. OBJECTIVE: This study aimed to obtain the perspectives of patients with bipolar disorder and their family members on the preferred and most effectively used information channels on bipolar disorder and the available treatment options. METHODS: We conducted nine focus groups in Montana, New Mexico, and California, in which we surveyed 84 individuals including patients with bipolar disorder and family members of patients with bipolar disorder. The participants were recruited using National Alliance on Mental Illness mailing lists and websites. Written verbatim responses to semistructured questionnaires were analyzed using summative content analysis based on grounded theory. Two annotators coded and analyzed the data on the sentence or phrase level to create themes. Relationships between demographics and information channel were also examined using the Chi-square and Fisher exact tests. RESULTS: The focus group participants mentioned a broad range of information channels that were successfully used in the past and could be recommended for future information dissemination. The majority of participants used providers (74%) and internet-based resources (75%) as their main information sources. There was no association between internet use and basic demographics such as age or geographical region of the focus groups. Patients considered time constraints and the fast pace in which an overwhelming amount of information is often presented by the provider as major barriers to successful provider-patient interactions. If Web-based channels were used, the participants perceived information obtained through Web-based channels as more helpful than information received in the provider's office (P<.05). CONCLUSIONS: Web-based resources are increasingly used by patients with bipolar disorder and their family members to educate themselves about the disease and its treatment. Although provider-patient interactions are frequently perceived to be burdened with time constraints, Web-based information sources are considered reliable and helpful. Future research should explore how high-quality websites could be used to empower patients and improve provider-patient interactions with the goal of enhancing shared decision making between patients and providers.

Comparison of 71 bipolar disorder pharmacotherapies for kidney disorder risk: The potential hazards of polypharmacy.

BACKGROUND: This study compared the largest set of bipolar disorder pharmacotherapies to date (71 drugs and drug combinations) for risk of kidney disorders (KDs). METHODS: This retrospective observational study used the IBM MarketScan® database to analyze data on 591,052 adults with bipolar disorder without prior nephropathy, for onset of KDs (of "moderate" or "high" severity) following psychopharmacotherapy (lithium, mood stabilizing anticonvulsants [MSAs], antipsychotics, antidepressants), or "No drug". Cox regression models included fixed pre-treatment covariates and time-varying drug exposure covariates to estimate the hazard ratio (HR) of each treatment versus "No drug". RESULTS: Newly observed KD occurred in 14,713 patients. No regimen had significantly lower risk of KDs than "No drug". The HR estimates ranged 0.86-2.66 for "all" KDs and 0.87-5.30 for "severe" KDs. As additional drugs were combined to compare more complex polypharmacies, higher HRs were consistently observed. Most regimens containing lithium, MSAs, or antipsychotics had a higher risk than "No drug" (p < 0.05). The risk for "all" and "severe" KDs was highest respectively on monoamine oxidase inhibitors (MAOIs) (HR = 2.66, p = 5.73 × 10-5), and a lithium-containing four-class combination (HR = 5.30, p = 2.46 × 10-9). The HR for lithium monotherapy was 1.82 (p = 4.73 × 10-17) for "severe" KDs. LIMITATIONS: The limitations inherent for an observational study were non-randomized assignment of patients to treatment groups, non-standardization of diagnostic decisions, and non-uniform quality of data collection. No correction was made for medication dosage. CONCLUSIONS: The findings support literature concerns about lithium nephrotoxicity and highlight the potential risks of MAOIs, MSAs, antipsychotics and psychotropic polypharmacy.

Trends in patient procurement of postoperative opioids and route of hysterectomy in the United States from 2004 through 2014.

BACKGROUND: The opioid epidemic in the United States is a public health emergency. Minimally invasive surgical technology has decreased length of hospital stay, improved postoperative recovery, and decreased postoperative pain. Hysterectomy is one of the most commonly performed surgeries in the United States. Increasing trends in minimally invasive gynecologic surgery are expected to reduce patients' postoperative pain. It is unclear whether this assumption has resulted in decreasing postoperative opioid prescriptions or patient need for these prescriptions, as prescribing patterns may be contributing to the current opioid public health emergency. OBJECTIVE: We sought to describe opioid prescribing and patient procurement practices for postoperative pain at time of discharge for benign hysterectomy from 2004 through 2014 using the Truven Health Analytics MarketScan Research Database. The trends of the route of hysterectomy over this time period were concomitantly described to reflect the movement toward more minimally invasive approaches. STUDY DESIGN: The Truven Health Analytics MarketScan Research Database including the Commercial Claims and Encounters Database, and the Medicare Supplemental and Coordination of Benefits Database were utilized. Current Procedural Terminology, 4th Edition, and International Classification of Diseases, Ninth Revision, codes identified all patients who underwent a hysterectomy for benign indications from 2004 through 2014. Hysterectomy routes were categorized into abdominal, laparoscopic, and vaginal. The MarketScan database captures prescriptions filled at a retail or mail-order pharmacy and does not capture prescriptions filled within the inpatient, hospital facility. The days of opioids procured by patients at the time of discharge were identified for each encounter. Descriptive statistics were used to summarize data within the entire study period. Although this article is purely descriptive, further analyses were conducted for exploratory purposes only. analysis of variance and χ2 analyses were used for continuous and categorical variables, respectively. Multiple linear regression models were used to describe associations between variables of interest and postoperative opioid prescriptions. RESULTS: We identified 793,016 patients who underwent a hysterectomy for benign indications from 2004 through 2014. Of these, 96% were identified from the Commercial Claims and Encounters Database. During the study period, the overall route of hysterectomy was categorized into 40.5% abdominal, 42.0% laparoscopic, and 17.5% vaginal hysterectomy. The route of hysterectomy changed from 60.2-25.6% (a decrease of Δ = 34.58; 95% confidence interval, 33.96-35.20) for abdominal, 17.0-61.9% (an increase of Δ = 44.83; 95% confidence interval, 44.21-45.44) for laparoscopic, and 22.8-12.6% (a decrease of Δ = 10.25; 95% confidence interval, 9.77-10.73) for vaginal. At discharge, the percentage of patients who were prescribed opioids and filled them increased from 25.6-82.1% (an increase of Δ = 56.50; 95% confidence interval, 55.88-57.13 with P < .001) from 2004 through 2014 for all hysterectomy routes. Additionally, the quantity of opioids prescribed also increased. CONCLUSION: Opioid prescriptions filled for postoperative pain after hysterectomy substantially increased from 2004 through 2014. Opioid prescription procurement has increased despite a concomitant increase in minimally invasive hysterectomy routes. In light of the current opioid epidemic, physicians must recognize that postoperative prescribing practices may contribute to chronic opioid use. Heightened awareness of opioid prescribing practices following surgery is critically important to decrease risk of development of chronic opioid dependence.

Comprehensive comparison of monotherapies for psychiatric hospitalization risk in bipolar disorders.

OBJECTIVES: This study compared 29 drugs for risk of psychiatric hospitalization in bipolar disorders, addressing the evidence gap on the >50 drugs used by US patients for treatment. METHODS: The Truven Health Analytics MarketScan® database was used to identify 190 894 individuals with bipolar or schizoaffective disorder who filled a prescription for one of 29 drugs of interest: lithium, first- or second-generation antipsychotics, mood-stabilizing anticonvulsants, and antidepressants. Competing risks regression survival analysis was used to compare drugs for risk of psychiatric hospitalization, adjusting for patient age, sex, comorbidities, and pretreatment medications. Other competing risks were ending monotherapy and non-psychiatric hospitalization. RESULTS: Three drugs were associated with significantly lower risk of psychiatric hospitalization than lithium: valproate (relative risk [RR] = 0.80, P = 3.20 × 10-4 ), aripiprazole (RR = 0.80, P = 3.50 × 10-4 ), and bupropion (RR = 0.80, P = 2.80 × 10-4 ). Eight drugs were associated with significantly higher risk of psychiatric hospitalization: haloperidol (RR = 1.57, P = 9.40 × 10-4 ), clozapine (RR = 1.52, P = .017), fluoxetine (RR = 1.17, P = 3.70 × 10-3 ), sertraline (RR = 1.17, P = 3.20 × 10-3 ), citalopram (RR = 1.14, P = .013), duloxetine (RR = 1.24, P = 5.10 × 10-4 ), venlafaxine (RR = 1.33; P = 1.00 × 10-6 ), and ziprasidone (RR = 1.25; P = 6.20 × 10-3 ). CONCLUSIONS: This largest reported retrospective observational study on bipolar disorders pharmacotherapy to date demonstrates that the majority of patients end monotherapy within 2 months after treatment start. The risk of psychiatric hospitalization varied almost two-fold across individual medications. The data add to the evidence favoring lithium and mood stabilizer use in short-term bipolar disorder management. The findings that the dopaminergic drugs aripiprazole and bupropion had better outcomes than other members of their respective classes and that antidepressant outcomes may vary by baseline mood polarity merit further investigation.

A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

Dysregulation of genetic pathways during human germ cell development leads to infertility. Here, we analysed bona fide human primordial germ cells (hPGCs) to probe the developmental genetics of human germ cell specification and differentiation. We examined the distribution of OCT4 occupancy in hPGCs relative to human embryonic stem cells (hESCs). We demonstrated that development, from pluripotent stem cells to germ cells, is driven by switching partners with OCT4 from SOX2 to PAX5 and PRDM1. Gain- and loss-of-function studies revealed that PAX5 encodes a critical regulator of hPGC development. Moreover, an epistasis analysis indicated that PAX5 acts upstream of OCT4 and PRDM1. The PAX5-OCT4-PRDM1 proteins form a core transcriptional network that activates germline and represses somatic programmes during human germ cell differentiation. These findings illustrate the power of combined genome editing, cell differentiation and engraftment for probing human developmental genetics that have historically been difficult to study.

Intensive herbicide use has selected for constitutively elevated levels of stress-responsive mRNAs and proteins in multiple herbicide-resistant Avena fatua L.

BACKGROUND: Intensive use of herbicides has led to the evolution of two multiple herbicide-resistant (MHR) Avena fatua (wild oat) populations in Montana that are resistant to members of all selective herbicide families available for A. fatua control in US small grain crops. We used transcriptome and proteome surveys to compare constitutive changes in MHR and herbicide-susceptible (HS) plants associated with non-target site resistance. RESULTS: Compared to HS plants, MHR plants contained constitutively elevated levels of differentially expressed genes (DEGs) with functions in xenobiotic catabolism, stress response, redox maintenance and transcriptional regulation that are similar to abiotic stress-tolerant phenotypes. Proteome comparisons identified similarly elevated proteins including biosynthetic and multifunctional enzymes in MHR plants. Of 25 DEGs validated by RT-qPCR assay, differential regulation of 21 co-segregated with flucarbazone-sodium herbicide resistance in F3 families, and a subset of 10 of these were induced or repressed in herbicide-treated HS plants. CONCLUSION: Although the individual and collective contributions of these DEGs and proteins to MHR remain to be determined, our results support the idea that intensive herbicide use has selected for MHR populations with altered, constitutively regulated patterns of gene expression that are similar to those in abiotic stress-tolerant plants. © 2017 Society of Chemical Industry.

Hypothyroidism risk compared among nine common bipolar disorder therapies in a large US cohort.

OBJECTIVES: Thyroid abnormalities in patients with bipolar disorder (BD) have been linked to lithium treatment for decades, yet other drugs have been less well studied. Our objective was to compare hypothyroidism risk for lithium versus the anticonvulsants and second-generation antipsychotics commonly prescribed for BD. METHODS: Administrative claims data on 24,574 patients with BD were analyzed with competing risk survival analysis. Inclusion criteria were (i) one year of no prior hypothyroid diagnosis nor BD drug treatment, (ii) followed by at least one thyroid test during BD monotherapy on lithium carbonate, mood-stabilizing anticonvulsants (lamotrigine, valproate, oxcarbazepine, or carbamazepine) or antipsychotics (aripiprazole, olanzapine, risperidone, or quetiapine). The outcome was cumulative incidence of hypothyroidism per drug, in the presence of the competing risk of ending monotherapy, adjusted for age, sex, physician visits, and thyroid tests. RESULTS: Adjusting for covariates, the four-year cumulative risk of hypothyroidism for lithium (8.8%) was 1.39-fold that of the lowest risk therapy, oxcarbazepine (6.3%). Lithium was non-statistically significantly different from quetiapine. While lithium conferred a higher risk when compared to all other treatments combined as a group, hypothyroidism risk error bars overlapped for all drugs. Treatment (p = 3.86e-3), age (p = 6.91e-10), sex (p = 3.93e-7), and thyroid testing (p = 2.79e-87) affected risk. Patients taking lithium were tested for hypothyroidism 2.26-3.05 times more frequently than those on other treatments. CONCLUSIONS: Thyroid abnormalities occur frequently in patients with BD regardless of treatment. Therefore, patients should be regularly tested for clinical or subclinical thyroid abnormalities on all therapies and treated as indicated to prevent adverse effects of hormone imbalances on mood.

Viral assemblage composition in Yellowstone acidic hot springs assessed by network analysis.

Understanding of viral assemblage structure in natural environments remains a daunting task. Total viral assemblage sequencing (for example, viral metagenomics) provides a tractable approach. However, even with the availability of next-generation sequencing technology it is usually only possible to obtain a fragmented view of viral assemblages in natural ecosystems. In this study, we applied a network-based approach in combination with viral metagenomics to investigate viral assemblage structure in the high temperature, acidic hot springs of Yellowstone National Park, USA. Our results show that this approach can identify distinct viral groups and provide insights into the viral assemblage structure. We identified 110 viral groups in the hot springs environment, with each viral group likely representing a viral family at the sub-family taxonomic level. Most of these viral groups are previously unknown DNA viruses likely infecting archaeal hosts. Overall, this study demonstrates the utility of combining viral assemblage sequencing approaches with network analysis to gain insights into viral assemblage structure in natural ecosystems.

ChIP-seq and in vivo transcriptome analyses of the Aspergillus fumigatus SREBP SrbA reveals a new regulator of the fungal hypoxia response and virulence.

The Aspergillus fumigatus sterol regulatory element binding protein (SREBP) SrbA belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA). How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs' complex role in infection site adaptation and fungal virulence.

Real-time digitization of metabolomics patterns from a living system using mass spectrometry.

The real-time quantification of changes in intracellular metabolic activities has the potential to vastly improve upon traditional transcriptomics and metabolomics assays for the prediction of current and future cellular phenotypes. This is in part because intracellular processes reveal themselves as specific temporal patterns of variation in metabolite abundance that can be detected with existing signal processing algorithms. Although metabolite abundance levels can be quantified by mass spectrometry (MS), large-scale real-time monitoring of metabolite abundance has yet to be realized because of technological limitations for fast extraction of metabolites from cells and biological fluids. To address this issue, we have designed a microfluidic-based inline small molecule extraction system, which allows for continuous metabolomic analysis of living systems using MS. The system requires minimal supervision, and has been successful at real-time monitoring of bacteria and blood. Feature-based pattern analysis of Escherichia coli growth and stress revealed cyclic patterns and forecastable metabolic trajectories. Using these trajectories, future phenotypes could be inferred as they exhibit predictable transitions in both growth and stress related changes. Herein, we describe an interface for tracking metabolic changes directly from blood or cell suspension in real-time.

In situ gene expression profiling of the thermoacidophilic alga Cyanidioschyzon in relation to visible and ultraviolet irradiance.

Ultraviolet and high-intensity visible radiation generate reactive intermediates that damage phototrophic microorganisms. In Yellowstone National Park, the thermoacidophilic alga Cyanidioschyzon exhibits an annual seasonal biomass fluctuation referred to as 'mat decline', where algal viability decreases as ultraviolet and visible irradiances increase during summer. We examined the role irradiance might play in mat decline using irradiance filters that uncouple ultraviolet and visible effects along with custom microarrays to study gene expression in situ. Of the 6507 genes, 88% showed no response to ultraviolet or visible, implying that at the biomolecular level, these algae inhabit a chemostat-like environment and is consistent with the near constant aqueous chemistry measured. The remaining genes exhibited expression changes linked to ultraviolet exposure, to increased visible radiation, or to the apparent combined effects of ultraviolet and visible. Expression of DNA repetitive elements was synchronized, being repressed by visible but also influenced by ultraviolet. At highest irradiance levels, these algae reduced transcription of genes encoding functions involved with DNA replication, photosynthesis and cell cycle progression but exhibited an uptick in activities related to repairing DNA damage. This corroborates known physiological responses to ultraviolet and visible radiation, and leads us to provisionally conclude that mat decline is linked to photoinhibition.

Comparative genomics of cryptosporidium.

Until recently, the apicomplexan parasites, Cryptosporidium hominis and C. parvum, were considered the same species. However, the two parasites, now considered distinct species, exhibit significant differences in host range, infectivity, and pathogenicity, and their sequenced genomes exhibit only 95-97% identity. The availability of the complete genome sequences of these organisms provides the potential to identify the genetic variations that are responsible for the phenotypic differences between the two parasites. We compared the genome organization and structure, gene composition, the metabolic and other pathways, and the local sequence identity between the genes of these two Cryptosporidium species. Our observations show that the phenotypic differences between C. hominis and C. parvum are not due to gross genome rearrangements, structural alterations, gene deletions or insertions, metabolic capabilities, or other obvious genomic alterations. Rather, the results indicate that these genomes exhibit a remarkable structural and compositional conservation and suggest that the phenotypic differences observed are due to subtle variations in the sequences of proteins that act at the interface between the parasite and its host.

Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum.

BACKGROUND: Phaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome has been sequenced (<30 Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis has been reported under different growth conditions. To elucidate P. tricornutum gene expression profiles during nutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P) and whole-genome transcripts were monitored over time via RNA-sequence determination. RESULTS: The specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, the increase in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphate was depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be an early trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genes associated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc7 and cyc10 were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulation after growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbon reduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbon assimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon (DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed (2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth. Alternative pathways that could utilize HCO3- were also suggested by the gene expression profiles (e.g., putative propionyl-CoA and methylmalonyl-CoA decarboxylases). CONCLUSIONS: The results indicate that P. tricornutum continued carbon dioxide reduction when population growth was arrested and different carbon-concentrating mechanisms were used dependent upon exogenous DIC levels. Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up of precursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actual enzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellular responses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.

Transcriptomic and proteomic analyses of the Aspergillus fumigatus hypoxia response using an oxygen-controlled fermenter.

BACKGROUND: Aspergillus fumigatus is a mold responsible for the majority of cases of aspergillosis in humans. To survive in the human body, A. fumigatus must adapt to microenvironments that are often characterized by low nutrient and oxygen availability. Recent research suggests that the ability of A. fumigatus and other pathogenic fungi to adapt to hypoxia contributes to their virulence. However, molecular mechanisms of A. fumigatus hypoxia adaptation are poorly understood. Thus, to better understand how A. fumigatus adapts to hypoxic microenvironments found in vivo during human fungal pathogenesis, the dynamic changes of the fungal transcriptome and proteome in hypoxia were investigated over a period of 24 hours utilizing an oxygen-controlled fermenter system. RESULTS: Significant increases in transcripts associated with iron and sterol metabolism, the cell wall, the GABA shunt, and transcriptional regulators were observed in response to hypoxia. A concomitant reduction in transcripts was observed with ribosome and terpenoid backbone biosynthesis, TCA cycle, amino acid metabolism and RNA degradation. Analysis of changes in transcription factor mRNA abundance shows that hypoxia induces significant positive and negative changes that may be important for regulating the hypoxia response in this pathogenic mold. Growth in hypoxia resulted in changes in the protein levels of several glycolytic enzymes, but these changes were not always reflected by the corresponding transcriptional profiling data. However, a good correlation overall (R(2) = 0.2, p < 0.05) existed between the transcriptomic and proteomics datasets for all time points. The lack of correlation between some transcript levels and their subsequent protein levels suggests another regulatory layer of the hypoxia response in A. fumigatus. CONCLUSIONS: Taken together, our data suggest a robust cellular response that is likely regulated both at the transcriptional and post-transcriptional level in response to hypoxia by the human pathogenic mold A. fumigatus. As with other pathogenic fungi, the induction of glycolysis and transcriptional down-regulation of the TCA cycle and oxidative phosphorylation appear to major components of the hypoxia response in this pathogenic mold. In addition, a significant induction of the transcripts involved in ergosterol biosynthesis is consistent with previous observations in the pathogenic yeasts Candida albicans and Cryptococcus neoformans indicating conservation of this response to hypoxia in pathogenic fungi. Because ergosterol biosynthesis enzymes also require iron as a co-factor, the increase in iron uptake transcripts is consistent with an increased need for iron under hypoxia. However, unlike C. albicans and C. neoformans, the GABA shunt appears to play an important role in reducing NADH levels in response to hypoxia in A. fumigatus and it will be intriguing to determine whether this is critical for fungal virulence. Overall, regulatory mechanisms of the A. fumigatus hypoxia response appear to involve both transcriptional and post-transcriptional control of transcript and protein levels and thus provide candidate genes for future analysis of their role in hypoxia adaptation and fungal virulence.

SREBP coordinates iron and ergosterol homeostasis to mediate triazole drug and hypoxia responses in the human fungal pathogen Aspergillus fumigatus.

Sterol regulatory element binding proteins (SREBPs) are a class of basic helix-loop-helix transcription factors that regulate diverse cellular responses in eukaryotes. Adding to the recognized importance of SREBPs in human health, SREBPs in the human fungal pathogens Cryptococcus neoformans and Aspergillus fumigatus are required for fungal virulence and susceptibility to triazole antifungal drugs. To date, the exact mechanism(s) behind the role of SREBP in these observed phenotypes is not clear. Here, we report that A. fumigatus SREBP, SrbA, mediates regulation of iron acquisition in response to hypoxia and low iron conditions. To further define SrbA's role in iron acquisition in relation to previously studied fungal regulators of iron metabolism, SreA and HapX, a series of mutants were generated in the ΔsrbA background. These data suggest that SrbA is activated independently of SreA and HapX in response to iron limitation, but that HapX mRNA induction is partially dependent on SrbA. Intriguingly, exogenous addition of high iron or genetic deletion of sreA in the ΔsrbA background was able to partially rescue the hypoxia growth, triazole drug susceptibility, and decrease in ergosterol content phenotypes of ΔsrbA. Thus, we conclude that the fungal SREBP, SrbA, is critical for coordinating genes involved in iron acquisition and ergosterol biosynthesis under hypoxia and low iron conditions found at sites of human fungal infections. These results support a role for SREBP-mediated iron regulation in fungal virulence, and they lay a foundation for further exploration of SREBP's role in iron homeostasis in other eukaryotes.

Physiology of Pseudomonas aeruginosa in biofilms as revealed by transcriptome analysis.

BACKGROUND: Transcriptome analysis was applied to characterize the physiological activities of Pseudomonas aeruginosa grown for three days in drip-flow biofilm reactors. Conventional applications of transcriptional profiling often compare two paired data sets that differ in a single experimentally controlled variable. In contrast this study obtained the transcriptome of a single biofilm state, ranked transcript signals to make the priorities of the population manifest, and compared rankings for a priori identified physiological marker genes between the biofilm and published data sets. RESULTS: Biofilms tolerated exposure to antibiotics, harbored steep oxygen concentration gradients, and exhibited stratified and heterogeneous spatial patterns of protein synthetic activity. Transcriptional profiling was performed and the signal intensity of each transcript was ranked to gain insight into the physiological state of the biofilm population. Similar rankings were obtained from data sets published in the GEO database http://www.ncbi.nlm.nih.gov/geo. By comparing the rank of genes selected as markers for particular physiological activities between the biofilm and comparator data sets, it was possible to infer qualitative features of the physiological state of the biofilm bacteria. These biofilms appeared, from their transcriptome, to be glucose nourished, iron replete, oxygen limited, and growing slowly or exhibiting stationary phase character. Genes associated with elaboration of type IV pili were strongly expressed in the biofilm. The biofilm population did not indicate oxidative stress, homoserine lactone mediated quorum sensing, or activation of efflux pumps. Using correlations with transcript ranks, the average specific growth rate of biofilm cells was estimated to be 0.08 h(-1). CONCLUSIONS: Collectively these data underscore the oxygen-limited, slow-growing nature of the biofilm population and are consistent with antimicrobial tolerance due to low metabolic activity.

Evolution of metabolic network organization.

BACKGROUND: Comparison of metabolic networks across species is a key to understanding how evolutionary pressures shape these networks. By selecting taxa representative of different lineages or lifestyles and using a comprehensive set of descriptors of the structure and complexity of their metabolic networks, one can highlight both qualitative and quantitative differences in the metabolic organization of species subject to distinct evolutionary paths or environmental constraints. RESULTS: We used a novel representation of metabolic networks, termed network of interacting pathways or NIP, to focus on the modular, high-level organization of the metabolic capabilities of the cell. Using machine learning techniques we identified the most relevant aspects of cellular organization that change under evolutionary pressures. We considered the transitions from prokarya to eukarya (with a focus on the transitions among the archaea, bacteria and eukarya), from unicellular to multicellular eukarya, from free living to host-associated bacteria, from anaerobic to aerobic, as well as the acquisition of cell motility or growth in an environment of various levels of salinity or temperature. Intuitively, we expect organisms with more complex lifestyles to have more complex and robust metabolic networks. Here we demonstrate for the first time that such organisms are not only characterized by larger, denser networks of metabolic pathways but also have more efficiently organized cross communications, as revealed by subtle changes in network topology. These changes are unevenly distributed among metabolic pathways, with specific categories of pathways being promoted to more central locations as an answer to environmental constraints. CONCLUSIONS: Combining methods from graph theory and machine learning, we have shown here that evolutionary pressures not only affects gene and protein sequences, but also specific details of the complex wiring of functional modules in the cell. This approach allows the identification and quantification of those changes, and provides an overview of the evolution of intracellular systems.

Phylogenetic distances are encoded in networks of interacting pathways.

MOTIVATION: Although metabolic reactions are unquestionably shaped by evolutionary processes, the degree to which the overall structure and complexity of their interconnections are linked to the phylogeny of species has not been evaluated in depth. Here, we apply an original metabolome representation, termed Network of Interacting Pathways or NIP, with a combination of graph theoretical and machine learning strategies, to address this question. NIPs compress the information of the metabolic network exhibited by a species into much smaller networks of overlapping metabolic pathways, where nodes are pathways and links are the metabolites they exchange. RESULTS: Our analysis shows that a small set of descriptors of the structure and complexity of the NIPs combined into regression models reproduce very accurately reference phylogenetic distances derived from 16S rRNA sequences (10-fold cross-validation correlation coefficient higher than 0.9). Our method also showed better scores than previous work on metabolism-based phylogenetic reconstructions, as assessed by branch distances score, topological similarity and second cousins score. Thus, our metabolome representation as network of overlapping metabolic pathways captures sufficient information about the underlying evolutionary events leading to the formation of metabolic networks and species phylogeny. It is important to note that precise knowledge of all of the reactions in these pathways is not required for these reconstructions. These observations underscore the potential for the use of abstract, modular representations of metabolic reactions as tools in studying the evolution of species. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.