In vitro effects of gamma-secretase inhibition in HPV-positive and HPV-negative head and neck squamous cell carcinoma.

BACKGROUND: New chemotherapy agents are warranted for head and neck squamous cell carcinoma (HNSCC), particularly for incidence-rising HPV-positive tumors. Based on the evidence of Notch pathway involvement in cancer promotion and progression, we aimed to gain insights into the in vitro antineoplastic effects of gamma-secretase inhibition in HPV-positive and -negative HNSCC models. METHODS: All in vitro experiments were conducted in two HPV-negative (Cal27 and FaDu) and one HPV-associated HNSCC cell line (SCC154). The influence of the gamma-secretase inhibitor PF03084014 (PF) on proliferation, migration, colony forming, and apoptosis was assessed. RESULTS: We observed significant anti-proliferative, anti-migratory, anti-clonogenic, and pro-apoptotic effects in all three HNSCC cell lines. Furthermore, synergistic effects with concomitant radiation were observable in the proliferation assay. Interestingly, effects were slightly more potent in the HPV-positive cells. CONCLUSION: We provided novel insights into the potential therapeutic relevance of gamma-secretase inhibition in HNSCC cell lines in vitro. Therefore, PF may become a viable treatment option for patients with HNSCC, particularly for patients with HPV-induced malignancy. Indeed, further in vitro and in vivo experiments should be conducted to validate our results and decipher the mechanism behind the observed anti-neoplastic effects.

Inhibition of beta-catenin shows therapeutic potential in head and neck squamous cell carcinoma in vitro.

Beta-catenin is known to be a vital component of the canonical Wnt signaling cascade, involved in the carcinogenesis of different solid tumors. We aimed to evaluate the effects of Beta-catenin inhibition in head and neck squamous cell carcinoma (HNSCC) in vitro. The small molecular compound MSAB was used to inhibit Wnt/Beta-catenin signaling in a human papillomavirus (HPV)-positive and HPV-negative cell line and its effects on cell proliferation, migration, colony formation, apoptosis, as well as radiosensitizing properties were assessed. Significant antineoplastic effects were observed in both cell lines. Interestingly, stronger anti-neoplastic and radiosensitizing effects were observed in the HPV-negative cell line, whereas stronger anti-migratory potential was detected in HPV-positive HNSCC cells. In conclusion, our findings suggest MSAB as a potential therapeutic agent for HNSCC. Further studies are warranted to unravel the mechanistic background of our findings.

Dissecting the energy metabolism in Mycoplasma pneumoniae through genome-scale metabolic modeling.

Mycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint-based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time-dependent changes, albeit using a static model. By performing an in silico knock-out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms.

No significant difference in the prognostic value of the 5th and 7th editions of AJCC staging for differentiated thyroid cancer.

OBJECTIVE: The seventh edition of the American Joint Committee on Cancer (AJCC) has more detailed staging categories for differentiated thyroid cancer (DTC) than the fifth edition. The aim was to compare potential alterations in the disease-specific (DSS), event-free (EFS) and overall survival (OS), after reclassification from the fifth to the seventh edition. METHODS: Data of 2460 patients with DTC referred to our centre were reclassified from the fifth to the seventh edition of AJCC. DSS, EFS and OS were calculated using the Kaplan-Meier method and compared by the log-rank test. The relative abilities of each edition to predict survival were calculated by the proportion of variance explained (PVE). RESULTS: After reclassification to the seventh edition, there was an increase in stage I and IV patients from 58·1% to 65·0% and from 6·2% to 10·1%, respectively, and a corresponding decrease in stage II and III patients from 22·4% to 12·5% and 13·3% to 12·4%, respectively. As to DSS, the seventh edition had only a marginally higher PVE value than the fifth edition. With respect to EFS, the predictability of the seventh edition was even inferior to that of the fifth edition. Similarly, with regard to OS, the PVE value was slightly better for the older edition. Furthermore, a comparison only for those patients affected by the reclassification revealed no differences for DSS, EFS or OS between classifications. CONCLUSION: When comparing the stages of the seventh with the fifth edition of the AJCC for DTC, there was no significant difference in predicting DSS, EFS and OS.

Cross-talk between phosphorylation and lysine acetylation in a genome-reduced bacterium.

Protein post-translational modifications (PTMs) represent important regulatory states that when combined have been hypothesized to act as molecular codes and to generate a functional diversity beyond genome and transcriptome. We systematically investigate the interplay of protein phosphorylation with other post-transcriptional regulatory mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae. Systematic perturbations by deletion of its only two protein kinases and its unique protein phosphatase identified not only the protein-specific effect on the phosphorylation network, but also a modulation of proteome abundance and lysine acetylation patterns, mostly in the absence of transcriptional changes. Reciprocally, deletion of the two putative N-acetyltransferases affects protein phosphorylation, confirming cross-talk between the two PTMs. The measured M. pneumoniae phosphoproteome and lysine acetylome revealed that both PTMs are very common, that (as in Eukaryotes) they often co-occur within the same protein and that they are frequently observed at interaction interfaces and in multifunctional proteins. The results imply previously unreported hidden layers of post-transcriptional regulation intertwining phosphorylation with lysine acetylation and other mechanisms that define the functional state of a cell.

Forecasting supply and demand in nursing professions: impacts of occupational flexibility and employment structure in Germany.

BACKGROUND: In light of Germany's ageing society, demand for nursing professionals is expected to increase in the coming years. This will pose a challenge for policy makers to increase the supply of nursing professionals. METHODOLOGY: To portray the different possible developments in the supply of nursing professionals, we projected the supply of formally trained nurses and the potential supply of persons who are able to work in a nursing profession. This potential supply of nursing professionals was calculated on the basis of empirical information on occupational mobility provided by the German Microcensus 2005 (Labour Force Survey). We also calculated how the supply of full-time equivalents (FTEs) will develop if current employment structures develop in the direction of employment behaviour in nursing professions in eastern and western Germany. We then compared these different supply scenarios with two demand projections ('status quo' and 'compression of morbidity' scenarios) from Germany's Federal Statistical Office. RESULTS: Our results show that, even as early as 2005, meeting demand for FTEs in nursing professions was not arithmetically possible when only persons with formal qualification in a nursing profession were taken into account on the supply side. When additional semi-skilled nursing professionals are included in the calculation, a shortage of labour in nursing professions can be expected in 2018 when the employment structure for all nursing professionals remains the same as the employment structure seen in Germany in 2005 (demand: 'status quo scenario'). Furthermore, given an employment structure as in eastern Germany, where more nursing professionals work on a full-time basis with longer working hours, a theoretical shortage of nursing professionals could be delayed until 2024. CONCLUSIONS: Our analysis of occupational flexibility in the nursing field indicates that additional potential supply could be generated by especially training more young people for a nursing profession as they tend to stay in their initial occupation. Furthermore, the number of FTEs in nursing professions could be increased by promoting more full-time contracts in Western Germany. Additionally, employment contracts for just a small number of weekly working hours (marginal employment) cannot be considered an adequate instrument for keeping formally trained nursing professionals employed in the nursing field.

ß-CATENIN is a positive prognostic marker for HPV-positive head and neck squamous cell carcinoma.

PURPOSE: The evolutionary-conserved Wnt/ß-CATENIN (WBC) pathway has been implicated in the pathogenesis of different solid malignant tumors. We evaluated the prognostic relevance of ß-CATENIN, a pivotal mediator of WBC activation, in patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC). METHODS: We analyzed if patients with HPV-positive HNSCC from the "The Cancer Genome Atlas" (TCGA cohort, n = 41) can be stratified based on their CTNNB1 mRNA expression. Moreover, in a tissue microarray (TMA) of primary tumor sections from HPV-positive HNSCC patients treated in a tertiary academic center (in-house cohort, n = 31), we evaluated the prognostic relevance of ß-CATENIN expression on protein level. RESULTS: In silico mining of CTNNB1 expression in HPV-positive HNSCC revealed that high CTNNB1 expression was linked to better overall survival (OS, p = 0.062). Moreover, high ß-CATENIN expression was significantly associated with a better OS in our in-house cohort (p = 0.035). CONCLUSION: Based on these findings, we postulate that ß-CATENIN expression could serve (potentially in conjunction with other WBC pathway members) as a marker for better survival outcomes in patients with HPV-positive HNSCC. However, it is evident that future studies on bigger cohorts are warranted.

Quantification of mRNA and protein and integration with protein turnover in a bacterium.

Biological function and cellular responses to environmental perturbations are regulated by a complex interplay of DNA, RNA, proteins and metabolites inside cells. To understand these central processes in living systems at the molecular level, we integrated experimentally determined abundance data for mRNA, proteins, as well as individual protein half-lives from the genome-reduced bacterium Mycoplasma pneumoniae. We provide a fine-grained, quantitative analysis of basic intracellular processes under various external conditions. Proteome composition changes in response to cellular perturbations reveal specific stress response strategies. The regulation of gene expression is largely decoupled from protein dynamics and translation efficiency has a higher regulatory impact on protein abundance than protein turnover. Stochastic simulations using in vivo data show how low translation efficiency and long protein half-lives effectively reduce biological noise in gene expression. Protein abundances are regulated in functional units, such as complexes or pathways, and reflect cellular lifestyles. Our study provides a detailed integrative analysis of average cellular protein abundances and the dynamic interplay of mRNA and proteins, the central biomolecules of a cell.

Targeting Wnt/Beta-Catenin Signaling in HPV-Positive Head and Neck Squamous Cell Carcinoma.

Wnt/Beta-Catenin signaling is involved in the carcinogenesis of different solid malignant tumors. The interaction of Creb-binding protein (CBP) with Beta-Catenin is a pivotal component of the Wnt/Beta-Catenin signaling pathway. The first aim of this study was to evaluate the association of CBP expression with survival in patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC). Second, the in vitro effects of the inhibition of CBP/Beta-Catenin interaction were analyzed. In particular, the effects of ICG-001, an inhibitor of CBP/Beta-Catenin interaction, on proliferation, cell death, modulation of Wnt/Beta-Catenin target expression, and cell migration were examined in vitro. High CBP expression is significantly associated with better survival on mRNA and protein levels. Furthermore, we observed cytotoxic as well as anti-migratory effects of ICG-001. These effects were particularly more potent in the HPV-positive than in the -negative cell line. Mechanistically, ICG-001 treatment induced apoptosis and led to a downregulation of CBP, c-MYC, and Cyclin D1 in HPV-positive cells, indicating inhibition of Wnt/Beta-Catenin signaling. In conclusion, high CBP expression is observed in HPV-positive HNSCC patients with a good prognosis, and ICG-001 showed a promising antineoplastic potential, particularly in HPV-positive HNSCC cells. Therefore, ICG-001 may potentially become an essential component of treatment de-escalation regimens for HPV-positive HNSCC. Further studies are warranted for additional assessment of the mechanistic background of our in vitro findings.

A more precise characterization of chaperonin substrates.

MOTIVATION: Molecular chaperones prevent the aggregation of their substrate proteins and thereby ensure that they reach their functional native state. The bacterial GroEL/ES chaperonin system is understood in great detail on a structural, mechanistic and functional level; its interactors in Escherichia coli have been identified and characterized. However, a long-standing question in the field is: What makes a protein a chaperone substrate? RESULTS: Here we identify, using a bioinformatics-based approach a simple set of quantities, which characterize the GroEL-substrate proteome. We define three novel parameters differentiating GroEL interactors from other cellular proteins: lower rate of evolution, hydrophobicity and aggregation propensity. Combining them with other known features to a simple Bayesian predictor allows us to identify known homologous and heterologous GroEL substrateproteins. We discuss our findings in relation to established mechanisms of protein folding and evolutionary buffering by chaperones.

The Potential Prognostic Value of a Novel Hematologic Marker Fibrinogen-to-Lymphocyte Ratio in Head and Neck Adenoid-Cystic Carcinoma.

Many patients with adenoid-cystic carcinoma (ACC) experience an indolent course of disease over many years but face late recurrence, and long-term survivors are rare. Due to its infrequent occurrence, it is hard to predict outcome in these patients. The fibrinogen-to-lymphocyte ratio (FLR) was recently proposed as an outcome prognosticator in different cancer entities. We aimed to investigate its prognostic relevance in patients with head and neck ACC. This retrospective analysis was performed including all patients treated for ACC between 1998 and 2020. The FLR ratio was calculated based on pretreatment values (0-7 days). The study cohort was dichotomized based on optimized threshold value and compared for differences in outcome (overall survival (OS) and disease-free survival (DFS)). In the cohort of 39 included patients, the OS was significantly longer in the low (n = 28) compared to the high pretreatment FLR group (n = 11) (median OS 150.5 months, 95% confidence intervals (CI) 85.3-215.7 months vs. 29.4 months, 95% CI not reached; p = 0.0093). Similarly, the DFS was significantly longer in the low FLR group (median DFS 74.5 months, 95% CI 30.6-118.4 months vs. 11.0 months, 95% CI 5.1-16.9 months; p = 0.019). The FLR is an easily obtainable and simple marker and may be a valuable outcome prognosticator in patients with ACC. Further studies are needed for validation of our results.

Untreated 'blow-in' fracture of the orbital floor causing a mucocele: report of an unusual late complication.

BACKGROUND: Several severe complications have been described with blow-in fractures. Therefore, immediate surgical treatment of these fractures has been recommended. To date, there is only minimal knowledge on long-term complications of blow-in fractures that have remained untreated. The present case report describes a late complication of an untreated blow-in fracture of the orbital floor. CASE: A 37-year-old male was involved in a car accident 16 years before. At that time, a non-dislocated midfacial fracture was diagnosed and remained untreated because of the lack of clinical symptoms. Four months before surgery an exophthalmos of the left globe began to develop. CT examination revealed a consolidated blow-in fracture of the left orbital floor and an opaque mass around the dislocated bony fragments. By an infraorbital approach the bony fragments and the surrounding mass were removed. Histological examination of the removed material revealed a cystic structure lined with respiratory epithelium. Therefore, the diagnosis 'post-traumatic mucocele in the orbit caused by dislocated respiratory epithelium from the maxillary sinus' was made. CONCLUSION: Even if blow-in fractures do not cause complications immediately after trauma, late complications like mucoceles can occur after several symptom-free years. Therefore, early reconstruction should be intended even in asymptomatic cases of blow-in fractures with minimal displacement of the bony fragments.

Differentiated thyroid cancer patients more than 60 years old paradoxically show an increased life expectancy.

UNLABELLED: The aim of this study was to compare the overall survival of a large, single-center cohort of patients who had differentiated thyroid cancer (DTC) with that of a matched general population. METHODS: We analyzed 2,428 consecutive patients who had DTC and underwent treatment from 1965 to 2013 at the Department of Nuclear Medicine, University Hospital of Münster, Münster, Germany, according to international standards. Patients were classified on the basis of the current, seventh edition of the American Joint Committee on Cancer/Union for International Cancer Control classification system. Additionally, a subgroup analysis with regard to age at diagnosis was performed. The overall survival of the patients was compared with the expected survival of the general population on the basis of age and sex, as provided by the Federal Statistical Office of Germany. RESULTS: Compared with the expected survival, the overall survival of patients with stage I disease paradoxically was significantly better (P < 0.001). In the subgroup analysis, a significantly lower mortality rate was observed in elderly patients (≥60 y old) with stage I disease. On the other hand, patients between 20 and 45 y of age and with distant metastases at diagnosis had a significantly increased standardized mortality rate. In contrast, other patients with stage II disease and more than 45 y old had a normal mortality rate. The mortality rate was significantly increased in all patients with stage IVC disease. CONCLUSION: Older patients with more limited disease paradoxically had better survival than would be expected on the basis of age and sex, whereas young adults as well as patients more than 45 y old and with distant metastases had increased mortality rates. For all other DTC patients, regardless of age or TNM stage, no significant survival difference was seen.

Relative en- and exophthalmometry in zygomatic fractures comparing optical non-contact, non-ionizing 3D imaging to the Hertel instrument and computed tomography.

AIM: It is the aim of the present study to introduce non-contact, non-invasive optical 3D imaging to relative exophthalmometry and to compare the resulting data to exophthalmometry values assessed by the Hertel instrument and computed tomography. PATIENTS AND METHODS: 20 patients (3 female, 17 male, 44.4+/-16.6 years) without orbital pathology, who were examined by computed tomography of head and neck for the exclusion of different diseases, and seven patients (1 female, 6 male, 40.1+/-14.4 years), who received routine orbital computed tomography because of zygomatic fractures, were included in the study. Optical 3D images of the facial surface were assessed and Hertel exophthalmometry was carried out to determine the relative globe position. In patients with zygomatic fractures the assessment of optical 3D images and Hertel values was repeated 5 days after surgery. RESULTS: For patients without orbital pathology relative exophthalmometry data were 1.4+/-1.1 mm for the Hertel instrument, 0.9+/-1.0 mm for computed tomography and 0.5+/-0.5 mm for optical 3D imaging. The values for Hertel exophthalmometry and computed tomography did not differ statistically significantly (p(Herteldifferencepreop/CTdifferencepreop)=0.284), while there was a significant difference between Hertel exophthalmometry and optical 3D imaging (p(Herteldifferencepreop/opticaldifferencepreop)=0.008). In the cases of zygomatic fractures, Hertel exophthalmometry revealed less pronounced relative differences in globe position than CT and optical 3D imaging data (Hertel 0.7+/-1.1 mm, CT 1.9+/-1.0 mm, optical 3D imaging 1.9+/-1.0 mm). Postoperatively, relative Hertel exophthalmometry showed an increased value revealing a more pronounced enophthalmos (1.7+/-1.0 mm), while the corresponding value of the optical 3D images decreased as a sign for normalization of the globe position (1.1+/-0.7 mm). CONCLUSION: Because of its reliance on the lateral orbital rims Hertel exophthalmometry can lead to an under- or overestimation of enophthalmos, when soft tissue oedema or a dislocation of the orbital rim are present. The combination of computed tomography as baseline measurement and optical 3D imaging for the follow-up examinations reveal more realistic data in cases of zygomatic fractures. Therefore, they should be preferred to the determination of Hertel values especially in more complex cases.

Validation of in vivo assessment of facial soft-tissue volume changes and clinical application in midfacial distraction: a technical report.

The purpose of this study was to validate the assessment of visible volume changes of the facial soft tissue with an optical three-dimensional sensor and to introduce new parameters for the evaluation of the soft-tissue shape achieved from three-dimensional data of selected cases of midfacial distraction. Images of a truncated cone of known volume were assessed repeatedly with an optical three-dimensional sensor based on phase-measuring triangulation to calculate the volume. Two cubic centimeters of anesthetic solution was injected into the right malar region of 10 volunteers who gave their informed consent. Three-dimensional images were assessed before and immediately after the injections for the assessment of the visible volume change. In five patients who underwent midfacial distraction after a high quadrangular Le Fort I osteotomy, three-dimensional scans were acquired before and 6 and 24 months after the operation. The visible soft-tissue volume change in the malar-midfacial area and the mean distance of the accommodation vector that transformed the preoperative into the postoperative surface were calculated. The volume of the truncated cone was 235.26 +/- 1.01 cc, revealing a measurement uncertainty of 0.4 percent. The injections of anesthetic solution into the malar area resulted in an average visible volume change of 2.06 +/- 0.06 cc. The measurement uncertainty was 3 percent. In the five patients, the average distance of maxillary advancement was 6.7 +/- 2.3 mm after 6 months and 5.4 +/- 3.0 mm after 2 years. It was accompanied by a mean visible volume increase of 8.92 +/- 5.95 cc on the right side and 9.54 +/- 4.39 cc on the left side after 6 months and 3.54 +/- 3.70 cc and 4.80 +/- 3.47 cc, respectively, after 2 years. The mean distance of the accommodation vector was 4.41 +/- 1.94 mm on the right side and 4.74 +/- 1.32 mm on the left side after 6 months and 1.62 +/- 1.96 mm and 2.16 +/- 1.52 mm, respectively, after 2 years. The assessment of visible volume changes by optical three-dimensional images can be carried out with considerable accuracy. The determination of volume changes and accompanying accommodation vectors completes the cephalometric analysis during the follow-up of patients undergoing midfacial distraction. The new parameters will help to assess normative soft-tissue data on the basis of three-dimensional imaging with a view to an improved three-dimensional prediction of the operative outcome of orthognathic surgery.

Integrative quantitation enables a comprehensive proteome comparison of two Mycoplasma pneumoniae genetic perturbations.

Quantitative proteomics is an essential tool in proteome research since it enables measuring changes in protein abundance in response to biological perturbations. During the last few years, different quantitative strategies have been developed in proteomics to compare different experimental conditions, including label-free and isobaric chemical labeling approaches. Here we show that different quantitation techniques have an important influence on detected sample variability, and we use the combination of six different quantitation strategies to perform a proteome comparison of three different Mycoplasma pneumoniae strains (ldh knockdown, Δprkc, and wild-type). The integration of the different datasets indicates that the ldh knockdown strongly affects the abundance of ribosomal proteins and enzymes involved in the regulation of the cellular redox state, whereas the prkc deletion affects key cellular physiological processes such as protein and DNA synthesis, and cytoadherence.

Large-scale metabolome analysis and quantitative integration with genomics and proteomics data in Mycoplasma pneumoniae.

Systems metabolomics, the identification and quantification of cellular metabolites and their integration with genomics and proteomics data, promises valuable functional insights into cellular biology. However, technical constraints, sample complexity issues and the lack of suitable complementary quantitative data sets prevented accomplishing such studies in the past. Here, we present an integrative metabolomics study of the genome-reduced bacterium Mycoplasma pneumoniae. We experimentally analysed its metabolome using a cross-platform approach. We explain intracellular metabolite homeostasis by quantitatively integrating our results with the cellular inventory of proteins, DNA and other macromolecules, as well as with available building blocks from the growth medium. We calculated in vivo catalytic parameters of glycolytic enzymes, making use of measured reaction velocities, as well as enzyme and metabolite pool sizes. A quantitative, inter-species comparison of absolute and relative metabolite abundances indicated that metabolic pathways are regulated as functional units, thereby simplifying adaptive responses. Our analysis demonstrates the potential for new scientific insight by integrating different types of large-scale experimental data from a single biological source.

Correlation of mRNA and protein in complex biological samples.

The correlation between mRNA and protein abundances in the cell has been reported to be notoriously poor. Recent technological advances in the quantitative analysis of mRNA and protein species in complex samples allow the detailed analysis of this pathway at the center of biological systems. We give an overview of available methods for the identification and quantification of free and ribosome-bound mRNA, protein abundances and individual protein turnover rates. We review available literature on the correlation of mRNA and protein abundances and discuss biological and technical parameters influencing the correlation of these central biological molecules.

Proteome organization in a genome-reduced bacterium.

The genome of Mycoplasma pneumoniae is among the smallest found in self-replicating organisms. To study the basic principles of bacterial proteome organization, we used tandem affinity purification-mass spectrometry (TAP-MS) in a proteome-wide screen. The analysis revealed 62 homomultimeric and 116 heteromultimeric soluble protein complexes, of which the majority are novel. About a third of the heteromultimeric complexes show higher levels of proteome organization, including assembly into larger, multiprotein complex entities, suggesting sequential steps in biological processes, and extensive sharing of components, implying protein multifunctionality. Incorporation of structural models for 484 proteins, single-particle electron microscopy, and cellular electron tomograms provided supporting structural details for this proteome organization. The data set provides a blueprint of the minimal cellular machinery required for life.

Impact of genome reduction on bacterial metabolism and its regulation.

To understand basic principles of bacterial metabolism organization and regulation, but also the impact of genome size, we systematically studied one of the smallest bacteria, Mycoplasma pneumoniae. A manually curated metabolic network of 189 reactions catalyzed by 129 enzymes allowed the design of a defined, minimal medium with 19 essential nutrients. More than 1300 growth curves were recorded in the presence of various nutrient concentrations. Measurements of biomass indicators, metabolites, and 13C-glucose experiments provided information on directionality, fluxes, and energetics; integration with transcription profiling enabled the global analysis of metabolic regulation. Compared with more complex bacteria, the M. pneumoniae metabolic network has a more linear topology and contains a higher fraction of multifunctional enzymes; general features such as metabolite concentrations, cellular energetics, adaptability, and global gene expression responses are similar, however.