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Metabolic efficiency profoundly influences organismal fitness. Nonphotosynthetic organisms, from yeast to mammals, derive usable energy primarily through glycolysis and respiration. Although respiration is more energy efficient, some cells favor glycolysis even when oxygen is available (aerobic glycolysis, Warburg effect). A leading explanation is that glycolysis is more efficient in terms of ATP production per unit mass of protein (that is, faster). Through quantitative flux analysis and proteomics, we find, however, that mitochondrial respiration is actually more proteome efficient than aerobic glycolysis. This is shown across yeast strains, T cells, cancer cells, and tissues and tumors in vivo. Instead of aerobic glycolysis being valuable for fast ATP production, it correlates with high glycolytic protein expression, which promotes hypoxic growth. Aerobic glycolytic yeasts do not excel at aerobic growth but outgrow respiratory cells during oxygen limitation. We accordingly propose that aerobic glycolysis emerges from cells maintaining a proteome conducive to both aerobic and hypoxic growth., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

This cross-sectional study investigated the antimicrobial resistance (AMR) patterns of gram-negative pathogens isolated from 4,789 hospitalized patients with lower respiratory tract infections (LRTIs). Of the collected specimens, 1,325 (27.7%) tested positive for gram-negative bacteria. Acinetobacter baumannii (38.6%), Pseudomonas aeruginosa (33.5%), Klebsiella pneumoniae (18.7%), Escherichia coli (5.6%), and Klebsiella aerogenes (3.5%) were the most prevalent isolates. AMR analysis revealed high resistance rates (79.9%-100%) of A. baumannii isolates to multiple classes of antibiotics except amikacin, trimethoprim/sulfamethoxazole, and colistin. P. aeruginosa displayed low resistance to colistin (< 10%) but high resistance to other antibiotics. K. pneumoniae displayed high resistance rates of 90.0%-100.0% to most penicillins, whereas resistance rates were notably lower for colistin (7.1%) and amikacin (16.7%). K. aerogenes exhibited high resistance to various antibiotics and sensitivity to amikacin (95.1%), ampicillin (100.0%), and colistin (100.0%). E. coli isolates exhibited resistance to ampicillin (96.9%) and maximum sensitivity to several antibiotics. Our study identified significant AMR trends and highlighted the prevalence of multidrug-resistant strains (93.6% for K. aerogenes and 69.1%-92.4% for other isolates). These findings emphasize the urgent need for appropriate antibiotic management practices to combat AMR in gram-negative pathogens associated with LRTIs.

Saccharomyces cerevisiae is an important model organism and a workhorse in bioproduction. Here, we reconstructed a compact and tractable genome-scale resource balance analysis (RBA) model (i.e., named scRBA) to analyze metabolic fluxes and proteome allocation in a computationally efficient manner. Resource capacity models such as scRBA provide the quantitative means to identify bottlenecks in biosynthetic pathways due to enzyme, compartment size, and/or ribosome availability limitations. ATP maintenance rate and in vivo apparent turnover numbers (k app ) were regressed from metabolic flux and protein concentration data to capture observed physiological growth yield and proteome efficiency and allocation, respectively. Estimated parameter values were found to vary with oxygen and nutrient availability. Overall, this work (i) provides condition-specific model parameters to recapitulate phenotypes corresponding to different extracellular environments, (ii) alludes to the enhancing effect of substrate channeling and post-translational activation on in vivo enzyme efficiency in glycolysis and electron transport chain, and (iii) reveals that the Crabtree effect is underpinned by specific limitations in mitochondrial proteome capacity and secondarily ribosome availability rather than overall proteome capacity., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The parameterization of kinetic models requires measurement of fluxes and/or metabolite levels for a base strain and a few genetic perturbations thereof. Unlike stoichiometric models that are mostly invariant to the specific strain, it remains unclear whether kinetic models constructed for different strains of the same species have similar or significantly different kinetic parameters. This important question underpins the applicability range and prediction limits of kinetic reconstructions. To this end, herein we parameterize two separate large-scale kinetic models using K-FIT with genome-wide coverage corresponding to two distinct strains of Saccharomyces cerevisiae: CEN.PK 113-7D strain (model k-sacce306-CENPK), and growth-deficient BY4741 (isogenic to S288c; model k-sacce306-BY4741). The metabolic network for each model contains 306 reactions, 230 metabolites, and 119 substrate-level regulatory interactions. The two models (for CEN.PK and BY4741) recapitulate, within one standard deviation, 77% and 75% of the fitted dataset fluxes, respectively, determined by 13 C metabolic flux analysis for wild-type and eight single-gene knockout mutants of each strain. Strain-specific kinetic parameterization results indicate that key enzymes in the TCA cycle, glycolysis, and arginine and proline metabolism drive the metabolic differences between these two strains of S. cerevisiae. Our results suggest that although kinetic models cannot be readily used across strains as stoichiometric models, they can capture species-specific information through the kinetic parameterization process., Competing Interests: Declaration of competing interest The authors declare no commercial or financial conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Flux balance analysis (FBA) and associated techniques operating on stoichiometric genome-scale metabolic models play a central role in quantifying metabolic flows and constraining feasible phenotypes. At the heart of these methods lie two important assumptions: (i) the biomass precursors and energy requirements neither change in response to growth conditions nor environmental/genetic perturbations, and (ii) metabolite production and consumption rates are equal at all times (i.e., steady-state). Despite the stringency of these two assumptions, FBA has been shown to be surprisingly robust at predicting cellular phenotypes. In this paper, we formally assess the impact of these two assumptions on FBA results by quantifying how uncertainty in biomass reaction coefficients, and departures from steady-state due to temporal fluctuations could propagate to FBA results. In the first case, conditional sampling of parameter space is required to re-weigh the biomass reaction so as the molecular weight remains equal to 1 g mmol -1 , and in the second case, metabolite (and elemental) pool conservation must be imposed under temporally varying conditions. Results confirm the importance of enforcing the aforementioned constraints and explain the robustness of FBA biomass yield predictions., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Nitrogen fixing-cyanobacteria can significantly improve the economic feasibility of cyanobacterial production processes by eliminating the requirement for reduced nitrogen. Anabaena sp. ATCC 33047 is a marine, heterocyst forming, nitrogen fixing cyanobacteria with a very short doubling time of 3.8 h. We developed a comprehensive genome-scale metabolic (GSM) model, i AnC892, for this organism using annotations and content obtained from multiple databases. i AnC892 describes both the vegetative and heterocyst cell types found in the filaments of Anabaena sp. ATCC 33047. i AnC892 includes 953 unique reactions and accounts for the annotation of 892 genes. Comparison of i AnC892 reaction content with the GSM of Anabaena sp. PCC 7120 revealed that there are 109 reactions including uptake hydrogenase, pyruvate decarboxylase, and pyruvate-formate lyase unique to i AnC892. i AnC892 enabled the analysis of energy production pathways in the heterocyst by allowing the cell specific deactivation of light dependent electron transport chain and glucose-6-phosphate metabolizing pathways. The analysis revealed the importance of light dependent electron transport in generating ATP and NADPH at the required ratio for optimal N 2 fixation. When used alongside the strain design algorithm, OptForce, i AnC892 recapitulated several of the experimentally successful genetic intervention strategies that over produced valerolactam and caprolactam precursors.

Kinetic formalisms of metabolism link metabolic fluxes to enzyme levels, metabolite concentrations and their allosteric regulatory interactions. Though they require the identification of physiologically relevant values for numerous parameters, kinetic formalisms uniquely establish a mechanistic link across heterogeneous omics datasets and provide an overarching vantage point to effectively inform metabolic engineering strategies. Advances in computational power, gene annotation coverage, and formalism standardization have led to significant progress over the past few years. However, careful interpretation of model predictions, limited metabolic flux datasets, and assessment of parameter sensitivity remain as challenges. In this review we highlight fundamental considerations which influence model quality and prediction, advances in methodologies, and success stories of deploying kinetic models to guide metabolic engineering., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Many platform chemicals can be produced from renewable biomass by microorganisms, with organic acids making up a large fraction. Intolerance to the resulting low pH growth conditions, however, remains a challenge for the industrial production of organic acids by microorganisms. Issatchenkia orientalis SD108 is a promising host for industrial production because it is tolerant to acidic conditions as low as pH 2.0. With the goal to systematically assess the metabolic capabilities of this non-model yeast, we developed a genome-scale metabolic model for I. orientalis SD108 spanning 850 genes, 1826 reactions, and 1702 metabolites. In order to improve the model's quantitative predictions, organism-specific macromolecular composition and ATP maintenance requirements were determined experimentally and implemented. We examined its network topology, including essential genes and flux coupling analysis and drew comparisons with the Yeast 8.3 model for Saccharomyces cerevisiae . We explored the carbon substrate utilization and examined the organism's production potential for the industrially-relevant succinic acid, making use of the OptKnock framework to identify gene knockouts which couple production of the targeted chemical to biomass production. The genome-scale metabolic model iIsor 850 is a data-supported curated model which can inform genetic interventions for overproduction., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

Rhodosporidium toruloides is a red, basidiomycetes yeast that can accumulate a large amount of lipids and produce carotenoids. To better assess this non-model yeast's metabolic capabilities, we reconstructed a genome-scale model of R. toruloides IFO0880's metabolic network ( iRhto 1108) accounting for 2204 reactions, 1985 metabolites and 1108 genes. In this work, we integrated and supplemented the current knowledge with in-house generated biomass composition and experimental measurements pertaining to the organism's metabolic capabilities. Predictions of genotype-phenotype relations were improved through manual curation of gene-protein-reaction rules for 543 reactions leading to correct recapitulations of 84.5% of gene essentiality data (sensitivity of 94.3% and specificity of 53.8%). Organism-specific macromolecular composition and ATP maintenance requirements were experimentally measured for two separate growth conditions: (i) carbon and (ii) nitrogen limitations. Overall, iRhto 1108 reproduced R. toruloides 's utilization capabilities for 18 alternate substrates, matched measured wild-type growth yield, and recapitulated the viability of 772 out of 819 deletion mutants. As a demonstration to the model's fidelity in guiding engineering interventions, the OptForce procedure was applied on iRhto 1108 for triacylglycerol overproduction. Suggested interventions recapitulated many of the previous successful implementations of genetic modifications and put forth a few new ones., (© 2019 The Authors.)

Conversion of renewable biomass to useful molecules in microbial cell factories can be approached in a rational and systematic manner using constraint-based reconstruction and analysis. Filtering for high confidence in silico designs is critical because in vivo construction and testing of strains is expensive and time consuming. As such, a workflow was devised to analyze the robustness of growth-coupled production when considering the biosynthetic costs of the proteome and variability in enzyme kinetic parameters using a genome-scale model of metabolism and gene expression (ME-model). A collection of 2632 unfiltered knockout designs in Escherichia coli was evaluated by the workflow. A ME-model was used in the workflow to test the designs' growth-coupled production in addition to a less complex genome-scale metabolic model (M-model). The workflow identified 634 M-model growth-coupled designs which met the filtering criteria and 42 robust designs, which met growth-coupled production criteria using both M and ME-models. Knockouts were found to follow a pattern of controlling intermediate metabolite consumption such as pyruvate consumption and high flux subsystems such as glycolysis. Kinetic parameter sampling using the ME-model revealed how enzyme efficiency and pathway tradeoffs can affect growth-coupled production phenotypes.

Tumor antigen (TA)-specific immunotherapy is an emerging approach for cancer treatment. Potent adjuvants are prerequisites to the immunotherapy for overcoming the low immunogenicity of TAs. We previously demonstrated that a bacterial flagellin, Vibrio vulnificus FlaB, has potent adjuvant activity in various vaccination models. In this study, we investigated whether the FlaB protein could be a potent adjuvant for a human papillomavirus 16 E6 and E7 (E6/E7) peptide-based anticancer immunotherapy. We used an E6/E7-expressing TC-1 carcinoma implantation animal model and tested TA-specific immunomodulation by FlaB. We co-administered the E6/E7 peptide either with or without FlaB into TC-1 tumor-bearing mice and then analyzed the antitumor activity of the peptide. FlaB significantly potentiated specific antitumor immune responses elicited by the peptide immunization, as evidenced by retarded in vivo tumor growth and significantly prolonged survival. We noticed that TC-1 cells do not express Toll-like receptor 5 (TLR5) on their surface and the TLR5 signaling pathway in TC-1 cells was not responsible for the antitumor effect of FlaB. FlaB potentiated the CTL activity and Ag-specific IFN-γ production of CD8(+) T cells from the draining lymph node and spleen. In addition, this antitumor activity was abrogated following the in vivo depletion of CD8(+) T cells and in TLR5 knockout (KO) or MyD88 KO mice. These results suggest that flagellin could enhance TA-specific CD8(+) CTL immune responses through TLR5 stimulation in cancer immunotherapy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We report a case of a 75-year-old male with indolent chronic lymphocytic leukaemia (CLL) for 8 years, who presented with a 6-month history of a painful, zosteriform eruption in a T3-4 distribution that evolved into an unusual crop of papular nodules. Upon biopsy and immunostaining of these lesions CLL was proven consistent with leukaemia cutis related to varicella-zoster virus reactivation. In the absence of other treatment indices, he was commenced on chlorambucil with successful resolution of both his pain and the lesions., (© 2012 The Authors; Internal Medicine Journal © 2012 Royal Australasian College of Physicians.)

Background: Recent gene profiling data suggest that, besides the anagen hair bulb, the epithelial stem cell region in the outer root sheath of hair follicles (HFs), termed the bulge, may also represent an area of relative immune privilege (IP)., Objectives: To investigate whether the human HF bulge is a site of relative IP within anagen VI HFs., Methods: Anagen VI HFs from normal human scalp skin were analysed using immunohistological staining techniques, quantitative histomorphometry and statistical analysis. For functional evidence we performed full-thickness human scalp skin organ cultures to investigate whether interferon (IFN)-gamma, a key inducer of IP collapse in hair bulbs, has a similar effect on the putative bulge IP., Results: Major histocompatibility complex (MHC) class Ia, beta(2)-microglobulin and MHC class II immunoreactivity are downregulated in the human bulge. The immunosuppressants alpha-melanocyte stimulating hormone, transforming growth factor-beta2, macrophage migration inhibitory factor and indoleamine-2,3-dioxygenase (IDO) are upregulated in the CD200+, stem cell-rich bulge region. These CD200+ cells also co-express HLA-E. Furthermore, IFN-gamma induces significant ectopic MHC class Ia expression in bulge cells of organ-cultured human scalp skin., Conclusions: These data suggest that the bulge of human anagen HFs represents a hitherto unrecognized site of relative IP in human skin. Simultaneously, we present the first evidence of IDO and HLA-E protein expression in normal human HFs. Bulge IP presumably protects the HF epithelial stem cell reservoir from autoaggressive immune attack whereas a loss of bulge IP may play a central role in the pathogenesis of cicatricial alopecias.

Purpose: To prospectively implement high-temporal-resolution cine magnetic resonance (MR) imaging protocol to compare cardiac preejection contraction (PEC) and prefilling relaxation (PFR) times between heart failure (HF) patients and healthy control subjects and to assess accuracy of PEC times to stratify HF patients, with ejection fraction (EF) and New York Heart Association (NYHA) symptom class as reference standards., Materials and Methods: Following institutional review board approval of this HIPAA-compliant study and written informed consent, 18 healthy volunteers (10 women, eight men; mean age, 43 years +/- 14 [standard deviation]) and 18 HF patients (five women, 13 men; mean age, 49.8 years +/- 3) were imaged (breath-hold true fast imaging with steady-state precession, with temporal resolution of 5.6 msec at 1.5 T). By using left ventricular (LV) outflow tract acquisition, PEC phase was defined as time at QRS trigger to immediately before aortic valve opening. PFR was defined as time from initial aortic valve closure to immediately before mitral valve opening. Group means were compared (unpaired Student t test). Accuracy of PEC parameters in stratifying participants with severe systolic HF on the basis of EF and NYHA symptom class was assessed (receiver operating characteristic curve analysis)., Results: Compared with control subjects, HF patients had prolonged mean PEC time (40.4 msec +/- 11.8 vs 91.3 msec +/- 26, P < .001) and mean PFR time (68.3 msec +/- 26.8 vs 103.7 msec +/- 41.8, P < .01). PEC time correlated with global EF (r = -0.73, P < .001) and LV mass (r = 0.69, P < .001). For identification of patients with severe LV systolic dysfunction (EF 2), PEC time had good accuracy (AUC, 0.875 [P < .001])., Conclusion: It is feasible to assess isovolumic PEC and PFR phases of the cardiac cycle with high-frame-rate cine MR images, and PEC time is a surrogate measure of moderate-to-severe systolic HF.

A 57-year-old woman developed repigmentation of white donor hairs 9 months after undergoing hair transplantation for an area of frontal scarring alopecia present since childhood. The hairs have remained repigmented more than 2 years post transplant. We discuss possible mechanisms underlying long-term follicular repigmentation of white hairs.

Background: Rituximab is a monoclonal antibody directed against the CD20 antigen expressed on B lymphocytes. There are reports of its efficacy in the treatment of autoimmune diseases, including pemphigus., Objectives: Prospectively to evaluate the efficacy of rituximab as adjuvant treatment for pemphigus vulgaris (PV)., Methods: Patients with PV were treated with intravenous rituximab (375 mg m(-2)) weekly for 4 weeks in this prospective open-label pilot study. Other concurrent immunosuppression was continued., Results: Of five patients, one achieved complete remission and was able to cease all medication, while two achieved clearance of clinical lesions but continued on systemic therapy. Two patients had progressive disease. Time to response was 2-8 months, with a 13- to 18-month response duration. Response was associated with reduction in serum antiepithelial antibodies. Two patients had significant infectious complications (one developed community-acquired pneumonia associated with delayed-onset neutropenia and the other developed cytomegalovirus infection)., Conclusions: Rituximab has shown efficacy in the treatment of PV. Patients on multiple immunosuppressives should be closely monitored for infectious complications.

Three patients suffering from classic dermatomyositis (juvenile and adult-onset) with prominent recalcitrant skin manifestations are described. All patients demonstrated good control of muscle symptoms on immunosuppressive medications, but their cutaneous disease persisted despite treatment with at least 4 different systemic treatments and topical agents. They were given rituximab, a monoclonal anti-CD20 antibody, achieving a response with minimal side effects. We document our experience with this medication for the cutaneous lesions of dermatomyositis.

Nitroglycerin and dipyridamole are two commonly available and well tolerated vasoactive medications. Their acute hemodynamic effects in patients with pulmonary arterial hypertension are not well defined in the current literature. The authors retrospectively analyzed the acute hemodynamic effects of IV nitroglycerin, dipyridamole, and epoprostenol in 59 patients with pulmonary arterial hypertension as determined by changes from baseline in systemic and pulmonary hemodynamic parameters. Statistical analysis was performed using the independent sample t test. A p value <0.05 was considered significant. Nitroglycerin is predominantly a vasodilator of the pulmonary vasculature with moderate systemic vasodilator effect, while dipyridamole is primarily a positive inotropic agent. Epoprostenol is a potent vasodilator of both pulmonary and systemic vessels and a strong positive inotropic agent. Nitroglycerin and dipyridamole may be useful in the acute management of pulmonary arterial hypertension.