Topical platelet-rich plasma as a possible treatment for olfactory dysfunction-A randomized controlled trial.

BACKGROUND: Olfactory dysfunction (OD) affects many survivors of COVID-19. Prior studies have investigated the use of platelet-rich plasma (PRP) injections for OD. We describe the first randomized controlled trial investigating topical PRP for OD treatment and contribute to existing literature illustrating PRP as an emerging therapeutic. METHODS: This is a single-blinded, randomized controlled trial conducted from July 2022 to December 2023. Adult patients with OD ≥6 months secondary to COVID-19 with Brief Smell Identification Test (BSIT) scores of ≤8/12 or SCENTinel odor intensity of ≤40/100 were included. Patients were randomized to three, monthly PRP or placebo-impregnated Surgifoam treatments into bilateral olfactory clefts. The BSIT, SCENTinel, and Questionnaire of Olfactory Disorders-Negative Statements (QOD-NS) were completed monthly through month 12. RESULTS: Of 104 patients screened, 83 participated. No significant differences in age, OD duration, BSIT, SCENTinel, or QOD-NS scores were found between PRP (n = 42) and placebo (n = 41) patients at baseline. PRP patients experienced a statistically significant increase in BSIT scores from baseline at months 5‒9, 11, and 12, while placebo patients did not (p < 0.05). However, total BSIT scores were similar between the two groups throughout the study. Neither the SCENTinel odor intensity scores nor the change from baseline were significantly different between the treatment groups. At month 12, PRP patients experienced minor improvement in OD-related quality-of-life compared with placebo. CONCLUSIONS: This study is the first to describe topical PRP as a safe, experimental treatment for OD in humans. PRP may impact odor identification in post-COVID-19 OD patients, although the lack of difference in total BSIT scores highlights the need for further study.

Stellate ganglion block for treating post-COVID-19 parosmia.

BACKGROUND: Post-COVID parosmia may be due to dysautonomia and sympathetic hyperresponsiveness, which can be attenuated by stellate ganglion block (SGB). This study evaluates SGB as a treatment for post-COVID olfactory dysfunction (OD). METHODS: Retrospective case series with prospective data of patients with post-COVID OD undergoing unilateral (UL) or bilateral (BL) SGB. Patients completed Brief Smell Identification Tests (BSIT) (12 points maximum) and post-procedure surveys including parosmia severity scores on a scale of 1 (absent) to 10 (severe). Scores were compared from before treatment (pre-SGB) to after first (SGB1) or second (SGB2) treatments in overall, UL, and BL cohorts. RESULTS: Forty-seven patients with post-COVID OD underwent SGB, including 23 UL and 24 BL. Twenty patients completed pre- and post-SGB BSITs (eight UL and 12 BL). Twenty-eight patients completed postprocedure surveys (11 UL and 17 BL). There were no differences in BSIT scores from pre-SGB to post-SGB1 or post-SGB2 for the overall (p = 0.098), UL (p = 0.168), or BL (p = 0.230) cohorts. Parosmia severity for the overall cohort improved from pre-SGB (8.82 ± 1.28) to post-SGB1 (6.79 ± 2.38) and post-SGB2 (5.41 ± 2.35), with significant differences from pre-SGB to post-SGB1 (p < 0.001) and pre-SGB to post-SGB2 (p < 0.001), but not post-SGB1 to post-SGB2 (p = 0.130). Number of parosmia triggers decreased for overall (p = 0.002), UL (p = 0.030) and BL (p = 0.024) cohorts. Quality of life (QOL) improved for all cohorts regarding food enjoyment, meal preparation, and socialization (p < 0.05). CONCLUSION: SGB may improve subjective parosmia and QOL for patients with post-COVID OD, however it may not affect odor identification. Further placebo-controlled studies are warranted.

Endoscopic endonasal repair of encephaloceles of the lateral sphenoid sinus: multiinstitution confirmation of a new classification.

OBJECTIVE: Encephaloceles of the lateral sphenoid sinus are rare. Originally believed to be due to defects in a patent lateral craniopharyngeal canal (Sternberg canal), they are now thought to originate more commonly from idiopathic intracranial hypertension, not unlike encephaloceles elsewhere in the skull base. A new classification of these encephaloceles was recently introduced, which divided them in relation to the foramen rotundum. Whether this classification can be applied to a larger cohort from multiple institutions and whether it might be useful in predicting outcome is unknown. Thus, the authors' goal was to divide a multiinstitutional cohort of patients with lateral sphenoid encephaloceles into four subtypes to determine their incidence and any correlation with surgical outcome. METHODS: A multicenter retrospective review of prospectively acquired databases was carried out across three institutions. Cases were categorized into one of four subtypes (type I, Sternberg canal; type II, medial to rotundum; type III, lateral to rotundum; and type IV, both medial and lateral with rotundum enlargement). Demographic and outcome metrics were collected. Kaplan-Meyer curves were used to determine the rate of recurrence after surgical repair. RESULTS: A total of 49 patients (71% female) were included. The average BMI was 32.8. All encephaloceles fell within the classification scheme. Type III was the most common (71.4%), followed by type IV (16.3%), type II (10.2%), and type I (2%). Cases were repaired endonasally, via a transpterygoidal approach. Lumbar drains were placed in 78% of cases. A variety of materials was used for closure, with a nasoseptal flap used in 65%. After a mean follow-up of 47 months, there were 4 (8%) CSF leak recurrences, all in patients with type III or type IV leaks and all within 1 year of the first repair. Two leaks were fixed with ventriculoperitoneal shunt and reoperation, 1 with ventriculoperitoneal shunt only, and 1 with a lumbar drain only. Of 45 patients in whom detailed information was available, there were 12 (26.7%) with postoperative dry eye or facial numbness, with facial numbness occurring in type III or type IV defects only. CONCLUSIONS: Endoscopic endonasal repair of lateral sphenoid wing encephaloceles is highly successful, but repair may lead to dry eye or facial numbness. True Sternberg (type I) leaks were uncommon. Failures and facial numbness occurred only in patients with type III and type IV leaks.

Interspecies interactions determine growth dynamics of biopolymer-degrading populations in microbial communities.

Microbial communities perform essential ecosystem functions such as the remineralization of organic carbon that exists as biopolymers. The first step in mineralization is performed by biopolymer degraders, which harbor enzymes that can break down polymers into constituent oligo- or monomeric forms. The released nutrients not only allow degraders to grow, but also promote growth of cells that either consume the degradation products, i.e., exploiters, or consume metabolites released by the degraders or exploiters, i.e., scavengers. It is currently not clear how such remineralizing communities assemble at the microscale-how interactions between the different guilds influence their growth and spatial distribution, and hence the development and dynamics of the community. Here, we address this knowledge gap by studying marine microbial communities that grow on the abundant marine biopolymer alginate. We used batch growth assays and microfluidics coupled to time-lapse microscopy to quantitatively investigate growth and spatial distribution of single cells. We found that the presence of exploiters or scavengers alters the spatial distribution of degrader cells. In general, exploiters and scavengers-which we collectively refer to as cross-feeder cells-slowed down the growth of degrader cells. In addition, coexistence with cross-feeders altered the production of the extracellular enzymes that break down polymers by degrader cells. Our findings reveal that ecological interactions by nondegrading community members have a profound impact on the functions of microbial communities that remineralize carbon biopolymers in nature.

Sleep Subdomain of the Sinonasal Outcome Test as a Potential Screening Tool for Sleep Apnea in Chronic Rhinosinusitis.

OBJECTIVES: Approximately 20% of patients with chronic rhinosinusitis (CRS) have comorbid obstructive sleep apnea (OSA). Patients with undiagnosed OSA are at high risk for perioperative complications. The Sinonasal Outcomes Test (SNOT-22) Questionnaire is commonly administered to CRS patients, whereas OSA screening tools are less routinely employed. This study compared SNOT-22 sleep subdomain (Sleep-SNOT) scores among non-OSA CRS versus OSA-CRS patients undergoing ESS, and assessed sensitivity, specificity, and diagnostic accuracy of the Sleep-SNOT for OSA screening. METHODS: Retrospective review of patients that underwent endoscopic sinus surgery (ESS) for CRS from 2012 to 2021. Patients either carried a reported OSA diagnosis and completed the SNOT-22, or had undocumented OSA status and completed both STOP-BANG and SNOT-22. Demographics, questionnaire scores, and OSA status were collected. A receiver operating characteristic (ROC) curve assessed cutoff scores, sensitivity, and specificity of the Sleep-SNOT for OSA screening. RESULTS: Of 600 patients reviewed, 109 were included. 41% had comorbid OSA. OSA patients had a higher BMI (32.1 ± 7.7 vs. 28.35 ± 6.7 kg/m2 ; p = 0.02), Sleep-SNOT (21.96 ± 12.1 vs. 16.8 ± 11.2; p = 0.021) and STOP-BANG (3.1 ± 1.44 vs. 2.06 ± 1.27; p = 0.038) scores. A Sleep-SNOT score of 17.5 had a sensitivity of 68.9%, specificity of 55.7%, and diagnostic accuracy of 63% for OSA detection (p = 0.022). CONCLUSIONS: Sleep-SNOT scores are greater for CRS-OSA patients. The Sleep-SNOT ROC curve demonstrates a high sensitivity, specificity, and accuracy for OSA screening in CRS patients. A Sleep-SNOT score of ≥17.5 should prompt further OSA evaluation. The Sleep-SNOT may be considered as a surrogate OSA screening tool when other validated tools are not employed. LEVEL OF EVIDENCE: Retrospective chart review, Level 3 Laryngoscope, 133:2029-2034, 2023.

A Comparison of Outcomes Using Combined Intra- and Extradural versus Extradural-Only Repair of Tegmen Defects.

Objective Tegmen tympani or tegmen mastoideum defects involve dehiscence of the temporal bone that can be a source of cerebrospinal fluid (CSF) otorrhea. Herein, we compare a combined intra-/extradural repair strategy with an extradural-only repair as it pertains to surgical and clinical outcomes. Design A retrospective review from our institution was performed of patients with tegmen defects requiring surgical intervention. Participants Patients with tegmen defects who underwent surgery (combined transmastoid and middle fossa craniotomy) for repair of tegmen defects between 2010 and 2020 were inclined in this study. Results A total of 60 patients with 40 intra-/extradural (mean follow-up time: 1,060 ± 1,103 days) and 20 extradural-only (mean follow-up time: 519 ± 369 days) repairs were identified. No major differences in demographic factors or presenting symptoms were identified between the two cohorts. There was no difference in hospital length of stay between the two patient cohorts (mean: 4.15 vs. 4.35 days, p = 0.8). In the extradural-only repair technique, synthetic bone cement was more frequently used (100 vs. 7.5%, p < 0.01), whereas in the combined intra-/extradural repair, synthetic dural substitute was used more often (80 vs. 35%, p < 0.01), with similar successful surgical outcomes achieved. Despite disparities in the techniques and materials used for repair, there were no differences in complication rates (wound infection, seizures, and ossicular fixation), 30-day readmission rates, or persistent CSF leak between the two treatment cohorts. Conclusion The results of this study suggest no difference in clinical outcomes between combined intra-/extradural versus extradural-only repair of tegmen defects. A simplified extradural-only repair strategy can be effective, and may reduce the morbidity of intradural reconstruction (seizures, stroke, and intraparenchymal hemorrhage).

Cell aggregation is associated with enzyme secretion strategies in marine polysaccharide-degrading bacteria.

Polysaccharide breakdown by bacteria requires the activity of enzymes that degrade polymers either intra- or extra-cellularly. The latter mechanism generates a localized pool of breakdown products that are accessible to the enzyme producers themselves as well as to other organisms. Marine bacterial taxa often show marked differences in the production and secretion of degradative enzymes that break down polysaccharides. These differences can have profound effects on the pool of diffusible breakdown products and hence on the ecological dynamics. However, the consequences of differences in enzymatic secretions on cellular growth dynamics and interactions are unclear. Here we study growth dynamics of single cells within populations of marine Vibrionaceae strains that grow on the abundant marine polymer alginate, using microfluidics coupled to quantitative single-cell analysis and mathematical modelling. We find that strains that have low extracellular secretions of alginate lyases aggregate more strongly than strains that secrete high levels of enzymes. One plausible reason for this observation is that low secretors require a higher cellular density to achieve maximal growth rates in comparison with high secretors. Our findings indicate that increased aggregation increases intercellular synergy amongst cells of low-secreting strains. By mathematically modelling the impact of the level of degradative enzyme secretion on the rate of diffusive oligomer loss, we find that enzymatic secretion capability modulates the propensity of cells within clonal populations to cooperate or compete with each other. Our experiments and models demonstrate that enzymatic secretion capabilities can be linked with the propensity of cell aggregation in marine bacteria that extracellularly catabolize polysaccharides.

Retrospective Review of Surgical Site Infections after Endoscopic Endonasal Sellar and Parasellar Surgery: Multicenter Quality Data from the North American Skull Base Society.

Introduction Transnasal access to the anterior skull base provides a minimally invasive approach for sellar and parasellar masses compared with its open counterparts. The unique microbiome of the sinonasal mucosa provides distinct challenges not encountered with other cranial approaches. The use of antibiotics in these cases has not been standardized, and data remain scarce regarding infectious outcomes. Methods We conducted a multicenter retrospective analysis of shared quality data points for the endoscopic endonasal approach (EEA) for pituitary adenomas, along with other sellar and parasellar region masses that were included by participating institutions. Patient and operative characteristics, perioperative and postoperative antibiotic regimens and their durations, intraoperative and postoperative cerebrospinal fluid leak, and onset of postoperative meningitis and sinusitis were compared. Results Fifteen institutions participated and provided 6 consecutive months' worth of case data. Five hundred ninety-three cases were included in the study, of which 564 were pituitary adenomectomies. The incidences of postoperative meningitis and sinusitis were low (0.67 and 2.87% for all pathologies, respectively; 0.35% meningitis for pituitary adenomas) and did not correlate with any specific antibiotic regimen. Immunocompromised status posed an increased odds of meningitis in pituitary adenomectomies (28.6, 95% confidence interval [1.72-474.4]). Conclusions The results show no clear benefit to postoperative antimicrobial use in EEA, with further larger studies needed.

Evaluation of early postoperative day 1 discharge after endoscopic endonasal pituitary adenoma resection.

OBJECTIVE: While multiple studies have evaluated the length of stay after endonasal transsphenoidal surgery (ETS) for pituitary adenoma, the potential for early discharge on postoperative day 1 (POD 1) remains unclear. The authors compared patients discharged on POD 1 with patients discharged on POD > 1 to better characterize factors that facilitate early discharge after ETS. METHODS: A retrospective chart review was performed for patients undergoing ETS for pituitary adenoma at a single tertiary care academic center from February 2005 to February 2020. Discharge on POD 1 was defined as a discharge within 24 hours of surgery. RESULTS: A total of 726 patients (mean age 55 years, 52% male) were identified, of whom 178 (24.5%) patients were discharged on POD 1. These patients were more likely to have pituitary incidentaloma (p = 0.001), require dural substitutes and DuraSeal (p = 0.0001), have fewer intraoperative CSF leaks (p = 0.02), and have lower postoperative complication rates (p = 0.006) compared with patients discharged on POD > 1. POD 1 patients also showed higher rates of macroadenomas (96.1% vs 91.4%, p = 0.03) and lower rates of functional tumors (p = 0.02). POD > 1 patients were more likely to have readmission within 30 days (p = 0.002), readmission after 30 days (p = 0.0001), nasal synechiae on follow-up (p = 0.003), diabetes insipidus (DI; 1.7% vs 9.8%, p = 0.0001), postoperative hypocortisolism (21.8% vs 12.1%, p = 0.01), and postoperative steroid usage (44.6% vs 59.7%, p = 0.003). The number of patients discharged on POD 1 significantly increased during each subsequent time epoch: 2005-2010, 2011-2015, and 2016-2020 (p = 0.0001). On multivariate analysis, DI (OR 7.02, 95% CI 2.01-24.57; p = 0.002) and intraoperative leak (OR 2.02, 95% CI 1.25-3.28; p = 0.004) were associated with increased risk for POD > 1 discharge, while operation epoch (OR 0.46, 95% CI 0.3-0.71; p = 0.0001) was associated with POD 1 discharge. CONCLUSIONS: This study demonstrates that discharge on POD 1 after ETS for pituitary adenomas was safe and feasible and without increased risk of 30-day readmission. On multivariate analysis, surgical epoch was associated with decreased risk of prolonged length of stay, while factors associated with increased risk of prolonged length of stay included DI and intraoperative CSF leak. These findings may help in selecting patients who are deemed reasonable for safe, early discharge after pituitary adenoma resection.

Short-range quorum sensing controls horizontal gene transfer at micron scale in bacterial communities.

In bacterial communities, cells often communicate by the release and detection of small diffusible molecules, a process termed quorum-sensing. Signal molecules are thought to broadly diffuse in space; however, they often regulate traits such as conjugative transfer that strictly depend on the local community composition. This raises the question how nearby cells within the community can be detected. Here, we compare the range of communication of different quorum-sensing systems. While some systems support long-range communication, we show that others support a form of highly localized communication. In these systems, signal molecules propagate no more than a few microns away from signaling cells, due to the irreversible uptake of the signal molecules from the environment. This enables cells to accurately detect micron scale changes in the community composition. Several mobile genetic elements, including conjugative elements and phages, employ short-range communication to assess the fraction of susceptible host cells in their vicinity and adaptively trigger horizontal gene transfer in response. Our results underscore the complex spatial biology of bacteria, which can communicate and interact at widely different spatial scales.

Nutrient complexity triggers transitions between solitary and colonial growth in bacterial populations.

Microbial populations often experience fluctuations in nutrient complexity in their natural environment such as between high molecular weight polysaccharides and simple monosaccharides. However, it is unclear if cells can adopt growth behaviors that allow individuals to optimally respond to differences in nutrient complexity. Here, we directly control nutrient complexity and use quantitative single-cell analysis to study the growth dynamics of individuals within populations of the aquatic bacterium Caulobacter crescentus. We show that cells form clonal microcolonies when growing on the polysaccharide xylan, which is abundant in nature and degraded using extracellular cell-linked enzymes; and disperse to solitary growth modes when the corresponding monosaccharide xylose becomes available or nutrients are exhausted. We find that the cellular density required to achieve maximal growth rates is four-fold higher on xylan than on xylose, indicating that aggregating is advantageous on polysaccharides. When collectives on xylan are transitioned to xylose, cells start dispersing, indicating that colony formation is no longer beneficial and solitary behaviors might serve to reduce intercellular competition. Our study demonstrates that cells can dynamically tune their behaviors when nutrient complexity fluctuates, elucidates the quantitative advantages of distinct growth behaviors for individual cells and indicates why collective growth modes are prevalent in microbial populations.

An interdisciplinary and application-oriented approach to teach microfluidics.

Microfluidics is a relatively novel interdisciplinary research area with broad applications in chemistry, physics, material science, and biology. Despite the rapid growth of the field, students' exposure to microfluidic technologies is still limited and often insufficient to appreciate the advantages over other commonly used technologies. To this end, we designed a five-day course, "Microfluidics for microbial ecology," in which students with very different backgrounds learn the basics of microfluidic technologies and sample a range of applications in microbial ecology. The course was created for Master and Ph.D. students interested in applying microfluidics to their research and, therefore, followed an application-oriented approach. The presentation of critical aspects of fluid flow phenomena at the microscale and an outline of the advantages and constraints of the technology provide students with the background to design and perform microfluidics-based experiments. In order to improve the effectiveness of learning in a class with diverse interests and backgrounds, two active learning exercises were implemented. The first comprised the design of an individualized microfluidics experiment in parallel with the lectures: students were guided to apply each module to their personalized application and discuss it in groups. The second was a group experimental activity, in which students jointly set up, performed, analyzed, and presented a microfluidics-based experiment. Given the multidisciplinary teaching context, the course was able to foster common conceptual ground and promote discussion among students. This application-oriented approach built upon experimental activities and in-class discussion is well suited to promote learning in a technology-related subject such as microfluidics.

Preoperative Screening for Obstructive Sleep Apnea Prior to Endoscopic Skull Base Surgery: A Survey of the North American Skull Base Society.

BACKGROUND: Obstructive sleep apnea (OSA) is a commonly seen comorbidity in patients undergoing endoscopic skull base surgery and its presence may influence perioperative decision-making. Current practice patterns for preoperative screening of OSA are poorly understood. OBJECTIVE: The objective of this study was to assess how endoscopic skull base surgeons screen for OSA, and how knowledge of OSA affects perioperative decision-making. METHODS: Seven question survey distributed to members of the North American Skull Base Society. RESULTS: Eighty-eight responses (10% response rate) were received. 60% of respondents were from academic centers who personally performed >50 cases per year. Most respondents noted that preoperative knowledge of OSA and its severity affected postoperative care and increased their concern for complications. Half of respondents noted that preoperative knowledge of OSA and its severity affects intraoperative skull base reconstruction decision-making. 70% of respondents did not have a preoperative OSA screening protocol. Body mass index and patient history were most frequently used by those who screened. Validated screening questionnaires were rarely used. 76% of respondents agreed or somewhat agreed that a preoperative polysomnogram should ideally be performed for patients with suspected OSA; however, 50% of respondents reported that <20% of their patients with suspected OSA are advised to obtain a preoperative polysomnogram. CONCLUSION: This study reveals that most endoscopic skull base surgeons agree that OSA affects postoperative patient care, but only a minority have a preoperative screening protocol in place. Additional study is needed to assess the most appropriate screening methods and protocols for OSA patients undergoing endoscopic skull base surgery.

Phenotypic variation in spatially structured microbial communities: ecological origins and consequences.

Microbial cells in nature live within dense multispecies conglomerates, forming a self-organizing ecosystem. In such assemblies, genotypes interact with each other in a myriad of ways, driving community dynamics and functionalities. The role of interactions between genotypes and their consequences for spatial structure and functional outcomes are being increasingly studied to understand the ecology and evolution of microbial communities. An increasing body of work with simple microbial populations has elucidated that phenotypic variation, that is, differences within isogenic cells can have important consequences for population dynamics and evolution. However, the role of individual level behavioral differences for community level dynamics is relatively unknown. I argue that it is necessary to study phenotypic variation and microscale processes in order to understand the emergence and consequences of interactions within microbial communities. I highlight possible explanations that can explain the emergence of variation in multi-genotypic assemblages and propose possible consequences on community dynamics.

Ecology and evolution of metabolic cross-feeding interactions in bacteria.

Literature covered: early 2000s to late 2017Bacteria frequently exchange metabolites with other micro- and macro-organisms. In these often obligate cross-feeding interactions, primary metabolites such as vitamins, amino acids, nucleotides, or growth factors are exchanged. The widespread distribution of this type of metabolic interactions, however, is at odds with evolutionary theory: why should an organism invest costly resources to benefit other individuals rather than using these metabolites to maximize its own fitness? Recent empirical work has shown that bacterial genotypes can significantly benefit from trading metabolites with other bacteria relative to cells not engaging in such interactions. Here, we will provide a comprehensive overview over the ecological factors and evolutionary mechanisms that have been identified to explain the evolution and maintenance of metabolic mutualisms among microorganisms. Furthermore, we will highlight general principles that underlie the adaptive evolution of interconnected microbial metabolic networks as well as the evolutionary consequences that result for cells living in such communities.

Experimental Evolution of Metabolic Dependency in Bacteria.

Bacteria frequently lose biosynthetic genes, thus making them dependent on an environmental uptake of the corresponding metabolite. Despite the ubiquity of this 'genome streamlining', it is generally unclear whether the concomitant loss of biosynthetic functions is favored by natural selection or rather caused by random genetic drift. Here we demonstrate experimentally that a loss of metabolic functions is strongly selected for when the corresponding metabolites can be derived from the environment. Serially propagating replicate populations of the bacterium Escherichia coli in amino acid-containing environments revealed that auxotrophic genotypes rapidly evolved in less than 2,000 generations in almost all replicate populations. Moreover, auxotrophs also evolved in environments lacking amino acids-yet to a much lesser extent. Loss of these biosynthetic functions was due to mutations in both structural and regulatory genes. In competition experiments performed in the presence of amino acids, auxotrophic mutants gained a significant fitness advantage over the evolutionary ancestor, suggesting their emergence was selectively favored. Interestingly, auxotrophic mutants derived amino acids not only via an environmental uptake, but also by cross-feeding from coexisting strains. Our results show that adaptive fitness benefits can favor biosynthetic loss-of-function mutants and drive the establishment of intricate metabolic interactions within microbial communities.

Metabolic network architecture and carbon source determine metabolite production costs.

Metabolism is essential to organismal life, because it provides energy and building block metabolites. Even though it is known that the biosynthesis of metabolites consumes a significant proportion of the resources available to a cell, the factors that determine their production costs remain less well understood. In this context, it is especially unclear how the nutritional environment affects the costs of metabolite production. Here, we use the amino acid metabolism of Escherichia coli as a model to show that the point at which a carbon source enters central metabolic pathways is a major determinant of individual metabolite production costs. Growth rates of auxotrophic genotypes, which in the presence of the required amino acid save biosynthetic costs, were compared to the growth rates that prototrophic cells achieved under the same conditions. The experimental results showed a strong concordance with computationally estimated biosynthetic costs, which allowed us, for the first time, to systematically quantify carbon source-dependent metabolite production costs. Thus, we demonstrate that the nutritional environment in combination with network architecture is an important but hitherto underestimated factor influencing biosynthetic costs and thus microbial growth. Our observations are highly relevant for the optimization of biotechnological processes as well as for understanding the ecology of microorganisms in their natural environments.

Plasticity and epistasis strongly affect bacterial fitness after losing multiple metabolic genes.

Many bacterial lineages lack seemingly essential metabolic genes. Previous work suggested selective benefits could drive the loss of biosynthetic functions from bacterial genomes when the corresponding metabolites are sufficiently available in the environment. However, the factors that govern this "genome streamlining" remain poorly understood. Here we determine the effect of plasticity and epistasis on the fitness of Escherichia coli genotypes from whose genome biosynthetic genes for one, two, or three different amino acids have been deleted. Competitive fitness experiments between auxotrophic mutants and prototrophic wild-type cells in one of two carbon environments revealed that plasticity and epistasis strongly affected the mutants' fitness individually and interactively. Positive and negative epistatic interactions were prevalent, yet on average cancelled each other out. Moreover, epistasis correlated negatively with the expected effects of combined auxotrophy-causing mutations, thus producing a pattern of diminishing returns. Moreover, computationally analyzing 1,432 eubacterial metabolic networks revealed that most pairs of auxotrophies co-occurred significantly more often than expected by chance, suggesting epistatic interactions and/or environmental factors favored these combinations. Our results demonstrate that both the genetic background and environmental conditions determine the adaptive value of a loss-of-biochemical-function mutation and that fitness gains decelerate, as more biochemical functions are lost.