DNA methylation signatures reveal that distinct combinations of transcription factors specify human immune cell epigenetic identity.

Epigenetic reprogramming underlies specification of immune cell lineages, but patterns that uniquely define immune cell types and the mechanisms by which they are established remain unclear. Here, we identified lineage-specific DNA methylation signatures of six immune cell types from human peripheral blood and determined their relationship to other epigenetic and transcriptomic patterns. Sites of lineage-specific hypomethylation were associated with distinct combinations of transcription factors in each cell type. By contrast, sites of lineage-specific hypermethylation were restricted mostly to adaptive immune cells. PU.1 binding sites were associated with lineage-specific hypo- and hypermethylation in different cell types, suggesting that it regulates DNA methylation in a context-dependent manner. These observations indicate that innate and adaptive immune lineages are specified by distinct epigenetic mechanisms via combinatorial and context-dependent use of key transcription factors. The cell-specific epigenomics and transcriptional patterns identified serve as a foundation for future studies on immune dysregulation in diseases and aging.

The Drosophila blood-brain barrier regulates sleep via Moody G protein-coupled receptor signaling.

Sleep is vital for most animals, yet its mechanism and function remain unclear. We found that permeability of the BBB (blood-brain barrier)-the organ required for the maintenance of homeostatic levels of nutrients, ions, and other molecules in the brain-is modulated by sleep deprivation (SD) and can cell-autonomously effect sleep changes. We observed increased BBB permeability in known sleep mutants as well as in acutely sleep-deprived animals. In addition to molecular tracers, SD-induced BBB changes also increased the penetration of drugs used in the treatment of brain pathologies. After chronic/genetic or acute SD, rebound sleep or administration of the sleeping aid gaboxadol normalized BBB permeability, showing that SD effects on the BBB are reversible. Along with BBB permeability, RNA levels of the BBB master regulator moody are modulated by sleep. Conversely, altering BBB permeability alone through glia-specific modulation of moody, gαo, loco, lachesin, or neuroglian-each a well-studied regulator of BBB function-was sufficient to induce robust sleep phenotypes. These studies demonstrate a tight link between BBB permeability and sleep and indicate a unique role for the BBB in the regulation of sleep.

Mathematical modeling and experimental validation for expression microdissection.

Using laser excitation, expression microdissection (xMD) can selectively heat cancer cells targeted via immunohistochemical staining to enable their selective retrieval from tumor tissue samples, thus reducing misdiagnoses caused by contamination of noncancerous cells. Several theoretical models have been validated for the photothermal effect in highly light absorbing and scattering media. However, these models are not generally applicable to the physics behind the process of xMD. In this study, we propose a thermal model that can analyze the transient temperature distribution and heat melt zone in an xMD sample medium composed of a thermoplastic film and a tumor tissue sample sandwiched between two glass slides. Furthermore, we experimentally examined the model using an ink layer with controllable optical properties to serve as a microscale-thin, tissue-mimicking phantom and found the experimentally measured film temperature is in good agreement with the model predictions. The validated model can help researchers to optimize cell retrieval by xMD for improved diagnostics of cancer and other diseases.

Has a Prescription-limiting Law in Rhode Island Helped to Reduce Opioid Use After Total Joint Arthroplasty?

BACKGROUND: In the United States, since 2016, at least 28 of 50 state legislatures have passed laws regarding mandatory prescribing limits for opioid medications. One of the earliest state laws (which was passed in Rhode Island in 2016) restricted the maximum morphine milligram equivalents provided in the first postoperative prescription for patients defined as opioid-naïve to 30 morphine milligram equivalents per day, 150 total morphine milligram equivalents, or 20 total doses. While such regulations are increasingly common in the United States, their effects on opioid use after total joint arthroplasty are unclear. QUESTIONS/PURPOSES: (1) Are legislative limitations to opioid prescriptions in Rhode Island associated with decreased opioid use in the immediate (first outpatient prescription postoperatively), 30-day, and 90-day periods after THA and TKA? (2) Is this law associated with similar changes in postoperative opioid use among patients who are opioid-naïve and those who are opioid-tolerant preoperatively? METHODS: Patients undergoing primary THA or TKA between January 1, 2016 and June 28, 2016 (before the law was passed on June 28, 2016) were retrospectively compared with patients undergoing surgery between June 1, 2017 and December 31, 2017 (after the law's implementation on April 17, 2017). The lapse between the pre-law and post-law periods was designed to avoid confounding from potential voluntary practice changes by physicians after the law was passed but before its mandatory implementation. Demographic and surgical details were extracted from a large multi-specialty orthopaedic group's surgical billing database using Current Procedural Terminology codes 27130 and 27447. Any patients undergoing revision procedures, same-day bilateral arthroplasties, or a second primary THA or TKA in the 3-month followup period were excluded. Secondary data were confirmed by reviewing individual electronic medical records in the associated hospital system which included three major hospital sites. We evaluated 1125 patients. In accordance with the state's department of health guidelines, patients were defined as opioid-tolerant if they had filled any prescription for an opioid medication in the 30-day preoperative period. Data on age, gender, and the proportion of patients who were defined as opioid tolerant preoperatively were collected and found to be no different between the pre-law and post-law groups. The state's prescription drug monitoring program database was used to collect data on prescriptions for all controlled substances filled between 30 days preoperatively and 90 days postoperatively. The primary outcomes were the mean morphine milligram equivalents of the initial outpatient postoperative opioid prescription after discharge and the mean cumulative morphine milligram equivalents at the 30- and 90-day postoperative intervals. Secondary analyses included subgroup analyses by procedure and by preoperative opioid tolerance. RESULTS: After the law was implemented, the first opioid prescriptions were smaller for patients who were opioid-naïve (mean 156 ± 106 morphine milligram equivalents after the law's passage versus 451 ± 296 before, mean difference 294 morphine milligram equivalents; p < 0.001) and those who were opioid-tolerant (263 ± 265 morphine milligram equivalents after the law's passage versus 534 ± 427 before, mean difference 271 morphine milligram equivalents; p < 0.001); however, for cumulative prescriptions in the first 30 days postoperatively, this was only true among patients who were previously opioid-naïve (501 ± 416 morphine milligram equivalents after the law's passage versus 796 ± 597 before, mean difference 295 morphine milligram equivalents; p < 0.001). Those who were opioid-tolerant did not have a decrease in the cumulative number of 30-day morphine milligram equivalents (1288 ± 1632 morphine milligram equivalents after the law's passage versus 1398 ± 1274 before, mean difference 110 morphine milligram equivalents; p = 0.066). CONCLUSIONS: The prescription-limiting law was associated with a decline in cumulative opioid prescriptions at 30 days postoperatively filled by patients who were opioid-naïve before total joint arthroplasty. This may substantially impact public health, and these policies should be considered an important tool for healthcare providers, communities, and policymakers who wish to combat the current opioid epidemic. However, given the lack of a discernible effect on cumulative opioids filled from 30 to 90 days postoperatively, further investigations are needed to evaluate more effective policies to prevent prolonged opioid use after total joint arthroplasty, particularly in patients who are opioid-tolerant preoperatively. LEVEL OF EVIDENCE: Level III, therapeutic study.

Opioid-Limiting Regulation: Effect on Patients Undergoing Knee and Shoulder Arthroscopy.

PURPOSE: To determine the effect prescription-limiting legislation passed in Rhode Island has had on opioids prescribed following arthroscopic knee and shoulder surgery at various time points, up to 90 days postoperatively. METHODS: All patients undergoing the 3 most common arthroscopic procedures at our institution (anterior cruciate ligament reconstruction, partial meniscectomy, and rotator cuff repair) were included. Patients were selected from 2 6-month study periods (prepassage and postimplementation of the law). The state's Prescription Drug Monitoring Program database was queried for controlled substances filled in the perioperative period (from 30 days preoperatively to 90 days postoperatively). Multiple logistic regressions were used to identify predictors of chronic (>30 days) opioid use. RESULTS: The morphine milligram equivalents (MMEs) prescribed in the initial postoperative script decreased from 319.04 (∼43 5-mg oxycodone tablets) in the prepassage to 152.45 MMEs (∼20 5-mg oxycodone tablets) in the postimplementation group (P < .001). The total MMEs filled in the first 30 days decreased from 520.93 to 299.94 MMEs (∼70 to ∼40 5-mg oxycodone tablets) (P < .001). MMEs filled between 30 and 90 days fell by 22.5% for all patients in this study; however, this change was not statistically significant (P = .263). Preoperative opioid use (odds ratio, 10.85; P < .001) and preoperative benzodiazepine use (odds ratio, 2.13; P = .005) predicted chronic opioid use postoperatively. CONCLUSIONS: State opioid-limiting legislation reduced cumulative MMEs following arthroscopic knee and shoulder surgery in the first 30 days. Further research assessing the impact of this legislation on postoperative pain control, patient satisfaction, and functional outcomes following surgery is warranted. LEVEL OF EVIDENCE: Level III, case-control study.

Middle Ear Histopathology Following Magnetic Delivery to the Cochlea of Prednisolone-loaded Iron Oxide Nanoparticles in Rats.

Delivery of therapy to the cochlea is a challenge and limits the efficacy of therapies meant to treat hearing loss, reverse tinnitus, and protect hearing from chemotherapy regimens. Magnetic injection is a technique that uses magnetic fields to inject nanoparticles from the middle ear into the cochlea, where they can then elute therapy to treat hearing disorders. To evaluate the safety of this treatment in the middle ear, 30 rats were subdivided into 6 groups and treated by single or multiple intratympanic injections of saline, prednisolone, nanoparticles, or nanoparticles loaded with prednisolone. A specially designed magnet array was used to magnetically inject the particles from the middle ear to the cochlea. Treatment began at study day 0, and animals were euthanized on study day 2, 30, or 90. Temporal bones were collected and prepared for histopathological examination. Intratympanic administration of magnetic nanoparticles and/or prednisolone resulted in minimal to mild inflammatory changes in all treated groups. The incidence and severity of the inflammatory changes observed appeared slightly increased in animals administered nanoparticles, with or without prednisolone, when compared to animals administered prednisolone alone. At study day 90, there was partial reversibility of the findings noted at study day 2 and 30. Repeat administration did not appear to cause greater inflammatory changes.

Nanostructure-Induced Distortion in Single-Emitter Microscopy.

Single-emitter microscopy has emerged as a promising method of imaging nanostructures with nanoscale resolution. This technique uses the centroid position of an emitter's far-field radiation pattern to infer its position to a precision that is far below the diffraction limit. However, nanostructures composed of high-dielectric materials such as noble metals can distort the far-field radiation pattern. Previous work has shown that these distortions can significantly degrade the imaging of the local density of states in metallic nanowires using polarization-resolved imaging. But unlike nanowires, nanoparticles do not have a well-defined axis of symmetry, which makes polarization-resolved imaging difficult to apply. Nanoparticles also exhibit a more complex range of distortions, because in addition to introducing a high dielectric surface, they also act as efficient scatterers. Thus, the distortion effects of nanoparticles in single-emitter microscopy remains poorly understood. Here we demonstrate that metallic nanoparticles can significantly distort the accuracy of single-emitter imaging at distances exceeding 300 nm. We use a single quantum dot to probe both the magnitude and the direction of the metallic nanoparticle-induced imaging distortion and show that the diffraction spot of the quantum dot can shift by more than 35 nm. The centroid position of the emitter generally shifts away from the nanoparticle position, which is in contradiction to the conventional wisdom that the nanoparticle is a scattering object that will pull in the diffraction spot of the emitter toward its center. These results suggest that dielectric distortion of the emission pattern dominates over scattering. We also show that by monitoring the distortion of the quantum dot diffraction spot we can obtain high-resolution spatial images of the nanoparticle, providing a new method for performing highly precise, subdiffraction spatial imaging. These results provide a better understanding of the complex near-field coupling between emitters and nanostructures and open up new opportunities to perform super-resolution microscopy with higher accuracy.

Scanning localized magnetic fields in a microfluidic device with a single nitrogen vacancy center.

Nitrogen vacancy (NV) color centers in diamond enable local magnetic field sensing with high sensitivity by optical detection of electron spin resonance (ESR). The integration of this capability with microfluidic technology has a broad range of applications in chemical and biological sensing. We demonstrate a method to perform localized magnetometry in a microfluidic device with a 48 nm spatial precision. The device manipulates individual magnetic particles in three dimensions using a combination of flow control and magnetic actuation. We map out the local field distribution of the magnetic particle by manipulating it in the vicinity of a single NV center and optically detecting the induced Zeeman shift with a magnetic field sensitivity of 17.5 µT Hz(-1/2). Our results enable accurate nanoscale mapping of the magnetic field distribution of a broad range of target objects in a microfluidic device.

Movement of magnetic nanoparticles in brain tissue: mechanisms and impact on normal neuronal function.

Magnetic nanoparticles (MNPs) have been used as effective vehicles for targeted delivery of theranostic agents in the brain. The advantage of magnetic targeting lies in the ability to control the concentration and distribution of therapy to a desired target region using external driving magnets. In this study, we investigated the behavior and safety of MNP motion in brain tissue. We found that MNPs move and form nanoparticle chains in the presence of a uniform magnetic field, and that this chaining is influenced by the applied magnetic field intensity and the concentration of MNPs in the tissue. Using electrophysiology recordings, immunohistochemistry and fluorescent imaging we assessed the functional health of neurons and neural circuits and found no adverse effects associated with MNP motion through brain tissue. FROM THE CLINICAL EDITOR: Much research has been done to test the use of nanocarriers for gaining access across the blood brain barrier (BBB). In this respect, magnetic nanoparticles (MNPs) are one of the most studied candidates. Nonetheless, the behavior and safety of MNP once inside brain tissue remains unknown. In this article, the authors thus studied this very important subject.

QUANTIFYING THE MOTION OF MAGNETIC PARTICLES IN EXCISED TISSUE: EFFECT OF PARTICLE PROPERTIES AND APPLIED MAGNETIC FIELD.

This article presents a method to investigate how magnetic particle characteristics affect their motion inside tissues under the influence of an applied magnetic field. Particles are placed on top of freshly excised tissue samples, a calibrated magnetic field is applied by a magnet underneath each tissue sample, and we image and quantify particle penetration depth by quantitative metrics to assess how particle sizes, their surface coatings, and tissue resistance affect particle motion. Using this method, we tested available fluorescent particles from Chemicell of four sizes (100 nm, 300 nm, 500 nm, and 1 µm diameter) with four different coatings (starch, chitosan, lipid, PEG/P) and quantified their motion through freshly excised rat liver, kidney, and brain tissues. In broad terms, we found that the applied magnetic field moved chitosan particles most effectively through all three tissue types (as compared to starch, lipid, and PEG/P coated particles). However, the relationship between particle properties and their resulting motion was found to be complex. Hence, it will likely require substantial further study to elucidate the nuances of transport mechanisms and to select and engineer optimal particle properties to enable the most effective transport through various tissue types under applied magnetic fields.

Fabrication of nanoassemblies using flow control.

Synthetic nanostructures, such as nanoparticles and nanowires, can serve as modular building blocks for integrated nanoscale systems. We demonstrate a microfluidic approach for positioning, orienting, and assembling such nanostructures into nanoassemblies. We use flow control combined with a cross-linking photoresist to position and immobilize nanostructures in desired positions and orientations. Immobilized nanostructures can serve as pivots, barriers, and guides for precise placement of subsequent nanostructures.

Unraveling the interplay of circadian rhythm and sleep deprivation on mood: A Real-World Study on first-year physicians.

The interplay between circadian rhythms, time awake, and mood remains poorly understood in the real-world. Individuals in high-stress occupations with irregular schedules or nighttime shifts are particularly vulnerable to depression and other mood disorders. Advances in wearable technology have provided the opportunity to study these interactions outside of a controlled laboratory environment. Here, we examine the effects of circadian rhythms and time awake on mood in first-year physicians using wearables. Continuous heart rate, step count, sleep data, and daily mood scores were collected from 2,602 medical interns across 168,311 days of Fitbit data. Circadian time and time awake were extracted from minute-by-minute wearable heart rate and motion measurements. Linear mixed modeling determined the relationship between mood, circadian rhythm, and time awake. In this cohort, mood was modulated by circadian timekeeping (p<0.001). Furthermore, we show that increasing time awake both deteriorates mood (p<0.001) and amplifies mood's circadian rhythm nonlinearly. These findings demonstrate the contributions of both circadian rhythms and sleep deprivation to underlying mood and show how these factors can be studied in real-world settings using Fitbits. They underscore the promising opportunity to harness wearables in deploying chronotherapies for psychiatric illness.

Activity-Based Nitric Oxide-Responsive Porphyrin for Site-Selective and Nascent Cancer Ablation.

Nitric oxide (NO) generated within the tumor microenvironment is an established driver of cancer progression and metastasis. Recent efforts have focused on leveraging this feature to target cancer through the development of diagnostic imaging agents and activatable chemotherapeutics. In this context, porphyrins represent an extraordinarily promising class of molecules, owing to their demonstrated use within both modalities. However, the remodeling of a standard porphyrin to afford a responsive chemical that can distinguish elevated NO from physiological levels has remained a significant research challenge. In this study, we employed a photoinduced electron transfer strategy to develop a panel of NO-activatable porphyrin photosensitizers (NOxPorfins) augmented with real-time fluorescence monitoring capabilities. The lead compound, NOxPorfin-1, features an o-phenylenediamine trigger that can effectively capture NO (via N2O3) to yield a triazole product that exhibits a 7.5-fold enhancement and a 70-fold turn-on response in the singlet oxygen quantum yield and fluorescence signal, respectively. Beyond demonstrating excellent in vitro responsiveness and selectivity toward NO, we showcase the potent photodynamic therapy (PDT) effect of NOxPorfin-1 in murine breast cancer and human non-small cellular lung cancer cells. Further, to highlight the in vivo efficacy, two key studies were executed. First, we utilized NOxPorfin-1 to ablate murine breast tumors in a site-selective manner without causing substantial collateral damage to healthy tissue. Second, we established a nascent human lung cancer model to demonstrate the unprecedented ability of NOxPorfin-1 to halt tumor growth and progression completely. The results of the latter study have tremendous implications for applying PDT to target metastatic lesions.

How bad could it be? Alcohol dampens stress responses to threat of uncertain intensity.

Stress response dampening is an important motive for alcohol use. However, stress reduction via alcohol (alcohol SRD) is observed inconsistently in the laboratory, and this has raised questions about the precise mechanisms and boundary conditions for these effects. Emerging evidence indicates that alcohol SRD may be observed selectively during uncertain but not certain threats. In a final sample of 89 participants, we measured stress response via potentiation of defensive startle reflex in response to threat of shock in blocks with certain (low and high) and uncertain shock intensity. Our alcohol-administration procedure produced blood alcohol concentrations (BACs) across a broad range (0.00%-0.12%) across participants. Increasing BACs were associated with linearly decreasing startle potentiation and self-reported anxiety. This SRD effect was greater during uncertain than certain threat. More broadly, these results suggest that distinct mechanisms are involved in response to threats of uncertain intensity and threats of certain intensity.

The Impact of religiosity/spirituality on slowing the progression of substance use: Based on the National Epidemiological Survey of Alcohol and Related Conditions (NESARC-III).

BACKGROUND: Religiosity has been suggested to be protective against substance use disorder (SUD) initiation but its impact of the progression of development is not known. AIMS: This study investigated the impact of religiosity/spirituality on the development of heavy use and SUD following substance use initiation (alcohol, cannabis, and tobacco) utilizing data from the 2012 to 2013 National Epidemiologic Survey on Alcohol and Related Conditions-III. METHOD: Individuals with a known age at onset of substance initiation were included (n = 30,590, n = 11,126, and n = 14,083; for alcohol, cannabis, or tobacco users, respectively). Religiosity was measured by importance of religious/spiritual beliefs and frequency of religious service attendance. The percentage of individuals who progressed to an SUD after substance initiation in each substance was estimated. Discrete-time analysis and survival analysis were used to measure the impact of religiosity on the progression from substance initiation to heavy use and from heavy use to SUD. RESULTS: After controlling for various variables, religious services attendance frequency was statistically associated with a slower progression from substance initiation to heavy use for all three substances: tobacco by 8% to 15%, cannabis by 5% to 26%, and alcohol 9% (p ⩽ .01). Religious importance was associated with slower progression to heavy use in cannabis users by 16% to 21% (p ⩽ .02). Religiosity (believes and attendance) was associated with slowed progression from heavy use to SUD development in alcohol users only. CONCLUSIONS: The findings illustrate strongest association between attending religious services and lower probabilities of progressing to heavy/daily use after substance use initiation for alcohol, tobacco, and cannabis users. This indicates the potential use of religious services as social support for individuals with risky substance use.

Dietary restriction of cysteine and methionine sensitizes gliomas to ferroptosis and induces alterations in energetic metabolism.

Ferroptosis is mediated by lipid peroxidation of phospholipids containing polyunsaturated fatty acyl moieties. Glutathione, the key cellular antioxidant capable of inhibiting lipid peroxidation via the activity of the enzyme glutathione peroxidase 4 (GPX-4), is generated directly from the sulfur-containing amino acid cysteine, and indirectly from methionine via the transsulfuration pathway. Herein we show that cysteine and methionine deprivation (CMD) can synergize with the GPX4 inhibitor RSL3 to increase ferroptotic cell death and lipid peroxidation in both murine and human glioma cell lines and in ex vivo organotypic slice cultures. We also show that a cysteine-depleted, methionine-restricted diet can improve therapeutic response to RSL3 and prolong survival in a syngeneic orthotopic murine glioma model. Finally, this CMD diet leads to profound in vivo metabolomic, proteomic and lipidomic alterations, highlighting the potential for improving the efficacy of ferroptotic therapies in glioma treatment with a non-invasive dietary modification.

Reducing chronic disease may just be a walk in the park.

Wearable technology allows the collection of real-world granular data at scales that would be impossible using traditional collection methods. Master et al. demonstrate the power of this technology to estimate the risk of disease based on daily step counts.1.

Bayesian Multi-View Clustering given complex inter-view structure.

Multi-view datasets are becoming increasingly prevalent. These datasets consist of different modalities that provide complementary characterizations of the same underlying system. They can include heterogeneous types of information with complex relationships, varying degrees of missingness, and assorted sample sizes, as is often the case in multi-omic biological studies. Clustering multi-view data allows us to leverage different modalities to infer underlying systematic structure, but most existing approaches are limited to contexts in which entities are the same across views or have clear one-to-one relationships across data types with a common sample size. Many methods also make strong assumptions about the similarities of clusterings across views. We propose a Bayesian multi-view clustering approach (BMVC) which can handle the realities of multi-view datasets that often have complex relationships and diverse structure. BMVC incorporates known and complex many-to-many relationships between entities via a probabilistic graphical model that enables the joint inference of clusterings specific to each view, but where each view informs the others. Additionally, BMVC estimates the strength of the relationships between each pair of views, thus moderating the degree to which it imposes dependence constraints. We benchmarked BMVC on simulated data to show that it accurately estimates varying degrees of inter-view dependence when inter-view relationships are not limited to one-to-one correspondence. Next, we demonstrated its ability to capture visually interpretable inter-view structure in a public health survey of individuals and households in Puerto Rico following Hurricane Maria. Finally, we showed that BMVC clusters integrate the complex relationships between multi-omic profiles of breast cancer patient data, improving the biological homogeneity of clusters and elucidating hypotheses for functional biological mechanisms. We found that BMVC leverages complex inter-view structure to produce higher quality clusters than those generated by standard approaches. We also showed that BMVC is a valuable tool for real-world discovery and hypothesis generation.

Limited Mechanistic Link Between the Monod Equation and Methanogen Growth: a Perspective from Metabolic Modeling.

The Monod equation has been widely applied as the general rate law of microbial growth, but its applications are not always successful. By drawing on the frameworks of kinetic and stoichiometric metabolic models and metabolic control analysis, the modeling reported here simulated the growth kinetics of a methanogenic microorganism and illustrated that different enzymes and metabolites control growth rate to various extents and that their controls peak at either very low, intermediate, or very high substrate concentrations. In comparison, with a single term and two parameters, the Monod equation only approximately accounts for the controls of rate-determining enzymes and metabolites at very high and very low substrate concentrations, but neglects the enzymes and metabolites whose controls are most notable at intermediate concentrations. These findings support a limited link between the Monod equation and methanogen growth, and unify the competing views regarding enzyme roles in shaping growth kinetics. The results also preclude a mechanistic derivation of the Monod equation from methanogen metabolic networks and highlight a fundamental challenge in microbiology: single-term expressions may not be sufficient for accurate prediction of microbial growth. IMPORTANCE The Monod equation has been widely applied to predict the rate of microbial growth, but its application is not always successful. Using a novel metabolic modeling approach, we simulated the growth of a methanogen and uncovered a limited mechanistic link between the Monod equation and the methanogen's metabolic network. Specifically, the equation provides an approximation to the controls by rate-determining metabolites and enzymes at very low and very high substrate concentrations, but it is missing the remaining enzymes and metabolites whose controls are most notable at intermediate concentrations. These results support the Monod equation as a useful approximation of growth rates and highlight a fundamental challenge in microbial kinetics: single-term rate expressions may not be sufficient for accurate prediction of microbial growth.