Precision engineering of biological function with large-scale measurements and machine learning.

As synthetic biology expands and accelerates into real-world applications, methods for quantitatively and precisely engineering biological function become increasingly relevant. This is particularly true for applications that require programmed sensing to dynamically regulate gene expression in response to stimuli. However, few methods have been described that can engineer biological sensing with any level of quantitative precision. Here, we present two complementary methods for precision engineering of genetic sensors: in silico selection and machine-learning-enabled forward engineering. Both methods use a large-scale genotype-phenotype dataset to identify DNA sequences that encode sensors with quantitatively specified dose response. First, we show that in silico selection can be used to engineer sensors with a wide range of dose-response curves. To demonstrate in silico selection for precise, multi-objective engineering, we simultaneously tune a genetic sensor's sensitivity (EC50) and saturating output to meet quantitative specifications. In addition, we engineer sensors with inverted dose-response and specified EC50. Second, we demonstrate a machine-learning-enabled approach to predictively engineer genetic sensors with mutation combinations that are not present in the large-scale dataset. We show that the interpretable machine learning results can be combined with a biophysical model to engineer sensors with improved inverted dose-response curves.

Method for reproducible automated bacterial cell culture and measurement.

Microbial cell culture is one of the most commonly performed protocols for synthetic biology, and laboratories are increasingly using 96-well plates and laboratory automation systems for cell culture. Here, we describe a method for reproducible microbial culture using laboratory automation systems, including automated liquid handling, automated plate sealing and de-sealing, automated incubation and measurement of growing cultures. We discuss the key considerations that, in our experience, are important for reproducibility and present statistical analyses of data from 150 automated microbial growth experiments performed over 27 months using our automated method.

The genotype-phenotype landscape of an allosteric protein.

Allostery is a fundamental biophysical mechanism that underlies cellular sensing, signaling, and metabolism. Yet a quantitative understanding of allosteric genotype-phenotype relationships remains elusive. Here, we report the large-scale measurement of the genotype-phenotype landscape for an allosteric protein: the lac repressor from Escherichia coli, LacI. Using a method that combines long-read and short-read DNA sequencing, we quantitatively measure the dose-response curves for nearly 105 variants of the LacI genetic sensor. The resulting data provide a quantitative map of the effect of amino acid substitutions on LacI allostery and reveal systematic sequence-structure-function relationships. We find that in many cases, allosteric phenotypes can be quantitatively predicted with additive or neural-network models, but unpredictable changes also occur. For example, we were surprised to discover a new band-stop phenotype that challenges conventional models of allostery and that emerges from combinations of nearly silent amino acid substitutions.

Population dynamics in river networks: analysis of steady states.

We study the population dynamics of an aquatic species in a river network. The habitat is viewed as a binary tree-like metric graph with the population density satisfying a reaction-diffusion-advection equation on each edge, along with the appropriate junction and boundary conditions. In the case of a linear reaction term and hostile downstream boundary condition, the question of persistence in such models was studied by Sarhad, Carlson and Anderson. We focus on the case of a nonlinear (logistic) reaction term and use an outflow downstream boundary condition. We obtain necessary and sufficient conditions for the existence and uniqueness of a positive steady state solution for a simple Y-shaped river network (with a single junction). We show that the existence of a positive steady state is equivalent to the persistence condition for the linearized model. The method can be generalized to a binary tree-like river network with an arbitrary number of segments.

Loss of the Cochlear Amplifier Prestin Reduces Temporal Processing Efficacy in the Central Auditory System.

Active mechanical amplification of sound occurs in cochlear outer hair cells (OHCs) that change their length with oscillations of their membrane potential. Such length changes are the proposed cellular source of the cochlear amplifier, and prestin is the motor protein responsible for OHC electromotility. Previous findings have shown that mice lacking prestin displayed a loss of OHC electromotility, subsequent loss of distortion-product otoacoustic emissions, and a 40-60 dB increase in hearing thresholds. In this study we were interested in studying the functional consequences of the complete loss of cochlear amplification on neural coding of frequency selectivity, tuning, and temporal processing in the auditory midbrain. We recorded near-field auditory evoked potentials and multi-unit activity from the inferior colliculus (IC) of prestin (-/-) null and prestin (+/+) wild-type control mice and determined frequency response areas (FRAs), tuning sharpness, and gap detection to tone bursts and silent gaps embedded in broadband noise. We were interested in determining if the moderate to severe sensorineural hearing loss associated with the loss of motor protein prestin would also impair auditory midbrain temporal-processing measures, or if compensatory mechanisms within the brainstem could compensate for the loss of prestin. In prestin knockout mice we observed that there are severe impairments in midbrain tuning, thresholds, excitatory drive, and gap detection suggesting that brainstem and midbrain processing could not overcome the auditory processing deficits afforded by the loss of OHC electromotility mediated by the prestin protein.

Evaluation of phenytoin serum levels following a loading dose in the acute hospital setting.

PURPOSE: Due to the complex pharmacokinetic profiles of phenytoin (PHT) and fosphenytoin (FOS), achieving sustained, targeted serum PHT levels in the first day of use is challenging. METHODS: A population based approach was used to analyze total serum PHT (tPHT) level within 2-24h of PHT/FOS loading with or without supplementary maintenance or additional loading doses among PHT-naïve patients in the acute hospital setting. Adequate tPHT serum level was defined as ≥20µg/mL. RESULTS: Among 494 patients with 545 tPHT serum levels obtained in the first 2-24h after the loading dose (LD), tPHT serum levels of either

Competition of Multiple Species in Advective Environments.

We study the effect of changes in flow speed on competition of an arbitrary number of species living in advective environments, such as streams and rivers. We begin with a spatial Lotka-Volterra model which is described by n reaction-diffusion-advection equations with Danckwerts boundary conditions. Using the dominant eigenvalue [Formula: see text] of the diffusion-advection operator subject to boundary conditions, we reduce the model to a system of ordinary differential equations. We impose a "transitive arrangement" of the competitors in terms of their interspecific coefficients and growth rates, which means that in the absence of advection, we have the following situation: for all [Formula: see text], species i out-competes species j, while species j has higher intrinsic growth rate than species i. Changing advection speed in the original spatial model corresponds to changing the value of [Formula: see text] in the spatially implicit model. Considering the cases of the odd and even n separately, we obtain explicit intervals of the values of [Formula: see text] that allow all n species to be present in the habitat (coexistence interval). Stability of this equilibrium is shown for [Formula: see text].

Supplemental Antithrombin Is Effective in Achieving Adequate Anticoagulation in Infants and Children With an Inadequate Response to Heparin.

OBJECTIVE: To demonstrate that supplemental antithrombin (AT) is effective in establishing adequate anticoagulation in infants and children with initially inadequate responses to heparin. DESIGN: Following institutional review board approval, a retrospective chart review was conducted on pediatric patients receiving AT during cardiac surgery requiring cardiopulmonary bypass. SETTING: A single institutional review in a hospital setting. PARTICIPANTS: Thirty-one pediatric patients with age ranging from 1 day to 36 months (median 12 weeks) receiving AT during the study period. INTERVENTIONS: As this was a retrospective chart review, no active interventions on patients were performed. MEASUREMENTS AND MAIN RESULTS: Data collected included: patient age, sex, weight, activated clotting time (ACT) values, as well as heparin and AT doses. Primary outcomes were the increase in the ACT from pre- to post-AT and the number of patients who achieved an ACT>480 seconds. The paired t-test was used to compare pre- and post-AT ACT. Mean dose of AT was 50 U/kg (standard deviation 6). Following administration of AT, 30 pediatric patients achieved an ACT of>480 seconds. The post-AT ACT was significantly higher than the pre-AT by a mean of 327 seconds (p<0.0001); 96% of patients achieved an adequate ACT to initiate cardiopulmonary bypass. No adverse events attributable to AT were recorded. CONCLUSION: AT was effective in achieving adequate anticoagulation in a small cohort of infants and children undergoing cardiac surgery who initially were poorly responsive to heparin. Further research to examine the utility of AT in improving clinical outcomes is warranted.

Analysis of spread and persistence for stream insects with winged adult stages.

Species such as stoneflies have complex life history details, with larval stages in the river flow and adult winged stages on or near the river bank. Winged adults often bias their dispersal in the upstream direction, and this bias provides a possible mechanism for population persistence in the face of unidirectional river flow. We use an impulsive reaction-diffusion equation with non-local impulse to describe the population dynamics of a stream-dwelling organism with a winged adult stage, such as stoneflies. We analyze this model from a variety of perspectives so as to understand the effect of upstream dispersal on population persistence. On the infinite domain we use the perspective of weak versus local persistence, and connect the concept of local persistence to positive up and downstream spreading speeds. These spreading speeds, in turn are connected to minimum travelling wave speeds for the linearized operator in upstream and downstream directions. We show that the conditions for weak and local persistence differ, and describe how weak persistence can give rise to a population whose numbers are growing but is being washed out because it cannot maintain a toe hold at any given location. On finite domains, we employ the concept of a critical domain size and dispersal success approximation to determine the ultimate fate of the populations. A simple, explicit formula for a special case allows us to quantify exactly the difference between weak and local persistence.

Aggregation and environmental transmission in Chronic Wasting Disease.

Disease transmission depends on the interplay between the infectious agent and the behavior of the host. Some diseases, such as Chronic Wasting Disease, can be transmitted directly between hosts as well as indirectly via the environment. The social behavior of hosts affects both of these pathways, and a successful intervention requires knowledge of the relative influence of the different etiological and behavioral aspects of the disease. We develop a strategic differential equation model for Chronic Wasting Disease and include direct and indirect transmission as well as host aggregation into our model. We calculate the basic reproduction number and perform a sensitivity analysis based on Latin hypercube sampling from published parameter values. We find conditions for the existence of an endemic equilibrium, and show that, under a certain mild assumption on parameters, the model does not exhibit a backward bifurcation or bistability. Hence, the basic reproduction number constitutes the disease elimination threshold. We find that the prevalence of the disease decreases with host aggregation and increases with the lifespan of the infectious agent in the environment.

Modeling seasonal behavior changes and disease transmission with application to chronic wasting disease.

Behavior and habitat of wildlife animals change seasonally according to environmental conditions. Mathematical models need to represent this seasonality to be able to make realistic predictions about the future of a population and the effectiveness of human interventions. Managing and modeling disease in wild animal populations requires particular care in that disease transmission dynamics is a critical consideration in the etiology of both human and animal diseases, with different transmission paradigms requiring different disease risk management strategies. Since transmission of infectious diseases among wildlife depends strongly on social behavior, mechanisms of disease transmission could also change seasonally. A specific consideration in this regard confronted by modellers is whether the contact rate between individuals is density-dependent or frequency-dependent. We argue that seasonal behavior changes could lead to a seasonal shift between density and frequency dependence. This hypothesis is explored in the case of chronic wasting disease (CWD), a fatal disease that affects deer, elk and moose in many areas of North America. Specifically, we introduce a strategic CWD risk model based on direct disease transmission that accounts for the seasonal change in the transmission dynamics and habitats occupied, guided by information derived from cervid ecology. The model is composed of summer and winter susceptible-infected (SI) equations, with frequency-dependent and density-dependent transmission dynamics, respectively. The model includes impulsive birth events with density-dependent birth rate. We determine the basic reproduction number as a weighted average of two seasonal reproduction numbers. We parameterize the model from data derived from the scientific literature on CWD and deer ecology, and conduct global and local sensitivity analyses of the basic reproduction number. We explore the effectiveness of different culling strategies for the management of CWD: although summer culling seems to be an effective disease eradication strategy, the total culling rate is limited by the requirement to preserve the herd.

How flow speed alters competitive outcome in advective environments.

Many species live in advective environments, such as rivers or streams. The community composition in such environments is shaped by the interplay between biotic interactions and hydrologic constraints. Lutscher et al. (Theor. Popul. Biol. 71:267-277, 2007) demonstrated by simulation that advective flow can shift competitive outcome from one species dominating to coexistence or even to the other species dominating. Here, we present a detailed analysis of the Lotka-Volterra advection-diffusion model underlying their simulations. We use variational techniques as well as a spatially implicit approximation to determine all possible advection-induced shifts in competitive outcome. We show that changes in advection follow relatively few and predictable paths. We illustrate our results in various bifurcation diagrams.

Ablation of mixed lineage kinase 3 (Mlk3) does not inhibit ototoxicity induced by acoustic trauma or aminoglycoside exposure.

Jun N-terminal kinase (JNK) is activated in cochlear hair cells following acoustic trauma or exposure to aminoglycoside antibiotics. Blockade of JNK activation using mixed lineage kinase (MLK) inhibitors prevents hearing loss and hair cell death following these stresses. Since current pharmacologic inhibitors of MLKs block multiple members of this kinase family, we examined the contribution of the major neuronal family member (MLK3) to stress-induced ototoxicity, usingMlk3(-/-) mice. Immunohistochemical staining revealed that MLK3 is expressed in cochlear hair cells of C57/BL6 mice (but not in Mlk3(-/-) animals). After exposure to acoustic trauma there was no significant difference in DPOAE and ABR values betweenMlk3(-/-) and wild-type mice at 48 h following exposure or 2 weeks later. Susceptibility of hair cells to aminoglycoside toxicity was tested by exposing explanted utricles to gentamicin. Gentamicin-induced hair cell death was equivalent in utricles from wild-type and Mlk3(-/-) mice. Blockade of JNK activation with the pharmacologic inhibitor SP600125 attenuated cell death in utricles from both wild-type and Mlk3(-/-) mice. These data show that MLK3 ablation does not protect against hair cell death following acoustic trauma or exposure to aminoglycoside antibiotics, suggesting that MLK3 is not the major upstream regulator of JNK-mediated hair cell death following these stresses. Rather, other MLK family members such as MLK1, which is also expressed in cochlea, may have a previously unappreciated role in noise- and aminoglycoside-induced ototoxicity.

Hormone replacement therapy diminishes hearing in peri-menopausal mice.

We recently discovered that progestin in hormone replacement therapy (HRT) for post-menopausal women has detrimental effects on the ear and central auditory system [Guimaraes, P., Frisina, S.T., Mapes, F., Tadros, S.F., Frisina, D.R., Frisina, R.D., 2006. Progestin negatively affects hearing in aged women. Proc. Natl. Acad. Sci. - PNAS 103, 14246-14249]. To start determining the generality and neural bases of these human findings, the present study examined the effects of combination HRT (estrogen+progestin) and estrogen alone on hearing in peri-menopausal mice. Specifically, auditory brainstem responses (ABRs-sensitivity of the auditory system) and distortion-product otoacoustic emissions (DPOAEs-cochlear outer hair cell system) were employed. Middle age female CBA mice received either a time-release, subcutaneous implanted pellet of estrogen+progestin, estrogen alone, or placebo. Longitudinal comparisons of ABR threshold data obtained at 4 months of treatment revealed statistically significant declines in auditory sensitivity over time for the combined estrogen+progestin treatment group, with the estrogen only group revealing milder changes at 3, 6 and 32 kHz. DPOAE testing revealed statistically significant differences for the estrogen+progestin treatment group in the high and middle frequency ranges (15-29 and 30-45 kHz) after as early as 2 months of treatment (p<0.01 and p<0.001, respectively). Statistically significant changes were also seen at 4 months of treatment across all frequencies for the combined HRT group. These data suggest that estrogen+progestin HRT therapy of 4 months duration impairs outer hair cell functioning and overall auditory sensitivity. These findings indicate that estrogen+progestin HRT may actually accelerate age-related hearing loss, relative to estrogen monotherapy; findings that are consistent with the clinical hearing loss observed in aging women that have taken combination HRT.

Interactions of hearing loss and diabetes mellitus in the middle age CBA/CaJ mouse model of presbycusis.

Recently, we characterized the more severe nature of hearing loss in aged Type 2 diabetic human subjects [Frisina, S.T., Mapes, F., Kim, S., Frisina, D.R., Frisina, R.D., 2006. Characterization of hearing loss in aged type II diabetics. Hear. Res. 211, 103-113]. The current study prospectively assessed hearing abilities in middle age CBA/CaJ mice with Type 1 diabetes mellitus (T1DM) (STZ injection) or Type 2 diabetes mellitus (T2DM) (high fat diet), for a period of 6 months. Blood glucose, body weight and auditory tests (Auditory Brainstem Response-ABR, Distortion Product Otoacoustic Emissions-DPOAE) were evaluated at baseline and every 2 months. Tone and broad-band noise-burst responses in the inferior colliculus were obtained at 6 months. Body weights of controls did not change over 6 months (approximately 32 g), but there was a significant (approximately 5 g) decline in the T1DM, while T2DM exhibited approximately 10 g weight gain. Blood glucose levels significantly increased: 3-fold for T1DM, 1.3-fold for T2DM; with no significant changes in controls. ABR threshold elevations were found for both types of diabetes, but were most pronounced in the T2DM, starting as early as 2 months after induction of diabetes. A decline of mean DPOAE amplitudes was observed in both diabetic groups at high frequencies, and for the T2DM at low frequencies. In contrast to ABR thresholds, tone and noise thresholds in the inferior colliculus were lower for both diabetic groups. Induction of diabetes in middle-aged CBA/CaJ mice promotes amplification of age-related peripheral hearing loss which makes it a suitable model for studying the interaction of age-related hearing loss and diabetes. On the other hand, initial results of effects from very high blood glucose level (T1DM) on the auditory midbrain showed disruption of central inhibition, increased response synchrony or enhanced excitation in the inferior colliculus.

Auditory efferent feedback system deficits precede age-related hearing loss: contralateral suppression of otoacoustic emissions in mice.

The C57BL/6J mouse has been a useful model of presbycusis, as it displays an accelerated age-related peripheral hearing loss. The medial olivocochlear efferent feedback (MOC) system plays a role in suppressing cochlear outer hair cell (OHC) responses, particularly for background noise. Neurons of the MOC system are located in the superior olivary complex, particularly in the dorsomedial periolivary nucleus (DMPO) and in the ventral nucleus of the trapezoid body (VNTB). We previously discovered that the function of the MOC system declines with age prior to OHC degeneration, as measured by contralateral suppression (CS) of distortion product otoacoustic emissions (DPOAEs) in humans and CBA mice. The present study aimed to determine the time course of age changes in MOC function in C57s. DPOAE amplitudes and CS of DPOAEs were collected for C57s from 6 to 40 weeks of age. MOC responses were observed at 6 weeks but were gone at middle (15-30 kHz) and high (30-45 kHz) frequencies by 8 weeks. Quantitative stereological analyses of Nissl sections revealed smaller neurons in the DMPO and VNTB of young adult C57s compared with CBAs. These findings suggest that reduced neuron size may underlie part of the noteworthy rapid decline of the C57 efferent system. In conclusion, the C57 mouse has MOC function at 6 weeks, but it declines quickly, preceding the progression of peripheral age-related sensitivity deficits and hearing loss in this mouse strain.