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Background: Intramuscular fat grafting in extremity muscles, especially the gastrocnemius, has become increasingly popular. However, while safety in truncal muscle fat grafting has been well-studied, research on extremity muscles is lacking., Objectives: In this study, we aimed to explore the anatomy of the gastrocnemius muscle and adjacent structures as intramuscular and subcutaneous recipient sites. Additionally, we sought to analyze pressure-volume relationships and fat migration patterns during posterior calf grafting., Methods: Eight cadaveric lower extremities were examined. A prosection was performed to better understand the vascular complex around the gastrocnemius. Ultrasound-guided fat injections were then performed into both the subcutaneous and intramuscular layers; dynamic pressures were measured with a manometer. Last, dyed-injectant was injected subcutaneously to better elucidate subcutaneous anatomy., Results: Anatomic prosection demonstrated the proximity of the gastrocnemius muscle to the popliteal venous system that arborized (>1 mm) intramuscularly. In the 3 specimens that underwent intramuscular fat grafting, the peak intramuscular pressures plateaued at 21 mmHg (19.5-23 mmHg); there was no observed extrafascial migration of the injectant. With subcutaneous injectant in 1 specimen, pressures in the subcutaneous space increased (125 mmHg) with additional injectant (240 mL) while pressures in the intramuscular space remained relatively constant (4 mmHg)., Conclusions: Intramuscular gastrocnemius fat grafting should be done with caution: its proximity to critical veins and sustained increased intramuscular pressures following grafting increases risks of embolus and thrombosis, respectively. Subcutaneous injection may be safer in that increased pressures are not communicated to deep structures. Last, we present a novel description of posterior calf subcutaneous compartments that may better allow surgeons to direct and predict subcutaneous injectant., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Aesthetic Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Experimental studies have shown that chromatin modifiers have a critical effect on cellular reprogramming, i.e., the conversion of differentiated cells to pluripotent stem cells. Here, we develop a model of the OCT4 gene regulatory network that includes genes expressing chromatin modifiers TET1 and JMJD2, and the chromatin modification circuit on which these modifiers act. We employ this model to compare three reprogramming approaches that have been considered in the literature with respect to reprogramming efficiency and latency variability. These approaches are overexpression of OCT4 alone, overexpression of OCT4 with TET1, and overexpression of OCT4 with JMJD2. Our results show more efficient and less variable reprogramming when also JMJD2 and TET1 are overexpressed, consistent with previous experimental data. Nevertheless, TET1 overexpression can lead to more efficient reprogramming compared to JMJD2 overexpression. This is the case when the recruitment of DNA methylation by H3K9me3 is weak and the methyl-CpG-binding domain (MBD) proteins are sufficiently scarce such that they do not hamper TET1 binding to methylated DNA. The model that we developed provides a mechanistic understanding of existing experimental results and is also a tool for designing optimized reprogramming approaches that combine overexpression of cell-fate specific transcription factors (TFs) with targeted recruitment of epigenetic modifiers., (© 2024. The Author(s).)

Reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs) is inefficient, with heterogeneity among transcription factor (TF) trajectories driving divergent cell states. Nevertheless, the impact of TF dynamics on reprogramming efficiency remains uncharted. We develop a system that accurately reports OCT4 protein levels in live cells and use it to reveal the trajectories of OCT4 in successful reprogramming. Our system comprises a synthetic genetic circuit that leverages noise to generate a wide range of OCT4 trajectories and a microRNA targeting endogenous OCT4 to set total cellular OCT4 protein levels. By fusing OCT4 to a fluorescent protein, we are able to track OCT4 trajectories with clonal resolution via live-cell imaging. We discover that a supraphysiological, stable OCT4 level is required, but not sufficient, for efficient iPSC colony formation. Our synthetic genetic circuit design and high-throughput live-imaging pipeline are generalizable for investigating TF dynamics for other cell fate programming applications.

Background: Although the Brazilian butt lift (BBL) is one of the most popular procedures in body contouring, it has been associated with the risk of pulmonary fat embolism when fat graft is injected into the gluteal muscles. The subcutaneous plane has been identified as a safe site for fat graft injection, but deaths from fat embolism continue to occur because there is no mechanism to confirm consistent subcutaneous placement., Objectives: The aim of this paper was to determine if real-time intraoperative ultrasound could accurately identify the subcutaneous gluteal anatomic landmarks and permit a single surgeon to consistently target fat graft placement in the subcutaneous space., Methods: In total, 4150 BBLs were performed with real-time intraoperative ultrasound being used to confirm the subcutaneous position of a static cannula during fat graft injection. Serial deposits of fat graft were performed in each buttock. Ultrasound confirmed that fat graft consistently remained above the deep gluteal fascia and migrated through the deep subcutaneous space. These fat graft deposits were then equalized with a moving cannula to correct any contour deformities. Operative times were recorded and compared with BBL performed by expansion vibration lipofilling without ultrasound., Results: Real-time intraoperative ultrasound allowed for the visual confirmation of consistent subcutaneous fat graft deposition and the targeting of fat graft into specific gluteal subcutaneous compartments., Conclusions: Real-time intraoperative ultrasound allows the surgeon to confirm a subcutaneous-only fat graft injection, target specific gluteal subcutaneous compartments, and take advantage of the unique architecture of the deep subcutaneous space to create gluteal projection and correct contour deformities., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Aesthetic Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Static injection, migration, and equalization allows for always subcutaneous-targeted fat grafting to either the deep or the superficial subcutaneous spaces., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Gluteal fat grafting is the fastest growing surgery in body contouring because of the powerful results that no other procedure can achieve. Efforts made to improve the safety of this procedure are reviewed., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: An increasing number of aesthetic surgery procedures are being performed in an ambulatory fashion in office-based settings. Postoperative monitoring of these patients has historically required paid private-duty nurses measuring vital signs, encouraging ambulation, and monitoring overall comfort level. Recently, advancements in nanotechnology have permitted high-acuity data acquisition of multiple clinical parameters that can be transmitted to the surgeon's mobile device in a continuous fashion., Objective: The aim of this study was to describe the authors' early experience with this emerging artificial intelligence technology in the postoperative setting., Methods: Twenty-three consecutive patients underwent radiofrequency-assisted liposuction and Brazilian butt lift surgery, and were placed in a monitoring garment, SimpleSense (Nanowear Technologies, Brooklyn, NY) postoperatively. The primary outcome was device usability, reflected by compliance with wearing the device and completeness of data collection., Results: During the first 48 hours, 91% of patients wore the device for more than 12 hours per day. Only 39% were compliant with postoperative positioning. No postoperative events were detected., Conclusions: The quality of data collected enables detection of clinical derangements and can alert the surgeon in real time, prompting interventions such as drug administration, position changes, or presentation to the emergency room., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Aesthetic Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A ratiometric response gives an output that is proportional to the ratio between the magnitudes of two inputs. Ratio computation has been observed in nature and is also needed in the development of smart probiotics and organoids. Here, we achieve ratiometric gene expression response in bacteria Escherichia coli with the incoherent merger network. In this network, one input molecule activates expression of the output protein while the other molecule activates an intermediate protein that enhances the output's degradation. When degradation rate is first order and faster than dilution, the output responds linearly to the ratio between the input molecules' levels over a wide range with R2 close to 1. Response sensitivity can be quantitatively tuned by varying the output's translation rate. Furthermore, ratiometric responses are robust to global perturbations in cellular components that influence gene expression because such perturbations affect the output through an incoherent feedforward loop. This work demonstrates a new molecular signal processing mechanism for multiplexed sense-and-respond circuits that are robust to intra-cellular context., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Continuous-time Markov chains are frequently used as stochastic models for chemical reaction networks, especially in the growing field of systems biology. A fundamental problem for these Stochastic Chemical Reaction Networks (SCRNs) is to understand the dependence of the stochastic behavior of these systems on the chemical reaction rate parameters. Towards solving this problem, in this paper we develop theoretical tools called comparison theorems that provide stochastic ordering results for SCRNs. These theorems give sufficient conditions for monotonic dependence on parameters in these network models, which allow us to obtain, under suitable conditions, information about transient and steady-state behavior. These theorems exploit structural properties of SCRNs, beyond those of general continuous-time Markov chains. Furthermore, we derive two theorems to compare stationary distributions and mean first passage times for SCRNs with different parameter values, or with the same parameters and different initial conditions. These tools are developed for SCRNs taking values in a generic (finite or countably infinite) state space and can also be applied for non-mass-action kinetics models. When propensity functions are bounded, our method of proof gives an explicit method for coupling two comparable SCRNs, which can be used to simultaneously simulate their sample paths in a comparable manner. We illustrate our results with applications to models of enzymatic kinetics and epigenetic regulation by chromatin modifications., (© 2023. The Author(s).)

Background: Fat grafting is the favored option for buttock augmentation by most surgeons, and buttock implants are mostly regarded as second choices. Accepted options for buttock implant pocket dissection are subfascial, intramuscular, and submuscular. To overcome the limitations of both intramuscular and submuscular pockets, and combine the benefits of both, the authors present a novel dual-plane pocket dissection. The dual-plane pocket involves a submuscular plane in the cranial half of the pocket and switching to an intramuscular plane in the caudal half. With this study, the authors describe their experience with this technique and analyze the dissection of the pocket on human cadavers., Methods: The authors reviewed 82 consecutive composite gluteal augmentation cases from March of 2019 to November of 2019. In all cases, the implant was placed in the "dual plane." The clinical study was supplemented by 10 hemigluteal dissections in five cadavers following the surgical technique, with assessment of the anatomical components of the implant pocket created., Results: Patients who underwent this technique showed excellent soft-tissue coverage over the implants. The main complications observed were seroma formation in five of 82 patients and temporary sciatic pain in four of the patients. The anatomical study confirmed double muscle coverage of both gluteus maximus and partial medius in the upper pocket pole and intramuscular gluteus maximus implant position in the lower pole., Conclusion: The authors present the dual-plane dissection technique as a valuable innovation to improve soft-tissue cover for the upper implant pole in buttock implant surgery., Competing Interests: Disclosure : The authors have no financial disclosures or conflicts of interest to report. No funding was received for this study., (Copyright © 2022 by the American Society of Plastic Surgeons.)

Background: An ever-increasing demand for liposuction and fat transplantation procedures in the United States reflects their continued safety and clinical effectiveness. Technical breakthroughs, such as the utilization of tumescent infiltration and fat separation techniques, have been instrumental in optimizing outcomes but add time to the surgical procedure. Simultaneous separation and tumescence (SST) is a new technique combining these innovations to further improve safety and efficiency in liposuction., Objectives: The authors describe their technique for utilizing SST on more than 1200 patients spanning a 6-year period. A split abdomen study was designed to compare the vasoconstrictive effectiveness of SST with traditional Klein infiltration techniques., Methods: A quantification perfusion analysis utilizing indocyanine green angiography was performed on 4 patients to illustrate the vasoconstrictive effectiveness of SST over traditional Klein infiltration techniques. A mean relative vasoconstriction score was calculated relative to the umbilicus for each technique., Results: Compared with traditional Klein tumescence techniques, SST has a similar complication rate, utilizes similar infiltrative volumes, and increases vasoconstriction (mean 89.6% SST hemiabdomen vs 48.1% Klein hemiabdomen at 2 minutes, P < 0.0001)., Conclusions: SST represents a novel, long-overdue departure from Klein tumescence, which has been a valuable mainstay for donor site preparation for over 3 decades. SST will pave the way for more efficient operative times and potentially higher volumes of fat being safely extracted., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Aesthetic Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Natural biological systems display complex regulation and synthetic biomolecular systems have been used to understand their natural counterparts and to parse sophisticated regulations into core design principles. At the same time, the engineering of biomolecular systems has unarguable potential to transform current and to enable new, yet-to-be-imagined, biotechnology applications. In this review, we discuss the progression of control systems design in synthetic biology, from the purpose of understanding the function of naturally occurring regulatory motifs to that of creating genetic circuits whose function is sufficiently robust for biotechnology applications., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Heterologous gene activation causes non-physiological burden on cellular resources that cells are unable to adjust to. Here, we introduce a feedforward controller that actuates growth rate upon activation of a gene of interest (GOI) to compensate for such a burden. The controller achieves this by activating a modified SpoT enzyme (SpoTH) with sole hydrolysis activity, which lowers ppGpp level and thus increases growth rate. An inducible RelA+ expression cassette further allows to precisely set the basal level of ppGpp, and thus nominal growth rate, in any bacterial strain. Without the controller, activation of the GOI decreased growth rate by more than 50%. With the controller, we could activate the GOI to the same level without growth rate defect. A cell strain armed with the controller in co-culture enabled persistent population-level activation of a GOI, which could not be achieved by a strain devoid of the controller. The feedforward controller is a tunable, modular, and portable tool that allows dynamic gene activation without growth rate defects for bacterial synthetic biology applications., (© 2022. The Author(s).)

On January 31, 2018, The Multi-Society Task Force for Safety in Gluteal Fat Grafting released a practice advisory regarding gluteal fat grafting. The task force was assembled in the wake of several highly publicized patient deaths involving Brazilian Butt Lift (BBL) and produced a second practice advisory in August 2019. In 2021, The Aesthetic Surgery President commissioned a Working Group on BBL Patient Safety, charging the group to address new guidelines affecting safety and welfare of BBL patients. The "Practice Advisory on Gluteal Fat Grafting" is the first advisory developed since the working group was formed. In addition to surgical technique as a major cause of fatal complications of BBL, the working group focused on micro-economic trends of operative time and regional BBL pricing and considered fatigue and distraction in formulating the current guidelines. In Florida, the majority of BBL deaths occur at the end of the week. Such a non-normalized distribution most likely represents the result of fatigue and/or distraction, which has been linked to surgical mortality in multiple published communications. In addition, mortality is likely due to uncertainty or lack of documentation as to the correct plane of fat injection. Therefore, the newest and most compelling recommendations from these guidelines include the utilization of ultrasound-guided documentation of cannula placement prior to and during fat injection, and the limitation of 3 BBL cases as a maximum amount of total operative cases per day. The authors thank members of the task force for the insights they brought to this process., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Aesthetic Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Information exchange is a critical process in all communication systems, including biological ones. Retroactivity describes the load that downstream modules apply to their upstream systems in biological circuits. The motivation behind this work is that of integrating retroactivity, a concept proper of biochemical circuits, with the metrics defined in Information Theory and Digital Communications. This paper focuses on studying the impact of retroactivity on different biological signaling system models, which present analogies with well-known telecommunication systems. The mathematical analysis is performed both in the high and low molecular counts regime, by mean of the Chemical Master Equation and the Linear Noise Approximation, respectively. The main goal of this work is to provide analytical tools to maximize the reliable information exchange across different biomolecular circuit models. Results highlight how, in general, retroactivity harms communication performance. This negative effect can be mitigated by adding to the signaling circuit an independent upstream system that connects with the same pool of downstream circuits.

Background: With the increase in popularity of fat transfer to the buttocks in body contouring surgery, new patient subsets are emerging. The gender confirmation patient has specific characteristics that must be addressed to achieve the best outcomes., Methods: The authors performed an observational prospective study including 36 consecutive patients who underwent body feminization as a gender affirming operation using large-volume fat transplantation with or without gluteal implants., Results: Thirty-six consecutive male-to-female transgender patients have undergone large-volume fat transplantation to the buttocks performed by the authors, between July of 2016 and January of 2019. In 12 of these 36 cases, large-volume fat grafting was supplemented by intramuscular placement of silicone implants. The mean waist-to-hip ratio before surgery was 1.11, and this improved to 0.81 after surgery (fat transfer-only group, 0.88; fat transfer plus implants group, 0.75)., Conclusions: The transgender patient is a unique patient requiring a specific set of surgeon's skills and intraoperative strategies to achieve good outcomes. Composite solutions with both implants and lipofilling lead to better outcomes in terms of waist-to-hip ratio in these patients., Clinical Question/level of Evidence: Therapeutic, IV., Competing Interests: Disclosure:The authors have no financial disclosures or conflicts of interest to report. No funding was received for this study., (Copyright © 2022 by the American Society of Plastic Surgeons.)

Epigenetic cell memory allows distinct gene expression patterns to persist in different cell types despite a common genotype. Although different patterns can be maintained by the concerted action of transcription factors (TFs), it was proposed that long-term persistence hinges on chromatin state. Here, we study how the dynamics of chromatin state affect memory, and focus on a biologically motivated circuit motif, among histones and DNA modifications, that mediates the action of TFs on gene expression. Memory arises from time-scale separation among three circuit's constituent processes: basal erasure, auto and cross-catalysis, and recruited erasure of modifications. When the two latter processes are sufficiently faster than the former, the circuit exhibits bistability and hysteresis, allowing active and repressed gene states to coexist and persist after TF stimulus removal. The duration of memory is stochastic with a mean value that increases as time-scale separation increases, but more so for the repressed state. This asymmetry stems from the cross-catalysis between repressive histone modifications and DNA methylation and is enhanced by the relatively slower decay rate of the latter. Nevertheless, TF-mediated positive autoregulation can rebalance this asymmetry and even confers robustness of active states to repressive stimuli. More generally, by wiring positively autoregulated chromatin modification circuits under time scale separation, long-term distinct gene expression patterns arise, which are also robust to failure in the regulatory links., Competing Interests: The authors have declared that no competing interests exist.

Engineered signaling networks can impart cells with new functionalities useful for directing differentiation and actuating cellular therapies. For such applications, the engineered networks must be tunable, precisely regulate target gene expression, and be robust to perturbations within the complex context of mammalian cells. Here, we use bacterial two-component signaling proteins to develop synthetic phosphoregulation devices that exhibit these properties in mammalian cells. First, we engineer a synthetic covalent modification cycle based on kinase and phosphatase proteins derived from the bifunctional histidine kinase EnvZ, enabling analog tuning of gene expression via its response regulator OmpR. By regulating phosphatase expression with endogenous miRNAs, we demonstrate cell-type specific signaling responses and a new strategy for accurate cell type classification. Finally, we implement a tunable negative feedback controller via a small molecule-stabilized phosphatase, reducing output expression variance and mitigating the context-dependent effects of off-target regulation and resource competition. Our work lays the foundation for establishing tunable, precise, and robust control over cell behavior with synthetic signaling networks., (© 2022. The Author(s).)

The design of genetic circuits typically relies on characterization of constituent modules in isolation to predict the behavior of modules' composition. However, it has been shown that the behavior of a genetic module changes when other modules are in the cell due to competition for shared resources. In order to engineer multimodule circuits that behave as intended, it is thus necessary to predict changes in the behavior of a genetic module when other modules load cellular resources. Here, we introduce two characteristics of circuit modules: the demand for cellular resources and the sensitivity to resource loading. When both are known for every genetic module in a circuit library, they can be used to predict any module's behavior upon addition of any other module to the cell. We develop an experimental approach to measure both characteristics for any circuit module using a resource sensor module. Using the measured resource demand and sensitivity for each module in a library, the outputs of the modules can be accurately predicted when they are inserted in the cell in arbitrary combinations. These resource competition characteristics may be used to inform the design of genetic circuits that perform as predicted despite resource competition.

The rise of systems biology has ushered a new paradigm: the view of the cell as a system that processes environmental inputs to drive phenotypic outputs. Synthetic biology provides a complementary approach, allowing us to program cell behavior through the addition of synthetic genetic devices into the cellular processor. These devices, and the complex genetic circuits they compose, are engineered using a design-prototype-test cycle, allowing for predictable device performance to be achieved in a context-dependent manner. Within mammalian cells, context effects impact device performance at multiple scales, including the genetic, cellular, and extracellular levels. In order for synthetic genetic devices to achieve predictable behaviors, approaches to overcome context dependence are necessary. Here, we describe control systems approaches for achieving context-aware devices that are robust to context effects. We then consider cell fate programing as a case study to explore the potential impact of context-aware devices for regenerative medicine applications., Competing Interests: Declaration of interests MIT has filed patents on behalf of R.D.J., R.W., and D.D.V. relating to the design of genetic controllers for use in mammalian cells. R.W. is a co-founder and has interest in Strand Therapeutics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

CRISPRi-mediated gene regulation allows simultaneous control of many genes. However, highly specific sgRNA-promoter binding is, alone, insufficient to achieve independent transcriptional regulation of multiple targets. Indeed, due to competition for dCas9, the repression ability of one sgRNA changes significantly when another sgRNA becomes expressed. To solve this problem and decouple sgRNA-mediated regulatory paths, we create a dCas9 concentration regulator that implements negative feedback on dCas9 level. This allows any sgRNA to maintain an approximately constant dose-response curve, independent of other sgRNAs. We demonstrate the regulator performance on both single-stage and layered CRISPRi-based genetic circuits, zeroing competition effects of up to 15-fold changes in circuit I/O response encountered without the dCas9 regulator. The dCas9 regulator decouples sgRNA-mediated regulatory paths, enabling concurrent and independent regulation of multiple genes. This allows predictable composition of CRISPRi-based genetic modules, which is essential in the design of larger scale synthetic genetic circuits.

Synthetic biology has the potential to bring forth advanced genetic devices for applications in healthcare and biotechnology. However, accurately predicting the behavior of engineered genetic devices remains difficult due to lack of modularity, wherein a device's output does not depend only on its intended inputs but also on its context. One contributor to lack of modularity is loading of transcriptional and translational resources, which can induce coupling among otherwise independently-regulated genes. Here, we quantify the effects of resource loading in engineered mammalian genetic systems and develop an endoribonuclease-based feedforward controller that can adapt the expression level of a gene of interest to significant resource loading in mammalian cells. Near-perfect adaptation to resource loads is facilitated by high production and catalytic rates of the endoribonuclease. Our design is portable across cell lines and enables predictable tuning of controller function. Ultimately, our controller is a general-purpose device for predictable, robust, and context-independent control of gene expression.

Multistability is a key property of dynamical systems modeling cellular regulatory networks implicated in cell fate decisions, where, different stable steady states usually represent distinct cell phenotypes. Monotone network motifs are highly represented in these regulatory networks. In this paper, we leverage the properties of monotone dynamical systems to provide theoretical results that guide the selection of inputs that trigger a transition, i.e., reprogram the network, to a desired stable steady state. We first show that monotone dynamical systems with bounded trajectories admit a minimum and a maximum stable steady state. Then, we provide input choices that are guaranteed to reprogram the system to these extreme steady states. For intermediate states, we provide an input space that is guaranteed to contain an input that reprograms the system to the desired state. We then provide implementation guidelines for finite-time procedures that search this space for such an input, along with rules to prune parts of the space during search. We demonstrate these results on simulations of two recurrent regulatory network motifs: self-activation within mutual antagonism and self-activation within mutual cooperation. Our results depend uniquely on the structure of the network and are independent of specific parameter values.

Intracellular spatial heterogeneity is frequently observed in bacteria, where the chromosome occupies part of the cell's volume and a circuit's DNA often localizes within the cell. How this heterogeneity affects core processes and genetic circuits is still poorly understood. In fact, commonly used ordinary differential equation (ODE) models of genetic circuits assume a well-mixed ensemble of molecules and, as such, do not capture spatial aspects. Reaction-diffusion partial differential equation (PDE) models have been only occasionally used since they are difficult to integrate and do not provide mechanistic understanding of the effects of spatial heterogeneity. In this paper, we derive a reduced ODE model that captures spatial effects, yet has the same dimension as commonly used well-mixed models. In particular, the only difference with respect to a well-mixed ODE model is that the association rate constant of binding reactions is multiplied by a coefficient, which we refer to as the binding correction factor (BCF). The BCF depends on the size of interacting molecules and on their location when fixed in space and it is equal to unity in a well-mixed ODE model. The BCF can be used to investigate how spatial heterogeneity affects the behavior of core processes and genetic circuits. Specifically, our reduced model indicates that transcription and its regulation are more effective for genes located at the cell poles than for genes located on the chromosome. The extent of these effects depends on the value of the BCF, which we found to be close to unity. For translation, the value of the BCF is always greater than unity, it increases with mRNA size, and, with biologically relevant parameters, is substantially larger than unity. Our model has broad validity, has the same dimension as a well-mixed model, yet it incorporates spatial heterogeneity. This simple-to-use model can be used to both analyze and design genetic circuits while accounting for spatial intracellular effects., Competing Interests: The authors have declared that no competing interests exist.

Modularity has been the subject of intense investigation in systems biology for more than two decades. Whether modularity holds in biological networks is a question that has attracted renewed attention in recent years with the advent of synthetic biology, mostly as a convenient property to perform bottom-up design of complex systems. A number of studies have appeared, which fundamentally challenge modularity as a property of engineered biological circuits. Here, we summarize some of these studies and potential engineering solutions that have been proposed to enable modular composition of biological circuits., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cellular reprogramming is traditionally accomplished through an open loop (OL) control approach, wherein key transcription factors (TFs) are injected in cells to steer the state of the pluripotency (PL) gene regulatory network (GRN), as encoded by TFs concentrations, to the pluripotent state. Due to the OL nature of this approach, the concentration of TFs cannot be accurately controlled. Recently, a closed loop (CL) feedback control strategy was proposed to overcome this problem with promising theoretical results. However, previous analyses of the controller were based on deterministic models. It is well known that cellular systems are characterized by substantial stochasticity, especially when molecules are in low copy number as it is the case in reprogramming problems wherein the gene copy number is usually one or two. Hence, in this paper, we analyze the Chemical Master Equation (CME) for the reaction model of the PL GRN with and without the feedback controller. We computationally and analytically investigate the performance of the controller in biologically relevant parameter regimes where stochastic effects dictate system dynamics. Our results indicate that the feedback control approach still ensures reprogramming even when both the PL GRN and the controller are stochastic.

Phenotypical variability in the absence of genetic variation often reflects complex energetic landscapes associated with underlying gene regulatory networks (GRNs). In this view, different phenotypes are associated with alternative states of complex nonlinear systems: stable attractors in deterministic models or modes of stationary distributions in stochastic descriptions. We provide theoretical and practical characterizations of these landscapes, specifically focusing on stochastic Slow Promoter Kinetics (SPK), a time scale relevant when transcription factor binding and unbinding are affected by epigenetic processes like DNA methylation and chromatin remodeling. In this case, largely unexplored except for numerical simulations, adiabatic approximations of promoter kinetics are not appropriate. In contrast to the existing literature, we provide rigorous analytic characterizations of multiple modes. A general formal approach gives insight into the influence of parameters and the prediction of how changes in GRN wiring, for example through mutations or artificial interventions, impact the possible number, location, and likelihood of alternative states. We adapt tools from the mathematical field of singular perturbation theory to represent stationary distributions of Chemical Master Equations for GRNs as mixtures of Poisson distributions and obtain explicit formulas for the locations and probabilities of metastable states as a function of the parameters describing the system. As illustrations, the theory is used to tease out the role of cooperative binding in stochastic models in comparison to deterministic models, and applications are given to various model systems, such as toggle switches in isolation or in communicating populations, a synthetic oscillator, and a trans-differentiation network., Competing Interests: The authors have declared that no competing interests exist.

The notion of reprogramming cell fate is a direct challenge to the traditional view in developmental biology that a cell's phenotypic identity is sealed after undergoing differentiation. Direct experimental evidence, beginning with the somatic cell nuclear transfer experiments of the twentieth century and culminating in the more recent breakthroughs in transdifferentiation and induced pluripotent stem cell (iPSC) reprogramming, have rewritten the rules for what is possible with cell fate transformation. Research is ongoing in the manipulation of cell fate for basic research in disease modeling, drug discovery, and clinical therapeutics. In many of these cell fate reprogramming experiments, there is often little known about the genetic and molecular changes accompanying the reprogramming process. However, gene regulatory networks (GRNs) can in some cases be implicated in the switching of phenotypes, providing a starting point for understanding the dynamic changes that accompany a given cell fate reprogramming process. In this chapter, we present a framework for computationally analyzing cell fate changes by mathematically modeling these GRNs. We provide a user guide with several tutorials of a set of techniques from dynamical systems theory that can be used to probe the intrinsic properties of GRNs as well as study their responses to external perturbations.

The behavior of genetic circuits is often poorly predictable. A gene's expression level is not only determined by the intended regulators, but also affected by changes in ribosome availability imparted by expression of other genes. Here we design a quasi-integral biomolecular feedback controller that enables the expression level of any gene of interest (GOI) to adapt to changes in available ribosomes. The feedback is implemented through a synthetic small RNA (sRNA) that silences the GOI's mRNA, and uses orthogonal extracytoplasmic function (ECF) sigma factor to sense the GOI's translation and to actuate sRNA transcription. Without the controller, the expression level of the GOI is reduced by 50% when a resource competitor is activated. With the controller, by contrast, gene expression level is practically unaffected by the competitor. This feedback controller allows adaptation of genetic modules to variable ribosome demand and thus aids modular construction of complicated circuits.

Multistable dynamical systems are ubiquitous in nature, especially in the context of regulatory networks controlling cell fate decisions, wherein stable steady states correspond to different cell phenotypes. In the past decade, it has become experimentally possible to "reprogram" the fate of a cell by suitable externally imposed input stimulations. In several of these reprogramming instances, the underlying regulatory network has a known structure and often it falls in the class of cooperative monotone dynamical systems. In this paper, we therefore leverage this structure to provide concrete guidance on the choice of inputs that reprogram a cooperative dynamical system to a desired target steady state. Our results are parameter-independent and therefore can serve as a practical guidance to cell-fate reprogramming experiments.

A central issue in the analysis of multi-stable systems is that of controlling the relative size of the basins of attraction of alternative states through suitable choices of system parameters. We are interested here mainly in the stochastic version of this problem, that of shaping the stationary probability distribution of a Markov chain so that various alternative modes become more likely than others. Although many of our results are more general, we were motivated by an important biological question, that of cell differentiation. In the mathematical modeling of cell differentiation, it is common to think of internal states of cells (quanfitied by activation levels of certain genes) as determining the different cell types. Specifically, we study here the "PU.1/GATA-1 circuit" which is involved in the control of the development of mature blood cells from hematopoietic stem cells (HSCs). All mature, specialized blood cells have been shown to be derived from multipotent HSCs. Our first contribution is to introduce a rigorous chemical reaction network model of the PU.1/GATA-1 circuit, which incorporates current biological knowledge. We then find that the resulting ODE model of these biomolecular reactions is incapable of exhibiting multistability, contradicting the fact that differentiation networks have, by definition, alternative stable steady states. When considering instead the stochastic version of this chemical network, we analytically construct the stationary distribution, and are able to show that this distribution is indeed capable of admitting a multiplicity of modes. Finally, we study how a judicious choice of system parameters serves to bias the probabilities towards different stationary states. We remark that certain changes in system parameters can be physically implemented by a biological feedback mechanism; tuning this feedback gives extra degrees of freedom that allow one to assign higher likelihood to some cell types over others.

Background: The gluteal lift ("Brazilian butt lift") has improved significantly in technique and in outcomes. Some postulate that fat embolism occurs because of damage to veins superficial to the gluteus muscles and that fat must be injected intramuscularly to achieve satisfactory aesthetic results. The purpose of this article is to present data where gluteal lift is performed under local anesthesia, address safety concerns, and clarify these speculations., Methods: All patients underwent gluteal lift under local anesthesia using oral medications and tumescent infiltration. Fat was harvested by closed-system liposuction, separated by gravity, injected using a peristaltic pump and reticulating basket cannulas., Results: Thirty-two female patients with an average age of 38.6 years and a body mass index of 24.8 kg/m underwent 47 gluteal lift operations under local anesthesia over 52 months. The average volume of injected fat was 359 ml per buttock. There were no deaths or complications., Conclusions: Recent concerns regarding buttock injections have been raised due to death from fat embolism. Despite numerous theories on the mechanism of entry, the fact that venous injury occurs with the introduction of boluses of fat in enough quantities to cause mechanical disruption of the cardiopulmonary circuit is not in dispute. The authors believe this cannot occur in the awake patient without the surgeon being acutely aware of a misguided cannula. These data suggest that buttock fat grafting under local anesthesia is a safe and effective procedure with an excellent safety profile and durable results., Clinical Question/level of Evidence: Therapeutic, IV.

This is the first work to introduce the use of blockchain technology for the electronic traceability of wood from standing tree to final user. Infotracing integrates the information related to the product quality with those related to the traceability [physical and digital documents (Radio Frequency IDentification-RFID-architecture)] within an online information system whose steps (transactions) can be made safe to evidence of alteration through the blockchain. This is a decentralized and distributed ledger that keeps records of digital transactions in such a way that makes them accessible and visible to multiple participants in a network while keeping them secure without the need of a centralized certification organism. This work implements a blockchain architecture within the wood chain electronic traceability. The infotracing system is based on RFID sensors and open source technology. The entire forest wood supply chain was simulated from standing trees to the final product passing through tree cutting and sawmill process. Different kinds of Internet of Things (IoT) open source devices and tags were used, and a specific app aiming the forest operations was engineered to collect and store in a centralized database information (e.g., species, date, position, dendrometric and commercial information).

Radiofrequency-assisted liposuction is a relatively new concept in energy-assisted body contouring techniques and has received instrument approval. This supplemental article reviews the clinical application of electromagnetic energy via the BodyTite (InMode Corporation, Toronto, Canada) device on soft tissues during suction lipectomy, its effect on soft tissue contraction, and its use in aesthetic body contouring in various clinical scenarios.

Background: Despite rapid growth, gluteal fat transplantation is an operation in search of science and a teachable technique. Long operating times, tedious syringe transfers, inability to shape the recipient site, and the risk of fat embolism all headline as impediments to clinical adoption of the procedure. Expansion vibration lipofilling is a syringe-free surgical strategy that is a logical extension of Separation, Aspiration, and Fat Equalization (SAFELipo). In expansion vibration lipofilling, there is simultaneous disruption of recipient-site connective tissue, internal expansion using exploded-tip cannulas, and backfilling of these spaces with roller pump-propelled fat., Methods: Two thousand four hundred nineteen consecutive cases of expansion vibration lipofilling fat transplantation to the buttocks were reviewed. Average follow-up was 12 months. The technique of expansion vibration lipofilling is dependent on the use of larger caliber cannulas attached to a roller pump and to an oscillatory power-assisted liposuction device, which is less labor-intensive, potentially allowing for better knowledge of cannula-tip location at all times during the procedure., Results: Operating times averaged 1 hour 40 minutes. The average volume of fat inserted was 1003 cc. Complications included donor-site seroma, infection, and one pulmonary embolism treated with anticoagulation. There were no cases of fat embolism or death., Conclusions: Expansion vibration lipofilling is a new method for large-volume fat transplantation. Avoidance of fatal fat emboli demands a surgeon's complete knowledge of cannula tip location at all times during the procedure. Syringe-free, larger caliber, and less flexible cannulas, combined with techniques requiring less operator upper extremity effort resulting in less fatigue, may contribute to avoidance of this dreadful complication., Clinical Question/level of Evidence: Therapeutic, IV.

A major problem in the design of synthetic genetic circuits is robustness to perturbations and uncertainty. Because of this, there have been significant efforts in recent years in finding approaches to implement integral control in genetic circuits. Integral controllers have the unique ability to make the output of a process adapt perfectly to disturbances. However, implementing an integral controller is challenging in living cells. This is because a key aspect of any integral controller is a 'memory' element that stores the accumulation (integral) of the error between the output and its desired set-point. The ability to realize such a memory element in living cells is fundamentally challenged by the fact that all biomolecules dilute as cells grow, resulting in a 'leaky' memory that gradually fades away. As a consequence, the adaptation property is lost. Here, we propose a general principle for designing integral controllers such that the performance is practically unaffected by dilution. In particular, we mathematically prove that if the reactions implementing the integral controller are all much faster than dilution, then the adaptation error due to integration leakiness becomes negligible. We exemplify this design principle with two synthetic genetic circuits aimed at reaching adaptation of gene expression to fluctuations in cellular resources. Our results provide concrete guidance on the biomolecular processes that are most appropriate for implementing integral controllers in living cells., (© 2018 The Author(s).)

A signaling pathway transmits information from an upstream system to downstream systems, ideally in a unidirectional fashion. A key obstacle to unidirectional transmission is retroactivity, the additional reaction flux that affects a system once its species interact with those of downstream systems. This raises the fundamental question of whether signaling pathways have developed specialized architectures that overcome retroactivity and transmit unidirectional signals. Here, we propose a general procedure based on mathematical analysis that provides an answer to this question. Using this procedure, we analyze the ability of a variety of signaling architectures to transmit one-way (from upstream to downstream) signals, as key biological parameters are tuned. We find that single stage phosphorylation and phosphotransfer systems that transmit signals from a kinase show a stringent design tradeoff that hampers their ability to overcome retroactivity. Interestingly, cascades of these architectures, which are highly represented in nature, can overcome this tradeoff and thus enable unidirectional transmission. By contrast, phosphotransfer systems, and single and double phosphorylation cycles that transmit signals from a substrate, are unable to mitigate retroactivity effects, even when cascaded, and hence are not well suited for unidirectional information transmission. These results are largely independent of the specific reaction-rate constant values, and depend on the topology of the architectures. Our results therefore identify signaling architectures that, allowing unidirectional transmission of signals, embody modular processes that conserve their input/output behavior across multiple contexts. These findings can be used to decompose natural signal transduction networks into modules, and at the same time, they establish a library of devices that can be used in synthetic biology to facilitate modular circuit design., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Synthetic gene expression is highly sensitive to intragenic compositional context (promoter structure, spacing regions between promoter and coding sequences, and ribosome binding sites). However, much less is known about the effects of intergenic compositional context (spatial arrangement and orientation of entire genes on DNA) on expression levels in synthetic gene networks. We compare expression of induced genes arranged in convergent, divergent, or tandem orientations. Induction of convergent genes yielded up to 400% higher expression, greater ultrasensitivity, and dynamic range than divergent- or tandem-oriented genes. Orientation affects gene expression whether one or both genes are induced. We postulate that transcriptional interference in divergent and tandem genes, mediated by supercoiling, can explain differences in expression and validate this hypothesis through modeling and in vitro supercoiling relaxation experiments. Treatment with gyrase abrogated intergenic context effects, bringing expression levels within 30% of each other. We rebuilt the toggle switch with convergent genes, taking advantage of supercoiling effects to improve threshold detection and switch stability., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A common approach to design genetic circuits is to compose gene expression cassettes together. While appealing, this modular approach is challenged by the fact that expression of each gene depends on the availability of transcriptional/translational resources, which is in turn determined by the presence of other genes in the circuit. This raises the question of how competition for resources by different genes affects a circuit's behavior. Here, we create a library of genetic activation cascades in E. coli bacteria, where we explicitly tune the resource demand by each gene. We develop a general Hill-function-based model that incorporates resource competition effects through resource demand coefficients. These coefficients lead to nonregulatory interactions among genes that reshape the circuit's behavior. For the activation cascade, such interactions result in surprising biphasic or monotonically decreasing responses. Finally, we use resource demand coefficients to guide the choice of ribosome binding site and DNA copy number to restore the cascade's intended monotonically increasing response. Our results demonstrate how unintended circuit's behavior arises from resource competition and provide a model-guided methodology to minimize the resulting effects.

To artificially reprogram cell fate, experimentalists manipulate the gene regulatory networks (GRNs) that maintain a cell's phenotype. In practice, reprogramming is often performed by constant overexpression of specific transcription factors (TFs). This process can be unreliable and inefficient. Here, we address this problem by introducing a new approach to reprogramming based on mathematical analysis. We demonstrate that reprogramming GRNs using constant overexpression may not succeed in general. Instead, we propose an alternative reprogramming strategy: a synthetic genetic feedback controller that dynamically steers the concentration of a GRN's key TFs to any desired value. The controller works by adjusting TF expression based on the discrepancy between desired and actual TF concentrations. Theory predicts that this reprogramming strategy is guaranteed to succeed, and its performance is independent of the GRN's structure and parameters, provided that feedback gain is sufficiently high. As a case study, we apply the controller to a model of induced pluripotency in stem cells., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Synthetic biology is increasingly used to develop sophisticated living devices for basic and applied research. Many of these genetic devices are engineered using multi-copy plasmids, but as the field progresses from proof-of-principle demonstrations to practical applications, it is important to develop single-copy synthetic modules that minimize consumption of cellular resources and can be stably maintained as genomic integrants. Here we use empirical design, mathematical modeling, and iterative construction and testing to build single-copy, bistable toggle switches with improved performance and reduced metabolic load that can be stably integrated into the host genome. Deterministic and stochastic models led us to focus on basal transcription to optimize circuit performance and helped to explain the resulting circuit robustness across a large range of component expression levels. The design parameters developed here provide important guidance for future efforts to convert functional multi-copy gene circuits into optimized single-copy circuits for practical, real-world use., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The past several years have witnessed an increased presence of control theoretic concepts in synthetic biology. This review presents an organized summary of how these control design concepts have been applied to tackle a variety of problems faced when building synthetic biomolecular circuits in living cells. In particular, we describe success stories that demonstrate how simple or more elaborate control design methods can be used to make the behaviour of synthetic genetic circuits within a single cell or across a cell population more reliable, predictable and robust to perturbations. The description especially highlights technical challenges that uniquely arise from the need to implement control designs within a new hardware setting, along with implemented or proposed solutions. Some engineering solutions employing complex feedback control schemes are also described, which, however, still require a deeper theoretical analysis of stability, performance and robustness properties. Overall, this paper should help synthetic biologists become familiar with feedback control concepts as they can be used in their application area. At the same time, it should provide some domain knowledge to control theorists who wish to enter the rising and exciting field of synthetic biology., (© 2016 The Author(s).)

Genetic circuits in living cells share transcriptional and translational resources that are available in limited amounts. This leads to unexpected couplings among seemingly unconnected modules, which result in poorly predictable circuit behavior. In this study, we determine these interdependencies between products of different genes by characterizing the economy of how transcriptional and translational resources are allocated to the production of proteins in genetic circuits. We discover that, when expressed from the same plasmid, the combinations of attainable protein concentrations are constrained by a linear relationship, which can be interpreted as an isocost line, a concept used in microeconomics. We created a library of circuits with two reporter genes, one constitutive and the other inducible in the same plasmid, without a regulatory path between them. In agreement with the model predictions, experiments reveal that the isocost line rotates when changing the ribosome binding site strength of the inducible gene and shifts when modifying the plasmid copy number. These results demonstrate that isocost lines can be employed to predict how genetic circuits become coupled when sharing resources and provide design guidelines for minimizing the effects of such couplings., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)