Perfect adaptation achieved by transport limitations governs the inorganic phosphate response in S. cerevisiae.

Cells cope with and adapt to ever-changing environmental conditions. Sophisticated regulatory networks allow cells to adjust to these fluctuating environments. One such archetypal system is the Saccharomyces cerevisiae Pho regulon. When external inorganic phosphate (Pi) concentration is low, the Pho regulon activates, expressing genes that scavenge external and internal Pi. However, the precise mechanism controlling this regulon remains elusive. We conducted a systems analysis of the Pho regulon on the single-cell level under well-controlled environmental conditions. This analysis identified a robust, perfectly adapted Pho regulon state in intermediate Pi conditions, and we identified an intermediate nuclear localization state of the transcriptional master regulator Pho4p. The existence of an intermediate nuclear Pho4p state unifies and resolves outstanding incongruities associated with the Pho regulon, explains the observed programmatic states of the Pho regulon, and improves our general understanding of how nature evolves and controls sophisticated gene regulatory networks. We further propose that robustness and perfect adaptation are not achieved through complex network-centric control but by simple transport biophysics. The ubiquity of multitransporter systems suggests that similar mechanisms could govern the function of other regulatory networks as well.

Systematic analysis of low-affinity transcription factor binding site clusters in vitro and in vivo establishes their functional relevance.

Binding to binding site clusters has yet to be characterized in depth, and the functional relevance of low-affinity clusters remains uncertain. We characterized transcription factor binding to low-affinity clusters in vitro and found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Furthermore, small clusters with binding sites an order of magnitude lower in affinity give rise to high mean occupancies at physiologically-relevant transcription factor concentrations. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters in a synthetic and native gene regulatory network in S. cerevisiae. In both systems, clusters of low-affinity binding sites generated transcriptional output comparable to single or even multiple consensus sites. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters.

Effects of Regular/Dilute Proparacaine Anesthetic Eye Drops in Combination with Ophthalmic Antibiotics on Corneal Wound Healing.

Purpose: Topical, local anesthetic eye drops in conjunction with antibiotics are commonly used to reduce ocular pain and treat patients in emergency clinics; however, their effects on corneal healing are poorly understood. This study examined whether regular or diluted proparacaine eye drops given in combination with common ophthalmic antibiotics affect corneal wound healing parameters using in vitro and in vivo models. Methods: Primary human corneal fibroblasts generated from donor corneas and New Zealand white rabbits were used. Regular (0.5%) and diluted (0.05%) proparacaine eye drops, twice daily for 3 days, were applied to cultures and rabbit eyes, with or without ophthalmic antibiotics (polymyxin B sulfate and trimethoprim). Trypan blue, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and scratch wound assays measured cellular viability, proliferation, and migration, respectively, in vitro. Slit lamp biomicroscopy, tonometry, fluorescein eye test, hematoxylin and eosin (H&E) staining, and 4',6-diamidino-2-phenylindole (DAPI) immunofluorescence were used for in vivo studies. Results: Both regular and diluted proparacaine affected wound healing response in the cornea in vitro and in vivo in a time-dependent manner. Adjunct antibiotic treatments had additive effects characterized by reduced corneal fibroblast viability, proliferation, and migration in vitro and corneal epithelial recovery in vivo. Regular proparacaine with antibiotics showed most pronounced effects on corneal wound healing parameters, and diluted proparacaine without antibiotics had minimal negative effects in vitro and in vivo. Conclusion: Both methods of regular (0.5%) and diluted (0.05%) proparacaine topical application to the cornea are safe, but impede corneal wound healing in vitro and in vivo. Adjunct antibiotic treatments had additive negative effects on corneal wound repair.

Independent control of mean and noise by convolution of gene expression distributions.

Gene expression noise can reduce cellular fitness or facilitate processes such as alternative metabolism, antibiotic resistance, and differentiation. Unfortunately, efforts to study the impacts of noise have been hampered by a scaling relationship between noise and expression level from individual promoters. Here, we use theory to demonstrate that mean and noise can be controlled independently by expressing two copies of a gene from separate inducible promoters in the same cell. We engineer low and high noise inducible promoters to validate this result in Escherichia coli, and develop a model that predicts the experimental distributions. Finally, we use our method to reveal that the response of a promoter to a repressor is less sensitive with higher repressor noise and explain this result using a law from probability theory. Our approach can be applied to investigate the effects of noise on diverse biological pathways or program cellular heterogeneity for synthetic biology applications.

Epidemiology of Thyroid Cancer: A Review of the National Cancer Database, 2000-2013.

Objective  To show the recent trends in thyroid cancer in the United States, elucidate the characteristics of stage IV thyroid cancer, and consider the effects of diagnostic testing on the rising incidence of thyroid cancer. Design  A retrospective population-based study conducted using the National Cancer Database from 2000-2013 (NCDB). Demographics of patients presenting with stage IV thyroid cancer were compared to patients presenting with all other stages using the chi-square testing. The incidence rates were examined with the trend graphs. Results  When compared to stages I-III, there was an increased incidence of stage IV thyroid cancer in: Medicare, lower level of education, lower income, advanced age, male sex, increased number of comorbidities, further distance from a treatment facility, and medullary/anaplastic histology. The incidence of thyroid cancer increased from 7.1 per 100,000 in 2000 to 17.6 per 100,000 in 2013. During this same time period, stage IV disease increased 1 per 100,000. The increase in the incidence of thyroid cancer was almost entirely due to an increase in papillary cancer. Conclusions  The United States has continued to see a rise in the incidence of thyroid cancer over the last decade, largely due to the detection of papillary cancers. During this same time, the incidence of stage IV thyroid cancer increased as well. Because early diagnosis and treatment of an increasing number of potentially lethal cancers should lead to a decrease in metastatic disease, we suggest that the increasing incidence of thyroid cancer in the United States is due to overdiagnosis and that more aggressive disease is not being removed by early detection.

Postpartum Invasive Group A Streptococcus Infection: Case Report and Mini-review.

The overall incidence of postpartum invasive group A streptococcal (GAS) disease is low in the United States. However, postpartum women are much more likely to develop GAS disease than nonpregnant women. Additionally, postpartum GAS has the potential to develop into a severe disease and a delay in diagnosis can have deadly consequences. This case describes a patient with invasive postpartum endometritis in the setting of diastases of the pubic symphysis. Sepsis secondary to the endometritis develops along with bilateral pneumonia. This case characterizes some of the typical and atypical symptoms a patient with invasive postpartum GAS can present with. Further, it outlines the timely identification of the disease and its appropriate treatment to prevent a potentially disastrous outcome.

Engineering an E. coli Near-Infrared Light Sensor.

Optogenetics is a technology wherein researchers combine light and genetically engineered photoreceptors to control biological processes with unrivaled precision. Near-infrared (NIR) wavelengths (>700 nm) are desirable optogenetic inputs due to their low phototoxicity and spectral isolation from most photoproteins. The bacteriophytochrome photoreceptor 1 (BphP1), found in several purple photosynthetic bacteria, senses NIR light and activates transcription of photosystem promoters by binding to and inhibiting the transcriptional repressor PpsR2. Here, we examine the response of a library of output promoters to increasing levels of Rhodopseudomonas palustris PpsR2 expression, and we identify that of Bradyrhizobium sp. BTAi1 crtE as the most strongly repressed in Escherichia coli. Next, we optimize Rps. palustris bphP1 and ppsR2 expression in a strain engineered to produce the required chromophore biliverdin IXα in order to demonstrate NIR-activated transcription. Unlike a previously engineered bacterial NIR photoreceptor, our system does not require production of a second messenger, and it exhibits rapid response dynamics. It is also the most red-shifted bacterial optogenetic tool yet reported by approximately 50 nm. Accordingly, our BphP1-PpsR2 system has numerous applications in bacterial optogenetics.

A photoconversion model for full spectral programming and multiplexing of optogenetic systems.

Optogenetics combines externally applied light signals and genetically engineered photoreceptors to control cellular processes with unmatched precision. Here, we develop a mathematical model of wavelength- and intensity-dependent photoconversion, signaling, and output gene expression for our two previously engineered light-sensing Escherichia coli two-component systems. To parameterize the model, we develop a simple set of spectral and dynamical calibration experiments using our recent open-source "Light Plate Apparatus" device. In principle, the parameterized model should predict the gene expression response to any time-varying signal from any mixture of light sources with known spectra. We validate this capability experimentally using a suite of challenging light sources and signals very different from those used during the parameterization process. Furthermore, we use the model to compensate for significant spectral cross-reactivity inherent to the two sensors in order to develop a new method for programming two simultaneous and independent gene expression signals within the same cell. Our optogenetic multiplexing method will enable powerful new interrogations of how metabolic, signaling, and decision-making pathways integrate multiple input signals.

An open-hardware platform for optogenetics and photobiology.

In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments.

Central Retinal Artery Occlusion: A Literature Review and the Rationale for Hyperbaric Oxygen Therapy.

Central retinal artery occlusion is visually devastating and has no proven treatments. The therapeutic interval between symptom onset and potentially sight-saving intervention is narrow. Traditional conservative approaches include digital massage, administration of systemic vasodilators and diuretics, and lowering of intraocular pressure. Systemic and targeted fibrinolytic therapy is under investigation but is associated with significant adverse reactions. We report a case in which hyperbaric oxygen therapy restored retinal perfusion, and the patient's vision was improved.

FlowCal: A User-Friendly, Open Source Software Tool for Automatically Converting Flow Cytometry Data from Arbitrary to Calibrated Units.

Flow cytometry is widely used to measure gene expression and other molecular biological processes with single cell resolution via fluorescent probes. Flow cytometers output data in arbitrary units (a.u.) that vary with the probe, instrument, and settings. Arbitrary units can be converted to the calibrated unit molecules of equivalent fluorophore (MEF) using commercially available calibration particles. However, there is no convenient, nonproprietary tool available to perform this calibration. Consequently, most researchers report data in a.u., limiting interpretation. Here, we report a software tool named FlowCal to overcome current limitations. FlowCal can be run using an intuitive Microsoft Excel interface, or customizable Python scripts. The software accepts Flow Cytometry Standard (FCS) files as inputs and is compatible with different calibration particles, fluorescent probes, and cell types. Additionally, FlowCal automatically gates data, calculates common statistics, and produces publication quality plots. We validate FlowCal by calibrating a.u. measurements of E. coli expressing superfolder GFP (sfGFP) collected at 10 different detector sensitivity (gain) settings to a single MEF value. Additionally, we reduce day-to-day variability in replicate E. coli sfGFP expression measurements due to instrument drift by 33%, and calibrate S. cerevisiae Venus expression data to MEF units. Finally, we demonstrate a simple method for using FlowCal to calibrate fluorescence units across different cytometers. FlowCal should ease the quantitative analysis of flow cytometry data within and across laboratories and facilitate the adoption of standard fluorescence units in synthetic biology and beyond.

Optogenetic characterization methods overcome key challenges in synthetic and systems biology.

Systems biologists aim to understand how organism-level processes, such as differentiation and multicellular development, are encoded in DNA. Conversely, synthetic biologists aim to program systems-level biological processes, such as engineered tissue growth, by writing artificial DNA sequences. To achieve their goals, these groups have adapted a hierarchical electrical engineering framework that can be applied in the forward direction to design complex biological systems or in the reverse direction to analyze evolved networks. Despite much progress, this framework has been limited by an inability to directly and dynamically characterize biological components in the varied contexts of living cells. Recently, two optogenetic methods for programming custom gene expression and protein localization signals have been developed and used to reveal fundamentally new information about biological components that respond to those signals. This basic dynamic characterization approach will be a major enabling technology in synthetic and systems biology.

Characterizing bacterial gene circuit dynamics with optically programmed gene expression signals.

Gene circuits are dynamical systems that regulate cellular behaviors, often using protein signals as inputs and outputs. Here we have developed an optogenetic 'function generator' method for programming tailor-made gene expression signals in live bacterial cells. We designed precomputed light sequences based on experimentally calibrated mathematical models of light-switchable two-component systems and used them to drive intracellular protein levels to match user-defined reference time courses. We used this approach to generate accelerated and linearized dynamics, sinusoidal oscillations with desired amplitudes and periods, and a complex waveform, all with unprecedented accuracy and precision. We also combined the function generator with a dual fluorescent protein reporter system, analogous to a dual-channel oscilloscope, to reveal that a synthetic repressible promoter linearly transforms repressor signals with an approximate 7-min delay. Our approach will enable a new generation of dynamical analyses of synthetic and natural gene circuits, providing an essential step toward the predictive design and rigorous understanding of biological systems.

Eye on statins: A comprehensive review.

Over the last 25 years, statins have demonstrated their safety from an ophthalmologic standpoint. Studies relating statin to cataract formation are insufficient to alter the usual and customary prescription of statins. If there is an association between statins and cataracts, it is weak and clinically insignificant. Prospective studies have not demonstrated a benefit of adding statin therapy in patients with age-related macular degeneration (ARMD), but these studies have not been adequately powered to detect moderate differences. A subset of patients with persistently elevated lipids despite taking statins may be at higher risk of developing wet ARMD. The use of statins for the prevention and/ or treatment of glaucoma patients warrants further prospective study. There is a possibility that statins may unmask or exacerbate myasthenia gravis.

Post-translational tools expand the scope of synthetic biology.

Synthetic biology is improving our understanding of and ability to control living organisms. To date, most progress has been made by engineering gene expression. However, computational and genetically encoded tools that allow protein activity and protein-protein interactions to be controlled on their natural time and length scales are emerging. These technologies provide a basis for the construction of post-translational circuits, which are capable of fast, robust and highly spatially resolved signal processing. When combined with their transcriptional and translational counterparts, synthetic post-translational circuits will allow better analysis and control of otherwise intractable biological processes such as cellular differentiation and the growth of tissues.

Stromal rejection following deep anterior lamellar keratoplasty: implications for postoperative care.

PURPOSE: To better characterize stromal rejection in the context of deep anterior lamellar keratoplasty (DALK) to improve early diagnosis and proper management. METHODS: The clinical records of 22 patients undergoing DALK by 2 surgeons between October 2006 and January 2008 were reviewed to identify patients who experienced stromal rejection. The diagnosis was made after the demonstration of acute stromal edema and/or stromal neovascularization in the absence of confounding preoperative conditions, such as herpetic keratitis. The incidence and clinical features of stromal rejection were compared with other descriptions found in the literature. RESULTS: Five of 20 eligible patients experienced stromal rejection within 12 months. Two patients were on low-dose corticosteroids when diagnosed. Four of the 5 patients were treated aggressively with q1-3 hourly prednisolone acetate 1% eye drops. The fifth was treated less aggressively with a maximum dose of only q6 hourly prednisolone acetate 1% and subsequently experienced a second rejection episode less than 5 months later. All episodes resolved completely with treatment. CONCLUSIONS: The incidence of stromal rejection in DALK is clinically significant, suggesting that these patients may benefit from corticosteroid regimens similar to those used in penetrating keratoplasty and implies that stromal rejection may be more common in penetrating keratoplasty than previously reported. If misdiagnosed or left untreated, stromal rejection can compromise graft clarity but prompt recognition and aggressive treatment can result in good anatomic and visual outcomes.