Exploring GPCR conformational dynamics using single-molecule fluorescence.

G protein-coupled receptors (GPCRs) are membrane proteins that transmit specific external stimuli into cells by changing their conformation. This conformational change allows them to couple and activate G-proteins to initiate signal transduction. A critical challenge in studying and inferring these structural dynamics arises from the complexity of the cellular environment, including the presence of various endogenous factors. Due to the recent advances in cell-expression systems, membrane-protein purification techniques, and labeling approaches, it is now possible to study the structural dynamics of GPCRs at a single-molecule level both in vitro and in live cells. In this review, we discuss state-of-the-art techniques and strategies for expressing, purifying, and labeling GPCRs in the context of single-molecule research. We also highlight four recent studies that demonstrate the applications of single-molecule microscopy in revealing the dynamics of GPCRs. These techniques are also useful as complementary methods to verify the results obtained from other structural biology tools like cryo-electron microscopy and x-ray crystallography.

Single-molecule visualization of human A2A adenosine receptor activation by a G protein and constitutively activating mutations.

Mutations that constitutively activate G protein-coupled receptors (GPCRs), known as constitutively activating mutations (CAMs), modify cell signaling and interfere with drugs, resulting in diseases with limited treatment options. We utilize fluorescence imaging at the single-molecule level to visualize the dynamic process of CAM-mediated activation of the human A2A adenosine receptor (A2AAR) in real time. We observe an active-state population for all CAMs without agonist stimulation. Importantly, activating mutations significantly increase the population of an intermediate state crucial for receptor activation, notably distinct from the addition of a partner G protein. Activation kinetics show that while CAMs increase the frequency of transitions to the intermediate state, mutations altering sodium sensitivity increase transitions away from it. These findings indicate changes in GPCR function caused by mutations may be predicted based on whether they favor or disfavor formation of an intermediate state, providing a framework for designing receptors with altered functions or therapies that target intermediate states.

Using conditional independence tests to elucidate causal links in cell cycle regulation in Escherichia coli.

How cells regulate their cell cycles is a central question for cell biology. Models of cell size homeostasis have been proposed for bacteria, archaea, yeast, plant, and mammalian cells. New experiments bring forth high volumes of data suitable for testing existing models of cell size regulation and proposing new mechanisms. In this paper, we use conditional independence tests in conjunction with data of cell size at key cell cycle events (birth, initiation of DNA replication, and constriction) in the model bacterium Escherichia coli to select between the competing cell cycle models. We find that in all growth conditions that we study, the division event is controlled by the onset of constriction at midcell. In slow growth, we corroborate a model where replication-related processes control the onset of constriction at midcell. In faster growth, we find that the onset of constriction is affected by additional cues beyond DNA replication. Finally, we also find evidence for the presence of additional cues triggering initiations of DNA replication apart from the conventional notion where the mother cells solely determine the initiation event in the daughter cells via an adder per origin model. The use of conditional independence tests is a different approach in the context of understanding cell cycle regulation and it can be used in future studies to further explore the causal links between cell events.

Are mRNA based transcriptomic signatures ready for diagnosing tuberculosis in the clinic? - A review of evidence and the technological landscape.

Advances in discovery and validation of diagnostic, prognostic and treatment-monitoring transcriptomic signatures of tuberculosis (TB) disease could accelerate the goal to end TB. We conducted a review to evaluate whether mRNA transcriptomics technologies are sufficiently mature to develop accurate next-generation TB diagnostic tests. Early studies tended to be limited in sample size, diversity of population groups, sample collection and processing methods, while recent prospective studies have addressed these limitations. Some of the existing signatures could be used for triage; however, high cost and complexity could limit their use. For a confirmatory test, setting an optimal cut-off to maintain specificity across populations and settings is a challenge. mRNA signatures have shown the potential to quantitatively monitor response to treatment. No prognostic signatures can accurately predict progression to active TB over 2 years while short term prediction is possible. The management strategy should be defined for individuals with positive prognostic tests. FUNDING: Development of this manuscript was supported by funding received from the Stop TB Partnership and USAID for the New Diagnostics Working Group. The funders had no role in paper design, article selection and review, interpretation, or writing of the paper.

Coupling between DNA replication, segregation, and the onset of constriction in Escherichia coli.

Escherichia coli cell cycle features two critical cell-cycle checkpoints: initiation of replication and the onset of constriction. While the initiation of DNA replication has been extensively studied, it is less clear what triggers the onset of constriction and when exactly it occurs during the cell cycle. Here, using high-throughput fluorescence microscopy in microfluidic devices, we determine the timing for the onset of constriction relative to the replication cycle in different growth rates. Our single-cell data and modeling indicate that the initiation of constriction is coupled to replication-related processes in slow growth conditions. Furthermore, our data suggest that this coupling involves the mid-cell chromosome blocking the onset of constriction via some form of nucleoid occlusion occurring independently of SlmA and the Ter linkage proteins. This work highlights the coupling between replication and division cycles and brings up a new nucleoid mediated control mechanism in E. coli.

Distinguishing different modes of growth using single-cell data.

Collection of high-throughput data has become prevalent in biology. Large datasets allow the use of statistical constructs such as binning and linear regression to quantify relationships between variables and hypothesize underlying biological mechanisms based on it. We discuss several such examples in relation to single-cell data and cellular growth. In particular, we show instances where what appears to be ordinary use of these statistical methods leads to incorrect conclusions such as growth being non-exponential as opposed to exponential and vice versa. We propose that the data analysis and its interpretation should be done in the context of a generative model, if possible. In this way, the statistical methods can be validated either analytically or against synthetic data generated via the use of the model, leading to a consistent method for inferring biological mechanisms from data. On applying the validated methods of data analysis to infer cellular growth on our experimental data, we find the growth of length in E. coli to be non-exponential. Our analysis shows that in the later stages of the cell cycle the growth rate is faster than exponential.

All cells ­ from bacteria to humans ­ tightly control their size as they grow and divide. Cells can also change the speed at which they grow, and the pattern of how fast a cell grows with time is called 'mode of growth'. Mode of growth can be 'linear', when cells increase their size at a constant rate, or 'exponential', when cells increase their size at a rate proportional to their current size. A cell's mode of growth influences its inner workings, so identifying how a cell grows can reveal information about how a cell will behave. Scientists can measure the size of cells as they age and identify their mode of growth using single cell imaging techniques. Unfortunately, the statistical methods available to analyze the large amounts of data generated in these experiments can lead to incorrect conclusions. Specifically, Kar et al. found that scientists had been using specific types of plots to analyze growth data that were prone to these errors, and may lead to misinterpreting exponential growth as linear and vice versa. This discrepancy can be resolved by ensuring that the plots used to determine the mode of growth are adequate for this analysis. But how can the adequacy of a plot be tested? One way to do this is to generate synthetic data from a known model, which can have a specific and known mode of growth, and using this data to test the different plots. Kar et al. developed such a 'generative model' to produce synthetic data similar to the experimental data, and used these data to determine which plots are best suited to determine growth mode. Once they had validated the best statistical methods for studying mode of growth, Kar et al. applied these methods to growth data from the bacterium Escherichia coli. This showed that these cells have a form of growth called 'super-exponential growth'. These findings identify a strategy to validate statistical methods used to analyze cell growth data. Furthermore, this strategy ­ the use of generative models to produce synthetic data to test the accuracy of statistical methods ­ could be used in other areas of biology to validate statistical approaches.

Occupational Inhalation Exposures to Nanoparticles at Six Singapore Printing Centers.

Laser printers emit high levels of nanoparticles (PM0.1) during operation. Although it is well established that toners contain multiple engineered nanomaterials (ENMs), little is known about inhalation exposures to these nanoparticles and work practices in printing centers. In this report, we present a comprehensive inhalation exposure assessment of indoor microenvironments at six commercial printing centers in Singapore, the first such assessment outside of the United States, using real-time personal and stationary monitors, time-integrated instrumentation, and multiple analytical methods. Extensive presence of ENMs, including titanium dioxide, iron oxide, and silica, was detected in toners and in airborne particles collected from all six centers studied. We document high transient exposures to emitted nanoparticles (peaks of ∼500 000 particles/cm3, lung-deposited surface area of up to 220 µm2/cm3, and PM0.1 up to 16 µg/m3) with complex PM0.1 chemistry that included 40-60 wt % organic carbon, 10-15 wt % elemental carbon, and 14 wt % trace elements. We also record 271.6-474.9 pmol/mg of Environmental Protection Agency-priority polycyclic aromatic hydrocarbons. These findings highlight the potentially high occupational inhalation exposures to nanoparticles with complex compositions resulting from widespread usage of nano-enabled toners in the printing industry, as well as inadequate ENM-specific exposure control measures in these settings.

Luminescence Properties of Self-Aggregating TbIII-DOTA-Functionalized Calix[4]arenes.

Self-aggregating calix[4]arenes carrying four DOTA ligands on the upper rim for stable complexation of paramagnetic GdIII-ions have already been proposed as MRI probes. In this work, we investigate the luminescence properties of TbIII-DOTA-calix[4]arene-4OPr containing four propyl-groups and compare them with those of the analog substituted with a phthalimide chromophore (TbIII-DOTA-calix[4]arene-3OPr-OPhth). We show that, given its four aromatic rings, the calix[4]arene core acts as an effective sensitizer of Tb-centered luminescence. Substituents on the lower rim can modulate the aggregation behavior, which in turn determines the luminescence properties of the compounds. In solid state, the quantum yield of the phthalimide derivative is almost three times as high as that of the propyl-functionalized analog demonstrating a beneficial role of the chromophore on Tb-luminescence. In solution, however, the effect of the phthalimide group vanishes, which we attribute to the large distance between the chromophore and the lanthanide, situated on the opposite rims of the calix[4]arene. Both quantum yields and luminescence lifetimes show clear concentration dependence in solution, related to the strong impact of aggregation on the luminescence behavior. We also evidence the variability in the values of the critical micelle concentration depending on the experimental technique. Such luminescent calix[4]arene platforms accommodating stable lanthanide complexes can be considered valuable building blocks for the design of dual MR/optical imaging probes.

The progression of replication forks at natural replication barriers in live bacteria.

Protein-DNA complexes are one of the principal barriers the replisome encounters during replication. One such barrier is the Tus-ter complex, which is a direction dependent barrier for replication fork progression. The details concerning the dynamics of the replisome when encountering these Tus-ter barriers in the cell are poorly understood. By performing quantitative fluorescence microscopy with microfuidics, we investigate the effect on the replisome when encountering these barriers in live Escherichia coli cells. We make use of an E. coli variant that includes only an ectopic origin of replication that is positioned such that one of the two replisomes encounters a Tus-ter barrier before the other replisome. This enables us to single out the effect of encountering a Tus-ter roadblock on an individual replisome. We demonstrate that the replisome remains stably bound after encountering a Tus-ter complex from the non-permissive direction. Furthermore, the replisome is only transiently blocked, and continues replication beyond the barrier. Additionally, we demonstrate that these barriers affect sister chromosome segregation by visualizing specific chromosomal loci in the presence and absence of the Tus protein. These observations demonstrate the resilience of the replication fork to natural barriers and the sensitivity of chromosome alignment to fork progression.

Correction of microstomia in an edentulous patient.

The problem of small oral aperture is big. Irrespective of the etiology, this problem may be overcome by adjunctive therapies in the form of prosthesis, surgery, or exercise. A patient is described with this problem, which was overcome by revisiting the 3 adjunctive therapies including a commissural stent designed with the patient's edentulous state in mind.

Fracture management of an edentulous mandible in a geriatric osteoporotic patient.

Fracture of an edentulous mandible is a difficult task primarily due to the absence of teeth. The management becomes even more difficult if the patient is geriatric and osteoporotic. A simple technique of using bite block splint, maxillomandibular fixation screws, and intermaxillary fixation has been presented to enable healing of fracture of mandible in such cases. The technique, which crosses the boundaries of conventional fracture management provides, promising results with minimum morbidity thus imparting optimum quality of life ahead for the patient.

Essential validation methods for E. coli strains created by chromosome engineering.

BACKGROUND: Chromosome engineering encompasses a collection of homologous recombination-based techniques that are employed to modify the genome of a model organism in a controlled fashion. Such techniques are widely used in both fundamental and industrial research to introduce multiple insertions in the same Escherichia coli strain. To date, λ-Red recombination (also known as recombineering) and P1 phage transduction are the most successfully implemented chromosome engineering techniques in E. coli. However, due to errors that can occur during the strain creation process, reliable validation methods are essential upon alteration of a strain's chromosome. RESULTS AND DISCUSSION: Polymerase chain reaction (PCR)-based methods and DNA sequence analysis are rapid and powerful methods to verify successful integration of DNA sequences into a chromosome. Even though these verification methods are necessary, they may not be sufficient in detecting all errors, imposing the requirement of additional validation methods. For example, as extraneous insertions may occur during recombineering, we highlight the use of Southern blotting to detect their presence. These unwanted mutations can be removed via transducing the region of interest into the wild type chromosome using P1 phages. However, in doing so one must verify that both the P1 lysate and the strains utilized are free from contamination with temperate phages, as these can lysogenize inside a cell as a large plasmid. Thus, we illustrate various methods to probe for temperate phage contamination, including cross-streak agar and Evans Blue-Uranine (EBU) plate assays, whereby the latter is a newly reported technique for this purpose in E. coli. Lastly, we discuss methodologies for detecting defects in cell growth and shape characteristics, which should be employed as an additional check. CONCLUSION: The simple, yet crucial validation techniques discussed here can be used to reliably verify any chromosomally engineered E. coli strains for errors such as non-specific insertions in the chromosome, temperate phage contamination, and defects in growth and cell shape. While techniques such as PCR and DNA sequence verification should standardly be performed, we illustrate the necessity of performing these additional assays. The discussed techniques are highly generic and can be easily applied to any type of chromosome engineering.

Transdermal diclofenac patches for control of post-extraction pain. Pilot randomized controlled double-blind study.

PURPOSE: The scope of transdermal analgesics have been limited to pain control following local trauma, muscle spasm, minor esthetic surgeries, or sports-related injuries. Its scope of application can be extended in the field of dentistry and minor oral surgical procedures. METHODS: A total of 40 healthy patients between the age of 18 and 50 years with deeply carious, nontender mandibular molar tooth deemed unsalvageable which was indicated for extraction were taken into study of which subjects were divided into cases and controls. RESULTS: The data were analyzed with chi-square test and Mann-Whitney U test; the chi-square test showed significant association with expected outcome in visual analog scale pain scores, the two groups at 6th and 12th hour. A Mann-Whitney U test was performed to identify strength of association between two groups where there were significant association between all parameters except for pain interference with routine activities and dental health status(0.022, 0.001); the differences between two groups in these parameters were significant (<0.05). CONCLUSION: Transdermal route seems to be comparable to oral diclofenac in post-extraction pain relief; further studies are required to confirm the results and safety for its use in various other dental situations.

Slow unloading leads to DNA-bound ß2-sliding clamp accumulation in live Escherichia coli cells.

The ubiquitous sliding clamp facilitates processivity of the replicative polymerase and acts as a platform to recruit proteins involved in replication, recombination and repair. While the dynamics of the E. coli ß2-sliding clamp have been characterized in vitro, its in vivo stoichiometry and dynamics remain unclear. To probe both ß2-clamp dynamics and stoichiometry in live E. coli cells, we use custom-built microfluidics in combination with single-molecule fluorescence microscopy and photoactivated fluorescence microscopy. We quantify the recruitment, binding and turnover of ß2-sliding clamps on DNA during replication. These quantitative in vivo results demonstrate that numerous ß2-clamps in E. coli remain on the DNA behind the replication fork for a protracted period of time, allowing them to form a docking platform for other enzymes involved in DNA metabolism.

Plasma cell mucositis with gingival enlargement and severe periodontitis.

Plasma cell mucositis (PCM) is a very rare, chronic, multifocal, idiopathic, non-neoplastic plasma cell proliferative disorder of the upper aerodigestive tract. The classic clinical presentation is an intensely erythematous mucosa with surface changes described variously as papillomatous, cobblestone, nodular or velvety. It is a very rare condition <50 cases reported in literature. A 72-year-old male patient complained of sore throat, stomatodynia, dysphagia, multiple oral ulcers, enlarged swollen bleeding gums and mobile teeth. There was chronic inflammatory enlargement of the gingiva and palate with severe periodontitis. Histopathological examination revealed a hyperplastic epithelium with a dense infiltrate of mature polyclonal plasma cells in the superficial layer of the lamina propria. PCM is a diagnosis of exclusion, to be differentiated from other infective, reactive, autoimmune, allergic and neoplastic disorders with plasma cell infiltrates. Management with surgical and immunosuppressive therapy is mostly ineffective with short remissions and frequent relapses.

Maxillo Mandibular Fixation in Edentulous Scenarios: Combined MMF Screws and Gunning Splints.

A fracture of the maxillary or mandibular bone requires the afflicted to undergo a maxillo mandibular fixation for the establishment of pre traumatic occlusion. This process is quiet tedious and consumes a considerable period of time before any surgical procedure can commence. Such a situation can be complicated in case the individual with maxillomandibular fracture has sparse or absent dentition; for such cases a splint is fabricated or an erstwhile existing denture is used for maintaining a vertical jaw proportion. Stabilizing such splints to the jaw requires various invasive approaches that can bring into harm's way, adjacent soft tissue vital structures. We describe here an innovative technique combining the time tested method of the "gunning splint" and the advanced minimally invasive MMF screws for obtaining closed reduction in edentulous jaw fractures.

Median facial dysplasia: A rare craniofacial syndrome and the surgical management of associated cleft lip.

Median facial dysplasia (MFD) is a distinct and unique disorder of the craniofacial region that is characteristic of deficient mid facial structures with the addition of a unilateral or bilateral cleft lip with or without a cleft palate. A cleft lip which is associated with MFD whether it is unilateral or bilateral does not represent a typical cleft lip and poses some challenges in reconstruction. The management of such cleft lip has been rarely discussed in previous literatures; the main obstacle comes in identifying the anatomical landmarks to establish an esthetic reconstruction of the lip. However, reasonably good result can be achieved when the adjacent and distant anatomical structures are correlated to aid the reconstruction.

Iatrogenically induced foreign body of the maxillary sinus and its surgical management: a unique situation.

The maxillary sinus is in close proximity of the maxillary dentition; due to the close interaction between these two, there is a possibility of any disease process affecting one that can spread or concomitantly affect the other. This proximity may lead to foreign bodies to displace into the maxillary sinus and lead to development of infection. The signs and symptoms of affecting one structure can superimpose on the other; this can lead to diagnostic dilemmas.