Anand K. Muthusamy

A general method to fine-tune fluorophores for live-cell and in vivo imaging

Pushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. We refined and extended this strategy, finding that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties of rhodamine dyes with unprecedented precision. This strategy allowed us to establish principles for fine-tuning the properties of fluorophores and to develop a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red. Our results demonstrate the versatility of these new dyes in cells, tissues and animals.

Nuclear microenvironments modulate transcription from low-affinity enhancers

Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. Here, we report that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogaster shavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation.

Shared Language and Moral Sensibility in Resolving Clinical Ethics Conflicts

Autumn Fiester’s “Neglected Ends: Clinical Ethics Consultation and the Prospects for Closure” (2015) demonstrates how a focus on recommendations in clinical ethics consultations (CECs) can fail to provide closure and affect moral residues for different stakeholders. Fiester convincingly argues for a shift to a dialogue-based process with an eye toward finding closure for all parties. Given that the American Medical Association (AMA) Code of Medical Ethics does not comment on the desired outcome of CECs and that empirical research on closure is somewhat limited, Fiester’s piece opens a needed discussion on the parameters and processes of CECs (AMA 1996; Epstein and Hamric 2009). As she anticipates, the main criticism of bioethics mediation is not its purpose or its intended outcome but rather the efficacy of the method. Revisiting the source of moral distress and moral emotions and how they shape interpersonal interactions can highlight the pressing areas for further investigation to improve the mediation process. Specifically, the mechanisms by which dialogue resolves moral residue should be defined rigorously. I argue that “shared language” and “moral sensibility” are at work in interpersonal ethical conflicts. Moreover, the addition of these two concepts to the CEC framework exposes how experience prior to the CEC shapes outcomes in the consultation. The discussion herein is focused on cases where the moral residue is a result of interpersonal ethical conflicts. To attain closure in a clinical encounter, all stakeholders must work together to address each other’s concerns, which can often go beyond satisfying rights and obligations. The mechanism by which closure is attained however deserves more investigation. As Fiester notes, it is not sufficient that the best outcome in the clinic was achieved. Rather, all stakeholders must feel as though they realized their justifiable interests and fulfilled their obligations without setbacks imposed by their own doubts or another’s competing choices. In this case, two prerequisites appear to be a shared language used to communicate personal experiences and moral thoughts, and a moral sensibility to reason and negotiate toward closure. The interplay of linguistics and ethics offers insights into the function of dialogue in consultations. This inquiry also demonstrates the dissolution of the boundary between the initial clinical encounter and the clinical consultation, opening space to think of new frameworks for dialogue. “Shared language” refers to the set of expressions used to make moral statements and share information about personal experiences and values. The CEC requires that physicians provide biomedical information about the patient’s status, their interpretation of their obligations to their practice and patient, and their suggested course of action, while the patients (or their surrogates) provide their subjective experience of their ailment, their overall health interests, and their own desired course of action. It is possible that other individuals are involved in the clinical encounter, but the kinds of information they present are already categorized in the preceding. Biomedical facts and the desired outcomes of all stakeholders can be more easily conveyed than the subjective experience of the patient. A major and long-standing obstacle in establishing a shared language in clinical consultations has been conveying the nature and degree of an individual’s suffering (Heath 1989). An especially challenging example is the experience of pain, which can often occur without externally verifiable indicators. If a patient and a physician have different conceptions of the status quo suffering and the impact of different medical options, not only is there a chance of picking a nonoptimal course of action but also this chasm lends itself to creating a moral residue. In this case, developing new and diverse means to express subjective states can add “vocabulary” to the patient–physician shared language, which in turn can increase the possibility of accessing closure. Recognizing that the clinic presents unique challenges to establishing a shared language is a first step toward improving the efficacy of dialogue in consultations. The complexities of the expression of suffering have been an ongoing study for bioethicists and medical anthropologists (Goubert, Craig, and Buysse 2007). A heavy focus on establishing shared language in the clinic may support Bergman’s (2013) claims that professionals assisting CECs need not be primarily bioethicists by training. “Moral sensibility” refers to one’s ethical framework in guiding one’s actions and negotiating conflicts with others. This concept is useful for thinking about what allows for dialogue to access closure. Given that ethical conflicts can give rise to moral distress and negative moral emotions for different classes of agents with different backgrounds, interests, and so on, it is a peculiar idea that closure can be

Live widefield JF635-HaloTag-Ubx(R3A N51A) stage 5 embryo

Wide field imaging in the anterior region of a live stage 5 Drosophila melanogaster embryo expressing HaloTag-Ubx(R3A N51A). This Ubx is DNA-binding deficient. The embryo is injected with JF635 linked to a HaloTag ligand 2 hours after egg-laying. The embryo is epi-illuminated with a 633 nm laser and videos are captured with an exposure time of 50 ms. Video is played at 10 frames per second.

Live widefield JF635-HaloTag-Ubx late stage 6 embryo

Wide field imaging in the anterior region of a live late stage 6 Drosophila melanogaster embryo expressing HaloTag-Ubx. The embryo is injected with JF635 linked to a HaloTag ligand 2 hours after egg-laying. The embryo is epi-illuminated with a 633 nm laser and videos are captured with an exposure time of 50 ms. Video is played at 10 frames per second.

Airyscan Alexa568-Ubx Alexa488-svb DAPI Embryo 01_01

Embryo 01 imaging area 01. Airyscan imaging in the a1 segment of a fixed stage 15 Drosophila melanogaster embryo. Channel 1: Ubx labeled with Alexa 568 using IF. Channel 2: Transcription sites of svb labeled with Alexa 488 using IF against DIG-RNA probes targeting the first intronic region of svb. Channel 3: DAPI.