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Dulac Lab.
PyMol model showing how dual-fluorescent nanobodies allow for molecularly defined exploration of electron microscopy based connectomics.
Brandon Logeman, Jeff Lichtman, Catherine Dulac
The relationship between adults and infants, although displayed in various ways, is apparent across many species. Two of the many behaviors include parenting and infanticide. Past studies have demonstrated that parental behavior is localized to the medial preoptic area (MPOA) whereas infanticidal behavior is localized to the perifornical area (PeFA). Circuit mapping techniques such as viral tracing have revealed sexual and state dependent connectivity alterations in both parental and infanticidal behaviors. Moreover, the MPOA and PeFA show rich local connectivity, which suggests that local circuit dynamics are important in the changes of parental and infanticidal behavior. The importance of dense local connectivity points toward a need for understanding the microarchitectures of these circuits.
A newly developed variant of correlative light-electron microscopy (CLEM), nanobody-assisted tissue immunostaining for volumetric EM (NATIVE), provides a potential method to understand microarchitecture through the use of fluorescently labeled, immunoreactive nanobodies. Nanobodies, derived from the antibodies of camelids (camels, llamas, alpacas, etc.), are unique in that the antigen binding region is composed of a single domain making them considerably smaller than traditional antibodies. By creating nanobodies against specific neuropeptides or receptors in neuronal circuits implicated in parenting and infanticide, we will be able to determine if local structural differences exist between male and female mice and are responsible for observed behavioral differences.
Alongside this biochemical approach, we are also utilizing advanced machine learning tools including DeepLabCut, SimBA, and Mask-RCNN to better classify and quantify sex-specific behavioral paradigms in the context of parenting and infanticide.
DeepLabCut allows for both single and multi-animal dynamic tracking.
Mask-RCNN can resolve changing nest shapes, a useful classifier for parental behavior.
Ion-selective electrodes provide a more efficient and versatile method to study the catalytic activity of butyrylcholinesterase.
Khaled Abdelazim, George Whitesides
Because of butyrylcholinesterase’s (BChE) function as a bioscavenger, namely, protecting the nervous system against a wide variety of potentially harmful chemicals, the accurate and affordable detection of BChE activity in human serum has large implications for potential therapeutic use.
The current method for determining BChE activity is via Ellman’s reagent assay, but this colorimetric method is both inefficient and ineffective as the reagent can only successfully detect two of BChE’s plethora of substrates. We investigate the development of a new tool, ion-selective electrodes (ISEs), which are better suited for determining the catalytic activity of BChE in response to a wide variety of potential targets.
Since the substrates of BChE all contain a positive charge, we can use ISEs to continuously record the changes in electric potential as BChE hydrolyzes its substrates over time. We report the ability of the ISE to measure the hydrolysis of six distinct substrates, acetylcholine, butyrylthiocholine, dimethocaine, and benzoylcholine, succinylcholine, and acetylthiocholine. Our results not only demonstrate the efficacy of the ISE as compared with the current standard, Ellman’s reagent assay, but also inform potential therapeutic use of BChE against neurotoxins.
Life cycle of T. cruzi, the parasite responsible for leishmaniasis.
Alex Carvalho, Santuza Teixeira
Thousands of cases of leishmaniasis are reported in Brazil each year, and although the molecular basis of the parasite is becoming well-understood, affordable test and treatment options are almost nonexistent. Rapid tests, which make use of specific antigens and dyes, are commercially available at a minimum of $1, or 4 Brazilian reais, but we hoped to create a product that would cost one-tenth of the price.
We relied on recombinant protein techniques and ELISA tests to identify candidate proteins that induce a strong immunologic response. The proteins that elicited a strong response were then purified to be incorporated into rapid test production. Moreover, because dogs are the primary reservoir of the parasite, we also optimized a lysis buffer canine DNA extraction protocol in conjunction with real-time PCR analysis to quickly and effectively test saliva samples from potentially afflicted domestic dogs.
Moreover, the tools we established can be applied to a variety of systems. Most recently, this real-time PCR protocol was employed to help diagnose COVID-19 cases.