Pharmacophore Oriented MP2 Characterization of Charge Distribution for Anti-SARS-CoV-2 Inhibitor Nirmatrelvir.

Quantum mechanical second order Møller-Plesset (MP2) perturbation theory and density functional theory (DFT) Becke, 3-parameter, Lee-Yang-Parr (B3LYP) and Minnesota 2006 local functional (M06L) calculations were performed to optimize structure of nirmatrelvir and compute the Merz-Kollman electrostatic potential (MK ESP), natural population analysis (NPA), Hirshfeld, charge model 5 (CM5), and mulliken partial charges. The mulliken partial charge distribution of nirmatrelvir exhibits a poor correlation with the MK ESP charges in MP2, B3LYP, and M06L calculations respectively. The NPA, Hirshfeld, and CM5 partial charge scheme of nirmatrelvir indicate a reasonable correlation with MK ESP charge assignments in B3LYP and M06L calculations. The above correlations were not improved by the inclusion of implicit solvation model. The MK ESP and CM5 partial charges show a strong correlation between the results of MP2 and two DFT methods. The three optimized structures present a certain degree of differences from the crystal bioactive conformation of nirmatrelvir, suggesting the nirmatrelvir-enzyme complex is formed in the induced-fit model. The Reactivity of warhead electrophilic nitrile is justified by the relatively weaker strength of π bonds in the MP2 calculations. The nirmatrelvir hydrogen bond acceptors consistently show strong delocalization of lone pair electrons in three calculations, whereas hydrogen bond donors are found to have high polarization on the heavy nitrogen atoms in MP2 computations. This work helps to parametrize the force field of nirmatrelvir and improve accuracy of molecular docking and rational inhibitor design.

Pandemic Teaching: Using the Allen Cell Types Database for Final Semester Projects in an Undergraduate Neurophysiology Lab Course.

We designed a final semester research project that allowed students to apply the electrophysiological concepts they learned in a lab course to propose and answer experimental questions without access to laboratory equipment. We created the activity based on lesson plans from Ashley Juavinett and the Allen Institute for Brain Science (AIBS) Allen SDK online examples. An interactive graphic interface was added for students to explore and easily quantify subtle neuronal voltage changes. Before starting the final project, students had experience with conventional extracellular and intracellular recording techniques to record and analyze extracellular action potential firing patterns and intracellular resting, action, and synaptic potentials. They demonstrated their understanding of neural signal transmission in required lab reports using data they gathered before the pandemic shutdown. After students left campus, they continued to analyze data and write lab reports focused on neuronal excitability in snail and fly neurons with data supplied by the instructors. For their final project, students were challenged to answer questions addressing neuronal excitability at both the single neuron and neuronal population level by analyzing and interpreting the open-access, patch clamp recording data from the Allen Cell Types Database using code we provided (Python/Jupyter Notebook). This virtual final semester project allowed students to ask real-world medical and scientific questions from "start to end". Through this project, students developed skills to navigate an extensive online database and gained experience with coding-based data analysis. They chose neuronal populations from human and mouse brains to compare passive properties and neuronal excitability between and within brain areas and across different species and disease states. Additionally, students learned to do simple manipulations of Python code, work remotely in teams, and polish their written scientific presentation skills. This activity could complement other remote learning options such as neuronal simulations. Few online sources offer such a wealth of neuroscience data that students can use for class assignments, and even for research and keystone projects. The activity extends the traditional material often taught in upper-level neuroscience courses, with or without a laboratory section, providing a deeper understanding of the range of excitability properties that neurons express.

Building Your Own Neuroscience Equipment: A Precision Micromanipulator and an Epi-fluorescence Microscope for Calcium Imaging.

A faculty member's ability to develop meaningful research-oriented laboratories in neurobiology is often hampered by the rapid pace of new technologies and the increasing cost of equipment. To help undergraduate neuroscience faculty meet these challenges, we introduce two important neuroscience research tools we designed and built. The first is a precision micromanipulator for neurophysiology applications costing less than $40 USD. We compare data generated using the DIY manipulator with commercial micromanipulators costing over $1000. The second tool is our newly designed 3D printed epi-fluorescence microscope. Commercial fluorescence imaging devices often cost over $20,000, but our 3D printed version is constructed for less than $1200. This epi-fluorescence microscope uses interchangeable LED light sources and filter sets to image static fluorescence in prepared slides and calcium imaging of neuronal activity in living Drosophila brains. This later technique uses transgenic flies with a genetically encoded calcium indicator, GCaMP, linked to green fluorescent protein (GFP). During an action potential, calcium ions (Ca2+) enter neurons and are observed as an increase in fluorescence intensity from a series of video images. These neuronal firing patterns can be assessed qualitatively and quantitatively to understand neural circuits leading to specific behaviors. We plan to develop curricula around the use of the epi-fluorescence microscope for calcium imaging in the next year, and to provide detailed parts sources and construction guides for the student and faculty DIY experience.

Reducing the Cost of Electrophysiology in the Teaching Laboratory.

Electrophysiology is a fundamental part of neuroscience and there are many published laboratory exercises suitable for undergraduates. However, the cost of equipping a lab is often a barrier to implementing these exercises. In this paper, we outline lab needs, suggest strategies for building a lab incrementally by adding equipment as budgets permit, and suggest specific areas for cost-cutting. We also point out instances in which it makes most sense to purchase or borrow research-grade equipment. A linked Google document lists specific items, prices, and purchase links.

Muscle Spindles and Our Sense of Physical Self: Kinesthetic Illusions of Limb Position and Posture.

This article describes three simple activities we presented at the 2017 FUN Faculty Workshop at Dominican University that demonstrate how proprioceptive information contributes to our mental image of physical self, and how artificially altering this information creates kinesthetic illusions. We focus on the muscle spindle contribution to limb positional sense and standing postural maintenance. We use a percussion stimulator to vibrate muscle spindles in several muscle groups, causing an artificially incorrect message to the CNS that a muscle has lengthened. This creates an illusion of limb position or standing posture change. Although descriptive data can suffice to engage students in these activities, we suggest quantitative measurements to add further depth. These activities are open for continued student-designed exploration. They lead directly to discussions of sensory physiology, central pathways for integration of sensory information and spinal pathways to execute motor commands. A broader context for the activities could include postural adaptations at sea and upon return to land, postural illusions experienced by astronauts and the postural and locomotor problems they experience upon return to Earth, and the effects of aging and disease on the proprioceptive control of limb position and posture.

Probing Synaptic Transmission and Behavior in Drosophila with Optogenetics: A Laboratory Exercise.

Optogenetics is possibly the most revolutionary advance in neuroscience research techniques within the last decade. Here, we describe lab modules, presented at a workshop for undergraduate neuroscience educators, using optogenetic control of neurons in the fruit fly Drosophila melanogaster. Drosophila is a genetically accessible model system that combines behavioral and neurophysiological complexity, ease of use, and high research relevance. One lab module utilized two transgenic Drosophila strains, each activating specific circuits underlying startle behavior and backwards locomotion, respectively. The red-shifted channelrhodopsin, CsChrimson, was expressed in neurons sharing a common transcriptional profile, with the expression pattern further refined by the use of a Split GAL4 intersectional activation system. Another set of strains was used to investigate synaptic transmission at the larval neuromuscular junction. These expressed Channelrhodopsin 2 (ChR2) in glutamatergic neurons, including the motor neurons. The first strain expressed ChR2 in a wild type background, while the second contained the SNAP-25ts mutant allele, which confers heightened evoked potential amplitude and greatly increased spontaneous vesicle release frequency at the larval neuromuscular junction. These modules introduced educators and students to the use of optogenetic stimulation to control behavior and evoked release at a model synapse, and establish a basis for students to explore neurophysiology using this technique, through recapitulating classic experiments and conducting independent research.

Biofilm carrier migration model describes reactor performance.

The accuracy of a biofilm reactor model depends on the extent to which physical system conditions (particularly bulk-liquid hydrodynamics and their influence on biofilm dynamics) deviate from the ideal conditions upon which the model is based. It follows that an improved capacity to model a biofilm reactor does not necessarily rely on an improved biofilm model, but does rely on an improved mathematical description of the biofilm reactor and its components. Existing biofilm reactor models typically include a one-dimensional biofilm model, a process (biokinetic and stoichiometric) model, and a continuous flow stirred tank reactor (CFSTR) mass balance that [when organizing CFSTRs in series] creates a pseudo two-dimensional (2-D) model of bulk-liquid hydrodynamics approaching plug flow. In such a biofilm reactor model, the user-defined biofilm area is specified for each CFSTR; thereby, Xcarrier does not exit the boundaries of the CFSTR to which they are assigned or exchange boundaries with other CFSTRs in the series. The error introduced by this pseudo 2-D biofilm reactor modeling approach may adversely affect model results and limit model-user capacity to accurately calibrate a model. This paper presents a new sub-model that describes the migration of Xcarrier and associated biofilms, and evaluates the impact that Xcarrier migration and axial dispersion has on simulated system performance. Relevance of the new biofilm reactor model to engineering situations is discussed by applying it to known biofilm reactor types and operational conditions.

The University of Ibadan/Grass Foundation Workshop in Neuroscience Teaching.

The University of Ibadan/Grass Foundation Workshop in Neuroscience Teaching (March 31st to April 2nd, 2017) in Ibadan, Nigeria was sponsored by the Grass Foundation as a "proof of principle" outreach program for young neuroscience faculty at Nigerian universities with limited educational and research resources. The workshop's goal was to introduce low cost equipment for student lab exercises and computational tutorials that could enhance the teaching and research capabilities of local neuroscience educators. Participant assessment of the workshop's activities was very positive and suggested that similar workshops for other faculty from institutions with limited resources could have a great impact on the quality of both the undergraduate and faculty experience.

Comparison of sidestream treatment technologies: post aerobic digestion and Anammox.

Post aerobic digestion (PAD) and anaerobic ammonium oxidation (Anammox) are sidestream treatment technologies which are both excellent options for the reduction of nitrogen recycled back to the liquid stream without the need for supplemental carbon or alkalinity. However, the achievement of this goal is where the similarities between the two technologies end. PAD is an advanced digestion process where aerobic digestion is designed to follow anaerobic digestion. Other benefits of PAD include volatile solids reduction, odor reduction, and struvite formation reduction. Anammox harnesses a specific species of autotrophic bacteria that can help achieve partial nitritation/deammonification. Other benefits of Anammox include lower energy consumption due to requiring less oxygen compared with conventional nitrification. This manuscript describes the unique benefits and challenges of each technology. Example installations are presented with a narrative of how and why the technology was selected. A whole plant simulator is used to compare and contrast the mass balances and net present value costs on an 'apples to apples' basis. The discussion includes descriptions of conditions under which each technology would potentially be the most beneficial and cost-effective against a baseline facility without sidestream treatment.

Light Activated Escape Circuits: A Behavior and Neurophysiology Lab Module using Drosophila Optogenetics.

The neural networks that control escape from predators often show very clear relationships between defined sensory inputs and stereotyped motor outputs. This feature provides unique opportunities for researchers, but it also provides novel opportunities for neuroscience educators. Here we introduce new teaching modules using adult Drosophila that have been engineered to express csChrimson, a red-light sensitive channelrhodopsin, in specific sets of neurons and muscles mediating visually guided escape behaviors. This lab module consists of both behavior and electrophysiology experiments that explore the neural basis of flight escape. Three preparations are described that demonstrate photo-activation of the giant fiber circuit and how to quantify these behaviors. One of the preparations is then used to acquire intracellular electrophysiology recordings from different flight muscles. The diversity of action potential waveforms and firing frequencies observed in the flight muscles make this a rich preparation to study the ionic basic of cellular excitability. By activating different cells within the giant fiber pathway we also demonstrate principles of synaptic transmission and neural circuits. Beyond conveying core neurobiological concepts it is also expected that using these cutting edge techniques will enhance student motivation and attitudes towards biological research. Data collected from students and educators who have been involved in development of the module are presented to support this notion.

QM/MM study of N501 involved intermolecular interaction between SARS-CoV-2 receptor binding domain and antibody of human origin.

Intermolecular interaction between key residue N501 of the epitope on SARS-CoV-2 RBD and screening antibody B38 was studied using the QM/MM and QM approach. The QM/MM optimized geometry shows that angle X-H---Y is 165° for O-H---O between mAb light chain S30 and RBD N501. High level MP2 calculations indicated the interaction between RBD N501 and S30 of B38 Fab light chain provide a relatively strong attractive force of - 3.32 kcal/mol, whereas the hydrogen bond between RBD Q498 and S30 was quantified as 0.10 kcal/mol. The decrease in ESP partial charge on hydrogen atom of hydroxyl group on S30 drops from 0.38 a.u. to 0.31 a.u., exhibiting the sharing of 0.07 a.u. from the lone pair electron oxygen of N501 due to hydrogen bond formation. The NBO occupancy of hydrogen atom also decreases from 25.79 % to 22.93 % in the hydroxyl H-O NBO bond of S30. However, the minor change of NBO hybridization of hydroxyl oxygen of S30 from sp3.00 to sp3.05 implies the rigidity of hydrogen bond tetrahedral geometry in the relative dynamic protein complex. The O-H---O angle is 165° which is close but not exactly linear. The structural requirement for sp3 hybridization of oxygen for hydroxyl group on S30 and dimension of protein likely prevent O-H---O from adopting linear geometry. The hydrogen bond strengths were also calculated using a variety of DFT methods, and the result of - 3.33 kcal/mol from the M06L method is the closest to that of the MP2 calculation. Results of this work may aid in the COVID-19 vaccine and drug screening.

Biological and physical selectors for mobile biofilms, aerobic granules, and densified-biological flocs in continuously flowing wastewater treatment processes: A state-of-the-art review.

There have been significant advances in the use of biological and physical selectors for the intensification of continuously flowing biological wastewater treatment (WWT) processes. Biological selection allows for the development of large biological aggregates (e.g., mobile biofilm, aerobic granules, and densified biological flocs). Physical selection controls the solids residence times of large biological aggregates and ordinary biological flocs, and is usually accomplished using screens or hydrocyclones. Large biological aggregates can facilitate different biological transformations in a single reactor and enhance liquid and solids separation. Continuous-flow WWT processes incorporating biological and physical selectors offer benefits that can include reduced footprint, lower costs, and improved WWT process performance. Thus, it is expected that both interest in and application of these processes will increase significantly in the future. This review provides a comprehensive summary of biological and physical selectors and their design and operation.

Neuromodulator-evoked synaptic metaplasticity within a central pattern generator network.

Synapses show short-term activity-dependent dynamics that alter the strength of neuronal interactions. This synaptic plasticity can be tuned by neuromodulation as a form of metaplasticity. We examined neuromodulator-induced metaplasticity at a graded chemical synapse in a model central pattern generator (CPG), the pyloric network of the spiny lobster stomatogastric ganglion. Dopamine, serotonin, and octopamine each produce a unique motor pattern from the pyloric network, partially through their modulation of synaptic strength in the network. We characterized synaptic depression and its amine modulation at the graded synapse from the pyloric dilator neuron to the lateral pyloric neuron (PD→LP synapse), driving the PD neuron with both long square pulses and trains of realistic waveforms over a range of presynaptic voltages. We found that the three amines can differentially affect the amplitude of graded synaptic transmission independently of the synaptic dynamics. Low concentrations of dopamine had weak and variable effects on the strength of the graded inhibitory postsynaptic potentials (gIPSPs) but reliably accelerated the onset of synaptic depression and recovery from depression independently of gIPSP amplitude. Octopamine enhanced gIPSP amplitude but decreased the amount of synaptic depression; it slowed the onset of depression and accelerated its recovery during square pulse stimulation. Serotonin reduced gIPSP amplitude but increased the amount of synaptic depression and accelerated the onset of depression. These results suggest that amine-induced metaplasticity at graded chemical synapses can alter the parameters of synaptic dynamics in multiple and independent ways.

The homemade alternative: teaching human neurophysiology with instrumentation made (almost) from scratch.

Recording human neurophysiological data in the teaching laboratory generally requires expensive instrumentation. From our experience in developing inexpensive equipment used in teaching neurophysiology laboratory exercises, we offer a strategy for the development of affordable and safe recording of human neurophysiological parameters. There are many resources available to guide the design and construction of electronic equipment that will record human biopotentials. An important consideration is subject safety, and the electrical characteristics of any equipment must meet strict galvanic isolation standards. Wireless data gathering offers the most complete isolation from 120VAC current. As an example, we present a homemade electrocardiogram recording circuit using only inexpensive and readily available components. We outline the feasibility of constructing equipment that meets the needs of the student laboratory for good data collection, and we consider the obstacles likely to be encountered in these projects. If students actively participate in the equipment design and construction, the process can also be a teaching tool. Students may gain a deeper understanding of the human neurobiology by making the electronic data acquisition and its presentation more transparent.