Antibody-mediated targeting of human microglial leukocyte Ig-like receptor B4 attenuates amyloid pathology in a mouse model.

Microglia help limit the progression of Alzheimer's disease (AD) by constraining amyloid-ß (Aß) pathology, effected through a balance of activating and inhibitory intracellular signals delivered by distinct cell surface receptors. Human leukocyte Ig-like receptor B4 (LILRB4) is an inhibitory receptor of the immunoglobulin (Ig) superfamily that is expressed on myeloid cells and recognizes apolipoprotein E (ApoE) among other ligands. Here, we find that LILRB4 is highly expressed in the microglia of patients with AD. Using mice that accumulate Aß and carry a transgene encompassing a portion of the LILR region that includes LILRB4, we corroborated abundant LILRB4 expression in microglia wrapping around Aß plaques. Systemic treatment of these mice with an anti-human LILRB4 monoclonal antibody (mAb) reduced Aß load, mitigated some Aß-related behavioral abnormalities, enhanced microglia activity, and attenuated expression of interferon-induced genes. In vitro binding experiments established that human LILRB4 binds both human and mouse ApoE and that anti-human LILRB4 mAb blocks such interaction. In silico modeling, biochemical, and mutagenesis analyses identified a loop between the two extracellular Ig domains of LILRB4 required for interaction with mouse ApoE and further indicated that anti-LILRB4 mAb may block LILRB4-mApoE by directly binding this loop. Thus, targeting LILRB4 may be a potential therapeutic avenue for AD.

Effect of sensor location for modifying center of pressure during gait using haptic feedback in people with chronic ankle instability.

BACKGROUND: Gait retraining using haptic biofeedback medially shifts the center of pressure (COP) while walking in orthopedic populations. However, the ideal sensor location needed to effectively shift COP medially has not been identified in people with chronic ankle instability (CAI). RESEARCH QUESTIONS: Can a heel sensor location feasibly be employed in people with CAI without negatively altering kinematics? Does a heel sensor placement relative to the 5th metatarsal head (5MH) impact COP location while walking in people with CAI? METHODS: In this exploratory crossover study, 10 participants with CAI walked on a treadmill with vibration feedback for 10 minutes with a plantar pressure sensor under the heel and 5MH. Separate 2×2 repeated measures analyses of covariances (rmANCOVAs) were used to compare the averaged COP location and 3-D lower extremity kinematics from the first 10% of stance before and after training and between sensor locations. Baseline measures served as covariates to adjust for baseline differences. RESULTS: Feedback triggered by a heel sensor resulted in 40% of participants avoiding a heel strike. There were no significant main effects or interactions between time and sensor location on COP location when controlling for baseline COP (p>0.05). However, with the 5MH placement, participants displayed less ankle internal rotation(IR) (5MH/Heel: -4.12±0.00º/ -6.43±0.62º), less forefoot abduction (-4.29±0.00º/ -5.14±1.01º), more knee flexion (3.40±0.32º/ 0.14±0.57º), less knee external rotation (-10.95±0.00º/-11.24±1.48º), less hip extension (-0.20±0.00º/-1.42±1.05º), and less hip external rotation (3.12±0.00º/3.75±1.98º). SIGNIFICANCE: A 5MH location may be more feasible based on difficulties maintaining heel strike when the sensor was under the heel. While no sensor location was statistically better at changing the COP, the 5MH location decreased proximal transverse plane motions making participants' gait more like controls. Individual response variations support comprehensive lower extremity assessments and the need to identify responder profiles using sensory feedback in people with CAI.

Epitope Mapping of Japanese Encephalitis Virus Neutralizing Antibodies by Native Mass Spectrometry and Hydrogen/Deuterium Exchange.

Japanese encephalitis virus (JEV) remains a global public health concern due to its epidemiological distribution and the existence of multiple strains. Neutralizing antibodies against this infection have shown efficacy in in vivo studies. Thus, elucidation of the epitopes of neutralizing antibodies can aid in the design and development of effective vaccines against different strains of JEV. Here, we describe a combination of native mass spectrometry (native-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to complete screening of eight mouse monoclonal antibodies (MAbs) against JEV E-DIII to identify epitope regions. Native-MS was used as a first pass to identify the antibodies that formed a complex with the target antigen, and it revealed that seven of the eight monoclonal antibodies underwent binding. Native mass spectra of a MAb (JEV-27) known to be non-binding showed broad native-MS peaks and poor signal, suggesting the protein is a mixture or that there are impurities in the sample. We followed native-MS with HDX-MS to locate the binding sites for several of the complex-forming antibodies. This combination of two mass spectrometry-based approaches should be generally applicable and particularly suitable for screening of antigen-antibody and other protein-protein interactions when other traditional approaches give unclear results or are difficult, unavailable, or need to be validated.

The effect of Tai Chi lower extremity exercise on the balance control of older adults in assistant living communities.

BACKGROUND: Although Tai Chi (TC) is an evidence-based fall prevention training for older adults, its effective movements remain unclear, which may limit the practice of TC. The purpose of this study was to compare the effectiveness of TC lower extremity exercise (TC LEE), the 8-form Tai Chi (8-form TC), and a stretching control intervention for improving balance and functional mobility among older adults. METHODS: This was a randomized controlled trial. A total of 102 participants (79 ± 6 years old) were recruited from assisted living facilities. All participants were randomly assigned to the TC LEE (n = 40), 8-form TC (n = 31), and stretching (n = 31) groups in which they received the respective interventions for 16 weeks. The Berg Balance Scale (BBS), Timed Up and Go (TUG) test, and center of pressure (COP) measurements during quiet stance were collected prior to and following the 16-week interventions. Comparisons on all measurements were conducted among all groups. RESULTS: Significant improvements were found in BBS (P = 0.002), TUG test (P = 0.001), root mean square amplitude of COP displacement in the anterior-posterior (P = 0.001) and medial-lateral (P = 0.001) directions, and average COP speed in the anterior-posterior (P = 0.001) and medial-lateral (P = 0.001) directions after training in the TC intervention groups compared with the stretching group. The upper limit of the 95% confidence interval (CI) of differences in change scores on the BBS (-0.8 - 1.3 points) between the TC LEE group and the 8-form TC group was within equivalence margins (1.8 points), while the upper limit of the 95% CI of differences in change scores on the TUG test (0.1 - 2.1 s) exceeded the equivalence margin (0.7 s) with the TC LEE group having the larger change scores. CONCLUSION: TC LEE can improve balance and functional mobility in older adults, and may have greater effect than the 8-form TC on improving functional mobility as measured by the TUG test. TRIAL REGISTRATION: ChiCTR2300070600 retrospectively registered.

The Use of Methylene Blue in Conjunction With Hydroxocobalamin and Multiple Pressors to Treat Severe Vasoplegia in a Patient Due to Calcium Channel Blocker Toxicity: A Case Report.

Vasoplegia, the demonstration of persistently low systemic vascular resistance (SVR) and resistant hypotension in the presence of a normal cardiac index despite aggressive resuscitation attempts, is a serious clinical diagnosis that requires prompt treatment to prevent patient morbidity and mortality. Currently, treatment of vasoplegia involves treatment with vasopressors such as vasopressin, norepinephrine, and hydroxocobalamin. However, some evidence suggests that in addition to this treatment regimen, the addition of methylene blue may result in a reduction in overall norepinephrine equivalent vasopressor requirements, increased mean arterial pressure, and an improved clinical course. Here, we report the case of a 64-year-old male patient who presented to the ED after being found unresponsive and covered in emesis at home. The patient's presentation was complicated by worsening dyspnea, hypotension, and hemodynamic instability, requiring intubation and admission to the ICU for management of undifferentiated shock of unclear etiology and acute respiratory failure. Urine studies were consistent with a diagnosis of vasoplegia due to dihydropyridine calcium channel blocker toxicity, which was confirmed by pill counting of his home medications in the setting of recent paranoia and depression. The patient was treated aggressively with vasopressors, including vasopressin, phenylephrine, and epinephrine, as well as a combination of hydroxocobalamin and methylene blue. He was also started on a calcium and insulin drip. Upon initiation of non-catecholamine agents for vasoplegia, his clinical course quickly improved, and he was weaned from all vasopressors. He regained hemodynamic stability, was successfully extubated, evaluated by psychiatry, and discharged from the hospital in a stable condition on day 15 with the continuation of outpatient psychiatric services.

Pesticides in small volume plasma samples: Method development and application to smallmouth bass (Micropterus dolomieu) from the Chesapeake Bay watershed, USA.

Nontarget organisms are exposed to pesticides following applications in agricultural and urban settings, potentially resulting in deleterious effects. Direct measurements of pesticides in biological tissues may aid in characterizing exposure, accumulation, and potential toxicity versus analyses in environmental media alone (e.g., water, soil, and air). Plasma represents a nonlethal sampling medium that can be used to assess recent exposures to contaminants. Herein, a method was developed to test the extraction of 210 pesticides and their transformation products in small volume plasma samples (100 µL). Plasma samples were protein precipitated with 0.5 % formic acid in acetonitrile added to the sample (ratio of 3.5:1). Pass-through solid phase extraction was used for sample matrix and lipid removal and samples were analyzed by liquid chromatography and gas chromatography with tandem mass spectrometry. Recoveries of 70.0-129.8 % were achieved for 182 pesticides and degradates across the low (25 ng mL-1), medium (100 ng mL-1), and high (250 ng mL-1) spike levels. Method detection levels ranged 0.4-13.0 ng mL-1. Following development, the method was applied to smallmouth bass (Micropterus dolomieu) plasma samples (n = 10) collected from adults in the Chesapeake Bay watershed. Individual plasma samples resulted in four to seven analytes detected with summed concentrations ranging 16.4-95.0 ng mL-1. Biological multiresidue pesticide methods help elucidate recent exposures of bioactive compounds to nontarget organisms.

Determinants of spring migration departure dates in a New World sparrow: Weather variables reign supreme.

Numerous factors influence the timing of spring migration in birds, yet the relative importance of intrinsic and extrinsic variables on migration initiation remains unclear. To test for interactions among weather, migration distance, parasitism, and physiology in determining spring departure date, we used the Dark-eyed Junco (Junco hyemalis) as a model migratory species known to harbor diverse and common haemosporidian parasites. Prior to spring migration departure from their wintering grounds in Indiana, USA, we quantified the intrinsic variables of fat, body condition (i.e., mass ~ tarsus residuals), physiological stress (i.e., ratio of heterophils to lymphocytes), cellular immunity (i.e., leukocyte composition and total count), migration distance (i.e., distance to the breeding grounds) using stable isotopes of hydrogen from feathers, and haemosporidian parasite intensity. We then attached nanotags to determine the timing of spring migration departure date using the Motus Wildlife Tracking System. We used additive Cox proportional hazard mixed models to test how risk of spring migratory departure was predicted by the combined intrinsic measures, along with meteorological predictors on the evening of departure (i.e., average wind speed and direction, relative humidity, and temperature). Model comparisons found that the best predictor of spring departure date was average nightly wind direction and a principal component combining relative humidity and temperature. Juncos were more likely to depart for spring migration on nights with largely southwestern winds and on warmer and drier evenings (relative to cooler and more humid evenings). Our results indicate that weather conditions at take-off are more critical to departure decisions than the measured physiological and parasitism variables.

Hydrogen/Deuterium Exchange Mass Spectrometry Provides Insights into the Role of Drosophila Testis-Specific Myosin VI Light Chain AndroCaM.

In Drosophila testis, myosin VI plays a special role, distinct from its motor function, by anchoring components to the unusual actin-based structures (cones) that are required for spermatid individualization. For this, the two calmodulin (CaM) light-chain molecules of myosin VI are replaced by androcam (ACaM), a related protein with 67% identity to CaM. Although ACaM has a similar bi-lobed structure to CaM, with two EF hand-type Ca2+ binding sites per lobe, only one functional Ca2+ binding site operates in the amino-terminus. To understand this light chain substitution, we used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to examine dynamic changes in ACaM and CaM upon Ca2+ binding and interaction with the two CaM binding motifs of myosin VI (insert2 and IQ motif). HDX-MS reveals that binding of Ca2+ to ACaM destabilizes its N-lobe but stabilizes the entire C-lobe, whereas for CaM, Ca2+ binding induces a pattern of alternating stabilization/destabilization throughout. The conformation of this stable holo-C-lobe of ACaM seems to be a "prefigured" version of the conformation adopted by the holo-C-lobe of CaM for binding to insert2 and the IQ motif of myosin VI. Strikingly, the interaction of holo-ACaM with either peptide converts the holo-N-lobe to its Ca2+-free, more stable, form. Thus, ACaM in vivo should bind the myosin VI light chain sites in an apo-N-lobe/holo-C-lobe state that cannot fulfill the Ca2+-related functions of holo-CaM required for myosin VI motor assembly and activity. These findings indicate that inhibition of myosin VI motor activity is a precondition for transition to an anchoring function.

Medical Sanctions Against Russia: Arresting Aggression or Abrogating Healthcare Rights?

Since 2022, the EU, US, and other nations have imposed medical sanctions on Russia to block the export of pharmaceuticals and medical devices and curtail clinical trials to degrade Russia's military capabilities. While international law proscribes sanctions that cause a humanitarian crisis, an outcome averted in Russia, the military effects of medical sanctions have been lean. Strengthening medical sanctions risks violating noncombatant and combatant rights to healthcare. Each group's claim is different. Noncombatants and severely injured soldiers who cannot return to duty enjoy the right to adequate health care that sanctions cannot undermine. Combatants falling captive enjoy the same medical care that adversaries provide their own troops. Combatants yet to renounce hostilities, however, have no claim to medical attention and remain subject to sanctions. Nevertheless, medical sanctions prove unworkable in this complex environment of conflicting rights and command no place in a sustainable sanction regime.

Treating the innocent victims of trolleys and war.

Both trolleys and war leave innocent victims to suffer death and injury. Trolley problems accounting for the injured, and not only the dead, tease out intuitions about liability that enhance our understanding of the obligation to provide compensation and medical care to civilian victims of war. Like many trolley victims, civilians in war may suffer justifiable, excusable, or negligent harms that demand compensation. Chief among these is collateral harm befalling civilians. Collateral harm is endemic to war and comprises permissible but unavoidable death or injury following necessary and proportionate military operations. Although state armies sometimes offer condolence payments for civilian death, injury, and property loss, they deny liability. Instead, they use compensation to enhance counterinsurgency efforts and assuage feelings of agent regret. As part of the medical rules of eligibility, Coalition forces in Iraq and Afghanistan also provided medical care to victims of collateral harm. However, they denied care to similarly sick or injured civilians. While compensation is often justified to cure the harm civilians suffer, the differential use of medical resources is not. Rather, medical care remains subject to the principle of beneficence and medical need. The duty to provide civilian healthcare in war, particularly in wars of humanitarian intervention, is far-reaching and imposes significant costs that military and medical ethics are yet to recognize.

Exploration of Resveratrol as a Potent Modulator of α-Synuclein Fibril Formation.

The molecular determinants of amyloid protein misfolding and aggregation are key for the development of therapeutic interventions in neurodegenerative disease. Although small synthetic molecules, bifunctional molecules, and natural products offer a potentially advantageous approach to therapeutics to remodel aggregation, their evaluation requires new platforms that are informed at the molecular level. To that end, we chose pulsed hydrogen/deuterium exchange mass spectrometry (HDX-MS) to discern the phenomena of aggregation modulation for a model system of alpha synuclein (αS) and resveratrol, an antiamyloid compound. We invoked, as a complement to HDX, advanced kinetic modeling described here to illuminate the details of aggregation and to determine the number of oligomeric populations by kinetically fitting the experimental data under conditions of limited proteolysis. The misfolding of αS is most evident within and nearby the nonamyloid-ß component region, and resveratrol significantly remodels that aggregation. HDX distinguishes readily a less solvent-accessible, more structured oligomer that coexists with a solvent-accessible, more disordered oligomer during aggregation. A view of the misfolding emerges from time-dependent changes in the fractional species across the protein with or without resveratrol, while details were determined through kinetic modeling of the protected species. A detailed picture of the inhibitory action of resveratrol with time and regional specificity emerges, a picture that can be obtained for other inhibitors and amyloid proteins. Moreover, the model reveals that new states of aggregation are sampled, providing new insights on amyloid formation. The findings were corroborated by circular dichroism and transmission electron microscopy.

Dispersion Engineering by Hybridizing the Back-Folded Soft Mode of Monomode Elastic Metamaterials with Stiff Acoustic Modes.

In many cases, the hybridization of two or more excitation modes in solids has led to new and useful dispersion relations of waves. Well-studied examples are phonon polaritons, plasmon polaritons, particle-plasmon polaritons, cavity polaritons, and magnetic resonances at optical frequencies. In all of these cases, the lowest propagating mode couples to a finite-frequency localized resonance. Herein, the unusual metamaterial phonon dispersion relations arising from the hybridization of an ordinary acoustical phonon mode with a back-folded soft or easy phonon mode of a monomode elastic metamaterial are discussed. Conceptually, the single easy mode can have strictly zero wave velocity. In reality, its wave velocity is very much smaller than that of all other modes. Considering polymeric three-dimensional printed elastic monomode metamaterials at ultrasound frequencies, it is shown theoretically and experimentally that the resulting pronounced avoided crossing, with a frequency splitting comparable to the mid-frequency, leads to backward-wave behavior for the lowest band over a broad frequency range, conceptually at zero loss.

Development of a High-Throughput Mass Spectrometry-Based SARS-CoV-2 Immunoassay.

The serious impact of the Covid-19 pandemic underscores the need for rapid, reliable, and high-throughput diagnosis methods for infection. Current analytical methods, either point-of-care or centralized detection, are not able to satisfy the requirements of patient-friendly testing, high demand, and reliability of results. Here, we propose a two-point separation on-demand diagnostic strategy that uses laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) and adopts a stable yet cleavable ionic probe as a mass reporter. The use of this reporter enables ultrasensitive, interruptible, storable, restorable, and high-throughput on-demand detection. We describe a demonstration of the concept whereby we (i) design and synthesize a laser-cleavable reporter (DTPA), (ii) conjugate the reporter onto an antibody and verify the function of the conjugate, (iii) detect with good turnaround and high sensitivity the conjugated reporter, (iv) analyze quantitatively by using a laser-cleavable internal standard, and (v) identify negative and positive samples containing the spike protein. The protocol has excellent sensitivity (amol for the SARS-CoV-2 Spike S1 subunit antibody) without any amplification. This strategy is also applicable for the detection of other disease antigens besides SARS-CoV-2.

A broadly reactive antibody targeting the N-terminal domain of SARS-CoV-2 spike confers Fc-mediated protection.

Most neutralizing anti-SARS-CoV-2 monoclonal antibodies (mAbs) target the receptor binding domain (RBD) of the spike (S) protein. Here, we characterize a panel of mAbs targeting the N-terminal domain (NTD) or other non-RBD epitopes of S. A subset of NTD mAbs inhibits SARS-CoV-2 entry at a post-attachment step and avidly binds the surface of infected cells. One neutralizing NTD mAb, SARS2-57, protects K18-hACE2 mice against SARS-CoV-2 infection in an Fc-dependent manner. Structural analysis demonstrates that SARS2-57 engages an antigenic supersite that is remodeled by deletions common to emerging variants. In neutralization escape studies with SARS2-57, this NTD site accumulates mutations, including a similar deletion, but the addition of an anti-RBD mAb prevents such escape. Thus, our study highlights a common strategy of immune evasion by SARS-CoV-2 variants and how targeting spatially distinct epitopes, including those in the NTD, may limit such escape.

Antibody Binding Captures High Energy State of an Antigen: The Case of Nsp1 SARS-CoV-2 as Revealed by Hydrogen-Deuterium Exchange Mass Spectrometry.

We describe an investigation using structural mass spectrometry (MS) of the impact of two antibodies, 15497 and 15498, binding the highly flexible SARS-CoV-2 Nsp1 protein. We determined the epitopes and paratopes involved in the antibody-protein interactions by using hydrogen-deuterium exchange MS (HDX-MS). Notably, the Fab (Fragment antigen binding) for antibody 15498 captured a high energy form of the antigen exhibiting significant conformational changes that added flexibility over most of the Nsp1 protein. The Fab for antibody 15497, however, showed usual antigen binding behavior, revealing local changes presumably including the binding site. These findings illustrate an unusual antibody effect on an antigen and are consistent with the dynamic nature of the Nsp1 protein. Our studies suggest that this interaction capitalizes on the high flexibility of Nsp1 to undergo conformational change and be trapped in a higher energy state by binding with a specific antibody.

Characterization of Higher Order Structural Changes of a Thermally Stressed Monoclonal Antibody via Mass Spectrometry Footprinting and Other Biophysical Approaches.

Characterizing changes in the higher order structure (HOS) of monoclonal antibodies upon stressed conditions is critical to gaining a better understanding of the product and process. One single biophysical approach may not be best suited to assess HOS comprehensively; thus, the synergy from multiple, complementary approaches improves characterization accuracy and resolution. In this study, we employed two mass spectrometry (MS )-based footprinting techniques, namely, fast photochemical oxidation of proteins (FPOP)-MS and hydrogen-deuterium exchange (HDX)-MS, supported by dynamic light scattering (DLS), differential scanning calorimetry (DSC), circular dichroism (CD), and nuclear magnetic resonance (NMR) to study changes to the HOS of a mAb upon thermal stress. The biophysical techniques report a nuanced characterization of the HOS in which CD detects no changes to the secondary or tertiary structure, yet DLS measurements show an increase in the hydrodynamic radius. DSC indicates that the stability decreases, and chemical or conformational changes accumulate with incubation time according to NMR. Furthermore, whereas HDX-MS does not indicate HOS changes, FPOP-MS footprinting reveals conformational changes at residue resolution for some amino acids. The local phenomena observed with FPOP-MS indicate that several residues show various patterns of degradation during thermal stress: no change, an increase in solvent exposure, and a biphasic response to solvent exposure. All evidences show that FPOP-MS efficiently resolves subtle structural changes and novel degradation pathways upon thermal stress treatment at residue-level resolution.

Precursor Reagent Hydrophobicity Affects Membrane Protein Footprinting.

Membrane proteins (MPs) play a crucial role in cell signaling, molecular transport, and catalysis and thus are at the heart of designing pharmacological targets. Although structural characterization of MPs at the molecular level is essential to elucidate their biological function, it poses a significant challenge for structural biology. Although mass spectrometry-based protein footprinting may be developed into a powerful approach for studying MPs, the hydrophobic character of membrane regions makes structural characterization difficult using water-soluble footprinting reagents. Herein, we evaluated a small series of MS-based photoactivated iodine reagents with different hydrophobicities. We used tip sonication to facilitate diffusion into micelles, thus enhancing reagent access to the hydrophobic core of MPs. Quantification of the modification extent in hydrophilic extracellular and hydrophobic transmembrane domains provides structurally sensitive information at the residue-level as measured by proteolysis and LC-MS/MS for a model MP, vitamin K epoxide reductase (VKOR). It also reveals a relationship between the reagent hydrophobicity and its preferential labeling sites in the local environment. The outcome should guide the future development of chemical probes for MPs and promote a direction for relatively high-throughput information-rich characterization of MPs in biochemistry and drug discovery.

Acromioclavicular joint mobilizations for the management of grade I sternoclavicular joint sprain: a case report.

OBJECTIVE: Injury to the sternoclavicular joint is a rare phenomenon that has implications for the shoulder complex. Limited literature exists on optimal physical therapy rehabilitation after a sternoclavicular ligament sprain. This case report details the physical therapy management and outcomes of a patient with a posterior sternoclavicular joint sprain. CASE DESCRIPTION: The patient was a 34-year-old female who sustained a grade I posteriorly directed sternoclavicular sprain during a motor vehicle accident. She received a combination of acromioclavicular joint mobilizations and therapeutic exercise for her shoulder complex for five sessions over six weeks. OUTCOMES: At discharge, the patient surpassed the minimally clinically important difference (MCID) and the minimal detectable change (MDC) in her Quick-DASH score. She surpassed the MDC and MCID in her Numerical Pain Rating Scale score. She was able to regain full functional use of her involved upper extremity and returned to her work and original exercise regimen with no further limitations. CONCLUSION: The outcomes suggest that a combination of acromioclavicular joint mobilizations and therapeutic exercise for the shoulder complex was a suitable option for the conservative management of this patient's grade I sternoclavicular sprain.

Fast updating feedback from piriform cortex to the olfactory bulb relays multimodal reward contingency signals during rule-reversal.

While animals readily adjust their behavior to adapt to relevant changes in the environment, the neural pathways enabling these changes remain largely unknown. Here, using multiphoton imaging, we investigated whether feedback from the piriform cortex to the olfactory bulb supports such behavioral flexibility. To this end, we engaged head-fixed mice in a multimodal rule-reversal task guided by olfactory and auditory cues. Both odor and, surprisingly, the sound cues triggered cortical bulbar feedback responses which preceded the behavioral report. Responses to the same sensory cue were strongly modulated upon changes in stimulus-reward contingency (rule reversals). The re-shaping of individual bouton responses occurred within seconds of the rule-reversal events and was correlated with changes in the behavior. Optogenetic perturbation of cortical feedback within the bulb disrupted the behavioral performance. Our results indicate that the piriform-to-olfactory bulb feedback carries reward contingency signals and is rapidly re-formatted according to changes in the behavioral context.

Hydrogen Deuterium Exchange and other Mass Spectrometry-based Approaches for Epitope Mapping.

Antigen-antibody interactions are a fundamental subset of protein-protein interactions responsible for the "survival of the fittest". Determining the interacting interface of the antigen, called an epitope, and that on the antibody, called a paratope, is crucial to antibody development. Because each antigen presents multiple epitopes (unique footprints), sophisticated approaches are required to determine the target region for a given antibody. Although X-ray crystallography, Cryo-EM, and nuclear magnetic resonance can provide atomic details of an epitope, they are often laborious, poor in throughput, and insensitive. Mass spectrometry-based approaches offer rapid turnaround, intermediate structural resolution, and virtually no size limit for the antigen, making them a vital approach for epitope mapping. In this review, we describe in detail the principles of hydrogen deuterium exchange mass spectrometry in application to epitope mapping. We also show that a combination of MS-based approaches can assist or complement epitope mapping and push the limit of structural resolution to the residue level. We describe in detail the MS methods used in epitope mapping, provide our perspective about the approaches, and focus on elucidating the role that HDX-MS is playing now and in the future by organizing a discussion centered around several improvements in prototype instrument/applications used for epitope mapping. At the end, we provide a tabular summary of the current literature on HDX-MS-based epitope mapping.