Spatial nonstationarity of image noise in widefield optical imaging and its effects on cluster-based inference for resting-state functional connectivity.

BACKGROUND: Resting-state functional connectivity (RSFC) analysis with widefield optical imaging (WOI) is a potentially powerful tool to develop imaging biomarkers in mouse models of disease before translating them to human neuroimaging with functional magnetic resonance imaging (fMRI). The delineation of such biomarkers depends on rigorous statistical analysis. However, statistical understanding of WOI data is limited. In particular, cluster-based analysis of neuroimaging data depends on assumptions of spatial stationarity (i.e., that the distribution of cluster sizes under the null is equal at all brain locations). Whether actual data deviate from this assumption has not previously been examined in WOI. NEW METHOD: In this manuscript, we characterize the effects of spatial nonstationarity in WOI RSFC data and adapt a "two-pass" technique from fMRI to correct cluster sizes and mitigate spatial bias, both parametrically and nonparametrically. These methods are tested on multi-institutional data. RESULTS AND COMPARISON WITH EXISTING METHODS: We find that spatial nonstationarity has a substantial effect on inference in WOI RSFC data with false positives much more likely at some brain regions than others. This pattern of bias varies between imaging systems, contrasts, and mouse ages, all of which could affect experimental reproducibility if not accounted for. CONCLUSIONS: Both parametric and nonparametric corrections for nonstationarity result in significant improvements in spatial bias. The proposed methods are simple to implement and will improve the robustness of inference in optical neuroimaging data.

Mycobacterium tuberculosis senses host Interferon-γ via the membrane protein MmpL10.

Mycobacterium tuberculosis (Mtb) is one of the most successful human pathogens. Several cytokines are known to increase virulence of bacterial pathogens, leading us to investigate whether Interferon-γ (IFN-γ), a central regulator of the immune defense against Mtb, has a direct effect on the bacteria. We found that recombinant and T-cell derived IFN-γ rapidly induced a dose-dependent increase in the oxygen consumption rate (OCR) of Mtb, consistent with increased bacterial respiration. This was not observed in attenuated Bacillus Calmette-Guérin (BCG), and did not occur for other cytokines tested, including TNF-α. IFN-γ binds to the cell surface of intact Mtb, but not BCG. Mass spectrometry identified mycobacterial membrane protein large 10 (MmpL10) as the transmembrane binding partner of IFN-γ, supported by molecular modelling studies. IFN-γ binding and the OCR response was absent in Mtb Δmmpl10 strain and restored by complementation with wildtype mmpl10. RNA-sequencing and RT-PCR of Mtb exposed to IFN-γ revealed a distinct transcriptional profile, including genes involved in virulence. In a 3D granuloma model, IFN-γ promoted Mtb growth, which was lost in the Mtb Δmmpl10 strain and restored by complementation, supporting the involvement of MmpL10 in the response to IFN-γ. Finally, IFN-γ addition resulted in sterilization of Mtb cultures treated with isoniazid, indicating clearance of phenotypically resistant bacteria that persist in the presence of drug alone. Together our data are the first description of a mechanism allowing Mtb to respond to host immune activation that may be important in the immunopathogenesis of TB and have use in novel eradication strategies.

Hinge disulfides in human IgG2 CD40 antibodies modulate receptor signaling by regulation of conformation and flexibility.

Antibodies protect from infection, underpin successful vaccines and elicit therapeutic responses in otherwise untreatable cancers and autoimmune conditions. The human IgG2 isotype displays a unique capacity to undergo disulfide shuffling in the hinge region, leading to modulation of its ability to drive target receptor signaling (agonism) in a variety of important immune receptors, through hitherto unexplained molecular mechanisms. To address the underlying process and reveal how hinge disulfide orientation affects agonistic activity, we generated a series of cysteine to serine exchange variants in the hinge region of the clinically relevant monoclonal antibody ChiLob7/4, directed against the key immune receptor CD40. We report how agonistic activity varies with disulfide pattern and is afforded by the presence of a disulfide crossover between F(ab) arms in the agonistic forms, independently of epitope, as observed in the determined crystallographic structures. This structural "switch" affects directly on antibody conformation and flexibility. Small-angle x-ray scattering and ensemble modeling demonstrated that the least flexible variants adopt the fewest conformations and evoke the highest levels of receptor agonism. This covalent change may be amenable for broad implementation to modulate receptor signaling in an epitope-independent manner in future therapeutics.

Agonistic CD27 antibody potency is determined by epitope-dependent receptor clustering augmented through Fc-engineering.

Agonistic CD27 monoclonal antibodies (mAb) have demonstrated impressive anti-tumour efficacy in multiple preclinical models but modest clinical responses. This might reflect current reagents delivering suboptimal CD27 agonism. Here, using a novel panel of CD27 mAb including a clinical candidate, we investigate the determinants of CD27 mAb agonism. Epitope mapping and in silico docking analysis show that mAb binding to membrane-distal and external-facing residues are stronger agonists. However, poor epitope-dependent agonism could partially be overcome by Fc-engineering, using mAb isotypes that promote receptor clustering, such as human immunoglobulin G1 (hIgG1, h1) with enhanced affinity to Fc gamma receptor (FcγR) IIb, or hIgG2 (h2). This study provides the critical knowledge required for the development of agonistic CD27 mAb that are potentially more clinically efficacious.

A Low-Cost, Wireless, Multi-Channel Deep Brain Stimulation System for Rodents.

We present a small (43mm x 24mm x 15mm), off-the-shelf wireless neurostimulator for rodent deep brain stimulation research. Our device enables researchers to wirelessly configure stimulator settings, such as amplitude, pulse width, channel selection, and frequency, via a phone app. The system uses impedance-independent current-mode stimulation and steers current to a selected channel. In addition to monophasic and biphasic stimulation, the system also supports arbitrary waveform stimulation using pre-stored lookup tables. The system uses a configurable grounding phase to clear residual charge and a stimulation compliance monitor to ensure safe operation. The compliance monitor wirelessly reports the current during stimulation, the amount of passive recharge current, and the DC voltage of the electrode interface. The 400mAh battery is easy to replace and can go over 40 hours between charges. The system can be built for less than $50 using easy-to-source components to support inexpensive, highly-parallel research applications.

Isotype Switching Converts Anti-CD40 Antagonism to Agonism to Elicit Potent Antitumor Activity.

Anti-CD40 monoclonal antibodies (mAbs) comprise agonists and antagonists, which display promising therapeutic activities in cancer and autoimmunity, respectively. We previously showed that epitope and isotype interact to deliver optimal agonistic anti-CD40 mAbs. The impact of Fc engineering on antagonists, however, remains largely unexplored. Here, we show that clinically relevant antagonists used for treating autoimmune conditions can be converted into potent FcγR-independent agonists with remarkable antitumor activity by isotype switching to hIgG2. One antagonist is converted to a super-agonist with greater potency than previously reported highly agonistic anti-CD40 mAbs. Such conversion is dependent on the unique disulfide bonding properties of the hIgG2 hinge. This investigation highlights the transformative capacity of the hIgG2 isotype for converting antagonists to agonists to treat cancer.