Integrated analysis of multimodal single-cell data.

The simultaneous measurement of multiple modalities represents an exciting frontier for single-cell genomics and necessitates computational methods that can define cellular states based on multimodal data. Here, we introduce "weighted-nearest neighbor" analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of 211,000 human peripheral blood mononuclear cells (PBMCs) with panels extending to 228 antibodies to construct a multimodal reference atlas of the circulating immune system. Multimodal analysis substantially improves our ability to resolve cell states, allowing us to identify and validate previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets and to interpret immune responses to vaccination and coronavirus disease 2019 (COVID-19). Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets and to look beyond the transcriptome toward a unified and multimodal definition of cellular identity.

Mechanisms of Endogenous HIV-1 Reactivation by Endocervical Epithelial Cells.

Pharmacological HIV-1 reactivation to reverse latent infection has been extensively studied. However, HIV-1 reactivation also occurs naturally, as evidenced by occasional low-level viremia ("viral blips") during antiretroviral treatment (ART). Clarifying where blips originate from and how they happen could provide clues to stimulate latency reversal more effectively and safely or to prevent viral rebound following ART cessation. We studied HIV-1 reactivation in the female genital tract, a dynamic anatomical target for HIV-1 infection throughout all disease stages. We found that primary endocervical epithelial cells from several women reactivated HIV-1 from latently infected T cells. The endocervical cells' HIV-1 reactivation capacity further increased upon Toll-like receptor 3 stimulation with poly(I·C) double-stranded RNA or infection with herpes simplex virus 2 (HSV-2). Notably, acyclovir did not eliminate HSV-2-induced HIV-1 reactivation. While endocervical epithelial cells secreted large amounts of several cytokines and chemokines, especially tumor necrosis factor alpha (TNF-α), CCL3, CCL4, and CCL20, their HIV-1 reactivation capacity was almost completely blocked by TNF-α neutralization alone. Thus, immunosurveillance activities by columnar epithelial cells in the endocervix can cause endogenous HIV-1 reactivation, which may contribute to viral blips during ART or rebound following ART interruption.IMPORTANCE A reason that there is no universal cure for HIV-1 is that the virus can hide in the genome of infected cells in the form of latent proviral DNA. This hidden provirus is protected from antiviral drugs until it eventually reactivates to produce new virions. It is not well understood where in the body or how this reactivation occurs. We studied HIV-1 reactivation in the female genital tract, which is often the portal of HIV-1 entry and which remains a site of infection throughout the disease. We found that the columnar epithelial cells lining the endocervix, the lower part of the uterus, are particularly effective in reactivating HIV-1 from infected T cells. This activity was enhanced by certain microbial stimuli, including herpes simplex virus 2, and blocked by antibodies against the inflammatory cytokine TNF-α. Avoiding HIV-1 reactivation could be important for maintaining a functional HIV-1 cure when antiviral therapy is stopped.

The demographic effects of functional traits: an integral projection model approach reveals population-level consequences of reproduction-defence trade-offs.

Quantitatively linking individual variation in functional traits to demography is a necessary step to advance our understanding of trait-based ecological processes. We constructed a population model for Asclepias syriaca to identify how functional traits affect vital rates and population growth and whether trade-offs in chemical defence and demography alter population growth. Plants with higher foliar cardenolides had lower fibre, cellulose and lignin levels, as well as decreased sexual and clonal reproduction. Average cardenolide concentrations had the strongest effect on population growth. In both the sexual and clonal pathway, the trade-off between reproduction and defence affected population growth. We found that both increasing the mean of the distribution of individual plant values for cardenolides and herbivory decreased population growth. However, increasing the variance in both defence and herbivory increased population growth. Functional traits can impact population growth and quantifying individual-level variation in traits should be included in assessments of population-level processes.

Functional Interactions between Mammalian Respiratory Rhythmogenic and Premotor Circuitry.

UNLABELLED: Breathing in mammals depends on rhythms that originate from the preBötzinger complex (preBötC) of the ventral medulla and a network of brainstem and spinal premotor neurons. The rhythm-generating core of the preBötC, as well as some premotor circuits, consist of interneurons derived from Dbx1-expressing precursors (Dbx1 neurons), but the structure and function of these networks remain incompletely understood. We previously developed a cell-specific detection and laser ablation system to interrogate respiratory network structure and function in a slice model of breathing that retains the preBötC, the respiratory-related hypoglossal (XII) motor nucleus and XII premotor circuits. In spontaneously rhythmic slices, cumulative ablation of Dbx1 preBötC neurons decreased XII motor output by ∼50% after ∼15 cell deletions, and then decelerated and terminated rhythmic function altogether as the tally increased to ∼85 neurons. In contrast, cumulatively deleting Dbx1 XII premotor neurons decreased motor output monotonically but did not affect frequency nor stop XII output regardless of the ablation tally. Here, we couple an existing preBötC model with a premotor population in several topological configurations to investigate which one may replicate the laser ablation experiments best. If the XII premotor population is a "small-world" network (rich in local connections with sparse long-range connections among constituent premotor neurons) and connected with the preBötC such that the total number of incoming synapses remains fixed, then the in silico system successfully replicates the in vitro laser ablation experiments. This study proposes a feasible configuration for circuits consisting of Dbx1-derived interneurons that generate inspiratory rhythm and motor pattern. SIGNIFICANCE STATEMENT: To produce a breathing-related motor pattern, a brainstem core oscillator circuit projects to a population of premotor interneurons, but the assemblage of this network remains incompletely understood. Here we applied network modeling and numerical simulation to discover respiratory circuit configurations that successfully replicate photonic cell ablation experiments targeting either the core oscillator or premotor network, respectively. If premotor neurons are interconnected in a so-called "small-world" network with a fixed number of incoming synapses balanced between premotor and rhythmogenic neurons, then our simulations match their experimental benchmarks. These results provide a framework of experimentally testable predictions regarding the rudimentary structure and function of respiratory rhythm- and pattern-generating circuits in the brainstem of mammals.

Biophysical changes in subcortical nuclei: the impact of diabetes and major depression.

Magnetization transfer (MT) is a neuroimaging technique that is frequently used to characterize the biophysical abnormalities in both gray and white matter regions of the brain. In our study, we used MT to examine the integrity of key nodes in frontal-subcortical circuits in four subject groups: patients diagnosed with type 2 diabetes with and without major depression (MDD), a healthy control group, and a group diagnosed with MDD without diabetes. In the MDD group, MT studies demonstrated lower magnetization transfer ratios (MTR), a marker of abnormalities in the macromolecular protein pool, in the thalami when compared with the control groups. The group with diabetes and MDD showed lower MTR in the globus pallidus when compared with the group with MDD. Biophysical measures, in subcortical nuclei, correlated inversely with measures of glycemic control, cerebrovascular burden and depression scores. These findings have broad implications for the underlying neuronal circuitry and neurobiology of mood disorders.

Mechanisms Leading to Rhythm Cessation in the Respiratory PreBötzinger Complex Due to Piecewise Cumulative Neuronal Deletions

The mammalian breathing rhythm putatively originates from Dbx1-derived interneurons in the preBötzinger complex (preBötC) of the ventral medulla. Cumulative deletion of ∼15% of Dbx1 preBötC neurons in an in vitro breathing model stops rhythmic bursts of respiratory-related motor output. Here we assemble in silico models of preBötC networks using random graphs for structure, and ordinary differential equations for dynamics, to examine the mechanisms responsible for the loss of spontaneous respiratory rhythm and motor output measured experimentally in vitro. Model networks subjected to cellular ablations similarly discontinue functionality. However, our analyses indicate that model preBötC networks remain topologically intact even after rhythm cessation, suggesting that dynamics coupled with structural properties of the underlying network are responsible for rhythm cessation. Simulations show that cumulative cellular ablations diminish the number of neurons that can be recruited to spike per unit time. When the recruitment rate drops below 1 neuron/ms the network stops spontaneous rhythmic activity. Neurons that play pre-eminent roles in rhythmogenesis include those that commence spiking during the quiescent phase between respiratory bursts and those with a high number of incoming synapses, which both play key roles in recruitment, i.e., recurrent excitation leading to network bursts. Selectively ablating neurons with many incoming synapses impairs recurrent excitation and stops spontaneous rhythmic activity and motor output with lower ablation tallies compared with random deletions. This study provides a theoretical framework for the operating mechanism of mammalian central pattern generator networks and their susceptibility to loss-of-function in the case of disease or neurodegeneration.

Disassociation of verbal learning and hippocampal volume in type 2 diabetes and major depression.

OBJECTIVE: The purpose of this study was to examine the relationship between verbal learning and memory performance and hippocampal volume in subjects with co-morbid type 2 diabetes and major depression compared with healthy control subjects and subjects with type 2 diabetes alone. METHODS: Twenty four subjects with type 2 diabetes and 20 subjects with type 2 diabetes and major depression were recruited from endocrinology clinics and were compared with 32 healthy control subjects recruited from the community. Subjects were scanned on a 1.5 T GE scanner, and hippocampal volumes were measured using Freesurfer. The California Verbal Learning Test assessed learning and memory. Significant predictors of verbal learning performance (e.g., age, gender, education, blood pressure, stroke risk, hemoglobin A1c, and hippocampal volume) were determined using a stepwise linear regression. RESULTS: Subjects with diabetes and depression had significantly worse performance on verbal list learning compared with healthy control subjects. Hippocampal volume was a strong predictor of performance in healthy control subjects, and age and hippocampal volume were strong predictors in subjects with type 2 diabetes alone. Age alone was a significant predictor of verbal learning performance in subjects with diabetes and depression. CONCLUSIONS: The relationship between hippocampal volume and performance on the California Verbal Learning Test is decoupled in subjects with type 2 diabetes and major depression and this decoupling may contribute to poor verbal learning and memory performance in this study population.

Laser ablation of Dbx1 neurons in the pre-Bötzinger complex stops inspiratory rhythm and impairs output in neonatal mice.

To understand the neural origins of rhythmic behavior one must characterize the central pattern generator circuit and quantify the population size needed to sustain functionality. Breathing-related interneurons of the brainstem pre-Bötzinger complex (preBötC) that putatively comprise the core respiratory rhythm generator in mammals are derived from Dbx1-expressing precursors. Here, we show that selective photonic destruction of Dbx1 preBötC neurons in neonatal mouse slices impairs respiratory rhythm but surprisingly also the magnitude of motor output; respiratory hypoglossal nerve discharge decreased and its frequency steadily diminished until rhythm stopped irreversibly after 85±20 (mean ± SEM) cellular ablations, which corresponds to ∼15% of the estimated population. These results demonstrate that a single canonical interneuron class generates respiratory rhythm and contributes in a premotor capacity, whereas these functions are normally attributed to discrete populations. We also establish quantitative cellular parameters that govern network viability, which may have ramifications for respiratory pathology in disease states.

Threshold Digraphs.

A digraph whose degree sequence has a unique vertex labeled realization is called threshold. In this paper we present several characterizations of threshold digraphs and their degree sequences, and show these characterizations to be equivalent. Using this result, we obtain a new, short proof of the Fulkerson-Chen theorem on degree sequences of general digraphs.

White-matter tract integrity in late-life depression: associations with severity and cognition.

BACKGROUND: Although significant changes in both gray and white matter have been noted in late-life depression (LLD), the pathophysiology of implicated white-matter tracts has not been fully described. In this study we examined the integrity of specific white-matter tracts in LLD versus healthy controls (HC). METHOD: Participants aged ⩾60 years were recruited from the community. The sample included 23 clinically diagnosed individuals with LLD and 23 HC. White-matter integrity metrics [fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD) and radial diffusivity (RD)] were calculated in the bilateral cingulum and uncinate fasciculus. Depression severity was measured using the Center for Epidemiological Studies Depression Scale (CESD). Composite scores for learning and memory and executive function were created using standardized neuropsychological assessments. RESULTS: White-matter integrity was lower in LLD versus HC in the bilateral cingulum and right uncinate fasciculus (p⩽0.05). In the whole sample, depression severity correlated with integrity in the bilateral cingulum and right uncinate fasciculus (p ⩽0.05). In patients, depression severity correlated with the integrity of the left uncinate fasciculus (p = 0.03); this tract also correlated with executive function (p = 0.02). Among HC, tract integrity did not correlate with depression scores; however, learning and memory correlated with integrity of the bilateral uncinate fasciculus and bilateral cingulum; executive function correlated with the right uncinate and left cingulum (p ⩽0.05). CONCLUSIONS: White-matter tract integrity was lower in LLD than in HC and was associated with depression severity across all participants. Tract integrity was associated with cognition in both groups but more robustly among HC.

Subcortical biophysical abnormalities in patients with mood disorders.

Cortical-subcortical circuits have been implicated in the pathophysiology of mood disorders. Structural and biochemical abnormalities have been identified in patients diagnosed with mood disorders using magnetic resonance imaging-related approaches. In this study, we used magnetization transfer (MT), an innovative magnetic resonance approach, to study biophysical changes in both gray and white matter regions in cortical-subcortical circuits implicated in emotional regulation and behavior. Our study samples comprised 28 patients clinically diagnosed with major depressive disorder (MDD) and 31 non-depressed subjects of comparable age and gender. MT ratio (MTR), representing the biophysical integrity of macromolecular proteins within key components of cortical-subcortical circuits-the caudate, thalamic, striatal, orbitofrontal, anterior cingulate and dorsolateral regions-was the primary outcome measure. In our study, the MTR in the head of the right caudate nucleus was significantly lower in the MDD group when compared with the comparison group. MTR values showed an inverse relationship with age in both groups, with more widespread relationships observed in the MDD group. These data indicate that focal biophysical abnormalities in the caudate nucleus may be central to the pathophysiology of depression and critical to the cortical-subcortical abnormalities that underlie mood disorders. Depression may also accentuate age-related changes in the biophysical properties of cortical and subcortical regions. These observations have broad implications for the neuronal circuitry underlying mood disorders across the lifespan.

Effect of type of cognitive task and walking speed on cognitive-motor interference during dual-task walking.

OBJECTIVE: We aimed to determine the effect of distinctly different cognitive tasks and walking speed on cognitive-motor interference of dual-task walking. METHODS: Fifteen healthy adults performed four cognitive tasks: visuomotor reaction time (VMRT) task, word list generation (WLG) task, serial subtraction (SS) task, and the Stroop (STR) task while sitting and during walking at preferred-speed (dual-task normal walking) and slow-speed (dual-task slow-speed walking). Gait speed was recorded to determine effect on walking. Motor and cognitive costs were measured. RESULTS: Dual-task walking had a significant effect on motor and cognitive parameters. At preferred-speed, the motor cost was lowest for the VMRT task and highest for the STR task. In contrast, the cognitive cost was highest for the VMRT task and lowest for the STR task. Dual-task slow walking resulted in increased motor cost and decreased cognitive cost only for the STR task. CONCLUSIONS: Results show that the motor and cognitive cost of dual-task walking depends heavily on the type and perceived complexity of the cognitive task being performed. Cognitive cost for the STR task was low irrespective of walking speed, suggesting that at preferred-speed individuals prioritize complex cognitive tasks requiring higher attentional and processing resources over walking. While performing VMRT task, individuals preferred to prioritize more complex walking task over VMRT task resulting in lesser motor cost and increased cognitive cost for VMRT task. Furthermore, slow walking can assist in diverting greater attention towards complex cognitive tasks, improving its performance while walking.

A Phase-1b study of tivantinib (ARQ 197) in adult patients with hepatocellular carcinoma and cirrhosis.

BACKGROUND: The mesenchymal-epithelial transition factor (MET) receptor is dysregulated in hepatocellular carcinoma (HCC), and tivantinib (ARQ 197) is an oral, selective, MET inhibitor. METHODS: This Phase-1b study assessed tivantinib safety as primary objective in patients with previously treated HCC and Child-Pugh A or B liver cirrhosis. Patients received oral tivantinib 360 mg twice daily until disease progression or unacceptable toxicity. RESULTS: Among 21 HCC patients, common drug-related adverse events (AEs) were neutropaenia, anaemia, asthenia, leucopaenia, anorexia, diarrhoea, and fatigue. No drug-related worsening of liver function or performance status occurred, but one Child-Pugh B patient experienced drug-related bilirubin increase. Four patients had drug-related serious AEs, including one neutropaenia-related death. Haematologic toxicities were more frequent than in previous tivantinib studies but were manageable with prompt therapy. Best response was stable disease (median, 5.3 months) in 9 of 16 evaluable patients (56%). Median time to progression was 3.3 months. CONCLUSION: Tivantinib demonstrated a manageable safety profile and preliminary antitumour activity in patients with HCC and Child-Pugh A or B cirrhosis.

Reduction of calcium release site models via moment fitting of phase-type distributions.

Models of calcium (Ca(2 +)) release sites derived from continuous-time Markov chain (CTMC) models of intracellular Ca(2 +) channels exhibit collective gating reminiscent of the experimentally observed phenomenon of Ca(2 +) puffs and sparks. In order to overcome the state-space explosion that occurs in compositionally defined Ca(2 +) release site models, we have implemented an automated procedure for model reduction that replaces aggregated states of the full release site model with much simpler CTMCs that have similar within-group phase-type sojourn times and inter-group transitions. Error analysis based on comparison of full and reduced models validates the method when applied to release site models composed of 20 three-state channels that are both activated and inactivated by Ca(2 +). Although inspired by existing techniques for fitting moments of phase-type distributions, the automated reduction method for compositional Ca(2 +) release site models is unique in several respects and novel in this biophysical context.

Effect of node-degree correlation on synchronization of identical pulse-coupled oscillators.

We explore the effect of correlations between the in and out degrees of random directed networks on the synchronization of identical pulse-coupled oscillators. Numerical experiments demonstrate that the proportion of initial conditions resulting in a globally synchronous state (prior to a large but finite time) is an increasing function of node-degree correlation. For those networks observed to globally synchronize, both the mean and standard deviation of time to synchronization are decreasing functions of node-degree correlation. Pulse-coupled oscillator networks with negatively correlated node degree often exhibit multiple coherent attracting states, with trajectories performing fast transitions between them. These effects of node-degree correlation on dynamics of pulse-coupled oscillators are consistent with aspects of network topology (e.g., the effect of node-degree correlation on the eigenvalues of the Laplacian matrix) that have been shown to affect synchronization in other contexts.

Markov chain models of coupled calcium channels: Kronecker representations and iterative solution methods.

Mathematical models of calcium release sites derived from Markov chain models of intracellular calcium channels exhibit collective gating reminiscent of the experimentally observed phenomenon of stochastic calcium excitability (i.e., calcium puffs and sparks). Calcium release site models are stochastic automata networks that involve many functional transitions, that is, the transition probabilities of each channel depend on the local calcium concentration and thus the state of the other channels. We present a Kronecker-structured representation for calcium release site models and perform benchmark stationary distribution calculations using both exact and approximate iterative numerical solution techniques that leverage this structure. When it is possible to obtain an exact solution, response measures such as the number of channels in a particular state converge more quickly using the iterative numerical methods than occupation measures calculated via Monte Carlo simulation. In particular, multi-level methods provide excellent convergence with modest additional memory requirements for the Kronecker representation of calcium release site models. When an exact solution is not feasible, iterative approximate methods based on the power method may be used, with performance similar to Monte Carlo estimates. This suggests approximate methods with multi-level iterative engines as a promising avenue of future research for large-scale calcium release site models.

Markov chain models of coupled intracellular calcium channels: Kronecker structured representations and benchmark stationary distribution calculations.

Mathematical models of calcium release sites derived from Markov chain models of intracellular calcium channels exhibit collective gating reminiscent of the experimentally observed phenomenon of stochastic calcium excitability (i.e., calcium puffs and sparks). We present a Kronecker structured representation for calcium release site models and perform benchmark stationary distribution calculations using numerical iterative solution techniques that leverage this structure. In this context we find multi-level methods and certain preconditioned projection methods superior to simple Gauss-Seidel type iterations. Response measures such as the number of channels in a particular state converge more quickly using these numerical iterative methods than occupation measures calculated via Monte Carlo simulation.

Modeling vocal fold motion with a hydrodynamic semicontinuum model.

Vocal fold (VF) motion is a fundamental process in voice production, and is also a challenging problem for numerical computation because the VF dynamics depend on nonlinear coupling of air flow with the response of elastic channels (VF), which undergo opening and closing, and induce internal flow separation. The traditional modeling approach makes use of quasisteady flow approximation or Bernoulli's law which ignores air compressibility, and is known to be invalid during VF opening. A hydrodynamic semicontinuum system for VF motion is presented. The airflow is modeled by a modified quasi-one-dimensional Euler system with coupling to VF velocity. The VF is modeled by a lumped two mass system with a built-in geometric condition on flow separation. The modified Euler system contains the Bernoulli's law as a special case, and is derivable from the two-dimensional compressible Navier-Stokes equations in the inviscid limit. The computational domain contains also solid walls next to VFs (flexible walls). It is shown numerically that several salient features of VFs are captured, especially transients such as the double peaks of the driving subglottal pressures at the opening and the closing stages of VF motion consistent with fully resolved two-dimensional direct simulations, and experimental data. The system is much simpler to compute than a VF model based on two-dimensional Navier-Stokes system.

Different underlying mechanisms for deficits in concept formation in dementia.

We investigated the different mechanisms that may underlie deficits in verbal concept formation among patients with Alzheimer's disease (AD) and ischaemic vascular dementia (IVD) associated with periventricular and deep white matter alterations. Concept formation was assessed with the WAIS-R Similarities subtest (SIM). Two types of errors were re-coded from the 0-point responses as scored by the WAIS-R manual. In set errors (e.g., dog-lion "they're alive") were coded when patients reported a very vague superordinate concept for the word pair. Out of set responses (e.g., dog-lion "the lion roars and the dog barks") were coded when a response was clearly out of mental set, i.e., when participants were unable to provide a superordinate concept for the word pair. Between-group comparisons demonstrated no difference in SIM test performance according to the scoring system described in the WAIS-R manual. Nonetheless, AD patients produced a greater proportion of in set errors, while IVD patients produced a greater proportion of out of set errors. Out of set errors were highly associated with measures of executive function, while in set errors were associated with measures related to delayed recognition memory and semantic intrusion errors. We conclude that the underlying deficits that contribute to poor concept formation differ between AD and IVD patients. In IVD impaired concept formation is related to deficits in the executive systems necessary to monitor responses and sustain mental set. In AD, by contrast, the deficit appears to be secondary to impaired verbal response selection.