Acute Neuropixels recordings in the marmoset monkey.

High-density linear probes, like Neuropixels, provide an unprecedented opportunity to understand how neural populations within specific laminar compartments contribute to behavior. Marmoset monkeys, unlike macaque monkeys, have a lissencephalic (smooth) cortex that enables recording perpendicular to the cortical surface, thus making them an ideal animal model for studying laminar computations. Here we present a method for acute Neuropixels recordings in the common marmoset (Callithrix jacchus). The approach replaces the native dura with an artificial silicon-based dura that grants visual access to the cortical surface, which is helpful in avoiding blood vessels, ensures perpendicular penetrations, and could be used in conjunction with optical imaging or optogenetic techniques. The chamber housing the artificial dura is simple to maintain with minimal risk of infection and could be combined with semi-chronic microdrives and wireless recording hardware. This technique enables repeated acute penetrations over a period of several months. With occasional removal of tissue growth on the pial surface, recordings can be performed for a year or more. The approach is fully compatible with Neuropixels probes, enabling the recording of hundreds of single neurons distributed throughout the cortical column.Significance statement The cerebral cortex of the macaque monkey is extensively folded, which poses a major problem for studying laminar computations in many cortical areas. Marmosets, however, have a smooth brain that allows for simultaneous recordings from all layers of the cortex in areas that are buried deep in sulci in the macaque. In this manuscript, we describe an artificial dura system that utilizes the state-of-the-art in high-density probes, Neuropixels. This system enables us to easily insert multiple Neuropixels into the marmoset cortex normal to the cortical surface permitting repeated laminar recordings for up to a year or more.

Controlled Stiffness of Direct-Write, Near-Field Electrospun Gelatin Fibers Generates Differences in Tenocyte Morphology and Gene Expression.

Tendinopathy is a leading cause of mobility issues. Currently, the cell-matrix interactions involved in the development of tendinopathy are not fully understood. In vitro tendon models provide a unique tool for addressing this knowledge gap as they permit fine control over biochemical, micromechanical, and structural aspects of the local environment to explore cell-matrix interactions. In this study, direct-write, near-field electrospinning of gelatin solution was implemented to fabricate micron-scale fibrous scaffolds that mimic native collagen fiber size and orientation. The stiffness of these fibrous scaffolds was found to be controllable between 1 MPa and 8 MPa using different crosslinking methods (EDC, DHT, DHT+EDC) or through altering the duration of crosslinking with EDC (1 h to 24 h). EDC crosslinking provided the greatest fiber stability, surviving up to 3 weeks in vitro. Differences in stiffness resulted in phenotypic changes for equine tenocytes with low stiffness fibers (∼1 MPa) promoting an elongated nuclear aspect ratio while those on high stiffness fibers (∼8 MPa) were rounded. High stiffness fibers resulted in the upregulation of matrix metalloproteinase (MMPs) and proteoglycans (possible indicators for tendinopathy) relative to low stiffness fibers. These results demonstrate the feasibility of direct-written gelatin scaffolds as tendon in vitro models and provide evidence that matrix mechanical properties may be crucial factors in cell-matrix interactions during tendinopathy formation.

Safety and Virologic Impact of Haploidentical NK Cells Plus Interleukin 2 or N-803 in HIV Infection.

BACKGROUND: Natural killer (NK) cells are dysfunctional in chronic human immunodeficiency virus (HIV) infection as they are not able to clear virus. We hypothesized that an infusion of NK cells, supported by interleukin 2 (IL-2) or IL-15, could decrease virus-producing cells in the lymphatic tissues. METHODS: We conducted a phase 1 pilot study in 6 persons with HIV (PWH), where a single infusion of haploidentical related donor NK cells was given plus either IL-2 or N-803 (an IL-15 superagonist). RESULTS: The approach was well tolerated with no unexpected adverse events. We did not pretreat recipients with cyclophosphamide or fludarabine to "make immunologic space," reasoning that PWH on stable antiretroviral treatment remain T-cell depleted in lymphatic tissues. We found donor cells remained detectable in blood for up to 8 days (similar to what is seen in cancer pretreatment with lymphodepleting chemotherapy) and in the lymph nodes and rectum up to 28 days. There was a moderate decrease in the frequency of viral RNA-positive cells in lymph nodes. CONCLUSIONS: There was a moderate decrease in HIV-producing cells in lymph nodes. Further studies are warranted to determine the impact of healthy NK cells on HIV reservoirs and if restoring NK-cell function could be part of an HIV cure strategy. Clinical Trials Registration. NCT03346499 and NCT03899480.

Horizontal cortical connections shape intrinsic traveling waves into feature-selective motifs that regulate perceptual sensitivity.

Intrinsic, ongoing fluctuations of cortical activity form traveling waves that modulate the gain of sensory-evoked responses and perceptual sensitivity. Several lines of evidence suggest that intrinsic traveling waves (iTWs) may arise, in part, from the coordination of synaptic activity through the recurrent horizontal connectivity within cortical areas, which include long range patchy connections that link neurons with shared feature preferences. In a spiking network model with anatomical topology that incorporates feature-selective patchy connections, which we call the Balanced Patchy Network (BPN), we observe repeated iTWs, which we refer to as motifs. In the model, motifs stem from fluctuations in the excitability of like-tuned neurons that result from shifts in E/I balance as action potentials traverse these patchy connections. To test if feature-selective motifs occur in vivo, we examined data previously recorded using multielectrode arrays in Area MT of marmosets trained to perform a threshold visual detection task. Using a newly developed method for comparing the similarity of wave patterns we found that some iTWs can be grouped into motifs. As predicted by the BPN, many of these motifs are feature selective, exhibiting direction-selective modulations in ongoing spiking activity. Further, motifs modulate the gain of the response evoked by a target and perceptual sensitivity to the target if the target matches the preference of the motif. These results provide evidence that iTWs are shaped by the patterns of horizontal fiber projections in the cortex and that patchy connections enable iTWs to regulate neural and perceptual sensitivity in a feature selective manner.

iPSC-derived natural killer cells expressing the FcγR fusion CD64/16A can be armed with antibodies for multitumor antigen targeting.

BACKGROUND: Antibody therapies can direct natural killer (NK) cells to tumor cells, tumor-associated cells, and suppressive immune cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). This antigen-specific effector function of human NK cells is mediated by the IgG Fc receptor CD16A (FcγRIIIA). Preclinical and clinical studies indicate that increasing the binding affinity and avidity of CD16A for antibodies improves the therapeutic potential of ADCC. CD64 (FcγRI), expressed by myeloid cells but not NK cells, is the only high affinity IgG Fc receptor and is uniquely capable of stably binding to free monomeric IgG as a physiological function. We have reported on the generation of the FcγR fusion CD64/16A, consisting of the extracellular region of CD64 and the transmembrane and cytoplasmic regions from CD16A, retaining its signaling and cellular activity. Here, we generated induced pluripotent stem cell (iPSC)-derived NK (iNK) cells expressing CD64/16A as a potential adoptive NK cell therapy for increased ADCC potency. METHODS: iPSCs were engineered to express CD64/16A as well as an interleukin (IL)-15/IL-15Rα fusion (IL-15RF) protein and differentiated into iNK cells. iNK cells and peripheral blood NK cells were expanded using irradiated K562-mbIL21-41BBL feeder cells and examined. NK cells, ovarian tumor cell lines, and therapeutic monoclonal antibodies were used to assess ADCC in vitro, performed by a DELFIA EuTDA assay or in real-time by IncuCyte assays, and in vivo. For the latter, we developed a xenograft mouse model with high circulating levels of human IgG for more physiological relevance. RESULTS: We demonstrate that (1) iNK-CD64/16A cells after expansion or thaw from cryopreservation can be coupled to therapeutic antibodies, creating armed iNK cells; (2) antibody-armed iNK-CD64/16A cells can be redirected by added antibodies to target new tumor antigens, highlighting additional potential of these cells; (3) cytokine-autonomous activity by iNK-CD64/16A cells engineered to express IL-15RF; and that (4) antibody-armed iNK-CD64/16A cells thawed from cryopreservation are capable of sustained and robust ADCC in vitro and in vivo, as determined by using a modified tumor xenograft model with high levels of competing human IgG. CONCLUSIONS: iNK cells expressing CD64/16A provide an off-the-shelf multiantigen targeting platform to address tumor heterogeneity and mitigate antigen escape.

Computational NMR investigation of mixed-metal (Al,Sc)-MIL-53 and its phase transitions.

Compositionally complex metal-organic frameworks (MOFs) have properties that depend on local structure that is often difficult to characterise. In this paper a density functional theory (DFT) computational study of mixed-metal (Al,Sc)-MIL-53, a flexible MOF with several different forms, was used to calculate the relative energetics of these forms and to predict NMR parameters that can be used to evaluate whether solid-state NMR spectroscopy can be used to differentiate, identify and characterise the forms adopted by mixed-metal MOFs of different composition. The NMR parameters can also be correlated with structural features in the different forms, giving fundamental insight into the nature and origin of the interactions that affect nuclear spins. Given the complexity of advanced NMR experiments required, and the potential need for expensive and difficult isotopic enrichment, the computational work is invaluable in predicting which experiments and approaches are likely to give the most information on the disorder, local structure and pore forms of these mixed-metal MOFs.

Intervertebral Disc Elastography to Relate Shear Modulus and Relaxometry in Compression and Bending.

Intervertebral disc degeneration is the most recognized cause of low back pain, characterized by the decline of tissue structure and mechanics. Image-based mechanical parameters (e.g., strain, stiffness) may provide an ideal assessment of disc function that is lost with degeneration but unfortunately remains underdeveloped. Moreover, it is unknown whether strain or stiffness of the disc may be predicted by MRI relaxometry (e.g. T1 or T2), an increasingly accepted quantitative measure of disc structure. In this study, we quantified T1 and T2 relaxation times and in-plane strains using displacement-encoded MRI within the disc under physiological levels of compression and bending. We then estimated shear modulus in orthogonal image planes and compared these values to relaxation times and strains within regions of the disc. Intratissue strain depended on the loading mode, and shear modulus in the nucleus pulposus was typically an order of magnitude lower than the annulus fibrosis, except in bending, where the apparent stiffness depended on the loading. Relative shear moduli estimated from strain data derived under compression generally did not correspond with those from bending experiments, with no correlations in the sagittal plane and only 4 of 15 regions correlated in the coronal plane, suggesting that future inverse models should incorporate multiple loading conditions. Strain imaging and strain-based estimation of material properties may serve as imaging biomarkers to distinguish healthy and diseased discs. Additionally, image-based elastography and relaxometry may be viewed as complementary measures of disc structure and function to assess degeneration in longitudinal studies.

The double-drift illusion biases the marmoset oculomotor system.

The double-drift illusion has two unique characteristics: The error between the perceived and physical position of the stimulus grows over time, and saccades to the moving target land much closer to the physical than the perceived location. These results suggest that the perceptual and saccade targeting systems integrate visual information over different time scales. Functional imaging studies in humans have revealed several potential cortical areas of interest, including the prefrontal cortex. However, we currently lack an animal model to study the neural mechanisms of location perception that underlie the double-drift illusion. To fill this gap, we trained two marmoset monkeys to fixate and then saccade to the double-drift stimulus. In line with human observers for radial double-drift trajectories with fast internal motion, we find that saccade endpoints show a significant bias that is, nevertheless, smaller than the bias seen in human perceptual reports. This bias is modulated by changes in the external and internal speeds of the stimulus. These results demonstrate that the saccade targeting system of the marmoset monkey is influenced by the double-drift illusion.

Exploring cation distribution in ion-exchanged Al,Ga-containing metal-organic frameworks using 17O NMR spectroscopy.

A mixed-metal metal-organic framework, (Al,Ga)-MIL-53, synthesised by post-synthetic ion exchange has been investigated using solid-state nuclear magnetic resonance (NMR) spectroscopy, microscopy and energy dispersive X-ray (EDX) spectroscopy. 17O enrichment during the ion-exchange process enables site specific information on the metal distribution to be obtained. Within this work two ion-exchange processes have been explored. In the first approach (exchange of metals in the framework with dissolved metal salts), 17O NMR spectroscopy reveals the formation of crystallites with a core-shell structure, where the cation exchange takes place on the surface of these materials forming a shell with a roughly equal ratio of Al3+ and Ga3+. For the second approach (exchange of metals between two frameworks), no core-shell structure is observed, and instead crystallites containing a majority of Al3+ are obtained with lower levels of Ga3+. Noticeably, these particles show little variation in the metal cation distribution between crystallites, a result not previously observed for bulk (Al,Ga)-MIL-53 materials. In all cases where ion exchange has taken place NMR spectroscopy reveals a slight preference for clustering of like cations.

Pressure-induced postsynthetic cluster anion substitution in a MIL-53 topology scandium metal-organic framework.

Postsynthetic modification of metal-organic frameworks (MOFs) has proven to be a hugely powerful tool to tune physical properties and introduce functionality, by exploiting reactive sites on both the MOF linkers and their inorganic secondary building units (SBUs), and so has facilitated a wide range of applications. Studies into the reactivity of MOF SBUs have focussed solely on removal of neutral coordinating solvents, or direct exchange of linkers such as carboxylates, despite the prevalence of ancillary charge-balancing oxide and hydroxide ligands found in many SBUs. Herein, we show that the µ2-OH ligands in the MIL-53 topology Sc MOF, GUF-1, are labile, and can be substituted for µ2-OCH3 units through reaction with pore-bound methanol molecules in a very rare example of pressure-induced postsynthetic modification. Using comprehensive solid-state NMR spectroscopic analysis, we show an order of magnitude increase in this cluster anion substitution process after exposing bulk samples suspended in methanol to a pressure of 0.8 GPa in a large volume press. Additionally, single crystals compressed in diamond anvil cells with methanol as the pressure-transmitting medium have enabled full structural characterisation of the process across a range of pressures, leading to a quantitative single-crystal to single-crystal conversion at 4.98 GPa. This unexpected SBU reactivity - in this case chemisorption of methanol - has implications across a range of MOF chemistry, from activation of small molecules for heterogeneous catalysis to chemical stability, and we expect cluster anion substitution to be developed into a highly convenient novel method for modifying the internal pore surface and chemistry of a range of porous materials.

Waves traveling over a map of visual space can ignite short-term predictions of sensory input.

Recent analyses have found waves of neural activity traveling across entire visual cortical areas in awake animals. These traveling waves modulate the excitability of local networks and perceptual sensitivity. The general computational role of these spatiotemporal patterns in the visual system, however, remains unclear. Here, we hypothesize that traveling waves endow the visual system with the capacity to predict complex and naturalistic inputs. We present a network model whose connections can be rapidly and efficiently trained to predict individual natural movies. After training, a few input frames from a movie trigger complex wave patterns that drive accurate predictions many frames into the future solely from the network's connections. When the recurrent connections that drive waves are randomly shuffled, both traveling waves and the ability to predict are eliminated. These results suggest traveling waves may play an essential computational role in the visual system by embedding continuous spatiotemporal structures over spatial maps.

Spike-phase coupling patterns reveal laminar identity in primate cortex.

The cortical column is one of the fundamental computational circuits in the brain. In order to understand the role neurons in different layers of this circuit play in cortical function it is necessary to identify the boundaries that separate the laminar compartments. While histological approaches can reveal ground truth they are not a practical means of identifying cortical layers in vivo. The gold standard for identifying laminar compartments in electrophysiological recordings is current-source density (CSD) analysis. However, laminar CSD analysis requires averaging across reliably evoked responses that target the input layer in cortex, which may be difficult to generate in less well-studied cortical regions. Further, the analysis can be susceptible to noise on individual channels resulting in errors in assigning laminar boundaries. Here, we have analyzed linear array recordings in multiple cortical areas in both the common marmoset and the rhesus macaque. We describe a pattern of laminar spike-field phase relationships that reliably identifies the transition between input and deep layers in cortical recordings from multiple cortical areas in two different non-human primate species. This measure corresponds well to estimates of the location of the input layer using CSDs, but does not require averaging or specific evoked activity. Laminar identity can be estimated rapidly with as little as a minute of ongoing data and is invariant to many experimental parameters. This method may serve to validate CSD measurements that might otherwise be unreliable or to estimate laminar boundaries when other methods are not practical.

Advances in NK cell therapy for brain tumors.

Despite advances in treatment regimens that comprise surgery, chemotherapy, and radiation, outcome of many brain tumors remains dismal, more so when they recur. The proximity of brain tumors to delicate neural structures often precludes complete surgical resection. Toxicity and long-term side effects of systemic therapy remain a concern. Novel therapies are warranted. The field of NK cell-based cancer therapy has grown exponentially and currently constitutes a major area of immunotherapy innovation. This provides a new avenue for the treatment of cancerous lesions in the brain. In this review, we explore the mechanisms by which the brain tumor microenvironment suppresses NK cell mediated tumor control, and the methods being used to create NK cell products that subvert immune suppression. We discuss the pre-clinical studies evaluating NK cell-based immunotherapies that target several neuro-malignancies and highlight advances in molecular imaging of NK cells that allow monitoring of NK cell-based therapeutics. We review current and ongoing NK cell based clinical trials in neuro-oncology.

Towards a standardized multi-tissue decellularization protocol for the derivation of extracellular matrix materials.

The goal of tissue decellularization is to efficiently remove unwanted cellular components, such as DNA and cellular debris, while retaining the complex structural and molecular milieu within the extracellular matrix (ECM). Decellularization protocols to date are centered on customized tissue-specific and lab-specific protocols that involve consecutive manual steps which results in variable and protocol-specific ECM material. The differences that result from the inconsistent protocols between decellularized ECMs affect consistency across batches, limit comparisons between results obtained from different laboratories, and could limit the transferability of the material for consistent laboratory or clinical use. The present study is the first proof-of-concept towards the development of a standardized protocol that can be used to derive multiple ECM biomaterials (powders and hydrogels) via a previously established automated system. The automated decellularization method developed by our group was used due to its short decellularization time (4 hours) and its ability to reduce batch-to-batch variability. The ECM obtained using this first iteration of a unified protocol was able to produce ECM hydrogels from skin, lung, muscle, tendons, cartilage, and laryngeal tissues. All hydrogels formed in this study were cytocompatible and showed gelation and rheological properties consistent with previous ECM hydrogels. The ECMs also showed unique proteomic composition. The present study represents the first step towards developing standardized protocols that can be used on multiple tissues in a fast, scalable, and reproducible manner.

Acute Neuropixels recordings in the marmoset monkey.

High-density linear probes, like Neuropixels, provide an unprecedented opportunity to understand how neural populations within specific laminar compartments contribute to behavior. Marmoset monkeys, unlike macaque monkeys, have a lissencephalic (smooth) cortex that enables recording perpendicular to the cortical surface, thus making them an ideal animal model for studying laminar computations. Here we present a method for acute Neuropixels recordings in the common marmoset (Callithrix jacchus). The approach replaces the native dura with an artificial silicon-based dura that grants visual access to the cortical surface, which is helpful in avoiding blood vessels, ensures perpendicular penetrations, and could be used in conjunction with optical imaging or optogenetic techniques. The chamber housing the artificial dura is simple to maintain with minimal risk of infection and could be combined with semi-chronic microdrives and wireless recording hardware. This technique enables repeated acute penetrations over a period of several months. With occasional removal of tissue growth on the pial surface, recordings can be performed for a year or more. The approach is fully compatible with Neuropixels probes, enabling the recording of hundreds of single neurons distributed throughout the cortical column.

Quadruple gene-engineered natural killer cells enable multi-antigen targeting for durable antitumor activity against multiple myeloma.

Allogeneic natural killer (NK) cell adoptive transfer is a promising treatment for several cancers but is less effective for the treatment of multiple myeloma. In this study, we report on quadruple gene-engineered induced pluripotent stem cell (iPSC)-derived NK cells designed for mass production from a renewable source and for dual targeting against multiple myeloma through the introduction of an NK cell-optimized chimeric antigen receptor (CAR) specific for B cell maturation antigen (BCMA) and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity when combined with therapeutic anti-CD38 antibodies. Additionally, these cells express a membrane-bound interleukin-15 fusion molecule to enhance function and persistence along with knock out of CD38 to prevent antibody-mediated fratricide and enhance NK cell metabolic fitness. In various preclinical models, including xenogeneic adoptive transfer models, quadruple gene-engineered NK cells consistently demonstrate durable antitumor activity independent of exogenous cytokine support. Results presented here support clinical translation of this off-the-shelf strategy for effective treatment of multiple myeloma.

Dual antigen-targeted off-the-shelf NK cells show durable response and prevent antigen escape in lymphoma and leukemia.

Substantial numbers of B cell leukemia and lymphoma patients relapse due to antigen loss or heterogeneity after anti-CD19 chimeric antigen receptor (CAR) T cell therapy. To overcome antigen escape and address antigen heterogeneity, we engineered induced pluripotent stem cell-derived NK cells to express both an NK cell-optimized anti-CD19 CAR for direct targeting and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity. In addition, we introduced a membrane-bound IL-15/IL-15R fusion protein to promote in vivo persistence. These engineered cells, termed iDuo NK cells, displayed robust CAR-mediated cytotoxic activity that could be further enhanced with therapeutic antibodies targeting B cell malignancies. In multiple in vitro and xenogeneic adoptive transfer models, iDuo NK cells exhibited robust anti-lymphoma activity. Furthermore, iDuo NK cells effectively eliminated both CD19+ and CD19- lymphoma cells and displayed a unique propensity for targeting malignant cells over healthy cells that expressed CD19, features not achievable with anti-CAR19 T cells. iDuo NK cells combined with therapeutic antibodies represent a promising approach to prevent relapse due to antigen loss and tumor heterogeneity in patients with B cell malignancies.

The Paradox of Time in Dynamic Causal Systems.

Recent work has shown that people use temporal information including order, delay, and variability to infer causality between events. In this study, we build on this work by investigating the role of time in dynamic systems, where causes take continuous values and also continually influence their effects. Recent studies of learning in these systems explored short interactions in a setting with rapidly evolving dynamics and modeled people as relying on simpler, resource-limited strategies to grapple with the stream of information. A natural question that arises from such an account is whether interacting with systems that unfold more slowly might reduce the systematic errors that result from these strategies. Paradoxically, we find that slowing the task indeed reduced the frequency of one type of error, albeit at the cost of increasing the overall error rate. To explain these results we posit that human learners analyze continuous dynamics into discrete events and use the observed relationships between events to draw conclusions about causal structure. We formalize this intuition in terms of a novel Causal Event Abstraction model and show that this model indeed captures the observed pattern of errors. We comment on the implications these results have for causal cognition.