In vitro production of infectious Plasmodium falciparum sporozoites.

An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.

Focused ultrasound excites action potentials in mammalian peripheral neurons in part through the mechanically gated ion channel PIEZO2.

Neurons of the peripheral nervous system (PNS) are tasked with diverse roles, from encoding touch, pain, and itch to interoceptive control of inflammation and organ physiology. Thus, technologies that allow precise control of peripheral nerve activity have the potential to regulate a wide range of biological processes. Noninvasive modulation of neuronal activity is an important translational application of focused ultrasound (FUS). Recent studies have identified effective strategies to modulate brain circuits; however, reliable parameters to control the activity of the PNS are lacking. To develop robust noninvasive technologies for peripheral nerve modulation, we employed targeted FUS stimulation and electrophysiology in mouse ex vivo skin-saphenous nerve preparations to record the activity of individual mechanosensory neurons. Parameter space exploration showed that stimulating neuronal receptive fields with high-intensity, millisecond FUS pulses reliably and repeatedly evoked one-to-one action potentials in all peripheral neurons recorded. Interestingly, when neurons were classified based on neurophysiological properties, we identified a discrete range of FUS parameters capable of exciting all neuronal classes, including myelinated A fibers and unmyelinated C fibers. Peripheral neurons were excited by FUS stimulation targeted to either cutaneous receptive fields or peripheral nerves, a key finding that increases the therapeutic range of FUS-based peripheral neuromodulation. FUS elicited action potentials with millisecond latencies compared with electrical stimulation, suggesting ion channel­mediated mechanisms. Indeed, FUS thresholds were elevated in neurons lacking the mechanically gated channel PIEZO2. Together, these results demonstrate that transcutaneous FUS drives peripheral nerve activity by engaging intrinsic mechanotransduction mechanisms in neurons [B. U. Hoffman, PhD thesis, (2019)].

Computation predicts rapidly adapting mechanotransduction currents cannot account for tactile encoding in Merkel cell-neurite complexes.

Distinct firing properties among touch receptors are influenced by multiple, interworking anatomical structures. Our understanding of the functions and crosstalk of Merkel cells and their associated neurites-the end organs of slowly adapting type I (SAI) afferents-remains incomplete. Piezo2 mechanically activated channels are required both in Merkel cells and in sensory neurons for canonical SAI responses in rodents; however, a central unanswered question is how rapidly inactivating currents give rise to sustained action potential volleys in SAI afferents. The computational model herein synthesizes mechanotransduction currents originating from Merkel cells and neurites, in context of skin mechanics and neural dynamics. Its goal is to mimic distinct spike firing patterns from wildtype animals, as well as Atoh1 knockout animals that completely lack Merkel cells. The developed generator function includes a Merkel cell mechanism that represents its mechanotransduction currents and downstream voltage-activated conductances (slower decay of current) and a neurite mechanism that represents its mechanotransduction currents (faster decay of current). To mimic sustained firing in wildtype animals, a longer time constant was needed than the 200 ms observed for mechanically activated membrane depolarizations in rodent Merkel cells. One mechanism that suffices is to introduce an ultra-slowly inactivating current, with a time constant on the order of 1.7 s. This mechanism may drive the slow adaptation of the sustained response, for which the skin's viscoelastic relaxation cannot account. Positioned within the sensory neuron, this source of current reconciles the physiology and anatomical characteristics of Atoh1 knockout animals.

The cellular basis of mechanosensation in mammalian tongue.

Mechanosensory neurons that innervate the tongue provide essential information to guide feeding, speech, and social grooming. We use in vivo calcium imaging of mouse trigeminal ganglion neurons to identify functional groups of mechanosensory neurons innervating the anterior tongue. These sensory neurons respond to thermal and mechanical stimulation. Analysis of neuronal activity patterns reveal that most mechanosensory trigeminal neurons are tuned to detect moving stimuli across the tongue. Using an unbiased, multilayer hierarchical clustering approach to classify pressure-evoked activity based on temporal response dynamics, we identify five functional classes of mechanosensory neurons with distinct force-response relations and adaptation profiles. These populations are tuned to detect different features of touch. Molecular markers of functionally distinct clusters are identified by analyzing cluster representation in genetically marked neuronal subsets. Collectively, these studies provide a platform for defining the contributions of functionally distinct mechanosensory neurons to oral behaviors crucial for survival in mammals.

Significant relaxation of SARS-CoV-2-targeted non-pharmaceutical interventions may result in profound mortality: A New York state modelling study.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is the most significant global health crisis of the 21st century. The aim of this study was to develop a model to simulate the effect of undocumented infections, seasonal infectivity, immunity, and non-pharmaceutical interventions (NPIs) on the transmission, morbidity, and mortality of SARS-CoV-2 in New York State (NYS) based on data collected between March 4 and April 28, 2020. Simulations predict that undocumented infections significantly contribute to infectivity, NPIs are effective in reducing morbidity and mortality, and relaxation >50% of NPIs from initial lock-down levels may result in tens-of-thousands more deaths. Endemic infection is likely to occur in the absence of sustained immunity. As a result, until an effective vaccine or other effective pharmaceutical intervention is developed, the risks of significantly reducing NPIs should be carefully considered. This study employs modelling to simulate fundamental characteristics of SARS-CoV-2 transmission, which can help policymakers navigate combating this virus in the coming years.

Significant Relaxation of SARS-CoV-2-Targeted Non-Pharmaceutical Interventions Will Result in Profound Mortality: A New York State Modelling Study.

Severe acute respiratory syndrome-coronavirus 2 (SARS CoV 2) is the most significant global health crisis of the 21st century. The aim of this study was to develop a model to estimate the effect of undocumented infections, seasonal infectivity, immunity, and non-pharmaceutical interventions (NPIs), such as social distancing, on the transmission, morbidity, and mortality of SARS-CoV-2 in New York State (NYS). Simulations revealed dramatic infectivity driven by undocumented infections, and a peak basic reproductive number in NYS of 5.7. NPIs have been effective, and relaxation >50% will result in tens-of-thousands more deaths. Endemic infection is likely to occur in the absence of profound sustained immunity. As a result, until an effective vaccine or other effective pharmaceutical intervention is developed, it will be critical to not reduce NPIs >50% below current levels. This study establishes fundamental characteristics of SARS CoV 2 transmission, which can help policymakers navigate combating this virus in the coming years.

Merkel Cells Activate Sensory Neural Pathways through Adrenergic Synapses.

Epithelial-neuronal signaling is essential for sensory encoding in touch, itch, and nociception; however, little is known about the release mechanisms and neurotransmitter receptors through which skin cells govern neuronal excitability. Merkel cells are mechanosensory epidermal cells that have long been proposed to activate neuronal afferents through chemical synaptic transmission. We employed a set of classical criteria for chemical neurotransmission as a framework to test this hypothesis. RNA sequencing of adult mouse Merkel cells demonstrated that they express presynaptic molecules and biosynthetic machinery for adrenergic transmission. Moreover, live-cell imaging directly demonstrated that Merkel cells mediate activity- and VMAT-dependent release of fluorescent catecholamine neurotransmitter analogs. Touch-evoked firing in Merkel-cell afferents was inhibited either by pre-synaptic silencing of SNARE-mediated vesicle release from Merkel cells or by neuronal deletion of ß2-adrenergic receptors. Together, these results identify both pre- and postsynaptic mechanisms through which Merkel cells excite mechanosensory afferents to encode gentle touch. VIDEO ABSTRACT.

The signaling lipid sphingosine 1-phosphate regulates mechanical pain.

Somatosensory neurons mediate responses to diverse mechanical stimuli, from innocuous touch to noxious pain. While recent studies have identified distinct populations of A mechanonociceptors (AMs) that are required for mechanical pain, the molecular underpinnings of mechanonociception remain unknown. Here, we show that the bioactive lipid sphingosine 1-phosphate (S1P) and S1P Receptor 3 (S1PR3) are critical regulators of acute mechanonociception. Genetic or pharmacological ablation of S1PR3, or blockade of S1P production, significantly impaired the behavioral response to noxious mechanical stimuli, with no effect on responses to innocuous touch or thermal stimuli. These effects are mediated by fast-conducting A mechanonociceptors, which displayed a significant decrease in mechanosensitivity in S1PR3 mutant mice. We show that S1PR3 signaling tunes mechanonociceptor excitability via modulation of KCNQ2/3 channels. Our findings define a new role for S1PR3 in regulating neuronal excitability and establish the importance of S1P/S1PR3 signaling in the setting of mechanical pain thresholds.

Plasmodium falciparum: effect of radiation on levels of gene transcripts in sporozoites.

Humans immunized by the bites of irradiated Plasmodium falciparum (Pf) sporozoite-infected mosquitoes are protected against malaria. Radiation attenuates the sporozoites preventing them from fully developing and replicating in hepatocytes, but the effects of radiation on gene expression in sporozoites are unknown. We used RT-PCR (35 cycles of PCR followed by densitometry) to assess the expression of ten genes in Pf sporozoites, and in sporozoites irradiated with 15,000cGy. Irradiation reduced expression substantially (>60%) of two DNA repair genes; moderately (30-60%) of PfUIS3, the Pf orthologue of PbUIS3, a gene up-regulated in Plasmodium berghei sporozoites and of a third DNA repair gene; and minimally (<30%) of the Pf18S ribosomal RNA, PfCSP, PfSSP2/TRAP, and PfCELTOS genes. Irradiation increased expression of PfSPATR minimally. PfLSA1 RNA was not detectable in sporozoites. These results establish that radiation of sporozoites affects gene expression levels and provide the foundation for studies to identify specific genes involved in attenuation and protective immunity.