Multiplexed bioluminescence microscopy via phasor analysis.

Bioluminescence imaging with luciferase-luciferin pairs is a well-established technique for visualizing biological processes across tissues and whole organisms. Applications at the microscale, by contrast, have been hindered by a lack of detection platforms and easily resolved probes. We addressed this limitation by combining bioluminescence with phasor analysis, a method commonly used to distinguish spectrally similar fluorophores. We built a camera-based microscope equipped with special optical filters to directly assign phasor locations to unique luciferase-luciferin pairs. Six bioluminescent reporters were easily resolved in live cells, and the readouts were quantitative and instantaneous. Multiplexed imaging was also performed over extended time periods. Bioluminescent phasor further provided direct measures of resonance energy transfer in single cells, setting the stage for dynamic measures of cellular and molecular features. The merger of bioluminescence with phasor analysis fills a long-standing void in imaging capabilities, and will bolster future efforts to visualize biological events in real time and over multiple length scales.

Perceived tactile intensity at a fixed primary afferent spike rate varies with the temporal pattern of spikes.

The perceived intensity of a vibrotactile stimulus is thought to depend on single-neuron firing rates (rate coding) and the number of active afferents (population coding). Unaddressed until now is whether the temporal relation of individual spikes also conveys information about tactile intensity. We used cutaneous electro-tactile stimulation to investigate how the temporal structure of a fixed number of spikes in a 1-s train influenced the perception of intensity. Four mean spike rates spanning the flutter and vibratory hum range (36 Hz, 60 Hz; 120 Hz, 180 Hz) were tested, with spikes grouped into a regular pattern, or bursts of 2-6 spikes spaced 3 ms apart. To link a putative neural code to perception, perceived intensity was assessed in 16 human participants (aged 20-45; 4 females) using the psychophysical paradigm of magnitude estimation. Compound sensory nerve action potentials were recorded to assess any stimulus variation in afferent recruitment. The temporal structuring of a fixed number of spikes into periodic bursts of multiple spikes altered perceived intensity as a function of burst spike count. The largest increase was seen at 36 Hz, where the bursts of six spikes were rated 2.1 times stronger than the regularly spaced spikes [95% confidence interval (CI): 1.9-2.3]. The true increase is likely larger as temporal structuring of spikes into bursts had some negative effect on afferent recruitment. We conclude that the perceived intensity can be modulated by changing temporal features of afferent discharge even when normalized for the number of recruited afferents.NEW & NOTEWORTHY Structuring a fixed number of spikes into temporal burst patterns evoke gradations of perceived intensity with burst spike count, emphasizing the importance of spike timing in primary afferents for shaping perception. This forms the basis for new strategies in communicating a range of intensity information to users of neural interfaces by simply varying the timing of spikes in nonspecific primary afferents using fixed-charge electric pulses, without requiring alterations in stimulation current or mean pulse frequency.

Generalized Bioluminescent Platform To Observe and Track Cellular Interactions.

Cell-to-cell communications are critical to biological processes ranging from embryonic development to cancer progression. Several imaging strategies have been developed to capture such interactions, but many are challenging to deploy in thick tissues and other complex environments. Here, we report a platform termed Luminescence to Observe and Track Intercellular Interactions (LOTIIS). The approach features split fragments of a luciferase enzyme that reassemble when target cells come into proximity. One fragment is secreted by "sender" cells, and the complementary piece is secreted by "receiver" cells. Split reporter assembly is facilitated by a single chain variable fragment (scFv)-peptide interaction on the receiver cell, resulting in localized light production. We demonstrate that LOTIIS can rapidly label cells in close proximity in a time- and distance-dependent fashion. The platform is also compatible with bioluminescence resonance energy transfer probes for multiplexed imaging. Collectively, these data suggest that LOTIIS will enable a variety of cellular interactions to be tracked in biological settings.

Burst gap code predictions for tactile frequency are valid across the range of perceived frequencies attributed to two distinct tactile channels.

Perceived frequency of vibrotactile stimuli can be divided into two distinctive cutaneous sensations-flutter (<60 Hz) and vibratory hum (>60 Hz), mediated by two different tactile afferent types [fast adapting type I (FA1) and fast adapting type II (FA2), respectively]. We recently demonstrated a novel form of neural coding in the human tactile system, where frequency perception of stimulus pulses grouped into periodic bursts in the flutter range depended on the duration of the silent gap between bursts, rather than the periodicity or mean impulse rate. Here, we investigated whether this interburst interval could also explain the perceived frequency of electrocutaneous pulse patterns delivered at frequencies above the flutter range. At stimulus rates of 50 to 190 pulses/s, the burst gap model correctly predicted the perceived frequency. This shows that the burst gap code represents a general coding strategy that spans the range of frequencies traditionally attributed to two different tactile channels.NEW & NOTEWORTHY We present evidence for a generalized frequency processing strategy on tactile afferent inputs that is shared across a broad range of frequencies extending beyond the flutter range, supporting the notion that spike timing has an important role in shaping tactile perception.

A Bioluminescent Sensor for Rapid Detection of PPEP-1, a Clostridioides difficile Biomarker.

Current assays for Clostridioides difficile in nonhospital settings are outsourced and time-intensive, resulting in both delayed diagnosis and quarantining of infected individuals. We designed a more rapid point-of-care assay featuring a "turn-on" bioluminescent readout of a C. difficile-specific protease, PPEP-1. NanoLuc, a bright and stable luciferase, was "caged" with a PPEP-1-responsive peptide tail that inhibited luminescence. Upon proteolytic cleavage, the peptide was released and NanoLuc activity was restored, providing a visible readout. The bioluminescent sensor detected PPEP-1 concentrations as low as 10 nM. Sensor uncaging was achieved within minutes, and signal was captured using a digital camera. Importantly, the sensor was also functional at ambient temperature and compatible with fecal material, suggesting that it can be readily deployed in a variety of settings.

Genetic Diversity and Demographic History of an Upper Hill Dipterocarp (Shorea platyclados): Implications for Conservation.

Southeast Asian rainforests at upper hill elevations are increasingly vulnerable to degradation because most lowland forest areas have been converted to different land uses. As such, understanding the genetics of upper hill species is becoming more crucial for their future management and conservation. Shorea platyclados is an important, widespread upper hill dipterocarp in Malaysia. To elucidate the genetic structure of S. platyclados and ultimately provide guidelines for a conservation strategy for this species, we carried out a comprehensive study of the genetic diversity and demographic history of S. platyclados. Twenty-seven populations of S. platyclados across its range in Malaysia were genotyped at 15 polymorphic microsatellite loci and sequenced at seven noncoding chloroplast DNA (cpDNA) regions. A total of 303 alleles were derived from the microsatellite loci, and 29 haplotypes were identified based on 2892 bp of concatenated cpDNA sequences. The populations showed moderately high genetic diversity (mean HE = 0.680 for microsatellite gene diversity and HT = 0.650 for total haplotype diversity) and low genetic differentiation (FST = 0.060). Bayesian clustering divided the studied populations into two groups corresponding to western and eastern Malaysia. Bottleneck analysis did not detect any recent bottleneck events. Extended Bayesian skyline analyses showed a model of constant size for the past population history of this species. Based on our findings, priority areas for in situ and ex situ conservation and a minimum population size are recommended for the sustainable utilization of S. platyclados.

Multiplexing intensity and frequency sensations for artificial touch by modulating temporal features of electrical pulse trains.

In neural prostheses, intensity modulation of a single channel (i.e., through a single stimulating electrode) has been achieved by increasing the magnitude or width of each stimulation pulse, which risks eliciting pain or paraesthesia; and by changing the stimulation rate, which leads to concurrent changes in perceived frequency. In this study, we sought to render a perception of tactile intensity and frequency independently, by means of temporal pulse train patterns of fixed magnitude, delivered non-invasively. Our psychophysical study exploits a previously discovered frequency coding mechanism, where the perceived frequency of stimulus pulses grouped into periodic bursts depends on the duration of the inter-burst interval, rather than the mean pulse rate or periodicity. When electrical stimulus pulses were organised into bursts, perceived intensity was influenced by the number of pulses within a burst, while perceived frequency was determined by the time between the end of one burst envelope and the start of the next. The perceived amplitude was modulated by 1.6× while perceived frequency was varied independently by 2× within the tested range (20-40 Hz). Thus, the sensation of intensity might be controlled independently from frequency through a single stimulation channel without having to vary the injected electrical current. This can form the basis for improving strategies in delivering more complex and natural sensations for prosthetic hand users.

Human Foot Outperforms the Hand in Mechanical Pain Discrimination.

Tactile discrimination has been extensively studied, but mechanical pain discrimination remains poorly characterized. Here, we measured the capacity for mechanical pain discrimination using a two-alternative forced choice paradigm, with force-calibrated indentation stimuli (Semmes-Weinstein monofilaments) applied to the hand and foot dorsa of healthy human volunteers. In order to characterize the relationship between peripheral nociceptor activity and pain perception, we recorded single-unit activity from myelinated (A) and unmyelinated (C) mechanosensitive nociceptors in the skin using microneurography. At the perceptual level, we found that the foot was better at discriminating noxious forces than the hand, which stands in contrast to that for innocuous force discrimination, where the hand performed better than the foot. This observation of superior mechanical pain discrimination on the foot compared to the hand could not be explained by the responsiveness of individual nociceptors. We found no significant difference in the discrimination performance of either the myelinated or unmyelinated class of nociceptors between skin regions. This suggests the possibility that other factors such as skin biophysics, receptor density or central mechanisms may underlie these regional differences.

Nuclear and chloroplast DNA phylogeography reveals Pleistocene divergence and subsequent secondary contact of two genetic lineages of the tropical rainforest tree species Shorea leprosula (Dipterocarpaceae) in South-East Asia.

Tropical rainforests in South-East Asia have been affected by climatic fluctuations during past glacial eras. To examine how the accompanying changes in land areas and temperature have affected the genetic properties of rainforest trees in the region, we investigated the phylogeographic patterns of a widespread dipterocarp species, Shorea leprosula. Two types of DNA markers were used: expressed sequence tag-based simple sequence repeats and chloroplast DNA (cpDNA) sequence variations. Both sets of markers revealed clear genetic differentiation between populations in Borneo and those in the Malay Peninsula and Sumatra (Malay/Sumatra). However, in the south-western part of Borneo, genetic admixture of the lineages was observed in the two marker types. Coalescent simulation based on cpDNA sequence variation suggested that the two lineages arose 0.28-0.09 million years before present and that following their divergence migration from Malay/Sumatra to Borneo strongly exceeded migration in the opposite direction. We conclude that the genetic structure of S. leprosula was largely formed during the middle Pleistocene and was subsequently modified by eastward migration across the subaerially exposed Sunda Shelf.

Phylogeographical pattern and evolutionary history of an important Peninsular Malaysian timber species, Neobalanocarpus heimii (Dipterocarpaceae).

Tectonic movements, climatic oscillations, and marine transgressions during the Cenozoic have had a dramatic effect on the biota of the tropical rain forest. This study aims to reveal the phylogeography and evolutionary history of a Peninsular Malaysian endemic tropical timber species, Neobalanocarpus heimii (Dipterocarpaceae). A total of 32 natural populations of N. heimii, with 8 samples from each population were investigated. Fifteen haplotypes were identified from five noncoding chloroplast DNA (cpDNA) regions. Overall, two major genealogical cpDNA lineages of N. heimii were elucidated: a widespread southern and a northern region. The species is predicted to have survived in multiple refugia during climatic oscillations: the northwestern region (R1), the northeastern region (R2), and the southern region (R3). These putative glacial refugia exhibited higher levels of genetic diversity, population differentiation, and the presence of unique haplotypes. Recolonization of refugia R1 and R2 could have first expanded into the northern region and migrated both northeastwards and northwestwards. Meanwhile, recolonization of N. heimii throughout the southern region could have commenced from refugia R3 and migrated toward the northeast and northwest, respectively. The populations of Tersang, Pasir Raja, and Rotan Tunggal exhibited remarkably high haplotype diversity, which could have been the contact zones that have received an admixture of gene pools from the northerly and also southerly regions. As a whole, the populations of N. heimii derived from glacial refugia and contact zones should be considered in the conservation strategies in order to safeguard the long-term survival of the species.

PIEZO2-dependent rapid pain system in humans and mice.

The PIEZO2 ion channel is critical for transducing light touch into neural signals but is not considered necessary for transducing acute pain in humans. Here, we discovered an exception - a form of mechanical pain evoked by hair pulling. Based on observations in a rare group of individuals with PIEZO2 deficiency syndrome, we demonstrated that hair-pull pain is dependent on PIEZO2 transduction. Studies in control participants showed that hair-pull pain triggered a distinct nocifensive response, including a nociceptive reflex. Observations in rare Aß deafferented individuals and nerve conduction block studies in control participants revealed that hair-pull pain perception is dependent on Aß input. Single-unit axonal recordings revealed that a class of cooling-responsive myelinated nociceptors in human skin is selectively tuned to painful hair-pull stimuli. Further, we pharmacologically mapped these nociceptors to a specific transcriptomic class. Finally, using functional imaging in mice, we demonstrated that in a homologous nociceptor, Piezo2 is necessary for high-sensitivity, robust activation by hair-pull stimuli. Together, we have demonstrated that hair-pulling evokes a distinct type of pain with conserved behavioral, neural, and molecular features across humans and mice.

The burst gap is a peripheral temporal code for pitch perception that is shared across audition and touch.

When tactile afferents were manipulated to fire in periodic bursts of spikes, we discovered that the perceived pitch corresponded to the inter-burst interval (burst gap) in a spike train, rather than the spike rate or burst periodicity as previously thought. Given that tactile frequency mechanisms have many analogies to audition, and indications that temporal frequency channels are linked across the two modalities, we investigated whether there is burst gap temporal encoding in the auditory system. To link this putative neural code to perception, human subjects (n = 13, 6 females) assessed pitch elicited by trains of temporally-structured acoustic pulses in psychophysical experiments. Each pulse was designed to excite a fixed population of cochlear neurons, precluding place of excitation cues, and to elicit desired temporal spike trains in activated afferents. We tested periodicities up to 150 Hz using a variety of burst patterns and found striking deviations from periodicity-predicted pitch. Like the tactile system, the duration of the silent gap between successive bursts of neural activity best predicted perceived pitch, emphasising the role of peripheral temporal coding in shaping pitch. This suggests that temporal patterning of stimulus pulses in cochlear implant users might improve pitch perception.

The Relationship Between Tactile Intensity Perception and Afferent Spike Count is Moderated by a Function of Frequency.

It has been suggested that tactile intensity perception can be explained by a linear function of spike rate weighted by afferent type. Other than relying on mathematical models, verifying this experimentally is difficult due to the frequency tuning of different afferent types and changes in population recruitment patterns with vibrotactile frequency. To overcome these complexities, we used pulsatile mechanical stimuli which activate the same afferent population regardless of the repetition rate (frequency), generating one action potential per pulse. We used trains of different frequencies (20-200 Hz) to investigate perceived intensity. Subjects' magnitude ratings increased with pulse rate up to ∼100 Hz and plateaued beyond this frequency. This was true regardless of pulse amplitude, from small pulses that exclusively activated Pacinian (PC) afferents, to pulses large enough to activate other afferents including slowly adapting. Electrical stimulation, which activates afferents indiscriminately, plateaued at a similar frequency, although not in all subjects. As the plateauing did not depend on indentation magnitude and hence on afferent weights, we propose that the contribution of spike count to intensity perception is weighted by a function of frequency. This may explain why fine textures evoking high frequency vibrations of a small magnitude do not feel disproportionally intense.

Auditory clicks elicit equivalent temporal frequency perception to tactile pulses: A cross-modal psychophysical study.

Both hearing and touch are sensitive to the frequency of mechanical oscillations-sound waves and tactile vibrations, respectively. The mounting evidence of parallels in temporal frequency processing between the two sensory systems led us to directly address the question of perceptual frequency equivalence between touch and hearing using stimuli of simple and more complex temporal features. In a cross-modal psychophysical paradigm, subjects compared the perceived frequency of pulsatile mechanical vibrations to that elicited by pulsatile acoustic (click) trains, and vice versa. Non-invasive pulsatile stimulation designed to excite a fixed population of afferents was used to induce desired temporal spike trains at frequencies spanning flutter up to vibratory hum (>50 Hz). The cross-modal perceived frequency for regular test pulse trains of either modality was a close match to the presented stimulus physical frequency up to 100 Hz. We then tested whether the recently discovered "burst gap" temporal code for frequency, that is shared by the two senses, renders an equivalent cross-modal frequency perception. When subjects compared trains comprising pairs of pulses (bursts) in one modality against regular trains in the other, the cross-sensory equivalent perceptual frequency best corresponded to the silent interval between the successive bursts in both auditory and tactile test stimuli. These findings suggest that identical acoustic and vibrotactile pulse trains, regardless of pattern, elicit equivalent frequencies, and imply analogous temporal frequency computation strategies in both modalities. This perceptual correspondence raises the possibility of employing a cross-modal comparison as a robust standard to overcome the prevailing methodological limitations in psychophysical investigations and strongly encourages cross-modal approaches for transmitting sensory information such as translating pitch into a similar pattern of vibration on the skin.

A human pluripotent stem cell-based model of SARS-CoV-2 infection reveals an ACE2-independent inflammatory activation of vascular endothelial cells through TLR4.

To date, the direct causative mechanism of SARS-CoV-2-induced endotheliitis remains unclear. Here, we report that human ECs barely express surface ACE2, and ECs express less intracellular ACE2 than non-ECs of the lungs. We ectopically expressed ACE2 in hESC-ECs to model SARS-CoV-2 infection. ACE2-deficient ECs are resistant to the infection but are more activated than ACE2-expressing ones. The virus directly induces endothelial activation by increasing monocyte adhesion, NO production, and enhanced phosphorylation of p38 mitogen-associated protein kinase (MAPK), NF-κB, and eNOS in ACE2-expressing and -deficient ECs. ACE2-deficient ECs respond to SARS-CoV-2 through TLR4 as treatment with its antagonist inhibits p38 MAPK/NF-κB/ interleukin-1ß (IL-1ß) activation after viral exposure. Genome-wide, single-cell RNA-seq analyses further confirm activation of the TLR4/MAPK14/RELA/IL-1ß axis in circulating ECs of mild and severe COVID-19 patients. Circulating ECs could serve as biomarkers for indicating patients with endotheliitis. Together, our findings support a direct role for SARS-CoV-2 in mediating endothelial inflammation in an ACE2-dependent or -independent manner.

Frequency of clinical and radiographic evidence of inflammation associated with retained tooth root fragments and the effects of tooth root fragment length and position on oral inflammation in dogs.

OBJECTIVE: To assess the frequency of clinical and radiographic evidence of inflammation (ie, evidence of inflammation) associated with retained tooth root fragments (RTRFs) in dogs and to determine whether evidence of inflammation was affected by RTRF length and position within the alveolar bone. SAMPLE: 148 RTRFs in 66 dogs. PROCEDURES: For each dog, demographic information was recorded, and full-mouth radiographs were obtained and reviewed for RTRFs. For each RTRF, the length of the fragment was measured on intraoral radiographic images, and its location and position relative to the alveolar bone margin were recorded. The presence or absence of evidence of inflammation in association with each RTRF was also recorded. Descriptive data were generated. Generalized linear mixed models were used to identify factors associated with evidence of inflammation around RTRFs. RESULTS: 81 of 148 (54.7%) RTRFs had evidence of inflammation. For every 1-mm increase in RTRF length, the odds of inflammation increased by 17% (OR, 1.17; 95% confidence interval [CI], 1.04 to 1.34; P = 0.009). Odds of inflammation for RTRFs that protruded from the alveolar bone margin were 2.98 (95% CI, 1.02 to 8.72; P = 0.046) and 7.58 (95% CI, 1.98 to 29.08; P = 0.001) times those for RTRFs that were buried and level with the alveolar bone margin, respectively. Tooth root fragment length was a poor predictor of inflammation. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that most RTRFs were associated with evidence of inflammation and supported the current recommendation for extraction of RTRFs whenever feasible.

Temporal patterns in electrical nerve stimulation: Burst gap code shapes tactile frequency perception.

We have previously described a novel temporal encoding mechanism in the somatosensory system, where mechanical pulses grouped into periodic bursts create a perceived tactile frequency based on the duration of the silent gap between bursts, rather than the mean rate or the periodicity. This coding strategy may offer new opportunities for transmitting information to the brain using various sensory neural prostheses and haptic interfaces. However, it was not known whether the same coding mechanisms apply when using electrical stimulation, which recruits a different spectrum of afferents. Here, we demonstrate that the predictions of the burst gap coding model for frequency perception apply to burst stimuli delivered with electrical pulses, re-emphasising the importance of the temporal structure of spike patterns in neural processing and perception of tactile stimuli. Reciprocally, the electrical stimulation data confirm that the results observed with mechanical stimulation do indeed depend on neural processing mechanisms in the central nervous system, and are not due to skin mechanical factors and resulting patterns of afferent activation.

Tapping Into the Language of Touch: Using Non-invasive Stimulation to Specify Tactile Afferent Firing Patterns.

The temporal pattern of action potentials can convey rich information in a variety of sensory systems. We describe a new non-invasive technique that enables precise, reliable generation of action potential patterns in tactile peripheral afferent neurons by brief taps on the skin. Using this technique, we demonstrate sophisticated coding of temporal information in the somatosensory system, that shows that perceived vibration frequency is not encoded in peripheral afferents as was expected by either their firing rate or the underlying periodicity of the stimulus. Instead, a burst gap or silent gap between trains of action potentials conveys frequency information. This opens the possibility of new encoding strategies that could be deployed to convey sensory information using mechanical or electrical stimulation in neural prostheses and brain-machine interfaces, and may extend to senses beyond artificial encoding of aspects of touch. We argue that a focus on appropriate use of effective temporal coding offers more prospects for rapid improvement in the function of these interfaces than attempts to scale-up existing devices.

Mandibular Carnassial Tooth Malformations in 6 Dogs-Micro-Computed Tomography and Histology Findings.

Objective: To document the clinical, radiographic, and histological characteristics of mandibular first molar teeth with developmental abnormalities previously attributed to dens invaginatus and enamel pearls in dogs. Materials and Methods: Affected mandibular first molar teeth from dogs were evaluated grossly and via intraoral radiography. Endodontically and/or periodontally compromised teeth were extracted and subjected to some combination of micro-computed tomography, histopathology, and immunohistochemistry with anti-amelogenin antibody. Results: Six dogs with developmental abnormalities of mandibular first molar teeth were identified, representing 11 affected teeth. The condition was bilateral in 5 dogs, while in 1 dog, only one mandibular first molar tooth was present. Patient weight ranged from 1.7 to 6 kg (median = 4.09 kg). On intraoral radiographs, root convergence or parallelism was noted in 6 of 11 teeth, and root dilaceration was noted in 3 of 11 teeth. Eight teeth required extraction due to periapical lucencies or periodontitis. On micro-CT, the abnormal teeth were characterized by the presence of abnormal, heterogenous hard tissue with beam attenuation characteristics midway between that of enamel and dentin. Enamel fissures were identified in 4 of 8 teeth, while ectopic radicular enamel was identified in 2 of 8 teeth. The abnormal tissue was traversed by channels measuring 20-40 µm in diameter. Channels communicated with the enamel fissures in 2/8 teeth, the furcation in 2/8 teeth and the pulp in 4/8 teeth. The abnormal tissue was frequently surrounded by disorganized dentin. Histologic features of enamel and dentin were absent from the abnormal tissue and immunohistochemistry to detect amelogenin in the abnormal tissue was negative in all samples. Conclusion: The dental abnormalities described here correspond to a previously unrecognized developmental abnormality involving the mandibular first molar teeth in dogs. The developmental origin of the abnormal tissue could not be ascertained, and further investigations are required to determine the mode of formation, origin of the abnormal tissue, and factors associated with development. These developmental abnormalities more closely resemble molar-incisor malformation, rather than dens invaginatus or enamel pearls as described in humans. The authors propose that affected mandibular first molar teeth simply be referred to as having carnassial tooth malformations.