Theory of tip structure-dependent microtubule catastrophes and damage-induced microtubule rescues.

Microtubules are essential cytoskeletal polymers that exhibit stochastic switches between tubulin assembly and disassembly. Here, we examine possible mechanisms for these switches, called catastrophes and rescues. We formulate a four-state Monte Carlo model, explicitly considering two biochemical and two conformational states of tubulin, based on a recently conceived view of microtubule assembly with flared ends. The model predicts that high activation energy barriers for lateral tubulin interactions can cause lagging of curled protofilaments, leading to a ragged appearance of the growing tip. Changes in the extent of tip raggedness explain some important but poorly understood features of microtubule catastrophe: weak dependence on tubulin concentration and an increase in its probability over time, known as aging. The model predicts a vanishingly rare frequency of spontaneous rescue unless patches of guanosine triphosphate tubulin are artificially embedded into microtubule lattice. To test our model, we used in vitro reconstitution, designed to minimize artifacts induced by microtubule interaction with nearby surfaces. Microtubules were assembled from seeds overhanging from microfabricated pedestals and thus well separated from the coverslip. This geometry reduced the rescue frequency and the incorporation of tubulins into the microtubule shaft compared with the conventional assay, producing data consistent with the model. Moreover, the rescue positions of microtubules nucleated from coverslip-immobilized seeds displayed a nonexponential distribution, confirming that coverslips can affect microtubule dynamics. Overall, our study establishes a unified theory accounting for microtubule assembly with flared ends, a tip structure-dependent catastrophe frequency, and a microtubule rescue frequency dependent on lattice damage and repair.

Monaural and dichotic forward masking in the dolphin's auditory system.

Short-latency auditory-evoked potentials (AEPs) were recorded non-invasively in the bottlenose dolphin Tursiops truncatus. The stimuli were two sound clicks that were played either monaurally (both clicks to one and the same acoustic window) or dichotically (the leading stimulus (masker) to one acoustic window and the delayed stimulus (test) to the other window). The ratio of the levels of the two stimuli was 0, 10, or 20 dB (at 10 and 20 dB, the leading stimulus was of a higher level). The inter-stimulus intervals (ISIs) varied from 0.15 to 10 ms. The test response magnitude was assessed by correlation analysis as a percentage of the control (non-masked) response. At monaural stimulation, the test response was of a constant magnitude (5-6% of the control) at ISIs of 0.15-0.3 ms and recovered at longer ISIs. At dichotic stimulation, the deepest suppression of the test response occurred at ISIs of 0.5-0.7 ms. The response was slightly suppressed at short ISIs (0.15-0.3 ms) and recovered at ISIs longer than 0.5-0.7 ms. The relation of parameters of the forward masking to echolocation in dolphins is discussed.

Forward masking in a bottlenose dolphin Tursiops truncatus: dependence on azimuthal positions of the masker and test sources.

Forward masking was investigated by the auditory evoked potentials (AEP) method in a bottlenose dolphin Tursiops truncatus using stimulation by two successive acoustic pulses (the masker and test) projected from spatially separated sources. The positions of the two sound sources either coincided with or were symmetrical relative to the head axis at azimuths from 0 to ± 90°. AEPs were recorded either from the vertex or from the lateral head surface next to the auditory meatus. In the last case, the test source was ipsilateral to the recording side, whereas the masker source was either ipsi- or contralateral. For lateral recording, AEP release from masking (recovery) was slower for the ipsi- than for the contralateral masker source position. For vertex recording, AEP recovery was equal both for the coinciding positions of the masker and test sources and for their symmetrical positions relative to the head axis. The data indicate that at higher levels of the auditory system of the dolphin, binaural convergence makes the forward masking nearly equal for ipsi- and contralateral positions of the masker and test.

The efficacy and safety of levilimab in severely ill COVID-19 patients not requiring mechanical ventilation: results of a multicenter randomized double-blind placebo-controlled phase III CORONA clinical study.

OBJECTIVE AND DESIGN: The aim of this double-blind, placebo-controlled, phase III CORONA clinical trial was to evaluate the efficacy and safety of IL-6 receptor inhibitor levilimab (LVL) in subjects with severe COVID-19. SUBJECTS: The study included 217 patients. The eligible were men and non-pregnant women aged 18 years or older, hospitalized for severe COVID-19 pneumonia. TREATMENT: 206 subjects were randomized (1:1) to receive single subcutaneous administration of LVL 324 mg or placebo, both in combination with standard of care (SOC). 204 patients received allocated therapy. After the LVL/placebo administration in case of deterioration of symptoms, the investigator could perform a single open-label LVL 324 mg administration as the rescue therapy. METHODS: The primary efficacy endpoint was the proportion of patients with sustained clinical improvement on the 7-category ordinal scale on Day 14. All efficacy data obtained after rescue therapy administration were considered missing. For primary efficacy analysis, all subjects with missing data were considered non-responders. RESULTS: 63.1% and 42.7% of patients in the LVL and in the placebo groups, respectively, achieved sustained clinical improvement on Day 14 (P = .0017). The frequency of adverse drug reactions was comparable between the groups. CONCLUSION: In patients with radiologically confirmed SARS-CoV-2 pneumonia, requiring or not oxygen therapy (but not ventilation) with no signs of other active infection administration of LVL + SOC results in an increase of sustained clinical improvement rate. TRAIL REGISTRATION: The trial is registered at the US National Institutes of Health (ClinicalTrials.gov; NCT04397562).

Large positive and negative Goos-Hänchen shifts near the surface plasmon resonance in subwavelength grating.

Diffraction of light of a visible spectral range by subwavelength metal gratings has been investigated experimentally and theoretically using rigorous electromagnetic calculations. It has been demonstrated that an effect of surface plasmon resonance (SPR), at which total absorption of light by metal grating can be observed, occurs under certain conditions. Large positive and negative Goos-Hänchen (GH) shifts occur near the SPR. It has been shown that the reflected beam is split into two parts, the relative powers of which depend on the incident beam width and the grating depth. The dependence of the GH shifts on the grating period and grating depth has been investigated for different incident beam widths. The high sensitivity of the GH shift on the incident angle of a light beam near the SPR has been demonstrated.

The rate of cochlear compression in a dolphin: a forward-masking evoked-potential study.

The "active" cochlear mechanism of hearing manifests in the cochlear compression. Investigations of compression in odontocetes help to determine the frequency limit of the active mechanism. The compression may be evaluated by comparison of low- and on-frequency masking. In a bottlenose dolphin, forward masking of auditory evoked potentials to tonal pips was investigated. Measurements were performed for test frequencies of 45 and 90 kHz. The low-frequency maskers were - 0.25 to - 0.75 oct relative the test. Masking efficiency was varied by masker-to-test delay variation from 2 to 20 ms, and masker levels at threshold (MLTs) were evaluated at each of the delays. It was assumed that low-frequency maskers were not subjected or little subjected to compression whereas on-frequency maskers were subjected equally to the test. Therefore, the compression rate was assessed as the slope of low-frequency MLT dependence on on-frequency MLT. For the 90-kHz test, the slopes were 0.63 and 0.18 dB/dB for masker of - 0.25 and - 0.5 oct, respectively. For the 45 kHz test, the slopes were 0.69 and 0.39 dB/dB for maskers of - 0.25 and - 0.5 oct. So, compression did not decay at the upper boundary of the hearing frequency range in the dolphin.

Spatial release from masking in a bottlenose dolphin Tursiops truncatus.

The impact of maskers on the receiving beam of a bottlenose dolphin, Tursiops truncatus, was investigated using the auditory evoked potential (AEP) method. The test signal was a train of tone pips with a 64 kHz carrier frequency. The stimulus produced AEPs as a sequence of waves replicating the pip rate: the rate following response (RFR). The masker was band-limited noise, with a passband of 45 to 90 kHz and a level of 105 dB re 1 µPa. Masker azimuths were at 0°, ±30°, ±60°, and ±90° relative to the head midline. The receiving beam was evaluated in terms of the RFR threshold dependence on the signal azimuth. The masked thresholds were higher than the baseline thresholds, which appeared mostly as a shift rather than a deformation in the receiving beam. The largest threshold shift appeared when the masker source was located in the most sensitive direction (zero azimuth); at lateral masker source positions, the threshold shift decreased. When the masker source was not at the head midline, the masked thresholds were higher at signal positions ipsilateral to the masker source than at positions contralateral to the masker source. The largest asymmetry was observed at the 30° masker azimuth in conjunction with the ±30° and ±120° signal azimuths; the asymmetries were 5.6 and 8.1 dB, respectively. This masking asymmetry was lower than expected from the previously found interaural intensity difference, which may be explained by the conflict between the test signal and the masker when it appeared at a binaural level of the auditory system.

Level-dependent masking of the auditory evoked responses in a dolphin: manifestation of the compressive nonlinearity.

At suprathreshold sound levels, interactions between masking noise and sound signals are liable to compressive nonlinearity in the auditory system. The compressive nonlinearity is a property of the "active" cochlear mechanism. It is not known whether this mechanism is capable to function at frequencies close to or above 100 kHz that are available to odontocetes (toothed whales, dolphins, and porpoises). This question may be answered by the use of the frequency-specific masking. Auditory evoked potentials to sound stimuli in a bottlenose dolphin, Tursiops truncatus, were recorded in the presence of simultaneous maskers. Stimulus frequencies were 45, 64, or 90 kHz. Maskers were on-frequency bandlimited noise or low-frequency noise of frequencies 0.25-1 oct below the stimulus frequency. The stimuli provoked responses as a series of brain-potential waves following the pip-train rate. For the on-frequency masker, the masker level at threshold dependence on the signal level was 1.1 dB/dB. For maskers of 1 oct below the stimulus, the dependence was 0.53-0.57 dB/dB. The data considered evidence for the compressive nonlinearity of responses to stimuli, and therefore, are indicative of the functioning of the active mechanism at frequencies up to 90 kHz.

Adaptation in the auditory system of a beluga whale: effect of adapting sound parameters.

The effects of adapting sounds (pip trains or pure tones) on auditory evoked potentials (the rate following response, RFR) were investigated in a beluga whale. During RFR acquisition, adapting signals lasting 128 ms each were alternated with test signals lasting 16 ms each; the test signal levels varied randomly. Adapting signals were trains of cosine-enveloped tone pips or pure tones. Pip rate varied with the envelope cosine cycle maintained at 0.125 of pip intervals and the cosine rise-fall time maintained at 0.0625 of pip intervals. Adapting signals shifted the amplitude-level function upward compared to the baseline (no adapting signal) function. The higher the adapting signal level was, the bigger the shift in the amplitude-level function was. The slower the pips were in the adapting signal, the smaller the adaptation effect was. A train of pips with a 0.0625-ms rise-fall time and 125 dB SPL shifted the function by 35-40 dB, whereas a train of pips with a 1-ms rise-fall time or a pure tone with the same SPL shifted the function by approximately 15 dB. The difference between the "fast" and "slow" adapting signals is supposed to be associated with their abilities to stimulate the auditory system in odontocetes.

Influence of background noise on auditory evoked responses to rippled-spectrum signals.

The resolution of spectral patterns in adaptation background noise was investigated in a beluga whale, Delphinapterus leucas, using the evoked-potential technique. The resolution of spectral patterns was investigated using rippled-spectrum test stimuli of various levels and ripple densities and recording the rhythmic evoked responses (the rate following response, RFR) to ripple phase reversals. In baseline (no adaptation background noise) experiments, the highest RFR magnitude was observed at signal sound pressure levels (SPLs) of 100-110 dB re 1 µPa; at SPLs both below the optimum (down to 80 dB re 1 µPa) and above the optimum (up to 140 dB re 1 µPa), the RFR magnitude decreased. For high signal levels (above 110 dB re 1 µPa), low-level adaptation background noise (from -10 to -20 dB re signal level) increased RFR magnitude compared to baseline. This effect is considered to be a result of the optimization of the sensation level of the high-SPL signals due to decreasing hearing sensitivity caused by the adaptation background noise.

Influence of fatiguing noise on auditory evoked responses to stimuli of various levels in a beluga whale, Delphinapterus leucas.

The negative impact of man-made noise on the hearing of odontocetes has attracted considerable recent attention. In the majority of studies, permanent or temporary reductions in sensitivity, known as permanent or temporary threshold shift (PTS or TTS, respectively), have been investigated. In the present study, the effects of a fatiguing sound on the hearing of a beluga whale, Delphinapterus leucas, within a wide range of levels of test signals was investigated. The fatiguing noise was half-octave band-limited noise centered at 32 kHz. Post-exposure effects of this noise on the evoked responses to test stimuli (rhythmic pip trains with a 45-kHz center frequency) at various levels (from threshold to 60 dB above threshold) were measured. For baseline (pre-exposure) responses, the magnitude-versus-level function featured a segment of steep magnitude dependence on level (up to 30 dB above threshold) that was followed by a plateau segment that featured little dependence on level (30 to 55 dB above threshold). Post-exposure, the function shifted upward along the level scale. The shift was 23 dB at the threshold and up to 33 dB at the supra-threshold level. Owing to the plateau in the magnitude-versus-level function, post-exposure suppression of responses depended on the stimulus level such that higher levels corresponded to less suppression. The experimental data may be modeled based on the compressive non-linearity of the cochlea. According to the model, post-exposure responses of the cochlea to high-level stimuli are minimally suppressed compared with the pre-exposure responses, despite a substantially increased threshold.

Frequency Tuning of Hearing in the Beluga Whale.

Data on frequency tuning in odontocetes are contradictory: different authors have reported filter qualities from 2 to almost 50. In this study, frequency tuning was measured in a beluga whale (Delphinapterus leucas) using a rippled-noise test stimulus in conjunction with the auditory evoked potential (AEP) technique. The response to ripple reversions was considered to indicate resolvability of the ripple pattern. The limit of ripple-pattern resolution ranged from 20 to 32 ripples per octave (rpo). A model of interaction of the ripple spectrum with frequency-tuned filters suggests that this resolution limit requires a filter quality of 29-46.

Auditory evoked potentials in the auditory system of a beluga whale Delphinapterus leucas to prolonged sound stimuli.

The effects of prolonged (up to 1500 s) sound stimuli (tone pip trains) on evoked potentials (the rate following response, RFR) were investigated in a beluga whale. The stimuli (rhythmic tone pips) were of frequencies of 45, 64, and 90 kHz at levels from 20 to 60 dB above threshold. Two experimental protocols were used: short- and long-duration. For the short-duration protocol, the stimuli were 500-ms-long pip trains that repeated at a rate of 0.4 trains/s. For the long-duration protocol, the stimuli were continuous pip successions lasting up to 1500 s. The RFR amplitude gradually decreased by three to seven times from 10 ms to 1500 s of stimulation. Decrease of response amplitude during stimulation was approximately proportional to initial (at the start of stimulation) response amplitude. Therefore, even for low stimulus level (down to 20 dB above the baseline threshold) the response was never suppressed completely. The RFR amplitude decay that occurred during stimulation could be satisfactorily approximated by a combination of two exponents with time constants of 30-80 ms and 3.1-17.6 s. The role of adaptation in the described effects and the impact of noise on the acoustic orientation of odontocetes are discussed.

Auditory sensitivity to local stimulation of the head surface in a beluga whale (Delphinapterus leucas).

Using the auditory evoked response technique, sensitivity to local acoustic stimulation of the ventro-lateral head surface was investigated in a beluga whale (Delphinapterus leucas). The stimuli were tone pip trains of carrier frequencies ranging from 16 to 128 kHz with a pip rate of 1 kHz. For higher frequencies (90-128 kHz), the low-threshold point was located next to the medial side of the middle portion of the lower jaw. For middle (32-64 kHz) and lower (16-22.5 kHz) frequencies, the low-threshold point was located at the lateral side of the middle portion of the lower jaw. For lower frequencies, there was an additional low-threshold point next to the bulla-meatus complex. Based on these data, several frequency-specific paths of sound conduction to the auditory bulla are suggested: (i) through an area on the lateral surface of the lower jaw and further through the intra-jaw fat-body channel (for a wide frequency range); (ii) through an area on the ventro-lateral head surface and further through the medial opening of the lower jaw and intra-jaw fat-body channel (for a high-frequency range); and (iii) through an area on the lateral (near meatus) head surface and further through the lateral fat-body channel (for a low-frequency range).

Evoked-potential recovery during double click stimulation in a beluga whale: implications for biosonar gain control.

Auditory evoked potentials (AEPs) were recorded in a beluga whale Delphinapterus leucas using a double-pulse stimulation paradigm, specifically measuring the recovery (release from masking) of the second (test) response as a function of delay after the first (conditioning) pulse at various levels of the conditioning and test stimuli. The conditioning/test stimulus level ratio influenced the recovery time (the higher the ratio, the longer the recovery). This interrelation was used to evaluate the intensity/time trade in release from forward masking. Trade was evaluated as 32.2 dB per time decade. Data were considered as simulating interactions between the transmitted pulse and echo during echolocation, assuming that a transmitted sonar pulse produces forward masking of the echo response. With increased target distance, the attenuation of the echo may be compensated by the release from masking. According to the model, the compensation results in substantial stabilization of the echo response even if the intensity/time trade of release from masking is not precisely equal to the rate of echo attenuation with distance.

Spectrum pattern resolution after noise exposure in a beluga whale, Delphinapterus leucas: Evoked potential study.

Temporary threshold shift (TTS) and the discrimination of spectrum patterns after fatiguing noise exposure (170 dB re 1 µPa, 10 min duration) was investigated in a beluga whale, Delphinapterus leucas, using the evoked potential technique. Thresholds were measured using rhythmic (1000/s) pip trains of varying levels and recording the rhythmic evoked responses. Discrimination of spectrum patterns was investigated using rippled-spectrum test stimuli of various levels and ripple densities, recording the rhythmic evoked responses to ripple phase reversals. Before noise exposure, the greatest responses to rippled-spectrum probes were evoked by stimuli with a low ripple density with a decrease in the response magnitude occurring with an increasing ripple density. After noise exposure, both a TTS and a reduction of the responses to rippled-spectrum probes appeared and recovered in parallel. The reduction of the responses to rippled-spectrum probes was maximal for high-magnitude responses at low ripple densities and was negligible for low-magnitude responses at high ripple densities. It is hypothesized that the impacts of fatiguing sounds are not limited by increased thresholds and decreased sensitivity results in reduced ability to discriminate fine spectral content with the greatest impact on the discrimination of spectrum content that may carry the most obvious information about stimulus properties.

The limits of applicability of the sound exposure level (SEL) metric to temporal threshold shifts (TTS) in beluga whales, Delphinapterus leucas.

The influence of fatiguing sound level and duration on post-exposure temporary threshold shift (TTS) was investigated in two beluga whales (Delphinapterus leucas). The fatiguing sound was half-octave noise with a center frequency of 22.5 kHz. TTS was measured at a test frequency of 32 kHz. Thresholds were measured by recording rhythmic evoked potentials (the envelope following response) to a test series of short (eight cycles) tone pips with a pip rate of 1000 s(-1). TTS increased approximately proportionally to the dB measure of both sound pressure (sound pressure level, SPL) and duration of the fatiguing noise, as a product of these two variables. In particular, when the noise parameters varied in a manner that maintained the product of squared sound pressure and time (sound exposure level, SEL, which is equivalent to the overall noise energy) at a constant level, TTS was not constant. Keeping SEL constant, the highest TTS appeared at an intermediate ratio of SPL to sound duration and decreased at both higher and lower ratios. Multiplication (SPL multiplied by log duration) better described the experimental data than an equal-energy (equal SEL) model. The use of SEL as a sole universal metric may result in an implausible assessment of the impact of a fatiguing sound on hearing thresholds in odontocetes, including under-evaluation of potential risks.

Subwavelength diffractive color beam combiner.

A high-efficiency subwavelength diffractive beam combiner operating in a visible spectral range is designed, fabricated, and demonstrated. Such a device combines red, green, and blue color beams into one output light beam. Diffraction efficiencies of different types of gratings are calculated for various materials, incidence angles, and polarizations of light. It is shown that the plasmon resonance via a grating coupling occurs at the determined conditions. Subwavelength gratings with a period of 400 nm are fabricated and tested using laser and laser diode sources.

Frequency tuning of hearing in the beluga whale: discrimination of rippled spectra.

Frequency tuning was measured in the beluga whale (Delphinapterus leucas) using rippled-noise test stimuli in conjunction with an auditory evoked potential (AEP) technique. The test stimulus was a 2-octave-wide rippled noise with frequency-proportional ripple spacing. The rippled-noise signal contained either a single reversal or rhythmic (1-kHz rate) reversals of the ripple phase. Single or rhythmic phase reversals evoked, respectively, a single auditory brainstem response (ABR) or a rhythmic AEP sequence-the envelope following response (EFR). The response was considered as an indication of resolvability of the ripple pattern. The rhythmic phase-reversal test with EFR recording revealed higher resolution than the single phase-reversal test with single ABR recording. The limit of ripple-pattern resolution with the single phase-reversal test ranged from 17 ripples per octave (rpo) at 32 kHz to 24 rpo at 45 to 64 kHz; for the rhythmic phase-reversal test, the limit ranged from 20 to 32 rpo. An interaction model of a ripple spectrum with frequency-tuned filters suggests that the ripple-pattern resolution limit of 20 to 32 rpo requires a filter quality Q of 29 to 46. Possible causes of disagreement of these estimates with several previously published data are discussed.

Hearing threshold shifts and recovery after noise exposure in beluga whales, Delphinapterus leucas.

Temporary threshold shift (TTS) after loud noise exposure was investigated in a male and a female beluga whale (Delphinapterus leucas). The thresholds were evaluated using the evoked-potential technique, which allowed for threshold tracing with a resolution of ~1 min. The fatiguing noise had a 0.5 octave bandwidth, with center frequencies ranging from 11.2 to 90 kHz, a level of 165 dB re. 1 µPa and exposure durations from 1 to 30 min. The effects of the noise were tested at probe frequencies ranging from -0.5 to +1.5 octaves relative to the noise center frequency. The effect was estimated in terms of both immediate (1.5 min) post-exposure TTS and recovery duration. The highest TTS with the longest recovery duration was produced by noises of lower frequencies (11.2 and 22.5 kHz) and appeared at a test frequency of +0.5 octave. At higher noise frequencies (45 and 90 kHz), the TTS decreased. The TTS effect gradually increased with prolonged exposures ranging from 1 to 30 min. There was a considerable TTS difference between the two subjects.