Chain-Length Dependence of Peptide-Lipid Bilayer Interaction Strength and Binding Kinetics: A Combined Theoretical and Experimental Approach.

Physical interactions between polypeptide chains and lipid membranes underlie critical cellular processes. Yet, despite fundamental importance, key mechanistic aspects of these interactions remain elusive. Bulk experiments have revealed a linear relationship between free energy and peptide chain length in a model system, but does this linearity extend to the interaction strength and to the kinetics of lipid binding? To address these questions, we utilized a combination of coarse-grained molecular dynamics (CG MD) simulations, analytical modeling, and atomic force microscopy (AFM)-based single molecule force spectroscopy. Following previous bulk experiments, we focused on interactions between short hydrophobic peptides (WLn, n = 1, ..., 5) with 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayers, a simple system that probes peptide primary structure effects. Potentials of mean force extracted from CG MD recapitulated the linearity of free energy with the chain length. Simulation results were quantitatively connected to bulk biochemical experiments via a single scaling factor of order unity, corroborating the methodology. Additionally, CG MD revealed an increase in the distance to the transition state, a result that weakens the dependence of the dissociation force on the peptide chain length. AFM experiments elucidated rupture force distributions and, through modeling, intrinsic dissociation rates. Taken together, the analysis indicates a rupture force plateau in the WLn-POPC system, suggesting that the final rupture event involves the last 2 or 3 residues. In contrast, the linear dependence on chain length was preserved in the intrinsic dissociation rate. This study advances the understanding of peptide-lipid interactions and provides potentially useful insights for the design of peptides with tailored membrane-interacting properties.

SARM1 regulates pro-inflammatory cytokine expression in human monocytes by NADase-dependent and -independent mechanisms.

SARM1 is a Toll-IL-1 receptor (TIR) domain-containing protein with roles in innate immunity and neuronal death in diverse organisms. Unlike other innate immune TIR proteins that function as adaptors for Toll-like receptors (TLRs), SARM1 has NADase activity, and this activity regulates murine neuronal cell death. However, whether human SARM1, and its NADase activity, are involved in innate immune regulation remains unclear. Here, we show that human SARM1 regulates proinflammatory cytokine expression in both an NADase-dependent and -independent manner in monocytes. SARM1 negatively regulated TLR4-dependent TNF mRNA induction independently of its NADase activity. In contrast, SARM1 inhibited IL-1ß secretion through both NADase-dependent inhibition of pro-IL-1ß expression, and NADase-independent suppression of the NLRP3 inflammasome and hence processing of pro-IL-1ß to mature IL-1ß. Our study reveals multiple mechanisms whereby SARM1 regulates pro-inflammatory cytokines in human monocytes and shows, compared to other mammalian TIR proteins, a distinct NADase-dependent role for SARM1 in innate immunity.

A genome-scale screen identifies sulfated glycosaminoglycans as pivotal in epithelial cell damage by Candida albicans.

Candidalysin is a cytolytic peptide produced by the opportunistic fungal pathogen Candida albicans. This peptide is a key virulence factor in mouse models of mucosal and hematogenously disseminated candidiasis. Despite intense interest in the role of candidalysin in C. albicans pathogenicity, its host cell targets have remained elusive. To fill this knowledge gap, we performed a genome-wide loss-of-function CRISPR screen in a human oral epithelial cell line to identify specific host factors required for susceptibility to candidalysin-induced cellular damage. Among the top hits were XYLT2, B3GALT6 and B3GAT3, genes that function in glycosaminoglycan (GAG) biosynthesis. Deletion of these genes led to the absence of GAGs such as heparan sulfate on the epithelial cell surface and increased resistance to damage induced by both candidalysin and live C. albicans. Biophysical analyses including surface plasmon resonance and atomic force and electron microscopy indicated that candidalysin physically binds to sulfated GAGs, facilitating its oligomerization or enrichment on the host cell surface. The addition of exogenous sulfated GAGs or the GAG analogue dextran sulfate protected cells against candidalysin-induced damage. Dextran sulfate, but not non-sulfated dextran, also inhibited epithelial cell endocytosis of C. albicans and fungal-induced epithelial cell cytokine and chemokine production. In a murine model of vulvovaginal candidiasis, topical dextran sulfate administration reduced host tissue damage and decreased intravaginal IL-1ß and neutrophil levels. Collectively, these data indicate that GAGs are epithelial cell targets of candidalysin and can be used therapeutically to protect cells from candidalysin-induced damage.

Personalising veteran healthcare: recognising barriers to access for minority and under-represented groups of veterans.

Veterans are not a demographically homogenous group, yet minority groups continue to be under-represented in research and report feeling less able to access clinical services to seek support. While veteran-specific healthcare has responded to the needs of the majority, the success of veteran mental health services is contingent on serving the whole veteran population. Key to the personalisation of healthcare is the question of access and a need to address specific inequalities and barriers to help-seeking behaviour. In this paper, we explore the issues of access to veteran healthcare at three levels: those barriers common to all veterans; those common to all minority groups of veterans; and those relevant to specific minority groups of veterans. Stigma, military attitudes and culture (eg, stoicism), and access to services and professionals with veteran-specific knowledge are universal barriers across veteran groups. Minority groups report a heightening of these barriers, alongside being 'othered' in veteran care settings, a lack of representation of them or their experiences in service descriptions and advertising, a lack of professional cultural competencies on specific issue, and the veteran environment potentially being retraumatising. Finally, barriers specific to individual groups are discussed. Attending to these is essential in developing holistic approaches to personalised healthcare that meets the needs of all veterans.

Functional benefits of continuous vs. categorical listening strategies on the neural encoding and perception of noise-degraded speech.

Acoustic information in speech changes continuously, yet listeners form discrete perceptual categories to ease the demands of perception. Being a more continuous/gradient as opposed to a discrete/categorical listener may be further advantageous for understanding speech in noise by increasing perceptual flexibility and resolving ambiguity. The degree to which a listener's responses to a continuum of speech sounds are categorical versus continuous can be quantified using visual analog scaling (VAS) during speech labeling tasks. Here, we recorded event-related brain potentials (ERPs) to vowels along an acoustic-phonetic continuum (/u/ to /a/) while listeners categorized phonemes in both clean and noise conditions. Behavior was assessed using standard two alternative forced choice (2AFC) and VAS paradigms to evaluate categorization under task structures that promote discrete (2AFC) vs. continuous (VAS) hearing, respectively. Behaviorally, identification curves were steeper under 2AFC vs. VAS categorization but were relatively immune to noise, suggesting robust access to abstract, phonetic categories even under signal degradation. Behavioral slopes were positively correlated with listeners' QuickSIN scores, suggesting a behavioral advantage for speech in noise comprehension conferred by gradient listening strategy. At the neural level, electrode level data revealed P2 peak amplitudes of the ERPs were modulated by task and noise; responses were larger under VAS vs. 2AFC categorization and showed larger noise-related delay in latency in the VAS vs. 2AFC condition. More gradient responders also had smaller shifts in ERP latency with noise, suggesting their neural encoding of speech was more resilient to noise degradation. Interestingly, source-resolved ERPs showed that more gradient listening was also correlated with stronger neural responses in left superior temporal gyrus. Our results demonstrate that listening strategy (i.e., being a discrete vs. continuous listener) modulates the categorical organization of speech and behavioral success, with continuous/gradient listening being more advantageous to speech in noise perception.

The Growing Class of Novel RNAi Therapeutics.

The clinical use of RNA interference (RNAi) molecular mechanisms has introduced a novel, growing class of RNA therapeutics capable of treating diseases by controlling target gene expression at the posttranscriptional level. With the newly approved nedosiran (Rivfloza), there are now six RNAi-based therapeutics approved by the United States Food and Drug Administration (FDA). Interestingly, five of the six FDA-approved small interfering RNA (siRNA) therapeutics [patisiran (Onpattro), lumasiran (Oxlumo), inclisiran (Leqvio), vutrisiran (Amvuttra), and nedosiran] were revealed to act on the 3'-untranslated regions of target mRNAs, instead of coding sequences, thereby following the common mechanistic action of genome-derived microRNAs (miRNA). Furthermore, three of the FDA-approved siRNA therapeutics [patisiran, givosiran (Givlaari), and nedosiran] induce target mRNA degradation or cleavage via near-complete rather than complete base-pair complementarity. These features along with previous findings confound the currently held characteristics to distinguish siRNAs and miRNAs or biosimilars, of which all converge in the RNAi regulatory pathway action. Herein, we discuss the RNAi mechanism of action and current criteria for distinguishing between miRNAs and siRNAs while summarizing the common and unique chemistry and molecular pharmacology of the six FDA-approved siRNA therapeutics. The term "RNAi" therapeutics, as used previously, provides a coherently unified nomenclature for broader RNAi forms as well as the growing number of therapeutic siRNAs and miRNAs or biosimilars that best aligns with current pharmacological nomenclature by mechanism of action. SIGNIFICANCE STATEMENT: The common and unique chemistry and molecular pharmacology of six FDA-approved siRNA therapeutics are summarized, in which nedosiran is newly approved. We point out rather a surprisingly mechanistic action as miRNAs for five siRNA therapeutics and discuss the differences and similarities between siRNAs and miRNAs that supports using a general and unified term "RNAi" therapeutics to align with current drug nomenclature criteria in pharmacology based on mechanism of action and embraces broader forms and growing number of novel RNAi therapeutics.

Polymerization mechanism of the Candida albicans virulence factor candidalysin.

Candida albicans is a commensal fungus that can cause epithelial infections and life-threatening invasive candidiasis. The fungus secretes candidalysin (CL), a peptide that causes cell damage and immune activation by permeation of epithelial membranes. The mechanism of CL action involves strong peptide assembly into polymers in solution. The free ends of linear CL polymers can join, forming loops that become pores upon binding to membranes. CL polymers constitute a therapeutic target for candidiasis, but little is known about CL self-assembly in solution. Here, we examine the assembly mechanism of CL in the absence of membranes using complementary biophysical tools, including a new fluorescence polymerization assay, mass photometry, and atomic force microscopy. We observed that CL assembly is slow, as tracked with the fluorescent marker C-laurdan. Single-molecule methods showed that CL polymerization involves a convolution of four processes. Self-assembly begins with the formation of a basic subunit, thought to be a CL octamer that is the polymer seed. Polymerization proceeds via the addition of octamers, and as polymers grow they can curve and form loops. Alternatively, secondary polymerization can occur and cause branching. Interplay between the different rates determines the distribution of CL particle types, indicating a kinetic control mechanism. This work elucidates key physical attributes underlying CL self-assembly which may eventually evoke pharmaceutical development.

Hearing in categories aids speech streaming at the "cocktail party".

Our perceptual system bins elements of the speech signal into categories to make speech perception manageable. Here, we aimed to test whether hearing speech in categories (as opposed to a continuous/gradient fashion) affords yet another benefit to speech recognition: parsing noisy speech at the "cocktail party." We measured speech recognition in a simulated 3D cocktail party environment. We manipulated task difficulty by varying the number of additional maskers presented at other spatial locations in the horizontal soundfield (1-4 talkers) and via forward vs. time-reversed maskers, promoting more and less informational masking (IM), respectively. In separate tasks, we measured isolated phoneme categorization using two-alternative forced choice (2AFC) and visual analog scaling (VAS) tasks designed to promote more/less categorical hearing and thus test putative links between categorization and real-world speech-in-noise skills. We first show that listeners can only monitor up to ~3 talkers despite up to 5 in the soundscape and streaming is not related to extended high-frequency hearing thresholds (though QuickSIN scores are). We then confirm speech streaming accuracy and speed decline with additional competing talkers and amidst forward compared to reverse maskers with added IM. Dividing listeners into "discrete" vs. "continuous" categorizers based on their VAS labeling (i.e., whether responses were binary or continuous judgments), we then show the degree of IM experienced at the cocktail party is predicted by their degree of categoricity in phoneme labeling; more discrete listeners are less susceptible to IM than their gradient responding peers. Our results establish a link between speech categorization skills and cocktail party processing, with a categorical (rather than gradient) listening strategy benefiting degraded speech perception. These findings imply figure-ground deficits common in many disorders might arise through a surprisingly simple mechanism: a failure to properly bin sounds into categories.

An autoantibody signature predictive for multiple sclerosis.

Although B cells are implicated in multiple sclerosis (MS) pathophysiology, a predictive or diagnostic autoantibody remains elusive. In this study, the Department of Defense Serum Repository (DoDSR), a cohort of over 10 million individuals, was used to generate whole-proteome autoantibody profiles of hundreds of patients with MS (PwMS) years before and subsequently after MS onset. This analysis defines a unique cluster in approximately 10% of PwMS who share an autoantibody signature against a common motif that has similarity with many human pathogens. These patients exhibit antibody reactivity years before developing MS symptoms and have higher levels of serum neurofilament light (sNfL) compared to other PwMS. Furthermore, this profile is preserved over time, providing molecular evidence for an immunologically active preclinical period years before clinical onset. This autoantibody reactivity was validated in samples from a separate incident MS cohort in both cerebrospinal fluid and serum, where it is highly specific for patients eventually diagnosed with MS. This signature is a starting point for further immunological characterization of this MS patient subset and may be clinically useful as an antigen-specific biomarker for high-risk patients with clinically or radiologically isolated neuroinflammatory syndromes.

Limitations of life-sustaining therapies in South Africa.

PURPOSE: Limitations of life sustaining therapies (LLST) are frequent in intensive care units (ICUs), but no previous studies have examined end-of-life (EOL) care and LLST in South Africa (SA). MATERIALS AND METHODS: This study evaluated LLST in SA from the data of a prospective, international, multicentre, observational study (Ethicus-2) and compared practices with countries in the rest of the world. RESULTS: LLST was relatively common in SA, and withholding was more frequent than withdrawing therapy. However, withdrawing and withholding therapy were less common, while failed CPR was more common, than in many other countries. No patients had an advance directive. Primary reasons for LLST in SA were poor quality of life, multisystem organ failure and patients' unresponsiveness to maximal therapy. Primary considerations for EOL decision-making were good medical practice and patients' best-interest, with the need for an ICU bed only rarely considered. CONCLUSIONS: Withholding was more common than withdrawing treatment both in SA and worldwide, although both were significantly less frequent in SA compared with the world average.

Novel RNA molecular bioengineering technology efficiently produces functional miRNA agents.

Genome-derived microRNAs (miRNAs or miRs) govern posttranscriptional gene regulation and play important roles in various cellular processes and disease progression. While chemo-engineered miRNA mimics or biosimilars made in vitro are widely available and used, miRNA agents produced in vivo are emerging to closely recapitulate natural miRNA species for research. Our recent work has demonstrated the success of high-yield, in vivo production of recombinant miRNAs by using human tRNA (htRNA) fused precursor miRNA (pre-miR) carriers. In this study, we aim to compare the production of bioengineered RNA (BioRNA) molecules with glycyl versus leucyl htRNA fused hsa-pre-miR-34a carriers, namely, BioRNAGly and BioRNALeu, respectively, and perform the initial functional assessment. We designed, cloned, overexpressed, and purified a total of 48 new BioRNA/miRNAs, and overall expression levels, final yields, and purities were revealed to be comparable between BioRNAGly and BioRNALeu molecules. Meanwhile, the two versions of BioRNA/miRNAs showed similar activities to inhibit non-small cell lung cancer cell viability. Interestingly, functional analyses using model BioRNA/miR-7-5p demonstrated that BioRNAGly/miR-7-5p exhibited greater efficiency to regulate a known target gene expression (EGFR) than BioRNALeu/miR-7-5p, consistent with miR-7-5p levels released in cells. Moreover, BioRNAGly/miR-7-5p showed comparable or slightly greater activities to modulate MRP1 and VDAC1 expression, compared with miRCURY LNA miR-7-5p mimic. Computational modeling illustrated overall comparable 3D structures for exemplary BioRNA/miRNAs with noticeable differences in htRNA species and payload miRNAs. These findings support the utility of hybrid htRNA/hsa-pre-miR-34a as reliable carriers for RNA molecular bioengineering, and the resultant BioRNAs serve as functional biologic RNAs for research and development.

Probing the Mechanisms Underlying the Transport of the Vinca Alkaloids by P-glycoprotein.

The efficacy of many cancer drugs is hindered by P-glycoprotein (Pgp), a cellular pump that removes drugs from cells. To improve chemotherapy, drugs capable of evading Pgp must be developed. Despite similarities in structure, vinca alkaloids (VAs) show disparate Pgp-mediated efflux ratios. ATPase activity and binding affinity studies show at least two binding sites for the VAs: high- and low-affinity sites that stimulate and inhibit the ATPase activity rate, respectively. The affinity for ATP from the ATPase kinetics curve for vinblastine (VBL) at the high-affinity site was 2- and 9-fold higher than vinorelbine (VRL) and vincristine (VCR), respectively. Conversely, VBL had the highest Km (ATP) for the low-affinity site. The dissociation constants (KDs) determined by protein fluorescence quenching were in the order VBL < VRL< VCR. The order of the KDs was reversed at higher substrate concentrations. Acrylamide quenching of protein fluorescence indicate that the VAs, either at 10 µM or 150 µM, predominantly maintain Pgp in an open-outward conformation. When 3.2 mM AMPPNP was present, 10 µM of either VBL, VRL, or VCR cause Pgp to shift to an open-outward conformation, while 150 µM of the VAs shifted the conformation of Pgp to an intermediate orientation, between opened inward and open-outward. However, the conformational shift induced by saturating AMPPNP and VCR condition was less than either VBL or VRL in the presence of AMPPNP. At 150 µM, atomic force microscopy (AFM) revealed that the VAs shift Pgp population to a predominantly open-inward conformation. Additionally, STDD NMR studies revealed comparable groups in VBL, VRL, and VCR are in contact with the protein during binding. Our results, when coupled with VAs-microtubule structure-activity relationship studies, could lay the foundation for developing next-generation VAs that are effective as anti-tumor agents. A model that illustrates the intricate process of Pgp-mediated transport of the VAs is presented.

Improvement in Hearing Loss Following Posterior Fossa Arachnoid Cyst Fenestration: A Case Study.

Arachnoid cysts are abnormal intradural collections of cerebrospinal fluid. For posterior fossa arachnoid cysts (PFACs), symptoms vary greatly, often relating to cranial nerve impingement and/or hydrocephalus. Literature on long-term symptomatic and radiographic follow-up of PFACs is lacking. This case study describes a 32-year-old man who presented with headaches and left-sided hearing loss and was found to have a large left-sided cerebellopontine angle arachnoid cyst with syrinx and ventriculomegaly. After PFAC fenestration and excision, his headaches resolved and his hearing markedly improved. At the one-year postoperative evaluation, symptom improvement persisted, and MRI demonstrated a stable decreased cyst and near-complete resolution of his syrinx.

A multivalent mRNA monkeypox virus vaccine (BNT166) protects mice and macaques from orthopoxvirus disease.

In response to the 2022 outbreak of mpox driven by unprecedented human-to-human monkeypox virus (MPXV) transmission, we designed BNT166, aiming to create a highly immunogenic, safe, accessible, and scalable next-generation vaccine against MPXV and related orthopoxviruses. To address the multiple viral forms and increase the breadth of immune response, two candidate multivalent mRNA vaccines were evaluated pre-clinically: a quadrivalent vaccine (BNT166a; encoding the MPXV antigens A35, B6, M1, H3) and a trivalent vaccine (BNT166c; without H3). Both candidates induced robust T cell responses and IgG antibodies in mice, including neutralizing antibodies to both MPXV and vaccinia virus. In challenge studies, BNT166a and BNT166c provided complete protection from vaccinia, clade I, and clade IIb MPXV. Furthermore, immunization with BNT166a was 100% effective at preventing death and at suppressing lesions in a lethal clade I MPXV challenge in cynomolgus macaques. These findings support the clinical evaluation of BNT166, now underway (NCT05988203).

Monitoring Disease Severity of Mild Cognitive Impairment from Single-Channel EEG Data Using Regression Analysis.

A deviation in the soundness of cognitive health is known as mild cognitive impairment (MCI), and it is important to monitor it early to prevent complicated diseases such as dementia, Alzheimer's disease (AD), and Parkinson's disease (PD). Traditionally, MCI severity is monitored with manual scoring using the Montreal Cognitive Assessment (MoCA). In this study, we propose a new MCI severity monitoring algorithm with regression analysis of extracted features of single-channel electro-encephalography (EEG) data by automatically generating severity scores equivalent to MoCA scores. We evaluated both multi-trial and single-trail analysis for the algorithm development. For multi-trial analysis, 590 features were extracted from the prominent event-related potential (ERP) points and corresponding time domain characteristics, and we utilized the lasso regression technique to select the best feature set. The 13 best features were used in the classical regression techniques: multivariate regression (MR), ensemble regression (ER), support vector regression (SVR), and ridge regression (RR). The best results were observed for ER with an RMSE of 1.6 and residual analysis. In single-trial analysis, we extracted a time-frequency plot image from each trial and fed it as an input to the constructed convolutional deep neural network (CNN). This deep CNN model resulted an RMSE of 2.76. To our knowledge, this is the first attempt to generate automated scores for MCI severity equivalent to MoCA from single-channel EEG data with multi-trial and single data.

High levels of metacercarial infestation (family: Diplostomidae) do not affect host energetics and swimming performance in the Epaulette goby (Coryogalops sordidus, Gobiidae).

Parasites have deleterious effects on their hosts, often resulting in altered host behavior or increased energy expenditure. When organisms are exposed to suboptimal environments, parasite loading may increase. Microbialite pools along the warm temperate South African coastline have been hypothesized as refugia for Epaulette gobies (Coryogalops sordidus, Gobiidae) when they are outside of their previously known subtropical distribution. The aim of this study was to determine if C. sordidus individuals infected with metacercarial cysts display higher metabolic rates or different swimming behavior compared to noninfected individuals. We measured each goby's swimming performance using a critical station-holding speed (Ucrit) test (n = 60) and visually scored their swimming behavior (n = 52) during these measurements. Also, we measured the metabolic rate of gobies using an intermittent flow respirometer system to determine standard metabolic rate (SMR) and maximum metabolic rate (MMR) from gobies at 21°C before and after swimming trials. Metacercarial load carried by infected gobies seemingly had no impact on the host's energetics (SMR or MMR), swimming ability (as repeated Ucrit tests), or swimming behavior compared to noninfected gobies. Thus, the metacercarial intensity observed in gobies in the current study appeared to have no impact on host swimming performance or behavior. Furthermore, the swimming capacity observed for C. sordidus, in general, suggests that this goby is a poor swimmer compared to other gobiid species.

Pseudogenes act as a neutral reference for detecting selection in prokaryotic pangenomes.

A long-standing question is to what degree genetic drift and selection drive the divergence in rare accessory gene content between closely related bacteria. Rare genes, including singletons, make up a large proportion of pangenomes (all genes in a set of genomes), but it remains unclear how many such genes are adaptive, deleterious or neutral to their host genome. Estimates of species' effective population sizes (Ne) are positively associated with pangenome size and fluidity, which has independently been interpreted as evidence for both neutral and adaptive pangenome models. We hypothesized that pseudogenes, used as a neutral reference, could be used to distinguish these models. We find that most functional categories are depleted for rare pseudogenes when a genome encodes only a single intact copy of a gene family. In contrast, transposons are enriched in pseudogenes, suggesting they are mostly neutral or deleterious to the host genome. Thus, even if individual rare accessory genes vary in their effects on host fitness, we can confidently reject a model of entirely neutral or deleterious rare genes. We also define the ratio of singleton intact genes to singleton pseudogenes (si/sp) within a pangenome, compare this measure across 668 prokaryotic species and detect a signal consistent with the adaptive value of many rare accessory genes. Taken together, our work demonstrates that comparing with pseudogenes can improve inferences of the evolutionary forces driving pangenome variation.

Effects of Stimulus Rate and Periodicity on Auditory Cortical Entrainment to Continuous Sounds.

The neural mechanisms underlying the exogenous coding and neural entrainment to repetitive auditory stimuli have seen a recent surge of interest. However, few studies have characterized how parametric changes in stimulus presentation alter entrained responses. We examined the degree to which the brain entrains to repeated speech (i.e., /ba/) and nonspeech (i.e., click) sounds using phase-locking value (PLV) analysis applied to multichannel human electroencephalogram (EEG) data. Passive cortico-acoustic tracking was investigated in N = 24 normal young adults utilizing EEG source analyses that isolated neural activity stemming from both auditory temporal cortices. We parametrically manipulated the rate and periodicity of repetitive, continuous speech and click stimuli to investigate how speed and jitter in ongoing sound streams affect oscillatory entrainment. Neuronal synchronization to speech was enhanced at 4.5 Hz (the putative universal rate of speech) and showed a differential pattern to that of clicks, particularly at higher rates. PLV to speech decreased with increasing jitter but remained superior to clicks. Surprisingly, PLV entrainment to clicks was invariant to periodicity manipulations. Our findings provide evidence that the brain's neural entrainment to complex sounds is enhanced and more sensitized when processing speech-like stimuli, even at the syllable level, relative to nonspeech sounds. The fact that this specialization is apparent even under passive listening suggests a priority of the auditory system for synchronizing to behaviorally relevant signals.

Atomic force microscope kymograph analysis: A case study of two membrane proteins.

Kymograph analysis is employed across the biological atomic force microscopy (AFM) community to boost temporal resolution. The method is well suited for revealing protein dynamics at the single molecule level in near-native conditions. Yet, kymograph analysis comes with limitations that depend on several factors including protein geometry and instrumental drift. This work focuses on conformational dynamics of difficult-to-study sparse distributions of membrane proteins. We compare and contrast AFM kymograph analysis for two proteins, one of which (SecDF) exhibits conformational dynamics primarily in the vertical direction (normal to the membrane surface) and the other (Pgp) exhibits a combination of lateral dynamics and vertical motion. Common experimental issues are analyzed including translational and rotational drift. Conformational transition detection is evaluated via kymograph simulations followed by state detection algorithms. We find that kymograph analysis is largely robust to lateral drift. Displacement of the AFM line scan trajectory away from the protein center of mass by a few nanometers, roughly half of the molecule diameter, does not significantly affect transition detection nor generate undue dwell time errors. On the other hand, for proteins like Pgp that exhibit significant azimuthal maximum height dependence, rotational drift can potentially produce artifactual transitions. Measuring the height of a membrane protein protrusion is generally superior to measurement of width, confirming intuition based on vertical resolution superiority. In low signal-to-noise scenarios, common state detection algorithms struggle with transition detection as opposed to infinite hidden Markov models. AFM kymography represents a valuable addition to the membrane biophysics toolkit; continued hardware and software improvements are poised to expand the method's impact in the field.