Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2 of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5-although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7-9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)10. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.

Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target.

Drugs that target histone deacetylase (HDAC) entered the pharmacopoeia in the 2000s. However, some enigmatic phenotypes suggest off-target engagement. Here, we developed a quantitative chemical proteomics assay using immobilized HDAC inhibitors and mass spectrometry that we deployed to establish the target landscape of 53 drugs. The assay covers 9 of the 11 human zinc-dependent HDACs, questions the reported selectivity of some widely-used molecules (notably for HDAC6) and delineates how the composition of HDAC complexes influences drug potency. Unexpectedly, metallo-ß-lactamase domain-containing protein 2 (MBLAC2) featured as a frequent off-target of hydroxamate drugs. This poorly characterized palmitoyl-CoA hydrolase is inhibited by 24 HDAC inhibitors at low nanomolar potency. MBLAC2 enzymatic inhibition and knockdown led to the accumulation of extracellular vesicles. Given the importance of extracellular vesicle biology in neurological diseases and cancer, this HDAC-independent drug effect may qualify MBLAC2 as a target for drug discovery.

Layer-specific stimulations of parvalbumin-positive cortical interneurons in mice entrain brain rhythms to different frequencies.

Neocortical interneurons provide inhibition responsible for organizing neuronal activity into brain oscillations that subserve cognitive functions such as memory, attention, or prediction. However, the interneuronal contribution to the entrainment of neocortical oscillations within and across different cortical layers was not described. Here, using layer-specific optogenetic stimulations with micro-Light-Emitting Diode arrays, directed toward parvalbumin-expressing (PV) interneurons in non-anesthetized awake mice, we found that supragranular layer stimulations of PV neurons were most efficient at entraining supragranular local field potential (LFP) oscillations at gamma frequencies (γ: 25-80 Hz), whereas infragranular layer stimulation of PV neurons better entrained the LFP at delta (δ: 2-5 Hz) and theta (θ: 6-10 Hz) frequencies. At the level of neuronal action potential activity, we observed that supragranular neurons better followed the imposed PV stimulation rhythm than their infragranular counterparts at most frequencies when the stimulation was delivered in their respective layer. Moreover, the neuronal entrainment evoked by local stimulation could propagate across layers, though with a lesser impact when the stimulation occurs in deep layers, suggesting a direction-specific laminar propagation. These results establish a layer-based framework for oscillations to entrain the primary somatosensory cortex in awake conditions.

Integration of the CA2 region in the hippocampal network during epileptogenesis.

The CA2 pyramidal cells are mostly resistant to cell death in mesial temporal lobe epilepsy (MTLE) with hippocampal sclerosis, but they are aberrantly integrated into the epileptic hippocampal network via mossy fiber sprouting. Furthermore, they show increased excitability in vitro in hippocampal slices obtained from human MTLE specimens or animal epilepsy models. Although these changes promote CA2 to contribute to epileptic activity (EA) in vivo, the role of CA2 in the epileptic network within and beyond the sclerotic hippocampus is still unclear. We used the intrahippocampal kainate mouse model for MTLE, which recapitulates most features of the human disease including pharmacoresistant epileptic seizures and hippocampal sclerosis, with preservation of dentate gyrus (DG) granule cells and CA2 pyramidal cells. In vivo recordings with electrodes in CA2 and the DG showed that EA occurs at high coincidence between the ipsilateral DG and CA2 and current source density analysis of silicon probe recordings in dorsal ipsilateral CA2 revealed CA2 as a local source of EA. Cell-specific viral tracing in Amigo2-icreERT2 mice confirmed the preservation of the axonal projection from ipsilateral CA2 pyramidal cells to contralateral CA2 under epileptic conditions and indeed, EA propagated from ipsi- to contralateral CA2 with increasing likelihood with time after KA injection, but always at lower intensity than within the ipsilateral hippocampus. Furthermore, we show that CA2 presents with local theta oscillations and like the DG, shows a pathological reduction of theta frequency already from 2 days after KA onward. The early changes in activity might be facilitated by the loss of glutamic acid decarboxylase 67 (Gad67) mRNA-expressing interneurons directly after the initial status epilepticus in ipsi- but not contralateral CA2. Together, our data highlight CA2 as an active player in the epileptic network and with its contralateral connections as one possible router of aberrant activity.

A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients.

State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm mass accuracy at sequencing speeds of tens of peptides per second with attomolar sensitivity. Here we describe a compact and robust quadrupole-orbitrap mass spectrometer equipped with a front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance of the Orbitrap Exploris 480 mass spectrometer is evaluated in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes in combination with FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal for DDA and DIA, respectively. Combining DIA with FAIMS using single CVs, the instrument surpasses 2500 peptides identified per minute. This enables quantification of >5000 proteins with short online LC gradients delivered by the Evosep One LC system allowing acquisition of 60 samples per day. The raw sensitivity of the instrument is evaluated by analyzing 5 ng of a HeLa digest from which >1000 proteins were reproducibly identified with 5 min LC gradients using DIA-FAIMS. To demonstrate the versatility of the instrument, we recorded an organ-wide map of proteome expression across 12 rat tissues quantified by tandem mass tags and label-free quantification using DIA with FAIMS to a depth of >10,000 proteins.

Protein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation.

Universal proteomics sample preparation is challenging because of the high heterogeneity of biological samples. Here we describe a novel mechanism that exploits the inherent instability of denatured proteins for nonspecific immobilization on microparticles by protein aggregation capture. To demonstrate the general applicability of this mechanism, we analyzed phosphoproteomes, tissue proteomes, and interaction proteomes as well as dilute secretomes. The findings present a practical, sensitive and cost-effective proteomics sample preparation method.

Towards the clinical translation of optogenetic skeletal muscle stimulation.

Paralysis is a frequent phenomenon in many diseases, and to date, only functional electrical stimulation (FES) mediated via the innervating nerve can be employed to restore skeletal muscle function in patients. Despite recent progress, FES has several technical limitations and significant side effects. Optogenetic stimulation has been proposed as an alternative, as it may circumvent some of the disadvantages of FES enabling cell type-specific, spatially and temporally precise stimulation of cells expressing light-gated ion channels, commonly Channelrhodopsin2. Two distinct approaches for the restoration of skeletal muscle function with optogenetics have been demonstrated: indirect optogenetic stimulation through the innervating nerve similar to FES and direct optogenetic stimulation of the skeletal muscle. Although both approaches show great promise, both have their limitations and there are several general hurdles that need to be overcome for their translation into clinics. These include successful gene transfer, sustained optogenetic protein expression, and the creation of optically active implantable devices. Herein, a comprehensive summary of the underlying mechanisms of electrical and optogenetic approaches is provided. With this knowledge in mind, we substantiate a detailed discussion of the advantages and limitations of each method. Furthermore, the obstacles in the way of clinical translation of optogenetic stimulation are discussed, and suggestions on how they could be overcome are provided. Finally, four specific examples of pathologies demanding novel therapeutic measures are discussed with a focus on the likelihood of direct versus indirect optogenetic stimulation.

Microfluidic chip to interface porous microneedles for ISF collection.

Porous microneedles (MNs) are expected to be applied for diagnostic microfluidic devices such as blood glucose monitoring as they enable a pain-free penetration of human skin and the extraction of interstitial fluids. However, conventional microfluidic systems require additional steps to separate the liquid from a porous structure used for fluid extraction. In this study, we developed a microfluidic system with a hydrodynamically designed interface between a porous MN array and microchannels to enable a direct analysis of liquids extracted by the porous MN array. The microfluidic chip with an interface for the MN array was successfully realized by standard MEMS processes, enabling a liquid flow through the whole microfluidic structure. The porous MN array was fabricated by the salt leaching and molding method, which was integrated with the chip and demonstrated the successful extraction of liquids from an agarose gel-based skin phantom.

Palaeoproteomic Profiling of Conservation Layers on a 14th Century Italian Wall Painting.

Ahead of display, a non-original layer was observed on the surface of a fragment of a wall painting by Ambrogio Lorenzetti (active 1319, died 1348/9). FTIR analysis suggested proteinaceous content. Mass spectrometry was used to better characterise this layer and revealed two protein components: sheep and cow glue and chicken and duck egg white. Analysis of post-translational modifications detected several photo-oxidation products, which suggest that the egg experienced prolonged exposure to UV light and was likely applied long before the glue layer. Additionally, glycation products detected may indicate naturally occurring glycoprotein degradation or reaction with a carbohydrate material such as starch, identified by ATR-FTIR in a cross-section of a sample taken from the painting. Palaeoproteomics is shown to provide detailed characterisation of organic layers associated with mural paintings and therefore aids reconstruction of the conservation history of these objects.

Hybrid intracerebral probe with integrated bare LED chips for optogenetic studies.

This article reports on the development, i.e., the design, fabrication, and validation of an implantable optical neural probes designed for in vivo experiments relying on optogenetics. The probes comprise an array of ten bare light-emitting diode (LED) chips emitting at a wavelength of 460 nm and integrated along a flexible polyimide-based substrate stiffened using a micromachined ladder-like silicon structure. The resulting mechanical stiffness of the slender, 250-µm-wide, 65-µm-thick, and 5- and 8-mm-long probe shank facilitates its implantation into neural tissue. The LEDs are encapsulated by a fluropolymer coating protecting the implant against the physiological conditions in the brain. The electrical interface to the external control unit is provided by 10-µm-thick, highly flexible polyimide cables making the probes suitable for both acute and chronic in vivo experiments. Optical and electrical properties of the probes are reported, as well as their in vivo validation in acute optogenetic studies in transgenic mice. The depth-dependent optical stimulation of both excitatory and inhibitory neurons is demonstrated by altering the brain activity in the cortex and the thalamus. Local network responses elicited by 20-ms-long light pulses of different optical power (20 µW and 1 mW), as well as local modulation of single unit neuronal activity to 1-s-long light pulses with low optical intensity (17 µW) are presented. The ability to modulate neural activity makes these devices suitable for a broad variety of optogenetic experiments.

Extending the Cortical Grasping Network: Pre-supplementary Motor Neuron Activity During Vision and Grasping of Objects.

Grasping relies on a network of parieto-frontal areas lying on the dorsolateral and dorsomedial parts of the hemispheres. However, the initiation and sequencing of voluntary actions also requires the contribution of mesial premotor regions, particularly the pre-supplementary motor area F6. We recorded 233 F6 neurons from 2 monkeys with chronic linear multishank neural probes during reaching-grasping visuomotor tasks. We showed that F6 neurons play a role in the control of forelimb movements and some of them (26%) exhibit visual and/or motor specificity for the target object. Interestingly, area F6 neurons form 2 functionally distinct populations, showing either visually-triggered or movement-related bursts of activity, in contrast to the sustained visual-to-motor activity displayed by ventral premotor area F5 neurons recorded in the same animals and with the same task during previous studies. These findings suggest that F6 plays a role in object grasping and extend existing models of the cortical grasping network.

Phosphor-based light conversion for miniaturized optical tools.

This paper describes the application of phosphor-based light conversion for its use in optogenetic experiments to tailor the wavelength of light emitted from implantable miniaturized light sources. Gallium-nitride-based blue light-emitting diodes are used in combination with orthosilicate phosphor immersed in an epoxy matrix and emitting in the yellow wavelength range. The miniaturization of the phosphor-containing polymer droplets toward diameters as small as 300 µm provides the compatibility with implantable optical probes. The parameter study applied here varied the concentration of the phosphor material in the polymer matrix as well as the droplet height in order to tailor the characteristics of blue-to-yellow light conversion.

Large-scale recording of thalamocortical circuits: in vivo electrophysiology with the two-dimensional electronic depth control silicon probe.

Recording simultaneous activity of a large number of neurons in distributed neuronal networks is crucial to understand higher order brain functions. We demonstrate the in vivo performance of a recently developed electrophysiological recording system comprising a two-dimensional, multi-shank, high-density silicon probe with integrated complementary metal-oxide semiconductor electronics. The system implements the concept of electronic depth control (EDC), which enables the electronic selection of a limited number of recording sites on each of the probe shafts. This innovative feature of the system permits simultaneous recording of local field potentials (LFP) and single- and multiple-unit activity (SUA and MUA, respectively) from multiple brain sites with high quality and without the actual physical movement of the probe. To evaluate the in vivo recording capabilities of the EDC probe, we recorded LFP, MUA, and SUA in acute experiments from cortical and thalamic brain areas of anesthetized rats and mice. The advantages of large-scale recording with the EDC probe are illustrated by investigating the spatiotemporal dynamics of pharmacologically induced thalamocortical slow-wave activity in rats and by the two-dimensional tonotopic mapping of the auditory thalamus. In mice, spatial distribution of thalamic responses to optogenetic stimulation of the neocortex was examined. Utilizing the benefits of the EDC system may result in a higher yield of useful data from a single experiment compared with traditional passive multielectrode arrays, and thus in the reduction of animals needed for a research study.

Fraxicon for Optical Applications with Aperture ∼1 mm: Characterisation Study.

Emerging applications of optical technologies are driving the development of miniaturised light sources, which in turn require the fabrication of matching micro-optical elements with sub-1 mm cross-sections and high optical quality. This is particularly challenging for spatially constrained biomedical applications where reduced dimensionality is required, such as endoscopy, optogenetics, or optical implants. Planarisation of a lens by the Fresnel lens approach was adapted for a conical lens (axicon) and was made by direct femtosecond 780 nm/100 fs laser writing in the SZ2080™ polymer with a photo-initiator. Optical characterisation of the positive and negative fraxicons is presented. Numerical modelling of fraxicon optical performance under illumination by incoherent and spatially extended light sources is compared with the ideal case of plane-wave illumination. Considering the potential for rapid replication in soft polymers and resists, this approach holds great promise for the most demanding technological applications.

Deep-time phylogenetic inference by paleoproteomic analysis of dental enamel.

In temperate and subtropical regions, ancient proteins are reported to survive up to about 2 million years, far beyond the known limits of ancient DNA preservation in the same areas. Accordingly, their amino acid sequences currently represent the only source of genetic information available to pursue phylogenetic inference involving species that went extinct too long ago to be amenable for ancient DNA analysis. Here we present a complete workflow, including sample preparation, mass spectrometric data acquisition and computational analysis, to recover and interpret million-year-old dental enamel protein sequences. During sample preparation, the proteolytic digestion step, usually an integral part of conventional bottom-up proteomics, is omitted to increase the recovery of the randomly degraded peptides spontaneously generated by extensive diagenetic hydrolysis of ancient proteins over geological time. Similarly, we describe other solutions we have adopted to (1) authenticate the endogenous origin of the protein traces we identify, (2) detect and validate amino acid variation in the ancient protein sequences and (3) attempt phylogenetic inference. Sample preparation and data acquisition can be completed in 3-4 working days, while subsequent data analysis usually takes 2-5 days. The workflow described requires basic expertise in ancient biomolecules analysis, mass spectrometry-based proteomics and molecular phylogeny. Finally, we describe the limits of this approach and its potential for the reconstruction of evolutionary relationships in paleontology and paleoanthropology.

Spread of activation and interaction between channels with multi-channel optogenetic stimulation in the mouse cochlea.

For individuals with severe to profound hearing loss resulting from irreversibly damaged hair cells, cochlear implants can be used to restore hearing by delivering electrical stimulation directly to the spiral ganglion neurons. However, current spread lowers the spatial resolution of neural activation. Since light can be easily confined, optogenetics is a technique that has the potential to improve the precision of neural activation, whereby visible light is used to stimulate neurons that are modified with light-sensitive opsins. This study compares the spread of neural activity across the inferior colliculus of the auditory midbrain during electrical and optical stimulation in the cochlea of acutely deafened mice with opsin-modified spiral ganglion neurons (H134R variant of the channelrhodopsin-2). Monopolar electrical stimulation was delivered via each of four 0.2 mm wide platinum electrode rings at 0.6 mm centre-to-centre spacing, whereas 453 nm wavelength light was delivered via each of five 0.22 × 0.27 mm micro-light emitting diodes (LEDs) at 0.52 mm centre-to-centre spacing. Channel interactions were also quantified by threshold changes during simultaneous stimulation by pairs of electrodes or micro-LEDs at different distances between the electrodes (0.6, 1.2 and 1.8 mm) or micro-LEDs (0.52, 1.04, 1.56 and 2.08 mm). The spread of activation resulting from single channel optical stimulation was approximately half that of monopolar electrical stimulation as measured at two levels of discrimination above threshold (p<0.001), whereas there was no significant difference between optical stimulation in opsin-modified deafened mice and pure tone acoustic stimulation in normal-hearing mice. During simultaneous micro-LED stimulation, there were minimal channel interactions for all micro-LED spacings tested. For neighbouring micro-LEDs/electrodes, the relative influence on threshold was 13-fold less for optical stimulation compared electrical stimulation (p<0.05). The outcomes of this study show that the higher spatial precision of optogenetic stimulation results in reduced channel interaction compared to electrical stimulation, which could increase the number of independent channels in a cochlear implant. Increased spatial resolution and the ability to activate more than one channel simultaneously could lead to better speech perception in cochlear implant recipients.

A deeper look at carrier proteome effects for single-cell proteomics.

Multiplexing approaches using tandem mass tags with a carrier proteome to boost sensitivity have advanced single cell proteomics by mass spectrometry (SCoPE-MS). Here, we probe the carrier proteome effects in single cell proteomics with mixed species TMTpro-labeled samples. We demonstrate that carrier proteomes, while increasing overall identifications, dictate which proteins are identified. We show that quantitative precision and signal intensity are limited at high carrier levels, hindering the recognition of regulated proteins. Guidelines for optimized mass spectrometry acquisition parameters and best practices for fold-change or protein copy number-based comparisons are provided.

Microfluidic chip connected to porous microneedle array for continuous ISF sampling.

Minimally invasive biosensing using microneedles (MNs) is a desirable technology for continuous healthcare monitoring. Among a wide range of MNs, porous MNs are expected to be applied for sampling of interstitial fluids (ISF) by connecting the internal tissue to external measurement devices. In order to realize a continuous measurement of biomarkers in ISF through porous MNs, their integration with a microfluidic chip is a promising approach due to its applicability to micro-total analysis system (µTAS) technology. In this study, we developed a fluidic system to directly interface porous MNs to a microfluidic chip consisting of a capillary pump for the continuous sampling of ISF. The porous and flexible MNs made of PDMS are connected to the microfluidic chip fabricated by standard microelectro-mechanical system (MEMS) processes, showing a continuous flow of phosphate buffered saline (PBS). The developed device will lead to the minimally invasive and continuous biosampling for long-term healthcare monitoring.

Multichannel optogenetics combined with laminar recordings for ultra-controlled neuronal interrogation.

Simultaneous large-scale recordings and optogenetic interventions may hold the key to deciphering the fast-paced and multifaceted dialogue between neurons that sustains brain function. Here we have taken advantage of thin, cell-sized, optical fibers for minimally invasive optogenetics and flexible implantations. We describe a simple procedure for making those fibers side-emitting with a Lambertian emission distribution. Here we combined those fibers with silicon probes to achieve high-quality recordings and ultrafast multichannel optogenetic inhibition. Furthermore, we developed a multi-channel optical commutator and general-purpose patch-cord for flexible experiments. We demonstrate that our framework allows to conduct simultaneous laminar recordings and multifiber stimulations, 3D optogenetic stimulation, connectivity inference, and behavioral quantification in freely moving animals. Our framework paves the way for large-scale photo tagging and controlled interrogation of rapid neuronal communication in any combination of brain areas.