Multi-Technique Characterization of 3.45 Ga Microfossils on Earth: A Key Approach to Detect Possible Traces of Life in Returned Samples from Mars.

The NASA Mars 2020 Perseverance rover is actively exploring Jezero crater to conduct analyses on igneous and sedimentary rock targets from outcrops located on the crater floor (Máaz and Séítah formations) and from the delta deposits, respectively. The rock samples collected during this mission will be recovered during the Mars Sample Return mission, which plans to bring samples back to Earth in the 2030s to conduct in-depth studies using sophisticated laboratory instrumentation. Some of these samples may contain traces of ancient martian life that may be particularly difficult to detect and characterize because of their morphological simplicity and subtle biogeochemical expressions. Using the volcanic sediments of the 3.45 Ga Kitty's Gap Chert (Pilbara, Australia), containing putative early life forms (chemolithotrophs) and considered as astrobiological analogues for potential early Mars organisms, we document the steps required to demonstrate the syngenicity and biogenicity of such biosignatures using multiple complementary analytical techniques to provide information at different scales of observation. These include sedimentological, petrological, mineralogical, and geochemical analyses to demonstrate macro- to microscale habitability. New approaches, some unavailable at the time of the original description of these features, are used to verify the syngenicity and biogenicity of the purported fossil chemolithotrophs. The combination of elemental (proton-induced X-ray emission spectrometry) and molecular (deep-ultraviolet and Fourier transform infrared) analyses of rock slabs, thin sections, and focused ion beam sections reveals that the carbonaceous matter present in the samples is enriched in trace metals (e.g., V, Cr, Fe, Co) and is associated with aromatic and aliphatic molecules, which strongly support its biological origin. Transmission electron microscopy observations of the carbonaceous matter documented an amorphous nanostructure interpreted to correspond to the degraded remains of microorganisms and their by-products (extracellular polymeric substances, filaments…). Nevertheless, a small fraction of carbonaceous particles has signatures that are more metamorphosed. They probably represent either reworked detrital biological or abiotic fragments of mantle origin. This study serves as an example of the analytical protocol that would be needed to optimize the detection of fossil traces of life in martian rocks.

Enhanced resonance energy transfer in gold nanoparticles bifunctionalized by tryptophan and riboflavin and its application in fluorescence bioimaging.

Gold nanoparticles were functionalized by amino acid tryptophan and vitamin riboflavin - a resonance energy transfer (RET) pair of biomolecules. The presence of the gold nanoparticles resulted in 65% increase in RET efficiency. Because of enhanced RET efficiency, the photobleaching dynamics of the fluorescent molecules at the surface of the nanoparticles is different from that of molecules in solution. The observed effect was used for detection of the functionalized nanoparticles within biological material rich with autofluorescent species. Synchrotron radiation deep-ultraviolet fluorescence microscopy is used to study the photobleaching dynamics of the fluorescence centers within human hepatocellular carcinoma Huh7.5.1 cells incubated with the nanoparticles. The fluorescent centers were classified according to their photobleaching dynamics, which enabled the discrimination of the cell areas where the accumulation of the nanoparticles takes place, even though the particles were smaller than the spatial resolution of the images.

Biological effects of the loss of homochirality in a multicellular organism.

Homochirality is a fundamental feature of all known forms of life, maintaining biomolecules (amino-acids, proteins, sugars, nucleic acids) in one specific chiral form. While this condition is central to biology, the mechanisms by which the adverse accumulation of non-L-α-amino-acids in proteins lead to pathophysiological consequences remain poorly understood. To address how heterochirality build-up impacts organism's health, we use chiral-selective in vivo assays to detect protein-bound non-L-α-amino acids (focusing on aspartate) and assess their functional significance in Drosophila. We find that altering the in vivo chiral balance creates a 'heterochirality syndrome' with impaired caspase activity, increased tumour formation, and premature death. Our work shows that preservation of homochirality is a key component of protein function that is essential to maintain homeostasis across the cell, tissue and organ level.

A framework for dissecting affinities of multidrug efflux transporter AcrB to fluoroquinolones.

Sufficient concentration of antibiotics close to their target is key for antimicrobial action. Among the tools exploited by bacteria to reduce the internal concentration of antibiotics, multidrug efflux pumps stand out for their ability to capture and expel many unrelated compounds out of the cell. Determining the specificities and efflux efficiency of these pumps towards their substrates would provide quantitative insights into the development of antibacterial strategies. In this light, we developed a competition efflux assay on whole cells, that allows measuring the efficacy of extrusion of clinically used quinolones in populations and individual bacteria. Experiments reveal the efficient competitive action of some quinolones that restore an active concentration of other fluoroquinolones. Computational methods show how quinolones interact with the multidrug efflux transporter AcrB. Combining experiments and computations unveils a key molecular mechanism acting in vivo to detoxify bacterial cells. The developed assay can be generalized to the study of other efflux pumps.

Application of Synchrotron Radiation-Based Micro-Analysis on Cadmium Yellows in Pablo Picasso's Femme.

The cultural heritage community is increasingly exploring synchrotron radiation (SR) based techniques for the study of art and archaeological objects. When considering heterogeneous and complex micro-samples, such as those from paintings, the combination of different SR X-ray techniques is often exploited to overcome the intrinsic limitations and sensitivity of the single technique. Less frequently, SR X-ray analyses are combined with SR micro-photoluminescence or micro-Fourier Transform Infrared spectroscopy, which provide complementary information on the molecular composition, offering a unique integrated analysis approach. Although the spatial correlation between the maps obtained with different techniques is not straightforward due to the different volumes probed by each method, the combination of the information provides a greater understanding and insight into the paint chemistry. In this work, we discuss the advantages and disadvantages of the combination of X-ray techniques and SR-based photoluminescence through the study of two paint micro-samples taken from Pablo Picasso's Femme (1907). The painting contains two cadmium yellow paints (based on CdS): one relatively intact and one visibly degraded. SR micro-analyses demonstrated that the two Cd-yellow paints differ in terms of structure, chemical composition, and photoluminescence properties. In particular, on the basis of the combination of different SR measurements, we hypothesize that the degraded yellow is based on nanocrystalline CdS with high presence of Cd(OH)Cl. These two characteristics have enhanced the reactivity of the paint and strongly influenced its stability.

BeStSel: webserver for secondary structure and fold prediction for protein CD spectroscopy.

Circular dichroism (CD) spectroscopy is widely used to characterize the secondary structure composition of proteins. To derive accurate and detailed structural information from the CD spectra, we have developed the Beta Structure Selection (BeStSel) method (PNAS, 112, E3095), which can handle the spectral diversity of ß-structured proteins. The BeStSel webserver provides this method with useful accessories to the community with the main goal to analyze single or multiple protein CD spectra. Uniquely, BeStSel provides information on eight secondary structure components including parallel ß-structure and antiparallel ß-sheets with three different groups of twist. It overperforms any available method in accuracy and information content, moreover, it is capable of predicting the protein fold down to the topology/homology level of the CATH classification. A new module of the webserver helps to distinguish intrinsically disordered proteins by their CD spectrum. Secondary structure calculation for uploaded PDB files will help the experimental verification of protein MD and in silico modelling using CD spectroscopy. The server also calculates extinction coefficients from the primary sequence for CD users to determine the accurate protein concentrations which is a prerequisite for reliable secondary structure determination. The BeStSel server can be freely accessed at https://bestsel.elte.hu.

Disordered-Ordered Protein Binary Classification by Circular Dichroism Spectroscopy.

Intrinsically disordered proteins lack a stable tertiary structure and form dynamic conformational ensembles due to their characteristic physicochemical properties and amino acid composition. They are abundant in nature and responsible for a large variety of cellular functions. While numerous bioinformatics tools have been developed for in silico disorder prediction in the last decades, there is a need for experimental methods to verify the disordered state. CD spectroscopy is widely used for protein secondary structure analysis. It is usable in a wide concentration range under various buffer conditions. Even without providing high-resolution information, it is especially useful when NMR, X-ray, or other techniques are problematic or one simply needs a fast technique to verify the structure of proteins. Here, we propose an automatized binary disorder-order classification method by analyzing far-UV CD spectroscopy data. The method needs CD data at only three wavelength points, making high-throughput data collection possible. The mathematical analysis applies the k-nearest neighbor algorithm with cosine distance function, which is independent of the spectral amplitude and thus free of concentration determination errors. Moreover, the method can be used even for strong absorbing samples, such as the case of crowded environmental conditions, if the spectrum can be recorded down to the wavelength of 212 nm. We believe the classification method will be useful in identifying disorder and will also facilitate the growth of experimental data in IDP databases. The method is implemented on a webserver and freely available for academic users.

Detection and localization of calcium oxalate in kidney using synchrotron deep ultraviolet fluorescence microscopy.

Renal oxalosis is a rare cause of renal failure whose diagnosis can be challenging. Synchrotron deep ultraviolet (UV) fluorescence was assayed to improve oxalosis detection on kidney biopsies spatial resolution and sensitivity compared with the Fourier transform infrared microspectroscopy gold standard. The fluorescence spectrum of synthetic mono-, di- and tri-hydrated calcium oxalate was investigated using a microspectrometer coupled to the synchrotron UV beamline DISCO, Synchrotron SOLEIL, France. The obtained spectra were used to detect oxalocalcic crystals in a case control study of 42 human kidney biopsies including 19 renal oxalosis due to primary (PHO, n = 11) and secondary hyperoxaluria (SHO, n = 8), seven samples from PHO patients who received combined kidney and liver transplants, and 16 controls. For all oxalocalcic hydrates samples, a fluorescence signal is detected at 420 nm. These spectra were used to identify standard oxalocalcic crystals in patients with PHO or SHO. They also revealed micrometric crystallites as well as non-aggregated oxalate accumulation in tubular cells. A nine-points histological score was established for the diagnosis of renal oxalosis with 100% specificity (76-100) and a 73% sensitivity (43-90). Oxalate tubular accumulation and higher histological score were correlated to lower estimated glomerular filtration rate and higher urinary oxalate over creatinine ratio.

Novel markers to early detect degradation on cellulose nitrate-based heritage at the submicrometer level using synchrotron UV-VIS multispectral luminescence.

Cellulose nitrate (CN) is an intrinsically unstable material that puts at risk the preservation of a great variety of objects in heritage collections, also posing threats to human health. For this reason, a detailed investigation of its degradation mechanisms is necessary to develop sustainable conservation strategies. To investigate novel probes of degradation, we implemented deep UV photoluminescence micro spectral-imaging, for the first time, to characterize a corpus of historical systems composed of cellulose nitrate. The analysis of cinematographic films and everyday objects dated from the nineteenth c./early twentieth c. (Perlov's collection), as well as of photo-aged CN and celluloid references allowed the identification of novel markers that correlate with different stages of CN degradation in artworks, providing insight into the role played by plasticizers, fillers, and other additives in stability. By comparison with photoaged references of CN and celluloid (70% CN and 30% camphor), it was possible to correlate camphor concentration with a higher rate of degradation of the cinematographic films. Furthermore, the present study investigates, at the sub-microscale, materials heterogeneity that correlates to the artworks' history, associating the different emission profiles of zinc oxide to specific color formulations used in the late nineteenth and early twentieth centuries.

PB1-F2 amyloid-like fibers correlate with proinflammatory signaling and respiratory distress in influenza-infected mice.

PB1-F2 is a virulence factor of influenza A virus known to increase viral pathogenicity in mammalian hosts. PB1-F2 is an intrinsically disordered protein displaying a propensity to form amyloid-like fibers. However, the correlation between PB1-F2 structures and the resulting inflammatory response is unknown. Here, we used synchrotron-coupled Fourier transform-IR and deep UV microscopies to determine the presence of PB1-F2 fibers in influenza A virus-infected mice. In order to study the correlation between PB1-F2 structure and the inflammatory response, transgenic mice expressing luciferase under the control of an NF-κB promotor, allowing in vivo monitoring of inflammation, were intranasally instilled with monomeric, fibrillated, or truncated forms of recombinant PB1-F2. Our intravital NF-κB imaging, supported by cytokine quantification, clearly shows the proinflammatory effect of PB1-F2 fibers compared with N-terminal region of PB1-F2 unable to fibrillate. It is noteworthy that instillation of monomeric PB1-F2 of H5N1 virus induced a stronger inflammatory response when compared with prefibrillated PB1-F2 of H1N1 virus, suggesting mechanisms of virulence depending on PB1-F2 sequence. Finally, using whole-body plethysmography to measure volume changes in the lungs, we quantified the effects of the different forms of PB1-F2 on respiratory parameters. Thus, we conclude that PB1-F2-induced inflammation and respiratory distress are tightly correlated with sequence polymorphism and oligomerization status of the protein.

Investigation by Synchrotron Radiation Circular Dichroism of the Secondary Structure Evolution of Pepsin under Oxidative Environment.

Food processing affects the structure and chemical state of proteins. In particular, protein oxidation occurs and may impair protein properties. These chemical reactions initiated during processing can develop during digestion. Indeed, the physicochemical conditions of the stomach (oxygen pressure, low pH) favor oxidation. In that respect, digestive proteases may be affected as well. Yet, very little is known about the link between endogenous oxidation of digestive enzymes, their potential denaturation, and, therefore, food protein digestibility. Thus, the objective of this study is to understand how oxidative chemical processes will impact the pepsin secondary structure and its hydrolytic activity. The folding and unfolding kinetics of pepsin under oxidative conditions was determined using Synchrotron Radiation Circular Dichroism. SRCD gave us the possibility to monitor the rapid kinetics of protein folding and unfolding in real-time, giving highly resolved spectral data. The proteolytic activity of control and oxidized pepsin was investigated by MALDI-TOF mass spectrometry on a meat protein model, the creatine kinase. MALDI-TOF MS allowed a rapid evaluation of the proteolytic activity through peptide fingerprint. This study opens up new perspectives by shifting the digestion paradigm taking into account the gastric digestive enzyme and its substrate.

Molecular changes tracking through multiscale fluorescence microscopy differentiate Meningioma grades and non-tumoral brain tissues.

Meningioma is the most common primary intracranial extra-axial tumor. Total surgical removal is the standard therapeutic method to treat this type of brain tumors. However, the risk of recurrence depends on the tumor grade and the extent of the resection including the infiltrated dura mater and, if necessary, the infiltrated bone. Therefore, proper resection of all invasive tumor borders without touching eloquent areas is of primordial in order to decrease the risk of recurrence. Nowadays, none of the intraoperative used tools is able to provide a precise real-time histopathological information on the tumor surrounding areas to help the surgeon to achieve a gross total removal. To respond to this problem, our team is developing a multimodal two-photon fluorescence endomicroscope, compatible with the surgeon tool, to better delimitate tumor boundaries, relying on the endogenous fluorescence of brain tissues. In this context, we are building a tissue database in order to specify each brain tissue, whether healthy or tumoral, with its specific optical signature. In this study, we present a multimodal and multiscale optical measurements on non-tumoral control brain tissue obtained in epilepsy surgery patients and several meningioma grades. We investigated tissue auto-fluorescence to track the molecular changes associated with the tumor grade from deep ultra-violet (DUV) to near infrared (NIR) excitation. Micro-spectroscopy, fluorescence lifetime imaging, two-photon fluorescence imaging and Second Harmonic Generation (SHG) imaging were performed. Several optically derived parameters such as collagen crosslinks fluorescence in DUV, SHG emission in NIR and long lifetime intensity fraction of Nicotinamide Adenine Dinucleotide and Flavins were correlated to discriminate cancerous tissue from control one. While collagen response managed to discriminate meningioma grades from control samples with a 100% sensitivity and 90% specificity through a 3D discriminative algorithm.

Microchemical analysis of Leonardo da Vinci's lead white paints reveals knowledge and control over pigment scattering properties.

Leonardo da Vinci (1452-1519) is a key artistic and scientific figure of the Renaissance. He is renowned for his science of art, taking advantage of his acute observations of nature to achieve striking pictorial results. This study describes the analysis of an exceptional sample from one of Leonardo's final masterpieces: The Virgin and Child with St. Anne (Musée du Louvre, Paris, France). The sample was analyzed at the microscale by synchrotron-based hyperspectral photoluminescence imaging and high-angular X-ray diffraction. The results demonstrate Leonardo's use of two subtypes of lead white pigment, thus revealing how he must have possessed a precise knowledge of his materials; carefully selecting them according to the aesthetical results he aimed at achieving in each painting. This work provides insights on how Leonardo obtained these grades of pigment and proposes new clues regarding the optical and/or working properties he may have tried to achieve.

Synchrotron multimodal imaging in a whole cell reveals lipid droplet core organization.

A lipid droplet (LD) core of a cell consists mainly of neutral lipids, triacylglycerols and/or steryl esters (SEs). The structuration of these lipids inside the core is still under debate. Lipid segregation inside LDs has been observed but is sometimes suggested to be an artefact of LD isolation and chemical fixation. LD imaging in their native state and in unaltered cellular environments appears essential to overcome these possible technical pitfalls. Here, imaging techniques for ultrastructural study of native LDs in cellulo are provided and it is shown that LDs are organized structures. Cryo soft X-ray tomography and deep-ultraviolet (DUV) transmittance imaging are showing a partitioning of SEs at the periphery of the LD core. Furthermore, DUV transmittance and tryptophan/tyrosine auto-fluorescence imaging on living cells are combined to obtain complementary information on cell chemical contents. This multimodal approach paves the way for a new label-free organelle imaging technique in living cells.

The challenge of intracellular antibiotic accumulation, a function of fluoroquinolone influx versus bacterial efflux.

With the spreading of antibiotic resistance, the translocation of antibiotics through bacterial envelopes is crucial for their antibacterial activity. In Gram-negative bacteria, the interplay between membrane permeability and drug efflux pumps must be investigated as a whole. Here, we quantified the intracellular accumulation of a series of fluoroquinolones in population and in individual cells of Escherichia coli according to the expression of the AcrB efflux transporter. Computational results supported the accumulation levels measured experimentally and highlighted how fluoroquinolones side chains interact with specific residues of the distal pocket of the AcrB tight monomer during recognition and binding steps.

Time resolved transient circular dichroism spectroscopy using synchrotron natural polarization.

Ultraviolet (UV) synchrotron radiation circular dichroism (SRCD) spectroscopy has made an important contribution to the determination and understanding of the structure of bio-molecules. In this paper, we report an innovative approach that we term time-resolved SRCD (tr-SRCD), which overcomes the limitations of current broadband UV SRCD setups. This technique allows accessing ultrafast time scales (down to nanoseconds), previously measurable only by other methods, such as infrared (IR), nuclear magnetic resonance (NMR), fluorescence and absorbance spectroscopies, and small angle X-ray scattering (SAXS). The tr-SRCD setup takes advantage of the natural polarization of the synchrotron radiation emitted by a bending magnet to record broadband UV CD faster than any current SRCD setup, improving the acquisition speed from 10 mHz to 130 Hz and the accessible temporal resolution by several orders of magnitude. We illustrate the new approach by following the isomer concentration changes of an azopeptide after a photoisomerization. This breakthrough in SRCD spectroscopy opens up a wide range of potential applications to the detailed characterization of biological processes, such as protein folding and protein-ligand binding.

Synchrotron Deep-UV Photoluminescence Imaging for the Submicrometer Analysis of Chemically Altered Zinc White Oil Paints.

Zinc oxide (ZnO) is a II-VI semiconductor that has been used for the last 150 years as an artists' pigment under the name of zinc white. Oil paints containing zinc white are known to be prone to the formation of zinc carboxylates, which can cause protrusions and mechanical failure. In this article, it is demonstrated how a multispectral synchrotron-based deep-UV photoluminescence microimaging technique can be used to show the distribution of zinc soaps on the submicrometer scale and how this information is used to further the understanding of zinc white degradation processes in oil paint. The technique is based on the luminescence of zinc soaps in the near-UV (∼3.65 eV) upon excitation in the deep-UV (4.51 eV), involving transitions that are argued to subsequently involve ligand-to-metal and metal-to-ligand charge transfer with intermediate structural reconfiguration. Because the primary emission peak lies at a higher energy than the band gap of ZnO (3.3 eV), the signal can easily be isolated from the pigment's very intense band gap and trap state emission by employing a multispectral acquisition approach. Moreover, analysis at such short wavelengths, in combination with a UV-transparent optical setup, allows for lateral resolution on the order of 200 nm to be obtained. The unprecedented capabilities of the microimaging technique are illustrated by showing its application to the study of a historical cross section from an early 20th century painting by Piet Mondrian. Revealing the submicrometer distribution of crystalline zinc soaps in this cross section provides new insights that suggest that microfissures, the starting points of paint delamination, are the result of an overall expansion of a heavily saponified zinc white layer.

Optical Signatures Derived From Deep UV to NIR Excitation Discriminates Healthy Samples From Low and High Grades Glioma.

Among all the tumors of the central nervous system (CNS), glioma are the most deadly and the most malignant. Surgical resection is the standard therapeutic method to treat this type of brain cancer. But the diffusive character of these tumors create many problems for surgeons during the operation. In fact, these tumors migrate outside the tumor solid zone and invade the surrounding healthy tissues. These infiltrative tissues have the same visual appearance as healthy tissues, making it very difficult for surgeons to distinguish the healthy ones from the diffused ones. The surgeon, therefore, cannot properly remove the tumor margins increasing the recurrence risk of the tumor. To resolve this problem, our team has developed a multimodal two-photon fibered endomicroscope, compatible with the surgeon trocar, to better delimitate tumor boundaries by relying on the endogenous fluorescence of brain tissues. In this context, and in order to characterize the optical signature of glioma tumors, this study offers multimodal and multi-scaled optical measurements from healthy tissues to high grade glioma. We can interrogate tissue from deep ultra-violet to near infrared excitation by working with spectroscopy, fluorescence lifetime imaging, two-photon fluorescene imaging and Second Harmonic Generation (SHG) imaging. Optically derived ratios such as the Tryptophan/Collagen ratio, the optical redox ratio and the long lifetime intensity fraction, discriminated diseased tissue from its normal counterparts when fitted by Gaussian ellipsoids and choosing a threshold for each. Additionally two-photon fluorescence and SHG images were shown to display similar histological features as Hematoxylin-Eosin stained images.

Mechanistic aspects of maltotriose-conjugate translocation to the Gram-negative bacteria cytoplasm.

Small molecule accumulation in Gram-negative bacteria is a key challenge to discover novel antibiotics, because of their two membranes and efflux pumps expelling toxic molecules. An approach to overcome this challenge is to hijack uptake pathways so that bacterial transporters shuttle the antibiotic to the cytoplasm. Here, we have characterized maltodextrin-fluorophore conjugates that can pass through both the outer and inner membranes mediated by components of the Escherichia coli maltose regulon. Single-channel electrophysiology recording demonstrated that the compounds permeate across the LamB channel leading to accumulation in the periplasm. We have also demonstrated that a maltotriose conjugate distributes into both the periplasm and cytoplasm. In the cytoplasm, the molecule activates the maltose regulon and triggers the expression of maltose binding protein in the periplasmic space indicating that the complete maltose entry pathway is induced. This maltotriose conjugate can (i) reach the periplasmic and cytoplasmic compartments to significant internal concentrations and (ii) auto-induce its own entry pathway via the activation of the maltose regulon, representing an interesting prototype to deliver molecules to the cytoplasm of Gram-negative bacteria.

Antibiotics and efflux: combined spectrofluorimetry and mass spectrometry to evaluate the involvement of concentration and efflux activity in antibiotic intracellular accumulation.

Background: In Gram-negative bacteria, passing through the double membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibiotic activity. Spectrofluorimetry has been developed to follow fluoroquinolone accumulation inside bacteria using intrinsic bacterial fluorescence as an internal standard. However, adaptation for non-fluorescent antibiotics is needed; quantitative methods based on MS offer the possibility of expanding the detection range obtained by spectrofluorimetry. Objectives: To validate, with spectrofluorimetry, the use of MS to measure antibiotic accumulation in cells and to determine the relationship between antibiotic concentrations and the amount of intrabacterial accumulation in different efflux backgrounds on the same batch of molecules. Methods: Spectrofluorimetry was performed in parallel with MS on the same samples to measure the ciprofloxacin and fleroxacin accumulation in cells expressing various efflux pump levels. A microplate protocol was set up to determine the antibiotic accumulation as a function of external antibiotic concentrations. Results: A correlation existed between the data obtained with spectrofluorimetry and MS, whatever the efflux pump or tested antibiotic. The results highlighted different dynamics of uptake between ciprofloxacin and fleroxacin as well as the relationship between the level of efflux activity and antibiotic accumulation. Conclusions: We have developed a microplate protocol and cross-validated two complementary methods: spectrofluorimetry, which contains a reliable internal standard; and MS, which allows detection of low antibiotic amounts. These assays allow study of the dose effect and the efflux impact on the intrabacterial accumulation of antibiotics.