ROAD: The Road Event Awareness Dataset for Autonomous Driving.

Humans drive in a holistic fashion which entails, in particular, understanding dynamic road events and their evolution. Injecting these capabilities in autonomous vehicles can thus take situational awareness and decision making closer to human-level performance. To this purpose, we introduce the ROad event Awareness Dataset (ROAD) for Autonomous Driving, to our knowledge the first of its kind. ROAD is designed to test an autonomous vehicle's ability to detect road events, defined as triplets composed by an active agent, the action(s) it performs and the corresponding scene locations. ROAD comprises videos originally from the Oxford RobotCar Dataset, annotated with bounding boxes showing the location in the image plane of each road event. We benchmark various detection tasks, proposing as a baseline a new incremental algorithm for online road event awareness termed 3D-RetinaNet. We also report the performance on the ROAD tasks of Slowfast and YOLOv5 detectors, as well as that of the winners of the ICCV2021 ROAD challenge, which highlight the challenges faced by situation awareness in autonomous driving. ROAD is designed to allow scholars to investigate exciting tasks such as complex (road) activity detection, future event anticipation and continual learning. The dataset is available at https://github.com/gurkirt/road-dataset; the baseline can be found at https://github.com/gurkirt/3D-RetinaNet.

Identifying small molecules for protecting chondrocyte function and matrix integrity after controlled compressive injury.

Objective: Articular cartilage injury is central for the development of post-traumatic osteoarthritis (PTOA). With few disease-modifying therapies successful at offsetting progressive osteoarthritis (OA), our goal is to use a high throughput screening platform of cartilage injury to identify novel chondroprotective compounds. Targeting articular cartilage damage immediately after injury remains a promising therapeutic strategy to overcome irreversible tissue damage. Method: We constructed a single impact-cartilage screening method using a multi-platen system that simultaneously impacts 48 samples and makes use of engineered cartilage tissue analogs (known as CTAs). Drug libraries were screened and assessed for their ability to alter two crucial biological responses to impact injuries, namely matrix degradation and cell stress. Results: Over 500 small molecules were screened for their ability to alter proteoglycan loss, matrix metalloproteinase activity, and cell stress or death. Fifty-five compounds passed through secondary screening and were from commercial libraries of natural and redox, stem cell related compounds, as well as protease, kinase and phosphatase inhibitors. Through secondary screening, 16 promising candidates exhibited activity on one or more critical function of chondrocytes. While many are mechanistically known compounds, their function in joint diseases is not known. Conclusion: This platform was validated for screening drug activity against a tissue engineered model of PTOA. Multiple compounds identified in this manner have potential application as early protective therapy for treating PTOA, and require further study. We propose this screening platform can identify novel molecules that act on early chondrocyte responses to injury and provide an invaluable tool for therapeutic development.

Non-Destructive Spectroscopic Assessment of High and Low Weight Bearing Articular Cartilage Correlates with Mechanical Properties.

OBJECTIVE: Autologous articular cartilage (AC) harvested for repair procedures of high weight bearing (HWB) regions of the femoral condyles is typically obtained from low weight bearing (LWB) regions, in part due to the lack of non-destructive techniques for cartilage composition assessment. Here, we demonstrate that infrared fiber optic spectroscopy can be used to non-destructively evaluate variations in compositional and mechanical properties of AC across LWB and HWB regions. DESIGN: AC plugs (N = 72) were harvested from the patellofemoral groove of juvenile bovine stifle joints, a LWB region, and femoral condyles, a HWB region. Near-infrared (NIR) and mid-infrared (MIR) fiber optic spectra were collected from plugs, and indentation tests were performed to determine the short-term and equilibrium moduli, followed by gravimetric water and biochemical analysis. RESULTS: LWB tissues had a significantly greater amount of water determined by NIR and gravimetric assay. The moduli generally increased in tissues from the patellofemoral groove to the condyles, with HWB condyle cartilage having significantly higher moduli. A greater amount of proteoglycan content was also found in HWB tissues, but no differences in collagen content. In addition, NIR-determined water correlated with short-term modulus and proteoglycan content (R = -0.40 and -0.31, respectively), and a multivariate model with NIR data was able to predict short-term modulus within 15% error. CONCLUSIONS: The properties of tissues from LWB regions differ from HWB tissues and can be determined non-destructively by infrared fiber optic spectroscopy. Clinicians may be able to use this modality to assess AC prior to harvesting osteochondral grafts for focal defect repair.

Spectroscopic Analysis of Human Tracheal Tissue during Decellularization.

OBJECTIVE: To use mid-infrared (IR) spectroscopy to assess changes in the cartilaginous framework of human trachea during decellularization. STUDY DESIGN: Laboratory-based study. SETTING: Research laboratory. METHODS: Six cadaveric human tracheas were decellularized using a detergent enzymatic method (DEM). Tissue samples were obtained from each specimen after 0, 1, 10, and 25 DEM cycles for histologic and spectroscopic analysis. Decellularization was confirmed using hematoxylin and eosin (H&E) and 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) staining. Changes in cartilaginous framework were examined using Fourier transform infrared imaging spectroscopy (FT-IRIS) and an attenuated total reflectance (ATR) probe in the mid-IR frequencies. Results were statistically analyzed using 1-way analysis of variance (ANOVA) and principal component analysis (PCA). RESULTS: Six decellularized tracheal scaffolds were successfully created using a DEM protocol. Histologic examination showed near-complete nuclear loss following 25 DEM cycles. As observed with FT-IRIS analysis, the collagen absorbance signal (1336 cm-1) was predominantly in the perichondria and remained stable after 25 DEM cycles ( P = .132), while the absorbance from sugar rings in proteoglycans and nucleic acids in hyaline cartilage (1080 cm-1) showed a significant decrease after 1 DEM cycle ( P = .0007). Examination of the luminal surface of the trachea with an ATR probe showed raw mid-IR spectra consistent with cartilage. PCA showed significant separation of spectra corresponding to treatment cycle along the principal components 1 and 2. CONCLUSION: Mid-IR spectroscopy is a viable method of monitoring changes in extracellular matrix components during the decellularization of human trachea.

Infrared Spectroscopic Quantification of Methacrylation of Hyaluronic Acid: A Scaffold for Tissue Engineering Applications.

Methacrylated hyaluronic acid (MeHA) has been used extensively in tissue engineering and drug delivery applications. The degree of methacrylation (DM) of HA impacts hydrogel crosslinking, which is of pivotal importance for cell interactions. The methacrylation reaction occurs over several hours, and DM is currently assessed post reaction and after dialysis of the solution, using nuclear magnetic resonance (1H NMR) data. Thus, there is little control over exact DM in a specific reaction. Here, infrared (IR) spectroscopy in attenuated total reflection (ATR) mode was investigated as an alternate modality for assessment of the DM of HA hydrogels, including during the reaction progression. Attenuated total reflection is a low-cost technique that is widely available in research and industry labs that can be used online during the reaction process. Strong correlations were achieved with IR-derived peak heights from dialyzed and lyophilized samples at 1708 cm-1 (from the methacrylic ester carbonyl vibration), and 1H NMR values ( R = 0.92, P = 6.56E-11). Additional IR peaks of importance were identified using principal component analysis and resulted in significant correlations with the 1H NMR DM parameter: 1454 cm-1 ( R = 0.85, P = 2.81E-8), 1300 cm-1 ( R = 0.95, P = 4.50E-14), 950 ( R = 0.85, P = 3.55E-8), 856 cm-1 ( R = 0.94, P = 1.20E-12), and 809 cm-1 ( R = 0.93, P = 3.54E-12). A multiple linear regression model to predict 1H NMR-derived DM using the 1708, 1300, and 1200 cm-1 peak heights as independent variables resulted in prediction with an error of 3.2% using dialyzed and lyophilized samples ( P < 0.001). Additionally, a multilinear regression model to predict the DM in undialyzed liquid MeHA samples obtained during the reaction process using similar peak height positions as independent variables resulted in a prediction error of 0.81% ( P < 0.05). Thus, IR spectroscopy can be utilized as an alternate modality to 1H NMR for quantification of the DM of MeHA while sampling either on-line during the methacrylation reaction as well as in post-lyophilized products. This could greatly simplify workflow for tissue engineering and other applications.

A novel mouse model of intervertebral disc degeneration shows altered cell fate and matrix homeostasis.

Intervertebral disc degeneration and associated low back and neck pain is a ubiquitous health condition that affects millions of people world-wide, and causes high incidence of disability and enormous medical/societal costs. However, lack of appropriate small animal models with spontaneous disease onset has impeded our ability to understand the pathogenetic mechanisms that characterize and drive the degenerative process. We report, for the first time, early onset spontaneous disc degeneration in SM/J mice known for their poor regenerative capacities compared to "super-healer" LG/J mice. In SM/J mice, degenerative process was marked by decreased nucleus pulposus (NP) cellularity and changes in matrix composition at P7, 4, and 8 weeks with increased severity by 17 weeks. Distinctions between NP and annulus fibrosus (AF) or endplate cartilage were lost, and NP and AF of SM/J mice showed higher histological grades. There was increased NP cell death in SM/J mice with decreased phenotypic marker expression. Polarized microscopy and FTIR spectroscopy demonstrated replacement of glycosaminoglycan-rich NP matrix with collagenous fibrous tissue. The levels of ARGxx were increased in, indicating higher aggrecan turnover. Furthermore, an aberrant expression of collagen X and MMP13 was observed in the NP of SM/J mice, along with elevated expression of Col10a1, Ctgf, and Runx2, markers of chondrocyte hypertrophy. Likewise, expression of Enpp1 as well as Alpl was higher, suggesting NP cells of SM/J mice promote dystrophic mineralization. There was also a decrease in several pathways necessary for NP cell survival and function including Wnt and VEGF signaling. Importantly, SM/J discs were stiffer, had decreased height, and poor vertebral bone quality, suggesting compromised motion segment mechanical functionality. Taken together, our results clearly demonstrate that SM/J mouse strain recapitulates many salient features of human disc degeneration, and serves as a novel small animal model.

Compositional Assessment of Human Tracheal Cartilage by Infrared Spectroscopy.

Objectives To assess the potential of infrared fiber-optic spectroscopy to evaluate the compositional properties of human tracheal cartilage. Study Design Laboratory-based study. Methods Twenty human cadaveric distal tracheas were harvested (age range 20-78 years; 6 females, 14 males) for compositional analysis. Histologic staining, Fourier transform infrared imaging spectroscopy data on collagen and proteoglycan (PG) content, and near-infrared (NIR) fiber-optic probe spectroscopic data that reflect protein and water content were evaluated. NIR fiber-optic probe data were also obtained from the proximal trachea in 4 human cadavers (age range 51-65 years; 2 females, 2 males) in situ for comparison to distal trachea spectral data. Results In the distal trachea cohort, the spectroscopic-determined ratio of PG/amide I, indicative of the relative amount of PG, was significantly higher in the tissues from the younger group compared to the older group (0.37 ± 0.08 vs 0.32 ± 0.05, P = .05). A principal component analysis of the NIR spectral data enabled separation of spectra based on tracheal location, likely due to differences in both protein and water content. The NIR-determined water content based on the 5200-cm-1 peak was significantly higher in the distal trachea compared to the proximal trachea ( P < .001). Conclusions Establishment of normative compositional values and further elucidating differences between the segments of trachea will enable more directed research toward appropriate compositional end points in regenerative medicine for tracheal repair.

Near-Infrared Spectroscopy Predicts Compositional and Mechanical Properties of Hyaluronic Acid-Based Engineered Cartilage Constructs.

Hyaluronic acid (HA) has been widely used for cartilage tissue engineering applications. However, the optimal time point to harvest HA-based engineered constructs for cartilage repair is still under investigation. In this study, we investigated the ability of a nondestructive modality, near-infrared spectroscopic (NIR) analysis, to predict compositional and mechanical properties of HA-based engineered cartilage constructs. NIR spectral data were collected from control, unseeded constructs, and twice per week by fiber optic from constructs seeded with chondrocytes during their development over an 8-week period. Constructs were harvested at 2, 4, 6, and 8 weeks, collagen and sulfated glycosaminoglycan content measured using biochemical assays, and the mechanical properties of the constructs evaluated using unconfined compression tests. NIR absorbances associated with the scaffold material, water, and engineered cartilage matrix, were identified. The NIR-determined matrix absorbance plateaued after 4 weeks of culture, which was in agreement with the biochemical assay results. Similarly, the mechanical properties of the constructs also plateaued at 4 weeks. A multivariate partial least square model based on NIR spectral input was developed to predict the moduli of the constructs, which resulted in a prediction error of 10% and R value of 0.88 for predicted versus actual values of dynamic modulus. Furthermore, the maximum increase in moduli was calculated from the first derivative of the curve fit of NIR-predicted and actual moduli values over time, and both occurred at ∼2 weeks. Collectively, these data suggest that NIR spectral data analysis could be an alternative to destructive biochemical and mechanical methods for evaluation of HA-based engineered cartilage construct properties.

Increase of Bacillus badius Phenylalanine dehydrogenase specificity towards phenylalanine substrate by site-directed mutagenesis.

Phenylalanine dehydrogenase (PheDH) is a key enzyme in medical diagnostic for determining the amount of phenylalanine to detect phenylketonuria (PKU) disease. However, determination of phenylalanine can be usually disturbed in presence of tyrosine in blood samples. Position N145 of B.sphaericus PheDH, has been previously showed a crucial role in substrate binding, which corresponded by position V144 in B. badius PheDH. In this study, the PheDH of B. badius due to reasonable activity was cloned and subjected to site-directed mutagenesis at mentioned position, followed by kinetic and structural studies to find more exclusive mutants. The results showed that the V144L mutant considerably increases specificity toward phenylalanine and decreases toward l-tyrosine, while in V144N mutant, the specificity reduces toward phenylalanine and increases toward tyrosine. Moreover, concerning the mutated V144D, significantly reduced kcat and also decreased km value for phenylalanine relative to that of wild type. The Phe/Tyr specificity constant in V144L increased more than 4-fold compared to wild type, makes it to be a suitable candidate for more specific identification of PKU. Finally, docking and molecular dynamic simulation on wild type and mutants clarified the structural basis behind more specificity of V144L mutant for phenylalanine substrate.

Bifunctional role of leucine 300 of firefly luciferase in structural rigidity.

Firefly luciferase is susceptible to thermal inactivation, thereby its intracellular half-life decreased. Previous reports indicated that L300R mutation (LRR mutant) in E354R/Arg356 double mutant (ERR mutant) from Lampyris turkestanicus luciferase has increased its thermal stability and rigidity through induction of some ionic bonds with Asp 270 and 271. Disruption of the deduced ionic bonds in an ultra-rigid mutant of firefly luciferase did not reverse the flexibility of the protein. In this study, we investigated the effects of this residue to find the truth behind an extraordinary increase in thermal stability and rigidity of luciferase after replacement of leucine 300 by arginine based on previous reports. For this purpose, L300R, L300K and L300E mutations were performed to compare the effects of these mutations on the native firefly luciferase. In spite of increase of intrinsic fluorescence of the mutants a slight increase in thermostability and retention of kinetic properties was observed. Based on our results, we can conclude that L300R mutation in LRR mutant accompanying with alteration in a flexible loop (352-359) increased thermostability and rigidity of luciferase.

Nondestructive Assessment of Engineered Cartilage Composition by Near Infrared Spectroscopy.

Tissue engineering presents a strategy to overcome the limitations of current tissue healing methods. Scaffolds, cells, external growth factors and mechanical input are combined in an effort to obtain constructs with properties that mimic native tissues. However, engineered constructs developed using similar culture environments can have very different matrix composition and biomechanical properties. Accordingly, a nondestructive technique to assess constructs during development such that appropriate compositional endpoints can be defined is desirable. Near infrared spectroscopy (NIRS) analysis is a modality being investigated to address the challenges associated with current evaluation techniques, which includes nondestructive compositional assessment. In the present study, cartilage tissue constructs were grown using chondrocytes seeded onto polyglycolic acid (PGA) scaffolds in similar environments in three separate tissue culture experiments and monitored using NIRS. Multivariate partial least squares (PLS) analysis models of NIR spectra were calculated and used to predict tissue composition, with biochemical assay information used as the reference data. Results showed that for combined data from all tissue culture experiments, PLS models were able to assess composition with significant correlations to reference values, including engineered cartilage water (at 5200 cm(-1), R = 0.68, p = 0.03), proteoglycan (at 4310 cm(-1), R = 0.82, p = 0.007), and collagen (at 4610 cm(-1), R = 0.84, p = 0.005). In addition, degradation of PGA was monitored using specific NIRS frequencies. These results demonstrate that NIR spectroscopy combined with multivariate analysis provides a nondestructive modality to assess engineered cartilage, which could provide information to determine the optimal time for tissue harvest for clinical applications.