Multi-Omic Temporal Landscape of Plasma and Synovial Fluid-Derived Extracellular Vesicles Using an Experimental Model of Equine Osteoarthritis.

Extracellular vesicles (EVs) contribute to osteoarthritis pathogenesis through their release into joint tissues and synovial fluid. Synovial fluid-derived EVs have the potential to be direct biomarkers in the causal pathway of disease but also enable understanding of their role in disease progression. Utilizing a temporal model of osteoarthritis, we defined the changes in matched synovial fluid and plasma-derived EV small non-coding RNA and protein cargo using sequencing and mass spectrometry. Data exploration included time series clustering, factor analysis and gene enrichment interrogation. Chondrocyte signalling was analysed using luciferase-based transcription factor activity assays. EV protein cargo appears to be more important during osteoarthritis progression than small non-coding RNAs. Cluster analysis revealed plasma-EVs represented a time-dependent response to osteoarthritis induction associated with supramolecular complexes. Clusters for synovial fluid-derived EVs were associated with initial osteoarthritis response and represented immune/inflammatory pathways. Factor analysis for plasma-derived EVs correlated with day post-induction and were primarily composed of proteins modulating lipid metabolism. Synovial fluid-derived EVs factors represented intermediate filament and supramolecular complexes reflecting tissue repair. There was a significant interaction between time and osteoarthritis for CRE, NFkB, SRE, SRF with a trend for osteoarthritis synovial fluid-derived EVs at later time points to have a more pronounced effect.

Leopard predation on wild Sichuan snub-nosed monkeys.

Predation is widely recognized as a powerful selective pressure on primate behavior and ecology, although knowledge of predator-prey relationships remains limited partly due to the rarity of directly observed attacks on primates. Here, we describe four confirmed or suspected instances of leopard (Panthera pardus) predation on free-ranging Sichuan (golden) snub-nosed monkeys (Rhinopithecus roxellana), a highly endangered colobine species endemic to China. We recorded predation events and the reactions of monkey group members. We suggest that the evolution of a multilevel society may be an adaptive response by Sichuan snub-nosed monkeys to the risk from leopards as well as other potential predators, one that balances the pressures of predation and intra-species competition and conflict.

Impact of Retinal Degeneration on Response of ON and OFF Cone Bipolar Cells to Electrical Stimulation.

In retinal degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), the photoreceptors become stressed and start to degenerate in the early stages of the disease. Retinal prosthetic devices have been developed to restore vision in patients by applying electrical stimulation to the surviving retinal cells. However, these devices provide limited visual perception as the therapeutic interventions are generally considered in the later stages of the disease when only inner retinal layer cells are left. A potential treatment option for retinal degenerative diseases in the early stages can be stimulating bipolar cells, which receive presynaptic signals from photoreceptors. In this work, we constructed computational models of healthy and degenerated (both ON and OFF-type) cone bipolar cells (CBCs) with realistic morphologies extracted from connectomes of the healthy and early-stage degenerated rabbit retina. We examined these cells' membrane potential and axon terminal calcium current differences when subjected to electrical stimulation. In addition, we investigated how differently healthy and degenerated cells behave with respect to various stimulation parameters, including pulse duration and cells' distance from the stimulating electrode. The results suggested that regardless of the position of the OFF CBCs in the retina model, there is not a significant difference between the membrane potential of healthy and degenerate cells when electrically stimulated. However, the healthy ON CBC axon terminal membrane potential rising time-constant is shorter (0.29 ± 0.03 ms) than the degenerated cells (0.8 ± 0.07 ms). Moreover, the ionic calcium channels at the axon terminals of the cells have a higher concentration and higher current in degenerated cells (32.24 ± 6.12 pA) than the healthy cells (13.64 ± 2.88 pA) independently of the cell's position.

Randomized, Double-Blind, Placebo-Controlled, Global Phase III Trial of Talimogene Laherparepvec Combined With Pembrolizumab for Advanced Melanoma.

PURPOSE: The combination of talimogene laherparepvec (T-VEC) and pembrolizumab previously demonstrated an acceptable safety profile and an encouraging complete response rate (CRR) in patients with advanced melanoma in a phase Ib study. We report the efficacy and safety from a phase III, randomized, double-blind, multicenter, international study of T-VEC plus pembrolizumab (T-VEC-pembrolizumab) versus placebo plus pembrolizumab (placebo-pembrolizumab) in patients with advanced melanoma. METHODS: Patients with stage IIIB-IVM1c unresectable melanoma, naïve to antiprogrammed cell death protein-1, were randomly assigned 1:1 to T-VEC-pembrolizumab or placebo-pembrolizumab. T-VEC was administered at ≤ 4 × 106 plaque-forming unit (PFU) followed by ≤ 4 × 108 PFU 3 weeks later and once every 2 weeks until dose 5 and once every 3 weeks thereafter. Pembrolizumab was administered intravenously 200 mg once every 3 weeks. The dual primary end points were progression-free survival (PFS) per modified RECIST 1.1 by blinded independent central review and overall survival (OS). Secondary end points included objective response rate per mRECIST, CRR, and safety. Here, we report the primary analysis for PFS, the second preplanned interim analysis for OS, and the final analysis. RESULTS: Overall, 692 patients were randomly assigned (346 T-VEC-pembrolizumab and 346 placebo-pembrolizumab). T-VEC-pembrolizumab did not significantly improve PFS (hazard ratio, 0.86; 95% CI, 0.71 to 1.04; P = .13) or OS (hazard ratio, 0.96; 95% CI, 0.76 to 1.22; P = .74) compared with placebo-pembrolizumab. The objective response rate was 48.6% for T-VEC-pembrolizumab (CRR 17.9%) and 41.3% for placebo-pembrolizumab (CRR 11.6%); the durable response rate was 42.2% and 34.1% for the arms, respectively. Grade ≥ 3 treatment-related adverse events occurred in 20.7% of patients in the T-VEC-pembrolizumab arm and in 19.5% of patients in the placebo-pembrolizumab arm. CONCLUSION: T-VEC-pembrolizumab did not significantly improve PFS or OS compared with placebo-pembrolizumab. Safety results of the T-VEC-pembrolizumab combination were consistent with the safety profiles of each agent alone.

Optical photothermal infrared spectroscopy can differentiate equine osteoarthritic plasma extracellular vesicles from healthy controls.

Equine osteoarthritis is a chronic degenerative disease of the articular joint, characterised by cartilage degradation resulting in pain and reduced mobility and thus is a prominent equine welfare concern. Diagnosis is usually at a late stage through clinical examination and radiographic imaging, whilst treatment is symptomatic not curative. Extracellular vesicles are nanoparticles that are involved in intercellular communication. The objective of this study was to investigate the feasibility of Raman and Optical Photothermal Infrared Spectroscopies to detect osteoarthritis using plasma-derived extracellular vesicles, specifically differentiating extracellular vesicles in diseased and healthy controls within the parameters of the techniques used. Plasma samples were derived from thoroughbred racehorses. A total of 14 samples were selected (control; n = 6 and diseased; n = 8). Extracellular vesicles were isolated using differential ultracentrifugation and characterised using nanoparticle tracking analysis, transmission electron microscopy, and human tetraspanin chips. Samples were then analysed using combined Raman and Optical Photothermal Infrared Spectroscopies. Infrared spectra were collected between 950-1800 cm-1. Raman spectra had bands between the wavelengths of 900-1800 cm-1 analysed. Spectral data for both Raman and Optical Photothermal Infrared Spectroscopy were used to generate clustering via principal components analysis and classification models were generated using partial least squared discriminant analysis in order to characterize the techniques' ability to distinguish diseased samples. Optical Photothermal Infrared Spectroscopy could differentiate osteoarthritic extracellular vesicles from healthy with good classification (93.4% correct classification rate) whereas Raman displayed poor classification (correct classification rate = -64.3%). Inspection of the infrared spectra indicated that plasma-derived extracellular vesicles from osteoarthritic horses contained increased signal for proteins, lipids and nucleic acids. For the first time we demonstrated the ability to use optical photothermal infrared spectroscopy combined with Raman spectroscopy to interrogate extracellular vesicles and osteoarthritis-related samples. Optical Photothermal Infrared Spectroscopy was superior to Raman in this study, and could distinguish osteoarthritis samples, suggestive of its potential use diagnostically to identify osteoarthritis in equine patients. This study demonstrates the potential of Raman and Optical Photothermal Infrared Spectroscopy to be used as a future diagnostic tool in clinical practice, with the capacity to detect changes in extracellular vesicles from clinically derived samples.

Female dogs evaluate levels of competence in humans.

Dogs are highly sensitive to human behavior, and they evaluate us using both their direct experiences and from a third-party perspective. Dogs pay attention to various aspects of our actions and make judgments about, for example, social vs. selfish acts. However, it is unclear if dogs judge human competence. To investigate this issue, we showed dogs two experimenters manipulating a transparent container: one was good at removing the lid to take an object out of the container (Competent person), whereas the other was unsuccessful at this task (Incompetent person). After demonstrating their actions twice with different containers, both experimenters simultaneously tried to open a third container which contained food (Food condition; 30 dogs) or was empty (Empty condition; 30 dogs). Dogs in the Food condition looked at the Competent person longer than the Incompetent one, and female dogs in particular were more likely to approach the Competent person. In contrast, dogs in the Empty condition showed no preferences. This result suggests that dogs can recognize different competence levels in humans, and that this ability influences their behavior according to the first situation. Our data also indicate that more attention should be given to potential sex differences in dogs' social evaluation abilities.

Small non-coding RNA landscape of extracellular vesicles from a post-traumatic model of equine osteoarthritis.

Extracellular vesicles comprise an as yet inadequately investigated intercellular communication pathway in the field of early osteoarthritis. We hypothesised that the small non-coding RNA expression pattern in synovial fluid and plasma would change during progression of experimental osteoarthritis. In this study, we conducted small RNA sequencing to provide a comprehensive overview of the temporal expression profiles of small non-coding transcripts carried by extracellular vesicles derived from plasma and synovial fluid for the first time in a posttraumatic model of equine osteoarthritis. Additionally, we characterised synovial fluid and plasma-derived extracellular vesicles with respect to quantity, size, and surface markers. The different temporal expressions of seven microRNAs in plasma and synovial fluid-derived extracellular vesicles, eca-miR-451, eca-miR-25, eca-miR-215, eca-miR-92a, eca-miR-let-7c, eca-miR-486-5p, and eca-miR-23a, and four snoRNAs, U3, snord15, snord46, and snord58, represent potential biomarkers for early osteoarthritis. Bioinformatics analysis of the differentially expressed microRNAs in synovial fluid highlighted that in early osteoarthritis these related to the inhibition of cell cycle, cell cycle progression, DNA damage and cell proliferation as well as increased cell viability and differentiation of stem cells. Plasma and synovial fluid-derived extracellular vesicle small non-coding signatures have been established for the first time in a temporal model of osteoarthritis. These could serve as novel biomarkers for evaluation of osteoarthritis progression or act as potential therapeutic targets.

Cannula cricothyroidotomy in the impalpable neck: An observational study of simulated 'can't intubate, can't oxygenate' scenarios by teams following a cannula-first algorithm in live anaesthetised pigs.

Live animal models can be used to train anaesthetists to perform emergency front-of-neck-access. Cannula cricothyroidotomy success reported in previous wet lab studies contradicts human clinical data. This prospective, observational study reports success of a cannula-first 'can't intubate, can't oxygenate' algorithm for impalpable anatomy during high fidelity team simulations using live, anaesthetised pigs.Forty-two trained anaesthesia teams were instructed to follow the Royal Perth Hospital can't intubate, can't oxygenate algorithm to re-oxygenate a desaturating pig with impalpable neck anatomy (mean (standard deviation, SD) 16.2 (3.5) kg); mean (SD) tracheal internal diameter 11 (1.4) mm. Teams were informed that failure would prompt veterinary-led euthanasia.All teams performed percutaneous cannula cricothyroidotomy as the initial technique, with a median (interquartile range, IQR (range)) start time of 42 (35-50 (24-93)) s. First-pass percutaneous cannula success was 29% to both insufflate tracheal oxygen and re-oxygenate. Insufflation success improved with repeated percutaneous attempts (up to three), but prolonged hypoxia time increasingly necessitated euthanasia (insufflation 57%; re-oxygenation 48%). First, second and third percutaneous attempts achieved insufflation at median (IQR (range)) 74 (64-91 (46-110)) s, 111 (95-136 (79-150)) s and 141 (127-159 (122-179)) s, respectively. Eighteen teams failed with percutaneous cannulae and performed scalpel techniques, predominantly dissection cannulation (n = 17) which achieved insufflation in all cases (insufflation 100%; re-oxygenation 47%). Scalpel attempts were started at median (IQR (range)) 142 (133-218 (97-293)) s and achieved insufflation at 232 (205-303 (152-344)) s.While percutaneous cannula cricothyroidotomy could rapidly re-oxygenate, the success rate was low and teams repeated attempts beyond the recommended 60 s time frame, delaying transition to the more successful dissection cannula technique. We recommend this 'cannula-first' can't intubate, can't oxygenate algorithm adopts a 'single best effort' strategy for percutaneous cannula, with failure prompting a scalpel technique.

Maternal caretaking behavior towards a dead juvenile in a wild, multi-level primate society.

Maternal caretaking and transport of dead infants are widespread among nonhuman primates, having been reported in numerous species of monkeys and apes. By contrast, accounts of such behaviors toward dead juveniles are scarce. Here, we describe responses by the mother and other group members to the death of a juvenile in a wild, multi-level group of Sichuan snub-nosed monkeys (Rhinopithecus roxellana). Following the juvenile's fatal accident, his mother transported and cared for the corpse for four days. Immature monkeys belonging to the same one-male unit, and some individuals from other social units also showed interest in and tended the corpse. Comparisons of this case with those involving the deaths of infants and an adult female in the same population highlight possible effects of physiological, psychological and emotional factors in primate thanatological responses, and provide an additional perspective on the origin and evolution of compassionate acts.

Mitochondrial Inorganic Polyphosphate (polyP) Is a Potent Regulator of Mammalian Bioenergetics in SH-SY5Y Cells: A Proteomics and Metabolomics Study.

Inorganic polyphosphate (polyP) is an ancient, ubiquitous, and well-conserved polymer which is present in all the studied organisms. It is formed by individual subunits of orthophosphate which are linked by structurally similar bonds and isoenergetic to those found in ATP. While the metabolism and the physiological roles of polyP have already been described in some organisms, including bacteria and yeast, the exact role of this polymer in mammalian physiology still remains poorly understood. In these organisms, polyP shows a co-localization with mitochondria, and its role as a key regulator of the stress responses, including the maintenance of appropriate bioenergetics, has already been demonstrated by our group and others. Here, using Wild-type (Wt) and MitoPPX (cells enzymatically depleted of mitochondrial polyP) SH-SY5Y cells, we have conducted a comprehensive study of the status of cellular physiology, using proteomics and metabolomics approaches. Our results suggest a clear dysregulation of mitochondrial physiology, especially of bioenergetics, in MitoPPX cells when compared with Wt cells. Moreover, the effects induced by the enzymatic depletion of polyP are similar to those present in the mitochondrial dysfunction that is observed in neurodegenerative disorders and in neuronal aging. Based on our findings, the metabolism of mitochondrial polyP could be a valid and innovative pharmacological target in these conditions.

LC-MS/MS-Based Serum Protein Profiling for Identification of Candidate Biomarkers in Pakistani Rheumatoid Arthritis Patients.

Rheumatoid arthritis is an autoimmune disorder of complex disease etiology. Currently available serological diagnostic markers lack in terms of sensitivity and specificity and thus additional biomarkers are warranted for early disease diagnosis and management. We aimed to screen and compare serum proteome profiles of rheumatoid arthritis serotypes with healthy controls in the Pakistani population for identification of potential disease biomarkers. Serum samples from rheumatoid arthritis patients and healthy controls were enriched for low abundance proteins using ProteoMinerTM columns. Rheumatoid arthritis patients were assigned to one of the four serotypes based on anti-citrullinated peptide antibodies and rheumatoid factor. Serum protein profiles were analyzed via liquid chromatography-tandem mass spectrometry. The changes in the protein abundances were determined using label-free quantification software ProgenesisQITM followed by pathway analysis. Findings were validated in an independent cohort of patients and healthy controls using an enzyme-linked immunosorbent assay. A total of 213 proteins were identified. Comparative analysis of all groups (false discovery rate < 0.05, >2-fold change, and identified with ≥2 unique peptides) identified ten proteins that were differentially expressed between rheumatoid arthritis serotypes and healthy controls including pregnancy zone protein, selenoprotein P, C4b-binding protein beta chain, apolipoprotein M, N-acetylmuramoyl-L-alanine amidase, catalytic chain, oncoprotein-induced transcript 3 protein, Carboxypeptidase N subunit 2, Apolipoprotein C-I and Apolipoprotein C-III. Pathway analysis predicted inhibition of liver X receptor/retinoid X receptor activation pathway and production of nitric oxide and reactive oxygen species pathway in macrophages in all serotypes. A catalogue of potential serum biomarkers for rheumatoid arthritis were identified. These biomarkers can be further evaluated in larger cohorts from different populations for their diagnostic and prognostic potential.

Mirror self-recognition in gorillas (Gorilla gorilla gorilla): a review and evaluation of mark test replications and variants.

Mirror self-recognition (MSR), widely regarded as an indicator of self-awareness, has not been demonstrated consistently in gorillas. We aimed to examine this issue by setting out a method to evaluate gorilla self-recognition studies that is objective, quantifiable, and easy to replicate. Using Suarez and Gallup's (J Hum Evol 10:175-183, 1981) study as a reference point, we drew up a list of 15 methodological criteria and assigned scores to all published studies of gorilla MSR for both methodology and outcomes. Key features of studies finding both mark-directed and spontaneous self-directed responses included visually inaccessible marks, controls for tactile and olfactory cues, subjects who were at least 5 years old, and clearly distinguishing between responses in front of versus away from the mirror. Additional important criteria include videotaping the tests, having more than one subject, subjects with adequate social rearing, reporting post-marking observations with mirror absent, and giving mirror exposure in a social versus individual setting. Our prediction that MSR studies would obtain progressively higher scores as procedures and behavioural coding practices improved over time was supported for methods, but not for outcomes. These findings illustrate that methodological rigour does not guarantee stronger evidence of self-recognition in gorillas; methodological differences alone do not explain the inconsistent evidence for MSR in gorillas. By implication, it might be suggested that, in general, gorillas do not show compelling evidence of MSR. We advocate that future MSR studies incorporate the same criteria to optimize the quality of attempts to clarify the self-recognition abilities of gorillas as well as other species.

Putting the cart before the horse: claims for mirror self-recognition in horses are unfounded.

The recent article by Baragli, Scopa, Maglieri, and Palagi (Anim Cogn https://doi.org/10.1007/s10071-021-01502-7 , 2021) that claims to demonstrate mirror self-recognition (MSR) in horses is not based on compelling evidence. We identify problems with their experimental procedures, data, and assertion about "demonstrating MSR at group level." Examples of these problems include incomplete experimental design, absence of important control conditions, inappropriate terminology, suboptimal mark application procedures and coding of videos, ambiguity of videos presented as supporting evidence, and inconsistencies in data presentation and interpretation. It is not the case that their study "marks a turning point in the analytical technique of MSR exploration."

Putrescine--a chemical cue of death-is aversive to chimpanzees.

As in many other species of nonhuman primates, chimpanzee mothers with a dead infant may continue to care for and transport the infant for days, weeks, or even longer. The bereaved females do this despite what humans perceive as the foul odour from the putrefying corpse. Putrescine is a major contributor to the "smell of death," and it elicits behaviours aimed at getting rid of the source of the smell, or escape responses in mammals including humans. However, it has never been shown that the odour of putrescine is aversive to chimpanzees. To address this question, we visually presented six adult chimpanzees with the corpse of a small bird, or a stuffed glove, in association with putrescine, ammonia, or water, and recorded the chimpanzees' reactions. The apes spent significantly less time near the object when it was paired with putrescine than the other substances, although they showed no signs of increased arousal or anxiety. We interpret the findings as evidence of an aversion to the smell of death in chimpanzees, discuss the implications for understanding the continued maternal-like behaviour of bereaved female chimpanzees, and suggest future research directions for the field of comparative evolutionary thanatology.

On experimental tests for studying altruism in capuchin monkeys.

Altruism is often considered as the ultimate form of prosociality and is defined as any act that benefits others without direct benefit to the actor. Many nonhuman species have been reported to express different forms of altruism, although their expression in experimental studies is highly dependent on the paradigm used. Tufted capuchin monkeys are one of the most studied species; however, the evidence for altruism in this species remains inconclusive. This study aimed to investigate whether two paradigms, adapted from those in which great apes have shown altruism, could be useful for revealing signs of altruistic capabilities in capuchins. Pairs of monkeys were tested in two experiments involving a similar mechanism but with different costs to acting altruistically. The first used a more costly operant sharing task in which an operator could unlock a door to allow a recipient to enter the room and share his food. The second consisted of a less costly helping task, in which the operator's food was secured but he could help the recipient to get other food that was in a locked container. The results suggest that capuchins, although apparently unwilling to share their food in a costly operant situation, might altruistically help selected recipients, in response to requesting by the latter. While our small sample size along with procedural limitations preclude firm conclusions, we discuss how further ameliorations of our tasks could further contribute to the study of altruistic capacities in primates.

Synaptic inputs to broad thorny ganglion cells in macaque retina.

In primates, broad thorny retinal ganglion cells are highly sensitive to small, moving stimuli. They have tortuous, fine dendrites with many short, spine-like branches that occupy three contiguous strata in the middle of the inner plexiform layer. The neural circuits that generate their responses to moving stimuli are not well-understood, and that was the goal of this study. A connectome from central macaque retina was generated by serial block-face scanning electron microscopy, a broad thorny cell was reconstructed, and its synaptic inputs were analyzed. It received fewer than 2% of its inputs from both ON and OFF types of bipolar cells; the vast majority of its inputs were from amacrine cells. The presynaptic amacrine cells were reconstructed, and seven types were identified based on their characteristic morphology. Two types of narrow-field cells, knotty bistratified Type 1 and wavy multistratified Type 2, were identified. Two types of medium-field amacrine cells, ON starburst and spiny, were also presynaptic to the broad thorny cell. Three types of wide-field amacrine cells, wiry Type 2, stellate wavy, and semilunar Type 2, also made synapses onto the broad thorny cell. Physiological experiments using a macaque retinal preparation in vitro confirmed that broad thorny cells received robust excitatory input from both the ON and the OFF pathways. Given the paucity of bipolar cell inputs, it is likely that amacrine cells provided much of the excitatory input, in addition to inhibitory input.

Model-based comparison of current flow in rod bipolar cells of healthy and early-stage degenerated retina.

Retinal degenerative diseases, such as retinitis pigmentosa, are generally thought to initiate with the loss of photoreceptors, though recent work suggests that plasticity and remodeling occurs prior to photoreceptor cell loss. This degeneration subsequently leads to death of other retinal neurons, creating functional alterations and extensive remodeling of retinal networks. Retinal prosthetic devices stimulate the surviving retinal cells by applying external current using implanted electrodes. Although these devices restore partial vision, the quality of restored vision is limited. Further knowledge about the precise changes in degenerated retina as the disease progresses is essential to understand how current flows in retinas undergoing degenerative disease and to improve the performance of retinal prostheses. We developed computational models that describe current flow from rod photoreceptors to rod bipolar cells (RodBCs) in the healthy and early-stage degenerated retina. Morphologically accurate models of retinal cells with their synapses are constructed based on retinal connectome datasets, created using serial section transmission electron microscopy (TEM) images of 70 nm-thick slices of either healthy (RC1) or early-stage degenerated (RPC1) rabbit retina. The passive membrane and active ion currents of each cell are implemented using conductance-based models in the Neuron simulation environment. In response to photocurrent input at rod photoreceptors, the simulated membrane potential at RodBCs in early degenerate tissue is approximately 10-20 mV lower than that of RodBCs of that observed in wild type retina. Results presented here suggest that although RodBCs in RPC1 show early, altered morphology compared to RC1, the lower membrane potential is primarily a consequence of reduced rod photoreceptor input to RodBCs in the degenerated retina. Frequency response and step input analyses suggest that individual cell responses of RodBCs in either healthy or early-degenerated retina, prior to substantial photoreceptor cell loss, do not differ significantly.