Incorporating uncertainty within dynamic interoceptive learning.

Introduction: Interoception, the perception of the internal state of the body, has been shown to be closely linked to emotions and mental health. Of particular interest are interoceptive learning processes that capture associations between environmental cues and body signals as a basis for making homeostatically relevant predictions about the future. One method of measuring respiratory interoceptive learning that has shown promising results is the Breathing Learning Task (BLT). While the original BLT required binary predictions regarding the presence or absence of an upcoming inspiratory resistance, here we extended this paradigm to capture continuous measures of prediction (un)certainty. Methods: Sixteen healthy participants completed the continuous version of the BLT, where they were asked to predict the likelihood of breathing resistances on a continuous scale from 0.0 to 10.0. In order to explain participants' responses, a Rescorla-Wagner model of associative learning was combined with suitable observation models for continuous or binary predictions, respectively. For validation, we compared both models against corresponding null models and examined the correlation between observed and modeled predictions. The model was additionally extended to test whether learning rates differed according to stimuli valence. Finally, summary measures of prediction certainty as well as model estimates for learning rates were considered against interoceptive and mental health questionnaire measures. Results: Our results demonstrated that the continuous model fits closely captured participant behavior using empirical data, and the binarised predictions showed excellent replicability compared to previously collected data. However, the model extension indicated that there were no significant differences between learning rates for negative (i.e. breathing resistance) and positive (i.e. no breathing resistance) stimuli. Finally, significant correlations were found between fatigue severity and both prediction certainty and learning rate, as well as between anxiety sensitivity and prediction certainty. Discussion: These results demonstrate the utility of gathering enriched continuous prediction data in interoceptive learning tasks, and suggest that the updated BLT is a promising paradigm for future investigations into interoceptive learning and potential links to mental health.

Extended blood stage sensitivity profiles of Plasmodium cynomolgi to doxycycline and tafenoquine, as a model for Plasmodium vivax.

Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which preclude determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax, we determined the physiologically significant delayed death effect induced by doxycycline [IC50(96 h), 1,401 ± 607 nM]. As expected, IC50(96 h) to chloroquine (20.4 nM), piperaquine (12.6 µM), and tafenoquine (1,424 nM) were not affected by extended exposure.

Functional and immunological mapping of domains of the reticulocyte binding protein, Plasmodium vivax PvRBP2a.

We previously described a novel Plasmodium vivax invasion mechanism into human reticulocytes via the PvRBP2a-CD98 receptor-ligand pair. We assessed the PvRBP2a epitopes involved in CD98 binding and recognised by antibodies from infected patients using linear epitope mapping. We identified two epitope clusters mediating PvRBP2a-CD98 interaction. One cluster named cluster B (PvRBP2a431-448, TAALKEKGKLLANLYNKL) was the target of antibody responses in P. vivax-infected humans. Peptides from each cluster were able to prevent live parasite invasion of human reticulocytes. These results provide new insights for development of a malaria blood stage vaccine against P. vivax.

Favorable Preclinical Pharmacological Profile of a Novel Antimalarial Pyrrolizidinylmethyl Derivative of 4-amino-7-chloroquinoline with Potent In Vitro and In Vivo Activities.

The 4-aminoquinoline drugs, such as chloroquine (CQ), amodiaquine or piperaquine, are still commonly used for malaria treatment, either alone (CQ) or in combination with artemisinin derivatives. We previously described the excellent in vitro activity of a novel pyrrolizidinylmethyl derivative of 4-amino-7-chloroquinoline, named MG3, against P. falciparum drug-resistant parasites. Here, we report the optimized and safer synthesis of MG3, now suitable for a scale-up, and its additional in vitro and in vivo characterization. MG3 is active against a panel of P. vivax and P. falciparum field isolates, either alone or in combination with artemisinin derivatives. In vivo MG3 is orally active in the P. berghei, P. chabaudi, and P. yoelii models of rodent malaria with efficacy comparable, or better, than that of CQ and of other quinolines under development. The in vivo and in vitro ADME-Tox studies indicate that MG3 possesses a very good pre-clinical developability profile associated with an excellent oral bioavailability, and low toxicity in non-formal preclinical studies on rats, dogs, and non-human primates (NHP). In conclusion, the pharmacological profile of MG3 is in line with those obtained with CQ or the other quinolines in use and seems to possess all the requirements for a developmental candidate.

The fate and movements of aquarium-released giant trevally Caranx ignobilis inferred from acoustic telemetry.

Movements of four adult giant trevally Caranx ignobilis were tracked using passive acoustic telemetry after being released from uShaka Sea World Aquarium in Durban, South Africa, where they had been kept on display for a period of 8 years. All four individuals were detected on a large network of deployed acoustic receivers for a minimum of 3 months to a maximum of over 6 years. Their movements were compared to 43 wild-caught and tagged C. ignobilis over a similar period and two individuals adopted movement behavior similar to that of their conspecifics, including repeated annual seasonal migrations to a known spawning aggregation site. This study shows that with good animal husbandry, indigenous fish kept in captivity can be released back into the wild and not only survive but adopt natural movement behavior and contribute to future generations of their species.

Integrative Genetic Manipulation of Plasmodium cynomolgi Reveals Multidrug Resistance-1 Y976F Associated With Increased In Vitro Susceptibility to Mefloquine.

The lack of a long-term in vitro culture method has severely restricted the study of Plasmodium vivax, in part because it limits genetic manipulation and reverse genetics. We used the recently optimized Plasmodium cynomolgi Berok in vitro culture model to investigate the putative P. vivax drug resistance marker MDR1 Y976F. Introduction of this mutation using clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) increased sensitivity to mefloquine, but had no significant effect on sensitivity to chloroquine, amodiaquine, piperaquine, and artesunate. To our knowledge, this is the first reported use of CRISPR-Cas9 in P. cynomolgi, and the first reported integrative genetic manipulation of this species.

Optical diffraction tomography and image reconstruction to measure host cell alterations caused by divergent Plasmodium species.

Malaria is a life-threatening infectious disease caused by parasites of the genus Plasmodium. Understanding the biological features of various parasite forms is important for the optical diagnosis and defining pathological states, which are often constrained by the lack of ambient visualization approaches. Here, we employ a label-free tomographic technique to visualize the host red blood cell (RBC) remodeling process and quantify changes in biochemical properties arising from parasitization. Through this, we provide a quantitative body of information pertaining to the influence of host cell environment on growth, survival, and replication of P. falciparum and P. vivax in their respective host cells: mature erythrocytes and young reticulocytes. These exquisite three-dimensional measurements of infected red cells demonstrats the potential of evolving 3D imaging to advance our understanding of Plasmodium biology and host-parasite interactions.

Erythrocyte tropism of malarial parasites: The reticulocyte appeal.

Erythrocytes are formed from the enucleation of erythroblasts in the bone marrow, and as erythrocytes develop from immature reticulocytes into mature normocytes, they undergo extensive cellular changes through their passage in the blood. During the blood stage of the malarial parasite life cycle, the parasite sense and invade susceptible erythrocytes. However, different parasite species display varying erythrocyte tropisms (i.e., preference for either reticulocytes or normocytes). In this review, we explore the erythrocyte tropism of malarial parasites, especially their predilection to invade reticulocytes, as shown from recent studies. We also discuss possible mechanisms mediating erythrocyte tropism and the implications of specific tropisms to disease pathophysiology. Understanding these allows better insight into the role of reticulocytes in malaria and provides opportunities for targeted interventions.

Evolving perspectives on rosetting in malaria.

The ability of the intraerythrocytic Plasmodium spp. to form spontaneous rosettes with uninfected red blood cells (URBCs) has been observed in the medically important malaria parasites. Since the discovery of rosettes in the late 1980s, different formation mechanisms and pathobiological roles have been postulated for rosetting; most of which have focused on Plasmodium falciparum. Recent breakthroughs, including new data from Plasmodium vivax, have highlighted the multifaceted roles of rosetting in the immunopathobiology and the development of drug resistance in human malaria. Here, we provide new perspectives on the formation and the role of rosetting in malaria rheopathobiology.

Geographical distribution and genetic diversity of Plasmodium vivax reticulocyte binding protein 1a correlates with patient antigenicity.

Plasmodium vivax is the most widespread cause of human malaria. Recent reports of drug resistant vivax malaria and the challenge of eradicating the dormant liver forms increase the importance of vaccine development against this relapsing disease. P. vivax reticulocyte binding protein 1a (PvRBP1a) is a potential vaccine candidate, which is involved in red cell tropism, a crucial step in the merozoite invasion of host reticulocytes. As part of the initial evaluation of the PvRBP1a vaccine candidate, we investigated its genetic diversity and antigenicity using geographically diverse clinical isolates. We analysed pvrbp1a genetic polymorphisms using 202 vivax clinical isolates from six countries. Pvrbp1a was separated into six regions based on specific domain features, sequence conserved/polymorphic regions, and the reticulocyte binding like (RBL) domains. In the fragmented gene sequence analysis, PvRBP1a region II (RII) and RIII (head and tail structure homolog, 152-625 aa.) showed extensive polymorphism caused by random point mutations. The haplotype network of these polymorphic regions was classified into three clusters that converged to independent populations. Antigenicity screening was performed using recombinant proteins PvRBP1a-N (157-560 aa.) and PvRBP1a-C (606-962 aa.), which contained head and tail structure region and sequence conserved region, respectively. Sensitivity against PvRBP1a-N (46.7%) was higher than PvRBP1a-C (17.8%). PvRBP1a-N was reported as a reticulocyte binding domain and this study identified a linear epitope with moderate antigenicity, thus an attractive domain for merozoite invasion-blocking vaccine development. However, our study highlights that a global PvRBP1a-based vaccine design needs to overcome several difficulties due to three distinct genotypes and low antigenicity levels.

Improving in vitro continuous cultivation of Plasmodium cynomolgi, a model for P. vivax.

The absence of a routine continuous in vitro cultivation method for Plasmodium vivax, an important globally distributed parasite species causing malaria in humans, has restricted investigations to field and clinical sampling. Such a method has recently been developed for the Berok strain of P. cynomolgi, a parasite of macaques that has long been used as a model for P. vivax, as these two parasites are nearly indistinguishable biologically and are genetically closely related. The availability of the P. cynomolgi Berok in routine continuous culture provides for the first time an opportunity to conduct a plethora of functional studies. However, the initial cultivation protocol proved unsuited for investigations requiring extended cultivation times, such as reverse genetics and drug resistance. Here we have addressed some of the critical obstacles to this, and we propose a set of modifications that help overcome them.

Rosetting Responses of Plasmodium-infected Erythrocytes to Antimalarials.

In malaria, rosetting is a phenomenon involving the cytoadherence of uninfected erythrocytes to infected erythrocytes (IRBC) harboring the late erythrocytic stage of Plasmodium spp. Recently, artesunate-stimulated rosetting has been demonstrated to confer a survival advantage to P. falciparum late-stage IRBC. This study investigated the rosetting response of P. falciparum and P. vivax clinical isolates to ex vivo antimalarial treatments. Brief exposure of IRBC to chloroquine, mefloquine, amodiaquine, quinine, and lumefantrine increased the rosetting rates of P. falciparum and P. vivax. Furthermore, the ex vivo combination of artesunate with mefloquine and piperaquine also resulted in increased the rosetting rates. Drug-mediated rosette-stimulation has important implications for the therapeutic failure of rapidly cleared drugs such as artesunate. However, further work is needed to establish the ramifications of increased rosetting rates by drugs with longer half-lifves, such as chloroquine, mefloquine, and piperaquine.

Perceptual and Ventilatory Responses to Hypercapnia in Athletes and Sedentary Individuals.

Purpose: Hypercapnic chemosensitivity traditionally captures the ventilatory response to elevated pressures of carbon dioxide in the blood. However, hypercapnia also contributes to subjective breathing perceptions, and previously we demonstrated a closer matching of perception to changes in ventilation in athletes compared to controls. Here we investigated any potential underlying hypercapnic chemosensitivity differences between groups, and explored whether these measures relate to ventilatory and perceptual responses during exercise as well as trait levels of affect. Methods: A hypercapnic challenge, incremental maximal exercise test and affective questionnaires were completed by 20 endurance athletes and 20 age-/sex-matched sedentary controls. The hypercapnic challenge involved elevating end-tidal PCO2 by 0.8% (6.1 mmHg) and 1.5% (11.2 mmHg) for 3 min each (randomised), with constant end-tidal oxygen. Ventilatory and perceptual responses to hypercapnia were compared between groups, and within each group the relationships between hypercapnic chemosensitivity (slope analyses) and exercising ventilation and perceptions were calculated using Spearman's non-parametric correlations. Results: While absolute ventilation differences during hypercapnia and exercise were observed, no group differences were found across hypercapnic chemosensitivity (slope) measures. Correlation analyses revealed the anxiety hypercapnic response was related to maximal exercise anxiety, but only in sedentary individuals. Conclusion: Ventilatory and perceptual hypercapnic chemosensitivity do not differ between athletes and sedentary individuals. However, ventilatory and anxiety hypercapnic chemosensitivities were related to ventilatory and anxiety responses during exercise in untrained individuals only. Athletes may employ additional strategies during exercise to reduce the influence of chemosensitivity on ventilatory and perceptual responses.

Investigation of the Optimal Parameters of Median Nerve Stimulation Using a Variety of Stimulation Methods and Its Effects on Heart Rate Variability: A Systematic Review.

OBJECTIVES: There is a wealth of literature supporting the use of median nerve stimulation (MNS) for modulating autonomic nervous system (ANS) dysfunction such as in hypoxia, recovery after heart valve replacement, ischemia, and cardiac contractibility. Heart rate variability (HRV) is considered a gold standard for measuring autonomic modulation and dynamic nonlinear ANS processes through the use of an electrocardiogram (ECG). Although the use of MNS on HRV in animals and humans has been documented, optimal stimulation parameters are yet to be outlined. MATERIALS AND METHODS: This review aims to synthesize findings of neurostimulation using MNS on animals and humans while observing HRV using an ECG. Using PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines with search parameters of "Median nerve stimulation," "Neiguan," "PC-6," "HRV," "Heart rate variability," and "Heart-rate variability" observing on animals and human subjects, we found a total of 17 eligible articles. RESULTS: In this review, changing two parameters, that is, stimulation frequency and side of stimulation, appears to be the most influential in effecting frequency-domain ECG analysis of HRV. However, it is evident from this review that to perform an effective comparison of the effects of MNS on HRV, more detailed reports of the studies are required. CONCLUSIONS: Finding the optimal stimulation parameters for MNS is crucial for improving HRV. This will in turn contribute to normalizing ANS function impaired in numerous clinical conditions, such as epilepsy or diabetes.

Plasmodium falciparum rosetting protects schizonts against artemisinin.

BACKGROUND: Artemisinin (ART) resistance in Plasmodium falciparum is thought to occur during the early stage of the parasite's erythrocytic cycle. Here, we identify a novel factor associated with the late stage parasite development that contributes to ART resistance. METHODS: Rosetting rates of clinical isolates pre- and post- brief (one hour) exposure to artesunate (AS, an ART derivative) were evaluated. The effects of AS-mediated rosetting on the post-AS-exposed parasite's replication and survival, as well as the extent of protection by AS-mediated rosetting on different parasite stages were investigated. The rosetting ligands, mechanisms, and gene mutations involved were studied. FINDINGS: Brief AS exposure stimulated rosetting, with AS-resistant isolates forming more rosettes in a more rapid manner. AS-mediated rosetting enabled infected erythrocytes (IRBC) to withstand AS exposure for several hours and protected the IRBC from phagocytosis. When their rosetting ability was blocked experimentally, the post-AS exposure survival advantage by the AS-resistant parasites was abrogated. Deletions in two genes coding for PfEMP1 exon 2 (PF3D7_0200300 and PF3D7_0223300) were found to be associated with AS-mediated rosetting, and these mutations were significantly selected through time in the parasite population under study, along with the K13 mutations, a molecular marker of ART-resistance. INTERPRETATION: Rapid ART parasite clearance is driven by the direct oxidative damages on IRBC by ART and the phagocytic destruction of the damaged IRBC. Rosetting serves as a rapid 'buying time' strategy that allows more parasites to complete schizont maturation, reinvasion and subsequent development into the intrinsically less ART-susceptible ring stage. FUNDING: A*STAR, NMRC-OF-YIRG, HRC e-ASIA, Wellcome.

Probing the distinct chemosensitivity of Plasmodium vivax liver stage parasites and demonstration of 8-aminoquinoline radical cure activity in vitro.

Improved control of Plasmodium vivax malaria can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of patients. We screened several compound libraries against P. vivax liver stages, including 1565 compounds against mature hypnozoites, resulting in one drug-like and several probe-like hits useful for investigating hypnozoite biology. Primaquine and tafenoquine, administered in combination with chloroquine, are currently the only FDA-approved antimalarials for radical cure, yet their activity against mature P. vivax hypnozoites has not yet been demonstrated in vitro. By developing an extended assay, we show both drugs are individually hypnozonticidal and made more potent when partnered with chloroquine, similar to clinically relevant combinations. Post-hoc analyses of screening data revealed excellent performance of ionophore controls and the high quality of single point assays, demonstrating a platform able to support screening of greater compound numbers. A comparison of P. vivax liver stage activity data with that of the P. cynomolgi blood, P. falciparum blood, and P. berghei liver stages reveals overlap in schizonticidal but not hypnozonticidal activity, indicating that the delivery of new radical curative agents killing P. vivax hypnozoites requires an independent and focused drug development test cascade.

Right frontal anxiolytic-sensitive EEG 'theta' rhythm in the stop-signal task is a theory-based anxiety disorder biomarker.

Psychiatric diagnoses currently rely on a patient's presenting symptoms or signs, lacking much-needed theory-based biomarkers. Our neuropsychological theory of anxiety, recently supported by human imaging, is founded on a longstanding, reliable, rodent 'theta' brain rhythm model of human clinical anxiolytic drug action. We have now developed a human scalp EEG homolog-goal-conflict-specific rhythmicity (GCSR), i.e., EEG rhythmicity specific to a balanced conflict between goals (e.g., approach-avoidance). Critically, GCSR is consistently reduced by different classes of anxiolytic drug and correlates with clinically-relevant trait anxiety scores (STAI-T). Here we show elevated GCSR in student volunteers divided, after testing, on their STAI-T scores into low, medium, and high (typical of clinical anxiety) groups. We then tested anxiety disorder patients (meeting diagnostic criteria) and similar controls recruited separately from the community. The patient group had higher average GCSR than their controls-with a mixture of high and low GCSR that varied with, but cut across, conventional disorder diagnosis. Consequently, GCSR scores should provide the first theoretically-based biomarker that could help diagnose, and so redefine, a psychiatric disorder.

Plasmodium cynomolgi Berok Growth Inhibition Assay by Thiol-reactive Probe Based Flow Cytometric Measurement.

The relapsing malaria species, Plasmodium vivax, is the most widely distributed and difficult-to-treat cause of human malaria. The merozoites of P. vivax preferentially invade ephemeral human CD71+ reticulocytes (nascent reticulocytes), thereby limiting the development of a robust continuous culture in vitro. Fortunately, P. vivax's sister species, P. cynomolgi Berok, can be cultured continuously, providing the ability to screen novel therapeutics drug and vaccine candidates in a reliable and high-throughput manner. Based on well-established growth inhibition activity (GIA) assays against P. falciparum and P. knowlesi, this protocol adopts the current flow cytometry assay methodology and investigates P. vivax inhibitory antibodies using the P. cynomolgi Berok invasion model based on the thiol-reactivity and DNA abundance of viable parasites in macaque erythrocytes. Established GIA assays screen antibodies at either a single concentration or high/low dose concentrations to provide quick insights for prioritizing potential antibodies capable of specifically interrupting parasite ligand and host receptor binding with minimal concentrations. Hence, this protocol expands on the existing GIA assay by using serially diluted antibodies and generating a dose-response curve to better quantify the inhibitory efficacy amongst selected vaccine candidates.

Plasmodium vivax binds host CD98hc (SLC3A2) to enter immature red blood cells.

More than one-third of the world's population is exposed to Plasmodium vivax malaria, mainly in Asia1. P. vivax preferentially invades reticulocytes (immature red blood cells)2-4. Previous work has identified 11 parasite proteins involved in reticulocyte invasion, including erythrocyte binding protein 2 (ref. 5) and the reticulocyte-binding proteins (PvRBPs)6-10. PvRBP2b binds to the transferrin receptor CD71 (ref. 11), which is selectively expressed on immature reticulocytes12. Here, we identified CD98 heavy chain (CD98), a heteromeric amino acid transporter from the SLC3 family (also known as SLCA2), as a reticulocyte-specific receptor for the PvRBP2a parasite ligand using mass spectrometry, flow cytometry, biochemical and parasite invasion assays. We characterized the expression level of CD98 at the surface of immature reticulocytes (CD71+) and identified an interaction between CD98 and PvRBP2a expressed at the merozoite surface. Our results identify CD98 as an additional host membrane protein, besides CD71, that is directly associated with P. vivax reticulocyte tropism. These findings highlight the potential of using PvRBP2a as a vaccine target against P. vivax malaria.

Longitudinal ex vivo and molecular trends of chloroquine and piperaquine activity against Plasmodium falciparum and P. vivax before and after introduction of artemisinin-based combination therapy in Papua, Indonesia.

Drug resistant Plasmodium parasites are a major threat to malaria control and elimination. After reports of high levels of multidrug resistant P. falciparum and P. vivax in Indonesia, in 2005, the national first-line treatment policy for uncomplicated malaria was changed in March 2006, to dihydroartemisinin-piperaquine against all species. This study assessed the temporal trends in ex vivo drug susceptibility to chloroquine (CQ) and piperaquine (PIP) for both P. falciparum and P. vivax clinical isolates collected between 2004 and 2018, by using schizont maturation assays, and genotyped a subset of isolates for known and putative molecular markers of CQ and PIP resistance by using Sanger and next generation whole genome sequencing. The median CQ IC50 values varied significantly between years in both Plasmodium species, but there was no significant trend over time. In contrast, there was a significant trend for increasing PIP IC50s in both Plasmodium species from 2010 onwards. Whereas the South American CQ resistant 7G8 pfcrt SVMNT isoform has been fixed since 2005 in the study area, the pfmdr1 86Y allele frequencies decreased and became fixed at the wild-type allele in 2015. In P. vivax isolates, putative markers of CQ resistance (no pvcrt-o AAG (K10) insertion and pvmdr1 Y967F and F1076L) were fixed at the mutant alleles since 2005. None of the putative PIP resistance markers were detected in P. falciparum. The ex vivo drug susceptibility and molecular analysis of CQ and PIP efficacy for P. falciparum and P. vivax after 12 years of intense drug pressure with DHP suggests that whilst the degree of CQ resistance appears to have been sustained, there has been a slight decline in PIP susceptibility, although this does not appear to have reached clinically significant levels. The observed decreasing trend in ex vivo PIP susceptibility highlights the importance of ongoing surveillance.