Discovery of a new predominant cytosine DNA modification that is linked to gene expression in malaria parasites.

DNA cytosine modifications are key epigenetic regulators of cellular processes in mammalian cells, with their misregulation leading to varied disease states. In the human malaria parasite Plasmodium falciparum, a unicellular eukaryotic pathogen, little is known about the predominant cytosine modifications, cytosine methylation (5mC) and hydroxymethylation (5hmC). Here, we report the first identification of a hydroxymethylcytosine-like (5hmC-like) modification in P. falciparum asexual blood stages using a suite of biochemical methods. In contrast to mammalian cells, we report 5hmC-like levels in the P. falciparum genome of 0.2-0.4%, which are significantly higher than the methylated cytosine (mC) levels of 0.01-0.05%. Immunoprecipitation of hydroxymethylated DNA followed by next generation sequencing (hmeDIP-seq) revealed that 5hmC-like modifications are enriched in gene bodies with minimal dynamic changes during asexual development. Moreover, levels of the 5hmC-like base in gene bodies positively correlated to transcript levels, with more than 2000 genes stably marked with this modification throughout asexual development. Our work highlights the existence of a new predominant cytosine DNA modification pathway in P. falciparum and opens up exciting avenues for gene regulation research and the development of antimalarials.

Severe Cloverleaf Skull Deformity in c.1061C>G (p.Ser354Cys) Mutated Fibroblast Growth Factor Receptor 2 Gene in Crouzon Syndrome.

ABSTRACT: Cloverleaf skull deformity (CSD), or Kleeblattschädel, is a condition with severe and unpatterned multisuture craniosynostosis, resulting in a trilobar-shaped skull. This deformity mainly comprises a cranio-orbito-facial malformation that leads to a spectrum of multidisciplinary issues. Several syndromes are associated with CSD, such as Crouzon syndrome (CS). Here, we report the case of an infant with CS and the pathogenic c.1061C>G (p.Ser354Cys) variant of the fibroblast growth factor receptor 2 (FGFR2) gene. The child presented with the severe form of CSD despite having a normal, mid-trimester, sonographic scan.

Vestibular modulation of muscle sympathetic nerve activity assessed over a 100-fold frequency range of sinusoidal galvanic vestibular stimulation.

We have previously shown that sinusoidal galvanic vestibular stimulation (sGVS), delivered at 0.2-2.0 Hz, evokes a partial entrainment of muscle sympathetic nerve activity (MSNA). Moreover, at lower frequencies of stimulation (0.08-0.18 Hz) sGVS produces two peaks of modulation: one (primary) peak associated with the positive peak of the sinusoidal stimulus and a smaller (secondary) peak associated with the trough. Here we assessed whether sGVS delivered at 0.05 Hz causes a more marked modulation of MSNA than at higher frequencies and tested the hypothesis that the primary and secondary peaks are of identical amplitude because of the longer cycle length. MSNA was recorded via tungsten microelectrodes inserted into the left peroneal nerve in 11 seated subjects. Bipolar binaural sGVS (±2 mA, 100 cycles) was applied to the mastoid processes at 0.05, 0.5, and 5.0 Hz (500 cycles). Cross-correlation analysis revealed two bursts of modulation of MSNA for each cycle at 0.05 and 0.5 Hz but only one at 5 Hz. There was a significant inverse linear relationship between vestibular modulation (primary peak) and frequency (P < 0.0001), with the amplitudes of the peaks being highest at 0.05 Hz. Moreover, the secondary peak at this frequency was not significantly different from the primary peak. These results indicate that vestibular modulation of MSNA operates over a large range of frequencies but is greater at lower frequencies of sGVS. We conclude that the vestibular apparatus, through its influence on muscle sympathetic outflow, preferentially contributes to the control of blood pressure at low frequencies. NEW & NOTEWORTHY Vestibulosympathetic reflexes have been documented in experimental animals and humans. Here we show that sinusoidal galvanic vestibular stimulation, a means of selectively exciting vestibular afferents in humans, induces greater modulation of muscle sympathetic nerve activity when delivered at a very low frequency (0.05 Hz) than at 0.5 or 5.0 Hz.

Changes in skin blood flow, respiration and blood pressure in participants reporting motion sickness during sinusoidal galvanic vestibular stimulation.

NEW FINDINGS: What is the central question of the study? We have previously shown that sinusoidal galvanic vestibular stimulation induces greater modulation of skin sympathetic nerve activity, but not muscle sympathetic nerve activity, in participants who report nausea during simulated motion, but the effects on skin blood flow and blood pressure are unknown. What is the main finding and its importance? During vestibular stimulation, nausea was associated with a greater increase in skin blood flow and a progressive reduction in skin sympathetic nerve activity, but no changes in muscle sympathetic nerve activity. This emphasizes the differential changes in sympathetic outflow to different tissues during nausea. ABSTRACT: We tested the hypothesis that galvanic vestibular stimulation, which produces illusions of side-to-side swaying, causes a greater reduction in skin blood flow in participants who report stimulation-induced nausea. A retrospective analysis was performed on data obtained in 30 participants. Bipolar sinusoidal galvanic vestibular stimulation (sGVS) was applied across the mastoid processes (±2 mA, 0.08 Hz) for 21 min. ECG, continuous blood pressure, respiration and skin blood flow were recorded. Muscle sympathetic nerve activity was recorded in 17 participants and skin sympathetic nerve activity in 12. Ten participants reported motion sickness, whereas 20 did not. Both groups showed an initial reduction in skin (finger) blood flow during sGVS, followed by a sustained increase and a subsequent return towards baseline levels throughout the stimulation; the increase was greater in those who experienced nausea. The increase fits with the progressive reduction in skin sympathetic nerve activity observed in the nauseous group. Mean blood pressure was significantly lower in those who experienced nausea and showed a much larger increase at the onset of sGVS, compared with those who did not. Moreover, the respiratory rate was higher at the outset for the subjects who experienced nausea, decreasing progressively during sGVS, whereas respiratory rate remained constant in those who did not experience nausea. Heart rate was more labile in the subjects who experienced nausea, showing a sustained increase towards the end of stimulation. We have shown that several autonomic parameters change during the nausea induced by vestibular stimulation, but a sustained decrease in skin blood flow is not a hallmark of incipient motion sickness.

Neck movement but not neck position modulates skin sympathetic nerve activity supplying the lower limbs of humans.

We previously showed that dynamic, but not static, neck displacement modulates muscle sympathetic nerve activity (MSNA) to lower limbs of humans. However, it is not known whether dynamic neck displacement modulates skin sympathetic nerve activity (SSNA). Tungsten microelectrodes inserted into the common peroneal nerve were used to record SSNA in 5 female and 4 male subjects lying supine on a table that fixed their head in space but allowed trapezoidal ramp (8.1 ± 1.2°/s) and hold (17.5° for 53 s) or sinusoidal (35° peak to peak at 0.33-0.46 Hz) horizontal displacement of the body about the head. SSNA recordings were made before, during, and after trapezoidal and sinusoidal displacements of the body. Spike frequency analysis of trapezoidal displacements and cross-correlation analysis during sinusoidal displacements revealed that SSNA was not changed by trapezoid body-only displacement but was cyclically modulated during sinusoidal angular displacements (median, 95% CI: 27.9%, 19.6-48.0%). The magnitude of this modulation was not statistically ( P > 0.05) different from that of cardiac and respiratory modulation at rest (47.1%, 18.7-56.3% and 48.6%, 28.4-59.3%, respectively) or during sinusoidal displacement (10.3%, 6.2-32.1% and 26.9%, 13.6-43.3%, respectively). Respiratory frequency was entrained above its resting rate (0.26 Hz, 0.2-0.29 Hz) during sinusoidal neck displacement; there was no significant difference ( P > 0.05) between respiratory frequency (0.38 Hz, 0.25-0.49 Hz) and sinusoidal displacement frequency (0.39 Hz, 0.35-0.42 Hz). This study provides evidence that SSNA is modulated during neck movement, raising the possibility that neck mechanoreceptors may contribute to the cutaneous vasoconstriction and sweat release associated with motion sickness. NEW & NOTEWORTHY This study demonstrates that dynamic, but not static, stretching of the neck modulates skin sympathetic nerve activity in the lower limbs.

Random-amplitude sinusoidal linear acceleration causes greater vestibular modulation of skin sympathetic nerve activity than constant-amplitude acceleration.

We tested the hypothesis that random variations in the magnitude of sinusoidal linear acceleration cause greater modulation of skin sympathetic nerve activity (SSNA), but not muscle sympathetic nerve activity (MSNA), than sinusoidal stimuli of the same frequency but constant amplitude. Subjects (n = 22) were seated in a sealed room mounted on a linear motor that could deliver peak sinusoidal accelerations of 30 mG in the antero-posterior direction. Subjects sat on a padded chair with their neck and head supported vertically, thereby minimizing somatosensory cues, facing the direction of motion in the anterior direction. Each block of sinusoidal motion was delivered at 0.2 Hz, either with a constant-amplitude (root mean square 14 mG) or randomly fluctuating amplitudes of the same mean amplitude. MSNA (n = 12) and SSNA (n = 10) were recorded via tungsten microelectrodes inserted into muscle or cutaneous fascicles of the common peroneal nerve. Cross-correlation analysis was used to measure the magnitude of vestibular modulation. The modulation index for SSNA was significantly higher during delivery of random vs constant-amplitude acceleration (31.4 ± 1.9 vs 24.5 ± 2.5%), but there was no significant difference in the modulation indices for MSNA (28.8 ± 2.9 vs 33.4 ± 4.1%). We conclude that the pattern of vestibular stimulation affects the magnitude of modulation of sympathetic outflow to skin but not to muscle. Presumably, this is related to the subperceptual development of nausea, which is known to be associated with greater vestibular modulation of SSNA but not MSNA.

Motion sickness is associated with an increase in vestibular modulation of skin but not muscle sympathetic nerve activity.

We have previously shown that sinusoidal galvanic vestibular stimulation (sGVS), delivered bilaterally at frequencies of 0.08-2.00 Hz, causes a pronounced modulation of muscle sympathetic nerve activity (MSNA) and skin sympathetic nerve activity (SSNA), together with robust frequency-dependent illusions of side-to-side motion. At low frequencies of sGVS (≤0.2 Hz), some subjects report nausea, so we tested the hypothesis that vestibular modulation of MSNA and SSNA is augmented in individuals reporting nausea. MSNA was recorded via tungsten microelectrodes inserted into the left common peroneal nerve in 22 awake, seated subjects; SSNA was recorded in 14 subjects. Bipolar binaural sGVS (±2 mA, 100 cycles) was applied to the mastoid processes at 0.08, 0.13, and 0.18 Hz. Nausea was reported by 21 out of 36 subjects (58 %), but across frequencies of sGVS there was no difference in the magnitude of the vestibular modulation of MSNA in subjects who reported nausea (27.1 ± 1.8 %) and those who did not (30.4 ± 2.9 %). This contrasts with the significantly greater vestibular modulation of SSNA with nausea (41.1 ± 2.0 vs. 28.7 ± 3.1 %) and indicates an organ-specific modulation of sympathetic outflow via the vestibular system during motion sickness.

Brainstem changes associated with increased muscle sympathetic drive in obstructive sleep apnoea.

Obstructive sleep apnoea (OSA) is associated with significantly increased bursts of muscle sympathetic nerve activity (MSNA), leading to hypertension and increased cardiovascular morbidity. The underlying mechanism responsible for this sympathoexcitation is unknown. The aim of this investigation was to determine brainstem sites that contribute to this increased on-going muscle vasoconstrictor drive. We measured regional grey matter volume using voxel-based morphometry of T1-weighted anatomical images in 20 subjects with OSA and 19 healthy age-matched controls. We also performed concurrent recordings of MSNA and Blood Oxygen Level Dependent (BOLD) signal intensity of the brainstem, using high-resolution functional magnetic resonance imaging, in 15 subjects with OSA and 15 controls. OSA subjects had significantly elevated MSNA, which was correlated to altered BOLD signal intensity changes in the dorsolateral pons, rostral ventrolateral medulla, medullary raphe and midbrain. The medullary raphe, rostroventrolateral medulla and dorsolateral pons also had significantly increased grey matter volumes in subjects with obstructive sleep apnoea compared with controls. Furthermore, we also found that obstructive sleep apnoea was associated with increases in grey matter volume in the region of the hypoglossal nucleus. These data suggest that the elevated muscle vasoconstrictor drive in obstructive sleep apnoea may result from functional and anatomical changes within the dorsolateral pons, rostroventrolateral medulla and medullary raphe. These brainstem regions are known to modulate sympathetic output either directly or indirectly via sympathetic preganglionic neurons within the spinal cord. In addition, the known increase in genioglossus muscle activity in OSA may reflect the increase in grey matter volume of the hypoglossal nucleus.

Vestibular modulation of muscle sympathetic nerve activity by the utricle during sub-perceptual sinusoidal linear acceleration in humans.

We assessed the capacity for the vestibular utricle to modulate muscle sympathetic nerve activity (MSNA) during sinusoidal linear acceleration at amplitudes extending from imperceptible to clearly perceptible. Subjects (n = 16) were seated in a sealed room, eliminating visual cues, mounted on a linear motor that could deliver peak sinusoidal accelerations of 30 mG in the antero-posterior direction. Subjects sat on a padded chair with their neck and head supported vertically, thereby minimizing somatosensory cues, facing the direction of motion in the anterior direction. Each block of sinusoidal motion was applied at a time unknown to subjects and in a random order of amplitudes (1.25, 2.5, 5, 10, 20 and 30 mG), at a constant frequency of 0.2 Hz. MSNA was recorded via tungsten microelectrodes inserted into muscle fascicles of the common peroneal nerve. Subjects used a linear potentiometer aligned to the axis of motion to indicate any perceived movement, which was compared with the accelerometer signal of actual room movement. On average, 67% correct detection of movement did not occur until 6.5 mG, with correct knowledge of the direction of movement at ~10 mG. Cross-correlation analysis revealed potent sinusoidal modulation of MSNA even at accelerations subjects could not perceive (1.25-5 mG). The modulation index showed a positive linear increase with acceleration amplitude, such that the modulation was significantly higher (25.3 ± 3.7%) at 30 mG than at 1.25 mG (15.5 ± 1.2%). We conclude that selective activation of the vestibular utricle causes a pronounced modulation of MSNA, even at levels well below perceptual threshold, and provides further evidence in support of the importance of vestibulosympathetic reflexes in human cardiovascular control.

Modulation of muscle sympathetic nerve activity by low-frequency physiological activation of the vestibular utricle in awake humans.

We recently showed that selective stimulation of one set of otolithic organs-those located in the utricle, sensitive to displacement in the horizontal axis-causes a marked entrainment of skin sympathetic nerve activity (SSNA). Here, we assessed whether muscle sympathetic nerve activity (MSNA) is similarly modulated. MSNA was recorded via tungsten microelectrodes inserted into cutaneous fascicles of the common peroneal nerve in 12 awake subjects, seated (head vertical, eyes closed) on a motorised platform. Slow sinusoidal accelerations-decelerations (±4 mG) were applied in the X (antero-posterior) or Y (medio-lateral) direction at 0.08 Hz. Cross-correlation analysis revealed partial entrainment of MSNA: vestibular modulation was 32 ± 3 % for displacements in the X-axis and 29 ± 3 % in the Y-axis; these were significantly smaller than those evoked in SSNA (97 ± 3 and 91 ± 5 %, respectively). For each sinusoidal cycle, there were two peaks of modulation-one associated with acceleration as the platform moved forward or to the side and one associated with acceleration in the opposite direction. We believe the two peaks reflect inertial displacement of the stereocilia within the utricle during sinusoidal acceleration, which evokes vestibulosympathetic reflexes that are expressed as vestibular modulation of MSNA as well as of SSNA. The smaller vestibular modulation of MSNA can be explained by the dominant modulation of MSNA by the arterial baroreceptors.

Early bone reformation after cranial vault remodelling for sagittal craniosynostosis: A retrospective 3D analysis.

This study aims to measure postoperative bone reformation percentage, rates and patterns after cranial vault remodelling (CVR) in isolated non-syndromic sagittal craniosynostosis. Volumetric bone measurements were performed starting from the DICOM files of previously available postoperative CT scans. The 3D images were then resampled into the master box, and 'Skull 3D models' were derived. The percentage of bone reformation was investigated using automated 3D analysis software. The intra-rater reliability analysis revealed high reliability (Intraclass correlation coefficient = 0.99, p < 0.001). The median bone reformation volume and rate were 11.2 ml and 1.98 ml/week, respectively. The median percentage of bone reformation was 56.7% when the median postoperative CT timing was 6.1 weeks. As a statistic model, the linear plateau showed the highest Pseudo R2 in both volume and percentage of bone reformation predicting patterns. By using the calculated model at 9 weeks postoperatively, the re-osteogenesis reaches 80% of the total cranial defect. After CVR, the early bone reformation pattern was demonstrated as a linear plateau model rather than logarithmic. This study gives a better understanding of the pattern and quantity of re-osteogenesis at cranial defects after CVR. The statistic model can facilitate healthcare practitioners to predict bone reformation and improve postoperative care protocol in sagittal craniosynostosis management.

Plasmodium falciparum Eukaryotic Translation Initiation Factor 3 is Stabilized by Quinazoline-Quinoline Bisubstrate Inhibitors.

Malaria drug resistance is hampering the fight against the deadliest parasitic disease affecting over 200 million people worldwide. We recently developed quinoline-quinazoline-based inhibitors (as compound 70) as promising new antimalarials. Here, we aimed to investigate their mode of action by using thermal proteome profiling (TPP). The eukaryotic translation initiation factor 3 (EIF3i) subunit I was identified as the main target protein stabilized by compound 70 in Plasmodium falciparum. This protein has never been characterized in malaria parasites. P. falciparum parasite lines were generated expressing either a HA tag or an inducible knockdown of the PfEIF3i gene to further characterize the target protein. PfEIF3i was stabilized in the presence of compound 70 in a cellular thermal shift Western blot assay, pointing that PfEIF3i indeed interacts with quinoline-quinazoline-based inhibitors. In addition, PfEIF3i-inducible knockdown blocks intra-erythrocytic development in the trophozoite stage, indicating that it has a vital function. We show that PfEIF3i is mostly expressed in late intra-erythrocytic stages and localizes in the cytoplasm. Previous mass spectrometry reports show that PfEIF3i is expressed in all parasite life cycle stages. Further studies will explore the potential of PfEIF3i as a target for the design of new antimalarial drugs active all along the life cycle of the parasite.

Low-frequency galvanic vestibular stimulation evokes two peaks of modulation in skin sympathetic nerve activity.

We have previously shown that sinusoidal galvanic vestibular stimulation (sGVS), delivered bilaterally at 0.2-2.0 Hz, evokes a potent entrainment of sympathetic outflow to muscle and skin. Most recently, we showed that stimulation at 0.08-0.18 Hz generates two bursts of modulation of muscle sympathetic nerve activity (MSNA), more pronounced at 0.08 Hz, which we interpreted as reflecting bilateral projections from the vestibular nuclei to the medullary nuclei responsible for the generation of MSNA. Here, we test the hypothesis that these very low frequencies of sGVS modulate skin sympathetic nerve activity (SSNA) in a similar fashion. SSNA was recorded via tungsten microelectrodes inserted into the left common peroneal nerve in 11 awake-seated subjects. Bipolar binaural sGVS (±2 mA, 100 cycles) was applied to the mastoid processes at 0.08, 0.13 and 0.18 Hz. As with MSNA, cross-correlation analysis revealed two bursts of modulation of SSNA for each cycle of stimulation but, unlike MSNA, this modulation was equally pronounced at all frequencies. These results further support our conclusion that bilateral sGVS causes cyclical modulation of the left and right vestibular nerves and a resultant modulation of sympathetic outflow that reflects the summed activity of bilateral projections from the vestibular nuclei onto, in this case, the primary output nuclei responsible for SSNA-the medullary raphé. Furthermore, these findings emphasise the role of the vestibular system in the control of skin sympathetic outflow, and the cutaneous expression of motion sickness: pallor and sweat release. Indeed, vestibular modulation of SSNA was higher in those subjects reporting nausea than in those who did not report nausea during this low-frequency sGVS.

Low-frequency physiological activation of the vestibular utricle causes biphasic modulation of skin sympathetic nerve activity in humans.

We have previously shown that sinusoidal galvanic vestibular stimulation, a means of selectively modulating vestibular afferent activity, can cause partial entrainment of sympathetic outflow to muscle and skin in human subjects. However, it influences the firing of afferents from the entire vestibular apparatus, including the semicircular canals. Here, we tested the hypothesis that selective stimulation of one set of otolithic organs-those located in the utricle, which are sensitive to displacement in the horizontal axis-could entrain sympathetic nerve activity. Skin sympathetic nerve activity (SSNA) was recorded via tungsten microelectrodes inserted into cutaneous fascicles of the common peroneal nerve in 10 awake subjects, seated (head vertical, eyes closed) on a motorised platform. Slow sinusoidal accelerations-decelerations (~4 mG) were applied in the X (antero-posterior) or Y (medio-lateral) direction at 0.08 Hz; composite movements in both directions were also applied. Subjects either reported feeling a vague sense of movement (with no sense of direction) or no movement at all. Nevertheless, cross-correlation analysis revealed a marked entrainment of SSNA for all types of movements: vestibular modulation was 97 ± 3 % for movements in the X axis and 91 ± 5 % for displacements in the Y axis. For each sinusoidal cycle, there were two major peaks of modulation-one associated with acceleration as the platform moved forward or to the side, and one associated with acceleration in the opposite direction. We interpret these observations as reflecting inertial displacement of the stereocilia within the utricle during acceleration, which causes a robust vestibulosympathetic reflex.

Evidence from bilateral recordings of sympathetic nerve activity for lateralisation of vestibular contributions to cardiovascular control.

Using low-frequency (0.08-0.18 Hz) sinusoidal galvanic vestibular stimulation (sGVS), we recently showed that two peaks of modulation of muscle sympathetic nerve activity (MSNA) and skin sympathetic nerve activity (SSNA) occurred for each cycle of stimulation: a large peak associated with the positive peak of the sinusoid (defined as the primary peak) and a smaller peak (defined as the secondary peak) related to the negative peak of the sinusoid. However, these recordings were only made from the left common peroneal nerve, so to investigate lateralisation of vestibulosympathetic reflexes, concurrent recordings were made from both sides of the body. Tungsten microelectrodes were inserted into muscle or cutaneous fascicles of the left and right common peroneal nerves in 17 healthy individuals. Bipolar binaural sinusoidal GVS (±2 mA, 100 cycles) was applied to the mastoid processes at 0.08 Hz. Cross-correlation analysis revealed that vestibular modulation of MSNA (10 bilateral recordings) and SSNA (6 bilateral recordings) on the left side was expressed as a primary peak related to the positive phase of the sinusoid and a secondary peak related to the negative phase of the sinusoid. Conversely, on the right side, the primary and secondary peaks were reversed: the secondary peak on the right coincided with the primary peak on the left and vice versa. Moreover, differences in pattern of outflow were apparent across sides. We believe the results support the conclusion that the left and right vestibular nuclei send both an ipsilateral and contralateral projection to the left and right medullary output nuclei from which MSNA and SSNA originate. This causes a "flip-flop" patterning between the two sympathetic outflows: when vestibular modulation of a burst is high on the left, it is low on the right, and when modulation is low on the left, it is high on the right.

Low-frequency sinusoidal galvanic stimulation of the left and right vestibular nerves reveals two peaks of modulation in muscle sympathetic nerve activity.

Studies previously performed in our laboratory have shown that sinusoidal galvanic vestibular stimulation (sGVS), a means of selectively modulating vestibular input without affecting other inputs, can cause partial entrainment of muscle sympathetic nerve activity (MSNA) at frequencies ranging from 0.2 to 2.0 Hz. Here we test the effect of sGVS on sympathetic outflow when stimulating the vestibular system at lower frequencies. MSNA was recorded via tungsten microelectrodes inserted into the left common peroneal nerve in 12 awake, seated subjects. Bipolar binaural sinusoidal GVS (±2 mA, 100 cycles) was applied to the mastoid processes at 0.08, 0.13 and 0.18 Hz. Cross-correlation analysis revealed two bursts of modulation of MSNA for each cycle of stimulation. We believe the primary peak is related to the positive phase of the sinusoid, in which the right vestibular nerve is hyperpolarised and the left vestibular nerve depolarised. Furthermore, we believe the secondary peak is related to the negative phase of the sinusoid (depolarisation of the right vestibular nerve and hyperpolarisation of the left vestibular nerve). This was never observed at higher frequencies of stimulation, presumably because at such frequencies there is insufficient time for a second peak to be expressed. The incidence of double peaks of MSNA was highest at 0.08 Hz and lowest at 0.18 Hz. These observations emphasise the role of the vestibular apparatus in the control of blood pressure, and further suggest convergence of bilateral inputs from vestibular nuclei onto the output nuclei from which MSNA originates, the rostral ventrolateral medulla (RVLM).

Malaria Parasite Stress Tolerance Is Regulated by DNMT2-Mediated tRNA Cytosine Methylation.

Malaria parasites need to cope with changing environmental conditions that require strong countermeasures to ensure pathogen survival in the human and mosquito hosts. The molecular mechanisms that protect Plasmodium falciparum homeostasis during the complex life cycle remain unknown. Here, we identify cytosine methylation of tRNAAsp (GTC) as being critical to maintain stable protein synthesis. Using conditional knockout (KO) of a member of the DNA methyltransferase family, called Pf-DNMT2, RNA bisulfite sequencing demonstrated the selective cytosine methylation of this enzyme of tRNAAsp (GTC) at position C38. Although no growth defect on parasite proliferation was observed, Pf-DNMT2KO parasites showed a selective downregulation of proteins with a GAC codon bias. This resulted in a significant shift in parasite metabolism, priming KO parasites for being more sensitive to various types of stress. Importantly, nutritional stress made tRNAAsp (GTC) sensitive to cleavage by an unknown nuclease and increased gametocyte production (>6-fold). Our study uncovers an epitranscriptomic mechanism that safeguards protein translation and homeostasis of sexual commitment in malaria parasites. IMPORTANCE P. falciparum is the most virulent malaria parasite species, accounting for the majority of the disease mortality and morbidity. Understanding how this pathogen is able to adapt to different cellular and environmental stressors during its complex life cycle is crucial in order to develop new strategies to tackle the disease. In this study, we identified the writer of a specific tRNA cytosine methylation site as a new layer of epitranscriptomic regulation in malaria parasites that regulates the translation of a subset of parasite proteins (>400) involved in different metabolic pathways. Our findings give insight into a novel molecular mechanism that regulates P. falciparum response to drug treatment and sexual commitment.

Procainamide-SAHA Fused Inhibitors of hHDAC6 Tackle Multidrug-Resistant Malaria Parasites.

Epigenetic post-translational modifications are essential for human malaria parasite survival and progression through its life cycle. Here, we present new functionalized suberoylanilide hydroxamic acid (SAHA) derivatives that chemically combine the pan-histone deacetylase inhibitor SAHA with the DNA methyltransferase inhibitor procainamide. A three- or four-step chemical synthesis was designed starting from cheap raw materials. Compared to the single drugs, the combined molecules showed a superior activity in Plasmodium and a potent inhibition against human HDAC6, exerting no cytotoxicity in human cell lines. These new compounds are fully active in multidrug-resistant Plasmodium falciparum Cambodian isolates. They target transmission of the parasite by inducing irreversible morphological changes in gametocytes and inhibiting exflagellation. The compounds are slow-acting and have an additive antimalarial effect in combination with fast-acting epidrugs and dihydroartemisinin. The lead compound decreases parasitemia in mice in a severe malaria model. Taken together, this novel fused molecule offers an affordable alternative to current failing antimalarial therapy.

DNA Methylation Bisubstrate Inhibitors Are Fast-Acting Drugs Active against Artemisinin-Resistant Plasmodium falciparum Parasites.

Malaria is the deadliest parasitic disease affecting over 200 million people worldwide. The increasing number of treatment failures due to multi-drug-resistant parasites in South-East Asia hinders the efforts for elimination. It is thus urgent to develop new antimalarials to contain these resistant parasites. Based on a previous report showing the presence of DNA methylation in Plasmodium, we generated new types of DNA methylation inhibitors against malaria parasites. The quinoline-quinazoline-based inhibitors kill parasites, including artemisinin-resistant field isolates adapted to culture, in the low nanomolar range. The compounds target all stages of the asexual cycle, including early rings, during a 6 h treatment period; they reduce DNA methylation in the parasite and show in vivo activity at 10 mg/kg. These potent inhibitors are a new starting point to develop fast-acting antimalarials that could be used in combination with artemisinins.

Vestibular Modulation of Sympathetic Nerve Activity to Muscle and Skin in Humans.

We review the existence of vestibulosympathetic reflexes in humans. While several methods to activate the human vestibular apparatus have been used, galvanic vestibular stimulation (GVS) is a means of selectively modulating vestibular afferent activity via electrodes over the mastoid processes, causing robust vestibular illusions of side-to-side movement. Sinusoidal GVS (sGVS) causes partial entrainment of sympathetic outflow to muscle and skin. Modulation of muscle sympathetic nerve activity (MSNA) from vestibular inputs competes with baroreceptor inputs, with stronger temporal coupling to the vestibular stimulus being observed at frequencies remote from the cardiac frequency; "super entrainment" was observed in some individuals. Low-frequency (<0.2 Hz) sGVS revealed two peaks of modulation per cycle, with bilateral recordings of MSNA or skin sympathetic nerve activity, providing evidence of lateralization of sympathetic outflow during vestibular stimulation. However, it should be noted that GVS influences the firing of afferents from the entire vestibular apparatus, including the semicircular canals. To identify the specific source of vestibular input responsible for the generation of vestibulosympathetic reflexes, we used low-frequency (<0.2 Hz) sinusoidal linear acceleration of seated or supine subjects to, respectively, target the utricular or saccular components of the otoliths. While others had discounted the semicircular canals, we showed that the contributions of the utricle and saccule to the vestibular modulation of MSNA are very similar. Moreover, that modulation of MSNA occurs at accelerations well below levels at which subjects are able to perceive any motion indicates that, like vestibulospinal control of posture, the vestibular system contributes to the control of blood pressure through potent reflexes in humans.