Noncovalent Peptide Stapling Using Alpha-Methyl-l-Phenylalanine for α-Helical Peptidomimetics.

Peptides and peptidomimetics are attractive drug candidates because of their high target specificity and low-toxicity profiles. Developing peptidomimetics using hydrocarbon (HC)-stapling or other stapling strategies has gained momentum because of their high stability and resistance to proteases; however, they have limitations. Here, we take advantage of the α-methyl group and an aromatic phenyl ring in a unique unnatural amino acid, α-methyl-l-phenylalanine (αF), and propose a novel, noncovalent stapling strategy to stabilize peptides. We utilized this strategy to create an α-helical B-chain mimetic of a complex insulin-like peptide, human relaxin-3 (H3 relaxin). Our comprehensive data set (in vitro, ex vivo, and in vivo) confirmed that the new high-yielding B-chain mimetic, H3B10-27(13/17αF), is remarkably stable in serum and fully mimics the biological function of H3 relaxin. H3B10-27(13/17αF) is an excellent scaffold for further development as a drug lead and an important tool to decipher the physiological functions of the neuropeptide G protein-coupled receptor, RXFP3.

Volumetric mapping of the functional neuroanatomy of the respiratory network in the perfused brainstem preparation of rats.

KEY POINTS: The functional neuroanatomy of the mammalian respiratory network is far from being understood since experimental tools that measure neural activity across this brainstem-wide circuit are lacking. Here, we use silicon multi-electrode arrays to record respiratory local field potentials (rLFPs) from 196-364 electrode sites within 8-10 mm3 of brainstem tissue in single arterially perfused brainstem preparations with respect to the ongoing respiratory motor pattern of inspiration (I), post-inspiration (PI) and late-expiration (E2). rLFPs peaked specifically at the three respiratory phase transitions, E2-I, I-PI and PI-E2. We show, for the first time, that only the I-PI transition engages a brainstem-wide network, and that rLFPs during the PI-E2 transition identify a hitherto unknown role for the dorsal respiratory group. Volumetric mapping of pontomedullary rLFPs in single preparations could become a reliable tool for assessing the functional neuroanatomy of the respiratory network in health and disease. ABSTRACT: While it is widely accepted that inspiratory rhythm generation depends on the pre-Bötzinger complex, the functional neuroanatomy of the neural circuits that generate expiration is debated. We hypothesized that the compartmental organization of the brainstem respiratory network is sufficient to generate macroscopic local field potentials (LFPs), and if so, respiratory (r) LFPs could be used to map the functional neuroanatomy of the respiratory network. We developed an approach using silicon multi-electrode arrays to record spontaneous LFPs from hundreds of electrode sites in a volume of brainstem tissue while monitoring the respiratory motor pattern on phrenic and vagal nerves in the perfused brainstem preparation. Our results revealed the expression of rLFPs across the pontomedullary brainstem. rLFPs occurred specifically at the three transitions between respiratory phases: (1) from late expiration (E2) to inspiration (I), (2) from I to post-inspiration (PI), and (3) from PI to E2. Thus, respiratory network activity was maximal at respiratory phase transitions. Spatially, the E2-I, and PI-E2 transitions were anatomically localized to the ventral and dorsal respiratory groups, respectively. In contrast, our data show, for the first time, that the generation of controlled expiration during the post-inspiratory phase engages a distributed neuronal population within ventral, dorsal and pontine network compartments. A group-wise independent component analysis demonstrated that all preparations exhibited rLFPs with a similar temporal structure and thus share a similar functional neuroanatomy. Thus, volumetric mapping of rLFPs could allow for the physiological assessment of global respiratory network organization in health and disease.

Modulation of hypercapnic respiratory response by cholinergic transmission in the commissural nucleus of the solitary tract.

The nucleus of the solitary tract (NTS) is an important area of the brainstem that receives and integrates afferent cardiorespiratory sensorial information, including those from arterial chemoreceptors and baroreceptors. It was described that acetylcholine (ACh) in the commissural subnucleus of the NTS (cNTS) promotes an increase in the phrenic nerve activity (PNA) and antagonism of nicotinic receptors in the same region reduces the magnitude of tachypneic response to peripheral chemoreceptor stimulation, suggesting a functional role of cholinergic transmission within the cNTS in the chemosensory control of respiratory activity. In the present study, we investigated whether cholinergic receptor antagonism in the cNTS modifies the sympathetic and respiratory reflex responses to hypercapnia. Using an arterially perfused in situ preparation of juvenile male Holtzman rats, we found that the nicotinic antagonist (mecamylamine, 5 mM), but not the muscarinic antagonist (atropine, 5 mM), into the cNTS attenuated the hypercapnia-induced increase of hypoglossal activity. Furthermore, mecamylamine in the cNTS potentiated the generation of late-expiratory (late-E) activity in abdominal nerve induced by hypercapnia. None of the cholinergic antagonists microinjected in the cNTS changed either the sympathetic or the phrenic nerve responses to hypercapnia. Our data provide evidence for the role of cholinergic transmission in the cNTS, acting on nicotinic receptors, modulating the hypoglossal and abdominal responses to hypercapnia.

[Hybrid stabilization technique with spinal fusion and interlaminar device to reduce the length of fusion and to protect symptomatic adjacent segments : Clinical long-term follow-up]. / Hybridversorgung mit Fusion und interlaminärem Implantat zur Verkürzung der Spondylodesestrecke und Protektion der symptomatischen Anschlussdegeneration : Klinisches Langzeit-Follow-up.

INTRODUCTION: Determination of the extent of spinal fusion for lumbar degenerative diseases is often difficult due to minor pathologies in the adjacent segment. Although surgical intervention is required, fusion seems to be an overtreatment. Decompression alone may be not enough as this segment is affected by multiple factors such as destabilization, low grade degeneration and an unfavorable biomechanical transition next to a rigid construct. An alternative surgical treatment is a hybrid construct, consisting of fusion and implantation of an interlaminar stabilization device at the adjacent level. The aim of this study was to compare long-term clinical outcome after lumbar fusion with a hybrid construct including an interlaminar stabilization device as "topping-off". MATERIALS AND METHODS: A retrospective analysis of 25 lumbar spinal fusions from 2003 to 2010 with additional interlaminar stabilization device was performed. Through a matched case controlled procedure 25 congruent patients who received lumbar spinal fusion in one or two levels were included as a control group. At an average follow-up of 43 months pre- and postoperative pain, ODI, SF-36 as well as clinical parameters, such as leg and back pain, walking distance and patient satisfaction were recorded. RESULTS: Pain relief, ODI improvement and patient satisfaction was significantly higher in the hybrid group compared to the control group. SF-36 scores improved in both groups but was higher in the hybrid group, although without significance. Evaluation of walking distance showed no significant differences. DISCUSSION: Many outcome parameters present significantly better long-term results in the hybrid group compared to sole spinal fusion. Therefore, in cases with a clear indication for lumbar spinal fusion with the need for decompression at the adjacent level due to spinal stenosis or moderate spondylarthrosis, support of this segment with an interlaminar stabilization device demonstrates a reasonable treatment option with good clinical outcome. Also, the length of the fusion construct can be reduced allowing for a softer and more harmonic transition.

Amyloid formation in human islets is enhanced by heparin and inhibited by heparinase.

Islet transplantation is a promising therapy for patients with diabetes, but its long-term success is limited by many factors, including the formation of islet amyloid deposits. Heparin is employed in clinical islet transplantation to reduce clotting but also promotes fibrillization of amyloidogenic proteins. We hypothesized that heparin treatment of islets during pre-transplant culture may enhance amyloid formation leading to beta cell loss and graft dysfunction. Heparin promoted the fibrillization of human islet amyloid polypeptide (IAPP) and enhanced its toxicity to INS-1 beta cells. Heparin increased amyloid deposition in cultured human islets, but surprisingly decreased islet cell apoptosis. Treatment of human islets with heparin prior to transplantation increased the likelihood of graft failure. Removal of islet heparan sulfate glycosaminoglycans, which localize with islet amyloid deposits in type 2 diabetes, by heparinase treatment decreased amyloid deposition and protected against islet cell death. These findings raise the possibility that pretransplant treatment of human islets with heparin could potentiate IAPP aggregation and amyloid formation and may be detrimental to subsequent graft function.

Differential modulation of sympathetic and respiratory activities by cholinergic mechanisms in the nucleus of the solitary tract in rats.

The contribution of cholinergic mechanisms of the nucleus of the solitary tract (NTS) to cardiorespiratory control is not completely clear. In the present study, we investigated the involvement of the cholinergic mechanisms in the intermediate NTS (iNTS) and commissural NTS (cNTS) on the control of sympathetic (SNA) and phrenic nerve activity (PNA). Decorticated, arterially perfused in situ preparations of male juvenile rats (60-100 g) were used. Acetylcholine (10 mm, 60 nl) injected into the iNTS reduced SNA (-54 ± 4%, versus vehicle -5 ± 3%; P < 0.001) and PNA (-30 ± 4%, versus vehicle -5 ± 6%; P < 0.001), whereas injections of ACh into the cNTS increased PNA (30 ± 6%, versus vehicle 5 ± 3%; P < 0.001), without changing SNA. Pretreatment with mecamylamine (nicotinic antagonist; 5 mm) abolished all the effects of ACh injected into the iNTS or the cNTS, whereas atropine (muscarinic antagonist; 5 mm) reduced only the effects of ACh injected into the cNTS. Mecamylamine injected into the cNTS also reduced the tachypnoea in response to peripheral chemoreflex activation. The baroreflex was unaltered by injections of atropine or mecamylamine into the NTS. The results suggest that ACh and mainly nicotinic receptors in the NTS are involved in the modulation of SNA and PNA, with distinct functions between the iNTS and the cNTS. An involvement of the nicotinic receptors in the cNTS in the tachypnoea in response to peripheral chemoreflex activation is also suggested.

Decreased inflammatory responses of human lung epithelial cells after ethanol exposure are mimicked by ethyl pyruvate.

BACKGROUND AND PURPOSE: Leukocyte migration into alveolar space plays a critical role in pulmonary inflammation resulting in lung injury. Acute ethanol (EtOH) exposure exerts anti-inflammatory effects. The clinical use of EtOH is critical due to its side effects. Here, we compared effects of EtOH and ethyl pyruvate (EtP) on neutrophil adhesion and activation of cultured alveolar epithelial cells (A549). EXPERIMENTAL APPROACH: Time course and dose-dependent release of interleukin- (IL-) 6 and IL-8 from A549 were measured after pretreatment of A549 with EtP (2.5-10 mM), sodium pyruvate (NaP, 10 mM), or EtOH (85-170 mM), and subsequent lipopolysaccharide or IL-1beta stimulation. Neutrophil adhesion to pretreated and stimulated A549 monolayers and CD54 surface expression were determined. KEY RESULTS: Treating A549 with EtOH or EtP reduced substantially the cytokine-induced release of IL-8 and IL-6. EtOH and EtP (but not NaP) reduced the adhesion of neutrophils to monolayers in a dose- and time-dependent fashion. CD54 expression on A549 decreased after EtOH or EtP treatment before IL-1beta stimulation. CONCLUSIONS AND IMPLICATIONS: EtP reduces secretory and adhesive potential of lung epithelial cells under inflammatory conditions. These findings suggest EtP as a potential treatment alternative that mimics the anti-inflammatory effects of EtOH in early inflammatory response in lungs.

The pre-Bötzinger complex is necessary for the expression of inspiratory and post-inspiratory motor discharge of the vagus.

The mammalian three-phase respiratory motor pattern of inspiration, post-inspiration and expiration is expressed in spinal and cranial motor nerve discharge and is generated by a distributed ponto-medullary respiratory pattern generating network. Respiratory motor pattern generation depends on a rhythmogenic kernel located within the pre-Bötzinger complex (pre-BötC). In the present study, we tested the effect of unilateral and bilateral inactivation of the pre-BötC after local microinjection of the GABAA receptor agonist isoguvacine (10 mM, 50 nl) on phrenic (PNA), hypoglossal (HNA) and vagal nerve (VNA) respiratory motor activities in an in situ perfused brainstem preparation of rats. Bilateral inactivation of the pre-BötC triggered cessation of phrenic (PNA), hypoglossal (HNA) and vagal (VNA) nerve activities for 15-20 min. Ipsilateral isoguvacine injections into the pre-BötC triggered transient (6-8 min) cessation of inspiratory and post-inspiratory VNA (p < 0.001) and suppressed inspiratory HNA by - 70 ± 15% (p < 0.01), while inspiratory PNA burst frequency increased by 46 ± 30% (p < 0.01). Taken together, these observations confirm the role of the pre-BötC as the rhythmogenic kernel of the mammalian respiratory network in situ and highlight a significant role for the pre-BötC in the transmission of vagal inspiratory and post-inspiratory pre-motor drive to the nucleus ambiguus.

Neuroanatomical frameworks for volitional control of breathing and orofacial behaviors.

Breathing is the only vital function that can be volitionally controlled. However, a detailed understanding how volitional (cortical) motor commands can transform vital breathing activity into adaptive breathing patterns that accommodate orofacial behaviors such as swallowing, vocalization or sniffing remains to be developed. Recent neuroanatomical tract tracing studies have identified patterns and origins of descending forebrain projections that target brain nuclei involved in laryngeal adductor function which is critically involved in orofacial behavior. These nuclei include the midbrain periaqueductal gray and nuclei of the respiratory rhythm and pattern generating network in the brainstem, specifically including the pontine Kölliker-Fuse nucleus and the pre-Bötzinger complex in the medulla oblongata. This review discusses the functional implications of the forebrain-brainstem anatomical connectivity that could underlie the volitional control and coordination of orofacial behaviors with breathing.

Euthanization methods influence cytokine mRNA expression levels in age 0 year Oncorhynchus mykiss.

Significant differences in cytokine transcription were found between Oncorhynchus mykiss euthanized using the pharmacological agents MS-222 v. benzocaine and also when contrasting death induced by carbon dioxide asphyxiation v. physical methods (cervical dislocation). This study highlights the need to consider the potentially confounding effect of euthanization method on gene expression data.

Forebrain projection neurons target functionally diverse respiratory control areas in the midbrain, pons, and medulla oblongata.

Eupnea is generated by neural circuits located in the ponto-medullary brainstem, but can be modulated by higher brain inputs which contribute to volitional control of breathing and the expression of orofacial behaviors, such as vocalization, sniffing, coughing, and swallowing. Surprisingly, the anatomical organization of descending inputs that connect the forebrain with the brainstem respiratory network remains poorly defined. We hypothesized that descending forebrain projections target multiple distributed respiratory control nuclei across the neuroaxis. To test our hypothesis, we made discrete unilateral microinjections of the retrograde tracer cholera toxin subunit B in the midbrain periaqueductal gray (PAG), the pontine Kölliker-Fuse nucleus (KFn), the medullary Bötzinger complex (BötC), pre-BötC, or caudal midline raphé nuclei. We quantified the regional distribution of retrogradely labeled neurons in the forebrain 12-14 days postinjection. Overall, our data reveal that descending inputs from cortical areas predominantly target the PAG and KFn. Differential forebrain regions innervating the PAG (prefrontal, cingulate cortices, and lateral septum) and KFn (rhinal, piriform, and somatosensory cortices) imply that volitional motor commands for vocalization are specifically relayed via the PAG, while the KFn may receive commands to coordinate breathing with other orofacial behaviors (e.g., sniffing, swallowing). Additionally, we observed that the limbic or autonomic (interoceptive) systems are connected to broadly distributed downstream bulbar respiratory networks. Collectively, these data provide a neural substrate to explain how volitional, state-dependent, and emotional modulation of breathing is regulated by the forebrain.

Reciprocal connectivity of the periaqueductal gray with the ponto-medullary respiratory network in rat.

Synaptic activities of the periaqueductal gray (PAG) can modulate or appropriate the respiratory motor activities in the context of behavior and emotion via descending projections to nucleus retroambiguus. However, alternative anatomical pathways for the mediation of PAG-evoked respiratory modulation via core nuclei of the brainstem respiratory network remains only partially described. We injected the retrograde tracer Cholera toxin subunit B (CT-B) in the pontine Kölliker-Fuse nucleus (KFn, n = 5), medullary Bötzinger (BötC, n = 3) and pre-Bötzinger complexes (pre-BötC; n = 3), and the caudal raphé nuclei (n = 3), and quantified the descending connectivity of the PAG targeting these brainstem respiratory regions. CT-B injections in the KFn, pre-BötC, and caudal raphé, but not in the BötC, resulted in CT-B-labeled neurons that were predominantly located in the lateral and ventrolateral PAG columns. In turn, CT-B injections in the lateral and ventrolateral PAG columns (n = 4) produced the highest numbers of CT-B-labeled neurons in the KFn and far fewer numbers of labeled neurons in the pre-BötC, BötC, and caudal raphé. Analysis of the relative projection strength revealed that the KFn shares the densest reciprocal connectivity with the PAG (ventrolateral and lateral columns, in particular). Overall, our data imply that the PAG may engage a distributed respiratory rhythm and pattern generating network beyond the nucleus retroambiguus to mediate downstream modulation of breathing. However, the reciprocal connectivity of the KFn and PAG suggests specific roles for synaptic interaction between these two nuclei that are most likely related to the regulation of upper airway patency during vocalization or other volitional orofacial behaviors.

Treatment with chemical coagulants at different dosing levels changes ecotoxicity of stormwater from the Tahoe basin, California, USA.

In recent decades, the transport of stormwater-associated fine particles and phosphorus into Lake Tahoe has led to decreased water clarity and related ecological changes. Polyaluminum chloride coagulants (PACs) have shown great promise in removing these constituents from stormwater before it enters the lake. However, the potential risks of coagulant treatment to aquatic organisms are not well understood. This study investigated stormwater and coagulant toxicity under non-dosed, optimally-dosed, and over-dosed conditions using the US EPA 3-species test through growth of green algae (Selenastrum capricornutum), zooplankton (waterflea, Ceriodaphnia dubia) mortality and reproduction, and larval fish (fathead minnow, Pimephales promelas) mortality and biomass. Stormwater samples were collected during a 2005 spring snowmelt runoff event from three sites representing various forms of developed regions around Lake Tahoe. Samples were dosed with two different coagulants (a chitosan and a PAC) at levels optimized with a streaming current detector (SCD). Non-treated highway runoff was toxic to zooplankton and fish. Optimal coagulant dosing increased algal growth and reduced zooplankton toxicity. Overdosing at two and three times the optimal level of a PAC decreased zooplankton reproduction and increased fish mortality. PAC-related toxicity was correlated with increasing total unfiltered aluminum and decreasing alkalinity, pH, and DOC. Because of toxicity risks, we recommend keeping PAC coagulant dosing at or below optimal levels in practice.

Relaxin-3 receptor (RXFP3) activation in the nucleus of the solitary tract modulates respiratory rate and the arterial chemoreceptor reflex in rat.

The neuropeptide relaxin-3 is expressed by the pontine nucleus incertus. Relaxin-3 and synthetic agonist peptides modulate arousal and cognitive processes via activation of the relaxin-family peptide 3 receptor (RXFP3). Despite the presence of RXFP3 in the nucleus of the solitary tract (NTS), the ability of RXFP3 to modulate NTS-mediated cardiorespiratory functions has not been explored. Therefore, we examined the effects of bilateral microinjections of the selective agonist, RXFP3-A2 (40 µM, 100 nL/side), into the NTS in perfused working-heart-brainstem-preparations from rats (n = 6), while recording phrenic, vagal, and thoracic sympathetic chain activity (PNA, VNA, t-SCA) and heart rate (HR). RXFP3-A2 significantly increased respiratory rate and shortened post-inspiratory VNA. RXFP3-A2 in the NTS also significantly enhanced arterial chemoreceptor reflex (a-CR)-mediated tachypnea. However, RXFP3-A2 had no significant effect on HR and t-SCA at baseline or during the a-CR. These data represent the first evidence that RXFP3 activation in the NTS can selectively modulate respiration at baseline and during reflex behaviour.

Coping with hypoxemia: Could erythropoietin (EPO) be an adjuvant treatment of COVID-19?

A very recent epidemiological study provides preliminary evidence that living in habitats located at 2500 m above sea level (masl) might protect from the development of severe respiratory symptoms following infection with the novel SARS-CoV-2 virus. This epidemiological finding raises the question of whether physiological mechanisms underlying the acclimatization to high altitude identifies therapeutic targets for the effective treatment of severe acute respiratory syndrome pivotal to the reduction of global mortality during the COVID-19 pandemic. This article compares the symptoms of acute mountain sickness (AMS) with those of SARS-CoV-2 infection and explores overlapping patho-physiological mechanisms of the respiratory system including impaired oxygen transport, pulmonary gas exchange and brainstem circuits controlling respiration. In this context, we also discuss the potential impact of SARS-CoV-2 infection on oxygen sensing in the carotid body. Finally, since erythropoietin (EPO) is an effective prophylactic treatment for AMS, this article reviews the potential benefits of implementing FDA-approved erythropoietin-based (EPO) drug therapies to counteract a variety of acute respiratory and non-respiratory (e.g. excessive inflammation of vascular beds) symptoms of SARS-CoV-2 infection.

Is a predominant left-to-right shunt associated with migraine?: A prospective atrial septal defect closure study.

BACKGROUND: A right-to-left shunt, as seen in patients with a patent foramen ovale, seems to be associated with migraine. An atrial septal defect (ASD), however, is characterized by a predominant left-to-right shunt (LRS). We prospectively evaluated the effect of percutaneous ASD closure on migraine METHODS: All 70 consecutive patients (>16 years) who underwent a percutaneous ASD closure between November 2003 and December 2005 in one of the two participating centers were included in the study. On the basis of standardized headache questionnaire, two independent neurologists diagnosed migraine with or without aura (MA+ and MA-, respectively) according to the International Headache Society criteria, before, 6 and 12 months after closure. RESULTS: Sixty-eight patients (97%; mean age 47.3 + or - 16.4 years; 22% men) agreed to participate in the study and completed the questionnaire. Before ASD closure, the overall prevalence of migraine was 34%, MA+ 22% and MA- 12%. At 6 months follow-up, the headache questionnaire was completed by 63 patients (93%) and the prevalence of overall migraine decreased to 19%, MA+ to 8% and MA- to 11% (Mc Nemar test, P = 0.08, P = 0.07, and P = 1.0, respectively). At 12 months, the prevalence of migraine decreased further to 12%, MA+ to 5% and MA- to 7% (McNemar test, P = 0.003, P = 0.04, and P = 0.29 versus at inclusion, respectively) based on a completed headache questionnaire of 57 patients (84%). CONCLUSION: We found a high prevalence of migraine in patients with an ASD, and observed prospectively a reduction in the occurrence of migraine, especially migraine with aura, 1 year after percutaneous closure.

Review of health safety aspects of nanotechnologies in food production.

Due to new, previously unknown, properties attributed to engineered nanoparticles many new products are introduced in the agro-food area. Nanotechnologies cover many aspects, such as disease treatment, food security, new materials for pathogen detection, packaging materials and delivery systems. As with most new and evolving technologies, potential benefits are emphasized, while little is known on safety of the application of nanotechnologies in the agro-food sector. This review gives an overview of scientific issues that need to be addressed with priority in order to improve the risk assessment for nanoparticles in food. The following research topics are considered to contribute pivotally to risk assessment of nanotechnologies and nanoparticles in food products. Set a definition for NPs to facilitate regulatory discussions, prioritization of research and exchange of study results. Develop analytical tools for the characterization of nanoparticles in complex biological matrices like food. Establish relevant dose metrics for nanoparticles used for both interpretation of scientific studies as well as regulatory frameworks. Search for deviant behavior (kinetics) and novel effects (toxicity) of nanoparticles and assess the validity of currently used test systems following oral exposure. Estimate the consumer exposure to nanoparticles.

Potential toxicity concerns from chemical coagulation treatment of stormwater in the Tahoe basin, California, USA.

Coagulant dosing of stormwater runoff with polyaluminum chlorides (PACs) is used in numerous waterbodies to improve water clarity, but the potential risks of PACs to aquatic organisms in Lake Tahoe, California are not fully understood. To assess these risks, the USEPA 3-species toxicity test and a non-standard fish test using Japanese medaka (Oryzias latipes) were used to determine the toxicity of PAC-treated and non-treated stormwater samples to aquatic species. Stormwater samples were collected from three sites representing runoff from different urbanized areas in May 2004; samples received coagulant dosing using three different coagulants (JC1720, PAX-XL9, Sumalchlor50) at levels optimized with jar testing. Raw stormwaters were toxic to algae and fathead minnows (mortality). Treatment with coagulants increased toxicity to zooplankton (reproduction) and had no consistent effects on the other toxicity metrics.