Synthesis of ionizable lipopolymers using split-Ugi reaction for pulmonary delivery of various size RNAs and gene editing.

We present an efficient approach for synthesizing cationic poly(ethylene imine) derivatives using the multicomponent split-Ugi reaction to rapidly create a library of complex functional ionizable lipopolymers. We synthesized a diverse library of 155 polymers, formulated them into polyplexes to establish structure-activity relationships crucial for endosomal escape and efficient transfection. After discovering a lead structure, lipopolymer-lipid hybrid nanoparticles are introduced to preferentially deliver to and elicit effective mRNA transfection in lung endothelium and immune cells, including T cells with low in vivo toxicity. The lipopolymer-lipid hybrid nanoparticles showed 300-fold improvement in systemic mRNA delivery to the lung compared to in vivo -JetPEI ® . Lipopolymer-lipid hybrid nanoparticles demonstrated efficient delivery of mRNA-based therapeutics for treatment of two different disease models. Lewis Lung cancer progression was significantly delayed after treatment with loaded IL-12 mRNA in U155@lipids after repeated i.v. administration. Systemic delivery of human CFTR (hCFTR) mRNA resulted in production of functional form of CFTR protein in the lungs. The functionality of hCFTR protein was confirmed by restoration of CFTR- mediated chloride secretion in conductive airway epithelia in CFTR knockout mice after nasal instillation of hCFTR mRNA loaded U155@lipids. We further showed that, U155@lipids nanoparticles can deliver complex CRISPR-Cas9 based RNA cargo to the lung, achieving 5.6 ± 2.4 % gene editing in lung tissue. Moreover, we demonstrated successful PD-1 gene knockout of T cells in vivo . Our results highlight a versatile delivery platform for systemic delivering of mRNA of various sizes for gene therapy for a variety of therapeutics.

Nasal continuous positive airway pressure practices in preterm infants: A survey of neonatal providers.

BACKGROUND: The standard of care for respiratory support of preterm infants is nasal continuous positive airway pressure (CPAP), yet practices are not standardized. Our aim was to survey CPAP practices in infants < 32 weeks gestation among the American Academy of Pediatrics Neonatal-Perinatal section. METHODS: A US, web-based survey inquired about the initiation, management, and discontinuation of CPAP, and chinstrap use and oral feedings on CPAP. RESULTS: 857 providers consented. Regarding criteria to discontinue/wean CPAP: 69% use specific respiratory stability criteria; 22% a specific post-menstrual age; 8% responded other. 64% did not have guidelines for CPAP discontinuation; 54% did not have guidelines for CPAP initiation. 66% believe chinstraps improve CPAP efficacy; however, 11% routinely apply a chinstrap. 22% allow oral feeds on CPAP in certain circumstances. CONCLUSION: There are meaningful variabilities in CPAP practices among neonatal providers across the US. Given the potential long-term implications this can have on the growth and development of the preterm lung, further evidence-based research is needed in relation to respiratory outcomes to optimize and standardize CPAP strategies.

Inhaled mRNA therapy for treatment of cystic fibrosis: Interim results of a randomized, double-blind, placebo-controlled phase 1/2 clinical study.

BACKGROUND: MRT5005, a codon-optimized CFTR mRNA, delivered by aerosol in lipid nanoparticles, was designed as a genotype-agnostic treatment for CF lung disease. METHODS: This was a randomized, double-blind, placebo-controlled Phase 1/2 study performed in the US. Adults with 2 severe class I and/or II CFTR mutations and baseline ppFEV1 values between 50 and 90% were randomized 3:1 (MRT5005: placebo). Six dose levels of MRT5005 (4, 8, 12, 16, 20, and 24 mg) or placebo (0.9% Sodium Chloride) were administered by nebulization. The single ascending dose cohort was treated over a range from 8 to 24 mg; the multiple ascending dose cohort received five weekly doses (range 8-20 mg); and the daily dosing cohort received five daily doses (4 mg). RESULTS: A total of 42 subjects were assigned to MRT5005 [31] or placebo [11]. A total of 14 febrile reactions were observed in 10 MRT5005-treated participants, which were mild [3] or moderate [11] in severity; two subjects discontinued related to these events. Additionally, two MRT5005-treated patients experienced hypersensitivity reactions, which were managed conservatively. The most common treatment emergent adverse events were cough and headache. No consistent effects on FEV1 were noted. CONCLUSIONS: MRT5005 was generally safe and well tolerated through 28 days of follow-up after the last dose, though febrile and hypersensitivity reactions were noted. The majority of these reactions resolved within 1-2 days with supportive care allowing continued treatment with MRT5005 and careful monitoring. In this small first-in-human study, FEV1 remained stable after treatment, but no beneficial effects on FEV1 were observed.

Pharmacokinetics of the anticonvulsant levetiracetam in neonatal foals.

BACKGROUND: Seizures are a common manifestation of neurological disease in the neonatal foal and are an important cause of morbidity and mortality in this population. Current antiepileptic options are effective, but often have undesirable adverse effects, short duration of action and high cost. Levetiracetam has an ideal safety and pharmacokinetic profile in multiple species, including the adult horse, and may be a safe and cost-effective alternative anticonvulsant in neonatal foals. Due to differences in drug disposition and clearance dosages in neonates, dosing recommendations in other species or adult horses cannot be extrapolated to foals. OBJECTIVE: To establish the pharmacokinetic profile of single-dose i.v. and intragastric administration of levetiracetam in healthy neonatal foals. STUDY DESIGN: Randomised crossover experimental study. METHODS: Levetiracetam was administered as a single dose to six healthy foals (ages 1-10 days) at a dose of 32 mg/kg bwt i.v. or intragastrically. Plasma levetiracetam concentrations were measured using a validated HPLC protocol. RESULTS: After i.v. administration to healthy foals, levetiracetam had a mean (±s.d.) elimination half-life of 7.76 ± 0.51 h, a mean systemic clearance of 61.67 ± 10.96 (mL/h/kg) and a mean apparent volume of distribution at steady state of 0.670 ± 0.124 (L/kg). Following intragastric administration, levetiracetam had a peak concentration of 38.34 ± 7.42 mg/L and time to achieve peak concentration was 0.875 (0.5-1.5) h. Mean bioavailability for IG administration was excellent (103.04 ± 14.51%). No significant differences in pharmacokinetic variables between routes and order of administration were observed. MAIN LIMITATIONS: Small sample size and single-dose administration. CONCLUSIONS: Levetiracetam has excellent intragastric bioavailability in foals and is predicted to maintain plasma concentrations at or above the proposed target concentration with twice daily i.v. or oral administration. Once-daily administration may be possible in some foals based on the therapeutic range recommended in other species.

Aqueous cigarette smoke extract induces a voltage-dependent inhibition of CFTR expressed in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel inhabits the apical membrane of airway epithelia, where its function is essential for mucus hydration, mucociliary clearance, and airway defense. Chronic obstructive pulmonary disease (COPD), most often a consequence of cigarette smoke (CS) exposure, affects 15 million persons in the US. Clinically, COPD is characterized by many of the salient features of cystic fibrosis lung disease, where CFTR is either absent or reduced in function. CS is an acidic aerosol (pH 5.3 to 6.3) reported to contain over 4,000 constituents. Acute CS exposure has been reported to decrease airway transepithelial voltage in vivo and short-circuit current in vitro; however, the mechanistic basis of these effects is uncertain. The goal of the studies described here was to develop a bioassay to characterize the effects of aqueous CS preparations on the channel function of CFTR. We studied aqueous CS extract (CSE) prepared in our laboratory, as well as commercial cigarette smoke condensate (CSC) in Xenopus oocytes expressing human CFTR. Application of CSE at pH 5.3 produced a reversible, voltage-dependent inhibition of CFTR conductance. CSE neutralized to pH 7.3 produced less inhibition of CFTR conductance. Serial dilution of CSE revealed a dose-dependent effect at acidic and neutral pH. In contrast, CSC did not inhibit CFTR conductance in oocytes. We conclude that one or more components of CSE inhibits CFTR in a manner similar to diphenylamine-2-carboxylate, a negatively charged, open-channel blocker.

Intracellular correlates of fast (>200 Hz) electrical oscillations in rat somatosensory cortex.

Oscillatory activity in excess of several hundred hertz has been observed in somatosensory evoked potentials (SEP) recorded in both humans and animals and is attracting increasing interest regarding its role in brain function. Currently, however, little is known about the cellular events underlying these oscillations. The present study employed simultaneous in-vivo intracellular and epipial field-potential recording to investigate the cellular correlates of fast oscillations in rat somatosensory cortex evoked by vibrissa stimulation. Two distinct types of fast oscillations were observed, here termed "fast oscillations" (FO) (200-400 Hz) and "very fast oscillations" (VFO) (400-600 Hz). FO coincided with the earliest slow-wave components of the SEP whereas VFO typically were later and of smaller amplitude. Regular spiking (RS) cells exhibited vibrissa-evoked responses associated with one or both types of fast oscillations and consisted of combinations of spike and/or subthreshold events that, when superimposed across trials, clustered at latencies separated by successive cycles of FO or VFO activity, or a combination of both. Fast spiking (FS) cells responded to vibrissae stimulation with bursts of action potentials that closely approximated the periodicity of the surface VFO. No cells were encountered that produced action potential bursts related to FO activity in an analogous fashion. We propose that fast oscillations define preferred latencies for action potential generation in cortical RS cells, with VFO generated by inhibitory interneurons and FO reflecting both sequential and recurrent activity of stations in the cortical lamina.

Focal stimulation of the thalamic reticular nucleus induces focal gamma waves in cortex.

Electrical stimulation of the thalamic reticular nucleus (TRN; 0.5-s trains of 500-Hz 0.5-ms pulses at 5-10 microA) evokes focal oscillations of cortical electrical potentials in the gamma frequency band ( approximately 35-55 Hz). These evoked oscillations are specific to either the somatosensory or auditory cortex and to subregions of the cortical receptotopic map, depending on what part of the TRN is stimulated. Focal stimulation of the internal capsule, however, evokes focal slow potentials, without gamma activity. Our results suggest that the TRN's role extends beyond that of general cortical arousal to include specific modality and submodality activation of the forebrain.

Volume and pressure modes of mechanical ventilation in pediatric patients.

There are few controlled pediatric studies comparing the various modes of ventilation in terms of patient outcomes. Thus at this time the choice of ventilator mode depends largely on the apparatus available, the patient's disease state, and personal preference based on one's experience. The next generation of ventilators may well allow the use of the best of both modes, setting both pressure and volume minimums and maximums, safely meeting ventilation targets. Today's challenges are to become familiar with the various modes of ventilators available, understand the developing physiology of the lung and lung disease pathophysiology, and incorporate all this into proper ventilator strategies to prevent ventilator-induced lung injury.

Thalamic modulation of high-frequency oscillating potentials in auditory cortex.

Perhaps the most widely recognized but least understood electrophysiological activity of the cerebral cortex is its characteristic electrical oscillations. Recently, there have been efforts to understand the mechanisms underlying high-frequency gamma oscillations(approximately 40 Hz) because they may coordinate sensory processing between populations of cortical cells. High-resolution cortical recordings show the gamma oscillations are constrained to sensory cortex, that they occur independently in auditory and somatosensory cortex, and that they are phase-locked between primary and secondary sensory cortex. As yet, the mechanism of their neurogenesis is unknown. Whereas cortical neurons can produce gamma oscillations without subcortical input, they may also be modulated by the thalamus and basal forebrain. Here we report that the neural generator of gamma oscillations in auditory cortex seems to be intracortical, serving to synchronize interactions between the primary and secondary areas. The acoustic thalamus directly modulates these oscillations, which are inhibited by stimulation of the dorsal and ventral divisions of the medial geniculate nucleus (MGd and MGv) and evoked by stimulation of the adjacent posterior intralaminar nucleus (PIL).

Inter- and intra-hemispheric spatiotemporal organization of spontaneous electrocortical oscillations.

1. Two 64-channel epipial electrode arrays were positioned on homologous locations of the right and left hemisphere, covering most of primary and secondary auditory and somatosensory cortex in eight lightly anesthetized rats. Array placement was verified with the use of cytochrome oxidase histochemistry. 2. Middle-latency auditory and somatosensory evoked potentials (MAEPs and MSEPs, respectively) and spontaneous oscillations in the frequency range of 20-40 Hz (gamma oscillations) were recorded and found to be spatially constrained to regions of granular cortex, suggesting that both phenomena are closely associated with sensory information processing. 3. The MAEP and MSEP consisted of an initial biphasic sharp wave in primary auditory and somatosensory cortex, respectively, and a similar biphasic sharp wave occurred approximately 4-8 ms later in secondary sensory cortex of the given modality. Averaged gamma oscillations also revealed asynchronous activation of sensory cortex, but with a shorter 2-ms delay between oscillations in primary and secondary regions. Although the long latency shift of the MAEP and MSEP may be due in part to asynchronous activation of parallel thalamocortical projections to primary and secondary sensory cortex, the much shorter shift of gamma oscillations in a given modality is consistent with intracortical coupling of these regions. 4. Gamma oscillations occurred independently in auditory and somatosensory cortex within a given hemisphere. Furthermore, time series averaging revealed that there was no phase-locking of oscillations between the sensory modalities. 5. Gamma oscillations were loosely coupled between hemispheres; oscillations occurring in auditory or somatosensory cortex of one hemisphere were often associated with lower-amplitude oscillations in homologous contralateral sensory cortex. Yet, the fact that time series averaging revealed no interhemispheric phase-locking suggests that the corpus callosum may not coordinate the bilateral gamma oscillations, and that a thalamic modulatory influence may be involved.

High frequency (gamma-band) oscillating potentials in rat somatosensory and auditory cortex.

An 8 x 8 multichannel electrode array was used to record epipial field potentials, spontaneous gamma oscillations, and the interaction between single trial evoked potentials and ongoing gamma activity in rat somatosensory and auditory Cortex. Array placement over both these cortical regions was verified using cytochrome oxidase histochemistry. Replicating earlier findings, the epipial middle latency auditory and somatosensory evoked potentials (MAEP and MSEP, respectively) consisted of a stereotyped pattern of activation characterized by a spatially confined biphasic sharp wave followed by more diffuse slow wave components whose areal distribution adhered closely to established boundaries of primary and secondary sensory cortex. Spontaneous gamma activity, while exhibiting far more spatiotemporal variation, was also centered on primary and secondary sensory cortex and was significantly attenuated at intercalated dysgranular regions. A modality specificity of gamma activity was also demonstrated in the present study, where spindles occurred independently in auditory and somatosensory cortex. Furthermore, following presentation of a single click or vibrissal displacement, spontaneous gamma activity was suppressed and subsequently enhanced only in the modality stimulated. We conclude that in the lightly anesthetized rodent, spontaneous gamma oscillations are not a global neocortical phenomena, but are instead restricted to the same areas of sensory cortex participating in evoked potentials. However, unlike the MAEP and MSEP which are dominated by systematic activation of parallel thalamocortical projections, the marked spatiotemporal variability of gamma spindles suggests a more complex neurogenesis, probably including dominant contributions from intracortical neural circuitry.

The genetic location of three mutations impairing penicillin production in Aspergillus nidulans.

Three mutations impairing penicillin production in Aspergillus nidulans, npeB, npeC and npeD, have been located on linkage groups III, IV and II, respectively, and positioned relative to other loci on these chromosomes.

Co-synthesis of penicillin following treatment of mutants of Aspergillus nidulans impaired in antibiotic production with lytic enzymes.

Mycelia from four mutants of Aspergillus nidulans impaired in penicillin production at separate genetic loci were treated with an enzyme complex capable of lysine cell walls, then mixed in all possible paired combinations and grown in osmotically buffered penicillin production media, containing 2-deoxyglucose and an unrefined mixture of polyoxins to prevent cell wall regeneration. The culture filtrates were assayed after 6 d and significant penicillin yields were observed in four of the six possible combinations. None of these pairs produced penicillin when grown together as normal mycelium, suggesting that intermediates of the penicillin biosynthetic pathway unable to diffuse from untreated mycelium could do so from enzyme-treated mycelium when cell wall regeneration was inhibited. A general method is thus available for examining biochemical pathways with mutants accumulating intermediates unable to cross the cell wall barrier.

A hydroxamic acid from Aspergillus nidulans with antibiotic activity against Proteus species.

An iron-complexing antibiotic with a narrow spectrum of biological activity was produced by several strains of Aspergillus nidulans when grown in a low-iron, chemically defined medium. Its chemical and biological properties closely resembled those of desferritriacetylfusigen, a metabolite of several other Aspergilli and Penicillia.