Safety, pharmacokinetics, and pharmacodynamics of LBP-EC01, a CRISPR-Cas3-enhanced bacteriophage cocktail, in uncomplicated urinary tract infections due to Escherichia coli (ELIMINATE): the randomised, open-label, first part of a two-part phase 2 trial.

BACKGROUND: The rate of antibiotic resistance continues to grow, outpacing small-molecule-drug development efforts. Novel therapies are needed to combat this growing threat, particularly for the treatment of urinary tract infections (UTIs), which are one of the largest contributors to antibiotic use and associated antibiotic resistance. LBP-EC01 is a novel, genetically enhanced, six-bacteriophage cocktail developed by Locus Biosciences (Morrisville, NC, USA) to address UTIs caused by Escherichia coli, regardless of antibiotic resistance status. In this first part of the two-part phase 2 ELIMINATE trial, we aimed to define a dosing regimen of LBP-EC01 for the treatment of uncomplicated UTIs that could advance to the second, randomised, controlled, double-blinded portion of the study. METHODS: This first part of ELIMINATE is a randomised, uncontrolled, open-label, phase 2 trial that took place in six private clinical sites in the USA. Eligible participants were female by self-identification, aged between 18 years and 70 years, and had an uncomplicated UTI at the time of enrolment, as well as a history of at least one drug-resistant UTI caused by E coli within the 12 months before enrolment. Participants were initially randomised in a 1:1:1 ratio into three treatment groups, but this part of the trial was terminated on the recommendation of the safety review committee after a non-serious tolerability signal was observed based on systemic drug exposure. A protocol update was then implemented, comprised of three new treatment groups. Groups A to C were dosed with intraurethral 2 × 1012 plaque-forming units (PFU) of LBP-EC01 on days 1 and 2 by catheter, plus one of three intravenous doses daily on days 1-3 of LBP-EC01 (1 mL of 1 × 1010 PFU intravenous bolus in group A, 1 mL of 1 × 109 PFU intravenous bolus in group B, and a 2 h 1 × 1011 PFU intravenous infusion in 100 mL of sodium lactate solution in group C). In all groups, oral trimethoprim-sulfamethoxazole (TMP-SMX; 160 mg and 800 mg) was given twice daily on days 1-3. The primary outcome was the level of LBP-EC01 in urine and blood across the treatment period and over 48 h after the last dose and was assessed in patients in the intention-to-treat (ITT) population who received at least one dose of LBP-EC01 and had concentration-time data available throughout the days 1-3 dosing period (pharmacokinetic population). Safety, a secondary endpoint, was assessed in enrolled patients who received at least one dose of study drug (safety population). As exploratory pharmacodynamic endpoints, we assessed E coli levels in urine and clinical symptoms of UTI in patients with at least 1·0 × 105 colony-forming units per mL E coli in urine at baseline who took at least one dose of study drug and completed their day 10 test-of-cure assessment (pharmacodynamic-evaluable population). This trial is registered with ClinicalTrials.gov, NCT05488340, and is ongoing. FINDINGS: Between Aug 22, 2022, and Aug 28, 2023, 44 patients were screened for eligibility, and 39 were randomly assigned (ITT population). Initially, eight participants were assigned to the first three groups. After the protocol was updated, 31 participants were allocated into groups A (11 patients), B (ten patients), and C (ten patients). One patient in group C withdrew consent on day 2 for personal reasons, but as she had received the first dose of the study drug was included in the modified ITT population. Maximum urine drug concentrations were consistent across intraurethral dosing, with a maximum mean concentration of 6·3 × 108 PFU per mL (geometric mean 8·8 log10 PFU per mL and geometric SD [gSD] 0·3). Blood plasma level of bacteriophages was intravenous dose-dependent, with maximum mean concentrations of 4·0 × 103 (geometric mean 3·6 log10 PFU per mL [gSD 1·5]) in group A, 2·5 × 103 (3·4 log10 PFU per mL [1·7]) in group B, and 8·0 × 105 (5·9 log10 PFU per mL [1·4]) in group C. No serious adverse events were observed. 44 adverse events were reported across 18 (46%) of the 39 participants in the safety population, with more adverse events seen with higher intravenous doses. Three patients in groups 1 to 3 and one patient in group C, all of whom received 1 × 1011 LBP-EC01 intravenously, had non-serious tachycardia and afebrile chills after the second intravenous dose. A rapid reduction of E coli in urine was observed by 4 h after the first treatment and maintained at day 10 in all 16 evaluable patients; these individuals had complete resolution of UTI symptoms by day 10. INTERPRETATION: A regimen consisting of 2 days of intraurethral LBP-EC01 and 3 days of concurrent intravenous LBP-EC01 (1 × 1010 PFU) and oral TMP-SMX twice a day was well tolerated, with consistent pharmacokinetic profiles in urine and blood. LBP-EC01 and TMP-SMX dosing resulted in a rapid and durable reduction of E coli, with corresponding elimination of clinical symptoms in evaluable patients. LBP-EC01 holds promise in providing an alternative therapy for uncomplicated UTIs, with further testing of the group A dosing regimen planned in the controlled, double-blind, second part of ELIMINATE. FUNDING: Federal funds from the US Department of Health and Human Services, Administration for Strategic Preparedness and Response, and Biomedical Advanced Research and Development Authority (BARDA).

Ketogenic diet enhances the effects of oxycodone in mice.

Opioids have been used to manage pain for thousands of years, but they have significant potential for abuse. Prescription opioids, like oxycodone, are associated with 32% of overdoses, that have reached a total of 75,673 deaths in 2021. A major challenge is maximizing their therapeutic potential while minimizing the negative side effects including opioid use disorder (OUD). The Ketogenic Diet (KD) has been reported to reduce pain and decrease the severity of alcohol use disorder, yet its effects on oxycodone responses remain unknown. KD mice displayed increased oxycodone-induced locomotor activity and enhanced antinociceptive effects of oxycodone, suggesting a dietary effect on opiate sensitivity. Male KD mice exposed to chronic oxycodone exhibited increased naloxone-induced jumps, suggesting a sex-specific effect of diet on opioid withdrawal. Consistent with this, male KD mice self-administered less oxycodone while female KD mice did not differ from controls. Finally, no effect of KD on motivation to obtain oxycodone was observed during a progressive ratio schedule. These data suggest sex-biased effects of KD on responses to opioids that should be considered and potentially leveraged in both clinical pain management and treatment of OUD.

Supermode noise mitigation and repetition rate control in harmonic mode-locked fiber laser implemented through the pulse train interaction with co-lased CW radiation.

We report on new, to the best of our knowledge, techniques enabling both the mitigation of supermode laser noise and highly precise setting of the pulse repetition rate (PRR) in a soliton harmonically mode-locked (HML) fiber laser employing nonlinear polarization evolution (NPE). The principle of operation relies on resonant interaction between the soliton pulses and a narrowband continuous wave (CW) component cooperatively generated within the same laser cavity. In contrast to our recent findings [Opt. Lett.46, 5747 (2021)10.1364/OL.441630 and Opt. Lett.46, 5687 (2021)10.1364/OL.443042], the new methods are implemented through the specific adjustment of the HML laser cavity only and do not require the use of an external tunable CW laser source.

Supermode noise mitigation and repetition rate control in a harmonic mode-locked fiber laser implemented through the pulse train interaction with co-lased CW radiation: publisher's note.

This publisher's note contains corrections to Opt. Lett.47, 5236 (2022)10.1364/OL.472780.

Brillouin-like amplification in rare-earth-doped optical fibers.

We present a theoretical formalism to describe the amplification of two monochromatic waves counter-propagating in a rare-earth-doped optical fiber amplifier. Interaction of the waves through a dynamical population inversion grating inscribed in the active fiber by the waves during their amplification results in a strong power transfer from one wave to another providing a preferable amplification of one wave at the expense of another. In this sense, the effect is similar to stimulated Brillouin scattering and is expected to be observed with both pumped and unpumped rare-earth-doped fibers possessing a finite polarizability difference between the excited and ground states.

Patrolling the boundaries of social domains: Neural activations to violations of expectations for romantic and work relationships.

According to the social domains hypothesis, we reduce the information-processing demands of complex social cues by classifying them into a limited number of domains, each with distinct sets of expectations. This requires rapid identification of violations of the boundaries between social domains. We hypothesized that these violations are likely to be associated with neural activation of the salience system. Using fMRI we compared responses of 20 adults to expected and unexpected everyday social scenarios in personal and work interactions. The vignettes exemplified different kinds of scenarios presented in the work setting, i.e., task-focused scenarios which are expected at work and scenarios with a personal focus, which are unexpected at work. The key contrast between task and personal focussed scenarios presented in the work setting was associated with fronto-insular activation. Perceived inappropriateness of the unexpected scenarios, and shorter response time to judgment of inappropriateness were also associated with fronto-insular activation, after controlling for unpleasantness. This study indicates specific neural responses to violations of expectations in different social situations. Our findings suggest that the fronto-insular region is implicated in rapid detection of behaviors that cross the boundaries of social domains, which are hypothesized to be necessary for efficient social information processing.

Seeded optical parametric generation in CdSiP2 pumped by a Raman fiber amplifier at 1.24 µm.

We report a seeded optical parametric generator (OPG) producing tunable radiation from 4.2-4.6 µm. The seeded OPG employs a 13 mm long CdSiP2 (CSP) crystal cut for non-critical phase-matching, pumped by a nanosecond-pulsed, MHz repetition rate Raman fiber amplifier system at 1.24 µm. A filtered, continuous-wave fiber supercontinuum source at 1.72 µm is used as the seed. The source generates up to 0.25 W of mid-infrared (MIR) idler power with a total pump conversion of 42% (combined signal and idler).

H3K9 Promotes Under-Replication of Pericentromeric Heterochromatin in Drosophila Salivary Gland Polytene Chromosomes.

Chromatin structure and its organization contributes to the proper regulation and timing of DNA replication. Yet, the precise mechanism by which chromatin contributes to DNA replication remains incompletely understood. This is particularly true for cell types that rely on polyploidization as a developmental strategy for growth and high biosynthetic capacity. During Drosophila larval development, cells of the salivary gland undergo endoreplication, repetitive rounds of DNA synthesis without intervening cell division, resulting in ploidy values of ~1350C. S phase of these endocycles displays a reproducible pattern of early and late replicating regions of the genome resulting from the activity of the same replication initiation factors that are used in diploid cells. However, unlike diploid cells, the latest replicating regions of polyploid salivary gland genomes, composed primarily of pericentric heterochromatic enriched in H3K9 methylation, are not replicated each endocycle, resulting in under-replicated domains with reduced ploidy. Here, we employ a histone gene replacement strategy in Drosophila to demonstrate that mutation of a histone residue important for heterochromatin organization and function (H3K9) but not mutation of a histone residue important for euchromatin function (H4K16), disrupts proper endoreplication in Drosophila salivary gland polyploid genomes thereby leading to DNA copy gain in pericentric heterochromatin. These findings reveal that H3K9 is necessary for normal levels of under-replication of pericentric heterochromatin and suggest that under-replication at pericentric heterochromatin is mediated through H3K9 methylation.

Nanosecond pulsed 620 nm source by frequency-doubling a phosphosilicate Raman fiber amplifier.

We demonstrate a nanosecond pulsed source at 620 nm with watt-level average power by frequency-doubling a 1240 nm phosphosilicate Raman fiber amplifier. A gain-switched laser diode operating at 1064 nm is amplified in an ytterbium fiber master oscillator power amplifier system and then converted to 1240 nm using a phosphosilicate Raman fiber amplifier with a conversion efficiency of up to 66%. The Raman fiber amplifier is seeded with a continuous-wave 1240 nm laser diode to obtain narrow-linewidth radiation, which is subsequently frequency-doubled in a periodically poled lithium tantalate crystal. A maximum average power of 1.5 W is generated at 620 nm, corresponding to a pulse energy of 300 nJ at a repetition rate of 5 MHz. The source has excellent beam quality (M2≤1.16) and an optical efficiency (1064 nm to 620 nm) of 20%, demonstrating an effective architecture for generating red pulsed light for biomedical imaging applications.

Chromatin conformation and transcriptional activity are permissive regulators of DNA replication initiation in Drosophila.

Chromatin structure has emerged as a key contributor to spatial and temporal control over the initiation of DNA replication. However, despite genome-wide correlations between early replication of gene-rich, accessible euchromatin and late replication of gene-poor, inaccessible heterochromatin, a causal relationship between chromatin structure and replication initiation remains elusive. Here, we combined histone gene engineering and whole-genome sequencing in Drosophila to determine how perturbing chromatin structure affects replication initiation. We found that most pericentric heterochromatin remains late replicating in H3K9R mutants, even though H3K9R pericentric heterochromatin is depleted of HP1a, more accessible, and transcriptionally active. These data indicate that HP1a loss, increased chromatin accessibility, and elevated transcription do not result in early replication of heterochromatin. Nevertheless, a small amount of pericentric heterochromatin with increased accessibility replicates earlier in H3K9R mutants. Transcription is de-repressed in these regions of advanced replication but not in those regions of the H3K9R mutant genome that replicate later, suggesting that transcriptional repression may contribute to late replication. We also explored relationships among chromatin, transcription, and replication in euchromatin by analyzing H4K16R mutants. In Drosophila, the X Chromosome gene expression is up-regulated twofold and replicates earlier in XY males than it does in XX females. We found that H4K16R mutation prevents normal male development and abrogates hyperexpression and earlier replication of the male X, consistent with previously established genome-wide correlations between transcription and early replication. In contrast, H4K16R females are viable and fertile, indicating that H4K16 modification is dispensable for genome replication and gene expression.

An Animal Model for Genetic Analysis of Multi-Gene Families: Cloning and Transgenesis of Large Tandemly Repeated Histone Gene Clusters.

Histone post-translational modifications (PTMs) are thought to participate in a range of essential molecular and cellular processes, including gene expression, replication, and nuclear organization. Importantly, histone PTMs are also thought to be prime candidates for carriers of epigenetic information across cell cycles and generations. However, directly testing the necessity of histone PTMs themselves in these processes by mutagenesis has been extremely difficult to carry out because of the highly repetitive nature of histone genes in animal genomes. We developed a transgenic system to generate Drosophila melanogaster genotypes in which the entire complement of replication-dependent histone genes is mutant at a residue of interest. We built a BAC vector containing a visible marker for lineage tracking along with the capacity to clone large (60-100 kb) inserts that subsequently can be site-specifically integrated into the D. melanogaster genome. We demonstrate that artificial tandem arrays of the core 5 kb replication-dependent histone repeat can be generated with relative ease. This genetic platform represents the first histone replacement system to leverage a single tandem transgenic insertion for facile genetics and analysis of molecular and cellular phenotypes. We demonstrate the utility of our system for directly preventing histone residues from being modified, and studying the consequent phenotypes. This system can be generalized to the cloning and transgenic insertion of any tandemly repeated sequence of biological interest.

Highly efficient nanosecond 560 nm source by SHG of a combined Yb-Raman fiber amplifier.

We demonstrate a nanosecond 560 nm pulse source based on frequency-doubling the output of a combined Yb-Raman fiber amplifier, achieving a pulse energy of 2.0 µJ with a conversion efficiency of 32% from the 976 nm pump light. By introducing a continuous-wave 1120 nm signal before the cladding pumped amplifier of a pulsed Yb:fiber master oscillator power amplifier system operating at 1064 nm, efficient conversion to 1120 nm occurs within the fiber amplifier due to stimulated Raman scattering. The output of the combined Yb-Raman amplifier is frequency-doubled to 560 nm using a periodically poled lithium tantalate crystal with a conversion efficiency of 47%, resulting in an average power of 3.0 W at a repetition rate of 1.5 MHz. The 560 nm pulse duration of 1.7 ns and the near diffraction-limited beam quality (M2≤1.18) make this source ideally suited to biomedical imaging applications such as optical-resolution photoacoustic microscopy and stimulated emission depletion microscopy.

Functional Redundancy of Variant and Canonical Histone H3 Lysine 9 Modification in Drosophila.

Histone post-translational modifications (PTMs) and differential incorporation of variant and canonical histones into chromatin are central modes of epigenetic regulation. Despite similar protein sequences, histone variants are enriched for different suites of PTMs compared to their canonical counterparts. For example, variant histone H3.3 occurs primarily in transcribed regions and is enriched for "active" histone PTMs like Lys9 acetylation (H3.3K9ac), whereas the canonical histone H3 is enriched for Lys9 methylation (H3K9me), which is found in transcriptionally silent heterochromatin. To determine the functions of K9 modification on variant vs. canonical H3, we compared the phenotypes caused by engineering H3.3K9R and H3K9R mutant genotypes in Drosophila melanogaster Whereas most H3.3K9R , and a small number of H3K9R , mutant animals are capable of completing development and do not have substantially altered protein-coding transcriptomes, all H3.3K9R H3K9R combined mutants die soon after embryogenesis and display decreased expression of genes enriched for K9ac. These data suggest that the role of K9ac in gene activation during development can be provided by either H3 or H3.3. Conversely, we found that H3.3K9 is methylated at telomeric transposons and that this mark contributes to repressive chromatin architecture, supporting a role for H3.3 in heterochromatin that is distinct from that of H3. Thus, our genetic and molecular analyses demonstrate that K9 modification of variant and canonical H3 have overlapping roles in development and transcriptional regulation, though to differing extents in euchromatin and heterochromatin.

Quantification of regional aerosol deposition patterns as a function of aerodynamic particle size in rhesus macaques using PET/CT imaging.

Aerosol aerodynamic particle size is known to affect deposition patterns of inhaled aerosol particles, as well as the virulence of inhaled bioaerosol particles. While a significant amount of work has been performed to describe the deposition of aerosol particles in the human respiratory tract, only a limited amount of work has been performed to describe the deposition of aerosol particles in the respiratory tract of nonhuman primates, an animal model commonly utilized in pharmacological and toxicological studies, especially in the biodefense field. In this study, anesthetized rhesus macaques inhaled radiolabeled aerosols with MMADs of 1.7, 3.6, 7.4 and 11.8 µm to characterize regional deposition patterns. The results demonstrate that the regional deposition pattern shifts as particle size increases, with greater deposition in more proximal regions of the respiratory tract and decreased deposition in the pulmonary region. The results of this study extend the findings of previous studies which demonstrated a similar shift in the deposition pattern as a function of particle size by providing greater resolution of deposition patterns. These data on regional deposition patterns provide a starting point to begin to explore potential mechanisms responsible for the differences in virulence of infectious bioaerosols as a function of particle size and deposition pattern reported in previous studies. Additionally, the data are useful to assess the performance of various deposition models that have been published in the literature.

Age-related delay in visual and auditory evoked responses is mediated by white- and grey-matter differences.

Slowing is a common feature of ageing, yet a direct relationship between neural slowing and brain atrophy is yet to be established in healthy humans. We combine magnetoencephalographic (MEG) measures of neural processing speed with magnetic resonance imaging (MRI) measures of white and grey matter in a large population-derived cohort to investigate the relationship between age-related structural differences and visual evoked field (VEF) and auditory evoked field (AEF) delay across two different tasks. Here we use a novel technique to show that VEFs exhibit a constant delay, whereas AEFs exhibit delay that accumulates over time. White-matter (WM) microstructure in the optic radiation partially mediates visual delay, suggesting increased transmission time, whereas grey matter (GM) in auditory cortex partially mediates auditory delay, suggesting less efficient local processing. Our results demonstrate that age has dissociable effects on neural processing speed, and that these effects relate to different types of brain atrophy.

High average power parametric wavelength conversion at 3.31-3.48 m in MgO:PPLN.

We present results of high average power mid-infrared (mid-IR) generation employing synchronized nanosecond pulsed ytterbium and erbium fiber amplifier systems using periodically poled lithium niobate. We generate greater than 6 W of mid-IR radiation tunable in wavelength between 3.31-3.48 µm, at power conversion efficiencies exceeding 75%, with near diffraction limited beam quality (M2 = 1.4). Numerical modeling is used to verify the experimental results in differing pump depletion regimes.

Direct interrogation of the role of H3K9 in metazoan heterochromatin function.

A defining feature of heterochromatin is methylation of Lys9 of histone H3 (H3K9me), a binding site for heterochromatin protein 1 (HP1). Although H3K9 methyltransferases and HP1 are necessary for proper heterochromatin structure, the specific contribution of H3K9 to heterochromatin function and animal development is unknown. Using our recently developed platform to engineer histone genes in Drosophila, we generated H3K9R mutant flies, separating the functions of H3K9 and nonhistone substrates of H3K9 methyltransferases. Nucleosome occupancy and HP1a binding at pericentromeric heterochromatin are markedly decreased in H3K9R mutants. Despite these changes in chromosome architecture, a small percentage of H3K9R mutants complete development. Consistent with this result, expression of most protein-coding genes, including those within heterochromatin, is similar between H3K9R and controls. In contrast, H3K9R mutants exhibit increased open chromatin and transcription from piRNA clusters and transposons, resulting in transposon mobilization. Hence, transposon silencing is a major developmental function of H3K9.

Highly efficient mid-infrared difference-frequency generation using synchronously pulsed fiber lasers.

We report the development of a high average power, picosecond-pulse, mid-infrared source based on difference-frequency generation (DFG) of two synchronous master oscillator power fiber amplifier systems. The generated idler can be tuned over the range 3.28-3.45 µm delivering greater than 3.4 W of average power, with a maximum pump to total DFG power conversion efficiency of 78%. The benefits of a synchronously pumped scheme, compared to CW seeding of DFG sources, are discussed.