Reactivation of Dormant Relay Pathways in Injured Spinal Cord by KCC2 Manipulations.

Many human spinal cord injuries are anatomically incomplete but exhibit complete paralysis. It is unknown why spared axons fail to mediate functional recovery in these cases. To investigate this, we undertook a small-molecule screen in mice with staggered bilateral hemisections in which the lumbar spinal cord is deprived of all direct brain-derived innervation, but dormant relay circuits remain. We discovered that a KCC2 agonist restored stepping ability, which could be mimicked by selective expression of KCC2, or hyperpolarizing DREADDs, in the inhibitory interneurons between and around the staggered spinal lesions. Mechanistically, these treatments transformed this injury-induced dysfunctional spinal circuit to a functional state, facilitating the relay of brain-derived commands toward the lumbar spinal cord. Thus, our results identify spinal inhibitory interneurons as a roadblock limiting the integration of descending inputs into relay circuits after injury and suggest KCC2 agonists as promising treatments for promoting functional recovery after spinal cord injury.

Deconstruction of Corticospinal Circuits for Goal-Directed Motor Skills.

Corticospinal neurons (CSNs) represent the direct cortical outputs to the spinal cord and play important roles in motor control across different species. However, their organizational principle remains unclear. By using a retrograde labeling system, we defined the requirement of CSNs in the execution of a skilled forelimb food-pellet retrieval task in mice. In vivo imaging of CSN activity during performance revealed the sequential activation of topographically ordered functional ensembles with moderate local mixing. Region-specific manipulations indicate that CSNs from caudal or rostral forelimb area control reaching or grasping, respectively, and both are required in the transitional pronation step. These region-specific CSNs terminate in different spinal levels and locations, therefore preferentially connecting with the premotor neurons of muscles engaged in different steps of the task. Together, our findings suggest that spatially defined groups of CSNs encode different movement modules, providing a logic for parallel-ordered corticospinal circuits to orchestrate multistep motor skills.

Harvesting and amplifying gene cassettes confers cross-resistance to critically important antibiotics.

Amikacin and piperacillin/tazobactam are frequent antibiotic choices to treat bloodstream infection, which is commonly fatal and most often caused by bacteria from the family Enterobacterales. Here we show that two gene cassettes located side-by-side in and ancestral integron similar to In37 have been "harvested" by insertion sequence IS26 as a transposon that is widely disseminated among the Enterobacterales. This transposon encodes the enzymes AAC(6')-Ib-cr and OXA-1, reported, respectively, as amikacin and piperacillin/tazobactam resistance mechanisms. However, by studying bloodstream infection isolates from 769 patients from three hospitals serving a population of 1.2 million people in South West England, we show that increased enzyme production due to mutation in an IS26/In37-derived hybrid promoter or, more commonly, increased transposon copy number is required to simultaneously remove these two key therapeutic options; in many cases leaving only the last-resort antibiotic, meropenem. These findings may help improve the accuracy of predicting piperacillin/tazobactam treatment failure, allowing stratification of patients to receive meropenem or piperacillin/tazobactam, which may improve outcome and slow the emergence of meropenem resistance.

Improving nitrofurantoin resistance prediction in Escherichia coli from whole-genome sequence by integrating NfsA/B enzyme assays.

Nitrofurantoin resistance in Escherichia coli is primarily caused by mutations damaging two enzymes, NfsA and NfsB. Studies based on small isolate collections with defined nitrofurantoin MICs have found significant random genetic drift in nfsA and nfsB, making it extremely difficult to predict nitrofurantoin resistance from whole-genome sequence (WGS) where both genes are not obviously disrupted by nonsense or frameshift mutations or insertional inactivation. Here, we report a WGS survey of 200 oqxAB-negative E. coli from community urine samples, of which 34 were nitrofurantoin resistant. We characterized individual non-synonymous mutations seen in nfsA and nfsB among this collection using complementation cloning and NfsA/B enzyme assays in cell extracts. We definitively identified R203C, H11Y, W212R, A112E, and A112T in NfsA and R121C, Q142H, F84S, P163H, W46R, K57E, and V191G in NfsB as amino acid substitutions that reduce enzyme activity sufficiently to cause resistance. In contrast, E58D, I117T, K141E, L157F, A172S, G187D, and A188V in NfsA and G66D, M75I, V93A, and A174E in NfsB are functionally silent in this context. We identified that 9/166 (5.4%) nitrofurantoin-susceptible isolates were "pre-resistant," defined as having loss of function mutations in nfsA or nfsB. Finally, using NfsA/B enzyme assays and proteomics, we demonstrated that 9/34 (26.5%) ribE wild-type nitrofurantoin-resistant isolates also carried functionally wild-type nfsB or nfsB/nfsA. In these cases, NfsA/B activity was reduced through downregulated gene expression. Our biological understanding of nitrofurantoin resistance is greatly improved by this analysis but is still insufficient to allow its reliable prediction from WGS data.

Randomized effectiveness-implementation trial of dialectical behavior therapy interventions for young people with borderline personality disorder symptoms.

OBJECTIVES: Dialectical behavior therapy (DBT) is an evidence-based treatment for people with emerging borderline personality disorder (BPD). In "real world" clinical settings, standard DBT is resource intensive. Emerging evidence suggests that group-based DBT skills training alone can lead to promising outcomes. This hybrid type 1 effectiveness-implementation trial directly compared the effectiveness of an 8-week group DBT-skills training program and a 16-week DBT-informed program including individual treatment and group-based skills training. METHODS: This pragmatic trial employed a staggered, parallel-groups design. We recruited 104 participants, aged 16-25 years, with emotion dysregulation or emerging BPD symptoms. Participants were randomized to receive either program at a youth mental health service located in the Gold Coast, Australia. Data was collected via online surveys at baseline, 8-week, 16-week, and 24-week follow-up. Mixed effect linear models compared groups on the primary outcomes of emotion dysregulation and BPD symptoms, and secondary outcomes of suicidal ideation, coping skills, depression, anxiety, and stress. RESULTS: Across groups there were significant and sustained improvements relating to emotion dysregulation, BPD symptoms, stress, depression, and emotion-focused coping; but not suicide risk, anxiety, or task-focused coping. There was no significant time by group differences between the 8-week and 16-week interventions on any primary or secondary outcome. CONCLUSION: The more intensive mode of delivering DBT was not more effective than the brief group-based skills training. Both interventions resulted in significant improvements across both primary and most secondary outcomes. These results have implications for clinical practice regarding length and intensity of DBT treatment in young people.

Zinc limitation triggers anticipatory adaptations in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) has complex and dynamic interactions with the human host, and subpopulations of Mtb that emerge during infection can influence disease outcomes. This study implicates zinc ion (Zn2+) availability as a likely driver of bacterial phenotypic heterogeneity in vivo. Zn2+ sequestration is part of "nutritional immunity", where the immune system limits micronutrients to control pathogen growth, but this defense mechanism seems to be ineffective in controlling Mtb infection. Nonetheless, Zn2+-limitation is an environmental cue sensed by Mtb, as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn2+-limited Mtb in vivo. Prolonged Zn2+ limitation leads to numerous physiological changes in vitro, including differential expression of certain antigens, alterations in lipid metabolism and distinct cell surface morphology. Furthermore, Mtb enduring limited Zn2+ employ defensive measures to fight oxidative stress, by increasing expression of proteins involved in DNA repair and antioxidant activity, including well described virulence factors KatG and AhpC, along with altered utilization of redox cofactors. Here, we propose a model in which prolonged Zn2+ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn2+-limited Mtb are more resistant to oxidative stress and exhibit increased survival and induce more severe pulmonary granulomas in mice. Considering that extracellular Mtb may transit through the Zn2+-limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn2+ availability likely plays a key role during early interactions with host cells.

Anti-inflammatory Quinoline Alkaloids from the Roots of Waltheria indica.

Sixteen new quinoline alkaloids (1a-7, 8a, 9, 10, 13-15, 17, and 21) and 10 known analogs (8b, 11, 12, 16, 18-20, and 22-24), along with three known cyclopeptide alkaloids (25-27), were isolated from the roots of Waltheria indica. The structures of the new compounds were elucidated by detailed NMR and circular dichroism with computational support and mass spectrometry data interpretation. Anti-inflammatory potential of isolates was evaluated based on inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production and tumor necrosis factor-alpha (TNF-α)-induced nuclear factor kappa B (NF-κB) activity with cell culture models. In the absence of cell growth inhibition, compounds 6, 8a, 9-11, 13, 21, and 24 reduced TNF-α-induced NF-κB activity with IC50 values ranging from 7.1 to 12.1 µM, comparable to the positive control (BAY 11-7082, IC50 = 9.7 µM). Compounds 6, 8a, 8b, and 11 showed significant NO-inhibitory activity with IC50 values ranging from 11.0 to 12.8 µM, being more active than the positive control (l-NMMA, IC50 = 22.7 µM). Structure-activity relationships indicated that NO inhibitory activity was significantly affected by C-8 substitution. Inhibition of LPS-induced nitric oxide synthase (iNOS) by 8b [(5S)-waltherione M, IC50 11.7 ± 0.8 µM] correlated with inhibition of iNOS mRNA expression. The biological potential of W. indica metabolites supports the traditional use of this plant for the treatment of inflammatory-related disorders.

New Nostocyclophanes from Nostoc linckia.

Six new nostocyclophanes and four known compounds have been isolated from Nostoc linckia (Nostocaceae) cyanobacterial strain UTEX B1932. The new compounds, nostocyclophanes E-J (1-6), were characterized by NMR and MS techniques. The known compounds were nostocyclophanes B-D, previously isolated from this strain, and dedichloronostocyclophane D. Structural modifications on the new [7.7]paracyclophane analogs 1-5, isolated from the 80% methanol fraction, range from simple changes such as the lack of methylation or halogenation to more unusual modifications such as those seen in nostocyclophane H (4), in which the exocyclic alkyl chains are of different length; this is the first time this modification has been observed in this family of natural products. In addition, nostocyclophane J (6) is a linear analog in which C-20 is chlorinated in preparation for the presumed enzymatic Friedel-Craft cyclization needed to form the final ring structure, analogous to the biosynthesis of the related cylindrocyclophanes. Nostocyclophane D, dedichloronostocyclophane D, and nostocyclophanes E-J demonstrated moderate to weak growth inhibition against MDA-MB-231 breast cancer cells.

Modelled-Microgravity Reduces Virulence Factor Production in Staphylococcus aureus through Downregulation of agr-Dependent Quorum Sensing.

Bacterial contamination during space missions is problematic for human health and damages filters and other vital support systems. Staphylococcus aureus is both a human commensal and an opportunistic pathogen that colonizes human tissues and causes acute and chronic infections. Virulence and colonization factors are positively and negatively regulated, respectively, by bacterial cell-to-cell communication (quorum sensing) via the agr (accessory gene regulator) system. When cultured under low-shear modelled microgravity conditions (LSMMG), S. aureus has been reported to maintain a colonization rather than a pathogenic phenotype. Here, we show that the modulation of agr expression via reduced production of autoinducing peptide (AIP) signal molecules was responsible for this behavior. In an LSMMG environment, the S. aureus strains JE2 (methicillin-resistant) and SH1000 (methicillin-sensitive) both exhibited reduced cytotoxicity towards the human leukemia monocytic cell line (THP-1) and increased fibronectin binding. Using S. aureus agrP3::lux reporter gene fusions and mass spectrometry to quantify the AIP concentrations, the activation of agr, which depends on the binding of AIP to the transcriptional regulator AgrC, was delayed in the strains with an intact autoinducible agr system. This was because AIP production was reduced under these growth conditions compared with the ground controls. Under LSMMG, S. aureus agrP3::lux reporter strains that cannot produce endogenous AIPs still responded to exogenous AIPs. Provision of exogenous AIPs to S. aureus USA300 during microgravity culture restored the cytotoxicity of culture supernatants for the THP-1 cells. These data suggest that microgravity does not affect AgrC-AIP interactions but more likely the generation of AIPs.

Flow Field-Flow Fractionation with a Thickness-Tapered Channel.

This study introduces the thickness-tapered channel design for flow field-flow fractionation (FlFFF) for the first time. In this design, the channel thickness linearly decreases along the channel axis such that the flow velocity increases down the channel. Channel thickness is an important variable for controlling retention time and resolution in field-flow fractionation. Especially, in the steric/hyperlayer mode of FlFFF, in which particles (>1 µm) migrate at elevated heights above the channel wall owing to hydrodynamic lift forces, the migration of long-retaining smaller-sized particles can be enhanced in a relatively thin channel or by increasing the migration flow rate; however, an upper size limit that can be resolved is simultaneously sacrificed. A thickness-tapered channel was constructed without a channel spacer by carving the surface of a channel block such that the channel inlet was deeper than the outlet (w = 400 → 200 µm). The performance of a thickness-tapered channel was evaluated using polystyrene standards and compared to that of a channel of uniform thickness (w = 300 µm) with a similar effective channel volume in terms of sample recovery, dynamic size range of separation, and steric transition under different flow rate conditions. The thickness-tapered channel can be an alternative to maintain the resolving power for particles with an upper large-diameter limit, faster separation of particles with a lower limit, and higher elution recovery without implementing the additional field-programming option.

Diagnostic MALDI-TOF MS can differentiate between high and low toxic Staphylococcus aureus bacteraemia isolates as a predictor of patient outcome.

Staphylococcus aureus bacteraemia (SAB) is a major cause of blood-stream infection (BSI) in both healthcare and community settings. While the underlying comorbidities of a patient significantly contributes to their susceptibility to and outcome following SAB, recent studies show the importance of the level of cytolytic toxin production by the infecting bacterium. In this study we demonstrate that this cytotoxicity can be determined directly from the diagnostic MALDI-TOF mass spectrum generated in a routine diagnostic laboratory. With further development this information could be used to guide the management and improve the outcomes for SAB patients.

Spirovetivane- and Eudesmane-Type Sesquiterpenoids Isolated from the Culture Media of Two Cyanobacterial Strains.

As part of a study examining polar metabolites produced by cyanobacterial strains, we examined media extracts of a Calothrix sp. (strain R-3-1) and a Scytonema sp. (strain U-3-3). The cell mass of each was separated from the media, and HP20 resin was added for adsorption of secreted metabolites, a relatively unexplored area of cyanobacterial chemistry. HPLC-UV-LCMS-guided isolation led to the discovery of seven sesquiterpenoid compounds with five new, one known, and one previously isolated as the methyl ester. Through a complement of 1D and 2D NMR spectroscopic techniques, the planar structures and relative configurations of the seven compounds were elucidated. Spironostoic acid (1), 11,12-didehydrospironostoic acid (2), and 12-hydroxy-2-oxo-11-epi-hinesol (4) are spirovetivane-type compounds from R-3-1, while stigolone (5), 11R,12-dihydroxystigolone (6), and 11S,12-dihydroxystigolone (7) are three eudesmane-type compounds from U-3-3. Circular dichroism was utilized to decipher the absolute configurations of new compounds 1, 2, 4, 5, 6, and 7. Due to the structural variety observed among the spirovetivane- and eudesmane-type compounds in the literature and often a lack of clarity in how determinations were made, computational spectra and model compounds were used to support the interpretation of ECD and NMR spectra. A straightforward process to determine the configuration of these systems is presented.

Coumarinolignans with Reactive Oxygen Species (ROS) and NF-κB Inhibitory Activities from the Roots of Waltheria indica.

Seven new coumarinolignans, walthindicins A-F (1a, 1b, 2-5, 7), along with five known analogs (6, 8-11), were isolated from the roots of Waltheria indica. The structures of the new compounds are determined by detailed nuclear magnetic resonance (NMR), circular dichroism (CD) with extensive computational support, and mass spectroscopic data interpretation. Compounds were tested for their antioxidant activity in Human Cervical Cancer cells (HeLa cells). Compounds 1a and 6 showed higher reactive oxygen species (ROS) inhibitory activity at 20 µg/mL when compared with other natural compound-based antioxidants such as ascorbic acid. Considering the role of ROS in nuclear-factor kappa B (NF-κB) activation, compounds 1a and 6 were evaluated for NF-κB inhibitory activity and showed a concentration-dependent inhibition in Human Embryonic Kidney 293 cells (Luc-HEK-293).

Trade-Offs between Antibacterial Resistance and Fitness Cost in the Production of Metallo-ß-Lactamases by Enteric Bacteria Manifest as Sporadic Emergence of Carbapenem Resistance in a Clinical Setting.

Meropenem is a clinically important antibacterial reserved for treatment of multiresistant infections. In meropenem-resistant bacteria of the family Enterobacterales, NDM-1 is considerably more common than IMP-1, despite both metallo-ß-lactamases (MBLs) hydrolyzing meropenem with almost identical kinetics. We show that blaNDM-1 consistently confers meropenem resistance in wild-type Enterobacterales, but blaIMP-1 does not. The reason is higher blaNDM-1 expression because of its stronger promoter. However, the cost of meropenem resistance is reduced fitness of blaNDM-1-positive Enterobacterales. In parallel, from a clinical case, we identified multiple Enterobacter spp. isolates carrying a plasmid-encoded blaNDM-1 having a modified promoter region. This modification lowered MBL production to a level associated with zero fitness cost, but, consequently, the isolates were not meropenem resistant. However, we identified a Klebsiella pneumoniae isolate from this same clinical case carrying the same blaNDM-1 plasmid. This isolate was meropenem resistant despite low-level NDM-1 production because of a ramR mutation reducing envelope permeability. Overall, therefore, we show how the resistance/fitness trade-off for MBL carriage can be resolved. The result is sporadic emergence of meropenem resistance in a clinical setting.

Uncoupling of neurogenesis and differentiation during retinal development.

Conventionally, neuronal development is regarded to follow a stereotypic sequence of neurogenesis, migration, and differentiation. We demonstrate that this notion is not a general principle of neuronal development by documenting the timing of mitosis in relation to multiple differentiation events for bipolar cells (BCs) in the zebrafish retina using in vivo imaging. We found that BC progenitors undergo terminal neurogenic divisions while in markedly disparate stages of neuronal differentiation. Remarkably, the differentiation state of individual BC progenitors at mitosis is not arbitrary but matches the differentiation state of post-mitotic BCs in their surround. By experimentally shifting the relative timing of progenitor division and differentiation, we provide evidence that neurogenesis and differentiation can occur independently of each other. We propose that the uncoupling of neurogenesis and differentiation could provide neurogenic programs with flexibility, while allowing for synchronous neuronal development within a continuously expanding cell pool.

Limited phylogenetic overlap between fluoroquinolone-resistant Escherichia coli isolated on dairy farms and those causing bacteriuria in humans living in the same geographical region.

BACKGROUND: Our primary aim was to test whether cattle-associated fluoroquinolone-resistant (FQ-R) Escherichia coli found on dairy farms are closely phylogenetically related to those causing bacteriuria in humans living in the same 50 × 50 km geographical region suggestive of farm-human sharing. Another aim was to identify risk factors for the presence of FQ-R E. coli on dairy farms. METHODS: FQ-R E. coli were isolated during 2017-18 from 42 dairy farms and from community urine samples. Forty-two cattle and 489 human urinary isolates were subjected to WGS, allowing phylogenetic comparisons. Risk factors were identified using a Bayesian regularization approach. RESULTS: Of 489 FQ-R human isolates, 255 were also third-generation-cephalosporin-resistant, with strong genetic linkage between aac(6')Ib-cr and blaCTX-M-15. We identified possible farm-human sharing for pairs of ST744 and ST162 isolates, but minimal core genome SNP distances were larger between farm-human pairs of ST744 and ST162 isolates (71 and 63 SNPs, respectively) than between pairs of isolates from different farms (7 and 3 SNPs, respectively). Total farm fluoroquinolone use showed a positive association with the odds of isolating FQ-R E. coli, while total dry cow therapy use showed a negative association. CONCLUSIONS: This work suggests that FQ-R E. coli found on dairy farms have a limited impact on community bacteriuria within the local human population. Reducing fluoroquinolone use may reduce the on-farm prevalence of FQ-R E. coli and this reduction may be greater when dry cow therapy is targeted to the ecology of resistant E. coli on the farm.

Droplet Microfluidic Optimisation Using Micropipette Characterisation of Bio-Instructive Polymeric Surfactants.

Droplet microfluidics can produce highly tailored microparticles whilst retaining monodispersity. However, these systems often require lengthy optimisation, commonly based on a trial-and-error approach, particularly when using bio-instructive, polymeric surfactants. Here, micropipette manipulation methods were used to optimise the concentration of bespoke polymeric surfactants to produce biodegradable (poly(d,l-lactic acid) (PDLLA)) microparticles with unique, bio-instructive surface chemistries. The effect of these three-dimensional surfactants on the interfacial tension of the system was analysed. It was determined that to provide adequate stabilisation, a low level (0.1% (w/v)) of poly(vinyl acetate-co-alcohol) (PVA) was required. Optimisation of the PVA concentration was informed by micropipette manipulation. As a result, successful, monodisperse particles were produced that maintained the desired bio-instructive surface chemistry.

Tolyporphins L-R: Unusual Tetrapyrroles from a Brasilonema sp. of Cyanobacterium.

Tolyporphins L-R (2-8) have been isolated from a mixed cyanobacterium-microbial culture. The structures of tolyporphins L and M have been revised to four constitutional isomers, isolated as two mixtures of dioxobacteriochlorins (2/3 and 4/5). In contrast, tolyporphin P (6) is a fully oxidized tetrapyrrole, while tolyporphins Q and R (7 and 8) are oxochlorins. X-ray structures are reported for the first time for tolyporphins A (1), R (8), and E (9), revealing unexpected stereochemical variation within the series.

6-Deoxy- and 11-Hydroxytolypodiols: Meroterpenoids from the Cyanobacterium HT-58-2.

Chemical investigation of cyanobacterial strain HT-58-2, which most closely aligns with the genus Brasilomena, has led to the isolation of two compounds related to tolypodiol. The structures and absolute configuration of 6-deoxytolypodiol (1) and 11-hydroxytolypodiol (2) were elucidated by spectroscopic and spectrometric analysis. While tolypodiol previously showed anti-inflammatory activity in a mouse ear edema assay, only 2 reduced in vitro thromboxane B2 and superoxide anion (O2-) generation from Escherichia coli lipopolysaccharide-activated rat neonatal microglia to any appreciable degree.