A Diels-Alder probe for discovery of natural products containing furan moieties.

Natural products (NPs) are fantastic sources of inspiration for novel pharmaceuticals, oftentimes showing unique bioactivity against interesting targets. Specifically, NPs containing furan moieties show activity against a variety of diseases including fungal infections, and cancers. However, it is challenging to discover and isolate these small molecules from cell supernatant. The work described herein showcases the development of a molecular probe that can covalently modify furan moieties via a [4 + 2] Diels-Alder cycloaddition, making them easily identifiable on liquid chromatography-mass spectrometry (LC-MS). The molecular probe, which undergoes this reaction with a variety of furans, was designed with both a UV-tag and a mass tag to enable easy identification. The probe has been tested with a variety of purified furans, including natural products, methylenomycin furan (MMF) hormones, and MMF derivatives. Moreover, the molecular probe has been tested in crude supernatants of various Streptomyces strains and enables identification of MMFs.

Actinomycetota bioprospecting from ore-forming environments.

Natural products from Actinomycetota have served as inspiration for many clinically relevant therapeutics. Despite early triumphs in natural product discovery, the rate of unearthing new compounds has decreased, necessitating inventive approaches. One promising strategy is to explore environments where survival is challenging. These harsh environments are hypothesized to lead to bacteria developing chemical adaptations (e.g. natural products) to enable their survival. This investigation focuses on ore-forming environments, particularly fluoride mines, which typically have extreme pH, salinity and nutrient scarcity. Herein, we have utilized metagenomics, metabolomics and evolutionary genome mining to dissect the biodiversity and metabolism in these harsh environments. This work has unveiled the promising biosynthetic potential of these bacteria and has demonstrated their ability to produce bioactive secondary metabolites. This research constitutes a pioneering endeavour in bioprospection within fluoride mining regions, providing insights into uncharted microbial ecosystems and their previously unexplored natural products.

Discovery of Streptomyces species CS-62, a novel producer of the Acinetobacter baumannii selective antibiotic factumycin.

Narrow-spectrum antibiotics are of great interest given their ability to spare the microbiome and decrease widespread antibiotic resistance compared to broad-spectrum antibiotics. Herein, we screened an in-house library of Actinobacteria strains for selective activity against Acinetobacter baumannii and successfully identified Streptomyces sp. CS-62 as a producer of a natural product with this valuable activity. Analysis of the cultures via high-resolution mass spectrometry and tandem mass spectrometry, followed by comparison with molecules in the Natural Product Atlas and the Global Natural Products Social Molecular Networking platform, suggested a novel natural product. Genome mining analysis initially supported the production of a novel kirromycin derivative. Isolation and structure elucidation via mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses revealed that the active natural product was the known natural product factumycin, exposing omissions and errors in the consulted databases. While public databases are generally very useful for avoiding rediscovery of known molecules, rediscovery remains a problem due to public databases either being incomplete or having errors that result in failed dereplication. Overall, the work describes the ongoing problem of dereplication and the continued need for public database curation.

Simulated GABA synaptic input and L-type calcium channels form functional microdomains in hypothalamic gonadotropin-releasing hormone neurons.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons integrate the multiple internal and external cues that regulate sexual reproduction. In contrast to other neurons that exhibit extensive dendritic arbors, GnRH neurons usually have a single dendrite with relatively little branching. This largely precludes the integration strategy in which a single dendritic branch serves as a unit of integration. In the present study, we identify a gradient in L-type calcium channels in dendrites of mouse GnRH neurons and its interaction with GABAergic and glutamatergic inputs. Higher levels of L-type calcium channels are in somata/proximal dendrites (i.e., 0-26 µm) and distal dendrites (∼130 µm dendrite length), but intervening midlengths of dendrite (∼27-130 µm) have reduced L-type calcium channels. Using uncaging of GABA, there is a decreasing GABAergic influence along the dendrite and the impact of GABA(A) receptors is dependent on activation of L-type calcium channels. This results in amplification of proximal GABAergic signals and attenuation of distal dendritic signals. Most interestingly, the intervening dendritic regions create a filter through which only relatively high-amplitude, low-frequency GABAergic signaling to dendrites elicits action potentials. The findings of the present study suggest that GnRH dendrites adopt an integration strategy whereby segments of single nonbranching GnRH dendrites create functional microdomains and thus serve as units of integration.

Dendritic action potential initiation in hypothalamic gonadotropin-releasing hormone neurons.

It is dogma that action potentials are initiated at the soma/axon hillock of neurons. However, dendrites often exhibit conductances necessary for spike generation and represent functionally independent processing compartments within neurons. GnRH neurons provide an interesting neuronal phenotype with simple, relatively unbranched, unipolar or bipolar dendrites of extensive lengths (>1000 microm) covered in spines. These neurons control fertility and must integrate a variety of internal homeostatic and external environmental cues. We used imaging, electrophysiological, and modeling studies to understand how they integrate and process information along dendrites. Simultaneous recordings from distal dendrites and somata of individual GnRH neurons indicate distal dendrites are the primary site of spike initiation in these cells. Compartmental modeling indicates that sites of spike initiation depend upon location of excitatory input and dendrite geometry. Together, these studies demonstrate a novel pattern of spike generation in mammalian neurons and indicate that afferent inputs within distal dendritic microdomains directly initiate action potentials.

Emerging methodologies for the study of hypothalamic gonadotropin-releasing-hormone (GnRH) neurons.

Gonadotropin-releasing-hormone (GnRH) neurons form part of a central neural oscillator that controls sexual reproduction through intermittent release of the GnRH peptide. Activity of GnRH neurons, and by extension release of GnRH, has been proposed to reflect intrinsic properties and synaptic input of GnRH neurons. To study GnRH neurons, we used traditional electrophysiology and computational methods. These emerging methodologies enhance the elucidation of processing in GnRH neurons. We used dynamic current-clamping to understand how living GnRH somata process input from glutamate and GABA, two key neurotransmitters in the neuroendocrine hypothalamus. In order to study the impact of synaptic integration in dendrites and neuronal morphology, we have developed full-morphology models of GnRH neurons. Using dynamic clamping, we have demonstrated that small-amplitude glutamatergic currents can drive repetitive firing in GnRH neurons. Furthermore, application of simulated GABAergic synapses with a depolarized reversal potential have revealed two functional subpopulations of GnRH neurons: one population in which GABA chronically depolarizes membrane potential (without inducing action potentials) and a second population in which GABAergic excitation results in slow spiking. Finally, when AMPA-type and GABA-type simulated inputs are applied together, action potentials occur when the AMPA-type conductance occurs during the descending phase of GABAergic excitation and at the nadir of GABAergic inhibition. Compartmental computer models have shown that excitatory synapses at >300 microns from somtata are unable to drive spiking with purely passive dendrites. In models with active dendrites, distal synapses are more efficient at driving spiking than somatic inputs. We then used our models to extend the results from dynamic current clamping at GnRH somata to distribute synaptic inputs along the dendrite. We show that propagation delays for dendritic synapses alter synaptic integration in GnRH neurons by widening the temporal window of interaction for the generation of action potentials. Finally, we have shown that changes in dendrite morphology can modulate the output of GnRH neurons by altering the efficacy of action potential generation in response to after-depolarization potentials (ADPs). Taken together, the methodologies of dynamic current clamping and multi-compartmental modeling can make major contributions to the study of synaptic integration and structure-function relationships in hypothalamic GnRH neurons. Use of these methodological approaches will continue to provide keen insights leading to conceptual advances in our understanding of reproductive hormone secretion in normal and pathological physiology and open the door to understanding whether the mechanisms of pulsatile GnRH release are conserved across species.