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Following spinal cord injury (SCI), prolonged muscle spasms are readily triggered by brief sensory stimuli. Animal and indirect human studies have shown that a substantial portion of the depolarization of motoneurons during a muscle spasm comes from the activation of persistent inward currents (PICs). The brief (single pulse) sensory stimuli that trigger the PICs and muscle spasms in chronically spinalized animals evoke excitatory post-synaptic potentials (EPSPs) that are broadened to more than 500 ms, the duration of depolarization required to activate a PIC in the motoneuron. Thus, in humans, we investigated if post-synaptic potentials (PSPs) evoked from brief (<20 ms) sensory stimulation are changed after SCI and if they are broadened to > or =500 ms to more readily activate motoneuron PICs and muscle spasms. To estimate both the shape and duration of PSPs in human subjects we used peristimulus frequencygrams (PSFs), which are plots of the instantaneous firing frequency of tonically active single motor units that are time-locked to the occurrence of the sensory stimulus. PSFs in response to cutaneomuscular stimulation of the medial arch or toe of the foot, a sensory stimulus that readily triggers muscle spasms, were compared between non-injured control subjects and in spastic subjects with chronic (>1 year), incomplete SCI. In non-injured controls, a single shock or brief (<20 ms) train of cutaneomuscular stimulation produced PSFs consisting of a 300 ms increase in firing rate above baseline with an interposed period of reduced firing. Parallel intracellular experiments in motoneurons of adult rats revealed that a 300 ms EPSP with a fast intervening inhibitory PSP (IPSP) reproduced the PSF recorded in non-injured subjects. In contrast, the same brief sensory stimulation in subjects with chronic SCI produced PSFs of comparatively long duration (1200 ms) with no evidence for IPSP activation, as reflected by a lack of reduced firing rates after the onset of the PSF. Thus, unlike non-injured controls, the motoneurons of subjects with chronic SCI are activated by very long periods of pure depolarization from brief sensory activation. It is likely that these second-long EPSPs securely recruit slowly activating PICs in motoneurons that are known to mediate, in large part, the many seconds-long activation of motoneurons during involuntary muscle spasms. [ABSTRACT FROM AUTHOR]

Cued recall is strongly affected by the strength of the preexisting connection between the test cue and the information to be recalled, the target. In all past work, preexisting cue-to-target strength has been measured by the probability that the cue produced the target in free association. This paper presents four experiments showing that this use of such norms underestimates the strength of the connection and that a more accurate estimate can be obtained by incorporating indirect as well as direct connections in the estimate. Experiments 1 and 2 showed that in extralist cued recall both the strength and number of two-step indirect connections facilitate recall. Experiment 3 showed that three-step connections have negligible effects. Experiment 4 used an intralist task in which cue and target are first studied together, and the results showed once again that indirect connections can affect recall. In all of these experiments, indirect connections had an effect on recall that was larger when direct cue-to-target strength was weak than when it was strong. Implications for using association norms in research are described, and an algorithm for using association norms to measure cue-to-target strength is proposed. [ABSTRACT FROM AUTHOR]

Oncogenic mutations in the RAS gene account for 30% of all human tumors; more than 60% of which present as KRAS mutations at the hotspot codon 12. After decades of intense pursuit, a covalent inhibition strategy has enabled selective targeting of this previously "undruggable" target. Herein, we disclose our journey toward the discovery of MK-1084, an orally bioavailable and low-dose KRAS G12C covalent inhibitor currently in phase I clinical trials (NCT05067283). We leveraged structure-based drug design to identify a macrocyclic core structure, and hypothesis-driven optimization of biopharmaceutical properties to further improve metabolic stability and tolerability.

Mutations in progranulin ( GRN ) cause frontotemporal dementia ( GRN -FTD) due to deficiency of the pleiotropic protein progranulin. GRN -FTD exhibits diverse pathologies including lysosome dysfunction, lipofuscinosis, microgliosis, and neuroinflammation. Yet, how progranulin loss causes disease remains unresolved. Here, we report that non-invasive retinal imaging of GRN -FTD patients revealed deficits in photoreceptors and the retinal pigment epithelium (RPE) that correlate with cognitive decline. Likewise, Grn -/- mice exhibit early RPE dysfunction, microglial activation, and subsequent photoreceptor loss. Super-resolution live imaging and transcriptomic analyses identified RPE mitochondria as an early driver of retinal dysfunction. Loss of mitochondrial fission protein 1 (MTFP1) in Grn -/- RPE causes mitochondrial hyperfusion and bioenergetic defects, leading to NF-kB-mediated activation of complement C3a-C3a receptor signaling, which drives further mitochondrial hyperfusion and retinal inflammation. C3aR antagonism restores RPE mitochondrial integrity and limits subretinal microglial activation. Our study identifies a previously unrecognized mechanism by which progranulin modulates mitochondrial integrity and complement-mediated neuroinflammation.

Motoneuron properties and their firing patterns undergo significant changes throughout development and in response to neuromodulators such as serotonin. Here, we examined the age-related development of self-sustained firing and general excitability of tibialis anterior motoneurons in a young development (7-17 years), young adult (18-28 years) and adult (32-53 years) group, as well as in a separate group of participants taking selective serotonin reuptake inhibitors (SSRIs, aged 11-28 years). Self-sustained firing, as measured by ΔF, was larger in the young development (∼5.8 Hz, n = 20) compared to the young adult (∼4.9 Hz, n = 13) and adult (∼4.8 Hz, n = 8) groups, consistent with a developmental decrease in self-sustained firing mediated by persistent inward currents (PIC). ΔF was also larger in participants taking SSRIs (∼6.5 Hz, n = 9) compared to their age-matched controls (∼5.3 Hz, n = 26), consistent with increased levels of spinal serotonin facilitating the motoneuron PIC. Participants in the young development and SSRI groups also had higher firing rates and a steeper acceleration in initial firing rates (secondary ranges), consistent with the PIC producing a steeper acceleration in membrane depolarization at the onset of motoneuron firing. In summary, both the young development and SSRI groups exhibited increased intrinsic motoneuron excitability compared to the adults, which, in the young development group, was also associated with a larger unsteadiness in the dorsiflexion torque profiles. We propose several intrinsic and extrinsic factors that affect both motoneuron PICs and cell discharge which vary during development, with a time course similar to the changes in motoneuron firing behaviour observed in the present study. KEY POINTS: Neurons in the spinal cord that activate muscles in the limbs (motoneurons) undergo increases in excitability shortly after birth to help animals stand and walk. We examined whether the excitability of human ankle flexor motoneurons also continues to change from child to adulthood by recording the activity of the muscle fibres they innervate. Motoneurons in children and adolescents aged 7-17 years (young development group) had higher signatures of excitability that included faster firing rates and more self-sustained activity compared to adults aged ≥18 years. Participants aged 11-28 years of age taking serotonin reuptake inhibitors had the highest measures of motoneuron excitability compared to their age-matched controls. The young development group also had more unstable contractions, which might partly be related to the high excitability of the motoneurons., (© 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

While neurodegeneration underlies the pathological basis for permanent disability in multiple sclerosis (MS), predictive biomarkers for progression are lacking. Using an animal model of chronic MS, we find that synaptic injury precedes neuronal loss and identify thinning of the inner plexiform layer (IPL) as an early feature of inflammatory demyelination-prior to symptom onset. As neuronal domains are anatomically segregated in the retina and can be monitored longitudinally, we hypothesize that thinning of the IPL could represent a biomarker for progression in MS. Leveraging our dataset with over 800 participants enrolled for more than 12 years, we find that IPL atrophy directly precedes progression and propose that synaptic loss is predictive of functional decline. Using a blood proteome-wide analysis, we demonstrate a strong correlation between demyelination, glial activation, and synapse loss independent of neuroaxonal injury. In summary, monitoring synaptic injury is a biologically relevant approach that reflects a potential driver of progression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genetic mutation of the leucine-rich repeat kinase 2 (LRRK2) protein has been associated with Parkinson's disease (PD), a disabling and progressive neurodegenerative disorder that is devoid of efficacious disease-modifying therapies. Herein, we describe the invention of an amidoisoquinoline (IQ)-derived LRRK2 inhibitor lead chemical series. Knowledge-, structure-, and property-based drug design in concert with rigorous application of in silico calculations and presynthesis predictions enabled the prioritization of molecules with favorable CNS "drug-like" physicochemical properties. This resulted in the discovery of compound 8 , which was profiled extensively before human ether-a-go-go (hERG) ion channel inhibition halted its progression. Strategic reduction of lipophilicity and basicity resulted in attenuation of hERG ion channel inhibition while maintaining a favorable CNS efflux transporter profile. Further structure- and property-based optimizations resulted in the discovery of preclinical candidate MK-1468 . This exquisitely selective LRRK2 inhibitor has a projected human dose of 48 mg BID and a preclinical safety profile that supported advancement toward GLP toxicology studies.

Glioblastoma multiforme (GBM) is the most aggressive type of brain cancer in adults, with an average life expectancy under treatment of approx. 15 months. GBM is characterised by a complex set of genetic alterations that results in significant disruption of receptor tyrosine kinase (RTK) signaling. We report here an exploration of the pyrazolo[3,4- d ]pyrimidine scaffold in search for antiproliferative compounds directed to GBM treatment. Small compound libraries were synthesised and screened against GBM cells to build up structure-antiproliferative activity-relationships (SAARs) and inform further rounds of design, synthesis and screening. 76 novel compounds were generated through this iterative process that found low micromolar potencies against selected GBM lines, including patient-derived stem cells. Phenomics analysis demonstrated preferential activity against glioma cells of the mesenchymal subtype, whereas kinome screening identified colony stimulating factor-1 receptor (CSF-1R) as the lead's target, a RTK implicated in the tumourigenesis and progression of different cancers and the immunoregulation of the GBM microenvironment., Competing Interests: The authors declare no conflicts of interest., (This journal is © The Royal Society of Chemistry.)

When a muscle is stretched, sensory feedback not only causes reflexes but also leads to a depolarization of sensory afferents throughout the spinal cord (primary afferent depolarization, PAD), readying the whole limb for further disturbances. This sensory-evoked PAD is thought to be mediated by a trisynaptic circuit, where sensory input activates first-order excitatory neurons that activate GABAergic neurons that in turn activate GABA A receptors on afferents to cause PAD, though the identity of these first-order neurons is unclear. Here, we show that these first-order neurons include propriospinal V3 neurons, as they receive extensive sensory input and in turn innervate GABAergic neurons that cause PAD, because optogenetic activation or inhibition of V3 neurons in mice mimics or inhibits sensory-evoked PAD, respectively. Furthermore, persistent inward sodium currents intrinsic to V3 neurons prolong their activity, explaining the prolonged duration of PAD. Also, local optogenetic activation of V3 neurons at one segment causes PAD in other segments, due to the long propriospinal tracts of these neurons, helping to explain the radiating nature of PAD. This in turn facilitates monosynaptic reflex transmission to motoneurons across the spinal cord. In addition, V3 neurons directly innervate proprioceptive afferents (including Ia), causing a glutamate receptor-mediated PAD (glutamate PAD). Finally, increasing the spinal cord excitability with either GABA A receptor blockers or chronic spinal cord injury causes an increase in the glutamate PAD. Overall, we show the V3 neuron has a prominent role in modulating sensory transmission, in addition to its previously described role in locomotion. NEW & NOTEWORTHY Locomotor-related propriospinal neurons depolarize sensory axons throughout the spinal cord by either direct glutamatergic axoaxonic contacts or indirect innervation of GABAergic neurons that themselves form axoaxonic contacts on sensory axons. This depolarization (PAD) increases sensory transmission to motoneurons throughout the spinal cord, readying the sensorimotor system for external disturbances. The glutamate-mediated PAD is particularly adaptable, increasing with either an acute block of GABA receptors or chronic spinal cord injury, suggesting a role in motor recovery.

The replacement of benzene rings with sp 3 -hybridized bioisosteres in drug candidates generally improves pharmacokinetic properties while retaining biological activity 1-5 . Rigid, strained frameworks such as bicyclo[1.1.1]pentane and cubane are particularly well suited as the ring strain imparts high bond strength and thus metabolic stability on their C-H bonds. Cubane is the ideal bioisostere as it provides the closest geometric match to benzene 6,7 . At present, however, all cubanes in drug design, like almost all benzene bioisosteres, act solely as substitutes for mono- or para-substituted benzene rings 1-7 . This is owing to the difficulty of accessing 1,3- and 1,2-disubstituted cubane precursors. The adoption of cubane in drug design has been further hindered by the poor compatibility of cross-coupling reactions with the cubane scaffold, owing to a competing metal-catalysed valence isomerization 8-11 . Here we report expedient routes to 1,3- and 1,2-disubstituted cubane building blocks using a convenient cyclobutadiene precursor and a photolytic C-H carboxylation reaction, respectively. Moreover, we leverage the slow oxidative addition and rapid reductive elimination of copper to develop C-N, C-C(sp 3 ), C-C(sp 2 ) and C-CF 3 cross-coupling protocols 12,13 . Our research enables facile elaboration of all cubane isomers into drug candidates, thus enabling ideal bioisosteric replacement of ortho-, meta- and para-substituted benzenes., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Suppression of the extensor H-reflex by flexor afferent conditioning is thought to be produced by a long-lasting inhibition of extensor Ia afferent terminals via GABA A receptor-activated primary afferent depolarization (PAD). Given the recent finding that PAD does not produce presynaptic inhibition of Ia afferent terminals, we examined in 28 participants if H-reflex suppression is instead mediated by post-activation depression of the extensor Ia afferents triggered by PAD-evoked spikes and/or by a long-lasting inhibition of the extensor motoneurons. A brief conditioning vibration of the flexor tendon suppressed both the extensor soleus H-reflex and the tonic discharge of soleus motor units out to 150 ms following the vibration, suggesting that part of the H-reflex suppression during this period was mediated by postsynaptic inhibition of the extensor motoneurons. When activating the flexor afferents electrically to produce conditioning, the soleus H-reflex was also suppressed but only when a short-latency reflex was evoked in the soleus muscle by the conditioning input itself. In mice, a similar short-latency reflex was evoked when optogenetic or afferent activation of GABAergic (GAD2 + ) neurons produced a large enough PAD to evoke orthodromic spikes in the test Ia afferents, causing post-activation depression of subsequent monosynaptic EPSPs. The long duration of this post-activation depression and related H-reflex suppression (seconds) was similar to rate-dependent depression that is also due to post-activation depression. We conclude that extensor H-reflex inhibition by brief flexor afferent conditioning is produced by both post-activation depression of extensor Ia afferents and long-lasting inhibition of extensor motoneurons, rather than from PAD inhibiting Ia afferent terminals. KEY POINTS: Suppression of extensor H-reflexes by flexor afferent conditioning was thought to be mediated by GABA A receptor-mediated primary afferent depolarization (PAD) shunting action potentials in the Ia afferent terminal. In line with recent findings that PAD has a facilitatory role in Ia afferent conduction, we show here that when large enough, PAD can evoke orthodromic spikes that travel to the Ia afferent terminal to evoke EPSPs in the motoneuron. These PAD-evoked spikes also produce post-activation depression of Ia afferent terminals and may mediate the short- and long-lasting suppression of extensor H-reflexes in response to flexor afferent conditioning. Our findings highlight that we must re-examine how changes in the activation of GABAergic interneurons and PAD following nervous system injury or disease affects the regulation of Ia afferent transmission to spinal neurons and ultimately motor dysfunction in these disorders., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Sensory and corticospinal tract (CST) pathways activate spinal GABAergic interneurons that have axoaxonic connections onto proprioceptive (Ia) afferents that cause long-lasting depolarizations (termed primary afferent depolarization, PAD). In rodents, sensory-evoked PAD is produced by GABA A receptors at nodes of Ranvier in Ia afferents, rather than at presynaptic terminals, and facilitates spike propagation to motoneurons by preventing branch-point failures, rather than causing presynaptic inhibition. We examined in 40 human participants whether putative activation of Ia-PAD by sensory or CST pathways can also facilitate Ia afferent activation of motoneurons via the H-reflex. H-reflexes in several leg muscles were facilitated by prior conditioning from low-threshold proprioceptive, cutaneous or CST pathways, with a similar long-lasting time course (∼200 ms) to phasic PAD measured in rodent Ia afferents. Long trains of cutaneous or proprioceptive afferent conditioning produced longer-lasting facilitation of the H-reflex for up to 2 min, consistent with tonic PAD in rodent Ia afferents mediated by nodal α5-GABA A receptors for similar stimulation trains. Facilitation of H-reflexes by this conditioning was likely not mediated by direct facilitation of the motoneurons because isolated stimulation of sensory or CST pathways did not alone facilitate the tonic firing rate of motor units. Furthermore, cutaneous conditioning increased the firing probability of single motor units (motoneurons) during the H-reflex without increasing their firing rate at this time, indicating that the underlying excitatory postsynaptic potential was more probable, but not larger. These results are consistent with sensory and CST pathways activating nodal GABA A receptors that reduce intermittent failure of action potentials propagating into Ia afferent branches. KEY POINTS: Controlled execution of posture and movement requires continually adjusted feedback from peripheral sensory pathways, especially those that carry proprioceptive information about body position, movement and effort. It was previously thought that the flow of proprioceptive feedback from Ia afferents was only reduced by GABAergic neurons in the spinal cord that sent axoaxonic projections to the terminal endings of sensory axons (termed GABA axo neurons). Based on new findings in rodents, we provide complementary evidence in humans to suggest that sensory and corticospinal pathways known to activate GABA axo neurons that project to dorsal parts of the Ia afferent also increase the flow of proprioceptive feedback to motoneurons in the spinal cord. These findings support a new role for spinal GABA axo neurons in facilitating afferent feedback to the spinal cord during voluntary or reflexive movements., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Postoperative hand therapy (HT) is important for regaining function and preventing complications in patients undergoing tendon repair of the hand and wrist. Loss to follow-up (LTFU) can hinder this process; so we sought to determine factors that predict attrition of these patients., Methods: Charts were retrospectively reviewed for patients who underwent extensor or flexor tendon repair of the hand, wrist, or forearm between 2014 and 2019. Demographic data, including age, sex, zip code, employment status, education level, and insurance type, were collected, and the rate of LTFU was calculated. Logistic regression was used to analyze factors., Results: A total of 149 patients were identified and analyzed. The rate of LTFU was 42%. Factors that predicted loss were younger age, male gender, lower educational degree, and a documented psychiatric history. Employment status, insurance type, and distance from the HT center did not predict attrition. The number of HT weeks recommended by the occupational therapist did not differ between those who were lost and those who were not. Lost patients completed, on average, 57% of their suggested HT course., Conclusions: The current study identified demographic factors associated with attrition in patients undergoing tendon repair of the distal upper extremity. Factors included patients who were younger, male gender, less educated, and had a documented psychiatric history. By identifying factors that predict LTFU, specific strategies can be developed to reduce attrition rates, particularly for at-risk populations, to improve patient care after tendon repair., Competing Interests: The authors have no financial interests to declare in relation to the content of this article., (Copyright © 2023 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The American Society of Plastic Surgeons.)

The replacement of aryl rings with saturated carbocyclic structures has garnered significant interest in drug discovery due to the potential for improved pharmacokinetic properties upon substitution. In particular, 1,3-difunctionalized bicyclo[1.1.1]pentanes (BCPs) have been widely adopted as bioisosteres for parasubstituted arene rings, appearing in a number of lead pharmaceutical candidates. However, despite the pharmaceutical value of 2-substituted BCPs as replacements for ortho- or meta-substituted arene rings, general and rapid syntheses of these scaffolds remain elusive. Current approaches to 2-substituted BCPs rely on installation of the bridge substituent prior to BCP core construction, leading to lengthy step counts and often nonmodular sequences. While challenging, direct functionalization of the strong bridge BCP C-H bonds would offer a more streamlined pathway to diverse 2-substituted BCPs. Here, we report a generalizable synthetic linchpin strategy for bridge functionalization via radical C-H abstraction of the BCP core. Through mild generation of a strong hydrogen atom abstractor, we rapidly synthesize novel 2-substituted BCP synthetic linchpins in one pot. These synthetic linchpins then serve as common precursors to complex 2-substituted BCPs, allowing one-step access to a number of previously inaccessible electrophile and nucleophile fragments at the 2-position via two new metallaphotoredox protocols. Altogether, this platform enables the expedient synthesis of four pharmaceutical analogues, all of which show similar or improved properties compared to their aryl-containing equivalents, demonstrating the potential of these 2-substituted BCPs in drug development.

Inhibition of leucine-rich repeat kinase 2 (LRRK2) kinase activity represents a genetically supported, chemically tractable, and potentially disease-modifying mechanism to treat Parkinson's disease. Herein, we describe the optimization of a novel series of potent, selective, central nervous system (CNS)-penetrant 1-heteroaryl-1 H -indazole type I (ATP competitive) LRRK2 inhibitors. Type I ATP-competitive kinase physicochemical properties were integrated with CNS drug-like properties through a combination of structure-based drug design and parallel medicinal chemistry enabled by sp 3 -sp 2 cross-coupling technologies. This resulted in the discovery of a unique sp 3 -rich spirocarbonitrile motif that imparted extraordinary potency, pharmacokinetics, and favorable CNS drug-like properties. The lead compound, 25 , demonstrated exceptional on-target potency in human peripheral blood mononuclear cells, excellent off-target kinase selectivity, and good brain exposure in rat, culminating in a low projected human dose and a pre-clinical safety profile that warranted advancement toward pre-clinical candidate enabling studies.

Background and Objectives: The timing of neurodegeneration in multiple sclerosis (MS) remains unclear. It is critical to understand the dynamics of neuroaxonal loss if we hope to prevent or forestall permanent disability in MS. We therefore used a deeply phenotyped longitudinal cohort to assess and compare rates of neurodegeneration in retina and brain throughout the MS disease course., Methods: We analyzed 597 patients with MS who underwent longitudinal optical coherence tomography imaging annually for 4.5 ± 2.4 years and 432 patients who underwent longitudinal MRI scans for 10 ± 3.4 years, quantifying macular ganglion cell-inner plexiform layer (GCIPL) volume and cortical gray matter (CGM) volume. The association between the slope of decline in the anatomical structure and the age of entry in the cohort (categorized by the MRI cohort's age quartiles) was assessed by hierarchical linear models., Results: The rate of CGM volume loss declined with increasing age of study entry (1.3% per year atrophy for the age of entry in the cohort younger than 35 years; 1.1% for older than 35 years and younger than 41; 0.97% for older than 41 years and younger than 49; 0.9% for older than 49 years) while the rate of GCIPL thinning was highest in patients in the youngest quartile, fell by more than 50% in the following age quartile, and then stabilized (0.7% per year thinning for the age of entry in the cohort younger than 35 years; 0.29% for age older than 35 and younger than 41 years; 0.34% for older than 41 and younger than 49 years; 0.33% for age older than 49 years)., Discussion: An age-dependent reduction in retinal and cortical volume loss rates during relapsing-remitting MS suggests deceleration in neurodegeneration in the earlier period of disease and further indicates that the period of greatest adaptive immune-mediated inflammatory activity is also the period with the greatest neuroaxonal loss., (© 2022 American Academy of Neurology.)

Movement and posture depend on sensory feedback that is regulated by specialized GABAergic neurons (GAD2 + ) that form axo-axonic contacts onto myelinated proprioceptive sensory axons and are thought to be inhibitory. However, we report here that activating GAD2 + neurons directly with optogenetics or indirectly by cutaneous stimulation actually facilitates sensory feedback to motor neurons in rodents and humans. GABA A receptors located at or near nodes of Ranvier of sensory axons cause this facilitation by preventing spike propagation failure at the many axon branch points, which is otherwise common without GABA. In contrast, GABA A receptors are generally lacking from axon terminals and so cannot inhibit transmitter release onto motor neurons, unlike GABA B receptors that cause presynaptic inhibition. GABAergic innervation near nodes and branch points allows individual branches to function autonomously, with GAD2 + neurons regulating which branches conduct, adding a computational layer to the neuronal networks generating movement and likely generalizing to other central nervous system axons., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Subcluster L3 bacteriophage Finnry was isolated from soil collected in Charleston, South Carolina, using Mycobacterium smegmatis mc 2 155 as a host. The genome of this temperate siphovirus is 75,632 bp long (130 predicted protein-coding genes, 9 tRNAs, and no transfer-messenger RNAs), and BLASTn alignment revealed 99.86% identity with the genome of L3 mycobacteriophage Samty.

3,3-Disubstituted oxetanes have been utilized as bioisosteres for gem-dimethyl and cyclobutane functionalities. We report the discovery of a novel class of oxetane indole-amine 2,3-dioxygenase (IDO1) inhibitors suitable for Q3W (once every 3 weeks) oral and parenteral dosing. A diamide class of IDO inhibitors was discovered through an automated ligand identification system (ALIS). Installation of an oxetane and fluorophenyl dramatically improved the potency. Identification of a biaryl moiety as an unconventional amide isostere addressed the metabolic liability of amide hydrolysis. Metabolism identification (Met-ID)-guided target design and the introduction of polarity resulted in the discovery of potent IDO inhibitors with excellent pharmacokinetic (PK) profiles in multiple species. To enable rapid synthesis of the key oxetane intermediate, a novel oxetane ring cyclization was also developed, as well as optimization of a literature route on kg scale. These IDO inhibitors may enable unambiguous proof-of-concept testing for the IDO1 inhibition mechanism for oncology.

Stereochemically and structurally complex cyclic dinucleotide-based stimulator of interferon genes (STING) agonists were designed and synthesized to access a previously unexplored chemical space. The assessment of biochemical affinity and cellular potency, along with computational, structural, and biophysical characterization, was applied to influence the design and optimization of novel STING agonists, resulting in the discovery of MK-1454 as a molecule with appropriate properties for clinical development. When administered intratumorally to immune-competent mice-bearing syngeneic tumors, MK-1454 exhibited robust tumor cytokine upregulation and effective antitumor activity. Tumor shrinkage in mouse models that are intrinsically resistant to single-agent therapy was further enhanced when treating the animals with MK-1454 in combination with a fully murinized antimouse PD-1 antibody, mDX400. These data support the development of STING agonists in combination with pembrolizumab (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 therapy.

Animal models of cervical spinal cord injury (SCI) have frequently utilized partial transection injuries to evaluate plasticity promoting treatments such as rehabilitation training of skilled reaching and grasping tasks. Though highly useful for studying the effects of cutting specific spinal tracts that are important for skilled forelimb motor function, cervical partial-transection SCI-models underappreciate the extensive spread of most human SCIs, thus offering poor predictability for the clinical setting. Conversely, moderate cervical contusion SCI models targeting the spinal tracts important for skilled reaching and grasping can better replicate the increased size of most human SCIs and are often considered more clinically relevant. However, it is unknown whether animals with moderate cervical contusion SCIs that damage key spinal motor tracts can train in skilled reaching and grasping tasks. In this study, we quantify the impact of injury size and distribution on recovery in a skilled motor task called the single pellet reaching, grasping and retrieval (SPRGR) task in rats with cervical unilateral contusion injuries (UCs), and compare to rats with a partial transection SCIs (i.e., dorsolateral quadrant transection; DLQ). We found that UCs damage key tracts important for performing skilled motor tasks, similar to DLQs, but UCs also produce more extensive grey matter damage and more ventral white matter damage than DLQs. We also compared forelimb functionality at 1, 3, and 5 weeks of rehabilitative motor training between trained and untrained rats and found a more severe drop in SPRGR performance than in DLQ SCIs. Nevertheless, despite more severe injuries and initially low SPRGR performance, rehabilitative training for contusion animals resulted in significant improvements in SPRGR performance and proportionally more recovery than DLQ rats. Our findings show that rehabilitative motor training can facilitate considerable amounts of motor recovery despite extensive spinal cord damage, especially grey matter damage, thus supporting the use of contusion or compression SCI models and showing that ventral grey and white matter damage are not necessarily detrimental to recovery after training., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence 1-3 . Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents 4,5 . Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic 6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes systemic antigen-specific CD8 + T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti-PD-1-resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 treatment by enhancing the anti-PD-1 immune profile., (©2021 American Association for Cancer Research.)

The leucine-rich repeat kinase 2 (LRRK2) protein has been genetically and functionally linked to Parkinson's disease (PD), a disabling and progressive neurodegenerative disorder whose current therapies are limited in scope and efficacy. In this report, we describe a rigorous hit-to-lead optimization campaign supported by structural enablement, which culminated in the discovery of brain-penetrant, candidate-quality molecules as represented by compounds 22 and 24 . These compounds exhibit remarkable selectivity against the kinome and offer good oral bioavailability and low projected human doses. Furthermore, they showcase the implementation of stereochemical design elements that serve to enable a potency- and selectivity-enhancing increase in polarity and hydrogen bond donor (HBD) count while maintaining a central nervous system-friendly profile typified by low levels of transporter-mediated efflux and encouraging brain penetration in preclinical models.

Arterial tortuosity describes variation via bending of the arterial wall and has been noted in several arteries throughout the body. Tortuous blood vessels can cause nerve compression, as well as present difficulties to surgeons and radiologists. Here we present an unusual case of multi-vessel arterial tortuosity discovered in 78-year-old Hispanic male cadaver, independent of systemic pathology. The left ulnar and right tibial arteries were dissected, and using calibrated digital callipers, their external and internal diameters were measured both at the origin site and at the site of greatest dilation. Both wall thickness and the number of inflection points were also measured. Six bends were noticed in the ulnar artery and its diameter measured 8.11 mm at its widest, with a wall thickness of 0.88 mm. On the lower extremity, the right tibial artery had three bends and its diameter measured 4.86 mm at its widest, with a wall thickness of 1.32 mm. This uncommon tortuosity is not only more prone to laceration during surgery, but the bending and thickening can be mistaken for tumours. Finally, fluid dynamics can be altered, resulting in an impact on blood pressure in the extremities. Thus, raising awareness is crucial to prevent both symptoms and iatrogenic complications.

Background: Extremity CT angiography (CTA) is frequently used to assess for vascular injury among patients with extremity trauma. The injured extremity index (IEI), defined as the ratio of systolic occlusion pressure between injured and uninjured extremities, has been implemented to screen patients being considered for CTA. Physical examination together with IEI is extremely sensitive for significant extremity vascular injury. Unfortunately, IEI cannot always be calculated. This study aimed to determine whether patients with normal pulse examinations and no hard signs of vascular injury benefitted from further imaging with CTA. We hypothesized that CTA has become overused among patients with extremity trauma, as determined by the outcome of vascular abnormalities that underwent vascular intervention but were missed by physical examination., Methods: The charts of traumatically injured patients who underwent extremity CTA were retrospectively reviewed. This study was performed at a level 1 trauma center for patients who presented as trauma activations from September 1, 2019 to September 1, 2020., Results: One hundred and thirty-six patients with 167 injured limbs were included. Eight limbs (4.8%) underwent an open vascular operation, whereas five limbs (3.0%) underwent an endovascular procedure. One of the 167 limbs (0.6%) had a vascular injury seen on CTA and underwent intervention that was not associated with a pulse abnormality or hard signs of vascular injury. This patient presented in a delayed fashion after an initially normal IEI and examination. Proximity injuries and fractures alone were not highly associated with vascular injuries., Discussion: Many patients with normal pulse examination and no hard signs of vascular injury underwent CTA; the vast majority of these patients did not then have a vascular intervention. Given the consequences of missed vascular injuries, further work is required to prospectively assess the utility of CTA among patients with extremity trauma., Level of Evidence: III., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

A novel series of IDO1 inhibitors have been identified with good IDO1 Hela cell and human whole blood activity. These inhibitors contain an indoline or a 3-azaindoline scaffold. Their structure-activity-relationship studies have been explored. Compounds 37 and 41 stood out as leads due to their good potency in IDO1 Hela assay, good IDO1 unbound hWB IC 50 s, reasonable unbound clearance, and good MRT in rat and dog PK studies., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Herein the discovery of potent IDO1 inhibitors with low predicted human dose is discussed. Metabolite identification (MetID) and structural data were used to strategically incorporate cyclopropane rings into this tetrahydronaphthyridine series of IDO1 inhibitors to improve their metabolic stability and potency. Enabling synthetic chemistry was developed to construct these unique fused cyclopropyl compounds, leading to inhibitors with improved pharmacokinetics and human whole blood potency and a predicted human oral dose as low as 9 mg once daily (QD)., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

Background: The great diversity in plant genome size and chromosome number is partly due to polyploidization (i.e. genome doubling events). The differences in genome size and chromosome number among diploid plant species can be a window into the intriguing phenomenon of past genome doubling that may be obscured through time by the process of diploidization. The genus Hibiscus L. (Malvaceae) has a wide diversity of chromosome numbers and a complex genomic history. Hibiscus is ideal for exploring past genomic events because although two ancient genome duplication events have been identified, more are likely to be found due to its diversity of chromosome numbers. To reappraise the history of whole-genome duplication events in Hibiscus, we tested three alternative scenarios describing different polyploidization events., Results: Using target sequence capture, we designed a new probe set for Hibiscus and generated 87 orthologous genes from four diploid species. We detected paralogues in > 54% putative single-copy genes. 34 of these genes were selected for testing three different genome duplication scenarios using gene counting. All species of Hibiscus sampled shared one genome duplication with H. syriacus, and one whole genome duplication occurred along the branch leading to H. syriacus., Conclusions: Here, we corroborated the independent genome doubling previously found in the lineage leading to H. syriacus and a shared genome doubling of this lineage and the remainder of Hibiscus. Additionally, we found a previously undiscovered genome duplication shared by the /Pavonia and /Malvaviscus clades (both nested within Hibiscus) with the occurrences of two copies in what were otherwise single-copy genes. Our results highlight the complexity of genomic diversity in some plant groups, which makes orthology assessment and accurate phylogenomic inference difficult.