A Platform for the Synthesis of Corynantheine-Type Corynanthe Alkaloids.

Corynantheine-type alkaloids are major components of the Mitragyna speciosa, also known as kratom, that exhibit unique pharmacological activity. However, no universal method to prepare these alkaloids has been reported. Disclosed herein is a catalytic, asymmetric platform that enables rapid access to corynantheine alkaloids. The first enantioselective total synthesis of (-)-corynantheidine pseudoindoxyl is described. The first asymmetric syntheses of (+)-corynoxine and (-)-corynoxine B were also achieved, along with enantioselective syntheses of (-)-corynantheidol and (-)-corynantheidine. Through this work, all series of corynantheine alkaloids including indole, spirooxindole, and pseudoindoxyl can now be accessed in the laboratory, enabling comprehensive biological investigation of kratom alkaloids to be undertaken.

Optimization of 1,2,4-Triazole-Based p97 Inhibitors for the Treatment of Cancer.

The AAA+ ATPase p97 (valosin-containing protein, VCP) is a master regulator of protein homeostasis and therefore represents a novel target for cancer therapy. Starting from a known allosteric inhibitor, NMS-873, we systematically optimized this scaffold, in particular, by applying a benzene-to-acetylene isosteric replacement strategy, specific incorporation of F, and eutomer/distomer identification, which led to compounds that exhibited nanomolar biochemical and cell-based potency. In cellular pharmacodynamic assays, robust effects on biomarkers of p97 inhibition and apoptosis, including increased levels of ubiquitinated proteins, CHOP and cleaved caspase 3, were observed. Compound (R)-29 (UPCDC-30766) represents the most potent allosteric inhibitor of p97 reported to date.

Enantioselective Syntheses of Yohimbine Alkaloids: Proving Grounds for New Catalytic Transformations.

The total synthesis of bioactive alkaloids is an enduring challenge and an indication of the state of the art of chemical synthesis. With the explosion of catalytic asymmetric methods over the past three decades, these compelling targets have been fertile proving grounds for enantioselective bond forming transformations. These activities are summarized herein both to highlight the power and versatility of these methods and to instill future inspiration for new syntheses of these privileged natural products.

A Concise, Enantioselective Approach for the Synthesis of Yohimbine Alkaloids.

We report a concise, enantioselective synthesis of the yohimbine alkaloids (-)-rauwolscine and (-)-alloyohimbane. The key transformation involves a highly enantio- and diastereoselective NHC-catalyzed dimerization and an amidation/N-acyliminium ion cyclization sequence to furnish four of the five requisite rings and three of the five stereocenters in two operations. This route also provides efficient access to all four diastereomeric arrangements of the core stereotriad of the yohimbine alkaloids from a common intermediate. This platform approach in combination with the ability to access both enantiomers from the carbene-catalyzed reaction is a powerful strategy that can produce a wide range of complex alkaloids and related structures for future biomedical investigations.

Oculomotor, Vestibular, and Reaction Time Effects of Sports-Related Concussion: Video-Oculography in Assessing Sports-Related Concussion.

OBJECTIVE: The purpose of the study was to test the ability of oculomotor, vestibular, and reaction time (OVRT) metrics to serve as a concussion assessment or diagnostic tool for general clinical use. SETTING AND PARTICIPANTS: Patients with concussion were high school-aged athletes clinically diagnosed in a hospital setting with a sports-related concussion (n = 50). Control subjects were previously recruited male and female high school student athletes from 3 local high schools (n = 170). DESIGN: Video-oculography was used to acquire eye movement metrics during OVRT tasks, combined with other measures. Measures were compared between groups, and a subset was incorporated into linear regression models that could serve as indicators of concussion. MEASURES: The OVRT test battery included multiple metrics of saccades, smooth pursuit tracking, nystagmoid movements, vestibular function, and reaction time latencies. RESULTS: Some OVRT metrics were significantly different between groups. Linear regression models distinguished control subjects from concussion subjects with high accuracy. Metrics included changes in smooth pursuit tracking, increased reaction time and reduced saccade velocity in a complex motor task, and decreased optokinetic nystagmus (OKN) gain. In addition, optokinetic gain was reduced and more variable in subjects assessed 22 or more days after injury. CONCLUSION: These results indicate that OVRT tests can be used as a reliable adjunctive tool in the assessment of concussion and that OKN results appear to be associated with a prolonged expression of concussion symptoms.

Transient and permanent arterial occlusions modeling poststroke epilepsy in aging rats.

The pathophysiological mechanisms of epileptogenesis following ischemic stroke in the aged brain are not well understood, largely due to limited developments in animal modeling of poststroke epilepsy (PSE). A recent study in our laboratory (Kelly et al., 2018) using transient (3 h) unilateral middle cerebral artery (MCA) and common carotid artery (CCA) occlusion (MCA/CCAo) in 4- and 20-month-old Fischer (F344) rats resulted in epileptic seizures in both age groups; age and infarction factors independently had effects on seizure frequency. We hypothesized that permanent unilateral MCA/CCAo, a simpler model, was capable of producing results comparable to those of transient MCA/CCAo. In this study, we performed permanent MCA/CCAo and compared it to transient MCA/CCAo in 76 4-, 12-, and 20-month-old F344 rats; 41 (54%) animals experienced early, unexpected mortality. The remaining 35 (46%) animals had depth electrodes implanted. Prior to implantation of depth electrodes, 9 (26%) of these 35 animals (26%) were monitored periodically by video alone before video-EEG monitoring (17,837 h total) to assess the potential development of PSE. No EEG recordings were obtained from 12- or 20-month-old transient occlusion or 20-month-old permanent occlusion animals due to premature deaths. Five animals (14%) demonstrated epileptic seizure activity after MCA/CCAo: one 4-month-old transient occlusion animal, one 4-month-old permanent occlusion animal, and three 12-month-old permanent occlusion animals. Of these 5 animals, all but the 4-month-old permanent animal demonstrated 1-4 Hz spike-wave discharges variably associated with inactivity or frank motor arrest, and 2 animals (4- and 12-month-old permanent) demonstrated generalized ictal EEG discharges associated with grade 5 convulsive activity. All animals monitored with video-EEG demonstrated generalized 7-9 Hz spike-wave discharges, innate in F344 animals and distinct from lesion-induced epileptic seizures. Gross inspection of brains revealed variability in lesion presence and size among age groups and occlusion types. Comparison of infarct volumes of permanent MCA/CCAo animals (2.9 ± 1.29 mm3, n = 6) with those of transient MCA/CCAo animals (1.7 ± 0.31 mm3, n = 3) was not significant (p = 0.44) due to the small sample size. Timm staining revealed no evidence of mossy fiber sprouting in 7 animals tested, only one of which was known to be epileptic (4-month-old transient). These results provide evidence of focal nonconvulsive electrographic ictal discharges and behavioral seizures in both permanent and transient MCA/CCAo animals lesioned at 4- or 12-months-of-age and support the use of arterial ligation as a viable method for modeling PSE.

Hippocampal neuropeptide Y protein expression following controlled cortical impact and posttraumatic epilepsy.

This study assessed neuropeptide Y (NPY) expression in the hippocampus after long-term survival following traumatic brain injury (TBI) induced by controlled cortical impact (CCI) with or without the development of posttraumatic epilepsy (PTE). We hypothesized that following long-term survival after CCI, the severity of tissue injury and the development of PTE would correlate with the degree of hippocampal neurodegeneration as reflected by NPY+ and neuronal nuclear antigen (NeuN)+ cell loss. Adult Sprague-Dawley rats of 2-3 months of age were lesioned in the right parietal cortex and monitored for seizure activity by video and/or video-EEG. Neuropeptide Y and NeuN immunoreactivities (IRs) were quantified by light microscopy and semiautomatic image analysis approaches for unbiased quantification. Severely injured animals, marked by extensive tissue loss in the ipsilateral neocortex and adjacent hippocampus, resulted in significantly lower NeuN+ hilar cell density and NPY+ cell loss in the contralateral Cornu Ammonis (CA)-3 and dentate hilus (DH). The degree of NPY+ cell loss was more severe in CCI-injured animals with PTE than those animals that did not develop PTE. Mildly injured animals demonstrated no significant change of NPY expression compared with control animals. Our findings of long-term alterations of NPY expression in the hippocampus of severely brain-injured animals can provide important insights into the cellular and molecular consequences of severe TBI and posttraumatic epileptogenesis.

Poststroke epilepsy following transient unilateral middle cerebral and common carotid artery occlusion in young adult and aged F344 rats.

The mechanisms of injured brain that establish poststroke seizures and epilepsy are not well understood, largely because animal modeling has had limited development. The main objective of this study was to determine whether an arterial occlusion model of cortical stroke in young adult and aged rats was capable of generating either focal or generalized epileptic seizures within 2 months of lesioning. Four- and 20-month-old male Fischer 344 (F344) sham-operated controls and those lesioned by transient (3 h) unilateral middle cerebral artery (MCA) and common carotid artery (CCA) occlusion (MCA/CCAo) were studied by video-EEG recordings up to 2 months post-procedure. The main findings were: 1) seizures (grade 3 and above) were recorded within 2 months in both young (4-month; 0.23/h) and aged (20-month; 1.93/h) MCA/CCAo rat groups; both MCA/CCAo rat groups had more seizures recorded than the respective control groups, i.e., no seizures in young controls and 0.52/h in old controls; 2) both age and infarction independently had effects on seizure frequency; however, there was no demonstrated interaction between the two factors; and 3) there was no difference in infarct volumes comparing 4- to 20-month-old MCA/CCAo animals. In addition, all lesioned and sham-operated animals demonstrated intermittent solitary myoclonic convulsions arising out of sleep. Morbidity and mortality of animals limited the extent to which the animals could be evaluated, especially 20-month-old animals. These results suggest that transient unilateral MCA/CCAo can result in poststroke epileptic seizures in both young adult and aged F344 rats within a relatively brief period of time following lesioning.

A Biocatalytic Route to Highly Enantioenriched ß-Hydroxydioxinones.

A novel biocatalytic system to access a wide variety of ß-hydroxydioxinones from ß-ketodioxinones employing commercial engineered ketoreductases has been developed. This practical system provides a remarkably straightforward solution to limitations in accessing certain chemical scaffolds common in ß-hydroxydioxinones that are of great interest due to their diversification capabilities. A few highlights of this system are that it is high yielding, highly enantioselective, and chromatography-free. We have demonstrated both a wide substrate scope and a high degree of scalability.

Posttraumatic seizures and epilepsy in adult rats after controlled cortical impact.

Posttraumatic epilepsy (PTE) has been modeled with different techniques of experimental traumatic brain injury (TBI) using mice and rats at various ages. We hypothesized that the technique of controlled cortical impact (CCI) could be used to establish a model of PTE in young adult rats. A total of 156 male Sprague-Dawley rats of 2-3 months of age (128 CCI-injured and 28 controls) was used for monitoring and/or anatomical studies. Provoked class 3-5 seizures were recorded by video monitoring in 7/57 (12.3%) animals in the week immediately following CCI of the right parietal cortex; none of the 7 animals demonstrated subsequent spontaneous convulsive seizures. Monitoring with video and/or video-EEG was performed on 128 animals at various time points 8-619 days beyond one week following CCI during which 26 (20.3%) demonstrated nonconvulsive or convulsive epileptic seizures. Nonconvulsive epileptic seizures of >10s were demonstrated in 7/40 (17.5%) animals implanted with 2 or 3 depth electrodes and usually characterized by an initial change in behavior (head raising or animal alerting) followed by motor arrest during an ictal discharge that consisted of high-amplitude spikes or spike-waves with frequencies ranging between 1 and 2Hz class 3-5 epileptic seizures were recorded by video monitoring in 17/88 (19%) and by video-EEG in 2/40 (5%) CCI-injured animals. Ninety of 156 (58%) animals (79 CCI-injured, 13 controls) underwent transcardial perfusion for gross and microscopic studies. CCI caused severe brain tissue loss and cavitation of the ipsilateral cerebral hemisphere associated with cell loss in the hippocampal CA1 and CA3 regions, hilus, and dentate granule cells, and thalamus. All Timm-stained CCI-injured brains demonstrated ipsilateral hippocampal mossy fiber sprouting in the inner molecular layer. These results indicate that the CCI model of TBI in adult rats can be used to study the structure-function relationships that underlie epileptogenesis and PTE.

Effects of age and cortical infarction on EEG dynamic changes associated with spike wave discharges in F344 rats.

Rodent models of absence seizures are used to investigate the network properties and regulatory mechanisms of the seizure's generalized spike and wave discharge (SWD). As rats age, SWDs occur more frequently, suggesting aging-related changes in the regulation of the corticothalamic mechanisms generating the SWD. We hypothesized that brain resetting mechanisms - how the brain "resets" itself to a more normal functional state following a transient period of abnormal function, e.g., a SWD - are impaired in aged animals and that brain infarction would further affect these resetting mechanisms. The main objective of this study was to determine the effects of aging, infarction, and their potential interaction on the resetting of EEG dynamics assessed by quantitative EEG (qEEG) measures of linear (signal energy measured by amplitude variation; signal frequency measured by mean zero-crossings) and nonlinear (signal complexity measured by the pattern match regularity statistic and the short-term maximum Lyapunov exponent) brain EEG dynamics in 4- and 20-month-old F344 rats with and without brain infarction. The main findings of the study were: 1) dynamic resetting of both linear and nonlinear EEG characteristics occurred following SWDs; 2) animal age significantly affected the degree of dynamic resetting in all four qEEG measures: SWDs in older rats exhibited a lower degree of dynamic resetting; 3) infarction significantly affected the degree of dynamic resetting only in terms of EEG signal complexity: SWDs in infarcted rats exhibited a lower degree of dynamic resetting; and 4) in all four qEEG measures, there was no significant interaction effect between age and infarction on dynamic resetting. We conclude that recovery of the brain to its interictal state following SWDs was better in young adult animals compared with aged animals, and to a lesser degree, in age-matched controls compared with infarction-injured animal groups, suggesting possible effects of brain resetting mechanisms and/or the disruption of the epileptogenic network that triggers SWDs.

Alterations of GABA(A) and glutamate receptor subunits and heat shock protein in rat hippocampus following traumatic brain injury and in posttraumatic epilepsy.

Traumatic brain injury (TBI) can result in the development of posttraumatic epilepsy (PTE). Recently, we reported differential alterations in tonic and phasic GABA(A) receptor (GABA(A)R) currents in hippocampal dentate granule cells 90 days after controlled cortical impact (CCI) (Mtchedlishvili et al., 2010). In the present study, we investigated long-term changes in the protein expression of GABA(A)R α1, α4, γ2, and δ subunits, NMDA (NR2B) and AMPA (GluR1) receptor subunits, and heat shock proteins (HSP70 and HSP90) in the hippocampus of Sprague-Dawley rats evaluated by Western blotting in controls, CCI-injured animals without PTE (CCI group), and CCI-injured animals with PTE (PTE group). No differences were found among all three groups for α1 and α4 subunits. Significant reduction of γ2 protein was observed in the PTE group compared to control. CCI caused a 194% and 127% increase of δ protein in the CCI group compared to control (p<0.0001), and PTE (p<0.0001) groups, respectively. NR2B protein was increased in CCI and PTE groups compared to control (p=0.0001, and p=0.011, respectively). GluR1 protein was significantly decreased in CCI and PTE groups compared to control (p=0.003, and p=0.001, respectively), and in the PTE group compared to the CCI group (p=0.036). HSP70 was increased in CCI and PTE groups compared to control (p=0.014, and p=0.005, respectively); no changes were found in HSP90 expression. These results provide for the first time evidence of long-term alterations of GABA(A) and glutamate receptor subunits and a HSP following CCI.

Classification-based approach for management of an adolescent with LBP, lower extremity pain, and a relevant postural deformity.

A 15-year-old female presented to physical therapy 2 months after a traumatic injury with severe low back pain, referred unilateral leg pain below the knee, and a lumbar lateral shift. A classification-based approach, mechanical diagnosis and therapy, was utilized to identify the patient with respect to a particular subgroup, and interventions were applied according to symptomatic response to loading strategies, sustained and repeated movements. Outcome measures included the Modified Oswestry Disability Questionnaire (MODQ) and a visual analog scale (VAS) for pain rating. After nine visits (4 weeks), there was a 60% improvement in MODQ score, 0/10 pain on the VAS, and the patient resumed participation in recreational activities. At the 1-year follow-up, there was a 78% overall improvement in MODQ score with continued 0/10 rated pain. Improvements after nine visits and at the 1-year follow-up both exceeded the minimal clinically important difference for the MODQ.

A robust spike and wave algorithm for detecting seizures in a genetic absence seizure model.

Animal Models are used extensively in basic epilepsy research. In many studies, there is a need to accurately score and quantify all epileptic spike and wave discharges (SWDs) as captured by electroencephalographic (EEG) recordings. Manual scoring of long term EEG recordings is a time-consuming and tedious task that requires inordinate amount of time of laboratory personnel and an experienced electroencephalographer. In this paper, we adapt a SWD detection algorithm, originally proposed by the authors for absence (petit mal) seizure detection in humans, to detect SWDs appearing in EEG recordings of Fischer 334 rats. The algorithm is robust with respect to the threshold parameters. Results are compared to manual scoring and the effect of different threshold parameters is discussed.