A Novel Gene Fusion YLPM1::PRKD1 Identified in a Cribriform Subtype of Polymorphous Adenocarcinoma.

Cribriform adenocarcinoma of the salivary gland (CASG) is an entity that is currently classified under polymorphous adenocarcinoma (PAC), cribriform subtype per the 2022 WHO classification of head and neck tumours. There is debate about whether CASG should be considered a separate diagnostic entity, as CASG differs from conventional PAC in anatomic site, clinical behaviors, and molecular patterns. Herein we describe a challenging and unique case which shares histologic and behavioral features between CASG and conventional PAC with a YLPM1::PRKD1 rearrangement not previously reported in the literature.

Rates of Cognitive Decline in 100 Patients With Alzheimer Disease.

Background: In the state of Louisiana, the prevalence of Alzheimer disease (AD) is projected to increase 26.4% by 2025 because of the rapidly increasing geriatric population. While significant research is available on risk factors for developing AD, less data are available regarding AD progression and the rate of change among patients with the disease. To date, no research has established the baseline cognitive decline of patients with AD residing in New Orleans, Louisiana. Methods: We evaluated 100 patients in the Ochsner Health system from September 2013 to December 2019 who had a diagnosis of AD and repeated Mini-Mental State Examination (MMSE) or Montreal Cognitive Assessment (MoCA) scores to determine annual rates of decline. Associated variables that were analyzed included race, age at diagnosis, social factors, and comorbidities. Results: The average annual rates of decline for MMSE and MoCA scores were 2.43 (SD 2.82) points and 2.39 (SD 1.88) points, respectively. Our results were significant for a faster rate of decline in MMSE scores among smokers (3.50 points, SD 3.20) vs nonsmokers (1.54 points, SD 2.07). We found no significant difference in MoCA scores for smokers vs nonsmokers, in addition to other demographic and lifestyle variables. Conclusion: The rate of decline seen in an urban population of patients with AD is lower than the average rate of decline reported in the literature, a finding that can help inform future interventional studies that use rate of decline as a primary outcome.

The Molecular Basis and Pathophysiology of Trigeminal Neuralgia.

Trigeminal neuralgia (TN) is a complex orofacial pain syndrome characterized by the paroxysmal onset of pain attacks in the trigeminal distribution. The underlying mechanism for this debilitating condition is still not clearly understood. Decades of basic and clinical evidence support the demyelination hypothesis, where demyelination along the trigeminal afferent pathway is a major driver for TN pathogenesis and pathophysiology. Such pathological demyelination can be triggered by physical compression of the trigeminal ganglion or another primary demyelinating disease, such as multiple sclerosis. Further examination of TN patients and animal models has revealed significant molecular changes, channelopathies, and electrophysiological abnormalities in the affected trigeminal nerve. Interestingly, recent electrophysiological recordings and advanced functional neuroimaging data have shed new light on the global structural changes and the altered connectivity in the central pain-related circuits in TN patients. The current article aims to review the latest findings on the pathophysiology of TN and cross-examining them with the current surgical and pharmacologic management for TN patients. Understanding the underlying biology of TN could help scientists and clinicians to identify novel targets and improve treatments for this complex, debilitating disease.

Ultrasound-guided regional anesthesia: feasibility and effectiveness of teaching via telesimulation in Ethiopia.

BACKGROUND: Acute pain management in resource-poor countries remains a challenge. Ultrasound-guided regional anesthesia is a cost-effective way of delivering analgesia in these settings. However, for financial and logistical reasons, educational workshops are inaccessible to many physicians in these environments. Telesimulation provides a way of teaching across distance by using simulators and video-conferencing software to connect instructors and students worldwide. We conducted a prospective study to determine the feasibility of ultrasound-guided regional anesthesia teaching via telesimulation in Ethiopia. METHODS: Eighteen Ethiopian orthopedic and emergency medicine house staff participated in telesimulation teaching of ultrasound-guided femoral nerve block. This consisted of four 90-min sessions, once per week. Week 1 consisted of a precourse test and a presentation on aspects of performing a femoral nerve block, weeks 2 and 3 were live teaching sessions on scanning and needling techniques, and in week 4, the house staff undertook a postcourse test. All participants were assessed using a validated Global Rating Scale and Checklist. RESULTS: Participants were provided with a validated checklist and global rating scale as a pretest and post-test. The participants showed significant improvement in their test scores, from a total mean of 51% in the pretest to 84% in their post-test. CONCLUSIONS: Teaching ultrasound-guided regional anesthesia of the femoral nerve remotely via telesimulation is feasible. Telesimulation can greatly improve the accessibility of ultrasound-guided regional anesthesia teaching to physicians in remote areas.

TET-mediated 5-methylcytosine oxidation in tRNA promotes translation.

Oxidation of 5-methylcytosine (5mC) in DNA by the ten-eleven translocation (TET) family of enzymes is indispensable for gene regulation in mammals. More recently, evidence has emerged to support a biological function for TET-mediated m5C oxidation in messenger RNA. Here, we describe a previously uncharacterized role of TET-mediated m5C oxidation in transfer RNA (tRNA). We found that the TET-mediated oxidation product 5-hydroxylmethylcytosine (hm5C) is specifically enriched in tRNA inside cells and that the oxidation activity of TET2 on m5C in tRNAs can be readily observed in vitro. We further observed that hm5C levels in tRNA were significantly decreased in Tet2 KO mouse embryonic stem cells (mESCs) in comparison with wild-type mESCs. Reciprocally, induced expression of the catalytic domain of TET2 led to an obvious increase in hm5C and a decrease in m5C in tRNAs relative to uninduced cells. Strikingly, we also show that TET2-mediated m5C oxidation in tRNA promotes translation in vitro. These results suggest TET2 may influence translation through impacting tRNA methylation and reveal an unexpected role for TET enzymes in regulating multiple nodes of the central dogma.

Information about contact force and surface texture is mixed in the firing rates of cutaneous afferent neurons.

Cutaneous mechanoreceptors in our hands gather information about the objects we handle. Tactile fibers encode mixed information about contact events and object properties. Neural coding in tactile afferents is typically studied by varying a single aspect of tactile stimuli, avoiding the confounds of real-world haptic interactions. We instead record responses of small populations of dorsal root ganglia (DRG) neurons to variable tactile stimuli and find that neurons primarily respond to force, though some texture information can be detected. Tactile nerve fibers convey information about many features of haptic interactions, including the force and speed of contact, as well as the texture and shape of the objects being handled. How we perceive these object features is relatively unaffected by the forces and movements we use when interacting with the object. Because signals related to contact events and object properties are mixed in the responses of tactile fibers, our ability to disentangle these different components of our tactile experience implies that they are demultiplexed as they propagate along the neuraxis. To understand how texture and contact mechanics are encoded together by tactile fibers, we studied the activity of multiple neurons recorded simultaneously in the cervical DRG of two anesthetized rhesus monkeys while textured surfaces were applied to the glabrous skin of the fingers and palm using a handheld probe. A transducer at the tip of the textured probe measured contact forces as tactile stimuli were applied at different locations on the finger-pads and palm. We examined how a sample population of DRG neurons encode force and texture and found that firing rates of individual neurons are modulated by both force and texture. In particular, slowly adapting (SA) neurons were more responsive to force than texture, and rapidly adapting (RA) neurons were more responsive to texture than force. Although force could be decoded accurately throughout the entire contact interval, texture signals were most salient during onset and offset phases of the contact interval.NEW & NOTEWORTHY Cutaneous mechanoreceptors in our hands gather information about the objects we handle. Tactile fibers encode mixed information about contact events and object properties. Neural coding in tactile afferents is typically studied by varying a single aspect of tactile stimuli, avoiding the confounds of real-world haptic interactions. We instead record responses of small populations of DRG neurons to variable tactile stimuli and find that neurons primarily respond to force, though some texture information can be detected.

Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency.

Active DNA demethylation via ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency.

A comparison of provider perspectives on cultural competency training: A mixed methods study.

BACKGROUND: We aimed to identify differences in training among colorectal cancer physicians and advanced practice providers with high and low cultural competency METHODS: Using explanatory sequential mixed methods, we surveyed providers and dichotomized into high and low cultural competency (CC) groups, conducted qualitative interviews, and analyzed verbatim transcripts using deductive and inductive codes to compared findings across groups using a joint display. RESULTS: Fifty-four of 92 providers (59%) responded; 10 respondents from each group (20/36 invited) completed semi-structured interviews about previous CC trainings. Low CC providers' training included explanations of cultural differences that, in practice, improved awareness and utilization of communication tools, but they also desired decision-making tools and cultural exposure. High CC providers' training included action-oriented toolkits. In practice, they admitted failures, improved communication, and attributed patient behaviors to external factors. High CC providers desired performance evaluations. CONCLUSIONS: Behaviorally-oriented CC training offered a robust foundation for culturally competent care.

Hindlimb motor responses evoked by microstimulation of the lumbar dorsal root ganglia during quiet standing.

Somatosensory afferent pathways have been a target for neural prostheses that seek to restore sensory feedback from amputated limbs and to recruit muscles paralyzed by neurological injury. These pathways supply inputs to spinal reflex circuits that are necessary for coordinating muscle activity in the lower limb. The dorsal root ganglia (DRG) is a potential site for accessing sensory neurons because DRG microstimulation selectively recruits major nerve branches of the cat hindlimb. Previous DRG microstimulation experiments have been performed in anesthetized animals, but effects on muscle recruitment and behavior in awake animals have not been examined. OBJECTIVE: The objective of the current study was to measure the effects of DRG microstimulation on evoking changes in hindlimb muscle activity during quiet standing. APPROACH: In this study, 32-channel penetrating microelectrode arrays were implanted chronically in the left L6 and L7 DRG of four cats. During each week of testing, one DRG electrode was selected to deliver microstimulation pulse-trains during quiet standing. Electromyographic (EMG) signals were recorded from intramuscular electrodes in ten hindlimb muscles, and ground-reaction forces (GRF) were measured under the foot of the implanted limb. MAIN RESULTS: DRG Microstimulation evoked a mix of excitatory and inhibitory responses across muscles. Response rates were highest when microstimulation was applied on the L7 array, producing more excitatory than inhibitory responses. Response rates for the L6 array were lower, and the composition of responses was more evenly balanced between excitation and inhibition. On approximately one third of testing weeks, microstimulation induced a transient unloading of the hindlimb as indicated by a decrease in GRF. Reciprocal inhibition at the knee was a prevalent response pattern across testing days which contributed to the unloading force on this subset of testing weeks. SIGNIFICANCE: Results show that single-channel microstimulation in the lumbar DRG evokes stereotyped patterns of muscle recruitment in awake animals, demonstrating that even limited sensory input can elicit hindlimb behavior. These findings imply that DRG microstimulation may have utility in neural prosthetic applications aimed at restoring somatosensory feedback and promoting motor function after neurological injury.

A wearable neural interface for detecting and decoding attempted hand movements in a person with tetraplegia.

We are developing a wearable neural interface based on high-density surface electromyography (HDEMG) for detecting and decoding signals from spared motor units in the forearms of people with tetraplegia after spinal cord injury (SCI). A lightweight, form-fitting garment containing 150 disc electrodes and covering the entire forearm was used to map the myoelectric activity of forearm muscles during a wide range of voluntary tasks of a person with chronic tetraplegia after SCI (C5 motor and C6 sensory American Spinal Injury Association Impairment Scale B spinal cord injury). Despite exhibiting no overt finger motion, myoelectric signals were detectable for attempted movements of individual digits and were highly discriminable. Motor unit decomposition was used to identify the activity of >30 motor neurons, active specifically during rotation, pronation of the wrist (4 units), and flexion of the elbow joint (7 units), and during attempted movements of individual hand digits (1-5 units). In addition, we performed a neural connectivity analysis based on the power of the common oscillations of the identified motor neurons in the delta (~5Hz), alpha (~6-12 Hz), and beta bands (~15-30 Hz). This analysis showed clear common synaptic inputs to the identified motor neurons in all the analyzed frequency bands. This neural interface offers a new potential for the control of assistive technologies, whereby the motor neurons spared after SCI may serve as a direct readout of motor intent that allows proportional control over several distinct degrees of freedom. Moreover, this framework can be used to study the reorganization and recovery of spinal networks after injury and rehabilitation.

Selectivity and Promiscuity in TET-Mediated Oxidation of 5-Methylcytosine in DNA and RNA.

Enzymes of the ten-eleven translocation (TET) family add diversity to the repertoire of nucleobase modifications by catalyzing the oxidation of 5-methylcytosine (5mC). TET enzymes were initially found to oxidize 5-methyl-2'-deoxycytidine in genomic DNA, yielding products that contribute to epigenetic regulation in mammalian cells, but have since been found to also oxidize 5-methylcytidine in RNA. Considering the different configurations of single-stranded (ss) and double-stranded (ds) DNA and RNA that coexist in a cell, defining the scope of TET's preferred activity and the mechanisms of substrate selectivity is critical to better understand the enzymes' biological functions. To this end, we have systematically examined the activity of human TET2 on DNA, RNA, and hybrid substrates in vitro. We found that, while ssDNA and ssRNA are well tolerated, TET2 is most proficient at dsDNA oxidation and discriminates strongly against dsRNA. Chimeric and hybrid substrates containing mixed DNA and RNA character helped reveal two main features by which the enzyme discriminates between substrates. First, the identity of the target nucleotide alone is the strongest reactivity determinant, with a preference for 5-methyldeoxycytidine, while both DNA or RNA are relatively tolerated on the rest of the target strand. Second, while a complementary strand is not required for activity, DNA is the preferred partner, and complementary RNA diminishes reactivity. Our biochemical analysis, complemented by molecular dynamics simulations, provides support for an active site optimally configured for dsDNA reactivity but permissive for various nucleic acid configurations, suggesting a broad range of plausible roles for TET-mediated 5mC oxidation in cells.

Characteristics of accidental injuries from power tools treated at two emergency departments in Queensland.

OBJECTIVE: Injuries are a major burden on the Australian healthcare system. Power tool usage is a common cause of accidental injury. A better understanding of the trends of power tool injuries will inform prevention strategies and potentially mitigate costs. METHODS: The ED databases from two level 1 hospitals were reviewed for presentations between 2005 and 2015 resulting from accidental injury with power tools. A subgroup of patients presenting to one hospital between 2016 and 2017 were interviewed about the activities and circumstances that led to their injuries, and followed up 3 months later to assess outcomes. RESULTS: A total of 4057 cases of accidental injury from power tool use were identified. Power saws and grinders contributed to 54% of injuries. Most injuries were located on an upper limb (48%) or the head and neck (30%). Over half (54%) of all head injuries were associated with metal and wood fragments to the eye from grinders, drills and saws. Hospital admission rates were highest for patients aged >60 years. Injuries to females were <5% of all presentations, but 40% of those caused by lawnmowers. Among the 200 patients interviewed, lapses in concentration during use, and modification and inappropriate use of a power tool were the main contributors to injury. Recovery periods >3 months were common. CONCLUSIONS: Accidental injuries from power tool use have a considerable impact on ED resources and can affect the long-term quality of life of those injured. Effective education about safe usage and protection may prevent many injuries.

Exploiting Substrate Promiscuity To Develop Activity-Based Probes for Ten-Eleven Translocation Family Enzymes.

Ten-eleven translocation (TET) enzymes catalyze repeated oxidations of 5-methylcytosine in genomic DNA. Because of the challenges of tracking reactivity within a complex DNA substrate, chemical tools to probe TET activity are limited, despite these enzyme's crucial role in epigenetic regulation. Here, building on precedents from related Fe(II)/α-ketoglutarate-dependent dioxygenases, we show that TET enzymes can promiscuously act upon cytosine bases with unnatural 5-position modifications. Oxidation of 5-vinylcytosine (vC) in DNA results in the predominant formation of a 5-formylmethylcytosine product that can be efficiently labeled to provide an end-point read-out for TET activity. The reaction with 5-ethynylcytosine (eyC), moreover, results in the formation of a high-energy ketene intermediate that can selectively trap any active TET isoform as a covalent enzyme-DNA complex, even in the complex milieu of a total cell lysate. Exploiting substrate promiscuity therefore offers a new and needed means to directly track TET activity in vitro or in vivo.

Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase.

Here we present APOBEC-coupled epigenetic sequencing (ACE-seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds on the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-seq yielded high-confidence 5hmC profiles with at least 1,000-fold less DNA input than conventional methods. Applying ACE-seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, we found that 5hmC was almost entirely confined to CG dinucleotides. The whole-genome map permitted cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and revealed genomic features that diverged from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods, thus expanding the scope of epigenome profiling to include scarce samples and opening new lines of inquiry regarding the role of cytosine modifications in genome biology.

OGT binds a conserved C-terminal domain of TET1 to regulate TET1 activity and function in development.

TET enzymes convert 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized derivatives. TETs stably associate with and are post-translationally modified by the nutrient-sensing enzyme OGT, suggesting a connection between metabolism and the epigenome. Here, we show for the first time that modification by OGT enhances TET1 activity in vitro. We identify a TET1 domain that is necessary and sufficient for binding to OGT and report a point mutation that disrupts the TET1-OGT interaction. We show that this interaction is necessary for TET1 to rescue hematopoetic stem cell production in tet mutant zebrafish embryos, suggesting that OGT promotes TET1's function during development. Finally, we show that disrupting the TET1-OGT interaction in mouse embryonic stem cells changes the abundance of TET2 and 5-methylcytosine, which is accompanied by alterations in gene expression. These results link metabolism and epigenetic control, which may be relevant to the developmental and disease processes regulated by these two enzymes.

Harnessing natural DNA modifying activities for editing of the genome and epigenome.

The introduction of site-specific DNA modifications to the genome or epigenome presents great opportunities for manipulating biological systems. Such changes are now possible through the combination of DNA-modifying enzymes with targeting modules, including dCas9, that can localize the enzymes to specific sites. In this review, we take a DNA modifying enzyme-centric view of recent advances. We highlight the variety of natural DNA-modifying enzymes-including DNA methyltransferases, oxygenases, deaminases, and glycosylases-that can be used for targeted editing and discuss how insights into the structure and function of these enzymes has further expanded editing potential by introducing enzyme variants with altered activities or by improving spatiotemporal control of modifications.