Global analysis of Poales diversification - parallel evolution in space and time into open and closed habitats.

Poales are one of the most species-rich, ecologically and economically important orders of plants and often characterise open habitats, enabled by unique suites of traits. We test six hypotheses regarding the evolution and assembly of Poales in open and closed habitats throughout the world, and examine whether diversification patterns demonstrate parallel evolution. We sampled 42% of Poales species and obtained taxonomic and biogeographic data from the World Checklist of Vascular Plants database, which was combined with open/closed habitat data scored by taxonomic experts. A dated supertree of Poales was constructed. We integrated spatial phylogenetics with regionalisation analyses, historical biogeography and ancestral state estimations. Diversification in Poales and assembly of open and closed habitats result from dynamic evolutionary processes that vary across lineages, time and space, most prominently in tropical and southern latitudes. Our results reveal parallel and recurrent patterns of habitat and trait transitions in the species-rich families Poaceae and Cyperaceae. Smaller families display unique and often divergent evolutionary trajectories. The Poales have achieved global dominance via parallel evolution in open habitats, with notable, spatially and phylogenetically restricted divergences into strictly closed habitats.

Quantum magnetic imaging of iron organelles within the pigeon cochlea.

The ability of pigeons to sense geomagnetic fields has been conclusively established despite a notable lack of determination of the underlying biophysical mechanisms. Quasi-spherical iron organelles previously termed "cuticulosomes" in the cochlea of pigeons have potential relevance to magnetoreception due to their location and iron composition; however, data regarding the magnetic susceptibility of these structures are currently limited. Here quantum magnetic imaging techniques are applied to characterize the magnetic properties of individual iron cuticulosomes in situ. The stray magnetic fields emanating from cuticulosomes are mapped and compared to a detailed analytical model to provide an estimate of the magnetic susceptibility of the individual particles. The images reveal the presence of superparamagnetic and ferrimagnetic domains within individual cuticulosomes and magnetic susceptibilities within the range 0.029 to 0.22. These results provide insights into the elusive physiological roles of cuticulosomes. The susceptibilities measured are not consistent with a torque-based model of magnetoreception, placing iron storage and stereocilia stabilization as the two leading putative cuticulosome functions. This work establishes quantum magnetic imaging as an important tool to complement the existing array of techniques used to screen for potential magnetic particle-based magnetoreceptor candidates.

Mesenchymal stromal cells-derived extracellular vesicles reprogramme macrophages in ARDS models through the miR-181a-5p-PTEN-pSTAT5-SOCS1 axis.

RATIONALE: A better understanding of the mechanism of action of mesenchymal stromal cells (MSCs) and their extracellular vesicles (EVs) is needed to support their use as novel therapies for acute respiratory distress syndrome (ARDS). Macrophages are important mediators of ARDS inflammatory response. Suppressor of cytokine signalling (SOCS) proteins are key regulators of the macrophage phenotype switch. We therefore investigated whether SOCS proteins are involved in mediation of the MSC effect on human macrophage reprogramming. METHODS: Human monocyte-derived macrophages (MDMs) were stimulated with lipopolysaccharide (LPS) or plasma samples from patients with ARDS (these samples were previously classified into hypo-inflammatory and hyper-inflammatory phenotype) and treated with MSC conditioned medium (CM) or EVs. Protein expression was measured by Western blot. EV micro RNA (miRNA) content was determined by miRNA sequencing. In vivo: LPS-injured C57BL/6 mice were given EVs isolated from MSCs in which miR-181a had been silenced by miRNA inhibitor or overexpressed using miRNA mimic. RESULTS: EVs were the key component of MSC CM responsible for anti-inflammatory modulation of human macrophages. EVs significantly reduced secretion of tumour necrosis factor-α and interleukin-8 by LPS-stimulated or ARDS plasma-stimulated MDMs and this was dependent on SOCS1. Transfer of miR-181a in EVs downregulated phosphatase and tensin homolog (PTEN) and subsequently activated phosphorylated signal transducer and activator of transcription 5 (pSTAT5) leading to upregulation of SOCS1 in macrophages. In vivo, EVs alleviated lung injury and upregulated pSTAT5 and SOCS1 expression in alveolar macrophages in a miR181-dependent manner. Overexpression of miR-181a in MSCs significantly enhanced therapeutic efficacy of EVs in this model. CONCLUSION: miR-181a-PTEN-pSTAT5-SOCS1 axis is a novel pathway responsible for immunomodulatory effect of MSC EVs in ARDS.

Mini-XT, a miniaturized tagmentation-based protocol for efficient sequencing of SARS-CoV-2.

BACKGROUND: The COVID-19 pandemic has highlighted the importance of whole genome sequencing (WGS) of SARS-CoV-2 to inform public health policy. By enabling definition of lineages it facilitates tracking of the global spread of the virus. The evolution of new variants can be monitored and knowledge of specific mutations provides insights into the mechanisms through which the virus increases transmissibility or evades immunity. To date almost 1 million SARS-CoV-2 genomes have been sequenced by members of the COVID-19 Genomics UK (COG-UK) Consortium. To achieve similar feats in a more cost-effective and sustainable manner in future, improved high throughput virus sequencing protocols are required. We have therefore developed a miniaturized library preparation protocol with drastically reduced consumable use and costs. RESULTS: We present the 'Mini-XT' miniaturized tagmentation-based library preparation protocol available on protocols.io ( https://doi.org/10.17504/protocols.io.bvntn5en ). SARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 multiplex RT-PCR protocol and purified using a conventional liquid handling system. Acoustic liquid transfer (Echo 525) was employed to reduce reaction volumes and the number of tips required for a Nextera XT library preparation. Sequencing was performed on an Illumina MiSeq. The final version of Mini-XT has been used to sequence 4384 SARS-CoV-2 samples from N. Ireland with a COG-UK QC pass rate of 97.4%. Sequencing quality was comparable and lineage calling consistent for replicate samples processed with full volume Nextera DNA Flex (333 samples) or using nanopore technology (20 samples). SNP calling between Mini-XT and these technologies was consistent and sequences from replicate samples paired together in maximum likelihood phylogenetic trees. CONCLUSIONS: The Mini-XT protocol maintains sequence quality while reducing library preparation reagent volumes eightfold and halving overall tip usage from sample to sequence to provide concomitant cost savings relative to standard protocols. This will enable more efficient high-throughput sequencing of SARS-CoV-2 isolates and future pathogen WGS.

Acoustomicrofluidic Concentration and Signal Enhancement of Fluorescent Nanodiamond Sensors.

Diamond nitrogen-vacancy (NV) centers constitute a promising class of quantum nanosensors owing to the unique magneto-optic properties associated with their spin states. The large surface area and photostability of diamond nanoparticles, together with their relatively low synthesis costs, make them a suitable platform for the detection of biologically relevant quantities such as paramagnetic ions and molecules in solution. Nevertheless, their sensing performance in solution is often hampered by poor signal-to-noise ratios and long acquisition times due to distribution inhomogeneities throughout the analyte sample. By concentrating the diamond nanoparticles through an intense microcentrifugation effect in an acoustomicrofluidic device, we show that the resultant dense NV ensembles within the diamond nanoparticles give rise to an order-of-magnitude improvement in the measured acquisition time. The ability to concentrate nanoparticles under surface acoustic wave (SAW) microcentrifugation in a sessile droplet is, in itself, surprising given the well-documented challenge of achieving such an effect for particles below 1 µm in dimension. In addition to a demonstration of their sensing performance, we thus reveal in this work that the reason why the diamond nanoparticles readily concentrate under the SAW-driven recirculatory flow can be attributed to their considerably higher density and hence larger acoustic contrast compared to those for typical particles and cells for which the SAW microcentrifugation flow has been shown to date.

Preferential coupling of diamond NV centres in step-index fibres.

Diamonds containing the negatively charged nitrogen-vacancy centre are a promising system for room-temperature magnetometry. The combination of nano- and micro-diamond particles with optical fibres provides an option for deploying nitrogen-vacancy magnetometers in harsh and challenging environments. Here we numerically explore the coupling efficiency from nitrogen-vacancy centres within a diamond doped at the core/clad interface across a range of commercially available fibre types so as to inform the design process for a diamond in fibre magnetometers. We determine coupling efficiencies from nitrogen-vacancy centres to the guided modes of a step-index fibre and predict the optically detected magnetic resonance (ODMR) generated by a ensemble of four nitrogen-vacancy centres in this hybrid fibre system. Our results show that the coupling efficiency is enhanced with a high refractive index difference between the fibre core and cladding and depends on the radial position of the nitrogen-vacancy centres in the fibre core. Our ODMR simulations show that due to the preferential coupling of the nitrogen-vacancy emission to the fibre guided modes, certain magnetometry features such as ODMR contrast can be enhanced and lead to improved sensitivity in such diamond-fibre systems, relative to conventional diamond only ensemble geometries.

Re-examining ferritin-bound iron: current and developing clinical tools.

Iron is a highly important metal ion cofactor within the human body, necessary for haemoglobin synthesis, and required by a wide range of enzymes for essential metabolic processes. Iron deficiency and overload both pose significant health concerns and are relatively common world-wide health hazards. Effective measurement of total iron stores is a primary tool for both identifying abnormal iron levels and tracking changes in clinical settings. Population based data is also essential for tracking nutritional trends. This review article provides an overview of the strengths and limitations associated with current techniques for diagnosing iron status, which sets a basis to discuss the potential of a new serum marker - ferritin-bound iron - and the improvement it could offer to iron assessment.

Single-cell transcriptomic profiling provides insights into retinal endothelial barrier properties.

Purpose: To better characterize retinal endothelial barrier properties through analysis of individual transcriptomes of primary bovine retinal microvascular endothelial cells (RMECs). Methods: Individual RMECs were captured on the Fluidigm C1 system, cDNA libraries were prepared using a Nextera XT kit, and sequencing was performed on a NextSeq system (Illumina). Data analysis was performed using R packages Scater, SC3, and Seurat, and the browser application Automated Single-cell Analysis Pipeline (ASAP). Alternative splicing events in single cells were quantified with Outrigger. Cytoscape was used for network analyses. Results: Application of a single-cell RNA sequencing (scRNA-seq) analysis workflow showed that RMECs form a relatively homogeneous population in culture, with the main differences related to proliferation status. Expression of markers from along the arteriovenous tree suggested that most cells originated from capillaries. Average gene expression levels across all cells were used to develop an in silico model of the inner blood-retina barrier incorporating junctional proteins not previously reported within the retinal vasculature. Correlation of barrier gene expression among individual cells revealed a subgroup of genes highly correlated with PECAM-1 at the center of the correlation network. Numerous alternative splicing events involving exons within microvascular barrier genes were observed, and in many cases, individual cells expressed one isoform exclusively. Conclusions: We optimized a workflow for single-cell transcriptomics in primary RMECs. The results provide fundamental insights into the genes involved in formation of the retinal-microvascular barrier.

A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma.

BACKGROUND: Circulating microRNAs (miRNAs) are attractive non-invasive biomarkers for a variety of conditions due to their stability and altered pathophysiological expression levels. Reliable detection of global expression profiles is required to maximise miRNA biomarker discovery. Although developments in small RNA-Seq technology have improved detection of plasma-based miRNAs, the low RNA content and sequencing bias introduced during library preparation remain challenging. In this study we compare commercially available RNA extraction methods using MagnaZol (Bioo Scientific) or miRNeasy (QIAGEN) and three library preparation methods - CleanTag (TriLink), NEXTflex (Bioo Scientific) and QIAseq (QIAGEN) - which aim to address one or both of these issues. RESULTS: Different RNA extractions and library preparation protocols result in differential detection of miRNAs. A greater proportion of reads mapped to miRNAs in libraries prepared with MagnaZol RNA than with miRNeasy RNA. Libraries prepared using QIAseq demonstrated the greatest miRNA diversity with many more very low abundance miRNAs detected (~ 2-3 fold more with < 10 reads), whilst CleanTag detected the fewest individual miRNAs and considerably over-represented miR-486-5p. Libraries prepared with QIAseq had the strongest correlation with RT-qPCR quantification. Analysis of unique molecular indices (UMIs) incorporated in the QIAseq protocol indicate that little PCR bias is introduced during small RNA library preparation. CONCLUSIONS: Small RNAs were consistently detected using all RNA extraction and library preparation protocols tested, but with some miRNAs at significantly different levels. Choice of the most suitable protocol should be informed by the relative importance of minimising the total sequencing required, detection of rare miRNAs or absolute quantification.

Quantum Sensing in a Physiological-Like Cell Niche Using Fluorescent Nanodiamonds Embedded in Electrospun Polymer Nanofibers.

Fluorescent nanodiamonds (fNDs) containing nitrogen vacancy (NV) centers are promising candidates for quantum sensing in biological environments. This work describes the fabrication and implementation of electrospun poly lactic-co-glycolic acid (PLGA) nanofibers embedded with fNDs for optical quantum sensing in an environment, which recapitulates the nanoscale architecture and topography of the cell niche. A protocol that produces uniformly dispersed fNDs within electrospun nanofibers is demonstrated and the resulting fibers are characterized using fluorescent microscopy and scanning electron microscopy (SEM). Optically detected magnetic resonance (ODMR) and longitudinal spin relaxometry results for fNDs and embedded fNDs are compared. A new approach for fast detection of time varying magnetic fields external to the fND embedded nanofibers is demonstrated. ODMR spectra are successfully acquired from a culture of live differentiated neural stem cells functioning as a connected neural network grown on fND embedded nanofibers. This work advances the current state of the art in quantum sensing by providing a versatile sensing platform that can be tailored to produce physiological-like cell niches to replicate biologically relevant growth environments and fast measurement protocols for the detection of co-ordinated endogenous signals from clinically relevant populations of electrically active neuronal circuits.

Rapid, High-Resolution Magnetic Microscopy of Single Magnetic Microbeads.

Magnetic microparticles or "beads" are used in a variety of research applications from cell sorting through to optical force traction microscopy. The magnetic properties of such particles can be tailored for specific applications with the uniformity of individual beads critical to their function. However, the majority of magnetic characterization techniques quantify the magnetic properties from large bead ensembles. Developing new magnetic imaging techniques to evaluate and visualize the magnetic fields from single beads will allow detailed insight into the magnetic uniformity, anisotropy, and alignment of magnetic domains. Here, diamond-based magnetic microscopy is applied to image and characterize individual magnetic beads with varying magnetic and structural properties: ferromagnetic and superparamagnetic/paramagnetic, shell (coated with magnetic material), and solid (magnetic material dispersed in matrix). The single-bead magnetic images identify irregularities in the magnetic profiles from individual bead populations. Magnetic simulations account for the varying magnetic profiles and allow to infer the magnetization of individual beads. Additionally, this work shows that the imaging technique can be adapted to achieve illumination-free tracking of magnetic beads, opening the possibility of tracking cell movements and mechanics in photosensitive contexts.

Proximity-Induced Artefacts in Magnetic Imaging with Nitrogen-Vacancy Ensembles in Diamond.

Magnetic imaging with ensembles of nitrogen-vacancy (NV) centres in diamond is a recently developed technique that allows for quantitative vector field mapping. Here we uncover a source of artefacts in the measured magnetic field in situations where the magnetic sample is placed in close proximity (a few tens of nm) to the NV sensing layer. Using magnetic nanoparticles as a test sample, we find that the measured field deviates significantly from the calculated field, in shape, amplitude and even in sign. By modelling the full measurement process, we show that these discrepancies are caused by the limited measurement range of NV sensors combined with the finite spatial resolution of the optical readout. We numerically investigate the role of the stand-off distance to identify an artefact-free regime, and discuss an application to ultrathin materials. This work provides a guide to predict and mitigate proximity-induced artefacts that can arise in NV-based wide-field magnetic imaging, and also demonstrates that the sensitivity of these artefacts to the sample can make them a useful tool for magnetic characterisation.

Nanomechanical Sensing Using Spins in Diamond.

Nanomechanical sensors and quantum nanosensors are two rapidly developing technologies that have diverse interdisciplinary applications in biological and chemical analysis and microscopy. For example, nanomechanical sensors based upon nanoelectromechanical systems (NEMS) have demonstrated chip-scale mass spectrometry capable of detecting single macromolecules, such as proteins. Quantum nanosensors based upon electron spins of negatively charged nitrogen-vacancy (NV) centers in diamond have demonstrated diverse modes of nanometrology, including single molecule magnetic resonance spectroscopy. Here, we report the first step toward combining these two complementary technologies in the form of diamond nanomechanical structures containing NV centers. We establish the principles for nanomechanical sensing using such nanospin-mechanical sensors (NSMS) and assess their potential for mass spectrometry and force microscopy. We predict that NSMS are able to provide unprecedented AC force images of cellular biomechanics and to not only detect the mass of a single macromolecule but also image its distribution. When combined with the other nanometrology modes of the NV center, NSMS potentially offer unparalleled analytical power at the nanoscale.

MicroRNA-containing extracellular vesicles released from endothelial colony-forming cells modulate angiogenesis during ischaemic retinopathy.

Endothelial colony-forming cells (ECFCs) are a defined subtype of endothelial progenitors that modulate vascular repair and promote perfusion in ischaemic tissues. Their paracrine activity on resident vasculature is ill-defined, but mediated, at least in part, by the transfer of extracellular vesicles (EVs). To evaluate the potential of isolated EVs to provide an alternative to cell-based therapies, we first performed a physical and molecular characterization of those released by ECFCs. Their effects upon endothelial cells in vitro and angiogenesis in vivo in a model of proliferative retinopathy were assessed. The EVs expressed typical markers CD9 and CD63 and formed a heterogeneous population ranging in size from ~60 to 1500 nm by electron microscopy. ECFC EVs were taken up by endothelial cells and increased cell migration. This was reflected by microarray analyses which showed significant changes in expression of genes associated with angiogenesis. Sequencing of small RNAs in ECFCs and their EVs showed that multiple microRNAs are highly expressed and concentrated in EVs. The functional categories significantly enriched for the predicted target genes of these microRNAs included angiogenesis. Intravitreally delivered ECFC EVs were associated with the vasculature and significantly reduced the avascular area in a mouse oxygen-induced retinopathy model. Our findings confirm the potential of isolated EVs to influence endothelial cell function and act as a therapy to modulate angiogenesis. The functions associated with the specific microRNAs detected in ECFC EVs support a role for microRNA transfer in mediating the observed effects.

Environmentally Mediated Coherent Control of a Spin Qubit in Diamond.

The coherent control of spin qubits forms the basis of many applications in quantum information processing and nanoscale sensing, imaging, and spectroscopy. Such control is conventionally achieved by direct driving of the qubit transition with a resonant global field, typically at microwave frequencies. Here we introduce an approach that relies on the resonant driving of nearby environment spins, whose localized magnetic field in turn drives the qubit when the environmental spin Rabi frequency matches the qubit resonance. This concept of environmentally mediated resonance (EMR) is explored experimentally using a qubit based on a single nitrogen-vacancy (NV) center in diamond, with nearby electronic spins serving as the environmental mediators. We demonstrate EMR driven coherent control of the NV spin state, including the observation of Rabi oscillations, free induction decay, and spin echo. This technique also provides a way to probe the nanoscale environment of spin qubits, which we illustrate by acquisition of electron spin resonance spectra from single NV centers in various settings.

Scanning Nanospin Ensemble Microscope for Nanoscale Magnetic and Thermal Imaging.

Quantum sensors based on solid-state spins provide tremendous opportunities in a wide range of fields from basic physics and chemistry to biomedical imaging. However, integrating them into a scanning probe microscope to enable practical, nanoscale quantum imaging is a highly challenging task. Recently, the use of single spins in diamond in conjunction with atomic force microscopy techniques has allowed significant progress toward this goal, but generalization of this approach has so far been impeded by long acquisition times or by the absence of simultaneous topographic information. Here, we report on a scanning quantum probe microscope which solves both issues by employing a nanospin ensemble hosted in a nanodiamond. This approach provides up to an order of magnitude gain in acquisition time while preserving sub-100 nm spatial resolution both for the quantum sensor and topographic images. We demonstrate two applications of this microscope. We first image nanoscale clusters of maghemite particles through both spin resonance spectroscopy and spin relaxometry, under ambient conditions. Our images reveal fast magnetic field fluctuations in addition to a static component, indicating the presence of both superparamagnetic and ferromagnetic particles. We next demonstrate a new imaging modality where the nanospin ensemble is used as a thermometer. We use this technique to map the photoinduced heating generated by laser irradiation of a single gold nanoparticle in a fluid environment. This work paves the way toward new applications of quantum probe microscopy such as thermal/magnetic imaging of operating microelectronic devices and magnetic detection of ion channels in cell membranes.

Enrichment of pathogenic alleles in the brittle cornea gene, ZNF469, in keratoconus.

Keratoconus, a common inherited ocular disorder resulting in progressive corneal thinning, is the leading indication for corneal transplantation in the developed world. Genome-wide association studies have identified common SNPs 100 kb upstream of ZNF469 strongly associated with corneal thickness. Homozygous mutations in ZNF469 and PR domain-containing protein 5 (PRDM5) genes result in brittle cornea syndrome (BCS) Types 1 and 2, respectively. BCS is an autosomal recessive generalized connective tissue disorder associated with extreme corneal thinning and a high risk of corneal rupture. Some individuals with heterozygous PRDM5 mutations demonstrate a carrier ocular phenotype, which includes a mildly reduced corneal thickness, keratoconus and blue sclera. We hypothesized that heterozygous variants in PRDM5 and ZNF469 predispose to the development of isolated keratoconus. We found a significant enrichment of potentially pathologic heterozygous alleles in ZNF469 associated with the development of keratoconus (P = 0.00102) resulting in a relative risk of 12.0. This enrichment of rare potentially pathogenic alleles in ZNF469 in 12.5% of keratoconus patients represents a significant mutational load and highlights ZNF469 as the most significant genetic factor responsible for keratoconus identified to date.

Detection of atomic spin labels in a lipid bilayer using a single-spin nanodiamond probe.

Magnetic field fluctuations arising from fundamental spins are ubiquitous in nanoscale biology, and are a rich source of information about the processes that generate them. However, the ability to detect the few spins involved without averaging over large ensembles has remained elusive. Here, we demonstrate the detection of gadolinium spin labels in an artificial cell membrane under ambient conditions using a single-spin nanodiamond sensor. Changes in the spin relaxation time of the sensor located in the lipid bilayer were optically detected and found to be sensitive to near-individual (4 ± 2) proximal gadolinium atomic labels. The detection of such small numbers of spins in a model biological setting, with projected detection times of 1 s [corresponding to a sensitivity of ∼5 Gd spins per Hz(1/2)], opens a pathway for in situ nanoscale detection of dynamical processes in biology.

Whole-mitochondrial genome sequencing in primary open-angle glaucoma using massively parallel sequencing identifies novel and known pathogenic variants.

PURPOSE: The aim of this study was to determine whether mutations in mitochondrial DNA play a role in high-pressure primary open-angle glaucoma (OMIM 137760) by analyzing new data from massively parallel sequencing of mitochondrial DNA. METHODS: Glaucoma patients with high-tension primary open-angle glaucoma and ethnically matched and age-matched control subjects without glaucoma were recruited. The entire human mitochondrial genome was amplified in two overlapping fragments by long-range polymerase chain reaction and used as a template for massively parallel sequencing on an Ion Torrent Personal Genome Machine. All variants were confirmed by conventional Sanger sequencing. RESULTS: Whole-mitochondrial genome sequencing was performed in 32 patients with primary open-angle glaucoma from India (n = 16) and Ireland (n = 16). In 16 of the 32 patients with primary open-angle glaucoma (50% of cases), there were 22 mitochondrial DNA mutations consisting of 7 novel mutations and 8 previously reported disease-associated sequence variants. Eight of 22 (36.4%) of the mitochondrial DNA mutations were in complex I mitochondrial genes. CONCLUSION: Massively parallel sequencing using the Ion Torrent Personal Genome Machine with confirmation by Sanger sequencing detected a pathogenic mitochondrial DNA mutation in 50% of the primary open-angle glaucoma cohort. Our findings support the emerging concept that mitochondrial dysfunction results in the development of glaucoma and, more specifically, that complex I defects play a significant role in primary open-angle glaucoma pathogenesis.

Guidelines for ethical behavior relating to clinical practice issues in neuromuscular and electrodiagnostic medicine.

The American Association of Neuromuscular & Electrodiagnostic Medicine (AANEM) developed guidelines to formalize the ethical standards that neuromuscular and electrodiagnostic (EDx) physicians should observe in their clinical and scientific activities. Neuromuscular and EDx medicine is a subspecialty of medicine that focuses on evaluation, diagnosis, and comprehensive medical management, including rehabilitation of individuals with neuromuscular disorders. Physicians working in this subspecialty focus on disorders of the motor unit, including muscle, neuromuscular junction, axon, plexus, nerve root, anterior horn cell, and the peripheral nerves (motor and sensory). The neuromuscular and EDx physician's goal is to diagnose and treat these conditions to mitigate their impact and improve the patient's quality of life. The guidelines are consistent with the Principles of Medical Ethics adopted by the American Medical Association and represent a revision of previous AANEM guidelines.