Pseudotyped virus infection of multiplexed ACE2 libraries reveals SARS-CoV-2 variant shifts in receptor usage.

Pairwise compatibility between virus and host proteins can dictate the outcome of infection. During transmission, both inter- and intraspecies variabilities in receptor protein sequences can impact cell susceptibility. Many viruses possess mutable viral entry proteins and the patterns of host compatibility can shift as the viral protein sequence changes. This combinatorial sequence space between virus and host is poorly understood, as traditional experimental approaches lack the throughput to simultaneously test all possible combinations of protein sequences. Here, we created a pseudotyped virus infection assay where a multiplexed target-cell library of host receptor variants can be assayed simultaneously using a DNA barcode sequencing readout. We applied this assay to test a panel of 30 ACE2 orthologs or human sequence mutants for infectability by the original SARS-CoV-2 spike protein or the Alpha, Beta, Gamma, Delta, and Omicron BA1 variant spikes. We compared these results to an analysis of the structural shifts that occurred for each variant spike's interface with human ACE2. Mutated residues were directly involved in the largest shifts, although there were also widespread indirect effects altering interface structure. The N501Y substitution in spike conferred a large structural shift for interaction with ACE2, which was partially recreated by indirect distal substitutions in Delta, which does not harbor N501Y. The structural shifts from N501Y greatly influenced the set of animal orthologs the variant spike was capable of interacting with. Out of the thirteen non-human orthologs, ten exhibited unique patterns of variant-specific compatibility, demonstrating that spike sequence changes during human transmission can toggle ACE2 compatibility and potential susceptibility of other animal species, and cumulatively increase overall compatibilities as new variants emerge. These experiments provide a blueprint for similar large-scale assessments of protein compatibility during entry by diverse viruses. This dataset demonstrates the complex compatibility relationships that occur between variable interacting host and virus proteins.

Mammalian Genomic Manipulation with Orthogonal Bxb1 DNA Recombinase Sites for the Functional Characterization of Protein Variants.

The Bxb1 bacteriophage serine DNA recombinase is an efficient tool for engineering recombinant DNA into the genomes of cultured cells. Generally, a single engineered "landing pad" site is introduced into the cell genome, permitting the integration of transgenic circuits or libraries of transgene variants. While sufficient for many studies, the extent of genetic manipulation possible with a single recombinase site is limiting and insufficient for more complex cell-based assays. Here, we harnessed two orthogonal Bxb1 recombinase sites to enable alternative avenues for using mammalian synthetic biology to characterize transgenic protein variants. By designing plasmids flanked by a second pair of auxiliary recombination sites, we demonstrate that we can avoid the genomic integration of undesirable bacterial DNA elements using the same starting cells engineered for whole-plasmid integration. We also created "double landing pad" cells simultaneously harboring two orthogonal Bxb1 recombinase sites at separate genomic loci, allowing complex cell-based genetic assays. Integration of a genetically encoded calcium indicator allowed for the real-time monitoring of intracellular calcium signaling dynamics, including kinetic perturbations that occur upon overexpression of the wild-type or variant version of the calcium signaling relay protein STIM1. A panel of missense mutants of the HIV-1 accessory protein Vif was paired with various paralogs within the human Apobec3 innate immune protein family to identify combinations capable or incapable of interacting within cells. These cells allow transgenic protein variant libraries to be readily paired with assay-specific protein partners or biosensors, enabling new functional readouts for large-scale genetic assays for protein function.

Expanded ACE2 dependencies of diverse SARS-like coronavirus receptor binding domains.

Viral spillover from animal reservoirs can trigger public health crises and cripple the world economy. Knowing which viruses are primed for zoonotic transmission can focus surveillance efforts and mitigation strategies for future pandemics. Successful engagement of receptor protein orthologs is necessary during cross-species transmission. The clade 1 sarbecoviruses including Severe Acute Respiratory Syndrome-related Coronavirus (SARS-CoV) and SARS-CoV-2 enter cells via engagement of angiotensin converting enzyme-2 (ACE2), while the receptor for clade 2 and clade 3 remains largely uncharacterized. We developed a mixed cell pseudotyped virus infection assay to determine whether various clades 2 and 3 sarbecovirus spike proteins can enter HEK 293T cells expressing human or Rhinolophus horseshoe bat ACE2 proteins. The receptor binding domains from BtKY72 and Khosta-2 used human ACE2 for entry, while BtKY72 and Khosta-1 exhibited widespread use of diverse rhinolophid ACE2s. A lysine at ACE2 position 31 appeared to be a major determinant of the inability of these RBDs to use a certain ACE2 sequence. The ACE2 protein from Rhinolophus alcyone engaged all known clade 3 and clade 1 receptor binding domains. We observed little use of Rhinolophus ACE2 orthologs by the clade 2 viruses, supporting the likely use of a separate, unknown receptor. Our results suggest that clade 3 sarbecoviruses from Africa and Europe use Rhinolophus ACE2 for entry, and their spike proteins appear primed to contribute to zoonosis under the right conditions.

Mutants of human ACE2 differentially promote SARS-CoV and SARS-CoV-2 spike mediated infection.

SARS-CoV and SARS-CoV-2 encode spike proteins that bind human ACE2 on the cell surface to enter target cells during infection. A small fraction of humans encode variants of ACE2, thus altering the biochemical properties at the protein interaction interface. These and other ACE2 coding mutants can reveal how the spike proteins of each virus may differentially engage the ACE2 protein surface during infection. We created an engineered HEK 293T cell line for facile stable transgenic modification, and expressed the major human ACE2 allele or 28 of its missense mutants, 24 of which are possible through single nucleotide changes from the human reference sequence. Infection with SARS-CoV or SARS-CoV-2 spike pseudotyped lentiviruses revealed that high ACE2 cell-surface expression could mask the effects of impaired binding during infection. Drastically reducing ACE2 cell surface expression revealed a range of infection efficiencies across the panel of mutants. Our infection results revealed a non-linear relationship between soluble SARS-CoV-2 RBD binding to ACE2 and pseudovirus infection, supporting a major role for binding avidity during entry. While ACE2 mutants D355N, R357A, and R357T abrogated entry by both SARS-CoV and SARS-CoV-2 spike proteins, the Y41A mutant inhibited SARS-CoV entry much more than SARS-CoV-2, suggesting differential utilization of the ACE2 side-chains within the largely overlapping interaction surfaces utilized by the two CoV spike proteins. These effects correlated well with cytopathic effects observed during SARS-CoV-2 replication in ACE2-mutant cells. The panel of ACE2 mutants also revealed altered ACE2 surface dependencies by the N501Y spike variant, including a near-complete utilization of the K353D ACE2 variant, despite decreased infection mediated by the parental SARS-CoV-2 spike. Our results clarify the relationship between ACE2 abundance, binding, and infection, for various SARS-like coronavirus spike proteins and their mutants, and inform our understanding for how changes to ACE2 sequence may correspond with different susceptibilities to infection.

Synthesis and assessment of ZD2-(68Ga-NOTA) specific to extradomain B fibronectin in tumor microenvironment for PET imaging of pancreatic cancer.

Patients diagnosed with pancreatic cancer at a late stage have a dismal survival rate. Accurate early detection of pancreatic cancer with a size of 10 mm or less could dramatically improve patient survival after timely treatments. We have developed a new PET probe ZD2-(68Ga-NOTA) specific to extradomain-B fibronectin (EDB-FN), an oncoprotein in tumor microenvironment, for sensitive molecular imaging and early diagnosis of pancreatic cancer. A targeted ligand ZD2-NOTA is synthesized by conjugation of a macrocyclic ligand NOTA via a 6-aminohexanoic acid spacer to a linear ZD2 peptide (Thr-Val-Arg-Thr-Ser-Ala-Asp). ZD2-(68Ga-NOTA) is synthesized by relabeling of ZD2-NOTA with 68GaCl3 in a high purity under GMP conditions. The expression of EDB-FN is demonstrated in BxPC3 and Capan-1 human pancreatic cancer cells and tumor xenografts in mice. ZD2-(68Ga-NOTA) results in significantly higher uptake in the both BxPC3 and Capan-1 tumor xenografts than normal organs and tissues, including the brain, heart, liver and muscle, at 1 hr postinjection in mice. The tumor to muscle uptake ratio is at least 5 folds for the tracer in both tumors. ZD2-(68Ga-NOTA) is able to clearly delineate the PaCa tumors with a size of 10 mm or less with minimal background noise in normal tissues, including the liver. Substantial tumor uptake is still visible at 2 hr post-injection. The results suggest that the ZD2 peptide targeted PET probe has a potential for sensitive molecular imaging of EDB-FN and early detection of pancreatic cancer to improve healthcare of the patients diagnosed with the disease.

Preparation and Evaluation of ZD2 Peptide 64Cu-DOTA Conjugate as a Positron Emission Tomography Probe for Detection and Characterization of Prostate Cancer.

Positron emission tomography (PET) is a sensitive modality for cancer molecular imaging. We aim to develop a PET probe for sensitive detection and risk stratification of prostate cancer by targeting an abundant microenvironment oncoprotein, extradomain-B fibronectin (EDB-FN). The probe consists of a small ZD2 peptide specific to EDB-FN and a 64Cu-DOTA chelate. The probe was synthesized using standard solid-phase peptide chemistry and chelated to 64Cu prior to imaging. PET images were acquired at 4 and 22 h after intravenously injecting a 200 µCi probe into mice bearing human PC3 and LNCaP tumors, which represent highly aggressive and slow-growing prostate tumors, respectively. At 4 and 22 h postinjection, tumors could be clearly identified in the PET images. A significant higher signal was observed in PC3 tumors than in LNCaP tumors at 22 h (p = 0.01). Probe accumulation was also higher in PC3 tumors at 24 h. These data demonstrated that PET molecular imaging of EDB-FN in the tumor microenvironment of prostate cancer allows efficient differentiation of PC3 and LNCaP tumors in vivo. The ZD2 peptide-targeted PET probe shows potential in the detection and characterization of high-risk prostate cancer to improve the clinical management of prostate cancer.

Optimization of ZD2 Peptide Targeted Gd(HP-DO3A) for Detection and Risk-Stratification of Prostate Cancer with MRI.

The aim of this work is to optimize a peptide targeted macrocyclic MRI contrast agent for detection and risk-stratification of aggressive prostate cancer. The optimized agent was prepared using click chemistry in the presence of CuSO4 and ascorbate at room temperature. The T1 and T2 relaxivities of ZD2-N3-Gd(HP-DO3A) are 5.44 and 7.10 mM-1 s-1 at 1.4 T, and 5.53 and 7.81 mM-1 s-1 at 7 T, respectively, higher than the previously reported ZD2-Gd(HP-DO3A). The specific tumor enhancement of the agent was investigated in male nude mice bearing aggressive PC3 human prostate cancer xenografts and slow-growing LNCaP tumor xenografts. Contrast enhanced MR images were acquired using a 2D spin-echo sequence and a 3D FLASH sequence with a 7 T small animal scanner. ZD2-N3-Gd(HP-DO3A) produced robust contrast enhancement in aggressive PC3 tumors and little enhancement in slow-growing LNCaP tumors. It produced 400% and 100% CNR increases in the T1-weighted 2D spin-echo MR images and 3D FLASH images of PC3 tumors, respectively, for at least 30 min at a dose of 0.1 mmol/kg. In contrast, less than 20% CNR increase was observed in the LNCaP tumors with both sequences. The optimized targeted contrast agent has higher relaxivities and are effective to detect aggressive PC3 tumors and differentiate the aggressive cancer from the slow-growing LNCaP prostate cancer in contrast enhanced MRI. ZD2-N3-Gd(HP-DO3A) has the promise for accurate detection and risk-stratification of aggressive prostate cancer.

Author Correction: Targeted gadofullerene for sensitive magnetic resonance imaging and risk-stratification of breast cancer.

In the original version of this Article, the penultimate sentence of the Abstract incorrectly read 'The dose of the contrast agent for effective molecular MRI is only slightly lower than that of ZD2-Cy5.5 (0.5 µmol kg-1) in fluorescence imaging.' The correct version states 'higher' in place of 'lower'. This error has been corrected in both the PDF and HTML versions of the Article.

Synthesis and Assessment of Peptide Gd-DOTA Conjugates Targeting Extradomain B Fibronectin for Magnetic Resonance Molecular Imaging of Prostate Cancer.

Contrast enhanced MRI is commonly used in imaging and treatment planning of prostate cancer. However, no tumor targeting contrast agent is commercially available for accurate detection and characterization of prostate cancer with MRI. Extradomain B fibronectin (EDB-FN), an oncoprotein present in aggressive tumors, is a promising molecular target for detection and stratification of high-risk prostate cancer. In this work, we have identified four small peptides (GVK, IGK, SGV, and ZD2) specific to EDB-FN for tumor targeting. In silico simulations of the binding patterns and affinities of peptides to the EDB protein fragment revealed different binding site to different peptide in the ligand-receptor interactions. Tumor specificity and organ distribution of the peptides were assessed using fluorescence imaging in male mice bearing PC-3 human prostate cancer xenografts. Targeted contrast agents were synthesized by conjugating tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to the peptides in the solid phase, followed by complexation with GdCl3. The contrast agents were characterized by MALDI-TOF mass spectrometry and relaxivity measurements. All four peptide Gd-DOTA conjugates resulted in robust tumor contrast enhancement in MR imaging of the PC3 mouse prostate cancer model. The peptide Gd-DOTA conjugates specific to EDB-FN are promising targeted small molecular macrocyclic contrast agents for MR molecular imaging of prostate cancer.

Targeted gadofullerene for sensitive magnetic resonance imaging and risk-stratification of breast cancer.

Molecular imaging of cancer biomarkers is critical for non-invasive accurate cancer detection and risk-stratification in precision healthcare. A peptide-targeted tri-gadolinium nitride metallofullerene, ZD2-Gd3N@C80, is synthesised for sensitive molecular magnetic resonance imaging of extradomain-B fibronectin in aggressive tumours. ZD2-Gd3N@C80 has superior r 1 and r 2 relaxivities of 223.8 and 344.7 mM-1 s-1 (1.5 T), respectively. It generates prominent contrast enhancement in aggressive MDA-MB-231 triple negative breast cancer in mice at a low dose (1.7 µmol kg-1, 1 T), but not in oestrogen receptor-positive MCF-7 tumours. Strong tumour contrast enhancement is consistently observed in other triple negative breast cancer models, but not in low-risk slow-growing tumours. The dose of the contrast agent for effective molecular MRI is only slightly lower than that of ZD2-Cy5.5 (0.5 µmol kg-1) in fluorescence imaging. These results demonstrate that high-sensitivity molecular magnetic resonance imaging with ZD2-Gd3N@C80 may provide accurate detection and risk-stratification of high-risk tumours for precision healthcare of breast cancer.Molecular MRI is a powerful clinical tool for non-invasive detection of cancer biomarkers. Here, the authors develop a targeted peptide gadofullerene contrast agent that can sensitively distinguish between breast cancers of different aggressiveness.

Targeted Contrast Agent Specific to an Oncoprotein in Tumor Microenvironment with the Potential for Detection and Risk Stratification of Prostate Cancer with MRI.

Accurate detection and risk stratification are paramount to the clinical management of prostate cancer. Current diagnostic methods, including prostate specific antigen (PSA) screening, are unable to differentiate high-risk tumors from low-risk tumors, resulting in overdiagnosis and overtreatment. A peptide targeted contrast agent, ZD2-Gd(HP-DO3A), specific to an oncoprotein in tumor microenvironment, EDB-FN, was synthesized for noninvasive detection and characterization of aggressive prostate cancer. EDB-FN, one of the subtypes of oncofetal fibronectin, is involved in tumor epithelial-to-mesenchymal transition (EMT), which is implicated in drug resistance and metastasis. The EDB-FN mRNA level in the metastatic PC3 cells was at least three times higher than that in non-metastatic LNCaP cells. In tumors, EDB-FN protein was highly expressed in PC3 tumor xenografts, but not in LNCaP tumors, as revealed by Western blot analysis. ZD2-Gd(HP-DO3A) produced over two times higher contrast-to-noise ratio in the PC3 tumors than in the LNCaP tumors in contrast-enhanced MRI during 30 min after injection. ZD2-Gd(HP-DO3A) possessed high chelate stability against transmetalation and minimal tissue accumulation. Our results demonstrate that molecular MRI of EDB-FN with ZD2-Gd(HP-DO3A) can potentially be used for noninvasive detection and risk stratification of human prostate cancer. Incorporation of this targeted contrast agent in the existing clinical contrast enhanced MRI procedures has the potential to improve diagnostic accuracy of prostate cancer.

Transcranial Direct Current Stimulation Targeting Primary Motor Versus Dorsolateral Prefrontal Cortices: Proof-of-Concept Study Investigating Functional Connectivity of Thalamocortical Networks Specific to Sensory-Affective Information Processing.

The pain matrix is comprised of an extensive network of brain structures involved in sensory and/or affective information processing. The thalamus is a key structure constituting the pain matrix. The thalamus serves as a relay center receiving information from multiple ascending pathways and relating information to and from multiple cortical areas. However, it is unknown how thalamocortical networks specific to sensory-affective information processing are functionally integrated. Here, in a proof-of-concept study in healthy humans, we aimed to understand this connectivity using transcranial direct current stimulation (tDCS) targeting primary motor (M1) or dorsolateral prefrontal cortices (DLPFC). We compared changes in functional connectivity (FC) with DLPFC tDCS to changes in FC with M1 tDCS. FC changes were also compared to further investigate its relation with individual's baseline experience of pain. We hypothesized that resting-state FC would change based on tDCS location and would represent known thalamocortical networks. Ten right-handed individuals received a single application of anodal tDCS (1 mA, 20 min) to right M1 and DLPFC in a single-blind, sham-controlled crossover study. FC changes were studied between ventroposterolateral (VPL), the sensory nucleus of thalamus, and cortical areas involved in sensory information processing and between medial dorsal (MD), the affective nucleus, and cortical areas involved in affective information processing. Individual's perception of pain at baseline was assessed using cutaneous heat pain stimuli. We found that anodal M1 tDCS and anodal DLPFC tDCS both increased FC between VPL and sensorimotor cortices, although FC effects were greater with M1 tDCS. Similarly, anodal M1 tDCS and anodal DLPFC tDCS both increased FC between MD and motor cortices, but only DLPFC tDCS modulated FC between MD and affective cortices, like DLPFC. Our findings suggest that M1 stimulation primarily modulates FC of sensory networks, whereas DLPFC stimulation modulates FC of both sensory and affective networks. Our findings when replicated in a larger group of individuals could provide useful evidence that may inform future studies on pain to differentiate between effects of M1 and DLPFC stimulation. Notably, our finding that individuals with high baseline pain thresholds experience greater FC changes with DLPFC tDCS implies the role of DLPFC in pain modulation, particularly pain tolerance.

The effect of motor overflow on bimanual asymmetric force coordination.

Motor overflow, typically described in the context of unimanual movements, refers to the natural tendency for a 'resting' limb to move during movement of the opposite limb and is thought to be influenced by inter-hemispheric interactions and intra-cortical networks within the 'resting' hemisphere. It is currently unknown, however, how motor overflow contributes to asymmetric force coordination task accuracy, referred to as bimanual interference, as there is need to generate unequal forces and corticospinal output for each limb. Here, we assessed motor overflow via motor evoked potentials (MEPs) and the regulation of motor overflow via inter-hemispheric inhibition (IHI) and short-intra-cortical inhibition (SICI) using transcranial magnetic stimulation in the presence of unimanual and bimanual isometric force production. All outcomes were measured in the left first dorsal interosseous (test hand) muscle, which maintained 30% maximal voluntary contraction (MVC), while the right hand (conditioning hand) was maintained at rest, 10, 30, or 70% of its MVC. We have found that as higher forces are generated with the conditioning hand, MEP amplitudes at the active test hand decreased and inter-hemispheric inhibition increased, suggesting reduced motor overflow in the presence of bimanual asymmetric forces. Furthermore, we found that subjects with less motor overflow (i.e., reduced MEP amplitudes in the test hemisphere) demonstrated poorer accuracy in maintaining 30% MVC across all conditions. These findings suggest that motor overflow may serve as an adaptive substrate to support bimanual asymmetric force coordination.

Post-exercise depression following submaximal and maximal isometric voluntary contraction.

It is well known that corticomotor excitability is altered during the post-exercise depression following fatigue within the primary motor cortex (M1). However, it is currently unknown whether corticomotor reorganization following muscle fatigue differs between magnitudes of force and whether corticomotor reorganization occurs measured with transcranial magnetic stimulation (TMS). Fifteen young healthy adults (age 23.8±1.4, 8 females) participated in a within-subjects, repeated measures design study, where they underwent three testing sessions separated by one-week each. Subjects performed separate sessions of each: low-force isometric contraction (30% maximal voluntary contraction [MVC]), high-force isometric contraction (95% MVC) of the first dorsal interosseous (FDI) muscle until self-perceived exhaustion, as well as one session of a 30-min rest as a control. We examined changes in corticomotor map area, excitability and location of the FDI representation in and around M1 using TMS. The main finding was that following low-force, but not high-force fatigue (HFF) corticomotor map area and excitability reduced [by 3cm(2) (t(14)=-2.94, p=0.01) and 56% respectively t(14)=-4.01, p<0.001)]. Additionally, the region of corticomotor excitability shifted posteriorly (6.4±2.5mm) (t(14)=-6.33, p=.019). Corticomotor output became less excitable particularly in regions adjoining M1. Overall, post-exercise depression is present in low-force, but not for HFF. Further, low-force fatigue (LFF) results in a posterior shift in corticomotor output. These changes may be indicative of increased sensory feedback from the somatosensory cortex during the recovery phase of fatigue.

Influence of Corticospinal Tracts from Higher Order Motor Cortices on Recruitment Curve Properties in Stroke.

BACKGROUND: Recruitment curves (RCs) acquired using transcranial magnetic stimulation are commonly used in stroke to study physiologic functioning of corticospinal tracts (CST) from M1. However, it is unclear whether CSTs from higher motor cortices contribute as well. OBJECTIVE: To explore whether integrity of CST from higher motor areas, besides M1, relates to CST functioning captured using RCs. METHODS: RCs were acquired for a paretic hand muscle in patients with chronic stroke. Metrics describing gain and overall output of CST were collected. CST integrity was defined by diffusion tensor imaging. For CST emerging from M1 and higher motor areas, integrity (fractional anisotropy) was evaluated in the region of the posterior limb of the internal capsule, the length of CST and in the region of the stroke lesion. RESULTS: We found that output and gain of RC was related to integrity along the length of CST emerging from higher motor cortices but not the M1. CONCLUSIONS: Our results suggest that RC parameters in chronic stroke infer function primarily of CST descending from the higher motor areas but not M1. RCs may thus serve as a simple, in-expensive means to assess re-mapping of alternate areas that is generally studied with resource-intensive neuroimaging in stroke.

Challenges in Recruitment for the Study of Noninvasive Brain Stimulation in Stroke: Lessons from Deep Brain Stimulation.

OBJECTIVE: Noninvasive brain stimulation (NIBS) can augment functional recovery following stroke; however, the technique lacks regulatory approval. Low enrollment in NIBS clinical trials is a key roadblock. Here, we pursued evidence to support the prevailing opinion that enrollment in trials of NIBS is even lower than enrollment in trials of invasive, deep brain stimulation (DBS). METHODS: We compared 2 clinical trials in stroke conducted within a single urban hospital system, one employing NIBS and the other using DBS, (1) to identify specific criteria that generate low enrollment rates for NIBS and (2) to devise strategies to increase recruitment with guidance from DBS. RESULTS: Notably, we found that enrollment in the NIBS case study was 5 times lower (2.8%) than the DBS trial (14.5%) (χ(2) = 20.815, P < .0001). Although the number of candidates who met the inclusion criteria was not different (χ(2) = .04, P < .841), exclusion rates differed significantly between the 2 studies (χ(2) = 21.354, P < .0001). Beyond lack of interest, higher exclusion rates in the NIBS study were largely due to exclusion criteria that were not present in the DBS study, including restrictions for recurrent strokes, seizures, and medications. CONCLUSIONS: Based on our findings, we conclude and suggest that by (1) establishing criteria specific to each NIBS modality, (2) adjusting exclusion criteria based on guidance from DBS, and (3) including patients with common contraindications based on a probability of risk, we may increase enrollment and hence significantly impact the feasibility and generalizability of NIBS paradigms, particularly in stroke.

Stimulation targeting higher motor areas in stroke rehabilitation: A proof-of-concept, randomized, double-blinded placebo-controlled study of effectiveness and underlying mechanisms.

PURPOSE: To demonstrate, in a proof-of-concept study, whether potentiating ipsilesional higher motor areas (premotor cortex and supplementary motor area) augments and accelerates recovery associated with constraint induced movement. METHODS: In a randomized, double-blinded pilot clinical study, 12 patients with chronic stroke were assigned to receive anodal transcranial direct current stimulation (tDCS) (n = 6) or sham (n = 6) to the ipsilesional higher motor areas during constraint-induced movement therapy. We assessed functional and neurophysiologic outcomes before and after 5 weeks of therapy. RESULTS: Only patients receiving tDCS demonstrated gains in function and dexterity. Gains were accompanied by an increase in excitability of the contralesional rather than the ipsilesional hemisphere. CONCLUSIONS: Our proof-of-concept study provides early evidence that stimulating higher motor areas can help recruit the contralesional hemisphere in an adaptive role in cases of greater ipsilesional injury. Whether this early evidence of promise translates to remarkable gains in functional recovery compared to existing approaches of stimulation remains to be confirmed in large-scale clinical studies that can reasonably dissociate stimulation of higher motor areas from that of the traditional primary motor cortices.

Reproducibility of transcranial magnetic stimulation metrics in the study of proximal upper limb muscles.

OBJECTIVE: Reproducibility of transcranial magnetic stimulation (TMS) metrics is essential in accurately tracking recovery and disease. However, majority of evidence pertains to reproducibility of metrics for distal upper limb muscles. We investigate for the first time, reliability of corticospinal physiology for a large proximal muscle - the biceps brachii and relate how varying statistical analyses can influence interpretations. METHODS: 14 young right-handed healthy participants completed two sessions assessing resting motor threshold (RMT), motor evoked potentials (MEPs), motor map and intra-cortical inhibition (ICI) from the left biceps brachii. Analyses included paired t-tests, Pearson's, intra-class (ICC) and concordance correlation coefficients (CCC) and Bland-Altman plots. RESULTS: Unlike paired t-tests, ICC, CCC and Pearson's were >0.6 indicating good reliability for RMTs, MEP intensities and locations of map; however values were <0.3 for MEP responses and ICI. CONCLUSIONS: Corticospinal physiology, defining excitability and output in terms of intensity of the TMS device, and spatial loci are the most reliable metrics for the biceps. MEPs and variables based on MEPs are less reliable since biceps receives fewer cortico-motor-neuronal projections. Statistical tests of agreement and associations are more powerful reliability indices than inferential tests. SIGNIFICANCE: Reliable metrics of proximal muscles when translated to a larger number of participants would serve to sensitively track and prognosticate function in neurological disorders such as stroke where proximal recovery precedes distal.