Drp1 controls complex II assembly and skeletal muscle metabolism by Sdhaf2 action on mitochondria.

The dynamin-related guanosine triphosphatase, Drp1 (encoded by Dnm1l), plays a central role in mitochondrial fission and is requisite for numerous cellular processes; however, its role in muscle metabolism remains unclear. Here, we show that, among human tissues, the highest number of gene correlations with DNM1L is in skeletal muscle. Knockdown of Drp1 (Drp1-KD) promoted mitochondrial hyperfusion in the muscle of male mice. Reduced fatty acid oxidation and impaired insulin action along with increased muscle succinate was observed in Drp1-KD muscle. Muscle Drp1-KD reduced complex II assembly and activity as a consequence of diminished mitochondrial translocation of succinate dehydrogenase assembly factor 2 (Sdhaf2). Restoration of Sdhaf2 normalized complex II activity, lipid oxidation, and insulin action in Drp1-KD myocytes. Drp1 is critical in maintaining mitochondrial complex II assembly, lipid oxidation, and insulin sensitivity, suggesting a mechanistic link between mitochondrial morphology and skeletal muscle metabolism, which is clinically relevant in combatting metabolic-related diseases.

Vitamin D and Marine n-3 Fatty Acids for Autoimmune Disease Prevention: Outcomes Two Years After Completion of a Double-Blind, Placebo-Controlled Trial.

OBJECTIVE: In the 5.3-year randomized, 2 × 2 factorial, double-blind, placebo-controlled Vitamin D and Omega-3 Trial (VITAL), vitamin D supplementation reduced autoimmune disease (AD) incidence (hazard ratio [HR] 0.78, 95% confidence interval [CI] 0.61-0.99). Omega-3 (n-3) fatty acid supplementation showed a statistically nonsignificant reduction (HR 0.85, 95% CI 0.67-1.08). We aimed to confirm further AD cases arising during and after randomization and assess sustained effects with two years of postintervention observation. METHODS: Of the 12,786 men aged ≥50 and 13,085 women aged ≥55 initially randomized, we observed surviving and willing participants for two more years. We continued to confirm annual participant-reported new AD by medical record review. Cox models calculated HRs for all confirmed incident AD, (and secondary endpoints, including probable cases, and individual ADs), during the observational and randomized periods. RESULTS: A total of 21,592 participants (83.5%) were observed for two more years; 514 participants developed incident confirmed AD (236 since prior report), of whom 255 had been randomized to vitamin D versus 259 to vitamin D placebo (HR 0.98 [95% CI 0.83-1.17] at 7 years). AD was confirmed in 234 participants initially randomized to n-3 fatty acids versus 280 randomized to its placebo (HR 0.83 [95% CI 0.70-0.99] at 7 years). Of newly confirmed cases, 65 had onset during randomization; their inclusion changed randomized results as follows: HR 0.85 (95% CI 0.70-1.04) for vitamin D and HR 0.87 (95% CI 0.71-1.06) for n-3 fatty acids. CONCLUSION: Two years after trial termination, the protective effects of 2000 IU/day of vitamin D dissipated, but 1,000 mg/day of n-3 fatty acids had a sustained effect in reducing AD incidence.

Pneumonic Plague Protection Induced by a Monophosphoryl Lipid A Decorated Yersinia Outer-Membrane-Vesicle Vaccine.

A new Yersinia pseudotuberculosis mutant strain, YptbS46, carrying the lpxE insertion and pmrF-J deletion is constructed and shown to exclusively produce monophosphoryl lipid A (MPLA) having adjuvant properties. Outer membrane vesicles (OMVs) isolated from YptbS46 harboring an lcrV expression plasmid, pSMV13, are designated OMV46-LcrV, which contained MPLA and high amounts of LcrV (Low Calcium response V) and displayed low activation of Toll-like receptor 4 (TLR4). Intramuscular prime-boost immunization with 30 µg of of OMV46-LcrV exhibited substantially reduced reactogenicity than the parent OMV44-LcrV and conferred complete protection to mice against a high-dose of respiratory Y. pestis challenge. OMV46-LcrV immunization induced robust adaptive responses in both lung mucosal and systemic compartments and orchestrated innate immunity in the lung, which are correlated with rapid bacterial clearance and unremarkable lung damage during Y. pestis challenge. Additionally, OMV46-LcrV immunization conferred long-term protection. Moreover, immunization with reduced doses of OMV46-LcrV exhibited further lower reactogenicity and still provided great protection against pneumonic plague. The studies strongly demonstrate the feasibility of OMV46-LcrV as a new type of plague vaccine candidate.

Pneumonic plague protection induced by a monophosphoryl lipid A decorated Yersinia outer-membrane-vesicle vaccine.

A newly constructed Yersinia pseudotuberculosis mutant (YptbS46) carrying the lpxE insertion and pmrF-J deletion exclusively synthesized an adjuvant form of lipid A, monophosphoryl lipid A (MPLA). Outer membrane vesicles (OMVs) isolated from YptbS46 harboring an lcrV expression plasmid, pSMV13, were designated OMV 46 -LcrV, which contained MPLA and high amounts of LcrV and displayed low activation of Toll-like receptor 4 (TLR4). Similar to the previous OMV 44 -LcrV, intramuscular prime-boost immunization with 30 µg of OMV 46 -LcrV exhibited substantially reduced reactogenicity and conferred complete protection to mice against a high-dose of respiratory Y. pestis challenge. OMV 46 -LcrV immunization induced robust adaptive responses in both lung mucosal and systemic compartments and orchestrated innate immunity in the lung, which were correlated with rapid bacterial clearance and unremarkable lung damage during Y. pestis challenge. Additionally, OMV 46 -LcrV immunization conferred long-term protection. Moreover, immunization with reduced doses of OMV 46 -LcrV exhibited further lower reactogenicity and still provided great protection against pneumonic plague. Our studies strongly demonstrate the feasibility of OMV 46 -LcrV as a new type of plague vaccine candidate.

MRI characteristics of canine prostatic neoplasia.

Magnetic resonance imaging (MRI) has been used to evaluate dogs with suspected prostatic neoplasia, however, published studies describing MRI characteristics of canine prostatic neoplasia are currently lacking. The aims of the current retrospective case series study were to describe MRI findings of the pelvic region in dogs with a histopathologic or cytologic diagnosis of prostatic neoplasia. Retrospective analysis of these images was then performed by a board-certified veterinary radiologist for shared imaging characteristics. The most consistent characteristics were heterogeneous hyperintensity of the tumor on T2-weighted images (10/10) and short tau inversion recovery images (10/10), prostatic capsular margin distortion by the tumor (10/10), cavitations (10/10), complete effacement of the prostatic architecture (9/10), neurovascular bundle (NVB) compression or invasion (9/10), heterogeneous isointensity of the tumor on T1-weighted images (9/10), and strong contrast enhancement of the tumor (8/10). Additional features included an overlying pattern of distorted radiating striations (7/10), regional lymphadenomegaly (5/10), mineralization within the mass (5/10), urinary bladder trigone involvement (6/10), and post-prostatic urethral involvement (7/10). These findings supported the use of MRI as an adjunct imaging modality for diagnosis and therapeutic planning of prostatic neoplasia and including prostatic neoplasia as a likely differential diagnosis for dogs with these MRI characteristics.

Kinetic Competition Screening of Yeast-Displayed Libraries for Isolating High Affinity Binders.

Yeast surface display is a robust platform for obtaining binders with high affinity. Kinetic competition screening is an effective method for maturing the affinity of binders with strong starting affinities, or when dissociation kinetics are a key consideration for the protein of interest. In this chapter, we describe detailed protocols for setting up and performing a kinetic competition screen. The duration of competition is determined based on the dissociation rate constant of the parental clone measured on the yeast surface. This methodology was successfully used to improve the affinity of a viral double-stranded RNA binding protein with starting affinity in the sub-nanomolar range.

What Is Your Diagnosis?

In collaboration with the American College of Veterinary Radiology.

Anthrax toxins regulate pain signaling and can deliver molecular cargoes into ANTXR2+ DRG sensory neurons.

Bacterial products can act on neurons to alter signaling and function. In the present study, we found that dorsal root ganglion (DRG) sensory neurons are enriched for ANTXR2, the high-affinity receptor for anthrax toxins. Anthrax toxins are composed of protective antigen (PA), which binds to ANTXR2, and the protein cargoes edema factor (EF) and lethal factor (LF). Intrathecal administration of edema toxin (ET (PA + EF)) targeted DRG neurons and induced analgesia in mice. ET inhibited mechanical and thermal sensation, and pain caused by formalin, carrageenan or nerve injury. Analgesia depended on ANTXR2 expressed by Nav1.8+ or Advillin+ neurons. ET modulated protein kinase A signaling in mouse sensory and human induced pluripotent stem cell-derived sensory neurons, and attenuated spinal cord neurotransmission. We further engineered anthrax toxins to introduce exogenous protein cargoes, including botulinum toxin, into DRG neurons to silence pain. Our study highlights interactions between a bacterial toxin and nociceptors, which may lead to the development of new pain therapeutics.

Nociceptive Sensory Neurons Mediate Inflammation Induced by Bacillus Anthracis Edema Toxin.

Bacterial products are able to act on nociceptive neurons during pathogenic infection. Neurogenic inflammation is an active part of pain signaling and has recently been shown to impact host-pathogen defense. Bacillus anthracis Edema Toxin (ET) produces striking edema in peripheral tissues, but the cellular mechanisms involved in tissue swelling are not completely understood. Here, we find that nociceptive neurons play a role in ET-induced edema and inflammation in mice. Subcutaneous footpad infection of B. anthracis Sterne caused ET-dependent local mechanical allodynia, paw swelling and body weight gain. Subcutaneous administration of ET induced paw swelling and vascular leakage, the early phases of which were attenuated in the absence of Trpv1+ or Nav1.8+ nociceptive neurons. Nociceptive neurons express the anthrax toxin receptor ANTXR2, but this did not mediate ET-induced edema. ET induced local cytokine expression and neutrophil recruitment, which were dependent in part on Trpv1+ nociceptive neurons. Ablation of Trpv1+ or Nav1.8+ nociceptive neurons also attenuated early increases in paw swelling and body weight gain during live B. anthracis infection. Our findings indicate that nociceptive neurons play an active role in inflammation caused by B. anthracis and Edema Toxin to potentially influence bacterial pathogenesis.

Divergence of Cardiovascular Biomarkers of Lipids and Subclinical Myocardial Injury Among Rheumatoid Arthritis Patients With Increased Inflammation.

OBJECTIVE: Patients with rheumatoid arthritis (RA) are 1.5 times more likely to develop cardiovascular disease (CVD) attributed to chronic inflammation. A decrease in inflammation in patients with RA is associated with increased low-density lipoprotein (LDL) cholesterol. This study was undertaken to prospectively evaluate the changes in lipid levels among RA patients experiencing changes in inflammation and determine the association with concomitant temporal patterns in markers of myocardial injury. METHODS: A total of 196 patients were evaluated in a longitudinal RA cohort, with blood samples and high-sensitivity C-reactive protein (hsCRP) levels measured annually. Patients were stratified based on whether they experienced either a significant increase in inflammation (an increase in hsCRP of ≥10 mg/liter between any 2 time points 1 year apart; designated the increased inflammation cohort [n = 103]) or decrease in inflammation (a decrease in hsCRP of ≥10 mg/liter between any 2 time points 1 year apart; designated the decreased inflammation cohort [n = 93]). Routine and advanced lipids, markers of inflammation (interleukin-6, hsCRP, soluble tumor necrosis factor receptor II), and markers of subclinical myocardial injury (high-sensitivity cardiac troponin T [hs-cTnT], N-terminal pro-brain natriuretic peptide) were measured. RESULTS: Among the patients in the increased inflammation cohort, the mean age was 59 years, 81% were women, and the mean RA disease duration was 17.9 years. The average increase in hsCRP levels was 36 mg/liter, and this increase was associated with significant reductions in LDL cholesterol, triglycerides, total cholesterol, apolipoprotein (Apo B), and Apo A-I levels. In the increased inflammation cohort at baseline, 45.6% of patients (47 of 103) had detectable circulating hs-cTnT, which further increased during inflammation (P = 0.02). In the decreased inflammation cohort, hs-cTnT levels remained stable despite a reduction in inflammation over follow-up. In both cohorts, hs-cTnT levels were associated with the overall estimated risk of CVD. CONCLUSION: Among RA patients who experienced an increase in inflammation, a significant decrease in routinely measured lipids, including LDL cholesterol, and an increase in markers of subclinical myocardial injury were observed. These findings highlight the divergence in biomarkers of CVD risk and suggest a role in future studies examining the benefit of including hs-cTnT for CVD risk stratification in RA.

A conformation-specific ON-switch for controlling CAR T cells with an orally available drug.

Molecular ON-switches in which a chemical compound induces protein-protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics.

EXTraction of EMR numerical data: an efficient and generalizable tool to EXTEND clinical research.

BACKGROUND: Electronic medical records (EMR) contain numerical data important for clinical outcomes research, such as vital signs and cardiac ejection fractions (EF), which tend to be embedded in narrative clinical notes. In current practice, this data is often manually extracted for use in research studies. However, due to the large volume of notes in datasets, manually extracting numerical data often becomes infeasible. The objective of this study is to develop and validate a natural language processing (NLP) tool that can efficiently extract numerical clinical data from narrative notes. RESULTS: To validate the accuracy of the tool EXTraction of EMR Numerical Data (EXTEND), we developed a reference standard by manually extracting vital signs from 285 notes, EF values from 300 notes, glycated hemoglobin (HbA1C), and serum creatinine from 890 notes. For each parameter of interest, we calculated the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and F1 score of EXTEND using two metrics. (1) completion of data extraction, and (2) accuracy of data extraction compared to the actual values in the note verified by chart review. At the note level, extraction by EXTEND was considered correct only if it accurately detected and extracted all values of interest in a note. Using manually-annotated labels as the gold standard, the note-level accuracy of EXTEND in capturing the numerical vital sign values, EF, HbA1C and creatinine ranged from 0.88 to 0.95 for sensitivity, 0.95 to 1.0 for specificity, 0.95 to 1.0 for PPV, 0.89 to 0.99 for NPV, and 0.92 to 0.96 in F1 scores. Compared to the actual value level, the sensitivity, PPV, and F1 score of EXTEND ranged from 0.91 to 0.95, 0.95 to 1.0 and 0.95 to 0.96. CONCLUSIONS: EXTEND is an efficient, flexible tool that uses knowledge-based rules to extract clinical numerical parameters with high accuracy. By increasing dictionary terms and developing new rules, the usage of EXTEND can easily be expanded to extract additional numerical data important in clinical outcomes research.

Interference of tumor necrosis factor inhibitor treatments on soluble tumor necrosis factor receptor 2 levels in rheumatoid arthritis.

OBJECTIVE: Soluble Tumor Necrosis Factor Receptor II (sTNFR2) is used as a biomarker to study cardiovascular disease (CVD) in diverse populations. TNF inhibitors (TNFi's) are a common treatment for inflammatory conditions. The objective of this study was to examine whether TNFi use impacts measured sTNFR2 levels. METHODS: We studied blood samples from a cohort of RA patients with clinical data and high sensitivity-C-reactive protein (hsCRP) measurements. To assess for interference, we tested the entire cohort for the expected positive correlation between sTNFR2 and TNFi using Pearson correlations. We then performed Pearson correlations between sTNFR2 and TNFi and sequentially removed subjects on adalimumab, etanercept, and infliximab; if interference was occurring, no correlation would be observed between hsCRP and sTNFR2, and correlation would be restored by removing subjects on the treatment causing the interference. RESULTS: We studied 190 subjects, 84.2% female, 73.4% anti-CCP positive. All subjects with sTNFR2 level exceeding measurable level were on etanercept. The expected positive correlation between hsCRP and sTNFR2 was not observed when assessing the entire cohort, r = 0.05, p = 0.51. However, the expected correlation was restored only after excluding subjects on etanercept, r = 0.46, p < 0.0001, and not adalimumab or infliximab. ELISA for sTNFR2 was performed using etanercept only and demonstrated direct binding to sTNFR2. CONCLUSIONS: Our data identified interference between etanercept and the TNFR2 assay. Of the TNFi's, only etanercept has a TNF-binding domain modeled after TNFR2. These data should be considered when designing studies using sTNFR2 in populations where etanercept is a treatment option.

Blocking Neuronal Signaling to Immune Cells Treats Streptococcal Invasive Infection.

The nervous system, the immune system, and microbial pathogens interact closely at barrier tissues. Here, we find that a bacterial pathogen, Streptococcus pyogenes, hijacks pain and neuronal regulation of the immune response to promote bacterial survival. Necrotizing fasciitis is a life-threatening soft tissue infection in which "pain is out of proportion" to early physical manifestations. We find that S. pyogenes, the leading cause of necrotizing fasciitis, secretes streptolysin S (SLS) to directly activate nociceptor neurons and produce pain during infection. Nociceptors, in turn, release the neuropeptide calcitonin gene-related peptide (CGRP) into infected tissues, which inhibits the recruitment of neutrophils and opsonophagocytic killing of S. pyogenes. Botulinum neurotoxin A and CGRP antagonism block neuron-mediated suppression of host defense, thereby preventing and treating S. pyogenes necrotizing infection. We conclude that targeting the peripheral nervous system and blocking neuro-immune communication is a promising strategy to treat highly invasive bacterial infections. VIDEO ABSTRACT.

Impact of Changes in Inflammation on Estimated Ten-Year Cardiovascular Risk in Rheumatoid Arthritis.

OBJECTIVE: Current validated cardiovascular (CV) risk estimates were developed in populations with relatively stable levels of inflammation, whereas patients with rheumatoid arthritis (RA) routinely experience significant changes in inflammation. This study was undertaken to test whether changes in inflammation affect estimated CV risk as measured using validated population-based risk calculators. METHODS: Participants in a prospective RA cohort who experienced a decrease or an increase of ≥10 mg/liter in the C-reactive protein (CRP) level at 2 consecutive time points 1 year apart (CRP decrease group and CRP increase group, respectively) were included in this study. We estimated 10-year CV risk using the following calculators: Framingham Risk Score, 2013 American College of Cardiology/American Heart Association Atherosclerotic Cardiovascular Disease Risk Score, Reynolds Risk Score (RRS), and QRISK2. Of these calculators, only the RRS includes a variable addressing the CRP level. Paired t-tests were performed to compare risk scores at baseline and 1-year follow-up. We calculated the correlations between the changes in risk scores and changes in pro-B-type natriuretic peptide (pro BNP), a surrogate marker of CV risk. RESULTS: One hundred eighty RA patients were included in the study (mean age 57.8 years, 84% female, 80% seropositive). Of the calculators studied, only the RRS was sensitive to changes in inflammation; an increase in inflammation was associated with increased estimated CV risk (P < 0.0001), and only the RRS was correlated with changes in proBNP (r = 0.17, P = 0.03). CONCLUSION: Our data showed no significant change in CV risk estimated using validated general population CV risk calculators except for the RRS. These findings suggest that CV risk may be modulated by changes in inflammation in RA, which is not typically considered when using existing CV risk calculators.

Microbial symbionts regulate the primary Ig repertoire.

The ability of immunoglobulin (Ig) to recognize pathogens is critical for optimal immune fitness. Early events that shape preimmune Ig repertoires, expressed on IgM+ IgD+ B cells as B cell receptors (BCRs), are poorly defined. Here, we studied germ-free mice and conventionalized littermates to explore the hypothesis that symbiotic microbes help shape the preimmune Ig repertoire. Ig-binding assays showed that exposure to conventional microbial symbionts enriched frequencies of antibacterial IgM+ IgD+ B cells in intestine and spleen. This enrichment affected follicular B cells, involving a diverse set of Ig-variable region gene segments, and was T cell-independent. Functionally, enrichment of microbe reactivity primed basal levels of small intestinal T cell-independent, symbiont-reactive IgA and enhanced systemic IgG responses to bacterial immunization. These results demonstrate that microbial symbionts influence host immunity by enriching frequencies of antibacterial specificities within preimmune B cell repertoires and that this may have consequences for mucosal and systemic immunity.

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins.

Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids.

Bacterial Signaling to the Nervous System through Toxins and Metabolites.

Mammalian hosts interface intimately with commensal and pathogenic bacteria. It is increasingly clear that molecular interactions between the nervous system and microbes contribute to health and disease. Both commensal and pathogenic bacteria are capable of producing molecules that act on neurons and affect essential aspects of host physiology. Here we highlight several classes of physiologically important molecular interactions that occur between bacteria and the nervous system. First, clostridial neurotoxins block neurotransmission to or from neurons by targeting the SNARE complex, causing the characteristic paralyses of botulism and tetanus during bacterial infection. Second, peripheral sensory neurons-olfactory chemosensory neurons and nociceptor sensory neurons-detect bacterial toxins, formyl peptides, and lipopolysaccharides through distinct molecular mechanisms to elicit smell and pain. Bacteria also damage the central nervous system through toxins that target the brain during infection. Finally, the gut microbiota produces molecules that act on enteric neurons to influence gastrointestinal motility, and metabolites that stimulate the "gut-brain axis" to alter neural circuits, autonomic function, and higher-order brain function and behavior. Furthering the mechanistic and molecular understanding of how bacteria affect the nervous system may uncover potential strategies for modulating neural function and treating neurological diseases.

Effects of repetitive transcranial magnetic stimulation combined with sensory cueing on unilateral neglect in subacute patients with right hemispheric stroke: a randomized controlled study.

OBJECTIVE: To compare the effects of rTMS combined with sensory cueing, rTMS alone, and conventional rehabilitation on unilateral neglect, hemiplegic arm functions and performance of activities of daily living. DESIGN: A single-blinded randomized controlled trial. SETTING: A convalescent hospital. SUBJECTS: Sixty inpatients with left unilateral neglect after stroke. INTERVENTIONS: Patients were randomly assigned to three groups: rTMS combined with sensory cueing, rTMS, and conventional rehabilitation alone. rTMS at 1 Hz was applied over P5 of the contralesional hemisphere while vibration cueing was emitted using a wristwatch device on the hemiplegic arm, five days per week for two weeks. The first two groups received the same dosage of conventional rehabilitation on top of their experimental interventions. Blinded assessments were administered at baseline, 2 weeks postintervention, and 6 weeks follow-up. MAIN MEASURES: Neglect and arm motor performance. RESULTS: Both rTMS combined with sensory cueing (99.6±33.0) and rTMS alone (88.2±28.7) significantly reduced unilateral neglect than conventional rehabilitation (72.7±33.1) when measured using the conventional subtests of the Behavioural Inattention Test, but the combination was better than rTMS alone. Hemiplegic arm functions and activities of daily living improved in all patients across the three groups but no significant differences were found between the groups. CONCLUSION: The combination of inhibitory P5-rTMS with sensory cueing was better than either rTMS or conventional rehabilitation alone in producing a stronger and long-lasting improvement in unilateral neglect, but the improvement was not associated with improved arm function or independence in activities of daily living.