Rapid Cytoskeletal Response of Epithelial Cells to Force Generation by Type IV Pili

Many bacterial pathogens interfere with cellular functions including phagocytosis and barrier integrity. The human pathogen Neissieria gonorrhoeae generates grappling hooks for adhesion, spreading, and induction of signal cascades that lead to formation cortical plaques containing f-actin and ezrin. It is unclear whether high mechanical forces generated by type IV pili (T4P) are a direct signal that leads to cytoskeletal rearrangements and at which time scale the cytoskeletal response occurs. Here we used laser tweezers to mimic type IV pilus mediated force generation by T4P-coated beads on the order of 100pN. We found that actin-EGFP and ezrin-EGFP accumulated below pilus-coated beads when force was applied. Within 2 min, accumulation significantly exceeded controls without force or without pili, demonstrating that T4P-generated force rapidly induces accumulation of plaque proteins. This finding adds mechanical force to the many strategies by which bacteria modulate the host cell cytoskeleton

Identification of Tmem10/Opalin as a novel marker for oligodendrocytes using gene expression profiling

<p>Abstract</p> <p>Background</p> <p>During the development of the central nervous system, oligodendrocytes generate large amounts of myelin, a multilayered insulating membrane that ensheathes axons, thereby allowing the fast conduction of the action potential and maintaining axonal integrity. Differentiation of oligodendrocytes to myelin-forming cells requires the downregulation of RhoA GTPase activity.</p> <p>Results</p> <p>To gain insights into the molecular mechanisms of oligodendrocyte differentiation, we performed microarray expression profiling of the oligodendroglial cell line, Oli-neu, treated with the Rho kinase (ROCK) inhibitor, Y-27632 or with conditioned neuronal medium. This resulted in the identification of the transmembrane protein 10 (Tmem10/Opalin), a novel type I transmembrane protein enriched in differentiating oligodendrocytes. In primary cultures, Tmem10 was abundantly expressed in O4-positive oligodendrocytes, but not in oligodendroglial precursor cells, astrocytes, microglia or neurons. In mature oligodendrocytes Tmem10 was enriched in the rims and processes of the cells and was only found to a lesser extent in the membrane sheets.</p> <p>Conclusion</p> <p>Together, our results demonstrate that Tmem10 is a novel marker for in vitro generated oligodendrocytes.</p

Upregulation of miRNA hsa-miR-342-3p in experimental and idiopathic prion disease

The aim of our study was to analyze the differential expression of miRNAs in the brains of BSE-infected cynomolgus macaques as a model for Creutzfeldt-Jakob disease (CJD). MicroRNAs (miRNAs) are small noncoding RNAs regulating gene expression by mRNA targeting. Among other functions they contribute to neuronal development and survival. Recently, the lack of miRNA processing has been shown to promote neurodegeneration and deregulation of several miRNAs has been reported to be associated with Scrapie in mice. Therefore, we hypothesized that miRNAs are also regulated in response to human prion disease. We have applied miRNA-microarrays to identify deregulated miRNA candidates in brains of BSE-infected macaques. Shock-frozen brain sections of six BSE-infected and five non-infected macaques were used to validate regulated miRNA candidates by two independent qRT-PCR-based methods. Our study revealed significant upregulation of hsa-miR-342-3p and hsa-miR-494 in the brains of BSE-infected macaques compared to non-infected animals. In a pilot study we could show that hsa-miR-342-3p was also upregulated in brain samples of human type 1 and type 2 sporadic CJD. With respect to the reported regulation of this miRNA in Scrapie-infected mice, we propose that upregulation of hsa-miR-342-3p may be a general phenomenon in late stage prion disease and might be used as a novel marker for animal and human TSEs

Pulmonary Hypertension in Patients With COPD : Results From the Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension (COMPERA)

Funding Information: FUNDING/SUPPORT: This work was supported by the German Center of Lung Research (DZL). COMPERA is funded by unrestricted grants from Acceleron , Actelion Pharmaceuticals , Bayer , OMT , and GSK . Funding Information: Financial/nonfinancial disclosures: The authors have reported to CHEST the following: C. D. V. has received fees for serving as a speaker, consultant, and an advisory board member from the following companies: Acceleron, Actelion, Bayer, Dompè, GSK, Janssen, MSD, Pfizer, and United Therapeutics. M. M. H. has received speaker fees, honoraria, or both for consultations from Acceleron, Actelion, Bayer, Janssen, MSD, and Pfizer. D. H. has received travel compensation from Actelion, Boehringer-Ingelheim, and Shire. D. P. has received fees for consultations from Actelion, Aspen, Biogen, Bayer, Boehringer Ingelheim, Johnson & Johnson, Novartis, Daiichi Sankyo, Sanofi, and Pfizer. N. B. received speaker fees from Bayer/MSD and Actelion/Janssen. K. M. O. has received speaker fees from Actelion, Bayer, and Lilly. H. A. G. has received honorariums for consultations, speaking at conferences, or both from Bayer HealthCare AG, Actelion, Encysive, Pfizer, Ergonex, Lilly, and Novartis. He is member of advisory boards for Bayer HealthCare AG, Pfizer, GSK, Actelion, Lilly, Merck, Encysive, and Ergonex. He also has received governmental grants from the German Research Foundation (DFG), Excellence Cluster Cardiopulmonary Research (ECCPS), State Government of Hessen (LOEWE), and the German Ministry for Education and Research (BMBF). M. Held has received speaker fees and honoraria for consultations from Actelion, Bayer, Boehringer Ingelheim Pharma, Encysive, Glaxo Smith Kline, Lilly, Janssen, Novartis, Pfizer, Nycomed, Roche, and Servier. H. K. has received speaker fees and honoraria for consultations from Actelion, Bayer, GSK, Lilly, Novartis, Pfizer, and United Therapeutics and research grants from Actelion. T. J. L. has received speaker fees, honoraria for consultations, and research funding from Actelion, Acceleron Pharma, Bayer, GSK, Janssen-Cilag, MSD, and Pfizer. S. R. has received honoraria for lectures, consultancy, or both from Actavis, Actelion, Bayer, GSK, Lilly, Novartis, Pfizer, and United Therapeutics. D. D. declares honoraria for lectures, consultancy, or both from Actelion, Bayer, GSK, Novartis, Pfizer, and Servier; participation in clinical trials for Actelion, Bayer, GSK, and Novartis; and research support to his institution from Actelion. R. B. has received fees from GSK, UT, Dompè, Bayer, Ferrer, MSD, and AOP Orphan Pharmaceuticals. M. C. has received fees for consulting from GSK and speaker fees from Bayer and Pfizer. M. Halank has received speaker fees and/or honoraria for consultations from Acceleron, Actelion, AstraZeneca, Bayer, BayerChemie, GSK, Janssen, MSD and Novartis. A. V.-N. reports receiving lecture fees from Actelion, Bayer, GlaxoSmithKline, Lilly, and Pfizer; serves on the advisory board of Actelion and Bayer; and serves on steering committees for Actelion, Bayer, GlaxoSmithKline, and Pfizer. D. S. received fees for lectures, consulting, research support, or a combination thereof to his institution from Actelion, Bayer, GSK, and Pfizer. R. E. has received speaker fees and honoraria for consultations from Actelion, Bayer, GSK, Lilly, Novartis, Pfizer, and United Therapeutics. J. S. R. G. has received speaker fees and honoraria for consultations from Acceleron, Actelion, Bayer, Complexa, GSK, MSD, Pfizer, and United Therapeutics. M. D. has received investigator, speaker, consultant, or steering committee member fees from Actelion, Aventis Pharmaceuticals, Bayer, Eli Lilly, Encysive, Gilead (Myogen), GlaxoSmithKline, Nippon Shyniaku, Novartis, Pfizer, Schering, and United Therapeutics; educational grants from Actelion, GlaxoSmithKline, Pfizer, and Therabel; and research grants from Actelion, Pfizer, and GlaxoSmithKline. She is holder of the Actelion Chair for Pulmonary Hypertension and of the GSK chair for research and education in pulmonary vascular pathology at the Catholic University of Leuven. J. C. has received fees for consultancies and lectures from Actelion, Bayer, GSK, United Therapeutics, and Pfizer as well as equipment and educational grants from Actelion. C. O. has received speaker fees and honoraria for consultations from Actelion, Bayer, GSK, Lilly, Novartis, and Pfizer. H. K. has received honoraria for lectures, consultancy, or both from Actelion-Janssen, Amicus Therapeutics, and Bristol Meyers Squibb. O. D. has or had consultancy relationships, has received research funding (last 3 years), or both from AbbVie, Actelion, Acceleron Pharma, Amgen, AnaMar, Baecon Discovery, Blade Therapeutics, Bayer, Boehringer Ingelheim, Catenion, Competitive Corpus, Drug Development International Ltd, CSL Behring, ChemomAb, Ergonex, Galapagos NV, Glenmark Pharmaceuticals, GSK, Horizon (Curzion) Pharmaceuticals, Inventiva, Italfarmaco, iQone, iQvia, Kymera Therapeutics, Lilly, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Novartis, Pfizer, Roche, Sanofi, Target Bio Science, and UCB in the area of potential treatments of scleroderma and its complications including PH. In addition, he has a patent mir-29 for the treatment of systemic sclerosis issued (US8247389, EP2331143). E. G. has received honoraria for consultations, speaking at conferences, or both from Bayer/MSD, Actelion/Janssen, GWT-TUD, and OMT/United Therapeutics. None declared (A. S.). Publisher Copyright: © 2021 The AuthorsBackground: Pulmonary hypertension (PH) in COPD is a poorly investigated clinical condition. Research Question: Which factors determine the outcome of PH in COPD? Study Design and Methods: We analyzed the characteristics and outcome of patients enrolled in the Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension (COMPERA) with moderate or severe PH in COPD as defined during the 6th PH World Symposium who received medical therapy for PH and compared them with patients with idiopathic pulmonary arterial hypertension (IPAH). Results: The population included incident patients with moderate PH in COPD (n = 68), with severe PH in COPD (n = 307), and with IPAH (n = 489). Patients with PH in COPD were older, predominantly male, and treated mainly with phosphodiesterase-5 inhibitors. Despite similar hemodynamic impairment, patients with PH in COPD achieved a worse 6-min walking distance (6MWD) and showed a more advanced World Health Organization functional class (WHO FC). Transplant-free survival rates at 1, 3, and 5 years were higher in the IPAH group than in the PH in COPD group (IPAH: 94%, 75%, and 55% vs PH in COPD: 86%, 55%, and 38%; P = .004). Risk factors for poor outcomes in PH in COPD were male sex, low 6MWD, and high pulmonary vascular resistance (PVR). In patients with severe PH in COPD, improvements in 6MWD by ≥ 30 m or improvements in WHO FC after initiation of medical therapy were associated with better outcomes. Interpretation: Patients with PH in COPD were functionally more impaired and had a poorer outcome than patients with IPAH. Predictors of death in the PH in COPD group were sex, 6MWD, and PVR. Our data raise the hypothesis that some patients with severe PH in COPD may benefit from PH treatment. Randomized controlled studies are necessary to explore this hypothesis further. Trial Registry: ClinicalTrials.gov; No.: NCT01347216; URL: www.clinicaltrials.govpublishersversionPeer reviewe

European guidelines on structure and qualification of general thoracic surgery

OBJECTIVE To update the recommendations for the structural characteristics of general thoracic surgery (GTS) in Europe in order to provide a document that can be used as a guide for harmonizing the general thoracic surgical practice in Europe. METHODS A task force was created to set the structural, procedural and qualification characteristics of a European GTS unit. These criteria were endorsed by the Executive Committee of the European Society of Thoracic Surgeons and by the Thoracic Domain of the European Association for Cardio-Thoracic Surgery and were validated by the European Board of Thoracic Surgery at European Union of Medical Specialists. RESULTS Criteria regarding definition and scope of GTS, structure and qualification of GTS unit, training and education and recommendations for subjects of particular interest (lung transplant, oesophageal surgery, minimally invasive thoracic surgery, quality surveillance) were developed. CONCLUSIONS This document will hopefully represent the first step of a process of revision of the modern thoracic surgeons' curricula, which need to be qualitatively rethought in the setting of the qualification process. The structural criteria highlighted in the present document are meant to help and tackle the challenge of cultural and language barriers as well as of widely varying national training programme

Medical treatment of pulmonary hypertension in adults with congenital heart disease : updated and extended results from the International COMPERA-CHD Registry

Funding Information: The authors are indebted to the COMPERA investigators and their staff. We explicitly thank Dr. Claudia S. Copeland for the professional editing of the final draft of the manuscript. Funding: COMPERA is funded by unrestricted grants from Acceleron, Actelion Pharmaceuticals (Janssen), Bayer, OMT and GSK. These companies were not involved in data analysis or the writing of this manuscript. Funding Information: ICMJE uniform disclosure form (available at https:// dx.doi.org/10.21037/cdt-21-351). The series “Current Management Aspects in Adult Congenital Heart Disease (ACHD): Part IV” was commissioned by the editorial office without any funding or sponsorship. Dr. DH reports non-financial support from Actelion, Boehringer-Ingelheim, and Shire, outside the submitted work; Dr. DP reports personal fees from Actelion, Biogen, Aspen, Bayer, Boehringer Ingelheim, Daiichi Sankyo, and Sanofi, outside the submitted work; Dr. MD reports personal fees from Actelion, Bayer, GSK and MSD, outside the submitted work; Dr. HAG reports personal fees from Actelion, Bayer, Gilead, GSK, MSD, Pfizer and United Therapeutics, outside the submitted work; Dr. MG reports personal fees from Actelion, Bayer and GSK, outside the submitted work; Dr. MMH reports personal fees from Acceleron, Actelion, Bayer, MSD and Pfizer, outside the submitted work; Dr. CDV reports personal fees from Actelion, Bayer, GSK, MSD, Pfizer, and United Therapeutics, outside the submitted work; Dr. RE reports personal fees from Actelion, Boehringer Ingelheim, OMT, Bayer, and Berlin Chemie; grants from Actelion and Boehringer Ingelheim, outside the submitted work; Dr. MH reports grants and personal fees from Actelion, personal fees from Bayer, Berlin Chemie, Boehringer Ingelheim, GSK, Janssen, Novartis and MSD, outside the submitted work; Dr. MH reports personal fees from Acceleron, Actelion, AstraZeneca, Bayer, BERLIN CHEMIE, GSK, MSD, Novartis and OMT, outside the submitted work; Dr. HW reports personal fees from Action, Bayer, Biotest, Boehringer, GSK, Pfizer, and Roche, outside the submitted work; Dr. DS reports personal fees from Actelion, Bayer, and GSK, outside the submitted work; Dr. LS reports personal fees from Actelion, Bayer, and MSD, outside the submitted work; Dr. SU reports grants from Swiss National Science Foundation, Zurich Lung, Swiss Lung, and Orpha Swiss, grants and personal fees from Actelion SA/Johnson & Johnson, Switzerland, and MSD Switzerland, outside the submitted work; Dr. TJL reports personal fees from Actelion, Janssen-Cilag, BMS, MSD, and OMT GmbH, outside the submitted work; Dr. LB reports personal fees from Actelion, outside the submitted work; Dr. MC reports personal fees from Boehringer Ingelheim Pharma GmbH, Roche Pharma, and Boehringer Ingelheim, outside the submitted work; Dr. HW reports personal fees from Boehringer Ingelheim, and Roche, outside the submitted work. Dr. EG reports personal fees from Actelion, Janssen, Bayer, MSD, Bial, OrPha Swiss GmbH, OMT and Medscape, outside the submitted work; Dr. SR reports personal fees from Actelion, Bayer, GSK, Pfizer, Novartis, Gilead, MSD, and United Therapeutics, outside the submitted work. The authors have no other conflicts of interest to declare. Publisher Copyright: © Cardiovascular Diagnosis and Therapy. All rights reserved.Background: Pulmonary arterial hypertension (PAH) is common in congenital heart disease (CHD). Because clinical-trial data on PAH associated with CHD (PAH-CHD) remain limited, registry data on the long-term course are essential. This analysis aimed to update information from the COMPERA-CHD registry on management strategies based on real-world data. Methods: The prospective international pulmonary hypertension registry COMPERA has since 2007 enrolled more than 10,000 patients. COMPERA-CHD is a sub-registry for patients with PAH-CHD Results: A total of 769 patients with PAH-CHD from 62 specialized centers in 12 countries were included into COMPERA-CHD from January 2007 through September 2020. At the last follow-up in 09/2020, patients [mean age 45.3±16.8 years; 512 (66%) female] had either post-tricuspid shunts (n=359; 46.7%), pre-tricuspid shunts (n=249; 32.4%), complex CHD (n=132; 17.2%), congenital left heart or aortic valve or aortic disease (n=9; 1.3%), or miscellaneous CHD (n=20; 2.6%). The mean 6-minute walking distance was 369±121 m, and 28.2%, 56.0%, and 3.8% were in WHO functional class I/II, III or IV, respectively (12.0% unknown). Compared with the previously published COMPERA-CHD data, after 21 months of followup, the number of included PAH-CHD patients increased by 91 (13.4%). Within this group the number of Eisenmenger patients rose by 39 (16.3%), the number of “Non-Eisenmenger PAH” patients by 45 (26.9%). Currently, among the 674 patients from the PAH-CHD group with at least one follow-up, 450 (66.8%) received endothelin receptor antagonists (ERA), 416 (61.7%) PDE-5 inhibitors, 85 (12.6%) prostacyclin analogues, and 36 (5.3%) the sGC stimulator riociguat. While at first inclusion in the COMPERA-CHD registry, treatment was predominantly monotherapy (69.3%), this has shifted to favoring combination therapy in the current group (53%). For the first time, the nature, frequency, and treatment of significant comorbidities requiring supportive care and medication are described. Conclusions: Analyzing “real life data” from the international COMPERA-CHD registry, we present a comprehensive overview about current management modalities and treatment concepts in PAH-CHD. There was an trend towards more aggressive treatment strategies and combination therapies. In the future, particular attention must be directed to the “Non-Eisenmenger PAH” group and to patients with complex CHD, including Fontan patients.publishersversionPeer reviewe

Idiopathic pulmonary arterial hypertension phenotypes determined by cluster analysis from the COMPERA registry

Funding Information: Marius M. Hoeper has received fees for lectures and/or consultations from Acceleron, Actelion, Bayer, MSD, and Pfizer. Nicola Benjamin has received fees for lectures and/or consultations from Actelion. Ekkehard Grünig has received fees for lectures and/or consultations from Actelion, Bayer, GSK, MSD, United Therapeutics, and Pfizer. Karen M. Olsson has received fees for lectures and/or consultations from Actelion, Bayer, United Therapeutics, GSK, and Pfizer. C. Dario Vizza has received fees from Actelion, Bayer, GSK, MSD, Pfizer, and United Therapeutics Europe. Anton Vonk-Noordegraaf has received fees for lectures and/or consultation from Actelion, Bayer, GSK, and MSD. Oliver Distler has/had a consultancy relationship with and/or has received research funding from 4-D Science, Actelion, Active Biotec, Bayer, Biogen Idec, Boehringer Ingelheim Pharma, BMS, ChemoAb, EpiPharm, Ergonex, espeRare foundation, GSK, Genentech/Roche, Inventiva, Lilly, medac, MedImmune, Mitsubishi Tanabe, Pharmacyclics, Pfizer, Sanofi, Serodapharm, and Sinoxa in the area of potential treatments of scleroderma and its complications including pulmonary arterial hypertension. In addition, Prof Distler has a patent for mir-29 for the treatment of systemic sclerosis licensed. Christian Opitz has received fees from Actelion, Bayer, GSK, Pfizer, and Novartis. J. Simon R. Gibbs has received fees for lectures and/or consultations from Actelion, Bayer, Bellerophon, GSK, MSD, and Pfizer. Marion Delcroix has received fees from Actelion, Bayer, GSK, and MSD. H. Ardeschir Ghofrani has received fees from Actelion, Bayer, Gilead, GSK, MSD, Pfizer, and United Therapeutics. Doerte Huscher has received fees for lectures and consultations from Actelion. David Pittrow has received fees for consultations from Actelion, Biogen, Aspen, Bayer, Boehringer Ingelheim, Daiichi Sankyo, and Sanofi. Stephan Rosenkranz has received fees for lectures and/or consultations from Actelion, Bayer, GSK, Pfizer, Novartis, Gilead, MSD, and United Therapeutics. Martin Claussen reports honoraria for lectures from Boehringer Ingelheim Pharma GmbH and Roche Pharma and for serving on advisory boards from Boehringer Ingelheim, outside the submitted work. Heinrike Wilkens reports personal fees from Boehringer and Roche during the conduct of the study and personal fees from Bayer, Biotest, Actelion, GSK, and Pfizer outside the submitted work. Juergen Behr received grants from Boehringer Ingelheim and personal fees for consultation or lectures from Actelion, Bayer, Boehringer Ingelheim, and Roche. Hubert Wirtz reports personal fees from Boehringer Ingelheim and Roche outside the submitted work. Hening Gall reports personal fees from Actelion, AstraZeneca, Bayer, BMS, GSK, Janssen-Cilag, Lilly, MSD, Novartis, OMT, Pfizer, and United Therapeutics outside the submitted work. Elena Pfeuffer-Jovic reports personal fees from Actelion, Boehringer Ingelheim, Novartis, and OMT outside the submitted work. Laura Scelsi reports personal fees from Actelion, Bayer, and MSD outside the submitted work. Siliva Ulrich reports grants from Swiss National Science Foundation, Zurich Lung, Swiss Lung, and Orpha Swiss, and grants and personal fees from Actelion SA/Johnson & Johnson Switzerland and MSD Switzerland outside the submitted work. The remaining authors have no conflicts of interest to disclose. Funding Information: This work was supported by the German Centre of Lung Research (DZL). COMPERA is funded by unrestricted grants from Acceleron , Actelion Pharmaceuticals , Bayer , OMT , and GSK . These companies were not involved in data analysis or the writing of this manuscript. Publisher Copyright: © 2020 The Authors Copyright: Copyright 2020 Elsevier B.V., All rights reserved.The term idiopathic pulmonary arterial hypertension (IPAH) is used to categorize patients with pre-capillary pulmonary hypertension of unknown origin. There is considerable variability in the clinical presentation of these patients. Using data from the Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension, we performed a cluster analysis of 841 patients with IPAH based on age, sex, diffusion capacity of the lung for carbon monoxide (DLCO; <45% vs ≥45% predicted), smoking status, and presence of comorbidities (obesity, hypertension, coronary heart disease, and diabetes mellitus). A hierarchical agglomerative clustering algorithm was performed using Ward's minimum variance method. The clusters were analyzed in terms of baseline characteristics; survival; and response to pulmonary arterial hypertension (PAH) therapy, expressed as changes from baseline to follow-up in functional class, 6-minute walking distance, cardiac biomarkers, and risk. Three clusters were identified: Cluster 1 (n = 106; 12.6%): median age 45 years, 76% females, no comorbidities, mostly never smokers, DLCO ≥45%; Cluster 2 (n = 301; 35.8%): median age 75 years, 98% females, frequent comorbidities, no smoking history, DLCO mostly ≥45%; and Cluster 3 (n = 434; 51.6%): median age 72 years, 72% males, frequent comorbidities, history of smoking, and low DLCO. Patients in Cluster 1 had a better response to PAH treatment than patients in the 2 other clusters. Survival over 5 years was 84.6% in Cluster 1, 59.2% in Cluster 2, and 42.2% in Cluster 3 (unadjusted p < 0.001 for comparison between all groups). The population of patients diagnosed with IPAH is heterogenous. This cluster analysis identified distinct phenotypes, which differed in clinical presentation, response to therapy, and survival.publishersversionPeer reviewe

Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex

Viral genetic tools to target specific brain cell types in humans and non-genetic model organisms will transform basic neuroscience and targeted gene therapy. Here we used comparative epigenetics to identify thousands of human neuronal subclass-specific putative enhancers to regulate viral tools, and 34% of these were conserved in mouse. We established an AAV platform to evaluate cellular specificity of functional enhancers by multiplexed fluorescent in situ hybridization (FISH) and single cell RNA sequencing. Initial testing in mouse neocortex yields a functional enhancer discovery success rate of over 30%. We identify enhancers with specificity for excitatory and inhibitory classes and subclasses including PVALB, LAMP5, and VIP/LAMP5 cells, some of which maintain specificity in vivo or ex vivo in monkey and human neocortex. Finally, functional enhancers can be proximal or distal to cellular marker genes, conserved or divergent across species, and could yield brain-wide specificity greater than the most selective marker genes