Asymmetric inheritance of centrosomes maintains stem cell properties in human neural progenitor cells.

During human forebrain development, neural progenitor cells (NPCs) in the ventricular zone (VZ) undergo asymmetric cell divisions to produce a self-renewed progenitor cell, maintaining the potential to go through additional rounds of cell divisions, and differentiating daughter cells, populating the developing cortex. Previous work in the embryonic rodent brain suggested that the preferential inheritance of the pre-existing (older) centrosome to the self-renewed progenitor cell is required to maintain stem cell properties, ensuring proper neurogenesis. If asymmetric segregation of centrosomes occurs in NPCs of the developing human brain, which depends on unique molecular regulators and species-specific cellular composition, remains unknown. Using a novel, recombination-induced tag exchange-based genetic tool to birthdate and track the segregation of centrosomes over multiple cell divisions in human embryonic stem cell-derived regionalised forebrain organoids, we show the preferential inheritance of the older mother centrosome towards self-renewed NPCs. Aberration of asymmetric segregation of centrosomes by genetic manipulation of the centrosomal, microtubule-associated protein Ninein alters fate decisions of NPCs and their maintenance in the VZ of human cortical organoids. Thus, the data described here use a novel genetic approach to birthdate centrosomes in human cells and identify asymmetric inheritance of centrosomes as a mechanism to maintain self-renewal properties and to ensure proper neurogenesis in human NPCs.

Towards clinical breakpoints for non-tuberculous mycobacteria - Determination of epidemiological cut off values for the Mycobacterium avium complex and Mycobacterium abscessus using broth microdilution.

OBJECTIVE: For non-tuberculous mycobacteria (NTM), minimum inhibitory concentration (MIC) distributions of wild-type isolates have not been systematically evaluated despite their importance for establishing antimicrobial susceptibility testing (AST) breakpoints. METHODS: We gathered MIC distributions for drugs used against the Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) obtained by commercial broth microdilution (SLOMYCOI and RAPMYCOI) from 12 laboratories. Epidemiological cut-off values (ECOFFs) and tentative ECOFFs (TECOFFs) were determined by EUCAST methodology including quality control (QC) strains. RESULTS: The clarithromycin ECOFF was 16 mg/L for M. avium (n = 1271) whereas TECOFFs were 8 mg/L for M. intracellulare (n = 415) and 1 mg/L for MAB (n = 1014) confirmed by analysing MAB subspecies without inducible macrolide resistance (n = 235). For amikacin, the ECOFFs were 64 mg/L for MAC and MAB. For moxifloxacin, the WT spanned >8 mg/L for both MAC and MAB. For linezolid, the ECOFF and TECOFF were 64 mg/L for M. avium and M. intracellulare, respectively. Current CLSI breakpoints for amikacin (16 mg/L), moxifloxacin (1 mg/L) and linezolid (8 mg/L) divided the corresponding WT distributions. For QC M. avium and M. peregrinum, ≥95% of MIC values were well within recommended QC ranges. CONCLUSION: As a first step towards clinical breakpoints for NTM, (T)ECOFFs were defined for several antimicrobials against MAC and MAB. Broad wild-type MIC distributions indicate a need for further method refinement which is now under development within the EUCAST subcommittee for anti-mycobacterial drug susceptibility testing. In addition, we showed that several CLSI NTM breakpoints are not consistent in relation to the (T)ECOFFs.

Intervención educativa dirigida al uso de métodos anticonceptivos en adolescentes / An educational intervention focused on the use of contraceptive methods in adolescents

El inicio de las relaciones sexuales en la adolescencia puede generar problemas de salud biopsi-cosociales, los que pueden ser más complejos sin el adecuado conocimiento acerca del uso opor-tuno de métodos anticonceptivos. La investigación tuvo el objetivo de valorar la efectividad de una intervención educativa dirigida a las conductas sexuales y reproductivas de riesgo en la adolescencia, en dos Consejos Populares del municipio de Quemado de Güines durante el perio-do 2017-2021. Se desarrolló un estudio preexperimental con una población constituida por 1501 adolescentes, de la que se seleccionó una muestra 102 individuos mediante un procedimiento no probabilístico atendiendo a criterios. En el estudio predominó: el sexo femenino (51%), aquellos con edades entre 13 y 15 años (67,7%) y los que no habían comenzado una vida sexual activa (54,9%). La intervención educativa produjo cambios estadísticamente significativos en el contexto de investigación en relación con la información sobre métodos anticonceptivos y la percepción del riesgo sobre su uso inadecuado.

The beginning of sexual relations in adolescence can generate biopsychosocial health problems, which can be more complex without adequate knowledge about the timely use of contraceptive methods. The research aimed to assess the effectiveness of an educational intervention aimed at sexual and reproductive risk behaviors in adolescents in two Popular Councils of the Quemado de Güines Hall during 2017-2021. A pre-experimental study was developed with a population of 1501 adolescents, from which a sample of 102 individuals was selected through a non-probabi-listic procedure according to criteria. The study predominated: the female gender (51%), the ages between 13 and 15 (67.7%), and those ones who had not started an active sexual life (54.9%). The educational intervention produced statistically significant changes in the research context concerning the information on contraceptive methods and the perception of risk regar-ding its inappropriate use

In vitro Evaluation of Isoniazid Derivatives as Potential Agents Against Drug-Resistant Tuberculosis.

The upsurge of multidrug-resistant tuberculosis has toughened the challenge to put an end to this epidemic by 2030. In 2020 the number of deaths attributed to tuberculosis increased as compared to 2019 and newly identified multidrug-resistant tuberculosis cases have been stably close to 3%. Such a context stimulated the search for new and more efficient antitubercular compounds, which culminated in the QSAR-oriented design and synthesis of a series of isoniazid derivatives active against Mycobacterium tuberculosis. From these, some prospective isonicotinoyl hydrazones and isonicotinoyl hydrazides are studied in this work. To evaluate if the chemical derivatizations are generating compounds with a good performance concerning several in vitro assays, their cytotoxicity against human liver HepG2 cells was determined and their ability to bind human serum albumin was thoroughly investigated. For the two new derivatives presented in this study, we also determined their lipophilicity and activity against both the wild type and an isoniazid-resistant strain of Mycobacterium tuberculosis carrying the most prevalent mutation on the katG gene, S315T. All compounds were less cytotoxic than many drugs in clinical use with IC50 values after a 72 h challenge always higher than 25 µM. Additionally, all isoniazid derivatives studied exhibited stronger binding to human serum albumin than isoniazid itself, with dissociation constants in the order of 10-4-10-5 M as opposed to 10-3 M, respectively. This suggests that their transport and half-life in the blood stream are likely improved when compared to the parent compound. Furthermore, our results are a strong indication that the N' = C bond of the hydrazone derivatives of INH tested is essential for their enhanced activity against the mutant strain of M. tuberculosis in comparison to both their reduced counterparts and INH.

Mammalian uterine morphogenesis and variations.

In eutherian and marsupial mammals, the site of embryo implantation and gestation is the uterus. Uterine morphologies vary between mammalian species. For example, laboratory mice have a bipartite uterus with two uterine horns and a single cervix, whereas humans have a simplex uterus with a single chamber and single cervix. The precursor tissue of the uterus, oviducts, and upper vagina is the Müllerian duct epithelium and its adjacent mesenchyme. Morphological variation between species is established during embryogenesis by species-specific differences in Müllerian duct fusion at the midline, growth, and differentiation. In humans, alterations in Müllerian duct development can lead to variations in uterine morphology that correlate with increased risks of miscarriage and infertility. Here we review the developmental genetic factors that regulate Müllerian duct development, uterine morphogenesis, and human uterine variation.

Ancient and recent differences in the intrinsic susceptibility of Mycobacterium tuberculosis complex to pretomanid.

OBJECTIVES: To develop a robust phenotypic antimicrobial susceptibility testing (AST) method with a correctly set breakpoint for pretomanid (Pa), the most recently approved anti-tuberculosis drug. METHODS: The Becton Dickinson Mycobacterial Growth Indicator Tube™ (MGIT) system was used at six laboratories to determine the MICs of a phylogenetically diverse collection of 356 Mycobacterium tuberculosis complex (MTBC) strains to establish the epidemiological cut-off value for pretomanid. MICs were correlated with WGS data to study the genetic basis of differences in the susceptibility to pretomanid. RESULTS: We observed ancient differences in the susceptibility to pretomanid among various members of MTBC. Most notably, lineage 1 of M. tuberculosis, which is estimated to account for 28% of tuberculosis cases globally, was less susceptible than lineages 2, 3, 4 and 7 of M. tuberculosis, resulting in a 99th percentile of 2 mg/L for lineage 1 compared with 0.5 mg/L for the remaining M. tuberculosis lineages. Moreover, we observed that higher MICs (≥8 mg/L), which probably confer resistance, had recently evolved independently in six different M. tuberculosis strains. Unlike the aforementioned ancient differences in susceptibility, these recent differences were likely caused by mutations in the known pretomanid resistance genes. CONCLUSIONS: In light of these findings, the provisional critical concentration of 1 mg/L for MGIT set by EMA must be re-evaluated. More broadly, these findings underline the importance of considering the global diversity of MTBC during clinical development of drugs and when defining breakpoints for AST.

Injection and electroporation of plasmid DNA into human cortical organoids.

Pluripotent stem cell-derived human cortical organoids allow for the analysis of stem cell behavior and neurogenesis in neural tissues. Delivery of plasmid DNA into organoids permits visualization of individual cells, characterization of cellular components, and manipulation of gene expression. We describe a protocol to transfect cells inside organoids with plasmid DNA using micro-injection and electroporation, allowing for DNA delivery to cells within cortical units. This protocol was optimized for cortical organoids; however, it may be adapted to other organoid models. For complete details on the use and execution of this protocol, please refer to Denoth-Lippuner et al. (2021).

Electrochemical Detection of Single-Nucleotide Polymorphism Associated with Rifampicin Resistance in Mycobacterium tuberculosis Using Solid-Phase Primer Elongation with Ferrocene-Linked Redox-Labeled Nucleotides.

Here, we report the electrochemical detection of single-point mutations using solid-phase isothermal primer elongation with redox-labeled oligonucleotides. A single-base mutation associated with resistance to rifampicin, an antibiotic commonly used for the treatment of Mycobacterium tuberculosis, was used as a model system to demonstrate a proof-of-concept of the approach. Four 5'-thiolated primers, designed to be complementary with the same fragment of the target sequence and differing only in the last base, addressing the polymorphic site, were self-assembled via chemisorption on individual gold electrodes of an array. Following hybridization with single-stranded DNA, Klenow (exo-) DNA polymerase-mediated primer extension with ferrocene-labeled 2'-deoxyribonucleoside triphosphates (dNFcTPs) was only observed to proceed at the electrode where there was full complementarity between the surface-tethered probe and the target DNA being interrogated. We tested all four ferrocenylethynyl-linked dNTPs and optimized the ratio of labeled/natural nucleotides to achieve maximum sensitivity. Following a 20 min hybridization step, Klenow (exo-) DNA polymerase-mediated primer elongation at 37 °C for 5 min was optimal for the enzymatic incorporation of a ferrocene-labeled nucleotide, achieving unequivocal electrochemical detection of a single-point mutation in 14 samples of genomic DNA extracted from Mycobacterium tuberculosis strains. The approach is rapid, cost-effective, facile, and can be extended to multiplexed electrochemical single-point mutation genotyping.

Designing new antitubercular isoniazid derivatives with improved reactivity and membrane trafficking abilities.

Isoniazid (INH) is one of the two most effective first-line antitubercular drugs and is still used at the present time as a scaffold for developing new compounds to fight TB. In a previous study, we have observed that an INH derivative, an hydrazide N'-substituted with a C10acyl chain, was able to counterbalance its smaller reactivity with a higher membrane permeability. This resulted in an improved performance against the most prevalent Mycobacterium tuberculosis (Mtb) resistant strain (S315T), compared to INH. In this work, we have designed two new series of INH derivatives (alkyl hydrazides and hydrazones) with promising in silico properties, namely membrane permeabilities and spontaneous IN* radical formation. The kinetics, cytotoxicity, and biological activity evaluations confirmed the in silico predictions regarding the very high reactivity of the alkyl hydrazides. The hydrazones, on the other hand, showed very similar behavior compared to INH, particularly in biological tests that take longer to complete, indicating that these compounds are being hydrolyzed back to INH. Despite their improved membrane permeabilities, the reactivities of these two series are too high, impairing their overall performance. Nevertheless, the systematic data gathered about these compounds have showed us the need to find a balance between lipophilicity and reactivity, which is paramount to devise better INH-based derivatives aimed at circumventing Mtb resistance.

Visualization of individual cell division history in complex tissues using iCOUNT.

The division potential of individual stem cells and the molecular consequences of successive rounds of proliferation remain largely unknown. Here, we developed an inducible cell division counter (iCOUNT) that reports cell division events in human and mouse tissues in vitro and in vivo. Analyzing cell division histories of neural stem/progenitor cells (NSPCs) in the developing and adult brain, we show that iCOUNT can provide novel insights into stem cell behavior. Further, we use single-cell RNA sequencing (scRNA-seq) of iCOUNT-labeled NSPCs and their progenies from the developing mouse cortex and forebrain-regionalized human organoids to identify functionally relevant molecular pathways that are commonly regulated between mouse and human cells, depending on individual cell division histories. Thus, we developed a tool to characterize the molecular consequences of repeated cell divisions of stem cells that allows an analysis of the cellular principles underlying tissue formation, homeostasis, and repair.

Allogeneic human neural stem cells for improved therapeutic delivery to peritoneal ovarian cancer.

BACKGROUND: Immortalized, clonal HB1.F3.CD 21 human neural stem/progenitor cells (NSCs), loaded with therapeutic cargo prior to intraperitoneal (IP) injection, have been shown to improve the delivery and efficacy of therapeutic agents in pre-clinical models of stage III ovarian cancer. In previous studies, the distribution and efficacy of the NSC-delivered cargo has been examined; however, the fate of the NSCs has not yet been explored. METHODS: To monitor NSC tropism, we used an unconventional method of quantifying endocytosed gold nanorods to overcome the weaknesses of existing cell-tracking technologies. RESULTS: Here, we report efficient tumor tropism of HB1.F3.CD 21 NSCs, showing that they primarily distribute to the tumor stroma surrounding individual tumor foci within 3 h after injection, reaching up to 95% of IP metastases without localizing to healthy tissue. Furthermore, we demonstrate that these NSCs are non-tumorigenic and non-immunogenic within the peritoneal setting. CONCLUSIONS: Their efficient tropism, combined with their promising clinical safety features and potential for cost-effective scale-up, positions this NSC line as a practical, off-the-shelf platform to improve the delivery of a myriad of peritoneal cancer therapeutics.

Multicentre testing of the EUCAST broth microdilution reference method for MIC determination on Mycobacterium tuberculosis.

OBJECTIVES: The first objective of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee for antimycobacterial susceptibility testing (AMST), launched in 2016, was to set a reference method for determining the MICs of antituberculous agents, since many protocols are used worldwide and a consensus one is needed for the determination of microbiological breakpoints. METHODS: During 2017 and 2018, MIC determination protocols were evaluated prospectively in a multicentre study within the four AMST laboratories. MIC results were obtained for isoniazid, levofloxacin and amikacin on the reference strain Mycobacterium tuberculosis H37Rv ATCC 27294. Broth microdilution (BMD) in Middlebrook 7H9 and solid medium dilution (SMD) in Middlebrook 7H10 were performed using two inoculum concentrations. MICs were interpreted with regard to visual and 99% inhibition after 7, 14 or 21 days of incubation for BMD and 21 days for SMD. RESULTS: Following the EUCAST reference protocol, intra- and inter-assay agreements were within ±1 MIC dilution for >95% of the observations for the three drugs in both methods. MIC values, presented as MIC mode (range) for BMD and SMD respectively, were: 0.03 (0.015-0.06) mg/L and 0.12 (0.06-0.25) mg/L for isoniazid, 0.25 mg/L (0.25-0.5) and 0.5 mg/L (0.12-0.5) for levofloxacin, and 0.5 mg/L (0.5-1.0) and 0.5 mg/L (0.5-1.0) for amikacin. CONCLUSIONS: Both SMD and BMD were reproducible and eligible as a reference method for MIC determination of the Mycobacterium tuberculosis complex (MTBC). BMD was finally selected as the EUCAST reference method. From now on it will be used to set epidemiological cut-off values and clinical breakpoints of new and old antituberculous agents.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis complex isolates - the EUCAST broth microdilution reference method for MIC determination.

SCOPE: Several methods are used worldwide for antibiotic susceptibility testing (AST) for the Mycobacterium tuberculosis complex (MTBC). The variability in the results obtained with these methods hampers setting epidemiological cut-off (ECOFF) values and clinical breakpoints according to EUCAST guidelines. Methods for susceptibility testing and determination of the minimal inhibitory concentrations (MICs) need to be standardized for MTBC isolates for old and new agents. Our objective was to establish a standardized reference method for MIC determination for MTBC. METHODS: The EUCAST antimycobacterial susceptibility testing subcommittee (AMST) compared protocols of MIC determination with regard to medium, inoculum preparation, antituberculous agent preparation, incubation, reading of the results and interpretation. RECOMMENDATIONS: The EUCAST reference method of MIC determination for MTBC is the broth microdilution method in Middlebrook 7H9-10% OADC medium. The final inoculum is a 105 CFU/mL suspension, obtained from a 10-2 dilution of a 0.5 McFarland suspension prepared after vortexing bacterial colonies with glass beads before suspending them in sterile water. The culture is maintained in a U-shaped 96-well polystyrene microtitre sterile plate with a lid incubated at 36° ± 1°C. Reading is done using an inverted mirror as soon as the 1:100 diluted control (i.e. 103 CFU/mL suspension) shows visual growth. The MIC, expressed in mg/L, is the lowest concentration that inhibits visual growth. Mycobacterium tuberculosis H37Rv ATCC 27294 is used as the reference strain and its targeted MIC values are within the range 0.03-0.12 for isoniazid, 0.12-0.5 for levofloxacin and 0.25-1 mg/L for amikacin. CONCLUSIONS: The EUCAST reference method for MTBC was endorsed by EUCAST after public consultation and will from now on be used to define EUCAST ECOFFs and clinical breakpoints. This reference method is not primarily intended to be used under routine conditions and the AST methods will need to be calibrated against this reference method to be used with EUCAST breakpoints.

Erratum: Enhanced Delivery of Oncolytic Adenovirus by Neural Stem Cells for Treatment of Metastatic Ovarian Cancer.

[This corrects the article DOI: 10.1016/j.omto.2018.12.003.].

Emergence of multidrug-resistant Mycobacterium tuberculosis of the Beijing lineage in Portugal and Guinea-Bissau: a snapshot of moving clones by whole-genome sequencing.

The Beijing genotype comprises a highly disseminated strain type that is frequently associated with multidrug resistant (MDR) tuberculosis (TB) and increased transmissibility but, countries such as Portugal and Guinea-Bissau fall outside the regions phylogeographically associated with this specific genotype. Nevertheless, recent data shows that this genotype might be gradually emerging in these two countries as an underlying cause of primary MDR-TB. Here, we describe the emergence of Mycobacterium tuberculosis Beijing strains associated with MDR-TB in Portugal and Guinea-Bissau demonstrating the presence of the well described superclusters 100-32 and 94-32 in Portugal and Guinea-Bissau, respectively. Genome-wide analysis and comparison with a global genomic dataset of M. tuberculosis Beijing strains, revealed the presence of two genomic clusters encompassing isolates from Portugal and Guinea-Bissau, GC1 (n = 121) and GC2 (n = 39), both of which bore SNP signatures compatible with the 100-32/B0/W148 and 94-32/Central Asia Outbreak clades, respectively. Moreover, GC2 encompasses a cross-border cluster between Portugal, Guinea-Bissau and Brazil thus supporting migration-associated introduction of MDR-TB and subsequent clonal expansion at the community-level. The comparison with global Beijing datasets demonstrates the global reach of the disease and its complex dissemination across multiple countries while in parallel there are clear microevolutionary trajectories towards extensively drug resistant TB.

The membrane periodic skeleton is an actomyosin network that regulates axonal diameter and conduction.

Neurons have a membrane periodic skeleton (MPS) composed of actin rings interconnected by spectrin. Here, combining chemical and genetic gain- and loss-of-function assays, we show that in rat hippocampal neurons the MPS is an actomyosin network that controls axonal expansion and contraction. Using super-resolution microscopy, we analyzed the localization of axonal non-muscle myosin II (NMII). We show that active NMII light chains are colocalized with actin rings and organized in a circular periodic manner throughout the axon shaft. In contrast, NMII heavy chains are mostly positioned along the longitudinal axonal axis, being able to crosslink adjacent rings. NMII filaments can play contractile or scaffolding roles determined by their position relative to actin rings and activation state. We also show that MPS destabilization through NMII inactivation affects axonal electrophysiology, increasing action potential conduction velocity. In summary, our findings open new perspectives on axon diameter regulation, with important implications in neuronal biology.

Design, synthesis, and evaluation of new 2-(quinoline-4-yloxy)acetamide-based antituberculosis agents.

Using a classical molecular simplification approach, a series of 36 quinolines were synthesized and evaluated as in vitro inhibitors of Mycobacterium tuberculosis (M. tuberculosis) growth. Structure-activity relationship (SAR) studies leaded to potent antitubercular agents, with minimum inhibitory concentration (MIC) values as low as 0.3 µM against M. tuberculosis H37Rv reference strain. Furthermore, the lead compounds were active against multidrug-resistant strains, without cross-resistance with some first- and second-line drugs. Testing the molecules against a spontaneous mutant strain containing a single mutation in the qcrB gene (T313A) indicated that the synthesized quinolines targeted the cytochrome bc1 complex. In addition, leading compounds were devoid of apparent toxicity to HepG2 and Vero cells and showed moderate elimination rates in human liver S9 fractions. Finally, the selected structures inhibited M. tuberculosis growth in a macrophage model of tuberculosis infection. Taken together, these data indicate that this class of compounds may furnish candidates for the future development of antituberculosis drugs.

Using genomics to understand the origin and dispersion of multidrug and extensively drug resistant tuberculosis in Portugal.

Portugal is a low incidence country for tuberculosis (TB) disease. Now figuring among TB low incidence countries, it has since the 1990s reported multidrug resistant and extensively drug resistant (XDR) TB cases, driven predominantly by two strain-types: Lisboa3 and Q1. This study describes the largest characterization of the evolutionary trajectory of M/XDR-TB strains in Portugal, spanning a time-period of two decades. By combining whole-genome sequencing and phenotypic susceptibility data for 207 isolates, we report the geospatial patterns of drug resistant TB, particularly the dispersion of Lisboa3 and Q1 clades, which underly 64.2% and 94.0% of all MDR-TB and XDR-TB isolates, respectively. Genomic-based similarity and a phylogenetic analysis revealed multiple clusters (n = 16) reflecting ongoing and uncontrolled recent transmission of M/XDR-TB, predominantly associated with the Lisboa3 and Q1 clades. These clades are now thought to be evolving in a polycentric mode across multiple geographical districts. The inferred evolutionary history is compatible with MDR- and XDR-TB originating in Portugal in the 70's and 80's, respectively, but with subsequent multiple emergence events of MDR and XDR-TB particularly involving the Lisboa3 clade. A SNP barcode was defined for Lisboa3 and Q1 and comparison with a phylogeny of global strain-types (n = 28 385) revealed the presence of Lisboa3 and Q1 strains in Europe, South America and Africa. In summary, Portugal displays an unusual and unique epidemiological setting shaped by >40 years of uncontrolled circulation of two main phylogenetic clades, leading to a sympatric evolutionary trajectory towards XDR-TB with the potential for global reach.

Profilin 1 delivery tunes cytoskeletal dynamics toward CNS axon regeneration.

After trauma, regeneration of adult CNS axons is abortive, causing devastating neurologic deficits. Despite progress in rehabilitative care, there is no effective treatment that stimulates axonal growth following injury. Using models with different regenerative capacities, followed by gain- and loss-of-function analysis, we identified profilin 1 (Pfn1) as a coordinator of actin and microtubules (MTs), powering axonal growth and regeneration. In growth cones, Pfn1 increased actin retrograde flow, MT growth speed, and invasion of filopodia by MTs, orchestrating cytoskeletal dynamics toward axonal growth. In vitro, active Pfn1 promoted MT growth in a formin-dependent manner, whereas localization of MTs to growth cone filopodia was facilitated by direct MT binding and interaction with formins. In vivo, Pfn1 ablation limited regeneration of growth-competent axons after sciatic nerve and spinal cord injury. Adeno-associated viral (AAV) delivery of constitutively active Pfn1 to rodents promoted axonal regeneration, neuromuscular junction maturation, and functional recovery of injured sciatic nerves, and increased the ability of regenerating axons to penetrate the inhibitory spinal cord glial scar. Thus, we identify Pfn1 as an important regulator of axonal regeneration and suggest that AAV-mediated delivery of constitutively active Pfn1, together with the identification of modulators of Pfn1 activity, should be considered to treat the injured nervous system.