Peer review perpetuates barriers for historically excluded groups.

Peer review is central to the scientific process and scientists' career advancement, but bias at various stages of the review process disadvantages some authors. Here we use peer review data from 312,740 biological sciences manuscripts across 31 studies to (1) examine evidence for differential peer review outcomes based on author demographics, (2) evaluate the efficacy of solutions to reduce bias and (3) describe the current landscape of peer review policies for 541 ecology and evolution journals. We found notably worse review outcomes (for example, lower overall acceptance rates) for authors whose institutional affiliations were in Asia, for authors whose country's primary language is not English and in countries with relatively low Human Development Indices. We found few data evaluating efficacy of interventions outside of reducing gender bias through double-blind review or diversifying reviewer/editorial boards. Despite evidence for review outcome gaps based on author demographics, few journals currently implement policies intended to mitigate bias (for example, 15.9% of journals practised double-blind review and 2.03% had reviewer guidelines that mentioned social justice issues). The lack of demographic equity signals an urgent need to better understand and implement evidence-based bias mitigation strategies.

Active learning narrows achievement gaps for underrepresented students in undergraduate science, technology, engineering, and math.

We tested the hypothesis that underrepresented students in active-learning classrooms experience narrower achievement gaps than underrepresented students in traditional lecturing classrooms, averaged across all science, technology, engineering, and mathematics (STEM) fields and courses. We conducted a comprehensive search for both published and unpublished studies that compared the performance of underrepresented students to their overrepresented classmates in active-learning and traditional-lecturing treatments. This search resulted in data on student examination scores from 15 studies (9,238 total students) and data on student failure rates from 26 studies (44,606 total students). Bayesian regression analyses showed that on average, active learning reduced achievement gaps in examination scores by 33% and narrowed gaps in passing rates by 45%. The reported proportion of time that students spend on in-class activities was important, as only classes that implemented high-intensity active learning narrowed achievement gaps. Sensitivity analyses showed that the conclusions are robust to sampling bias and other issues. To explain the extensive variation in efficacy observed among studies, we propose the heads-and-hearts hypothesis, which holds that meaningful reductions in achievement gaps only occur when course designs combine deliberate practice with inclusive teaching. Our results support calls to replace traditional lecturing with evidence-based, active-learning course designs across the STEM disciplines and suggest that innovations in instructional strategies can increase equity in higher education.

The Origins and Diversification of the Exceptionally Rich Gemsnakes (Colubroidea: Lamprophiidae: Pseudoxyrhophiinae) in Madagascar.

Processes leading to spectacular diversity of both form and species on islands have been well-documented under island biogeography theory, where distance from source and island size are key factors determining immigration and extinction resistance. But far less understood are the processes governing in situ diversification on the world's mega islands, where large and isolated land masses produced morphologically distinct radiations from related taxa on continental regions. Madagascar has long been recognized as a natural laboratory due to its isolation, lack of influence from adjacent continents, and diversification of spectacular vertebrate radiations. However, only a handful of studies have examined rate shifts of in situ diversification for this island. Here, we examine rates of diversification in the Malagasy snakes of the family Pseudoxyrhophiinae (gemsnakes) to understand if rates of speciation were initially high, enhanced by diversification into distinct biomes, and associated with key dentition traits. Using a genomic sequence-capture data set for 366 samples, we determine that all previously described and newly discovered species are delimitable and therefore useful candidates for understanding diversification trajectories through time. Our analysis detected no shifts in diversification rate between clades or changes in biome or dentition type. Remarkably, we demonstrate that rates of diversification of the gemsnake radiation, which originated in Madagascar during the early Miocene, remained steady throughout the Neogene. However, we do detect a significant slowdown in diversification during the Pleistocene. We also comment on the apparent paradox where most living species originated in the Pleistocene, despite diversification rates being substantially higher during the earlier 15 myr.

Phylogenomics of phrynosomatid lizards: conflicting signals from sequence capture versus restriction site associated DNA sequencing.

Sequence capture and restriction site associated DNA sequencing (RADseq) are popular methods for obtaining large numbers of loci for phylogenetic analysis. These methods are typically used to collect data at different evolutionary timescales; sequence capture is primarily used for obtaining conserved loci, whereas RADseq is designed for discovering single nucleotide polymorphisms (SNPs) suitable for population genetic or phylogeographic analyses. Phylogenetic questions that span both "recent" and "deep" timescales could benefit from either type of data, but studies that directly compare the two approaches are lacking. We compared phylogenies estimated from sequence capture and double digest RADseq (ddRADseq) data for North American phrynosomatid lizards, a species-rich and diverse group containing nine genera that began diversifying approximately 55 Ma. Sequence capture resulted in 584 loci that provided a consistent and strong phylogeny using concatenation and species tree inference. However, the phylogeny estimated from the ddRADseq data was sensitive to the bioinformatics steps used for determining homology, detecting paralogs, and filtering missing data. The topological conflicts among the SNP trees were not restricted to any particular timescale, but instead were associated with short internal branches. Species tree analysis of the largest SNP assembly, which also included the most missing data, supported a topology that matched the sequence capture tree. This preferred phylogeny provides strong support for the paraphyly of the earless lizard genera Holbrookia and Cophosaurus, suggesting that the earless morphology either evolved twice or evolved once and was subsequently lost in Callisaurus.

Genetic modifier screens to identify components of a redox-regulated cell adhesion and migration pathway.

Under normal physiological conditions, cells use oxidants, particularly H2O2, for signal transduction during processes such as proliferation and migration. Though recent progress has been made in determining the precise role H2O2 plays in these processes, many gaps still remain. To further understand this, we describe the use of a dominant enhancer screen to identify novel components of a redox-regulated cell migration and adhesion pathway in Drosophila melanogaster. Here, we discuss our methodology and progress as well as the benefits and limitations of applying such an approach to study redox-regulated pathways. Depending on the nature of these pathways, unbiased genetic modifier screens may prove a productive way to identify novel redox-regulated signaling components.

Endogenous transforming growth factor beta 1 suppresses inflammation and promotes survival in adult CNS.

Transforming growth factor beta1 (TGFbeta1) is a pleiotropic cytokine with potent neurotrophic and immunosuppressive properties that is upregulated after injury, but also expressed in the normal nervous system. In the current study, we examined the regulation of TGFbeta1 and the effects of TGFbeta1 deletion on cellular response in the uninjured adult brain and in the injured and regenerating facial motor nucleus. To avoid lethal autoimmune inflammation within 3 weeks after birth in TGFbeta1-deficient mice, this study was performed on a T- and B-cell-deficient RAG2-/- background. Compared with wild-type siblings, homozygous deletion of TGFbeta1 resulted in an extensive inflammatory response in otherwise uninjured brain parenchyma. Astrocytes increased in GFAP and CD44 immunoreactivity; microglia showed proliferative activity, expression of phagocytosis-associated markers [alphaXbeta2, B7.2, and MHC1 (major histocompatibility complex type 1)], and reduced branching. Ultrastructural analysis revealed focal blockade of axonal transport, perinodal damming of axonal organelles, focal demyelination, and myelin debris in granule-rich, phagocytic microglia. After facial axotomy, absence of TGFbeta1 led to a fourfold increase in neuronal cell death (52 vs 13%), decreased central axonal sprouting, and significant delay in functional recovery. It also interfered with the microglial response, resulting in a diminished expression of early activation markers [ICAM1 (intercellular adhesion molecule 1), alpha6beta1, and alphaMbeta2] and reduced proliferation. In line with axonal and glial findings in the otherwise uninjured CNS, absence of endogenous TGFbeta1 also caused an approximately 10% reduction in the number of normal motoneurons, pointing to an ongoing and potent trophic role of this anti-inflammatory cytokine in the normal as well as in the injured brain.

Fibrinogen inhibits neurite outgrowth via beta 3 integrin-mediated phosphorylation of the EGF receptor.

Changes in the molecular and cellular composition of the CNS after injury or disease result in the formation of an inhibitory environment that inhibits the regeneration of adult mammalian CNS neurons. Although a dramatic change in the CNS environment after traumatic injury or disease is hemorrhage because of vascular rupture or leakage of the blood-brain barrier, the potential role for blood proteins in repair processes remains unknown. Here we show that the blood protein fibrinogen is an inhibitor of neurite outgrowth that is massively deposited in the spinal cord after injury. We show that fibrinogen acts as a ligand for beta3 integrin and induces the transactivation of EGF receptor (EGFR) in neurons. Fibrinogen-mediated inhibition of neurite outgrowth is reversed by blocking either beta3 integrin or phoshorylation of EGFR. Inhibition of Src family kinases that mediate the cross-talk between integrin and growth factor receptors rescue the fibrinogen-induced phosphorylation of EGFR. These results identify fibrinogen as the first blood-derived inhibitor of neurite outgrowth and suggest fibrinogen-induced EGFR transactivation on neuronal cells as a molecular link between vascular and neuronal damage in the CNS after injury.

Axon regeneration through scars and into sites of chronic spinal cord injury.

Cellular and extracellular inhibitors are thought to restrict axon growth after chronic spinal cord injury (SCI), confronting the axon with a combination of chronic astrocytosis and extracellular matrix-associated inhibitors that collectively constitute the chronic "scar." To examine whether the chronically injured environment is strongly inhibitory to axonal regeneration, we grafted permissive autologous bone marrow stromal cells (MSCs) into mid-cervical SCI sites of adult rats, 6 weeks post-injury without resection of the "chronic scar." Additional subjects received MSCs genetically modified to express neurotrophin-3 (NT-3), providing a further local stimulus to axon growth. Anatomical analysis 3 months post-injury revealed extensive astrocytosis surrounding the lesion site, together with dense deposition of the inhibitory extracellular matrix molecule NG2. Despite this inhibitory environment, axons penetrated the lesion site through the chronic scar. Robust axonal regeneration occurred into chronic lesion cavities expressing NT-3. Notably, chronically regenerating axons preferentially associated with Schwann cell surfaces expressing both inhibitory NG2 substrates and the permissive substrates L1 and NCAM in the lesion site. Collectively, these findings indicate that inhibitory factors deposited at sites of chronic SCI do not create impenetrable boundaries and that inhibition can be balanced by local and diffusible signals to generate robust axonal growth even without resecting chronic scar tissue.

Neurotrophin-3 gradients established by lentiviral gene delivery promote short-distance axonal bridging beyond cellular grafts in the injured spinal cord.

Neurotrophic factor delivery to sites of spinal cord injury (SCI) promotes axon growth into but not beyond lesion sites. We tested the hypothesis that sustained growth factor gradients beyond regions of SCI will promote significant axonal bridging into and beyond lesions. Adult rats underwent C3 lesions to transect ascending dorsal column sensory axons, and autologous bone marrow stromal cells were grafted into the lesion to provide a cellular bridge for growth into the injured region. Concurrently, lentiviral vectors expressing neurotrophin-3 (NT-3) or green fluorescent protein (GFP) (controls) were injected into the host cord rostral to the lesion to promote axon extension beyond the graft/lesion. Four weeks later, NT-3 gradients beyond the lesion were detectable by ELISA in animals that received NT-3-expressing lentiviral vectors, with highest average NT-3 levels located near the rostral vector injection site. Significantly more ascending sensory axons extended into tissue rostral to the lesion site in animals injected with NT-3 vectors compared with GFP vectors, but only if the zone of NT-3 vector transduction extended continuously from the injection site to the graft; any "gap" in NT-3 expression from the graft to rostral tissue resulted in axon bridging failure. Despite axon bridging beyond the lesion, regenerating axons did not continue to grow over very long distances, even in the presence of a continuing growth factor gradient beyond the lesion. These findings indicate that a localized and continuous gradient of NT-3 can achieve axonal bridging beyond the glial scar, but growth for longer distances is not sustainable simply with a trophic stimulus.

Combinatorial therapy with neurotrophins and cAMP promotes axonal regeneration beyond sites of spinal cord injury.

Previous attempts to promote regeneration after spinal cord injury have succeeded in stimulating axonal growth into or around lesion sites but rarely beyond them. We tested whether a combinatorial approach of stimulating the neuronal cell body with cAMP and the injured axon with neurotrophins would propel axonal growth into and beyond sites of spinal cord injury. A preconditioning stimulus to sensory neuronal cell bodies was delivered by injecting cAMP into the L4 dorsal root ganglion, and a postinjury stimulus to the injured axon was administered by injecting neurotrophin-3 (NT-3) within and beyond a cervical spinal cord lesion site grafted with autologous bone marrow stromal cells. One to 3 months later, long-projecting dorsal-column sensory axons regenerated into and beyond the lesion. Regeneration beyond the lesion did not occur after treatment with cAMP or NT-3 alone. Thus, clear axonal regeneration beyond spinal cord injury sites can be achieved by combinatorial approaches that stimulate both the neuronal soma and the axon, representing a major advance in strategies to enhance spinal cord repair.

Axonal regeneration through regions of chondroitin sulfate proteoglycan deposition after spinal cord injury: a balance of permissiveness and inhibition.

Increased expression of certain extracellular matrix (ECM) molecules after CNS injury is believed to restrict axonal regeneration. The chondroitin sulfate proteoglycans (CSPGs) are one such class of ECM molecules that inhibit neurite outgrowth in vitro and are upregulated after CNS injury. We examined growth responses of several classes of axons to this inhibitory environment in the presence of a cellular fibroblast bridge in a spinal cord lesion site and after a growth factor stimulus at the lesion site (fibroblasts genetically modified to secrete NGF). Immunohistochemical analysis showed dense labeling of the CSPGs NG2, brevican, neurocan, versican, and phosphacan at the host-lesion interface after spinal cord injury (SCI). Furthermore, robust expression of NG2, and to a lesser extent versican, was also observed throughout grafts of control and NGF-secreting fibroblasts. Despite this inhibitory milieu, several axonal classes penetrated control fibroblast grafts, including dorsal column sensory, rubrospinal, and nociceptive axons. Axon growth was amplified more in the presence of NGF-secreting grafts. Confocal microscopy demonstrated that axon growth was, paradoxically, preferentially associated with NG2-rich substrates in both graft types. NG2 expression also increased after sciatic nerve injury, wherein axons successfully regenerate. Cellular sources of NG2 in SCI and peripheral nerve lesion sites included Schwann cells and endothelial cells. Notably, these same cellular sources in lesion sites produced the cell adhesion molecules L1 and laminin, and these molecules all colocalized. Thus, axons grow along substrates coexpressing both inhibitory and permissive molecules, suggesting that regeneration is successful when local permissive signals balance and exceed inhibitory signals.

The chondroitin sulfate proteoglycans neurocan, brevican, phosphacan, and versican are differentially regulated following spinal cord injury.

Chondroitin sulfate proteoglycans (CSPGs) are extracellular matrix (ECM) molecules that are widely expressed throughout the developing and adult CNS. In vitro studies demonstrate their potential to restrict neurite outgrowth, and it is believed that CSPGs also inhibit axonal regeneration after CNS injury in vivo. Previous studies demonstrated that CSPGs are generally upregulated after spinal cord injury, and more recent reports have begun to identify individual proteoglycans that may play dominant roles in limiting axonal regeneration. The current study systematically examined the extended deposition patterns after CNS injury of four putatively inhibitory CSPGs that have not been extensively investigated previously in vivo: neurocan, brevican, phosphacan, and versican. After spinal cord injury, neurocan, brevican, and versican immunolabeling increased within days in injured spinal cord parenchyma surrounding the lesion site and peaked at 2 weeks. Neurocan and versican were persistently elevated for 4 weeks postinjury, and brevican expression persisted for at least 2 months. On the other hand, phosphacan immunolabeling decreased in the same region immediately following injury but later recovered and then peaked after 2 months. Combined glial fibrillary acidic protein (GFAP) immunohistochemistry and in situ hybridization demonstrated that GFAP astrocytes constituted a source of neurocan production after spinal cord injury. Thus, the production of several CSPG family members is differentially affected by spinal cord injury, overall establishing a CSPG-rich matrix that persists for up to 2 months following injury. Optimization of strategies to reduce CSPG expression to enhance regeneration may need to target several different family members over an extended period following injury.

Lymphocyte infiltration in the injured brain: role of proinflammatory cytokines.

Studies using mouse axotomised facial motoneuron model show a strong and highly selective entry of CD3+ lymphocytes into the affected nucleus, with a maximum at Day 14, which coincides with the peak of neuronal cell death, microglial phagocytosis, and increased synthesis of interleukin-1 beta (IL1beta), tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma). We explored the possible involvement of these cytokines during the main phase of lymphocyte recruitment into the axotomised facial motor nucleus 7-21 days after nerve cut using mice homozygously deficient for IL1 receptor type 1 (IL1R1-/-), TNF receptor type 1 (TNFR1-/-), type 2 (TNFR2-/-) and type 1 and 2 (TNFR1&2-/-), IFNgamma receptor type 1 (IFNgammaR1-/-), and the appropriate controls for the genetic background. Transgenic deletion of IL1R1 led to a 54% decrease and that of TNFR2 to a 44% reduction in the number of CD3+ T-cells in the axotomised facial motor nucleus, with a similar relative decrease at Day 7, 14, and 21. Deletion of TNFR1 or IFNgammaR1 had no significant effect. Deletion of both TNFR1 and 2 (TNFR1&2-/-) caused a somewhat stronger, 63% decrease than did TNFR2 deletion alone, but this could be due to an almost complete inhibition of neuronal cell death. No mutations seemed to inhibit aggregation of CD3+ T-cells around glial nodules consisting of Ca-ion binding adaptor protein-1 (IBA1)+ phagocytotic microglia and neuronal debris. Altogether, the current data show the importance of IL1R1 and TNFR2 as the key players during the main phase of lymphocyte recruitment to the damaged part of the central nervous system.

NT-3 gene delivery elicits growth of chronically injured corticospinal axons and modestly improves functional deficits after chronic scar resection.

Nervous system growth factors promote axonal growth following acute spinal cord injury. In the present experiment, we examined whether delivery of neurotrophic factors after chronic spinal cord injury would also promote axonal growth and influence functional outcomes. Adult Fischer 344 rats underwent mid-thoracic spinal cord dorsal hemisection lesions. Three months later, primary fibroblasts genetically modified to express human neurotrophin-3 (NT-3) were placed in, and distal to, the lesion cavity. Upon sacrifice 3 months later (6 months following the initial lesion), NT-3-grafted animals exhibited significant growth of corticospinal axons up to 15 mm distal to the lesion site and showed a modest but significant 1.5-point improvement in locomotor scores (P < 0.05) on the BBB scale, compared to control-grafted animals. Thus, growth factor gene delivery can elicit growth of corticospinal axons in chronic stages of injury and improves functional outcomes compared to non-growth-factor-treated animals.

Spontaneous and augmented growth of axons in the primate spinal cord: effects of local injury and nerve growth factor-secreting cell grafts.

Little is known about molecular and cellular responses to spinal cord injury in primates. In this study, the normal milieu of the primate spinal cord was disturbed by multiple needle penetrations and cell injections in the mid-thoracic spinal cord; subsequent effects on local axons and expression of extracellular matrix (ECM) molecules were examined, together with effects of cellular delivery of nerve growth factor (NGF) to the injured region. Four adult rhesus monkeys each received injections of two grafts of autologous fibroblasts genetically modified to secrete human NGF, and, in control injection sites, two separate grafts of autologous fibroblasts transduced to express the reporter gene, beta-galactosidase. Three months later, Schwann cells extensively infiltrated the region of localized injury and penetrated both NGF and control fibroblast grafts. Marked upregulation of several ECM molecules occurred, including chondroitin and heparan sulfate proteoglycans and type IV collagen, in or adjacent to all injection sites. Schwann cells were an apparent source of some ECM expression. Spinal cord sensory axons and putative coerulospinal axons extended into both graft types, but they penetrated NGF grafts to a significantly greater extent. Many of these axons expressed the cell adhesion molecule L1. Thus, extensive cellular and molecular changes occur at sites of localized primate spinal cord injury and grafting, attributable in part to migrating Schwann cells, and are accompanied by spontaneous axonal plasticity. These molecular and cellular events closely resemble those observed in the rodent spinal cord after injury. Furthermore, as in rodent studies, cellular delivery of a trophic factor significantly augments axonal plasticity in the primate spinal cord.

Spinal cord injury elicits expression of keratan sulfate proteoglycans by macrophages, reactive microglia, and oligodendrocyte progenitors.

Keratan sulfate proteoglycans (KSPGs) are extracellular matrix molecules that appear to establish boundaries for axonal growth in the developing brain and spinal cord. In vitro studies confirm that KSPGs define inhibitory boundaries to extending neurites. The aim of the current study was to investigate whether KSPGs are expressed after spinal cord injury (SCI) and thereby might act as potential inhibitors of axonal growth. Adult Fischer 344 rats were subjected to spinal cord lesions, and the temporal and spatial expression of KSPGs was examined using the 5D4 monoclonal anti-KSPG antibody. In the intact spinal cord, a subpopulation of microglia expressed 5D4-KSPG throughout the white and gray matter. Within 24 hr of injury, 5D4-KSPG immunoreactivity substantially increased and appeared on cellular profiles in close proximity to the spinal cord lesion site, peaking 3 d after injury. Double immunolabeling revealed that 5D4-KSPG expression arose from multiple cell types at the lesion site, including reactive microglia, macrophages, and oligodendrocyte progenitors. Astrocytes were not identified as a source of 5D4-KSPG. The robust and extensive production of 5D4-KSPG at sites of SCI precedes the expression of other putatively inhibitory proteoglycan molecules such as chondroitin sulfate proteoglycans. This is the first demonstration that KSPGs are expressed after SCI in a temporal and spatial relationship that could exert an early and important role in modulating axonal growth after SCI.

NG2 is a major chondroitin sulfate proteoglycan produced after spinal cord injury and is expressed by macrophages and oligodendrocyte progenitors.

Several extracellular matrix (ECM) molecules have been identified as potent inhibitors of neurite outgrowth in vitro and are believed to limit axonal growth after CNS injury. Recent studies have shown that different members of the chondroitin sulfate proteoglycan (CSPG) class of putatively inhibitory ECM molecules are expressed after a number of CNS injuries. The purpose of this study was to evaluate the relative amounts of individual CSPGs expressed after spinal cord injury (SCI) and identify their cells of origin. Evaluation of total soluble CSPGs 2 weeks after dorsal column lesion in the rat demonstrated that NG2 is highly upregulated and is a major CSPG species. Immunocytochemical analysis further demonstrated that NG2 expression is upregulated within 24 hr of injury, peaks at 1 week, and remains elevated for at least an additional 7 weeks. NG2 expression results from a multicellular response to injury, including both reactive macrophages and oligodendrocyte progenitors; astrocytes were not identified as a major source of NG2. Immunocytochemical analysis of other CSPG family members 7 d after injury showed moderate upregulation of versican, brevican, and neurocan, and downregulation of phosphacan. Axonal tracing experiments demonstrated dense NG2 labeling adjacent to the forward processes of transected corticospinal tract axons in a spatial profile that could restrict axonal growth. Thus, NG2 is a major component of this putatively inhibitory class of ECM molecules expressed at sites of SCI and may restrict axonal regeneration.