Raman spectroscopic analysis of skin as a diagnostic tool for Human African Trypanosomiasis.

Human African Trypanosomiasis (HAT) has been responsible for several deadly epidemics throughout the 20th century, but a renewed commitment to disease control has significantly reduced new cases and motivated a target for the elimination of Trypanosoma brucei gambiense-HAT by 2030. However, the recent identification of latent human infections, and the detection of trypanosomes in extravascular tissues hidden from current diagnostic tools, such as the skin, has added new complexity to identifying infected individuals. New and improved diagnostic tests to detect Trypanosoma brucei infection by interrogating the skin are therefore needed. Recent advances have improved the cost, sensitivity and portability of Raman spectroscopy technology for non-invasive medical diagnostics, making it an attractive tool for gambiense-HAT detection. The aim of this work was to assess and develop a new non-invasive diagnostic method for T. brucei through Raman spectroscopy of the skin. Infections were performed in an established murine disease model using the animal-infective Trypanosoma brucei brucei subspecies. The skin of infected and matched control mice was scrutinized ex vivo using a confocal Raman microscope with 532 nm excitation and in situ at 785 nm excitation with a portable field-compatible instrument. Spectral evaluation and Principal Component Analysis confirmed discrimination of T. brucei-infected from uninfected tissue, and a characterisation of biochemical changes in lipids and proteins in parasite-infected skin indicated by prominent Raman peak intensities was performed. This study is the first to demonstrate the application of Raman spectroscopy for the detection of T. brucei by targeting the skin of the host. The technique has significant potential to discriminate between infected and non-infected tissue and could represent a unique, non-invasive diagnostic tool in the goal for elimination of gambiense-HAT as well as for Animal African Trypanosomiasis (AAT).

Phylogenetic history of vascular plant metabolism revealed using a macroevolutionary common garden.

While the fundamental biophysics of C3 photosynthesis is highly conserved across plants, substantial leaf structural and enzymatic variation translates into variability in rates of carbon assimilation. Although this variation is well documented, it remains poorly understood how photosynthetic rates evolve, and whether macroevolutionary changes are related to the evolution of leaf morphology and biochemistry. A substantial challenge in large-scale comparative studies is disentangling evolutionary adaptation from environmental acclimation. We overcome this by using a 'macroevolutionary common garden' approach in which we measured metabolic traits (Jmax and Vcmax) from 111 phylogenetically diverse species in a shared environment. We find substantial phylogenetic signal in these traits at moderate phylogenetic timescales, but this signal dissipates quickly at deeper scales. Morphological traits exhibit phylogenetic signal over much deeper timescales, suggesting that these are less evolutionarily constrained than metabolic traits. Furthermore, while morphological and biochemical traits (LMA, Narea and Carea) are weakly predictive of Jmax and Vcmax, evolutionary changes in these traits are mostly decoupled from changes in metabolic traits. This lack of tight evolutionary coupling implies that it may be incorrect to use changes in these functional traits in response to global change to infer that photosynthetic strategy is also evolving.

Macroevolutionary history predicts flowering time but not phenological sensitivity to temperature in grasses.

PREMISE: Long-term observations show that flowering phenology has shifted in many lineages in response to climate change. However, it remains unclear whether these results can be generalized to predict the presence, direction, or magnitude of responses in lineages for which we lack long time-series data. If phenological responses are phylogenetically conserved, we can extrapolate from species for which we have data to predict the responses of close relatives. While several studies have found that closely related species flower at similar times, fewer have evaluated whether phylogenetically proximal species respond to environmental change similarly. METHODS: We paired flowering time data from 3161 manually scored herbarium specimens of 72 species of grasses (Poaceae) with historical climate data and analyzed the phylogenetic signal and phylogenetic half-life of phenological sensitivity. We also ran these analyses on a subset of species showing statistically significant sensitivities, in order to assess the role of sampling bias on phylogenetic signal. RESULTS: Closely related grass species tend to flower at similar times, but flowering times respond to temperature changes in species-specific ways. We also show that only including species for which there is strong evidence of phenological shifts results in overestimating phylogenetic signal. CONCLUSIONS: In agreement with other recent studies, our results suggest caution in extrapolating from evidence of phylogenetic similarity to predicting shared responses in this ecologically relevant trait. Future work is needed to better understand the discrepancy between the phylogenetic signal in observed phenological shifts and absence of such signal in sensitivity.

Herbarium records indicate variation in bloom-time sensitivity to temperature across a geographically diverse region.

Anthropogenic warming's effects on phenology across environmental and temporal gradients are well recognized. Long-term phenological monitoring data are often limited in duration and geographic scope, but recent efforts to digitize herbaria collections make it possible to reliably reconstruct historic flowering phenology across broad geographic scales and multiple species, lending to an increased understanding of community response to climate change. In this study, we examined collection dates (1901 to 2015) of 8540 flowering specimens from 39 native species in the Pacific Northwest (PNW) region of North America. We hypothesized that flowering phenology would be sensitive to temperature but that sensitivity would vary depending on blooming season and geographic range position. As expected, we found that early-season bloomers are more sensitive to temperature than later-season bloomers. Sensitivity to temperature was significantly greater at low elevations and in the maritime (western) portion of the PNW than at higher elevations and in the eastern interior, respectively. The elevational and longitudinal effects on flowering sensitivity reflect spring "arriving" earlier at low elevations and in the maritime portion of the PNW. These results demonstrate that phenological responses to warming vary substantially across climatically diverse regions, warranting careful and nuanced consideration of climate warming's effects on plant phenology.

Delineating neuroinflammation, parasite CNS invasion, and blood-brain barrier dysfunction in an experimental murine model of human African trypanosomiasis.

Although Trypanosoma brucei spp. was first detected by Aldo Castellani in CSF samples taken from sleeping sickness patients over a century ago there is still a great deal of debate surrounding the timing, route and effects of transmigration of the parasite from the blood to the CNS. In this investigation, we have applied contrast-enhance magnetic resonance imaging (MRI) to study the effects of trypanosome infection on the blood-brain barrier (BBB) in the well-established GVR35 mouse model of sleeping sickness. In addition, we have measured the trypanosome load present in the brain using quantitative Taqman PCR and assessed the severity of the neuroinflammatory reaction at specific time points over the course of the infection. Contrast enhanced-MRI detected a significant degree of BBB impairment in mice at 14days following trypanosome infection, which increased in a step-wise fashion as the disease progressed. Parasite DNA was present in the brain tissue on day 7 after infection. This increased significantly in quantity by day 14 post-infection and continued to rise as the infection advanced. A progressive increase in neuroinflammation was detected following trypanosome infection, reaching a significant level of severity on day 14 post-infection and rising further at later time-points. In this model stage-2 disease presents at 21days post-infection. The combination of the three methodologies indicates that changes in the CNS become apparent prior to the onset of established stage-2 disease. This could in part account for the difficulties associated with defining specific criteria to distinguish stage-1 and stage-2 infections and highlights the need for improved staging diagnostics.

A sensitive and reproducible in vivo imaging mouse model for evaluation of drugs against late-stage human African trypanosomiasis.

OBJECTIVES: To optimize the Trypanosoma brucei brucei GVR35 VSL-2 bioluminescent strain as an innovative drug evaluation model for late-stage human African trypanosomiasis. METHODS: An IVIS® Lumina II imaging system was used to detect bioluminescent T. b. brucei GVR35 parasites in mice to evaluate parasite localization and disease progression. Drug treatment was assessed using qualitative bioluminescence imaging and real-time quantitative PCR (qPCR). RESULTS: We have shown that drug dose-response can be evaluated using bioluminescence imaging and confirmed quantification of tissue parasite load using qPCR. The model was also able to detect drug relapse earlier than the traditional blood film detection and even in the absence of any detectable peripheral parasites. CONCLUSIONS: We have developed and optimized a new, efficient method to evaluate novel anti-trypanosomal drugs in vivo and reduce the current 180 day drug relapse experiment to a 90 day model. The non-invasive in vivo imaging model reduces the time required to assess preclinical efficacy of new anti-trypanosomal drugs.

Plant traits and associated data from a warming experiment, a seabird colony, and along elevation in Svalbard.

The Arctic is warming at a rate four times the global average, while also being exposed to other global environmental changes, resulting in widespread vegetation and ecosystem change. Integrating functional trait-based approaches with multi-level vegetation, ecosystem, and landscape data enables a holistic understanding of the drivers and consequences of these changes. In two High Arctic study systems near Longyearbyen, Svalbard, a 20-year ITEX warming experiment and elevational gradients with and without nutrient input from nesting seabirds, we collected data on vegetation composition and structure, plant functional traits, ecosystem fluxes, multispectral remote sensing, and microclimate. The dataset contains 1,962 plant records and 16,160 trait measurements from 34 vascular plant taxa, for 9 of which these are the first published trait data. By integrating these comprehensive data, we bridge knowledge gaps and expand trait data coverage, including on intraspecific trait variation. These data can offer insights into ecosystem functioning and provide baselines to assess climate and environmental change impacts. Such knowledge is crucial for effective conservation and management in these vulnerable regions.

Microarray profiling predicts early neurological and immune phenotypic traits in advance of CNS disease during disease progression in Trypanosoma. b. brucei infected CD1 mouse brains.

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a major cause of mortality and morbidity in sub-Saharan Africa. We hypothesised that recent findings of neurological features and parasite brain infiltration occurring at much earlier stages in HAT than previously thought could be explained by early activation of host genetic programmes controlling CNS disease. Accordingly, a transcriptomal analysis was performed on brain tissue at 0, 7, 14, 21 and 28dpi from the HAT CD1/GVR35 mouse model. Up to 21dpi, most parasites are restricted to the blood and lymphatic system. Thereafter the trypanosomes enter the brain initiating the encephalitic stage. Analysis of ten different time point Comparison pairings, revealed a dynamic transcriptome comprising four message populations. All 7dpi Comparisons had by far more differentially expressed genes compared to all others. Prior to invasion of the parenchyma, by 7dpi, ~2,000 genes were up-regulated, denoted [7dpi↑] in contrast to a down regulated population [7dpi↓] also numbering ~2,000. However, by 14dpi both patterns had returned to around the pre-infected levels. The third, [28dpi↑] featured over three hundred transcripts which had increased modestly up to14dpi, thereafter were significantly up-regulated and peaked at 28dpi. The fourth, a minor population, [7dpi↑-28dpi↑], had similar elevated levels at 7dpi and 28dpi. KEGG and GO enrichment analysis predicted a diverse phenotype by 7dpi with changes to innate and adaptive immunity, a Type I interferon response, neurotransmission, synaptic plasticity, pleiotropic signalling, circadian activity and vascular permeability without disruption of the blood brain barrier. This key observation is consistent with recent rodent model neuroinvasion studies and clinical reports of Stage 1 HAT patients exhibiting CNS symptoms. Together, these findings challenge the strict Stage1/Stage2 phenotypic demarcation in HAT and show that that significant neurological, and immune changes can be detected prior to the onset of CNS disease.

Kynurenine pathway inhibition reduces central nervous system inflammation in a model of human African trypanosomiasis.

Human African trypanosomiasis, or sleeping sickness, is caused by the protozoan parasites Trypanosoma brucei rhodesiense or Trypanosoma brucei gambiense, and is a major cause of systemic and neurological disability throughout sub-Saharan Africa. Following early-stage disease, the trypanosomes cross the blood-brain barrier to invade the central nervous system leading to the encephalitic, or late stage, infection. Treatment of human African trypanosomiasis currently relies on a limited number of highly toxic drugs, but untreated, is invariably fatal. Melarsoprol, a trivalent arsenical, is the only drug that can be used to cure both forms of the infection once the central nervous system has become involved, but unfortunately, this drug induces an extremely severe post-treatment reactive encephalopathy (PTRE) in up to 10% of treated patients, half of whom die from this complication. Since it is unlikely that any new and less toxic drug will be developed for treatment of human African trypanosomiasis in the near future, increasing attention is now being focussed on the potential use of existing compounds, either alone or in combination chemotherapy, for improved efficacy and safety. The kynurenine pathway is the major pathway in the metabolism of tryptophan. A number of the catabolites produced along this pathway show neurotoxic or neuroprotective activities, and their role in the generation of central nervous system inflammation is well documented. In the current study, Ro-61-8048, a high affinity kynurenine-3-monooxygenase inhibitor, was used to determine the effect of manipulating the kynurenine pathway in a highly reproducible mouse model of human African trypanosomiasis. It was found that Ro-61-8048 treatment had no significant effect (P = 0.4445) on the severity of the neuroinflammatory pathology in mice during the early central nervous system stage of the disease when only a low level of inflammation was present. However, a significant (P = 0.0284) reduction in the severity of the neuroinflammatory response was detected when the inhibitor was administered in animals exhibiting the more severe, late central nervous system stage, of the infection. In vitro assays showed that Ro-61-8048 had no direct effect on trypanosome proliferation suggesting that the anti-inflammatory action is due to a direct effect of the inhibitor on the host cells and not a secondary response to parasite destruction. These findings demonstrate that kynurenine pathway catabolites are involved in the generation of the more severe inflammatory reaction associated with the late central nervous system stages of the disease and suggest that Ro-61-8048 or a similar drug may prove to be beneficial in preventing or ameliorating the PTRE when administered as an adjunct to conventional trypanocidal chemotherapy.

Teachers' Perceptions of Word Callers and Related Literacy Concepts.

The purpose of the study was to investigate teachers' perceptions of word callers as they relate to the concepts of reading fluency and reading comprehension. To this end, second grade students (N = 408) completed a series of reading fluency and reading comprehension assessments, and their teachers (N = 31) completed word caller nominations and a questionnaire regarding their concepts surrounding these issues. Our findings suggested that teachers often over nominated children as word callers. Further, questionnaire data indicated a great deal of ambiguity and inconsistency exists regarding teachers' understanding and use of the term word caller. By contrast, teachers seemed to possess a veridical understanding of the related terms reading fluency and reading comprehension.

The blood-brain barrier significantly limits eflornithine entry into Trypanosoma brucei brucei infected mouse brain.

Drugs to treat African trypanosomiasis are toxic, expensive and subject to parasite resistance. New drugs are urgently being sought. Although the existing drug, eflornithine, is assumed to reach the brain in high concentrations, little is known about how it crosses the healthy and infected blood-brain barrier. This information is essential for the design of drug combinations and new drugs. This study used novel combinations of animal models to address these omissions. Eflornithine crossed the healthy blood-CNS interfaces poorly, but this could be improved by co-administering suramin, but not nifurtimox, pentamidine or melarsoprol. Work using a murine model of sleeping sickness demonstrated that Trypanosoma brucei brucei crossed the blood-CNS interfaces, which remained functional, early in the course of infection. Concentrations of brain parasites increased during the infection and this resulted in detectable blood-brain barrier, but not choroid plexus, dysfunction at day 28 post-infection with resultant increases in eflornithine brain delivery. Barrier integrity was never restored and the animals died at day 37.9 +/- 1.2. This study indicates why an intensive treatment regimen of eflornithine is required (poor blood-brain barrier penetration) and suggests a possible remedy (combining eflornithine with suramin). The blood-brain barrier retains functionality until a late, possibly terminal stage, of trypanosoma infection.

Combination chemotherapy with a substance P receptor antagonist (aprepitant) and melarsoprol in a mouse model of human African trypanosomiasis.

Drug therapy for late-stage (encephalitic) human African trypanosomiasis (HAT) is currently very unsatisfactory with the most commonly used drug, melarsoprol, having a 5% overall mortality. There is evidence in a mouse model of HAT that Substance P (SP) receptor antagonism reduces the neuroinflammatory reaction to CNS trypanosome infection. In this study we investigated the effects of combination chemotherapy with melarsoprol and a humanised SP receptor antagonist aprepitant (EMEND) in this mouse model. The melarsoprol/aprepitant drug combination did not produce any clinical signs of illness in mice with CNS trypanosome infection. This lack of any additional or unexpected CNS toxicity in the mouse model of CNS HAT provides valuable safety data for the future possible use of this drug combination in patients with late-stage HAT.

Trypanosoma brucei Invasion and T-Cell Infiltration of the Brain Parenchyma in Experimental Sleeping Sickness: Timing and Correlation with Functional Changes.

BACKGROUND: The timing of Trypanosoma brucei entry into the brain parenchyma to initiate the second, meningoencephalitic stage of human African trypanosomiasis or sleeping sickness is currently debated and even parasite invasion of the neuropil has been recently questioned. Furthermore, the relationship between neurological features and disease stage are unclear, despite the important diagnostic and therapeutic implications. METHODOLOGY: Using a rat model of chronic Trypanosoma brucei brucei infection we determined the timing of parasite and T-cell neuropil infiltration and its correlation with functional changes. Parasite DNA was detected using trypanosome-specific PCR. Body weight and sleep structure alterations represented by sleep-onset rapid eye movement (SOREM) periods, reported in human and experimental African trypanosomiasis, were monitored. The presence of parasites, as well as CD4+ and CD8+ T-cells in the neuropil was assessed over time in the brain of the same animals by immunocytochemistry and quantitative analyses. PRINCIPAL FINDINGS: Trypanosome DNA was present in the brain at day 6 post-infection and increased more than 15-fold by day 21. Parasites and T-cells were observed in the parenchyma from day 9 onwards. Parasites traversing blood vessel walls were observed in the hypothalamus and other brain regions. Body weight gain was reduced from day 7 onwards. SOREM episodes started in most cases early after infection, with an increase in number and duration after parasite neuroinvasion. CONCLUSION: These findings demonstrate invasion of the neuropil over time, after an initial interval, by parasites and lymphocytes crossing the blood-brain barrier, and show that neurological features can precede this event. The data thus challenge the current clinical and cerebrospinal fluid criteria of disease staging.

Meningoencephalitic African trypanosomiasis: Brain IL-10 and IL-6 are associated with protection from neuro-inflammatory pathology.

The relationship of neuropathology to CNS inflammatory and counter-inflammatory cytokine production in African trypanosome-infected mice was studied using an infection model with a defined disease progression. The initial phase of CNS infection by trypanosomes, where only mild neuropathology is evident, was characterised by high levels of IL-10 and IL-6. In the later phase of CNS infection and in a post-drug treatment model, moderate to severe neuropathology was associated with high levels of IFN-gamma and TNF-alpha. The relationship of these cytokines to neuropathological grade suggests that IL-10 and IL-6 protect the CNS from inflammatory pathology when parasites first enter the brain and the data reconcile previously contradictory clinical measurements of CSF cytokines in meningoencephalitic patients with post-mortem histopathology observations.

Central Nervous System Parasitosis and Neuroinflammation Ameliorated by Systemic IL-10 Administration in Trypanosoma brucei-Infected Mice.

Invasion of the central nervous system (CNS) by African trypanosomes represents a critical step in the development of human African trypanosomiasis. In both clinical cases and experimental mouse infections it has been demonstrated that predisposition to CNS invasion is associated with a type 1 systemic inflammatory response. Using the Trypanosoma brucei brucei GVR35 experimental infection model, we demonstrate that systemic delivery of the counter-inflammatory cytokine IL-10 lowers plasma IFN-γ and TNF-α concentrations, CNS parasitosis and ameliorates neuro-inflammatory pathology and clinical symptoms of disease. The results provide evidence that CNS invasion may be susceptible to immunological attenuation.

Human African trypanosomiasis: potential therapeutic benefits of an alternative suramin and melarsoprol regimen.

Treatment of late-stage human African trypanosomiasis is complicated by the presence of trypanosomes within the central nervous system (CNS). The regimen commonly prescribed to treat CNS-stage disease involves the use of the trypanocidal drugs suramin and melarsoprol. Suramin does not cross the blood-brain barrier efficiently and therefore, at normal dosages, will not cure CNS-stage infections. An initial treatment with suramin is given to eliminate the parasites from the peripheral tissues. This is followed by a course of intravenous melarsoprol, which can enter the CNS. However, melarsoprol not only produces severe adverse reactions but also is extremely painful to administer. One possible method to help alleviate these problems is to reduce the total amount of melarsoprol in the treatment regimen. This study indicates a synergism between suramin and melarsoprol and demonstrates that experimental murine CNS-trypanosomiasis can be cured with a single intraperitoneal dose of 20 mg/kg suramin followed almost immediately by 0.05 ml (4.5 micromol) topical melarsoprol. These dosages will not cure the infection when administered as monotherapies. Moreover, the timing of the drug administration appears to be crucial to the successful outcome of the regimen. If the interval between injection of suramin and application of topical melarsoprol is extended from 15 min to 3 or 7 days, the infections are not cured. Although extended relapse times occur following these regimens when compared with monotherapy approaches. Thus, there is strong evidence that injected suramin and topical melarsoprol should be given almost simultaneously to achieve the most effective combination of the two drugs.

Magnetic resonance imaging to assess blood-brain barrier damage in murine trypanosomiasis.

The ability of trypanosomes to invade the brain and induce an inflammatory reaction is well-recognized. This study uses magnetic resonance imaging (MRI) in conjunction with a murine model of central nervous system (CNS) stage trypanosomiasis to investigate this phenomenon at the level of the blood-brain barrier (BBB). Mice were scanned before and after administration of the contrast agent. Signal enhancement maps were generated, and the percentage signal change was calculated. The severity of the neuroinflammation was also assessed. Statistical analysis of the signal change data revealed a significantly (P = 0.028) higher signal enhancement in mice at 28 days post-infection (least squares mean = 26.709) compared with uninfected animals (6.298), indicating the presence of BBB impairment. Leukocytes were found in the meninges and perivascular space of some blood vessels in the infected mice. This study shows that the integrity of the BBB is compromised during CNS stage trypanosomiasis and that the impairment does not correlate with inflammatory cell infiltration.

Melarsoprol cyclodextrin inclusion complexes as promising oral candidates for the treatment of human African trypanosomiasis.

Human African trypanosomiasis (HAT), or sleeping sickness, results from infection with the protozoan parasites Trypanosoma brucei (T. b.) gambiense or T. b. rhodesiense and is invariably fatal if untreated. There are 60 million people at risk from the disease throughout sub-Saharan Africa. The infection progresses from the haemolymphatic stage where parasites invade the blood, lymphatics and peripheral organs, to the late encephalitic stage where they enter the central nervous system (CNS) to cause serious neurological disease. The trivalent arsenical drug melarsoprol (Arsobal) is the only currently available treatment for CNS-stage T. b. rhodesiense infection. However, it must be administered intravenously due to the presence of propylene glycol solvent and is associated with numerous adverse reactions. A severe post-treatment reactive encephalopathy occurs in about 10% of treated patients, half of whom die. Thus melarsoprol kills 5% of all patients receiving it. Cyclodextrins have been used to improve the solubility and reduce the toxicity of a wide variety of drugs. We therefore investigated two melarsoprol cyclodextrin inclusion complexes; melarsoprol hydroxypropyl-ß-cyclodextrin and melarsoprol randomly-methylated-ß-cyclodextrin. We found that these compounds retain trypanocidal properties in vitro and cure CNS-stage murine infections when delivered orally, once per day for 7-days, at a dosage of 0.05 mmol/kg. No overt signs of toxicity were detected. Parasite load within the brain was rapidly reduced following treatment onset and magnetic resonance imaging showed restoration of normal blood-brain barrier integrity on completion of chemotherapy. These findings strongly suggest that complexed melarsoprol could be employed as an oral treatment for CNS-stage HAT, delivering considerable improvements over current parenteral chemotherapy.

Myth and Reality of the Word Caller: The Relation Between Teacher Nominations and Prevalence Among Elementary School Children.

The purpose of the study was to investigate (a) the prevalence of word callers in elementary school, (b) the accuracy of teachers' word caller nominations, and (c) teachers' conceptualization of reading fluency and reading comprehension. To this end, 2 cross-sectional studies of second- and third- (N = 868) and of third- and fifth-grade (N = 202) children were conducted. Our findings suggest that word callers occur infrequently in the primary grades but that they are more prevalent in late elementary school. Regardless of grade level, teachers often overnominated children as word callers. Furthermore, a great deal of ambiguity and inconsistency seems to exist regarding teachers' understanding and use of the term. These findings suggest that the term should be used relatively rarely and that reading educators should be cautious about their identification of word callers in early elementary school.

Teaching Children to Become Fluent and Automatic Readers.

The purpose of the study was to examine the effects of two instructional approaches designed to improve the reading fluency of 2nd-grade children. The first approach was based on Stahl and Heubach's (2005) fluency-oriented reading instruction (FORI) and involved the scaffolded, repeated reading of grade-level texts over the course of each week. The second was a wide-reading approach that also involved scaffolded instruction. hut that incorporated the reading of 3 different grade-level texts each week and provicled significantly less opportunity for repetition. By the end of the school year. FORI and wide-reading approaches showed similar benefits for standardized measures of word reading efficiency and reading comprehension skills compared to control approachcs. although the benefits of the wide-reading approach emerged earlier and included oral text reading fluency skill. Thus, we conclude that fluency instruction that emphasizes extensive oral reading of grade-level text using scaffolded approaches is effective for promoting reading development in young learners.