Streptococcus and Prevotella species between the small and large intestine, suggesting a decompartmentalization of gut microbial communities. Our studies provide valuable insight into the outcomes of diarrheal diseases and growth faltering not attainable in humans and lays the groundwork to test interventions in a controlled and reproducible setting.Despite the impact of childhood diarrhea on morbidity and mortality, our understanding of its sequelae has been significantly hampered by the lack of studies that examine samples across the entire intestinal tract. Infant rhesus macaques are naturally susceptible to human enteric pathogens and recapitulate the hallmarks of diarrheal disease such as intestinal inflammation and growth faltering. Here, we examined intestinal biopsies, lamina propria leukocytes, luminal contents, and fecal samples from healthy infants and those experiencing growth faltering with distant acute or chronic active diarrhea. We show that growth faltering in the presence or absence of active diarrhea is associated with a heightened systemic and mucosal pro-inflammatory state centered in the colon. Moreover, polyclonal stimulation of colonic lamina propria leukocytes resulted in a dampened cytokine response, indicative of immune exhaustion. We also detected a functional and taxonomic shift in the luminal microbiome across multiple gut sites including the migration of  In addition to high mortality rates, diarrhea early in life can have a lasting effect on host physiology2. Specifically, diarrhea results in a dangerous feedback loop of malnutrition6 and intestinal damage8, which ultimately leads to growth faltering, poor responses to oral vaccination12, and diminished resilience to subsequent enteric infections13. This cycle can occur even in the absence of overt diarrhea15. Indeed, growth faltering children in developing countries have been shown to have a significant enteric pathogen burden without acute disease17. These subclinical infections are believed to perpetuate intestinal damage and restrict healthy growth18. Moreover, unknown and emerging etiological agents are thought to play an underappreciated role in childhood diarrheal disease and growth faltering. Specifically, multiple large-scale international studies have found that less than 50% of diarrheal cases can be attributed to a specific pathogen20. This pattern is likely a combination of high pathogen burden in the absence of symptoms21 and the use of clinical tests that limit detection of novel pathogens not routinely detected23. More recent studies have begun to identify a set of non-traditional pathogens, especially in the genus Campylobacter, that are missed by clinical tests but highly abundant in developing countries26. Additional studies are needed to determine the role of these newly identified pathogens in the cycle of diarrheal disease, malnutrition, and growth faltering.Despite significant improvements in healthcare delivery and sanitation, over 500,000 children under the age of 5 die of diarrheal disease every year9. While this study reported an association between growth faltering and damage to the upper small intestine, the involvement of other gut sites remains unknown. More importantly, the interpretation of these observations is complicated by the lack of comparison to samples obtained from asymptomatic children living under the same conditions. Transcriptional analysis of fecal samples obtained from infants with environmental enteric dysfunction revealed increased expression of immune genes27; however, the cellular source of these expression changes remains unclear.In addition, few studies have investigated diarrheal disease-induced damage within the gut itself. One study reported increased gut permeability and inflammation markers in duodenal biopsies obtained from Gambian infants with growth faltering compared to those obtained from healthy children living in the United KingdomAlterations in mucosal immune cell frequency and function across the GI tract due to growth faltering/diarrheal disease have yet to be identified due to the challenges associated with the collection of intestinal biopsies from healthy infants and those experiencing growth faltering while controlling for key variables such as geographic location, diet, and socioeconomic status. A deeper understanding of the pleiotropic impact of diarrhea on GI immunological and microbial community composition is key to successfully identify sub-clinical cases and developing targeted treatments against growth faltering and malnutrition.30, as well as the persistence of an immature gut microbiome state32. Moreover, growth faltering is associated with the decompartmentalization of microbial communities as illustrated by the recent report of potential infiltration of nasopharyngeal microbes into the GI tract33. Interestingly, levels of enteric pathogens such as Escherichia coli and Campylobacter spp. are elevated in the fecal microbiome of children with malnutrition and growth faltering regardless of diarrhea35. However, the vast majority of clinical findings associated with diarrheal disease and growth faltering are centered around the small intestine, while the majority of microbiome data is generated from fecal samples. This dichotomy may conceal clinically relevant information and no studies to date have interrogated microbial changes within the multiple intestinal tract sections at the taxonomic or functional level.Malnutrition, growth faltering, and diarrhea have all been associated with dysbiosis of the gut microbiome at a taxonomic level using fecal samplesCampylobacter and Shigella being the primary pathogens isolated at clinic admission38. In addition, infant macaques that experience diarrhea suffer growth faltering37. Rhesus macaques also display a high pathogen burden even in the absence of diarrheal disease, as described in infants19. Moreover, we recently reported that the gut microbiome of infant rhesus macaques closely resembles that of children in the developing world where diarrheal disease and growth faltering is most prevalent29.Answering these questions would be greatly facilitated by the availability of an animal model that was susceptible to human enteric pathogens and displayed the hallmarks of growth faltering. Rhesus macaques are the gold standard model for the study of several infectious diseases due to their genetic and physiological similarity to humans. Rhesus macaques housed at the Oregon National Primate Research Center (ONPRC) and California National Primate Research Center (CNPRC) naturally develop the diarrheal disease at a predictable yearly rate with Campylobacter strains in animals with growth faltering. Together, our findings suggest that the pathology of growth faltering extends beyond the small intestine and may be more prominent in the large intestine.In this study, we leveraged the infant macaque model to characterize the impact of growth faltering with distant or active chronic diarrhea on the mucosal immune system and microbial communities throughout the intestinal tract. Our data revealed immunological and microbial dysregulation that was more pronounced in the colon. Specifically, we detected alterations in the frequency of immune cell populations and their response to polyclonal stimulation, as well as shifts in microbial communities along the entire GI tract, as well as the migration of microbes between gut sites suggestive of decompartmentalization. Shotgun metagenomics of luminal contents revealed key differences in the functional potential of luminal communities and led to the assembly of novel n = 7) grew at or above the average growth trajectory with only 1 of the 7 infants experiencing diarrhea ~3.3 months before necropsy (Supp. Table n = 5) group, 3 of 5 of the infants had experienced clinical diarrhea but had not had an episode of diarrhea for at least one 1-month before necropsy  all had a history of active chronic diarrhea and had diarrhea at the time of necropsy , Growth Faltering with acute distance or no history of diarrhea (GF), and Growth Faltering with active chronic diarrhea (GF-DX) based on growth rate and clinical status at the time of necropsy , macrophages, or NK cells at either gut site Fig. . The freTo explore the impact of growth faltering/diarrhea on the function of LPLs, cells were stimulated with PMA/ionomycin (PMAi) for 16 h and the production of 33 immune mediators was measured using Luminex. No significant differences in the production of immune mediators by ileal LPLs were detected between the three groups of infants with or without PMAi stimulation as illustrated by the overlap of samples in the PCA plot  and fecal samples obtained from the same HG, GF, and GF-DX infants using 16S rRNA gene amplicon sequencing. Gut site, rather than host status or individual, was the major determinant of community composition for weighted UniFrac dissimilarity  with microbial communities of the small intestine  clustering separately from those of the colon and fecal materials  detected in at least two samples at greater than 0.01% relative abundance were included in this analysis Fig. . A totalnts Fig. . Similarius Fig. . The larira Fig. . On the SVs Fig. . Finallylla Fig. .Streptococcus ASV 124, which is normally relegated to the small intestine luminal microbiome in HG animals, was abundant in the large intestine and fecal microbiome of GF/GF-DX  was higher in the small intestine of GF/GF-DX infants  correlations between immune cell populations and the luminal microbiome    or bacterial translocation  in GF and GF/DX infants. However, our analysis revealed higher levels of systemic inflammatory markers in both groups of growth faltering infants. In addition, we found a stark increase in the levels of BDNF which was detectable in 2/6 HG infants, 3/4 GF infants, and 4/4 GF-DX infants. BDNF can be produced by multiple cell types in the gut45 and is important for the regulation of tight junctions46. Moreover, systemic levels of BDNF have been positively correlated with visceral hypersensitivity (pain) in irritable bowel syndrome (IBS)47. Our data suggest that plasma levels of BDNF could be used as an alternative marker of intestinal injury. Interestingly, plasma levels of IL-1β, IFNα, IFNγ, and IL-15 were higher in GF infants, indicating that distant history of diarrhea can have long-lasting ramifications on the host immune status. High levels of systemic IL-1β have been linked to poor neurodevelopment in Bangladeshi children48, while IFNγ has been associated with increased microbial translocation in growth faltering infants49. This enhanced systemic inflammation was accompanied by a dampened IL-17 and TNFα response of peripheral CD4 T cells to polyclonal stimulation, potentially an indication of an immunoregulatory phenotype.In contrast to what has been reported in humans50, in both faltering groups. This loss could be one of the factors contributing to the reduced immune response to oral Rotavirus and Polio vaccines observed in children with growth faltering and diarrheal diseases52. In addition, we detected a higher relative frequency of the non-classical colonic CD16+ macrophages in GF infants, indicative of higher inflammation even in the absence of active diarrhea. Indeed, CD16+ macrophages in the gut have been shown to mediate fibrosis in inflammatory bowel disease53. We also observed an increase in the percentage of terminally differentiated colonic memory CD4+ T, suggesting an accelerated differentiation. CD4+ T cells are the most abundant LPL population and play important roles in tissue maintenance and anti-microbial responses56. In healthy infants, tissue-resident T cells are typically naive, and regulatory and resident effector memory cells do not develop until adulthood58. High pathogen load or repeated exposure to antigen could drive the observed shifts in memory CD4 T cell populations. Terminally differentiated memory T cells are usually associated with increased inflammatory responses. However, colonic LPLs from GF-DX animals generated a dampened response to polyclonal stimulation relative to colonic LPLs from HG infants failing to produce canonical cytokines IL-2, IL-17, and IFNγ. This dampened response was also evident at the transcriptional level. Interestingly colonic LPLs from GF infants generated a larger transcriptional response to polyclonal stimulation in line with increased plasma levels of inflammatory cytokines. Together these observations suggest that GF with a limited diarrhea history is associated with exacerbated inflammation while GF with chronic diarrhea is associated with immune exhaustion. However, further measurement of exhaustion markers on LPL such as PD-1 and CTLA-4 in the colon is needed to confirm this.Significant differences in immune cell frequencies and response to polyclonal stimulation between HG and GF-DX animals were observed in colonic, but not ileal, LPLs. Specifically, we observed a reduction in the relative frequency of colonic mDCs, a major antigen-presenting cell subset59 in GF-DX compared to GF infants. This suggests that GF in the absence of active diarrhea is associated with increased B cell activity in the ileum. B cells have been shown to increase in abundance and aggregate in the small intestine of growth faltering infants in the Gambia49. This chronic immune activation may contribute to systemic inflammation and inhibit the host from adequately responding to mucosal vaccines/infection. In contrast, the transcriptional landscape in the colon was indicative of enhanced immune activation in GF-DX infants compared to GF infants. Many of the genes upregulated in the colon of GF-DX animals were associated with T cell and macrophage activation in agreement with our flow cytometry data, as well as granulocytes (not included in our immunophenotyping). Finally, histological examination of tissue biopsies from the mid-ileum and traverse colon confirmed that GF is associated with a heightened inflammatory landscape in the colon. Taken together, our phenotypic, functional, transcriptional, and histological data strongly suggest that GF, especially when coupled with chronic active diarrhea, primarily impacts the colon rather than the small intestine. While more recent human studies have focused on the small intestine, earlier studies identified mucosal abnormalities in rectal biopsies of volunteers in southern India which they termed tropical colonopathy60.Our transcriptomic analysis of ileal and colonic biopsies revealed the downregulation of genes important for immune activation and highly expressed by gut-associated B cells61, the luminal microbiome of the small and large intestines are distinct. Phylogenetic diversity in the small intestinal was higher in GF-DX infants. On the other hand, and as described in human infants with, malnutrition32 and growth faltering31, the alpha diversity in the fecal samples of GF/GF-DX macaques was reduced compared to HG macaques. Furthermore, inter-sample variability was higher in microbial communities from GF/GF-DX animals, indicative of microbiome instability or a more immature state as has been shown in the fecal microbiome of malnourished children32.This study provided the first insight into the microbial communities within luminal contents along the intestinal tract of infant macaques. As previously reported for adult macaquesLactobacillus salivarius, a core member of the oral microbiome in humans62, were increased in the lumen of both the small and large intestine of GF-DX infants. L. salivarius has been reported to be enriched in the fecal samples of growth-stunted children33. Moreover, Streptococcus, also part of the core oral microbiome62 and normally relegated to the small intestine in healthy animals, was detected in colonic luminal contents. This observation is similar to recent reports describing the detection of nasopharyngeal taxa to the gut in children experiencing growth stunting33. This loss of strict compartmentalization could be due to a reduced ability of the host immune system to tightly control microbial growth.In addition, multiple bacterial taxa normally sequestered to either the small or large intestine in HG infants were detected in several gut sites in GF/GF-DX infants, suggesting the loss of strict compartmentalization between gut-sites with growth faltering. Specifically, the relative abundance of 63. However, SCFA has also been reported to skew T cells towards anti-inflammatory IL-10 production and play a role in preventing colitis and other inflammatory conditions64. In addition, the SCFA butyrate can modulate the function of innate immune cells by reducing the expression and nuclear translocation of NF-κB in intestinal macrophages66. Therefore, the loss of SCFA production potential in GF and GF-DX animals may contribute to the heightened inflammatory state we observed.We also observed differences in the functional potential in both the ileal and colonic luminal microbiome. Specifically, we observed a reduced capacity of short-chain fatty acid (SCFA) production in GF and GF-DX infants in both ileal and colonic microbial communities. The microbial production of SCFA has been closely linked to the activation of mucosal Th1, Th17, and TregsLactobacillus abundance and Ileal Macrophages. Lactobacillus strains have also been shown to activate macrophages in the small intestine68. The increased abundance of Lactobacillus salivarius in the small and large intestine of GF-DX infants coupled with increased frequency of activated macrophages suggest an infiltration of oral microbes into the small intestine resulting in activation of the innate immune system. In the colon, we found an inverse relationship between Prevotella and Campylobacter hyointestinalis with colonic CD4+ T cell subsets. While gut Prevotella has been linked to systemic inflammation and reduced systemic CD4+ T cells in the western world, particularly in HIV infection71. It is important to note that Prevotella is the most abundant microbe in the gut of humans with a non-westernized lifestyle and is largely believed to be beneficial in this context73. However, the positive correlation of CD4+ EM T cells with Campylobacter hyointestinalis may indicate recurrent infection and the expansion of a pathogen-specific T cell subset.We also observed correlations between intestinal immune cell frequencies and the relative abundance of microbes. In the Ileum this included a positive correlation between Campylobacter hyointestinalis and Escherichia-Shigella in fecal samples collected from GF infants, indicative of an increased pathogen burden, as reported in multiple human studies75. Diversity within the Campylobacter genus is often missed in 16S amplicon surveys due to their highly conserved 16S rRNA gene76; therefore, we resorted to metagenomic genome assembly. We constructed 3 Campylobacter genomes only from the colonic luminal contents of faltering infants. Moreover, a higher number of short reads mapped to these three genomes in colonic luminal contents of faltering infants. One of our assembled Campylobacter genomes, as well as some of our previously described29. Campylobacter assemblies from infant rhesus macaque feces were most closely related to the newly described “Candidatus Campylobacter infanis”25. Non-coli/jejuni Campylobacter has recently been implicated in diarrheal diseases and childhood mortality in the developing world77.We detected an increased abundance of Helicobacter genomes. These data differ from our recent report of a decreased abundance of Helicobacter in the fecal microbiome of infants that experienced diarrhea and received antibiotics. This difference could be potentially be explained by the fact that Helicobacter is tightly associated with gut mucosa and greatly reduced in the lumen61. We also assembled several new Prevotella genomes mostly from GF animals. These new genomes differed from the cluster of Prevotella genomes we recently identified from the fecal microbiome of 1-month old infants that would later experience diarrhea29. Interestingly, the frequency of reads mapping to these genomes did not differ between the three groups even though 90% of the genomes were assembled from GF/GF-DX animals.In contrast, we did not observe differences in the abundance of our assembled 78 including the prophylactic administration of azithromycin79, micronutrients80, probiotics (Lactobacillus species)81. However, many of these interventions have resulted in little to no improvement in growth or other primary endpoints78. Potential new strategies could include broad-acting phage therapy against Campylobacter, probiotic treatment with commensals that are relevant to children who live in the developing world, SCFA supplementation to reduce inflammation and improve gut immune fitness, and immune-modulatory drugs that can improve gut integrity. Infant macaques provide a highly relevant translational animal model to test these novel interventions and evaluate their impact on tissue inflammation, immune function, and microbial communities across all sections of the gut.In summary, this study provides the first comprehensive look at the association of growth faltering and diarrheal disease with luminal microbiomes and local immune dysfunction that would not be possible in humans. Together, our data suggest that the colon is the major site of immune dysfunction during active diarrhea. We also found that immune dysfunction in GF and GF-DX is associated with a decompartmentalization of the luminal microbiome. Data presented in this manuscript highlight the need for novel multifaceted approaches that can address the complex etiology of GF and diarrheal disease with the goal of reducing pathogen burden, restoring gut integrity, and rejuvenating the intestinal immune system. Previous interventions aimed at reducing childhood mortality due to growth faltering and enteric diseaseThe Guide for the Care and Use of Laboratory Animals. All animals were tested for simian viruses  and received a tuberculin test semi-annually.Samples were obtained from a total of 17 6–12-month old infant macaques , soluble CD14 (sCD14) , and internal fatty acid-binding protein (iFABP)  in plasma samples were measured using an enzyme-immunoassay technique (ELISA) following the manufacturer’s protocol. Plasma levels of free bacterial endotoxin were measured using a chromogenic Limulus amebocyte lysate assay  following the manufacturer’s protocol. Plasma levels of circulating immune factors were measured using a Magnetic 33-plex assay  and a MAGPIX instrument . Bubble plots were generated using ggplot2 R package with concentration (pg/ml).82. Cells were cryopreserved in FetalPlex Animal Serum Complex  and DMSO.Tissue biopsies were collected from the ileum and descending colon into Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum, streptomycin/penicillin, and L-glutamine (RP10). Lamina propria leukocytes (LPLs) were isolated using collagenase IX/DNAse digestion followed by centrifugation over a Percoll gradient  as previously described6 LPLs were stained using antibodies against CD3, CD4, CD8b, CD95, CD28, CCR7, and CD20 to delineate naïve and memory CD4 and CD8 T cells, as well as CD20 B cells. The cells were subsequently fixed and permeabilized before the addition of anti-Ki-67 to measure proliferation. T cells were then divided into Naive , central memory , transitional effector memory , and Effector memory , and. The second tube of 1 × 106 LPLs was stained using antibodies against: CD3, CD20, HLA-DR, CD14, CD11c, CD123, CD16, and CD8a to delineate macrophages (CD3− CD20− CD14 + HLA-DR+), dendritic cells , and natural killer  cell subsets. Macrophages were further subdivided into classical (CD16−) and non-classical macrophages (CD16+). DCs were further subdivided into myeloid DC  and plasmacytoid DC . All flow cytometry samples were acquired using Attune NxT  and analyzed using FlowJo .1 × 105/well) were thawed and incubated for 16 h in the absence (unstimulated) or presence (stimulated) of 5 ng/ml PMA and 1 μg/ml Ionomycin  at 37 °C in a humidified incubator (5% CO2). At the end of the incubation, cells were centrifuged for 5 min at 2000 rpm. Supernatants were collected to measure the production of immune mediators using a Magnetic 33-plex assay  and a MAGPIX instrument . Absolute change of immune mediators was calculated by subtracting the basal level of production from the level post-stimulation in a pairwise fashion. Bubble plots were generated using ggplot2 R package with concentration (pg/ml).LPLs . rRNA-depleted RNA was fragmented and converted to double-stranded cDNA. Adapters were ligated, and ~300 base pair fragments were amplified by PCR and selected by size exclusion using Ampure beads XP . Each library was labeled with a unique barcode for multiplexing. To ensure proper sizing, quantitation, and quality before sequencing, libraries were analyzed on the Agilent 2100 Bioanalyzer. Multiplexed libraries were sequenced (100 base pair single-end) using the HiSeq 4000 platform.83. Quality filtration and trimming were performed using Trimmomatic with an average phred score cutoff of 30 and a minimum length of 75 bp. Reads were mapped with the splice-aware aligner suite Bowtie2/Tophat2 against the Macaca mulatta genome (Macaca_mulatta.MMUL_1.78). Raw count values were generated with summarizeOverlaps function. Only reads overlapping exonic regions of genes were counted, discarding reads mapping to ambiguous regions of exons from overlapping genes. Analysis of differentially expressed genes (DEGs) was performed using edgeR package83. DEGs were defined as those with a fold change of ≥2 and a Benjamini-Hochberg–controlled false discovery rate of <0.05. Functional enrichment analysis was performed to identify significant Gene Ontology (GO) terms and using Metascape84. Immquant was used to infer alteration in immune cell frequency based on tissue transcriptional profiles85.Data analysis was performed with the RNA sequencing workflow module of the systemPiperR package available on BioconductorTissue samples from each animal were collected from the mid ileum, and descending colon. The tissues were placed in 10% neutral buffered formalin at room temperature for 48–72 h and processed in a Tissue Tek VIP 5 Tissue Processor. The blocks were paraffin-embedded, cut, and stained with hematoxylin and eosin. Representative images were captured at ×10 magnification by a blinded pathologist.86. PCR reactions contained 12.5 μl GoTaq mastermix, 9.5 μl nuclease-free H20, 1 μl template DNA, and 1 μl 10 μM primer mix. Thermal cycling parameters were 94 °C for 5 min, 35 cycles of 94 °C for 20 s, 50 °C for 20 s, 72 °C for 30 s, followed by 72 °C for 5 min. PCR products were purified using a MinElute 96 UF PCR Purification Kit . Libraries were sequenced (2 ×300 bases) using an Illumina MiSeq.Total DNA was extracted from ~0.25 g of luminal contents using the PowerSoil DNA Isolation Kit . Next, we amplified the hypervariable V4-V5 region of the 16S rRNA gene using PCR primers (515 F/926 R with the forward primers including a 12-bp barcode)87. Briefly, sequences were demultiplexed and the quality filtered using DADA288, which filters chimeric sequences and generates an amplicon sequence variant (ASV) table equivalent to an operational taxonomic unit (OTU) table at 100% sequence similarity. Sequence variants were then aligned using MAFFT89. and a phylogenetic tree was constructed using FastTree290. Taxonomy was assigned to sequence variants using q2-feature-classifier91. against the SILVA database (release 119)92. To prevent sequencing depth bias samples were rarified to 13,000 sequences per sample before alpha and beta diversity analysis. QIIME 2 was also used to generate the following alpha diversity metrics: richness (as observed taxonomic units), Shannon evenness, and phylogenetic diversity. Beta diversity was estimated in QIIME 2 using weighted and unweighted UniFrac distances93.Raw FASTQ 16S rRNA gene amplicon sequences were uploaded and processed using the QIIME2 analysis pipeline94, and potential host reads were removed by aligning trimmed reads to the Macaca mulata genome (Mmul 8.0.1) using BowTie295. Trimmed and decontaminated reads were then annotated using the HUMAnN296. pipeline using default settings with the UniRef50 database and assigned to Metacyc pathways. Functional annotations were normalized using copies per million (CPM) reads before statistical analysis99. Species-level taxonomy was assigned to quality-controlled short reads using Metaphlan2100. Genome assemblies were generated for each sample. Trimmed and decontaminated reads were assembled into contigs using meta-SPAdes with default parameters101. and binned into putative genomes using MetaBat102. Genome completeness/contamination was tested using CheckM39, and all bins with a completeness >80% and contamination <2% were annotated using PATRIC103. Taxonomy of draft genomes was determined using PATRICs’ similar genome finder.Shotgun metagenomic libraries were prepared from 50 ng of gDNA using the Nextera library prep  per Illumina’s recommended protocol and sequenced on an Illumina HiSeq 4000 2 × 100. Raw demultiplexed reads were quality filtered using Trimmomatic104. Bray Curtis dissimilarity matrices were constructed for both species-level relative abundance and normalized gene annotations using the vegdist function in the R package Vegan. Principal coordinate analysis (PcoA) was conducted using the R function cmdscale. PERMANOVAs were performed using the Vegan function ADONIS. Unpaired t-test, 1-way, and 2-way ANOVA were implemented using PRISM8 were noted to generate p-values and utilizing the corresponding post-hoc-test when the initial ANOVA was significant. The LEfSe algorithm was used to identify differentially abundant taxa and pathways between groups with logarithmic Linear discriminant analysis (LDA) score cutoff of 299.All statistical analyses were conducted using PRISM (V8) or the R package Veganp-values105. These values were then plotted using the R package corrplot106.Spearman rank correlations were used to investigate the relationship between Lamina propria immune cell populations and the taxonomy of the luminal contents collected from the same site. The “rcorr” function from the R package Hmisc to calculate Spreaman rho and Supplementary InformationSupplementary Table1Supplementary Table1–7


Broca’s area in the posterior half of the left inferior frontal gyrus has traditionally been considered an important node in the speech production network. Nevertheless, recovery of speech production has been reported, to different degrees, within a few months of damage to Broca’s area. Importantly, contemporary evidence suggests that, within Broca’s area, its posterior part (i.e. pars opercularis) plays a more prominent role in speech production than its anterior part (i.e. pars triangularis). In this study, we therefore investigated the brain activation patterns that underlie accurate speech production following stroke damage to the opercular part of Broca’s area. By combining functional MRI and 13 tasks that place varying demands on speech production, brain activation was compared in (i) seven patients of interest with damage to the opercular part of Broca’s area; (ii) 55 neurologically intact controls; and (iii) 28 patient controls with left-hemisphere damage that spared Broca’s area. When producing accurate overt speech responses, the patients with damage to the left pars opercularis activated a substantial portion of the normal bilaterally distributed system. Within this system, there was a lesion-site-dependent effect in a specific part of the right cerebellar Crus I where activation was significantly higher in the patients with damage to the left pars opercularis compared to both neurologically intact and patient controls. In addition, activation in the right pars opercularis was significantly higher in the patients with damage to the left pars opercularis relative to neurologically intact controls but not patient controls (after adjusting for differences in lesion size). By further examining how right Crus I and right pars opercularis responded across a range of conditions in the neurologically intact controls, we suggest that these regions play distinct roles in domain-general cognitive control. Finally, we show that enhanced activation in the right pars opercularis cannot be explained by release from an inhibitory relationship with the left pars opercularis (i.e. dis-inhibition) because right pars opercularis activation was positively related to left pars opercularis activation in neurologically intact controls. Our findings motivate and guide future studies to investigate (i) how exactly right Crus I and right pars opercularis support accurate speech production after damage to the opercular part of Broca’s area and (ii) whether non-invasive neurostimulation to one or both of these regions boosts speech production recovery after damage to the opercular part of Broca’s area.  By combining functional neuroimaging and a wide range of tasks that place varying demands on speech production, Lorca-Puls et al. reveal that right cerebellar Crus I and right pars opercularis are likely to play a particularly important role in supporting successful speech production following damage to Broca’s area. Paul Broca’s seminal work in the 1860s attributed a critical role in speech production to the posterior half of the left inferior frontal gyrus.In the present fMRI study, we investigated which brain regions support accurate overt speech production in stroke survivors with relatively circumscribed left frontal damage involving the opercular part of Broca’s area. This is in contrast to the vast majority of previous functional neuroimaging studies in which patients with heterogeneous lesion locations were pooled together. Instead, our work draws from several emerging sources of evidence indicating that the brain activation patterns underlying normal behavioural responses after stroke depend, among other factors, on the location of the lesion.,Our interest in investigating the brain activation patterns that support accurate overt speech production after LpOp damage stems from the following three observations. First, an inability to accurately produce overt speech is one of the most disabling behavioural consequences of stroke, affecting the capacity of the patient to verbally express their feelings, thoughts and needs. Second, the association between damage to Broca’s area and speech production impairments has been reported to weaken with time post-stroke,,,,,What specific tasks should be used to identify the brain regions that support accurate overt speech production following LpOp damage? Previous studies adopted a variety of speech production tasks to probe the function of LpOp, consistent with the fact that speech production is a multifaceted human ability that requires the interaction of multiple levels of processing, such as phonetic, phonological, lexical-semantic and morphosyntactic processing. LpOp function has been assessed, for example, with the production of single words and connected speech.n = 6 and 12, respectively).To the best of our knowledge, only two previous functional imaging studies, by Rosen et al.n = 55) to better estimate the range of normal brain responses and increase statistical power; (vi) included a group of patients with lesions elsewhere in the left hemisphere (n = 28) to elucidate if any differences in brain activation between patients with LpOp damage and neurologically intact controls were lesion-site-dependent; (vii) characterized inter-patient variability in brain activation within the identified compensatory regions; and (viii) capitalized on the flexibility afforded by our multi-task fMRI paradigm to infer the type of function that the identified potentially compensatory regions may normally subserve.With the aim of building, improving and extending upon the work of Rosen et al.,,,Based on previous research findings, we hypothesized that accurate overt speech production in patients with LpOp damage would recruit a bilaterally distributed set of brain areas similar to that observed in neurologically intact individuals.In summary, the primary goal of the present fMRI study was to generate new insights into the neural mechanisms supporting accurate overt speech production following damage to the opercular part of Broca’s area.1-weighted whole-brain image was visually inspected to confirm the presence of damage to LpOp. Finally, patients whose lesions extended into posterior speech production areas in lateral inferior parietal and superior temporal association cortices were excluded from the selected sample. Other inclusion criteria were as follows: (i) more than 50% accuracy on at least one of the two fMRI tasks of interest (described below) and no less than 25% accuracy on the other; (ii) >6 months since stroke onset; (iii) aged over 18 years at stroke onset; (iv) no history of neurological or psychiatric illness (other than stroke); (v) native speaker of English; and (vi) right-handed pre-morbidly. A binary classification of the patients’ speech production abilities into impaired or unimpaired performance was not part of the inclusion criteria because even those patients with impaired speech production abilities will sometimes be able to produce accurate overt speech responses. By focussing on these accurate overt speech responses, we should therefore be able to reveal the brain regions that support accurate overt speech production in stroke survivors with relatively circumscribed left frontal damage involving LpOp, a brain region that has repeatedly been implicated in speech production in the prior literature. In other words, the absence of impaired speech production abilities (immediately after stroke onset or at the time of testing) in the presence of damage to a speech production region makes our research even more relevant.We searched the Predicting Language Outcome and Recovery After Stroke (PLORAS)1-weighted whole-brain image of two of these patients (P1 and P4).A total of seven patients met our strict criteria and were included in the study. All were more than one-year post-stroke (i.e. in the chronic phase), and none had previously suffered another stroke. The type of stroke was ischaemic in six cases and haemorrhagic in one case (i.e. P2). Moreover, in all seven cases, damage to LpOp co-occurred with damage to other neighbouring frontal regions that previous studies have associated with speech production such as the adjacent ventrolateral premotor cortex and the underlying white matter in the vicinity of the arcuate fasciculus see . Regardin = 55), who were all right-handed native English speakers, served as a normative group. A third group of 28 left-hemisphere stroke patients was also included to investigate whether brain activation differences between patients with LpOp damage and neurologically intact controls were lesion-site-dependent. These ‘patient controls’ were selected based on the absence of damage to LpOp but otherwise with the same inclusion criteria (1–6 specified above) as the patients of interest with LpOp damage. All 28 patient controls were more than one-year post-stroke (i.e. in the chronic phase), and none had previously suffered another stroke. Regarding the type of stroke, 25 patient controls suffered an ischaemic insult and three patient controls suffered a haemorrhagic insult. In relation to the presence of changes in the periventricular white matter consistent with leukoaraiosis, these were seen on the T1-weighted whole-brain image of five of these patients. As shown in The neurologically intact participants (P = 0.005), but well matched to the patient controls (without LpOp damage) for age at scan, age at stroke and time post-stroke . Therefore, any differences in brain activation between the patients with LpOp damage and controls  could not be explained by any of these factors. As the patients with LpOp damage had significantly larger lesions than the patient controls (P = 0.029), we considered the potential influence of lesion size on our results. For all three participant groups, in-scanner behavioural performance is summarized in Summary demographic, clinical and lesion information for the three participant groups is provided in The study was approved by the London Queen Square Research Ethics Committee. All participants gave written informed consent prior to enrolment and were compensated £10 per hour for their time. The experimental procedures were in accordance with the Declaration of Helsinki.All participants performed the same fMRI paradigm comprising 13 different tasks, in two consecutive experiments (Experiment 1 and Experiment 2). The flexibility afforded by our multi-task fMRI paradigm therefore allows a wide range of research questions to be investigated by focussing on a specific combination of tasks. In this study, the paradigm was used to investigate the function of regions that showed decreased/increased activation during accurate pseudoword production following LpOp damage relative to neurologically intact and patient controls.Our tasks of interest were pseudoword reading and repetition. Data from the remaining 11 tasks in Experiment 1 and 2 are reported to aid the interpretation of the results obtained from the pseudoword tasks of interest. The first experiment with five tasks (Experiment 1) has already been fully described, including stimuli characteristics, in Sanjuán et al.Together with pseudoword reading and pseudoword repetition, Experiment 2 comprised a 2×2×2 factorial design that tested speech production in response to stimuli that varied in sensory modality , semantic content (words and objects > pseudowords and baselines) and phonological content (words and pseudowords > objects and baselines). This allowed us to tease apart activation related to perceptual, semantic and phonological processing in terms of behavioural and neural response. The word production tasks involved reading or repeating aloud familiar written or spoken object names, respectively. The object naming tasks entailed either naming objects from pictures  or from the sounds they make . The baseline tasks entailed either naming the colour of meaningless patterns or naming the gender of the voice humming a meaningless unfamiliar rhythm with no speech or semantics. Accuracy scores and response times (RTs) from the eight tasks in Experiment 2 were analysed with repeated measures ANOVAs see  to segreThe five tasks in Experiment 1 presented pictures of two objects or pairs of spoken object names. The four tasks that presented pictures of two objects involved: (i) naming the two objects; (ii) producing a short sentence to describe how the two objects were interacting (e.g. ‘The cat is drinking from a jug’); (iii) naming the verb describing the action between the two objects (‘drinking’); or (iv) silently making semantic matching decisions (related versus unrelated objects) on the pictures. The fifth task presented pairs of spoken object names and entailed silently making semantic matching decisions (related versus unrelated objects) on the heard object names. This combination of tasks enables us to identify activation related to noun versus verb naming, sentence production and semantic associations while controlling for the perceptual content of the stimuli.All participants completed the tasks in the same order and the stimuli used for each task were kept constant across participants. This ensures that any group differences cannot be explained by differences in experimental variables (tasks and stimuli).Pseudowords (e.g. ‘vop’) were created using a nonword generator.Prior to scanning, each participant was trained on all tasks using a separate set of stimuli until they felt comfortable with the task. Once inside the fMRI scanner, participants were asked to produce overt speech while minimizing the amount of head motion as much as possible. They were also instructed to fixate their eyes on a cross at the centre of the screen. This was monitored with eye-tracking.Each task comprised a separate scan run with four blocks of stimuli. In Experiment 2 (which included the pseudoword reading and repetition tasks), each block presented 10 stimuli at a rate of one every 2.5 s. In Experiment 1, pairs of items were presented at half the rate (one every 5 s) resulting in 10 stimuli per block in all tasks. Every stimulus block, in all 13 tasks, was followed by 16-s resting periods, during which the participant fixated on a cross centred on the screen. These resting-with-fixation periods allowed activation to return to baseline between blocks, therefore ensuring maximum sensitivity to all effects of interest.Immediately before the start of each stimulus block, a written instruction was displayed (e.g. ‘Read unfamiliar words’) lasting for the length of one inter-scan interval (i.e. 3.08 s), which reminded the subject of the task. Within the pseudoword reading and pseudoword repetition tasks, novel  one-syllable and two-syllable pseudowords were presented in the first and third blocks, respectively. The second and fourth blocks presented identical stimuli as the preceding block but in a different order.TM Optoacoustics, Or-Yehuda, Israel), and transcribed manually for offline analysis.Visual stimuli were presented via an LCD projector, and an adjustable head-coil mirror, onto a screen that was clearly visible to the subject (1024×768 resolution). Text for the reading tasks was displayed in lower case Helvetica with a visual angle ranging from 1.47 to 4.41 degrees. Auditory stimuli were presented via MRI compatible headphones , which filtered ambient in-scanner noise. Volume levels were adjusted for each subject prior to scanning. Spoken responses were recorded via a noise-cancelling MRI microphone (FOMRI IIISpoken responses were transcribed online and scored offline. Each response was categorized as either ‘correct’ (i.e. when the response matched the target) or ‘incorrect’ . For spoken pseudoword responses, deviations of up to one phoneme were scored as ‘correct’, because an item analysis indicated that this amount of variability in pronunciation was frequently seen in neurologically intact participants.RTs for spoken responses were obtained from the audio files, using an adaptive moving window filter. The optimal window length  was based on a portion of the respective audio file collected during rest. After smoothing the whole time series, we defined the onset of speech as a rise in the absolute amplitude of the smoothed audio stream of at least three standard deviations from the mean.The median RT for each task per subject was calculated based on correct responses only and submitted to behavioural data analyses , which wFunctional and structural data were collected using the same acquisition sequences on two 3 T Trio scanners  with a 12-channel head coil. To minimize movement during scanning, a careful head fixation procedure with foam padding was adopted when positioning each participant’s head in the 12-channel head coil. Total scanning time was approximately 1 h and 30 min per subject, including set up and the acquisition of a structural scan.3. The TR was chosen to maximize whole-brain coverage (44 slices) and allowed us to asynchronise the slice acquisition with stimulus onset to allow for distributed sampling of stimulus onset across slices in each task.Functional images were acquired using a gradient-echo EPI sequence: TR/TE = 3080 ms/30 ms, Flip angle = 90°, matrix size = 64×64, FOV = 192×192, slice thickness = 2 mm, inter-slice gap = 1 mm, voxel size = 3×3×3 mm3.Structural images were acquired after the EPIs, using a 3D modified driven equilibrium Fourier transform sequence2* signal. The structural T1-weighted image was co-registered to the mean EPI image generated during the realignment step and then spatially normalized to MNI space using the new unified normalization-segmentation routine in SPM12. To spatially register all realigned EPI scans to MNI space, we applied the deformation field parameters that were obtained during the normalization of the structural T1-weighted image. The original resolution of the structural and functional images was maintained during normalization . After the normalization procedure, functional images were spatially smoothed with a 6 mm full-width-half-maximum isotropic Gaussian kernel to compensate for residual anatomical variability and to permit application of Gaussian random field theory for statistical inference.We performed fMRI data pre-processing and statistical analysis in SPM12 , running in MATLAB . Functional volumes were spatially realigned to the first EPI volume and unwarped. We used the unwarping procedure in preference to including the realignment parameters as covariates of no interest in the first level analysis because unwarping accounts for non-linear distortions by modelling the interaction between head movement and any inhomogeneity in the T1-weighted image for each stroke patient was converted into a 3D image of the lesion in standard MNI space following automated procedures described in Seghier et al.In addition, the structural TIn the first level statistical analysis, each pre-processed functional volume was entered into a subject specific fixed-effect analysis using the general linear model.P < 0.001 uncorrected) in at least four out of seven patients with LpOp damage during pseudoword reading and/or repetition at the first level. The second contrast tested for brain regions where activation during both pseudoword tasks was lower in patients with LpOp damage than neurologically intact controls (i.e. [−1 −1 1 1]) and higher than rest in neurologically intact controls (using inclusive masks [0 0 1 0] and [0 0 0 1]). Reduced activation in spared brain tissue in the patients could be interpreted as a marker of distant lesion effects. Finally, the third contrast tested for brain regions where activation during both pseudoword tasks was higher in patients with LpOp damage than neurologically intact controls (i.e. [1 1 −1 −1]) and higher than rest in patients with LpOp damage (using inclusive masks [1 0 0 0] and [0 1 0 0]). Increased activation in spared brain tissue in the patients could be interpreted as a marker of functional reorganization and adaptive neuroplasticity. As with contrast 1, we inclusively masked contrast 3 with the first level functional overlap map that included activation from at least four of the seven patients with LpOp damage.In the second level statistical analysis, the contrast images from the first level analysis (comparing correct responses against rest) were submitted to a two-way mixed ANOVA with group  and task (pseudoword reading and repetition) as the factors. There were three contrasts of interest. The first contrast tested for brain regions where activation during both pseudoword tasks was common to patients with LpOp damage and neurologically intact controls (i.e. [1 1 1 1]) and higher than rest in patients with LpOp damage (using inclusive masks [1 0 0 0] and [0 1 0 0]) and neurologically intact controls (using inclusive masks [0 0 1 0] and [0 0 0 1]). This allowed us to identify the regions within the normal system that support accurate behaviour in the patients. In addition, to minimize the possibility that activation in patients with LpOp damage was driven by only one or two outliers, we inclusively masked contrast 1 with a functional overlap map which comprised voxels activated (using a voxel-wise threshold of P < 0.05 after family-wise error (FWE) correction for multiple comparisons across the whole brain. Where significant differences between patients with LpOp damage and neurologically intact controls were found , region-of-interest analyses comparing activation  in patients with LpOp damage and patient controls (without LpOp damage) were carried out in IBM SPSS Statistics for Windows, Version 26.0  to test if the identified effects were lesion-site-dependent, while adjusting for differences in lesion size. This was necessary because the patients with LpOp damage had, on average, larger lesions than the patient controls  was set at c.j.price@ucl.ac.uk) upon reasonable request.The data that support the findings of this study are available from the senior author . Despite this self-reported improvement, however, none of the patients with LpOp damage felt that their speech production abilities had completely returned to premorbid levels by 1 year after stroke onset. See With the assistance of a speech and language therapist and their carer, each of the patients with LpOp damage retrospectively rated their speech production abilities at 1 week, 1 month and 1 year post-stroke, using an in-house ordinal scale ranging from 1 (= unable to attempt) to 7  that is administered to all patients upon entry to the PLORAS study. All seven patients reported that their speech production abilities had (i) been affected by the stroke and (ii) improved from 1 month to 1 year after stroke onset. In contrast, there was greater inter-patient variability in self-reported improvement from 1 week to 1 month post-stroke. Consequently, a Wilcoxon signed-rank test only captured a statistically significant positive change in self-rated speech production abilities between 1 month and 1 year post-stroke  been affected by the stroke; (ii) improved within the first-year post-stroke; and (iii) not returned to premorbid levels by 1 year after stroke onset. Third, the self-rated speech production abilities of the patients with LpOp damage were consistently worse than those of the patient controls across the three time points: 1 week , 1 month  and 1 year post-stroke . See As a group, the patient controls also reported that their speech production abilities had improved within the first year after stroke onset. However, there were three notable differences relative to the patients with LpOp damage. First, the patient controls’ self-reported improvement in speech production abilities between 1 week and 1 month post-stoke was significant at t(60) = 2.05, P = 0.044]. Critically, however, there were no significant differences in RTs or accuracy scores between patients with LpOp damage and patient controls (both P > 0.6).For pseudoword repetition, the normal range of accuracy was between 83% and 100% (mean = 98%). All but 2 of the patients with LpOp damage had normal or near-to-normal accuracy . The 2 exceptions achieved 73% accuracy (P7) and 68% accuracy (P4). RTs for pseudoword repetition were significantly slower in the patients with LpOp damage than neurologically intact controls , with a (non-significant) trend in the same direction when patients with LpOp damage were compared to patient controls . We argue that any group differences in activation that are common to pseudoword reading and repetition cannot be explained by differences in RTs, because RTs during pseudoword repetition were well equated for patients with LpOp damage and patient controls (see above).For pseudoword reading, the normal range of accuracy was between 85% and 100% (mean = 98%). All but one of the patients with LpOp damage had normal or near-to-normal accuracy . The exception (P6) was correct on 43% of trials. Patient controls attained similar accuracy (on average) as the patients with LpOp damage . The location of LS1/M1 relative to the lesion of each patient with LpOp damage is illustrated in During accurate pseudoword production, patients with LpOp damage activated the majority of the intact part of the normal speech production system, including bilateral mid superior temporal cortices, bilateral SMA/pre-SMA, right ventral pars opercularis and right sensorimotor cortex .The effect of most interest was that patients with LpOp damage showed increased activation within right cerebellar Crus I  was higher in patients with LpOp damage compared to neurologically intact controls in the right ventral pars opercularis .Consistent with Rosen et al.At the individual subject level, all but one (i.e. P3) of the seven patients with LpOp damage showed increased activation during pseudoword production in either RCrusI, RpOp or both, compared to neurologically intact controls; see The focus of the results above has been on two tasks (i.e. reading and repeating pseudowords) that were expected to robustly engage LpOp on the basis of prior findings. In addition, all our participants completed 11 other tasks . By examining the pattern of responses across all 13 tasks (from the two experiments), we can infer the type of function that LS1/M1, RCrusI and RpOp subserve in the neurologically intact brain. These region-of-interest analyses  were carried out in SPSS.P < 0.05 for all comparisons) between each speech production task and each sematic matching task (see P < 0.001) indicated that the reduction in LS1/M1 activation affected pseudoword reading/repetition more than word reading/repetition. The responses of LS1/M1 in each patient with LpOp damage during each fMRI task can be found in LS1/M1 activation in neurologically intact controls occurred during the 11 speech production tasks (3 in Experiment 1 and 8 in Experiment 2) but not during the two silent semantic matching tasks (Experiment 1), with a statistically significant difference . In contrast, during the two silent semantic matching tasks (Experiment 1), RCrusI activation was reduced, rather than enhanced, in patients with LpOp damage compared to neurologically intact controls (P < 0.05 for both comparisons). This resulted in a significant interaction (P = 0.001) between group  and task (naming pictures of 2 objects > semantic decisions on pictures of 2 objects); see RCrusI activation in neurologically intact controls occurred during all 13 tasks (across Experiments 1 and 2), irrespective of the demands on speech production, phonological processing, semantic processing or perceptual processing see . In patiP = 0.001).RpOp activation in neurologically intact controls occurred during all 13 tasks (across Experiments 1 and 2), with stronger responses during all speech production tasks than silent semantic matching tasks see ; but witBy focussing on the fMRI data from neurologically intact controls, we found that, during pseudoword reading and repetition, RCrusI activation co-varied positively with activation in LpOp  but not LS1/M1 . The strength of covariance with RCrusI was also significantly higher for LpOp than LS1/M1 see .Z-score = 5.14, P = 0.007 FWE-corrected across the whole brain, K = 4 voxels). The more ventral pOp peak was located at  ; see Within LpOp, covariance with RCrusI activation during pseudoword reading and repetition peaked in two distinct parts of the pars opercularis (pOp). The more dorsal pOp peak was located at  . In contrast, the ventral pOp region did not track the demands on perceptual, semantic or phonological processing during speech production ; see When we examined how the responses in the dorsal and ventral parts of pOp were influenced by the type of speech production task (Experiment 2), we found that the dorsal pOp region was sensitive to the demands on phonological processing during speech production  = 0.34, P = 0.011] and pseudoword repetition .There was no evidence that the enhanced activation within RpOp in patients with LpOp damage could be explained by dis-inhibition (i.e. an inhibitory relationship between LpOp and RpOp that was lost following LpOp damage). To the contrary, we found that in neurologically intact controls, there was positive (not negative) covariance between the responses of the RpOp region that showed the highest activation in patients with LpOp damage  and responses of the homologous LpOp region . This was observed during both pseudoword reading [The goal of this study was to investigate the brain activation patterns that underlie accurate overt speech production in chronic stroke patients with relatively circumscribed left frontal lobe damage involving the posterior part of Broca’s area: left pars opercularis (LpOp). All seven patients with LpOp damage included in this study reported, using an in-house ordinal scale, that their speech production abilities had improved in the first-year post-stroke compared to how they were initially after stroke onset. By focussing on accurate overt speech responses, our study has yielded the following three novel observations: (i) activation in a specific region within the left sensorimotor cortex  was significantly lower in patients with LpOp damage relative to neurologically intact controls during pseudoword production but not other tasks, suggesting that the relationship between LpOp and LS1/M1 is task-sensitive; (ii) activation in a specific part of right cerebellar Crus I (RCrusI) was significantly higher in patients with LpOp damage relative to both neurologically intact controls and patients without LpOp damage, suggesting that RCrusI may play a particularly important role in compensating for the effect of LpOp damage on speech production; and (iii) higher activation in the right ventral pars opercularis (RpOp) co-varied with higher activation in its left-hemisphere homologue in neurologically intact controls, suggesting that increased RpOp activation following LpOp damage cannot be explained in terms of dis-inhibition.Below we discuss these findings in light of prior literature to highlight the novel insights they provide for understanding how accurate speech production is achieved after damage to the opercular part of Broca’s area and the functional anatomy of speech production.Although patients with LpOp damage recruited most of the intact part of the speech production system as normal, we identified a region within the left sensorimotor cortex  that showed reduced activation relative to neurologically intact controls. Overall, the response pattern of this LS1/M1 region across tasks was similar for neurologically intact controls and patients with LpOp damage. In both groups, LS1/M1 activation was greater during overt speech production than silent semantic matching, with activation during silent semantic matching not raising above resting baseline levels. This is consistent with a role for LS1/M1 in the sensorimotor control of speech. Interestingly, reduced LS1/M1 activation in patients with LpOp damage compared to neurologically intact controls was task-specific, affecting pseudoword reading/repetition but not other tasks. Although the current dataset does not allow us to unequivocally elucidate the underlying cause of such a response profile, in what follows we offer three tentative explanations to motivate further research, with the one we favour considered last.,1-weighted images of the patients with LpOp damage does raise questions about whether or not LS1/M1 activation in P6 fell within compromised tissue (see P < 0.001) and patient controls . Third, the task-specific reduction in LS1/M1 activation might be the consequence of a loss of excitatory inputs from LpOp to LS1/M1 during pseudoword production following LpOp damage. In other words, this explanation suggests that the relationship between LpOp and LS1/M1 is task-sensitive, consistent with the notion of dynamic diaschisis where abnormal evoked responses in a structurally intact region depend on task-relevant neuronal interactions with a damaged region.First, the reduction in LS1/M1 activation during pseudoword reading and repetition might be the consequence of altered cerebral perfusion in tissue neighbouring the lesion but without evident structural damage.ssue see . HoweverFinally, the fact that patients with LpOp damage were able to produce accurate overt speech responses during pseudoword reading/repetition indicates that reduced LS1/M1 activation was either sufficient for task performance or these patients were using alternative neural pathways to bypass LS1/M1. Future effective connectivity studies are needed to investigate these hypotheses directly, by examining, for example, how LpOp and LS1/M1 influence one another and how these influences depend on the task (e.g. pseudoword versus word production).,Unlike the two previous functional imaging studies that investigated overt speech production in chronic stroke patients with left frontal lobe damage involving Broca’s area,,,,Importantly, the RCrusI region we have identified does not correspond to the areas of the cerebellum in bilateral lobules I–VI and VIII that have previously been associated with sensorimotor processing,Does increased RCrusI activation during accurate speech production reflect a compensatory mechanism triggered by damage to the opercular part of Broca’s area? Although further research is needed, four lines of evidence (three from our study and one from previous work) support an affirmative answer. First, increased RCrusI activation was observed in patients with, but not without, LpOp damage, indicating a lesion-site-dependent effect. Second, in neurologically intact controls, higher RCrusI activation co-varied with higher activation in LpOp but not LS1/M1 and the specificity of this inter-regional coupling was confirmed by a significant difference in the strength of covariance between RCrusI and LpOp versus RCrusI and LS1/M1. Third, no other speech production region within the left frontal lobe (apart from LpOp) exhibited significant positive covariance with RCrusI activation in neurologically intact controls. Fourth, prior reports have shown that (i) RCrusI is co-activated with LpOp but not, for example, primary motor cortex during task performance and (ii) a transient reduction in blood flow and metabolism in the right lateral cerebellum can result from damage in and around Broca’s area.,The two previous functional imaging studies of overt speech production in chronic stroke patients with left frontal lobe damage involving Broca’s area, both reported increased activation within the right posterior inferior frontal gyrus (RpIFG) relative to neurologically intact controls.Regarding the role of RpOp in accurate speech production, we examined its response over a wide range of tasks. This indicated that, in neurologically intact controls, RpOp (i) responded during all 13 tasks but with greater strength during overt speech production than silent semantic matching (Experiment 1) and (ii) was insensitive to the demands on perceptual, semantic or phonological processing during speech production (Experiment 2). We propose that such a response profile is consistent with a role for RpOp in domain-general cognitive control that is enhanced by, but not specific to, speech production. Further light on the role of RpOp is shed by prior studies that have largely associated this area with a specific component of cognitive control, namely response inhibition that is needed to suppress errors.We have shown that patients with damage to the opercular part of Broca’s area are able to recruit most of the intact part of the normal system to support accurate overt speech production. Within this system, activation in two specific regions within the right cerebellar Crus I (RCrusI) and right pars opercularis (RpOp) was increased following LpOp damage compared to controls. We have discussed how these regions might help to compensate for the effect of damage to the opercular part of Broca’s area. Our findings thus motivate and guide future studies with larger patient numbers to investigate: (i) how exactly RCrusI and RpOp support accurate speech production after damage to the opercular part of Broca’s area; (ii) how RCrusI and RpOp activation changes over time as a function of speech production recovery after damage to the opercular part of Broca’s area; (iii) what lesion and non-lesion factors explain inter-patient variability in RCrusI and RpOp activation after damage to the opercular part of Broca’s area; and (iv) whether non-invasive neurostimulation to RCrusI and/or RpOp boosts speech production recovery in patients with, versus without, damage to the opercular part of Broca’s area.Brain Communications online.fcab230_Supplementary_DataClick here for additional data file.


MnO2/extended graphite exhibited an increase in sp2 carbon bonds in comparison with that of extended graphite. It can be expected to display better electrical conductivity and further promote electron/ion transport kinetics for boosting the electrochemical performance in supercapacitors and glucose sensing. In supercapacitors, MnO2/extended graphite delivered an areal capacitance value of 20.4 mF cm−2 at 0.25 mA cm−2 current densities and great cycling stability (capacitance retention of 83% after 1000 cycles). In glucose sensing, MnO2/extended graphite exhibited a good linear relationship in glucose concentration up to about 5 mM, sensitivity of 43 μA mM−1cm−2, and the limit of detection of 0.081 mM. It is further concluded that MnO2/extended graphite could be a good candidate for the future design of synergistic multifunctional materials in electrochemical techniques.A simple, efficient, and cost-effective extended graphite as a supporting platform further supported the MnO Over the years, fossil fuel consumption and public health problems have posed serious issues, including global warming and substantial human diseases, that have caused significant negative economic and human health impacts. The relationship expressed above indicated that key technological changes and suitable implementation strategies might be necessary for promoting opportunities to reduce fossil fuel dependency, and, simultaneously, implement health promotion/disease prevention. Supercapacitors serve as intermediate energy storage systems between traditional capacitors and batteries, due to their simple construction, short charging time, safety, high power density, and excellent lifetime, which could replace fossil fuel use to meet energy requirements for reducing the fossil fuel energy consumption and further relaxing environmental pollution.World-class public health is another globally important issue and challenge. The International Diabetes Federation (IDF) estimated that the number of people worldwide with diabetes (aged 20–79 years) would increase to 700 million in 2045 . In Taiw2 nanostructures with well-controlled morphology and crystal phase are expected to exhibit improved electrochemical performance when being used in supercapacitors and electrochemical sensing. More advantages for the use of MnO2 as the electrode material were explained in previous reports. Yang et al. reported that MnO2 had great advantages in terms of high theoretical specific capacity (up to 1370 F g−1), low cost, natural abundance, and multiple crystallographic phases to be one of the most promising candidates for supercapacitors and glucose sensing. The structural transformation of multiple MnO2 polymorphic forms composed of MnO6 octahedrons by edge and/or corner sharing made differences in the intrinsic channel dimensions of MnO2 to possess different electrochemical activities. MnO2 based composites for supercapacitor applications with enhanced capacitive behaviors were attributed to electrochemically induced polymorphic phase and structural transformations of MnO2, thus affecting the surface area and interfacial resistance . F. F2/gra-aterials ,45,46. T2/gra-H in practical applications, the recovery tests of glucose are carried out by adding a known concentration (1 mM) to samples, which is then evaluated by analyzing the amperometric response to glucose by 3-time repeated measurements. The results of the recovery tests obtained by the proposed amperometric method are in good agreement with the results obtained by the reference method (mass spectrometry), and the comparison results are given in 2/gra-H makes satisfactory recovery of the added glucose concentrations.To further demonstrate the potential of the proposed MnO2/extended graphite displayed high bifunctional electro-activity performances in electrochemical multifunctional applications (including supercapacitors and glucose sensing). Thanks to the excellent surface modification of gra-H through electrochemically activated treatment of graphite inducing extended defects on the surface of graphite, providing a new set of growth platforms to form hierarchical flower-like MnO2, and thus a perfect graphitic crystalline structure present in MnO2/gra-H, which provide a continuous conductive network to optimize electron/ion transport kinetics for boosting the electrochemical performances of supercapacitors  and glucose sensing . Because of its electrochemical synergistic effects, MnO2/extended graphite is a promising candidate for the development of electrochemical multifunctional applications.In this study, MnO


The lack of scar-free healing and regeneration in many adult human tissues imposes severe limitations on the recovery of function after injury. In stark contrast, salamanders can functionally repair a range of clinically relevant tissues throughout adult life. The impressive ability to regenerate whole limbs after amputation, or regenerate following cardiac injury, is critically dependent on the recruitment of (myeloid) macrophage white blood cells to the site of injury. Amputation in the absence of macrophages results in regeneration failure and scar tissue induction. Identifying the exact hematopoietic source or reservoir of myeloid cells supporting regeneration is a necessary step in characterizing differences in macrophage phenotypes regulating scarring or regeneration across species. Mammalian wounds are dominated by splenic-derived monocytes that originate in the bone marrow and differentiate into macrophages within the wound. Unlike mammals, adult axolotls do not have functional bone marrow but instead utilize liver and spleen tissues as major sites for adult hematopoiesis. To interrogate leukocyte identity, tissue origins, and modes of recruitment, we established several transgenic axolotl hematopoietic tissue transplant models and flow cytometry protocols to study cell migration and identify the source of pro-regenerative macrophages. We identified that although bidirectional trafficking of leukocytes can occur between spleen and liver tissues, the liver is the major source of leukocytes recruited to regenerating limbs. Recruitment of leukocytes and limb regeneration occurs in the absence of the spleen, thus confirming the dependence of liver-derived myeloid cells in regeneration and that splenic maturation is dispensable for the education of pro-regenerative macrophages. This work provides an important foundation for understanding the hematopoietic origins and education of myeloid cells recruited to, and essential for, adult tissue regeneration. Salamanders are the only vertebrate to regenerate limbs as adults, a trait that is lost in mammals and related amphibians . Adult sIn mammals, the bone marrow is the major site of adult hematopoiesis, with the spleen also serving as an extramedullary site for monocyte production in some circumstances . In salaWe previously described the requirement for salamander macrophages in the success of limb regeneration  and a baQuantitative RT-PCR analysis using marker genes associated with distinct cell types showed high levels of purity in the five sorted populations . The typ+ myeloid and lymphoid B cells from peripheral blood and inject these into naïve hosts to profile the early time course of wounding for myeloid vs. lymphoid recruitment  confirmed macrophage recruitment between 1 and 7 dpa. The CD18negIB4+ putative T-cell population showed a small number of GFP+ donor cells arriving at 1 dpa and no major accumulation up to 7 dpa (negIB4negPanIgneg) population that has yet to be identified did show some recruitment by 18 h post-amputation and was still visible up to 14 dpa  in the axolotl , the exat tissue , which at tissue . Similar+ spleen . These gthe skin . In contost skin . Very liat 4 dpa . These e 6 weeks . These e+ liver or spleen tissue grafts, we next tested the potential for dissociated GFP+ liver and spleen-derived single cells to migrate to amputated limbs  that is consistent with a mixture of neutrophils and macrophages  as well as the putative T cell populations (CD18negIB4+PanIgneg) appear relatively stable over 24 months in the liver and spleen . Phagocytic myeloid cells are critical during the early phases of regeneration and so we used a functional marker for phagocytic macrophages (uptake of fluorescent DiI liposomes ) that ald 10 dpa . B cell d 10 dpa . The trid spleen . Taken t+ adoptive cell transplants to examine inter-organ trafficking and potential homing biases (N = 4) or spleen-origin (N = 4) donor cells, delivered to alternative recipient (GFP–) cohorts (N = 4 + 4)  into new (GFP–) terminal recipients (N = 4 + 4 + 4 + 4). After another week, both spleens and livers were collected as paired samples from each of the 16 animals. To assess the effect of cross organ exposure (education) on liver-origin or spleen-origin GFP cells, we used flow cytometry to count GFP+ cells in the terminal recipients of paired liver and spleen tissue. We then calculated the ratio of GFP cells between liver and spleen per recipient animal where a 1:1 ratio would reflect that the GFP+ cell mixture that was adoptively transferred had an equal chance of trafficking to either the liver or spleen. Those animals with ratios higher than 1 would reflect a GFP+ donor cell population with a preference for liver. Those with a ratio lower than 1 represent donor GFP+ cells with a preference to home to spleens. The results  . After 1 results  indicatent model , it has + donor on white leucistic (d/d) animals  with lower lymphocyte counts than rodents (5–25% granulocytes and 75–90% lymphocytes) . InteresWhilst several non-mammalian model organisms are utilized in regenerative biology, amphibians such as anurans (frogs) and urodeles  offer a unique perspective to study the contributions of the innate immune system as they share a majority of the key immune tissues present in mammals . The notIn addition to functioning as sites for hematopoiesis, our results indicate that the liver and spleen also serve as reservoirs for myeloid cells and B cells. This is in line with historical histological findings in several salamander species, which observed granulocytes primarily residing in the sub-capsular zone of the liver and lymphocytes predominantly inhabiting the white pulp regions of the spleen . During A recent study conducted by Regeneration of the mouse heart is observed during a narrow temporal window during development and is gradually lost between 1 and 7 days of postnatal life . This loSustained myeloid cell recruitment into the site of injury is a conserved process between salamander and mammalian tissues. Blockade of this process in the axolotl results in the formation of fibrotic tissue similar to that induced in the injured mouse heart characterized by the abnormal appearance of myofibroblasts and an irregular build-up of thick collagen I and IV fibers in the place of thin collagen III . Our finAll scientific procedures involving animals were undertaken in line with Animal Ethics Committee guidelines for Monash University or the MDI Biological laboratory.Ambystoma mexicanum (Mexican axolotl) animals  were obtained from the Ambystoma Genetic Stock Centre (AGSC), Lexington, KY, United States, and captive bred. Animals were individually housed in carbon-filtered tap water tanks on a 12-h light, 12-h dark cycle. Juvenile and adult animals were used for all experiments and were between 4 and 24 months old. Prior to animal surgeries , animals were anesthetized using 0.1% ethyl-3-aminobenzoate methanesulfonate salt . Forelimb and tail amputation procedures were carried out as previously described . Liver and spleen grafts were performed on 9–12-month-old animals (snout to tail length of 15–20 cm) where 25% of the rostral side of the host organ was resected and then equal volume of donor tissue was grafted onto the host. For live imaging procedures, harvested organs or anesthetized animals were imaged using a Zeiss Lumar V12 fluorescent stereo microscope. Spleens were removed via keyhole surgery. The remaining vessels were cauterized to prevent any blood loss and the incision into the skin was sutured. The control cohorts for these experiments underwent sham surgery.® winged infusion set  and was dispensed into 50-ml tubes containing ice-cold 0.7X HBSS-5 mM EDTA. Cells were then centrifuged at 200 × g at 4°C for 20 min (with no decelerating brakes) to remove platelets. Contaminating red blood cells were then removed by layering the cell suspension onto Ficoll Paque Plus  and centrifuging the sample for 5 min at 400 × g at room temperature (no decelerating brakes). All liquid phases except the red blood cell phase were collected and washed. Liver and spleens were harvested from euthanized animals and briefly rinsed in ice-cold 1X HBSS/5 mM EDTA and placed onto a 10-mm tissue culture dish. Tissues were then processed with surgical scissors and minced into fine chunks of approximately 25 mm in cubic size then processed through a 70-μM cell-strainer with the aid of a 3-mL syringe plunger. Cells were then centrifuged twice at 200 × g at 4°C for 20 min with no decelerating brakes to remove tissue debris, and their numbers were counted using the Countess Automated Cell Counter .Peripheral blood was isolated as previously described  with minTM Red 780  following manufacturer’s instructions. Cell viability for FACS experiments was assessed using DAPI. FACS was performed using either the BD symphony A6 or BD Influx Cell sorters. Flow cytometry quantification was performed on LSRII Flow Cytometers . Compensation of fluorescence spectral overlap was used with UltraComp eBeads  according to manufacturer’s instructions. FCS 3.0 files generated by flow cytometry were analyzed using FlowJo Software v10. For list of antibodies used in these experiments and their working dilutions, see Single cell suspensions were blocked with ice-cold 1 × HBSS/5 mM EDTA/1X DNase1/2% goat serum for at least 20 min on ice as previously described . PrimaryL-lysine slides utilizing a cytospin centrifuge and then fixed in 4% PFA. Working dilution May–Grunwald stain was applied to cells for 5 min and then washed after which working dilution Giemsa stain was applied for 15 min. Following staining, slides were then rinsed multiple times with distilled water, air-dried, and preserved in DEPEX and imaged under a light microscope.Cytospins and staining was performed as described in ® reagents (Thermo Fisher Scientific). RNA was purified using Direct-ZolTM RNA MiniPrep  according to manufacturer’s instructions. RNA quality was assessed by spectrophotometry using a NanoDrop ND-1000 (NanoDrop). Reverse transcription was performed using SuperScript® VILOTM cDNA synthesis kit (Thermo Fisher Scientific). Quantitative polymerase chain reaction (qPCR) assays were performed using LightCycler® 480 SYBR green . Gene expression levels were calculated using the 2–ΔΔCt method. Sample gene expression was normalized to the geometric mean of three housekeeping genes and either expressed as relative fold change or log2 fold change. Primer sequences used in qPCR assays are listed in Cell samples were collected into TRIzolt-test, one-way ANOVA for cell counts, or a two-way ANOVA with Turkey’s multiple comparisons test for RT-PCR comparisons between groups as indicated in figure legends.Statistical analyses were performed using the software Prism 9 . Data are always shown as mean values ± SEM. Analyses of significant differences between means were performed using unpaired Student’s The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Monash University Animal Use and Care Committee MDI Biological Laboratory IACUC.RD and JG conceived and designed the experiments, and wrote the manuscript. RD, AH, and GJ performed the experiments and analyzed the data. NR provided funding, infrastructure, and editorial support for the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Patients often report complaining of fractured or decayed teeth with severe morphological deformities. However, all these clinical scenarios require the same level of care and consideration to rehabilitate form, function and esthetics. Some cases havesufficient clinical crown height while others often require an interdisciplinary approach in the form of orthodontic/surgical extrusion or surgical periodontal options. A common factor delaying treatment is soft tissue regrowth after crown lengthening whichdelays the impression required for final prosthesis. Therefore, it is of interest to compare the prevalence of soft tissue regrowth a week after different crown lengthening techniques including laser gingivectomy, electrocautery gingivectomy, modified Widmanflap and apically repositioned. The parameters assessed included 1-week postoperative soft tissue regrowth after crown lengthening, age of patients and gender. It was observed that laser and electrocautery-assisted gingivectomy had a higher rate of soft tissueregrowth as compared to surgical techniques. It was further noted that laser and electrocautery assisted gingivectomy had a higher frequency of soft tissue rebound growth compared to surgical crown lengthening using modified widman flap and apically repositionedflap, which was statistically insignificant. Patients within the age groups of 26-60 years were found to have a higher tendency of soft tissue regrowth, which was found to be clinically and statistically significant (p<0.05). Crown lengthening is a surgical procedure designed to increase the extent of supragingival tooth structure for restorative or esthetic purposes by apically positioning the gingival margin removing supporting bone or both .This prThe current study was performed as a single centered retrospective university design, using dental information archiving software for the comparison of the entire patient outflow of Saveetha Dental College and Hospital, Chennai from 1st June 2019 till 1stMarch 2020. Two blinded investigators screened data, where inter-examiner agreement of ninety percent was reached prior to inclusion of individuals into the study. The segregation of data was initiated after ethical approval from Saveetha university scientificreview board where patients included were individuals who underwent crown lengthening using laser gingivectomy, electrocautery gingivectomy, modified widman flap and apically repositioned flap.Age group of patients was subdivided for analysis and interpretation into group A (14-25 years), group B (26-40 years), groups C (41-60 years) and group D (above 60 years). Similarly the region involved in the crown lengthening was subdivided into group AUpper arch, group B Lower arch, group C sextant 5 and group D sextant 2.Individuals excluded from this study were (i) pregnant women or lactating mothers (ii) smokers (iii) patients with uncontrolled systemic disease specifically affecting collagen metabolism(iv) patients under medication (v) Incomplete data collection . The parameters assessed in this study included 1-week postoperative soft tissue regrowth after crown lengthening.All values obtained from this study were analysed using SPSS software version 23  where p values < 0.05 were considered to bestatistically significant.A total of 373 patients were included in this study 187 males and 186 females with different age groups 18-25 years (96), 26-40 years (167), 41-60 years (88) and above 60 years (22). In this study group we observed that lasergingivectomy and electrocautery had a higher soft tissue regrowth after 1 week postoperative examination as compared to surgical techniques . It was Among all findings obtained intragroup analysis was found to be statistically insignificant (p>0.05) using Chi square while parameters such as the relation between different age groups and soft tissue regrowth along with the crown lengthening techniqueemploys and the presence of soft tissue regrowth 1 week postoperative were found to be significant with (p<0.05). We live in a day and age where aesthetic is considered as one of the key features that defines an individual's personality and bring outconfidence, in terms of crown lengthening the use of procedures that leave scars such as conventional flap surgery are considered unaesthetic while laser assisted procedures, comprehensive procedures like lip repositioning and implant based full mouthrehabilitation in aggressive periodontitis cases are considered advantageous -18.Soft tissue regrowth was seen with a higher tendency in laser gingivectomy and electrocautery gingivectomy groups.(Table 2 - see PDF) Both groups conserve bone leaving biological width as a sacrificeable tissue to obtain required clinical crown height. It isdefined as dimension of space of root surface coronal to alveolar crest to which the junctional epithelium and connective tissue are attached (2.04 mm). Violation of biological width is associated with persistent gingival inflammation and tissue regrowth tocorrect this space occurring spontaneously within 3 months ,19. SimiThe second finding of age groups 26-60 years having a higher frequency of soft tissue rebound regrowth could only be related to better wound healing after incomplete removal of connective tissue during the initial crown lengthening procedure.All liteThe limitations of this study include no exact measurement value of soft tissue regrowth to relate to demographic data or technique of crown lengthening. Another variable not included in the present study was the extent of crown lenghtening required forprosthetic rehabilitation of teeth, which changes depending on treatment planned and clinical visible crown height. The use of potential periodontal biomarkers of healing such as fibroblast growth factor 2 levels, cathepsin levels, endothelin 1 levels could havebeen correlated to obtained findings.-24The prIt was observed that laser and electrocautery assisted gingivectomy had a higher frequency of soft tissue rebound growth related to the age groups of 26-60 years as compared to surgical crown lengthening using modified widman flap and apically repositionedflap which was statistically significant (p<0.05).