Pro- and synbiotics have been reported to ameliorate the adverse (dysbiotic) effects of antibiotics on the gut microbial architecture, but little is known how synbiotics and antibiotics interact with each other in shaping the gut microbiota. To explore this mutual interaction we examined, first, the effect of a multi-strain synbiotic on antibiotic-induced dysbiosis and, second, the dysbiotic effect of antibiotics followed by prolonged synbiotic exposure.The synbiotic containing nine bacterial strains was administered to male mice via the drinking water, while the antibiotic mix containing bacitracin, meropenem, neomycin, and vancomycin was administered via oral gavage. Two experimental protocols were used. In protocol 1, mice were administered placebo or synbiotic for 3 weeks prior to and during an 11-day vehicle or antibiotic treatment. In protocol 2 the synbiotic was administered for a prolonged period of time, starting 3 weeks prior and continuing for 12 weeks after an 11-day vehicle or antibiotic treatment. Subsequently, the fecal microbiome was analyzed by 16S rRNA sequencing using oligonucleotide primers 16s_515_S3_fwd: GATTGCCAGCAGCCGCGGTAA and 16s_806_S2_rev: GGACTACCAGGGTATCTAAT followed by sequencing using the Ion Torrent One. The final sequence files were analyzed by QIIME 1.8 workflow scripts.Antibiotic treatment markedly decreased the bacterial richness and diversity of the fecal microbiota. Synbiotic administration for 3 weeks prior to and during an 11-day antibiotic treatment preserved the Lactobacillales and expanded the Verrucomicrobiales and Bifidobacteriales order, but did not prevent the depletion of Bacteroidales and the short-term proliferation of Enterobacteriales. When the synbiotic administration was continued for 12 weeks after the end of antibiotic treatment, the rise of Verrucomicrobiales was maintained, whereas the preservation of Lactobacillales and boost of Bifidobacteriales was lost. The abundance of Clostridiales was enhanced by long-term synbiotic treatment after short-term exposure to antibiotics, while the antibiotic-depleted Bacteroidales underwent a delayed recovery.There are complex synergistic and antagonistic interactions of synbiotics and antibiotics in influencing distinct bacterial orders of the fecal microbiota. The impact of a short-term antibiotic exposure is profoundly different when analyzed after synbiotic pretreatment or following prolonged synbiotic administration in the post-antibiotic period. Bacteria represent a major constituent of the gut microbiota, the major phyla in the mammalian intestine being Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria and Fusobacteria , 2. The Lactobacillus and Bifidobacterium. Prebiotics are dietary components that aid the growth of beneficial microorganisms [Lactobacillus have a beneficial effect in experimental models of colitis [Against this background, pre-, pro- and synbiotics have gained increasing attention for their potential in the management of microbiome-related diseases. Probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” , 20. Prerganisms , 21 whilrganisms , 22–24.  colitis  as well  colitis , 27.Probiotics have long been considered to represent an adjunct therapy to ameliorate the adverse effects of antibiotic treatment on the gut microbiota, which may result in dysbiosis and antibiotic-associated diarrhea. Although this promise has not been fulfilled in some human studies , an overCollectively, these findings highlight a lack of information about the mutual interaction between antibiotics and probiotics in shaping the architecture of the gut microbiota. It is therefore difficult to make full use of the therapeutic potential of probiotics/synbiotics in the prevention or treatment of antibiotic-induced adverse reactions such as antibiotic-associated diarrhea. Specifically, little is known as to how, on the one hand, synbiotics modify the ability of antibiotics to cause dysbiosis and whether, on the other hand, any short- or long-term effect of antibiotics is influenced by the presence of probiotics or synbiotics. The aim of our study was to address these questions in mice, using a combination of four non-absorbable antibiotics  administered via gastric gavage and a synbiotic containing nine strains of probiotic bacteria administered via the drinking water. In protocol 1, the effect of this multi-strain synbiotic to alleviate antibiotic-induced dysbiosis was examined, whereas in protocol 2 the effect of the antibiotics in the presence of the synbiotic was tested. Using 16S rDNA sequencing, we obtained an unbiased view of the fecal microbiome profile, its perturbation by gut-directed antibiotic treatment and its modification by the synbiotic.® STRESS Repair and Ecologic®825) has thus far been studied in humans only. Oral intake of the synbiotic by healthy volunteers for 4 weeks failed to change microbial diversity in stool samples, but enlarged the abundance of Bacteroides sp. and Alistipes sp. [Lactobacillaceae was increased [The synbiotic chosen for this study , and conducted according to the Directive of the European Parliament and of the Council of September 22, 2010 (2010/63/EU). The experiments were designed in such a way that both the number of animals used and their suffering was minimized.The experiments were carried out with adult, 8 weeks old, male C57BL/6N mice obtained from Charles River Laboratories . Throughout the experiments the mice were kept in groups of two. The housing conditions including lighting , temperature (set point 22 °C), and relative air humidity (set point 50%) were tightly controlled. Tap water and standard laboratory chow were provided ad libitum.® STRESS Repair by Institut Allergosan, Graz, Austria, and as Ecologic®825 by Winclove, Amsterdam, The Netherlands) and placebo were kindly provided as water-soluble preparations by Winclove. The synbiotic consisted of 9 bacterial strains . Besides the bacterial strains (1.9%), the synbiotic contained maize starch (62.0%), maltodextrin (15.5%), inulin P7 (13.8%), potassium chloride (3.4%), magnesium sulfate (1.6%), fructooligosaccharides P7 (1.2%), enzymes (0.5%) and manganese sulfate (0.0035%). The placebo added to the drinking water included maize starch (76%), maltodextrin (19%) as the main carriers [9 CFU/l.The synbiotic , in which the intake of the synbiotic and placebo solution per cage was monitored for 3 weeks, showed that the intake of the synbiotic solution was nominally, but not significantly, higher than the intake of the placebo solution. The synbiotic and placebo solutions were prepared fresh on Mondays, Wednesdays and Fridays. The survival of the probiotic strains after 48 h in tap water was confirmed in two cell count/growth tests by plating the synbiotic at 1:10 000 dilution on De Man, Rogosa and Sharpe agar. In the first test the CFU/plate count increased from 128 to 176 and in the second test the CFU/plate count increased from 160 to 240 during the 48-h test period.Bacillus licheniformis, catalogue number 11702, Sigma-Aldrich, Vienna, Austria), meropenem , neomycin  and vancomycin . The ability of this antibiotic combination plus ampicillin to deplete the gut microbiota has previously been demonstrated [A combination of four non-absorbable antibiotics with a spectrum of action that affects a broad range of intestinal bacteria was used to deplete the gut microbiota. The antibiotics used included bacitracin .The interaction between synbiotic and antibiotic treatment in modifying the gut microbiota was investigated with two experimental protocols Figs. , 2. In bIn protocol 1, mice were administered placebo or the synbiotic for 3 weeks prior to the administration of vehicle or the antibiotic combination for 11 days  according to the manufacturer’s instructions with slight modifications. Using the lysis buffer, stool samples were homogenized on a MagNA Lyser Instrument using MagNA Lyser Green Beads . After homogenization, the samples were treated with 2.5 mg/ml lysozyme  for 30 min at 37 °C, followed by digestion with 1 mg/ml proteinase K for 60 min at 56 °C. The enzyme was inactivated by exposure to 95 °C for 10 min, after which 600 µl of lysate was used for DNA isolation in the Maxwell RSC. The DNA concentration was measured by Picogreen fluorescence.Bacterial 16S rRNA was amplified with the Mastermix 16S Complete PCR kit  according to the manufacturer’s instructions using a 0.4 µM final concentration of primers and 57 °C as annealing temperature for 25 cycles. The first PCR reaction product was subjected to a second round of PCR with primers fusing the 16S primer sequence to the A and P adapters necessary for Ion Torrent sequencing, while additionally a molecular barcode sequence was included to allow multiplexing of up to 96 samples simultaneously. PCR products were subjected to agarose gel electrophoresis, and the band of the expected length (350 nt) was excised from the gel and purified using the QiaQuick  gel extraction system. The DNA concentration of the final PCR product was measured by Picogreen fluorescence.Amplicons from up to 60 samples were pooled at equimolar amounts and subjected to emulsion PCR using the Ion Torrent One Touch 2.0 Kit according to the manufacturer’s protocols. After emulsion PCR the beads were purified on the Ion ES station and loaded onto Ion Torrent 318 chips for sequencing. Sequencing reactions were performed on Ion Torrent PGM using the Ion 400BP Sequencing Kit running for 1000 flows . Sequences were split by barcode and transferred to the Torrent Suite server. Unmapped bam files were used as input for bioinformatics.All sequences were initially trimmed by a sliding window quality filter with a width of 20 nt and a cutoff of Q20. Reads shorter than 100 nucleotides and reads mapping to the human genome were removed using DeconSeq . The res® Spotfire® (v 7.0.0) or Prism 8 GraphPad. For microbiome analysis OTUs were visualized as OTU tables, bar charts and PCoA plots using the QIIME core microbiome script, and subsequently color-coded and formatted in Inkscape. Additionally, groupings supplied in the mapping file were tested for statistical significance using the QIIME implementation of the Adonis test. The significance of individual bacterial strains was determined by Kruskal–Wallis test. Linear discriminant analysis (LDA) effect size (LEfSe) analysis [p values < 0.05 were regarded as significant.Results were statistically evaluated either with SPSS 22 , R (R Development Core Team 2011)  using Tibcoanalysis  was perfp < 0.001 by Adonis test). In contrast, there was little separation between vehicle-treated mice receiving placebo or synbiotic , antibiotic treatment followed by continued presence of the synbiotic altered the abundance of particular bacterial orders to a marked extent Fig. . Thus, aIn contrast, the recovery of Clostridiales was boosted in antibiotic/synbiotic-treated animals, as the abundance of Clostridiales at T3 already exceeded that seen in vehicle/synbiotic-treated mice Fig. b. The reLactobacillus and Bifidobacterium strains were components of the synbiotic administered. In fact, the relative abundance of Lactobacillales measured in antibiotic/synbiotic-treated animals fell below that seen in vehicle/synbiotic-treated mice during the post-antibiotic period. In contrast, the beneficial effect of synbiotic pretreatment on the Verrucomicrobiales during antibiotic treatment was maintained under prolonged synbiotic administration, while the relative abundance of Clostridiales was enhanced only with some delay in the post-antibiotic period under synbiotic treatment.Analysis of the effects of antibiotic and synbiotic treatment on the murine fecal microbiome in two complementary experimental protocols revealed unexpected interactions between the two interventions. In particular, the impact of antibiotics on distinct bacterial orders is modified by concomitant synbiotic treatment in a time-dependent manner. Specifically, the effects seen during short-term exposure to antibiotics after synbiotic pretreatment are profoundly different from those seen following prolonged synbiotic administration in the post-antibiotic period. For instance, synbiotic pretreatment preserved the Lactobacillales population and boosted the relative abundance of Bifidobacteriales during antibiotic treatment. This effect was completely lost during continued synbiotic administration, despite the fact that both Diversity in its composition enables the gut microbiota to resist adverse changes in its environment (resistance) and to return to equilibrium following perturbation (resilience) . In the In experimental protocol 1, we examined how the microbial community is affected by gut-directed antibiotic treatment and analyzed the resistance of the microbial community in response to antibiotic and synbiotic challenge. Although pro- and synbiotics are in general thought to ameliorate the adverse effect of antibiotics on the gut microbiota , 29–32, Citrobacter rodentium when gastrointestinal inflammation has become manifest [Placebo- and synbiotic-treated animals did not appreciably differ in bacterial richness and diversity from each other, which is in keeping with the reported resistance of the murine intestinal microbiota to probiotic colonization . In contmanifest .Lactobacillus and Bifidobacterium species. The dynamic alterations in Lactobacillales and Bifidobacteriales abundance during and after antibiotic treatment in the presence of a multi-strain synbiotic as found here is likely to have a bearing on other components of the microbiota, since members of the Lactobacillales and Bifidobacteriales order are known to modulate the activity and vitality of other gut microbes [Lactobacillus are able to inhibit the growth of the human fecal microbiota in vitro by reducing the number of observed species and modulating the community structure [Antibiotic treatment likewise enhanced the relative abundance of Lactobacillales and Bifidobacteriales as noted at the end of the antibiotic treatment period in protocols 1 and 2. Since this antibiotic effect was observed only following synbiotic pretreatment, it is conceivable that it reflects intestinal colonization of Lactobacillales and Bifidobacteriales strains present in the synbiotic. However, this argument is questioned by the observation that the increase in relative abundance of Lactobacillales and Bifidobacteriales was not maintained during the post-antibiotic period despite the continued administration of the synbiotic. We hypothesize that changes in the microbiome architecture during the post-antibiotic period occluded the ecologic niche in which the synbiotic-derived Lactobacillales and Bifidobacteriales found a favorable habitat to colonize during antibiotic treatment. This contention is supported by similar findings reported by Suez et al.  during tmicrobes –56. Spectructure .Akkermansia muciniphila, the sole identified member of this order [Akkermansia abundance seen at the end of the antibiotic treatment period was maintained when synbiotic administration was continued during the post-antibiotic period. As in the case of Lactobacillales and Bifidobacteriales, antibiotic treatment appeared to clear a niche in which the synbiotic could induce Akkermansia to proliferate for a prolonged period of time. This finding may be of physiological significance, given that Akkermansia is a mucin-degrading bacterium that resides in the mucus layer of the gut [Akkermansia muciniphila is thought to be a candidate probiotic with a potential benefit in pathologies such as obesity, diabetes, liver injury and cardiovascular disorders [Akkermansia muciniphila can also be increased by prebiotic supplementation with oligofructose although, in vitro, Akkermansia muciniphila does not grow on oligofructose-enriched media [Another bacterial taxon that specifically increased in relative abundance following combined synbiotic and antibiotic treatment is the order of Verrucomicrobiales, specifically is order , 58. The the gut , strengt the gut , 58. In isorders , 58, 59.ed media . The synAnalysis of the post-antibiotic period revealed that the fecal microbiome did not fully recover in the continued presence of the synbiotic. This outcome needs to be seen in conjunction, on the one hand, with the reported resistance of the murine intestinal microbiota to probiotic colonization  and, on These findings illustrate the dynamics of the intestinal microbial ecosystem that comes to light when its integrity is disturbed by antibiotic treatment. In analyzing the complex interactions that we observed between the synbiotic and antibiotic treatment, a number of limitations need be considered. These interactions will depend on the antibiotic sensitivity of particular bacterial taxa in the native microbiota and in the synbiotic as well as on a multitude of positive and negative interactions between distinct taxa of the gut microbiota and the synbiotic. The interactions may also be influenced by factors of the host organism, which we wanted to avoid to a certain extent by using only non-absorbable antibiotics. In interpreting the observed changes in bacterial taxa, it needs to be taken into account that alterations of the relative abundance of a given taxon will increase or decrease if the relative abundance of other taxa decreases or increases, respectively. Finally, it needs to be borne in mind that the effects of the synbiotic under study may be the net effect of its pre- and probiotic ingredients, whose contributions remain to be analyzed.Lactobacillus can also secrete molecules that inhibit microbial proliferation [In conclusion, our data show that the synbiotic under study per se had an insignificant influence on the microbial community but was able to modulate the impact of antibiotic treatment on the fecal microbiota in a time-dependent fashion because the antibiotic-induced microbial perturbations underwent distinct changes when the administration of the synbiotic was continued during the post-antibiotic period. The pertinent observations disclose dynamic interactions between synbiotic and antibiotic interventions in shaping the intestinal microbiota. These interactions need to be construed in light of the resistance and resilience of the microbial community to challenges of its integrity. In addition, it is important to bear in mind that the baseline composition of the intestinal microbiota is a factor that determines colonization of the gut by bacterial components  of the sferation . These cferation , 61, 62.


Neonatal maternal separation (NMS), as an early-life stress (ELS), is a risk factor to develop emotional disorders. However, the exact mechanisms remain to be defined. In the present study, we investigated the mechanisms involved in developing emotional disorders caused by NMS. First, we confirmed that NMS provoked impulsive behavior, orienting and nonselective attention-deficit, abnormal grooming, and depressive-like behaviors in adolescence. Excitatory amino acid carrier 1 (EAAC1) is an excitatory amino acid transporter expressed specifically by neurons and is the route for the neuronal uptake of glutamate/aspartate/cysteine. Compared with that in the normal control group, EAAC1 expression was remarkably reduced in the ventral hippocampus and cerebral cortex in the NMS group. Additionally, EAAC1 expression was reduced in parvalbumin-positive hippocampal GABAergic neurons in the NMS group. We also found that EAAC1-knockout (EAAC1−/−) mice exhibited impulsive-like, nonselective attention-deficit, and depressive-like behaviors compared with WT mice in adolescence, characteristics similar to those of the NMS behavior phenotype. Taken together, our results revealed that ELS induced a reduction in EAAC1 expression, suggesting that reduced EAAC1 expression is involved in the pathophysiology of attention-deficit and depressive behaviors in adolescence caused by NMS. Elucidating the mechanism of the correlation between early-life manipulations and subsequent disease is difficult to be proven in humans2. In animal models, early developmental manipulations involving maternal care has been identified as an ethologically relevant stressor that induces cognitive and emotional dysfunction throughout life3. Neonatal maternal separation (NMS) enhances responses to aversive and appetitive stimuli more cautiously toward fear signals in the environment5. The postnatal period is accompanied by significant maturation of neuronal systems. Experiencing stressful events results in increased neurochemical, neurobehavioral, and immune-inflammatory abnormalities in subsequent years6.Early-life stressful events have detrimental effects on the brain and are risk factors for behaviors that are associated with the etiology of several psychiatric disorders. Childhood adversities are associated with maladaptive family functioning that has been related to one-third of adult psychopathology7. Dysregulation of glutamatergic neurotransmission is related to stress- and depressive-like behaviors9. Excitatory amino acid carrier 1  is one subtype of the excitatory amino acid transporter (EAAT) family10. Compared with other subtypes, including the glutamate aspartate transporter , glutamate transporter-1 , EAAT4 and EAAT5, EAAC1 plays a more significant role in cysteine transport in the brain11. EAAC1 was first described as a neuronal glutamate transporter10, although it has now been shown to play only a minor role in glutamate removal from the extracellular space, as this task is primarily performed by astrocyte glutamate transporters such as GLT1 and GLAST13. EAAC1 is expressed at presynaptic GABAergic terminals, where the uptake of glutamate could contribute to GABA synthesis16. The loss of brain EAAC1 expression interferes with GABA synthesis and results in epilepsy18. EAAC1 expression is altered under pathological conditions and stimuli, such as epilepsy, hypoxia, multiple sclerosis, bipolar, schizophrenia, H2O2, retinoids, and neuregulin-123. Moreover, genetic studies implicate Slc1a1, a gene encoding EAAC1, in obsessive-compulsive disorder25.Glutamate is the primary excitatory neurotransmitter in the central nervous system and plays a central role in the neurotransmission of ~80% of synapses26, and the transporter is expressed before both EAAT1 and EAAT2 expression in vitro28 and in vivo29. Early expression of EAAC1 suggests a role for EAAC1 in the neuroprotection of CNS cells during brain development.The expression of EAAC1 varies during brain developmentHere, we show that EAAC1 is reduced or lost in NMS and EAAC1 (−/−) rats, respectively, in which nonselective attention-deficit and depressive behaviors were shown in adolescence. These results suggest that EAAC1 exerts a regulatory role in neuromodulation and that the reduction in EAAC1 expression contributes to the pathogenesis of depression.Several previous studies have shown that early-life stress alters emotional behavior in adults and adolescents. However, whether NMS leads to increased or decreased fearful/anxiety-like behaviors in adolescence remains unclear. In this study, we used an NMS model of daily separation from mother and siblings for 3 h/day individually Fig. .Fig. 1ScIn the first step, all the rats were investigated for their developmental milestones from PND1 to PND21  divided into the edge zone, side zone, and inner zone on the captured image Fig. . When ann = 21; NMS, 10.71 ± 2.00, n = 20; t(39) = 2.231, P < 0.05) and wall-leaning  = 4.829, P < 0.001) in the open field. Moreover, compared with CON rats, NMS rats showed decreased stretching numbers  = 2.362, P < 0.05; Fig. n = 15; NMS, 5.21 ± 0.45, n = 15; t(28) = 3.570, P < 0.01; Fig. To verify these impulsive-like behaviors, we tested CON and NMS rats for orienting behavior in adolescence. As shown in Fig. n = 21; NMS, 129.74 ± 10.17, n = 24; t(43) = 4.391, P < 0.001; Fig. n = 21; NMS, 60.54 ± 3.83, n = 24; t(43) = 7.128, P < 0.001; Fig. We next analyzed the immobility and climbing time using the forced swimming test, which measures despair and depressive behaviors. We revealed that NMS induced depressive behaviors in adolescence. We found that NMS rats showed a significant increase in the duration of immobility  = 8.933 in the DG, P < 0.001; CON, 1.00 ± 0.05; NMS, 0.62 ± 0.04; t(21) = 5.798 in the CA1; P < 0.001; CON, 1.00 ± 0.05; NMS, 0.63 ± 0.05; t(18) = 5.158 in the CA3; P < 0.001; Fig. t(20) = 5.200 in the DG, P < 0.001; CON, 1.00 ± 0.03; NMS, 0.63 ± 0.08; t(22) = 4.374 in the CA1, P < 0.001; CON, 1.00 ± 0.04; NMS, 0.61 ± 0.08; t(22) = 4.486 in the CA3, P < 0.001; Fig. We next analyzed the expression of EAAC1 in the ventral hippocampus in adolescence. The ventral region is related to stress, emotion and affectt(18) = 7.222 in the layer III, P < 0.001; CON, 1.00 ± 0.09; NMS, 0.55 ± 0.05; t(20) = 4.641 in the layer V, P < 0.001; CON, 1.00 ± 0.03; NMS, 0.65 ± 0.05; t(20) = 6.256 in the layer VI, P < 0.001; Fig. t(28) = 2.745, P < 0.05; Fig. t(20) = 5.962, P < 0.001; Fig. Similar to the ventral hippocampus, we stained the cerebral cortex sections in adolescence. We found that the EAAC1 protein level was remarkably reduced in the cerebral cortex of NMS rats  = 9.777, P < 0.001; Fig. t(30) = 8.238, P < 0.001; Fig. We checked the immunoreactivity of EAAC1 and PV in NMS rats to investigate the involvement of the GABAergic pathway. Previously, we reported that EAAC1 is expressed in GABAergic neurons in the prefrontal cortex PV Fig. . Additio PV Fig. . However PV Fig. , consistn = 20; EAAC1 (−/−), 71.92 ± 5.83, n = 15; t(33) = 2.94, P < 0.01; Fig. n = 20; EAAC1 (−/−), 483.886 ± 19.06; t(33) = 3.135, n = 15, P < 0.01; Fig. t(15) = 2.543, P < 0.05) and wall-leaning , 32.57 ± 1.05; t(15) = 2.998, P < 0.01) in the open field. EAAC1 (−/−) mice showed an increased duration of self-grooming , 17.53 ± 3.00; t(33) = 2.163, P < 0.05)  mice display deficits in orienting and nonselective attention behavior.We next assessed exploration in EAAC1 (−/−) mice because impulsive-like behavior was shown in NMS rats. Interestingly, EAAC1 (−/−) mice showed impulsive-like exploration, similar to NMS rats  Fig. , corrobon = 10; EAAC1 (−/−), 127.40 ± 18.34, n = 7; t(15) = 2.208, P < 0.05; Fig. n = 14; EAAC1 (−/−), 208.15 ± 16.09, n = 11; t(23) = 6.275, P < 0.001; Fig. EAAC1 (−/−) mice showed an increased time of immobility using the forced swimming test  can cause far-reaching consequences. ELS has been established as a major risk factor for major depression and suicidal behavior along with other psychiatric illnesses in adolescence and adulthood39. Prenatal restraint stress offspring rats showed significantly induced depressive-like behavior and decreased EAAT2 and EAAT3 mRNA expression in the hippocampus40. In contrast, the transcript levels of EAAC1 were not significantly altered in learned helpless rats, an animal model of depression41.In the present study, we found that EAAC1 protein expression was significantly decreased in the hippocampus and cerebral cortex of adolescent NMS rats. However, NMS did not affect the mRNA level of EAAC1 in the hippocampus in adolescence. These results may indicate regulation of the EAAC1 protein degradation pathway. Additionally, we did not observe significantly reduced EAAT1 and EAAT2 expression in the hippocampus of adolescent NMS rats. Previous human postmortem studies reported reduced expression of EAAC1 (EAAT3) and EAAT4 mRNA in the striatum in bipolar disorder42. Male NMS rats spent less time in the periphery (edge and side zone), suggesting that they were more impulsive than CON rats43. Moreover, we found for the first time that NMS rats showed hyperactivity only in the inner zone. Rats normally prefer to stay beside the wall where no predators may attack from the back. Furthermore, we found that EAAC1 (−/−) mice exhibited impulsive-like behavior as NMS rats. The initially reported Slclal/EAAT3-null mice showed decreased activity in the open field44, whereas other investigators have reported no significant changes in EAAC1 (−/−) mice46.Our data revealed that male NMS rats showed increased impulsive behavior in adolescence. During the open field task, male NMS rats spent a significantly longer time exploring a longer distance in the inner zone Fig. . These f38. Both rearing and leaning  to novelty have been associated with the distribution of hippocampal mossy fibers in rats48. Additionally, rearing activity was differentially affected by hippocampal cholinergic and dynorphinergic systems, inhibition of nitric oxide synthesis, and blockade of NMDA glutamate receptors51. The NMS rats showed significantly less rearing and wall-leaning episodes than CON rats, indicating that they may have a deficit in nonselective attention and orienting. Here, we report that EAAC1 (−/−) mice also showed decreased rearing and wall-leaning activity.Rearing consists of stopping ambulation and standing on the hindlimbs and is used as a measure of orienting behavior because orienting or nonselective attention is associated with the duration of rearing episodes, where longer rearing indicates more orienting behavior53; thus, a high frequency of these postures indicates a higher level of anxiety. We observed that NMS rats showed decreased stretching numbers in adolescence. These findings correlate with an increase in spending time in the inner zone; NMS rats showed a decrease in spending time in the edge zone.Moreover, stretch attend postures occur a risk-assessment behavior indicating that the rodent is hesitant to move from its present location to a new position56. Our data revealed increased self-grooming behavior in NMS rats and EAAC1 (−/−) mice in adolescence. Interestingly, several independent genetic studies consistently implicate the Slc1al gene, which encodes EAAC1, a candidate gene for obsessive-compulsive disorder in both males and females59. The sequential patterned strokes performed during grooming are reminiscent of the excessive and repetitive hand-washing behaviors of patients with OCD60. Regarding the different grooming behavior phenotypes, Slc1al/EAAC1-null mice demonstrated no significant changes in 8- to 16-wk-old mice45, and others have reported increased grooming activity in EAAC1 knock-out mice (P14-P35)33. Additionally, other reports have published evidence for a role of EAAC1 in schizophrenia. Increased Slc1al/EAAC1 transcripts and proteins were reported in schizophrenic subjects21. Interestingly, we previously found that NRG1/ErbB4 (susceptibility genes of schizophrenia) signaling is critical for EAAC1 expression and function16.Acute stressors, including exposure to a novel environment, can potently modulate self-grooming, often increasing the frequency or total duration of episodes61. Some investigations have demonstrated that applying pharmacological treatments during adolescence has beneficial effects on the mitigation of depressive-like behaviors induced by early-life stress62. However, no previous studies have investigated EAAC1 in early-life stress circuits or concerning depressive-like behaviors. Here, we reported that EAAC1 (−/−) mice significantly show depressive-like behavior in adolescence. Moreover, we found that EAAC1 expression was reduced in PV-positive hippocampal GABAergic neurons in the NMS group. Depression and stress decrease both glutamate and GABA neurotransmitter circuits in the limbic and cortical regions64. The loss of EAAC1 expression may cause an abnormality in glutamate conversion to GABA in presynaptic GABAergic terminals. Further research is needed to clarify the underlying mechanism of EAAC1 in these phenotypes. EAAC1 can transport not only extracellular glutamate but also cysteine into the neurons.Our data showed that NMS rats provoke depressive-like behavior in adolescence. Notably, EAAC1 (−/−) mice also showed depressive-like behavior in adolescence. Adolescence correlates with developmental changes in the brain circuits in the limbic areas, such as the hippocampusOur data clarified that early-life stress, such as NMS, can provoke impulsive-like behavior, orienting and nonselective attention deficit, abnormal grooming behavior, and depressive-like behaviors in adolescence. Furthermore, we identified a novel function of EAAC1: EAAC1 (−/−) mice show similar abnormal behaviors to NMS in adolescence, which may be associated with decreased EAAC1 expression. These results suggest that early-life stress can cause changes in EAAC1 expression, which may participate in the pathogenesis of nonselective attention deficit and depression in adolescence. These results may provide new insights into EAAC1 signaling in the pathophysiology of depression and shed light on the development strategies of new antidepressants.Antibodies were supplied by Santa Cruz Biotechnology Inc.  , Cell Signaling Technology  , Abcam  , ab11427), Synaptic System  , Sigma  , and Millipore Corporation  (EAAT3(EAAC1), MAB1587).42. During separation sessions, the pups were removed from the mother for periods of 3 h per day. From PND1, the pups were weighed daily, and their overall health was assessed . Daily, each pup was assessed for the achievement of species-specific developmental milestones as described in Additional file 2: Table S266. EAAC1 (−/−) mice were also housed in a regulated environment. EAAC1 (−/−) mice were descendants of the strain established by Peghinni et al.44, in which exon 1 was disrupted by a neomycin resistance cassette. EAAC1 (−/−) mice were outbred to wild-type (WT) CD1 mice for more than 10 generations prior to these studies. A WT colony was maintained using the WT offspring from the latter outcrosses. We used only males for this experiment.Experiments with animals were performed following the institutional and Eulji University guidelines. Pregnant Sprague-Dawley (SD) rats were obtained at 15–17 days of gestation from a laboratory animal supplier (Samtako bio Korea) and were housed individually in cages under standard laboratory conditions with a 12-h light/12-h dark cycle. On postnatal day (PND) 1, the litters were culled to 6–10 pups at a constant sex ratio of 1:1 to avoid sex-based maternal behavioral biasest-test was used for comparisons of the means between two groups of cells in a single experiment. P < 0.05 was considered statistically significant.The data are presented as the means ± SEM of three or more independent experiments. Student’s paired The Supplemental Materials and Methods are provided in additional file 1: Table Additional files figure legendsAdditional file 1 : Table S1Additional file 2. Table S2Additional file 3 : Figure S1Additional file 4 : Figure S2Additional file 5 : Video 1


Compared to postcontrast CTHU, serum IgG4 levels had a larger area under the ROC curve : 0.674–1.000, p = 0.005] vs. 0.766 ), higher sensitivity (0.889 [95% CI: 0.518–0.997] vs. 0.75 [95% CI: 0.509–0.913]), higher specificity (0.813 [95% CI: 0.544–0.960] vs. 0.778 [95% CI: 0.578–0.914]) and a higher cutoff value (≥132.5 mg/dL [milligrams per deciliter] vs. ≥89.5). Conclusions: IgG4-ROD showed distinct CT features and elevated serum IgG4 (≥132.5 mg/dL), which could help distinguish IgG4-ROD from OL.Background: Benign immunoglobulin G4 (IgG4)-related orbital disease (IgG4-ROD)—characterized as tumors mimicking malignant orbital lymphoma (OL)—responds well to steroids, instead of chemotherapy, radiotherapy and/or surgery of OL. The objective of this study was to report the differences in computed tomography (CT) features and- serum IgG4 levels of IgG4-ROD and OL. Methods: This study retrieved records for patients with OL and IgG4-ROD from a pathology database during an eight-year-and-five-month period. We assessed the differences between 16 OL patients with 27 lesions and nine IgG4-ROD patients with 20 lesions according to prebiopsy CT features of lesions and prebiopsy serum IgG4 levels and immunoglobulin G (IgG) levels This study also established the receiver-operating curves (ROC) of precontrast and postcontrast CT Hounsfield unit scales (CTHU), serum IgG4 levels, serum IgG levels and their ratios. Results: Significantly related to IgG4-ROD (all  Orbital space-occupying lesions comprise a wide range of benign and malignant masses . The topImmunoglobulin G4–related disease—which can involve any organs including orbital structures—is a systemic fibroinflammatory condition due to tissue infiltration by immunoglobulin G4 (IgG4) plasma cells . IgG4-reIgG4-related disease diagnostic criteria commonly follow: a serum IgG4 concentration higher than 135 mg/dL (milligrams per deciliter) , the ratFurthermore, orbital neoplasm rupture via biopsy may lead to tumor seeding and poor prognosis . IntegraThe Institutional Review Board of Taipei Veterans General Hospital (TVGH) approved this study to waive informed consent because of the retrospective nature of the research.A doctor (SYY) blinded from the research hypothesis searched pathologic results from the pathology database at TVGH using the keywords “orbit” or “orbital” for cases from 1 January 2010, to 31 May 2018.The research returned 178 patients with orbital lesions and pathologic results. Thirteen (7%) of 178 patients had multiple orbital lesions (≥2): one (7.7%) of the thirteen patients with lung carcinoid tumors and multiple metastases in the right orbital cavity and 12 (92.3%) patients with lymphoplasmacyte-rich lesions . The other 165 (93%) of the 178 patients showed only one lesion in the orbital cavity or eyelid. Sixteen (9%) of the 178 patients had orbital lymphoma (OL) and 9 (5%) patients had IgG4-related orbital disease (IgG4-ROD).Among these patients, this study only considered patients who had OL or IgG4-ROD with prebiopsy precontrast and postcontrast orbital CT scans, serum IgG4 levels and serum IgG levels and excluded those were younger than 20 years of age or pregnant cases or those lacked prebiopsy CT and serologic data.As a result, 25 patients pathologically diagnosed as OL (16 patients) or IgG4-ROD (9 patients) met the inclusion criteria and had prebiopsy orbital CT scans, serum IgG4 levels and serum IgG levels. All patients met the eligibility criteria. We enrolled 25 patients to collect and analyze demographic data, symptoms and signs, past medical histories, CT qualitative and quantitative (Hounsfield unit density) features of orbital lesions, serum IgG levels and serum IgG4 levels of patients with IgG4-ROD from those of patients with OL.n = 5), Philips Healthcare, Cleveland, OH, USA, Somatom Sensation 16 , Siemens Healthcare, Forchheim, Germany, ECLOS Hitachi Medical Corporation , Tokyo, Japan, and Aquilion 64, Toshiba Medical Systems , Tochigi, Japan. Orbital CT scans were obtained with or without an intravenous contrast medium, which included iobitridol  and iohexol . The data records showed that twenty-five patients underwent an intravenous power injection as a bolus of 1.2-mL/kg (kilogram) iodine-based contrast medium at 1 mL/second (s). Postcontrast CT images were performed after the complete injection of contrast medium. The axial sections of precontrast and postcontrast orbital CT images scanned along the transaxial direction with the sections parallel to the optic nerve along a line from the inferior border of the maxillary sinus to the middle part of the frontal region. A Hitachi CT scanner took the slice thickness for image viewing of axial images at 1.25 mm (mm) and other MDCT scanners at 2–4 mm. The coronal and sagittal sections of postcontrast orbital CT images were reformatted with 2–4 mm in slice thickness. The reconstruction matrix for MDCT scans of orbit was 512 × 512.This study examined orbital CT images taken by a multiple-detector computed tomography (MDCT) scanner for the selected 25 patients. MDCT scanners of orbit or face included iCT 256  reanalyzed orbital lesions of the 25 patients on orbital CT images with axial, sagittal and/or coronal images together without knowledge of pathologic diagnosis of orbital tumors. The consensus from the two radiologists served as the final interpretation of images. If the two radiologists could not reach an agreement on any features from orbital CT scans, a third experienced radiologist (HCH) mediated the disagreement.This study analyzed the following orbital CT features of each lesion or of each patient: maximum diameter of a lesion, lesion borders, homogeneity of CT density, a lesion involving extraocular muscle tendons, the lacrimal sac, lacrimal gland, preseptal space, extraconal, conal or intraconal orbital compartments, the optic nerve, infraorbital nerve, presence or absence of bone remodeling, single tumor or multiple lesions and single or bilateral orbital involvement, single or bilateral lacrimal gland involvement. This study also measured the mean values of the precontrast and postcontrast CT Hounsfield unit scales (CTHU) of each orbital lesion among the 25 patients.A regular border of an orbital tumor on CT scans indicated the contour of a lesion from the surrounding tissue was smooth for more than 75% of the lesion. An irregular border of a lesion showed microlobulated, microangulated or indistinct contour from the surrounding tissue with ≥25% of the lesion. Lesion involvement indicated lesion infiltration, invasion or encasement on orbital CT scans.This study measured CTHU for all 47 orbital lesions of 25 patients on both pre and postcontrast prebiopsy CT scans. The region of interest (ROI) maker in an oval shape was placed in the center of each lesion to cover 50% of the largest tumor area on CT axial images, avoiding the inclusion of bone and blood vessels . This stAn experienced pathologist (AFYL) with 29 years of experience in pathology diagnosis reviewed the pathologic and immunohistochemical sections of the specimens of the 25 patients to confirm pathologic results of OL and IgG4-ROD. The two main pathologic criteria of IgG4-ROD included (1) IgG4+/IgG+ ratio > 40%, and/or (2) IgG4+ cells > 10/high-powered field (HPF) in histopathologic examination .The radiologist (WHY) integrated demographic data, patient symptoms, signs and past histories, prebiopsy serum IgG4 levels and IgG levels and CT imaging interpretations and the mean values of CTHU measurement results of the 25 patients to evaluate the differences in CT qualitative and quantitative features, serum IgG and IgG4 levels between OL and IgG4-ROD.p < 0.05. Receiver operating characteristic (ROC) curve analysis calculated the area under the ROC curve to identify diagnostic values of CTHU, serum IgG4 levels and serum IgG levels of IgG4-ROD. This study assessed the findings based on sensitivity, specificity and accuracy with a 95% confidence interval (95% CI).This study used SPSS version 19.0 software  for data analysis. Specially, we applied the Mann–Whitney U test to compare continuous variables because of the small sample size and the χ2 or Fisher’s exact test for categorical variables at the level of significance of p = 0.934, Mann–Whitney U test). Of the 25 patients, 17 (68%) were male and 8 (32%) were female. Twelve (12 or 71%) of the 17 male patients were OL and 5 (29%) were IgG4-ROD; four (50%) of 8 females were OL patients and 4 (50%) were IgG4-ROD .The median age  of the selected 25 patients was 59 . The median age  of 16 patients with OL was 60.5  and that of 9 patients with IgG4-ROD was 58  .The 25 patients showed proptosis, palpable mass and/or eyelid swelling—none of the 25 patients suffered from orbital pain or tender palpable mass. Six (6 or 24%) of the 25 patients had malignancy histories. Five (83%) of the 6 patients with malignant histories had OL: one with renal cell carcinoma, one with prostatic cancer and soft palate follicular lymphoma, one with squamous cell carcinoma of the tongue, one with follicular lymphoma involving lung, neck lymph nodes and bone marrow and one with chronic lymphocytic leukemia. Only one (17%) of the 6 patients with malignant history was an IgG4-ROD patient who had ovarian cancer. Patient malignant histories of the two groups had no significant difference (A pathologist (AFYL) reviewed the pathologic sections of the 25 patients. The pathologic review concluded 13 patients with extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), 1 with low-grade B cell lymphoma with plasmacytic differentiation, 1 with diffuse large B cell lymphoma, 1 with follicular lymphoma and 9 with IgG4-ROD. The histopathologic findings of the 9 patients with IgG4-ROD showed diffuse lymphoplasmacytic infiltration, IgG4-positive (IgG4+) plasma cells, IgG-positive (IgG+) plasma cells and various degree fibrosis. Seven (78%) of the nine IgG4-ROD patients showed IgG4+ cells > 100 cells/HPF and IgG4+/IgG+ ratio > 40% . AnotherFurthermore, CT images indicated a total of 47 orbital tumors among the 25 patients: 27 lesions were OL and 20 lesions were IgG4-ROD. Of 47 orbital tumors, none appeared inside the eyeball.p = 0.0069), multiple tumors (p = 0.0414), lacrimal gland involvement (p = 0.0085), lesions involving bilateral lacrimal glands and bilateral orbital cavities  had a solitary tumor in an orbital cavity or at eyelids, 1 (6%) had 2 tumors, 2 (13%) had 3 and 2 (13%) had 4. The other 9 out of the 25 patients had IgG4-ROD: 2 (22%) with 1 tumor; 5 (56%) with 2 tumors, 1 (11%) with 3 tumors and 1 (11%) with 5 tumors. CT features statistically significantly associated with IgG4-ROD included lesions with regular borders . Two IgG4-ROD patients and 6 OL patients had a solitary tumor involving the preseptal space .p = 0.484), 0.766  and 0.670 . According to p = 0.005), 0.684  and 0.819 , respectively.Compared with above data, the AUC using postcontrast CTHU (= 0.766) and serum IgG4 levels (= 0.847) was moderately accurate for the diagnostic yield of IgG4-ROD because both AUC measures fell between 0.7 and 0.9. The largest Jordon index 0.528 [(sensitivity−[1−specificity] = 0.528)] suggested a cutoff value of postcontrast CTHU equal to 89.5. The sensitivity and specificity were 0.75 (95% CI: 0.509–0.913) and 0.778 (95% CI: 0.578–0.914), respectively. As to serum IgG4 level, the largest Jordon index (0.701) suggested a cutoff value equal to 132.5 mg/dL, which resulted in sensitivity of 0.889 (95% CI: 0.518–0.997) and specificity of 0.813 (95% CI: 0.544–0.960), respectively.p = 0.0414, Fisher’s exact test). For serum IgG4 levels ≥ 132.5 mg/dL, 8 (73%) were IgG4-ROD patients and 3 (27%) OL (p = 0.0021).For patients with postcontrast CTHU ≥ 89.5 in at least one orbital nodule in the two groups, 7 (58%) were IgG4-ROD patients and 5 (42%) were OL patients  in 3 OL patients and 1 IgG4-ROD case; lower postcontrast CTHU (<89.5) in 8 OL and 1 IgG4-ROD; and lower serum IgG4 levels (<132.5) in all 11 OL patients and 1 IgG4-ROD case with higher CTHU. In addition, one IgG4-ROD patient with a lower postcontrast CTHU showed a higher serum IgG4 level ≥ 132.5.The postcontrast CTHU and serum IgG4 level for patients with multiple orbital lesions in the two groups were as follows: lower postcontrast CTHU (<89.5) were noted in 3 OL patients and in 1 IgG4-ROD case; higher postcontrast CTHU (≥89.5) were found in 2 OL patients and in 6 IgG4-ROD cases; higher serum IgG4 levels (≥132.5 mg/dL) were found in 3 OL patients and in 7 IgG4-ROD cases; lower serum IgG4 levels (<132.5 mg/dL) appeared in 2 OL patients. Two of the three OL patients with lower postcontrast CTHU showed OL involving bilateral lacrimal glands, who had different serum IgG4 levels: 51.3 and 339.5, respectively. Concurrent higher postcontrast CTHU and a higher serum IgG4 level were found in 1 OL patient  with multiple tumors in the left orbital cavity and in 6 IgG4-ROD cases with tumors involving bilateral lacrimal glands. Lower postcontrast CTHU and higher serum IgG4 level were noted in only one IgG4-ROD  case, who showed tumors mainly in intraconal spaces of bilateral orbits.p = 0.0022), “bilateral lacrimal gland involvement and a higher serum IgG4 level (≥132.5 mg/dL) (p = 0.0005)” or “bilateral lacrimal gland involvement and higher postcontrast CTHU  (p = 0.00047)” or “higher postcontrast CTHU and a higher serum IgG4 level” (p = 0.0029) as helpful test tools for diagnosis of IgG4-ROD . Postcontrast CTHU ≥ 89.5 showed 0.75 sensitivity and 0.778 specificity with the AUC = 0.766 ; serum IgG4 levels ≥ 132.5 mg/dL had 0.889 sensitivity and 0.813 specificity, with the AUC = 0.847 . Lesions with regular borders, multiple orbital tumors, lacrimal gland involvement, lesions simultaneously involving bilateral lacrimal glands and bilateral orbital cavities and higher postcontrast CTHU (≥89.5) on orbital CT scans were significantly associated with IgG4-ROD . The difference in extraconal, conal and intraconal compartments of orbital lesions between OL and IgG4-ROD groups was not statistically significant (p = 0.4813). In addition to CT, magnetic resonance imaging (MRI) also helps in further diagnostic workup of orbital tumors and provides ocular anatomy for lesions involvement, perineural spread and intracranial extension [Patient’s age, standard imaging features and localizing orbital lesions to intraconal, conal or extraconal compartments help limit the differential diagnosis . Our stuxtension . Both rextension ,18. Ninextension ,18. For xtension ,18. In cxtension . The mosxtension . Cavernoxtension . Maligna−3 mm2/ sec and an ADC ratio < 1.2 are optimal for predicting orbital malignant tumors [−3 mm2/ sec and ADC ratio < 1.2 to differentiate orbital lymphoma from benign OP and IgG4-ROD with more than 95% accuracy [Multiple or multicompartmental orbital masses include venolymphatic malformations (VLM), rhabdomyosarcoma (RMS), plexiform neurofibroma, thyroid ophthalmopathy (TO), orbital pseudotumor (OP), lymphoma, metastases and IgG4-ROD . The firt tumors . Prior saccuracy . Howeveraccuracy . OP maniaccuracy ,18. Noneaccuracy , which cAn effective clinical diagnosis or appropriate disease classification for IgG4-related disease needs the integration of clinical findings, radiological features and serologic or pathologic data . None ofThis study had only two IgG4-ROD patients with a solitary tumor, which was a small sample and lacked specific CT features. Prebiopsy diagnosis of a solitary IgG4-ROD could depend on a painless orbital mass, postcontrast CTHU ≥ 89.5 and serum IgG4 level ≥ 132.5 mg/dL. Tissue proof is an ultimate diagnostic way. However, biopsy is not always suitable for orbital lesions. The best medical option for a benign mixed tumor or malignant mass of the lacrimal gland may be excision en bloc without biopsy once clinical and imaging diagnosis. However, incomplete excision or ruptures of neoplasms via biopsy may result in tumor recurrence, malignant transformation of a mixed tumor and poor prognosis . In clinp = 0.0022). Neither Sato et al. nor this study had patients with IgG4-ROD originating from conjunctival or subconjunctival tissue. However, prior research suggested that IgG4-ROD can also develop at conjunctival tissue [Sato et al.  reportedl tissue ,24.This study has several limitations. First, this retrospective research had a small sample of patients. Second, the patients in this study received different brands of contrast agents and CT machine, which could produce potential bias in measurement of CTHU. Last, incomplete data records ruled out the possibility of deciphering a detailed correlation between CT features and clinical presentations of OL and IgG4 ROD.p < 0.05). For diagnosis of IgG4-ROD, postcontrast CTHU ≥ 89.5 and serum IgG4 level ≥ 132.5 mg/dL provided moderate diagnostic accuracy, AUC = 0.776 and 0.847, respectively, which were higher than those of postcontrast HU/precontrast HU and serum IgG4/IgG level ratio. The special CT features and elevated serum IgG4 levels could help differentiate IgG4-ROD from OL. Prebiopsy diagnosis of the uncommon solitary type of IgG4-ROD could also depend on a painless orbital mass and elevated CTHU ≥ 89.5 and serum IgG4 level ≥ 132.5 mg/dL.This study compared prebiopsy precontrast and postcontrast CT features, serum IgG4 and serum IgG levels of IgG4-ROD with those of OL. The key findings showed that IgG4-ROD had high correlation with the presence of lesions with regular borders, presence of multiple lesions, lesions involving the lacrimal gland, both lacrimal glands and bilateral orbital cavities on CT scans, higher values of postcontrast CTHU, postcontrast CTHU/precontrast CTHU ratios, serum IgG4 levels and serum IgG4/IgG level ratios (all


Reinforced concrete is the most widely used building material in history. However, alternative natural and synthetic materials are being investigated for reinforcing concrete structures, given the limited availability of steel in developing countries, the rising costs of steel as the main reinforcement material, the amount of energy required by the production of steel, and the sensitivity of steel to corrosion. This paper reports on a unique use of bamboo as a sustainable alternative to synthetic fibers for production of bamboo fiber-reinforced polymer composite as reinforcement for structural-concrete beams. The aim of this study is to evaluate the feasibility of using this novel bamboo composite reinforcement system for reinforced structural-concrete beams. The bond strength with concrete matrix, as well as durability properties, including the water absorption and alkali resistance of the bamboo composite reinforcement, are also investigated in this study. The results of this study indicate that bamboo composite reinforced concrete beams show comparable ultimate loads with regards to fiber reinforced polymer (FRP) reinforced concrete beams according to the ACI standard. Furthermore, the results demonstrate the potential of the newly developed bamboo composite material for use as a new type of element for non-deflection-critical applications of reinforced structural-concrete members. The design guidelines that are stated in ACI 440.1R-15 for fiber reinforced polymer (FRP) reinforcement bars are also compared with the experimental results that were obtained in this study. The American Concrete Institute (ACI) design guidelines are suitable for non-deflection-critical design and construction of bamboo-composite reinforced-concrete members. This study demonstrates that there is significant potential for practical implementation of the bamboo-composite reinforcement described in this paper. The results of this study can be utilized for construction of low-cost and low-rise housing units where the need for ductility is low and where secondary-element failure provides adequate warning of collapse. Reinforced concrete is described as the “most fruitful and generous of all building materials” by Pier Luigi Nervi, an engineer and contractor and one of the greatest 20th-century structural designers in reinforced concrete ,2. Over The most common type of reinforcing bar is steel reinforcement. Steel reinforcement has been used in a variety of structural concrete applications over the past decades. However, the susceptibility to steel corrosion is the major drawback. Corrosion is often initiated by either concrete carbonation or exposure of the concrete element to chloride ions, as discussed in many studies ,4,5. TheVarious methods have been investigated by researchers worldwide to overcome the corrosion of the steel reinforcement in concrete. Improving the quality of concrete, employing protective coating on reinforcement bars, and the application of non-ferrous and non-metallic reinforcement materials are some of the many ways to reduce the problems that are associated with corrosion of steel reinforcement bars in concrete ,11,12,13Steel reinforcement is not only costly; problems that are associated with corrosion and the additional investment required to minimize corrosion have led to many challenges, particularly in developing countries. For instance, very few developing countries have the required resources of steel for development of their infrastructure. According to the report that was published by World Steel Association in 2015, out of 54 African nations, only two produce steel in noticeable quantities . TherefoSteel reinforcement is not without alternatives. There is an alternative renewable natural resource that grows in the tropical zone of earth that coincides with the location of developing countries—bamboo. Bamboo belongs to the botanical family of grasses and it has relatively high tensile capacity, thus making it a potential reinforcement alternative for steel ,18. SomeThe idea of employing bamboo for concrete applications is not new. In 1914, Hou-kun Chow, at MIT tested for the very first time small diameter raw bamboo as reinforcement materials for concrete members . Later oBetween 1995 and 2005 more recent research in the field of bamboo as reinforcement for concrete, as carried out in Brazil ,23,24. SUnfortunately, the long-term behavior of bamboo in concrete structures has since remained a challenge for many researchers. When natural bamboo is exposed to the concrete matrix, through time, it absorbs water from the concrete. This will result in the swelling of bamboo elements. Repetitive swelling and shrinkage of the natural bamboo has often led to sudden de-bonding of the bamboo element from the concrete matrix. This caused a near-complete loss in the structural load-bearing capacity of the reinforced concrete member.The results from studies on the application of natural bamboo from 1914 till today have indicated that raw bamboo has potential for replacing steel in reinforced concrete beams ,28,29,30In an earlier study by the authors, a new technology was developed to process bamboo culms into fibers that are suitable for application in the form a novel bamboo composite material . AdvantaThe main objective of this paper is to report on an investigation into the possibility of using this newly developed bamboo composite material for reinforcement in structural-concrete beams. The development of the bamboo composite reinforcement system and the durability properties, such as water absorption, swelling, shrinking, and chemical resistance are evaluated. Additionally, challenges that are related to thermal expansion and the bonding mechanism are studied through an extensive series of tests. Finally, comparisons of the design parameters with reference to the design guidelines stated in ACI (American Concrete Institute) 440.1R-15 for FRP (Fiber Reinforced Polymer) reinforcement in concrete  help to Dendrocalamus asper, known as Petung Putih bamboo, was selected from a bamboo forest on the Java island of Indonesia. Dendrocalamus asper is widely available in Java and is being used for small housing projects. The bamboo samples used in this study had an average culm length of 20 m. The outer diameter of the selected culms was between 70 mm and 160 mm. In an earlier study that was carried out by the authors, the mechanical properties of Dendrocalamus asper bamboo from Indonesia with respect to culm physical properties, including culm diameter, wall thickness, height, moisture content, and specific density, were investigated and correlations between mechanical properties, including tensile strength, modulus of rupture and modulus of elasticity in flexure and tension, and culm physical properties have been studied . For theThere are various types of matrix that can be used for composite fabrication. The role of the matrix is to hold the dispersed fibers together and transfer the load to the fibers. The matrix phase of any fiber reinforced polymer composite can be classified as either thermoset or thermoplastic. Among the various thermosetting matrices, the epoxy system is considered to be one of the high-performance systems used mostly for advanced composites with high qualities. A commercially available two-part epoxy resin system was chosen based on three main criteria;-Low Volatile Organic Compounds (VOCs) content;-acceptable mechanical properties;-sustainable sources.® epoxy systems are produced by Entropy Resins Inc. in the US and according to the manufacturer, these contain up to 20% bio-based and renewable contents sourced from waste materials, such as wood pulp and bio-fuels production.SUPER SAPThe bamboo composite samples were produced following a patented processing technology that was developed by the authors . PatenteBamboo-composite reinforcement systems for reinforcing concrete beams of up to 1300 mm in length were fabricated following the details that are explained in The longitudinal bamboo composite reinforcement elements were produced by using a steel mold that was employed within the hot press machine that had a width of 150 mm and length of at least 1200 mm. As mentioned earlier in this section, the length of the concrete beams was 1300 mm and, therefore, the additional 100 mm was designed as the cover of the reinforcements on the both ends of the reinforced concrete beams. The impregnated bamboo fiber layers were placed into the rectangular mold of hot-press and pressed at a temperature of 100 °C and a pressure of 20 MPa for 30/30 min. of curing/post-curing duration. The final composite board was cured for an additional 48 h in a curing oven at a temperature of 50 °C after removing from the hot press to ensure that the full cross-linking of the epoxy resin could take place. The final board had a length of 1,200 mm, a width of 150 mm, and a thickness of 10 mm. The final bamboo composite boards were then cut into reinforcement of size 10 × 10 × 1200 mm for reinforcing the concrete beams. The square cross section was the result of the production process of the bamboo composite materials and the rectangular steel mold of the hot press. The stirrups were placed perpendicular to the long axis of the beam to resist the shear forces and provide sufficient anchorage to the longitudinal reinforcements. Therefore, the load transfer within the bamboo fibers in the bamboo composite samples for stirrup production should be directed in a continuous loop, unlike in longitudinal reinforcement, in which only a unidirectional load transfer in one axis was required. To do so, a U-shape mold was fabricated from heat-treated steel for the production of the bamboo composite stirrups in this study. The cross-section of the mold was designed according to the preliminary reinforced concrete beam design before the casting and production of the concrete samples by considering the shear design concept of reinforced concrete member. Furthermore, the wide-angle corners of the cross sections of the mold gave the corners and sides of the stirrups sufficient pressure from the hot-press compression molding machine during production. This design ensured sufficient epoxy penetration into the bamboo fiber layers at the corners and along the sides of the composite samples. The stirrups were prepared by aligning the impregnated stacks of bamboo fibers along the short axis of the U-shape mold covering the two sides and bottom section of the mold. The fiber direction of the veneer layers was maintained along the sides of the steel mold, as shown in Maintaining the fiber direction along the shorter axis of the steel mold had the advantage of achieving the highest axial strength  of the stirrups along the fiber direction by utilizing the unidirectional properties, which would give the bamboo composite stirrups their maximum mechanical properties. The final bamboo composite board of stirrup was then cut along the fiber direction into pieces with cross sections of 15 mm in width and 6mm in thickness, as shown in A closed loop shape would provide sufficient development length along both vertical  sides of the stirrups, according to the American Concrete Institute ACI 318 “Building Code Requirements for Structural Concrete and Commentary” . TherefoBefore preparing the reinforcement bars from the final bamboo composite boards, the tensile and flexural properties, including strength and modulus of elasticity, were measured according to ASTM D3039-08, “Standard Test Method for Tensile Properties of Polymer Matrix Composite Materials” and ASTM D7264, “Standard Test Method for Flexural Properties of Polymer Matrix Composite Materials”, respectively. The compressive strength of the boards was obtained according to ASTM D6641, “Standard Test Method for Compressive Properties of Polymer Matrix Composite Materials”, along the fiber direction. For each series of tests, five samples were prepared from each composite board. A total of 10 boards were prepared for the longitudinal reinforcement and at least 50 samples were tested for each property. The samples were taken from various locations along the boards to obtain average values that represented the composite properties along the full length and width of the boards.There are various factors that are involved in the design and proportioning of concrete beams reinforced with the newly developed bamboo composite reinforcement. The bond between bamboo composite reinforcement and the concrete matrix and the effect of water penetration as well as alkaline environment of concrete on the mechanical properties of bamboo composite reinforcement are investigated in this study.Acceptable bond between bamboo composite reinforcement and the concrete matrix ensures a smooth stress transfer between the two materials and, therefore, contributes to the overall load bearing capacity of the reinforced concrete element. In the event of weak bonding between reinforcement and concrete, the stress transfer between reinforcement and concrete matrix will be disrupted and, thus, failure of the member will follow, especially in the tension zone of the concrete element. Therefore, ensuring perfect and smooth bond between concrete and bamboo composite reinforcement would contribute to higher ultimate load-bearing capacity of the reinforced concrete member by activating the maximum mechanical capacities of the bamboo reinforcement through providing an interfacial microstructure that would ensure continuous tensile stress transfer between the two materials.A detailed study was previously carried out by the authors where a series of pull-out tests were designed to find the most suitable method to enhance the bonding between the two materials to understand the bonding mechanism between bamboo composite reinforcement and concrete, . In the Therefore, a two-component, water-based epoxy system was chosen in combination with sand particles. The waterproof system would block the moisture and water from the interface of the concrete and bamboo composite reinforcement, thus the stress transfer between concrete and reinforcement would be enhanced. Furthermore, the addition of sand particles could improve the surface roughness of reinforcement, which results in an improved bonding strength with the concrete matrix through creating physical and mechanical interlocking systems with the aggregates of the existing concrete matrix .3 concrete.Normal strength concrete with an average compressive strength of 20 MPa was used to prepare the concrete samples for the beam and pull-out tests. Half dog-bone shaped bars with a cross section size of 10 mm × 10 mm were prepared from the bamboo composite boards that were produced in this study. The dog-bone shape for the grip of the UTM machine helped to eliminate the risk of slippage during the tests. An embedment length of 200 mm was used in this study, which showed the highest bending strength in the earlier work that was carried out by the authors . Cylindea is the embedment length in mm, τ is the bond strength in MPa, and a and b are the cross-sectional dimensions of the reinforcement in mm.Average tensile strength and elastic modulus in compression of the concrete samples were 6.8 MPa and 26,100 Mpa, respectively, after 28 days of curing. For the purpose of the study’s pull out tests have been carried out by preparing a custom-made insert for the Shimadzu UTM machine of type AG-IC 100 kN following the works that were described by . The setEquilibrium in From Equation (1), the bond strength, τ, is determined while using Equation (2):A total of 20 pull-out specimens were tested in this study.The alkaline environment of concrete could potentially affect the properties of bamboo composite reinforcement when used in concrete. There are several factors that might have negative impacts on the physical and mechanical properties of bamboo composite reinforcement in concrete. Water penetration occurs over time through the concrete matrix when it is exposed to humid air and rain. In this study, the effect of water penetration and alkaline environment of concrete on the mechanical properties of bamboo composite reinforcement is investigated through a series of laboratory tests.The samples of 10 × 10 × 10mm in size were prepared from bamboo composite boards for water absorption tests. The samples were immersed in distilled water with a temperature of 23 °C and 60 °C. The higher temperature was chosen to evaluate the effect of water uptake at higher temperature on the properties of bamboo composite reinforcement in comparison with room temperature water absorption. The water absorption tests were carried out in accordance with ASTM D5229M-14 “Standard Test Method for Moisture Absorption Properties and Equilibrium Conditioning of Polymer Matrix Composite Materials”. The weight changes were recorded every 2 h during the first week of the experiment when the water uptake rate was relatively high; subsequently, measurements were only carried out once per day for up to three months’ immersion in water (2160 h) when the rate of water absorption was reduced. 10 samples were prepared for each testing condition. At various stages of soaking, the percentage of the weight gain was calculated by measuring the difference in weight between the dry condition and the weight after water immersion2, 0.90 g of NaOH, and 4.2 g of KOH in one liter of tap water with an initial pH value of 12.6 to 13.0, similar to the pH value of the pore water inside the concrete matrix. The samples were immersed in the alkaline solution at 60 °C for a period of up to three months (2160 h). At different intervals, the samples were removed from the alkaline solution and carefully washed with tap water to remove the excess alkaline solution before measuring their tensile strength and modulus of elasticity in tension. The tensile capacity retention was calculated according to Equation (3).etR = tensile capacity retention (%)tu0F = initial tensile capacity before immersion in alkaline solution, in Newtontu1F = tensile capacity after immersion in alkaline solution, in NewtonThe alkaline resistance of bamboo composite reinforcement in concrete was investigated according to ASTM D7705-12 “Standard Test Method for Alkali Resistance of Fiber Reinforced Polymer (FRP) Matrix Composite Bars used in Concrete Construction”. The tensile test samples were prepared according to ASTM D3039-08 and coated with the water-based epoxy coating before being subjected to an alkaline solution consisting of 118.5 g of Ca(OH)The Bamboo composite reinforcement system has different behavior when compared with steel reinforcement in terms of the physical and mechanical properties. The behavior of bamboo composite reinforcement systems is similar to typical FRP reinforcement that is produced with carbon fiber (CFRP) or glass fiber (GFRP). Bamboo composite materials display an elastic behavior up to the ultimate failure point and do not yield like steel.In general, bamboo composite reinforcement displays low stiffness when compared with traditional construction materials. Therefore, it is difficult to compete in the mass construction market, where, for instance, the deflections of the building can be critical for medium to high rise structures. Such structures would likely develop cracks due to the lower elastic modulus of the bamboo composite reinforcement. However, there are applications where deflections are not critical in the design, such as low rise, low cost housing solutions for developing countries where the demand for ductility is low and the failure of secondary elements will provide adequate warning of collapse.ACI 440.1R-15 “Guide for the design and construction of structural concrete reinforced with Fiber Reinforced Polymer (FRP) bars” has provided design guides for the application of FRP materials as reinforcement in concrete. These guides account for the lower ductility of the FRP reinforced concrete when compared with steel reinforced concrete members.Given the consideration of ductility by ACI 440.1R-15, this design guide was the primary and most relevant guide to be used in this study for evaluating the performance of bamboo composite reinforced concrete beams. The design guides provided by ACI 440.1R-15 are based on the concepts of equilibrium and compatibility . The ultimate strength design method was preferred over the working stress design approach to achieve comparable results with the methods employed by other standards, such as ACI 318 for steel reinforced concrete design, to design the concrete beams reinforced with bamboo composite reinforcement.The serviceability limit state could govern the design of concrete members reinforced with bamboo composite materials to prevent excessive deflections as a result of lower ductility given the lower modulus of elasticity of bamboo composite reinforcement compared to steel. Furthermore, ACI 440.1R-15 recommends applying reduction factors to the guaranteed tensile strength to include the effect of various environmental conditions on the properties of FRP reinforcement.In this study, a conservative reduction factor of 0.80, similar to that for GFRP reinforcement not exposed to earth or extreme weather conditions, has been used for designing the concrete beams for laboratory conditions. No reduction factor has been recommended by ACI 440.1R-15 for the modulus of elasticity, thus a similar reduction factor (0.80) has been used for the design modulus of elasticity of the bamboo composite reinforcement. A similar reduction factor (0.80) was also employed for the rupture strain to calculate the design rupture strain for the determination of the serviceability limit states of design. The average guaranteed rupture strain measured during the tensile tests was 1.35% for bamboo composite reinforcement.In the case of bamboo composite reinforced concrete member, no ductile behavior is observed when compared to steel reinforcement. Significant elastic elongation is expected for the concrete beams that were reinforced with bamboo composite reinforcements. According to ACI 440.1R-15 and depending on the reinforcement ratio, two modes of failure are expected for bamboo composite reinforced concrete members: bamboo composite reinforcement rupture and concrete crushing according. If the reinforcement ratio is less than the balanced reinforcement ratio, the bamboo composite reinforcement rupture occurs first; otherwise, concrete crushing controls the failure mode.fbf: is the design tensile strength of the bend portion of FRP reinforcement (MPa),br: is the radius of the bend (mm),bd: is the diameter of reinforcing bar (mm), andfuf: is the design tensile strength of FRP reinforcing bar after employing the reduction factor (MPa).ACI 440.1R-15 has recommended using the following formulae to calculate the design tensile strength of FRP bars at a bend and in this study for the bamboo composite stirrups.ff is the stress in reinforcement in tension in MPa and can be computed according to the recommendation of ACI 440.1R-15.The nominal flexural strength of FRP or bamboo composite reinforced concrete member can be calculated while using the following formula that was recommended by ACI 440.1R-15 only when the concrete crushing limit state controls the failure.In the case of bamboo composite reinforcement rupture mode of failure, the nominal flexural strength can be determined by using the following formulae as per the recommendation of ACI 440.1R-15.This formula involves the unknown parameter (c), which is the depth to the neutral axis of the concrete cross section. A minimum amount of reinforcement is required for the section during the design process to ensure adequate resistance is provided to the minimum tensile stresses developed as a result of external loadings to prevent sudden failure of the reinforced member due to concrete crushing. The minimum amount of reinforcement for FRP reinforced concrete members according can be found according to ACI 440.1R-15.crM) and evaluation of the flexural moment when the member has cracked after deflection, given the lower modulus of elasticity of FRP. ACI 440.1R-15 recommends using the cracked moment of inertia  to estimate the deflection of FRP reinforced concrete beams, according to the following formulae.aM is the maximum service load moment and ϒ accounts for the variation in stiffness along the length of the concrete member and it can be calculated by the following formulae.Given the variation in stiffness along the length of the reinforced concrete member, ACI 440.1R-15 recommends using an effective moment of inertia  between the stirrups along the reinforced concrete beam can be computed by following formulae.Furthermore, ACI 440.1R-15 recommends using the following to check the tensile strength of the FRP stirrups for shear design.The design and calculations of the concrete beams reinforced with bamboo composite reinforcement are carried out according to ACI 440.1R-15, as it is the only relevant design standard available for fiber reinforced composite materials used as reinforcement in concrete, as stated earlier for the purpose of this study.In this study, the cross section of the concrete beams was designed according to the dimensions of the produced bamboo composite reinforcement system and the limitation of the UTM and its capacity, as explained earlier. Closed-loop testing under deformation control was performed on bamboo composite beam having dimensions of 130 × 130 mm. A concrete cover of 15mm around the stirrups following the recommendations of ACI 440.1R-15 was chosen, which results in a concrete cross section of 160 × 160 mm, as shown in A total of 16 concrete beams were prepared based on three parameters affecting their design, including the number of longitudinal bars at the bottom of the beam, spacing of the stirrups, and the distance between the load introduction points in the middle section of the beam. The four-point loading set-up was used in this study to allow for a zero shear zone along the middle section of the bamboo composite reinforced concrete beam. For comparison purposes, four concrete beams without reinforcement were also tested in this study.Petung Putih bamboo, used in this study have been previously reported by the authors, as shown in Tensile and flexural properties, including strength and modulus of elasticity of the bamboo composite reinforcement, were measured according to ASTM D3039-08, while the compressive strength of the samples was obtained according to ASTM D6641. The average properties of the Dendrocalamus asper, known as The average mechanical properties of bamboo composite reinforcement materials are higher than the average mechanical properties of raw bamboo, except in the case of transverse reinforcement, where the tensile strength is slightly lower than the upper limit of the strength of the raw bamboo, as shown in As explained in earlier with regards to the fabrication of the stirrups, the inclined side legs of the stirrups received less pressure when compared with the bottom flat sections laid on the bottom side of the steel mold due to the wide angle chosen for the mold on the two sides due to the complexity of the shape required for the steel mold during the hot-press compression molding process. The reduced pressure on the inclined sides of the steel mold subsequently led to lower penetration and infusion of the epoxy matrix into the veneer layers. Thus, the interface between the epoxy and fibers was not completely developed to its full strength due to the lack of complete infusion of the epoxy matrix into the various layers of bamboo fibers. As a result, the weak interfacial bond strength between bamboo fibers and epoxy system subsequently led to lower mechanical properties. However, the results in In this study a two-component, water-based epoxy system was chosen in combination with sand particles to enhance the bonding mechanism between bamboo composite reinforcement and concrete, while an embedment length of 200mm was used following the earlier work that was carried out by the authors, as discussed in The results show that similar bond strength values were obtained through the techniques used in this study, as reported by . The bonWater absorption tests were carried out in accordance with ASTM D5229M-14 while the weight changes were recorded every 2 h during the first week of the experiment and, subsequently, measurements were only carried out once per day for up to three months’ immersion in water (2160 h). The water absorption of the bamboo composite reinforcement samples reached a maximum of 0.5% of the weight of the dry sample. The low rate of water absorption indicates the high resistance of the bamboo composite samples to water and moisture ingress, even at extreme conditions. A quasi-equilibrium state was achieved in both 23 °C and 60 °C temperatures after 170 h of immersion in water. However, the water absorption values for samples that were immersed in water at a temperature of 60 °C were slightly higher than samples that were immersed in room temperature water for a similar period of time. This difference could be attributed to the moisture diffusion behaviour into the composites, in which the polymer matrices are affected by the temperature. At higher temperatures, the diffusion rate tends to be influenced by the faster movement of the individual molecules when compared to lower temperatures; therefore, the moisture or water absorption would increase as a result of a higher diffusion rate.Nevertheless, water-based coating was applied on the surface of all reinforcements when used in concrete to minimise humidity infusion and water absorption effects on the integrity of the bamboo composite reinforcement. The water-based coating had two functions; firstly, enhancing the bonding strength of the bamboo composite reinforcement and, secondly, deterring the water from infusing into the bamboo composite reinforcement while in concrete.etR) for the tensile strength and modulus of elasticity of the samples. The reduction in both tensile properties followed a similar trend. The modulus of elasticity was shown to be more influenced by the exposure to alkaline solution over time. However, it has been observed that tensile properties reach a steady state after one month’s exposure to the alkaline solution.The alkaline resistance of bamboo composite reinforcement in concrete was investigated according to ASTM D7705-12. The tensile capacity retention was calculated according to Equation (3). A total of 16 concrete beams were prepared based on three parameters affecting their design, including the number of longitudinal bars at the bottom of the beam, the spacing of the stirrups, and the distance between the load introduction points in the middle section of the beam. The initial cracking load where the first crack was observed in the concrete beam started to increase by the increase in the number of bottom reinforcement while maintaining the same load introduction points. The lowest initial cracking load was observed for non-reinforced concrete beams and the largest initial cracking load was observed with beam design C samples with four reinforcement bars at the tension face of the concrete cross section and four stirrups on each side of the beams. It was observed that, by reducing the spacing between the stirrups, initial cracking and ultimate failure load were both improved. This is the result of the enhancement in shear span of the beam by preventing the shear failure and, thus, the bamboo composite longitudinal reinforcement could be activated up to its ultimate tensile capacity.In general, three modes of failure were observed: rupture of bamboo composite longitudinal reinforcement in tension, crushing of concrete in compression, and shear failure of the beams. In concrete beam series of “A” with a stirrup spacing of 70 mm and two bottom tension reinforcement, two concrete beams experienced shear failure, while two concrete beams had a concrete crushing mode of failure. By increasing the number of bottom longitudinal bars with similar spacing of stirrups or with an increase in spacing while maintaining the four bars at the bottom side of the beam the shear capacity was improved to the point that no shear failure was observed within the samples of concrete beams belonging to the “B”, “C”, and “D” series.The concrete crushing mode of failure was caused by the high amount of compressive stresses at the distance between the load introduction points. The concrete beams belonging to the “B”, “C”, and “D” series experienced crushing mode of failure in concrete. Furthermore, the concrete crushing was accompanied by the tensile rupture of the bamboo composite reinforcement at the tension face of the beam. The various spacing of stirrups that were used in this study and the failure modes observed showed the importance of stirrups in preventing the shear failure and the effectiveness of newly developed bamboo composite stirrups in enhancing the shear capacity of the concrete beams when used with appropriate spacing.Furthermore, when the maximum mid-span deflection of the beams was measured during the tests, it was observed that the mid-span deflection decreased by increasing the amount of tensile reinforcement A series of calculations were also carried out to estimate the failure load and deflection limits to compare the results obtained from the tests on the ultimate failure load and the maximum deflection as well as the mode of failure with the calculations specified in ACI 440.1R-15.Concrete beams from design “A” Series display the lowest failure load that was obtained both from the experiments and ACI design guide. However, the cracking loads obtained from the ACI design guide remain the same for beams from design “A”, “B”, and “C” series, while beams from design “D” series display the lowest ACI cracking load. This indicates the effect of reinforcement ratio on the ultimate failure load and the cracking load. Furthermore, the interaction of longitudinal reinforcement and stirrups helps to enhance the shear capacity of the bamboo composite reinforced concrete beams. The shear capacity of the beams plays an important role in determining the ultimate failure modes. The ACI 440.1R-15 standard enforces the required spacing of the stirrups to prevent any potential shear failure of the beam and to activate either concrete crushing or reinforcement rupture as the desirable modes of failure. Only beams from design “A” series have experienced shear failure mode while no shear failure was observed within the other design series, as described earlier.This finding points to the importance of the safety factors and strength reduction factors in the design and evaluation of bamboo composite reinforced concrete beams, which are in the scope of the ACI standard. The safety factors and strength reduction factors allow the engineer to include uncertainty in materials properties, including tensile and flexural strength. In the case of bamboo composite reinforcement, where a natural-based fiber (bamboo) composite material was employed as the main load-bearing element, the safety factors and strength reduction factors should also be incorporated in the design, similar to the recommendation that is given in ACI 440.1R-15 for FRP reinforcement.The deflection results, as shown in Flexural test results demonstrate the suitability of the ACI 440.1R-15 guidelines for the application of newly developed bamboo composite reinforcement in structural concrete beams.The results of the concrete beam test series carried out in this study validate the expected performance that has been indicated from small-specimen tests of tensile and pull-out properties of bamboo composite materials.The results of this study can be utilized for the design of low-cost and low-rise housing units where reinforcing steel is hard to obtain, where the demand for ductility is low, and where secondary-element failure provides adequate warning of collapse.Earlier studies on the application of natural bamboo from 1914 have indicated that raw bamboo has the potential for replacing steel in reinforced concrete beams, However, problems that are associated with durability have impeded the widespread use of bamboo in the construction industry. This research demonstrates the potential of the newly developed bamboo composite material for use as a new type of element for non-deflection-critical applications of reinforced structural-concrete members. Durability is greatly enhanced since fibers are embedded in epoxy. The results of this study lead to the following additional conclusions: